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| United States Patent Application |
20170355779
|
| Kind Code
|
A1
|
|
Wickman; Grant Raymond
; et al.
|
December 14, 2017
|
METHODS OF USING BISPECIFIC ANTIGEN-BINDING CONSTRUCTS TARGETING HER2
Abstract
Described herein methods of using antigen-binding constructs to treat
HER2+ tumors in a subject such as breast, lung, or head and neck tumors.
In some aspects, the tumor volume in the subject after receiving at least
seven doses of the antigen binding construct is less than the tumor
volume of a control subject receiving an equivalent amount of
trastuzumab. In some aspects, the survival of the subject receiving the
antigen binding construct is increased as compared to a control subject
receiving an equivalent amount of a non-specific control antibody or as
compared to a control subject not receiving treatment.
| Inventors: |
Wickman; Grant Raymond; (Vancouver, CA)
; Ng; Gordon Yiu Kon; (Vancouver, CA)
; Weisser; Nina E.; (Delta, CA)
|
| Applicant: | | Name | City | State | Country | Type |
Zymeworks Inc. |
Vancouver | |
CA |
| |
|---|
| Family ID:
|
56073259
|
| Appl. No.:
|
15/526888
|
| Filed:
|
November 26, 2015 |
| PCT Filed:
|
November 26, 2015 |
| PCT NO:
|
PCT/CA2015/051238 |
| 371 Date:
|
May 15, 2017 |
Related U.S. Patent Documents
| | | | |
|---|
|
| Application Number | Filing Date | Patent Number |
|---|
| | PCT/CA2014/051140 | Nov 27, 2014 | |
| | 15526888 | | |
| | 62166844 | May 27, 2015 | |
|
|
| Current U.S. Class: |
1/1 |
| Current CPC Class: |
C07K 2317/55 20130101; C07K 2317/622 20130101; A61K 39/395 20130101; C07K 2317/526 20130101; C07K 2317/77 20130101; C07K 2317/72 20130101; A61K 47/6879 20170801; C07K 2317/732 20130101; A61K 47/6803 20170801; C07K 16/32 20130101; C07K 2317/64 20130101; A61K 47/6869 20170801; C07K 2317/92 20130101; A61P 35/00 20180101; C07K 2317/31 20130101; C07K 2317/73 20130101; C07K 16/28 20130101; A61K 47/6855 20170801; C07K 2317/522 20130101; A61K 2039/505 20130101; C07K 2317/41 20130101; C07K 2317/24 20130101; C07K 2317/94 20130101; C07K 2317/76 20130101 |
| International Class: |
C07K 16/32 20060101 C07K016/32; A61K 39/395 20060101 A61K039/395; A61K 47/68 20060101 A61K047/68; C07K 16/28 20060101 C07K016/28 |
Claims
1. A method of treating a subject having a tumor; inhibiting, reducing or
blocking HER2 signaling; or killing or inhibiting the growth of a
HER2-expressing tumor cell, the method comprising administering an
effective amount of an antigen binding construct comprising: a first
antigen-binding polypeptide construct which monovalently and specifically
binds a HER2 (human epidermal growth factor receptor 2) ECD2
(extracellular domain 2) antigen on a HER2-expressing cell; a second
antigen-binding polypeptide construct which monovalently and specifically
binds a HER2 ECD4 (extracellular domain 4) antigen on a HER2-expressing
cell; first and second linker polypeptides, wherein the first linker
polypeptide is operably linked to the first antigen-binding polypeptide
construct, and the second linker polypeptide is operably linked to the
second antigen-binding polypeptide construct; wherein the linker
polypeptides are capable of forming a covalent linkage with each other,
wherein one or both of the first or the second antigen binding
polypeptide construct is an scFv, wherein the dissociation constant
(K.sub.D) of the antigen binding construct to murine HER2 extracellular
domain as measured by surface plasmon resonance (SPR) is equal to or less
than the dissociation constant of a monospecific anti-HER2 ECD4 antibody
(v506; SEQ ID NO:1 and SEQ ID NO:317) to murine HER2 extracellular domain
as measured by surface plasmon resonance (SPR), and wherein tumor growth
is decreased as compared to a control receiving an equivalent amount of a
non-specific control antibody, as compared to a control receiving an
equivalent amount of Herceptin/trastuzumab, or as compared to a control
not receiving treatment.
2. The method of claim 1 wherein the antigen binding construct comprises
the full length sequences set forth in SEQ ID NOs 97, 295, and 69
(v10000), and optionally wherein the dissociation constant (K.sub.D) of
the construct to murine HER2 extracellular domain as measured by surface
plasmon resonance (SPR) is approximately 0.6 nM.
3. A method of treating a subject having a tumor; inhibiting, reducing or
blocking HER2 signaling; or killing or inhibiting the growth of a
HER2-expressing tumor cell, the method comprising administering an
effective amount of an antigen binding construct comprising: a first
antigen-binding polypeptide construct which monovalently and specifically
binds a HER2 (human epidermal growth factor receptor 2) ECD2
(extracellular domain 2) antigen on a HER2-expressing cell, wherein the
first antigen-binding polypeptide construct comprises a first variable
light-chain (VL1) domain and a first variable heavy-chain (VH1) domain,
wherein the first antigen-binding polypeptide construct comprises VH1 and
VL1 CDR sequences that are at least 90, 91, 92, 93, 94, 95, 96, 97, 98,
or 99% identical to the VH1 and VL1 CDR sequences of v7091 (SEQ ID NOs
223, 225, 227, 37, 39, and 41), and wherein the VL1 domain comprises 1,
2, 3, 4, or 5 amino acid substitutions and/or the VH1 domain comprises 1,
2, 3, 4, or 5 amino acid substitutions; a second antigen-binding
polypeptide construct which monovalently and specifically binds a HER2
ECD4 (extracellular domain 4) antigen on a HER2-expressing cell; first
and second linker polypeptides, wherein the first linker polypeptide is
operably linked to the first antigen-binding polypeptide construct, and
the second linker polypeptide is operably linked to the second
antigen-binding polypeptide construct; wherein one or both of the first
or the second antigen binding polypeptide construct is an scFv, wherein
the linker polypeptides are capable of forming a covalent linkage with
each other, and wherein tumor growth is decreased as compared to a
control receiving an equivalent amount of a non-specific control
antibody, as compared to a control receiving an equivalent amount of
Herceptin/trastuzumab, or as compared to a control not receiving
treatment.
4. The method of any of the above claims, wherein the binding affinity of
the antigen binding construct to murine HER2 extracellular domain as
measured by surface plasmon resonance (SPR) is greater than the binding
affinity of v7091 (SEQ ID NOs 33, 219, and 295) to murine HER2
extracellular domain as measured by surface plasmon resonance (SPR),
optionally wherein the antigen binding construct and v7091 bind the same
epitope, optionally wherein the antigen binding construct binds the same
epitope as pertuzamab, optionally wherein the antigen binding construct
has a greater Bmax than v7091, and optionally wherein the antigen binding
construct is internalized to a greater extent upon cell surface binding
relative to v7091.
5. The method of any of the above claims, wherein the binding affinity of
the antigen binding construct to murine HER2 extracellular domain as
measured by surface plasmon resonance (SPR) is equal to or greater than
the binding affinity of a monospecific anti-HER2 ECD4 antibody (v506; SEQ
ID NO:1 and SEQ ID NO:317) to murine HER2 extracellular domain as
measured by surface plasmon resonance (SPR).
6. The method of any of the above claims, wherein the first
antigen-binding polypeptide construct comprises the VH1 and VL1 CDR
sequences of v7091 (SEQ ID NOs 223, 225, 227, 37, 39, and 41), wherein
the VL1 domain comprises 1, 2, 3, 4, or 5 amino acid substitutions and/or
the VH1 domain comprises 1, 2, 3, 4, or 5 amino acid substitutions,
optionally wherein the first antigen-binding polypeptide construct
comprises a substitution at Y96 in the VL1 domain (SEQ ID NO:35),
optionally wherein the first antigen-binding polypeptide construct
comprises a Y96A substitution in the VL1 domain (SEQ ID NO:35),
optionally wherein the first antigen-binding polypeptide construct
comprises substitutions at T30, A49, and/or L69 in the VH1 domain (SEQ ID
NO:221), optionally wherein the first antigen-binding polypeptide
construct comprises T30A, A49G, and/or L69F substitution(s) in the VH1
domain (SEQ ID NO:221), and optionally wherein the first antigen-binding
polypeptide construct comprises T30A, A49G, and L69F substitution(s) in
the VH1 domain (SEQ ID NO:221).
7. The method of any of the above claims, wherein the second
antigen-binding polypeptide construct comprises VH2 and VL2 CDR sequences
that are at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to
the VH2 and VL2 CDR sequences of v10000 (SEQ ID NOs 299, 301, 303, 307,
309, and 311), optionally wherein the second antigen-binding polypeptide
construct comprises the VH2 and VL2 CDR sequences of v10000 (SEQ ID NOs
299, 301, 303, 307, 309, and 311).
8. The method of any of the above claims, wherein the antigen binding
construct comprises the variable domain sequences set forth in SEQ ID NOs
71 and/or 99, the variable domain sequences set forth in SEQ ID NOs 297
and/or 305, or the variable domain sequences set forth in SEQ ID NOs 71,
99, 297, and 305.
9. The method of any of the above claims, wherein the antigen binding
construct comprises the full length sequence set forth in SEQ ID NO 97,
the full length sequence set forth in SEQ ID NO 295, the full length
sequence set forth in SEQ ID NO 69, or the full length sequences set
forth in SEQ ID NOs 97, 295, and 69 (v10000).
10. The method of any of the above claims, wherein the first and second
linker polypeptide each comprise an immunoglobulin hinge region
polypeptide selected from an IgG1, IgG2 or IgG4 hinge region.
11. The method of any of the above claims, wherein the first and second
linker polypeptides are operably linked to a scaffold, optionally an Fc.
12. The method of any of the above claims, wherein the first and second
linker polypeptides are operably linked to a dimeric Fc comprising first
and second Fc polypeptides each comprising a CH3 sequence, wherein the
first Fc polypeptide is operably linked to the first linker polypeptide
and the second Fc polypeptide is operably linked to the second linker
polypeptide.
13. The method of any of the above claims, wherein (i) the first antigen
binding polypeptide construct is an scFv and the second antigen binding
polypeptide construct is a Fab; or (ii) the first antigen binding
polypeptide construct is a Fab and the second antigen binding polypeptide
construct is an scFv; or (iii) both the first antigen binding polypeptide
construct and the second antigen binding polypeptide construct are scFvs.
14. The method of any of the above claims, wherein i. the first
antigen-binding polypeptide construct is a Fab and comprises a. a first
heavy chain variable polypeptide VH1 comprising the VH of the pertuzumab
arm of v5019, v 5020 v7091, v6717 or v10000 (SEQ ID NOs 221, 149, 221,
259, and 99, respectively), and b. a first variable light chain
polypeptide VL1 comprising the VL of the pertuzumab arm of v5019, v 5020
v7091, v6717 or v10000 (SEQ ID NOs 35, 35, and 71 for v5019, v7091, and
v10000, respectively); and the second antigen-binding polypeptide
construct is an scFv and comprises (a) a second variable heavy chain
polypeptide VH2 comprising the VH of the trastuzumab arm of v5019, v 5020
v7091, v6717 or v10000 (SEQ ID NOs 171, 205, 297, 171, and 297,
respectively), and (b) a second variable light chain polypeptide VL2
comprising the VL of the trastzumab arm of v5019, v 5020 v7091, v6717 or
v10000 (SEQ ID NO:35 for v5020); or ii. the first antigen-binding
polypeptide construct is an scFv and comprises (a) a first variable heavy
chain polypeptide VH1 comprising the VH of the pertuzumab arm of v5019, v
5020 v7091, v6717 or v10000 (SEQ ID NOs 221, 149, 221, 259, and 99,
respectively), and (b) a first variable light chain polypeptide VL1
comprising the VL of the pertuzumab arm of v5019, v 5020 v7091, v6717 or
v10000 (SEQ ID NOs 35, 35, and 71 for v5019, v7091, and v10000,
respectively), and the second antigen-binding polypeptide construct is an
Fab and comprises (a) a second heavy chain variable polypeptide VH2
comprising the VH of the trastuzumab arm of v5019, v 5020 v7091, v6717 or
v10000 (SEQ ID NOs 171, 205, 297, 171, and 297, respectively), and (b) a
second variable light chain polypeptide VL2 comprising the VL of the
trastuzumab arm of v5019, v 5020 v7091, v6717 or v10000 (SEQ ID NO:35 for
v5020); or iii. the first antigen-binding polypeptide construct is an
scFv and comprises (a) a first heavy chain variable polypeptide VH1
comprising the VH of the pertuzumab arm of v5019, v 5020 v7091, v6717 or
v10000 (SEQ ID NOs 221, 149, 221, 259, and 99, respectively), and (b) a
first variable light chain polypeptide VL1 comprising the VL of the
pertuzumab arm of v5019, v 5020 v7091, v6717 or v10000 (SEQ ID NOs 35,
35, and 71 for v5019, v7091, and v10000, respectively), and the second
antigen-binding polypeptide construct is an scFv and comprises (a) a
second heavy chain variable polypeptide VH2 comprising the VH of the
trastuzumab arm of v5019, v 5020 v7091, v6717 or v10000 (SEQ ID NOs 171,
205, 297, 171, and 297, respectively), and (b) a second variable light
chain polypeptide VL2 comprising the VL of the trastuzumab arm of v5019,
v 5020 v7091, v6717 or v10000 (SEQ ID NO:35 for v5020).
15. The method of any of the above claims, wherein the first
antigen-binding polypeptide construct is selected from: i. a polypeptide
construct comprising three VH CDR sequences comprising the amino acid
sequences SEQ ID NO: 335, SEQ ID NO:336 and SEQ ID NO:337, or SEQ ID
NO:335, SEQ ID NO:336, and SEQ ID NO:348; ii. a polypeptide construct
comprising three VH CDR sequences comprising amino acid sequences that
are at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the
three VH CDR sequences of SEQ ID NO: 335, SEQ ID NO:336 and SEQ ID
NO:337, or SEQ ID NO:335, SEQ ID NO:336, and SEQ ID NO:348; iii. a
polypeptide construct comprising three VL CDR sequences comprising the
amino acid sequences of the three VL CDR sequences of SEQ ID NO: 338, SEQ
ID NO:339 and SEQ ID NO:340, or SEQ ID NO:338, SEQ ID NO:347, and SEQ ID
NO:340; iv. a polypeptide construct comprising three VL CDR sequences
that are at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to
the amino acid sequences of the three VL CDR sequences are at least 90,
91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 338, SEQ
ID NO:339 and SEQ ID NO:340, or SEQ ID NO:338, SEQ ID NO:347, and SEQ ID
NO:340; v. a polypeptide construct comprising six CDR sequences
comprising the amino acid sequences of the six CDR sequences of SEQ ID
NO: 335, SEQ ID NO:336, SEQ ID NO:337, SEQ ID NO: 338, SEQ ID NO:339 and
SEQ ID NO:340; or SEQ ID NO:335, SEQ ID NO:336, SEQ ID NO:348, SEQ ID
NO:338, SEQ ID NO:347, and SEQ ID NO:340; or vi. a polypeptide construct
comprising six CDR sequences comprising the amino acid sequences that are
at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the six
CDR sequences of SEQ ID NO: 335, SEQ ID NO:336, SEQ ID NO:337, SEQ ID NO:
338, SEQ ID NO:339 and SEQ ID NO:340; or SEQ ID NO:335, SEQ ID NO:336,
SEQ ID NO:348, SEQ ID NO:338, SEQ ID NO:347, and SEQ ID NO:340; and the
second antigen-binding polypeptide is selected from vii. a polypeptide
construct comprising three VH CDR sequences comprising the amino acid
sequences of the three VH CDR sequences of SEQ ID NO: 341, SEQ ID NO:342
and SEQ ID NO:343; viii. a polypeptide construct comprising three VH CDR
sequences comprising amino acids sequences that are at least 90, 91, 92,
93, 94, 95, 96, 97, 98, or 99% identical to the three VH CDR sequences of
SEQ ID NO: 341, SEQ ID NO:342 and SEQ ID NO:343; ix. a polypeptide
construct comprising three VL CDR sequences comprising the amino acid
sequences of the three VL CDR sequences of SEQ ID NO: 344, SEQ ID NO:345
and SEQ ID NO:346; x. a polypeptide construct comprising three VL CDR
sequences that are at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%
identical to the amino acid sequences of the three VL CDR sequences of
SEQ ID NO: 344, SEQ ID NO:345 and SEQ ID NO:346; xi. a polypeptide
construct comprising six CDR sequences comprising the amino acid
sequences of the six CDR sequences of SEQ ID NO: 341, SEQ ID NO:342, SEQ
ID NO:343, SEQ ID NO: 344, SEQ ID NO:345 and SEQ ID NO:346; or xii. a
polypeptide construct comprising six CDR sequences comprising the amino
acid sequences that are at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or
99% identical to the six CDR sequences of SEQ ID NO: 341, SEQ ID NO:342,
SEQ ID NO:343, SEQ ID NO: 344, SEQ ID NO:345 and SEQ ID NO:346.
16. The method of any of the above claims wherein the first antigen
binding polypeptide construct: (i) blocks by 50% or greater the binding
of pertuzumab to ECD2, and/or (ii) the second antigen binding polypeptide
blocks by 50% or greater the binding of trastuzumab to ECD4.
17. The method according to any preceding claim wherein the first antigen
binding polypeptide construct comprises one of the v5019, v10000, v7091,
v5020 or v6717 antigen binding polypeptide constructs specific for HER2
ECD2, and the second antigen binding polypeptide construct comprises one
of the v5019, v10000, v7091, v5020 or v6717 antigen-binding polypeptide
constructs specific for HER2 ECD4.
18. The method according to any preceding claim, wherein the first
antigen-binding polypeptide construct comprises an amino acid sequence at
least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the v5019,
v10000, v7091, v5020 or v6717 antigen-binding polypeptide construct
specific for HER2 ECD2 and the second antigen-binding polypeptide
construct comprises an amino acid sequence at least 80%, 90%, 95%, 96%,
97%, 98%, or 99% identical to the v5019, v10000, v7091, v5020 or v6717
antigen-binding polypeptide construct specific for HER2 ECD4.
19. The method according to any preceding claim, selected from v5019,
v10000, v7091, v5020 and v6717.
20. The method according to any preceding claim, wherein the first
antigen binding polypeptide construct is an Fab and the second antigen
binding polypeptide construct is an scFv, and wherein the antigen binding
construct (i) induces increased receptor internalization in HER2 3+ cells
and/or (ii) displays higher potency in an ADCC (antibody directed
cellular cytotoxicity) assay against HER2 1+ cells, and/or (iii)
comprises one or more of the characteristics described in one or more of
the Examples, Tables, and Figures, as compared to a reference biparatopic
antigen binding construct having two Fabs.
21. The method according to according to any preceding claim, wherein the
first and second antigen binding polypeptide constructs are scFvs, and
wherein the antigen binding construct induces increased receptor
internalization in HER2 1+, 2+ and 3+ cells as compared to a reference
antigen binding construct having two Fabs.
22. The method according to any preceding claim, wherein the
antigen-binding construct comprises an Fc, optionally wherein the Fc is a
heterodimeric Fc.
23. The method according to any preceding claim, wherein the
antigen-binding construct comprises a heterodimeric Fc, wherein the
dimerized CH3 sequences have a melting temperature (Tm) of about 68, 69,
70, 71, 72, 73, 74, 75, 76, 77, 77.5, 78, 79, 80, 81, 82, 83, 84, or
85.degree. C. or higher.
24. The method according to any preceding claim, wherein the
antigen-binding construct comprises a heterodimeric Fc formed with a
purity greater than about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% when expressed.
25. The method according to any preceding claim, wherein the
antigen-binding construct comprises a heterodimeric Fc formed with a
purity greater than about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% when expressed via
a single cell.
26. The method according to any preceding claim, wherein the
antigen-binding construct comprises a heterodimeric Fc comprising one or
more modifications in at least one of the CH3 sequences.
27. The method according to any preceding claim, wherein the
antigen-binding construct comprises a heterodimeric Fc comprising one or
more modifications in at least one of the CH3 sequences that promote the
formation of a heterodimer with stability comparable to a wild-type
homodimeric Fc.
28. The method according to any preceding claim, wherein the
antigen-binding construct comprises: i. a heterodimeric IgG1 Fc having
the modifications L351Y_F405A_Y407V in the first Fc polypeptide, and the
modifications T366L_K392M_T394W in the second polypeptide; ii. a
heterodimeric IgG1 Fc having the modifications L351Y_F405A_Y407V in the
first Fc polypeptide, and the modifications T366L_K392L_T394W in the
second Fc polypeptide; iii. a heterodimeric IgG1 Fc having the
modifications T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and
the modifications T350V_T366L_K392L_T394W in the second Fc polypeptide;
iv. a heterodimeric IgG1 Fc having the modifications
T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the
modifications T350V_T366L_K392M_T394W in the second Fc polypeptide; v. a
heterodimeric IgG1 Fc having the modifications
T350V_L351Y_S400E_F405A_Y407V in the first Fc polypeptide, and the
modifications T350V_T366L_N390R_K392M_T394W in the second Fc polypeptide,
vi. a heterodimeric IgG1 Fc having the modifications
T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the
modifications T366I_N390R_K392M_T394W in the second Fc polypeptide; or
vii. a heterodimeric IgG1 Fc having the modifications
L351Y_S400E_F405A_Y405V in the first Fc polypeptide, and the
modifications T350V_T366L_K392L_T394W in the second Fc polypeptide;
according to EU numbering compared to a wild-type homodimeric Fc.
29. The method according to any preceding claim, wherein the
antigen-binding construct comprises a heterodimeric Fc comprising at
least one CH2 domain.
30. The method according to claim 29, wherein the CH2 domain(s) of the
heterodimeric Fc comprises one or more modifications.
31. The method according to any preceding claim, wherein the
antigen-binding construct comprises a heterodimeric Fc comprising one or
more modifications to promote selective binding of Fc-gamma receptors.
32. The method according to any preceding claim, wherein the
antigen-binding construct comprises at least one modification, and
wherein the modification is afucosylation.
33. The method according to any preceding claim, wherein the
antigen-binding construct is conjugated to a drug.
34. The method construct according to claim 33, wherein the drug is
maytansine (DM1).
35. The method according to claim 34, wherein the construct is conjugated
to DM1 through an SMCC linker.
36. The method according to any preceding claim, wherein the
antigen-binding construct is formulated in a pharmaceutical composition
with a pharmaceutical carrier.
37. The method claim 36, wherein the pharmaceutical carrier comprises a
buffer, an antioxidant, a low molecular weight molecule, a drug, a
protein, an amino acid, a carbohydrate, a lipid, a chelating agent, a
stabilizer, or an excipient.
38. The method according to any preceding claim, wherein the result of
the treatment is shrinking the tumor, inhibiting growth of the tumor,
increasing time to progression of the tumor, prolonging disease-free
survival of the subject, decreasing metastases, increasing the
progression-free survival of the subject, or increasing overall survival
of the subject.
39. The method according to any preceding claim, wherein the tumor
comprises cells that express an average of 10,000 or more copies of HER2
per tumor cell, optionally wherein the tumor is HER2 gene-amplified.
40. The method according to any preceding claim, wherein the tumor is
HER2 1+, HER2 2+ or HER2 3+ as determined by immunohistochemistry (IHC).
41. The method according to any preceding claim, wherein the tumor
expresses HER2 at a level of 2+ or lower as determined by IHC.
42. The method according to any preceding claim, wherein the HER2+ tumor
is a breast cancer that expresses HER2 at a 2+ level or lower, as
determined by immunohistochemistry (IHC).
43. The method according to any preceding claim, wherein the tumor is a
lung tumor, optionally wherein the tumor is a non-squamous non-small cell
lung tumor that is HER2-low, non-HER2 gene amplified.
44. The method of claim 43, wherein the tumor is HER3+.
45. The method of claim 43 or 44, wherein the tumor is EGFR low.
46. The method of claim 43, 44, or 45, wherein the tumor is moderately
sensitive to Cisplatin at the MTD.
47. The method according to any preceding claim, wherein the tumor is a
head and neck tumor, optionally wherein the tumor is a squamous cell
tumor of the head and neck that is HER2 low, non-HER2 gene amplified.
48. The method of claim 47, wherein the tumor is HER3+ low.
49. The method of claim 47 or 48, wherein the tumor is EGFR+.
50. The method of claim 47, 48, or 49, wherein the tumor is highly
sensitive to Cisplatin at the MTD.
51. The method according to any preceding claim, wherein the tumor is a
breast tumor, optionally wherein the tumor is a ER+/PR- breast cancer
with a luminal B molecular classification.
52. The method according to any preceding claim, wherein the tumor is a
pancreatic tumor, optionally wherein the pancreatic tumor is HER2
negative as determined by IHC.
53. The method according to any preceding claim, wherein the tumor is a
gastric tumor, optionally wherein the gastric tumor is HER2 3+.
54. The method according to any preceding claim, wherein the subject has
not previously been treated with an anti-HER2 antibody.
55. The method according to any preceding claim, wherein the tumor is
resistant or refractory to pertuzumab, trastuzumab and/or TDM1.
56. The method according to any preceding claim, wherein the subject has
previously been treated with pertuzumab, trastuzumab and/or TDM1.
57. The method of any one of claims 1-41, wherein the tumor is (i) a HER2
3+ estrogen receptor negative (ER-), progesterone receptor negative
(PR-), trastuzumab resistant, chemotherapy resistant invasive ductal
breast cancer, (ii) a HER2 3+ ER-, PR-, trastuzumab resistant
inflammatory breast cancer, (iii) a HER2 3+, ER-, PR-, invasive ductal
carcinoma or (iv) a HER2 2+ HER2 gene amplified trastuzumab and
pertuzumab resistant breast cancer.
58. The method any one of claims 1-41 wherein the tumor cell is a HER2 1+
or 2+ human pancreatic carcinoma cell, a HER2 3+ human lung carcinoma
cell, a HER2 2+ human Caucasian bronchioaveolar carcinoma cell, a human
pharyngeal carcinoma cell, a HER2 2+ human tongue squamous cell carcinoma
cell, a HER2 2+ squamous cell carcinoma cell of the pharynx, a HER2 1+ or
2+ human colorectal carcinoma cell, a HER2 3+ human gastric carcinoma
cell, a HER2 1+ human breast ductal ER+ (estrogen receptor-positive)
carcinoma cell, a HER2 2+/3+ human ER+, HER2-amplified breast carcinoma
cell, a HER2 0+/1+ human triple negative breast carcinoma cell, a HER2 2+
human endometrioid carcinoma cell, a HER2 1+ lung-metastatic malignant
melanoma cell, a HER2 1+ human cervix carcinoma cell, Her2 1+ human renal
cell carcinoma cell, or a HER2 1+ human ovary carcinoma cell.
59. The method of any one of claims 1-41 wherein the tumor cell is a HER2
1+ or 2+ or 3+ human pancreatic carcinoma cell, a HER2 2+ metastatic
pancreatic carcinoma cell, a HER2 0+/1+, +3+ human lung carcinoma cell, a
HER2 2+ human Caucasian bronchioaveolar carcinoma cell, a HER2 0+
anaplastic lung carcinoma, a human non-small cell lung carcinoma cell, a
human pharyngeal carcinoma cell, a HER2 2+ human tongue squamous cell
carcinoma cell, a HER2 2+ squamous cell carcinoma cell of the pharynx, a
HER2 1+ or 2+ human colorectal carcinoma cell, a HER2 0+, 1+ or 3+ human
gastric carcinoma cell, a HER2 1+ human breast ductal ER+ (estrogen
receptor-positive) carcinoma cell, a HER2 2+/3+ human ER+, HER2-amplified
breast carcinoma cell, a HER2 0+/1+ human triple negative breast
carcinoma cell, a HER2 0+ human breast ductal carcinoma (Basal B,
Mesenchymal-like triple negative) cell, a HER2 2+ER+ breast carcinoma, a
HER2 0+ human metastatic breast carcinoma cell (ER-, HER2- amplified,
luminal A, TN), a human uterus mesodermal tumor (mixed grade III) cell, a
2+ human endometrioid carcinoma cell, a HER2 1+ human skin epidermoid
carcinoma cell, a HER2 1+ lung-metastatic malignant melanoma cell, a HER2
1+ malignant melanoma cell, a human cervix epidermoid carcinoma vcell, a
HER2 1+ human urinary bladder carcinoma cell, a HER2 1+ human cervix
carcinoma cell, Her2 1+ human renal cell carcinoma cell, or a HER2 1+, 2+
or 3+ human ovary carcinoma cell, and wherein the antigen-binding
construct is conjugated to maytansine (DM1).
60. The method of any one of claims 1-41 wherein the tumor cell is
selected from a HER2 2/3+, gene amplified ovarian cancer cell, a HER2
0+/1+ triple negative breast cancer cell; an ER+, HER2 1+ breast cancer
cell; a trastuzumab resistant HER2 2+ breast cancer cell; an ER+, HER2+
breast cancer cell; or a HER2 3+ breast cancer cell.
61. The method according to any preceding claim, wherein the construct is
selected from v5019, v10000, v7091, v5020 or v6717.
62. The method according to any preceding claim, wherein administering is
done by injection or infusion, optionally wherein the administering is
intravenous.
63. The method according to any preceding claim, further comprising
administering to the subject an additional agent, optionally a
chemotherapeutic agent.
64. The method of claim 63, wherein the additional agent is one or more
of bleomycin, carboplatin, cisplatin, nab-paclitaxel, docetaxel,
doxorubicin, erlotinib, fluorouracil, gemcitabine, methotrexate,
pemetrexed, topotecan, vinorelbine, capecitabine, navelbine, or
paclitaxel.
65. The method of claim 63, wherein i. the tumor is non-small cell lung
cancer, and the additional agent is one or more of cisplatin,
carboplatin, paclitaxel, albumin-bound paclitaxel, nab-paclitaxel,
docetaxel, gemcitabine, vinorelbine, irinotecan, etoposide, vinblastine,
capecitabine, navelbine or pemetrexed; or ii. the tumor is head and neck
cancer, and the additional agent is one or more of paclitaxel,
carboplatin, doxorubicin or cisplatin; or iii. the tumor is a estrogen
and/or progesterone positive breast cancer, and the additional agent is
one or more of doxorubicin, epirubicin, paclitaxel, nab-paclitaxel,
docetaxel, fluorouracil, cyclophosphamide, carboplatin, letrozole,
mifepristone, capecitabine, gemcitabine, vinorelbine or tamoxifen; or iv.
the tumor is a pancreatic tumor and the additional agent is
nab-paclitaxel, capecitabine, gemcitabine, navelbine or paclitaxel.
66. The method according to any preceding claim, wherein the subject is a
human.
67. The method according to any preceding claim, wherein the method
comprises inhibiting, reducing or blocking HER2 signaling.
68. The method according to any preceding claim, wherein the method
comprises killing or inhibiting the growth of a HER2-expressing tumor
cell.
69. The method according to any preceding claim, wherein the subject is
administered at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, or 20 doses.
70. The method according to any preceding claim, wherein the amount of at
least one of the plurality of doses is at least 0.3, 0.5, 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/kg.
71. The method according to any preceding claim, wherein the amount of
each of the plurality of doses is at least 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/kg.
72. The method according to any preceding claim, wherein each dose is
administered at least daily, weekly, or monthly.
73. The method according to any preceding claim, wherein each dose is
administered at least every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20 days.
74. The method according to any preceding claim, wherein treatment
continues for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days;
at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, or 20 weeks; or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20 months.
75. The method according to any preceding claim, wherein the mean tumor
volume in the subject after receiving at least 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 doses is less than the mean
tumor volume of a control subject receiving an equivalent amount of
trastuzumab.
76. The method according to any preceding claim, wherein overall survival
of the subject is significantly increased as compared to a control
subject receiving an equivalent amount of a non-specific control antibody
or as compared to a control subject not receiving treatment; or wherein
the growth of tumor is significantly decreased as compared to a control
subject receiving an equivalent amount of a non-specific control
antibody, as compared to a control subject receiving an equivalent amount
of Herceptin, or as compared to a control subject not receiving
treatment.
77. The method of claim 76, wherein the significance is measured by a log
rank test.
78. The method of claim 76, wherein the p value is less than 0.5, 0.01,
or 0.001.
79. The method according to any preceding claim, wherein overall survival
of the subject is more significantly increased as compared to a control
subject receiving an equivalent amount of trastuzumab.
80. The method of claim 79, wherein the antigen-binding construct p value
is less than 0.001 and wherein the trastuzumab p value is greater than
0.001.
81. The method according to any preceding claim, wherein the p value of
the significance of the increase relative to the control subject
receiving an equivalent amount of a non-specific control antibody is less
than the p value of an increase in survival of a second control receiving
an equivalent amount of trastuzumab as compared to the control subject
receiving an equivalent amount of a non-specific control antibody.
82. The method of claim 81, wherein the antigen-binding construct p value
is less than 0.001 and wherein the trastuzumab p value is greater than
0.001.
83. The method according to any preceding claim, wherein overall survival
of the subject after receiving a combination of the antigen-binding
construct and an additional agent is significantly increased as compared
to a control subject receiving an equivalent amount of trastuzumab alone.
84. The method according to any preceding claim, wherein overall survival
of the subject is significantly increased as compared to a control
subject receiving a lesser amount of trastuzumab.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of PCT/CA2014/051140, filed
Nov. 27, 2014, and 62/166,844, filed May 27, 2015; each of which is
herein incorporated by reference, in its entirety, for all purposes.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which will be
submitted via EFS-Web and is hereby incorporated by reference in its
entirety. Said ASCII copy, created on Nov. 24, 2015, is named
32565PCT_sequencelisting.txt, and is 275,091 bytes in size.
BACKGROUND
[0003] The majority of current marketed antibody therapeutics are bivalent
monospecific antibodies optimized and selected for high affinity binding
and avidity conferred by the two antigen-binding domains. Afucosylation
or enhancement of FcgR binding by mutagenesis have been employed to
render antibodies more efficacious via antibody Fc dependent cell
cytotoxicity mechanisms. Afucyosylated antibodies or antibodies with
enhanced FcgR binding still suffer from incomplete therapeutic efficacy
in clinical testing and marketed drug status has yet to be achieved for
any of these antibodies. Typical bivalent antibodies conjugated to toxins
(antibody drug conjugates) are more efficacious but broader clinical
utility is limited by dose-limiting toxicity.
[0004] Therapeutic antibodies would ideally possess certain minimal
characteristics, including target specificity, biostability,
bioavailability and biodistribution following administration to a subject
patient, and sufficient target binding affinity and high target occupancy
to maximize antibody dependent therapeutic effects. Typically therapeutic
antibodies are monospecific. Monospecific targeting however does not
address other target epitopes that may be relevant in signaling and
disease pathogenesis, allowing for drug resistance and escape mechanism.
Some of the current therapeutic paradigms call for the use of combination
of two therapeutic monospecific antibodies targeting two different
epitopes of the same target antigen. One example is the use of a
combination of Trastuzumab and Pertuzumab, both targeting the HER2
receptor protein on the surface of some cancer cells, but patients still
progress with disease while others with lower HER2 receptor levels (HER2
<3+ by Hercept test) show no therapeutic benefit. Therapeutic
antibodies targeting HER2 are disclosed in WO 2012/143523 to GenMab and
WO 2009/154651 to Genentech. Antibodies are also described in WO
2009/068625 and WO 2009/068631.
[0005] Co-owned patent application number PCT/CA2014/051140 describes HER2
antibodies. Co-owned patent application number PCT/US2014/037401 (WO
2014/182970) describes HER2 antibodies. Co-owned patent application
number PCT/CA2013/050358 (WO 2013/166604) describes single arm monovalent
antibodies. Co-owned patent applications PCT/CA2011/001238, filed Nov. 4,
2011, PCT/CA2012/050780, filed Nov. 2, 2012, PCT/CA2013/00471, filed May
10, 2013, and PCT/CA2013/050358, filed May 8, 2013 describe therapeutic
antibodies. Each is hereby incorporated by reference in their entirety
for all purposes.
SUMMARY
[0006] Described herein are methods of using one or more antigen-binding
constructs to treat tumors in a subject, e.g., such as gastric,
pancreatic, breast, lung, or head and neck tumors. The one or more
antigen-binding constructs can comprise a first antigen-binding
polypeptide construct which monovalently and specifically binds a HER2
(human epidermal growth factor receptor 2) ECD2 (extracellular domain 2)
antigen on a HER2-expressing cell and a second antigen-binding
polypeptide construct which monovalently and specifically binds a HER2
ECD4 (extracellular domain 4) antigen on a HER2-expressing cell, first
and second linker polypeptides, wherein the first linker polypeptide is
operably linked to the first antigen-binding polypeptide construct, and
the second linker polypeptide is operably linked to the second
antigen-binding polypeptide construct; wherein the linker polypeptides
are capable of forming a covalent linkage with each other, wherein at
least one of the ECD2- or the ECD4-binding polypeptide constructs is an
scFv. In certain embodiments, the ECD2-binding polypeptide construct is
an scFv, and the ECD2-binding polypeptide construct is a Fab. In certain
embodiments, the ECD2-binding polypeptide construct is a Fab and the ECD4
binding polypeptide construct is an scFv. In some embodiments, both the
ECD2- and ECD4-binding polypeptide constructs are scFvs. In some
embodiments, the antigen-binding constructs have a dimeric Fc comprising
a CH3 sequence. In some embodiments, the Fc is a heterodimer having one
or more modifications in the CH3 sequence that promote the formation of a
heterodimer with stability comparable to a wild-type homodimeric Fc. In
some embodiments, the heterodimeric CH3 sequence has a melting
temperature (Tm) of 68.degree. C. or higher.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1A depicts the structure of a biparatopic antibody in a
Fab-Fab format. FIGS. 1B to 1E depict the structure of possible versions
of a biparatopic antibody in an scFv-Fab format. In FIG. 1B,
antigen-binding domain 1 is an scFv, fused to Chain A, while
antigen-binding domain 2 is a Fab, fused to Chain B. In FIG. 1C,
antigen-binding domain 1 is a Fab, fused to Chain A, while
antigen-binding domain 2 is an scFv, fused to Chain B. In FIG. 1D,
antigen-binding domain 2 is a Fab, fused to Chain A, while
antigen-binding domain 1 is an scFv, fused to Chain B. In FIG. 1E,
antigen-binding domain 2 is an scFv, fused to Chain A, while
antigen-binding domain 1 is a Fab, fused to Chain B. In FIG. 1F, both
antigen-binding domains are scFvs.
[0008] FIG. 2 depicts the characterization of expression and purification
of exemplary anti-HER2 biparatopic antibodies. FIG. 2A and FIG. 2B depict
the SEC chromatograph of the protein A purified antibody, and
non-reducing SDS-PAGE analysis of 10 L expression and purification of
v5019. FIG. 2C depicts the SDS-PAGE analysis of a 25 L expression and
purification of v10000.
[0009] FIG. 3 depicts the results of UPLC-SEC analysis of exemplary
anti-HER2 biparatopic antibodies purified by protein A and SEC. FIG. 3A
shows the results for v5019, where the upper panel shows the results of
the purification and the lower panel shows the same result with an
expanded scale for the y-axis. A summary of the data obtained is provided
below the UPLC-SEC results. FIG. 3B shows the results for v10000.
[0010] FIG. 4 depicts LCMS analysis of the heterodimer purity of exemplary
anti-HER2 biparatopic antibodies. FIG. 4A depicts results from LC-MS
analysis of the pooled SEC fractions of v5019. FIG. 4B depicts the
results from LC-MS analysis of the pooled protein A fractions of v10000.
[0011] FIG. 5 depicts analysis of a 25 L-scale preparation of an exemplary
anti-HER2 biparatopic antibody. FIG. 5A depicts the SDS-PAGE profile of
an exemplary anti-HER2 biparatopic following MabSelect.TM. and HiTrap.TM.
SP FF purification. FIG. 5B depicts LCMS analysis of the purified
antibody.
[0012] FIG. 6 compares the ability of an exemplary biparatopic anti-HER2
antibodies to bind to HER2+ whole cells displaying different HER2
receptor density compared to control antibodies, as measured by FACS.
FIG. 6A and FIG. 6E depict binding to SKOV3 cells;
[0013] FIG. 6B depicts binding to JIMT1 cells; FIG. 6C and FIG. 6F depict
binding to MCF7 cells; FIG. 6D depicts binding to MDA-MB-231 cells; and
FIG. 6G depicts binding to WI-38 cells.
[0014] FIG. 7 depicts the ability of exemplary anti-HER2 biparatopic
antibodies to inhibit the growth of HER2+ cells. FIG. 7A and FIG. 7D
shows growth inhibition in SKOV3 cells; FIG. 7B shows growth inhibition
in BT-474 cells; FIG. 7C shows growth inhibition in SKBR3 cells, and FIG.
7E shows growth inhibition in JIMT-1 cells.
[0015] FIG. 8 depicts the SPR binding data relating to the paratopes of an
exemplary anti-HER2 biparatopic antibodies. FIG. 8A illustrates the
K.sub.D values (nM) of a monovalent anti-Her2 antibody (v1040;
representing the antigen-binding domain on CH--B of exemplary anti-Her2
biparatopic antibody), for binding to immobilized Her2 ECD or dimeric
Her2-Fc. FIG. 8B illustrates the K.sub.D values (nM) of a monovalent
anti-Her2 antibody (v4182; representing the antigen-binding domain on
CH-A of exemplary anti-Her2 biparatopic antibody) for binding to
immobilized Her2 ECD or dimeric Her2-Fc.
[0016] FIG. 9 depicts the ability of exemplary anti-HER2 biparatopic
antibody to internalize in HER2+ cells. FIG. 9A depicts internalization
in BT-474 cells, while FIG. 9b depicts internalization in JIMT-1 cells.
[0017] FIG. 10 depicts surface binding and internalization of exemplary
anti-HER2 biparatopic antibodies. FIG. 10A (v5019) depicts the result in
BT-474 cells; FIG. 10B (v5019) and FIG. 10F (v5019 and v10000) depict the
result in JIMT1 cells; FIG. 10C (v5019) and FIG. 10E (v5019 and v10000)
depict the result in SKOV3 cells, and FIG. 10D (v5019) depicts the result
in MCF7 cells.
[0018] FIG. 11 depicts the ability of an exemplary anti-HER2 biparatopic
antibody to mediate ADCC in SKOV3 cells. In FIG. 11A, the assay was
carried out using an effector to target cell ratio of 5:1; in FIG. 11B,
the assay was carried out using an effector to target cell ratio of 3:1;
and in FIG. 11C, the assay was carried out using an effector to target
cell ratio of 1.1.
[0019] FIG. 12 depicts the characterization of affinity and binding
kinetics of monovalent anti-HER2 (v630 and v4182) and an exemplary
biparatopic anti-Her2 antibody (v5019) to recombinant human HER2. FIG.
12A shows the measurement of ka (1/Ms). FIG. 12B shows the measurement of
kd (1/s). FIG. 12C shows the measurement of K.sub.D (M).
[0020] FIG. 13 depicts affinity and binding characteristics of an
exemplary biparatopic anti-HER2 antibody to recombinant human HER2 over a
range of antibody capture levels. FIG. 13A depicts the measurement of kd
(1/s) to HER2 ECD determined over a range of antibody capture levels for
exemplary biparatopic anti-Her2 antibody (v5019). FIG. 13B depicts the
measurement of kd (1/s) to HER2 ECD determined over a range of antibody
capture levels for monovalent anti-Her2 antibody (v4182). FIG. 13C
depicts the measurement of kd (1/s) to HER2 ECD determined over a range
of antibody capture levels for monovalent anti-Her2 antibody (v630).
[0021] FIG. 14 shows a comparison of the mechanism of binding of a
monospecific anti-ECD4 HER2 antibody (left), and a Fab-scFv biparatopic
anti-ECD2.times.ECD4 HER2 antibody (right). The monospecific anti-ECD4
HER2 antibody is capable of binding one antibody molecule to two HER2
molecules; whereas the biparatopic anti-ECD2.times.ECD4 HER2 antibody is
capable of binding one antibody to two HER2 molecule, as well as 2
antibodies to one HER2 molecule and combinations therein which results in
HER2 receptor cross-linking and lattice formation followed by downstream
biological effects such as internalization and/or growth inhibition as
indicated by the arrows. IEC represents "immune effector cells." The four
extracellular domains of HER2 are numbered as 1, 2, 3, or 4 where 1=ECD1,
2=ECD2, 3=ECD3, and 4=ECD4.
[0022] FIG. 15 depicts the effect of an exemplary anti-HER2 biparatopic
antibody on AKT phosphorylation in BT-474 cells.
[0023] FIG. 16 depicts the effect of an exemplary anti-HER2 biparatopic
antibody on cardiomyocyte viability. FIG. 16A depicts the effect of v5019
and the corresponding ADC v6363 on cardiomyocyte viability; FIG. 16B
depicts the effect of v5019, v7091, and v10000 and corresponding ADCs
v6363, 7148, 10553 on cardiomyocyte viability, and FIG. 16C depicts the
effect of v5019, v7091, and v10000 and corresponding ADCs v6363, 7148,
10553 on the viability of doxorubicin-pretreated cardiomyocytes.
[0024] FIG. 17 depicts the ability of exemplary anti-HER2 biparatopic
antibody drug conjugates to inhibit the growth of HER2+ cells. FIG. 17A
shows the ability of the ADC v6363 to inhibit the growth of JIMT1 cells.
FIG. 17B shows the ability of the ADC v6363 to inhibit the growth of
SKOV3 cells. FIG. 17C shows the ability of the ADC v6363 to inhibit the
growth of MCF7 cells. FIG. 17D shows the ability of the ADC v6363 to
inhibit the growth of MDA-MB-231 cells. FIG. 17E shows the ability of
ADCs v6363, v10553, and v1748 to inhibit the growth of SKOV3 cells. FIG.
17F shows the ability of ADCs v6363, v10553, and v1748 to inhibit the
growth of JIMT-1 cells. FIG. 17G shows the ability of ADCs v6363, v10553,
and v1748 to inhibit the growth of NCI-N87 cells.
[0025] FIG. 18 depicts the effect of a biparatopic anti-HER2 antibody in a
human ovarian cancer line xenograft model (SKOV3). FIG. 18A shows the
effect of the antibody on mean tumor volume. FIG. 18B shows the effect of
the antibody on percent survival of the animals.
[0026] FIG. 19 depicts the effect of a biparatopic anti-HER2 antibody drug
conjugate (ADC) in a human ovarian cancer line xenograft model (SKOV3).
FIG. 19A shows the effect of the antibody on mean tumor volume. FIG. 19B
shows the effect of the antibody on percent survival of the animals.
[0027] FIG. 20 depicts the effect of a biparatopic anti-HER2 antibody drug
conjugate (ADC) on mean tumour volume in a human breast primary cell
xenograft model (HBCx-13b).
[0028] FIG. 21 depicts the effect of a biparatopic anti-HER2 antibody drug
conjugate (ADC) on mean tumour volume in a human breast primary cell
xenograft model (T226).
[0029] FIG. 22 depicts the effect of a biparatopic anti-HER2 antibody drug
conjugate (ADC) on mean tumour volume in a human breast primary cell
xenograft model (HBCx-5).
[0030] FIG. 23 depicts the effect of a biparatopic anti-HER2 antibody drug
conjugate (ADC) on anti-HER2 treatment resistant tumors in a human cell
line xenograft model (SKOV3).
[0031] FIG. 24 depicts the effect of a biparatopic anti-HER2 antibody drug
conjugate (ADC) to anti-HER2 treatment resistant tumors in human primary
cell xenograft model (HBCx-13b).
[0032] FIG. 25 depicts the thermal stability of exemplary anti-HER2
biparatopic antibodies. FIG. 25A depicts the thermal stability of v5019.
FIG. 25B depicts the thermal stability of v10000. FIG. 25C depicts the
thermal stability of v7091.
[0033] FIG. 26 depicts the thermal stability of exemplary anti-HER2
biparatopic antibody drug conjugates. FIG. 26A depicts the thermal
stability of v6363. FIG. 26B depicts the thermal stability of v10553.
FIG. 26C depicts the thermal stability of v7148.
[0034] FIG. 27 depicts the ability of anti-HER2 biparatopic antibodies to
mediate ADCC in HER2+ cells. The legend shown in FIG. 27C applies to FIG.
27A and FIG. 27B. FIG. 27A depicts this ability in SKBR3 cells; FIG. 27B
depicts this ability in JIMT-1 cells; FIG. 27C depicts this ability in
MDA-MB-231 cells; and FIG. 27D depicts this ability in WI-38 cells.
[0035] FIG. 28 depicts the effect of afucosylation on the ability of
anti-HER2 biparatopic antibodies to mediate ADCC. The legend shown in
FIG. 28B applies to FIG. 28A as well. FIG. 28A compares the ability of an
afucosylated version of v5019 to mediate ADCC to that of Herceptin.TM. in
SKOV3 cells. FIG. 28B compares the ability of an afucosylated version of
v5019 to mediate ADCC to that of Herceptin.TM. in MDA-MB-231 cells.
[0036] FIG. 28C compares the ability of v10000 and an afucosylated version
of v10000 to mediate ADCC against that of Herceptin.TM. in ZR-75-1 cells.
[0037] FIG. 29 depicts the ability of v5019 to inhibit growth of BT-474
cells in the presence or absence of growth-stimulatory ligands.
[0038] FIG. 30 depicts the effect of an afucosylated version of v5019
(v7187) on tumor volume in a human breast cancer xenograft model
(HBCx13B).
[0039] FIG. 31 depicts the ability of anti-HER2 biparatopic antibodies and
anti-HER2 biparatopic-ADCs to bind to HER2+ tumor cells. FIG. 31A
compares the binding of v6363 to a T-DM1 analog, v6246, in SKOV3 cells.
FIG. 31B compares the binding of v6363 to a T-DM1 analog, v6246, in
JIMT-1 cells. FIG. 31C compares the binding of several exemplary
anti-HER2 biparatopic antibodies and anti-HER2 biparatopic-ADCs to
controls, in SKOV3 cells. FIG. 31D compares the binding of several
exemplary anti-HER2 biparatopic antibodies and anti-HER2 biparatopic-ADCs
to controls, in JIMT-1 cells.
[0040] FIG. 32 depicts Dose-Dependent Tumour Growth Inhibition of an
exemplary anti-HER2 biparatopic-ADC in a HER2 3+ (ER-PR negative) patient
derived xenograft model (HBCx13b). FIG. 32A shows the effect of v6363 on
tumor volume, while FIG. 32B shows the effect on percent survival.
[0041] FIG. 33 depicts the effect of Biparatopic anti-HER2-ADC v6363
compared to Standard of Care Combinations in a Trastuzumab Resistant PDX
HBCx-13b xenograft model. FIG. 33A depicts the effect of treatment on
tumor volume, while FIG. 33B depicts the effect of treatment on survival.
[0042] FIG. 34 depicts the efficacy of a biparatopic anti-HER2-ADC in
HER2+ trastuzumab-resistant breast cancer cell derived tumour xenograft
model (JIMT-1).
[0043] FIG. 35 depicts the efficacy of exemplary anti-HER2 biparatopic
antibodies in vivo in a trastuzumab sensitive ovarian cancer cell derived
tumour xenograft model (SKOV3). FIG. 35A depicts the effect of treatment
on tumor volume, while FIG. 35B depicts the effect of treatment on
survival.
[0044] FIG. 36 depicts the dose-dependent efficacy of exemplary anti-HER2
biparatopic antibodies in vivo in a trastuzumab sensitive ovarian cancer
cell derived tumour xenograft model (SKOV3).
[0045] FIG. 37 depicts the ability of an anti-HER2 biparatopic antibody
and an anti-HER2 biparatopic-ADC to inhibit growth of cell lines
expressing HER2, and EGFR and/or HER3 at the 3+, 2+ or 1+ levels. FIG.
37A depicts the ability of v10000 to inhibit growth selected cell lines.
FIG. 37B depicts the ability of v10553 to inhibit growth of selected cell
lines.
[0046] FIG. 38 depicts a summary of the ability of v10000 and v10553 to
inhibit growth in a panel of cell lines. Hyphenated values (e.g. 1/2)
indicate discrepant erbb receptor levels as reported in the literature;
Erbb IHC values were obtained internally or from the literature. Where no
value is reported the receptor quantities are unknown and/or not
reported. * IHC level estimate based on erBb2 gene expression data (Crown
BioSciences). Numbered references are described below.
[0047] FIG. 39 depicts the ability of v10000 to mediate ADCC in HER2+
cells. FIG. 39A depicts the results in FaDu cells. FIG. 39B depicts the
results in A549 cells. FIG. 39C depicts the results in BxPC3 cells. FIG.
39D depicts the results in MiaPaca2 cells.
[0048] FIG. 40 depicts the ability of anti-HER2 biparatopic antibodies to
mediate ADCC in HER2+ cells. FIG. 40A depicts the results in A549 cells.
FIG. 40B depicts the results in NCI-N87 cells. FIG. 40C depicts the
results in HCT-116 cells.
[0049] FIG. 41 depicts the effect of anti-HER2 biparatopic antibody format
on binding HER2+ cells. FIG. 41A depicts the effect of format on binding
to BT-474 cells. FIG. 41B depicts the effect of format on binding to
JIMT-1 cells. FIG. 41C depicts the effect of format on binding to MCF7
cells. FIG. 41D depicts the effect of format on binding to MDA-MB-231
cells.
[0050] FIG. 42 depicts the effect of anti-HER2 biparatopic antibody format
on internalization of antibody in HER2+ cells. FIG. 42A depicts the
effect on internalization in BT-474 cells. FIG. 42B depicts the effect on
internalization in JIMT-1 cells. FIG. 42C depicts the effect on
internalization in MCF7 cells.
[0051] FIG. 43 depicts the effect of anti-HER2 biparatopic antibody format
on the ability to mediate ADCC in HER2+ cells. FIG. 43A depicts the
effect in JIMT-1 cells. FIG. 43B depicts the effect in MCF7 cells. FIG.
43C depicts the effect in HER2 0/1+ MDA-MB-231 breast tumor cells.
[0052] FIG. 44 depicts the effect of anti-HER2 biparatopic antibody format
on the ability of the antibodies to inhibit HER2+ tumor cell growth in
BT-474 cells in the presence or absence of growth-stimulatory ligands.
[0053] FIG. 45 depicts the effect of anti-HER2 biparatopic antibody format
on the ability of the antibodies to inhibit growth of SKBR3 cells.
[0054] FIG. 46 depicts the effect of anti-HER2 biparatopic antibody format
on the ability of antibodies to inhibit growth of HER2+ tumor cells. FIG.
46A depicts growth inhibition in SKOV3 cells. FIG. 46B depicts growth
inhibition in JIMT-1 cells. FIG. 46C depicts growth inhibition in MCF7
cells.
[0055] FIG. 47 depicts a comparison of binding characteristics of
anti-HER2 biparatopic antibodies of differing format as measured by SPR.
FIG. 47A depicts the plot and linear regression analysis for the kd (1/s)
at different antibody capture levels with v6903 and v7091. FIG. 47B
depicts the plot and linear regression analysis for the KD (M) at
different antibody capture levels with v6903 and v7091.
[0056] References found in FIG. 38 are as follows: 1. Labouret et al.
2012, Neoplasia 14:121-130; 2. Ghasemi et al. 2014, Oncogenesis
doi:10.1038/oncsis.2014.31; 3. Gaborit et al. 2011 J Bio Chem,
286:1133-11345; 4. Kimura et al. 2006, Clin Cancer Res; 12:4925-4932; 5.
Komoto et al. 2009, Canc Sci; 101:468-473; 6. Cretella et al. 2014,
Molecular Cancer 13:143-155; 7. Bunn et al. 2001, Clin Cancer Res;
7:3239-3250; 8. Lewis Phillips et al. 2013, Clin Cancer Res, 20:456-468;
9. McDonagh et al. 2012, 11:582-593; 10. Coldren et al. 2006, Mol Cancer
Res: 521-528; 11. Cavazzoni et al. 2012 Mol Cancer, 11:91-115; 12. Li et
al. 2014, Mol Cancer Res, doi:10.1158/1541-7786.MCR-13-0396; 13.
Chmielewski et al. 2004, Immunology, 173:7647-7653; 14. Kuwada et al.
2004, Int J Cancer, 109:291-301; 15. Fujimoto-Ouchi et al. 2007, Clin
Chemother Pharmacol, 59:795-805; 16. Chavez-Blanco et al. 2004, BMC
Cancer, 4:59; 17. Campiglio et al. 2004, J Cellular Physiology.
198:259-268; 18. Lehmann et al. 2011, J Clin Investigation,
121:2750-2767; 19. Collins et al. 2011, Annals Oncology, 23:1788-1795;
20. Takai et al. 2005, Cancer, 104:2701-2708; 21. Rusnack et al. 2007,
Cell Prolif, 40:580-594; 22. Ma et al. 2013, PLOS ONE, 8:e73261-e73261;
23. Meira et al. 2009, British J Cancer, 101:782-791; 24. Hayashi MP28-14
poster; 25. Wang et al. 2005 J Huazhong Univ Sci Technolog Med Sci.
25:326-8; 26. Makhja et al. 2010. J Clinc Oncolo 28:1215-1223.
[0057] FIG. 48A-B depicts the effect of a biparatopic anti-HER2 antibody
in a xenograft model of HER2-low, non-small cell lung cancer. FIG. 48A
shows the effect of the antibody on tumor volume. FIG. 48B shows the
effect of the antibody on percent survival of the animals.
[0058] FIG. 49A-B depicts the effect of a biparatopic anti-HER2 antibody
in a xenograft model of HER2-low, head and neck squamous cell carcinoma.
FIG. 49A shows the effect of the antibody on tumor volume. FIG. 49B shows
the effect of the antibody on percent survival of the animals.
[0059] FIG. 50A-B depicts the effect of a biparatopic anti-HER2 antibody
in a xenograft model of HER2-low, ER+ breast cancer. FIG. 50A shows the
effect of the antibody on tumor volume. FIG. 50B shows the effect of the
antibody on percent survival of the animals.
[0060] FIG. 51A-B shows tumor volume and survival in a xenograft model of
pancreatic cancer.
[0061] FIG. 52 shows tumor volume in a xenograft model of gastric cancer.
DETAILED DESCRIPTION
[0062] Described herein are methods of using bispecific antigen-binding
constructs that bind HER2.
Antigen-Binding Constructs
[0063] Provided herein are antigen-binding constructs, e.g., antibodies,
that bind HER2. The antigen-binding constructs include at least one
antigen-binding polypeptide construct binding a HER2 ECD2 antigen. In
some embodiments, antigen-binding constructs include a second
antigen-binding polypeptide construct binding a second antigen, e.g., a
HER2 ECD4 antigen or the HER2 ECD2 antigen. As described in more detail
below, the antigen-binding polypeptide constructs can be, but are not
limited to, protein constructs such as Fab (fragment antigen-binding),
scFv (single chain Fv) and sdab (single domain antibody). In some
embodiments, the antigen-binding construct includes a scaffold, e.g, an
Fc.
[0064] The term "antigen-binding construct" refers to any agent, e.g.,
polypeptide or polypeptide complex capable of binding to an antigen. In
some aspects an antigen-binding construct is a polypeptide that
specifically binds to an antigen of interest. An antigen-binding
construct can be a monomer, dimer, multimer, a protein, a peptide, or a
protein or peptide complex; an antibody, an antibody fragment, or an
antigen-binding fragment thereof; an scFv and the like. An
antigen-binding construct can be monospecific, bispecific, or
multispecific. In some aspects, an antigen-binding construct can include,
e.g., one or more antigen-binding polypeptide constructs (e.g., Fabs or
scFvs) linked to one or more Fc. Further examples of antigen-binding
constructs are described below and provided in the Examples.
[0065] In some embodiments, the antigen-binding construct is monospecific.
A monospecific antigen-binding construct refers to an antigen-binding
construct with one binding specificity. In other words, the
antigen-binding polypeptide construct binds to the same epitope on the
same antigen. Examples of monospecific antigen-binding constructs include
trastuzumab and pertuzumab.
[0066] A bispecific antigen binding construct has two antigen binding
polypeptide constructs, each with a unique binding specificity. For
example, a first antigen binding polypeptide construct binds to an
epitope on a first antigen, and a second antigen binding polypeptide
construct binds to an epitope on a second antigen. The term "biparatopic"
as used herein, refers to a bispecific antibody where the first antigen
binding moiety and the second antigen binding moiety bind to different
epitopes on the same antigen.
[0067] An antigen-binding construct can be an antibody or antigen-binding
portion thereof. As used herein, an "antibody" or "immunoglobulin" refers
to a polypeptide substantially encoded by an immunoglobulin gene or
immunoglobulin genes, or fragments thereof, which specifically bind and
recognize an analyte (e.g., antigen). The recognized immunoglobulin genes
include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant
region genes, as well as the myriad immunoglobulin variable region genes.
Light chains are classified as either kappa or lambda. The "class" of an
antibody or immunoglobulin refers to the type of constant domain or
constant region possessed by its heavy chain. There are five major
classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these
may be further divided into subclasses (isotypes), e.g., IgG1, IgG.sub.2,
IgG.sub.3, IgG.sub.4, IgA1, and IgA.sub.2. The heavy chain constant
domains that correspond to the different classes of immunoglobulins are
called .alpha., .delta., .epsilon., .gamma., and respectively.
[0068] An exemplary immunoglobulin (antibody) structural unit is composed
of two pairs of polypeptide chains, each pair having one "light" (about
25 kD) and one "heavy" chain (about 50-70 kD). The N-terminal domain of
each chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms variable
light chain (VL) and variable heavy chain (VH) refer to these light and
heavy chain domains respectively. The IgG1 heavy chain comprises of the
VH, CH1, CH2 and CH3 domains respectively from the N to C-terminus. The
light chain comprises of the VL and CL domains from N to C terminus. The
IgG1 heavy chain comprises a hinge between the CH1 and CH2 domains.
[0069] The term "hypervariable region" or "HVR", as used herein, refers to
each of the regions of an antibody variable domain which are
hypervariable in sequence and/or form structurally defined loops
("hypervariable loops"). Generally, native four-chain antibodies comprise
six HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3).
HVRs generally comprise amino acid residues from the hypervariable loops
and/or from the complementarity determining regions (CDRs), the latter
being of highest sequence variability and/or involved in antigen
recognition. With the exception of CDR1 in VH, CDRs generally comprise
the amino acid residues that form the hypervariable loops. Hypervariable
regions (HVRs) are also referred to as "complementarity determining
regions" (CDRs), and these terms are used herein interchangeably in
reference to portions of the variable region that form the
antigen-binding regions. This particular region has been described by
Kabat et al., U.S. Dept. of Health and Human Services, Sequences of
Proteins of Immunological Interest (1983) and by Chothia et al., J Mol
Biol 196:901-917 (1987), where the definitions include overlapping or
subsets of amino acid residues when compared against each other.
Nevertheless, application of either definition to refer to a CDR of an
antibody or variants thereof is intended to be within the scope of the
term as defined and used herein. The exact residue numbers which
encompass a particular CDR will vary depending on the sequence and size
of the CDR. Those skilled in the art can routinely determine which
residues comprise a particular CDR given the variable region amino acid
sequence of the antibody.
[0070] "Humanized" forms of non-human (e.g., rodent) antibodies are
chimeric antibodies that contain minimal sequence derived from non-human
immunoglobulin. For the most part, humanized antibodies are human
immunoglobulins (recipient antibody) in which residues from a
hypervariable region of the recipient are replaced by residues from a
hypervariable region of a non-human species (donor antibody) such as
mouse, rat, rabbit or nonhuman primate having the desired specificity,
affinity, and capacity. In some instances, framework region (FR) residues
of the human immunoglobulin are replaced by corresponding non-human
residues. Furthermore, humanized antibodies may comprise residues that
are not found in the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of at
least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of a
non-human immunoglobulin and all or substantially all of the FRs are
those of a human immunoglobulin sequence. The humanized antibody
optionally also will comprise at least a portion of an immunoglobulin
constant region (Fc), typically that of a human immunoglobulin. For
further details, see Jones et al., Nature 321:522-525 (1986); Riechmann
et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.
2:593-596 (1992).
[0071] Humanized HER2 antibodies include huMAb4D5-1, huMAb4D5-2,
huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8
or Trastuzumab (HERCEPTIN.RTM.) as described in Table 3 of U.S. Pat. No.
5,821,337 expressly incorporated herein by reference; humanized 520C9
(WO93/21319) and humanized 2C4 antibodies as described in US Patent
Publication No. 2006/0018899.
Antigen-Binding Polypeptide Construct
[0072] The antigen-binding constructs described herein comprise at least
one antigen-binding polypeptide construct that each binds to a HER2 ECD2
antigen. In some embodiments, the antigen-binding constructs described
herein include a second antigen-binding polypeptide construct that binds
to, e.g., a HER2 ECD2 antigen or a HER2 ECD4 antigen. In some embodiments
the antigen-binding polypeptide construct comprises a sequence that is
disclosed in the examples below, e.g., the VH or VL or CDRs of v5019,
v5020, v7091, v10000, or v6717.
[0073] The antigen-binding polypeptide construct is typically monovalent,
i.e. can bind only one epitope. In some embodiments, however, the
antigen-binding polypeptide construct can be bivalent (binding to two
epitopes) or multivalent.
[0074] Either antigen-binding polypeptide construct can be, e.g., a Fab,
or an scFv, depending on the application. In some embodiments, the
antigen binding construct includes two antigen-binding polypeptide
constructs. The format of the antigen-binding construct may be Fab-Fab,
scFv-scFv, or Fab-scFv or scFv-Fab (first antigen-binding polypeptide
construct-second antigen-binding polypeptide respectively).
[0075] A Fab (also referred to as fragment antigen-binding) contains the
constant domain (CL) of the light chain and the first constant domain
(CH1) of the heavy chain along with the variable domains VL and VH on the
light and heavy chains respectively. The variable domains comprise the
complementarity determining loops (CDR, also referred to as hypervariable
region) that are involved in antigen-binding. Fab' fragments differ from
Fab fragments by the addition of a few residues at the carboxy terminus
of the heavy chain CH1 domain including one or more cysteines from the
antibody hinge region.
[0076] A "single-chain Fv" or "scFv" includes the VH and VL domains of an
antibody, wherein these domains are present in a single polypeptide
chain. In one embodiment, the Fv polypeptide further comprises a
polypeptide linker between the VH and VL domains which enables the scFv
to form the desired structure for antigen-binding. For a review of scFv
see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
HER2 antibody scFv fragments are described in WO93/16185; U.S. Pat. No.
5,571,894; and U.S. Pat. No. 5,587,458.
[0077] A "single domain antibody" or "sdAb" format is an individual
immunoglobulin domain. SdAbs are fairly stable and easy to express as
fusion partner with the Fc chain of an antibody (Harmsen MM, De Haard HJ
(2007). "Properties, production, and applications of camelid
single-domain antibody fragments". Appl. Microbiol Biotechnol. 77(1):
13-22).
[0078] In some embodiments the antigen binding polypeptide construct is
derived from an antibody, a fibronectin, an affibody, anticalin, cysteine
knot protein, DARPin, avimer, Kunitz domain or variant or derivative
thereof.
[0079] The antigen binding polypeptide constructs described herein can be
converted to different formats. For example, a Fab can be converted to an
scFv or an scFv can be converted to a Fab. Methods of converting between
types of antigen-binding domains are known in the art (see for example
methods for converting an scFv to a Fab format described at, e.g., Zhou
et al (2012) Mol Cancer Ther 11:1167-1476. The methods described therein
are incorporated by reference.).
[0080] The antigen binding constructs described herein specifically bind
HER2. "Specifically binds", "specific binding" or "selective binding"
means that the binding is selective for the antigen and can be
discriminated from unwanted or non-specific interactions. The ability of
an antigen-binding construct to bind to a specific antigenic determinant
can be measured either through an enzyme-linked immunosorbent assay
(ELISA) or other techniques familiar to one of skill in the art, e.g.
surface plasmon resonance (SPR) technique (analyzed on a BIAcore
instrument) (Liljeblad et al, Glyco J 17, 323-329 (2000)), and
traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).
[0081] In one embodiment, the extent of binding of an antigen-binding
moiety to an unrelated protein is less than about 10% of the binding of
the antigen-binding construct to the antigen as measured, e.g., by SPR.
HER2
[0082] The antigen-binding constructs described herein include an
antigen-binding polypeptide construct that binds to the ECD2 of HER2.
[0083] The expressions "ErbB2" and "HER2" are used interchangeably herein
and refer to human HER2 protein described, for example, in Semba et al.,
PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al. Nature 319:230-234
(1986) (Genebank accession number X03363). The term "erbB2" and "neu"
refers to the gene encoding human ErbB2 protein. p185 or p185neu refers
to the protein product of the neu gene.
[0084] HER2 is a HER receptor. A "HER receptor" is a receptor protein
tyrosine kinase which belongs to the human epidermal growth factor
receptor (HER) family and includes EGFR, HER2, HER3 and HER4 receptors. A
HER receptor will generally comprise an extracellular domain, which may
bind an HER ligand; a lipophilic transmembrane domain; a conserved
intracellular tyrosine kinase domain; and a carboxyl-terminal signaling
domain harboring several tyrosine residues which can be phosphorylated.
By "HER ligand" is meant a polypeptide which binds to and/or activates an
HER receptor.
[0085] The extracellular (ecto) domain of HER2 comprises four domains,
Domain I (ECD1, amino acid residues from about 1-195), Domain II (ECD2,
amino acid residues from about 196-319), Domain III (ECD3, amino acid
residues from about 320-488), and Domain IV (ECD4, amino acid residues
from about 489-630) (residue numbering without signal peptide). See
Garrett et al. Mol. Cell. 11: 495-505 (2003), Cho et al. Nature 421:
756-760 (2003), Franklin et al. Cancer Cell 5:317-328 (2004), Tse et al.
Cancer Treat Rev. 2012 April; 38(2):133-42 (2012), or Plowman et al.
Proc. Natl. Acad. Sci. 90:1746-1750 (1993).
[0086] The sequence of HER2 is as follows; ECD boundaries are Domain I:
1-165; Domain II: 166-322; Domain III: 323-488; Domain IV: 489-607.
TABLE-US-00001
(SEQ ID NO: 349)
1 tqvctgtdmk lrlpaspeth ldmlrhlyqg cqvvqgnlel tylptnasls flgdigevqg
61 yvliahnqvr qvplqrlriv rgtqlfedny alavldngdp lnnttpvtga spgglrelql
121 rslteilkgg vliqrnpq1c yqdtilwkdi fhknnqlalt lidtnrsrac hpcspmckgs
181 rcwgessedc qsltrtvcag gcarckgplp tdccheqcaa gctgpkhsdc laclhfnhsg
241 icelhcpalv tyntdtfesm pnpegrytfg ascvtacpyn ylstdvgsct lvcplhnqev
301 taedgtqrce kcskpcarvc yglgmehlre vravtsaniq efagckkifg slaflpesfd
361 gdpasntapl gpeqlqvfet leeitgylyi sawpdslpdl svfqnlqvir grilhngays
421 ltlqglgisw lglrslrelg sglalihhnt hlcfvhtvpw dqlfrnphqa llhtanrped
481 ecvgeglach qlcarghcwg pgptqcvncs qflrggecve ecrvlqglpr eyvnarhclp
541 chpecqpqng svtcfgpead qcvacahykd ppfcvarcps gvkpdlsymp iwkfpdeega
601 cqpcpin
[0087] The "epitope 2C4" is the region in the extracellular domain of HER2
to which the antibody 2C4 binds. Epitope 2C4 comprises residues from
domain II in the extracellular domain of HER2. 2C4 and Pertuzumab bind to
the extracellular domain of HER2 at the junction of domains I, II and
III. Franklin et al. Cancer Cell 5:317-328 (2004). In order to screen for
antibodies which bind to the 2C4 epitope, a routine cross-blocking assay
such as that described in Antibodies, A Laboratory Manual, Cold Spring
Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed.
Alternatively, epitope mapping can be performed to assess whether the
antibody binds to the 2C4 epitope of HER2 using methods known in the art
and/or one can study the antibody-HER2 structure (Franklin et al. Cancer
Cell 5:317-328 (2004)) to see what domain(s) of HER2 is/are bound by the
antibody.
[0088] The "epitope 4D5" is the region in the extracellular domain of HER2
to which the antibody 4D5 (ATCC CRL 10463) and Trastuzumab bind. This
epitope is close to the transmembrane domain of HER2, and within Domain
IV of HER2. To screen for antibodies which bind to the 4D5 epitope, a
routine cross-blocking assay such as that described in Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David
Lane (1988), can be performed. Alternatively, epitope mapping can be
performed to assess whether the antibody binds to the 4D5 epitope of HER2
(e.g. any one or more residues in the region from about residue 529 to
about residue 625, inclusive, see FIG. 1 of US Patent Publication No.
2006/0018899).
Exemplary Anti-HER2 Antigen Binding Constructs
[0089] Exemplary anti-HER2 antibodies (or antigen-binding constructs) and
controls are provided herein. Representations of exemplary biparatopic
formats are shown in FIG. 1. In all of the formats shown in FIG. 1, the
heterodimeric Fc is depicted with one chain (Chain A) shown in black and
the other (Chain B) shown in grey, while one antigen-binding domain (1)
is shown in hatched fill and the other antigen-binding domain (2) is
shown in white.
[0090] FIG. 1A depicts the structure of a biparatopic antibody in a
Fab-Fab format. FIGS. 1B to 1E depict the structure of possible versions
of a biparatopic antibody in an scFv-Fab format. In FIG. 1B,
antigen-binding domain 1 is an scFv, fused to Chain A, while
antigen-binding domain 2 is a Fab, fused to Chain B. In FIG. 1C,
antigen-binding domain 1 is a Fab, fused to Chain A, while
antigen-binding domain 2 is an scFv, fused to Chain B. In FIG. 1D,
antigen-binding domain 2 is a Fab, fused to Chain A, while
antigen-binding domain 1 is an scFv, fused to Chain B. In FIG. 1E,
antigen-binding domain 2 is an scFv, fused to Chain A, while
antigen-binding domain 1 is a Fab, fused to Chain B. In FIG. 1F, both
antigen-binding domains are scFvs.
[0091] The sequences of the following variants are provided in the
Sequence Table found after the Examples. CDR regions were identified
using a combination of the Kabat and Chothia methods. Regions may vary
slightly based on method used for identification.
[0092] Exemplary Anti-HER2 Biparatopic Antibodies
[0093] Exemplary anti-HER2 biparatopic antibodies are shown in Table 1.
TABLE-US-00002
TABLE 1
Exemplary anti-HER2 biparatbopic antibodies
Variant Chain A Chain B
5019 domain ECD2 ECD4
containing
the epitope
Format Fab scFv
Antibody Pertuzumab Trastuzumab
name
CH3 T350V_L351Y_F405A_Y407V T366I_N390R_K392M_T394W
sequence
substitutions
5020 domain ECD4 ECD2
containing
the epitope
format scFv Fab
Antibody Trastuzumab Pertuzumab
name
CH3 L351Y_S400E_F405A_Y407V T350V_T366L_K392L_T394W
sequence
substitutions
7091 domain ECD2 ECD4
containing
the epitope
format Fab scFv
Antibody Pertuzumab Trastuzumab
name
CH3 T350V_L351Y_F405A_Y407V T350V_T366L_K392L_T394W
sequence
substitutions
10000 domain ECD2 ECD4
containing
the epitope
format Fab scFv
Antibody Pertuzumab - with Y96A in VL Trastuzumab
name region and T30A/A49G/L69F in
VH region
CH3 T350V_L351Y_F405A_Y407V T350V_T366L_K392L_T394W
sequence
substitutions
6902 domain ECD2 ECD4
containing
the epitope
format Fab Fab
Antibody Trastuzumab Pertuzumab
name
Fab HC: L143E_K145T HC: D146G_Q179K
substitutions LC: Q124R LC: Q124E_Q160E_T180E
CH3 T350V_L351Y_F405A_Y407V T350V_T366L_K392L_T394W
sequence
substitutions
6903 domain ECD2 ECD4
containing
the epitope
format Fab Fab
Fab HC: L143E_K145T HC: D146G_Q179K
substitutions LC: Q124R_Q1160K_T178R LC: Q124E_Q160E_T180E
Antibody Trastuzumab Pertuzumab
name
CH3 T350V_L351Y_F405A_Y407V T350V_T366L_K392L_T394W
sequence
substitutions
6717 domain ECD4 ECD2
containing
the epitope
format scFv scFv
Antibody Pertuzumab Trastuzumab
name
CH3 T350V_L351Y_F405A_Y407V T366I_N390R_K392M_T394W
sequence
substitutions
Notes:
CH3 numbering according to EU index as in Kabat referring to the numbering
of the EU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:
78-85);
Fab or variable domain numbering according to Kabat (Kabat and Wu, 1991;
Kabat et al, Sequences of proteins of immunological interest. 5th Edition
- US Department of Health and Human Services, NIH publication n.sup.o
91-3242, p 647 (1991))
"domain containing the epitope" = domain of HER2 to which antigen-binding
moiety binds;
"Antibody name" = antibody from which antigen-binding moiety is derived,
includes substitutions compared to wild-type when present;
"Fab substitutions" = substitutions in Fab that promote correct light
chain pairing;
"CH3 sequence substitutions" = substitutions in CH3 domain that promote
formation of heterodimeric Fc
[0094] Exemplary Anti-HER2 Monovalent Control Antibodies
[0095] v1040: a monovalent anti-HER2 antibody, where the HER2 binding
domain is a Fab derived from trastuzumab on chain A, and the Fc region is
a heterodimer having the mutations T350V_L351Y_F405A_Y407V in Chain A,
T350V_T366L_K392L_T394W in Chain B, and the hinge region of Chain B
having the mutation C226S; the antigen-binding domain binds to domain 4
of HER2.
