The present invention relates, in general, to polypeptides capable of
binding and neutralizing transforming growth factor beta (TGF-beta)
ligands, and uses of these polypeptides for treating disorders related to
TGF-beta expression or activation (e.g. cancer and fibrotic diseases),
and methods of making such molecules.
| Inventors: |
Zwaagstra; John C.; (Laval, CA)
; Sulea; Traian; (Kirkland, CA)
; Jaramillo; Maria; (Beaconsfield, CA)
; O'Connor; Maureen D.; (Beaconsfield, CA)
; Lenferink; Anne E.G.; (Lorraine, CA)
|
| Applicant: | | Name | City | State | Country | Type |
National Research Council of Canada |
Ottawa | |
CA | | |
|---|
| Assignee: |
National Research Council of Canada
Ottawa
ON
|
| Family ID:
|
58186979
|
| Appl. No.:
|
15/755595
|
| Filed:
|
August 31, 2016 |
| PCT Filed:
|
August 31, 2016 |
| PCT NO:
|
PCT/IB2016/055204 |
| 371 Date:
|
February 27, 2018 |
Related U.S. Patent Documents
| | | | |
|---|
|
| Application Number | Filing Date | Patent Number |
|---|
| | 62212058 | Aug 31, 2015 | |
|
|
| Current U.S. Class: |
1/1 |
| Current CPC Class: |
C07K 2319/30 20130101; A61P 35/00 20180101; C07K 2317/94 20130101; C07K 14/71 20130101; C07K 19/00 20130101; C07K 16/22 20130101; C12N 15/62 20130101; A61K 2039/505 20130101; C07K 16/30 20130101 |
| International Class: |
C07K 14/71 20060101 C07K014/71; C07K 16/22 20060101 C07K016/22; A61P 35/00 20060101 A61P035/00 |
Claims
1. A polypeptide construct comprising: a first portion comprising the
second constant domain (C.sub.H2) and/or third constant domain (C.sub.H3)
of an antibody heavy chain, and a second portion comprising at least two
TGF-.beta. superfamily receptor ectodomains (T.beta.SR-ED) linked in
tandem, wherein the N-terminus of the second portion is linked to the
C-terminus of the first portion.
2. A polypeptide construct comprising: a first portion comprising the
second constant domain (CH2) and/or third constant domain (CH3) of an
antibody heavy chain, and a second portion comprising at least one
TGF-.beta. superfamily receptor ectodomains (T.beta.SR-ED), wherein the
N-terminus of the second portion is linked to the C-terminus of the first
portion, and further wherein the first portion does not further comprise
an antibody that binds to an antigen that is PD-L1, EGFR1, Her-2, CD4,
CD6, CD20, CD25, MUC-1, IL-2, IL-6, or CTLA-4.
3. A polypeptide construct comprising: a first portion comprising the
second constant domain (C.sub.H2) and/or third constant domain (C.sub.H3)
of an antibody heavy chain, and a second portion comprising at least one
TGF-.beta. superfamily receptor ectodomain (T.beta.SR-ED), wherein the
N-terminus of the second portion is directly fused to the C-terminus of
the first portion.
4. A polypeptide construct comprising a first portion comprising the
second constant domain (C.sub.H2) and/or third constant domain (C.sub.H3)
of an antibody heavy chain, and a second portion comprising at least one
TGF-.beta. superfamily receptor ectodomain (T.beta.SR-ED), wherein the
N-terminus of the second portion is linked to the C-terminus of the first
portion, and wherein the polypeptide construct neutralizes TGF-.beta.
with at least 100-fold more potency than the T.beta.SR-ED alone.
5. The polypeptide construct of claims 2-4, wherein the second portion
comprises one T.beta.SR-ED.
6. The polypeptide construct of claim 5, wherein the second portion
comprises two T.beta.SR-EDs.
7. The polypeptide construct according to claims 1-6, wherein the
T.beta.SR-ED is a TGF-.beta. receptor type II ectodomain
(T.beta.R-II-ED).
8. The polypeptide construct of claims 1-6, wherein the T.beta.SR-ED
comprises a sequence selected from the group consisting of SEQ ID NO:35,
SEQ ID NO:69, SEQ ID NO:75, SEQ ID NO:81, and a sequence substantially
identical thereto.
9. The polypeptide construct of claims 1-8, wherein the second portion
comprises a sequence selected from the group consisting of SEQ ID
NO:43-SEQ ID NO:51, SEQ ID NO:61-SEQ ID NO:68, SEQ ID NO:73, SEQ ID
NO:74, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:85, SEQ ID NO:86, SEQ ID
NO:88, and a sequence substantially identical thereto.
10. The polypeptide construct of any one of claims 1-8, wherein the first
portion further comprises a C.sub.H1, a C.sub.H1 and V.sub.H, or C.sub.H1
and scFv.
11. The polypeptide construct of any one of claims 1-10, wherein the
antibody heavy chain is of human origin.