[0096] v630--a monovalent anti-HER2 antibody, where the HER2 binding
domain is an scFv derived from trastuzumab on Chain A, and the Fc region
is a heterodimer having the mutations L351Y_S400E_F405A_Y407V in Chain A,
T366I_N390R_K392M_T394W in Chain B; and the hinge region having the
mutation C226S (EU numbering) in both chains; the antigen-binding domain
binds to domain 4 of HER2.
[0097] v4182: a monovalent anti-HER2 antibody, where the HER2 binding
domain is a Fab derived from pertuzumab on chain A, and the Fc region is
a heterodimer having the mutations T350V_L351Y_F405A_Y407V in Chain A,
T350V_T366L_K392L_T394W in Chain B, and the hinge region of Chain B
having the mutation C226S; the antigen-binding domain binds to domain 2
of HER2.
[0098] Exemplary Anti-HER2 Monospecific Bivalent Antibody Controls
(Full-Sized Antibodies, FSAs)
[0099] v506 is a wild-type anti HER2 produced in-house in Chinese Hamster
Ovary (CHO) cells, as a control. Both HER2 binding domains are derived
from trastuzumab in the Fab format and the Fc is a wild type homodimer;
the antigen-binding domain binds to domain 4 of HER2. This antibody is
also referred to as a trastuzumab analog.
[0100] v792, is wild-type trastuzumab with a IgG1 hinge, where both HER2
binding domains are derived from trastuzumab in the Fab format, and the
and the Fc region is a heterodimer having the mutations
T350V_L351Y_F405A_Y407V in Chain A, and T350V_T366L_K392L_T394W Chain B;
the antigen-binding domain binds to domain 4 of HER2. This antibody is
also referred to as a trastuzumab analog.
[0101] v4184, a bivalent anti-HER2 antibody, where both HER2 binding
domains are derived from pertuzumab in the Fab format, and the Fc region
is a heterodimer having the mutations T350V_L351Y_F405A_Y407V in Chain A,
and T350V_T366L_K392L_T394W Chain B. The antigen-binding domain binds to
domain 2 of HER2. This antibody is also referred to as a pertuzumab
analog.
[0102] Exemplary Anti-HER2 Biparatopic Antibody Drug Conjugates (ADCs)
[0103] The following are exemplary anti-HER2 biparatopic antibody drug
conjugates (anti-HER2 biparatopic-ADCs). ADCs of variants 5019, 7091,
10000 and 506 are identified as follows: [0104] v6363 (v5019 conjugated
to DM1) [0105] v7148 (v7091 conjugated to DM1) [0106] v10553 (v10000
conjugated to DM1) [0107] v6246 (v506 conjugated to DM1, analogous to
T-DM1, trastuzumab-emtansine) [0108] v6249 (human IgG conjugated to DM1)
Fc of Antigen-Binding Constructs.
[0109] In some embodiments, the antigen-binding constructs described
herein comprise an Fc, e.g., a dimeric Fc. A dimeric Fc can be
homodimeric or heterodimeric
[0110] The term "Fc domain" or "Fc region" herein is used to define a
C-terminal region of an immunoglobulin heavy chain that contains at least
a portion of the constant region. The term includes native sequence Fc
regions and variant Fc regions. Unless otherwise specified herein,
numbering of amino acid residues in the Fc region or constant region is
according to the EU numbering system, also called the EU index, as
described in Kabat et al, Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of Health,
Bethesda, Md., 1991. An "Fc polypeptide" of a dimeric Fc as used herein
refers to one of the two polypeptides forming the dimeric Fc domain, i.e.
a polypeptide comprising C-terminal constant regions of an immunoglobulin
heavy chain, capable of stable self-association. For example, an Fc
polypeptide of a dimeric IgG Fc comprises an IgG CH2 and an IgG CH3
constant domain sequence.
[0111] An Fc domain comprises either a CH3 domain or a CH3 and a CH2
domain. The CH3 domain comprises two CH3 sequences, one from each of the
two Fc polypeptides of the dimeric Fc. The CH2 domain comprises two CH2
sequences, one from each of the two Fc polypeptides of the dimeric Fc.
[0112] In some aspects, the Fc comprises at least one or two CH3
sequences. In some aspects, the Fc is coupled, with or without one or
more linkers, to a first antigen-binding construct and/or a second
antigen-binding construct. In some aspects, the Fc is a human Fc. In some
aspects, the Fc is a human IgG or IgG1 Fc. In some aspects, the Fc is a
heterodimeric Fc. In some aspects, the Fc comprises at least one or two
CH2 sequences.
[0113] In some aspects, the Fc comprises one or more modifications in at
least one of the CH3 sequences. In some aspects, the Fc comprises one or
more modifications in at least one of the CH2 sequences. In some aspects,
an Fc is a single polypeptide. In some aspects, an Fc is multiple
peptides, e.g., two polypeptides.
[0114] In some aspects, an Fc is an Fc described in patent applications
PCT/CA2011/001238, filed Nov. 4, 2011 or PCT/CA2012/050780, filed Nov. 2,
2012, the entire disclosure of each of which is hereby incorporated by
reference in its entirety for all purposes.
[0115] Modified CH3 Domains
[0116] In some aspects, the antigen-binding construct described herein
comprises a heterodimeric Fc comprising a modified CH3 domain that has
been asymmetrically modified. The heterodimeric Fc can comprise two heavy
chain constant domain polypeptides: a first Fc polypeptide and a second
Fc polypeptide, which can be used interchangeably provided that Fc
comprises one first Fc polypeptide and one second Fc polypeptide.
Generally, the first Fc polypeptide comprises a first CH3 sequence and
the second Fc polypeptide comprises a second CH3 sequence.
[0117] Two CH3 sequences that comprise one or more amino acid
modifications introduced in an asymmetric fashion generally results in a
heterodimeric Fc, rather than a homodimer, when the two CH3 sequences
dimerize. As used herein, "asymmetric amino acid modifications" refers to
any modification where an amino acid at a specific position on a first
CH3 sequence is different from the amino acid on a second CH3 sequence at
the same position, and the first and second CH3 sequence preferentially
pair to form a heterodimer, rather than a homodimer. This
heterodimerization can be a result of modification of only one of the two
amino acids at the same respective amino acid position on each sequence;
or modification of both amino acids on each sequence at the same
respective position on each of the first and second CH3 sequences. The
first and second CH3 sequence of a heterodimeric Fc can comprise one or
more than one asymmetric amino acid modification.
[0118] Table A provides the amino acid sequence of the human IgG1 Fc
sequence, corresponding to amino acids 231 to 447 of the full-length
human IgG1 heavy chain. The CH3 sequence comprises amino acid 341-447 of
the full-length human IgG1 heavy chain.
[0119] Typically an Fc can include two contiguous heavy chain sequences (A
and B) that are capable of dimerizing. In some aspects, one or both
sequences of an Fc include one or more mutations or modifications at the
following locations: L351, F405, Y407, T366, K392, T394, T350, S400,
and/or N390, using EU numbering. In some aspects, an Fc includes a mutant
sequence shown in Table X. In some aspects, an Fc includes the mutations
of Variant 1 A-B. In some aspects, an Fc includes the mutations of
Variant 2 A-B. In some aspects, an Fc includes the mutations of Variant 3
A-B. In some aspects, an Fc includes the mutations of Variant 4 A-B. In
some aspects, an Fc includes the mutations of Variant 5 A-B.
TABLE-US-00003
TABLE A
IgG1 Fc sequences
Human IgG1 Fc APELLGGPSVFLFPPKPKDTLMISRTP
sequence 231-447 EVTCVVVDVSHEDPEVKFNWYVDGVEV
(EU-numbering) HNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSRDELTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPG
K (SEQ ID NO: 350)
Variant IgG1
Fc sequence
(231-447) Chain Mutations
1 A L351Y_F405A_Y407V
1 B T366L_K392M_T394W
2 A L351Y_F405A_Y407V
2 B T366L_K392L_T394W
3 A T350V_L351Y_F405A_Y407V
3 B T350V_T366L_K392L_T394W
4 A T350V_L351Y_F405A_Y407V
4 B T350V_T366L_K392M_T394W
5 A T350V_L351Y_S400E_F405A_
Y407V
5 B T350V_T366L_N390R_K392M_
T394W
[0120] The first and second CH3 sequences can comprise amino acid
mutations as described herein, with reference to amino acids 231 to 447
of the full-length human IgG1 heavy chain. In one embodiment, the
heterodimeric Fc comprises a modified CH3 domain with a first CH3
sequence having amino acid modifications at positions F405 and Y407, and
a second CH3 sequence having amino acid modifications at position T394.
In one embodiment, the heterodimeric Fc comprises a modified CH3 domain
with a first CH3 sequence having one or more amino acid modifications
selected from L351Y, F405A, and Y407V, and the second CH3 sequence having
one or more amino acid modifications selected from T366L, T366I, K392L,
K392M, and T394W.
[0121] In one embodiment, a heterodimeric Fc comprises a modified CH3
domain with a first CH3 sequence having amino acid modifications at
positions L351, F405 and Y407, and a second CH3 sequence having amino
acid modifications at positions T366, K392, and T394, and one of the
first or second CH3 sequences further comprising amino acid modifications
at position Q347, and the other CH3 sequence further comprising amino
acid modification at position K360. In another embodiment, a
heterodimeric Fc comprises a modified CH3 domain with a first CH3
sequence having amino acid modifications at positions L351, F405 and
Y407, and a second CH3 sequence having amino acid modifications at
position T366, K392, and T394, one of the first or second CH3 sequences
further comprising amino acid modifications at position Q347, and the
other CH3 sequence further comprising amino acid modification at position
K360, and one or both of said CH3 sequences further comprise the amino
acid modification T350V.
[0122] In one embodiment, a heterodimeric Fc comprises a modified CH3
domain with a first CH3 sequence having amino acid modifications at
positions L351, F405 and Y407, and a second CH3 sequence having amino
acid modifications at positions T366, K392, and T394 and one of said
first and second CH3 sequences further comprising amino acid modification
of D399R or D399K and the other CH3 sequence comprising one or more of
T411E, T411D, K409E, K409D, K392E and K392D. In another embodiment, a
heterodimeric Fc comprises a modified CH3 domain with a first CH3
sequence having amino acid modifications at positions L351, F405 and
Y407, and a second CH3 sequence having amino acid modifications at
positions T366, K392, and T394, one of said first and second CH3
sequences further comprises amino acid modification of D399R or D399K and
the other CH3 sequence comprising one or more of T411E, T411D, K409E,
K409D, K392E and K392D, and one or both of said CH3 sequences further
comprise the amino acid modification T350V.
[0123] In one embodiment, a heterodimeric Fc comprises a modified CH3
domain with a first CH3 sequence having amino acid modifications at
positions L351, F405 and Y407, and a second CH3 sequence having amino
acid modifications at positions T366, K392, and T394, wherein one or both
of said CH3 sequences further comprise the amino acid modification of
T350V.
[0124] In one embodiment, a heterodimeric Fc comprises a modified CH3
domain comprising the following amino acid modifications, where "A"
represents the amino acid modifications to the first CH3 sequence, and
"B" represents the amino acid modifications to the second CH3 sequence:
A:L351Y_F405A_Y407V, B:T366L_K392M_T394W, A:L351Y_F405A_Y407V,
B:T366L_K392L_T394W, A:T350V_L351Y_F405A_Y407V,
B:T350V_T366L_K392L_T394W, A:T350V_L351Y_F405A_Y407V,
B:T350V_T366L_K392M_T394W, A:T350V_L351Y_S400E_F405A_Y407V, and/or
B:T350V_T366L_N390R_K392M_T394W.
[0125] The one or more asymmetric amino acid modifications can promote the
formation of a heterodimeric Fc in which the heterodimeric CH3 domain has
a stability that is comparable to a wild-type homodimeric CH3 domain. In
an embodiment, the one or more asymmetric amino acid modifications
promote the formation of a heterodimeric Fc domain in which the
heterodimeric Fc domain has a stability that is comparable to a wild-type
homodimeric Fc domain. In an embodiment, the one or more asymmetric amino
acid modifications promote the formation of a heterodimeric Fc domain in
which the heterodimeric Fc domain has a stability observed via the
melting temperature (Tm) in a differential scanning calorimetry study,
and where the melting temperature is within 4.degree. C. of that observed
for the corresponding symmetric wild-type homodimeric Fc domain. In some
aspects, the Fc comprises one or more modifications in at least one of
the C.sub.H3 sequences that promote the formation of a heterodimeric Fc
with stability comparable to a wild-type homodimeric Fc.
[0126] In one embodiment, the stability of the CH3 domain can be assessed
by measuring the melting temperature of the CH3 domain, for example by
differential scanning calorimetry (DSC). Thus, in a further embodiment,
the CH3 domain has a melting temperature of about 68.degree. C. or
higher. In another embodiment, the CH3 domain has a melting temperature
of about 70.degree. C. or higher. In another embodiment, the CH3 domain
has a melting temperature of about 72.degree. C. or higher. In another
embodiment, the CH3 domain has a melting temperature of about 73.degree.
C. or higher. In another embodiment, the CH3 domain has a melting
temperature of about 75.degree. C. or higher. In another embodiment, the
CH3 domain has a melting temperature of about 78.degree. C. or higher. In
some aspects, the dimerized CH3 sequences have a melting temperature (Tm)
of about 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 77.5, 78, 79, 80, 81,
82, 83, 84, or 85.degree. C. or higher.
[0127] In some embodiments, a heterodimeric Fc comprising modified CH3
sequences can be formed with a purity of at least about 75% as compared
to homodimeric Fc in the expressed product. In another embodiment, the
heterodimeric Fc is formed with a purity greater than about 80%. In
another embodiment, the heterodimeric Fc is formed with a purity greater
than about 85%. In another embodiment, the heterodimeric Fc is formed
with a purity greater than about 90%. In another embodiment, the
heterodimeric Fc is formed with a purity greater than about 95%. In
another embodiment, the heterodimeric Fc is formed with a purity greater
than about 97%. In some aspects, the Fc is a heterodimer formed with a
purity greater than about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% when expressed. In
some aspects, the Fc is a heterodimer formed with a purity greater than
about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, or 99% when expressed via a single cell.
[0128] Additional methods for modifying monomeric Fc polypeptides to
promote heterodimeric Fc formation are described in International Patent
Publication No. WO 96/027011 (knobs into holes), in Gunasekaran et al.
(Gunasekaran K. et al. (2010) J Biol Chem. 285, 19637-46, electrostatic
design to achieve selective heterodimerization), in Davis et al. (Davis,
J H. et al. (2010) Prot Eng Des Sel; 23(4): 195-202, strand exchange
engineered domain (SEED) technology), and in Labrijn et al [Efficient
generation of stable bispecific IgG1 by controlled Fab-arm exchange.
Labrijn A F, Meesters J I, de Goeij B E, van den Bremer E T, Neijssen J,
van Kampen M D, Strumane K, Verploegen S, Kundu A, Gramer M J, van Berkel
P H, van de Winkel J G, Schuurman J, Parren P W. Proc Natl Acad Sci USA.
2013 Mar. 26; 110(13):5145-50.
[0129] CH2 Domains
[0130] In some embodiments, the Fc of the antigen-binding construct
comprises a CH2 domain. One example of an CH2 domain of an Fc is amino
acid 231-340 of the sequence shown in Table A. Several effector functions
are mediated by Fc receptors (FcRs), which bind to the Fc of an antibody.
[0131] The terms "Fc receptor" and "FcR" are used to describe a receptor
that binds to the Fc region of an antibody. For example, an FcR can be a
native sequence human FcR. Generally, an FcR is one which binds an IgG
antibody (a gamma receptor) and includes receptors of the Fc.gamma.RI,
Fc.gamma.RII, and Fc.gamma.RIII subclasses, including allelic variants
and alternatively spliced forms of these receptors. Fc.gamma.RII
receptors include Fc.gamma.RIIA (an "activating receptor") and
Fc.gamma.RIIB (an "inhibiting receptor"), which have similar amino acid
sequences that differ primarily in the cytoplasmic domains thereof.
Immunoglobulins of other isotypes can also be bound by certain FcRs (see,
e.g., Janeway et al., Immuno Biology: the immune system in health and
disease, (Elsevier Science Ltd., NY) (4th ed., 1999)). Activating
receptor Fc.gamma.RIIA contains an immunoreceptor tyrosine-based
activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition motif
(ITIM) in its cytoplasmic domain (reviewed in Daeron, Annu. Rev. Immunol.
15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev.
Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and
de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs,
including those to be identified in the future, are encompassed by the
term "FcR" herein. The term also includes the neonatal receptor, FcRn,
which is responsible for the transfer of maternal IgGs to the fetus
(Guyer et al., J. Immunol. 117:587 (1976); and Kim et al., J. Immunol.
24:249 (1994)).
[0132] Modifications in the CH2 domain can affect the binding of FcRs to
the Fc. A number of amino acid modifications in the Fc region are known
in the art for selectively altering the affinity of the Fc for different
Fcgamma receptors. In some aspects, the Fc comprises one or more
modifications to promote selective binding of Fc-gamma receptors.
[0133] Exemplary mutations that alter the binding of FcRs to the Fc are
listed below:
[0134] S298A/E333A/K334A, S298A/E333A/K334A/K326A (Lu Y, Vernes J M,
Chiang N, et al. J Immunol Methods. 2011 Feb. 28; 365(1-2):132-41);
[0135] F243L/R292P/Y300L/V305I/P396L, F243L/R292P/Y300L/L235V/P396L
(Stavenhagen J B, Gorlatov S, Tuaillon N, et al. Cancer Res. 2007 Sep.
15; 67(18):8882-90; Nordstrom J L, Gorlatov S, Zhang W, et al. Breast
Cancer Res. 2011 Nov. 30; 13(6):R123);
[0136] F243L (Stewart R, Thom G, Levens M, et al. Protein Eng Des Sel.
2011 September; 24(9):671-8.), S298A/E333A/K334A (Shields R L, Namenuk A
K, Hong K, et al. J Biol Chem. 2001 Mar. 2; 276(9):6591-604);
[0137] S239D/I332E/A330L, S239D/I332E (Lazar G A, Dang W, Karki S, et al.
Proc Natl Acad Sci USA. 2006 Mar. 14; 103(11):4005-10);
[0138] S239D/S267E, S267E/L328F (Chu S Y, Vostiar I, Karki S, et al. Mol
Immunol. 2008 September; 45(15):3926-33);
[0139] S239D/D265S/S298A/I332E, S239E/S298A/K326A/A327H, G237F/S298A/A33
0L/I332E, S239D/I332E/S298A, S239D/K326E/A330L/I332E/S298A,
G236A/S239D/D270L/I3 32E, S239E/S267E/H268D, L234F/S267E/N325L,
G237F/V266L/S267D and other mutations listed in WO2011/120134 and
WO2011/120135, herein incorporated by reference. Therapeutic Antibody
Engineering (by William R. Strohl and Lila M. Strohl, Woodhead Publishing
series in Biomedicine No 11, ISBN 1 907568 37 9, October 2012) lists
mutations on page 283.
[0140] In some embodiments an antigen-binding construct described herein
comprises an antigen-binding polypeptide construct which binds an
antigen; and a dimeric Fc that has superior biophysical properties like
stability and ease of manufacture relative to an antigen-binding
construct which does not include the same dimeric Fc. In some embodiments
a CH2 domain comprises one or more asymmetric amino acid modifications.
Exemplary asymmetric mutations are described in International Patent
Application No. PCT/CA2014/050507.
[0141] Additional Modifications to Improve Effector Function.
[0142] In some embodiments an antigen-binding construct described herein
includes modifications to improve its ability to mediate effector
function. Such modifications are known in the art and include
afucosylation, or engineering of the affinity of the Fc towards an
activating receptor, mainly FCGR3a for ADCC, and towards C1q for CDC. The
following Table B summarizes various designs reported in the literature
for effector function engineering.
[0143] Methods of producing antigen-binding constructs with little or no
fucose on the Fc glycosylation site (Asn 297 EU numbering) without
altering the amino acid sequence are well known in the art. The
GlymaX.RTM. technology (ProBioGen AG) is based on the introduction of a
gene for an enzyme which deflects the cellular pathway of fucose
biosynthesis into cells used for antigen-binding construct production.
This prevents the addition of the sugar "fucose" to the N-linked antibody
carbohydrate part by antigen-binding construct-producing cells. (von
Horsten et al. (2010) Glycobiology. 2010 December; 20 (12):1607-18.
Another approach to obtaining antigen-binding constructs with lowered
levels of fucosylation can be found in U.S. Pat. No. 8,409,572, which
teaches selecting cell lines for antigen-binding construct production for
their ability to yield lower levels of fucosylation on antigen-binding
constructs Antigen-binding constructs can be fully afucosylated (meaning
they contain no detectable fucose) or they can be partially afucosylated,
meaning that the isolated antibody contains less than 95%, less than 85%,
less than 75%, less than 65%, less than 55%, less than 45%, less than
35%, less than 25%, less than 15% or less than 5% of the amount of fucose
normally detected for a similar antibody produced by a mammalian
expression system.
[0144] Thus, in one embodiment, an antigen-binding construct described
herein can include a dimeric Fc that comprises one or more amino acid
modifications as noted in Table B that confer improved effector function.
In another embodiment, the antigen-binding construct can be afucosylated
to improve effector function.
TABLE-US-00004
TABLE B
CH2 domains and effector function engineering.
Reference Mutations Effect
Lu, 2011, Afucosylated Increased ADCC
Ferrara 2011,
Mizushima 2011
Lu, 2011 S298A/E333A/K334A Increased ADCC
Lu, 2011 S298A/E333A/K334A/K326A Increased ADCC
Stavenhagen, F243L/R292P/Y300L/V305I/ Increased ADCC
2007 P396L
Nordstrom, 2011 F243L/R292P/Y300L/L235V/ Increased ADCC
P396L
Stewart, 2011 F243L Increased ADCC
Shields, 2001 S298A/E333A/K334A Increased ADCC
Lazar, 2006 S239D/I332E/A330L Increased ADCC
Lazar, 2006 S239D/I332E Increased ADCC
Bowles, 2006 AME-D, not specified mutations Increased ADCC
Heider, 2011 37.1, mutations not disclosed Increased ADCC
Moore, 2010 S267E/H268F/S324T Increased CDC
[0145] Fc modifications reducing Fc.gamma.R and/or complement binding
and/or effector function are known in the art. Recent publications
describe strategies that have been used to engineer antibodies with
reduced or silenced effector activity (see Strohl, W R (2009), Curr Opin
Biotech 20:685-691, and Strohl, W R and Strohl L M, "Antibody Fc
engineering for optimal antibody performance" In Therapeutic Antibody
Engineering, Cambridge: Woodhead Publishing (2012), pp 225-249). These
strategies include reduction of effector function through modification of
glycosylation, use of IgG2/IgG4 scaffolds, or the introduction of
mutations in the hinge or CH2 regions of the Fc. For example, US Patent
Publication No. 2011/0212087 (Strohl), International Patent Publication
No. WO 2006/105338 (Xencor), US Patent Publication No. 2012/0225058
(Xencor), US Patent Publication No. 2012/0251531 (Genentech), and Strop
et al ((2012) J. Mol. Biol. 420: 204-219) describe specific modifications
to reduce Fc.gamma.R or complement binding to the Fc.
[0146] Specific, non-limiting examples of known amino acid modifications
to reduce Fc.gamma.R or complement binding to the Fc include those
identified in the following table:
TABLE-US-00005
TABLE C
modifications to reduce Fc.gamma.R or complement binding to the Fc
Company Mutations
GSK N297A
Ortho Biotech L234A/L235A
Protein Design labs IGG2 V234A/G237A
Wellcome Labs IGG4 L235A/G237A/E318A
GSK IGG4 S228P/L236E
Alexion IGG2/IGG4combo
Merck IGG2 H268Q/V309L/A330S/A331S
Bristol-Myers C220S/C226S/C229S/P238S
Seattle Genetics C226S/C229S/E3233P/L235V/L235A
Amgen E. coli production, non glyco
Medimune L234F/L235E/P331S
Trubion Hinge mutant, possibly C226S/P230S
[0147] In one embodiment, the Fc comprises at least one amino acid
modification identified in the above table. In another embodiment the Fc
comprises amino acid modification of at least one of L234, L235, or D265.
In another embodiment, the Fc comprises amino acid modification at L234,
L235 and D265. In another embodiment, the Fc comprises the amino acid
modification L234A, L235A and D265S.
Linkers and Linker Polypeptides
[0148] In some embodiments, the antigen-binding constructs described
herein include two antigen-binding polypeptide constructs. In these
embodiments, the antigen-binding polypeptide constructs are each
operatively linked to a linker polypeptide wherein the linker
polypeptides are capable of forming a complex or interface with each
other. In some embodiments, the linker polypeptides are capable of
forming a covalent linkage with each other. The spatial conformation of
the antigen-binding construct comprising a first and second
antigen-binding polypeptide constructs with the linker polypeptides is
similar to the relative spatial conformation of the paratopes of a
F(ab')2 fragment generated by papain digestion, albeit in the context of
an antigen-binding construct with 2 antigen-binding polypeptide
constructs.
[0149] In some embodiments, the linker polypeptides are selected such that
they maintain the relative spatial conformation of the paratopes of a
F(ab') fragment, and are capable of forming a covalent bond equivalent to
the disulphide bond in the core hinge of IgG. Suitable linker
polypeptides include IgG hinge regions such as, for example those from
IgG1, IgG2, or IgG4. Modified versions of these exemplary linkers can
also be used. For example, modifications to improve the stability of the
IgG4 hinge are known in the art (see for example, Labrijn et al. (2009)
Nature Biotechnology 27, 767-771).
[0150] In one embodiment, the linker polypeptides are operatively linked
to a scaffold as described here, for example an Fc. In some aspects, an
Fc is coupled to the one or more antigen-binding polypeptide constructs
with one or more linkers. In some aspects, Fc is coupled to the heavy
chain of each antigen-binding polypeptide by a linker.
[0151] In other embodiments, the linker polypeptides are operatively
linked to scaffolds other than an Fc. A number of alternate protein or
molecular domains are know in the art and can be used to form selective
pairs of two different antigen-binding polypeptides. An example is the
leucine zipper domains such as Fos and Jun that selectively pair together
[S A Kostelny, M S Cole, and J Y Tso. Formation of a bispecific antibody
by the use of leucine zippers. J Immunol 1992 148:1547-53; Bernd J.
Wranik, Erin L. Christensen, Gabriele Schaefer, Janet K. Jackman, Andrew
C. Vendel, and Dan Eaton. LUZ-Y, a Novel Platform for the Mammalian Cell
Production of Full-length IgG-bispecific AntibodiesJ. Biol. Chem. 2012
287: 43331-43339]. Alternately, other selectively pairing molecular pairs
such as the barnase barstar pair [Deyev, S. M., Waibel, R., Lebedenko, E.
N., Schubiger, A. P., and Pluckthun, A. (2003). Design of multivalent
complexes using the barnase*barstar module. Nat Biotechnol 21,
1486-1492], DNA strand pairs [Zahida N. Chaudri, Michael Bartlet-Jones,
George Panayotou, Thomas Klonisch, Ivan M. Roitt, Torben Lund, Peter J.
Delves, Dual specificity antibodies using a double-stranded
oligonucleotide bridge, FEBS Letters, Volume 450, Issues 1-2, 30 Apr.
1999, Pages 23-26], split fluorescent protein pairs [Ulrich Brinkmann,
Alexander Haas. Fluorescent antibody fusion protein, its production and
use, WO 2011135040 A1] can also be employed.
Affinity
[0152] In some embodiments, affinity is determined by SPR (surface plasmon
resonance) and/or FACS (fluorescence activated cell sorting). In some
embodiments, affinity is determined by SPR and/or FACS as described
below.
Dissociation Constant (K) and Maximal Binding (Bmax)
[0153] In some embodiments, an antigen-binding construct is described by
functional characteristics including but not limited to a dissociation
constant and a maximal binding.
[0154] The term "dissociation constant (K.sub.D)" as used herein, is
intended to refer to the equilibrium dissociation constant of a
particular ligand-protein interaction. As used herein, ligand-protein
interactions refer to, but are not limited to protein-protein
interactions or antibody-antigen interactions. The K.sub.D measures the
propensity of two proteins (e.g. AB) to dissociate reversibly into
smaller components (A+B), and is define as the ratio of the rate of
dissociation, also called the "off-rate (k.sub.off)", to the association
rate, or "on-rate (k.sub.on)". Thus, K.sub.D equals k.sub.off/k.sub.on
and is expressed as a molar concentration (M). It follows that the
smaller the K.sub.D, the stronger the affinity of binding. Therefore, a
K.sub.D of 1 mM indicates weak binding affinity compared to a K.sub.D of
1 nM. K.sub.D values for antigen-binding constructs can be determined
using methods well established in the art. One method for determining the
K.sub.D of an antigen-binding construct is by using surface plasmon
resonance (SPR), typically using a biosensor system such as a
Biacore.RTM. system. Isothermal titration calorimetry (ITC) is another
method that can be used to determine.
[0155] The binding characteristics of an antigen-binding construct can be
determined by various techniques. One of which is the measurement of
binding to target cells expressing the antigen by flow cytometry (FACS,
Fluorescence-activated cell sorting). Typically, in such an experiment,
the target cells expressing the antigen of interest are incubated with
antigen-binding constructs at different concentrations, washed, incubated
with a secondary agent for detecting the antigen-binding construct,
washed, and analyzed in the flow cytometer to measure the median
fluorescent intensity (MFI) representing the strength of detection signal
on the cells, which in turn is related to the number of antigen-binding
constructs bound to the cells. The antigen-binding construct
concentration vs. MFI data is then fitted into a saturation binding
equation to yield two key binding parameters, Bmax and apparent K.sub.D.
[0156] Apparent K.sub.D, or apparent equilibrium dissociation constant,
represents the antigen-binding construct concentration at which half
maximal cell binding is observed. Evidently, the smaller the K.sub.D
value, the smaller antigen-binding construct concentration is required to
reach maximum cell binding and thus the higher is the affinity of the
antigen-binding construct. The apparent K.sub.D is dependent on the
conditions of the cell binding experiment, such as different receptor
levels expressed on the cells and incubation conditions, and thus the
apparent K.sub.D is generally different from the K.sub.D values
determined from cell-free molecular experiments such as SPR and ITC.
However, there is generally good agreement between the different methods.
[0157] The term "Bmax", or maximal binding, refers to the maximum
antigen-binding construct binding level on the cells at saturating
concentrations of antigen-binding construct. This parameter can be
reported in the arbitrary unit MFI for relative comparison, or converted
into an absolute value corresponding to the number of antigen-binding
constructs bound to the cell with the use of a standard curve.
Testing of Antigen-Binding Constructs: HER2 Binding
[0158] The antigen-binding constructs or pharmaceutical compositions
described herein are tested in vitro, and then in vivo for the desired
therapeutic or prophylactic activity, prior to use in humans. For
example, in vitro assays to demonstrate the therapeutic or prophylactic
utility of a compound or pharmaceutical composition include, the effect
of a compound on a cell line or a patient tissue sample. The effect of
the compound or composition on the cell line and/or tissue sample can be
determined utilizing techniques known to those of skill in the art
including, but not limited to, rosette formation assays and cell lysis
assays. In accordance with the invention, in vitro assays which can be
used to determine whether administration of a specific antigen-binding
construct is indicated, include in vitro cell culture assays, or in vitro
assays in which a patient tissue sample is grown in culture, and exposed
to or otherwise administered antigen-binding construct, and the effect of
such antigen-binding construct upon the tissue sample is observed.
[0159] Candidate antigen-binding constructs can be assayed using cells,
e.g., breast cancer cell lines, expressing HER2. The following Table D
describes the expression level of HER2 in several representative cancer
cell lines.
TABLE-US-00006
TABLE D
Relative expression levels of HER2 in cell lines of interest.
IHC HER2
Cell Line Description scoring receptors/cell
NCI-N87 Human gastric carcinoma 3+ Not assessed
A549 Human lung alveolar 0/1+ Not assessed
carcinoma (non-small
cell lung cancer)
BxPC-3 Human pancreatic 1+ Not assessed
adenocarcinoma
MIA Human pancreatic ductal 2+ Not assessed
PaCa-2 adenocarcinoma
FaDu Human pharyngeal 2+ Not assessed
squamous cell
carcinoma
HCT-116 Human colorectal 1+ Not assessed
epithelial carcinoma
WI-38 Normal fetal lung 0 1.0 .times. 10E4
MDA- Human triple negative 0/1+ 1.7 .times. 10E4-2.3 .times. 10E4
MB-231 breast epithelial
adenocarcinoma
MCF-7 Human estrogen receptor 1+ 4 .times. 10E4-7 .times. 10E4
positive breast
epithelial
adenocarcinoma
JIMT-1 Trastuzumab resistant 2+ 2 .times. 10E5-8 .times. 10E5
breast epithelial
carcinoma, amplified
HER2 oncogene,
insensitive to
HER2-inhibiting
drugs (i.e. Herceptin .TM.)
ZR-75-1 Estrogen receptor 2+ 3 .times. 10E5
positive breast ductal
carcinoma
SKOV-3 Human ovarian epithelial 2/3+ 5 .times. 10E5-1 .times. 10E6
adenocarcinoma, HER2
gene amplified
SK-BR-3 Human breast epithelial 3+ >1 .times. 10E6
adenocarcinoma
BT-474 Human breast epithelial 3+ >1 .times. 10E6
ductal carcinoma,
[0160] McDonagh et al Mol Cancer Ther. 2012 March; 11(3):582-93; Subik et
al. (2010) Breast Cancer: Basic Clinical Research: 4; 35-41; Carter et
al. PNAS, 1994:89; 4285-4289; Yarden 2000, HER2: Basic Research,
Prognosis and Therapy; Hendricks et al Mol Cancer Ther 2013; 12:1816-28.
[0161] As is known in the art, a number of assays may be employed in order
to identify antigen-binding constructs suitable for use in the methods
described herein. These assays can be carried out in cancer cells
expressing HER2. Examples of suitable cancer cells are identified in
Table A5. Examples of assays that may be carried out are described as
follows.
[0162] For example, to identify growth inhibitory candidate
antigen-binding constructs that bind HER2, one may screen for antibodies
which inhibit the growth of cancer cells which express HER2. In one
embodiment, the candidate antigen-binding construct of choice is able to
inhibit growth of cancer cells in cell culture by about 20-100% and
preferably by about 50-100% at compared to a control antigen-binding
construct.
[0163] To select for candidate antigen-binding constructs which induce
cell death, loss of membrane integrity as indicated by, e.g., PI
(phosphatidylinositol), trypan blue or 7AAD uptake may be assessed
relative to control.
[0164] In order to select for candidate antigen-binding constructs which
induce apoptosis, an annexin binding assay may be employed. In addition
to the annexin binding assay, a DNA staining assay may also be used.
[0165] In one embodiment, the candidate antigen-binding construct of
interest may block heregulin dependent association of ErbB2 with ErbB3 in
both MCF7 and SK-BR-3 cells as determined in a co-immunoprecipitation
experiment substantially more effectively than monoclonal antibody 4D5,
and preferably substantially more effectively than monoclonal antibody
7F3.
[0166] To screen for antigen-binding constructs which bind to an epitope
on ErbB2 bound by an antibody of interest, a routine cross-blocking assay
such as that described in Antibodies, A Laboratory Manual, Cold Spring
Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed.
Alternatively, or additionally, epitope mapping can be performed by
methods known in the art.
[0167] Competition between antigen-binding constructs can be determined by
an assay in which an antigen-binding construct under test inhibits or
blocks specific binding of a reference antigen-binding construct to a
common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990;
Fendly et al. Cancer Research 50: 1550-1558; U.S. Pat. No. 6,949,245). A
test antigen-binding construct competes with a reference antigen-binding
construct if an excess of a test antigen-binding construct (e.g., at
least 2.times., 5.times., 10.times., 20.times., or 100.times.) inhibits
or blocks binding of the reference antigen-binding construct by, e.g., at
least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as measured in a
competitive binding assay. Antigen-binding constructs identified by
competition assay (competing antigen-binding construct) include
antigen-binding constructs binding to the same epitope as the reference
antigen-binding construct and antigen-binding constructs binding to an
adjacent epitope sufficiently proximal to the epitope bound by the
reference antigen-binding construct for steric hindrance to occur. For
example, a second, competing antigen-binding construct can be identified
that competes for binding to HER2 with a first antigen-binding construct
described herein. In certain instances, the second construct can block or
inhibit binding of the first construct by, e.g., at least 50%, 60%, 70%,
75%, 80%, 85%, 90%, 95%, or 99% as measured in a competitive binding
assay. In certain instances, the second construct can displace the first
construct by greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.
[0168] In some embodiments, antigen-binding constructs described herein
are assayed for function in vivo, e.g., in animal models. In some
embodiments, the animal models are those described in Table E. In some
embodiments, the animal models are those described in the Examples. In
some embodiments, the antigen-binding constructs display an increase in
efficacy of treatment in an animal model compared to a reference
antigen-binding construct.
TABLE-US-00007
TABLE E
Animal models for testing HER2 binding antigen-binding constructs
Xenograft Model Description Reference
SKOV3 human HER2+/3+, gene amplified, Rhodes et al. 2002. American Journal
of
ovarian cancer moderately sensitive to trastuzumab Pathology 118: 408-417;
Sims et al. 2012.
British Journal of Cancer 106: 1779-1789
HBCx-13b human HER2 3+, estrogen receptor negative, Marangoni et al. 2007.
Clinical Cancer
metastatic progesterone receptor negative; Research 13: 3989-3998; Reyal
et al. 2012.
breast cancer Invasive ductal breast carcinoma; Breast Cancer Research 14:
R11
Chemotherapy resistant, Trastuzumab
resistant
T226 human HER2 3+, estrogen receptor negative,
breast cancer progesterone receptor negative;
Inflammatory breast cancer;
Trastuzumab resistant, Docetaxel and
capecitabine moderately sensitive,
Adriamycin/cyclophosphamide
sensitive
HBCx-5 human HER2 3+, estrogen receptor negative, Marangoni et al. 2007.
Clinical Cancer
breast cancer progesterone receptor negative; Research 13: 3989-3998;
Reyal et al. 2012.
Invasive ductal carcinoma, luminal B; Breast Cancer Research 14: R11
Trastuzumab resistant, Docetaxel
moderately sensitive, Capecitabine,
Adriamycin/Cyclophosphamide
sensitive
JIMT-1 human HER2 2+, HER2 gene amplified, Tanner et al. 2004. Molecular
Cancer
breast cancer Trastuzumab and pertuzumab Therapeutics 3: 1585-1592
resistant
Reference Antigen-Binding Construct
[0169] In some embodiments, the functional characteristics of the
antigen-binding constructs described herein are compared to those of a
reference antigen-binding construct. The identity of the reference
antigen-binding construct depends on the functional characteristic being
measured or the distinction being made. For example, when comparing the
functional characteristics of antigen-binding constructs described
herein, the reference antigen-binding construct may be a trastuzumab (for
example v6336), or analog thereof, or may be a control IgG, for example a
non-specific polyclonal human antibody.
Antigen-Binding Constructs and Antibody Drug Conjugates (ADC)
[0170] In certain embodiments an antigen-binding construct is conjugated
to a drug, e.g., a toxin, a chemotherapeutic agent, an immune modulator,
or a radioisotope. Several methods of preparing ADCs (antibody drug
conjugates or antigen-binding construct drug conjugates) are known in the
art and are described below.
[0171] In some embodiments, the drug is selected from a maytansine,
auristatin, calicheamicin, or derivative thereof. In other embodiments,
the drug is a maytansine selected from DM1 and DM4. Further examples are
described below.
[0172] In some embodiments the drug is conjugated to the isolated
antigen-binding construct with an SMCC linker (DM1), or an SPDB linker
(DM4). Additional examples are described below. The
drug-to-antigen-binding protein ratio (DAR) can be, e.g., 1.0 to 6.0 or
3.0 to 5.0 or 3.5-4.2.
[0173] In some embodiments the antigen-binding construct is conjugated to
a cytotoxic agent. The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or causes
destruction of cells. The term is intended to include radioactive
isotopes (e.g. At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32,
and Lu177), chemotherapeutic agents, and toxins such as small molecule
toxins or enzymatically active toxins of bacterial, fungal, plant or
animal origin, including fragments and/or variants thereof. Further
examples are described below.
[0174] Drugs
[0175] Non-limiting examples of drugs or payloads used in various
embodiments of ADCs include DM1 (maytansine,
N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)- or
N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine), mc-MMAD
(6-maleimidocaproyl-monomethylauristatin-D or
N-methyl-L-valyl-N-[(1S,2R)-2-methoxy-4-[(2S)-2-[(1R,2R)-1-methoxy-2-meth-
yl-3-oxo-3-[[(1S)-2-phenyl-1-(2-thiazolyl)ethyl]amino]propyl]-1-pyrrolidin-
yl]-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-(9Cl)-L-valinamide),
mc-MMAF (maleimidocaproyl-monomethylauristatin F or
N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-N-methyl-L-valyl--
L-valyl-(3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanoyl-(.alpha.R,
.beta.R,2S)-.beta.-methoxy-.alpha.-methyl-2-pyrrolidinepropanoyl-L-phenyl-
alanine) and mc-Val-Cit-PABA-MMAE
(6-maleimidocaproyl-ValcCit-(p-aminobenzyloxycarbonyl)-monomethylauristat-
in E or N-[[[4-[[N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-L-
-valyl-N5-(aminocarbonyl)-L-ornithyl]amino]phenyl]methoxy]carbonyl]-N-meth-
yl-L-valyl-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2--
phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-metho-
xy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide). DM1 is a
derivative of the tubulin inhibitor maytansine while MMAD, MMAE, and MMAF
are auristatin derivatives.
[0176] Maytansinoid Drug Moieties
[0177] As indicated above, in some embodiments the drug is a maytansinoid.
Exemplary maytansinoids include DM1, DM3
(N.sup.2'-deacetyl-N.sup.2'-(4-mercapto-1-oxopentyl) maytansine), and DM4
(N.sup.2'-deacetyl-N.sup.2'-(4-methyl-4-mercapto-1-oxopentyl)methylmaytan-
sine) (see US20090202536).
[0178] Many positions on maytansine compounds are known to be useful as
the linkage position, depending upon the type of link. For example, for
forming an ester linkage, the C-3 position having a hydroxyl group, the
C-14 position modified with hydroxymethyl, the C-15 position modified
with a hydroxyl group and the C-20 position having a hydroxyl group are
all suitable.
[0179] All stereoisomers of the maytansinoid drug moiety are contemplated
for the ADCs described herein, i.e. any combination of R and S
configurations at the chiral carbons of D.
[0180] Auristatins
[0181] In some embodiments, the drug is an auristatin, such as auristatin
E (also known in the art as a derivative of dolastatin-10) or a
derivative thereof. The auristatin can be, for example, an ester formed
between auristatin E and a keto acid. For example, auristatin E can be
reacted with paraacetyl benzoic acid or benzoylvaleric acid to produce
AEB and AEVB, respectively. Other typical auristatins include AFP, MMAF,
and MMAE. The synthesis and structure of exemplary auristatins are
described in U.S. Pat. Nos. 6,884,869, 7,098,308, 7,256,257, 7,423,116,
7,498,298 and 7,745,394, each of which is incorporated by reference
herein in its entirety and for all purposes.
[0182] Chemotherapeutic Agents
[0183] In some embodiments the antigen-binding construct is conjugated to
a chemotherapeutic agent. Examples include but are not limited to
Cisplantin and Lapatinib. A "chemotherapeutic agent" is a chemical
compound useful in the treatment of cancer.
[0184] Examples of chemotherapeutic agents include alkylating agents such
as thiotepa and cyclosphosphamide (CYTOXAN.TM.); alkyl sulfonates such as
busulfan, improsulfan and piposulfan; aziridines such as benzodopa,
carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine, trietylenephosphoramide,
triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards
such as chlorambucil, chlomaphazine, cholophosphamide, estramustine,
ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,
melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins,
actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins,
dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin,
mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic
acid analogues such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,
doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid; aceglatone;
aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
elfornithine; elliptinium acetate; etoglucid; gallium nitrate;
hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol;
nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid;
2-ethylhydrazide; procarbazine; PSK7; razoxane; sizofiran;
spirogermanium; tenuazonic acid; triaziquone; 2,
2',2'=-trichlorotriethylamine; urethan; vindesine; dacarbazine;
mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;
arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, e.g.
paclitaxel (TAXOL.RTM., Bristol-Myers Squibb Oncology, Princeton, N.J.)
and doxetaxel (TAXOTERE.RTM., Rhone-Poulenc Rorer, Antony, France);
chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;
platinum analogs such as cisplatin and carboplatin; vinblastine;
platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;
vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;
aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS
2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins;
capecitabine; and pharmaceutically acceptable salts, acids or derivatives
of any of the above. Also included in this definition are anti-hormonal
agents that act to regulate or inhibit hormone action on tumors such as
anti-estrogens including for example tamoxifen, raloxifene, aromatase
inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,
LY117018, onapristone, and toremifene (Fareston); and anti-androgens such
as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and
pharmaceutically acceptable salts, acids or derivatives of any of the
above.
[0185] Conjugate Linkers
[0186] In some embodiments, the drug is linked to the antigen-binding
construct, e.g., antibody, by a linker. Attachment of a linker to an
antibody can be accomplished in a variety of ways, such as through
surface lysines, reductive-coupling to oxidized carbohydrates, and
through cysteine residues liberated by reducing interchain disulfide
linkages. A variety of ADC linkage systems are known in the art,
including hydrazone-, disulfide- and peptide-based linkages.
[0187] Suitable linkers include, for example, cleavable and non-cleavable
linkers. A cleavable linker is typically susceptible to cleavage under
intracellular conditions. Suitable cleavable linkers include, for
example, a peptide linker cleavable by an intracellular protease, such as
lysosomal protease or an endosomal protease. In exemplary embodiments,
the linker can be a dipeptide linker, such as a valine-citrulline
(val-cit), a phenylalanine-lysine (phe-lys) linker, or
maleimidocapronic-valine-citruline-p-aminobenzyloxycarbonyl
(mc-Val-Cit-PABA) linker. Another linker is
Sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC).
Sulfo-smcc conjugation occurs via a maleimide group which reacts with
sulfhydryls (thiols, --SH), while its Sulfo-NHS ester is reactive toward
primary amines (as found in Lysine and the protein or peptide
N-terminus). Yet another linker is maleimidocaproyl (MC). Other suitable
linkers include linkers hydrolyzable at a specific pH or a pH range, such
as a hydrazone linker. Additional suitable cleavable linkers include
disulfide linkers. The linker may be covalently bound to the antibody to
such an extent that the antibody must be degraded intracellularly in
order for the drug to be released e.g. the MC linker and the like.
[0188] Preparation of ADCs
[0189] The ADC may be prepared by several routes, employing organic
chemistry reactions, conditions, and reagents known to those skilled in
the art, including: (1) reaction of a nucleophilic group or an
electrophilic group of an antibody with a bivalent linker reagent, to
form antibody-linker intermediate Ab-L, via a covalent bond, followed by
reaction with an activated drug moiety D; and (2) reaction of a
nucleophilic group or an electrophilic group of a drug moiety with a
linker reagent, to form drug-linker intermediate D-L, via a covalent
bond, followed by reaction with the nucleophilic group or an
electrophilic group of an antibody. Conjugation methods (1) and (2) may
be employed with a variety of antibodies, drug moieties, and linkers to
prepare the antibody-drug conjugates described here.
[0190] Several specific examples of methods of preparing ADCs are known in
the art and are described in U.S. Pat. No. 8,624,003 (pot method), U.S.
Pat. No. 8,163,888 (one-step), and U.S. Pat. No. 5,208,020 (two-step
method).
Methods of Preparation of Antigen-Binding Constructs
[0191] Antigen-binding constructs described herein may be produced using
recombinant methods and compositions, e.g., as described in U.S. Pat. No.
4,816,567.
[0192] In one embodiment, isolated nucleic acid encoding an
antigen-binding construct described herein is provided. Such nucleic acid
may encode an amino acid sequence comprising the VL and/or an amino acid
sequence comprising the VH of the antigen-binding construct (e.g., the
light and/or heavy chains of the antigen-binding construct). In a further
embodiment, one or more vectors (e.g., expression vectors) comprising
such nucleic acid are provided. In one embodiment, the nucleic acid is
provided in a multicistronic vector. In a further embodiment, a host cell
comprising such nucleic acid is provided. In one such embodiment, a host
cell comprises (e.g., has been transformed with): (1) a vector comprising
a nucleic acid that encodes an amino acid sequence comprising the VL of
the antigen-binding construct and an amino acid sequence comprising the
VH of the antigen-binding polypeptide construct, or (2) a first vector
comprising a nucleic acid that encodes an amino acid sequence comprising
the VL of the antigen-binding polypeptide construct and a second vector
comprising a nucleic acid that encodes an amino acid sequence comprising
the VH of the antigen-binding polypeptide construct. In one embodiment,
the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell, or
human embryonic kidney (HEK) cell, or lymphoid cell (e.g., Y0, NS0, Sp20
cell). In one embodiment, a method of making an antigen-binding construct
is provided, wherein the method comprises culturing a host cell
comprising nucleic acid encoding the antigen-binding construct, as
provided above, under conditions suitable for expression of the
antigen-binding construct, and optionally recovering the antigen-binding
construct from the host cell (or host cell culture medium).
[0193] For recombinant production of the antigen-binding construct,
nucleic acid encoding an antigen-binding construct, e.g., as described
above, is isolated and inserted into one or more vectors for further
cloning and/or expression in a host cell. Such nucleic acid may be
readily isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the antigen-binding
construct).
[0194] The term "substantially purified" refers to a construct described
herein, or variant thereof that may be substantially or essentially free
of components that normally accompany or interact with the protein as
found in its naturally occurring environment, i.e. a native cell, or host
cell in the case of recombinantly produced heteromultimer that in certain
embodiments, is substantially free of cellular material includes
preparations of protein having less than about 30%, less than about 25%,
less than about 20%, less than about 15%, less than about 10%, less than
about 5%, less than about 4%, less than about 3%, less than about 2%, or
less than about 1% (by dry weight) of contaminating protein. When the
heteromultimer or variant thereof is recombinantly produced by the host
cells, the protein in certain embodiments is present at about 30%, about
25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about
2%, or about 1% or less of the dry weight of the cells. When the
heteromultimer or variant thereof is recombinantly produced by the host
cells, the protein, in certain embodiments, is present in the culture
medium at about 5 g/L, about 4 g/L, about 3 g/L, about 2 g/L, about 1
g/L, about 750 mg/L, about 500 mg/L, about 250 mg/L, about 100 mg/L,
about 50 mg/L, about 10 mg/L, or about 1 mg/L or less of the dry weight
of the cells. In certain embodiments, "substantially purified"
heteromultimer produced by the methods described herein, has a purity
level of at least about 30%, at least about 35%, at least about 40%, at
least about 45%, at least about 50%, at least about 55%, at least about
60%, at least about 65%, at least about 70%, specifically, a purity level
of at least about 75%, 80%, 85%, and more specifically, a purity level of
at least about 90%, a purity level of at least about 95%, a purity level
of at least about 99% or greater as determined by appropriate methods
such as SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.
[0195] Suitable host cells for cloning or expression of antigen-binding
construct-encoding vectors include prokaryotic or eukaryotic cells
described herein.
[0196] A "recombinant host cell" or "host cell" refers to a cell that
includes an exogenous polynucleotide, regardless of the method used for
insertion, for example, direct uptake, transduction, f-mating, or other
methods known in the art to create recombinant host cells. The exogenous
polynucleotide may be maintained as a nonintegrated vector, for example,
a plasmid, or alternatively, may be integrated into the host genome.
[0197] As used herein, the term "eukaryote" refers to organisms belonging
to the phylogenetic domain Eucarya such as animals (including but not
limited to, mammals, insects, reptiles, birds, etc.), ciliates, plants
(including but not limited to, monocots, dicots, algae, etc.), fungi,
yeasts, flagellates, microsporidia, protists, etc.
[0198] As used herein, the term "prokaryote" refers to prokaryotic
organisms. For example, a non-eukaryotic organism can belong to the
Eubacteria (including but not limited to, Escherichia coli, Thermus
thermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens,
Pseudomonas aeruginosa, Pseudomonas putida, etc.) phylogenetic domain, or
the Archaea (including but not limited to, Methanococcus jannaschii,
Methanobacterium thermoautotrophicum, Halobacterium such as Haloferax
volcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus,
Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pemix, etc.)
phylogenetic domain.
[0199] For example, antigen-binding construct may be produced in bacteria,
in particular when glycosylation and Fc effector function are not needed.
For expression of antigen-binding construct fragments and polypeptides in
bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523.
(See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo,
ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, describing
expression of antibody fragments in E. coli.) After expression, the
antigen-binding construct may be isolated from the bacterial cell paste
in a soluble fraction and can be further purified.
[0200] In addition to prokaryotes, eukaryotic microbes such as filamentous
fungi or yeast are suitable cloning or expression hosts for
antigen-binding construct-encoding vectors, including fungi and yeast
strains whose glycosylation pathways have been "humanized," resulting in
the production of an antigen-binding construct with a partially or fully
human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414
(2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
[0201] Suitable host cells for the expression of glycosylated
antigen-binding constructs are also derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells include
plant and insect cells. Numerous baculoviral strains have been identified
which may be used in conjunction with insect cells, particularly for
transfection of Spodoptera frugiperda cells.
[0202] Plant cell cultures can also be utilized as hosts. See, e.g., U.S.
Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429
(describing PLANTIBODIES.TM. technology for producing antigen-binding
constructs in transgenic plants).
[0203] Vertebrate cells may also be used as hosts. For example, mammalian
cell lines that are adapted to grow in suspension may be useful. Other
examples of useful mammalian host cell lines are monkey kidney CV1 line
transformed by SV40 (COS-7); human embryonic kidney line (293 or 293
cells as described, e.g., in Graham et al., J Gen Virol. 36:59 (1977));
baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as
described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey
kidney cells (CV1); African green monkey kidney cells (VERO-76); human
cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat
liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep
G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in
Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and
FS4 cells. Other useful mammalian host cell lines include Chinese hamster
ovary (CHO) cells, including DHFR CHO cells (Urlaub et al., Proc. Natl.
Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0
and Sp2/0. For a review of certain mammalian host cell lines suitable for
antigen-binding construct production, see, e.g., Yazaki and Wu, Methods
in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa,
N.J.), pp. 255-268 (2003).
[0204] In one embodiment, the antigen-binding constructs described herein
are produced in stable mammalian cells, by a method comprising:
transfecting at least one stable mammalian cell with: nucleic acid
encoding the antigen-binding construct, in a predetermined ratio; and
expressing the nucleic acid in the at least one mammalian cell. In some
embodiments, the predetermined ratio of nucleic acid is determined in
transient transfection experiments to determine the relative ratio of
input nucleic acids that results in the highest percentage of the
antigen-binding construct in the expressed product.
[0205] In some embodiments is the method of producing a antigen-binding
construct in stable mammalian cells as described herein wherein the
expression product of the at least one stable mammalian cell comprises a
larger percentage of the desired glycosylated antigen-binding construct
as compared to the monomeric heavy or light chain polypeptides, or other
antibodies.
[0206] In some embodiments is the method of producing a glycosylated
antigen-binding construct in stable mammalian cells described herein,
said method comprising identifying and purifying the desired glycosylated
antigen-binding construct. In some embodiments, the said identification
is by one or both of liquid chromatography and mass spectrometry.
[0207] If required, the antigen-binding constructs can be purified or
isolated after expression. Proteins may be isolated or purified in a
variety of ways known to those skilled in the art. Standard purification
methods include chromatographic techniques, including ion exchange,
hydrophobic interaction, affinity, sizing or gel filtration, and
reversed-phase, carried out at atmospheric pressure or at high pressure
using systems such as FPLC and HPLC. Purification methods also include
electrophoretic, immunological, precipitation, dialysis, and
chromatofocusing techniques. Ultrafiltration and diafiltration
techniques, in conjunction with protein concentration, are also useful.
As is well known in the art, a variety of natural proteins bind Fc and
antibodies, and these proteins can find use in the present invention for
purification of antigen-binding constructs. For example, the bacterial
proteins A and G bind to the Fc region. Likewise, the bacterial protein L
binds to the Fab region of some antibodies. Purification can often be
enabled by a particular fusion partner. For example, antibodies may be
purified using glutathione resin if a GST fusion is employed, Ni.sup.+2
affinity chromatography if a His-tag is employed, or immobilized
anti-flag antibody if a flag-tag is used. For general guidance in
suitable purification techniques, see, e.g. incorporated entirely by
reference Protein Purification: Principles and Practice, 3.sup.rd Ed.,
Scopes, Springer-Verlag, N.Y., 1994, incorporated entirely by reference.
The degree of purification necessary will vary depending on the use of
the antigen-binding constructs. In some instances no purification is
necessary.
[0208] In certain embodiments the antigen-binding constructs are purified
using Anion Exchange Chromatography including, but not limited to,
chromatography on Q-sepharose, DEAE sepharose, poros HQ, poros DEAF,
Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource/Source Q and DEAE,
Fractogel Q and DEAE columns.
[0209] In specific embodiments the proteins described herein are purified
using Cation Exchange Chromatography including, but not limited to,
SP-sepharose, CM sepharose, poros HS, poros CM, Toyopearl SP, Toyopearl
CM, Resource/Source S and CM, Fractogel S and CM columns and their
equivalents and comparables.
[0210] In addition, antigen-binding constructs described herein can be
chemically synthesized using techniques known in the art (e.g., see
Creighton, 1983, Proteins: Structures and Molecular Principles, W. H.
Freeman & Co., N.Y and Hunkapiller et al., Nature, 310:105-111 (1984)).
For example, a polypeptide corresponding to a fragment of a polypeptide
can be synthesized by use of a peptide synthesizer. Furthermore, if
desired, nonclassical amino acids or chemical amino acid analogs can be
introduced as a substitution or addition into the polypeptide sequence.
Non-classical amino acids include, but are not limited to, to the
D-isomers of the common amino acids, 2,4diaminobutyric acid, alpha-amino
isobutyric acid, 4aminobutyric acid, Abu, 2-amino butyric acid, g-Abu,
e-Ahx, 6amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino
propionic acid, ornithine, norleucine, norvaline, hydroxyproline,
sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine,
t-butylalanine, phenylglycine, cyclohexylalanine, .quadrature.-alanine,
fluoro-amino acids, designer amino acids such as .quadrature.-methyl
amino acids, C.quadrature.-methyl amino acids, N.quadrature.-methyl amino
acids, and amino acid analogs in general. Furthermore, the amino acid can
be D (dextrorotary) or L (levorotary).
[0211] Post-Translational Modifications:
[0212] In certain embodiments antigen-binding constructs described herein
are differentially modified during or after translation.
[0213] The term "modified," as used herein refers to any changes made to a
given polypeptide, such as changes to the length of the polypeptide, the
amino acid sequence, chemical structure, co-translational modification,
or post-translational modification of a polypeptide. The form
"(modified)" term means that the polypeptides being discussed are
optionally modified, that is, the polypeptides under discussion can be
modified or unmodified.
[0214] The term "post-translationally modified" refers to any modification
of a natural or non-natural amino acid that occurs to such an amino acid
after it has been incorporated into a polypeptide chain. The term
encompasses, by way of example only, co-translational in vivo
modifications, co-translational in vitro modifications (such as in a
cell-free translation system), post-translational in vivo modifications,
and post-translational in vitro modifications.
[0215] In some embodiments, the modification is at least one of:
glycosylation, acetylation, phosphorylation, amidation, derivatization by
known protecting/blocking groups, proteolytic cleavage and linkage to an
antibody molecule or antigen-binding construct or other cellular ligand.
In some embodiments, the antigen-binding construct is chemically modified
by known techniques, including but not limited, to specific chemical
cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease,
NaBH.sub.4; acetylation, formylation, oxidation, reduction; and metabolic
synthesis in the presence of tunicamycin.
[0216] Additional post-translational modifications of antigen-binding
constructs described herein include, for example, N-linked or O-linked
carbohydrate chains, processing of N-terminal or C-terminal ends),
attachment of chemical moieties to the amino acid backbone, chemical
modifications of N-linked or O-linked carbohydrate chains, and addition
or deletion of an N-terminal methionine residue as a result of
procaryotic host cell expression. The antigen-binding constructs
described herein are modified with a detectable label, such as an
enzymatic, fluorescent, isotopic or affinity label to allow for detection
and isolation of the protein. In certain embodiments, examples of
suitable enzyme labels include horseradish peroxidase, alkaline
phosphatase, beta-galactosidase, or acetylcholinesterase; examples of
suitable prosthetic group complexes include streptavidin biotin and
avidin/biotin; examples of suitable fluorescent materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an
example of a luminescent material includes luminol; examples of
bioluminescent materials include luciferase, luciferin, and aequorin; and
examples of suitable radioactive material include iodine, carbon, sulfur,
tritium, indium, technetium, thallium, gallium, palladium, molybdenum,
xenon, fluorine.
[0217] In specific embodiments, antigen-binding constructs described
herein are attached to macrocyclic chelators that associate with
radiometal ions.
[0218] In some embodiments, the antigen-binding constructs described
herein are modified by either natural processes, such as
post-translational processing, or by chemical modification techniques
which are well known in the art. In certain embodiments, the same type of
modification may be present in the same or varying degrees at several
sites in a given polypeptide. In certain embodiments, polypeptides from
antigen-binding constructs described herein are branched, for example, as
a result of ubiquitination, and in some embodiments are cyclic, with or
without branching. Cyclic, branched, and branched cyclic polypeptides are
a result from posttranslation natural processes or made by synthetic
methods. Modifications include acetylation, acylation, ADP-ribosylation,
amidation, covalent attachment of flavin, covalent attachment of a heme
moiety, covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent attachment
of phosphotidylinositol, cross-linking, cyclization, disulfide bond
formation, demethylation, formation of covalent cross-links, formation of
cysteine, formation of pyroglutamate, formylation, gamma-carboxylation,
glycosylation, GPI anchor formation, hydroxylation, iodination,
methylation, myristylation, oxidation, pegylation, proteolytic
processing, phosphorylation, prenylation, racemization, selenoylation,
sulfation, transfer-RNA mediated addition of amino acids to proteins such
as arginylation, and ubiquitination. (See, for instance,
PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton,
W. H. Freeman and Company, New York (1993); POST-TRANSLATIONAL COVALENT
MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York,
pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990);
Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
[0219] In certain embodiments, antigen-binding constructs described herein
are attached to solid supports, which are particularly useful for
immunoassays or purification of polypeptides that are bound by, that bind
to, or associate with proteins described herein. Such solid supports
include, but are not limited to, glass, cellulose, polyacrylamide, nylon,
polystyrene, polyvinyl chloride or polypropylene.
Pharmaceutical Compositions
[0220] Also provided herein are pharmaceutical compositions comprising an
antigen-binding construct described herein. Pharmaceutical compositions
comprise the construct and a pharmaceutically acceptable carrier.
[0221] The term "pharmaceutically acceptable" means approved by a
regulatory agency of the Federal or a state government or listed in the
U.S. Pharmacopeia or other generally recognized pharmacopeia for use in
animals, and more particularly in humans. The term "carrier" refers to a
diluent, adjuvant, excipient, or vehicle with which the therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids, such
as water and oils, including those of petroleum, animal, vegetable or
synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame
oil and the like. In some aspects, the carrier is a man-made carrier not
found in nature. Water can be used as a carrier when the pharmaceutical
composition is administered intravenously. Saline solutions and aqueous
dextrose and glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. Suitable pharmaceutical excipients
include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,
chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol
and the like. The composition, if desired, can also contain minor amounts
of wetting or emulsifying agents, or pH buffering agents. These
compositions can take the form of solutions, suspensions, emulsion,
tablets, pills, capsules, powders, sustained-release formulations and the
like. The composition can be formulated as a suppository, with
traditional binders and carriers such as triglycerides. Oral formulation
can include standard carriers such as pharmaceutical grades of mannitol,
lactose, starch, magnesium stearate, sodium saccharine, cellulose,
magnesium carbonate, etc. Examples of suitable pharmaceutical carriers
are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
Such compositions will contain a therapeutically effective amount of the
compound, preferably in purified form, together with a suitable amount of
carrier so as to provide the form for proper administration to the
patient. The formulation should suit the mode of administration.
[0222] In certain embodiments, the composition comprising the construct is
formulated in accordance with routine procedures as a pharmaceutical
composition adapted for intravenous administration to human beings.
Typically, compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition may
also include a solubilizing agent and a local anesthetic such as
lignocaine to ease pain at the site of the injection. Generally, the
ingredients are supplied either separately or mixed together in unit
dosage form, for example, as a dry lyophilized powder or water free
concentrate in a hermetically sealed container such as an ampoule or
sachette indicating the quantity of active agent. Where the composition
is to be administered by infusion, it can be dispensed with an infusion
bottle containing sterile pharmaceutical grade water or saline. Where the
composition is administered by injection, an ampoule of sterile water for
injection or saline can be provided so that the ingredients may be mixed
prior to administration.
[0223] In certain embodiments, the compositions described herein are
formulated as neutral or salt forms. Pharmaceutically acceptable salts
include those formed with anions such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with
cations such as those derived from sodium, potassium, ammonium, calcium,
ferric hydroxide isopropylamine, triethylamine, 2-ethylamino ethanol,
histidine, procaine, etc.
Methods of Treatment
[0224] In certain embodiments, provided is a method of treating a disease
or disorder comprising administering to a subject in which such
treatment, prevention or amelioration is desired, an antigen-binding
construct described herein, in an amount effective to treat, prevent or
ameliorate the disease or disorder.
[0225] "Disorder" refers to any condition that would benefit from
treatment with an antigen-binding construct or method described herein.
This includes chronic and acute disorders or diseases including those
pathological conditions which predispose the mammal to the disorder in
question. In some embodiments, the disorder is cancer, as described in
more detail below.
[0226] The term "subject" refers to an animal, in some embodiments a
mammal, which is the object of treatment, observation or experiment. An
animal may be a human, a non-human primate, a companion animal (e.g.,
dogs, cats, and the like), farm animal (e.g., cows, sheep, pigs, horses,
and the like) or a laboratory animal (e.g., rats, mice, guinea pigs, and
the like).
[0227] The term "mammal" as used herein includes but is not limited to
humans, non-human primates, canines, felines, murines, bovines, equines,
and porcines.
[0228] "Treatment" refers to clinical intervention in an attempt to alter
the natural course of the individual or cell being treated, and can be
performed either for prophylaxis or during the course of clinical
pathology. Desirable effects of treatment include preventing occurrence
or recurrence of disease, alleviation of symptoms, diminishing of any
direct or indirect pathological consequences of the disease, preventing
metastasis, decreasing the rate of disease progression, amelioration or
palliation of the disease state, and remission or improved prognosis. In
some embodiments, antigen-binding constructs described herein are used to
delay development of a disease or disorder. In one embodiment,
antigen-binding constructs and methods described herein effect tumor
regression. In one embodiment, antigen-binding constructs and methods
described herein effect inhibition of tumor/cancer growth.
[0229] Desirable effects of treatment include, but are not limited to,
preventing occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of the
disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, improved
survival, and remission or improved prognosis. In some embodiments,
antigen-binding constructs described herein are used to delay development
of a disease or to slow the progression of a disease.
[0230] The term "effective amount" as used herein refers to that amount of
construct being administered, which will accomplish the goal of the
recited method, e.g., relieve to some extent one or more of the symptoms
of the disease, condition or disorder being treated. The amount of the
composition described herein which will be effective in the treatment,
inhibition and prevention of a disease or disorder associated with
aberrant expression and/or activity of a therapeutic protein can be
determined by standard clinical techniques. In addition, in vitro assays
may optionally be employed to help identify optimal dosage ranges. The
precise dose to be employed in the formulation will also depend on the
route of administration, and the seriousness of the disease or disorder,
and should be decided according to the judgment of the practitioner and
each patient's circumstances. Effective doses are extrapolated from
dose-response curves derived from in vitro or animal model test systems.
[0231] The antigen-binding construct is administered to the subject.
Various delivery systems are known and can be used to administer an
antigen-binding construct formulation described herein, e.g.,
encapsulation in liposomes, microparticles, microcapsules, recombinant
cells capable of expressing the compound, receptor-mediated endocytosis
(see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction
of a nucleic acid as part of a retroviral or other vector, etc. Methods
of introduction include but are not limited to intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,
epidural, and oral routes. The compounds or compositions may be
administered by any convenient route, for example by infusion or bolus
injection, by absorption through epithelial or mucocutaneous linings
(e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be
administered together with other biologically active agents.
Administration can be systemic or local. In addition, in certain
embodiments, it is desirable to introduce the antigen-binding construct
compositions described herein into the central nervous system by any
suitable route, including intraventricular and intrathecal injection;
intraventricular injection may be facilitated by an intraventricular
catheter, for example, attached to a reservoir, such as an Ommaya
reservoir. Pulmonary administration can also be employed, e.g., by use of
an inhaler or nebulizer, and formulation with an aerosolizing agent.
[0232] In a specific embodiment, it is desirable to administer the
antigen-binding constructs, or compositions described herein locally to
the area in need of treatment; this may be achieved by, for example, and
not by way of limitation, local infusion during surgery, topical
application, e.g., in conjunction with a wound dressing after surgery, by
injection, by means of a catheter, by means of a suppository, or by means
of an implant, said implant being of a porous, non-porous, or gelatinous
material, including membranes, such as sialastic membranes, or fibers.
Preferably, when administering a protein, including an antigen-binding
construct, described herein, care must be taken to use materials to which
the protein does not absorb.
[0233] In another embodiment, the antigen-binding constructs or
composition can be delivered in a vesicle, in particular a liposome (see
Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler
(eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp.
317-327; see generally ibid.)
[0234] In yet another embodiment, the antigen-binding constructs or
composition can be delivered in a controlled release system. In one
embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref.
Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);
Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment,
polymeric materials can be used (see Medical Applications of Controlled
Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974);
Controlled Drug Bioavailability, Drug Product Design and Performance,
Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J.,
Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al.,
Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard
et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a
controlled release system can be placed in proximity of the therapeutic
target, e.g., the brain, thus requiring only a fraction of the systemic
dose (see, e.g., Goodson, in Medical Applications of Controlled Release,
vol. 2, pp. 115-138 (1984)).
[0235] In a specific embodiment comprising a nucleic acid encoding
antigen-binding constructs decribed herein, the nucleic acid can be
administered in vivo to promote expression of its encoded protein, by
constructing it as part of an appropriate nucleic acid expression vector
and administering it so that it becomes intracellular, e.g., by use of a
retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection,
or by use of microparticle bombardment (e.g., a gene gun; Biolistic,
Dupont), or coating with lipids or cell-surface receptors or transfecting
agents, or by administering it in linkage to a homeobox-like peptide
which is known to enter the nucleus (see e.g., Joliot et al., Proc. Natl.
Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid
can be introduced intracellularly and incorporated within host cell DNA
for expression, by homologous recombination.
[0236] In certain embodiments an antigen-binding construct described
herein is administered as a combination with antigen-binding constructs
with non-overlapping binding target epitopes.
[0237] The amount of the antigen-binding construct which will be effective
in the treatment, inhibition and prevention of a disease or disorder can
be determined by standard clinical techniques. In addition, in vitro
assays may optionally be employed to help identify optimal dosage ranges.
The precise dose to be employed in the formulation will also depend on
the route of administration, and the seriousness of the disease or
disorder, and should be decided according to the judgment of the
practitioner and each patient's circumstances. Effective doses are
extrapolated from dose-response curves derived from in vitro or animal
model test systems.
[0238] The antigen-binding constructs described herein may be administered
alone or in combination with other types of treatments (e.g., radiation
therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor
agents). Generally, administration of products of a species origin or
species reactivity (in the case of antibodies) that is the same species
as that of the patient is preferred. Thus, in an embodiment, human
antigen-binding constructs, fragments derivatives, analogs, or nucleic
acids, are administered to a human patient for therapy or prophylaxis.
Methods of Treating Cancers
[0239] Described herein are methods of treating a HER2+ cancer or a tumor
in a subject, and methods of inhibiting the growth of a HER2+ tumor cell
or killing a HER2+ tumor cell using the antigen-binding constructs
described herein.
[0240] By a HER2+ cancer is meant a cancer that expresses HER2 such that
the antigen-binding constructs described herein are able to bind to the
cancer. As is known in the art, HER2+ cancers express HER2 at varying
levels. To determine ErbB, e.g. ErbB2 (HER2) expression in the cancer,
various diagnostic/prognostic assays are available. In one embodiment,
ErbB2 overexpression may be analyzed by IHC, e.g. using the
HERCEPTEST.RTM. (Dako). Paraffin embedded tissue sections from a tumor
biopsy may be subjected to the IHC assay and accorded a ErbB2 protein
staining intensity criteria as follows:
[0241] Score 0 no staining is observed or membrane staining is observed in
less than 10% of tumor cells.
[0242] Score 1+ a faint/barely perceptible membrane staining is detected
in more than 10% of the tumor cells. The cells are only stained in part
of their membrane.
[0243] Score 2+ a weak to moderate complete membrane staining is observed
in more than 10% of the tumor cells.
[0244] Score 3+ a moderate to strong complete membrane staining is
observed in more than 10% of the tumor cells.
[0245] Those tumors with 0 or 1+ scores for ErbB2 overexpression
assessment may be characterized as not overexpressing ErbB2, whereas
those tumors with 2+ or 3+ scores may be characterized as overexpressing
ErbB2.
[0246] Alternatively, or additionally, fluorescence in situ hybridization
(FISH) assays such as the INFORM.TM. (sold by Ventana, Ariz.) or
PATHVISION.TM. (Vysis, Ill.) may be carried out on formalin-fixed,
paraffin-embedded tumor tissue to determine the extent (if any) of ErbB2
overexpression in the tumor. In comparison with IHC assay, the FISH
assay, which measures HER2 gene amplification, seems to correlate better
with response of patients to treatment with HERCEPTIN.RTM., and is
currently considered to be the preferred assay to identify patients
likely to benefit from HERCEPTIN.RTM. treatment.
[0247] Table D describes the expression level of HER2 on several
representative breast cancer and other cancer cell lines (Subik et al.
(2010) Breast Cancer: Basic Clinical Research: 4; 35-41; Prang et a.
(2005) British Journal of Cancer Research: 92; 342-349). As shown in the
table, MCF-7 and MDA-MB-231 cells are considered to be low HER2
expressing cells; JIMT-1, and ZR-75-1 cells are considered to be medium
HER2 expressing cells, and SKBR3 and BT-474 cells are considered to be
high HER2 expressing cells. SKOV3 (ovarian cancer) cells are considered
to be medium HER2 expressing cells.
[0248] Described herein are methods of treating a subject having a HER2+
cancer or a tumor comprising providing to the subject an effective amount
of a pharmaceutical composition comprising an antigen-binding construct
described herein.
[0249] Also described herein is the use of an HER2 antigen-binding
construct described herein for the manufacture of a medicament for
treating a cancer or a tumor. Also described herein are HER2
antigen-binding constructs for use in the treatment of cancer or a tumor.
[0250] In some embodiments, the subject being treated has pancreatic
cancer, head and neck cancer, gastric cancer, colorectal cancer, breast
cancer, renal cancer, cervical cancer, ovarian cancer, brain cancer,
endometrial cancer, bladder cancer, non-small cell lung cancer or an
epidermal-derived cancer. In some embodiments, the tumor is metastatic.
[0251] In general, the tumor in the subject being treated expresses an
average of 10,000 or more copies of HER2 per tumor cell. In certain
embodiments the tumor is HER2 0-1+, 1+, HER2 2+ or HER2 3+ as determined
by IHC. In some embodiments the tumor is HER2 2+ or lower, or HER2 1+ or
lower. In some embodiments, the tumor has an amplified HER2 gene. In some
embodiments the HER2 gene is non-amplified.
[0252] In some embodiments, the tumor of the subject being treated with
the antigen-binding constructs is a breast cancer. In some embodiments,
the breast cancer expresses HER2 at a 3+ level. In some embodiments the
breast cancer expresses HER2 at less than a 3+ level. In a specific
embodiment, the breast cancer expresses HER2 at a 2+ level or lower. In a
specific embodiment, the breast cancer expresses HER2 at a 1+ level or
lower. In some embodiments, the breast cancer expresses estrogen
receptors (ER+) and/or progesterone receptors (PR+). In some embodiments,
the breast cancer is ER- and or PR-. In some embodiments the breast
cancer has an amplified HER2 gene. In some embodiments the HER2 gene is
non-amplified. In some embodiments, the breast cancer is a HER2 3+
estrogen receptor negative (ER-), progesterone receptor negative (PR-),
trastuzumab resistant, chemotherapy resistant invasive ductal breast
cancer. In another embodiment, the breast cancer is a HER2 3+ER-, PR-,
trastuzumab resistant inflammatory breast cancer. In another embodiment,
the breast cancer is a HER2 3+, ER-, PR-, invasive ductal carcinoma. In
another embodiment, the breast cancer is a HER2 2+ HER2 gene amplified
trastuzumab and pertuzumab resistant breast cancer. In some embodiments,
the breast cancer is triple negative (ER-, PR- and low HER2-expressing).
In some embodiments the breast cancer is resistant or refractory to
trastuzumab, pertuzumab and/or trastuzumab conjugated to DM1
(ado-trastuzumab emtansine or T-DM1).
[0253] In one embodiment, the tumor is an HER2 2/3+ ovarian epithelial
adenocarcinoma having an amplified HER2 gene.
[0254] Provided herein are methods for treating a subject having a HER2+
tumor that is resistant or becomes resistant to other standard-of-care
therapies comprising administering to the subject a pharmaceutical
composition comprising the antigen-binding constructs described herein.
In certain embodiments the antigen-binding constructs described herein
are provided to subjects that are unresponsive to current therapies,
optionally in combination with one or more current anti-HER2 therapies.
In some embodiments the current anti-HER2 therapies include, but are not
limited to, anti-HER2 or anti-HER3 monospecific bivalent antibodies,
trastuzumab, pertuzumab, T-DM1, a bi-specific HER2/HER3 scFv, or
combinations thereof. In some embodiments, the cancer is resistant to
various chemotherapeutic agents such as taxanes. In some embodiments the
cancer is resistant to trastuzumab. In some embodiment the cancer is
resistant to pertuzumab. In one embodiment, the cancer is resistant or
refractory to TDM1 (trastuzumab conjugated to DM1). In some embodiments,
the subject has previously been treated with an anti-HER2 antibody such
as trastuzumab, pertuzumab or DM1. In some embodiments, the subject has
not been previously treated with an anti-HER2 antibody. In one
embodiment, the antigen-binding construct is provided to a subject for
the treatment of metastatic cancer when the patient has progressed on
previous anti-HER2 therapy.
[0255] Provided herein are methods of treating a subject having a HER2+
tumor comprising providing an effective amount of a pharmaceutical
composition comprising an antigen-binding construct described herein in
conjunction with an additional anti-tumor agent. The additional
anti-tumor agent may be a therapeutic antibody as noted above, or a
chemotherapeutic agent. Chemotherapeutic agents useful for use in
combination with the antigen-binding constructs of the invention include
cisplatin, carboplatin, paclitaxel, albumin-bound paclitaxel,
nab-paclitaxel, docetaxel, gemcitabine, vinorelbine, irinotecan,
etoposide, vinblastine, pemetrexed, 5-fluorouracil (with or without
folinic acid), capecitabine, carboplatin, epirubicin, oxaliplatin,
folfirinox, abraxane, navelbine and cyclophosphamide, capecitabine,
gemcitabine, navelbine, paclitaxel, nab-paclitaxel.
[0256] In some embodiments, the tumor is non-small cell lung cancer, and
the additional agent is one or more of cisplatin, carboplatin,
paclitaxel, albumin-bound paclitaxel, nab-paclitaxel, capecitabine,
navelbine, docetaxel, gemcitabine, vinorelbine, irinotecan, etoposide,
vinblastine or pemetrexed. In embodiments, the tumor is gastric or
stomach cancer, and the additional agent is one or more of 5-fluorouracil
(with or without folinic acid), capecitabine, carboplatin, cisplatin,
docetaxel, epirubicin, irinotecan, oxaliplatin, nab-paclitaxel or
paclitaxel. In other embodiments the tumor is pancreatic cancer, and the
additional agent is one or more of nab-paclitaxel, capecitabine,
navelbine, gemcitabine, folfirinox, abraxane, or 5-fluorouracil. In other
embodiments the tumor is a estrogen and/or progesterone positive breast
cancer, and the additional agent is one or more of paclitaxel,
capecitabine, navelbine, gemcitabine, paclitaxel or nab-paclitaxel or a
combination of (a) doxorubicin and epirubicin, (b) a combination of
paclitaxel and docetaxel, or (c) a combination of 5-fluorouracil,
cyclophosphamide and carboplatin. In other embodiments, the tumor is head
and neck cancer, and the additional agent is one or more of paclitaxel,
capecitabine, navelbine, gemcitabine or nab-paclitaxel carboplatin,
doxorubicin or cisplatin. In other embodiments, the tumor is ovarian
cancer and the additional agent may be one or more of capecitabine,
navelbine, gemcitabine, nab-paclitaxel, cisplatin, carboplatin, or a
taxane such as paclitaxel or docetaxel.
[0257] The additional agents may be administered to the subject being
treated concurrently with the antigen-binding constructs or sequentially.
[0258] The subject being treated with the antigen-binding constructs may
be a human, a non-human primate or other mammal such as a mouse.
[0259] In some embodiments, the result of providing an effective amount of
the antigen-binding construct to a subject having a tumor is shrinking
the tumor, inhibiting growth of the tumor, increasing time to progression
of the tumor, prolonging disease-free survival of the subject, decreasing
metastases, increasing the progression-free survival of the subject, or
increasing overall survival of the subject or increasing the overall
survival of a group of subjects receiving the treatment.
[0260] Also described herein are methods of killing or inhibiting the
growth of a HER2-expressing tumor cell comprising contacting the cell
with the antigen-binding construct provided herein.
[0261] In various embodiments, a tumor cell may be a HER2 1+ or 2+ human
pancreatic carcinoma cell, a HER2 3+ human lung carcinoma cell, a HER2 2+
human Caucasian bronchioaveolar carcinoma cell, a human pharyngeal
carcinoma cell, a HER2 2+ human tongue squamous cell carcinoma cell, a
HER2 2+ squamous cell carcinoma cell of the pharynx, a HER2 1+ or 2+
human colorectal carcinoma cell, a HER2 3+ human gastric carcinoma cell,
a HER2 1+ human breast ductal ER+ (estrogen receptor-positive) carcinoma
cell, a HER2 2+/3+ human ER+, HER2-amplified breast carcinoma cell, a
HER2 0+/1+ human triple negative breast carcinoma cell, a HER2 2+ human
endometrioid carcinoma cell, a HER2 1+ lung-metastatic malignant melanoma
cell, a HER2 1+ human cervix carcinoma cell, Her2 1+ human renal cell
carcinoma cell, or a HER2 1+ human ovary carcinoma cell.
[0262] In embodiments in which the antigen-binding constructs are
conjugated to DM1, the tumor cell may be a HER2 1+ or 2+ or 3+ human
pancreatic carcinoma cell, a HER2 2+ metastatic pancreatic carcinoma
cell, a HER2 0+/1+, +3+ human lung carcinoma cell, a HER2 2+ human
Caucasian bronchioaveolar carcinoma cell, a HER2 0+ anaplastic lung
carcinoma, a human non-small cell lung carcinoma cell, a human pharyngeal
carcinoma cell, a HER2 2+ human tongue squamous cell carcinoma cell, a
HER2 2+ squamous cell carcinoma cell of the pharynx, a HER2 1+ or 2+
human colorectal carcinoma cell, a HER2 0+, 1+ or 3+ human gastric
carcinoma cell, a HER2 1+ human breast ductal ER+ (estrogen
receptor-positive) carcinoma cell, a HER2 2+/3+ human ER+, HER2-amplified
breast carcinoma cell, a HER2 0+/1+ human triple negative breast
carcinoma cell, a HER2 0+ human breast ductal carcinoma (Basal B,
Mesenchymal-like triple negative) cell, a HER2 2+ER+ breast carcinoma, a
HER2 0+ human metastatic breast carcinoma cell (ER-, HER2-amplified,
luminal A, TN), a human uterus mesodermal tumor (mixed grade III) cell, a
2+ human endometrioid carcinoma cell, a HER2 1+ human skin epidermoid
carcinoma cell, a HER2 1+ lung-metastatic malignant melanoma cell, a HER2
1+ malignant melanoma cell, a human cervix epidermoid carcinoma vcell, a
HER2 1+ human urinary bladder carcinoma cell, a HER2 1+ human cervix
carcinoma cell, Her2 1+ human renal cell carcinoma cell, or a HER2 1+, 2+
or 3+ human ovary carcinoma cell.
[0263] In some embodiments the tumor cell may be one or more of the
following cell lines: pancreatic tumor cell lines BxPC3, Capan-1,
MiaPaca2; lung tumor cell lines Calu-3, NCI-H322; head and neck tumor
cells lines Detroit 562, SCC-25, FaDu; colorectal tumor cell lines HT29,
SNU-C2B; gastric tumor cell line NCI-N87; breast tumor cell lines MCF-7,
MDA-MB-175, MDA-MB-361, MDA-MB-231, BT-20, JIMT-1, SkBr3, BT-474; uterine
tumor cell line TOV-112D; skin tumor cell line Malme-3M; cervical tumor
cell lines Caski, MS751; bladder tumor cell line T24, ovarian tumor cell
lines CaOV3, and SKOV3.
[0264] In some embodiments in which the antigen-binding constructs are
conjugated to DM1, the tumor cell may be one or more of the following
cell lines: pancreatic tumor cell lines BxPC3, Capan-1, MiaPaca2, SW
1990, Pancl; lung tumor cell lines A549, Calu-3, Calu-6, NCI-H2126,
NCI-H322; head and neck tumor cells lines Detroit 562, SCC-15, SCC-25,
FaDu; colorectal tumor cell lines Colo201, DLD-1, HCT116, HT29, SNU-C2B;
gastric tumor cell lines SNU-1, SNU-16, NCI-N87; breast tumor cell lines
SkBr3, MCF-7, MDA-MB-175, MDA-MB-361, MDA-MB-231, ZR-75-1, BT-20, BT549,
BT-474, CAMA-1, MDA-MB-453, JIMT-1, T47D; Uterine tumor cell lines
SK-UT-1, TOV-112D; skin tumor cell lines A431, Malme-3M, SKEMEL28;
cervical tumor cell lines Caski, MS751; bladder tumor cell line T24,
renal tumor cell line ACHN; ovarian tumor cell lines CaOV3, Ovar-3, and
SKOV3.
[0265] Also described herein are methods of treating a subject having a
HER2 expressing (HER2+) tumor such as a HER2+ lung, head and neck, or
breast tumor by administering an antigen binding construct disclosed
herein. In some aspects, the tumor volume in the subject after receiving
at least seven doses of the antigen binding construct is less than the
tumor volume of a control subject receiving an equivalent amount of
trastuzumab. In some aspects, the survival of the subject receiving the
antigen binding construct is increased as compared to a control subject
receiving an equivalent amount of a non-specific control antibody or as
compared to a control subject not receiving treatment.
[0266] In some aspects, the tumor is a lung tumor, optionally wherein the
tumor is a non-squamous non-small cell lung tumor that is HER2-low,
non-HER2 gene amplified. In some aspects, the tumor is HER3+. In some
aspects, the tumor is EGFR low. In some aspects, the tumor is moderately
sensitive to Cisplatin at the MTD.
[0267] In some aspects, the tumor is a head and neck tumor, optionally
wherein the tumor is a squamous cell tumor of the head and neck that is
HER2 low, non-HER2 gene amplified. In some aspects, the tumor is HER3+
low. In some aspects, the tumor is EGFR+. In some aspects, the tumor is
highly sensitive to Cisplatin at the MTD.
[0268] In some aspects, the tumor is a breast tumor, optionally wherein
the tumor is a ER+/PR- breast cancer with a luminal B molecular
classification.
[0269] In some aspects, the subject is administered at least 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 doses. In some
aspects, the amount of at least one of the plurality of doses is at least
0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, or 20 mg/kg. In some aspects, the amount of each of the plurality of
doses is at least 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20 mg/kg. In some aspects, each dose is
administered at least daily, weekly, or monthly. In some aspects, each
dose is administered at least every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, or 31 days. In some aspects, treatment continues for at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, or 31 days; at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks; or at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
months.
[0270] In some aspects, the mean tumor volume in the subject after
receiving at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
doses is less than the mean tumor volume of a control subject receiving
an equivalent amount of trastuzumab.
[0271] In some aspects, overall survival of the subject is significantly
increased as compared to a control subject receiving an equivalent amount
of a non-specific control antibody or as compared to a control subject
not receiving treatment. In some aspects, the significance is measured by
a log rank test. In some aspects, the p value is less than 0.5, 0.01, or
0.001.
[0272] In some aspects, overall survival of the subject is more
significantly increased as compared to a control subject receiving an
equivalent amount of trastuzumab. In some aspects, the antigen-binding
construct p value is less than 0.001 and wherein the trastuzumab p value
is greater than 0.001.
[0273] In some aspects, the p value of the significance of the increase
relative to the control subject receiving an equivalent amount of a
non-specific control antibody is less than the p value of an increase in
survival of a second control receiving an equivalent amount of
trastuzumab as compared to the control subject receiving an equivalent
amount of a non-specific control antibody. In some aspects, the
antigen-binding construct p value is less than 0.001 and wherein the
trastuzumab p value is greater than 0.001.
[0274] In some aspects, overall survival of the subject after receiving a
combination of the antigen-binding construct and an additional agent is
significantly increased as compared to a control subject receiving an
equivalent amount of trastuzumab alone.
[0275] In some aspects, overall survival of the subject is significantly
increased as compared to a control subject receiving a lesser amount of
trastuzumab.
Kits and Articles of Manufacture
[0276] Also described herein are kits comprising one or more
antigen-binding construct described herein. Individual components of the
kit would be packaged in separate containers and, associated with such
containers, can be a notice in the form prescribed by a governmental
agency regulating the manufacture, use or sale of pharmaceuticals or
biological products, which notice reflects approval by the agency of
manufacture, use or sale. The kit may optionally contain instructions or
directions outlining the method of use or administration regimen for the
antigen-binding construct.
[0277] When one or more components of the kit are provided as solutions,
for example an aqueous solution, or a sterile aqueous solution, the
container means may itself be an inhalant, syringe, pipette, eye dropper,
or other such like apparatus, from which the solution may be administered
to a subject or applied to and mixed with the other components of the
kit.
[0278] The components of the kit may also be provided in dried or
lyophilized form and the kit can additionally contain a suitable solvent
for reconstitution of the lyophilized components. Irrespective of the
number or type of containers, the kits described herein also may comprise
an instrument for assisting with the administration of the composition to
a patient. Such an instrument may be an inhalant, nasal spray device,
syringe, pipette, forceps, measured spoon, eye dropper or similar
medically approved delivery vehicle.
[0279] In another aspect described herein, an article of manufacture
containing materials useful for the treatment, prevention and/or
diagnosis of the disorders described above is provided. The article of
manufacture comprises a container and a label or package insert on or
associated with the container. Suitable containers include, for example,
bottles, vials, syringes, IV solution bags, etc. The containers may be
formed from a variety of materials such as glass or plastic. The
container holds a composition which is by itself or combined with another
composition effective for treating, preventing and/or diagnosing the
condition and may have a sterile access port (for example the container
may be an intravenous solution bag or a vial having a stopper pierceable
by a hypodermic injection needle). At least one active agent in the
composition is a T cell activating antigen-binding construct described
herein. The label or package insert indicates that the composition is
used for treating the condition of choice. Moreover, the article of
manufacture may comprise (a) a first container with a composition
contained therein, wherein the composition comprises an antigen-binding
construct described herein; and (b) a second container with a composition
contained therein, wherein the composition comprises a further cytotoxic
or otherwise therapeutic agent. The article of manufacture in this
embodiment described herein may further comprise a package insert
indicating that the compositions can be used to treat a particular
condition. Alternatively, or additionally, the article of manufacture may
further comprise a second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water for
injection (BWFI), phosphate-buffered saline, Ringer's solution and
dextrose solution. It may further include other materials desirable from
a commercial and user standpoint, including other buffers, diluents,
filters, needles, and syringes.
Polypeptides and Polynucleotides
[0280] The antigen-binding constructs described herein comprise at least
one polypeptide. Also described are polynucleotides encoding the
polypeptides described herein. The antigen-binding constructs are
typically isolated.
[0281] As used herein, "isolated" means an agent (e.g., a polypeptide or
polynucleotide) that has been identified and separated and/or recovered
from a component of its natural cell culture environment. Contaminant
components of its natural environment are materials that would interfere
with diagnostic or therapeutic uses for the antigen-binding construct,
and may include enzymes, hormones, and other proteinaceous or
non-proteinaceous solutes. Isolated also refers to an agent that has been
synthetically produced, e.g., via human intervention.
[0282] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid residues. That
is, a description directed to a polypeptide applies equally to a
description of a peptide and a description of a protein, and vice versa.
The terms apply to naturally occurring amino acid polymers as well as
amino acid polymers in which one or more amino acid residues is a
non-naturally encoded amino acid. As used herein, the terms encompass
amino acid chains of any length, including full length proteins, wherein
the amino acid residues are linked by covalent peptide bonds.
[0283] The term "amino acid" refers to naturally occurring and
non-naturally occurring amino acids, as well as amino acid analogs and
amino acid mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally encoded amino acids are the 20 common
amino acids (alanine, arginine, asparagine, aspartic acid, cysteine,
glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,
lysine, methionine, phenylalanine, praline, serine, threonine,
tryptophan, tyrosine, and valine) and pyrrolysine and selenocysteine.
Amino acid analogs refers to compounds that have the same basic chemical
structure as a naturally occurring amino acid, i.e., an a carbon that is
bound to a hydrogen, a carboxyl group, an amino group, and an R group,
such as, homoserine, norleucine, methionine sulfoxide, methionine methyl
sulfonium. Such analogs have modified R groups (such as, norleucine) or
modified peptide backbones, but retain the same basic chemical structure
as a naturally occurring amino acid. Reference to an amino acid includes,
for example, naturally occurring proteogenic L-amino acids; D-amino
acids, chemically modified amino acids such as amino acid variants and
derivatives; naturally occurring non-proteogenic amino acids such as
.beta.-alanine, ornithine, etc.; and chemically synthesized compounds
having properties known in the art to be characteristic of amino acids.
Examples of non-naturally occurring amino acids include, but are not
limited to, a-methyl amino acids (e.g. a-methyl alanine), D-amino acids,
histidine-like amino acids (e.g., 2-amino-histidine,
.beta.-hydroxy-histidine, homohistidine), amino acids having an extra
methylene in the side chain ("homo" amino acids), and amino acids in
which a carboxylic acid functional group in the side chain is replaced
with a sulfonic acid group (e.g., cysteic acid). The incorporation of
non-natural amino acids, including synthetic non-native amino acids,
substituted amino acids, or one or more D-amino acids into the proteins
of the present invention may be advantageous in a number of different
ways. D-amino acid-containing peptides, etc., exhibit increased stability
in vitro or in vivo compared to L-amino acid-containing counterparts.
Thus, the construction of peptides, etc., incorporating D-amino acids can
be particularly useful when greater intracellular stability is desired or
required. More specifically, D-peptides, etc., are resistant to
endogenous peptidases and proteases, thereby providing improved
bioavailability of the molecule, and prolonged lifetimes in vivo when
such properties are desirable. Additionally, D-peptides, etc., cannot be
processed efficiently for major histocompatibility complex class
II-restricted presentation to T helper cells, and are therefore, less
likely to induce humoral immune responses in the whole organism.
[0284] Amino acids may be referred to herein by either their commonly
known three letter symbols or by the one-letter symbols recommended by
the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,
may be referred to by their commonly accepted single-letter codes.
[0285] Also included in the invention are polynucleotides encoding
polypeptides of the antigen-binding constructs. The term "polynucleotide"
or "nucleotide sequence" is intended to indicate a consecutive stretch of
two or more nucleotide molecules. The nucleotide sequence may be of
genomic, cDNA, RNA, semisynthetic or synthetic origin, or any combination
thereof.
[0286] The term "nucleic acid" refers to deoxyribonucleotides,
deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers
thereof in either single- or double-stranded form. Unless specifically
limited, the term encompasses nucleic acids containing known analogues of
natural nucleotides which have similar binding properties as the
reference nucleic acid and are metabolized in a manner similar to
naturally occurring nucleotides. Unless specifically limited otherwise,
the term also refers to oligonucleotide analogs including PNA
(peptidonucleic acid), analogs of DNA used in antisense technology
(phosphorothioates, phosphoroamidates, and the like). Unless otherwise
indicated, a particular nucleic acid sequence also implicitly encompasses
conservatively modified variants thereof (including but not limited to,
degenerate codon substitutions) and complementary sequences as well as
the sequence explicitly indicated. Specifically, degenerate codon
substitutions may be achieved by generating sequences in which the third
position of one or more selected (or all) codons is substituted with
mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.
19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985);
Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
[0287] "Conservatively modified variants" applies to both amino acid and
nucleic acid sequences. With respect to particular nucleic acid
sequences, "conservatively modified variants" refers to those nucleic
acids which encode identical or essentially identical amino acid
sequences, or where the nucleic acid does not encode an amino acid
sequence, to essentially identical sequences. Because of the degeneracy
of the genetic code, a large number of functionally identical nucleic
acids encode any given protein. For instance, the codons GCA, GCC, GCG
and GCU all encode the amino acid alanine. Thus, at every position where
an alanine is specified by a codon, the codon can be altered to any of
the corresponding codons described without altering the encoded
polypeptide. Such nucleic acid variations are "silent variations," which
are one species of conservatively modified variations. Every nucleic acid
sequence herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of ordinary skill in the art
will recognize that each codon in a nucleic acid (except AUG, which is
ordinarily the only codon for methionine, and TGG, which is ordinarily
the only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic acid
which encodes a polypeptide is implicit in each described sequence.
[0288] As to amino acid sequences, one of ordinary skill in the art will
recognize that individual substitutions, deletions or additions to a
nucleic acid, peptide, polypeptide, or protein sequence which alters,
adds or deletes a single amino acid or a small percentage of amino acids
in the encoded sequence is a "conservatively modified variant" where the
alteration results in the deletion of an amino acid, addition of an amino
acid, or substitution of an amino acid with a chemically similar amino
acid. Conservative substitution tables providing functionally similar
amino acids are known to those of ordinary skill in the art. Such
conservatively modified variants are in addition to and do not exclude
polymorphic variants, interspecies homologs, and alleles described
herein.
[0289] Conservative substitution tables providing functionally similar
amino acids are known to those of ordinary skill in the art. The
following eight groups each contain amino acids that are conservative
substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic
acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4)
Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M),
Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine
(S), Threonine (T); and [0139] 8) Cysteine (C), Methionine (M) (see,
e.g., Creighton, Proteins: Structures and Molecular Properties (W H
Freeman & Co.; 2nd edition (December 1993)
[0290] The terms "identical" or percent "identity," in the context of two
or more nucleic acids or polypeptide sequences, refer to two or more
sequences or subsequences that are the same. Sequences are "substantially
identical" if they have a percentage of amino acid residues or
nucleotides that are the same (i.e., about 60% identity, about 65%, about
70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity
over a specified region), when compared and aligned for maximum
correspondence over a comparison window, or designated region as measured
using one of the following sequence comparison algorithms (or other
algorithms available to persons of ordinary skill in the art) or by
manual alignment and visual inspection. This definition also refers to
the complement of a test sequence. The identity can exist over a region
that is at least about 50 amino acids or nucleotides in length, or over a
region that is 75-100 amino acids or nucleotides in length, or, where not
specified, across the entire sequence of a polynucleotide or polypeptide.
A polynucleotide encoding a polypeptide of the present invention,
including homologs from species other than human, may be obtained by a
process comprising the steps of screening a library under stringent
hybridization conditions with a labeled probe having a polynucleotide
sequence described herein or a fragment thereof, and isolating
full-length cDNA and genomic clones containing said polynucleotide
sequence. Such hybridization techniques are well known to the skilled
artisan.
[0291] For sequence comparison, typically one sequence acts as a reference
sequence, to which test sequences are compared. When using a sequence
comparison algorithm, test and reference sequences are entered into a
computer, subsequence coordinates are designated, if necessary, and
sequence algorithm program parameters are designated. Default program
parameters can be used, or alternative parameters can be designated. The
sequence comparison algorithm then calculates the percent sequence
identities for the test sequences relative to the reference sequence,
based on the program parameters.
[0292] A "comparison window", as used herein, includes reference to a
segment of any one of the number of contiguous positions selected from
the group consisting of from 20 to 600, usually about 50 to about 200,
more usually about 100 to about 150 in which a sequence may be compared
to a reference sequence of the same number of contiguous positions after
the two sequences are optimally aligned. Methods of alignment of
sequences for comparison are known to those of ordinary skill in the art.
Optimal alignment of sequences for comparison can be conducted, including
but not limited to, by the local homology algorithm of Smith and Waterman
(1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of
Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for
similarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci.
USA 85:2444, by computerized implementations of these algorithms (GAP,
BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection (see, e.g., Ausubel et al., Current
Protocols in Molecular Biology (1995 supplement)).
[0293] One example of an algorithm that is suitable for determining
percent sequence identity and sequence similarity are the BLAST and BLAST
2.0 algorithms, which are described in Altschul et al. (1997) Nuc. Acids
Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,
respectively. Software for performing BLAST analyses is publicly
available through the National Center for Biotechnology Information
available at the World Wide Web at ncbi.nlm.nih.gov. The BLAST algorithm
parameters W, T, and X determine the sensitivity and speed of the
alignment. The BLASTN program (for nucleotide sequences) uses as defaults
a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a
comparison of both strands. For amino acid sequences, the BLASTP program
uses as defaults a wordlength of 3, and expectation (E) of 10, and the
BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl.
Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands. The BLAST algorithm is
typically performed with the "low complexity" filter turned off
[0294] The BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin and Altschul (1993)
Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity
provided by the BLAST algorithm is the smallest sum probability (P(N)),
which provides an indication of the probability by which a match between
two nucleotide or amino acid sequences would occur by chance. For
example, a nucleic acid is considered similar to a reference sequence if
the smallest sum probability in a comparison of the test nucleic acid to
the reference nucleic acid is less than about 0.2, or less than about
0.01, or less than about 0.001.
[0295] The phrase "selectively (or specifically) hybridizes to" refers to
the binding, duplexing, or hybridizing of a molecule only to a particular
nucleotide sequence under stringent hybridization conditions when that
sequence is present in a complex mixture (including but not limited to,
total cellular or library DNA or RNA).
[0296] The phrase "stringent hybridization conditions" refers to
hybridization of sequences of DNA, RNA, or other nucleic acids, or
combinations thereof under conditions of low ionic strength and high
temperature as is known in the art. Typically, under stringent conditions
a probe will hybridize to its target subsequence in a complex mixture of
nucleic acid (including but not limited to, total cellular or library DNA
or RNA) but does not hybridize to other sequences in the complex mixture.
Stringent conditions are sequence-dependent and will be different in
different circumstances. Longer sequences hybridize specifically at
higher temperatures. An extensive guide to the hybridization of nucleic
acids is found in Tijssen, Laboratory Techniques in Biochemistry and
Molecular Biology--Hybridization with Nucleic Probes, "Overview of
principles of hybridization and the strategy of nucleic acid assays"
(1993).
[0297] As used herein, the terms "engineer, engineered, engineering", are
considered to include any manipulation of the peptide backbone or the
post-translational modifications of a naturally occurring or recombinant
polypeptide or fragment thereof. Engineering includes modifications of
the amino acid sequence, of the glycosylation pattern, or of the side
chain group of individual amino acids, as well as combinations of these
approaches. The engineered proteins are expressed and produced by
standard molecular biology techniques.
[0298] By "isolated nucleic acid molecule or polynucleotide" is intended a
nucleic acid molecule, DNA or RNA, which has been removed from its native
environment. For example, a recombinant polynucleotide encoding a
polypeptide contained in a vector is considered isolated. Further
examples of an isolated polynucleotide include recombinant
polynucleotides maintained in heterologous host cells or purified
(partially or substantially) polynucleotides in solution. An isolated
polynucleotide includes a polynucleotide molecule contained in cells that
ordinarily contain the polynucleotide molecule, but the polynucleotide
molecule is present extrachromosomally or at a chromosomal location that
is different from its natural chromosomal location. Isolated RNA
molecules include in vivo or in vitro RNA transcripts, as well as
positive and negative strand forms, and double-stranded forms. Isolated
polynucleotides or nucleic acids described herein, further include such
molecules produced synthetically, e.g., via PCR or chemical synthesis. In
addition, a polynucleotide or a nucleic acid, in certain embodiments,
include a regulatory element such as a promoter, ribosome binding site,
or a transcription terminator.
[0299] The term "polymerase chain reaction" or "PCR" generally refers to a
method for amplification of a desired nucleotide sequence in vitro, as
described, for example, in U.S. Pat. No. 4,683,195. In general, the PCR
method involves repeated cycles of primer extension synthesis, using
oligonucleotide primers capable of hybridising preferentially to a
template nucleic acid.
[0300] By a nucleic acid or polynucleotide having a nucleotide sequence at
least, for example, 95% "identical" to a reference nucleotide sequence of
the present invention, it is intended that the nucleotide sequence of the
polynucleotide is identical to the reference sequence except that the
polynucleotide sequence may include up to five point mutations per each
100 nucleotides of the reference nucleotide sequence. In other words, to
obtain a polynucleotide having a nucleotide sequence at least 95%
identical to a reference nucleotide sequence, up to 5% of the nucleotides
in the reference sequence may be deleted or substituted with another
nucleotide, or a number of nucleotides up to 5% of the total nucleotides
in the reference sequence may be inserted into the reference sequence.
These alterations of the reference sequence may occur at the 5' or 3'
terminal positions of the reference nucleotide sequence or anywhere
between those terminal positions, interspersed either individually among
residues in the reference sequence or in one or more contiguous groups
within the reference sequence. As a practical matter, whether any
particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%,
97%, 98% or 99% identical to a nucleotide sequence of the present
invention can be determined conventionally using known computer programs,
such as the ones discussed above for polypeptides (e.g. ALIGN-2).
[0301] A derivative, or a variant of a polypeptide is said to share
"homology" or be "homologous" with the peptide if the amino acid
sequences of the derivative or variant has at least 50% identity with a
100 amino acid sequence from the original peptide. In certain
embodiments, the derivative or variant is at least 75% the same as that
of either the peptide or a fragment of the peptide having the same number
of amino acid residues as the derivative. In certain embodiments, the
derivative or variant is at least 85% the same as that of either the
peptide or a fragment of the peptide having the same number of amino acid
residues as the derivative. In certain embodiments, the amino acid
sequence of the derivative is at least 90% the same as the peptide or a
fragment of the peptide having the same number of amino acid residues as
the derivative. In some embodiments, the amino acid sequence of the
derivative is at least 95% the same as the peptide or a fragment of the
peptide having the same number of amino acid residues as the derivative.
In certain embodiments, the derivative or variant is at least 99% the
same as that of either the peptide or a fragment of the peptide having
the same number of amino acid residues as the derivative.
[0302] The term "modified," as used herein refers to any changes made to a
given polypeptide, such as changes to the length of the polypeptide, the
amino acid sequence, chemical structure, co-translational modification,
or post-translational modification of a polypeptide. The form
"(modified)" term means that the polypeptides being discussed are
optionally modified, that is, the polypeptides under discussion can be
modified or unmodified.
[0303] In some aspects, an antigen-binding construct comprises an amino
acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% identical to a relevant amino acid sequence or fragment
thereof set forth in the Table(s) or accession number(s) disclosed
herein. In some aspects, an isolated antigen-binding construct comprises
an amino acid sequence encoded by a polynucleotide that is at least 80,
85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a
relevant nucleotide sequence or fragment thereof set forth in Table(s) or
accession number(s) disclosed herein.
[0304] It is to be understood that this invention is not limited to the
particular protocols; cell lines, constructs, and reagents described
herein and as such may vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the present
invention
[0305] All publications and patents mentioned herein are incorporated
herein by reference for the purpose of describing and disclosing, for
example, the constructs and methodologies that are described in the
publications, which might be used in connection with the presently
described invention. The publications discussed herein are provided
solely for their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of prior
invention or for any other reason.
EXAMPLES
[0306] Below are examples of specific embodiments for carrying out the
present invention. The examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present invention in
any way. Efforts have been made to ensure accuracy with respect to
numbers used (e.g., amounts, temperatures, etc.), but some experimental
error and deviation should, of course, be allowed for.
[0307] The practice of the present invention will employ, unless otherwise
indicated, conventional methods of protein chemistry, biochemistry,
recombinant DNA techniques and pharmacology, within the skill of the art.
Such techniques are explained fully in the literature. See, e.g., T. E.
Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman
and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers,
Inc., current addition); Sambrook, et al., Molecular Cloning: A
Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick
and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical
Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990);
Carey and Sundberg Advanced Organic Chemistry 3.sup.rd Ed. (Plenum Press)
Vols A and B (1992).
Example 1: Preparation of Exemplary Anti-HER2 Bispecific Antibodies and
Controls
[0308] A number of exemplary anti-HER2 biparatopic antibodies (or
antigen-binding constructs) and controls were prepared as described
below. The antibodies and controls have been prepared in different
formats, and representations of exemplary biparatopic formats are shown
in FIG. 1. In all of the formats shown in FIG. 1, the heterodimeric Fc is
depicted with one chain (Chain A) shown in black and the other (Chain B)
shown in grey, while one antigen-binding domain (1) is shown in hatched
fill, while the other antigen-binding domain (2) is shown in white.
[0309] FIG. 1A depicts the structure of a biparatopic antibody in a
Fab-Fab format. FIGS. 1B to 1E depict the structure of possible versions
of a biparatopic antibody in an scFv-Fab format. In FIG. 1B,
antigen-binding domain 1 is an scFv, fused to Chain A, while
antigen-binding domain 2 is a Fab, fused to Chain B. In FIG. 1C,
antigen-binding domain 1 is a Fab, fused to Chain A, while
antigen-binding domain 2 is an scFv, fused to Chain B. In FIG. 1D,
antigen-binding domain 2 is a Fab, fused to Chain A, while
antigen-binding domain 1 is an scFv, fused to Chain B. In FIG. 1E,
antigen-binding domain 2 is an scFv, fused to Chain A, while
antigen-binding domain 1 is a Fab, fused to Chain B. In FIG. 1F, both
antigen-binding domains are scFvs.
[0310] The sequences of the following variants are provided in the
Sequence Table found after the Examples. CDR regions were identified
using a combination of the Kabat and Chothia methods. Regions may vary
slightly based on method used for identification.
[0311] Exemplary Anti-HER2 Biparatopic Antibodies
[0312] Exemplary anti-HER2 biparatopic antibodies were prepared as shown
in Table 1.
TABLE-US-00008
TABLE 1
Exemplary anti-HER2 biparatbopic antibodies
Variant Chain A Chain B
5019 domain ECD2 ECD4
containing
the epitope
Format Fab scFv
Antibody Pertuzumab Trastuzumab
name
CH3 T350V_L351Y_F405A_Y407V T366I_N390R_K392M_T394W
sequence
substitutions
5020 domain ECD4 ECD2
containing
the epitope
format scFv Fab
Antibody Trastuzumab Pertuzumab
name
CH3 L351Y_S400E_F405A_Y407V T350V_T366L_K392L_T394W
sequence
substitutions
7091 domain ECD2 ECD4
containing
the epitope
format Fab scFv
Antibody Pertuzumab Trastuzumab
name
CH3 T350V_L351Y_F405A_Y407V T350V_T366L_K392L_T394W
sequence
substitutions
10000 domain ECD2 ECD4
containing
the epitope
format Fab scFv
Antibody Pertuzumab - with Y96A in VL Trastuzumab
name region and T30A/A49G/L69F in
VH region
CH3 T350V_L351Y_F405A_Y407V T350V_T366L_K392L_T394W
sequence
substitutions
6902 domain ECD2 ECD4
containing
the epitope
format Fab Fab
Antibody Trastuzumab Pertuzumab
name
Fab HC: L143E_K145T HC: D146G_Q179K
substitutions LC: Q124R LC: Q124E_Q160E_T180E
CH3 T350V_L351Y_F405A_Y407V T350V_T366L_K392L_T394W
sequence
substitutions
6903 domain ECD2 ECD4
containing
the epitope
format Fab Fab
Fab HC: L143E_K145T HC: D146G_Q179K
substitutions LC: Q124R_Q1160K_T178R LC: Q124E_Q160E_T180E
Antibody Trastuzumab Pertuzumab
name
CH3 T350V_L351Y_F405A_Y407V T350V_T366L_K392L_T394W
sequence
substitutions
6717 domain ECD4 ECD2
containing
the epitope
format scFv scFv
Antibody Pertuzumab Trastuzumab
name
CH3 T350V_L351Y_F405A_Y407V T366I_N390R_K392M_T394W
sequence
substitutions
Notes:
CH3 numbering according to EU index as in Kabat referring to the numbering
of the EU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:
78-85);
Fab or variable domain numbering according to Kabat (Kabat and Wu, 1991;
Kabat et al, Sequences of proteins of immunological interest. 5th Edition
- US Department of Health and Human Services, NIH publication no 91-3242,
p 647 (1991))
"domain containing the epitope" = domain of HER2 to which antigen-binding
moiety binds;
"Antibody name" = antibody from which antigen-binding moiety is derived,
includes substitutions compared to wild-type when present;
"Fab substitutions" = substitutions in Fab that promote correct light
chain pairing;
"CH3 sequence substitutions" = substitutions in CH3 domain that promote
formation of heterodimeric Fc
[0313] Exemplary Anti-HER2 Monovalent Control Antibodies
[0314] v1040: a monovalent anti-HER2 antibody, where the HER2 binding
domain is a Fab derived from trastuzumab on chain A, and the Fc region is
a heterodimer having the mutations T350V_L351Y_F405A_Y407V in Chain A,
T350V_T366L_K392L_T394W in Chain B, and the hinge region of Chain B
having the mutation C226S; the antigen-binding domain binds to domain 4
of HER2.
[0315] v630--a monovalent anti-HER2 antibody, where the HER2 binding
domain is an scFv derived from trastuzumab on Chain A, and the Fc region
is a heterodimer having the mutations L351Y_S400E_F405A_Y407V in Chain A,
T366I_N390R_K392M_T394W in Chain B; and the hinge region having the
mutation C226S (EU numbering) in both chains; the antigen-binding domain
binds to domain 4 of HER2.
[0316] v4182: a monovalent anti-HER2 antibody, where the HER2 binding
domain is a Fab derived from pertuzumab on chain A, and the Fc region is
a heterodimer having the mutations T350V_L351Y_F405A_Y407V in Chain A,
T350V_T366L_K392L_T394W in Chain B, and the hinge region of Chain B
having the mutation C226S; the antigen-binding domain binds to domain 2
of HER2.
[0317] Exemplary Anti-HER2 Monospecific Bivalent Antibody Controls
(Full-Sized Antibodies, FSAs)
[0318] v506 is a wild-type anti HER2 produced in-house in Chinese Hamster
Ovary (CHO) cells, as a control. Both HER2 binding domains are derived
from trastuzumab in the Fab format and the Fc is a wild type homodimer;
the antigen-binding domain binds to domain 4 of HER2. This antibody is
also referred to as a trastuzumab analog.
[0319] v792, is wild-type trastuzumab with a IgG1 hinge, where both HER2
binding domains are derived from trastuzumab in the Fab format, and the
and the Fc region is a heterodimer having the mutations
T350V_L351Y_F405A_Y407V in Chain A, and T350V_T366L_K392L_T394W Chain B;
the antigen-binding domain binds to domain 4 of HER2. This antibody is
also referred to as a trastuzumab analog.
[0320] v4184, a bivalent anti-HER2 antibody, where both HER2 binding
domains are derived from pertuzumab in the Fab format, and the Fc region
is a heterodimer having the mutations T350V_L351Y_F405A_Y407V in Chain A,
and T350V_T366L_K392L_T394W Chain B. The antigen-binding domain binds to
domain 2 of HER2. This antibody is also referred to as a pertuzumab
analog.
[0321] hIgG, is a commercial non-specific polyclonal antibody control
(Jackson ImmunoResearch, #009-000-003).
[0322] These antibodies and controls (other than human IgG) were cloned
and expressed as follows. The genes encoding the antibody heavy and light
chains were constructed via gene synthesis using codons optimized for
human/mammalian expression. The Trastuzumab Fab sequence was generated
from a known HER2/neu domain 4 binding antibody (Carter P. et al. (1992)
Humanization of an anti p185 HER2 antibody for human cancer therapy. Proc
Natl Acad Sci 89, 4285.) And the Fc was an IgG1 isotype. The scFv
sequence was generated from the VH and VL domains of Trastuzumab using a
glycine-serine linker (Carter P. et al. (1992) Humanization of an anti
p185 her2 antibody for human cancer therapy. Proc Natl Acad Sci 89,
4285.). The Pertuzumab Fab sequence was generated from a known HER2/neu
domain 2 binding Ab (Adams C W et al. (2006) Humanization of a
recombinant monoclonal antibody to produce a therapeutic her dimerization
inhibitor, Pertuzumab. Cancer Immunol Immunother. 2006; 55(6):717-27).
[0323] The final gene products were sub-cloned into the mammalian
expression vector PTT5 (NRC-BRI, Canada) and expressed in CHO cells
(Durocher, Y., Perret, S. & Kamen, A. High-level and high-throughput
recombinant protein production by transient transfection of
suspension-growing CHO cells. Nucleic acids research 30, e9 (2002)).
[0324] The CHO cells were transfected in exponential growth phase (1.5 to
2 million cells/ml) with aqueous 1 mg/ml 25 kDa polyethylenimine (PEI,
polysciences) at a PEI:DNA ratio of 2.5:1. (Raymond C. et al. A
simplified polyethylenimine-mediated transfection process for large-scale
and high-throughput applications. Methods. 55(1):44-51 (2011)). To
determine the optimal concentration range for forming heterodimers, the
DNA was transfected in optimal DNA ratios of the heavy chain a (HC-A),
light chain (LC), and heavy chain B (HC-B) that allow for heterodimer
formation (e.g. HC-A/HC-B/LC ratios=30:30:40 (v5019). Transfected cells
were harvested after 5-6 days with the culture medium collected after
centrifugation at 4000 rpm and clarified using a 0.45 .mu.m filter.
[0325] The clarified culture medium was loaded onto a MabSelect SuRe (GE
Healthcare) protein-A column and washed with 10 column volumes of PBS
buffer at pH 7.2. The antibody was eluted with 10 column volumes of
citrate buffer at pH 3.6 with the pooled fractions containing the
antibody neutralized with TRIS at pH 11.
[0326] The protein-A antibody eluate was further purified by gel
filtration (SEC). For gel filtration, 3.5 mg of the antibody mixture was
concentrated to 1.5 mL and loaded onto a Sephadex 200 HiLoad 16/600 200
pg column (GE Healthcare) via an AKTA Express FPLC at a flow-rate of 1
mL/min. PBS buffer at pH 7.4 was used at a flow-rate of 1 mL/min.
Fractions corresponding to the purified antibody were collected,
concentrated to .about.1 mg/mL.
[0327] Exemplary anti-HER2 ECD2.times.ECD4 biparatopic antibodies with
different molecular formats (e.g. v6717, scFv-scFv IgG1; v6903 and v6902
Fab-Fab IgG1; v5019, v7091 and v10000 Fab-scFv IgG1) were cloned,
expressed and purified as described above.
[0328] To quantify antibody purity and to determine the amount of target
heterodimer protein and possible homodimer and/or half antibody and/or
mispaired light chain contaminant, LC-MS intact mass analysis was
performed. The LC-MS intact mass analysis was performed as described in
Example 2, excluding DAR analysis calculations used for ADC molecules.
[0329] The data is shown in Table 2. Table 2 shows that expression and
purification of these biparatopic antibodies resulted in 100% of the
desired product for v6717, 91% of the desired heterodimeric product for
v6903, and 62% of the desired product for v6902. The numbers in brackets
indicate the quantities of the main peak plus a side peak of +81 Da. This
side peak is typically detected with variants that contain C-terminal HA
tags (such of v6903 and v6902). Adding the main and side peaks yields
heterodimer purities of approximately 98% and 67% for v6903 and v6903.
Based on the high heterodimer purity, v6903 was identified as the
representative Fab-Fab anti-HER2 biparatopic variant for direct
comparison to the scFv-scFv and Fab-scFv formats. v6903 was included in
all format comparison assays.
TABLE-US-00009
TABLE 2
Expression and purification of antibodies
Variant Desired heterodimer species (+side peak)
6717 100.0
6903 90.9 (97.7)
6902 62.4 (67.4)
Example 2: Preparation of Exemplary Anti-HER2 Biparatopic Antibody Drug
Conjugates (ADCs)
[0330] The following anti-HER2 biparatopic antibody drug conjugates
(anti-HER2 biparatopic-ADCs) were prepared. ADCs of variants 5019, 7091,
10000 and 506 were prepared. These ADCs are identified as follows:
[0331] v6363 (v5019 conjugated to DM1) [0332] v7148 (v7091 conjugated to
DM1) [0333] v10553 (v10000 conjugated to DM1) [0334] v6246 (v506
conjugated to DM1, analogous to T-DM1, trastuzumab-emtansine) [0335]
v6249 (human IgG conjugated to DM1)
[0336] The ADCs were prepared via direct coupling to maytansine.
Antibodies purified by Protein A and SEC, as described in Example 1
(>95% purity), were used in the preparation of the ADC molecules. ADCs
were conjugated following the method described in Kovtun Y V, Audette C
A, Ye Y, et al. Antibody-drug conjugates designed to eradicate tumors
with homogeneous and heterogeneous expression of the target antigen.
Cancer Res 2006; 66:3214-21. The ADCs had an average molar ratio of 3.0
maytansinoid molecules per antibody as determined by LC/MS and described
below.
[0337] Details of the reagents used in the ADC conjugation reaction are as
follows: Conjugation Buffer 1: 50 mM Potassium Phosphate/50 mM Sodium
Chloride, pH 6.5, 2 mM EDTA. Conjugation Buffer 2: 50 mM Sodium
Succinate, pH 5.0. ADC formulation buffer: 20 mM Sodium Succinate, 6%
(w/v) Trehalose, 0.02% polysorbate 20, pH 5.0. Dimethylacetamide (DMA);
10 mM SMCC in DMA (prepared before conjugation), 10 mM DM1-SH in DMA
(prepared before conjugation), 1 mM DTNB in PBS, 1 mM Cysteine in buffer,
20 mM Sodium Succinate, pH 5.0. UV-VIS spectrophotometer (Nano drop 100
from Fisher Scientific), PD-10 columns (GE Healthcare).
[0338] The ADCs were prepared as follows. The starting antibody solution
was loaded onto the PD-10 column, previously equilibrated with 25 mL of
Conjugation Buffer 1, followed by 0.5 ml Conjugation Buffer 1. The
antibody eluate was collect and the concentration measured at A.sub.280
and the concentration was adjusted to 20 mg/mL. The 10 mM SMCC-DM1
solution in DMA was prepared. A 7.5 molar equivalent of SMCC-DM1 to
antibody was added to the antibody solution and DMA was added to a final
DMA volume of 10% v/v. The reaction was briefly mixed and incubated at RT
for 2 h. A second PD-10 column was equilibrated with 25 ml of Conjugation
Buffer 1 and the antibody-MCC-DM1 solution was added to the column follow
by 0.5 ml of Buffer 1. The antibody-MCC-DM1 eluate was collected and the
A.sub.252 and A.sub.280 of antibody solution was measured. The
Antibody-MCC-DM1 concentration was calculated (.quadrature.=1.45
mg.sup.-1cm.sup.-1, or 217500 M.sup.-1cm.sup.-1). The ADCs were analyzed
on a SEC-HPLC column for high MW analysis (SEC-HPLC column TOSOH,
G3000-SWXL, 7.8 mm.times.30 cm, Buffer, 100 mM Sodium phosphate, 300 mM
Sodium Chloride, pH 7.0, flow rate: 1 ml/min).
[0339] ADC drug to antibody ratio (DAR) was analysed by HIC-HPLC_using the
Tosoh TSK gel Butyl-NPR column (4.6 mm.times.3.5 mm.times.2.5 mm).
Elution was performed at 1 ml/min using a gradient of 10-90% buffer B
over 25 min followed by 100% buffer B for 4 min. Buffer A comprises 20 mM
sodium phosphate, 1.5 M ammonium sulphate, pH 7.0. Buffer B comprises 20
mM sodium phosphate, 25% v/v isopropanol, pH 7.0.
[0340] ADC drug to antibody ratio (DAR) was determined by LC-MS by the
following method. The antibodies were deglycosylated with PNGase F prior
to loading on the LC-MS. Liquid chromatography was carried out on an
Agilent 1100 Series HPLC under the following conditions:
[0341] Flow rate: 1 mL/min split post column to 100 uL/min to MS.
Solvents: A=0.1% formic acid in ddH2O, B=65% acetonitrile, 25% THF, 9.9%
ddH2O, 0.1% formic acid. Column: 2.1.times.30 mm PorosR2. Column
Temperature: 80.degree. C.; solvent also pre-heated. Gradient: 20% B (0-3
min), 20-90% B (3-6 min), 90-20% B (6-7 min), 20% B (7-9 min).
[0342] Mass Spectrometry (MS) was subsequently carried out on an
LTQ-Orbitrap XL mass spectrometer under the following conditions:
Ionization method using Ion Max Electrospray. Calibration and Tuning
Method: 2 mg/mL solution of CsI is infused at a flowrate of 104/min. The
Orbitrap was tuned on m/z 2211 using the Automatic Tune feature (overall
CsI ion range observed: 1690 to 2800). Cone Voltage: 40V; Tube Lens:
115V; FT Resolution: 7,500; Scan range m/z 400-4000; Scan Delay: 1.5 min.
A molecular weight profile of the data was generated using Thermo's
Promass deconvolution software. Average DAR of the sample was determined
as a function of DAR observed at each fractional peak (using the
calculation: (DAR.times.fractional peak intensity)).
[0343] Table 3 summarizes the average DAR for the ADC molecules. The
average DAR for the exemplary anti-HER2 biparatopic antibody and control
was approximately 3.
TABLE-US-00010
TABLE 3
Average DAR for ADCs
DAR (LC-MS) DAR (HIC) n
v6246 2.9 3.0 5
v6363 2.6 3.3 5
v7148 3.4 3.9 1
v10553 4.0 4.0 1
Example 3: Expression and Bench-Scale Purification of Anti-HER2
Biparatopic Antibody
[0344] The anti-HER2 biparatopic antibodies (v5019, v7091 and v10000)
described in Example 1 were expressed in 10 and/or 25 L volumes and
purified by protein A and size exclusion chromatography (SEC) as follows.
[0345] The clarified culture medium was loaded onto a MabSelect SuRe (GE
Healthcare) protein-A column and washed with 10 column volumes of PBS
buffer at pH 7.2. The antibody was eluted with 10 column volumes of
citrate buffer at pH 3.6 with the pooled fractions containing the
antibody neutralized with Tris at pH 11.
[0346] The protein-A antibody eluate was further purified by gel
filtration (SEC). For gel filtration, 3.5 mg of the antibody mixture was
concentrated to 1.5 mL and loaded onto a Sephadex 200 HiLoad 16/600 200
pg column (GE Healthcare) via an AKTA Express FPLC at a flow-rate of 1
mL/min. PBS buffer at pH 7.4 was used at a flow-rate of 1 mL/min.
Fractions corresponding to the purified antibody were collected,
concentrated to .about.1 mg/mL. The purified proteins were analyzed by
LC-MS as described in Example 2.
[0347] The results of the 10 L expression and bench-scale protein A and
SEC purification are shown in FIGS. 2A and 2B. FIG. 2A shows the SEC
chromatograph of the protein A purified v5019 and FIG. 2B shows the
non-reducing SDS-PAGE gel that compares the relative purity of a protein
A pooled fraction as well as SEC fractions 15 and 19 and pooled SEC
fractions 16-18. These results show that the anti-HER2 biparatopic
antibody was expressed and that purification by protein A and SEC yielded
a pure protein sample. Further quantification was performed by UPLC-SEC
and LC-MS analysis and is described in Example 4.
[0348] The results of the 25 L expression and bench-scale protein A
purification is shown in FIG. 2C. FIG. 2C shows SDS-PAGE gel that
compares the relative purity of a protein A purified v10000. Lane M
contains: protein marker; lane 1 contains: v10000 under reducing
conditions; lane 2 contains v10000 under non-reducing conditions. The
SDS-PAGE gel shows that v10000 is pure and runs at the correct predicted
MW of approximately 125 kDa under non-reducing conditions. Under reducing
conditions two heavy chains bands are visible corresponding to the CH-A
heavy chain (approximately 49 kDa) and the CH-B heavy chain
(approximately 52.5 kDa); the CH-A light chain is visible and runs at the
correct predicted mass of approximately 23.5 kDa. These results show that
the anti-HER2 biparatopic antibody was expressed and that one-step
purification by protein A yielded a pure protein sample. Further
quantification was performed by UPLC-SEC and LC-MS analysis and is
described in Example 4.
Example 4: Analysis of Biparatopic Anti-HER2 Antibody Purity by UPLC-SEC
and LC-MS
[0349] The purity and percent aggregation of exemplary protein A and SEC
purified biparatopic anti-HER2 heteromultimers was determined by UPLC-SEC
by the method described.
[0350] UPLC-SEC analysis was performed using a Waters BEH200 SEC column
set to 30.degree. C. (2.5 mL, 4.6.times.150 mm, stainless steel, 1.7
.mu.m particles) at 0.4 ml/min. Run times consisted of 7 min and a total
volume per injection of 2.8 mL with running buffers of 25 mM sodium
phosphate, 150 mM sodium acetate, pH 7.1; and, 150 mM sodium phosphate,
pH 6.4-7.1. Detection by absorbance was facilitated at 190-400 nm and by
fluorescence with excitation at 280 nm and emission collected from
300-360 nm. Peak integration was analyzed by Empower 3 software.
[0351] UPLC-SEC results of the pooled v5019 SEC fractions are shown in
FIG. 3A. These results indicate that the exemplary anti-HER2 biparatopic
antibody was purified to >99% purity with less than 1% HMW species by
protein A and SEC chromatography.
[0352] UPLC-SEC results of the v10000 pooled Protein A fractions are shown
in FIG. 3B. These results indicate that the exemplary anti-HER2
biparatopic antibody was purified to >96% purity with less than 1% HMW
species by protein A chromatography.
[0353] The purity of exemplary biparatopic anti-HER2 antibodies was
determined using LC-MS under standard conditions by the method described
in Example 2. Results from LC-MS analysis of the pooled SEC fractions of
v5019 are shown in FIG. 4A. This data shows that the exemplary
biparatopic anti-HER2 heterodimer has a heterodimer purity of 100%.
Results from LC-MS analysis of the pooled protein A fractions of v10000
are shown in FIG. 4B. This data shows that the exemplary biparatopic
anti-HER2 heterodimer has a heterodimer purity of 98% following a
one-step protein A purification.
[0354] Antibodies purified by protein A chromatography and/or protein A
and SEC were used for the assays described in the following Examples.
Example 5. Large-Scale Expression and Manufacturability Assessment of
Biparatopic Anti-HER2 Antibody Purified by Protein A and CEX
Chromatography
[0355] The exemplary anti-HER2 biparatopic antibody v5019 described in
Example 1 was expressed in a 25 L scale and purified as follows.
[0356] Antibody was obtained from supernatant followed by a two-step
purification method that consisted of Protein A purification
(MabSelect.TM. resin; GE Healthcare) followed by cation exchange
chromatography (HiTrap.TM. SP FF resin; GE Healthcare) by the protocol
described.
[0357] CHO-3E7 cells were maintained in serum-free Freestyle CHO
expression medium (Invitrogen, Carlsbad, Calif., USA) in Erlenmeyer
Flasks at 37.degree. C. with 5% CO2 (Corning Inc., Acton, Mass.) on an
orbital shaker (VWR Scientific, Chester, Pa.). Two days before
transfection, the cells were seeded at an appropriate density in a 50 L
CellBag with a volume of 25 L using the Wave Bioreactor System 20/50 (GE
Healthcare Bio-Science Corp). On the day of transfection, DNA and PEI
(Polysciences, Eppelheim, Germany) were mixed at an optimal ratio and
added to the cells using the method described in Example 1. Cell
supernatants collected on day 6 was used for further purification.
[0358] Cell culture broth was centrifuged and filtered before loading onto
30 mL Mabselect.TM. resin packed in XK26/20 (GE Healthcare, Uppsala,
Sweden) at 10.0 mL/min. After washing and elution with appropriate
buffer, the fractions were collected and neutralized with 1 M Tris-HCl,
pH 9.0. The target protein was further purified via 20 mL SP FF resin
packed in XK16/20 (GE Healthcare, Uppsala, Sweden). MabSelect.TM.
purified sample was diluted with 20 mM NaAC, pH5.5 to adjust the
conductivity to <5 ms/cm and 50 mM citrate acid (pH3.0) was added
adjust the sample pH value to 5.5. Sample was loaded at a 1 mL/min onto
the HiTrap.TM. SP FF resin (GE Healthcare) and washed with 20 mM NaAC.
Protein was eluted using a gradient elution 0-100% of 20 mM NaAC, 1 M
NaCl, pH5.5, 10 CV at 1 mL/min.
[0359] The purified protein was analyzed by SDS-PAGE as described in
Example 1, and LC-MS for heterodimer purity by the method described in
example 4. The results are shown in FIGS. 5A and 5B. FIG. 5A shows the
SDS-PAGE results of v5019 following MabSelect.TM. and HiTrap.TM. SP FF
purification; lane M contains: protein marker; lane 1: v5019 under
reducing conditions (3 .mu.g); Lane 2: v5019 under non-reducing
conditions (2.5 .mu.g). The SDS-PAGE gel shows that v5019 is relatively
pure following MabSelect.TM. and HiTrap.TM. SP FF purification and, under
non-reducing conditions, runs at the correct predicted MW of
approximately 125 kDa. Under reducing conditions two heavy chains bands
are visible corresponding to the CH-A heavy chain (approximately 49 kDa)
and the CH-B heavy chain (approximately 52.5 kDa); the CH-A light chain
is visible and runs at the correct predicted mass of approximately 23.5
kDa.
[0360] LC-MS analysis of the MabSelect.TM. and HiTrap.TM. SP FF purified
v5019 was performed to determine heterodimer purity using the method
described in Example 4. Results from the LC-MS analysis are shown in FIG.
5B. These results show that v5019 purification using MabSelect.TM. and
HiTrap.TM. SP FF yields protein with >99% heterodimer purity and with
little (<1%) or undetectable homodimer or half antibody contamination.
Example 6: Comparison of Bmax of a Biparatopic Anti-HER2 Antibody Against
Bmax of Controls in Cell Lines Expressing Low to High Levels of HER2
[0361] The following experiment was performed to measure the ability of an
exemplary biparatopic anti-HER2 antibody to bind to cells expressing
varying levels of HER2 in comparison to controls. The cell lines used
were SKOV3 (HER2 2+/3+), JIMT-1 (HER2 2+), MDA-MB-231 (HER2 0/1+), and
MCF7 (HER2 1+). The biparatopic anti-HER2 antibodies tested include
v5019, v7091 and v10000. The ability of the biparatopic anti-HER2
antibodies to bind to the HER2 expressing (HER2+) cells was determined as
described below, with specific measurement of B.sub.max and apparent
K.sub.D (equilibrium dissociation constant).
[0362] Binding of the test antibodies to the surface of HER2+ cells was
determined by flow cytometry. Cells were washed with PBS and resuspended
in DMEM at 1.times.10.sup.5 cells/100 .mu.l. 100 .mu.l cell suspension
was added into each microcentrifuge tube, followed by 10 .mu.l/tube of
the antibody variants. The tubes were incubated for 2 hr 4.degree. C. on
a rotator. The microcentrifuge tubes were centrifuged for 2 min 2000 RPM
at room temperature and the cell pellets washed with 500 .mu.l media.
Each cell pellet was resuspended 100 .mu.l of fluorochrome-labelled
secondary antibody diluted in media to 2 .mu.g/sample. The samples were
then incubated for 1 hr at 4.degree. C. on a rotator. After incubation,
the cells were centrifuged for 2 min at 2000 rpm and washed in media. The
cells were resuspended in 500 .mu.l media, filtered in tube containing 5
.mu.l propidium iodide (PI) and analyzed on a BD LSR II flow cytometer
according to the manufacturer's instructions. The K.sub.D of exemplary
biparatopic anti-HER2 heterodimer antibody and control antibodies were
assessed by FACS with data analysis and curve fitting performed in
GraphPad Prism.
[0363] The results are shown in FIGS. 6A-6G. These results demonstrate
that exemplary biparatopic anti-HER2 antibodies (v5019, v7091 and v10000)
can bind to HER2+ cells with approximately a 1.5-fold higher Bmax
compared to an anti-HER2 FSA (v506). The results in FIG. 6A-6G also show
that biparatopic anti-HER2 antibodies (v5019, v7091 and v10000) can bind
to HER2+ cells with a similar Bmax compared to a combination of two
anti-HER2 FSAs (v506+v4184).
[0364] The binding results for HER2+ SKOV3 cells (HER2 2/3+) are shown in
FIGS. 6A, 6E and Table 4 and Table 5. The results in FIG. 6A and Table 4
show that exemplary biparatopic anti-HER2 antibody (v5019) displays
approximately a 1.5-fold higher Bmax in binding to SKOV3 cells compared
to two different anti-HER2 FSAs (v506 or v4184). The results also show
that exemplary biparatopic anti-HER2 antibody (v5019) displays equivalent
Bmax compared to the combination of two anti-HER2 FSAs (v506+v4184). The
apparent K.sub.D of v5019 for binding to SKOV3 was approximately 2 to
4-fold higher compared to either anti-HER2 FSA alone (v506 or v4184), or
the combination of two anti-HER2 FSAs (v506+v4184).
TABLE-US-00011
TABLE 4
Binding to SKOV3 cells
Antibody variant K.sub.D (nM) Bmax
v506 2.713 29190
v4184 4.108 29204
v5019 8.084 47401
v506 + v4184 4.414 49062
[0365] The results in FIG. 6E and Table 5 show that exemplary biparatopic
anti-HER2 antibodies (v5019, 7091 and v10000) display approximately a 1.5
to 1.6-fold higher Bmax in binding to SKOV3 cells compared to two
different anti-HER2 FSAs (v506 or v4184). The results also show that
exemplary biparatopic anti-HER2 antibodies (v5019, 7091 and v10000)
display equivalent Bmax compared to the combination of two anti-HER2 FSAs
(v506+v4184). The apparent K.sub.D of v5019, v7091, v10000 and the
combination of two anti-HER2 FSAs (v506+v4184) for binding to SKOV3 was
approximately 2 to 3-fold higher compared to either anti-HER2 FSA alone
(v506 or v4184).
TABLE-US-00012
TABLE 5
Binding to SKOV3
Antibody Variant K.sub.D (nM) Bmax
v506 4.8 30007
v4184 5.6 27628
v506 + v4184 10.0 49014
v5019 13.6 47693
v7091 14.5 44737
v10000 10.3 48054
[0366] Binding curves in the JIMT-1 cell line (HER2 2+) are shown in FIG.
6B and Table 6. These results show that exemplary biparatopic anti-HER2
antibody (v5019) displays approximately a 1.5-fold higher Bmax in binding
to JIMT-1 cells compared to an anti-HER2 FSAs (v506). The results also
show that exemplary biparatopic anti-HER2 antibody (v5019) displays
equivalent Bmax compared to the combination of two anti-HER2 FSAs
(v506+v4184). The apparent K.sub.D of v5019 for binding to JIMT-1 was
approximately 2-fold higher compared to the anti-HER2 FSA (v506), and was
similar (approximately 1.2 fold greater) compared to the combination of
two anti-HER2 FSAs (v506+v4184).
TABLE-US-00013
TABLE 6
Binding to JIMT-1 cells
Antibody variant K.sub.D (nM) Bmax
v506 1.875 4905
v5019 4.317 7203
v506 + v4184 5.057 7200
[0367] Binding curves in the MCF7 cell line (HER2 1+) are shown in FIG.
6C, 6F and Tables 7 and 8. These results show that exemplary biparatopic
anti-HER2 antibodies (v5019, 7091 and v10000) display approximately a
1.5-fold higher Bmax in binding to MCF7 cells compared to an anti-HER2
FSAs (v506). The results in FIG. 6C also show that exemplary biparatopic
anti-HER2 antibody (v5019) displays equivalent Bmax compared to the
combination of two anti-HER2 FSAs (v506+v4184). The apparent K.sub.D of
v5019 for binding to MCF7 was similar to the anti-HER2 FSA (v506) and the
combination of two anti-HER2 FSAs (v506+v4184).
TABLE-US-00014
TABLE 7
Binding to MCF7 cells
Antibody variant K.sub.D (nM) Bmax
v506 1.301 542
v5019 1.506 872
v506 + v4184 2.095 903
The results in FIG. 6F and Table 8 show that exemplary biparatopic
anti-HER2 antibodies (v5019, v7091 and v10000) display approximately 1.6
to 1.7-fold greater Bmax compared to the FSA monospecific v506. The
apparent K.sub.D of v5019, v7091 and v10000 was similar to the anti-HER2
FSA (v506).
TABLE-US-00015
TABLE 8
Binding to MCF7 cells
Antibody Variant K.sub.D (nM) Bmax
v506 3.5 571
v5019 5.6 968
v7091 6.5 918
v10000 3.7 915
[0368] Binding curves in the MDA-MB-231 cell line (HER2 0/1+) are shown in
FIG. 6D and Table 9. These results show that exemplary biparatopic
anti-HER2 antibody (v5019) displays approximately a 1.5-fold higher Bmax
in binding to MDA-MB-231 cells compared to an anti-HER2 FSA (v506). The
results also show that exemplary biparatopic anti-HER2 antibody (v5019)
displays equivalent Bmax compared to the combination of two anti-HER2
FSAs (v506+v4184). The apparent K.sub.D of v5019 for binding to
MDA-MB-231 was approximately 2.4-fold lower compared to the anti-HER2 FSA
(v506) and was approximately 1.7-fold higher compared to the combination
of two anti-HER2 FSAs (v506+v4184).
TABLE-US-00016
TABLE 9
Binding to MDA-MB-231 cells
Antibody variant K.sub.D (nM) Bmax
v506 8.364 0.9521
v5019 3.543 1.411
v506 + v4184 2.040 1.542
[0369] Binding curves in the WI-38 lung fibroblast cell line are shown in
FIG. 6G and Table 10. The WI-38 cell line is a normal lung epithelium
that expresses basal levels (HER2 0+, .about.10,000 receptors/cell) of
HER2 (Carter et al. 1992, PNAS, 89:4285-4289; Yarden 2000, HER2: Basic
Research, Prognosis and Therapy). These results show that exemplary
biparatopic anti-HER2 antibodies (v5019, v7091, v10000) displays
equivalent cell surface decoration (Bmax) in binding to WI-38 cells
compared to an anti-HER2 FSAs (v506); however, note that binding for v506
did not appear to reach saturation, and thus KD could not be determined.
The apparent K.sub.D among the exemplary biparatopic anti-HER2 antibodies
was equivalent.
TABLE-US-00017
TABLE 10
Binding to WI-38 cells
Antibody Variant K.sub.D (nM) Bmax
v506 Not determined ~366
v5019 7.0 380
v7091 8.3 371
v10000 8.4 418
[0370] These results show that an exemplary biparatopic anti-HER2 antibody
can bind to HER2 1+, 2+ and 3+ tumor cells to levels that are
approximately 1.5 to 1.6-fold greater than an anti-HER2 monospecific FSA,
and that exemplary biparatopic anti-HER2 antibodies can bind to HER2 1+,
2+ and 3+ tumor cells to equivalent levels compared to the combination of
two unique monospecific anti-HER2 FSAs with different epitope
specificities. These results also show that the biparatopic anti-HER2
antibodies do not show increased binding (i.e. compared to monospecific
anti-HER2 antibody, v506) to basal HER2 expressing cells that express
approximately 10,000 HER2 receptors/cell or less, and that a threshold
for increased cell surface binding to the biparatopic anti-HER2
antibodies occurs when the HER2 receptor level is approximately
>10,000 receptors/cell. Based on this data it would be expected that
the exemplary biparatopic anti-HER2 antibodies would have increased cell
surface binding to HER2 3+, 2+ and 1+ tumor cells but would not have
increased cell surface binding to non-tumor cells that express basal
levels of the HER2 receptor at approximately 10,000 receptors or less.
Example 7: Ability of Biparatopic Anti-HER2 Antibody to Inhibit Growth of
HER2+ Cells
[0371] The ability of an exemplary biparatopic anti-HER2 antibody to
inhibit growth of cells expressing HER2 at the 3+ and 2+ level was
measured. The experiment was carried out in the HER2 3+ cell lines
BT-474, SKBr3, SKOV3, and HER2 2+ JIMT-1. The biparatopic anti-HER2
antibodies v5019, v7091 and v10000 were tested. The ability of the
biparatopic anti-HER2 antibodies to inhibit the growth of BT-474 cells
(200 nM antibody); SKOV3, SKBr3 and JIMT-1 cells (300 nM antibody) was
measured as described below.
[0372] Test antibodies were diluted in media and added to the cells at 10
.mu.l/well in triplicate. The plates were incubated for 3 days 37.degree.
C. Cell viability was measured using either AlamarBlue.TM. (Biosource #
dal1100), or CelltiterGlo.RTM. and absorbance read as per the
manufacturer's instructions. Data was normalized to untreated control and
analysis was performed in GraphPad prism.
[0373] The growth inhibition results are shown in FIG. 7A-E. A summary of
the results is provided in Tables 11A and 11B. The results FIGS. 7A-B and
Table 11A indicate that exemplary anti-HER2 biparatopic (v5019) is
capable of growth inhibition of HER2+ SKOV3 and BT-474 cell lines. FIG.
10A shows that anti-HER2 biparatopic antibody mediated the greatest
growth inhibition of SKOV3 when compared to anti-HER2 FSA (v506) and when
compared to the combination of two anti-HER2 FSA antibodies (v506+v4184).
TABLE-US-00018
TABLE 11A
Growth Inhibition of HER2 3+ Cancer Cells
% Survival
Treatment SKOV3 HER2 2+/3+ BT-474 HER2 3+
v506 88 37
v506 + v4184 96 32
v5019 77 43
[0374] The results in FIGS. 7C-E and Table 11B indicate that exemplary
anti-HER2 biparatopic antibodies (v5019, v7091 and v10000) can inhibit
growth of HER2 3+ SKBR3, HER2 2+/3+ SKOV3, and HER2 2+ JIMT-1 tumor cell
lines. FIG. 7C shows that anti-HER2 biparatopic antibodies v7091 and
v10000 mediated the greatest growth inhibition of HER2 3+ SKBr3 breast
tumor cells. FIG. 7D shows that anti-HER2 biparatopic antibodies (v7091
and v10000) mediated the greatest growth inhibition of HER2 3+ SKOV3
ovarian tumor cells. FIG. 7E shows that anti-HER2 biparatopic antibodies
(v7091 and v10000) mediated the greatest growth inhibition of HER2 2+
Herceptin-resistant JIMT-1 tumor cells. In all cell lines tested,
exemplary anti-HER2 biparatopic antibodies (v7091 and v10000) mediated
greater growth inhibition compared to the anti-HER2 FSA monospecific
antibody (v506).
TABLE-US-00019
TABLE 11B
Growth inhibition of HER2 3+ Cancer Cells
% Survival
Treatment SKBr3 HER2 3+ SKOV3 HER2 2+/3+ JIMT-1 HER2 2+
v506 52 107 107
v5019 59 83 106
v7091 35 79 85
v10000 34 73 84
[0375] These results show that exemplary saturating concentrations of
biparatopic anti-HER2 antibodies can growth inhibit HER2 3+ and 2+ breast
and ovarian and HER2 2+ Trastuzumab resistant tumor cells approximately
20% greater than a FSA anti-HER2 monospecific antibody.
Example 8: Preferential Binding of Paratopes of Biparatopic Anti-HER2
Antibodies to Dimeric HER2 Compared to HER2 ECD
[0376] This experiment was performed to determine the ability of the
individual paratopes of exemplary biparatopic anti-HER2 antibodies to
bind to dimeric HER2 and the HER2 ECD as a surrogate for differential
binding between membrane bound HER2 (HER2-Fc) and the shed HER2 ECD. The
experiment was carried out as follows.
[0377] Surface plasmon resonance (SPR) analysis: affinity of monovalent
anti-HER2 antibodies (v1040 or v4182) for binding to the HER2
extracellular domain (sHER-2, Ebioscience BMS362, encoding amino acid
23-652 of the full length protein) and HER2-Fc (dimeric HER2-Fc fusion
encoding the amino acid 1-652 of the extracellular domain; Sino
Biological Inc., 10004-H02H) was measured by SPR using the T200 system
from Biacore (GE Healthcare). Binding to the HER2 ECD was determined by
the following method. HER2 ECD in 10 mm Hepes pH 6.8, was immobilized on
CMS chip through amine coupling to a level of 44 RU (response units).
Monovalent anti-HER2 antibodies were passed over the surface of the HER2
immobilized chip at concentrations ranging from 0.76-60 nM. Binding to
the HER2-Fc was determined by the following method. HER2-Fc in 10 mm
Hepes pH 6.8, was immobilized on CMS chip through amine coupling to a
level of 43 RU. Monovalent anti-HER2 antibodies were passed over the
surface of the HER2 immobilized chip at concentrations ranging from
0.76-60 nM. Antibody concentrations were analyzed for binding in
triplicate. Equilibrium dissociation binding constants (K.sub.D) and
kinetics (ka and kd) were determined using the single cycle kinetics
method. Sensograms were fit globally to a 1:1 Langmuir binding model. All
experiments were conducted at room temperature.
[0378] Results are shown in FIG. 8A, FIG. 8B, Table 11C and Table 11D. The
results in FIG. 8A and Table 11C show SPR binding data of the monovalent
anti-HER2 antibody (v1040; representing the antigen-binding domain on
CH-B of exemplary anti-HER2 biparatopic antibody). FIG. 8A illustrates
the K.sub.D values (nM) of v1040 binding to immobilized HER2 ECD or
HER2-Fc and shows that monovalent anti-HER2 antibody has a lower K.sub.D
for binding to the HER2-Fc compared to the HER2 ECD. Table 11C shows the
ka (1/M s) and kd (1/s) values of the monovalent anti-HER2 antibody (OA)
compared to the full-sized anti-HER2 antibody (FSA) in binding to the
HER2 ECD and HER2-FC (`HER2 mem`). This data shows comparable on (ka) and
off (kd) rates of the OA and FSA for binding to the HER2 ECD and HER2-Fc.
TABLE-US-00020
TABLE 11C
ka (1/M s) and kd (1/s) values of the monovalent anti-HER2
antibody (OA) compared to the full-sized anti-HER2 antibody
(FSA) in binding to the HER2 ECD and HER2-FC (`HER2 mem`
ka (1/Ms) kd (1/s)
OA vs. HER2 ECD 2.00E+05 6.15E-05
FSA vs. HER2 ECD 4.14E+05 2.01E-05
OA vs. HER2 mem 1.88E+05 4.38E-05
FSA vs. HER2 mem 3.41E+05 4.94E-06*
[0379] Results in FIG. 8B and Table 11D show the SPR binding data of the
monovalent anti-HER2 antibody (v4182; representing the antigen-binding
domain on CH-A of exemplary anti-HER2 biparatopic antibody). FIG. 8B
illustrates the K.sub.D values (nM) of v4182 binding to immobilized HER2
ECD or HER2-Fc and shows that monovalent anti-HER2 antibody has a lower
K.sub.D for binding to the HER2-Fc compared to the HER2 ECD. Table 11D
shows the ka (1/M s) and kd (1/s) values of the monovalent anti-HER2
antibody (OA) compared to the full-sized anti-HER2 antibody (FSA) in
binding to the HER2 ECD and HER2-FC (`HER2 mem`). This data shows
comparable on rates (ka) and off rates (kd) of the OA and FSA for binding
to the HER2 ECD and HER2-Fc.
TABLE-US-00021
TABLE 11D
ka (1/Ms) kd (1/s)
OA vs. HER2 ECD 9.08E+04 6.17E-04
FSA vs. HER2 ECD 9.55E+04 3.93E-04
OA vs. HER2 mem 1.39E+05 2.04E-04
FSA vs. HER2 mem 1.77E+05 6.84E-05
[0380] These data show that each of the paratopes of the exemplary
anti-HER2 biparatopic antibody have lower K.sub.D values for binding to
the dimeric HER2 antigen, a representative of membrane bound HER2, as
compared to the HER2 ECD. Based on this data it would be expected that
the exemplary anti-HER2 antibody would have a higher binding affinity for
the membrane bound HER2 antigen as compared to the shed HER2 ECD that is
present in the serum of diseased patients and can act as a sink for the
therapeutic antibody (Brodowicz T, et al. Soluble HER-2/neu neutralizes
biologic effects of anti-HER-2/neu antibody on breast cancer cells in
vitro. Int J Cancer. 1997; 73:875-879). For example, baseline HER2 ECD
levels.ltoreq.15 ng/mL; whereas patients with progressive disease have
HER2 ECD.gtoreq.38 ng/mL.