12. The polypeptide construct of any one of claims 1-11, wherein the
antibody heavy chain is selected from the group consisting of a human
IgG1, IgG2, IgG3, or IgG4 heavy chain.
13. The polypeptide construct of any one of claims 1-12, wherein the
antibody heavy chain is a human IgG1.
14. The polypeptide construct of claim 4, wherein the polypeptide
construct shows longer in vivo half-life compared to the half-life of the
second portion alone.
15. The polypeptide construct of any one of claims 1-14, wherein the
polypeptide construct is a single chain polypeptide.
16. The polypeptide construct of any one of claims 1-15, wherein the
polypeptide construct forms a dimeric polypeptide.
17. The polypeptide construct of claims 1-16, wherein the polypeptide
construct is heterodimeric.
18. A polypeptide construct selected from the group consisting of any one
of SEQ ID NO:91 to SEQ ID NO:120, and a sequence substantially identical
thereto.
19. A polypeptide construct according to claims 1-16, wherein the
construct comprises an antibody, antigen binding fragment thereof, or a
targeting moiety.
20. A polypeptide construct according to claim 19, comprising the
antibody, antigen binding fragment, or targeting moiety at the N-terminus
of the first portion.
21. A polypeptide construct according to claim 19, wherein the antigen
binding fragment may be selected from the group consisting of a Fv, scFv,
Fab, or sdAb.
22. A polypeptide construct according to claim 19, wherein the antigen
binding fragment binds to an antigen that is not PD-L1, EGFR1, Her-2,
CD4, CD6, CD20, CD25, MUC-1, IL-2, IL-6, or CTLA-4.
23. A polypeptide construct according to claim 19, wherein the antibody
is selected from the group consisting of Cetuximab, Avastin, Herceptin,
Synagis, and FC5.
24. A polypeptide construct according to claim 23, wherein the antibody
is Cetuximab.
25. A polypeptide construct according to claim 19, wherein the targeting
moiety comprises a poly-aspartate sequence motif for bone targeting.
26. A polypeptide construct according to claim 25, wherein the targeting
moiety comprises D10.
27. A polypeptide construct according to any preceding claim wherein the
construct is a dimeric polypeptide.
28. A polypeptide construct according to claim 27, wherein the dimeric
polypeptide comprises: a first single chain polypeptide comprising a
first portion comprising the second constant domain (C.sub.H2) and third
constant domain (C.sub.H3) of an antibody heavy chain, and a heavy chain
variable region of a given antibody; a second portion comprising one or
more TGF-.beta. superfamily receptor ectodomains (T.beta.SR-ED), wherein
the N-terminus of the second portion is linked to the C-terminus of the
first portion, and a second single chain polypeptide comprising a first
portion comprising the second constant domain (C.sub.H2) and third
constant domain (C.sub.H3) of an antibody heavy chain, and a light chain
variable region of said given antibody; a second portion comprising one
or more TGF-.beta. superfamily receptor ectodomain (T.beta.SR-ED) which
is the same or different from the ectodomain(s) in the first polypeptide,
wherein the N-terminus of the second portion is linked to the C-terminus
of the first portion.
29. A nucleic acid molecule encoding the polypeptide construct of any
preceding claim.
30. A vector comprising the nucleic acid molecule of claim 29.
31. A composition comprising one or more than one independently selected
polypeptide construct of any one of claims 1 to 30 and a
pharmaceutically-acceptable carrier, diluent, or excipient.
32. A transgenic cellular host comprising the nucleic acid molecule of
claim 29 or a vector of claim 30.
33. The transgenic cellular host of claim 32, further comprising a second
nucleic acid molecule or a second vector encoding a second polypeptide
construct different from the first polypeptide construct.
34. The use of a polypeptide construct according to any one of claims
1-28, for treatment of a medical condition, disease or disorder.
35. The use according to claim 34, wherein the medical condition, disease
or disorder comprises cancer, ocular diseases, fibrotic diseases, or
genetic disorders of connective tissue.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to TGF-.beta. superfamily receptor
ectodomain fusion molecules and uses thereof. More specifically, the
present invention relates to TGF-.beta. superfamily receptor ectodomain
fusion molecules and their use in TGF-.beta. superfamily ligand
neutralization.
BACKGROUND OF THE INVENTION
[0002] TGF-.beta. is part of a superfamily of over 30 ligands that
regulate several physiological processes, including cell proliferation,
migration and differentiation. Perturbation of their levels and/or
signaling gives rise to significant pathological effects. For instance,
TGF-.beta. and activin ligands play critical pathogenic roles in many
diseases including cancer (Hawinkels & Ten Dijke, 2011; Massague et al,
2000; Rodgarkia-Dara et al, 2006). TGF-.beta., in particular, is
considered as a critical regulator of tumor progression and is
overexpressed by most tumor types. It favors tumorigenesis in part by
inducing an epithelial-mesenchymal transition (EMT) in the epithelial
tumor cells, leading to aggressive metastasis (Thiery et al, 2009).