Example 9: Whole Cell Loading and Internalization of Biparatopic Anti-HER2
Antibody in HER2+ Cells
[0381] This experiment was performed to assess the ability of an exemplary
biparatopic anti-HER2 antibody to be internalized in HER2 2+ cells. The
direct internalization method was followed according to the protocol
detailed in Schmidt, M. et al., Kinetics of anti-carcinoembryonic antigen
antibody internalization: effects of affinity, bivalency, and stability.
Cancer Immunol Immunother (2008) 57:1879-1890. Specifically, the
antibodies were directly labeled using the AlexaFluor.RTM. 488 Protein
Labeling Kit (Invitrogen, cat. no. A10235), according to the
manufacturer's instructions.
[0382] For the internalization assay, 12 well plates were seeded with
1.times.10.sup.5 cells/well and incubated overnight at 37.degree. C.+5%
CO2. The following day, the labeled antibodies were added at 200 nM in
DMEM+10% FBS and incubated 24 hours at 37.degree. C.+5% CO2. Under dark
conditions, media was aspirated and wells were washed 2.times.500 .mu.L
PBS. To harvest cells, cell dissociation buffer was added (250 .mu.L) at
37.degree. C. Cells were pelleted and resuspended in 100 .mu.L DMEM+10%
FBS without or with anti-Alexa Fluor 488, rabbit IgG fraction (Molecular
Probes, A11094) at 50 .mu.g/mL, and incubated on ice for 30 min. Prior to
analysis 300 .mu.L DMEM+10% FBS the samples filtered 4 .mu.l propidium
iodide was added. Samples were analyzed using the LSRII flow cytometer.
[0383] The ability of exemplary anti-HER2 biparatopic antibody to
internalize in HER2+ cells is shown in FIG. 9A and FIG. 9B. FIG. 9A shows
the results of detectable surface and internal antibody in BT-474 cells
following 24 h incubation with the exemplary anti-HER2 biparatopic
antibody and anti-HER2 FSA control. These results show that incubation
with exemplary anti-HER2 biparatopic antibody (v5019) results in
approximately 2-fold more internalized antibody in BT-474 cells compared
to the anti-HER2 FSA control. FIG. 9B shows the results of detectable
surface and internal antibody in JIMT-1 cells following 24 h incubation
with the exemplary anti-HER2 biparatopic antibody and anti-HER2 FSA
control. These results show that incubation with exemplary anti-HER2
biparatopic antibody (v5019) results in approximately 2-fold more
internalized antibody in JIMT-1 cells compared to the anti-HER2 FSA
control. The amount of surface staining post 24 h was comparable among
the biparatopic anti-HER2 and anti-HER2 FSA in both BT-474 and JIMT-1
cells.
[0384] The results in FIG. 10A-F show a comparison of detectable antibody
bound to the surface of whole cells after 2 h at 4.degree. C., compared
to antibody bound to the surface following incubation for 24 h at
37.degree. C.; in addition to the amount of internalized antibody
following 24 h at 37.degree. C. FIG. 10A shows the results in BT-474
cells following incubation with the exemplary anti-HER2 biparatopic
antibody and anti-HER2 FSA control. These results show that incubation of
exemplary anti-HER2 biparatopic antibody with BT-474 cells for 24 h
results in approximately a 15% reduction of antibody detected on the
surface of whole cells. FIG. 10A also shows that incubation with
exemplary anti-HER2 biparatopic antibody (v5019) results in approximately
2-fold more internalized antibody in BT-474 cells compared to the
anti-HER2 FSA control.
[0385] FIG. 10B shows the results in JIMT-1 cells following incubation
with the exemplary anti-HER2 biparatopic antibody and anti-HER2 FSA
control. FIG. 10B is a repeat of the experiment shown in FIG. 9B with the
addition of surface staining following 2 h at 4.degree. C. These results
show that incubation of exemplary anti-HER2 biparatopic antibody with
JIMT-1 cells for 24 h results in approximately a 57% reduction of
antibody detected on the surface of whole cells. FIG. 10B also shows that
incubation with exemplary anti-HER2 biparatopic antibody (v5019) results
more internalized antibody in BT-474 cells following 24 incubation at
37.degree. C., compared to the anti-HER2 FSA control.
[0386] FIG. 10C shows the results in SKOV3 cells following incubation with
the exemplary anti-HER2 biparatopic antibody. These results show that
incubation of exemplary anti-HER2 biparatopic antibody with SKOV3 cells
for 24 h results in approximately a 32% reduction of antibody detected on
the surface of whole cells.
[0387] FIG. 10D shows the results in MCF7 cells following incubation with
the exemplary anti-HER2 biparatopic antibody. These results show that
incubation of exemplary anti-HER2 biparatopic antibody with MCF7 cells
for 24 h results in approximately a 45% reduction of antibody detected on
the surface of whole cells.
[0388] FIG. 10E shows the results in SKOV3 cells following incubation with
the exemplary anti-HER2 biparatopic antibodies, v5019, v7091 and v10000.
These results show that incubation of exemplary anti-HER2 biparatopic
antibodies results in 1.5 to 1.8-fold more internalized antibody with
SKOV3 cells compared to the anti-HER2 FSA control. Incubation with the
anti-HER2 FSA control for 24 h resulted in the greatest reduction
(.about.77%) of antibody detected on the surface of whole cells.
[0389] FIG. 10F shows the results in JIMT-1 cells following incubation
with the exemplary anti-HER2 biparatopic antibodies, v5019, v7091 and
v10000. These results show that incubation of exemplary anti-HER2
biparatopic antibodies results in 1.4 to 1.8-fold more internalized
antibody with JIMT-1 cells compared to the anti-HER2 FSA control.
Incubation with the anti-HER2 biparatopic antibodies (v5019 and v10000)
for 24 h resulted in the greatest reduction (.about.64%) of antibody
detected on the surface of whole cells.
[0390] These results show that exemplary anti-HER2 biparatopic antibodies
have superior internalization properties in HER2+ cells compared to a
monospecific anti-HER2 FSA. The reduction of surface antibody detected
following 24 h incubation at 37.degree. C. shows that an exemplary
anti-HER2 biparatopic antibody is capable of reducing the amount of cell
surface HER2 receptor following incubation in HER2+ cells and that
surface HER2 reduction post incubation is greatest in HER2 2+ tumor
cells.
Example 10: Cellular Staining and Location of an Anti-HER2 Biparatopic
Antibody Following Incubation with HER2+ Cells at 1, 3 and 16 Hours
[0391] This experiment was performed to analyze internalization of the
exemplary anti-HER2 biparatopic antibody in HER2+ JIMT-1 cells at
different time points and as an orthogonal method to that presented in
Example 9 to analyze whole cell loading and internalization.
[0392] JIMT-1 cells were incubated with the antibody (v506, v4184, v5019,
or a combination of v506 and v4184) at 200 nM in serum-free DMEM,
37.degree. C.+5% CO.sub.2 for 1h, 3h and 16h. Cells were gently washed
two times with warmed sterile PBS (500 ml/well). Cells were fixed with
250 ml of 10% formalin/PBS solution for 10 min at RT. The fixed cells
were washed three times with PBS (500 .mu.l/well), permeabilized with 250
.mu.l/well of PBS containing 0.2% Triton X-100 for 5 min, and washed
three times with 500 .mu.l/well PBS. Cells were blocked with 500
.mu.l/well of PBS+5% goat serum for 1 h at RT. Blocking buffer was
removed, and 300 .mu.l/well secondary antibody (Alexa Fluor
488-conjugated AffiniPure Fab Fragment Goat anti-Human IgG (H+L); Jackson
ImmunoResearch Laboritories, Inc.; 109-547-003) was incubated for 1 h at
RT. Cells were washed three times with 500 .mu.l/well of PBS and the
coverslips containing fixed cells were then mounted on a slide using
Prolong gold anti-fade with DAPI (Life Technologies; #P36931). 60.times.
single images were acquired using Olympus FV1000 Confocal microscope.
[0393] The results indicated that the exemplary anti-HER2 biparatopic
antibody (v5019) was internalized into JIMT-1 cells at 3 h and was
primarily located close to the nuclei. Comparing images at the 3h
incubation showed a greater amount of internal staining associated with
the anti-HER2 biparatopic antibody compared to the combination of two
anti-HER2 FSAs (v506+v4184) and compared to the individual anti-HER2 FSA
(v506 or v4184). Differences in the cellular location of antibody
staining were seen when the anti-HER2 biparatopic antibody (v5019)
results were compared with the anti-HER2 FSA (v4184); where the anti-HER2
FSA (v4184) showed pronounced plasma membrane staining at the 1, 3 and 16
h time points. The amount of detectable antibody was reduced at the 16 h
for the anti-HER2 FSA (v506), the combination of two anti-HER2 FSAs
(v506+v4184) and anti-HER2 biparatopic antibody treatments (data not
shown).
[0394] These results show that the exemplary anti-HER2 biparatopic
antibody v5019 was internalized in HER2+ cells and the internalized
antibody was detectable after 3 h incubation. These results are
consistent with the results presented in Example 9 that show exemplary
anti-HER2 biparatopic antibody can internalize to greater amounts in
HER2+ cells compared to an anti-HER2 FSA.
Example 11: ADCC of HER2+ Cells Mediated by Biparatopic Anti-HER2 Antibody
Compared to Controls
[0395] This experiment was performed in order to measure the ability of an
exemplary biparatopic anti-HER2 antibody to mediate ADCC in SKOV3 cells
(ovarian cancer, HER2 2+/3+).
[0396] Target cells were pre-incubated with test antibodies (10-fold
descending concentrations from 45 .mu.g/ml) for 30 min followed by adding
effector cells with effector/target cell ratio of 5:1 and the incubation
continued for 6 hours at 37.degree. C.+5% CO.sub.2. Samples were tested
with 8 concentrations, 10 fold descending from 45 .mu.g/ml. LDH release
was measured using LDH assay kit.
[0397] Dose-response studies were performed with various concentrations of
the samples with a effector/target (E/T) ratios of 5:1. 3:1 and 1:1. Half
maximal effective concentration (EC.sub.50) values were analyzed with the
sigmoidal dose-response non-linear regression fit using GraphPad prism.
[0398] Cells were maintained in McCoy's 5a complete medium at 37.degree.
C./5% CO.sub.2 and regularly sub-cultured with suitable medium
supplemented with 10% FBS according to protocol from ATCC. Cells with
passage number fewer than p10 were used in the assays. The samples were
diluted to concentrations between 0.3-300 nM with phenol red free DMEM
medium supplemented with 1% FBS and 1% pen/strep prior to use in the
assay.
[0399] The ADCC results in HER2+ SKOV3 cells at an effector to target cell
ratio of 5:1 are shown in FIG. 11A and Table 12. These results show that
the exemplary biparatopic anti-HER2 antibody (v5019) mediated the
greatest percentage of maximum target cell lysis by ADCC when compared to
the anti-HER2 FSA (v792) and combination of two different anti-HER2 FSAs
(v792+v4184). The difference in maximum cell lysis mediated by the
exemplary biparatopic anti-HER2 antibody was approximately 1.6-fold
greater compared to the anti-HER2 FSA, and approximately 1.2-fold greater
compared to a combination of two different anti-HER2 FSAs (v792+v4184).
TABLE-US-00022
TABLE 12
Antibody variant EC.sub.50 (nM) % Max Cell Lysis
v792 ~0.032 17.82
v5019 ~0.164 28.57
v792 + v4184 ~0.042 23.85
[0400] The ADCC results in HER2+ SKOV3 cells at an effector to target cell
ratio of 3:1 are shown in FIG. 11B and Table 13. These results show that
the exemplary biparatopic anti-HER2 antibody (v5019) mediated the
greatest percentage of maximum target cell lysis by ADCC when compared to
the anti-HER2 FSA (v792) and combination of two different anti-HER2 FSAs
(v792+v4184). The difference in maximum cell lysis mediated by the
exemplary biparatopic anti-HER2 antibody was approximately 1.3-fold
greater compared to the anti-HER2 FSA, and approximately 1.8-fold greater
compared to a combination of two different anti-HER2 FSAs (v792+v4184).
TABLE-US-00023
TABLE 13
Antibody variant EC.sub.50 (nM) % Max Cell Lysis
v792 1.064 16.9
v5019 ~0.4608 22.3
v792 + v4184 ~1.078 12.3
[0401] The ADCC results in HER2+ SKOV3 cells at an effector to target cell
ratio of 1:1 are shown in FIG. 11C and Table 14. These results show that
the exemplary biparatopic anti-HER2 antibody (v5019) mediated the
greatest percentage of maximum target cell lysis by ADCC when to compared
to the anti-HER2 FSA (v792) and combination of two different anti-HER2
FSAs (v792+v4184). The difference in maximum cell lysis mediated by the
exemplary biparatopic anti-HER2 antibody was approximately 1.8-fold
greater compared to the anti-HER2 FSA, and approximately 1.13-fold
greater compared to a combination of two different anti-HER2 FSAs
(v792+v4184).
TABLE-US-00024
TABLE 14
Antibody variant EC.sub.50 (nM) % Max Cell Lysis
v792 1.429 7.529
v5019 ~1.075 13.29
v792 + v4184 ~0.1121 11.73
[0402] The results in FIG. 11 and Tables 12-14 show that the exemplary
biparatopic HER2 antibody mediates the greatest ADCC of SKOV3 cells at
different E:T ratios when compared to an anti-HER2 FSA and combination of
two anti-HER2 FSAs. The observation of increased ADCC mediated by the
anti-HER2 biparatopic antibody would be expected in HER2+ diseased
patients who express variable and/or reduced circulating effector cells
following chemotherapy (Suzuki E. et al. Clin Cancer Res 2007;
13:1875-1882). The observations in FIG. 11 are consistent with the whole
cell binding Bmax data presented in Example 6, that shows an approximate
1.5-fold increase in cell binding to the exemplary anti-HER2 biparatopic
antibody compared to the anti-HER2 FSA.
Example 12: Ability of Exemplary Anti-HER2 Antibody to Bind to HER2 ECD
[0403] An SPR assay was used to evaluate the mechanism by which an
exemplary anti-HER2 biparatopic antibody binds to HER2 ECD; specifically,
to understand whether both paratopes of one biparatopic antibody molecule
can bind to one HER2 ECD (Cis binding; 1:1 antibody to HER2 molecules) or
if each paratope of one biparatopic antibody can bind two different HER2
ECDs (Trans binding; 1:2 antibody to HER2 molecules). A representation of
cis vs. trans binding is illustrated in FIG. 14. The correlation between
a reduced (slower) off-rate with increasing antibody capture levels
(surface density) is an indication of Trans binding (i.e. one antibody
molecule binding to two HER2 molecules.
[0404] Affinity and binding kinetics of the exemplary biparatopic
anti-HER2 antibody (v5019) to recombinant human HER2 were measured and
compared to that of monovalent anti-HER2 antibodies (v630 or v4182;
comprising the individual paratopes of v5019) was measured by SPR using
the T200 system from Biacore (GE Healthcare). Between 2000 and 4000 RU of
anti-human Fc injected at concentration between 5 and 10 .mu.g/ml was
immobilized on a CMS chip using standard amine coupling. Monovalent
anti-HER2 antibody (v630 or v4182) and exemplary biparatopic anti-HER2
antibody (v5019) were captured on the anti-human Fc (injected at
concentration ranging 0.08 to 8 .mu.g/ml in PBST, 1 min at 10 ul/min) at
response levels ranging from 350-15 RU. Recombinant human HER2 was
diluted in PBST and injected at starting concentration of either 120 nM,
200 nM or 300 nM with 3-fold dilutions and injected at a flow rate of 50
.mu.l/min for 3 minutes, followed by dissociation for another 30 minutes
at the end of the last injection. HER2 dilutions were analyzed in
duplicate. Sensograms were fit globally to a 1:1 Langmuir binding model.
All experiments were conducted at 25.degree. C.
[0405] The results are shown in FIG. 12 and FIG. 13.
[0406] The results in FIG. 12A show the ka (1/Ms) of monovalent anti-HER2
(v630 and v4182) and exemplary biparatopic anti-HER2 antibody (v5019) for
binding to recombinant human HER2 over a range of injected and captured
antibody concentrations on the surface of the chip. These results show
that ka does not change when for v630, v4182 and v5019 at different
antibody capture levels.
[0407] The results in FIG. 12B show the kd (1/s) of monovalent anti-HER2
(v630 and v4182) and exemplary biparatopic anti-HER2 antibody (v5019) for
binding to recombinant human HER2 over a range of injected and captured
antibody concentrations on the surface of the chip. These results show
that kd decreased only for the exemplary anti-HER2 biparatopic antibody
(v5019) at increasing antibody capture levels.
[0408] The results in FIG. 12C show the K.sub.D (M) of monovalent
anti-HER2 (v630 and v4182) and exemplary biparatopic anti-HER2 antibody
(v5019) for binding to recombinant human HER2 over a range of injected
and captured antibody concentrations on the surface of the chip. These
results show that K.sub.D decreased only for the exemplary anti-HER2
biparatopic antibody (v5019) at increasing antibody capture levels. This
result correlated to the decreasing kd values shown in FIG. 15B.
[0409] The results in FIG. 13A show the kd (1/s) of exemplary biparatopic
anti-HER2 antibody (v5019) for binding to recombinant human HER2 over a
range of antibody capture levels. These results show kd values are
inversely proportional to higher RUs of antibody captured on the surface
of the chip (i.e slower off-rates at higher antibody capture levels). The
results indicate that exemplary biparatopic anti-HER2 antibody (v5019) is
capable of binding HER2 ECD2 and HER2 ECD4 on two separate HER2 molecules
(i.e. trans binding) as is evidenced by the reduction in off-rate at
higher antibody capture levels. This data is supported by a similar
experiment presented in FIG. 47 and discussed in Example 43, where
bivalent monospecific anti-HER2 FSA (v506) demonstrated Cis binding (1:1
antibody to HER2) where the kd (1/s) and K.sub.D (M) values remained
constant at increasing antibody capture levels as is expected for this
molecule.
[0410] The results in FIG. 13B show the kd (1/s) of monovalent anti-HER2
antibody (v4182) for binding to recombinant human HER2 over a range of
antibody capture levels. These results show no change in kd values over
the range of different antibody RUs captured on the surface of the chip.
These results show that monovalent anti-HER2 antibody (v4182) is binding
monovalently 1:1 (cis binding).
[0411] The results in FIG. 13C show the kd (1/s) of monovalent anti-HER2
antibody (v630) for binding to recombinant human HER2 over a range of
antibody capture levels. These results show no change in kd values over
the range of different antibody RUs captured on the surface of the chip.
These results show that monovalent anti-HER2 antibody (v630) is binding
monovalently 1:1 (cis binding). This data is supported by the experiment
presented in FIG. 47 and discussed in Example 43X, where the bivalent
monospecific anti-HER2 FSA (v506) showed no change in kd (1/s).
[0412] The results in FIG. 12, and FIG. 13 indicate that exemplary
biparatopic anti-HER2 antibody (v5019) is capable of simultaneously
binding to two HER2 molecules in trans (antibody to HER2 ratio 1:2). The
trans mechanism of binding detected by SPR is consistent with the higher
cell surface saturation binding data (Bmax), presented in Example 6, in
combination with the internalization data presented in Examples 9 and 10.
Example 13: Effect of Exemplary Biparatopic Anti-HER2 Antibody Incubation
on AKT Phosphorylation in BT-474 Cells
[0413] The ability of an exemplary anti-HER2 biparatopic antibody to
reduce pAKT signaling in BT-474 cells was tested using the AKT
Colorimetric In-Cell ELISA Kit (Thermo Scientific; cat no. 62215)
according to the manufacturer's instructions with the following
modifications. Cells were seeded at 5.times.10.sup.3/well and incubated
24 h at 37.degree. C.+5% CO.sub.2. Cells were incubated with 100 nM
antibody for with 30 min followed by a 15 min incubation with rhHRG-f31.
Cells were washed, fixed, and permeabilized according to the
instructions. Secondary antibodies (1:5000; Jackson ImmunoReasearch,
HRP-donkey anti-mouse IgG, JIR, Cat#715-036-150, HRP-donkey anti-rabbit
IgG, JIR, Cat#711-036-452) were added and the assay processed according
to the manufacturer's instructions.
[0414] The results in FIG. 15 show that incubation with exemplary
anti-HER2 biparatopic antibody mediated an approximate 1.2-fold reduction
in p-Akt levels in the presence of HRG.beta.1 relative to the human IgG
control (CTL). The combination of two anti-HER2 FSAs (v506+v4184)
mediated the greatest reduction in p-Akt levels in the presence
HRG.beta.1 that was approximately 1.5-fold less compared to the human IgG
control. A modest reduction in p-Akt was detected with the exemplary
anti-HER2 biparatopic antibody in the absence of ligand (HRG.beta.1)
compared to the human IgG control antibody.
[0415] These data show that exemplary anti-HER2 biparatopic antibody can
block ligand-activated signaling in HER2+ cells.
Example 14: Effect of Biparatopic Anti-HER2 Antibody on Cardiomyocyte
Viability
[0416] The effect of exemplary biparatopic anti-HER2 antibodies and ADCs
on cardiomyocyte viability was measured in order to obtain a preliminary
indication of potentially cardiotoxic effects.
[0417] iCell cardiomyocytes (Cellular Dynamics International,
CMC-100-010), that express basal levels of the HER2 receptor, were grown
according the manufacturer's instructions and used as target cells to
assess cardiomyocyte health following antibody treatment. The assay was
performed as follows. Cells were seeded in 96-well plates (15,000
cells/well) and maintained for 48 h. The cell medium was replaced with
maintenance media and cells were maintained for 72h. To access the
effects of antibody-induced cardiotoxicity, cells were treated for 72 h
with 10 and 100 nM of, variants alone or in combinations. To access the
effects of anthracycline-induced cardiotoxicity (alone or in combination
with the exemplary biparatopic anti-HER2 antibodies), cells were treated
with 3 uM (.about.IC.sub.20) of doxorubicin for 1 hr followed by 72 h
with 10 and 100 nM of, antibody variants alone or in combinations. Cell
viability was assessed by quantitating cellular ATP levels with the
CellTiter-Glo.RTM. Luminescent Cell Viability Assay (Promega, G7570)
and/or Sulphorhodamine (Sigma 230162-5G) as per the manufacturer's
instructions.
[0418] The results are shown in FIG. 16A-C. The results in FIG. 16A show
that incubation of the cardiomyocytes with therapeutically relevant
concentrations of exemplary anti-HER2 biparatopic antibody (v5019) and
exemplary anti-HER2 biparatopic-ADC (v6363), did not affect cardiomyocyte
viability relative to the untreated control (`mock`).
[0419] The results in FIG. 16B show that incubation of the cardiomyocytes
with therapeutically relevant concentrations of exemplary anti-HER2
biparatopic antibodies (v5019, v7091 and v10000), and exemplary anti-HER2
biparatopic-ADCs (v6363, v7148 and v10553), had no effect on
cardiomyocyte viability relative to the untreated control (`mock`). Based
on the results in FIGS. 16A and 16B it is expected that exemplary
anti-HER2 biparatopic antibodies and exemplary anti-HER2 biparatopic-ADCs
should not induce cardiomyopathy, for example through mitochondrial
dysfunction, as is reported with other anti-HER2 targeting antibodies
(Grazette L. P. et al. Inhibition of ErbB2 Causes Mitochondrial
Dysfunction in Cardiomyocytes; Journal of the American College of
Cardiology: 2004; 44:11).
[0420] The results in FIG. 16C show that pretreatment of the
cardiomyocytes with doxorubicin followed by incubation with
therapeutically relevant concentrations of exemplary anti-HER2
biparatopic antibodies (v5019, v7091 and v10000) and exemplary anti-HER2
biparatopic-ADCs (v6363, v7148 and v10553), had no effect on
cardiomyocyte viability relative to the untreated control+doxorubicin
(`Mock+Dox`). Based on the results in FIG. 16C it is expected that
exemplary anti-HER2 biparatopic antibodies and exemplary anti-HER2
biparatopic-ADCs should not result in an increased risk of cardiac
dysfunction in patients receiving concurrent anthracycline treatment
(Seidman A, Hudis C, Pierri M K, et al. Cardiac dysfunction in the
trastuzumab clinical trials experience. J Clin Oncol (2002)
20:1215-1221).
[0421] FIGS. 16A-C show that incubation of cardiomyocytes with the
anti-HER2 biparatopic antibodies and ADCs had equivalent effects compared
to monospecific anti-HER2 FSA antibody (v506), anti-HER2 FSA combination
(v506+v4184) and ADC (v6246) when treated either alone, or in combination
with doxorubicin. Based on these results, it is expected that exemplary
anti-HER2 biparatopic antibodies and ADCs would not have greater
cardiotoxic effects compared to anti-monospecific anti-HER2 FSA,
trastuzumab or ADC, T-DM1.
Example 15: Cytotoxicity of Exemplary Biparatopic Anti-HER2-ADCs in HER2+
Cells
[0422] The ability of exemplary biparatopic anti-HER2-ADC antibodies
(v6363, v7148 and v10553) to mediate cellular cytotoxicity in HER2+ cells
was measured. Human IgG conjugated to DM1 (v6249) was used as a control
in some cases. The experiment was carried out in HER2+ breast tumor cell
lines JIMT-1, MCF7, MDA-MB-231, the HER2+ ovarian tumor cell line SKOV3,
and HER2+ gastric cell line NCI-N87. The cytotoxicity of exemplary
biparatopic anti-HER2-ADC antibodies in HER2+ cells was evaluated and
compared to the monospecific anti-HER2 FSA-ADC (v6246) and
anti-HER2-FSA-ADC+ anti-HER2-FSA controls (v6246+v4184). The method was
conducted as described in Example 7 with the following modifications. The
anti-HER2 ADCs were incubated with the target SKOV3 and JIMT-1 (FIGS. 17A
and B) cells for 24 h, cells washed, media replaced and cell survival was
evaluated after 5 day incubation at 37.degree. C. The anti-HER2 ADCs were
incubated with target MCF7 and MDA-MB-231 target cells for 6 h (FIGS. 17C
and D), cells washed media replaced and cell survival was evaluated at 5
days incubation at 37.degree. C. In FIG. 17E-G, anti-HER2 ADCs were
incubated continuously with target SKOV3, JIMT-1, NCI-N87 cells for 5
days. Cell viability was measured as described in Example 7 using either
AlamarBlue.TM. (FIGS. 17A-D) or Celltiter-Glo.RTM. (FIGS. 17E-G).
[0423] The results are shown in FIG. 17A-G and the data is summarized in
Tables 15 and 16.
[0424] The results in FIG. 17A and Table 15 and 16 show that exemplary
anti-HER2 biparatopic-ADC (v6363) is more cytotoxic in JIMT-1 compared to
the anti-HER2-FSA-ADC (v6246) and the combination of anti-HER2-FSA-ADC+
anti-HER2 FSA (v6246+v4184). The exemplary anti-HER2 biparatopic-ADC had
a superior EC.sub.50 that was approximately 13-fold lower compared to the
anti-HER2 FSA-ADC control.
[0425] The results in FIG. 17B and Table 15 show that exemplary anti-HER2
biparatopic-ADC (v6363) is more cytotoxic in SKOV3 compared to the
anti-HER2-FSA-ADC (v6246) and the combination of anti-HER2-FSA-ADC+
anti-HER2 FSA (v6246+v4184). The exemplary anti-HER2 biparatopic-ADC had
a superior EC.sub.50 that was approximately 5-fold lower compared to the
anti-HER2 FSA-ADC control.
[0426] The results in FIG. 17C and Table 15 show that exemplary anti-HER2
biparatopic-ADC (v6363) is more cytotoxic in MCF7 compared to the
anti-HER2-FSA-ADC (v6246) and the combination of anti-HER2-FSA-ADC+
anti-HER2 FSA (v6246+v4184). The exemplary anti-HER2 biparatopic-ADC had
a superior EC.sub.50 that was approximately 2-fold lower compared to the
anti-HER2 FSA-ADC control.
[0427] The results in FIG. 17D and Table 15 show that exemplary anti-HER2
biparatopic-ADC (v6363) is more cytotoxic in MDA-MB-231 compared to the
anti-HER2-FSA-ADC (v6246) and the combination of anti-HER2-FSA-ADC+
anti-HER2 FSA (v6246+v4184). The exemplary anti-HER2 biparatopic-ADC had
a superior EC.sub.50 that was approximately 2-fold lower compared to the
anti-HER2 FSA-ADC control.
TABLE-US-00025
TABLE 15
v6246 0.9225 5.942 122.0 ~1075
v6246 + 4184 3.146 12.68 ~24432 136.4
v6363 0.1776 0.4443 58.55 141.0
indicates data missing or illegible when filed
[0428] The results in FIG. 17E and Table 16 show that exemplary anti-HER2
biparatopic-ADCs (v6363, v7148 and v10553) are more cytotoxic in SKOV3
ovarian tumor cells compared to the anti-HER2-FSA-ADC (v6246). The
exemplary anti-HER2 biparatopic-ADCs had a superior EC.sub.50 values that
were approximately 2 to 7-fold lower compared to the anti-HER2 FSA-ADC
control.
[0429] The results in FIG. 17F and Table 16 show that exemplary anti-HER2
biparatopic-ADCs (v6363, v7148 and v10553) are more cytotoxic in JIMT-1
breast tumor cells compared to the anti-HER2-FSA-ADC (v6246). The
exemplary anti-HER2 biparatopic-ADCs had a superior EC.sub.50 values were
approximately 6 to 9-fold lower compared to the anti-HER2 FSA-ADC
control.
[0430] The results in FIG. 17G and Table 16 show that exemplary anti-HER2
biparatopic-ADCs (v6363, v7148 and v10553) are cytotoxic in NCI-N87
gastric tumor cells. The exemplary anti-HER2 biparatopic-ADCs had has
approximately equivalent EC.sub.50 values compared to the anti-HER2
FSA-ADC control.
TABLE-US-00026
TABLE 16
Antibody EC.sub.50 (nM)
variant SKOV3 JIMT-1 NCI-N87
v6246 0.22 3.52 1.04
v6363 0.03 0.56 1.33
v7148 0.06 0.56 2.74
v10553 0.09 0.39 1.69
These results show that exemplary anti-HER2 biparatopic-ADCs (v6363,
v7148 and v10553) are more cytotoxic compared to anti-HER-FSA-ADC control
in HER2 3+, 2+, and 1+ breast tumor cells. These results also show that
exemplary anti-HER2 biparatopic-ADCs (v6363, v7148 and v10553) are
cytotoxic in HER2 2/3+ gastric tumor cells. These results are consistent
with the internalization results presented in Example 9.
Example 16: Effect of a Biparatopic Anti-HER2 Antibody in a Human Ovarian
Cancer Cell Xenograft Model
[0431] The established human ovarian cancer cell derived xenograft model
SKOV3 was used to assess the anti-tumor efficacy of an exemplary
biparatopic anti-HER2 antibody.
[0432] Female athymic nude mice were inoculated with the tumor via the
insertion of a 1 mm.sup.3 tumor fragment subcutaneously. Tumors were
monitored until they reached an average volume of 220 mm.sup.3; animals
were then randomized into 3 treatment groups: IgG control, anti-HER2 FSA
(v506), and biparatopic anti-HER2 antibody (v5019).
[0433] Fifteen animals were included in each group. Dosing for each group
is as follows:
[0434] A) IgG control was dosed intravenously with a loading dose of 30
mg/kg on study day 1 then with maintenance doses of 20 mg/kg twice per
week to study day 39.
[0435] B) Anti-HER2 FSA (v506) was dosed intravenously with a loading dose
of 15 mg/kg on study day 1 then with maintenance doses of 10 mg/kg twice
per week to study day 18. On days 22 through 39, 5 mg/kg anti-HER2 FSA
was dosed intravenously twice per week. Anti-HER2 FSA (v4184) was dosed
simultaneously at 5 mg/kg intraperitoneally twice per week.
[0436] C) Biparatopic anti-HER2 antibody was dosed intravenously with a
loading dose of 15 mg/kg on study day 1 then with maintenance doses of 10
mg/kg twice per week to study day 39.
[0437] Tumor volume was measured twice weekly over the course of the
study, number of responders and median survival was assessed at day 22.
The results are shown in FIG. 18 and Table 17.
[0438] The biparatopic anti-HER2 and anti-HER2 FSA demonstrated superior
tumor growth inhibition compared to IgG control. The biparatopic
anti-HER2 antibody induced superior tumor growth inhibition compared to
anti-HER2 FSA combination (FIG. 18A). The biparatopic anti-HER2 antibody
was associated with an increase in the number of responding tumors
compared to anti-HER2 FSA v506 at day 22 (11 and 5, respectively)(Table
17). The exemplary biparatopic anti-HER2 antibody and anti-HER2 FSA
demonstrated superior survival compared to IgG control. The biparatopic
anti-HER2 antibody had a superior median survival (61 days) compared to
anti-HER2 FSA (36 days)(FIG. 18B and Table 17). On study day 22 a second
anti-HER2 FSA (v4184) was added in combination to the anti-HER2 FSA
(v506). The combination of two anti-HER2 FSAs induced a further tumour
growth inhibition compared to anti-HER2 FSA (v506) alone.
TABLE-US-00027
TABLE 17
n = 15, Day 22 IgG v506 v5019
Mean TV 1908 (+766%) 1291 (+486%) 697 (+217%)
(mm3) (% change from
Baseline)
% TGI 0 32 63
Responders 0/15 5/15 11/15
(TV <50% of control)
Median Survival (days) 22 36 61
Example 17: Effect of a Biparatopic Anti-HER2 Antibody Drug Conjugate
(ADC) in a Human Ovarian Cancer Cell Line Xenograft Model
[0439] The established human ovarian cancer cell derived xenograft model
SKOV3 was used to assess the anti-tumor efficacy of an exemplary
biparatopic anti-HER2 antibody conjugated to DM1 (v6363).
[0440] Female athymic nude mice were inoculated with the tumor via the
insertion of a 1 mm.sup.3 tumor fragment subcutaneously. Tumors were
monitored until they reached an average volume of 220 mm.sup.3; animals
were then randomized into 3 treatment groups: IgG control, anti-HER2
FSA-ADC, and a biparatopic anti-HER2-ADC.
[0441] Fifteen animals were included in each group. Dosing for each group
is as follows:
[0442] A) IgG control was dosed intravenously with a loading dose of 30
mg/kg on study day 1 then with maintenance doses of 20 mg/kg twice per
week to study day 39.
[0443] B) Anti-HER2 FSA-ADC (v6246) was dosed intravenously with a loading
dose of 10 mg/kg on study day 1 then with a maintenance dose of 5 mg/kg
on day 15 and 29.
[0444] C) Biparatopic anti-HER2 antibody-ADC (v6363) was dosed
intravenously with a loading dose of 10 mg/kg on study day 1 then with a
maintenance dose of 5 mg/kg on day 15 and 29.
[0445] Tumor volume was measured throughout the study, and the number of
responders and median survival was assessed at day 22. The results are
shown in FIG. 19. A summary of the results is shown in Table 18.
[0446] The biparatopic anti-HER2-ADC and anti-HER2 FSA-ADC inhibited tumor
growth better than IgG control (FIG. 19A and Table 18). The biparatopic
anti-HER2-ADC inhibited tumor growth to a greater degree than did the
anti-HER2 FSA-ADC. The biparatopic anti-HER2-ADC group was associated
with an increase in the number of responding tumors compared to anti-HER2
FSA-ADC (11 and 9, respectively). The biparatopic anti-HER2-ADC and
anti-HER2 FSA-ADC groups demonstrated superior survival compared to IgG
control (FIG. 19B and Table 18). The biparatopic anti-HER2 antibody group
demonstrated median survival of 61 days compared to the anti-HER2 FSA-ADC
which had a median survival of 36 days (FIG. 19B and Table 18).
TABLE-US-00028
TABLE 18
n = 15, Day 22 IgG v6246 v6363
Mean TV 1908 (+766%) 873 (+297%) 632 (+187%)
(mm3) (% change from
Baseline)
% TGI 0 54% 67%
Responders 0/15 9/15 11/15
(TV <50% of control)
Median survival (days) 22 36 61
Example 18: Effect of a Biparatopic Anti-HER2 Antibody Drug Conjugate
(ADC) in a Human Primary Cell Xenograft Model (HBCx-13b)
[0447] The trastuzumab resistant patient derived xenograft model from
human breast cancer, HBCx-13B, was used to assess the anti-tumor efficacy
of an exemplary biparatopic anti-HER2 antibody conjugated to DM1.
[0448] Female athymic nude mice were inoculated with the tumor via the
insertion of a 20 mm.sup.3 tumor fragment subcutaneously. Tumors were
monitored until they reached an average volume of 100 mm.sup.3; animals
were then randomized into 3 treatment groups: anti-HER2 FSA (v506),
anti-HER2 FSA-ADC (v6246), and the biparatopic anti-HER2-ADC (v6363).
Seven animals were included in each group. Dosing for each group was as
follows:
[0449] A) Anti-HER2 FSA was dosed intravenously with a loading dose of 15
mg/kg on study day 1 and maintenance doses of 10 mg/kg administered on
study days 4, 8, 11, 15, 18, 22, and 25.
[0450] B) Anti-HER2 FSA-ADC was dosed intravenously with a loading dose of
10 mg/kg on study day 1 then with a maintenance dose of 5 mg/kg on day
22.
[0451] C) Biparatopic anti-HER2 antibody-ADC was dosed intravenously with
a loading dose of 10 mg/kg on study day 1 then with a maintenance dose of
5 mg/kg on day 22.
[0452] Tumor volume was measured throughout the study, and mean tumor
volume, complete response, and zero residual disease parameters were
assessed at Day 50. The results are shown in FIG. 20. A summary of the
results is shown in Table 19.
[0453] The biparatopic anti-HER2-ADC and anti-HER2 FSA-ADC demonstrated
greater tumor growth inhibition compared to an anti-HER2 FSA (v506). The
biparatopic anti-HER2-ADC inhibited tumor growth better than the
anti-HER2 FSA-ADC. The biparatopic anti-HER2-ADC group as compared to the
anti-HER2 FSA-ADC group was associated with an increase in the number of
tumors showing complete responses (more than a 10% decrease below
baseline), 7 and 4 respectively, and showing zero residual disease, 5 and
2 respectively.
TABLE-US-00029
TABLE 19
n = 7, Day 50 v506 v6246 v6363
Mean TV 1149 (+1018%) 262 (+153%) 26 (-75%)
(mm3) (% change from
Baseline)
% TGI 0% 77% 98%
Complete response 0 4/7 7/7
(>10% baseline regression)
Zero residual disease 0 2/7 5/7
(TV <20 mm3)
Example 19: Effect of a Biparatopic Anti-HER2 Antibody Drug Conjugate
(ADC) in a Human Primary Cell Xenograft Model (T226)
[0454] The patient derived trastuzumab resistant xenograft model from
human breast cancer, T226, was used to assess the anti-tumor efficacy of
an exemplary biparatopic anti-HER2-ADC.
[0455] Female athymic nude mice were inoculated with the tumor via the
insertion of a 20 mm.sup.3 tumor fragment subcutaneously. Tumors were
monitored until they reached an average volume of 100 mm.sup.3; animals
were then randomized into 4 treatment groups: IgG control (n=15),
anti-HER2 FSA (v506; n=15), anti-HER2 FSA-ADC (v6246; n=16), and the
biparatopic anti-HER2-ADC conjugate (v6363; n=16). Dosing for each group
was as follows:
[0456] A) IgG control was dosed intravenously with a loading dose of 15
mg/kg on study day 1 and maintenance doses of 10 mg/kg administered on
study days 4, 8, 11, 15, 18, 22, and 25
[0457] B) Anti-HER2 FSA was dosed intravenously with a loading dose of 15
mg/kg on study day 1 and maintenance doses of 10 mg/kg administered on
study days 4, 8, 11, 15, 18, 22, and 25
[0458] C) Anti-HER2 FSA-ADC was dosed intravenously with 5 mg/kg on study
days 1 and 15
[0459] D) Biparatopic anti-HER2-ADC conjugate was dosed intravenously with
5 mg/kg on study days 1 and 15.
[0460] Tumor volume was measured throughout the course of the study, and
mean tumor volume and complete response parameters were assessed at day
31. The results are shown in FIG. 21. A summary of the results is shown
in Table 20.
[0461] The biparatopic anti-HER2-ADC and anti-HER2 FSA-ADC demonstrated
better tumor growth inhibition compared to the anti-HER2 FSA (v506) and
IgG control. The exemplary biparatopic anti-HER2-ADC induced equivalent
tumor growth inhibition and complete baseline regression compared to
anti-HER2 FSA-ADC (FIG. 21 and Table 20) in this model.
TABLE-US-00030
TABLE 20
v506 v6363
Day 31 IgG (n = 13) (n = 13) v6246 (n = 16) (n = 16)
Mean TV 1797 1611 422 572
(mm3) (% change (+1728%) (+1573) (+332%) (+483%)
from Baseline)
% TGI (vs. hIgG) 0% 11% 77% 68%
Complete response 0/13 0/14 1/16 1/16
(>10% baseline
regression)
Example 20: Effect of a Biparatopic Anti-HER2 Antibody Drug Conjugate
(ADC) in a Human Primary Cell Xenograft Model (HBCx-5)
[0462] The patient derived trastuzumab resistant xenograft model from
human breast cancer, HBCx-5 (invasive ductal carcinoma, luminal B), was
used to assess the anti-tumor efficacy of an exemplary biparatopic
anti-HER2-ADC.
[0463] Female athymic nude mice were inoculated with the tumor via the
insertion of a 20 mm.sup.3 tumor fragment subcutaneously. Tumors were
monitored until they reached an average volume of 100 mm.sup.3; animals
were then randomized into 4 treatment groups: IgG control (n=15),
anti-HER2 FSA (v506; n=15), anti-HER2 FSA-ADC (v6246; n=16), and the
biparatopic anti-HER2-ADC (v6363; n=16). Dosing for each group was as
follows:
[0464] A) IgG control was dosed intravenously with a loading dose of 15
mg/kg on study day 1 and maintenance doses of 10 mg/kg administered on
study days 4, 8, 11, 15, 18, 22, and 25
[0465] B) Anti-HER2 FSA was dosed intravenously with a loading dose of 15
mg/kg on study day 1 and maintenance doses of 10 mg/kg administered on
study days 4, 8, 11, 15, 18, 22, and 25
[0466] C) Anti-HER2 FSA-ADC was dosed intravenously with 10 mg/kg on study
days 1 and 15, 22, 29, 36
[0467] D) Biparatopic anti-HER2-ADC was dosed intravenously with 10 mg/kg
on study days 1 and 15, 22, 29, 36.
[0468] Tumor volume was measured throughout the course of the study, and
the mean tumor volume, T/C ratio, number of responders, complete
response, and zero residual disease parameters were assessed at day 43.
The results are shown in FIG. 22. A summary of the results is shown in
Table 21.
[0469] The biparatopic anti-HER2-ADC and anti-HER2 FSA-ADC demonstrated
better tumor growth inhibition compared to an anti-HER2 FSA (v506) and
IgG control. The exemplary biparatopic anti-HER2-ADC induced equivalent
tumor growth inhibition and had an increased number of responders
compared to anti-HER2 FSA-ADC (FIG. 22 and Table 21) in the trastuzumab
resistant HBCx-5 human breast cancer xenograft model.
TABLE-US-00031
TABLE 21
IgG Herceptin T-DM1 6363
Day 43 (n = 4) (n = 5) (n = 7) (n = 7)
Mean TV 922 815 193 241
(mm3) (% change (+693%) (+598%) (+65%) (+106%)
from Baseline)
T/C (IgG) ratio 1 0.88 0.21 0.26
Responders 0/4 1/5 6/7 7/7
(TV<50% of control)
Complete response 0/4 0/5 1/7 0/7
(>10% baseline regression)
Zero residual disease 0/4 0/5 0/7 0/7
(TV <20 mm3)
Example 21: Effect of a Biparatopic Anti-HER2 Antibody Drug Conjugate
(ADC) to Anti-HER2 Treatment Resistant Tumors in a Human Cell Line
Xenograft Model (SKOV3)
[0470] The established human ovarian cancer cell derived xenograft model
SKOV3, described in Example 17, was used to assess the anti-tumor
efficacy of an exemplary biparatopic anti-HER2-ADC in anti-HER2 treatment
resistant tumors.
[0471] The methods were followed as described in Example 17 with the
following modifications. A cohort of animals was dosed with an anti-HER2
antibody intravenously with 15 mg/kg on study day 1 and with 10 mg/kg on
day 4, 8, 15; however, this treatment failed to demonstrate an
efficacious response by day 15 in this model. This treatment group was
then converted to treatment with the exemplary biparatopic anti-HER2
antibody drug conjugate (v6363) and was dosed with 5 mg/kg and on study
day 19 and 27 and 15 mg/kg on study day 34, 41 and 48.
[0472] Tumor volume was measured twice weekly throughout the course of the
experiment.
[0473] The results are shown in FIG. 23 and indicate that the group
treated with exemplary biparatopic anti-HER2-ADC (v6363) showed tumor
regression to a mean tumor volume less than the initial mean starting
volume of 220 mm.sup.3.
Example 22: Effect of a Biparatopic Anti-HER2 Antibody Drug Conjugate
(ADC) on Anti-HER2 Treatment Resistant Tumors in Human Primary Cell
Xenograft Model (HBCx-13b)
[0474] The trastuzumab resistant patient derived xenograft model from
human breast cancer, HBCx-13B, was used to assess the anti-tumor efficacy
of an exemplary biparatopic anti-HER2 antibody conjugated to DM1.
[0475] The methods were followed as described in Example 18 with the
following modifications. A cohort of animals was dosed with a bi-specific
anti-ErbB family targeting antibody intravenously with 15 mg/kg on study
day 1 and with 10 mg/kg on day 4, 8, 15, 18, 22, and 25; however, this
treatment failed to demonstrate an efficacious response. This treatment
group was then converted to treatment with the exemplary biparatopic
anti-HER2 antibody drug conjugate (v6363) and was dosed with 10 mg/kg on
days 31, 52 and with 5 mg/kg on day 45. Tumor volume was measured
throughout the duration of the study.
[0476] The results are shown in FIG. 24. These results show that the
exemplary biparatopic anti-HER2-ADC (v6363) prevented tumour progression.
From the first dose to day 57 the tumour volume of the v6363 treated
group increased by less than 2% while in the same interval the v506
treated group grew by more than 110%.
Example 23: Analysis of Fucose Content of an Exemplary Biparatopic
Anti-HER2 Antibody
[0477] Glycopeptide analysis was performed to quantify the fucose content
of the N-linked glycan of the exemplary biparatopic anti-HER2 antibodies
(v5019, v7091 and v10000).
[0478] The glycopeptide analysis was performed as follows. Antibody
samples were reduced with 10 mM DTT at 56.degree. C. 1 h and alkylated
with 55 mM iodoacetamide at RT 1 h and digested in-solution with trypsin
in 50 mM ammonium bicarbonate overnight at 37.degree. C. Tryptic digests
were analyzed by nanoLC-MS/MS on a QTof-Ultima. The NCBI database was
searched with Mascot to identify protein sequences. MaxEnt3 (MassLynx)
was used to deconvolute the glycopeptide ions and to quantify the
different glycoforms.
[0479] A summary of the glycopeptide analysis results is in Table 22. The
N-linked glycans of exemplary biparatopic anti-HER2 antibodies (v5019,
v7091 and v10000) are, approximately 90% fucosylated (10% N-linked
glycans without fucose). The N-linked glycans of monospecific anti-HER2
FSA (v506) are, approximately 96% fucosylated (4% N-linked glycans
without fucose) and Herceptin.RTM. is approximately 87% fucosylated (4%
N-linked glycans without fucose).
TABLE-US-00032
TABLE 22
Fc N-linked Glycopeptide Analysis
Average
Antibody % of Glycopeptides Average % of Glycopeptides
Variant Observed With Fucose Observed Without Fucose n
v506 96.4 3.6 5
Herceptin .RTM. 86.5 13.4 4
v5019 90.5 9.4 6
v7091 89.9 26.9 3
v10000 89.2 10.7 5
[0480] These results show that biparatopic anti-HER2 antibodies (with a
heterodimeric Fc), expressed transiently in CHO cells, have approximately
3% higher fucose content in the N-glycan compared to commercial
Herceptin.RTM.. The homodimeric anti-HER2 FSA (v506), expressed
transiently in CHO cells, has the highest fucose content of approximately
96%.
Example 24: Thermal Stability of an Exemplary Biparatopic Anti-HER2
Antibody
[0481] Thermal stability of exemplary biparatopic anti-HER2 antibodies
(v5019, v7091 and v10000) and ADCs (v6363, v7148 and v10533) was measured
by DSC as described below.
[0482] DSC was performed in the MicroCal.TM. VP-Capillary DSC (GE
Healthcare) using a purified protein sample (anti-HER2 biparatopic
antibodies and anti-HER2 biparatopic-ADCs) adjusted to about 0.3 mg/ml in
PBS. The sample was scanned from 20 to 100.degree. C. at a 60.degree.
C./hr rate, with low feedback, 8 sec filter, 5 min preTstat, and 70 psi
nitrogen pressure. The resulting thermogram was analyzed using Origin 7
software.
[0483] The thermal stability results of exemplary biparatopic anti-HER2
antibodies (v5019, v7091 and v10000) are shown in FIG. 25A-C. FIG. 25A
shows the thermogram for v5019; the Fc and chain A Fab of each have a
T.sub.m of 75.degree. Celsius and the chain B scFv of 5019 has a T.sub.m
of 69.degree. Celsius. FIG. 25B shows the thermogram for v10000; the Fc
CH3 domain has a T.sub.m 82.degree. Celsius, Fab chain A has T.sub.m of
76.5.degree. Celsius and the chain B scFv has a T.sub.m of 69.5.degree.
Celsius. FIG. 25C shows the thermogram for v7091; the Fc CH3 domain has a
T.sub.m 82.degree. Celsius, Fab chain A has T.sub.m of 76.7.degree.
Celsius and the chain B scFv has a T.sub.m of 69.5.degree. Celsius.
[0484] The thermal stability results of exemplary biparatopic anti-HER2
ADCs (v6363, v7148 and v10533) are shown in FIG. 26A-C. FIG. 26A shows
the thermogram for v6363; the Fc has a T.sub.m of 75.degree. Celsius and
the chain A Fab and Fc CH3 domain have a T.sub.m of 75.degree. Celsius.
The chain B scFv of 6363 has a T.sub.m of 69.degree. Celsius. FIG. 26B
shows the thermogram for v10553; the Fc CH3 domain has a T.sub.m of
83.degree. Celsius, the chain A Fab has a T.sub.m of 75.7.degree. Celsius
and the chain B scFv has a T.sub.m of 66.2.degree. Celsius. FIG. 26C
shows the thermogram for v7148; the Fc CH3 domain has a T.sub.m of
82.6.degree. Celsius, the chain A Fab has a T.sub.m of 74.8.degree.
Celsius and the chain B scFv has a T.sub.m of 66.6.degree. Celsius.
[0485] The exemplary biparatopic antibodies and ADCs have thermal
stability comparable to wildtype IgG.
Example 25: Ability of an Exemplary Biparatopic Anti-HER2 Antibody to
Elicit ADCC of Breast Tumor Cells Expressing Varying Levels of HER2
[0486] The ability of exemplary biparatopic antibody (v5019) to elicit
dose-dependent ADCC of HER2 positive 3+, 2+, and 0/1+ HER2 expressing
(triple-negative) breast cancer cell lines was examined. The ADCC
experiments were performed as described in Example 11 with the exception
that NK effector cell to target cell ratio remained constant at 5:1.
[0487] The ADCC results are shown in FIG. 27 and Table 23. The results in
FIG. 27A-C show that exemplary biparatopic antibody (v5019) elicits
approximately 1.2 to 1.3-fold greater maximum cell lysis of HER2 positive
3+, 2+ and 0/1+ HER2 expressing breast cancer cells compared to
Herceptin.RTM.. The results also show that v5019 (90% N-glycans with
fucose) more effectively mediates ADCC of HER2 positive 3+, 2+ and 0/1+
HER2 expressing breast cancer despite having approximately a 4% higher
fucose content in the N-glycan (resulting in lower binding affinity to
CD16 on NK cells) compared to Herceptin.RTM. (86% N-glycans with fucose;
Example 23). The higher target cell killing elicited by v5019 is
presumably due to increased tumor cell decoration as described in Example
6.
TABLE-US-00033
TABLE 23
ADCC of HER2 3+, 2+ and 0/1+ HER2 expressing breast cancer cells
SKBr3
HER2 3+ JIMT-1 HER2 2+
Max % Max % MDA-MB-231 HER2 0/1+
Target Cell EC.sub.50 Target EC.sub.50 Max % Target EC.sub.50
Treatment Lysis (nM) Cell Lysis (nM) Cell Lysis (nM)
v5019 30 ~0.9 60 0.001 53 0.9
Herceptin .RTM. 23 ~0.9 51 0.002 44 0.9
[0488] The ADCC results in FIG. 27D show that exemplary biparatopic
antibodies (v7091 and v10000) elicit similar maximal cell lysis compared
to Herceptin.RTM. in the basal HER2 expressing WI-38 cell line. The ADCC
results support the cell binding data (Example 6), showing that a
threshold for increased binding and ADCC occurs when the HER2 receptor
levels are greater than 10,000 HER2/cell. Based on this data it would be
expected that the exemplary biparatopic anti-HER2 antibodies would have
increased cell surface binding and ADCC of HER2 3+, 2+ and 1+ tumor cells
but would not have increase cell surface binding and ADCC of non-tumor
cells that express basal levels of the HER2 receptor at approximately
10,000 receptors or less.
Example 26: Effect of Antibody Afucosylation on ADCC
[0489] The ability of afucosylated exemplary biparatopic antibodies
(v5019-afuco, 10000-afuco) to elicit dose-dependent ADCC of HER2 positive
2/3+, 2+ and 0/1+ HER2 expressing (triple-negative) breast cancer cell
lines, was examined. ADCC experiments were performed as described in
Example 11, in SKOV3 cells, MDA-MB-231 cells and ZR75-1 cells with the
exception that a constant NK effector cell or PBMC effector to target
(E:T) cell ratio of 5:1 was used. Afucosylated exemplary biparatopic
antibodies were produced transiently in CHO cells as described in Example
1, using the transiently expressed RMD enzyme as described in von Horsten
et al. 2010 Glycobiology 20:1607-1618. The fucose content of v5019-afuco
and v10000-afuco were measured as described in Example 23 and determined
to be less <2% fucosylated (data not shown). Data using NK effector
cells is shown in FIG. 28A-B, while data using PBMCs is shown in FIG.
28C.
[0490] FIG. 28A, FIG. 28B and Table 24 show that afucosylated v5019
(v5019-afuco) elicits ADCC of HER 2/3+ and 0/1+ HER2 expressing breast
cancer cells with approximately 1.5 to 1.7-fold higher maximum cell lysis
than Herceptin.RTM..
TABLE-US-00034
TABLE 24
ADCC of HER2 2/3+ and basal HER2 expressing (triple-negative) breast
cancer cells
MDA-
SKOV3 HER2 2+/3+ MD-231 HER2 0/1+
Max % Target Max % Target
Treatment Cell Lysis EC.sub.50 (nM) Cell Lysis EC.sub.50 (nM)
v5019- 24 ~0.6 58 ~0.6
afucosylated
Herceptin .RTM. 14 ~0.6 40 ~0.3
[0491] The results in FIG. 28C and Table 25 show that v10000 elicits ADCC
of HER2 2+ ZR-75-1 breast cancer cells with approximately 1.3-fold
greater maximal cell lysis than Herceptin.RTM., and v10000-afuco elicits
approximately 1.5-fold greater maximal cell lysis than Herceptin.RTM..
TABLE-US-00035
TABLE 25
ADCC of HER2 2/3+ breast cancer cells
ZR-751 HER2 2+
Max % Target
Treatment Cell Lysis EC.sub.50 (nM)
v10000 28 ~0.06
v10000-afucosylated 32 ~0.7
Herceptin .RTM. 21 ~0.5
[0492] The ADCC results show that the exemplary afucosylated biparatopic
antibodies (v5019-afuco, v10000-afuco) elicit approximately 15-25%
greater maximum cell lysis compared to the fucosylated antibodies (v5019
Example 25, v10000) when Herceptin.RTM. is used as a benchmark. These
results show that reducing the fucose content of the Fc N-glycan results
in increased maximal cell lysis by ADCC.
Example 27: Ability of Exemplary Biparatopic Anti-HER2 Antibody to Inhibit
Growth of HER2 3+ Breast Cancer Cells in the Presence of Exogenous
Growth-Stimulatory Ligands (EGF and HRG)
[0493] The ability of 5019 to inhibit growth of HER2 3+ breast cancer
cells in the presence of exogenous growth-stimulatory ligands (EGF and
HRG) was examined.
[0494] Test antibodies and exogenous ligand (10 ng/mL HRG or 50 ng/mL EGF)
were added to the target BT-474 HER2 3+ cells in triplicate and incubated
for 5 days at 37.degree. C. Cell viability was measured using
AlamarBlue.TM. (37.degree. C. for 2 hr), absorbance read at 530/580 nm.
Data was normalised to untreated control and analysis was performed using
GraphPad Prism.
[0495] The results are shown in FIG. 29 and Table 26. The results show
that exemplary biparatopic antibody v5019 inhibits the growth of HER2 3+
breast cancer cells in the absence of growth stimulatory ligand (70%
inhibition), as well as in the presence of EGF (40% inhibition) or HRG
(.about.10% inhibition). The anti-HER2 monospecific FSA (v506) does not
block EGF or HRG induced tumor cell growth via other erbB receptors EGFR
and HER3. v5019 is superior to v506 in inhibiting HER2 and
ligand-dependent dimerization and growth via other companion erbB
receptors.
TABLE-US-00036
TABLE 26
Growth Inhibition of HER2 3+ Cancer Cells
% Survival
Treatment Antibody only +EGF +HRG
Mock 100 122 110
v506 41 114 129
v5019 31 56 92
[0496] These results show that exemplary biparatopic antibody is capable
of reducing ligand-dependent growth of HER2+ cells, presumably due
binding of the anti-ECD2 chain A Fab arm and subsequent blocking of
ligand stimulated receptor homo- and heterodimerization, and erbB
signaling.
Example 28: Effect of a Biparatopic Anti HER2 Antibody in a
Trastuzumab-Resistant and Chemotherapy Resistant HER2 3+ Patient-Derived
(PDX) Metastatic Breast Cancer Xenograft Model of Invasive Ductal Breast
Carcinoma
[0497] The HER2 3+ (ER-PR negative) patient derived xenograft model from
invasive ductal human breast cancer, HBCx-13B, was used to assess the
anti-tumor efficacy of an exemplary biparatopic anti-HER2 antibody,
v7187. v7187 is an afucosylated version of v5019. The model is resistant
to single agent trastuzumab, the combination of trastuzumab and
pertuzumab (see example 31), capecitabine, docetaxel, and
adriamycin/cyclophosphamide.
[0498] Female athymic nude mice were inoculated subcutaneously with a 20
mm.sup.3 tumor fragment. Tumors were then monitored until reaching an
average volume of 140 mm3. Animals were then randomized into 2 treatment
groups: vehicle control and v7187 with eight animals in each group. IV
Dosing was as follows. Vehicle control was dosed intravenously with 5
ml/kg of formulation buffer twice per week to study day 43. v7187 was
dosed intravenously with 10 mg/kg twice per week to study day 43. Tumor
volume was measured throughout the study, and other parameters assessed
at day 43 as shown in Table 27.
[0499] The results are shown in FIG. 30 and Table 27. The results show
that tumors treated with vehicle control showed continual progression and
exceeded 1600 mm.sup.3 by study day 43. Mice treated with v7187 showed
significantly greater tumor growth inhibition (T/C-0.44) with a mean
tumor volume of 740 mm.sup.3 on day 43. v7187 induced responses in 5/8
tumors with a single tumor showing complete regression with zero residual
disease on study day 43. Animals treated with v7187 had a superior
response rate with 5/8 tumors responding to therapy compared to 0/8 mice
treated with vehicle control. In addition, treatment with v7187
significantly delayed tumor progression compared to vehicle control with
doubling times of 19 and 11 days respectively.
TABLE-US-00037
TABLE 27
Tumour Response Vehicle V7087
Day 43 Mean TV (mm3) 1683 740
(% Change from (+1079%) (+422%)
Baseline)
T/C ratio 1 0.44
Responders 0/8 5/8
(TV < 50%
of control)
PR (>10% 0/8 1/8
baseline regression)
ZRD (TV < 20 mm3) 0/8 1/8
Time to Doubling 11 19
progression time (days)
[0500] These data show that the exemplary anti-HER2 biparatopic (v7187) is
efficacious in a Trastuzumab+Pertuzumab resistant HER2 3+ metastatic
breast cancer tumor xenograft model. V7187 treatment has a high response
rate and can significantly impair tumor progression of standard of care
treatment resistant HER2 3+ breast cancers.
Example 29: Assessment of Biparatopic Anti-HER2 ADC Binding to HER2+ Tumor
Cell Lines
[0501] The ability of exemplary biparatopic anti-HER2 ADCs to bind and
saturate HER2 positive 3+, 2+, breast and ovarian tumor cell lines was
analyzed by FACS as described in Example 6.
[0502] The data is shown in FIG. 31. FIG. 31A shows v6363 binding to SKOV3
tumor cell lines with approximately a 2.0-fold greater Bmax (MFI) than
T-DM1 (v6246) at saturating concentrations. FIG. 31B shows v6363 binds to
JIMT-1 tumor cell lines with approximately a 1.6-fold greater Bmax (MFI)
than T-DM1 (v6246) at saturating concentrations. These data show that
v6363 (ADC) has similar tumor cell binding properties of increased cell
surface binding compared to the parent unconjugated v5019 antibody
(Example 6). Conjugation of v5019 with SMCC-DM1 (v6363) does not alter
the antigen-binding properties of the antibody.
[0503] The FACS binding assay was repeated to include direct comparison to
the exemplary biparatopic antibodies (v5019, v7091 and v10000) and ADCs
(v6363, v7148 and v10553). The data is shown in FIG. 31C and FIG. 31D.
The exemplary biparatopic anti-HER2 ADCs (v6363, v7148 and v10553) have
equivalent cell surface saturation (Bmax) compared to the unlabeled
biparatopic antibodies (v5019, v7091 and v10000).
[0504] These data show that conjugation of exemplary biparatopic
antibodies (v5019, v7091 and v10000) with SMCC-DM1 does not alter the
binding properties. The exemplary anti-HER2 biparatopic anti-HER2 ADCs
(v6363, v7148 and v10553) have approximately 1.5-fold (or greater)
increased cell surface binding compared to a monospecific anti-HER2 ADC
(v6246, T-DM1).
Example 30: Dose-Dependent Tumour Growth Inhibition of an Exemplary
Anti-HER2 Biparatopic-ADC in a HER2 3+ (ER-PR Negative) Patient Derived
Xenograft Model
[0505] The HER2 3+ (ER-PR negative) patient derived xenograft model from
invasive ductal human breast cancer, HBCx-13B, was used to assess the
anti-tumor efficacy of an exemplary biparatopic anti-HER2 ADC, v6363. The
model is resistant to single agent trastuzumab, the combination of
trastuzumab and pertuzumab (see example 31), capecitabine, docetaxel, and
adriamycin/cyclophosphamide.
[0506] Female athymic nude mice were inoculated with the tumor via the
subcutaneous insertion of a 20 mm.sup.3 tumor fragment. Tumors were
monitored until they reached an average volume of 160 mm.sup.3; animals
were then randomized into 5 treatment groups: non-specific human IgG
control, and 4 escalating doses of v6363. 8-10 animals were included in
each group. Dosing for each group was as follows. IgG control was dosed
intravenously with 10 mg/kg twice per week to study day 29. v6363 was
dosed intravenously with 0.3, 1, 3, or 10 mg/kg on study days 1, 15, and
29. Tumor volume was assessed throughout the study and parameters
assessed as indicated in Table 29.
[0507] The results are shown in FIG. 32 and Table 28. These results show
that the exemplary anti-HER2 biparatopic ADC (v6363) mediated
dose-dependent tumor growth inhibition in the Trastuzumab-resistant
HBCx-13b PDX model (FIG. 32A). In addition, v6363 improved overall
survival in a dose-dependent manner, with median survival time of more
than 63 days for 3 mg/kg and 10 mg/kg doses compared to 43 days for IgG
control (FIG. 32B and Table 28). The 3 mg/kg dose was associated with an
increased response rate (5/10) compared to control (0/8). All mice
treated with v6363 at 10 mg/kg dose not only responded to therapy (9/9)
but also showed prevention of tumor progression. Moreover, the majority
of tumors had objective partial responses (7/9) and, at the end of the
study, many had zero residual disease (6/9). v6363 was well tolerated at
all doses, no adverse events were observed and no body weight loss was
observed.
TABLE-US-00038
TABLE 28
6363 6363 6363 6363
0.3 1 3 10
Tumour Response IgG mg/kg mg/kg mg/kg mg/kg
Day 43 Mean TV 1963 1916 1613 1268 84
(mm3) (% (+1119%) (+1073%) (+895%) (+682%) (-49%)
change
from
Baseline)
T/C (IgG) 1 0.97 0.82 0.64 0.04
ratio
Re- 0/8 0/10 2/10 5/10 9/9
sponders
(TV <
50% of
control)
PR 0/8 0/10 0/10 0/10 7/9
(>10%
baseline
re-
gression)
ZRD 0/8 0/10 0/10 0/10 6/9
(TV < 20
mm3)
Time to Tumor 9 9 14 17 52
pro- doubling
gression time
(days)
Survival Median 43 41 50 >63 >63
Re- Survival
sponse (Days)
Body % Change +10% +10% +9% +5% +0%
Weight from
Baseline
[0508] These data show that the exemplary anti-HER2 biparatopic ADC
(v6363) is efficacious in a Trastuzumab+Pertuzumab resistant HER2 3+
metastatic breast cancer tumor xenograft model. v6363 treatment is
associated with a high response rate, significantly impairs tumor
progression, and prolongs survival in a standard of care resistant HER2
3+ breast cancers.
Example 31: Biparatopic Anti-HER2-ADC Compared to Standard of Care
Combinations in the Trastuzumab Resistant PDX HBCx-13b
[0509] The efficacy of v6363 in a HER2 3+, ER-PR negative Trastuzumab
resistant patient-derived breast cancer xenograft model (HBCx-13b), was
evaluated and compared to to the combination of:
Herceptin.TM.+Perjeta.TM.; and Herceptin.TM.+Docetaxel.
[0510] Female athymic nude mice were inoculated with the tumor via the
subcutaneous insertion of a 20 mm3 tumor fragment. Tumors were monitored
until they reached an average volume of 100 mm3; animals were then
randomized into 4 treatment groups (8-10 animals/group): non-specific
human IgG control, Herceptin.TM.+Docetaxel, Herceptin.TM.+Perjeta.TM.,
and v6363. Dosing for each group was as follow. IgG control was dosed
intravenously with 10 mg/kg twice per week to study day 29.
Herceptin.TM.+Docetaxel combination Herceptin.TM. was dosed intravenously
with 10 mg/kg IV twice weekly to study day 29 and Docetaxel was dosed
intraperitoneally with 20 mg/kg on study day 1 and 22.
Herceptin.TM.+Perjeta.TM. combination Herceptin was dosed intravenously
with 5 mg/kg twice per week to study day 29 and Perjeta.TM. was dosed
intravenously with 5 mg/kg twice per week to study day 29. The dosing of
Herceptin.TM. and Perjeta.TM. was concurrent. v6363 was dosed
intravenously with 10 mg/kg on study day 1, 15, and 29.
[0511] The results are shown in FIG. 33 and Table 29. FIG. 33A shows tumor
volume over time, and FIG. 33B shows a survival plot. These results show
that the combination of Herceptin.TM.+Perjeta.TM. did not produce any
tumor growth inhibition compared to control IgG and exceeded 1800
mm.sup.3 on day 39. The combination of Herceptin.TM.+Docetaxel did not
significantly reduce tumor growth but did prolong median survival to 53
days compared to 43 days for IgG control. v6363 produced significant
tumor growth inhibition (T/C-0.04), where, all tumors responded to
therapy and 7/10 tumors experienced complete regressions (zero residual
disease). v6363 significantly prolonged survival compared to both
combination therapies. Body weights across cohorts were not significantly
affected by treatments.
TABLE-US-00039
TABLE 29
HerceptinTM + HerceptinTM + v6363
Tumour Response IgG PerjetaTM Docetaxel 10 mg/kg
Day 39 Mean TV (mm3) 1809 1975 1328 76
(% change from (+1023%) (+1085%) (+714%) (-54%)
Baseline)
T/C (IgG) ratio 1.0 1.10 0.73 0.04
Responders 0/8 0/8 1/10 9/9
(TV < 50% of
control)
PR 0/8 0/8 0/10 8/9
(>10% baseline
regression)
ZRD 0/8 0/8 0/10 6/9
(TV < 20 mm3)
Survival Median Survival 43 39 53 >63
Response (days)
Body % Change from +10% +7% +3% -2%
Weight Baseline
[0512] These results show that exemplary anti-HER2 biparatopic ADC (v6363)
is superior to standard of care combinations with respect to all
parameters tested in this xenograft model.
Example 32: Efficacy of a Biparatopic Anti-HER2-ADC in HER2+
Trastuzumab-Resistant Breast Cancer Cell Derived Tumour Xenograft Model
[0513] The efficacy of v6363 in a HER2 3+ Trastuzumab resistant breast
cancer cell-derived (JIMT-1, HER2 2+) xenograft model was evaluated
(Tanner et al. 2004. Molecular Cancer Therapeutics 3: 1585-1592).
[0514] Female RAG2 mice were inoculated with the tumor subcutaneously.
Tumors were monitored until they reached an average volume of 115
mm.sup.3; animals were then randomized into 2 treatment groups:
Trastuzumab (n=10) and v6363. Dosing for each group was as follows.
Trastuzumab was dosed intravenously with 15 mg/kg on study day 1 and 10
mg/kg twice per week to study day 26. v6363 was dosed intravenously with
5 mg/kg on study days 1 and 15 and with 10 mg/kg on day 23 and 30 and 9
mg/kg on day 37 and 44.
[0515] The results are shown in FIG. 34 and Table 30. These results show
that v6363 significantly inhibited tumor growth (T/C-0.74) compared to
Trastuzumab on study day 36. v6363 and Trastuzumab treatment did not
significantly change body weight. v6363 serum exposure was 17.9 .mu.g/ml
7 days after the first 10 mg/kg dose.
TABLE-US-00040
TABLE 30
Tumour Response Trastuzumab 6363
Day 36 Mean TV (mm3) 718 532
(% change from (+541) (+335%)
Baseline)
T/C (Tras) ratio 1 0.74
Responders 1/10 2/13
(TV < 50% of
control)
PR 0/10 0/13
(>10% baseline
regression)
ZRD 0/10 0/13
(TV < 20 mm3)
Body % Change from +5.8% +3.1%
Weight Baseline
Drug Mean Serum 187.2 17.9
Exposure Concentration
(day 7) (ug/ml)
[0516] These results show that exemplary anti-HER2 biparatopic ADC (v6363)
is efficacious in a Trastuzumab-resistant breast cancer and has a
potential utility in treating breast cancers that are resistant to
current standards of care.
Example 33: Fc.gamma.R Binding to Heterodimeric Fc of Anti-HER2
Biparatopic Antibodies and Anti-HER2 Biparatopic-ADCs
[0517] The binding of anti-HER2 biparatopic antibody (v5019, v7019 v10000)
and ADC (v6363, v7148 and v10553) having a heterodimeric Fc, to human
Fc.gamma.Rs was assessed and compared to anti-HER2 FSA (v506) and ADC
(v6246) having a homodimeric Fc.
[0518] Affinity of Fc.gamma.R to antibody Fc region was measured by SPR
using a ProteOn XPR36 (BIO-RAD). HER2 was immobilized (3000 RU) on CMS
chip by standard amine coupling. Antibodies were antigen captured on the
HER2 surface. Purified Fc.gamma.R was injected various concentration
(20-30 .mu.l/min) for 2 minutes, followed by 4 minute dissociation.
Sensograms were fit globally to a 1:1 Langmuir binding model. Experiments
were conducted at 25.degree. C.
[0519] The results are shown in Table 31. The exemplary heterodimeric
anti-HER2 biparatopic antibodies and ADCs bound to CD16aF, CD16aV158,
CD32aH, CD32aR131, CD32bY163 and CD64A with comparable affinities.
Conjugation of the antibodies with SMCC-DM1 does not negatively affect
Fc.gamma.R binding. The heterodimeric anti-HER2 biparatopic antibodies
have approximately 1.3 to 2-fold higher affinity to CD16aF, CD32aR131,
CD32aH compared to homodimeric anti-HER2 FSA (v506) and ADC (v6246).
These results show that the heterodimeric anti-HER2 biparatopic
antibodies and ADCs bind different polymorphic forms of Fc.gamma.Rs on
immune effector cells with similar or greater affinity than a WT
homodimeric IgG1.
TABLE-US-00041
TABLE 31
Human Fc7R Binding by SPR
10 uM 10 uM 10 uM 10 uM 10 uM
CD16a v158 CD16aF CD32aR131 CD32aH CD32b Y163 100 nM CD64A
Variant KD Ave SD KD Ave SD KD Ave SD KD Ave SD KD Ave SD KD Ave SD
v506 1.5E-07 2E-08 7.1E-07 1.E-08 7.6E-07 1.E-07 6.3E-07 2E-08 2.4E-06
1.E-07 8.64E-10 4.33E-10
v6246 1.6E-07 2E-08 7.0E-07 9.E-09 7.4E-07 7.E-08 6.3E-07 2E-08 2.1E-06
7.E-08 1.08E-09 5.13E-10
v10000 1.2E-07 1E-08 4.8E-07 2.E-08 5.1E-07 9.E-08 4.6E-07 2E-08 1.5E-06
7.E-08 8.41E-10 4.74E-10
v10553 1.2E-07 2E-08 4.9E-07 2.E-07 3.5E-07 1.E-07 3.6E-07 4E-09 1.2E-06
7E-08 4.95E-10 1.41E-10
v7091 1.2E-07 1E-08 5.1E-07 2.E-08 5.6E-07 9.E-08 5.0E-07 3E-08 1.7E-06
8E-08 9.68E-10 5.05E-10
v7148 1.2E-07 2E-08 5.4E-07 2.E-07 3.7E-07 1.E-07 4.2E-07 1E-08 1.5E-06
1.E-07 5.77E-10 2.02E-10
v5019 1.3E-07 1E-08 5.2E-07 1.E-08 5.6E-07 6.E-08 4.7E-07 2E-08 1.6E-06
2.E-07 8.44E-10 4.88E-10
v6363 1.2E-07 2E-08 4.5E-07 1.E-07 3.5E-07 1.E-07 3.4E-07 1E-08 1.2E-06
5.E-08 4.58E-10 1.13E-10
Example 34: Efficacy of Exemplary Anti-HER2 Biparatopic Antibodies In Vivo
in a Trastuzumab Sensitive Ovarian Cancer Cell Derived Tumour Xenograft
Model
[0520] The established human ovarian cancer cell derived xenograft model
SKOV3, described in Example 17, was used to assess the anti-tumor
efficacy of the exemplary biparatopic anti-HER2 antibodies, v5019, v7091
and v10000.
[0521] Female athymic nude mice were inoculated with a tumor suspension of
325,000 cells in HBSS subcutaneously on the left flank. Tumors were
monitored until they reached an average volume of 190 mm.sup.3 and
enrolled in a randomized and staggered fashion into 4 treatment groups:
non-specific human IgG control, v5019, v7091, and v10000. Dosing for each
group was as follows. Non-specific human IgG was dosed intravenously with
10 mg/kg starting on study day 1 twice per week to study day 26. V5019,
v7091, and v10000 were dosed intravenously with 3 mg/kg starting on study
day 1 twice per week to study day 26. Tumor volume was measured
throughout the study, and the parameters listed in Table 32 were measured
at day 29.
[0522] The data are presented in FIG. 35A (tumor growth), FIG. 35B
(survival plot) and Table 32 and show that treatment with v5019, v7091
and v10000 resulted in comparable tumor growth inhibition (T/C:
0.53-0.71), number of responding tumors, time to progression, and
survival on study day 29 compared to IgG control. The serum exposure of
v5019, v7091, and v10000 was similar (31-41 microg/ml) on study day 7.
TABLE-US-00042
TABLE 32
IgG v5019 V7091 V10000
Tumour Response (n = 8) (n = 11) (n = 11) (n = 11)
Day 29 Mean TV 1903 1001 1354 1114
(mm3) (% (+899%) (+416%) (+618%) (+503%)
change from
Baseline)
T/C (Tras) ratio 1 0.53 0.71 0.58
Responders 1/8 5/11 4/11 6/11
(TV < 50% of
control)
PR 0/8 1/11 0/11 0/11
(>10% baseline
regression)
ZRD 0/8 0/11 0/11 0/11
(TV < 20 mm3)
Time to Tumor doubling 12 15 16 15
progression time (days)
Survival Median survival 29 Na 37 41
(days)
Drug Mean Serum na 31.2 41.0 31.2
Exposure Concentration
(day 7) (ug/ml)
[0523] These results show that the exemplary anti-HER2 biparatopic
antibodies, v5019, v7091, and v10000) have potential utility in treating
moderately Trastuzumab sensitive HER2 overexpressing ovarian cancers.