TGF-.beta. also promotes tumorigenesis by acting as a powerful suppressor
of the immune response in the tumor microenvironment (Li et al, 2006). In
fact, TGF-.beta. is recognized as one of the most potent
immunosuppressive factors present in the tumor microenvironment.
TGF-.beta. interferes with the differentiation, proliferation and
survival of many immune cell types, including dendritic cells,
macrophages, NK cells, neutrophils, B-cells and T-cells; thus, it
modulates both innate and adaptive immunity (Santarpia et al, 2015; Yang
et al, 2010). The importance of TGF-beta in the tumor microenvironment is
highlighted by evidence showing that, in several tumor types (including
melanoma, lung, pancreatic, colorectal, hepatic and breast), elevated
levels of TGF-.beta. ligand are correlated with disease progression and
recurrence, metastasis, and mortality. Hence, significant effort has been
invested in devising anti-tumor therapeutic approaches that involve
TGF-.beta. inhibition (Arteaga, 2006; Mourskaia et al, 2007;
Wojtowicz-Praga, 2003).
[0003] One approach to developing therapeutic agents that inhibit
TGF-.beta. function has been to use antibodies or soluble decoy receptors
(also termed receptor ectodomain (ED)-based ligand traps) to bind and
sequester ligand, thereby blocking access of ligand to its normal cell
surface receptors (Zwaagstra et al, 2012). In general, receptor ED-based
traps are a class of therapeutic agents that are able to sequester a wide
range of ligands and that can be optimized using protein engineering
approaches (Economides et al, 2003; Holash et al, 2002; Jin et al, 2009).
[0004] Previously, a novel protein engineering design strategy was used to
generate single-chain, bivalent traps that are able to potently
neutralize members of the TGF-.beta. superfamily of ligands due to
avidity effects (Zwaagstra et al, 2012) [WO 2008/113185; WO 2010/031168].
In this case, bivalency was achieved via covalent linkage of two
T.beta.RII ectodomains using portions of the intrinsically disordered
regions (IDR) that flank the structured, ligand-binding domain of
T.beta.RII-ED. One example of these single-chain bivalent traps, T22d35,
exhibited TGF-.beta. neutralization potencies .about.100-fold higher than
the monovalent non-engineered T.beta.RII ectodomain, though it did not
neutralize the TGF-.beta.2 isoform and had a relatively short circulating
half-life.
[0005] While research to date indicates that single-chain TGF-.beta. traps
have promising therapeutic potential, their circulating half-lives and
manufacturability present challenges to the commercial application.
SUMMARY OF THE INVENTION
[0006] The present invention relates to TGF-.beta. superfamily receptor
ectodomain fusion molecules and uses thereof. More specifically, the
present invention relates to TGF-.beta. superfamily receptor ectodomain
fusion molecules and their use in TGF-.beta. superfamily ligand
neutralization.
[0007] In some aspects, the invention relates to TGF-.beta. superfamily
receptor ectodomain-based polypeptides that are similar to typical Fc
fusions in design, in that the ectodomain is fused to a dimeric antibody
constant domain. In particular, with respect to the present polypeptides,
the Fc portion occupies the N-terminal position. Fc fusions in the prior
art typically provide the Fc portion at the C-terminal end of the fusion.
As will be evident from the results presented herein, this difference in
orientation provides a number of significant advantages.
[0008] In other aspects, the present polypeptides incorporate at least two
TGF-.beta. superfamily receptor ectodomains that are linked in tandem to
the C-terminus of an antibody constant domain.
[0009] Thus, there is provided a polypeptide construct comprising: a first
portion comprising the second constant domain (C.sub.H2) and/or third
constant domain (C.sub.H3) of an antibody heavy chain, and a second
portion comprising at least two TGF-.beta. superfamily receptor
ectodomains (T.beta.SR-ED) linked in tandem; wherein the N-terminus of
the second portion is linked to the C-terminus of the first portion.
[0010] There is also provided a polypeptide construct comprising: a first
portion comprising the second constant domain (CH2) and/or third constant
domain (CH3) of an antibody heavy chain, and a second portion comprising
at least one TGF-.beta. superfamily receptor ectodomains (T.beta.SR-ED),
wherein the N-terminus of the second portion is linked to the C-terminus
of the first portion, and further wherein the first portion does not
further comprise an antibody that binds to an antigen that is PD-L1,
EGFR1, Her-2, CD4, CD6, CD20, CD25, MUC-1, IL-2, IL-6, or CTLA-4.
[0011] There is provided a polypeptide construct comprising: a first
portion comprising the second constant domain (C.sub.H2) and/or third
constant domain (C.sub.H3) of an antibody heavy chain, and a second
portion comprising at least one TGF-.beta. superfamily receptor
ectodomain (T.beta.SR-ED), wherein the N-terminus of the second portion
is directly fused to the C-terminus of the first portion.
[0012] In an embodiment, there is provided a polypeptide construct
comprising a first portion comprising the second constant domain
(C.sub.H2) and/or third constant domain (C.sub.H3) of an antibody heavy
chain, and a second portion comprising at least one TGF-.beta.
superfamily receptor ectodomain (T.beta.SR-ED), wherein the N-terminus of
the second portion is linked to the C-terminus of the first portion, and
wherein the polypeptide construct neutralizes TGF-.beta. with at least
100-fold more potency than the T.beta.SR-ED alone.
[0013] In a preferred embodiment, the second portion comprises one, two,
or multiple TGF-.beta. superfamily receptor ectodomain (T.beta.SR-ED). In
a preferred embodiment, the T.beta.SR-ED is a TGF-.beta. receptor type II
ectodomain (TI.beta.R-II-ED). In a preferred embodiment, the T.beta.SR-ED
comprises a sequence selected from the group consisting of SEQ ID NO:35,
SEQ ID NO:69, SEQ ID NO:75, SEQ ID NO:81, and a sequence substantially
identical thereto.
[0014] The second portion may comprise a sequence selected from the group
consisting of SEQ ID NO:43-SEQ ID NO:51, SEQ ID NO:61-SEQ ID NO:68, SEQ
ID NO:73, SEQ ID NO:74, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:85, SEQ ID
NO:86, SEQ ID NO:88, and a sequence substantially identical thereto.
[0015] In a preferred embodiment, the first portion of a polypeptide
construct of the present invention further comprises a C.sub.H1, a
C.sub.H1 and V.sub.H, or C.sub.H1 and scFv.
[0016] There is provided a polypeptide construct of the present invention
wherein the antibody heavy chain is of human origin. In a preferred
embodiment, the antibody heavy chain is selected from the group
consisting of a human IgG1, IgG2, IgG3, or IgG4 heavy chain. More
preferably, the antibody heavy chain is a human IgG1.
[0017] In accordance with the present invention, the polypeptide construct
shows longer in vivo half-life compared to the half-life of the second
portion alone.
[0018] There is provided a polypeptide construct of the present invention,
wherein the polypeptide construct is a single chain polypeptide.
[0019] In an embodiment, the polypeptide construct forms a dimeric
polypeptide. In another embodiment, the polypeptide construct is
heterodimeric.
[0020] There is provided a polypeptide construct selected from the group
consisting of any one of SEQ ID NO:91 to SEQ ID NO:120, and a sequence
substantially identical thereto.
[0021] There is provided a polypeptide construct according to the present
invention, wherein the construct comprises an antibody, antigen binding
fragment thereof, or a targeting moiety. In a preferred embodiment, the
antibody, the antigen binding fragment, or the targeting moiety is at the
N-terminus of the first portion.
[0022] In a preferred embodiment, the antigen binding fragment may be
selected from the group consisting of a Fv, scFv, Fab, or sdAb. In a
preferred embodiment, the antigen binding fragment binds to any antigen,
provided that it is not PD-L1, EGFR1, Her-2, CD4, CD6, CD20, CD25, MUC-1,
IL-2, IL-6, or CTLA-4.
[0023] In a preferred embodiment, a polypeptide construct of the present
invention comprises an antibody selected from the group consisting of
Cetuximab, Avastin, Herceptin, Synagis, and FC5. In a preferred
embodiment, the antibody is Cetuximab.
[0024] In a preferred embodiment, a polypeptide construct of the present
invention comprises a targeting moiety, wherein the targeting moiety
comprises a poly-aspartate sequence motif for bone targeting. In a
preferred embodiment, the targeting moiety comprises D10.
[0025] There is provided a polypeptide construct according to the present
invention wherein the construct is a dimeric polypeptide; wherein the
dimeric polypeptide comprises: a first single chain polypeptide
comprising a first portion comprising the second constant domain
(C.sub.H2) and third constant domain (C.sub.H3) of an antibody heavy
chain, and a heavy chain variable region of a given antibody; a second
portion comprising one or more TGF-.beta. superfamily receptor
ectodomains (T.beta.SR-ED), wherein the N-terminus of the second portion
is linked to the C-terminus of the first portion, and a second single
chain polypeptide comprising a first portion comprising the second
constant domain (C.sub.H2) and third constant domain (C.sub.H3) of an
antibody heavy chain, and a light chain variable region of said given
antibody; a second portion comprising one or more TGF-.beta. superfamily
receptor ectodomain (T.beta.SR-ED) which is the same or different from
the ectodomain(s) in the first polypeptide, wherein the N-terminus of the
second portion is linked to the C-terminus of the first portion.
[0026] There is also provided a nucleic acid molecule encoding the
polypeptide construct of the present invention. There is also provided a
vector comprising the nucleic acid molecule of claim the present
invention.
[0027] There is also provided a composition comprising one or more than
one independently selected polypeptide construct of the present invention
and a pharmaceutically-acceptable carrier, diluent, or excipient.
[0028] There is also provided a transgenic cellular host comprising the
nucleic acid molecule or a vector of the present invention. The
transgenic cellular host further comprising a second nucleic acid
molecule or a second vector encoding a second polypeptide construct
different from the first polypeptide construct.