Example 35: Exemplary Biparatopic Anti-Her2 Antibodies Dose-Dependently
Inhibit Tumour Growth in the Trastuzumab-Sensitive Ovarian Cancer Cell
Derived Tumour Xenograft
[0524] The established human ovarian cancer cell derived xenograft model
SKOV3, described in Example 17, was used to assess the dose-dependent
efficacy of an exemplary biparatopic anti-HER2 antibody, v10000.
[0525] Female athymic nude mice were inoculated with a tumor suspension of
325,000 cells in HBSS subcutaneously on the left flank. Tumors were
monitored until they reached an average volume of 190 mm.sup.3 and
enrolled in a randomized and staggered fashion into 6 treatment groups:
non-specific human IgG control and 5 escalating doses of v10000. 9-13
animals were included in each group. Dosing for each group was as
follows. IgG control was dosed intravenously with 10 mg/kg twice per week
to study day 26. V10000 was dosed intravenously with 0.1, 0.3, 1, 3, or
10 mg/kg twice per week.
[0526] The data are presented in FIG. 36 and Table 33 and show that
treatment with v10000 dose dependently induces tumor growth inhibition
(T/C: 0.28-0.73) compared to control IgG. In addition, v10000 was
dose-dependently associated with responding tumors (7/9 at 10 mg/kg and
3/11 at 0.1 mg/kg) increased time to progression (24 days at 10 mg/kg and
12 days at 0.1 mg/kg) on study day 29. The serum exposure of v10000 on
day 7 was dose dependent and increased from 0.46 microg/ml with a 0.1
mg/kg dose to 79.3 microg/ml with a 10 mg/kg dose.
TABLE-US-00043
TABLE 33
V10000, V10000, V10000, V10000,
V10000, 10 3 mg/kg 1 mg/kg 0.3 mg/kg 0.1 mg/kg
Tumor Response IgG (n = 8) mg/kg (n = 9) (n = 11) (n = 11) (n = 13) (n =
11)
Day 29 Mean TV (mm3) 1903 (+899%) 543 1114 1534 1535 1385
(% change from (+281%) (+503%) (+688%) (+694%) (+643%)
Baseline)
T/C ratio 1 0.28 0.58 0.81 0.81 0.73
Responders 1/8 7/9 6/11 2/11 3/13 3/11
(TV < 50% of
control)
PR 0/8 1/9 0/11 0/11 0/13 0/11
(>10% baseline
regression)
ZRD 0/8 0/9 0/11 0/11 0/13 0/11
(TV < 20 mm3)
Time to Tumor doubling 12 24 15 14 12 12
Progression time (days)
Drug Exposure Mean Serum na 79.3 31.2 4.7 1.5 0.46
(Day 7) Concentration
(ug/ml)
[0527] These results show that the exemplary anti-HER2 biparatopic
antibody, v10000, inhibits tumor progression in a dose-dependent manner.
Example 36: Ability of Anti-HER2 Biparatopic Antibody and Anti-HER2
Biparatopic-ADC to Inhibit Growth of Cell Lines Expressing HER2, and EGFR
and/or HER3 at the 3+, 2+ or 1+ Levels
[0528] The following experiment was performed to measure the ability of an
exemplary biparatopic anti-HER2 antibody (v10000) and corresponding
biparatopic anti-HER2 ADC (v10553) to inhibit growth of a selection of
breast, colorectal, gastric, lung, skin, ovarian, renal, pancreatic, head
and neck, uterine and bladder tumor cell lines that express HER2, and
EGFR and/or HER3 at the 3+, 2+, 1+ or 0+ level as defined by IHC.
[0529] The experiment was conducted as follows. The optimal seeding
density for each cell line was uniquely determined to identify a seeding
density that yielded approximately 60-90% confluency after the 72 hr
duration of the assay. Each cell line was seeded at the optimal seeding
density, in the appropriate growth medium per cell line, in a 96-well
plate and incubated for 24.degree. C. at 36.degree. C. and 5% CO.sub.2.
Antibodies were added at three concentrations (v10000 at 300, 30 and 0.3
nM; v10553 at 300, 1, 0.1 nM), along with the positive and vehicle
controls. The positive control chemococktail drug combination of 5-FU
(5-fluorouracil), paclitaxel, cisplatin, etoposide (25 microM), the
vehicle control consisted of PBS. The antibody treatments and controls
were incubated with the cells for 72 h in a cell culture incubator at
36.degree. C. and 5% CO.sub.2. The plates were centrifuged at 1200 RPM
for 10 min and culture medium completely removed by aspiration. RPMI SFM
medium (200 microL) and MTS (20 microL) was added to each well and
incubated at 36.degree. C. and 5% CO.sub.2 for 3 h. Optical density was
read at 490 nM and percent growth inhibition was determined relative to
the vehicle control.
[0530] The results are shown in FIG. 37 and a summary of all test results
are shown in FIG. 38. FIG. 37A shows the growth inhibition results of
v10000. These results show that v10000 can inhibit growth of breast,
colorectal, gastric, lung, skin, ovarian, renal, pancreatic, head and
neck, uterine, and endometrial tumor cell lines that express HER2 and
coexpress EGFR and/or HER3 at the 3+, 2+, 1+ or 0+ level. The activity of
v10000 and v10553 at 300 nM is summarized in FIG. 38, where `+` indicates
cell lines that showed a reduction in cell viability at 300 nM that was
>5% of the vehicle control, and `-` indicates .ltoreq.5% viability of
the vehicle control.
[0531] FIG. 37B shows the growth inhibition results of v10553. These
results show that v10553 can inhibit growth of breast, colorectal,
gastric, lung, skin, ovarian, renal, pancreatic, head and neck, uterine
and bladder tumor cell lines that express HER2 and coexpress EGFR and/or
HER3 at the 3+, 2+, 1+ or 0+ level (see also FIG. 38). The results
plotted in FIG. 37B are defined by cell lines that showed a minimum of
dose-dependent growth inhibition at 300 and 1 nM, and where the growth
inhibition at 1 nM is equal or greater than 5% (FIG. 37B).
[0532] These results show that exemplary biparatopic antibody v10000 and
ADC v10553 can inhibit growth of tumor cells originating from breast,
colorectal, gastric, lung, skin, ovarian, renal, pancreatic, head and
neck, uterine and bladder histologies that express HER2 at the 3+, 2/3+,
2+, 1+ and 0/1+ levels and that coexpress EGFR and/or HER3 at the 2+, 1+
levels.
Example 37: Ability of Anti-HER2 Biparatopic Antibodies to Mediate ADCC of
HER2 2+, 1+ and 0/1+ Cancer Cells
[0533] The following experiment was conducted to determine the ability of
anti-HER2 biparatopic antibodies to mediate ADCC of tumor cells that
express HER2 at the 2+, 1+ and/or 0/1+ levels and that coexpress EGFR
and/or HER3 at the 2+ or 1+ level. The anti-HER2 biparatopic antibodies
tested were 5019, 10000, and 10154 (an afucosylated version of v10000),
with Herceptin.TM. and v506 as controls.
[0534] The ADCC experiment was conducted as described in Example 11 and
Example 25 with E/T: 5:1 with NK-92 effector cells (FIG. 39), and as
described in Example 26 with E/T 30:1 with PBMC effector cells.
[0535] The results are shown in FIG. 39 (NK-92 effector cells) and FIG. 40
(PBMC effector cells). FIG. 39A shows the ADCC results of the HER2 2+
head and neck tumor cell line (hypopharyngeal carcinoma), FaDu, where the
anti-HER2 biparatopic elicits approximately 15% maximal cell lysis. FIG.
39C shows the ADCC results of the HER2 1+BxPC3 pancreatic tumor cell
line, and FIG. 39D the results of the HER2 2+ MiaPaca2 pancreatic tumor
cell line. FIG. 39B shows the ADCC results of the HER2 0/1+A549 NSCLC
(non-small cell lung cancer) tumor cell line. In the BxPC3, MiaPaca2 and
A549 tumor cell lines, v10000 mediated approximately 5% maximal tumor
cell lysis.
[0536] FIG. 40 shows the ADCC results in A549, NCI-N87, and HCT-116 cells,
where PBMCs were used as the effector cells. FIG. 40A shows the ADCC
results of the HER2 0/1+A549 NSCLC tumor cell line, where v10000 elicited
.about.28% maximum cell lysis and this was comparable to Herceptin.TM.
that has equivalent level of fucose content in the N-linked glycan. The
exemplary 100% afucosylated (0% fucose) biparatopic v10154 shows an
increase in maximal cell lysis (40% maximum cell lysis) and increased
potency compared to v10000 and Herceptin that have approximately 88%
fucose in the N-linked glycan.
[0537] FIG. 40B shows the ADCC results of the HER2 3+ gastric tumor cell
line, NCI-N87. FIG. 40B shows that exemplary biparatopic v5019
(approximately 88% fucosylated) mediates approximately 23% maximal cell
lysis and has a lower EC50 compared to Trastuzumab v506 (approximately
98% fucosylated).
[0538] FIG. 40C shows the ADCC results of the HER2 1+ HCT-116 colorectal
tumor cell line. FIG. 40C shows that exemplary biparatopic v5019
(approximately 88% fucosylated) mediates approximately 25% maximal cell
lysis and is more potent compared to Trastuzumab v506 (approximately 98%
fucosylated).
[0539] These results show that exemplary anti-HER2 biparatopic antibodies
can elicit ADCC of HER2 01/+, 2+ and 3+ tumor cells that originate from
head and neck, gastric, NSCLC, and pancreatic tumor histologies. ADCC in
the presence of NK-92 cells as the effector cells had an apparent HER2 2+
receptor level requirement (i.e. 2+ or greater) to show higher (>5%)
percentage of maximum cell lysis. However, when PBMC cells were used as
effector cells higher levels of maximum cell lysis were achieved (>5%
and up to 28% or 40%; v10000 and v10154, respectively) and were
independent of HER2 receptor density as ADCC >5% was seen at the 0/1+,
1+ and 3+ HER2 receptor density levels.
Example 38: HER2 Binding Affinity and Kinetics as Measured by SPR
[0540] As indicated in Example 1, anti-HER2 biparatopic antibodies having
different antigen-binding moiety formats were constructed, as described
in Table 1. The formats included scFv-scFv format (v6717), Fab-Fab format
(v6902 and v6903), along with Fab-scFv format (v5019, v7091, and v10000).
The following experiment was conducted to compare HER2 binding affinity
and kinetics of these exemplary anti-HER2 biparatopic antibody formats.
[0541] Affinity and binding kinetics to murine HER2 ECD (Sino Biological
50714-M08H) was measured by single cycle kinetics with the T200 SPR
system from Biacore (GE Healthcare). Between 2000-4000 RU of anti-human
Fc was immobilized on a CMS chip using standard amine coupling. 5019 was
captured on the anti-human Fc surface at 50 RU. Recombinant HER2 ECD
(1.8-120 nM) was injected at 50 .mu.1/min for 3 minutes, followed by a 30
minute dissociation after the last injection. HER2 dilutions were
analyzed in duplicate. Sensorgrams were fit globally to a 1:1 Langmuir
binding model. All experiments were conducted at room temperature,
25.degree. C.
[0542] The results in Table 34 show that Fab-scFv biparatopic antibodies
(v5019 and v7091), Fab-Fab variants (v6902 and v6903) and the scFv-scFv
variant (v6717) have comparable binding affinity (1-4 nM). The Fab-scFv
variant v10000 had higher binding affinity (lower KD) of approximately
0.6 nM. The monspecific anti-HER2 ECD4 antibody (v506) and anti-HER2 ECD2
antibody (v4184) were included in the assay as controls. These results
indicate that the molecular formats including v6717, v6902, v6903, v5019
and/or v7091 have equivalent binding affinities, and thus differences in
function between these antibodies may be considered to result from
differences in format.
TABLE-US-00044
TABLE 34
Anti- STD DEV
body AVERAGE Ka
Variant Ka (1/Ms) Kd (1/s) KD (M) (1/Ms) Kd (1/s) KD (M)
v506 7.34E+04 4.08E-05 5.56E-10 1.13.E+ 3.04E- 3.28E-
03 06 11
v4184 3.61E+04 5.46E-04 1.56E-08 7.78.E + 2.80E- 4.12E-
03 05 09
v5019 6.01E+04 7.77E-05 1.29E-09 1.30.E+ 8.56E- 4.24E-
03 07 11
v7091 5.17E+04 1.19E-04 2.31E-09 2.70.E + 1.49E- 4.09E-
03 05 10
v10000 6.44E+04 3.69E-05 5.79E-10 6.18.E+ 6.72.E- 1.42.E-
03 06 10
v6902 6.83E+04 1.72E-04 2.72E-09 1.93E+ 4.49E- 1.43E-
04 05 09
v6903 7.10E+04 1.71E-04 2.75E-09 3.60E+ 3.96E- 1.34E-
04 06 09
v6717 1.50E4-05 5.33E-04 4.45E-09 1.28E+ 2.54E- 2.11E-
05 04 09
Example 39: Effect of Anti-HER2 Biparatopic Antibody Format on Binding to
HER2+ Tumor Cells
[0543] The following experiment was conducted to compare the whole cell
binding properties (Bmax and apparent K.sub.D) of exemplary anti-HER2
ECD2.times.ECD4 biparatopic antibodies that have different molecular
formats (e.g. v6717, scFv-scFv IgG1; v6903 and v6902 Fab-Fab IgG1; v5019,
v7091 and v10000 Fab-scFv IgG1).
[0544] The experiment was conducted as described in Example 6. The results
are shown in FIG. 41 and Tables 35-38. FIG. 41A and Table 35 shows the
FACS binding results of the exemplary biparatopic antibodies to the BT474
HER2 3+ breast tumor cell line. The results show that all anti-HER2
antibodies have a higher Bmax (1.5 to 1.7-fold greater) when compared to
the monospecific bivalent anti-HER2 antibody v506. The Fab-scFv (v5019,
v7091 and v10000) and the Fab-Fab (v6903) formats had approximately a
1.7-fold increased Bmax and the scFv-scFv format (v6717) had a 1.5-fold
increased Bmax compared to v506. An equimolar combination of FSAs v506
and v4184 resulted in a 1.7-fold increase in Bmax. The apparent K.sub.D
of the exemplary anti-HER2 biparatopic antibodies was approximately 2 to
3-fold higher compared to the monospecific v506.
TABLE-US-00045
TABLE 35
FACS binding BT-474
Antibody Variant K.sub.D (nM) Bmax
v506 9.0 23536
v10000 16 39665
v506 + v4184 16 40320
v5019 21 39727
v7091 22 36718
v6717 30 36392
v6903 31 40321
[0545] FIG. 41B and Table 36 shows the FACS binding results to the JIMT-1
HER2 2+ breast tumor cell line. The results show that all anti-HER2
antibodies have a higher Bmax (1.5 to 1.8-fold greater) when compared to
the monospecific bivalent anti-HER2 antibody v506. The Fab-scFv (v7091
and v10000) and the Fab-Fab (v6903) formats had approximately a 1.7-fold
increased Bmax, the scFv-scFv format (v6717) had a 1.5-fold increased
Bmax and the Fab-scFv (v5019) and FSA combination (v506+v4184) had a
1.8-fold increased Bmax compared to v506. The apparent K.sub.D of the
exemplary anti-HER2 biparatopic Fab-scFv antibodies was approximately 2
to 4-fold higher compared to the monospecific v506; whereas the K.sub.D
of the Fab-Fab (v6903) and scFv-scFv (v6717) were approximately 8-fold
higher compared to v506.
TABLE-US-00046
TABLE 36
FACS Binding JIMT-1
Antibody Variant K.sub.D (nM) Bmax
v506 3.5 2574
v10000 7.6 4435
v506 + v4184 8.0 4617
v5019 12 4690
v7091 14 4456
v6717 26 3769
v6903 28 4452
[0546] FIG. 41C and Table 37 shows the FACS binding results of the
exemplary biparatopic antibodies to the HER2 1+ MCF7 breast tumor cell
line. The results show that anti-HER2 antibody v10000 and FSA combination
(v506+v4184) have a 1.6-fold higher Bmax compared to the monospecific
bivalent anti-HER2 antibody v506. The Fab-scFv (v5019, v7091) had
approximately a 1.4-fold; the scFv-scFv format (v6717) a 1.3-fold, and
the Fab-Fab format (v6903) had a 1.2-fold increased Bmax compared to
v506. The apparent K.sub.D of the exemplary anti-HER2 biparatopic
Fab-scFv, Fab-Fab (v6903) and FSA combination (v506+v4184) was
approximately 2 to 3-fold lower compared to v506; whereas the K.sub.D of
the scFv-scFv (v6717) was approximately 3-fold higher compared to v506.
TABLE-US-00047
TABLE 37
FACS Binding MCF7
Antibody Variant K.sub.D (nM) Bmax
v506 + v4184 4.5 1410
v7091 6.1 1216
v5019 6.3 1201
v10000 6.8 1381
v6903 7.1 1105
v506 12 889
v6717 32 1167
[0547] FIG. 41D and Table 38 shows the FACS binding results of the
exemplary biparatopic antibodies to the HER2 0/1+ MDA-MD-231 breast tumor
cell line. The results show that exemplary biparatopic anti-HER2
antibodies had approximately 1.3 to 1.4-fold increased Bmax compared to
the monospecific bivalent anti-HER2 antibody v506. The FSA combination
(v506+v4184) had a 1.7-fold increased Bmax The apparent K.sub.D of the
exemplary anti-HER2 biparatopic Fab-scFv antibodies (v5019, v7091,
v10000) and FSA combination (v506+v4184) had an approximate equivalent KD
compared to v506; whereas Fab-Fab (v6903) and scFv-scFv (v6717) was
approximately 4 and 16-fold higher K.sub.D respectively, compared to
v506.
TABLE-US-00048
TABLE 38
FACS Binding MDA-MB-231
Antibody Variant K.sub.D (nM) Bmax
v506 4.8 395
v10000 5.6 558
v506 + v4184 7.3 662
v7091 7.9 525
v5019 8.7 548
v6903 17 534
v6717 77 524
[0548] The tumor cell binding results show that anti-HER2 biparatopic
antibodies with different molecular formats have an increased Bmax on
HER2 3+, 2+, 1+ and 0/1+ tumor cells compared to a bivalent monospecific
anti-HER2 antibody. Of the different anti-HER2 biparatopic antibodies,
the scFv-scFv format had the lowest Bmax gain relative to v506 on HER2
3+, 2+, 1+ and 0/1+ tumor cells These results also show that scFv-scFv
and Fab-Fab formats have the greatest increase in K.sub.D on HER2 3+, 2+,
1+ and 0/1+ tumor cells compared monospecific v506 (3 to 16-fold
increase) and the biparatopic Fab-scFv formats (approximately 2-fold or
greater). The increase in K.sub.D is an indication of a reduction in avid
binding and suggests that different biparatopic formats have unique
mechanisms of binding to HER2 on the cell surface.
Example 40: Effect of Anti-HER2 Biparatopic Antibody Format on
Internalization in HER2+ Cells
[0549] The following experiment was conducted to compare the ability of
exemplary anti-HER2 ECD2.times.ECD4 biparatopic antibodies that have
different molecular formats (e.g. v6717, scFv-scFv IgG1; v6903 and v6902
Fab-Fab IgG1; v5019, v7091 and v10000 Fab-scFv IgG1) to internalize in
HER2+ cells expressing HER2 at varying levels.
[0550] The experiment was conducted as detailed in Example 9. The results
are shown in FIG. 42 and Tables 39-41. FIG. 42A and Table 39 show the
internalization results in HER2 3+BT-474. These results show that the
Fab-scFv format (v10000) and the FSA combination (v506+v4184) have
2.2-fold greater quantities of intracellular antibody, compared to the
monospecific anti-HER2 v506. The scFv-scFv format (v6717) had 1.9-fold
greater; the Fab-scFv formats (v5019 and v7091) had 1.5 to 1.7-fold
greater; and the Fab-Fab formats (v6902 and v6903) had 1.2 to 1.3-fold
greater quantities of intracellular antibody accumulation compared to
v506.
TABLE-US-00049
TABLE 39
Internalization BT-474
Antibody Variant Surface 4.degree. C. Surface37.degree. C. Internal
37.degree. C.
v506 2156 1590 3453
v6902 2407 2077 4035
v6903 2717 986 4573
v7091 2759 2227 5111
v5019 2867 2675 5710
v6717 2006 1212 6498
v10000 3355 2851 7528
v506 + v4184 3998 2326 7569
[0551] FIG. 42B and Table 40 show the internalization results in HER2 2+
JIMT-1. These results show that the Fab-scFv format (v10000) and the FSA
combination (v506+v4184) have respectively 1.8 and 1.9-fold greater
quantities of intracellular antibody, compared to the monospecific
anti-HER2 v506. The scFv-scFv (v6717) and the Fab-scFv formats (v5019)
have 1.4-fold greater; and the Fab-scFv (v7091) and Fab-Fab formats
(v6902 and v6903) had 1.2-fold greater quantities of intracellular
antibody accumulation compared to v506.
TABLE-US-00050
TABLE 40
Internalization JIMT-1
Antibody Variant Surface 4.degree. C. Surface 37.degree. C. Internal
37.degree. C.
v506 337 -7.1 759
v6902 389 152 926
v7091 426 102 935
v6903 392 130 945
v5019 437 5.2 1035
v6717 247 31 1082
v10000 474 103 1375
v506 + v4184 583 89 1449
[0552] FIG. 42C and Table 41 show the internalization results in HER2 1+
MCF7. These results show that the scFv-scFv format and Fab-scFv formats
have 3.0 and 2.8-fold greater quantities of intracellular antibody,
compared to the monospecific anti-HER2 v506. The Fab-scFv format (v10000)
and the FSA combination (v506+v4184) have approximately 2.0-fold; the
Fab-scFv (v7091) and Fab-Fab (v6903) formats have 1.8-fold greater
quantities of intracellular antibody accumulation compared to v506.
TABLE-US-00051
TABLE 41
Internalization MCF7
Antibody Variant Surface 4.degree. C. Surface 37.degree. C. Internal
37.degree. C.
v506 48 10 48
v7091 77 27 87
v6903 81 35 89
v10000 78 20 96
v506 + v4184 87 19 103
v5019 81 17 134
v6717 48 31 145
[0553] These results show that anti-HER2 biparatopic antibodies with
different molecular formats have unique degrees of internalization in
HER2 3+, 2+ and 1+ tumor cells that varies with respect to the structure
and format of the antigen-binding domains. In general, the monospecific
FSA combination of v506 and v4184, the Fab-scFv (v10000, v7091 and v5019)
and the scFv-scFv (v6717) biparatopic formats had the higher
internalization values in the HER2 3+, 2+ and 1+ tumor cells. Whereas,
the Fab-Fab biparatopic formats (v6902 and v6903) had the lowest
internalization values in the HER2 3+, 2+ and 1+ tumor cells. These data
suggest that the molecular format and geometric spacing of the
antigen-binding domains has an influence on the ability of the
biparatopic antibodies to cross-link HER2 receptors, and subsequently to
internalize in HER2+ tumor cells. The Fab-Fab biparatopic format, having
the greatest distance between the two antigen-binding domains, resulted
in the lowest degree of internalization, whereas the Fab-scFv and
scFv-scFv formats, having shorter distances between the antigen-binding
domains, had greater internalization in HER2+ cells. This is consistent
with the correlation of potency and shorter linker length as described in
Jost et al 2013, Structure 21, 1979-1991).
Example 41: Effect of Anti-HER2 Biparatopic Antibody Format on ADCC in
HER2+ Cells
[0554] The following experiment was conducted to compare the ability of
exemplary anti-HER2 ECD2.times.ECD4 biparatopic antibodies that have
different molecular formats (e.g. v6717, scFv-scFv IgG1; v6903 and v6902
Fab-Fab IgG1; v5019, v7091 and v10000 Fab-scFv IgG1) to mediate ADCC in
HER2+ cells expressing HER2 at varying levels.
[0555] Prior to performing the ADCC assay, glycopeptide analysis was
performed on the antibody samples to quantify the fucose content in the
N-linked glycopeptide. The method was followed as described in Example
23. The results are shown in Table 42; the data shows that exemplary
biparatopic variants v5019, v6717, v6903 have equivalent fucose content
in the N-linked glycan (91-93%). Antibody samples with equivalent levels
of fucose in the N-glycan were selected for the ADCC assay to normalize
for fucose content in the interpretation of the ADCC assay results.
TABLE-US-00052
TABLE 42
LC-MS Tryptic peptide analysis
Percentage of Percentage of
Glycopeptides Observed Glycopeptides Observed
Variant WITH Fucose WITHOUT Fucose
v6903 90.7 9.3
v6717 92.8 7.2
v5019 91.3 8.7
[0556] The ADCC experiment was conducted as described in Example 11 with
E/T: 5:1 with NK-92 effector cells. The ADCC results are shown in FIG. 43
and Tables 43-45. FIG. 43A and Table 43 show the ADCC results in HER2 2+
JIMT-1 breast tumor cells. These data show that v5019, v6717 and v6903
elicit similar levels of maximum cell lysis and that the scFv-scFv format
(v6717) is less potent compared to v5019 and v6903 when HER2 2+ tumor
cells are targets.
TABLE-US-00053
TABLE 43
JIMT-1 ADCC
Antibody variant EC50 (nM) % Max Cell Lysis
v6903 ~0.03 48
v5019 ~0.16 47
v6717 ~0.72 51
[0557] FIG. 43B and Table 44 show the ADCC results in HER2 1+ MCF7 breast
tumor cells. These data show that v5019 and v6717 have slightly higher
maximum cell lysis (27-30%) compared to v6903 (24%). These data also show
that v6717 is the least potent, followed by v6903 and v5019, which have
lower EC50 values.
TABLE-US-00054
TABLE 44
MCF7 ADCC
Antibody variant EC.sub.50 (nM) % Max Cell Lysis
v5019 ~0.69 27
v6717 109 30
v6903 0.94 24
[0558] FIG. 43C and Table 45 show the ADCC results in HER2 0/1+ MDA-MB-231
breast tumor cells. These data show that v5019 shows slightly higher
maximum cell lysis (77%) compared to v6903 (62%) and v6717 (63%). These
data also show that v6717 is the least potent, followed by v6903 and
v5019, which have lower EC.sub.50 values.
TABLE-US-00055
TABLE 45
MDA-MB-231 ADCC
% Max Cell Lysis
Antibody variant EC.sub.50 (nM) (top only)
v5019 0.20 71
v6717 10 63
v6903 0.79 62
[0559] These data show that exemplary anti-HER2 ECD2.times.ECD4
biparatopic antibodies elicit similar levels of maximum cell lysis by
ADCC in HER2 2+ and 1+ tumor cells. Despite similarities in maximal cell
lysis, these data also show that the different molecular formats have
unique ADCC potencies. The scFv-scFv was the least potent (greatest
EC.sub.50 values) in the HER2 2+ and HER2 1+. Differential potencies
among the three formats was seen in the ADCC data targeting HER2 1+
cells, where the EC50 values for v6717>v6903>v5019. These data are
consistent with the observations presented in Example 40 (FACS binding),
where an increase in K.sub.D (reduced affinity) was seen with the Fab-Fab
and scFv-scFv formats.
Example 42: Effect of Anti-HER2 Biparatopic Antibody Format on Growth of
HER2+ Tumor Cells
[0560] The following experiment was conducted to compare the effect of
anti-HER2 biparatopic antibody format on growth of HER2 3+, 2+ and 1+
tumor cells, either basal growth or ligand-stimulated. Basal growth was
measured as described in Example 15, while ligand-stimulated growth was
measured as described in Example 27. In both types of experiments, growth
was measured as % survival with respect to control treatment.
[0561] FIG. 44 and Table 46 show the effect of exemplary anti-HER2
ECD2.times.ECD4 biparatopic antibodies on growth of HER2 3+ breast cancer
cells (BT-474) in the presence of exogenous growth-stimulatory ligands
(EGF and HRG). In the absence of EGF or HRG, the anti-HER2 biparatopic
antibodies were able to inhibit growth of BT-474 cells, where % survival
of each treatment group ranked as follows:
v6903<v506+v4184<506<v7091<v5019<v10000<v6717. In the
presence of HRG, growth inhibition relative to the mock control was
achieved only with the FSA combination of v506+v4184. In the presence of
EGF, growth inhibition relative to the mock control was achieved, where %
survival of each treatment group ranked as follows:
v6903<v506+v4184<7091<v10000<5019.
TABLE-US-00056
TABLE 46
% Survival
Antibody
Treatment only +HRG +EGF
Mock 100 143 131
v6717 113 126 129
v10000 70 118 78
v5019 67 133 81
v7091 61 119 61
v506 53 141 118
v506 + v4184 43 89 45
v6903 32 120 39
[0562] FIG. 45 shows the dose-dependent effect of the anti-HER2
biparatopic antibody formats on growth inhibition of the SKBr3 HER2 3+
cell line. The data is consistent with the results presented in FIG. 44,
where the rank order potency/efficacy of the biparatopic formats is as
follows Fab-Fab>Fab-scFv>scFv-scFv in HER2 3+ tumor cells.
[0563] The effect of anti-HER2 biparatopic antibody formats on survival of
HER2+ cells is shown in FIG. 46, where FIG. 46A shows the result in the
Trastuzumab sensitive SKOV3 HER2 2+/3+ cell line at 300 nM; FIG. 46B
shows the result in JIMT-1 HER2 2+(Trastuzumab resistant) cells at 300
nM, and FIG. 46C shows the result in MCF7 HER2 1+ cell line at 300 nM. In
the SKOV3 cell line, little difference was observed among the biparatopic
formats in the extent of growth inhibition, and no growth inhibition was
observed by any of the test antibodies in JIMT-1 and MCF7 cells.
[0564] The data in FIG. 44 and FIG. 45 show that anti-HER2 ECD2.times.ECD4
biparatopic antibodies with the Fab-scFv and Fab-Fab formats (v5019,
v7091, v10000, v6903) are capable of growth inhibition HER2 3+ tumor
cells in the absence, and presence of EGF or HRG. In the HER2 3+ cell
lines BT-474 and SKBR3, growth inhibition relative to the mock control
rank ordered as follows, where
v506+v4184>v6903>v7091>v10000>v5019>v506>v6717. The
distance between antigen-binding domains
(Fab-Fab>Fab-scFv>scFv-scFv) correlates with the rank order of
growth inhibition in the HER2 3+ tumor cells. Based on the data in
trastuzumab-sensitive tumor cells, BT-474, and SKBr3, it may be expected
that the growth inhibition difference among formats is significant at the
HER2 3+ level but less so at the HER2 2+ or HER2 1+ levels.
Example 43: Evaluation of HER2 Binding Affinity and Kinetic at Varying
Antibody Capture Levels
[0565] The following experiment was conducted to compare HER2 binding
kinetics (kd, off-rate) of exemplary anti-HER2 ECD2.times.ECD4
biparatopic antibodies when captured at varying surface densities by SPR.
The correlation between a reduced (slower) off-rate with increasing
antibody capture levels (surface density) is an indication of Trans
binding (i.e. one antibody molecule binding to two HER2 molecules,
described in Example 12). In this experiment the Fab-Fab format (v6903)
was compared to the Fab-scFv format (v7091) to determine potential
difference in Trans binding among the variants. Due to the larger spatial
distance between antigen-binding domains, it is hypothesized that the
Fab-Fab format may be capable of Cis binding (engaging ECD 2 and 4 on one
HER2 molecule); whereas, the Fab-scFv would not capable of Cis binding
due to the shorter distance between the it's antigen-binding domains. The
anti-HER2 monospecific v506 was included as a control.
[0566] The experiment was conducted by SPR as described in Example 12. The
data are shown in FIG. 47. FIG. 47A shows the plot and linear regression
analysis for the kd (1/s) at different antibody capture levels with v6903
and v7091. Both v7091 and v6093 show a trend for decreasing off-rate with
increasing surface capture levels; however, the correlation is
significant with the Fab-scFv variant (v7091; P value=0.023) but not the
Fab-Fab format (v6093; P value=0.053). The off-rate remained unchanged
with varying antibody capture levels for the anti-HER2 monospecific
control, v506.
[0567] FIG. 47B shows the plot and linear regression analysis for the
K.sub.D (M) at different antibody capture levels with v6903 and v7091.
Similar to the off-rate comparison, both v7091 and v6093 show a trend for
increasing affinity (lower K.sub.D value) with increasing surface capture
levels. However, the correlation is significant with the Fab-scFv variant
(v7091; P value=0.04) but not the Fab-Fab format (v6093; P value=0.51).
The K.sub.D remained unchanged with varying antibody capture levels for
the anti-HER2 monospecific control, v506. The data in FIG. 47 shows that
the Fab-Fab and Fab-scFv anti-HER2 biparatopic antibody formats show
trends of decreasing off-rates with increasing antibody surface capture
levels; these trends are unique compared to a monospecific anti-Her2
antibody.
Example 44: Affinity and Stability Engineering of the Pertuzumab Fab
[0568] As indicated in Table 1, one variant (v10000) contains mutations in
the Pertuzumab Fab. This Fab was derived from affinity and stability
engineering in silico efforts, which were measured experimentally as
monovalent or One-Armed Antibodies (OAAs).
[0569] Variant 9996: a monovalent anti-HER2 antibody, where the HER2
binding domain is a Fab derived from pertuzumab on chain A, with Y96A in
VL region and T30A/A49G/L69F in VH region (Kabat numbering) and the Fc
region is a heterodimer having the mutations T350V_L351Y_F405A_Y407V (EU
numbering) in Chain A, T350V_T366L_K392L_T394W (EU numbering) in Chain B,
and the hinge region of Chain B having the mutation C226S; the
antigen-binding domain binds to domain 4 of HER2.
[0570] Variant 10014: a monovalent anti-HER2 antibody, where the HER2
binding domain is a Fab derived from pertuzumab on chain A, with Y96A in
VL region and T30A in VH region (Kabat numbering) and the Fc region is a
heterodimer having the mutations T350V_L351Y_F405A_Y407V (EU numbering)
in Chain A, T350V_T366L_K392L_T394W (EU numbering) in Chain B, and the
hinge region of Chain B having the mutation C226S; the antigen-binding
domain binds to domain 4 of HER2.
[0571] Variant 10013: a monovalent anti-HER2 antibody, where the HER2
binding domain is a Fab derived from wild type pertuzumab on chain A, and
the Fc region is a heterodimer having the mutations T350V_L35
1Y_F405A_Y407V (EU numbering) in Chain A, T350V_T366L_K392L_T394W (EU
numbering) in Chain B, and the hinge region of Chain B having the
mutation C226S; the antigen-binding domain binds to domain 4 of HER2.
[0572] The following experiments were conducted to compare HER2 binding
affinity and stability of the engineered Pertuzumab variants.
[0573] OAA variants were cloned and expressed as described in Example 1.
[0574] OAA were purified by protein A chromatography and Size Exclusion
Chromatography, as described in Example 1.
[0575] Heterodimer purity (i.e. amount of OAA with a heterodimeric Fc) was
assessed by non-reducing High Throughput Protein Express assay using
Caliper LabChip GXII (Perkin Elmer #760499). Procedures were carried out
according to HT Protein Express LabChip User Guide version2 LabChip GXII
User Manual, with the following modifications. Heterodimer samples, at
either 2 .mu.l or 5 .mu.l (concentration range 5-2000 ng/.mu.1), were
added to separate wells in 96 well plates (BioRad # HSP9601) along with 7
.mu.l of HT Protein Express Sample Buffer (Perkin Elmer #760328). The
heterodimer samples were then denatured at 70.degree. C. for 15 mins. The
LabChip instrument is operated using the HT Protein Express Chip (Perkin
Elmer #760499) and the Ab-200 assay setting. After use, the chip was
cleaned with MilliQ water and stored at 4.degree. C.
[0576] The stability of the samples was assessed by measuring melting
temperature or Tm, as determined by DSC with the protocol shown in
example 24. The DSC was measured before and after SEC purification.
[0577] The affinity towards HER2 ECD of the samples was measured by SPR
following the protocol from example 12. The SPR was measured before and
after SEC purification. As summarized in Table 47A and 47B, the mutations
in the variable domain have increased the HER2 affinity of the Fab
compared to wild type pertuzumab, while maintaining WT stability. (.sup.1
Purity determined by Caliper LabChip; .sup.2 KD(WT)/KD(mut)
TABLE-US-00057
TABLE 47A
SPR pre-SEC Het SPR post-SEC
Pr-A KD Fold purity KD Fold
OAA Fab HC Yield KD AVE STDEV wrt post- KD AVE STDEV wrt
variant mutations LC mut (mg/L) (nM) (nM) n WT.sup.2 SEC.sup.1 (nM) (nM) n
WT
v9996 T30A/A49G/ Y96A 22 1.7E-09 1.7E-10 5 9.6 93% 1.8E-09 1.6E-11 2 8.4
L69F
v10014 T30A Y96A 20 2.0E-09 3.1E-10 4 8.1 81% 2.1E-09 5.2E-10 3 7.0
v10013 WT WT 18 1.6E-08 5.1E-09 16 1.0 91% 1.5E-08 3.5E-09 4 1.0
TABLE-US-00058
TABLE 47B
DSC DSC
pre-SEC post-SEC
.DELTA.Tm .DELTA.Tm
wrt wrt
OAA Tm WT Tm WT
variant (C.) (C.) (C.) (C.)
v9996 77.2 -0.2 77.2 -0.7
v10014 75.5 -1.9 75.5 -2.4
v10013 77.4 0.0 77.9 0.0
Example 45: Effect of v10000 on Survival and Tumor Growth in a Xenograft
Model of HER2-Low, Non-Small Cell Lung Cancer (NSCLC)
[0578] This experiment was performed to assess efficacy of v10000 compared
to control IgG (v6908) in an A549 xenograft model of lung cancer. A549
cells are derived from non-squamous non-small cell lung cancer that is
HER2-low, non-HER2 gene amplified, HER3+, EGFR-low and moderately
sensitive to Cisplatin at the MTD (maximum tolerated dose). The study was
carried out as described below.
[0579] Tumor cell suspensions were implanted subcutaneously into athymic
nude mice. When tumors reached 158 mm.sup.3 the animals were randomly
assigned to groups as shown in Table A1, and treatment began in a blinded
and controlled study. Animals were treated according to Regimen 1 on Day
1, followed by treatment according to Regimen 2 on subsequent days as
indicated in Table A1.
TABLE-US-00059
TABLE A1
Study Design
Regimen 1 Regimen 2
Group Dosage Dosage
(n) Agent (mg/kg) Route Schedule Agent (mg/kg) Route Schedule
1 (20) v6908 15 iv Day 1 v6908 10 iv Days 4, 8, 11,
15, 18, 22 and
25
2 (20) v10000 15 iv Day 1 v10000 10 iv Days 4, 8, 11,
15, 18, 22 and
25
[0580] Tumor volume was measured by calipers twice weekly. The study
duration was 66 days with survival as the primary endpoint. Additional
tumor response criteria were measured and are shown in Table A2. Mice
were euthanized when tumor volume exceeded 800 mm.sup.3, the surviving
percentage versus study day was plotted on a Kaplan-Meier and was
statistically assessed using a log-rank test. Serum concentration of
v10000 was determined by HER2 ELISA on study day 7.
[0581] The results are shown in FIG. 48A (tumor volume) and FIG. 48B
(Kaplan-Meier survival). Variant 10000 reduced tumor growth compared to
v6908 treated controls and significantly prolonged survival by log-rank
test (FIG. 48B and Table A3). Animals treated with v10000 had a median
survival of greater than 66 days while those treated with v6908 had a
median survival of 25.78 days (FIG. 48B and Table A2). Tumor volume on
study day 30 was 461 mm3 and 810 mm3 for v10000 and v6908 treated groups
respectively (FIG. 48A and Table A2). Serum exposure was 140.9 microg/mL
on study day 7, indicating that the anticipated serum concentration was
achieved.
[0582] These results show that treatment with v10000 was able to reduce
tumor growth and prolong survival compared to treatment with a control
hIgG in this HER2-low non-gene amplified NSCLC model.
TABLE-US-00060
TABLE A2
A549 Tumor Response Profile
6908 10000
Tumor Response on Day 30
Mean TV (mm.sup.3) (% .DELTA. from base line) 810 (413%) 461 (191%)
Treatment/Control Ratio 1.00 0.57
RECIST Scores
CR (TV <20 mm.sup.3) 0/20 0/20
PR (>30% baseline regression) 0/20 1/20
PD (>20% baseline growth) 20/20 19/20
SD (neither PD or PR) 0/20 0/20
Median Time to Progression (days) 3.30 2.31
Survival Response
Median Survival (days) 25.78 >66
CR--Complete Response
PR--Partial Response
PD--Progressive Disease
SD--Stable Disease
TABLE-US-00061
TABLE A3
Log Rank Summary
Group 6908
6908 --
10000 .star-solid..star-solid..star-solid.
Legend:
ns = not significant,
.star-solid. = P < 0.05.
.star-solid..star-solid. = P < 0.01,
.star-solid..star-solid..star-solid. = P < 0.001
Example 46: Effect of v10000 on Survival and Tumor Growth in a Xenograft
Model of HER2-Low, Head and Neck Squamous Cell Carcinoma
[0583] This experiment was performed to assess efficacy of v10000 compared
to Herceptin.TM. (v6336) and control human IgG (v6908) in the FaDu
xenograft model of head and neck cancer. FaDu cells are derived from
squamous cell cancer of the head and neck that is HER2 low, non-HER2 gene
amplified, HER3+, EGFR+ and highly sensitive to Cisplatin at the MTD. The
study was carried out as described below.
[0584] Tumor cell suspensions were implanted subcutaneously into athymic
nude mice. When tumors reached 121 mm.sup.3 the animals were randomly
assigned to groups as shown in Table A4, and treatment began in a blinded
and controlled study. Cisplatin was purchased and provided for the study
by Charles River Laboratories (Morrisville, N.C.). Animals were treated
according to Regimen 1 at Day 1, followed by Regimen 2 on subsequent days
as noted in Table A4.
TABLE-US-00062
TABLE A4
Study Design
Regimen 1 Regimen 2
Group Dosage Dosage
(n) Agent (mg/kg) Route Schedule Agent (mg/kg) Route Schedule
1 (15) v6908 15 iv Day 1 v6908 10 iv Days 4, 8, 11,
15, 18, 22 and
25
2 (15) v6336 15 iv Day 1 v6336 10 iv Days 4, 8, 11,
15, 18, 22 and
25
3 (15) v10000 15 iv Day 1 v10000 10 iv Days 4, 8, 11,
15, 18, 22 and
25
4 (15) Cisplatin 2 ip Day 1, 3, 5,
7, 9, 11
5 (15) v10000 15 iv Day 1 v10000 10 iv Days 4, 8, 11,
15, 18, 22 and
25
Cisplatin 2 ip Day 1, 3, 5,
7, 9, 11
[0585] Tumor volume was measured by calipers twice weekly. The study
duration was 59 days with survival as the primary endpoint. Additional
tumor response criteria were measured and are shown in Table A5. Mice
were euthanized when tumor volume exceeded 2000 mm.sup.3, the surviving
percentage versus study day was plotted on a Kaplan-Meier and was
statistically assessed using a log-rank test. Serum concentration of
v10000 and v6336 was determined by HER2 ELISA on study day 7.
[0586] The results are shown in FIG. 49A (tumor volume) and FIG. 49B
(Kaplan-Meier survival). Variant 10000 reduced tumor growth compared to
v6908 treated controls and v6336, as well as significantly prolonged
survival by log-rank test compared to v6908 (FIG. 48B and Table A3).
Animals treated with v10000 had a median survival of greater than 46 days
while those treated with v6908 and v6336 had median survivals of 25 and
40 days, respectively (FIG. 49B and Table A5). Tumor volume on study day
25 was 1025, 1979, 1257 mm.sup.3 for v10000, v6908 and v6336 treated
groups respectively (FIG. 49A and Table A5). Serum exposure was 116.6
microg/mL for v10000, 119.9 microg/mL for v6336, and 107.2 microg/mL for
v10000+Cisplatin on study day 7, indicating that the anticipated serum
concentration was achieved for each test article.
[0587] These results show that treatment with v10000 as a monotherapy was
able to decrease tumor volume and prolong survival, compared to treatment
with control IgG in this model of HER2-low non-gene amplified head and
neck cancer. Overall, v10000 showed a trend towards decreasing tumor
volume compared to v6336 (Herceptin.TM.).
[0588] Variant 10000 was also tested in combination with cisplatin. The
combination of v10000 and cisplatin significantly prolonged survival
compared to v6908, v6336, and single agent cisplatin (Table A5). The
median survival of the v10000 and cisplatin combination was 53 days while
the median survival of v6908, v6336, and single agent cisplatin was 25,
40, and 40 days, respectively.
[0589] These results demonstrate that treatment with v10000 in combination
with cisplatin was able to decrease tumor growth and prolong survival
compared to v6908 and v6336, in this model of head and neck cancer.
TABLE-US-00063
TABLE A5
FaDu Tumor Response Profile
6908 6336 10000 cisplatin 10000 + cisplatin
Tumor Response on Day 25
Mean TV (mm.sup.3) (% .DELTA. from base 1979 1257 1025 1070 816
line) (1532%) (929%) (782%) (782%) (573%)
Treatment/Control Ratio 1.00 0.63 0.52 0.54 0.41
RECIST Scores
CR (TV <20 mm.sup.3) 0/15 0/14 0/15 0/15 0/15
PR (>30% baseline regression) 0/15 0/14 0/15 0/15 0/15
PD (>20% baseline growth) 15/15 14/14 15/15 15/15 15/15
SD (neither PD or PR) 0/15 0/15 0/15 0/15 0/15
Median Time to Progression 5.9 7.6 7.8 8.4 10.8
(days)
Survival Response
Median Survival (days) 25 40 46 40 53
CR--Complete Response
PR--Partial Response
PD--Progressive Disease
SD--Stable Disease
TABLE-US-00064
TABLE A6
Log Rank Summary
Group 6908 6336 10000 Cisplatin
6908 -- -- -- --
6336 .star-solid..star-solid. -- -- --
10000 .star-solid..star-solid..star-solid. n/s -- --
Cisplatin .star-solid..star-solid..star-solid. n/s .star-solid. --
10000 + Cisplatin .star-solid..star-solid..star-solid. .star-solid. n/s
.star-solid..star-solid..star-solid.
Legend:
ns = not significant,
.star-solid. = P < 0.05.
.star-solid..star-solid. = P < 0.01,
.star-solid..star-solid..star-solid. = P < 0.001
Example 47: Effect of v10000 on Survival and Tumor Growth Inhibition in a
Xenograft Model of HER2 1+, ER+ Breast Cancer
[0590] This experiment was performed to assess efficacy of v10000 compared
to a control IgG (v6908) or Herceptin.TM. (v6336) in the ST1337B
xenograft model of breast cancer. ST1337B is a patient derived xenograft
(PDX) established in nude mice from an ER+/PR- breast cancer with a
luminal B molecular classification. ST1337 is HER2 1+ as measured by IHC.
The study was carried out as described below.
[0591] Tumor fragments were implanted subcutaneously into athymic nude
mice. When tumors reached 180 mm.sup.3 the animals were randomly assigned
to groups as shown in Table A7 and treatment began in a blinded and
controlled study. Animals were treated according to Regimen 1 as shown in
Table A7
TABLE-US-00065
TABLE A7
Study Design
Regimen 1
Group Dosage
(n) Agent (mg/kg) Route Schedule
1 (15) v6908 30 iv Days 1,4, 8, 11, 15, 18, 22, 25, 28,
and 32
2 (15) V6336 10 iv Days 1, 4, 8, 11, 15, 18, 22, 25, 28,
and 32
3 (15) v10000 3 iv Days 1, 4, 8, 11, 15, 18, 22, 25, 28,
and 32
4 (15) v10000 10 iv Days 1, 4, 8, 11, 15, 18, 22, 25, 28,
and 32
5 (15) v10000 30 iv Days 1, 4, 8, 11, 15, 18, 22, 25, 28,
and 32
[0592] Tumor volume was measured by calipers twice weekly. The study
duration was 63 days with survival as the primary endpoint. Additional
tumor response criteria were measured and are shown in Table A8. Mice
were euthanized when tumor volume exceeded 2000 mm.sup.3, the surviving
percentage versus study day was plotted on a Kaplan-Meier and was
statistically assessed using a log-rank test. Serum concentration of
v10000 and v6336 was determined by HER2 ELISA on study day 7 and on day
36, 4 days following the last dose on day 32.
[0593] The results are shown in FIG. 50A (tumor volume) and FIG. 50B
(Kaplan-Meier survival). Treatment with variant 10000 at all doses tested
reduced tumor growth compared to treatment with v6908 and significantly
prolonged survival by log-rank test compared to v6908 (FIG. 50B and Table
A9). In addition, treatment with v10000 at 30 mg/kg significantly
prolonged survival compared to treatment with v6336 at 10 mg/kg (FIG. 50B
and Table A8). Animals treated with v10000 had median survivals of 49,
59, and 59 days for the 3, 10 and 30 mg/kg doses respectively (FIG. 50B
and Table A8). Tumor volume on study day 29 for treatment with v10000 at
3, 10 and 30 mg/kg was 1010, 1016, and 931 mm3, respectively. Tumor
volumes for v6908 and v6336 on study day 29 was 1898 and 1264 mm3
respectively (FIG. 50A and Table A8). The serum exposure of v6336 and
v10000 is shown in Table A10. These results confirm that increasing the
dosage of v10000 results in an increase in serum concentration of v10000,
and that similar doses of v10000 and v6336 result in similar serum
concentrations of antibody.
[0594] These results indicate that treatment with v10000 is able to
decrease tumor volume and prolong survival in this model of HER2-low ER+
breast cancer, when compared to the IgG control and to Herceptin.TM..
TABLE-US-00066
TABLE A8
ST1337b Tumor Response Profile
6908, 6336, 10000, 10000,
30 mg/kg 10 mg/kg 3 mg/kg 10 mg/kg 10000, 30 mg/kg
Tumor Response on Day 29
Mean TV (mm.sup.3) (% .DELTA. from base 1898 1264 1010 1016 931
line) (953%) (601%) (460%) (457%) (411%)
Treatment/Control Ratio 1.00 0.66 0.53 0.53 0.49
RECIST Scores
CR (TV <20 mm.sup.3) 0/15 0/15 0/15 0/15 0/15
PR (>30% baseline regression) 0/15 0/15 0/15 0/15 0/15
PD (>20% baseline growth) 15/15 15/15 15/15 15/15 15/15
SD (neither PD or PR) 0/15 0/15 0/15 0/15 0/15
Median Time to Progression 11 10 14 26 13
(days)
Survival Response
Median Survival (days) 29 43 49 59 59
CR--Complete Response
PR--Partial Response
PD--Progressive Disease
SD--Stable Disease
TABLE-US-00067
TABLE A9
Log Rank Summary
6908, 6336, 10000, 10000, 10000,
Group 30 mg/kg 10 mg/kg 3 mg/kg 10 mg/kg 30 mg/kg
6908, -- -- -- --
30 mg/kg
6336, .star-solid..star-solid. -- -- -- --
10 mg/kg
10000, .star-solid..star-solid. n/s -- -- --
3 mg/kg
10000, .star-solid..star-solid..star-solid. n/s n/s -- --
10 mg/kg
10000, .star-solid..star-solid..star-solid. .star-solid. n/s n/s --
30 mg/kg
Legend:
ns = not significant,
.star-solid. = P < 0.05.
.star-solid..star-solid. = P < 0.01,
.star-solid..star-solid..star-solid. = P < 0.001
TABLE-US-00068
TABLE A10
Serum Exposure Summary
10000, 10000,
Sample Day 6336, 10 mg/kg 10000, 3 mg/kg 10 mg/kg 30 mg/kg
7 133.0 30.7 101.7 286.6
36 135.2 46.0 186.3 279.7
Example 48: Effect of v10000 on Survival and Tumor Growth Inhibition in a
Xenograft Model of HER2 Negative Pancreatic Cancer
[0595] This experiment was performed to assess efficacy of v10000 compared
to a control IgG (v12470), Herceptin.TM. (v6336), and nab-paclitaxel as
single agents and v10000 in combination with nab-paclitaxel (Abraxane.TM.
Celgene) in the ST803 xenograft model of pancreatic cancer. ST803 is a
patient-derived xenograft (PDX) of pancreatic cancer (South Texas
Accelerated Research Therapeutics, San Antonio, Tex. 78229) that is HER2
negative as measured by IHC. The study was carried out as described
below.
[0596] Tumor fragments were implanted subcutaneously into athymic nude
mice. When tumors reached 170 mm.sup.3 the animals were randomly assigned
to groups as shown in Table All and treatment began in a blinded and
controlled study. Animals were treated according to Regimen 1 and 2 as
shown in Table A11. All treatments were administered intravenously.
TABLE-US-00069
TABLE A11
Study Design
Regimen 1 Regimen 2
Group Dosage Dosage Sched-
(n) Agent (mg/kg) Schedule Agent (mg/kg) ule
1 (20) v12470 30 Twice weekly
for four weeks
2 (20) V6336 30 Twice weekly
for four weeks
3 (20) v10000 30 Twice weekly
for four weeks
4 (20) v12470 30 Twice weekly nab- 30 Days 2,
for four weeks paclitaxel 9, 16
5 (20) v10000 30 Twice weekly nab- 30 Days 2,
for four weeks paclitaxel 9, 16
[0597] Tumor volume was measured by calipers twice weekly. The study
duration was 71 days with survival as the primary endpoint. Additional
tumor response criteria were measured and are shown in Table A12. Mice
were euthanized when tumor volume exceeded 2000 mm.sup.3; the surviving
percentage versus study day was plotted on a Kaplan-Meier and was
statistically assessed using a log-rank test. Serum concentration in
groups dosed with v10000 and v6336 was determined by HER2 ELISA on study
day 7.
[0598] The results are shown in FIG. 51A (tumor volume) and FIG. 51B
(Kaplan-Meier survival). Only treatment with variant 10000 in combination
with nab-paclitaxel reduced tumor growth and significantly prolonged
survival by log-rank test compared to treatment with control IgG (v12470)
(FIG. 51B and Table A13). In addition, treatment with v10000 in
combination with nab-paclitaxel significantly prolonged survival compared
to treatment with nab-paclitaxel plus control IgG (FIG. 51B and Table
A13). The median survival of v10000 in combination with nab-paclitaxel
was greater than 71 days while the median survival of v12470, v6336,
v10000, and nab-paclitaxel as single agents was 58.8, 65.9, 69.3, and
60.6 days respectively. Mean tumor volume on study day 54 for treatment
with v10000 in combination with nab-paclitaxel was 1073 mm3. Tumor
volumes for v12470, v6336, v10000, and nab-paclitaxel as single agents on
study day 54 was 1663, 1494, 1305, and 1365 mm3 respectively (FIG. 51A
and Table A12). The serum exposure of v6336 and v10000 from day 14 serum
samples is shown in Table A14.
[0599] These results indicate that treatment with v10000 in combination
with nab-paclitaxel is able to decrease tumor volume and prolong survival
in this model of HER2 negative pancreatic cancer, when compared to the
IgG control, Herceptin.TM., and single agent v10000.
TABLE-US-00070
TABLE A12
ST803 Tumor Response Profile
12470 + nab- 12470 + nab-
12470 6336 10000 pac* pac*
Tumor Response on Day 54
Mean TV (mm.sup.3) (% .DELTA. from base 1663 1494 1305 1365 1073
line) (+888%) (+806%) (+659%) (+693%) (+522%)
Treatment/Control Ratio 1.00 0.90 0.78 0.82 0.64
RECIST Scores
CR (TV <20 mm.sup.3) 0/18 0/17 0/20 0/16 0/19
PR (>30% baseline regression) 0/18 0/17 0/20 0/16 0/19
PD (>20% baseline growth) 18/18 17/17 20/20 16/16 19/19
SD (neither PD or PR) 0/18 0/17 0/20 0/16 0/19
Median Time to Progression 4.4 3.6 3.6 4.4 5.6
(days)
Survival Response
Median Survival (days) 58.8 65.9 69.3 60.6 >71
CR--Complete Response
PR--Partial Response
PD--Progressive Disease
SD--Stable Disease
*nab-paclitaxel
TABLE-US-00071
TABLE A13
Log Rank Summary
12470 +
nab- 10000 + nab-
Group 12470 6336 10000 pac* pac*
12470 -- -- -- --
6336 ns -- -- -- --
10000 ns ns -- -- --
12470 + nab- ns -- Ns -- --
pac
10000, + nab- .star-solid..star-solid. -- Ns .star-solid..star-solid. --
pac
Legend:
ns = not significant,
.star-solid. = P < 0.05.
.star-solid..star-solid. = P < 0.01,
.star-solid..star-solid..star-solid. = P < 0.001
*nab-paclitaxel
TABLE-US-00072
TABLE A14
Serum Exposure Summary
10000
(microg/mL) +
6336 10000 nab-
Sample Day (microg/mL) (microg/mL) paclitaxel
14 426.7 279 391
Example 49: Effect of v10000 on Tumor Growth Inhibition in a Xenograft
Model of HER2 3+ Gastric Cancer
[0600] This experiment was performed to assess efficacy of v10000 compared
to a control IgG (v12470) and Herceptin.TM. (v6336) as single agents in
the GXA3054 xenograft model of gastric cancer. GXA3054 is a patient
derived xenograft (PDX) of gastric cancer that is HER2 3+ (Oncotest GmbH,
Am Flughafen 12-14, 79108 Freiburg, Germany). The study was carried out
as described below.
[0601] Tumor fragments were implanted subcutaneously into athymic nude
mice. When tumors reached 144 mm.sup.3 the animals were randomly assigned
to groups as shown in Table A15 and treatment began in a blinded and
controlled study. Animals were treated according to Regimen 1 as shown in
Table A15.
TABLE-US-00073
TABLE A15
Study Design
Regimen 1
Dosage
Group (n) Agent (mg/kg) Route Schedule
1 (10) v12470 30 IV Twice weekly for five weeks
2 (10) V6336 30 IV Twice weekly for five weeks
3 (10) v10000 30 IV Twice weekly for five weeks
[0602] Tumor volume was measured by calipers twice weekly. The study
duration was 59 days with tumor growth inhibition as the primary
endpoint. Additional tumor response criteria were measured and are shown
in Table A16. Mice were euthanized when tumor volume exceeded 2000
mm.sup.3.
[0603] The results are shown in FIG. 52 (tumor volume). Treatment with
variant 10000 and v6336 reduced tumor growth compared to treatment with
control IgG (v12470) (FIG. 52 and Table A16). In addition, treatment with
v10000 reduced tumor growth compared to treatment with v6336 (FIG. 52 and
Table A16). Mean tumor volume on study day 35 for treatment with control
IgG, v10000 and v6336 was 1340, 236, and 7.8 mm3, respectively. Tumor
growth inhibition on day 35 for v10000 and v6336 was 111 and 92%,
respectively (Table A16). On day 35 tumors treated with v10000 showed
greater responses (7/10 complete and 3/10 partial responses) compared to
tumors treated with v6336 (0/10 complete and 1/10 partial response)
(Table A16). At the completion of the study, on day 59, 9/10 tumors
treated with v10000 had complete responses with no evidence of recurrent
tumor, while for v6336 treated tumors only 1/10 tumors had a complete
response.
[0604] These results indicate that treatment with v10000 can regress
tumors in this model of HER2 3+ gastric cancer. The tumor growth
inhibition of v10000 was superior to IgG control and Herceptin.TM..
TABLE-US-00074
TABLE A16
GXA3054 Tumor Response Profile
12470 6336 10000
Tumor Response on Day 35 Na 92 111
Tumor Growth Inhibition (%)
RECIST Scores
CR (.ltoreq.-95%) 0/10 0/10 7/10
PR (>-95% and <-66%) 0/10 1/10 3/10
SD (.gtoreq.-66% and .ltoreq.+73%) 0/10 5/10 0/10
PD (>+73%) 10/10 4/10 0/10
CR--Complete Response
PR--Partial Response
PD--Progressive Disease
SD--Stable Disease
[0605] The reagents employed in the examples are generally commercially
available or can be prepared using commercially available
instrumentation, methods, or reagents known in the art. The foregoing
examples illustrate various aspects described herein and practice of the
methods described herein. The examples are not intended to provide an
exhaustive description of the many different embodiments of the
invention. Thus, although the forgoing invention has been described in
some detail by way of illustration and example for purposes of clarity of
understanding, those of ordinary skill in the art will realize readily
that many changes and modifications can be made thereto without departing
from the spirit or scope of the appended claims.
[0606] All publications, patents and patent applications mentioned in this
specification are herein incorporated by reference into the specification
to the same extent as if each individual publication, patent or patent
application was specifically and individually indicated to be
incorporated herein by reference.
TABLE-US-00075
SEQUENCE TABLE
Variant Ht clone name H2 clone name L1 clone name L2 clone name
792 1011 1015 -2 -2
5019 3057 720 1811 NA
5020 719 3041 NA 1811
7091 3057 5244 1811 NA
10000 6586 5244 3382 NA
6903 5065 3468 5037 3904
6902 5065 3468 5034 3904
6717 3317 720 NA NA
1040 4560 4553 NA 4561
630 719 716 NA NA
4182 4560 3057 NA 1811
506 642 642 -2 -2
4184 3057 3041 1811 1811
9996 4372 6586 NA 3382
SEQ
ID
NO. Clone Desc. Sequence (amino acid or
1 642 Full EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADS-
VKGRFTISADT
SKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL-
GCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE-
PKS
CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK-
PRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS-
LTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ-
KSL
SLSPGK
2 642 Full GAGGTGCAGCTGGTGGAAAGCGGAGGAGGACTGGTGCAGCCAGGAGGATCTCTGCGACTGAGT-
TGCGCCGCTTC
AGGATTCAACATCAAGGACACCTACATTCACTGGGTGCGACAGGCTCCAGGAAAAGGACTGGAGTGGGTGG-
CTC
GAATCTATCCCACTAATGGATACACCCGGTATGCCGACTCCGTGAAGGGGAGGTTTACTATTAGCGCCGAT-
ACA
TCCAAAAACACTGCTTACCTGCAGATGAACAGCCTGCGAGCCGAAGATACCGCTGTGTACTATTGCAGTCG-
ATG
GGGAGGAGACGGATTCTACGCTATGGATTATTGGGGACAGGGGACCCTGGTGACAGTGAGCTCCGCCTCTA-
CCA
AGGGCCCCAGTGTGTTTCCCCTGGCTCCTTCTAGTAAATCCACCTCTGGAGGGACAGCCGCTCTGGGATGT-
CTG
GTGAAGGACTATTTCCCCGAGCCTGTGACCGTGAGTTGGAACTCAGGCGCCCTGACAAGCGGAGTGCACAC-
TTT
TCCTGCTGTGCTGCAGTCAAGCGGGCTGTACTCCCTGTCCTCTGTGGTGACAGTGCCAAGTTCAAGCCTGG-
GCA
CACAGACTTATATCTGCAACGTGAATCATAAGCCCTCAAATACAAAAGTGGACAAGAAAGTGGAGCCCAAG-
AGC
TGTGATAAGACCCACACCTGCCCTCCCTGTCCAGCTCCAGAACTGCTGGGAGGACCTAGCGTGTTCCTGTT-
TCC
CCCTAAGCCAAAAGACACTCTGATGATTTCCAGGACTCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCTC-
ACG
AGGACCCCGAAGTGAAGTTCAACTGGTACGTGGATGGCGTGGAAGTGCATAATGCTAAGACAAAACCAAGA-
GAG
GAACAGTACAACTCCACTTATCGCGTCGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGGAA-
GGA
GTATAAGTGCAAAGTCAGTAATAAGGCCCTGCCTGCTCCAATCGAAAAAACCATCTCTAAGGCCAAAGGCC-
AGC
CAAGGGAGCCCCAGGTGTACACACTGCCACCCAGCAGAGACGAACTGACCAAGAACCAGGTGTCCCTGACA-
TGT
CTGGTGAAAGGCTTCTATCCTAGTGATATTGCTGTGGAGTGGGAATCAAATGGACAGCCAGAGAACAATTA-
CAA
GACCACACCTCCAGTGCTGGACAGCGATGGCAGCTTCTTCCTGTATTCCAAGCTGACAGTGGATAAATCTC-
GAT
GGCAGCAGGGGAACGTGTTTAGTTGTTCAGTGATGCATGAAGCCCTGCACAATCATTACACTCAGAAGAGC-
CTG
TCCCTGTCTCCCGGCAAA
3 642 VH EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVK-
GRFTISADT
SKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
4 642 VH GAGGTGCAGCTGGTGGAAAGCGGAGGAGGACTGGTGCAGCCAGGAGGATCTCTGCGACTGAGTTG-
CGCCGCTTC
AGGATTCAACATCAAGGACACCTACATTCACTGGGTGCGACAGGCTCCAGGAAAAGGACTGGAGTGGGTGG-
CTC
GAATCTATCCCACTAATGGATACACCCGGTATGCCGACTCCGTGAAGGGGAGGTTTACTATTAGCGCCGAT-
ACA
TCCAAAAACACTGCTTACCTGCAGATGAACAGCCTGCGAGCCGAAGATACCGCTGTGTACTATTGCAGTCG-
ATG
GGGAGGAGACGGATTCTACGCTATGGATTATTGGGGACAGGGGACCCTGGTGACAGTGAGCTCC
5 642 H1 GFNIKDTY
6 642 H1 GGATTCAACATCAAGGACACCTAC
7 642 H3 SRWGGDGFYAMDY
8 642 H3 AGTCGATGGGGAGGAGACGGATTCTACGCTATGGATTAT
9 642 H2 IYPTNGYT
10 642 H2 ATCTATCCCACTAATGGATACACC
11 642 CH1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY-
SLSSVVTVPSS
SLGTQTYICNVNHKPSNTKVDKKV
12 642 CH1 GCCTCTACCAAGGGCCCCAGTGTGTTTCCCCTGGCTCCTTCTAGTAAATCCACCTCTGGAGGG-
ACAGCCGCTCT
GGGATGTCTGGTGAAGGACTATTTCCCCGAGCCTGTGACCGTGAGTTGGAACTCAGGCGCCCTGACAAGCG-
GAG
TGCACACTTTTCCTGCTGTGCTGCAGTCAAGCGGGCTGTACTCCCTGTCCTCTGTGGTGACAGTGCCAAGT-
TCA
AGCCTGGGCACACAGACTTATATCTGCAACGTGAATCATAAGCCCTCAAATACAAAAGTGGACAAGAAAGT-
G
13 642 CH2 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE-
EQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
14 642 CH2 GCTCCAGAACTGCTGGGAGGACCTAGCGTGTTCCTGTTTCCCCCTAAGCCAAAAGACACTCTG-
ATGATTTCCAG
GACTCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCTCACGAGGACCCCGAAGTGAAGTTCAACTGGTACG-
TGG
ATGGCGTGGAAGTGCATAATGCTAAGACAAAACCAAGAGAGGAACAGTACAACTCCACTTATCGCGTCGTG-
AGC
GTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGGAAGGAGTATAAGTGCAAAGTCAGTAATAAGGCCCT-
GCC
TGCTCCAATCGAAAAAACCATCTCTAAGGCCAAA
15 642 CH3 GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS-
FFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
16 642 CH3 GGCCAGCCAAGGGAGCCCCAGGTGTACACACTGCCACCCAGCAGAGACGAACTGACCAAGAAC-
CAGGTGTCCCT
GACATGTCTGGTGAAAGGCTTCTATCCTAGTGATATTGCTGTGGAGTGGGAATCAAATGGACAGCCAGAGA-
ACA
ATTACAAGACCACACCTCCAGTGCTGGACAGCGATGGCAGCTTCTTCCTGTATTCCAAGCTGACAGTGGAT-
AAA
TCTCGATGGCAGCAGGGGAACGTGTTTAGTTGTTCAGTGATGCATGAAGCCCTGCACAATCATTACACTCA-
GAA
GAGCCTGTCCCTGTCTCCCGGC
17 3468 Full
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGCSIYNQRFKGRFTLSVD-
R
SKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG-
CLV
KGYFPEPVTVSWNSGALTSGVHTFPAVLKSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP-
KSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP-
REE
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSL-
LCL
VKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK-
SLS
LSPG
18 3468 Full
GAAGTGCAGCTGGTCGAATCTGGAGGAGGACTGGTGCAGCCAGGAGGGTCCCTGCGCCTGTCTTGCGCCGCTA-
G
TGGCTTCACTTTTACCGACTACACCATGGATTGGGTGCGACAGGCACCTGGAAAGGGCCTGGAGTGGGTCG-
CCG
ATGTGAACCCAAATAGCGGAGGCTCCATCTACAACCAGCGGTTCAAGGGCCGGTTCACCCTGTCAGTGGAC-
CGG
AGCAAAAACACCCTGTATCTGCAGATGAATAGCCTGCGAGCCGAAGATACTGCTGTGTACTATTGCGCCCG-
GAA
TCTGGGGCCCTCCTTCTACTTTGACTATTGGGGGCAGGGAACTCTGGTCACCGTGAGCTCCGCCTCCACCA-
AGG
GACCTTCTGTGTTCCCACTGGCTCCCTCTAGTAAATCCACATCTGGGGGAACTGCAGCCCTGGGCTGTCTG-
GTG
AAGGGCTACTTCCCAGAGCCCGTCACAGTGTCTTGGAACAGTGGCGCTCTGACTTCTGGGGTCCACACCTT-
TCC
TGCAGTGCTGAAGTCAAGCGGGCTGTACAGCCTGTCCTCTGTGGTCACCGTGCCAAGTTCAAGCCTGGGAA-
CAC
AGACTTATATCTGCAACGTGAATCACAAGCCATCCAATACAAAAGTCGACAAGAAAGTGGAACCCAAGTCT-
TGT
GATAAAACCCATACATGCCCCCCTTGTCCTGCACCAGAGCTGCTGGGAGGACCAAGCGTGTTCCTGTTTCC-
ACC
CAAGCCTAAAGATACACTGATGATTAGTAGGACCCCAGAAGTCACATGCGTGGTCGTGGACGTGAGCCACG-
AGG
ACCCCGAAGTCAAGTTTAACTGGTACGTGGACGGCGTCGAGGTGCATAATGCCAAGACTAAACCCAGGGAG-
GAA
CAGTACAACAGTACCTATCGCGTCGTGTCAGTCCTGACAGTGCTGCATCAGGATTGGCTGAACGGGAAAGA-
GTA
TAAGTGCAAAGTGAGCAATAAGGCTCTGCCCGCACCTATCGAGAAAACAATTTCCAAGGCAAAAGGACAGC-
CTA
GAGAACCACAGGTGTACGTGCTGCCTCCATCAAGGGATGAGCTGACAAAGAACCAGGTCAGCCTGCTGTGT-
CTG
GTGAAAGGATTCTATCCCTCTGACATTGCTGTGGAGTGGGAAAGTAATGGCCAGCCTGAGAACAATTACCT-
GAC
CTGGCCCCCTGTGCTGGACTCAGATGGCAGCTTCTTTCTGTATAGCAAGCTGACCGTCGACAAATCCCGGT-
GGC
AGCAGGGGAATGTGTTTAGTTGTTCAGTCATGCACGAGGCACTGCACAACCATTACACCCAGAAGTCACTG-
TCA
CTGTCACCAGGG
19 3468 VH EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGCSIYNQR-
FKGRFTLSVDR
SKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS
20 3468 VH GAAGTGCAGCTGGTCGAATCTGGAGGAGGACTGGTGCAGCCAGGAGGGTCCCTGCGCCTGTCT-
TGCGCCGCTAG
TGGCTTCACTTTTACCGACTACACCATGGATTGGGTGCGACAGGCACCTGGAAAGGGCCTGGAGTGGGTCG-
CCG
ATGTGAACCCAAATAGCGGAGGCTCCATCTACAACCAGCGGTTCAAGGGCCGGTTCACCCTGTCAGTGGAC-
CGG
AGCAAAAACACCCTGTATCTGCAGATGAATAGCCTGCGAGCCGAAGATACTGCTGTGTACTATTGCGCCCG-
GAA
TCTGGGGCCCTCCTTCTACTTTGACTATTGGGGGCAGGGAACTCTGGTCACCGTGAGCTCC
21 3468 H1 GFTFTDYT
22 3468 H1 GGCTTCACTTTTACCGACTACACC
23 3468 H3 ARNLGPSFYFDY
24 3468 H3 GCCCGGAATCTGGGGCCCTCCTTCTACTTTGACTAT
25 3468 H2 VNPNSGGS
26 3468 H2 GTGAACCCAAATAGCGGAGGCTCC
27 3468 CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKGYFPEPVTVSWNSGALTSGVHTFPAVLKSSGLYSLSSVVTVPS-
S
SLGTQTYICNVNHKPSNTKVDKKV
28 3468 CH1
GCCTCCACCAAGGGACCTTCTGTGTTCCCACTGGCTCCCTCTAGTAAATCCACATCTGGGGGAACTGCAGCCC-
T
GGGCTGTCTGGTGAAGGGCTACTTCCCAGAGCCCGTCACAGTGTCTTGGAACAGTGGCGCTCTGACTTCTG-
GGG
TCCACACCTTTCCTGCAGTGCTGAAGTCAAGCGGGCTGTACAGCCTGTCCTCTGTGGTCACCGTGCCAAGT-
TCA
AGCCTGGGAACACAGACTTATATCTGCAACGTGAATCACAAGCCATCCAATACAAAAGTCGACAAGAAAGT-
G
29 3468 CH2
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV-
S
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
30 3468 CH2
GCACCAGAGCTGCTGGGAGGACCAAGCGTGTTCCTGTTTCCACCCAAGCCTAAAGATACACTGATGATTAGTA-
G
GACCCCAGAAGTCACATGCGTGGTCGTGGACGTGAGCCACGAGGACCCCGAAGTCAAGTTTAACTGGTACG-
TGG
ACGGCGTCGAGGTGCATAATGCCAAGACTAAACCCAGGGAGGAACAGTACAACAGTACCTATCGCGTCGTG-
TCA
GTCCTGACAGTGCTGCATCAGGATTGGCTGAACGGGAAAGAGTATAAGTGCAAAGTGAGCAATAAGGCTCT-
GCC
CGCACCTATCGAGAAAACAATTTCCAAGGCAAAA
31 3468 CH3
GQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVD-
K
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
32 3468 CH3
GGACAGCCTAGAGAACCACAGGTGTACGTGCTGCCTCCATCAAGGGATGAGCTGACAAAGAACCAGGTCAGCC-
T
GCTGTGTCTGGTGAAAGGATTCTATCCCTCTGACATTGCTGTGGAGTGGGAAAGTAATGGCCAGCCTGAGA-
ACA
ATTACCTGACCTGGCCCCCTGTGCTGGACTCAGATGGCAGCTTCTTTCTGTATAGCAAGCTGACCGTCGAC-
AAA
TCCCGGTGGCAGCAGGGGAATGTGTTTAGTTGTTCAGTCATGCACGAGGCACTGCACAACCATTACACCCA-
GAA
GTCACTGTCACTGTCACCAGGG
33 1811 Full
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTL-
T
ISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK-
VQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
34 1811 Full
GATATTCAGATGACCCAGTCCCCAAGCTCCCTGAGTGCCTCAGTGGGCGACCGAGTCACCATCACATGCAAGG-
C
TTCCCAGGATGTGTCTATTGGAGTCGCATGGTACCAGCAGAAGCCAGGCAAAGCACCCAAGCTGCTGATCT-
ATA
GCGCCTCCTACCGGTATACCGGCGTGCCCTCTAGATTCTCTGGCAGTGGGTCAGGAACAGACTTTACTCTG-
ACC
ATCTCTAGTCTGCAGCCTGAGGATTTCGCTACCTACTATTGCCAGCAGTACTATATCTACCCATATACCTT-
TGG
CCAGGGGACAAAAGTGGAGATCAAGAGGACTGTGGCCGCTCCCTCCGTCTTCATTTTTCCCCCTTCTGACG-
AAC
AGCTGAAAAGTGGCACAGCCAGCGTGGTCTGTCTGCTGAACAATTTCTACCCTCGCGAAGCCAAAGTGCAG-
TGG
AAGGTCGATAACGCTCTGCAGAGCGGCAACAGCCAGGAGTCTGTGACTGAACAGGACAGTAAAGATTCAAC-
CTA
TAGCCTGTCAAGCACACTGACTCTGAGCAAGGCAGACTACGAGAAGCACAAAGTGTATGCCTGCGAAGTCA-
CAC
ATCAGGGGCTGTCCTCTCCTGTGACTAAGAGCTTTAACAGAGGAGAGTGT
35 1811 VL DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFS-
GSGSGTDFTLT
ISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK
36 1811 VL GATATTCAGATGACCCAGTCCCCAAGCTCCCTGAGTGCCTCAGTGGGCGACCGAGTCACCATC-
ACATGCAAGGC
TTCCCAGGATGTGTCTATTGGAGTCGCATGGTACCAGCAGAAGCCAGGCAAAGCACCCAAGCTGCTGATCT-
ATA
GCGCCTCCTACCGGTATACCGGCGTGCCCTCTAGATTCTCTGGCAGTGGGTCAGGAACAGACTTTACTCTG-
ACC
ATCTCTAGTCTGCAGCCTGAGGATTTCGCTACCTACTATTGCCAGCAGTACTATATCTACCCATATACCTT-
TGG
CCAGGGGACAAAAGTGGAGATCAAG
37 1811 L1 QDVSIG
38 1811 L1 CAGGATGTGTCTATTGGA
39 1811 L3 QQYYIYPYT
40 1811 L3 CAGCAGTACTATATCTACCCATATACC
41 1811 L2 SAS
42 1811 L2 AGCGCCTCC
43 1811 CL RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD-
STYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
44 1811 CL AGGACTGTGGCCGCTCCCTCCGTCTTCATTTTTCCCCCTTCTGACGAACAGCTGAAAAGTGGC-
ACAGCCAGCGT
GGTCTGTCTGCTGAACAATTTCTACCCTCGCGAAGCCAAAGTGCAGTGGAAGGTCGATAACGCTCTGCAGA-
GCG
GCAACAGCCAGGAGTCTGTGACTGAACAGGACAGTAAAGATTCAACCTATAGCCTGTCAAGCACACTGACT-
CTG
AGCAAGGCAGACTACGAGAAGCACAAAGTGTATGCCTGCGAAGTCACACATCAGGGGCTGTCCTCTCCTGT-
GAC
TAAGAGCTTTAACAGAGGAGAGTGT
45 5034 Full
DYKDDDDKDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGS-
R
SGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDERLKSGTASVVCLLN-
NFY
PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR-
GEC
46 5034 Full
GACTACAAAGACGACGATGACAAAGATATCCAGATGACCCAGTCCCCTAGCTCCCTGTCCGCTTCTGTGGGCG-
A
TAGGGTCACTATTACCTGCCGCGCATCTCAGGACGTGAACACCGCAGTCGCCTGGTACCAGCAGAAGCCTG-
GGA
AAGCTCCAAAGCTGCTGATCTACAGTGCATCATTCCTGTATTCAGGAGTGCCCAGCCGGTTTAGCGGCAGC-
AGA
TCTGGCACCGATTTCACACTGACTATTTCTAGTCTGCAGCCTGAGGACTTTGCCACATACTATTGCCAGCA-
GCA
CTATACCACACCCCCTACTTTCGGCCAGGGGACCAAAGTGGAGATCAAGCGAACTGTGGCCGCTCCAAGTG-
TCT
TCATTTTTCCACCCAGCGATGAAAGACTGAAGTCCGGCACAGCTTCTGTGGTCTGTCTGCTGAACAATTTT-
TAC
CCCAGAGAGGCCAAAGTGCAGTGGAAGGTCGACAACGCTCTGCAGAGTGGCAACAGCCAGGAGAGCGTGAC-
AGA
ACAGGATTCCAAAGACTCTACTTATAGTCTGTCAAGCACCCTGACACTGAGCAAGGCAGACTACGAAAAGC-
ATA
AAGTGTATGCCTGTGAGGTCACACATCAGGGGCTGTCATCACCAGTCACCAAATCATTCAATCGGGGGGAG-
TGC
47 5034 VL DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFS-
GSRSGTDFTLT
ISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK
48 5034 VL GATATCCAGATGACCCAGTCCCCTAGCTCCCTGTCCGCTTCTGTGGGCGATAGGGTCACTATT-
ACCTGCCGCGC
ATCTCAGGACGTGAACACCGCAGTCGCCTGGTACCAGCAGAAGCCTGGGAAAGCTCCAAAGCTGCTGATCT-
ACA
GTGCATCATTCCTGTATTCAGGAGTGCCCAGCCGGTTTAGCGGCAGCAGATCTGGCACCGATTTCACACTG-
ACT
ATTTCTAGTCTGCAGCCTGAGGACTTTGCCACATACTATTGCCAGCAGCACTATACCACACCCCCTACTTT-
CGG
CCAGGGGACCAAAGTGGAGATCAAG
49 5034 L1 QDVNTA
50 5034 L1 CAGGACGTGAACACCGCA
51 5034 L3 QQHYTTPPT
52 5034 L3 CAGCAGCACTATACCACACCCCCTACT
53 5034 L2 SAS
54 5034 L2 AGTGCATCA
55 5034 CL RTVAAPSVFIFPPSDERLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD-
STYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
56 5034 CL CGAACTGTGGCCGCTCCAAGTGTCTTCATTTTTCCACCCAGCGATGAAAGACTGAAGTCCGGC-
ACAGCTTCTGT
GGTCTGTCTGCTGAACAATTTTTACCCCAGAGAGGCCAAAGTGCAGTGGAAGGTCGACAACGCTCTGCAGA-
GTG
GCAACAGCCAGGAGAGCGTGACAGAACAGGATTCCAAAGACTCTACTTATAGTCTGTCAAGCACCCTGACA-
CTG
AGCAAGGCAGACTACGAAAAGCATAAAGTGTATGCCTGTGAGGTCACACATCAGGGGCTGTCATCACCAGT-
CAC
CAAATCATTCAATCGGGGGGAGTGC
57 5037 Full
DYKDDDDKDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGS-
R
SGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDERLKSGTASVVCLLN-
NFY
PREAKVQWKVDNALQSGNSKESVTEQDSKDSTYSLSSRLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR-
GEC
58 5037 Full
GACTACAAAGACGACGATGACAAAGATATCCAGATGACCCAGTCCCCTAGCTCCCTGTCCGCTTCTGTGGGCG-
A
TAGGGTCACTATTACCTGCCGCGCATCTCAGGACGTGAACACCGCAGTCGCCTGGTACCAGCAGAAGCCTG-
GGA
AAGCTCCAAAGCTGCTGATCTACAGTGCATCATTCCTGTATTCAGGAGTGCCCAGCCGGTTTAGCGGCAGC-
AGA
TCTGGCACCGATTTCACACTGACTATTTCTAGTCTGCAGCCTGAGGACTTTGCCACATACTATTGCCAGCA-
GCA
CTATACCACACCCCCTACTTTCGGCCAGGGGACCAAAGTGGAGATCAAGCGAACTGTGGCCGCTCCAAGTG-
TCT
TCATTTTTCCACCCAGCGATGAAAGACTGAAGTCCGGCACAGCTTCTGTGGTCTGTCTGCTGAACAATTTT-
TAC
CCCAGAGAGGCCAAAGTGCAGTGGAAGGTCGACAACGCTCTGCAGAGTGGCAACAGCAAGGAGAGCGTGAC-
AGA
ACAGGATTCCAAAGACTCTACTTATAGTCTGTCAAGCAGACTGACACTGAGCAAGGCAGACTACGAAAAGC-
ATA
AAGTGTATGCCTGTGAGGTCACACATCAGGGGCTGTCATCACCAGTCACCAAATCATTCAATCGGGGGGAG-
TGC
59 5037 VL DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFS-
GSRSGTDFTLT
ISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK
60 5037 VL GATATCCAGATGACCCAGTCCCCTAGCTCCCTGTCCGCTTCTGTGGGCGATAGGGTCACTATT-
ACCTGCCGCGC
ATCTCAGGACGTGAACACCGCAGTCGCCTGGTACCAGCAGAAGCCTGGGAAAGCTCCAAAGCTGCTGATCT-
ACA
GTGCATCATTCCTGTATTCAGGAGTGCCCAGCCGGTTTAGCGGCAGCAGATCTGGCACCGATTTCACACTG-
ACT
ATTTCTAGTCTGCAGCCTGAGGACTTTGCCACATACTATTGCCAGCAGCACTATACCACACCCCCTACTTT-
CGG
CCAGGGGACCAAAGTGGAGATCAAG
61 5037 L1 QDVNTA
62 5037 L1 CAGGACGTGAACACCGCA
63 5037 L3 QQHYTTPPT
64 5037 L3 CAGCAGCACTATACCACACCCCCTACT
65 5037 L2 SAS
66 5037 L2 AGTGCATCA
67 5037 CL RTVAAPSVFIFPPSDERLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSKESVTEQDSKD-
STYSLSSRLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
68 5037 CL CGAACTGTGGCCGCTCCAAGTGTCTTCATTTTTCCACCCAGCGATGAAAGACTGAAGTCCGGC-
ACAGCTTCTGT
GGTCTGTCTGCTGAACAATTTTTACCCCAGAGAGGCCAAAGTGCAGTGGAAGGTCGACAACGCTCTGCAGA-
GTG
GCAACAGCAAGGAGAGCGTGACAGAACAGGATTCCAAAGACTCTACTTATAGTCTGTCAAGCAGACTGACA-
CTG
AGCAAGGCAGACTACGAAAAGCATAAAGTGTATGCCTGTGAGGTCACACATCAGGGGCTGTCATCACCAGT-
CAC
CAAATCATTCAATCGGGGGGAGTGC
69 3382 Full
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTL-
T
ISSLQPEDFATYYCQQYYIYPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK-
VQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
70 3382 Full
GATATTCAGATGACCCAGTCCCCAAGCTCCCTGAGTGCCTCAGTGGGCGACCGAGTCACCATCACATGCAAGG-
C
TTCCCAGGATGTGTCTATTGGAGTCGCATGGTACCAGCAGAAGCCAGGCAAAGCACCCAAGCTGCTGATCT-
ATA
GCGCCTCCTACCGGTATACCGGCGTGCCCTCTAGATTCTCTGGCAGTGGGTCAGGAACAGACTTTACTCTG-
ACC
ATCTCTAGTCTGCAGCCTGAGGATTTCGCTACCTACTATTGCCAGCAGTACTATATCTACCCAGCCACCTT-
TGG
CCAGGGGACAAAAGTGGAGATCAAGAGGACTGTGGCCGCTCCCTCCGTCTTCATTTTTCCCCCTTCTGACG-
AAC
AGCTGAAAAGTGGCACAGCCAGCGTGGTCTGTCTGCTGAACAATTTCTACCCTCGCGAAGCCAAAGTGCAG-
TGG
AAGGTCGATAACGCTCTGCAGAGCGGCAACAGCCAGGAGTCTGTGACTGAACAGGACAGTAAAGATTCAAC-
CTA
TAGCCTGTCAAGCACACTGACTCTGAGCAAGGCAGACTACGAGAAGCACAAAGTGTATGCCTGCGAAGTCA-
CAC
ATCAGGGGCTGTCCTCTCCTGTGACTAAGAGCTTTAACAGAGGAGAGTGT
71 3382 VL DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFS-
GSGSGTDFTLT
ISSLQPEDFATYYCQQYYIYPATFGQGTKVEIK
72 3382 VL GATATTCAGATGACCCAGTCCCCAAGCTCCCTGAGTGCCTCAGTGGGCGACCGAGTCACCATC-
ACATGCAAGGC
TTCCCAGGATGTGTCTATTGGAGTCGCATGGTACCAGCAGAAGCCAGGCAAAGCACCCAAGCTGCTGATCT-
ATA
GCGCCTCCTACCGGTATACCGGCGTGCCCTCTAGATTCTCTGGCAGTGGGTCAGGAACAGACTTTACTCTG-
ACC
ATCTCTAGTCTGCAGCCTGAGGATTTCGCTACCTACTATTGCCAGCAGTACTATATCTACCCAGCCACCTT-
TGG
CCAGGGGACAAAAGTGGAGATCAAG
73 3382 L1 QDVSIG
74 3382 L1 CAGGATGTGTCTATTGGA
75 3382 L3 QQYYIYPAT
76 3382 L3 CAGCAGTACTATATCTACCCAGCCACC
77 3382 L2 SAS
78 3382 L2 AGCGCCTCC
79 3382 CL RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD-
STYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
80 3382 CL AGGACTGTGGCCGCTCCCTCCGTCTTCATTTTTCCCCCTTCTGACGAACAGCTGAAAAGTGGC-
ACAGCCAGCGT
GGTCTGTCTGCTGAACAATTTCTACCCTCGCGAAGCCAAAGTGCAGTGGAAGGTCGATAACGCTCTGCAGA-
GCG
GCAACAGCCAGGAGTCTGTGACTGAACAGGACAGTAAAGATTCAACCTATAGCCTGTCAAGCACACTGACT-
CTG
AGCAAGGCAGACTACGAGAAGCACAAAGTGTATGCCTGCGAAGTCACACATCAGGGGCTGTCCTCTCCTGT-
GAC
TAAGAGCTTTAACAGAGGAGAGTGT
81 5065 Full
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAD-
T
SKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL-
GCE
VTDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE-
PKS
CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK-
PRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVS-
LTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ-
KSL
SLSPG
82 5065 Full
GAGGTGCAGCTGGTCGAAAGCGGAGGAGGACTGGTGCAGCCAGGAGGGTCACTGCGACTGAGCTGCGCAGCTT-
C
CGGCTTCAACATCAAGGACACCTACATTCACTGGGTCCGCCAGGCTCCTGGAAAAGGCCTGGAGTGGGTGG-
CAC
GAATCTATCCAACTAATGGATACACCCGGTATGCCGACTCCGTGAAGGGCCGGTTCACCATTTCTGCAGAT-
ACA
AGTAAAAACACTGCCTACCTGCAGATGAACAGCCTGCGAGCCGAAGATACAGCCGTGTACTATTGCAGCCG-
ATG
GGGAGGCGACGGCTTCTACGCTATGGATTATTGGGGGCAGGGAACCCTGGTCACAGTGAGCTCCGCATCAA-
CAA
AGGGGCCTAGCGTGTTTCCACTGGCCCCCTCTAGTAAATCCACCTCTGGGGGAACAGCAGCCCTGGGATGT-
GAG
GTGACCGACTACTTCCCAGAGCCCGTCACTGTGAGCTGGAACTCCGGCGCCCTGACATCTGGGGTCCATAC-
TTT
TCCTGCTGTGCTGCAGTCAAGCGGCCTGTACAGCCTGTCCTCTGTGGTCACTGTGCCAAGTTCAAGCCTGG-
GGA
CTCAGACCTATATCTGCAACGTGAATCACAAGCCATCCAATACCAAAGTCGACAAGAAAGTGGAACCCAAG-
TCT
TGTGATAAAACACATACTTGCCCCCCTTGTCCTGCACCAGAGCTGCTGGGAGGACCAAGCGTGTTCCTGTT-
TCC
ACCCAAGCCTAAAGACACCCTGATGATTAGTAGGACTCCAGAAGTCACCTGCGTGGTCGTGGACGTGAGCC-
ACG
AGGACCCCGAAGTCAAGTTCAACTGGTACGTGGATGGCGTCGAGGTGCATAATGCCAAGACAAAACCCAGG-
GAG
GAACAGTACAACTCCACTTATCGCGTCGTGTCTGTCCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAA-
GGA
GTATAAGTGCAAAGTGAGCAATAAGGCTCTGCCCGCACCTATCGAGAAAACAATTTCCAAGGCTAAAGGGC-
AGC
CTAGAGAACCACAGGTGTACGTGTACCCTCCATCTAGGGACGAGCTGACCAAGAACCAGGTCAGTCTGACA-
TGT
CTGGTGAAAGGGTTCTATCCCAGCGATATCGCAGTGGAGTGGGAATCCAATGGACAGCCTGAGAACAATTA-
CAA
GACCACACCCCCTGTGCTGGACTCTGATGGAAGTTTCGCCCTGGTGAGTAAGCTGACCGTCGATAAATCAC-
GGT
GGCAGCAGGGCAACGTGTTCAGCTGTTCAGTGATGCACGAAGCACTGCACAACCACTACACCCAGAAAAGC-
CTG
TCCCTGTCCCCCGGC
83 5065 VH EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADS-
VKGRFTISADT
SKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
84 5065 VH GAGGTGCAGCTGGTCGAAAGCGGAGGAGGACTGGTGCAGCCAGGAGGGTCACTGCGACTGAGC-
TGCGCAGCTTC
CGGCTTCAACATCAAGGACACCTACATTCACTGGGTCCGCCAGGCTCCTGGAAAAGGCCTGGAGTGGGTGG-
CAC
GAATCTATCCAACTAATGGATACACCCGGTATGCCGACTCCGTGAAGGGCCGGTTCACCATTTCTGCAGAT-
ACA
AGTAAAAACACTGCCTACCTGCAGATGAACAGCCTGCGAGCCGAAGATACAGCCGTGTACTATTGCAGCCG-
ATG
GGGAGGCGACGGCTTCTACGCTATGGATTATTGGGGGCAGGGAACCCTGGTCACAGTGAGCTCC
85 5065 H1 GFNIKDTY
86 5065 H1 GGCTTCAACATCAAGGACACCTAC
87 5065 H3 SRWGGDGFYAMDY
88 5065 H3 AGCCGATGGGGAGGCGACGGCTTCTACGCTATGGATTAT
89 5065 H2 IYPTNGYT
90 5065 H2 ATCTATCCAACTAATGGATACACC
91 5065 CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCEVTDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS-
S
SLGTQTYICNVNHKPSNTKVDKKV
92 5065 CH1
GCATCAACAAAGGGGCCTAGCGTGTTTCCACTGGCCCCCTCTAGTAAATCCACCTCTGGGGGAACAGCAGCCC-
T
GGGATGTGAGGTGACCGACTACTTCCCAGAGCCCGTCACTGTGAGCTGGAACTCCGGCGCCCTGACATCTG-
GGG
TCCATACTTTTCCTGCTGTGCTGCAGTCAAGCGGCCTGTACAGCCTGTCCTCTGTGGTCACTGTGCCAAGT-
TCA
AGCCTGGGGACTCAGACCTATATCTGCAACGTGAATCACAAGCCATCCAATACCAAAGTCGACAAGAAAGT-
G
93 5065 CH2
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV-
S
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
94 5065 CH2
GCACCAGAGCTGCTGGGAGGACCAAGCGTGTTCCTGTTTCCACCCAAGCCTAAAGACACCCTGATGATTAGTA-
G
GACTCCAGAAGTCACCTGCGTGGTCGTGGACGTGAGCCACGAGGACCCCGAAGTCAAGTTCAACTGGTACG-
TGG
ATGGCGTCGAGGTGCATAATGCCAAGACAAAACCCAGGGAGGAACAGTACAACTCCACTTATCGCGTCGTG-
TCT
GTCCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTATAAGTGCAAAGTGAGCAATAAGGCTCT-
GCC
CGCACCTATCGAGAAAACAATTTCCAAGGCTAAA
95 5065 CH3
GQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVD-
K
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
96 5065 CH3
GGGCAGCCTAGAGAACCACAGGTGTACGTGTACCCTCCATCTAGGGACGAGCTGACCAAGAACCAGGTCAGTC-
T
GACATGTCTGGTGAAAGGGTTCTATCCCAGCGATATCGCAGTGGAGTGGGAATCCAATGGACAGCCTGAGA-
ACA
ATTACAAGACCACACCCCCTGTGCTGGACTCTGATGGAAGTTTCGCCCTGGTGAGTAAGCTGACCGTCGAT-
AAA
TCACGGTGGCAGCAGGGCAACGTGTTCAGCTGTTCAGTGATGCACGAAGCACTGCACAACCACTACACCCA-
GAA
AAGCCTGTCCCTGTCCCCCGGC
97 6586 Full
EVQLVESGGGLVQPGGSLRLSCAASGFTFADYTMDWVRQAPGKGLEWVGDVNPNSGCSIYNQRFKGRFTFSVD-
R
SKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG-
CLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP-
KSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP-
REE
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSL-
TCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK-
SLS
LSPG
98 6586 Full
GAGGTGCAGCTGGTGGAATCAGGAGGGGGCCTGGTGCAGCCCGGAGGGTCTCTGCGACTGTCATGTGCCGCTT-
C
TGGGTTCACTTTCGCAGACTACACAATGGATTGGGTGCGACAGGCCCCCGGAAAGGGACTGGAGTGGGTGG-
GCG
ATGTCAACCCTAATTCTGGCGGGAGTATCTACAACCAGCGGTTCAAGGGGAGATTCACTTTTTCAGTGGAC-
AGA
AGCAAAAACACCCTGTATCTGCAGATGAACAGCCTGAGGGCCGAAGATACCGCTGTCTACTATTGCGCTCG-
CAA
TCTGGGCCCCAGTTTCTACTTTGACTATTGGGGGCAGGGAACCCTGGTGACAGTCAGCTCCGCTAGCACTA-
AGG
GGCCTTCCGTGTTTCCACTGGCTCCCTCTAGTAAATCCACCTCTGGAGGCACAGCTGCACTGGGATGTCTG-
GTG
AAGGATTACTTCCCTGAACCAGTCACAGTGAGTTGGAACTCAGGGGCTCTGACAAGTGGAGTCCATACTTT-
TCC
CGCAGTGCTGCAGTCAAGCGGACTGTACTCCCTGTCCTCTGTGGTCACCGTGCCTAGTTCAAGCCTGGGCA-
CCC
AGACATATATCTGCAACGTGAATCACAAGCCATCAAATACAAAAGTCGACAAGAAAGTGGAGCCCAAGAGC-
TGT
GATAAAACTCATACCTGCCCACCTTGTCCGGCGCCAGAACTGCTGGGAGGACCAAGCGTGTTCCTGTTTCC-
ACC
CAAGCCTAAAGACACCCTGATGATTTCCCGGACTCCTGAGGTCACCTGCGTGGTCGTGGACGTGTCTCACG-
AGG
ACCCCGAAGTCAAGTTCAACTGGTACGTGGATGGCGTCGAAGTGCATAATGCCAAGACCAAACCCCGGGAG-
GAA
CAGTACAACTCTACCTATAGAGTCGTGAGTGTCCTGACAGTGCTGCACCAGGACTGGCTGAATGGGAAGGA-
GTA
TAAGTGTAAAGTGAGCAACAAAGCCCTGCCCGCCCCAATCGAAAAAACAATCTCTAAAGCAAAAGGACAGC-
CTC
GCGAACCACAGGTCTACGTCTACCCCCCATCAAGAGATGAACTGACAAAAAATCAGGTCTCTCTGACATGC-
CTG
GTCAAAGGATTCTACCCTTCCGACATCGCCGTGGAGTGGGAAAGTAACGGCCAGCCCGAGAACAATTACAA-
GAC
CACACCCCCTGTCCTGGACTCTGATGGGAGTTTCGCTCTGGTGTCAAAGCTGACCGTCGATAAAAGCCGGT-
GGC
AGCAGGGCAATGTGTTTAGCTGCTCCGTCATGCACGAAGCCCTGCACAATCACTACACACAGAAGTCCCTG-
AGC
CTGAGCCCTGGC
99 6586 VH EVQLVESGGGLVQPGGSLRLSCAASGFTFADYTMDWVRQAPGKGLEWVGDVNPNSGCSIYNQR-
FKGRFTFSVDR
SKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS
100 6586 VH
GAGGTGCAGCTGGTGGAATCAGGAGGGGGCCTGGTGCAGCCCGGAGGGTCTCTGCGACTGTCATGTGCCGCTT-
C
TGGGTTCACTTTCGCAGACTACACAATGGATTGGGTGCGACAGGCCCCCGGAAAGGGACTGGAGTGGGTGG-
GCG
ATGTCAACCCTAATTCTGGCGGGAGTATCTACAACCAGCGGTTCAAGGGGAGATTCACTTTTTCAGTGGAC-
AGA
AGCAAAAACACCCTGTATCTGCAGATGAACAGCCTGAGGGCCGAAGATACCGCTGTCTACTATTGCGCTCG-
CAA
TCTGGGCCCCAGTTTCTACTTTGACTATTGGGGGCAGGGAACCCTGGTGACAGTCAGCTCC
101 6586 H1 GFTFADYT
102 6586 H1 GGGTTCACTTTCGCAGACTACACA
103 6586 H3 ARNLGPSFYFDY
104 6586 H3 GCTCGCAATCTGGGCCCCAGTTTCTACTTTGACTAT
105 6586 H2 VNPNSGGS
106 6586 H2 GTCAACCCTAATTCTGGCGGGAGT
107 6586 CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS-
S
SLGTQTYICNVNHKPSNTKVDKKV
108 6586 CH1
GCTAGCACTAAGGGGCCTTCCGTGTTTCCACTGGCTCCCTCTAGTAAATCCACCTCTGGAGGCACAGCTGCAC-
T
GGGATGTCTGGTGAAGGATTACTTCCCTGAACCAGTCACAGTGAGTTGGAACTCAGGGGCTCTGACAAGTG-
GAG
TCCATACTTTTCCCGCAGTGCTGCAGTCAAGCGGACTGTACTCCCTGTCCTCTGTGGTCACCGTGCCTAGT-
TCA
AGCCTGGGCACCCAGACATATATCTGCAACGTGAATCACAAGCCATCAAATACAAAAGTCGACAAGAAAGT-
G
109 6586 CH2
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV-
S
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
110 6586 CH2
GCGCCAGAACTGCTGGGAGGACCAAGCGTGTTCCTGTTTCCACCCAAGCCTAAAGACACCCTGATGATTTCCC-
G
GACTCCTGAGGTCACCTGCGTGGTCGTGGACGTGTCTCACGAGGACCCCGAAGTCAAGTTCAACTGGTACG-
TGG
ATGGCGTCGAAGTGCATAATGCCAAGACCAAACCCCGGGAGGAACAGTACAACTCTACCTATAGAGTCGTG-
AGT
GTCCTGACAGTGCTGCACCAGGACTGGCTGAATGGGAAGGAGTATAAGTGTAAAGTGAGCAACAAAGCCCT-
GCC
CGCCCCAATCGAAAAAACAATCTCTAAAGCAAAA
111 6586 CH3
GQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVD-
K
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
112 6586 CH3
GGACAGCCTCGCGAACCACAGGTCTACGTCTACCCCCCATCAAGAGATGAACTGACAAAAAATCAGGTCTCTC-
T
GACATGCCTGGTCAAAGGATTCTACCCTTCCGACATCGCCGTGGAGTGGGAAAGTAACGGCCAGCCCGAGA-
ACA
ATTACAAGACCACACCCCCTGTCCTGGACTCTGATGGGAGTTTCGCTCTGGTGTCAAAGCTGACCGTCGAT-
AAA
AGCCGGTGGCAGCAGGGCAATGTGTTTAGCTGCTCCGTCATGCACGAAGCCCTGCACAATCACTACACACA-
GAA
GTCCCTGAGCCTGAGCCCTGGC
113 3904 Full
YPYDVPDYATGSDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSR-
F
SGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEELKSGTASVV-
CLL
NNFYPREAKVQWKVDNALQSGNSEESVTEQDSKDSTYSLSSTLELSKADYEKHKVYACEVTHQGLSSPVTK-
SFN
RGEC
114 3904 Full
TATCCCTACGATGTGCCTGACTACGCTACTGGCTCCGATATCCAGATGACCCAGTCTCCAAGCTCCCTGAGTG-
C
ATCAGTGGGGGACCGAGTCACCATCACATGCAAGGCTTCCCAGGATGTGTCTATTGGAGTCGCATGGTACC-
AGC
AGAAGCCAGGCAAAGCACCCAAGCTGCTGATCTACAGCGCCTCCTACCGGTATACTGGGGTGCCTTCCAGA-
TTC
TCTGGCAGTGGGTCAGGAACCGACTTTACTCTGACCATCTCTAGTCTGCAGCCCGAGGATTTCGCCACCTA-
CTA
TTGCCAGCAGTACTATATCTACCCTTATACCTTTGGCCAGGGGACAAAAGTGGAGATCAAGAGGACAGTGG-
CCG
CTCCAAGTGTCTTCATTTTTCCCCCTTCCGACGAAGAGCTGAAAAGTGGAACTGCTTCAGTGGTCTGTCTG-
CTG
AACAATTTCTACCCCCGCGAAGCCAAAGTGCAGTGGAAGGTCGATAACGCTCTGCAGAGCGGCAATTCCGA-
GGA
GTCTGTGACAGAACAGGACAGTAAAGATTCAACTTATAGCCTGTCAAGCACACTGGAGCTGTCTAAGGCAG-
ACT
ACGAGAAGCACAAAGTGTATGCCTGCGAAGTCACCCATCAGGGGCTGTCCTCTCCCGTGACAAAGAGCTTT-
AAC
AGAGGAGAGTGT
115 3904 VL
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTL-
T
ISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK
116 3904 VL
GATATCCAGATGACCCAGTCTCCAAGCTCCCTGAGTGCATCAGTGGGGGACCGAGTCACCATCACATGCAAGG-
C
TTCCCAGGATGTGTCTATTGGAGTCGCATGGTACCAGCAGAAGCCAGGCAAAGCACCCAAGCTGCTGATCT-
ACA
GCGCCTCCTACCGGTATACTGGGGTGCCTTCCAGATTCTCTGGCAGTGGGTCAGGAACCGACTTTACTCTG-
ACC
ATCTCTAGTCTGCAGCCCGAGGATTTCGCCACCTACTATTGCCAGCAGTACTATATCTACCCTTATACCTT-
TGG
CCAGGGGACAAAAGTGGAGATCAAG
117 3904 L1 QDVSIG
118 3904 L1 CAGGATGTGTCTATTGGA
119 3904 L3 QQYYIYPYT
120 3904 L3 CAGCAGTACTATATCTACCCTTATACC
121 3904 L2 SAS
122 3904 L2 AGCGCCTCC
123 3904 CL
RTVAAPSVFIFPPSDEELKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSEESVTEQDSKDSTYSLSSTLE-
L
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
124 3904 CL
AGGACAGTGGCCGCTCCAAGTGTCTTCATTTTTCCCCCTTCCGACGAAGAGCTGAAAAGTGGAACTGCTTCAG-
T
GGTCTGTCTGCTGAACAATTTCTACCCCCGCGAAGCCAAAGTGCAGTGGAAGGTCGATAACGCTCTGCAGA-
GCG
GCAATTCCGAGGAGTCTGTGACAGAACAGGACAGTAAAGATTCAACTTATAGCCTGTCAAGCACACTGGAG-
CTG
TCTAAGGCAGACTACGAGAAGCACAAAGTGTATGCCTGCGAAGTCACCCATCAGGGGCTGTCCTCTCCCGT-
GAC
AAAGAGCTTTAACAGAGGAGAGTGT
125 4553 Full
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAD-
T
SKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL-
GCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE-
PKS
CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK-
PRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVS-
LTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ-
KSL
SLSPGK
126 4553 Full
GAAGTCCAGCTGGTCGAAAGCGGAGGAGGACTGGTGCAGCCAGGAGGGTCTCTGCGACTGAGTTGCGCCGCTT-
C
AGGCTTCAACATCAAGGACACCTACATTCACTGGGTGCGCCAGGCTCCTGGAAAAGGCCTGGAGTGGGTGG-
CAC
GAATCTATCCAACTAATGGATACACCCGGTATGCAGACAGCGTGAAGGGCCGGTTCACCATTAGCGCAGAT-
ACA
TCCAAAAACACTGCCTACCTGCAGATGAACAGCCTGCGAGCCGAAGATACTGCTGTGTACTATTGCAGTCG-
GTG
GGGAGGCGACGGCTTCTACGCTATGGATTATTGGGGGCAGGGAACCCTGGTCACAGTGAGCTCCGCATCTA-
CAA
AGGGGCCTAGTGTGTTTCCACTGGCCCCCTCTAGTAAATCCACCTCTGGGGGAACAGCAGCCCTGGGATGT-
CTG
GTGAAGGACTATTTCCCAGAGCCCGTCACTGTGAGTTGGAACTCAGGCGCCCTGACATCCGGGGTCCATAC-
TTT
TCCTGCTGTGCTGCAGTCAAGCGGCCTGTACTCTCTGTCCTCTGTGGTCACCGTGCCAAGTTCAAGCCTGG-
GGA
CTCAGACCTATATCTGCAACGTGAATCACAAGCCAAGCAATACAAAAGTCGACAAGAAAGTGGAACCCAAG-
AGC
TGTGATAAAACACATACTTGCCCCCCTTGTCCTGCACCAGAGCTGCTGGGAGGACCATCCGTGTTCCTGTT-
TCC
ACCCAAGCCTAAAGACACCCTGATGATTTCCAGGACTCCAGAAGTCACCTGCGTGGTCGTGGACGTGTCTC-
ACG
AGGACCCCGAAGTCAAGTTCAACTGGTACGTGGATGGCGTCGAGGTGCATAATGCCAAGACAAAACCCAGG-
GAG
GAACAGTACAACTCAACTTATCGCGTCGTGAGCGTCCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAA-
GGA
GTATAAGTGCAAAGTGAGCAATAAGGCTCTGCCCGCACCTATCGAGAAAACCATTAGCAAGGCCAAAGGGC-
AGC
CTAGAGAACCACAGGTCTACGTGTATCCTCCAAGCAGGGACGAGCTGACCAAGAACCAGGTCTCCCTGACA-
TGT
CTGGTGAAAGGGTTTTACCCCAGTGATATCGCTGTGGAGTGGGAATCAAATGGACAGCCTGAAAACAATTA-
TAA
GACCACACCCCCTGTGCTGGACAGCGATGGCAGCTTCGCTCTGGTCTCCAAGCTGACTGTGGATAAATCTC-
GGT
GGCAGCAGGGCAACGTCTTTAGTTGTTCAGTGATGCATGAGGCACTGCACAATCATTACACCCAGAAGAGC-
CTG
TCCCTGTCTCCCGGCAAA
127 4553 VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAD-
T
SKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
128 4553 VH
GAAGTCCAGCTGGTCGAAAGCGGAGGAGGACTGGTGCAGCCAGGAGGGTCTCTGCGACTGAGTTGCGCCGCTT-
C
AGGCTTCAACATCAAGGACACCTACATTCACTGGGTGCGCCAGGCTCCTGGAAAAGGCCTGGAGTGGGTGG-
CAC
GAATCTATCCAACTAATGGATACACCCGGTATGCAGACAGCGTGAAGGGCCGGTTCACCATTAGCGCAGAT-
ACA
TCCAAAAACACTGCCTACCTGCAGATGAACAGCCTGCGAGCCGAAGATACTGCTGTGTACTATTGCAGTCG-
GTG
GGGAGGCGACGGCTTCTACGCTATGGATTATTGGGGGCAGGGAACCCTGGTCACAGTGAGCTCC
129 4553 H1 GFNIKDTY
130 4553 H1 GGCTTCAACATCAAGGACACCTAC
131 4553 H3 SRWGGDGFYAMDY
132 4553 H3 AGTCGGTGGGGAGGCGACGGCTTCTACGCTATGGATTAT
133 4553 H2 IYPTNGYT
134 4553 H2 ATCTATCCAACTAATGGATACACC
135 4553 CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS-
S
SLGTQTYICNVNHKPSNTKVDKKV
136 4553 CH1
GCATCTACAAAGGGGCCTAGTGTGTTTCCACTGGCCCCCTCTAGTAAATCCACCTCTGGGGGAACAGCAGCCC-
T
GGGATGTCTGGTGAAGGACTATTTCCCAGAGCCCGTCACTGTGAGTTGGAACTCAGGCGCCCTGACATCCG-
GGG
TCCATACTTTTCCTGCTGTGCTGCAGTCAAGCGGCCTGTACTCTCTGTCCTCTGTGGTCACCGTGCCAAGT-
TCA
AGCCTGGGGACTCAGACCTATATCTGCAACGTGAATCACAAGCCAAGCAATACAAAAGTCGACAAGAAAGT-
G
137 4553 CH2
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV-
S
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
138 4553 CH2
GCACCAGAGCTGCTGGGAGGACCATCCGTGTTCCTGTTTCCACCCAAGCCTAAAGACACCCTGATGATTTCCA-
G
GACTCCAGAAGTCACCTGCGTGGTCGTGGACGTGTCTCACGAGGACCCCGAAGTCAAGTTCAACTGGTACG-
TGG
ATGGCGTCGAGGTGCATAATGCCAAGACAAAACCCAGGGAGGAACAGTACAACTCAACTTATCGCGTCGTG-
AGC
GTCCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTATAAGTGCAAAGTGAGCAATAAGGCTCT-
GCC
CGCACCTATCGAGAAAACCATTAGCAAGGCCAAA
139 4553 CH3
GQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVD-
K
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
140 4553 CH3
GGGCAGCCTAGAGAACCACAGGTCTACGTGTATCCTCCAAGCAGGGACGAGCTGACCAAGAACCAGGTCTCCC-
T
GACATGTCTGGTGAAAGGGTTTTACCCCAGTGATATCGCTGTGGAGTGGGAATCAAATGGACAGCCTGAAA-
ACA
ATTATAAGACCACACCCCCTGTGCTGGACAGCGATGGCAGCTTCGCTCTGGTCTCCAAGCTGACTGTGGAT-
AAA
TCTCGGTGGCAGCAGGGCAACGTCTTTAGTTGTTCAGTGATGCATGAGGCACTGCACAATCATTACACCCA-
GAA
GAGCCTGTCCCTGTCTCCCGGC
141 716 Full
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK-
T
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK-
NQV
SLICLVKGFYPSDIAVEWESNGQPENRYMTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN-
HYT
QKSLSLSPGK
142 716 Full
GAGCCCAAGAGCAGCGATAAGACCCACACCTGCCCTCCCTGTCCAGCTCCAGAACTGCTGGGAGGACCTAGCG-
T
GTTCCTGTTTCCCCCTAAGCCAAAAGACACTCTGATGATTTCCAGGACTCCCGAGGTGACCTGCGTGGTGG-
TGG
ACGTGTCTCACGAGGACCCCGAAGTGAAGTTCAACTGGTACGTGGATGGCGTGGAAGTGCATAATGCTAAG-
ACA
AAACCAAGAGAGGAACAGTACAACTCCACTTATCGCGTCGTGAGCGTGCTGACCGTGCTGCACCAGGACTG-
GCT
GAACGGGAAGGAGTATAAGTGCAAAGTCAGTAATAAGGCCCTGCCTGCTCCAATCGAAAAAACCATCTCTA-
AGG
CCAAAGGCCAGCCAAGGGAGCCCCAGGTGTACACACTGCCACCCAGCAGAGACGAACTGACCAAGAACCAG-
GTG
TCCCTGATCTGTCTGGTGAAAGGCTTCTATCCTAGTGATATTGCTGTGGAGTGGGAATCAAATGGACAGCC-
AGA
GAACAGATACATGACCTGGCCTCCAGTGCTGGACAGCGATGGCAGCTTCTTCCTGTATTCCAAGCTGACAG-
TGG
ATAAATCTCGATGGCAGCAGGGGAACGTGTTTAGTTGTTCAGTGATGCATGAAGCCCTGCACAATCATTAC-
ACT
CAGAAGAGCCTGTCCCTGTCTCCCGGCAAA
143 716 CH2
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV-
S
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
144 716 CH2
GCTCCAGAACTGCTGGGAGGACCTAGCGTGTTCCTGTTTCCCCCTAAGCCAAAAGACACTCTGATGATTTCCA-
G
GACTCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCTCACGAGGACCCCGAAGTGAAGTTCAACTGGTACG-
TGG
ATGGCGTGGAAGTGCATAATGCTAAGACAAAACCAAGAGAGGAACAGTACAACTCCACTTATCGCGTCGTG-
AGC
GTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGGAAGGAGTATAAGTGCAAAGTCAGTAATAAGGCCCT-
GCC
TGCTCCAATCGAAAAAACCATCTCTAAGGCCAAA
145 716 CH3
GQPREPQVYTLPPSRDELTKNQVSLICLVKGFYPSDIAVEWESNGQPENRYMTWPPVLDSDGSFFLYSKLTVD-
K
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
146 716 CH3
GGCCAGCCAAGGGAGCCCCAGGTGTACACACTGCCACCCAGCAGAGACGAACTGACCAAGAACCAGGTGTCCC-
T
GATCTGTCTGGTGAAAGGCTTCTATCCTAGTGATATTGCTGTGGAGTGGGAATCAAATGGACAGCCAGAGA-
ACA
GATACATGACCTGGCCTCCAGTGCTGGACAGCGATGGCAGCTTCTTCCTGTATTCCAAGCTGACAGTGGAT-
AAA
TCTCGATGGCAGCAGGGGAACGTGTTTAGTTGTTCAGTGATGCATGAAGCCCTGCACAATCATTACACTCA-
GAA
GAGCCTGTCCCTGTCTCCCGGC
147 719 Full
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTL-
T
ISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGSGGGSGGGSGGGSGGGSGEVQLVESGGGLVQPGGSL-
RLS
CAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTA-
VYY
CSRWGGDGFYAMDYWGQGTLVTVSSAAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV-
TCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE-
KTI
SKAKGQPREPQVYTYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDEDGSFALV-
SKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPCK
148 719 Full
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGG-
C
AAGTCAGGACGTTAACACCGCTGTAGCTTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCT-
ATT
CTGCATCCTTTTTGTACAGTGGGGTCCCATCAAGGTTCAGTGGCAGTCGATCTGGGACAGATTTCACTCTC-
ACC
ATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGCATTACACTACCCCACCCACTTT-
CGG
CCAAGGGACCAAAGTGGAGATCAAAGGTGGTTCTGGTGGTGGTTCTGGTGGTGGTTCTGGTGGTGGTTCTG-
GTG
GTGGTTCTGGTGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCGGGTCCCTGAGACTC-
TCC
TGTGCAGCCTCTGGATTCAACATTAAAGATACTTATATCCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCT-
GGA
GTGGGTCGCACGTATTTATCCCACAAATGGTTACACACGGTATGCGGACTCTGTGAAGGGCCGATTCACCA-
TCT
CCGCAGACACTTCCAAGAACACCGCGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCGTTTAT-
TAC
TGTTCAAGATGGGGCGGAGACGGTTTCTACGCTATGGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTC-
CTC
AGCCGCCGAGCCCAAGAGCAGCGATAAGACCCACACCTGCCCTCCCTGTCCAGCTCCAGAACTGCTGGGAG-
GAC
CTAGCGTGTTCCTGTTTCCCCCTAAGCCAAAAGACACTCTGATGATTTCCAGGACTCCCGAGGTGACCTGC-
GTG
GTGGTGGACGTGTCTCACGAGGACCCCGAAGTGAAGTTCAACTGGTACGTGGATGGCGTGGAAGTGCATAA-
TGC
TAAGACAAAACCAAGAGAGGAACAGTACAACTCCACTTATCGCGTCGTGAGCGTGCTGACCGTGCTGCACC-
AGG
ACTGGCTGAACGGGAAGGAGTATAAGTGCAAAGTCAGTAATAAGGCCCTGCCTGCTCCAATCGAAAAAACC-
ATC
TCTAAGGCCAAAGGCCAGCCAAGGGAGCCCCAGGTGTACACATACCCACCCAGCAGAGACGAACTGACCAA-
GAA
CCAGGTGTCCCTGACATGTCTGGTGAAAGGCTTCTATCCTAGTGATATTGCTGTGGAGTGGGAATCAAATG-
GAC
AGCCAGAGAACAATTACAAGACCACACCTCCAGTGCTGGACGAGGATGGCAGCTTCGCCCTGGTGTCCAAG-
CTG
ACAGTGGATAAATCTCGATGGCAGCAGGGGAACGTGTTTAGTTGTTCAGTGATGCATGAAGCCCTGCACAA-
TCA
TTACACTCAGAAGAGCCTGTCCCTGTCTCCCGGCAAA
149 719 VL DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFS-
GSRSGTDFTLT
ISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK
150 719 VL GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATC-
ACTTGCCGGGC
AAGTCAGGACGTTAACACCGCTGTAGCTTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCT-
ATT
CTGCATCCTTTTTGTACAGTGGGGTCCCATCAAGGTTCAGTGGCAGTCGATCTGGGACAGATTTCACTCTC-
ACC
ATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGCATTACACTACCCCACCCACTTT-
CGG
CCAAGGGACCAAAGTGGAGATCAAA
151 719 L1 QDVNTA
152 719 L1 CAGGACGTTAACACCGCT
153 719 L3 QQHYTTPPT
154 719 L3 CAACAGCATTACACTACCCCACCCACT
155 719 L2 SAS
156 719 L2 TCTGCATCC
157 719 VH EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADS-
VKGRFTISADT
SKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
158 719 VH GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCGGGTCCCTGAGACTCTCC-
TGTGCAGCCTC
TGGATTCAACATTAAAGATACTTATATCCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCG-
CAC
GTATTTATCCCACAAATGGTTACACACGGTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCGCAGAC-
ACT
TCCAAGAACACCGCGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCGTTTATTACTGTTCAAG-
ATG
GGGCGGAGACGGTTTCTACGCTATGGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCA
159 719 H1 GFNIKDTY
160 719 H1 GGATTCAACATTAAAGATACTTAT
161 719 H3 SRWGGDGFYAMDY
162 719 H3 TCAAGATGGGGCGGAGACGGTTTCTACGCTATGGACTAC
163 719 H2 IYPTNGYT
164 719 H2 ATTTATCCCACAAATGGTTACACA
165 719 CH2
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV-
S
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
166 719 CH2
GCTCCAGAACTGCTGGGAGGACCTAGCGTGTTCCTGTTTCCCCCTAAGCCAAAAGACACTCTGATGATTTCCA-
G
GACTCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCTCACGAGGACCCCGAAGTGAAGTTCAACTGGTACG-
TGG
ATGGCGTGGAAGTGCATAATGCTAAGACAAAACCAAGAGAGGAACAGTACAACTCCACTTATCGCGTCGTG-
AGC
GTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGGAAGGAGTATAAGTGCAAAGTCAGTAATAAGGCCCT-
GCC
TGCTCCAATCGAAAAAACCATCTCTAAGGCCAAA
167 719 CH3
GQPREPQVYTYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDEDGSFALVSKLTVD-
K
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
168 719 CH3
GGCCAGCCAAGGGAGCCCCAGGTGTACACATACCCACCCAGCAGAGACGAACTGACCAAGAACCAGGTGTCCC-
T
GACATGTCTGGTGAAAGGCTTCTATCCTAGTGATATTGCTGTGGAGTGGGAATCAAATGGACAGCCAGAGA-
ACA
ATTACAAGACCACACCTCCAGTGCTGGACGAGGATGGCAGCTTCGCCCTGGTGTCCAAGCTGACAGTGGAT-
AAA
TCTCGATGGCAGCAGGGGAACGTGTTTAGTTGTTCAGTGATGCATGAAGCCCTGCACAATCATTACACTCA-
GAA
GAGCCTGTCCCTGTCTCCCGGC
169 720 Full
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTL-
T
ISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGSGGGSGGGSGGGSGGGSGEVQLVESGGGLVQPGGSL-
RLS
CAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTA-
VYY
CSRWGGDGFYAMDYWGQGTLVTVSSAAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV-
TCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE-
KTI
SKAKGQPREPQVYTLPPSRDELTKNQVSLICLVKGFYPSDIAVEWESNGQPENRYMTWPPVLDSDGSFFLY-
SKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPCK
170 720 Full
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGG-
C
AAGTCAGGACGTTAACACCGCTGTAGCTTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCT-
ATT
CTGCATCCTTTTTGTACAGTGGGGTCCCATCAAGGTTCAGTGGCAGTCGATCTGGGACAGATTTCACTCTC-
ACC
ATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGCATTACACTACCCCACCCACTTT-
CGG
CCAAGGGACCAAAGTGGAGATCAAAGGTGGTTCTGGTGGTGGTTCTGGTGGTGGTTCTGGTGGTGGTTCTG-
GTG
GTGGTTCTGGTGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCGGGTCCCTGAGACTC-
TCC
TGTGCAGCCTCTGGATTCAACATTAAAGATACTTATATCCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCT-
GGA
GTGGGTCGCACGTATTTATCCCACAAATGGTTACACACGGTATGCGGACTCTGTGAAGGGCCGATTCACCA-
TCT
CCGCAGACACTTCCAAGAACACCGCGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCGTTTAT-
TAC
TGTTCAAGATGGGGCGGAGACGGTTTCTACGCTATGGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTC-
CTC
AGCCGCCGAGCCCAAGAGCAGCGATAAGACCCACACCTGCCCTCCCTGTCCAGCTCCAGAACTGCTGGGAG-
GAC
CTAGCGTGTTCCTGTTTCCCCCTAAGCCAAAAGACACTCTGATGATTTCCAGGACTCCCGAGGTGACCTGC-
GTG
GTGGTGGACGTGTCTCACGAGGACCCCGAAGTGAAGTTCAACTGGTACGTGGATGGCGTGGAAGTGCATAA-
TGC
TAAGACAAAACCAAGAGAGGAACAGTACAACTCCACTTATCGCGTCGTGAGCGTGCTGACCGTGCTGCACC-
AGG
ACTGGCTGAACGGGAAGGAGTATAAGTGCAAAGTCAGTAATAAGGCCCTGCCTGCTCCAATCGAAAAAACC-
ATC
TCTAAGGCCAAAGGCCAGCCAAGGGAGCCCCAGGTGTACACACTGCCACCCAGCAGAGACGAACTGACCAA-
GAA
CCAGGTGTCCCTGATCTGTCTGGTGAAAGGCTTCTATCCTAGTGATATTGCTGTGGAGTGGGAATCAAATG-
GAC
AGCCAGAGAACAGATACATGACCTGGCCTCCAGTGCTGGACAGCGATGGCAGCTTCTTCCTGTATTCCAAG-
CTG
ACAGTGGATAAATCTCGATGGCAGCAGGGGAACGTGTTTAGTTGTTCAGTGATGCATGAAGCCCTGCACAA-
TCA
TTACACTCAGAAGAGCCTGTCCCTGTCTCCCGGCAAA
171 720 VL DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFS-
GSRSGTDFTLT
ISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK
172 720 VL GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATC-
ACTTGCCGGGC
AAGTCAGGACGTTAACACCGCTGTAGCTTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCT-
ATT
CTGCATCCTTTTTGTACAGTGGGGTCCCATCAAGGTTCAGTGGCAGTCGATCTGGGACAGATTTCACTCTC-
ACC
ATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGCATTACACTACCCCACCCACTTT-
CGG
CCAAGGGACCAAAGTGGAGATCAAA
173 720 L1 QDVNTA
174 720 L1 CAGGACGTTAACACCGCT
175 720 L3 QQHYTTPPT
176 720 L3 CAACAGCATTACACTACCCCACCCACT
177 720 L2 SAS
178 720 L2 TCTGCATCC
179 720 VH EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADS-
VKGRFTISADT
S SKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS
180 720 VH GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCGGGTCCCTGAGACTCTCC-
TGTGCAGCCTC
TGGATTCAACATTAAAGATACTTATATCCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCG-
CAC
GTATTTATCCCACAAATGGTTACACACGGTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCGCAGAC-
ACT
TCCAAGAACACCGCGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCGTTTATTACTGTTCAAG-
ATG
GGGCGGAGACGGTTTCTACGCTATGGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCA
181 720 H1 GFNIKDTY
182 720 H1 GGATTCAACATTAAAGATACTTAT
183 720 H3 SRWGGDGFYAMDY
184 720 H3 TCAAGATGGGGCGGAGACGGTTTCTACGCTATGGACTAC
185 720 H2 IYPTNGYT
186 720 H2 ATTTATCCCACAAATGGTTACACA
187 720 CH2
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV-
S
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
188 720 CH2
GCTCCAGAACTGCTGGGAGGACCTAGCGTGTTCCTGTTTCCCCCTAAGCCAAAAGACACTCTGATGATTTCCA-
G
GACTCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCTCACGAGGACCCCGAAGTGAAGTTCAACTGGTACG-
TGG
ATGGCGTGGAAGTGCATAATGCTAAGACAAAACCAAGAGAGGAACAGTACAACTCCACTTATCGCGTCGTG-
AGC
GTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGGAAGGAGTATAAGTGCAAAGTCAGTAATAAGGCCCT-
GCC
TGCTCCAATCGAAAAAACCATCTCTAAGGCCAAA
189 720 CH3
GQPREPQVYTLPPSRDELTKNQVSLICLVKGFYPSDIAVEWESNGQPENRYMTWPPVLDSDGSFFLYSKLTVD-
K
PG SRWQQGNVFSCSVMHEALHNHYTQKSLSLS
190 720 CH3
GGCCAGCCAAGGGAGCCCCAGGTGTACACACTGCCACCCAGCAGAGACGAACTGACCAAGAACCAGGTGTCCC-
T
GATCTGTCTGGTGAAAGGCTTCTATCCTAGTGATATTGCTGTGGAGTGGGAATCAAATGGACAGCCAGAGA-
ACA
GATACATGACCTGGCCTCCAGTGCTGGACAGCGATGGCAGCTTCTTCCTGTATTCCAAGCTGACAGTGGAT-
AAA
TCTCGATGGCAGCAGGGGAACGTGTTTAGTTGTTCAGTGATGCATGAAGCCCTGCACAATCATTACACTCA-
GAA
GAGCCTGTCCCTGTCTCCCGGC
191 4561 Full
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTL-
T
ISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK-
VQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
192 4561 Full
GATATTCAGATGACCCAGTCCCCTAGCTCCCTGTCCGCTTCTGTGGGCGACAGGGTCACTATCACCTGCCGCG-
C
ATCTCAGGATGTGAACACCGCAGTCGCCTGGTACCAGCAGAAGCCTGGGAAAGCTCCAAAGCTGCTGATCT-
ACA
GTGCATCATTCCTGTATTCAGGAGTGCCCAGCCGGTTTAGCGGCAGCAGATCTGGCACCGACTTCACACTG-
ACT
ATCTCTAGTCTGCAGCCTGAGGATTTTGCCACATACTATTGCCAGCAGCACTATACCACACCCCCTACTTT-
CGG
CCAGGGGACCAAAGTGGAGATCAAGCGAACTGTGGCCGCTCCAAGTGTCTTCATTTTTCCACCCAGCGACG-
AAC
AGCTGAAATCCGGCACAGCTTCTGTGGTCTGTCTGCTGAACAACTTCTACCCCAGAGAGGCCAAAGTGCAG-
TGG
AAGGTCGATAACGCTCTGCAGAGTGGCAACAGCCAGGAGAGCGTGACAGAACAGGACTCCAAAGATTCTAC-
TTA
TAGTCTGTCAAGCACCCTGACACTGAGCAAGGCAGACTACGAAAAGCATAAAGTGTATGCCTGTGAGGTGA-
CCC
ATCAGGGGCTGTCTTCTCCCGTGACCAAGTCTTTCAACCGAGGCGAATGT
193 4561 VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTL-
T
ISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK
194 4561 VL
GATATTCAGATGACCCAGTCCCCTAGCTCCCTGTCCGCTTCTGTGGGCGACAGGGTCACTATCACCTGCCGCG-
C
ATCTCAGGATGTGAACACCGCAGTCGCCTGGTACCAGCAGAAGCCTGGGAAAGCTCCAAAGCTGCTGATCT-
ACA
GTGCATCATTCCTGTATTCAGGAGTGCCCAGCCGGTTTAGCGGCAGCAGATCTGGCACCGACTTCACACTG-
ACT
ATCTCTAGTCTGCAGCCTGAGGATTTTGCCACATACTATTGCCAGCAGCACTATACCACACCCCCTACTTT-
CGG
CCAGGGGACCAAAGTGGAGATCAAG
195 4561 L1 QDVNTA
196 4561 L1 CAGGATGTGAACACCGCA
197 4561 L3 QQHYTTPPT
198 4561 L3 CAGCAGCACTATACCACACCCCCTACT
199 4561 L2 SAS
200 4561 L2 AGTGCATCA
201 4561 CL
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT-
L
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
202 4561 CL
CGAACTGTGGCCGCTCCAAGTGTCTTCATTTTTCCACCCAGCGACGAACAGCTGAAATCCGGCACAGCTTCTG-
T
GGTCTGTCTGCTGAACAACTTCTACCCCAGAGAGGCCAAAGTGCAGTGGAAGGTCGATAACGCTCTGCAGA-
GTG
GCAACAGCCAGGAGAGCGTGACAGAACAGGACTCCAAAGATTCTACTTATAGTCTGTCAAGCACCCTGACA-
CTG
AGCAAGGCAGACTACGAAAAGCATAAAGTGTATGCCTGTGAGGTGACCCATCAGGGGCTGTCTTCTCCCGT-
GAC
CAAGTCTTTCAACCGAGGCGAATGT
203 3041 Full
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGCSIYNQRFKGRFTLSVD-
R
SKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG-
CLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP-
KSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP-
REE
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSL-
LCL
VKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK-
SLS
LSPG
204 3041 Full
GAAGTGCAGCTGGTCGAATCTGGAGGAGGACTGGTGCAGCCAGGAGGGTCCCTGCGCCTGTCTTGCGCCGCTA-
G
TGGCTTCACTTTTACCGACTACACCATGGATTGGGTGCGACAGGCACCTGGAAAGGGCCTGGAGTGGGTCG-
CCG
ATGTGAACCCAAATAGCGGAGGCTCCATCTACAACCAGCGGTTCAAGGGCCGGTTCACCCTGTCAGTGGAC-
CGG
AGCAAAAACACCCTGTATCTGCAGATGAATAGCCTGCGAGCCGAAGATACTGCTGTGTACTATTGCGCCCG-
GAA
TCTGGGGCCCTCCTTCTACTTTGACTATTGGGGGCAGGGAACTCTGGTCACCGTGAGCTCCGCCTCCACCA-
AGG
GACCTTCTGTGTTCCCACTGGCTCCCTCTAGTAAATCCACATCTGGGGGAACTGCAGCCCTGGGCTGTCTG-
GTG
AAGGACTACTTCCCAGAGCCCGTCACAGTGTCTTGGAACAGTGGCGCTCTGACTTCTGGGGTCCACACCTT-
TCC
TGCAGTGCTGCAGTCAAGCGGGCTGTACAGCCTGTCCTCTGTGGTCACCGTGCCAAGTTCAAGCCTGGGAA-
CAC
AGACTTATATCTGCAACGTGAATCACAAGCCATCCAATACAAAAGTCGACAAGAAAGTGGAACCCAAGTCT-
TGT
GATAAAACCCATACATGCCCCCCTTGTCCTGCACCAGAGCTGCTGGGAGGACCAAGCGTGTTCCTGTTTCC-
ACC
CAAGCCTAAAGATACACTGATGATTAGTAGGACCCCAGAAGTCACATGCGTGGTCGTGGACGTGAGCCACG-
AGG
ACCCCGAAGTCAAGTTTAACTGGTACGTGGACGGCGTCGAGGTGCATAATGCCAAGACTAAACCCAGGGAG-
GAA
CAGTACAACAGTACCTATCGCGTCGTGTCAGTCCTGACAGTGCTGCATCAGGATTGGCTGAACGGGAAAGA-
GTA
TAAGTGCAAAGTGAGCAATAAGGCTCTGCCCGCACCTATCGAGAAAACAATTTCCAAGGCAAAAGGACAGC-
CTA
GAGAACCACAGGTGTACGTGCTGCCTCCATCAAGGGATGAGCTGACAAAGAACCAGGTCAGCCTGCTGTGT-
CTG
GTGAAAGGATTCTATCCCTCTGACATTGCTGTGGAGTGGGAAAGTAATGGCCAGCCTGAGAACAATTACCT-
GAC
CTGGCCCCCTGTGCTGGACTCAGATGGCAGCTTCTTTCTGTATAGCAAGCTGACCGTCGACAAATCCCGGT-
GGC
AGCAGGGGAATGTGTTTAGTTGTTCAGTCATGCACGAGGCACTGCACAACCATTACACCCAGAAGTCACTG-
TCA
CTGTCACCAGGG
205 3041 VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGCSIYNQRFKGRFTLSVD-
R
SKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS
206 3041 VH
GAAGTGCAGCTGGTCGAATCTGGAGGAGGACTGGTGCAGCCAGGAGGGTCCCTGCGCCTGTCTTGCGCCGCTA-
G
TGGCTTCACTTTTACCGACTACACCATGGATTGGGTGCGACAGGCACCTGGAAAGGGCCTGGAGTGGGTCG-
CCG
ATGTGAACCCAAATAGCGGAGGCTCCATCTACAACCAGCGGTTCAAGGGCCGGTTCACCCTGTCAGTGGAC-
CGG
AGCAAAAACACCCTGTATCTGCAGATGAATAGCCTGCGAGCCGAAGATACTGCTGTGTACTATTGCGCCCG-
GAA
TCTGGGGCCCTCCTTCTACTTTGACTATTGGGGGCAGGGAACTCTGGTCACCGTGAGCTCC
207 3041 H1 GFTFTDYT
208 3041 H1 GGCTTCACTTTTACCGACTACACC
209 3041 H3 ARNLGPSFYFDY
210 3041 H3 GCCCGGAATCTGGGGCCCTCCTTCTACTTTGACTAT
211 3041 H2 VNPNSGGS
212 3041 H2 GTGAACCCAAATAGCGGAGGCTCC
213 3041 CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS-
S
SLGTQTYICNVNHKPSNTKVDKKV
214 3041 CH1
GCCTCCACCAAGGGACCTTCTGTGTTCCCACTGGCTCCCTCTAGTAAATCCACATCTGGGGGAACTGCAGCCC-
T
GGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTCACAGTGTCTTGGAACAGTGGCGCTCTGACTTCTG-
GGG
TCCACACCTTTCCTGCAGTGCTGCAGTCAAGCGGGCTGTACAGCCTGTCCTCTGTGGTCACCGTGCCAAGT-
TCA
AGCCTGGGAACACAGACTTATATCTGCAACGTGAATCACAAGCCATCCAATACAAAAGTCGACAAGAAAGT-
G
215 3041 CH2
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV-
S
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
216 3041 CH2
GCACCAGAGCTGCTGGGAGGACCAAGCGTGTTCCTGTTTCCACCCAAGCCTAAAGATACACTGATGATTAGTA-
G
GACCCCAGAAGTCACATGCGTGGTCGTGGACGTGAGCCACGAGGACCCCGAAGTCAAGTTTAACTGGTACG-
TGG
ACGGCGTCGAGGTGCATAATGCCAAGACTAAACCCAGGGAGGAACAGTACAACAGTACCTATCGCGTCGTG-
TCA
GTCCTGACAGTGCTGCATCAGGATTGGCTGAACGGGAAAGAGTATAAGTGCAAAGTGAGCAATAAGGCTCT-
GCC
CGCACCTATCGAGAAAACAATTTCCAAGGCAAAA
217 3041 CH3
GQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVD-
K
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
218 3041 CH3
GGACAGCCTAGAGAACCACAGGTGTACGTGCTGCCTCCATCAAGGGATGAGCTGACAAAGAACCAGGTCAGCC-
T
GCTGTGTCTGGTGAAAGGATTCTATCCCTCTGACATTGCTGTGGAGTGGGAAAGTAATGGCCAGCCTGAGA-
ACA
ATTACCTGACCTGGCCCCCTGTGCTGGACTCAGATGGCAGCTTCTTTCTGTATAGCAAGCTGACCGTCGAC-
AAA
TCCCGGTGGCAGCAGGGGAATGTGTTTAGTTGTTCAGTCATGCACGAGGCACTGCACAACCATTACACCCA-
GAA
GTCACTGTCACTGTCACCAGGG
219 3057 Full
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGCSIYNQRFKGRFTLSVD-
R
SKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG-
CLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP-
KSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP-
REE
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSL-
TCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK-
SLS
LSPG
220 3057 Full
GAAGTGCAGCTGGTCGAATCTGGAGGAGGACTGGTGCAGCCAGGAGGGTCCCTGCGCCTGTCTTGCGCCGCTA-
G
TGGCTTCACTTTTACCGACTACACCATGGATTGGGTGCGACAGGCACCTGGAAAGGGCCTGGAGTGGGTCG-
CCG
ATGTGAACCCAAATAGCGGAGGCTCCATCTACAACCAGCGGTTCAAGGGCCGGTTCACCCTGTCAGTGGAC-
CGG
AGCAAAAACACCCTGTATCTGCAGATGAATAGCCTGCGAGCCGAAGATACTGCTGTGTACTATTGCGCCCG-
GAA
TCTGGGGCCCTCCTTCTACTTTGACTATTGGGGGCAGGGAACTCTGGTCACCGTGAGCTCCGCCTCCACCA-
AGG
GACCTTCTGTGTTCCCACTGGCTCCCTCTAGTAAATCCACATCTGGGGGAACTGCAGCCCTGGGCTGTCTG-
GTG
AAGGACTACTTCCCAGAGCCCGTCACAGTGTCTTGGAACAGTGGCGCTCTGACTTCTGGGGTCCACACCTT-
TCC
TGCAGTGCTGCAGTCAAGCGGGCTGTACAGCCTGTCCTCTGTGGTCACCGTGCCAAGTTCAAGCCTGGGAA-
CAC
AGACTTATATCTGCAACGTGAATCACAAGCCATCCAATACAAAAGTCGACAAGAAAGTGGAACCCAAGTCT-
TGT
GATAAAACCCATACATGCCCCCCTTGTCCTGCACCAGAGCTGCTGGGAGGACCAAGCGTGTTCCTGTTTCC-
ACC
CAAGCCTAAAGATACACTGATGATTAGTAGGACCCCAGAAGTCACATGCGTGGTCGTGGACGTGAGCCACG-
AGG
ACCCCGAAGTCAAGTTTAACTGGTACGTGGACGGCGTCGAGGTGCATAATGCCAAGACTAAACCCAGGGAG-
GAA
CAGTACAACAGTACCTATCGCGTCGTGTCAGTCCTGACAGTGCTGCATCAGGATTGGCTGAACGGGAAAGA-
GTA
TAAGTGCAAAGTGAGCAATAAGGCTCTGCCCGCACCTATCGAGAAAACAATTTCCAAGGCAAAAGGACAGC-
CTA
GAGAACCACAGGTGTACGTGTATCCTCCATCAAGGGATGAGCTGACAAAGAACCAGGTCAGCCTGACTTGT-
CTG
GTGAAAGGATTCTATCCCTCTGACATTGCTGTGGAGTGGGAAAGTAATGGCCAGCCTGAGAACAATTACAA-
GAC
CACACCCCCTGTGCTGGACTCAGATGGCAGCTTCGCGCTGGTGAGCAAGCTGACCGTCGACAAATCCCGGT-
GGC
AGCAGGGGAATGTGTTTAGTTGTTCAGTCATGCACGAGGCACTGCACAACCATTACACCCAGAAGTCACTG-
TCA
CTGTCACCAGGG
221 3057 VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGCSIYNQRFKGRFTLSVD-
R
SKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS
222 3057 VH
GAAGTGCAGCTGGTCGAATCTGGAGGAGGACTGGTGCAGCCAGGAGGGTCCCTGCGCCTGTCTTGCGCCGCTA-
G
TGGCTTCACTTTTACCGACTACACCATGGATTGGGTGCGACAGGCACCTGGAAAGGGCCTGGAGTGGGTCG-
CCG
ATGTGAACCCAAATAGCGGAGGCTCCATCTACAACCAGCGGTTCAAGGGCCGGTTCACCCTGTCAGTGGAC-
CGG
AGCAAAAACACCCTGTATCTGCAGATGAATAGCCTGCGAGCCGAAGATACTGCTGTGTACTATTGCGCCCG-
GAA
TCTGGGGCCCTCCTTCTACTTTGACTATTGGGGGCAGGGAACTCTGGTCACCGTGAGCTCC
223 3057 H1 GFTFTDYT
224 3057 H1 GGCTTCACTTTTACCGACTACACC
225 3057 H3 ARNLGPSFYFDY
226 3057 H3 GCCCGGAATCTGGGGCCCTCCTTCTACTTTGACTAT
227 3057 H2 VNPNSGGS
228 3057 H2 GTGAACCCAAATAGCGGAGGCTCC
229 3057 CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS-
S
SLGTQTYICNVNHKPSNTKVDKKV
230 3057 CH1
GCCTCCACCAAGGGACCTTCTGTGTTCCCACTGGCTCCCTCTAGTAAATCCACATCTGGGGGAACTGCAGCCC-
T
GGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTCACAGTGTCTTGGAACAGTGGCGCTCTGACTTCTG-
GGG
TCCACACCTTTCCTGCAGTGCTGCAGTCAAGCGGGCTGTACAGCCTGTCCTCTGTGGTCACCGTGCCAAGT-
TCA
AGCCTGGGAACACAGACTTATATCTGCAACGTGAATCACAAGCCATCCAATACAAAAGTCGACAAGAAAGT-
G
231 3057 CH2
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV-
S
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
232 3057 CH2
GCACCAGAGCTGCTGGGAGGACCAAGCGTGTTCCTGTTTCCACCCAAGCCTAAAGATACACTGATGATTAGTA-
G
GACCCCAGAAGTCACATGCGTGGTCGTGGACGTGAGCCACGAGGACCCCGAAGTCAAGTTTAACTGGTACG-
TGG
ACGGCGTCGAGGTGCATAATGCCAAGACTAAACCCAGGGAGGAACAGTACAACAGTACCTATCGCGTCGTG-
TCA
GTCCTGACAGTGCTGCATCAGGATTGGCTGAACGGGAAAGAGTATAAGTGCAAAGTGAGCAATAAGGCTCT-
GCC
CGCACCTATCGAGAAAACAATTTCCAAGGCAAAA
233 3057 CH3
GQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVD-
K
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
234 3057 CH3
GGACAGCCTAGAGAACCACAGGTGTACGTGTATCCTCCATCAAGGGATGAGCTGACAAAGAACCAGGTCAGCC-
T
GACTTGTCTGGTGAAAGGATTCTATCCCTCTGACATTGCTGTGGAGTGGGAAAGTAATGGCCAGCCTGAGA-
ACA
ATTACAAGACCACACCCCCTGTGCTGGACTCAGATGGCAGCTTCGCGCTGGTGAGCAAGCTGACCGTCGAC-
AAA
TCCCGGTGGCAGCAGGGGAATGTGTTTAGTTGTTCAGTCATGCACGAGGCACTGCACAACCATTACACCCA-
GAA
GTCACTGTCACTGTCACCAGGG
235 1011 Full
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAD-
T
SKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL-
GCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE-
PKS
CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK-
PRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVS-
LTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ-
KSL
SLSPGK
236 1011 Full
GAGGTGCAGCTGGTGGAAAGCGGAGGAGGACTGGTGCAGCCAGGAGGATCTCTGCGACTGAGTTGCGCCGCTT-
C
AGGATTCAACATCAAGGACACCTACATTCACTGGGTGCGACAGGCTCCAGGAAAAGGACTGGAGTGGGTGG-
CTC
GAATCTATCCCACTAATGGATACACCCGGTATGCCGACTCCGTGAAGGGGAGGTTTACTATTAGCGCCGAT-
ACA
TCCAAAAACACTGCTTACCTGCAGATGAACAGCCTGCGAGCCGAAGATACCGCTGTGTACTATTGCAGTCG-
ATG
GGGAGGAGACGGATTCTACGCTATGGATTATTGGGGACAGGGGACCCTGGTGACAGTGAGCTCCGCCTCTA-
CCA
AGGGCCCCAGTGTGTTTCCCCTGGCTCCTTCTAGTAAATCCACCTCTGGAGGGACAGCCGCTCTGGGATGT-
CTG
GTGAAGGACTATTTCCCCGAGCCTGTGACCGTGAGTTGGAACTCAGGCGCCCTGACAAGCGGAGTGCACAC-
TTT
TCCTGCTGTGCTGCAGTCAAGCGGGCTGTACTCCCTGTCCTCTGTGGTGACAGTGCCAAGTTCAAGCCTGG-
GCA
CACAGACTTATATCTGCAACGTGAATCATAAGCCCTCAAATACAAAAGTGGACAAGAAAGTGGAGCCCAAG-
AGC
TGTGATAAGACCCACACCTGCCCTCCCTGTCCAGCTCCAGAACTGCTGGGAGGACCTAGCGTGTTCCTGTT-
TCC
CCCTAAGCCAAAAGACACTCTGATGATTTCCAGGACTCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCTC-
ACG
AGGACCCCGAAGTGAAGTTCAACTGGTACGTGGATGGCGTGGAAGTGCATAATGCTAAGACAAAACCAAGA-
GAG
GAACAGTACAACTCCACTTATCGCGTCGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGGAA-
GGA
GTATAAGTGCAAAGTCAGTAATAAGGCCCTGCCTGCTCCAATCGAAAAAACCATCTCTAAGGCCAAAGGCC-
AGC
CAAGGGAGCCCCAGGTGTACGTGTACCCACCCAGCAGAGACGAACTGACCAAGAACCAGGTGTCCCTGACA-
TGT
CTGGTGAAAGGCTTCTATCCTAGTGATATTGCTGTGGAGTGGGAATCAAATGGACAGCCAGAGAACAATTA-
CAA
GACCACACCTCCAGTGCTGGACAGCGATGGCAGCTTCGCCCTGGTGTCCAAGCTGACAGTGGATAAATCTC-
GAT
GGCAGCAGGGGAACGTGTTTAGTTGTTCAGTGATGCATGAAGCCCTGCACAATCATTACACTCAGAAGAGC-
CTG
TCCCTGTCTCCCGGCAAA
237 1011 VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAD-
T
SKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
238 1011 VH
GAGGTGCAGCTGGTGGAAAGCGGAGGAGGACTGGTGCAGCCAGGAGGATCTCTGCGACTGAGTTGCGCCGCTT-
C
AGGATTCAACATCAAGGACACCTACATTCACTGGGTGCGACAGGCTCCAGGAAAAGGACTGGAGTGGGTGG-
CTC
GAATCTATCCCACTAATGGATACACCCGGTATGCCGACTCCGTGAAGGGGAGGTTTACTATTAGCGCCGAT-
ACA
TCCAAAAACACTGCTTACCTGCAGATGAACAGCCTGCGAGCCGAAGATACCGCTGTGTACTATTGCAGTCG-
ATG
GGGAGGAGACGGATTCTACGCTATGGATTATTGGGGACAGGGGACCCTGGTGACAGTGAGCTCC
239 1011 H1 GFNIKDTY
240 1011 H1 GGATTCAACATCAAGGACACCTAC
241 1011 H3 SRWGGDGFYAMDY
242 1011 H3 AGTCGATGGGGAGGAGACGGATTCTACGCTATGGATTAT
243 1011 H2 IYPTNGYT
244 1011 H2 ATCTATCCCACTAATGGATACACC
245 1011 CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS-
S
SLGTQTYICNVNHKPSNTKVDKKV
246 1011 CH1
GCCTCTACCAAGGGCCCCAGTGTGTTTCCCCTGGCTCCTTCTAGTAAATCCACCTCTGGAGGGACAGCCGCTC-
T
GGGATGTCTGGTGAAGGACTATTTCCCCGAGCCTGTGACCGTGAGTTGGAACTCAGGCGCCCTGACAAGCG-
GAG
TGCACACTTTTCCTGCTGTGCTGCAGTCAAGCGGGCTGTACTCCCTGTCCTCTGTGGTGACAGTGCCAAGT-
TCA
AGCCTGGGCACACAGACTTATATCTGCAACGTGAATCATAAGCCCTCAAATACAAAAGTGGACAAGAAAGT-
G
247 1011 CH2
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV-
S
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
248 1011 CH2
GCTCCAGAACTGCTGGGAGGACCTAGCGTGTTCCTGTTTCCCCCTAAGCCAAAAGACACTCTGATGATTTCCA-
G
GACTCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCTCACGAGGACCCCGAAGTGAAGTTCAACTGGTACG-
TGG
ATGGCGTGGAAGTGCATAATGCTAAGACAAAACCAAGAGAGGAACAGTACAACTCCACTTATCGCGTCGTG-
AGC
GTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGGAAGGAGTATAAGTGCAAAGTCAGTAATAAGGCCCT-
GCC
TGCTCCAATCGAAAAAACCATCTCTAAGGCCAAA
249 1011 CH3
GQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVD-
K
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
250 1011 CH3
GGCCAGCCAAGGGAGCCCCAGGTGTACGTGTACCCACCCAGCAGAGACGAACTGACCAAGAACCAGGTGTCCC-
T
GACATGTCTGGTGAAAGGCTTCTATCCTAGTGATATTGCTGTGGAGTGGGAATCAAATGGACAGCCAGAGA-
ACA
ATTACAAGACCACACCTCCAGTGCTGGACAGCGATGGCAGCTTCGCCCTGGTGTCCAAGCTGACAGTGGAT-
AAA
TCTCGATGGCAGCAGGGGAACGTGTTTAGTTGTTCAGTGATGCATGAAGCCCTGCACAATCATTACACTCA-
GAA
GAGCCTGTCCCTGTCTCCCGGC
251 4560 Full
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK-
T
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTK-
NQV
SLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN-
HYT
QKSLSLSPGK
252 4560 Full
GAACCTAAAAGCAGCGACAAGACCCACACATGCCCCCCTTGTCCAGCTCCAGAACTGCTGGGAGGACCAAGCG-
T
GTTCCTGTTTCCACCCAAGCCCAAAGATACACTGATGATCAGCCGAACTCCCGAGGTCACCTGCGTGGTCG-
TGG
ACGTGTCCCACGAGGACCCCGAAGTCAAGTTCAACTGGTACGTGGACGGCGTCGAAGTGCATAATGCAAAG-
ACT
AAACCACGGGAGGAACAGTACAACTCTACATATAGAGTCGTGAGTGTCCTGACTGTGCTGCATCAGGATTG-
GCT
GAACGGCAAAGAGTATAAGTGCAAAGTGTCTAATAAGGCCCTGCCTGCTCCAATCGAGAAAACTATTAGTA-
AGG
CAAAAGGGCAGCCCAGGGAACCTCAGGTCTACGTGCTGCCTCCAAGTCGCGACGAGCTGACCAAGAACCAG-
GTC
TCACTGCTGTGTCTGGTGAAAGGATTCTATCCTTCCGATATTGCCGTGGAGTGGGAATCTAATGGCCAGCC-
AGA
GAACAATTACCTGACCTGGCCCCCTGTGCTGGACAGCGATGGGTCCTTCTTTCTGTATTCAAAGCTGACAG-
TGG
ACAAAAGCAGATGGCAGCAGGGAAACGTCTTTAGCTGTTCCGTGATGCACGAAGCCCTGCACAATCATTAC-
ACC
CAGAAGTCTCTGAGTCTGTCACCTGGCAAA
253 4560 CH2
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV-
S
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
254 4560 CH2
GCTCCAGAACTGCTGGGAGGACCAAGCGTGTTCCTGTTTCCACCCAAGCCCAAAGATACACTGATGATCAGCC-
G
AACTCCCGAGGTCACCTGCGTGGTCGTGGACGTGTCCCACGAGGACCCCGAAGTCAAGTTCAACTGGTACG-
TGG
ACGGCGTCGAAGTGCATAATGCAAAGACTAAACCACGGGAGGAACAGTACAACTCTACATATAGAGTCGTG-
AGT
GTCCTGACTGTGCTGCATCAGGATTGGCTGAACGGCAAAGAGTATAAGTGCAAAGTGTCTAATAAGGCCCT-
GCC
TGCTCCAATCGAGAAAACTATTAGTAAGGCAAAA
255 4560 CH3
GQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVD-
K
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
256 4560 CH3
GGGCAGCCCAGGGAACCTCAGGTCTACGTGCTGCCTCCAAGTCGCGACGAGCTGACCAAGAACCAGGTCTCAC-
T
GCTGTGTCTGGTGAAAGGATTCTATCCTTCCGATATTGCCGTGGAGTGGGAATCTAATGGCCAGCCAGAGA-
ACA
ATTACCTGACCTGGCCCCCTGTGCTGGACAGCGATGGGTCCTTCTTTCTGTATTCAAAGCTGACAGTGGAC-
AAA
AGCAGATGGCAGCAGGGAAACGTCTTTAGCTGTTCCGTGATGCACGAAGCCCTGCACAATCATTACACCCA-
GAA
GTCTCTGAGTCTGTCACCTGGC
257 3317 Full
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTL-
T
ISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCA-
ASG
FTFTDYTMDWVRQAPGKGLEWVADVNPNSGCSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCA-
RNL
GPSFYFDYWGQGTLVTVSSAAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD-
VSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA-
KGQ
PREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVD-
KSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPCK
258 3317 Full
GACATTCAGATGACCCAGAGCCCTAGCTCCCTGAGTGCCTCAGTCGGGGACAGGGTGACTATCACCTGCAAGG-
C
TTCACAGGATGTCAGCATTGGCGTGGCATGGTACCAGCAGAAGCCAGGGAAAGCACCCAAGCTGCTGATCT-
ATA
GCGCCTCCTACAGGTATACAGGCGTGCCATCCCGCTTCTCTGGCAGTGGGTCAGGAACTGACTTTACACTG-
ACT
ATTTCTAGTCTGCAGCCCGAAGATTTCGCCACATACTATTGCCAGCAGTACTATATCTACCCTTATACTTT-
TGG
CCAGGGGACCAAAGTGGAGATTAAGGGCGGAGGAGGCTCCGGAGGAGGAGGGTCTGGAGGAGGAGGAAGTG-
AGG
TCCAGCTGGTGGAATCTGGAGGAGGACTGGTGCAGCCAGGAGGGTCCCTGAGGCTGTCTTGTGCCGCTAGT-
GGC
TTCACCTTTACAGACTACACAATGGATTGGGTGCGCCAGGCACCAGGAAAGGGACTGGAATGGGTCGCTGA-
TGT
GAACCCTAATAGCGGAGGCTCCATCTACAACCAGCGGTTCAAAGGACGGTTCACCCTGTCAGTGGACCGGA-
GCA
AGAACACCCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGATACTGCTGTGTACTATTGCGCCAGGAAT-
CTG
GGCCCAAGCTTCTACTTTGACTATTGGGGGCAGGGAACACTGGTCACTGTGTCAAGCGCAGCCGAACCCAA-
ATC
CTCTGATAAGACTCACACCTGCCCACCTTGTCCAGCTCCAGAGCTGCTGGGAGGACCTAGCGTGTTCCTGT-
TTC
CACCCAAGCCAAAAGACACTCTGATGATTTCTAGAACCCCTGAAGTGACATGTGTGGTCGTGGACGTCAGT-
CAC
GAGGACCCCGAAGTCAAATTCAACTGGTACGTGGATGGCGTCGAGGTGCATAATGCCAAGACCAAACCCCG-
AGA
GGAACAGTACAACTCAACCTATCGGGTCGTGAGCGTCCTGACAGTGCTGCATCAGGACTGGCTGAACGGCA-
AGG
AGTATAAGTGCAAAGTGAGCAACAAGGCTCTGCCTGCACCAATCGAGAAGACCATTTCCAAGGCTAAAGGG-
CAG
CCCCGCGAACCTCAGGTCTACGTGTATCCTCCAAGCCGAGATGAGCTGACAAAAAACCAGGTCTCCCTGAC-
TTG
TCTGGTGAAGGGATTTTACCCAAGTGACATCGCAGTGGAGTGGGAATCAAATGGCCAGCCCGAAAACAATT-
ATA
AGACCACACCCCCTGTGCTGGACTCTGATGGGAGTTTCGCACTGGTCTCCAAACTGACCGTGGACAAGTCT-
CGG
TGGCAGCAGGGAAACGTCTTTAGCTGTTCCGTGATGCACGAGGCCCTGCACAATCATTACACACAGAAATC-
TCT
GAGTCTGTCACCTGGCAAG
259 3317 VL
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTL-
T
ISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK
260 3317 VL
GACATTCAGATGACCCAGAGCCCTAGCTCCCTGAGTGCCTCAGTCGGGGACAGGGTGACTATCACCTGCAAGG-
C
TTCACAGGATGTCAGCATTGGCGTGGCATGGTACCAGCAGAAGCCAGGGAAAGCACCCAAGCTGCTGATCT-
ATA
GCGCCTCCTACAGGTATACAGGCGTGCCATCCCGCTTCTCTGGCAGTGGGTCAGGAACTGACTTTACACTG-
ACT
ATTTCTAGTCTGCAGCCCGAAGATTTCGCCACATACTATTGCCAGCAGTACTATATCTACCCTTATACTTT-
TGG
CCAGGGGACCAAAGTGGAGATTAAG
261 3317 L1 QDVSIG
262 3317 L1 CAGGATGTCAGCATTGGC
263 3317 L3 QQYYIYPYT
264 3317 L3 CAGCAGTACTATATCTACCCTTATACT
265 3317 L2 SAS
266 3317 L2 AGCGCCTCC
267 3317 VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGCSIYNQRFKGRFTLSVD-
R
SKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS
268 3317 VH
GAGGTCCAGCTGGTGGAATCTGGAGGAGGACTGGTGCAGCCAGGAGGGTCCCTGAGGCTGTCTTGTGCCGCTA-
G
TGGCTTCACCTTTACAGACTACACAATGGATTGGGTGCGCCAGGCACCAGGAAAGGGACTGGAATGGGTCG-
CTG
ATGTGAACCCTAATAGCGGAGGCTCCATCTACAACCAGCGGTTCAAAGGACGGTTCACCCTGTCAGTGGAC-
CGG
AGCAAGAACACCCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGATACTGCTGTGTACTATTGCGCCAG-
GAA
TCTGGGCCCAAGCTTCTACTTTGACTATTGGGGGCAGGGAACACTGGTCACTGTGTCAAGC
269 3317 H1 GFTFTDYT
270 3317 H1 GGCTTCACCTTTACAGACTACACA
271 3317 H3 ARNLGPSFYFDY
272 3317 H3 GCCAGGAATCTGGGCCCAAGCTTCTACTTTGACTAT
273 3317 H2 VNPNSGGS
274 3317 H2 GTGAACCCTAATAGCGGAGGCTCC
275 3317 CH2
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV-
S
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
276 3317 CH2
GCTCCAGAGCTGCTGGGAGGACCTAGCGTGTTCCTGTTTCCACCCAAGCCAAAAGACACTCTGATGATTTCTA-
G
AACCCCTGAAGTGACATGTGTGGTCGTGGACGTCAGTCACGAGGACCCCGAAGTCAAATTCAACTGGTACG-
TGG
ATGGCGTCGAGGTGCATAATGCCAAGACCAAACCCCGAGAGGAACAGTACAACTCAACCTATCGGGTCGTG-
AGC
GTCCTGACAGTGCTGCATCAGGACTGGCTGAACGGCAAGGAGTATAAGTGCAAAGTGAGCAACAAGGCTCT-
GCC
TGCACCAATCGAGAAGACCATTTCCAAGGCTAAA
277 3317 CH3
GQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVD-
K
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
278 3317 CH3
GGGCAGCCCCGCGAACCTCAGGTCTACGTGTATCCTCCAAGCCGAGATGAGCTGACAAAAAACCAGGTCTCCC-
T
GACTTGTCTGGTGAAGGGATTTTACCCAAGTGACATCGCAGTGGAGTGGGAATCAAATGGCCAGCCCGAAA-
ACA
ATTATAAGACCACACCCCCTGTGCTGGACTCTGATGGGAGTTTCGCACTGGTCTCCAAACTGACCGTGGAC-
AAG
TCTCGGTGGCAGCAGGGAAACGTCTTTAGCTGTTCCGTGATGCACGAGGCCCTGCACAATCATTACACACA-
GAA
ATCTCTGAGTCTGTCACCTGGC
279 1015 Full
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAD-
T
SKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL-
GCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE-
PKS
CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK-
PRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVS-
LLC
LVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ-
KSL
SLSPGK
280 1015 Full
GAGGTGCAGCTGGTGGAAAGCGGAGGAGGACTGGTGCAGCCAGGAGGATCTCTGCGACTGAGTTGCGCCGCTT-
C
AGGATTCAACATCAAGGACACCTACATTCACTGGGTGCGACAGGCTCCAGGAAAAGGACTGGAGTGGGTGG-
CTC
GAATCTATCCCACTAATGGATACACCCGGTATGCCGACTCCGTGAAGGGGAGGTTTACTATTAGCGCCGAT-
ACA
TCCAAAAACACTGCTTACCTGCAGATGAACAGCCTGCGAGCCGAAGATACCGCTGTGTACTATTGCAGTCG-
ATG
GGGAGGAGACGGATTCTACGCTATGGATTATTGGGGACAGGGGACCCTGGTGACAGTGAGCTCCGCCTCTA-
CCA
AGGGCCCCAGTGTGTTTCCCCTGGCTCCTTCTAGTAAATCCACCTCTGGAGGGACAGCCGCTCTGGGATGT-
CTG
GTGAAGGACTATTTCCCCGAGCCTGTGACCGTGAGTTGGAACTCAGGCGCCCTGACAAGCGGAGTGCACAC-
TTT
TCCTGCTGTGCTGCAGTCAAGCGGGCTGTACTCCCTGTCCTCTGTGGTGACAGTGCCAAGTTCAAGCCTGG-
GCA
CACAGACTTATATCTGCAACGTGAATCATAAGCCCTCAAATACAAAAGTGGACAAGAAAGTGGAGCCCAAG-
AGC
TGTGATAAGACCCACACCTGCCCTCCCTGTCCAGCTCCAGAACTGCTGGGAGGACCTAGCGTGTTCCTGTT-
TCC
CCCTAAGCCAAAAGACACTCTGATGATTTCCAGGACTCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCTC-
ACG
AGGACCCCGAAGTGAAGTTCAACTGGTACGTGGATGGCGTGGAAGTGCATAATGCTAAGACAAAACCAAGA-
GAG
GAACAGTACAACTCCACTTATCGCGTCGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGGAA-
GGA
GTATAAGTGCAAAGTCAGTAATAAGGCCCTGCCTGCTCCAATCGAAAAAACCATCTCTAAGGCCAAAGGCC-
AGC
CAAGGGAGCCCCAGGTGTACGTGCTGCCACCCAGCAGAGACGAACTGACCAAGAACCAGGTGTCCCTGCTG-
TGT
CTGGTGAAAGGCTTCTATCCTAGTGATATTGCTGTGGAGTGGGAATCAAATGGACAGCCAGAGAACAATTA-
CCT
GACCTGGCCTCCAGTGCTGGACAGCGATGGCAGCTTCTTCCTGTATTCCAAGCTGACAGTGGATAAATCTC-
GAT
GGCAGCAGGGGAACGTGTTTAGTTGTTCAGTGATGCATGAAGCCCTGCACAATCATTACACTCAGAAGAGC-
CTG
TCCCTGTCTCCCGGCAAA
281 1015 VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAD-
T
SKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
282 1015 VH
GAGGTGCAGCTGGTGGAAAGCGGAGGAGGACTGGTGCAGCCAGGAGGATCTCTGCGACTGAGTTGCGCCGCTT-
C
AGGATTCAACATCAAGGACACCTACATTCACTGGGTGCGACAGGCTCCAGGAAAAGGACTGGAGTGGGTGG-
CTC
GAATCTATCCCACTAATGGATACACCCGGTATGCCGACTCCGTGAAGGGGAGGTTTACTATTAGCGCCGAT-
ACA
TCCAAAAACACTGCTTACCTGCAGATGAACAGCCTGCGAGCCGAAGATACCGCTGTGTACTATTGCAGTCG-
ATG
GGGAGGAGACGGATTCTACGCTATGGATTATTGGGGACAGGGGACCCTGGTGACAGTGAGCTCC
283 1015 H1 GFNIKDTY
284 1015 H1 GGATTCAACATCAAGGACACCTAC
285 1015 H3 SRWGGDGFYAMDY
286 1015 H3 AGTCGATGGGGAGGAGACGGATTCTACGCTATGGATTAT
287 1015 H2 IYPTNGYT
288 1015 H2 ATCTATCCCACTAATGGATACACC
289 1015 CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS-
S
SLGTQTYICNVNHKPSNTKVDKKV
290 1015 CH1
GCCTCTACCAAGGGCCCCAGTGTGTTTCCCCTGGCTCCTTCTAGTAAATCCACCTCTGGAGGGACAGCCGCTC-
T
GGGATGTCTGGTGAAGGACTATTTCCCCGAGCCTGTGACCGTGAGTTGGAACTCAGGCGCCCTGACAAGCG-
GAG
TGCACACTTTTCCTGCTGTGCTGCAGTCAAGCGGGCTGTACTCCCTGTCCTCTGTGGTGACAGTGCCAAGT-
TCA
AGCCTGGGCACACAGACTTATATCTGCAACGTGAATCATAAGCCCTCAAATACAAAAGTGGACAAGAAAGT-
G
291 1015 CH2
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV-
S
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
292 1015 CH2
GCTCCAGAACTGCTGGGAGGACCTAGCGTGTTCCTGTTTCCCCCTAAGCCAAAAGACACTCTGATGATTTCCA-
G
GACTCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCTCACGAGGACCCCGAAGTGAAGTTCAACTGGTACG-
TGG
ATGGCGTGGAAGTGCATAATGCTAAGACAAAACCAAGAGAGGAACAGTACAACTCCACTTATCGCGTCGTG-
AGC
GTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGGAAGGAGTATAAGTGCAAAGTCAGTAATAAGGCCCT-
GCC
TGCTCCAATCGAAAAAACCATCTCTAAGGCCAAA
293 1015 CH3
GQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVD-
K
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
294 1015 CH3
GGCCAGCCAAGGGAGCCCCAGGTGTACGTGCTGCCACCCAGCAGAGACGAACTGACCAAGAACCAGGTGTCCC-
T
GCTGTGTCTGGTGAAAGGCTTCTATCCTAGTGATATTGCTGTGGAGTGGGAATCAAATGGACAGCCAGAGA-
ACA
ATTACCTGACCTGGCCTCCAGTGCTGGACAGCGATGGCAGCTTCTTCCTGTATTCCAAGCTGACAGTGGAT-
AAA
TCTCGATGGCAGCAGGGGAACGTGTTTAGTTGTTCAGTGATGCATGAAGCCCTGCACAATCATTACACTCA-
GAA
GAGCCTGTCCCTGTCTCCCGGC
295 5244 Full
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTL-
T
ISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGSGGGSGGGSGGGSGGGSGEVQLVESGGGLVQPGGSL-
RLS
CAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTA-
VYY
CSRWGGDGFYAMDYWGQGTLVTVSSAAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV-
TCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE-
KTI
SKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLY-
SKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
296 5244 Full
GACATTCAGATGACACAGAGCCCCAGCTCCCTGAGTGCTTCAGTCGGCGACAGGGTGACTATCACCTGCCGCG-
C
ATCCCAGGATGTCAACACCGCTGTGGCATGGTACCAGCAGAAGCCTGGAAAAGCCCCAAAGCTGCTGATCT-
ACA
GCGCTTCCTTCCTGTATTCTGGCGTGCCAAGTCGGTTTTCTGGAAGTAGATCAGGCACTGACTTCACACTG-
ACT
ATCTCTAGTCTGCAGCCCGAAGATTTTGCCACCTACTATTGCCAGCAGCACTATACCACACCCCCTACATT-
CGG
ACAGGGCACTAAAGTGGAGATTAAGGGCGGGTCAGGCGGAGGGAGCGGAGGAGGGTCCGGAGGAGGGTCTG-
GAG
GAGGGAGTGGAGAGGTCCAGCTGGTGGAATCTGGAGGAGGACTGGTGCAGCCTGGAGGCTCACTGCGACTG-
AGC
TGTGCCGCTTCCGGCTTTAACATCAAAGACACATACATTCATTGGGTCAGGCAGGCACCAGGGAAGGGACT-
GGA
ATGGGTGGCCCGCATCTATCCCACAAATGGGTACACTCGATATGCCGACAGCGTGAAAGGACGGTTTACCA-
TTT
CTGCTGATACCAGTAAGAACACAGCATACCTGCAGATGAACAGCCTGCGCGCAGAGGATACAGCCGTGTAC-
TAT
TGCAGTCGATGGGGGGGAGACGGCTTCTACGCCATGGATTATTGGGGCCAGGGGACTCTGGTCACCGTGTC-
AAG
CGCAGCCGAACCTAAATCCTCTGACAAGACCCACACATGCCCACCCTGTCCTGCTCCAGAGCTGCTGGGAG-
GAC
CATCCGTGTTCCTGTTTCCTCCAAAGCCTAAAGATACACTGATGATTAGCCGCACTCCCGAAGTCACCTGT-
GTG
GTCGTGGACGTGTCCCACGAGGACCCCGAAGTCAAGTTCAACTGGTACGTGGACGGCGTCGAGGTGCATAA-
TGC
CAAGACTAAACCAAGAGAGGAACAGTACAATTCAACCTATAGGGTCGTGAGCGTCCTGACAGTGCTGCATC-
AGG
ATTGGCTGAACGGCAAGGAGTATAAGTGCAAAGTGTCTAACAAGGCCCTGCCCGCTCCTATCGAGAAGACT-
ATT
AGCAAGGCAAAAGGGCAGCCACGGGAACCCCAGGTCTACGTGCTGCCCCCTAGCAGAGACGAGCTGACCAA-
AAA
CCAGGTCTCCCTGCTGTGTCTGGTGAAGGGCTTTTATCCTAGTGATATCGCTGTGGAGTGGGAATCAAATG-
GGC
AGCCAGAAAACAATTACCTGACATGGCCACCCGTGCTGGACAGCGATGGGTCCTTCTTTCTGTATTCCAAA-
CTG
ACTGTGGACAAGTCTAGATGGCAGCAGGGAAACGTCTTCAGCTGTTCCGTGATGCACGAGGCCCTGCACAA-
TCA
TTACACCCAGAAGTCTCTGAGTCTGTCACCCGGC
297 5244 VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTL-
T
ISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK
298 5244 VL
GACATTCAGATGACACAGAGCCCCAGCTCCCTGAGTGCTTCAGTCGGCGACAGGGTGACTATCACCTGCCGCG-
C
ATCCCAGGATGTCAACACCGCTGTGGCATGGTACCAGCAGAAGCCTGGAAAAGCCCCAAAGCTGCTGATCT-
ACA
GCGCTTCCTTCCTGTATTCTGGCGTGCCAAGTCGGTTTTCTGGAAGTAGATCAGGCACTGACTTCACACTG-
ACT
ATCTCTAGTCTGCAGCCCGAAGATTTTGCCACCTACTATTGCCAGCAGCACTATACCACACCCCCTACATT-
CGG
ACAGGGCACTAAAGTGGAGATTAAG
299 5244 L1 QDVNTA
300 5244 L1 CAGGATGTCAACACCGCT
301 5244 L3 QQHYTTPPT
302 5244 L3 CAGCAGCACTATACCACACCCCCTACA
303 5244 L2 SAS
304 5244 L2 AGCGCTTCC
305 5244 VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAD-
T
SKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
306 5244 VH
GAGGTCCAGCTGGTGGAATCTGGAGGAGGACTGGTGCAGCCTGGAGGCTCACTGCGACTGAGCTGTGCCGCTT-
C
CGGCTTTAACATCAAAGACACATACATTCATTGGGTCAGGCAGGCACCAGGGAAGGGACTGGAATGGGTGG-
CCC
GCATCTATCCCACAAATGGGTACACTCGATATGCCGACAGCGTGAAAGGACGGTTTACCATTTCTGCTGAT-
ACC
AGTAAGAACACAGCATACCTGCAGATGAACAGCCTGCGCGCAGAGGATACAGCCGTGTACTATTGCAGTCG-
ATG
GGGGGGAGACGGCTTCTACGCCATGGATTATTGGGGCCAGGGGACTCTGGTCACCGTGTCAAGC
307 5244 H1 GFNIKDTY
308 5244 H1 GGCTTTAACATCAAAGACACATAC
309 5244 H3 SRWGGDGFYAMDY
310 5244 H3 AGTCGATGGGGGGGAGACGGCTTCTACGCCATGGATTAT
311 5244 H2 IYPTNGYT
312 5244 H2 ATCTATCCCACAAATGGGTACACT
313 5244 CH2
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV-
S
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
314 5244 CH2
GCTCCAGAGCTGCTGGGAGGACCATCCGTGTTCCTGTTTCCTCCAAAGCCTAAAGATACACTGATGATTAGCC-
G
CACTCCCGAAGTCACCTGTGTGGTCGTGGACGTGTCCCACGAGGACCCCGAAGTCAAGTTCAACTGGTACG-
TGG
ACGGCGTCGAGGTGCATAATGCCAAGACTAAACCAAGAGAGGAACAGTACAATTCAACCTATAGGGTCGTG-
AGC
GTCCTGACAGTGCTGCATCAGGATTGGCTGAACGGCAAGGAGTATAAGTGCAAAGTGTCTAACAAGGCCCT-
GCC
CGCTCCTATCGAGAAGACTATTAGCAAGGCAAAA
315 5244 CH3
GQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVD-
K
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
316 5244 CH3
GGGCAGCCACGGGAACCCCAGGTCTACGTGCTGCCCCCTAGCAGAGACGAGCTGACCAAAAACCAGGTCTCCC-
T
GCTGTGTCTGGTGAAGGGCTTTTATCCTAGTGATATCGCTGTGGAGTGGGAATCAAATGGGCAGCCAGAAA-
ACA
ATTACCTGACATGGCCACCCGTGCTGGACAGCGATGGGTCCTTCTTTCTGTATTCCAAACTGACTGTGGAC-
AAG
TCTAGATGGCAGCAGGGAAACGTCTTCAGCTGTTCCGTGATGCACGAGGCCCTGCACAATCATTACACCCA-
GAA
GTCTCTGAGTCTGTCACCCGGC
317 -2 Full
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTL-
T
ISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK-
VQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
318 -2 Full
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGG-
C
AAGTCAGGACGTTAACACCGCTGTAGCTTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCT-
ATT
CTGCATCCTTTTTGTACAGTGGGGTCCCATCAAGGTTCAGTGGCAGTCGATCTGGGACAGATTTCACTCTC-
ACC
ATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGCATTACACTACCCCACCCACTTT-
CGG
CCAAGGGACCAAAGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATG-
AGC
AGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAG-
TGG
AAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAAGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCAC-
CTA
CAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCA-
CCC
ATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT
319 -2 VL DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSG-
SRSGTDFTLT
ISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK
320 -2 VL GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCA-
CTTGCCGGGC
AAGTCAGGACGTTAACACCGCTGTAGCTTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCT-
ATT
CTGCATCCTTTTTGTACAGTGGGGTCCCATCAAGGTTCAGTGGCAGTCGATCTGGGACAGATTTCACTCTC-
ACC
ATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGCATTACACTACCCCACCCACTTT-
CGG
CCAAGGGACCAAAGTGGAGATCAAA
321 -2 L1 QDVNTA
322 -2 L1 CAGGACGTTAACACCGCT
323 -2 L3 QQHYTTPPT
324 -2 L3 CAACAGCATTACACTACCCCACCCACT
325 -2 L2 SAS
326 -2 L2 TCTGCATCC
327 -2 CL RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS-
TYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
328 -2 CL CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAA-
CTGCCTCTGT
TGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAAT-
CGG
GTAACTCCCAAGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACG-
CTG
AGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGT-
CAC
AAAGAGCTTCAACAGGGGAGAGTGT
329 4372 Full
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK-
T
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTK-
NQV
SLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN-
HYT
QKSLSLSPG
330 4372 Full
GAACCTAAATCCAGCGACAAGACCCACACATGCCCCCCTTGTCCAGCTCCAGAACTGCTGGGAGGACCAAGCG-
T
GTTCCTGTTTCCACCCAAGCCCAAAGATACACTGATGATCAGCCGAACTCCCGAGGTCACCTGCGTGGTCG-
TGG
ACGTGTCCCACGAGGACCCCGAAGTCAAGTTCAACTGGTACGTGGACGGCGTCGAAGTGCATAATGCAAAG-
ACT
AAACCACGGGAGGAACAGTACAACTCTACATATAGAGTCGTGAGTGTCCTGACTGTGCTGCATCAGGATTG-
GCT
GAACGGCAAAGAGTATAAGTGCAAAGTGTCTAATAAGGCCCTGCCTGCTCCAATCGAGAAAACTATTAGTA-
AGG
CAAAAGGGCAGCCCAGGGAACCTCAGGTCTACGTGCTGCCTCCAAGTCGCGACGAGCTGACCAAGAACCAG-
GTC
TCACTGCTGTGTCTGGTGAAAGGATTCTATCCTTCCGATATTGCCGTGGAGTGGGAATCTAATGGCCAGCC-
AGA
GAACAATTACCTGACCTGGCCCCCTGTGCTGGACAGCGATGGGTCCTTCTTTCTGTATTCAAAGCTGACAG-
TGG
ACAAAAGCAGATGGCAGCAGGGAAACGTCTTTAGCTGTTCCGTGATGCACGAAGCCCTGCACAATCATTAC-
ACC
CAGAAGTCTCTGAGTCTGTCACCTGGC
331 4372 CH2
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV-
S
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
332 4372 CH2
GCTCCAGAACTGCTGGGAGGACCAAGCGTGTTCCTGTTTCCACCCAAGCCCAAAGATACACTGATGATCAGCC-
G
AACTCCCGAGGTCACCTGCGTGGTCGTGGACGTGTCCCACGAGGACCCCGAAGTCAAGTTCAACTGGTACG-
TGG