[0029] There is also provided the use of a polypeptide construct according
to the present invention for treatment of a medical condition, disease or
disorder; wherein the medi medical condition, disease or disorder
comprises, but is not limited to, cancer, ocular diseases, fibrotic
diseases, or genetic disorders of connective tissue.
[0030] In a preferred embodiment, there therefore provided a polypeptide
construct comprising: [0031] a first portion comprising the second
constant domain (CH2) and/or third constant domain (CH3) of an antibody
heavy chain, and [0032] a second portion comprising at least two
TGF-.beta. superfamily receptor ectodomains (T.beta.SR-ED), [0033]
wherein the N-terminus of the second portion is linked to the C-terminus
of the first portion.
[0034] The antibody constant domain can further comprise, either linked
thereto or formed integrally therewith, a binding agent such as a full
size antibody, a ligand or any other protein of interest. In the
alternative, the antibody constant domain comprises only the CH2 and/or
CH3 regions, and not a full size antibody. In these and other types of
constructs, the CH2 and/or CH3 region can be altered by deleting or
substituting amino acids including one or more of the cysteines that
provide cross-linking when the present constructs are provided as dimeric
constructs.
[0035] In other aspects of the present invention, there is provided a
polypeptide construct that incorporates one or more such ectodomains.
When the constructs comprise only one ectodomain linked to the antibody
constant domain, then the construct is further characterized by at least
one of the following: (1) when the constant domain further comprises a
full sized antibody, that antibody does not bind effectively to PD-L1 or
to an immunoregulatory antigen selected, (2) the constant domain
comprises only the CH2 and/or CH3 regions, (3) the constant domain
comprises an amino acid alteration relative to a wild type counterpart,
such as a cysteine residue alteration; and (4) the first portion is
linked to the second portion directly and without intervening amino
acids.
[0036] In another of its aspects, the present invention provides a
polypeptide construct comprising [0037] a first portion comprising the
second constant domain (C.sub.H2) and/or third constant domain (C.sub.H3)
of an antibody heavy chain, and [0038] a second portion comprising at
least one TGF-.beta. superfamily receptor ectodomain (T.beta.SR-ED),
wherein
[0039] the N-terminus of the second portion is linked to the C-terminus of
the first portion. These polypeptide constructs can neutralize
TGF-.beta., and with at least 100-fold more potency than the T.beta.SR-ED
alone.
[0040] The second portion of the polypeptide construct of the present
invention may comprise one or two or more T.beta.SR-ED. In a preferred
embodiment the construct comprises two or more independently selected
ectodomains linked in tandem and to the C-terminus of the constant
domain. The T.beta.SR-ED may be selected from the group consisting of a
TGF-.beta. receptor type II ectodomain (T.beta.RII-ED), a bone
morphogenetic protein receptor type la ectodomain (BMPR-ED), an activin
receptor type IIa ectodomain (ActRIIA-ED), and an activin receptor type
IIb ectodomain (ActRIIb-ED). In another preferred embodiment, the
ectodomain is a TI.beta.R-II ectodomain.
[0041] In the polypeptide construct described herein, the first portion
further may comprise a C.sub.H1, a C.sub.H1 and V.sub.H, or a C.sub.H1
and scFv. It may constitute an Fc region, an antibody, or any ligand
binding agent or moiety.
[0042] The polypeptide construct of the present invention may comprise a
C.sub.H2 and C.sub.H3 from an antibody heavy chain that is of human or
mouse origin. For example, and without wishing to be limiting, the
antibody heavy chain may be selected from the group consisting of a human
IgG1, IgG2, IgG3, or IgG4 heavy chain. In embodiments, the constant
domain in the constructs is CH2 per se, or CH3 per se or CH2-CH3.
[0043] The polypeptide construct described herein may show longer in vivo
half-life compared to the half-life of T.beta.SR-ED alone.
[0044] In one example, the polypeptide construct of the present invention
may be a single chain polypeptide. The polypeptide construct as described
herein may also form a dimeric polypeptide. This dimeric polypeptide may
be heterodimeric.
[0045] The present invention further provides a polypeptide construct
comprising [0046] a first portion comprising the second constant domain
(C.sub.H2) and/or third constant domain (C.sub.H3) of an antibody heavy
chain, and [0047] a second portion comprising at least one TGF-.beta.
superfamily receptor ectodomain (T.beta.SR-ED),
[0048] wherein the N-terminus of the second portion is linked to the
C-terminus of the first portion; additionally, in the construct as just
described, the first portion is not derived from certain antibodies
discussed infra.