ACGGCGTCGAAGTGCATAATGCAAAGACTAAACCACGGGAGGAACAGTACAACTCTACATATAGAGTCGTG-
AGT
GTCCTGACTGTGCTGCATCAGGATTGGCTGAACGGCAAAGAGTATAAGTGCAAAGTGTCTAATAAGGCCCT-
GCC
TGCTCCAATCGAGAAAACTATTAGTAAGGCAAAA
333 4372 CH3
GQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVD-
K
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
334 4372 CH3
GGGCAGCCCAGGGAACCTCAGGTCTACGTGCTGCCTCCAAGTCGCGACGAGCTGACCAAGAACCAGGTCTCAC-
T
GCTGTGTCTGGTGAAAGGATTCTATCCTTCCGATATTGCCGTGGAGTGGGAATCTAATGGCCAGCCAGAGA-
ACA
ATTACCTGACCTGGCCCCCTGTGCTGGACAGCGATGGGTCCTTCTTTCTGTATTCAAAGCTGACAGTGGAC-
AAA
AGCAGATGGCAGCAGGGAAACGTCTTTAGCTGTTCCGTGATGCACGAAGCCCTGCACAATCATTACACCCA-
GAA
GTCTCTGAGTCTGTCACCTGGC
SEQ ID NO: Pertuzumab WT CDR sequences
335 CDR-H2 VNPNSGGS
336 CDR-H3 ARNLGPSFYFDY
337 CDR-H1 GFTFTDYT
338 CDR-L2 SAS
339 CDR-L3 QQYYIYPYT
340 CDR-L1 QDVSIG
SEQ ID NO: Trastuzumab WT CDR sequences
341 CDR-H2 IYPTNGYT
342 CDR-H3 SRWGGDGFYAMDY
343 CDR-H1 GFNIKDTY
344 CDR-L2 SAS
345 CDR-L3 QQHYTTPPT
346 CDR-L1 QDVNTA
Pertuzumab variant CDR-L3: QQYYIYPAT
Clone 3382, variant 10000 (SEQ ID NO: 347)
Pertuzumab variant CDR-H1: GFTFADYT
Clone 6586, variant 10000 (SEQ ID NO: 348)
Sequence CWU
1
1
3501450PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 1Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30 Tyr Ile His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr
Thr Arg Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn
Thr Ala Tyr 65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ser Arg Trp Gly
Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val 115 120
125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala 130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145
150 155 160 Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165
170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro 180 185
190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys 195 200 205 Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210
215 220 Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230
235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 245 250
255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270 Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275
280 285 Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295
300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys 305 310 315
320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335 Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340
345 350 Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu 355 360
365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp 370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385
390 395 400 Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405
410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His 420 425
430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro 435 440 445
Gly Lys 450 21350DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 2gaggtgcagc tggtggaaag cggaggagga
ctggtgcagc caggaggatc tctgcgactg 60agttgcgccg cttcaggatt caacatcaag
gacacctaca ttcactgggt gcgacaggct 120ccaggaaaag gactggagtg ggtggctcga
atctatccca ctaatggata cacccggtat 180gccgactccg tgaaggggag gtttactatt
agcgccgata catccaaaaa cactgcttac 240ctgcagatga acagcctgcg agccgaagat
accgctgtgt actattgcag tcgatgggga 300ggagacggat tctacgctat ggattattgg
ggacagggga ccctggtgac agtgagctcc 360gcctctacca agggccccag tgtgtttccc
ctggctcctt ctagtaaatc cacctctgga 420gggacagccg ctctgggatg tctggtgaag
gactatttcc ccgagcctgt gaccgtgagt 480tggaactcag gcgccctgac aagcggagtg
cacacttttc ctgctgtgct gcagtcaagc 540gggctgtact ccctgtcctc tgtggtgaca
gtgccaagtt caagcctggg cacacagact 600tatatctgca acgtgaatca taagccctca
aatacaaaag tggacaagaa agtggagccc 660aagagctgtg ataagaccca cacctgccct
ccctgtccag ctccagaact gctgggagga 720cctagcgtgt tcctgtttcc ccctaagcca
aaagacactc tgatgatttc caggactccc 780gaggtgacct gcgtggtggt ggacgtgtct
cacgaggacc ccgaagtgaa gttcaactgg 840tacgtggatg gcgtggaagt gcataatgct
aagacaaaac caagagagga acagtacaac 900tccacttatc gcgtcgtgag cgtgctgacc
gtgctgcacc aggactggct gaacgggaag 960gagtataagt gcaaagtcag taataaggcc
ctgcctgctc caatcgaaaa aaccatctct 1020aaggccaaag gccagccaag ggagccccag
gtgtacacac tgccacccag cagagacgaa 1080ctgaccaaga accaggtgtc cctgacatgt
ctggtgaaag gcttctatcc tagtgatatt 1140gctgtggagt gggaatcaaa tggacagcca
gagaacaatt acaagaccac acctccagtg 1200ctggacagcg atggcagctt cttcctgtat
tccaagctga cagtggataa atctcgatgg 1260cagcagggga acgtgtttag ttgttcagtg
atgcatgaag ccctgcacaa tcattacact 1320cagaagagcc tgtccctgtc tcccggcaaa
13503120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
3Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20
25 30 Tyr Ile His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp
Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65
70 75 80 Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala
Met Asp Tyr Trp Gly Gln 100 105
110 Gly Thr Leu Val Thr Val Ser Ser 115
120 4360DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 4gaggtgcagc tggtggaaag cggaggagga
ctggtgcagc caggaggatc tctgcgactg 60agttgcgccg cttcaggatt caacatcaag
gacacctaca ttcactgggt gcgacaggct 120ccaggaaaag gactggagtg ggtggctcga
atctatccca ctaatggata cacccggtat 180gccgactccg tgaaggggag gtttactatt
agcgccgata catccaaaaa cactgcttac 240ctgcagatga acagcctgcg agccgaagat
accgctgtgt actattgcag tcgatgggga 300ggagacggat tctacgctat ggattattgg
ggacagggga ccctggtgac agtgagctcc 36058PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Gly
Phe Asn Ile Lys Asp Thr Tyr 1 5
624DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 6ggattcaaca tcaaggacac ctac
24713PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 7Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala
Met Asp Tyr 1 5 10
839DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 8agtcgatggg gaggagacgg attctacgct atggattat
3998PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 9Ile Tyr Pro Thr Asn Gly Tyr Thr 1
5 1024DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 10atctatccca
ctaatggata cacc
241198PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 11Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45 Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr 65 70 75
80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95 Lys Val
12294DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 12gcctctacca agggccccag tgtgtttccc ctggctcctt
ctagtaaatc cacctctgga 60gggacagccg ctctgggatg tctggtgaag gactatttcc
ccgagcctgt gaccgtgagt 120tggaactcag gcgccctgac aagcggagtg cacacttttc
ctgctgtgct gcagtcaagc 180gggctgtact ccctgtcctc tgtggtgaca gtgccaagtt
caagcctggg cacacagact 240tatatctgca acgtgaatca taagccctca aatacaaaag
tggacaagaa agtg 29413110PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 13Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5
10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 20 25
30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr 35 40 45 Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50
55 60 Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His 65 70
75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90
95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
100 105 110 14330DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
14gctccagaac tgctgggagg acctagcgtg ttcctgtttc cccctaagcc aaaagacact
60ctgatgattt ccaggactcc cgaggtgacc tgcgtggtgg tggacgtgtc tcacgaggac
120cccgaagtga agttcaactg gtacgtggat ggcgtggaag tgcataatgc taagacaaaa
180ccaagagagg aacagtacaa ctccacttat cgcgtcgtga gcgtgctgac cgtgctgcac
240caggactggc tgaacgggaa ggagtataag tgcaaagtca gtaataaggc cctgcctgct
300ccaatcgaaa aaaccatctc taaggccaaa
33015106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 15Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp 1 5 10
15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
20 25 30 Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35
40 45 Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55
60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly 65 70 75
80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95 Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 100 105
16318DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 16ggccagccaa gggagcccca ggtgtacaca ctgccaccca
gcagagacga actgaccaag 60aaccaggtgt ccctgacatg tctggtgaaa ggcttctatc
ctagtgatat tgctgtggag 120tgggaatcaa atggacagcc agagaacaat tacaagacca
cacctccagt gctggacagc 180gatggcagct tcttcctgta ttccaagctg acagtggata
aatctcgatg gcagcagggg 240aacgtgttta gttgttcagt gatgcatgaa gccctgcaca
atcattacac tcagaagagc 300ctgtccctgt ctcccggc
31817448PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 17Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Thr Asp Tyr 20 25
30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala
Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50
55 60 Lys Gly Arg Phe Thr Leu
Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110 Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115
120 125 Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp 145 150 155
160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175 Lys Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180
185 190 Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro 195 200
205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys 210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225
230 235 240 Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245
250 255 Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp 260 265
270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn 275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290
295 300 Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310
315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys 325 330
335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Val 340 345 350 Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Leu 355
360 365 Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375
380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Leu Thr
Trp Pro Pro Val Leu 385 390 395
400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415 Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420
425 430 Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445 181344DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 18gaagtgcagc tggtcgaatc
tggaggagga ctggtgcagc caggagggtc cctgcgcctg 60tcttgcgccg ctagtggctt
cacttttacc gactacacca tggattgggt gcgacaggca 120cctggaaagg gcctggagtg
ggtcgccgat gtgaacccaa atagcggagg ctccatctac 180aaccagcggt tcaagggccg
gttcaccctg tcagtggacc ggagcaaaaa caccctgtat 240ctgcagatga atagcctgcg
agccgaagat actgctgtgt actattgcgc ccggaatctg 300gggccctcct tctactttga
ctattggggg cagggaactc tggtcaccgt gagctccgcc 360tccaccaagg gaccttctgt
gttcccactg gctccctcta gtaaatccac atctggggga 420actgcagccc tgggctgtct
ggtgaagggc tacttcccag agcccgtcac agtgtcttgg 480aacagtggcg ctctgacttc
tggggtccac acctttcctg cagtgctgaa gtcaagcggg 540ctgtacagcc tgtcctctgt
ggtcaccgtg ccaagttcaa gcctgggaac acagacttat 600atctgcaacg tgaatcacaa
gccatccaat acaaaagtcg acaagaaagt ggaacccaag 660tcttgtgata aaacccatac
atgcccccct tgtcctgcac cagagctgct gggaggacca 720agcgtgttcc tgtttccacc
caagcctaaa gatacactga tgattagtag gaccccagaa 780gtcacatgcg tggtcgtgga
cgtgagccac gaggaccccg aagtcaagtt taactggtac 840gtggacggcg tcgaggtgca
taatgccaag actaaaccca gggaggaaca gtacaacagt 900acctatcgcg tcgtgtcagt
cctgacagtg ctgcatcagg attggctgaa cgggaaagag 960tataagtgca aagtgagcaa
taaggctctg cccgcaccta tcgagaaaac aatttccaag 1020gcaaaaggac agcctagaga
accacaggtg tacgtgctgc ctccatcaag ggatgagctg 1080acaaagaacc aggtcagcct
gctgtgtctg gtgaaaggat tctatccctc tgacattgct 1140gtggagtggg aaagtaatgg
ccagcctgag aacaattacc tgacctggcc ccctgtgctg 1200gactcagatg gcagcttctt
tctgtatagc aagctgaccg tcgacaaatc ccggtggcag 1260caggggaatg tgtttagttg
ttcagtcatg cacgaggcac tgcacaacca ttacacccag 1320aagtcactgt cactgtcacc
aggg 134419119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
19Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20
25 30 Thr Met Asp Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln
Arg Phe 50 55 60
Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe
Asp Tyr Trp Gly Gln Gly 100 105
110 Thr Leu Val Thr Val Ser Ser 115
20357DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 20gaagtgcagc tggtcgaatc tggaggagga ctggtgcagc
caggagggtc cctgcgcctg 60tcttgcgccg ctagtggctt cacttttacc gactacacca
tggattgggt gcgacaggca 120cctggaaagg gcctggagtg ggtcgccgat gtgaacccaa
atagcggagg ctccatctac 180aaccagcggt tcaagggccg gttcaccctg tcagtggacc
ggagcaaaaa caccctgtat 240ctgcagatga atagcctgcg agccgaagat actgctgtgt
actattgcgc ccggaatctg 300gggccctcct tctactttga ctattggggg cagggaactc
tggtcaccgt gagctcc 357218PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 21Gly Phe Thr Phe Thr Asp Tyr
Thr 1 5 2224DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 22ggcttcactt
ttaccgacta cacc
242312PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 23Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr 1
5 10 2436DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 24gcccggaatc
tggggccctc cttctacttt gactat
36258PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 25Val Asn Pro Asn Ser Gly Gly Ser 1 5
2624DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 26gtgaacccaa atagcggagg ctcc
242798PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 27Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys 1 5 10
15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
Gly Tyr 20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Lys Ser Ser Gly Leu Tyr Ser 50
55 60 Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr 65 70
75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90
95 Lys Val 28294DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 28gcctccacca agggaccttc tgtgttccca
ctggctccct ctagtaaatc cacatctggg 60ggaactgcag ccctgggctg tctggtgaag
ggctacttcc cagagcccgt cacagtgtct 120tggaacagtg gcgctctgac ttctggggtc
cacacctttc ctgcagtgct gaagtcaagc 180gggctgtaca gcctgtcctc tgtggtcacc
gtgccaagtt caagcctggg aacacagact 240tatatctgca acgtgaatca caagccatcc
aatacaaaag tcgacaagaa agtg 29429110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
29Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1
5 10 15 Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20
25 30 Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr 35 40
45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu 50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65
70 75 80 Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85
90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys 100 105 110
30330DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 30gcaccagagc tgctgggagg accaagcgtg ttcctgtttc
cacccaagcc taaagataca 60ctgatgatta gtaggacccc agaagtcaca tgcgtggtcg
tggacgtgag ccacgaggac 120cccgaagtca agtttaactg gtacgtggac ggcgtcgagg
tgcataatgc caagactaaa 180cccagggagg aacagtacaa cagtacctat cgcgtcgtgt
cagtcctgac agtgctgcat 240caggattggc tgaacgggaa agagtataag tgcaaagtga
gcaataaggc tctgcccgca 300cctatcgaga aaacaatttc caaggcaaaa
33031106PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 31Gly Gln Pro Arg Glu Pro
Gln Val Tyr Val Leu Pro Pro Ser Arg Asp 1 5
10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Leu Cys
Leu Val Lys Gly Phe 20 25
30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu 35 40 45 Asn
Asn Tyr Leu Thr Trp Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50
55 60 Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70
75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr 85 90
95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 100
105 32318DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 32ggacagccta gagaaccaca ggtgtacgtg
ctgcctccat caagggatga gctgacaaag 60aaccaggtca gcctgctgtg tctggtgaaa
ggattctatc cctctgacat tgctgtggag 120tgggaaagta atggccagcc tgagaacaat
tacctgacct ggccccctgt gctggactca 180gatggcagct tctttctgta tagcaagctg
accgtcgaca aatcccggtg gcagcagggg 240aatgtgttta gttgttcagt catgcacgag
gcactgcaca accattacac ccagaagtca 300ctgtcactgt caccaggg
31833214PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
33Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr
Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20
25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala 100 105
110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
Gly 115 120 125 Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130
135 140 Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150
155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170
175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190 Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195
200 205 Phe Asn Arg Gly Glu Cys
210 34642DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 34gatattcaga tgacccagtc cccaagctcc
ctgagtgcct cagtgggcga ccgagtcacc 60atcacatgca aggcttccca ggatgtgtct
attggagtcg catggtacca gcagaagcca 120ggcaaagcac ccaagctgct gatctatagc
gcctcctacc ggtataccgg cgtgccctct 180agattctctg gcagtgggtc aggaacagac
tttactctga ccatctctag tctgcagcct 240gaggatttcg ctacctacta ttgccagcag
tactatatct acccatatac ctttggccag 300gggacaaaag tggagatcaa gaggactgtg
gccgctccct ccgtcttcat ttttccccct 360tctgacgaac agctgaaaag tggcacagcc
agcgtggtct gtctgctgaa caatttctac 420cctcgcgaag ccaaagtgca gtggaaggtc
gataacgctc tgcagagcgg caacagccag 480gagtctgtga ctgaacagga cagtaaagat
tcaacctata gcctgtcaag cacactgact 540ctgagcaagg cagactacga gaagcacaaa
gtgtatgcct gcgaagtcac acatcagggg 600ctgtcctctc ctgtgactaa gagctttaac
agaggagagt gt 64235107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
35Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr
Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20
25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105 36321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
36gatattcaga tgacccagtc cccaagctcc ctgagtgcct cagtgggcga ccgagtcacc
60atcacatgca aggcttccca ggatgtgtct attggagtcg catggtacca gcagaagcca
120ggcaaagcac ccaagctgct gatctatagc gcctcctacc ggtataccgg cgtgccctct
180agattctctg gcagtgggtc aggaacagac tttactctga ccatctctag tctgcagcct
240gaggatttcg ctacctacta ttgccagcag tactatatct acccatatac ctttggccag
300gggacaaaag tggagatcaa g
321376PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 37Gln Asp Val Ser Ile Gly 1 5
3818DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 38caggatgtgt ctattgga
18399PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 39Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr 1
5 4027DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 40cagcagtact
atatctaccc atatacc
27413PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 41Ser Ala Ser 1 429DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 42agcgcctcc
943107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
43Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1
5 10 15 Gln Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20
25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln 35 40
45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser 50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65
70 75 80 Lys His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85
90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 100 105 44321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
44aggactgtgg ccgctccctc cgtcttcatt tttccccctt ctgacgaaca gctgaaaagt
60ggcacagcca gcgtggtctg tctgctgaac aatttctacc ctcgcgaagc caaagtgcag
120tggaaggtcg ataacgctct gcagagcggc aacagccagg agtctgtgac tgaacaggac
180agtaaagatt caacctatag cctgtcaagc acactgactc tgagcaaggc agactacgag
240aagcacaaag tgtatgcctg cgaagtcaca catcaggggc tgtcctctcc tgtgactaag
300agctttaaca gaggagagtg t
32145222PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 45Asp Tyr Lys Asp Asp Asp Asp Lys Asp Ile Gln
Met Thr Gln Ser Pro 1 5 10
15 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg
20 25 30 Ala Ser
Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro 35
40 45 Gly Lys Ala Pro Lys Leu Leu
Ile Tyr Ser Ala Ser Phe Leu Tyr Ser 50 55
60 Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly
Thr Asp Phe Thr 65 70 75
80 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
85 90 95 Gln Gln His
Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val 100
105 110 Glu Ile Lys Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro 115 120
125 Ser Asp Glu Arg Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu 130 135 140
Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 145
150 155 160 Ala Leu Gln Ser
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 165
170 175 Lys Asp Ser Thr Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala 180 185
190 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly 195 200 205
Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
215 220 46666DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
46gactacaaag acgacgatga caaagatatc cagatgaccc agtcccctag ctccctgtcc
60gcttctgtgg gcgatagggt cactattacc tgccgcgcat ctcaggacgt gaacaccgca
120gtcgcctggt accagcagaa gcctgggaaa gctccaaagc tgctgatcta cagtgcatca
180ttcctgtatt caggagtgcc cagccggttt agcggcagca gatctggcac cgatttcaca
240ctgactattt ctagtctgca gcctgaggac tttgccacat actattgcca gcagcactat
300accacacccc ctactttcgg ccaggggacc aaagtggaga tcaagcgaac tgtggccgct
360ccaagtgtct tcatttttcc acccagcgat gaaagactga agtccggcac agcttctgtg
420gtctgtctgc tgaacaattt ttaccccaga gaggccaaag tgcagtggaa ggtcgacaac
480gctctgcaga gtggcaacag ccaggagagc gtgacagaac aggattccaa agactctact
540tatagtctgt caagcaccct gacactgagc aaggcagact acgaaaagca taaagtgtat
600gcctgtgagg tcacacatca ggggctgtca tcaccagtca ccaaatcatt caatcggggg
660gagtgc
66647107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 47Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30 Val Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Ser Ala Ser Phe Leu Tyr
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95 Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 105
48321DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 48gatatccaga tgacccagtc ccctagctcc ctgtccgctt
ctgtgggcga tagggtcact 60attacctgcc gcgcatctca ggacgtgaac accgcagtcg
cctggtacca gcagaagcct 120gggaaagctc caaagctgct gatctacagt gcatcattcc
tgtattcagg agtgcccagc 180cggtttagcg gcagcagatc tggcaccgat ttcacactga
ctatttctag tctgcagcct 240gaggactttg ccacatacta ttgccagcag cactatacca
caccccctac tttcggccag 300gggaccaaag tggagatcaa g
321496PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 49Gln Asp Val Asn Thr Ala 1
5 5018DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 50caggacgtga acaccgca
18519PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 51Gln
Gln His Tyr Thr Thr Pro Pro Thr 1 5
5227DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 52cagcagcact ataccacacc ccctact
27533PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 53Ser Ala Ser 1
549DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 54agtgcatca
955107PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 55Arg Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu 1 5 10
15 Arg Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe 20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45 Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50
55 60 Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu 65 70
75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser 85 90
95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100
105 56321DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 56cgaactgtgg ccgctccaag
tgtcttcatt tttccaccca gcgatgaaag actgaagtcc 60ggcacagctt ctgtggtctg
tctgctgaac aatttttacc ccagagaggc caaagtgcag 120tggaaggtcg acaacgctct
gcagagtggc aacagccagg agagcgtgac agaacaggat 180tccaaagact ctacttatag
tctgtcaagc accctgacac tgagcaaggc agactacgaa 240aagcataaag tgtatgcctg
tgaggtcaca catcaggggc tgtcatcacc agtcaccaaa 300tcattcaatc ggggggagtg c
32157222PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
57Asp Tyr Lys Asp Asp Asp Asp Lys Asp Ile Gln Met Thr Gln Ser Pro 1
5 10 15 Ser Ser Leu Ser
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg 20
25 30 Ala Ser Gln Asp Val Asn Thr Ala Val
Ala Trp Tyr Gln Gln Lys Pro 35 40
45 Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu
Tyr Ser 50 55 60
Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr 65
70 75 80 Leu Thr Ile Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 85
90 95 Gln Gln His Tyr Thr Thr Pro Pro Thr Phe
Gly Gln Gly Thr Lys Val 100 105
110 Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro 115 120 125 Ser
Asp Glu Arg Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 130
135 140 Asn Asn Phe Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn 145 150
155 160 Ala Leu Gln Ser Gly Asn Ser Lys Glu Ser Val
Thr Glu Gln Asp Ser 165 170
175 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Arg Leu Thr Leu Ser Lys Ala
180 185 190 Asp Tyr
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 195
200 205 Leu Ser Ser Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 210 215
220 58666DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 58gactacaaag acgacgatga caaagatatc
cagatgaccc agtcccctag ctccctgtcc 60gcttctgtgg gcgatagggt cactattacc
tgccgcgcat ctcaggacgt gaacaccgca 120gtcgcctggt accagcagaa gcctgggaaa
gctccaaagc tgctgatcta cagtgcatca 180ttcctgtatt caggagtgcc cagccggttt
agcggcagca gatctggcac cgatttcaca 240ctgactattt ctagtctgca gcctgaggac
tttgccacat actattgcca gcagcactat 300accacacccc ctactttcgg ccaggggacc
aaagtggaga tcaagcgaac tgtggccgct 360ccaagtgtct tcatttttcc acccagcgat
gaaagactga agtccggcac agcttctgtg 420gtctgtctgc tgaacaattt ttaccccaga
gaggccaaag tgcagtggaa ggtcgacaac 480gctctgcaga gtggcaacag caaggagagc
gtgacagaac aggattccaa agactctact 540tatagtctgt caagcagact gacactgagc
aaggcagact acgaaaagca taaagtgtat 600gcctgtgagg tcacacatca ggggctgtca
tcaccagtca ccaaatcatt caatcggggg 660gagtgc
66659107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
59Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20
25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 60321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
60gatatccaga tgacccagtc ccctagctcc ctgtccgctt ctgtgggcga tagggtcact
60attacctgcc gcgcatctca ggacgtgaac accgcagtcg cctggtacca gcagaagcct
120gggaaagctc caaagctgct gatctacagt gcatcattcc tgtattcagg agtgcccagc
180cggtttagcg gcagcagatc tggcaccgat ttcacactga ctatttctag tctgcagcct
240gaggactttg ccacatacta ttgccagcag cactatacca caccccctac tttcggccag
300gggaccaaag tggagatcaa g
321616PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 61Gln Asp Val Asn Thr Ala 1 5
6218DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 62caggacgtga acaccgca
18639PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 63Gln Gln His Tyr Thr Thr Pro Pro Thr 1
5 6427DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 64cagcagcact
ataccacacc ccctact
27653PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 65Ser Ala Ser 1 669DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 66agtgcatca
967107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
67Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1
5 10 15 Arg Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20
25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln 35 40
45 Ser Gly Asn Ser Lys Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser 50 55 60
Thr Tyr Ser Leu Ser Ser Arg Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65
70 75 80 Lys His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85
90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 100 105 68321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
68cgaactgtgg ccgctccaag tgtcttcatt tttccaccca gcgatgaaag actgaagtcc
60ggcacagctt ctgtggtctg tctgctgaac aatttttacc ccagagaggc caaagtgcag
120tggaaggtcg acaacgctct gcagagtggc aacagcaagg agagcgtgac agaacaggat
180tccaaagact ctacttatag tctgtcaagc agactgacac tgagcaaggc agactacgaa
240aagcataaag tgtatgcctg tgaggtcaca catcaggggc tgtcatcacc agtcaccaaa
300tcattcaatc ggggggagtg c
32169214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 69Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly
20 25 30 Val Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Ser Ala Ser Tyr Arg Tyr
Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Ala
85 90 95 Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100
105 110 Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu Ala 130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145
150 155 160 Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165
170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185
190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205
Phe Asn Arg Gly Glu Cys 210 70642DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
70gatattcaga tgacccagtc cccaagctcc ctgagtgcct cagtgggcga ccgagtcacc
60atcacatgca aggcttccca ggatgtgtct attggagtcg catggtacca gcagaagcca
120ggcaaagcac ccaagctgct gatctatagc gcctcctacc ggtataccgg cgtgccctct
180agattctctg gcagtgggtc aggaacagac tttactctga ccatctctag tctgcagcct
240gaggatttcg ctacctacta ttgccagcag tactatatct acccagccac ctttggccag
300gggacaaaag tggagatcaa gaggactgtg gccgctccct ccgtcttcat ttttccccct
360tctgacgaac agctgaaaag tggcacagcc agcgtggtct gtctgctgaa caatttctac
420cctcgcgaag ccaaagtgca gtggaaggtc gataacgctc tgcagagcgg caacagccag
480gagtctgtga ctgaacagga cagtaaagat tcaacctata gcctgtcaag cacactgact
540ctgagcaagg cagactacga gaagcacaaa gtgtatgcct gcgaagtcac acatcagggg
600ctgtcctctc ctgtgactaa gagctttaac agaggagagt gt
64271107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 71Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly
20 25 30 Val Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Ser Ala Ser Tyr Arg Tyr
Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Ala
85 90 95 Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 105
72321DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 72gatattcaga tgacccagtc cccaagctcc ctgagtgcct
cagtgggcga ccgagtcacc 60atcacatgca aggcttccca ggatgtgtct attggagtcg
catggtacca gcagaagcca 120ggcaaagcac ccaagctgct gatctatagc gcctcctacc
ggtataccgg cgtgccctct 180agattctctg gcagtgggtc aggaacagac tttactctga
ccatctctag tctgcagcct 240gaggatttcg ctacctacta ttgccagcag tactatatct
acccagccac ctttggccag 300gggacaaaag tggagatcaa g
321736PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 73Gln Asp Val Ser Ile Gly 1
5 7418DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 74caggatgtgt ctattgga
18759PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 75Gln
Gln Tyr Tyr Ile Tyr Pro Ala Thr 1 5
7627DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 76cagcagtact atatctaccc agccacc
27773PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 77Ser Ala Ser 1
789DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 78agcgcctcc
979107PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 79Arg Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu 1 5 10
15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe 20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45 Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50
55 60 Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu 65 70
75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser 85 90
95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100
105 80321DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 80aggactgtgg ccgctccctc
cgtcttcatt tttccccctt ctgacgaaca gctgaaaagt 60ggcacagcca gcgtggtctg
tctgctgaac aatttctacc ctcgcgaagc caaagtgcag 120tggaaggtcg ataacgctct
gcagagcggc aacagccagg agtctgtgac tgaacaggac 180agtaaagatt caacctatag
cctgtcaagc acactgactc tgagcaaggc agactacgag 240aagcacaaag tgtatgcctg
cgaagtcaca catcaggggc tgtcctctcc tgtgactaag 300agctttaaca gaggagagtg t
32181449PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
81Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20
25 30 Tyr Ile His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp
Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65
70 75 80 Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala
Met Asp Tyr Trp Gly Gln 100 105
110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val 115 120 125 Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130
135 140 Leu Gly Cys Glu Val Thr
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150
155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val 165 170
175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190 Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195
200 205 Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215
220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly 225 230 235
240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255 Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260
265 270 Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His 275 280
285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg 290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305
310 315 320 Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325
330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr 340 345
350 Val Tyr Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu 355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370
375 380 Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390
395 400 Leu Asp Ser Asp Gly Ser Phe Ala Leu Val
Ser Lys Leu Thr Val Asp 405 410
415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His 420 425 430 Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435
440 445 Gly
821347DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 82gaggtgcagc tggtcgaaag cggaggagga ctggtgcagc
caggagggtc actgcgactg 60agctgcgcag cttccggctt caacatcaag gacacctaca
ttcactgggt ccgccaggct 120cctggaaaag gcctggagtg ggtggcacga atctatccaa
ctaatggata cacccggtat 180gccgactccg tgaagggccg gttcaccatt tctgcagata
caagtaaaaa cactgcctac 240ctgcagatga acagcctgcg agccgaagat acagccgtgt
actattgcag ccgatgggga 300ggcgacggct tctacgctat ggattattgg gggcagggaa
ccctggtcac agtgagctcc 360gcatcaacaa aggggcctag cgtgtttcca ctggccccct
ctagtaaatc cacctctggg 420ggaacagcag ccctgggatg tgaggtgacc gactacttcc
cagagcccgt cactgtgagc 480tggaactccg gcgccctgac atctggggtc catacttttc
ctgctgtgct gcagtcaagc 540ggcctgtaca gcctgtcctc tgtggtcact gtgccaagtt
caagcctggg gactcagacc 600tatatctgca acgtgaatca caagccatcc aataccaaag
tcgacaagaa agtggaaccc 660aagtcttgtg ataaaacaca tacttgcccc ccttgtcctg
caccagagct gctgggagga 720ccaagcgtgt tcctgtttcc acccaagcct aaagacaccc
tgatgattag taggactcca 780gaagtcacct gcgtggtcgt ggacgtgagc cacgaggacc
ccgaagtcaa gttcaactgg 840tacgtggatg gcgtcgaggt gcataatgcc aagacaaaac
ccagggagga acagtacaac 900tccacttatc gcgtcgtgtc tgtcctgacc gtgctgcacc
aggactggct gaacggcaag 960gagtataagt gcaaagtgag caataaggct ctgcccgcac
ctatcgagaa aacaatttcc 1020aaggctaaag ggcagcctag agaaccacag gtgtacgtgt
accctccatc tagggacgag 1080ctgaccaaga accaggtcag tctgacatgt ctggtgaaag
ggttctatcc cagcgatatc 1140gcagtggagt gggaatccaa tggacagcct gagaacaatt
acaagaccac accccctgtg 1200ctggactctg atggaagttt cgccctggtg agtaagctga
ccgtcgataa atcacggtgg 1260cagcagggca acgtgttcag ctgttcagtg atgcacgaag
cactgcacaa ccactacacc 1320cagaaaagcc tgtccctgtc ccccggc
134783120PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 83Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Asn Ile Lys Asp Thr 20 25
30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala
Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile
Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110 Gly Thr
Leu Val Thr Val Ser Ser 115 120
84360DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 84gaggtgcagc tggtcgaaag cggaggagga ctggtgcagc
caggagggtc actgcgactg 60agctgcgcag cttccggctt caacatcaag gacacctaca
ttcactgggt ccgccaggct 120cctggaaaag gcctggagtg ggtggcacga atctatccaa
ctaatggata cacccggtat 180gccgactccg tgaagggccg gttcaccatt tctgcagata
caagtaaaaa cactgcctac 240ctgcagatga acagcctgcg agccgaagat acagccgtgt
actattgcag ccgatgggga 300ggcgacggct tctacgctat ggattattgg gggcagggaa
ccctggtcac agtgagctcc 360858PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 85Gly Phe Asn Ile Lys Asp Thr
Tyr 1 5 8624DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 86ggcttcaaca
tcaaggacac ctac
248713PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 87Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr 1
5 10 8839DNAArtificial
SequenceDescription of Artificial Sequence Synthetic oligonucleotide
88agccgatggg gaggcgacgg cttctacgct atggattat
39898PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 89Ile Tyr Pro Thr Asn Gly Tyr Thr 1 5
9024DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 90atctatccaa ctaatggata cacc
249198PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 91Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys 1 5 10
15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Glu Val Thr
Asp Tyr 20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50
55 60 Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr 65 70
75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90
95 Lys Val 92294DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 92gcatcaacaa aggggcctag cgtgtttcca
ctggccccct ctagtaaatc cacctctggg 60ggaacagcag ccctgggatg tgaggtgacc
gactacttcc cagagcccgt cactgtgagc 120tggaactccg gcgccctgac atctggggtc
catacttttc ctgctgtgct gcagtcaagc 180ggcctgtaca gcctgtcctc tgtggtcact
gtgccaagtt caagcctggg gactcagacc 240tatatctgca acgtgaatca caagccatcc
aataccaaag tcgacaagaa agtg 29493110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
93Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1
5 10 15 Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20
25 30 Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr 35 40
45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu 50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65
70 75 80 Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85
90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys 100 105 110
94330DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 94gcaccagagc tgctgggagg accaagcgtg ttcctgtttc
cacccaagcc taaagacacc 60ctgatgatta gtaggactcc agaagtcacc tgcgtggtcg
tggacgtgag ccacgaggac 120cccgaagtca agttcaactg gtacgtggat ggcgtcgagg
tgcataatgc caagacaaaa 180cccagggagg aacagtacaa ctccacttat cgcgtcgtgt
ctgtcctgac cgtgctgcac 240caggactggc tgaacggcaa ggagtataag tgcaaagtga
gcaataaggc tctgcccgca 300cctatcgaga aaacaatttc caaggctaaa
33095106PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 95Gly Gln Pro Arg Glu Pro
Gln Val Tyr Val Tyr Pro Pro Ser Arg Asp 1 5
10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe 20 25
30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu 35 40 45 Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50
55 60 Ala Leu Val Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70
75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr 85 90
95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 100
105 96318DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 96gggcagccta gagaaccaca ggtgtacgtg
taccctccat ctagggacga gctgaccaag 60aaccaggtca gtctgacatg tctggtgaaa
gggttctatc ccagcgatat cgcagtggag 120tgggaatcca atggacagcc tgagaacaat
tacaagacca caccccctgt gctggactct 180gatggaagtt tcgccctggt gagtaagctg
accgtcgata aatcacggtg gcagcagggc 240aacgtgttca gctgttcagt gatgcacgaa
gcactgcaca accactacac ccagaaaagc 300ctgtccctgt cccccggc
31897448PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
97Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asp Tyr 20
25 30 Thr Met Asp Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40
45 Gly Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln
Arg Phe 50 55 60
Lys Gly Arg Phe Thr Phe Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe
Asp Tyr Trp Gly Gln Gly 100 105
110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe 115 120 125 Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130
135 140 Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150
155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195
200 205 Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215
220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro 225 230 235
240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255 Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260
265 270 Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn 275 280
285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val 290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305
310 315 320 Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325
330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Val 340 345
350 Tyr Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu Thr 355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370
375 380 Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390
395 400 Asp Ser Asp Gly Ser Phe Ala Leu Val Ser
Lys Leu Thr Val Asp Lys 405 410
415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu 420 425 430 Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435
440 445 981344DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
98gaggtgcagc tggtggaatc aggagggggc ctggtgcagc ccggagggtc tctgcgactg
60tcatgtgccg cttctgggtt cactttcgca gactacacaa tggattgggt gcgacaggcc
120cccggaaagg gactggagtg ggtgggcgat gtcaacccta attctggcgg gagtatctac
180aaccagcggt tcaaggggag attcactttt tcagtggaca gaagcaaaaa caccctgtat
240ctgcagatga acagcctgag ggccgaagat accgctgtct actattgcgc tcgcaatctg
300ggccccagtt tctactttga ctattggggg cagggaaccc tggtgacagt cagctccgct
360agcactaagg ggccttccgt gtttccactg gctccctcta gtaaatccac ctctggaggc
420acagctgcac tgggatgtct ggtgaaggat tacttccctg aaccagtcac agtgagttgg
480aactcagggg ctctgacaag tggagtccat acttttcccg cagtgctgca gtcaagcgga
540ctgtactccc tgtcctctgt ggtcaccgtg cctagttcaa gcctgggcac ccagacatat
600atctgcaacg tgaatcacaa gccatcaaat acaaaagtcg acaagaaagt ggagcccaag
660agctgtgata aaactcatac ctgcccacct tgtccggcgc cagaactgct gggaggacca
720agcgtgttcc tgtttccacc caagcctaaa gacaccctga tgatttcccg gactcctgag
780gtcacctgcg tggtcgtgga cgtgtctcac gaggaccccg aagtcaagtt caactggtac
840gtggatggcg tcgaagtgca taatgccaag accaaacccc gggaggaaca gtacaactct
900acctatagag tcgtgagtgt cctgacagtg ctgcaccagg actggctgaa tgggaaggag
960tataagtgta aagtgagcaa caaagccctg cccgccccaa tcgaaaaaac aatctctaaa
1020gcaaaaggac agcctcgcga accacaggtc tacgtctacc ccccatcaag agatgaactg
1080acaaaaaatc aggtctctct gacatgcctg gtcaaaggat tctacccttc cgacatcgcc
1140gtggagtggg aaagtaacgg ccagcccgag aacaattaca agaccacacc ccctgtcctg
1200gactctgatg ggagtttcgc tctggtgtca aagctgaccg tcgataaaag ccggtggcag
1260cagggcaatg tgtttagctg ctccgtcatg cacgaagccc tgcacaatca ctacacacag
1320aagtccctga gcctgagccc tggc
134499119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 99Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asp Tyr
20 25 30 Thr Met
Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Gly Asp Val Asn Pro Asn Ser
Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55
60 Lys Gly Arg Phe Thr Phe Ser Val Asp Arg Ser Lys
Asn Thr Leu Tyr 65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Asn
Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100
105 110 Thr Leu Val Thr Val Ser Ser
115 100357DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 100gaggtgcagc
tggtggaatc aggagggggc ctggtgcagc ccggagggtc tctgcgactg 60tcatgtgccg
cttctgggtt cactttcgca gactacacaa tggattgggt gcgacaggcc 120cccggaaagg
gactggagtg ggtgggcgat gtcaacccta attctggcgg gagtatctac 180aaccagcggt
tcaaggggag attcactttt tcagtggaca gaagcaaaaa caccctgtat 240ctgcagatga
acagcctgag ggccgaagat accgctgtct actattgcgc tcgcaatctg 300ggccccagtt
tctactttga ctattggggg cagggaaccc tggtgacagt cagctcc
3571018PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 101Gly Phe Thr Phe Ala Asp Tyr Thr 1 5
10224DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 102gggttcactt tcgcagacta caca
2410312PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 103Ala Arg Asn Leu Gly Pro Ser
Phe Tyr Phe Asp Tyr 1 5 10
10436DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 104gctcgcaatc tgggccccag tttctacttt gactat
361058PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 105Val Asn Pro Asn Ser Gly Gly Ser 1
5 10624DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 106gtcaacccta
attctggcgg gagt
2410798PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 107Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45 Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr 65 70 75
80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95 Lys Val
108294DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 108gctagcacta aggggccttc cgtgtttcca ctggctccct
ctagtaaatc cacctctgga 60ggcacagctg cactgggatg tctggtgaag gattacttcc
ctgaaccagt cacagtgagt 120tggaactcag gggctctgac aagtggagtc catacttttc
ccgcagtgct gcagtcaagc 180ggactgtact ccctgtcctc tgtggtcacc gtgcctagtt
caagcctggg cacccagaca 240tatatctgca acgtgaatca caagccatca aatacaaaag
tcgacaagaa agtg 294109110PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 109Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5
10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 20 25
30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr 35 40 45 Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50
55 60 Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His 65 70
75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90
95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
100 105 110 110330DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
110gcgccagaac tgctgggagg accaagcgtg ttcctgtttc cacccaagcc taaagacacc
60ctgatgattt cccggactcc tgaggtcacc tgcgtggtcg tggacgtgtc tcacgaggac
120cccgaagtca agttcaactg gtacgtggat ggcgtcgaag tgcataatgc caagaccaaa
180ccccgggagg aacagtacaa ctctacctat agagtcgtga gtgtcctgac agtgctgcac
240caggactggc tgaatgggaa ggagtataag tgtaaagtga gcaacaaagc cctgcccgcc
300ccaatcgaaa aaacaatctc taaagcaaaa
330111106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 111Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr
Pro Pro Ser Arg Asp 1 5 10
15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
20 25 30 Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35
40 45 Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55
60 Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly 65 70 75
80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95 Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 100 105
112318DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 112ggacagcctc gcgaaccaca ggtctacgtc taccccccat
caagagatga actgacaaaa 60aatcaggtct ctctgacatg cctggtcaaa ggattctacc
cttccgacat cgccgtggag 120tgggaaagta acggccagcc cgagaacaat tacaagacca
caccccctgt cctggactct 180gatgggagtt tcgctctggt gtcaaagctg accgtcgata
aaagccggtg gcagcagggc 240aatgtgttta gctgctccgt catgcacgaa gccctgcaca
atcactacac acagaagtcc 300ctgagcctga gccctggc
318113226PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 113Tyr Pro Tyr Asp Val Pro
Asp Tyr Ala Thr Gly Ser Asp Ile Gln Met 1 5
10 15 Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly Asp Arg Val Thr 20 25
30 Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly Val Ala Trp
Tyr 35 40 45 Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser 50
55 60 Tyr Arg Tyr Thr Gly Val
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 65 70
75 80 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro Glu Asp Phe Ala 85 90
95 Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly Gln
100 105 110 Gly Thr
Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe 115
120 125 Ile Phe Pro Pro Ser Asp Glu
Glu Leu Lys Ser Gly Thr Ala Ser Val 130 135
140 Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
Lys Val Gln Trp 145 150 155
160 Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Glu Glu Ser Val Thr
165 170 175 Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Glu 180
185 190 Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys Val Tyr Ala Cys Glu Val 195 200
205 Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
Asn Arg Gly 210 215 220
Glu Cys 225 114678DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 114tatccctacg atgtgcctga
ctacgctact ggctccgata tccagatgac ccagtctcca 60agctccctga gtgcatcagt
gggggaccga gtcaccatca catgcaaggc ttcccaggat 120gtgtctattg gagtcgcatg
gtaccagcag aagccaggca aagcacccaa gctgctgatc 180tacagcgcct cctaccggta
tactggggtg ccttccagat tctctggcag tgggtcagga 240accgacttta ctctgaccat
ctctagtctg cagcccgagg atttcgccac ctactattgc 300cagcagtact atatctaccc
ttataccttt ggccagggga caaaagtgga gatcaagagg 360acagtggccg ctccaagtgt
cttcattttt cccccttccg acgaagagct gaaaagtgga 420actgcttcag tggtctgtct
gctgaacaat ttctaccccc gcgaagccaa agtgcagtgg 480aaggtcgata acgctctgca
gagcggcaat tccgaggagt ctgtgacaga acaggacagt 540aaagattcaa cttatagcct
gtcaagcaca ctggagctgt ctaaggcaga ctacgagaag 600cacaaagtgt atgcctgcga
agtcacccat caggggctgt cctctcccgt gacaaagagc 660tttaacagag gagagtgt
678115107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
115Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr
Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20
25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105 116321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
116gatatccaga tgacccagtc tccaagctcc ctgagtgcat cagtggggga ccgagtcacc
60atcacatgca aggcttccca ggatgtgtct attggagtcg catggtacca gcagaagcca
120ggcaaagcac ccaagctgct gatctacagc gcctcctacc ggtatactgg ggtgccttcc
180agattctctg gcagtgggtc aggaaccgac tttactctga ccatctctag tctgcagccc
240gaggatttcg ccacctacta ttgccagcag tactatatct acccttatac ctttggccag
300gggacaaaag tggagatcaa g
3211176PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 117Gln Asp Val Ser Ile Gly 1 5
11818DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 118caggatgtgt ctattgga
181199PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 119Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr 1
5 12027DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 120cagcagtact
atatctaccc ttatacc
271213PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 121Ser Ala Ser 1 1229DNAArtificial
SequenceDescription of Artificial Sequence Synthetic oligonucleotide
122agcgcctcc
9123107PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 123Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu 1 5 10 15
Glu Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30 Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35
40 45 Ser Gly Asn Ser Glu Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser 50 55
60 Thr Tyr Ser Leu Ser Ser Thr Leu Glu Leu Ser Lys Ala
Asp Tyr Glu 65 70 75
80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95 Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 100 105
124321DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 124aggacagtgg ccgctccaag tgtcttcatt tttccccctt
ccgacgaaga gctgaaaagt 60ggaactgctt cagtggtctg tctgctgaac aatttctacc
cccgcgaagc caaagtgcag 120tggaaggtcg ataacgctct gcagagcggc aattccgagg
agtctgtgac agaacaggac 180agtaaagatt caacttatag cctgtcaagc acactggagc
tgtctaaggc agactacgag 240aagcacaaag tgtatgcctg cgaagtcacc catcaggggc
tgtcctctcc cgtgacaaag 300agctttaaca gaggagagtg t
321125450PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 125Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Asn Ile Lys Asp Thr 20 25
30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala
Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile
Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110 Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115
120 125 Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135
140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser 145 150 155
160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175 Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180
185 190 Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys 195 200
205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp 210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225
230 235 240 Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245
250 255 Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu 260 265
270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His 275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290
295 300 Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310
315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu 325 330
335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr 340 345 350 Val
Tyr Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355
360 365 Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375
380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val 385 390 395
400 Leu Asp Ser Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp
405 410 415 Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420
425 430 Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445 Gly Lys 450 1261350DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
126gaagtccagc tggtcgaaag cggaggagga ctggtgcagc caggagggtc tctgcgactg
60agttgcgccg cttcaggctt caacatcaag gacacctaca ttcactgggt gcgccaggct
120cctggaaaag gcctggagtg ggtggcacga atctatccaa ctaatggata cacccggtat
180gcagacagcg tgaagggccg gttcaccatt agcgcagata catccaaaaa cactgcctac
240ctgcagatga acagcctgcg agccgaagat actgctgtgt actattgcag tcggtgggga
300ggcgacggct tctacgctat ggattattgg gggcagggaa ccctggtcac agtgagctcc
360gcatctacaa aggggcctag tgtgtttcca ctggccccct ctagtaaatc cacctctggg
420ggaacagcag ccctgggatg tctggtgaag gactatttcc cagagcccgt cactgtgagt
480tggaactcag gcgccctgac atccggggtc catacttttc ctgctgtgct gcagtcaagc
540ggcctgtact ctctgtcctc tgtggtcacc gtgccaagtt caagcctggg gactcagacc
600tatatctgca acgtgaatca caagccaagc aatacaaaag tcgacaagaa agtggaaccc
660aagagctgtg ataaaacaca tacttgcccc ccttgtcctg caccagagct gctgggagga
720ccatccgtgt tcctgtttcc acccaagcct aaagacaccc tgatgatttc caggactcca
780gaagtcacct gcgtggtcgt ggacgtgtct cacgaggacc ccgaagtcaa gttcaactgg
840tacgtggatg gcgtcgaggt gcataatgcc aagacaaaac ccagggagga acagtacaac
900tcaacttatc gcgtcgtgag cgtcctgacc gtgctgcacc aggactggct gaacggcaag
960gagtataagt gcaaagtgag caataaggct ctgcccgcac ctatcgagaa aaccattagc
1020aaggccaaag ggcagcctag agaaccacag gtctacgtgt atcctccaag cagggacgag
1080ctgaccaaga accaggtctc cctgacatgt ctggtgaaag ggttttaccc cagtgatatc
1140gctgtggagt gggaatcaaa tggacagcct gaaaacaatt ataagaccac accccctgtg
1200ctggacagcg atggcagctt cgctctggtc tccaagctga ctgtggataa atctcggtgg
1260cagcagggca acgtctttag ttgttcagtg atgcatgagg cactgcacaa tcattacacc
1320cagaagagcc tgtccctgtc tcccggcaaa
1350127120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 127Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30 Tyr Ile
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Arg Ile Tyr Pro Thr Asn
Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr 65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ser Arg Trp
Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 128360DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 128gaagtccagc
tggtcgaaag cggaggagga ctggtgcagc caggagggtc tctgcgactg 60agttgcgccg
cttcaggctt caacatcaag gacacctaca ttcactgggt gcgccaggct 120cctggaaaag
gcctggagtg ggtggcacga atctatccaa ctaatggata cacccggtat 180gcagacagcg
tgaagggccg gttcaccatt agcgcagata catccaaaaa cactgcctac 240ctgcagatga
acagcctgcg agccgaagat actgctgtgt actattgcag tcggtgggga 300ggcgacggct
tctacgctat ggattattgg gggcagggaa ccctggtcac agtgagctcc
3601298PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 129Gly Phe Asn Ile Lys Asp Thr Tyr 1 5
13024DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 130ggcttcaaca tcaaggacac ctac
2413113PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 131Ser Arg Trp Gly Gly Asp Gly
Phe Tyr Ala Met Asp Tyr 1 5 10
13239DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 132agtcggtggg gaggcgacgg cttctacgct atggattat
391338PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 133Ile Tyr Pro Thr Asn Gly Tyr Thr 1
5 13424DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 134atctatccaa
ctaatggata cacc
2413598PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 135Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45 Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr 65 70 75
80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95 Lys Val
136294DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 136gcatctacaa aggggcctag tgtgtttcca ctggccccct
ctagtaaatc cacctctggg 60ggaacagcag ccctgggatg tctggtgaag gactatttcc
cagagcccgt cactgtgagt 120tggaactcag gcgccctgac atccggggtc catacttttc
ctgctgtgct gcagtcaagc 180ggcctgtact ctctgtcctc tgtggtcacc gtgccaagtt
caagcctggg gactcagacc 240tatatctgca acgtgaatca caagccaagc aatacaaaag
tcgacaagaa agtg 294137110PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 137Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5
10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 20 25
30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr 35 40 45 Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50
55 60 Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His 65 70
75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90
95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
100 105 110 138330DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
138gcaccagagc tgctgggagg accatccgtg ttcctgtttc cacccaagcc taaagacacc
60ctgatgattt ccaggactcc agaagtcacc tgcgtggtcg tggacgtgtc tcacgaggac
120cccgaagtca agttcaactg gtacgtggat ggcgtcgagg tgcataatgc caagacaaaa
180cccagggagg aacagtacaa ctcaacttat cgcgtcgtga gcgtcctgac cgtgctgcac
240caggactggc tgaacggcaa ggagtataag tgcaaagtga gcaataaggc tctgcccgca
300cctatcgaga aaaccattag caaggccaaa
330139106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 139Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr
Pro Pro Ser Arg Asp 1 5 10
15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
20 25 30 Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35
40 45 Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55
60 Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly 65 70 75
80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95 Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 100 105
140318DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 140gggcagccta gagaaccaca ggtctacgtg tatcctccaa
gcagggacga gctgaccaag 60aaccaggtct ccctgacatg tctggtgaaa gggttttacc
ccagtgatat cgctgtggag 120tgggaatcaa atggacagcc tgaaaacaat tataagacca
caccccctgt gctggacagc 180gatggcagct tcgctctggt ctccaagctg actgtggata
aatctcggtg gcagcagggc 240aacgtcttta gttgttcagt gatgcatgag gcactgcaca
atcattacac ccagaagagc 300ctgtccctgt ctcccggc
318141232PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 141Glu Pro Lys Ser Ser Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5
10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro 20 25
30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val 35 40 45 Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50
55 60 Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70
75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln 85 90
95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110 Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115
120 125 Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135
140 Lys Asn Gln Val Ser Leu Ile Cys Leu Val Lys Gly
Phe Tyr Pro Ser 145 150 155
160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Arg Tyr
165 170 175 Met Thr Trp
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180
185 190 Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe 195 200
205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys 210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys 225 230
142696DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 142gagcccaaga gcagcgataa gacccacacc tgccctccct
gtccagctcc agaactgctg 60ggaggaccta gcgtgttcct gtttccccct aagccaaaag
acactctgat gatttccagg 120actcccgagg tgacctgcgt ggtggtggac gtgtctcacg
aggaccccga agtgaagttc 180aactggtacg tggatggcgt ggaagtgcat aatgctaaga
caaaaccaag agaggaacag 240tacaactcca cttatcgcgt cgtgagcgtg ctgaccgtgc
tgcaccagga ctggctgaac 300gggaaggagt ataagtgcaa agtcagtaat aaggccctgc
ctgctccaat cgaaaaaacc 360atctctaagg ccaaaggcca gccaagggag ccccaggtgt
acacactgcc acccagcaga 420gacgaactga ccaagaacca ggtgtccctg atctgtctgg
tgaaaggctt ctatcctagt 480gatattgctg tggagtggga atcaaatgga cagccagaga
acagatacat gacctggcct 540ccagtgctgg acagcgatgg cagcttcttc ctgtattcca
agctgacagt ggataaatct 600cgatggcagc aggggaacgt gtttagttgt tcagtgatgc
atgaagccct gcacaatcat 660tacactcaga agagcctgtc cctgtctccc ggcaaa
696143110PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 143Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5
10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 20 25
30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr 35 40 45 Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50
55 60 Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His 65 70
75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90
95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
100 105 110 144330DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
144gctccagaac tgctgggagg acctagcgtg ttcctgtttc cccctaagcc aaaagacact
60ctgatgattt ccaggactcc cgaggtgacc tgcgtggtgg tggacgtgtc tcacgaggac
120cccgaagtga agttcaactg gtacgtggat ggcgtggaag tgcataatgc taagacaaaa
180ccaagagagg aacagtacaa ctccacttat cgcgtcgtga gcgtgctgac cgtgctgcac
240caggactggc tgaacgggaa ggagtataag tgcaaagtca gtaataaggc cctgcctgct
300ccaatcgaaa aaaccatctc taaggccaaa
330145106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 145Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp 1 5 10
15 Glu Leu Thr Lys Asn Gln Val Ser Leu Ile Cys Leu Val Lys Gly Phe
20 25 30 Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35
40 45 Asn Arg Tyr Met Thr Trp Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55
60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly 65 70 75
80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95 Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 100 105
146318DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 146ggccagccaa gggagcccca ggtgtacaca ctgccaccca
gcagagacga actgaccaag 60aaccaggtgt ccctgatctg tctggtgaaa ggcttctatc
ctagtgatat tgctgtggag 120tgggaatcaa atggacagcc agagaacaga tacatgacct
ggcctccagt gctggacagc 180gatggcagct tcttcctgta ttccaagctg acagtggata
aatctcgatg gcagcagggg 240aacgtgttta gttgttcagt gatgcatgaa gccctgcaca
atcattacac tcagaagagc 300ctgtccctgt ctcccggc
318147481PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 147Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Val Asn Thr Ala 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr
Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Arg Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
Tyr Thr Thr Pro Pro 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Ser Gly Gly
100 105 110 Gly Ser
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Glu 115
120 125 Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly Ser 130 135
140 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile
Lys Asp Thr Tyr 145 150 155
160 Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala
165 170 175 Arg Ile Tyr
Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys 180
185 190 Gly Arg Phe Thr Ile Ser Ala Asp
Thr Ser Lys Asn Thr Ala Tyr Leu 195 200
205 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Ser 210 215 220
Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly 225
230 235 240 Thr Leu Val Thr
Val Ser Ser Ala Ala Glu Pro Lys Ser Ser Asp Lys 245
250 255 Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro 260 265
270 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser 275 280 285
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 290
295 300 Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 305 310
315 320 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 325 330
335 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu 340 345 350 Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 355
360 365 Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 370 375
380 Tyr Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr 385 390 395
400 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
405 410 415 Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 420
425 430 Asp Glu Asp Gly Ser Phe Ala
Leu Val Ser Lys Leu Thr Val Asp Lys 435 440
445 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu 450 455 460
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465
470 475 480 Lys
1481443DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 148gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca ggacgttaac accgctgtag
cttggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctattct gcatcctttt
tgtacagtgg ggtcccatca 180aggttcagtg gcagtcgatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg caacttacta ctgtcaacag cattacacta
ccccacccac tttcggccaa 300gggaccaaag tggagatcaa aggtggttct ggtggtggtt
ctggtggtgg ttctggtggt 360ggttctggtg gtggttctgg tgaagtgcag ctggtggagt
ctgggggagg cttggtacag 420cctggcgggt ccctgagact ctcctgtgca gcctctggat
tcaacattaa agatacttat 480atccactggg tccggcaagc tccagggaag ggcctggagt
gggtcgcacg tatttatccc 540acaaatggtt acacacggta tgcggactct gtgaagggcc
gattcaccat ctccgcagac 600acttccaaga acaccgcgta tctgcaaatg aacagtctga
gagctgagga cacggccgtt 660tattactgtt caagatgggg cggagacggt ttctacgcta
tggactactg gggccaaggg 720accctggtca ccgtctcctc agccgccgag cccaagagca
gcgataagac ccacacctgc 780cctccctgtc cagctccaga actgctggga ggacctagcg
tgttcctgtt tccccctaag 840ccaaaagaca ctctgatgat ttccaggact cccgaggtga
cctgcgtggt ggtggacgtg 900tctcacgagg accccgaagt gaagttcaac tggtacgtgg
atggcgtgga agtgcataat 960gctaagacaa aaccaagaga ggaacagtac aactccactt
atcgcgtcgt gagcgtgctg 1020accgtgctgc accaggactg gctgaacggg aaggagtata
agtgcaaagt cagtaataag 1080gccctgcctg ctccaatcga aaaaaccatc tctaaggcca
aaggccagcc aagggagccc 1140caggtgtaca catacccacc cagcagagac gaactgacca
agaaccaggt gtccctgaca 1200tgtctggtga aaggcttcta tcctagtgat attgctgtgg
agtgggaatc aaatggacag 1260ccagagaaca attacaagac cacacctcca gtgctggacg
aggatggcag cttcgccctg 1320gtgtccaagc tgacagtgga taaatctcga tggcagcagg
ggaacgtgtt tagttgttca 1380gtgatgcatg aagccctgca caatcattac actcagaaga
gcctgtccct gtctcccggc 1440aaa
1443149107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 149Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Val Asn Thr Ala 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr
Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Arg Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
Tyr Thr Thr Pro Pro 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 150321DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 150gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggcaagtca ggacgttaac accgctgtag cttggtatca gcagaaacca 120gggaaagccc
ctaagctcct gatctattct gcatcctttt tgtacagtgg ggtcccatca 180aggttcagtg
gcagtcgatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg
caacttacta ctgtcaacag cattacacta ccccacccac tttcggccaa 300gggaccaaag
tggagatcaa a
3211516PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 151Gln Asp Val Asn Thr Ala 1 5
15218DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 152caggacgtta acaccgct
181539PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 153Gln Gln His Tyr Thr Thr Pro Pro Thr 1
5 15427DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 154caacagcatt
acactacccc acccact
271553PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 155Ser Ala Ser 1 1569DNAArtificial
SequenceDescription of Artificial Sequence Synthetic oligonucleotide
156tctgcatcc
9157120PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 157Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30 Tyr Ile His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr
Thr Arg Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn
Thr Ala Tyr 65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ser Arg Trp Gly
Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 158360DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 158gaagtgcagc
tggtggagtc tgggggaggc ttggtacagc ctggcgggtc cctgagactc 60tcctgtgcag
cctctggatt caacattaaa gatacttata tccactgggt ccggcaagct 120ccagggaagg
gcctggagtg ggtcgcacgt atttatccca caaatggtta cacacggtat 180gcggactctg
tgaagggccg attcaccatc tccgcagaca cttccaagaa caccgcgtat 240ctgcaaatga
acagtctgag agctgaggac acggccgttt attactgttc aagatggggc 300ggagacggtt
tctacgctat ggactactgg ggccaaggga ccctggtcac cgtctcctca
3601598PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 159Gly Phe Asn Ile Lys Asp Thr Tyr 1 5
16024DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 160ggattcaaca ttaaagatac ttat
2416113PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 161Ser Arg Trp Gly Gly Asp Gly
Phe Tyr Ala Met Asp Tyr 1 5 10
16239DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 162tcaagatggg gcggagacgg tttctacgct atggactac
391638PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 163Ile Tyr Pro Thr Asn Gly Tyr Thr 1
5 16424DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 164atttatccca
caaatggtta caca
24165110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 165Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys 1 5 10
15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30 Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35
40 45 Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55
60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His 65 70 75
80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95 Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100
105 110 166330DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 166gctccagaac
tgctgggagg acctagcgtg ttcctgtttc cccctaagcc aaaagacact 60ctgatgattt
ccaggactcc cgaggtgacc tgcgtggtgg tggacgtgtc tcacgaggac 120cccgaagtga
agttcaactg gtacgtggat ggcgtggaag tgcataatgc taagacaaaa 180ccaagagagg
aacagtacaa ctccacttat cgcgtcgtga gcgtgctgac cgtgctgcac 240caggactggc
tgaacgggaa ggagtataag tgcaaagtca gtaataaggc cctgcctgct 300ccaatcgaaa
aaaccatctc taaggccaaa
330167106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 167Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Tyr
Pro Pro Ser Arg Asp 1 5 10
15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
20 25 30 Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35
40 45 Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Glu Asp Gly Ser Phe 50 55
60 Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly 65 70 75
80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95 Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 100 105
168318DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 168ggccagccaa gggagcccca ggtgtacaca tacccaccca
gcagagacga actgaccaag 60aaccaggtgt ccctgacatg tctggtgaaa ggcttctatc
ctagtgatat tgctgtggag 120tgggaatcaa atggacagcc agagaacaat tacaagacca
cacctccagt gctggacgag 180gatggcagct tcgccctggt gtccaagctg acagtggata
aatctcgatg gcagcagggg 240aacgtgttta gttgttcagt gatgcatgaa gccctgcaca
atcattacac tcagaagagc 300ctgtccctgt ctcccggc
318169481PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 169Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Val Asn Thr Ala 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr
Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Arg Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
Tyr Thr Thr Pro Pro 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Ser Gly Gly
100 105 110 Gly Ser
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Glu 115
120 125 Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly Ser 130 135
140 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile
Lys Asp Thr Tyr 145 150 155
160 Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala
165 170 175 Arg Ile Tyr
Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys 180
185 190 Gly Arg Phe Thr Ile Ser Ala Asp
Thr Ser Lys Asn Thr Ala Tyr Leu 195 200
205 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Ser 210 215 220
Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly 225
230 235 240 Thr Leu Val Thr
Val Ser Ser Ala Ala Glu Pro Lys Ser Ser Asp Lys 245
250 255 Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro 260 265
270 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser 275 280 285
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 290
295 300 Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 305 310
315 320 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 325 330
335 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu 340 345 350 Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 355
360 365 Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 370 375
380 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Ile 385 390 395
400 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
405 410 415 Ser Asn
Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu 420
425 430 Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys 435 440
445 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu 450 455 460
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465
470 475 480 Lys
1701443DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 170gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca ggacgttaac accgctgtag
cttggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctattct gcatcctttt
tgtacagtgg ggtcccatca 180aggttcagtg gcagtcgatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg caacttacta ctgtcaacag cattacacta
ccccacccac tttcggccaa 300gggaccaaag tggagatcaa aggtggttct ggtggtggtt
ctggtggtgg ttctggtggt 360ggttctggtg gtggttctgg tgaagtgcag ctggtggagt
ctgggggagg cttggtacag 420cctggcgggt ccctgagact ctcctgtgca gcctctggat
tcaacattaa agatacttat 480atccactggg tccggcaagc tccagggaag ggcctggagt
gggtcgcacg tatttatccc 540acaaatggtt acacacggta tgcggactct gtgaagggcc
gattcaccat ctccgcagac 600acttccaaga acaccgcgta tctgcaaatg aacagtctga
gagctgagga cacggccgtt 660tattactgtt caagatgggg cggagacggt ttctacgcta
tggactactg gggccaaggg 720accctggtca ccgtctcctc agccgccgag cccaagagca
gcgataagac ccacacctgc 780cctccctgtc cagctccaga actgctggga ggacctagcg
tgttcctgtt tccccctaag 840ccaaaagaca ctctgatgat ttccaggact cccgaggtga
cctgcgtggt ggtggacgtg 900tctcacgagg accccgaagt gaagttcaac tggtacgtgg
atggcgtgga agtgcataat 960gctaagacaa aaccaagaga ggaacagtac aactccactt
atcgcgtcgt gagcgtgctg 1020accgtgctgc accaggactg gctgaacggg aaggagtata
agtgcaaagt cagtaataag 1080gccctgcctg ctccaatcga aaaaaccatc tctaaggcca
aaggccagcc aagggagccc 1140caggtgtaca cactgccacc cagcagagac gaactgacca
agaaccaggt gtccctgatc 1200tgtctggtga aaggcttcta tcctagtgat attgctgtgg
agtgggaatc aaatggacag 1260ccagagaaca gatacatgac ctggcctcca gtgctggaca
gcgatggcag cttcttcctg 1320tattccaagc tgacagtgga taaatctcga tggcagcagg
ggaacgtgtt tagttgttca 1380gtgatgcatg aagccctgca caatcattac actcagaaga
gcctgtccct gtctcccggc 1440aaa
1443171107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 171Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Val Asn Thr Ala 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr
Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Arg Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
Tyr Thr Thr Pro Pro 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 172321DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 172gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggcaagtca ggacgttaac accgctgtag cttggtatca gcagaaacca 120gggaaagccc
ctaagctcct gatctattct gcatcctttt tgtacagtgg ggtcccatca 180aggttcagtg
gcagtcgatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg
caacttacta ctgtcaacag cattacacta ccccacccac tttcggccaa 300gggaccaaag
tggagatcaa a
3211736PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 173Gln Asp Val Asn Thr Ala 1 5
17418DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 174caggacgtta acaccgct
181759PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 175Gln Gln His Tyr Thr Thr Pro Pro Thr 1
5 17627DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 176caacagcatt
acactacccc acccact
271773PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 177Ser Ala Ser 1 1789DNAArtificial
SequenceDescription of Artificial Sequence Synthetic oligonucleotide
178tctgcatcc
9179120PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 179Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30 Tyr Ile His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr
Thr Arg Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn
Thr Ala Tyr 65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ser Arg Trp Gly
Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 180360DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 180gaagtgcagc
tggtggagtc tgggggaggc ttggtacagc ctggcgggtc cctgagactc 60tcctgtgcag
cctctggatt caacattaaa gatacttata tccactgggt ccggcaagct 120ccagggaagg
gcctggagtg ggtcgcacgt atttatccca caaatggtta cacacggtat 180gcggactctg
tgaagggccg attcaccatc tccgcagaca cttccaagaa caccgcgtat 240ctgcaaatga
acagtctgag agctgaggac acggccgttt attactgttc aagatggggc 300ggagacggtt
tctacgctat ggactactgg ggccaaggga ccctggtcac cgtctcctca
3601818PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 181Gly Phe Asn Ile Lys Asp Thr Tyr 1 5
18224DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 182ggattcaaca ttaaagatac ttat
2418313PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 183Ser Arg Trp Gly Gly Asp Gly
Phe Tyr Ala Met Asp Tyr 1 5 10
18439DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 184tcaagatggg gcggagacgg tttctacgct atggactac
391858PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 185Ile Tyr Pro Thr Asn Gly Tyr Thr 1
5 18624DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 186atttatccca
caaatggtta caca
24187110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 187Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys 1 5 10
15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30 Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35
40 45 Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55
60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His 65 70 75
80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95 Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100
105 110 188330DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 188gctccagaac
tgctgggagg acctagcgtg ttcctgtttc cccctaagcc aaaagacact 60ctgatgattt
ccaggactcc cgaggtgacc tgcgtggtgg tggacgtgtc tcacgaggac 120cccgaagtga
agttcaactg gtacgtggat ggcgtggaag tgcataatgc taagacaaaa 180ccaagagagg
aacagtacaa ctccacttat cgcgtcgtga gcgtgctgac cgtgctgcac 240caggactggc
tgaacgggaa ggagtataag tgcaaagtca gtaataaggc cctgcctgct 300ccaatcgaaa
aaaccatctc taaggccaaa
330189106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 189Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp 1 5 10
15 Glu Leu Thr Lys Asn Gln Val Ser Leu Ile Cys Leu Val Lys Gly Phe
20 25 30 Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35
40 45 Asn Arg Tyr Met Thr Trp Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55
60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly 65 70 75
80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95 Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 100 105
190318DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 190ggccagccaa gggagcccca ggtgtacaca ctgccaccca
gcagagacga actgaccaag 60aaccaggtgt ccctgatctg tctggtgaaa ggcttctatc
ctagtgatat tgctgtggag 120tgggaatcaa atggacagcc agagaacaga tacatgacct
ggcctccagt gctggacagc 180gatggcagct tcttcctgta ttccaagctg acagtggata
aatctcgatg gcagcagggg 240aacgtgttta gttgttcagt gatgcatgaa gccctgcaca
atcattacac tcagaagagc 300ctgtccctgt ctcccggc
318191214PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 191Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Val Asn Thr Ala 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr
Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Arg Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
Tyr Thr Thr Pro Pro 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110 Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115
120 125 Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln 145 150 155
160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175 Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180
185 190 Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 195 200
205 Phe Asn Arg Gly Glu Cys 210
192642DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 192gatattcaga tgacccagtc ccctagctcc ctgtccgctt
ctgtgggcga cagggtcact 60atcacctgcc gcgcatctca ggatgtgaac accgcagtcg
cctggtacca gcagaagcct 120gggaaagctc caaagctgct gatctacagt gcatcattcc
tgtattcagg agtgcccagc 180cggtttagcg gcagcagatc tggcaccgac ttcacactga
ctatctctag tctgcagcct 240gaggattttg ccacatacta ttgccagcag cactatacca
caccccctac tttcggccag 300gggaccaaag tggagatcaa gcgaactgtg gccgctccaa
gtgtcttcat ttttccaccc 360agcgacgaac agctgaaatc cggcacagct tctgtggtct
gtctgctgaa caacttctac 420cccagagagg ccaaagtgca gtggaaggtc gataacgctc
tgcagagtgg caacagccag 480gagagcgtga cagaacagga ctccaaagat tctacttata
gtctgtcaag caccctgaca 540ctgagcaagg cagactacga aaagcataaa gtgtatgcct
gtgaggtgac ccatcagggg 600ctgtcttctc ccgtgaccaa gtctttcaac cgaggcgaat
gt 642193107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 193Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Val Asn Thr Ala 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr
Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Arg Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
Tyr Thr Thr Pro Pro 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 194321DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 194gatattcaga
tgacccagtc ccctagctcc ctgtccgctt ctgtgggcga cagggtcact 60atcacctgcc
gcgcatctca ggatgtgaac accgcagtcg cctggtacca gcagaagcct 120gggaaagctc
caaagctgct gatctacagt gcatcattcc tgtattcagg agtgcccagc 180cggtttagcg
gcagcagatc tggcaccgac ttcacactga ctatctctag tctgcagcct 240gaggattttg
ccacatacta ttgccagcag cactatacca caccccctac tttcggccag 300gggaccaaag
tggagatcaa g
3211956PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 195Gln Asp Val Asn Thr Ala 1 5
19618DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 196caggatgtga acaccgca
181979PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 197Gln Gln His Tyr Thr Thr Pro Pro Thr 1
5 19827DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 198cagcagcact
ataccacacc ccctact
271993PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 199Ser Ala Ser 1 2009DNAArtificial
SequenceDescription of Artificial Sequence Synthetic oligonucleotide
200agtgcatca
9201107PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 201Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu 1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30 Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35
40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser 50 55
60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu 65 70 75
80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95 Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 100 105
202321DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 202cgaactgtgg ccgctccaag tgtcttcatt tttccaccca
gcgacgaaca gctgaaatcc 60ggcacagctt ctgtggtctg tctgctgaac aacttctacc
ccagagaggc caaagtgcag 120tggaaggtcg ataacgctct gcagagtggc aacagccagg
agagcgtgac agaacaggac 180tccaaagatt ctacttatag tctgtcaagc accctgacac
tgagcaaggc agactacgaa 240aagcataaag tgtatgcctg tgaggtgacc catcaggggc
tgtcttctcc cgtgaccaag 300tctttcaacc gaggcgaatg t
321203448PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 203Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Thr Asp Tyr 20 25
30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala
Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50
55 60 Lys Gly Arg Phe Thr Leu
Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110 Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115
120 125 Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp 145 150 155
160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175 Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180
185 190 Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro 195 200
205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys 210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225
230 235 240 Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245
250 255 Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp 260 265
270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn 275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290
295 300 Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310
315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys 325 330
335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Val 340 345 350 Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Leu 355
360 365 Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375
380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Leu Thr
Trp Pro Pro Val Leu 385 390 395
400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415 Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420
425 430 Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445 2041344DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 204gaagtgcagc
tggtcgaatc tggaggagga ctggtgcagc caggagggtc cctgcgcctg 60tcttgcgccg
ctagtggctt cacttttacc gactacacca tggattgggt gcgacaggca 120cctggaaagg
gcctggagtg ggtcgccgat gtgaacccaa atagcggagg ctccatctac 180aaccagcggt
tcaagggccg gttcaccctg tcagtggacc ggagcaaaaa caccctgtat 240ctgcagatga
atagcctgcg agccgaagat actgctgtgt actattgcgc ccggaatctg 300gggccctcct
tctactttga ctattggggg cagggaactc tggtcaccgt gagctccgcc 360tccaccaagg
gaccttctgt gttcccactg gctccctcta gtaaatccac atctggggga 420actgcagccc
tgggctgtct ggtgaaggac tacttcccag agcccgtcac agtgtcttgg 480aacagtggcg
ctctgacttc tggggtccac acctttcctg cagtgctgca gtcaagcggg 540ctgtacagcc
tgtcctctgt ggtcaccgtg ccaagttcaa gcctgggaac acagacttat 600atctgcaacg
tgaatcacaa gccatccaat acaaaagtcg acaagaaagt ggaacccaag 660tcttgtgata
aaacccatac atgcccccct tgtcctgcac cagagctgct gggaggacca 720agcgtgttcc
tgtttccacc caagcctaaa gatacactga tgattagtag gaccccagaa 780gtcacatgcg
tggtcgtgga cgtgagccac gaggaccccg aagtcaagtt taactggtac 840gtggacggcg
tcgaggtgca taatgccaag actaaaccca gggaggaaca gtacaacagt 900acctatcgcg
tcgtgtcagt cctgacagtg ctgcatcagg attggctgaa cgggaaagag 960tataagtgca
aagtgagcaa taaggctctg cccgcaccta tcgagaaaac aatttccaag 1020gcaaaaggac
agcctagaga accacaggtg tacgtgctgc ctccatcaag ggatgagctg 1080acaaagaacc
aggtcagcct gctgtgtctg gtgaaaggat tctatccctc tgacattgct 1140gtggagtggg
aaagtaatgg ccagcctgag aacaattacc tgacctggcc ccctgtgctg 1200gactcagatg
gcagcttctt tctgtatagc aagctgaccg tcgacaaatc ccggtggcag 1260caggggaatg
tgtttagttg ttcagtcatg cacgaggcac tgcacaacca ttacacccag 1320aagtcactgt
cactgtcacc aggg
1344205119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 205Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr
20 25 30 Thr Met
Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Asp Val Asn Pro Asn Ser
Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55
60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys
Asn Thr Leu Tyr 65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Asn
Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100
105 110 Thr Leu Val Thr Val Ser Ser
115 206357DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 206gaagtgcagc
tggtcgaatc tggaggagga ctggtgcagc caggagggtc cctgcgcctg 60tcttgcgccg
ctagtggctt cacttttacc gactacacca tggattgggt gcgacaggca 120cctggaaagg
gcctggagtg ggtcgccgat gtgaacccaa atagcggagg ctccatctac 180aaccagcggt
tcaagggccg gttcaccctg tcagtggacc ggagcaaaaa caccctgtat 240ctgcagatga
atagcctgcg agccgaagat actgctgtgt actattgcgc ccggaatctg 300gggccctcct
tctactttga ctattggggg cagggaactc tggtcaccgt gagctcc
3572078PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 207Gly Phe Thr Phe Thr Asp Tyr Thr 1 5
20824DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 208ggcttcactt ttaccgacta cacc
2420912PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 209Ala Arg Asn Leu Gly Pro Ser
Phe Tyr Phe Asp Tyr 1 5 10
21036DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 210gcccggaatc tggggccctc cttctacttt gactat
362118PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 211Val Asn Pro Asn Ser Gly Gly Ser 1
5 21224DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 212gtgaacccaa
atagcggagg ctcc
2421398PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 213Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45 Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr 65 70 75
80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95 Lys Val
214294DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 214gcctccacca agggaccttc tgtgttccca ctggctccct
ctagtaaatc cacatctggg 60ggaactgcag ccctgggctg tctggtgaag gactacttcc
cagagcccgt cacagtgtct 120tggaacagtg gcgctctgac ttctggggtc cacacctttc
ctgcagtgct gcagtcaagc 180gggctgtaca gcctgtcctc tgtggtcacc gtgccaagtt
caagcctggg aacacagact 240tatatctgca acgtgaatca caagccatcc aatacaaaag
tcgacaagaa agtg 294215110PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 215Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5
10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 20 25
30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr 35 40 45 Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50
55 60 Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His 65 70
75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90
95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
100 105 110 216330DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
216gcaccagagc tgctgggagg accaagcgtg ttcctgtttc cacccaagcc taaagataca
60ctgatgatta gtaggacccc agaagtcaca tgcgtggtcg tggacgtgag ccacgaggac
120cccgaagtca agtttaactg gtacgtggac ggcgtcgagg tgcataatgc caagactaaa
180cccagggagg aacagtacaa cagtacctat cgcgtcgtgt cagtcctgac agtgctgcat
240caggattggc tgaacgggaa agagtataag tgcaaagtga gcaataaggc tctgcccgca
300cctatcgaga aaacaatttc caaggcaaaa
330217106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 217Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Leu
Pro Pro Ser Arg Asp 1 5 10
15 Glu Leu Thr Lys Asn Gln Val Ser Leu Leu Cys Leu Val Lys Gly Phe
20 25 30 Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35
40 45 Asn Asn Tyr Leu Thr Trp Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55
60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly 65 70 75
80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95 Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 100 105
218318DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 218ggacagccta gagaaccaca ggtgtacgtg ctgcctccat
caagggatga gctgacaaag 60aaccaggtca gcctgctgtg tctggtgaaa ggattctatc
cctctgacat tgctgtggag 120tgggaaagta atggccagcc tgagaacaat tacctgacct
ggccccctgt gctggactca 180gatggcagct tctttctgta tagcaagctg accgtcgaca
aatcccggtg gcagcagggg 240aatgtgttta gttgttcagt catgcacgag gcactgcaca
accattacac ccagaagtca 300ctgtcactgt caccaggg
318219448PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 219Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Thr Asp Tyr 20 25
30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala
Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50
55 60 Lys Gly Arg Phe Thr Leu
Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110 Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115
120 125 Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp 145 150 155
160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175 Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180
185 190 Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro 195 200
205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys 210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225
230 235 240 Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245
250 255 Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp 260 265
270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn 275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290
295 300 Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310
315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys 325 330
335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Val 340 345 350 Tyr
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355
360 365 Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375
380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu 385 390 395
400 Asp Ser Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys
405 410 415 Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420
425 430 Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445 2201344DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 220gaagtgcagc
tggtcgaatc tggaggagga ctggtgcagc caggagggtc cctgcgcctg 60tcttgcgccg
ctagtggctt cacttttacc gactacacca tggattgggt gcgacaggca 120cctggaaagg
gcctggagtg ggtcgccgat gtgaacccaa atagcggagg ctccatctac 180aaccagcggt
tcaagggccg gttcaccctg tcagtggacc ggagcaaaaa caccctgtat 240ctgcagatga
atagcctgcg agccgaagat actgctgtgt actattgcgc ccggaatctg 300gggccctcct
tctactttga ctattggggg cagggaactc tggtcaccgt gagctccgcc 360tccaccaagg
gaccttctgt gttcccactg gctccctcta gtaaatccac atctggggga 420actgcagccc
tgggctgtct ggtgaaggac tacttcccag agcccgtcac agtgtcttgg 480aacagtggcg
ctctgacttc tggggtccac acctttcctg cagtgctgca gtcaagcggg 540ctgtacagcc
tgtcctctgt ggtcaccgtg ccaagttcaa gcctgggaac acagacttat 600atctgcaacg
tgaatcacaa gccatccaat acaaaagtcg acaagaaagt ggaacccaag 660tcttgtgata
aaacccatac atgcccccct tgtcctgcac cagagctgct gggaggacca 720agcgtgttcc
tgtttccacc caagcctaaa gatacactga tgattagtag gaccccagaa 780gtcacatgcg
tggtcgtgga cgtgagccac gaggaccccg aagtcaagtt taactggtac 840gtggacggcg
tcgaggtgca taatgccaag actaaaccca gggaggaaca gtacaacagt 900acctatcgcg
tcgtgtcagt cctgacagtg ctgcatcagg attggctgaa cgggaaagag 960tataagtgca
aagtgagcaa taaggctctg cccgcaccta tcgagaaaac aatttccaag 1020gcaaaaggac
agcctagaga accacaggtg tacgtgtatc ctccatcaag ggatgagctg 1080acaaagaacc
aggtcagcct gacttgtctg gtgaaaggat tctatccctc tgacattgct 1140gtggagtggg
aaagtaatgg ccagcctgag aacaattaca agaccacacc ccctgtgctg 1200gactcagatg
gcagcttcgc gctggtgagc aagctgaccg tcgacaaatc ccggtggcag 1260caggggaatg
tgtttagttg ttcagtcatg cacgaggcac tgcacaacca ttacacccag 1320aagtcactgt
cactgtcacc aggg
1344221119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 221Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr
20 25 30 Thr Met
Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Asp Val Asn Pro Asn Ser
Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55
60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys
Asn Thr Leu Tyr 65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Asn
Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100
105 110 Thr Leu Val Thr Val Ser Ser
115 222357DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 222gaagtgcagc
tggtcgaatc tggaggagga ctggtgcagc caggagggtc cctgcgcctg 60tcttgcgccg
ctagtggctt cacttttacc gactacacca tggattgggt gcgacaggca 120cctggaaagg
gcctggagtg ggtcgccgat gtgaacccaa atagcggagg ctccatctac 180aaccagcggt
tcaagggccg gttcaccctg tcagtggacc ggagcaaaaa caccctgtat 240ctgcagatga
atagcctgcg agccgaagat actgctgtgt actattgcgc ccggaatctg 300gggccctcct
tctactttga ctattggggg cagggaactc tggtcaccgt gagctcc
3572238PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 223Gly Phe Thr Phe Thr Asp Tyr Thr 1 5
22424DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 224ggcttcactt ttaccgacta cacc
2422512PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 225Ala Arg Asn Leu Gly Pro Ser
Phe Tyr Phe Asp Tyr 1 5 10
22636DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 226gcccggaatc tggggccctc cttctacttt gactat
362278PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 227Val Asn Pro Asn Ser Gly Gly Ser 1
5 22824DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 228gtgaacccaa
atagcggagg ctcc
2422998PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 229Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45 Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr 65 70 75
80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95 Lys Val
230294DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 230gcctccacca agggaccttc tgtgttccca ctggctccct
ctagtaaatc cacatctggg 60ggaactgcag ccctgggctg tctggtgaag gactacttcc
cagagcccgt cacagtgtct 120tggaacagtg gcgctctgac ttctggggtc cacacctttc
ctgcagtgct gcagtcaagc 180gggctgtaca gcctgtcctc tgtggtcacc gtgccaagtt
caagcctggg aacacagact 240tatatctgca acgtgaatca caagccatcc aatacaaaag
tcgacaagaa agtg 294231110PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 231Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5
10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 20 25
30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr 35 40 45 Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50
55 60 Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His 65 70
75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90
95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
100 105 110 232330DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
232gcaccagagc tgctgggagg accaagcgtg ttcctgtttc cacccaagcc taaagataca
60ctgatgatta gtaggacccc agaagtcaca tgcgtggtcg tggacgtgag ccacgaggac
120cccgaagtca agtttaactg gtacgtggac ggcgtcgagg tgcataatgc caagactaaa
180cccagggagg aacagtacaa cagtacctat cgcgtcgtgt cagtcctgac agtgctgcat
240caggattggc tgaacgggaa agagtataag tgcaaagtga gcaataaggc tctgcccgca
300cctatcgaga aaacaatttc caaggcaaaa
330233106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 233Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr
Pro Pro Ser Arg Asp 1 5 10
15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
20 25 30 Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35
40 45 Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55
60 Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly 65 70 75
80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95 Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 100 105
234318DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 234ggacagccta gagaaccaca ggtgtacgtg tatcctccat
caagggatga gctgacaaag 60aaccaggtca gcctgacttg tctggtgaaa ggattctatc
cctctgacat tgctgtggag 120tgggaaagta atggccagcc tgagaacaat tacaagacca
caccccctgt gctggactca 180gatggcagct tcgcgctggt gagcaagctg accgtcgaca
aatcccggtg gcagcagggg 240aatgtgttta gttgttcagt catgcacgag gcactgcaca
accattacac ccagaagtca 300ctgtcactgt caccaggg
318235450PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 235Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Asn Ile Lys Asp Thr 20 25
30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala
Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile
Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110 Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115
120 125 Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135
140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser 145 150 155
160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175 Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180
185 190 Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys 195 200
205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp 210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225
230 235 240 Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245
250 255 Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu 260 265
270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His 275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290
295 300 Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310
315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu 325 330
335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr 340 345 350 Val
Tyr Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355
360 365 Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375
380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val 385 390 395
400 Leu Asp Ser Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp
405 410 415 Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420
425 430 Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445 Gly Lys 450 2361350DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
236gaggtgcagc tggtggaaag cggaggagga ctggtgcagc caggaggatc tctgcgactg
60agttgcgccg cttcaggatt caacatcaag gacacctaca ttcactgggt gcgacaggct
120ccaggaaaag gactggagtg ggtggctcga atctatccca ctaatggata cacccggtat
180gccgactccg tgaaggggag gtttactatt agcgccgata catccaaaaa cactgcttac
240ctgcagatga acagcctgcg agccgaagat accgctgtgt actattgcag tcgatgggga
300ggagacggat tctacgctat ggattattgg ggacagggga ccctggtgac agtgagctcc
360gcctctacca agggccccag tgtgtttccc ctggctcctt ctagtaaatc cacctctgga
420gggacagccg ctctgggatg tctggtgaag gactatttcc ccgagcctgt gaccgtgagt
480tggaactcag gcgccctgac aagcggagtg cacacttttc ctgctgtgct gcagtcaagc
540gggctgtact ccctgtcctc tgtggtgaca gtgccaagtt caagcctggg cacacagact
600tatatctgca acgtgaatca taagccctca aatacaaaag tggacaagaa agtggagccc
660aagagctgtg ataagaccca cacctgccct ccctgtccag ctccagaact gctgggagga
720cctagcgtgt tcctgtttcc ccctaagcca aaagacactc tgatgatttc caggactccc
780gaggtgacct gcgtggtggt ggacgtgtct cacgaggacc ccgaagtgaa gttcaactgg
840tacgtggatg gcgtggaagt gcataatgct aagacaaaac caagagagga acagtacaac
900tccacttatc gcgtcgtgag cgtgctgacc gtgctgcacc aggactggct gaacgggaag
960gagtataagt gcaaagtcag taataaggcc ctgcctgctc caatcgaaaa aaccatctct
1020aaggccaaag gccagccaag ggagccccag gtgtacgtgt acccacccag cagagacgaa
1080ctgaccaaga accaggtgtc cctgacatgt ctggtgaaag gcttctatcc tagtgatatt
1140gctgtggagt gggaatcaaa tggacagcca gagaacaatt acaagaccac acctccagtg
1200ctggacagcg atggcagctt cgccctggtg tccaagctga cagtggataa atctcgatgg
1260cagcagggga acgtgtttag ttgttcagtg atgcatgaag ccctgcacaa tcattacact
1320cagaagagcc tgtccctgtc tcccggcaaa
1350237120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 237Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30 Tyr Ile
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Arg Ile Tyr Pro Thr Asn
Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr 65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ser Arg Trp
Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 238360DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 238gaggtgcagc
tggtggaaag cggaggagga ctggtgcagc caggaggatc tctgcgactg 60agttgcgccg
cttcaggatt caacatcaag gacacctaca ttcactgggt gcgacaggct 120ccaggaaaag
gactggagtg ggtggctcga atctatccca ctaatggata cacccggtat 180gccgactccg
tgaaggggag gtttactatt agcgccgata catccaaaaa cactgcttac 240ctgcagatga
acagcctgcg agccgaagat accgctgtgt actattgcag tcgatgggga 300ggagacggat
tctacgctat ggattattgg ggacagggga ccctggtgac agtgagctcc
3602398PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 239Gly Phe Asn Ile Lys Asp Thr Tyr 1 5
24024DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 240ggattcaaca tcaaggacac ctac
2424113PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 241Ser Arg Trp Gly Gly Asp Gly
Phe Tyr Ala Met Asp Tyr 1 5 10
24239DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 242agtcgatggg gaggagacgg attctacgct atggattat
392438PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 243Ile Tyr Pro Thr Asn Gly Tyr Thr 1
5 24424DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 244atctatccca
ctaatggata cacc
2424598PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 245Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45 Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr 65 70 75
80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95 Lys Val
246294DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 246gcctctacca agggccccag tgtgtttccc ctggctcctt
ctagtaaatc cacctctgga 60gggacagccg ctctgggatg tctggtgaag gactatttcc
ccgagcctgt gaccgtgagt 120tggaactcag gcgccctgac aagcggagtg cacacttttc
ctgctgtgct gcagtcaagc 180gggctgtact ccctgtcctc tgtggtgaca gtgccaagtt
caagcctggg cacacagact 240tatatctgca acgtgaatca taagccctca aatacaaaag
tggacaagaa agtg 294247110PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 247Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5
10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 20 25
30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr 35 40 45 Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50
55 60 Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His 65 70
75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90
95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
100 105 110 248330DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
248gctccagaac tgctgggagg acctagcgtg ttcctgtttc cccctaagcc aaaagacact
60ctgatgattt ccaggactcc cgaggtgacc tgcgtggtgg tggacgtgtc tcacgaggac
120cccgaagtga agttcaactg gtacgtggat ggcgtggaag tgcataatgc taagacaaaa
180ccaagagagg aacagtacaa ctccacttat cgcgtcgtga gcgtgctgac cgtgctgcac
240caggactggc tgaacgggaa ggagtataag tgcaaagtca gtaataaggc cctgcctgct
300ccaatcgaaa aaaccatctc taaggccaaa
330249106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 249Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr
Pro Pro Ser Arg Asp 1 5 10
15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
20 25 30 Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35
40 45 Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55
60 Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly 65 70 75
80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95 Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 100 105
250318DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 250ggccagccaa gggagcccca ggtgtacgtg tacccaccca
gcagagacga actgaccaag 60aaccaggtgt ccctgacatg tctggtgaaa ggcttctatc
ctagtgatat tgctgtggag 120tgggaatcaa atggacagcc agagaacaat tacaagacca
cacctccagt gctggacagc 180gatggcagct tcgccctggt gtccaagctg acagtggata
aatctcgatg gcagcagggg 240aacgtgttta gttgttcagt gatgcatgaa gccctgcaca
atcattacac tcagaagagc 300ctgtccctgt ctcccggc
318251232PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 251Glu Pro Lys Ser Ser Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5
10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro 20 25
30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val 35 40 45 Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50
55 60 Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70
75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln 85 90
95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110 Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115
120 125 Arg Glu Pro Gln Val Tyr Val
Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135
140 Lys Asn Gln Val Ser Leu Leu Cys Leu Val Lys Gly
Phe Tyr Pro Ser 145 150 155
160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175 Leu Thr Trp
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180
185 190 Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe 195 200
205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys 210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys 225 230
252696DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 252gaacctaaaa gcagcgacaa gacccacaca tgcccccctt
gtccagctcc agaactgctg 60ggaggaccaa gcgtgttcct gtttccaccc aagcccaaag
atacactgat gatcagccga 120actcccgagg tcacctgcgt ggtcgtggac gtgtcccacg
aggaccccga agtcaagttc 180aactggtacg tggacggcgt cgaagtgcat aatgcaaaga
ctaaaccacg ggaggaacag 240tacaactcta catatagagt cgtgagtgtc ctgactgtgc
tgcatcagga ttggctgaac 300ggcaaagagt ataagtgcaa agtgtctaat aaggccctgc
ctgctccaat cgagaaaact 360attagtaagg caaaagggca gcccagggaa cctcaggtct
acgtgctgcc tccaagtcgc 420gacgagctga ccaagaacca ggtctcactg ctgtgtctgg
tgaaaggatt ctatccttcc 480gatattgccg tggagtggga atctaatggc cagccagaga
acaattacct gacctggccc 540cctgtgctgg acagcgatgg gtccttcttt ctgtattcaa
agctgacagt ggacaaaagc 600agatggcagc agggaaacgt ctttagctgt tccgtgatgc
acgaagccct gcacaatcat 660tacacccaga agtctctgag tctgtcacct ggcaaa
696253110PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 253Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5
10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 20 25
30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr 35 40 45 Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50
55 60 Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His 65 70
75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90
95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
100 105 110 254330DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
254gctccagaac tgctgggagg accaagcgtg ttcctgtttc cacccaagcc caaagataca
60ctgatgatca gccgaactcc cgaggtcacc tgcgtggtcg tggacgtgtc ccacgaggac
120cccgaagtca agttcaactg gtacgtggac ggcgtcgaag tgcataatgc aaagactaaa
180ccacgggagg aacagtacaa ctctacatat agagtcgtga gtgtcctgac tgtgctgcat
240caggattggc tgaacggcaa agagtataag tgcaaagtgt ctaataaggc cctgcctgct
300ccaatcgaga aaactattag taaggcaaaa
330255106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 255Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Leu
Pro Pro Ser Arg Asp 1 5 10
15 Glu Leu Thr Lys Asn Gln Val Ser Leu Leu Cys Leu Val Lys Gly Phe
20 25 30 Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35
40 45 Asn Asn Tyr Leu Thr Trp Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55
60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly 65 70 75
80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95 Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 100 105
256318DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 256gggcagccca gggaacctca ggtctacgtg ctgcctccaa
gtcgcgacga gctgaccaag 60aaccaggtct cactgctgtg tctggtgaaa ggattctatc
cttccgatat tgccgtggag 120tgggaatcta atggccagcc agagaacaat tacctgacct
ggccccctgt gctggacagc 180gatgggtcct tctttctgta ttcaaagctg acagtggaca
aaagcagatg gcagcaggga 240aacgtcttta gctgttccgt gatgcacgaa gccctgcaca
atcattacac ccagaagtct 300ctgagtctgt cacctggc
318257475PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 257Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln
Asp Val Ser Ile Gly 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr
Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Tyr Ile Tyr Pro Tyr 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110 Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu 115
120 125 Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly Ser Leu Arg Leu Ser Cys 130 135
140 Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr Thr Met
Asp Trp Val Arg 145 150 155
160 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Asp Val Asn Pro Asn
165 170 175 Ser Gly Gly
Ser Ile Tyr Asn Gln Arg Phe Lys Gly Arg Phe Thr Leu 180
185 190 Ser Val Asp Arg Ser Lys Asn Thr
Leu Tyr Leu Gln Met Asn Ser Leu 195 200
205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asn
Leu Gly Pro 210 215 220
Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 225
230 235 240 Ser Ala Ala Glu
Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro 245
250 255 Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro 260 265
270 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr 275 280 285
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 290
295 300 Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 305 310
315 320 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val 325 330
335 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser 340 345 350 Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 355
360 365 Gly Gln Pro Arg Glu Pro
Gln Val Tyr Val Tyr Pro Pro Ser Arg Asp 370 375
380 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe 385 390 395
400 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
405 410 415 Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 420
425 430 Ala Leu Val Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly 435 440
445 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr 450 455 460
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470
475 2581425DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 258gacattcaga
tgacccagag ccctagctcc ctgagtgcct cagtcgggga cagggtgact 60atcacctgca
aggcttcaca ggatgtcagc attggcgtgg catggtacca gcagaagcca 120gggaaagcac
ccaagctgct gatctatagc gcctcctaca ggtatacagg cgtgccatcc 180cgcttctctg
gcagtgggtc aggaactgac tttacactga ctatttctag tctgcagccc 240gaagatttcg
ccacatacta ttgccagcag tactatatct acccttatac ttttggccag 300gggaccaaag
tggagattaa gggcggagga ggctccggag gaggagggtc tggaggagga 360ggaagtgagg
tccagctggt ggaatctgga ggaggactgg tgcagccagg agggtccctg 420aggctgtctt
gtgccgctag tggcttcacc tttacagact acacaatgga ttgggtgcgc 480caggcaccag
gaaagggact ggaatgggtc gctgatgtga accctaatag cggaggctcc 540atctacaacc
agcggttcaa aggacggttc accctgtcag tggaccggag caagaacacc 600ctgtatctgc
agatgaacag cctgagagcc gaggatactg ctgtgtacta ttgcgccagg 660aatctgggcc
caagcttcta ctttgactat tgggggcagg gaacactggt cactgtgtca 720agcgcagccg
aacccaaatc ctctgataag actcacacct gcccaccttg tccagctcca 780gagctgctgg
gaggacctag cgtgttcctg tttccaccca agccaaaaga cactctgatg 840atttctagaa
cccctgaagt gacatgtgtg gtcgtggacg tcagtcacga ggaccccgaa 900gtcaaattca
actggtacgt ggatggcgtc gaggtgcata atgccaagac caaaccccga 960gaggaacagt
acaactcaac ctatcgggtc gtgagcgtcc tgacagtgct gcatcaggac 1020tggctgaacg
gcaaggagta taagtgcaaa gtgagcaaca aggctctgcc tgcaccaatc 1080gagaagacca
tttccaaggc taaagggcag ccccgcgaac ctcaggtcta cgtgtatcct 1140ccaagccgag
atgagctgac aaaaaaccag gtctccctga cttgtctggt gaagggattt 1200tacccaagtg
acatcgcagt ggagtgggaa tcaaatggcc agcccgaaaa caattataag 1260accacacccc
ctgtgctgga ctctgatggg agtttcgcac tggtctccaa actgaccgtg 1320gacaagtctc
ggtggcagca gggaaacgtc tttagctgtt ccgtgatgca cgaggccctg 1380cacaatcatt
acacacagaa atctctgagt ctgtcacctg gcaag
1425259107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 259Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly
20 25 30 Val Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Ser Ala Ser Tyr Arg Tyr
Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr
85 90 95 Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 105
260321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 260gacattcaga tgacccagag ccctagctcc
ctgagtgcct cagtcgggga cagggtgact 60atcacctgca aggcttcaca ggatgtcagc
attggcgtgg catggtacca gcagaagcca 120gggaaagcac ccaagctgct gatctatagc
gcctcctaca ggtatacagg cgtgccatcc 180cgcttctctg gcagtgggtc aggaactgac
tttacactga ctatttctag tctgcagccc 240gaagatttcg ccacatacta ttgccagcag
tactatatct acccttatac ttttggccag 300gggaccaaag tggagattaa g
3212616PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 261Gln
Asp Val Ser Ile Gly 1 5 26218DNAArtificial
SequenceDescription of Artificial Sequence Synthetic oligonucleotide
262caggatgtca gcattggc
182639PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 263Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr 1 5
26427DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 264cagcagtact atatctaccc ttatact
272653PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 265Ser
Ala Ser 1 2669DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 266agcgcctcc
9267119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
267Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20
25 30 Thr Met Asp Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln
Arg Phe 50 55 60
Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe
Asp Tyr Trp Gly Gln Gly 100 105
110 Thr Leu Val Thr Val Ser Ser 115
268357DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 268gaggtccagc tggtggaatc tggaggagga ctggtgcagc
caggagggtc cctgaggctg 60tcttgtgccg ctagtggctt cacctttaca gactacacaa
tggattgggt gcgccaggca 120ccaggaaagg gactggaatg ggtcgctgat gtgaacccta
atagcggagg ctccatctac 180aaccagcggt tcaaaggacg gttcaccctg tcagtggacc
ggagcaagaa caccctgtat 240ctgcagatga acagcctgag agccgaggat actgctgtgt
actattgcgc caggaatctg 300ggcccaagct tctactttga ctattggggg cagggaacac
tggtcactgt gtcaagc 3572698PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 269Gly Phe Thr Phe Thr Asp Tyr
Thr 1 5 27024DNAArtificial
SequenceDescription of Artificial Sequence Synthetic oligonucleotide
270ggcttcacct ttacagacta caca
2427112PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 271Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr 1
5 10 27236DNAArtificial
SequenceDescription of Artificial Sequence Synthetic oligonucleotide
272gccaggaatc tgggcccaag cttctacttt gactat
362738PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 273Val Asn Pro Asn Ser Gly Gly Ser 1 5
27424DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 274gtgaacccta atagcggagg ctcc
24275110PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 275Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5
10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val 20 25
30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr 35 40 45 Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50
55 60 Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His 65 70
75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90
95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
100 105 110 276330DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
276gctccagagc tgctgggagg acctagcgtg ttcctgtttc cacccaagcc aaaagacact
60ctgatgattt ctagaacccc tgaagtgaca tgtgtggtcg tggacgtcag tcacgaggac
120cccgaagtca aattcaactg gtacgtggat ggcgtcgagg tgcataatgc caagaccaaa
180ccccgagagg aacagtacaa ctcaacctat cgggtcgtga gcgtcctgac agtgctgcat
240caggactggc tgaacggcaa ggagtataag tgcaaagtga gcaacaaggc tctgcctgca
300ccaatcgaga agaccatttc caaggctaaa
330277106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 277Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr
Pro Pro Ser Arg Asp 1 5 10
15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
20 25 30 Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35
40 45 Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55
60 Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly 65 70 75
80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95 Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 100 105
278318DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 278gggcagcccc gcgaacctca ggtctacgtg tatcctccaa
gccgagatga gctgacaaaa 60aaccaggtct ccctgacttg tctggtgaag ggattttacc
caagtgacat cgcagtggag 120tgggaatcaa atggccagcc cgaaaacaat tataagacca
caccccctgt gctggactct 180gatgggagtt tcgcactggt ctccaaactg accgtggaca
agtctcggtg gcagcaggga 240aacgtcttta gctgttccgt gatgcacgag gccctgcaca
atcattacac acagaaatct 300ctgagtctgt cacctggc
318279450PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 279Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Asn Ile Lys Asp Thr 20 25
30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala
Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile
Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110 Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115
120 125 Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135
140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser 145 150 155
160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175 Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180
185 190 Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys 195 200
205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp 210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225
230 235 240 Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245
250 255 Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu 260 265
270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His 275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290
295 300 Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310
315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu 325 330
335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr 340 345 350 Val
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355
360 365 Leu Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375
380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Leu
Thr Trp Pro Pro Val 385 390 395
400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415 Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420
425 430 Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445 Gly Lys 450 2801350DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
280gaggtgcagc tggtggaaag cggaggagga ctggtgcagc caggaggatc tctgcgactg
60agttgcgccg cttcaggatt caacatcaag gacacctaca ttcactgggt gcgacaggct
120ccaggaaaag gactggagtg ggtggctcga atctatccca ctaatggata cacccggtat
180gccgactccg tgaaggggag gtttactatt agcgccgata catccaaaaa cactgcttac
240ctgcagatga acagcctgcg agccgaagat accgctgtgt actattgcag tcgatgggga
300ggagacggat tctacgctat ggattattgg ggacagggga ccctggtgac agtgagctcc
360gcctctacca agggccccag tgtgtttccc ctggctcctt ctagtaaatc cacctctgga
420gggacagccg ctctgggatg tctggtgaag gactatttcc ccgagcctgt gaccgtgagt
480tggaactcag gcgccctgac aagcggagtg cacacttttc ctgctgtgct gcagtcaagc
540gggctgtact ccctgtcctc tgtggtgaca gtgccaagtt caagcctggg cacacagact
600tatatctgca acgtgaatca taagccctca aatacaaaag tggacaagaa agtggagccc
660aagagctgtg ataagaccca cacctgccct ccctgtccag ctccagaact gctgggagga
720cctagcgtgt tcctgtttcc ccctaagcca aaagacactc tgatgatttc caggactccc
780gaggtgacct gcgtggtggt ggacgtgtct cacgaggacc ccgaagtgaa gttcaactgg
840tacgtggatg gcgtggaagt gcataatgct aagacaaaac caagagagga acagtacaac
900tccacttatc gcgtcgtgag cgtgctgacc gtgctgcacc aggactggct gaacgggaag
960gagtataagt gcaaagtcag taataaggcc ctgcctgctc caatcgaaaa aaccatctct
1020aaggccaaag gccagccaag ggagccccag gtgtacgtgc tgccacccag cagagacgaa
1080ctgaccaaga accaggtgtc cctgctgtgt ctggtgaaag gcttctatcc tagtgatatt
1140gctgtggagt gggaatcaaa tggacagcca gagaacaatt acctgacctg gcctccagtg
1200ctggacagcg atggcagctt cttcctgtat tccaagctga cagtggataa atctcgatgg
1260cagcagggga acgtgtttag ttgttcagtg atgcatgaag ccctgcacaa tcattacact
1320cagaagagcc tgtccctgtc tcccggcaaa
1350281120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 281Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30 Tyr Ile
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Arg Ile Tyr Pro Thr Asn
Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr 65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ser Arg Trp
Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 282360DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 282gaggtgcagc
tggtggaaag cggaggagga ctggtgcagc caggaggatc tctgcgactg 60agttgcgccg
cttcaggatt caacatcaag gacacctaca ttcactgggt gcgacaggct 120ccaggaaaag
gactggagtg ggtggctcga atctatccca ctaatggata cacccggtat 180gccgactccg
tgaaggggag gtttactatt agcgccgata catccaaaaa cactgcttac 240ctgcagatga
acagcctgcg agccgaagat accgctgtgt actattgcag tcgatgggga 300ggagacggat
tctacgctat ggattattgg ggacagggga ccctggtgac agtgagctcc
3602838PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 283Gly Phe Asn Ile Lys Asp Thr Tyr 1 5
28424DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 284ggattcaaca tcaaggacac ctac
2428513PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 285Ser Arg Trp Gly Gly Asp Gly
Phe Tyr Ala Met Asp Tyr 1 5 10
28639DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 286agtcgatggg gaggagacgg attctacgct atggattat
392878PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 287Ile Tyr Pro Thr Asn Gly Tyr Thr 1
5 28824DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 288atctatccca
ctaatggata cacc
2428998PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 289Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45 Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr 65 70 75
80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95 Lys Val
290294DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 290gcctctacca agggccccag tgtgtttccc ctggctcctt
ctagtaaatc cacctctgga 60gggacagccg ctctgggatg tctggtgaag gactatttcc
ccgagcctgt gaccgtgagt 120tggaactcag gcgccctgac aagcggagtg cacacttttc
ctgctgtgct gcagtcaagc 180gggctgtact ccctgtcctc tgtggtgaca gtgccaagtt
caagcctggg cacacagact 240tatatctgca acgtgaatca taagccctca aatacaaaag
tggacaagaa agtg 294291110PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 291Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5
10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 20 25
30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr 35 40 45 Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50
55 60 Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His 65 70
75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90
95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
100 105 110 292330DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
292gctccagaac tgctgggagg acctagcgtg ttcctgtttc cccctaagcc aaaagacact
60ctgatgattt ccaggactcc cgaggtgacc tgcgtggtgg tggacgtgtc tcacgaggac
120cccgaagtga agttcaactg gtacgtggat ggcgtggaag tgcataatgc taagacaaaa
180ccaagagagg aacagtacaa ctccacttat cgcgtcgtga gcgtgctgac cgtgctgcac
240caggactggc tgaacgggaa ggagtataag tgcaaagtca gtaataaggc cctgcctgct
300ccaatcgaaa aaaccatctc taaggccaaa
330293106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 293Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Leu
Pro Pro Ser Arg Asp 1 5 10
15 Glu Leu Thr Lys Asn Gln Val Ser Leu Leu Cys Leu Val Lys Gly Phe
20 25 30 Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35
40 45 Asn Asn Tyr Leu Thr Trp Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55
60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly 65 70 75
80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95 Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 100 105
294318DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 294ggccagccaa gggagcccca ggtgtacgtg ctgccaccca
gcagagacga actgaccaag 60aaccaggtgt ccctgctgtg tctggtgaaa ggcttctatc
ctagtgatat tgctgtggag 120tgggaatcaa atggacagcc agagaacaat tacctgacct
ggcctccagt gctggacagc 180gatggcagct tcttcctgta ttccaagctg acagtggata
aatctcgatg gcagcagggg 240aacgtgttta gttgttcagt gatgcatgaa gccctgcaca
atcattacac tcagaagagc 300ctgtccctgt ctcccggc
318295480PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 295Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Val Asn Thr Ala 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr
Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Arg Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
Tyr Thr Thr Pro Pro 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Ser Gly Gly
100 105 110 Gly Ser
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Glu 115
120 125 Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly Ser 130 135
140 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile
Lys Asp Thr Tyr 145 150 155
160 Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala
165 170 175 Arg Ile Tyr
Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys 180
185 190 Gly Arg Phe Thr Ile Ser Ala Asp
Thr Ser Lys Asn Thr Ala Tyr Leu 195 200
205 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Ser 210 215 220
Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly 225
230 235 240 Thr Leu Val Thr
Val Ser Ser Ala Ala Glu Pro Lys Ser Ser Asp Lys 245
250 255 Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro 260 265
270 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser 275 280 285
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 290
295 300 Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 305 310
315 320 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 325 330
335 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu 340 345 350 Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 355
360 365 Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Val 370 375
380 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Leu 385 390 395
400 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
405 410 415 Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Leu Thr Trp Pro Pro Val Leu 420
425 430 Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys 435 440
445 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu 450 455 460
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465
470 475 480
2961440DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 296gacattcaga tgacacagag ccccagctcc ctgagtgctt
cagtcggcga cagggtgact 60atcacctgcc gcgcatccca ggatgtcaac accgctgtgg
catggtacca gcagaagcct 120ggaaaagccc caaagctgct gatctacagc gcttccttcc
tgtattctgg cgtgccaagt 180cggttttctg gaagtagatc aggcactgac ttcacactga
ctatctctag tctgcagccc 240gaagattttg ccacctacta ttgccagcag cactatacca
caccccctac attcggacag 300ggcactaaag tggagattaa gggcgggtca ggcggaggga
gcggaggagg gtccggagga 360gggtctggag gagggagtgg agaggtccag ctggtggaat
ctggaggagg actggtgcag 420cctggaggct cactgcgact gagctgtgcc gcttccggct
ttaacatcaa agacacatac 480attcattggg tcaggcaggc accagggaag ggactggaat
gggtggcccg catctatccc 540acaaatgggt acactcgata tgccgacagc gtgaaaggac
ggtttaccat ttctgctgat 600accagtaaga acacagcata cctgcagatg aacagcctgc
gcgcagagga tacagccgtg 660tactattgca gtcgatgggg gggagacggc ttctacgcca
tggattattg gggccagggg 720actctggtca ccgtgtcaag cgcagccgaa cctaaatcct
ctgacaagac ccacacatgc 780ccaccctgtc ctgctccaga gctgctggga ggaccatccg
tgttcctgtt tcctccaaag 840cctaaagata cactgatgat tagccgcact cccgaagtca
cctgtgtggt cgtggacgtg 900tcccacgagg accccgaagt caagttcaac tggtacgtgg
acggcgtcga ggtgcataat 960gccaagacta aaccaagaga ggaacagtac aattcaacct
atagggtcgt gagcgtcctg 1020acagtgctgc atcaggattg gctgaacggc aaggagtata
agtgcaaagt gtctaacaag 1080gccctgcccg ctcctatcga gaagactatt agcaaggcaa
aagggcagcc acgggaaccc 1140caggtctacg tgctgccccc tagcagagac gagctgacca
aaaaccaggt ctccctgctg 1200tgtctggtga agggctttta tcctagtgat atcgctgtgg
agtgggaatc aaatgggcag 1260ccagaaaaca attacctgac atggccaccc gtgctggaca
gcgatgggtc cttctttctg 1320tattccaaac tgactgtgga caagtctaga tggcagcagg
gaaacgtctt cagctgttcc 1380gtgatgcacg aggccctgca caatcattac acccagaagt
ctctgagtct gtcacccggc 1440297107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 297Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Val Asn Thr Ala 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr
Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Arg Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
Tyr Thr Thr Pro Pro 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 298321DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 298gacattcaga
tgacacagag ccccagctcc ctgagtgctt cagtcggcga cagggtgact 60atcacctgcc
gcgcatccca ggatgtcaac accgctgtgg catggtacca gcagaagcct 120ggaaaagccc
caaagctgct gatctacagc gcttccttcc tgtattctgg cgtgccaagt 180cggttttctg
gaagtagatc aggcactgac ttcacactga ctatctctag tctgcagccc 240gaagattttg
ccacctacta ttgccagcag cactatacca caccccctac attcggacag 300ggcactaaag
tggagattaa g
3212996PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 299Gln Asp Val Asn Thr Ala 1 5
30018DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 300caggatgtca acaccgct
183019PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 301Gln Gln His Tyr Thr Thr Pro Pro Thr 1
5 30227DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 302cagcagcact
ataccacacc ccctaca
273033PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 303Ser Ala Ser 1 3049DNAArtificial
SequenceDescription of Artificial Sequence Synthetic oligonucleotide
304agcgcttcc
9305120PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 305Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30 Tyr Ile His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr
Thr Arg Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn
Thr Ala Tyr 65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ser Arg Trp Gly
Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 306360DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 306gaggtccagc
tggtggaatc tggaggagga ctggtgcagc ctggaggctc actgcgactg 60agctgtgccg
cttccggctt taacatcaaa gacacataca ttcattgggt caggcaggca 120ccagggaagg
gactggaatg ggtggcccgc atctatccca caaatgggta cactcgatat 180gccgacagcg
tgaaaggacg gtttaccatt tctgctgata ccagtaagaa cacagcatac 240ctgcagatga
acagcctgcg cgcagaggat acagccgtgt actattgcag tcgatggggg 300ggagacggct
tctacgccat ggattattgg ggccagggga ctctggtcac cgtgtcaagc
3603078PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 307Gly Phe Asn Ile Lys Asp Thr Tyr 1 5
30824DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 308ggctttaaca tcaaagacac atac
2430913PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 309Ser Arg Trp Gly Gly Asp Gly
Phe Tyr Ala Met Asp Tyr 1 5 10
31039DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 310agtcgatggg ggggagacgg cttctacgcc atggattat
393118PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 311Ile Tyr Pro Thr Asn Gly Tyr Thr 1
5 31224DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 312atctatccca
caaatgggta cact
24313110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 313Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys 1 5 10
15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30 Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35
40 45 Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55
60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His 65 70 75
80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95 Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100
105 110 314330DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 314gctccagagc
tgctgggagg accatccgtg ttcctgtttc ctccaaagcc taaagataca 60ctgatgatta
gccgcactcc cgaagtcacc tgtgtggtcg tggacgtgtc ccacgaggac 120cccgaagtca
agttcaactg gtacgtggac ggcgtcgagg tgcataatgc caagactaaa 180ccaagagagg
aacagtacaa ttcaacctat agggtcgtga gcgtcctgac agtgctgcat 240caggattggc
tgaacggcaa ggagtataag tgcaaagtgt ctaacaaggc cctgcccgct 300cctatcgaga
agactattag caaggcaaaa
330315106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 315Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Leu
Pro Pro Ser Arg Asp 1 5 10
15 Glu Leu Thr Lys Asn Gln Val Ser Leu Leu Cys Leu Val Lys Gly Phe
20 25 30 Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35
40 45 Asn Asn Tyr Leu Thr Trp Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55
60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly 65 70 75
80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95 Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 100 105
316318DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 316gggcagccac gggaacccca ggtctacgtg ctgcccccta
gcagagacga gctgaccaaa 60aaccaggtct ccctgctgtg tctggtgaag ggcttttatc
ctagtgatat cgctgtggag 120tgggaatcaa atgggcagcc agaaaacaat tacctgacat
ggccacccgt gctggacagc 180gatgggtcct tctttctgta ttccaaactg actgtggaca
agtctagatg gcagcaggga 240aacgtcttca gctgttccgt gatgcacgag gccctgcaca
atcattacac ccagaagtct 300ctgagtctgt cacccggc
318317214PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 317Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Val Asn Thr Ala 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr
Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Arg Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
Tyr Thr Thr Pro Pro 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110 Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115
120 125 Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln 145 150 155
160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175 Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180
185 190 Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 195 200
205 Phe Asn Arg Gly Glu Cys 210
318642DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 318gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca ggacgttaac accgctgtag
cttggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctattct gcatcctttt
tgtacagtgg ggtcccatca 180aggttcagtg gcagtcgatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg caacttacta ctgtcaacag cattacacta
ccccacccac tttcggccaa 300gggaccaaag tggagatcaa acgaactgtg gctgcaccat
ctgtcttcat cttcccgcca 360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt
gcctgctgaa taacttctat 420cccagagagg ccaaagtaca gtggaaggtg gataacgccc
tccaatcggg taactcccaa 480gagagtgtca cagagcagga cagcaaggac agcacctaca
gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa gtctacgcct
gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt
gt 642319107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 319Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Val Asn Thr Ala 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr
Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Arg Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
Tyr Thr Thr Pro Pro 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 320321DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 320gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggcaagtca ggacgttaac accgctgtag cttggtatca gcagaaacca 120gggaaagccc
ctaagctcct gatctattct gcatcctttt tgtacagtgg ggtcccatca 180aggttcagtg
gcagtcgatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg
caacttacta ctgtcaacag cattacacta ccccacccac tttcggccaa 300gggaccaaag
tggagatcaa a
3213216PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 321Gln Asp Val Asn Thr Ala 1 5
32218DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 322caggacgtta acaccgct
183239PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 323Gln Gln His Tyr Thr Thr Pro Pro Thr 1
5 32427DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 324caacagcatt
acactacccc acccact
273253PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 325Ser Ala Ser 1 3269DNAArtificial
SequenceDescription of Artificial Sequence Synthetic oligonucleotide
326tctgcatcc
9327107PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 327Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu 1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30 Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35
40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser 50 55
60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu 65 70 75
80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95 Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 100 105
328321DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 328cgaactgtgg ctgcaccatc tgtcttcatc ttcccgccat
ctgatgagca gttgaaatct 60ggaactgcct ctgttgtgtg cctgctgaat aacttctatc
ccagagaggc caaagtacag 120tggaaggtgg ataacgccct ccaatcgggt aactcccaag
agagtgtcac agagcaggac 180agcaaggaca gcacctacag cctcagcagc accctgacgc
tgagcaaagc agactacgag 240aaacacaaag tctacgcctg cgaagtcacc catcagggcc
tgagctcgcc cgtcacaaag 300agcttcaaca ggggagagtg t
321329231PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 329Glu Pro Lys Ser Ser Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5
10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro 20 25
30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val 35 40 45 Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50
55 60 Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70
75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln 85 90
95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110 Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115
120 125 Arg Glu Pro Gln Val Tyr Val
Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135
140 Lys Asn Gln Val Ser Leu Leu Cys Leu Val Lys Gly
Phe Tyr Pro Ser 145 150 155
160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175 Leu Thr Trp
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180
185 190 Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe 195 200
205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys 210 215 220
Ser Leu Ser Leu Ser Pro Gly 225 230
330693DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 330gaacctaaat ccagcgacaa gacccacaca tgcccccctt
gtccagctcc agaactgctg 60ggaggaccaa gcgtgttcct gtttccaccc aagcccaaag
atacactgat gatcagccga 120actcccgagg tcacctgcgt ggtcgtggac gtgtcccacg
aggaccccga agtcaagttc 180aactggtacg tggacggcgt cgaagtgcat aatgcaaaga
ctaaaccacg ggaggaacag 240tacaactcta catatagagt cgtgagtgtc ctgactgtgc
tgcatcagga ttggctgaac 300ggcaaagagt ataagtgcaa agtgtctaat aaggccctgc
ctgctccaat cgagaaaact 360attagtaagg caaaagggca gcccagggaa cctcaggtct
acgtgctgcc tccaagtcgc 420gacgagctga ccaagaacca ggtctcactg ctgtgtctgg
tgaaaggatt ctatccttcc 480gatattgccg tggagtggga atctaatggc cagccagaga
acaattacct gacctggccc 540cctgtgctgg acagcgatgg gtccttcttt ctgtattcaa
agctgacagt ggacaaaagc 600agatggcagc agggaaacgt ctttagctgt tccgtgatgc
acgaagccct gcacaatcat 660tacacccaga agtctctgag tctgtcacct ggc
693331110PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 331Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5
10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 20 25
30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr 35 40 45 Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50
55 60 Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His 65 70
75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90
95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
100 105 110 332330DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
332gctccagaac tgctgggagg accaagcgtg ttcctgtttc cacccaagcc caaagataca
60ctgatgatca gccgaactcc cgaggtcacc tgcgtggtcg tggacgtgtc ccacgaggac
120cccgaagtca agttcaactg gtacgtggac ggcgtcgaag tgcataatgc aaagactaaa
180ccacgggagg aacagtacaa ctctacatat agagtcgtga gtgtcctgac tgtgctgcat
240caggattggc tgaacggcaa agagtataag tgcaaagtgt ctaataaggc cctgcctgct
300ccaatcgaga aaactattag taaggcaaaa
330333106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 333Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Leu
Pro Pro Ser Arg Asp 1 5 10
15 Glu Leu Thr Lys Asn Gln Val Ser Leu Leu Cys Leu Val Lys Gly Phe
20 25 30 Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35
40 45 Asn Asn Tyr Leu Thr Trp Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55
60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly 65 70 75
80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95 Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 100 105
334318DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 334gggcagccca gggaacctca ggtctacgtg ctgcctccaa
gtcgcgacga gctgaccaag 60aaccaggtct cactgctgtg tctggtgaaa ggattctatc
cttccgatat tgccgtggag 120tgggaatcta atggccagcc agagaacaat tacctgacct
ggccccctgt gctggacagc 180gatgggtcct tctttctgta ttcaaagctg acagtggaca
aaagcagatg gcagcaggga 240aacgtcttta gctgttccgt gatgcacgaa gccctgcaca
atcattacac ccagaagtct 300ctgagtctgt cacctggc
3183358PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 335Val Asn Pro Asn Ser Gly Gly
Ser 1 5 33612PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 336Ala
Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr 1 5
10 3378PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 337Gly Phe Thr Phe Thr Asp Tyr Thr 1
5 3383PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 338Ser Ala Ser 1
3399PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 339Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr 1 5
3406PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 340Gln Asp Val Ser Ile Gly 1 5
3418PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 341Ile Tyr Pro Thr Asn Gly Tyr Thr 1 5
34213PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 342Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp
Tyr 1 5 10 3438PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 343Gly
Phe Asn Ile Lys Asp Thr Tyr 1 5
3443PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 344Ser Ala Ser 1 3459PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 345Gln
Gln His Tyr Thr Thr Pro Pro Thr 1 5
3466PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 346Gln Asp Val Asn Thr Ala 1 5
3479PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 347Gln Gln Tyr Tyr Ile Tyr Pro Ala Thr 1 5
3488PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 348Gly Phe Thr Phe Ala Asp Tyr Thr 1
5 349607PRTHomo sapiens 349Thr Gln Val Cys Thr Gly Thr Asp
Met Lys Leu Arg Leu Pro Ala Ser 1 5 10
15 Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln
Gly Cys Gln 20 25 30
Val Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser
35 40 45 Leu Ser Phe Leu
Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu Ile 50
55 60 Ala His Asn Gln Val Arg Gln Val
Pro Leu Gln Arg Leu Arg Ile Val 65 70
75 80 Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu
Ala Val Leu Asp 85 90
95 Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro
100 105 110 Gly Gly Leu
Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu Lys 115
120 125 Gly Gly Val Leu Ile Gln Arg Asn
Pro Gln Leu Cys Tyr Gln Asp Thr 130 135
140 Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn Gln Leu
Ala Leu Thr 145 150 155
160 Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro Met
165 170 175 Cys Lys Gly Ser
Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser 180
185 190 Leu Thr Arg Thr Val Cys Ala Gly Gly
Cys Ala Arg Cys Lys Gly Pro 195 200
205 Leu Pro Thr Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys
Thr Gly 210 215 220
Pro Lys His Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly 225
230 235 240 Ile Cys Glu Leu His
Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr 245
250 255 Phe Glu Ser Met Pro Asn Pro Glu Gly Arg
Tyr Thr Phe Gly Ala Ser 260 265
270 Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly
Ser 275 280 285 Cys
Thr Leu Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp 290
295 300 Gly Thr Gln Arg Cys Glu
Lys Cys Ser Lys Pro Cys Ala Arg Val Cys 305 310
315 320 Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val
Arg Ala Val Thr Ser 325 330
335 Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser Leu
340 345 350 Ala Phe
Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr Ala 355
360 365 Pro Leu Gln Pro Glu Gln Leu
Gln Val Phe Glu Thr Leu Glu Glu Ile 370 375
380 Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser
Leu Pro Asp Leu 385 390 395
400 Ser Val Phe Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His Asn
405 410 415 Gly Ala Tyr
Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu Gly 420
425 430 Leu Arg Ser Leu Arg Glu Leu Gly
Ser Gly Leu Ala Leu Ile His His 435 440
445 Asn Thr His Leu Cys Phe Val His Thr Val Pro Trp Asp
Gln Leu Phe 450 455 460
Arg Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu Asp 465
470 475 480 Glu Cys Val Gly
Glu Gly Leu Ala Cys His Gln Leu Cys Ala Arg Gly 485
490 495 His Cys Trp Gly Pro Gly Pro Thr Gln
Cys Val Asn Cys Ser Gln Phe 500 505
510 Leu Arg Gly Gln Glu Cys Val Glu Glu Cys Arg Val Leu Gln
Gly Leu 515 520 525
Pro Arg Glu Tyr Val Asn Ala Arg His Cys Leu Pro Cys His Pro Glu 530
535 540 Cys Gln Pro Gln Asn
Gly Ser Val Thr Cys Phe Gly Pro Glu Ala Asp 545 550
555 560 Gln Cys Val Ala Cys Ala His Tyr Lys Asp
Pro Pro Phe Cys Val Ala 565 570
575 Arg Cys Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr Met Pro Ile
Trp 580 585 590 Lys
Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys Pro Ile Asn 595
600 605 350217PRTHomo sapiens 350Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1
5 10 15 Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20
25 30 Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr 35 40
45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu 50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65
70 75 80 Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85
90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln 100 105
110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu 115 120 125 Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130
135 140 Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150
155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu 165 170
175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190 Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195
200 205 Lys Ser Leu Ser Leu Ser Pro
Gly Lys 210 215
* * * * *