[0049] The present invention also provides a polypeptide construct,
comprising: [0050] a first single chain polypeptide comprising a first
portion comprising the second constant domain (C.sub.H2) and/or third
constant domain (C.sub.H3) of an antibody heavy chain, and a heavy chain
variable region of a given antibody; and a second portion comprising one
or more TGF-.beta. superfamily receptor ectodomains (T.beta.SR-ED),
[0051] wherein the N-terminus of the second portion is linked to the
C-terminus of the first portion, and [0052] a second single chain
polypeptide comprising a first portion comprising the second constant
domain (C.sub.H2) and/or third constant domain (C.sub.H3) of an antibody
heavy chain, and a light chain variable region of said given antibody;
and a second portion comprising one or more TGF-.beta. superfamily
receptor ectodomain (T.beta.SR-ED) which is the same or different from
the ectodomain(s) in the first polypeptide, wherein the N-terminus of the
second portion is linked to the C-terminus of the first portion.
[0053] In alternative constructs of the present invention, the polypeptide
construct comprises an antibody Fc fragment linked at the C-terminus of
each heavy chain to at least one TGF-.beta. superfamily receptor
ectodomain (T.beta.SR-ED), as described above. In embodiments the
receptor ectodomain portion comprises two independently selected
ectodomains that are linked in tandem, i.e., in a linear manner. In some
embodiments, the ectodomains are the same in sequence, or least the same
with respect to their target ligand. The construct may further comprise a
binding fragment or moiety at the N-terminus of the Fc; the binding
fragment may be selected from the group consisting of a Fv, scFv, Fab, or
sdAb, or any other binding moiety such as a motif for bone targeting,
also as described above. In the polypeptide constructs as described
above, the TGF-.beta. receptor ectodomain does not interfere in the
native function or specificity of the binding fragment.
[0054] The present invention also provides a nucleic acid molecule
encoding the polypeptide constructs as described herein. A vector
comprising the nucleic acid molecule just described is also encompassed
by the invention. The invention also includes a transgenic cellular host
comprising the nucleic acid molecule or a vector as described herein; the
cellular host may further include a second nucleic acid molecule or a
second vector encoding a second polypeptide construct different from the
first polypeptide construct. Systems used to produce the present
polypeptides can be secretion systems, particularly in the case where
dimerization through disulfide bridges is required, and the expression
polynucleotides thus encode secretion signals that are cleaved by the
host upon secretion into the culturing medium.
[0055] Compositions comprising one or more than one independently selected
polypeptide construct described herein and a pharmaceutically-acceptable
carrier, diluent, or excipient are also encompassed by the present
invention.
[0056] Additional aspects and advantages of the present invention will be
apparent in view of the following description. The detailed descriptions
and examples, while indicating preferred embodiments of the invention,
are given by way of illustration only, as various changes and
modifications within the scope of the invention will become apparent to
those skilled in the art in light of the teachings of this invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] These and other features of the invention will now be described by
way of example, with reference to the appended drawings, wherein:
[0058] FIG. 1A is a schematic diagram showing TGF-.beta. Type II receptor
ectodomain (T.beta.RII-ED)-based molecules T2m and T22d35 along with
their sequences (SEQ ID NO:43 and 46, respectively). Natural linker
sequences (SEQ ID NO:36, 39 and 40) are underlined and depicted as dark
grey lines; the sequence of the TI.beta.R-II-ED structured domain (SEQ ID
NO:35) is shown in bold, and the domain labeled and depicted in dark
grey; the site of the fusion of natural linkers is depicted by a slash.
FIGS. 1B-D are schematic diagrams of IgG Fc-based scaffolds: an IgG Fc
region (FIG. 1B), a V.sub.HH-IgG Fc (comprising a V.sub.HH single domain
antibody fused to the N-terminus of an Fc region; FIG. 1C), and a
full-size antibody (FIG. 1D).
[0059] FIG. 2 is a schematic representation of TGF-.beta. superfamily
receptor-ectodomain-based fusion constructs of the present invention. (A)
represents constructs in which T22d35 (dark grey) is fused to the
C-terminus of IgG Fc regions (IgG isoforms 1, 2, 3 or 4) with no Fab or
other functional binding moiety at the N-terminus (Fc-T22d35, A), (D)
represents constructs in which T22d35 (dark grey) is fused to the
C-terminus of the IgG Fc region of full-size antibodies with heavy and
light chain Fabs (FSA-T22d35, D), (E) represents constructs in which
T22d35 (dark grey) is fused to the C-terminus of IgG Fc regions that have
a non-Fab binding/localization moiety at the N-terminus, such as the
variable region of a camelid V.sub.HH antibody (V.sub.HH-Fc-T22d35) or a
deca-aspartate motif for bone targeting (D10-Fc-T22d35). Similarly, (B),
(C) and (F) and (G) represent constructs in which T2m (the TGF-.beta.
Type II receptor ectodomain, T.beta.RII-ED--dark grey) is fused at the
N-terminus of an IgG Fc (T2m-Fc, B) or the C-terminus of IgG Fc regions
with no Fab or other functional binding moiety at the N-terminus (Fc-T2m,
C), or the C-terminus of full-size antibodies with heavy and light chain
Fabs (FSA-T2m, F), or the C-terminus of IgG Fc regions that have a
non-Fab binding/localization moiety at the N-terminus, such as the
variable region of a camelid V.sub.HH antibody (V.sub.HH-Fc-T2m, G) or a
deca-aspartate motif for bone targeting (D10-Fc-T2m).
[0060] FIG. 3 presents (ProtA)-affinity column elution profiles, size
exclusion (SEC) purification profiles, SDS-PAGE gels, and UPLC-SEC
profiles of representatives of constructs type C and D in FIG. 2. FIG. 3A
is a (ProtA)-affinity column elution profile for T22d35 fused to the
Cetuximab FSA (Cet-T22d35--a representative of construct D in FIG. 2).
FIG. 3B is the size exclusion (SEC) purification profile of the
Cet-T22d35. FIG. 3C show 4-15% SDS-PAGE gels of ProtA-purified Cet-T22d35
under reducing (left panel) and non-reducing (right panel) conditions
(CetHC-T22d35, Cetuximab heavy chain fused to T22d35; CetLC, Cetuximab
light chain). Lanes 1 are the pooled Prot-A eluted fractions, while lanes
2 are the pooled SEC fractions. FIG. 3D shows the UPLC-SEC profile of
ProtA-purified Cet-T22d35. FIG. 3E shows the (ProtA)-affinity column
elution profile for hIgG1Fc.DELTA.K(C)-T2m (a construct with T2m fused to
an Fc region with no functional binding moiety at the N-terminus; a
representative of construct D in FIG. 2). FIG. 3F, G, H show the UPLC-SEC
profile before SEC (F), the UPLC-SEC profile after SEC (G) and the
SDS-PAGE (NR & R) (H) of hIgG1Fc.DELTA.K(C)-T2m.
[0061] FIG. 4A shows graphs depicting the efficient inhibition of
TGF-.beta.1 (top panel), TGF-.beta.3 (middle panel) and TGF-.beta.2
(bottom panel) signaling in Mv1 Lu luciferase reporter cells by Cet-T2m
(a representative of construct F in FIG. 2) and Cet-T22d35 (a
representative of construct D in FIG. 2), compared to the significantly
lower inhibition potency of non-Fc-fused T22d35.
[0062] FIG. 4B shows graphs and a summary table depicting the efficient
inhibition of TGF-.beta.1 signaling in an A549/IL-11 cell-based assay by
several representatives of FSA-T22d35 constructs (Type D construct from
FIG. 2), compared to the lower inhibition potency of Fc-T2m (Type C
construct) and non-Fc-fused T22d35.
[0063] FIG. 4C shows graphs and a summary table depicting the efficient
inhibition of TGF-.beta.1 signaling in an A549/IL-11 release cell-based
assay by several representatives of "headless"-T2m constructs (Type C
construct from FIG. 2), compared to the lower inhibition potency of
non-Fc-fused T22d35.
[0064] FIG. 4D is a graph showing competitive SPR analysis of binding of
Cetuximab-fusion constructs to TGF-.beta. isoforms in solution, compared
to T22d35.
[0065] FIG. 5A is a SDS-PAGE gel showing the inhibition of EGFR
phosphorylation in A549 cells by Cetuximab-fusion constructs. FIG. 5B is
a graph showing Cet-T22d35 (triangles) cytotoxicity in MDA-MB-468 and
HaCat cells compared to Cetuximab (circles) and T22d35 (squares).
[0066] FIG. 6 is a bar graph showing the apparent permeability coefficient
(P.sub.app) values, as a measure of transport of FC5-Fc, FC5-Fc-fusion
constructs, T22d35, and T2m across a human brain endothelial cell barrier
in vitro, relative to a non-transporting V.sub.HH control (A20.1).
[0067] FIG. 7 demonstrates the Cet-T22d35 inhibition of EGF+TGF-.beta.1
induced EMT in A549 cells. FIG. 7A shows pictures of cultured A549 cells
showing their morphologies before treatment (left panel A) and after
treatment with EGF+TGF-.beta.1 (right panel B). FIG. 7B shows a western
blot of whole cell lysates of A549 cells treated with EGF+TGF-.beta.1 in
the presence or absence of various concentrations of Cetuximab (Cetux),
Cet-T22d35 or T22d35, probed for the epithelial marker E-Cadherin, while
FIG. 7C is the densitometer quantification of the E-cadherin bands in the
Western blot. Results show that Cet-T22d35 is much more potent than
T22d35 alone or Cetuximab alone in upregulating E-cadherin, i.e. blocking
EMT. FIG. 7D shows the inhibition of EGF+TGF-.beta.-induced EMT by
Cetuximab (Cetux), Cet-T22d35, T22d35 or Cet-T22d35 plus T22d35 as
measured by flow cytometry detection of the epithelial E-Cadherin (top
panel) and mesenchymal N-Cadherin (bottom panel) markers.
[0068] FIG. 8A represents the pharmacokinetic (PK) profile of Cet-T22d35
in the serum collected from BALB/C mice that were injected with a single
dose of Cet-T22d35. The fusion construct appears to be cleaved in vivo;
the terminal half-life of the T22d35 potion of the construct was
determined to be 45.8 hours, while the terminal half-life of the
Cetuximab portion of the construct was determined to be 262.5 hours. FIG.
8B represents the PK profiles and data table (serum half-lives in bold)
for constructs in which the lysine at the C-terminus of the Fc region was
removed, i.e. is not present at the fusion joint between the Fc region
and T2m (Cet.DELTA.K-T2m, hIgG1Fc.DELTA.K(SS)-T2m,
hIgG1Fc.DELTA.K(.DELTA.C)-T2m, and hIg2GFc.DELTA.K(SS)-T2m). The data
demonstrate that the removal of the lysine prevents cleavage of the
constructs in vivo.
[0069] FIGS. 9A, 9B and 9C present graphs showing the effect of "headless"
Fc-T2m constructs (representatives of construct C in FIG. 2) and a
FSA-T2m construct (a representative of construct F in FIG. 2) on tumor
growth and T-cell function in an immune-competent syngeneic triple
negative breast cancer (4T1) model (for comparison, the effects of the
1D11 antibody and non-Fc-fused T22d35 are also shown). The results
demonstrate the improved efficacy on T-cell function of two headless-T2m
constructs relative to the FSA-T2m construct, and relative to 1D11 and
non-Fc-fused T22d35.
[0070] FIG. 10 shows data illustrating enhanced bone localization of two
constructs containing a deca-aspartate motif for bone targeting at the
N-terminus of the Fc region (D10-hIgG1Fc.DELTA.K(CC)-T2m (SEQ ID NO:136)
and D10-GSL-hIgG1Fc.DELTA.K(CC)-T2m (SEQ ID NO:137)--representatives of
construct G in FIG. 2). FIGS. 10A and B show results from an A549/IL-11
release cell-based assay demonstrating that the addition of D10 at the
N-terminus did not affect TGF-.beta. neutralization potency. FIGS. 10C
and D show images demonstrating significant enhancement of the
accumulation of the D10-containing constructs in bones relative to a
construct without D10.
DETAILED DESCRIPTION OF THE INVENTION
[0071] The present invention relates to TGF-.beta. superfamily receptor
ectodomain fusion molecules and uses thereof. More specifically, the
present invention relates to TGF-.beta. superfamily receptor ectodomain
fusion molecules and their use in TGF-.beta. superfamily ligand
neutralization.
[0072] The present invention provides polypeptide constructs, comprising
[0073] a first portion comprising the second constant domain (C.sub.H2)
and/or third constant domain (C.sub.H3) of an antibody heavy chain, and
[0074] a second portion comprising at least one TGF-.beta. superfamily
receptor ectodomain (T.beta.SR-ED), wherein the N-terminus of the second
portion (ectodomain) is linked to the C-terminus of the first portion (Fc
region), and wherein the polypeptide construct neutralizes TGF-.beta.
with at least 100-fold more potency than the T.beta.SR-ED alone. The
polypeptide construct referred to herein is a synthetic polypeptide
produced via protein engineering. It comprises two protein "portions" (or
"parts") that are linked to form the chimeric polypeptide construct. When
the polypeptide construct is expressed, two polypeptide chains dimerize,
such that the C.sub.H2 and C.sub.H3 domains form an antibody Fc region.
[0075] In specific embodiments of the present invention, descriptions of
which are elaborated further herein, there are provided polypeptide
constructs in which TGF-.beta. superfamily receptor-ectodomains were
fused to IgG Fc regions. Specifically, the T2m (single ectodomain) or
T22d35 (double ectodomain) moieties were linked (fused) to the C-terminal
end of the Fc region. It was observed that fusion constructs of this type
have advantages relative to several other versions of receptor-ectodomain
based molecules, including non-Fc fused monovalent or multivalent
TGF-.beta. receptor ectodomain constructs (such as T2m and T22d35) and
constructs in which a receptor ectodomain is fused to the N-terminus of
an Fc region. In particular, the constructs have improved
manufacturability due to the presence of the Fc region (for example,
purification can be accomplished using protein A chromatography). The Fc
region also allows for improved circulating half-lives. Importantly, the
present constructs have substantially higher TGF-.beta. neutralization
potencies compared to T2m and T22d35 alone or to constructs where a
receptor ectodomain is fused to the N-terminus of an Fc region. Thus, an
advantage of the present invention is unexpected high potency TGF-.beta.
superfamily ligand neutralization, including some degree of
neutralization of TGF-.beta.2, which was not observed for the non-Fc
fused constructs T2m and T22d35. Finally, constructs in accordance with
embodiments of the present invention, that is where the ectodomain(s)
is/are fused to the C-terminus of an Fc region of an antibody, allows for
preservation of the structure and function of the natural N-terminal
regions/domains of an antibody; as such, antigen binding to the antibody
CDR regions is not perturbed. This leads to the generation of a
bifunctional construct able to interact with the target of the antibody
while interacting with, and neutralizing, members of the TGF-.beta.
superfamily of ligands.
[0076] The invention relates not only to bifunctional constructs, but also
to constructs that are monofunctional, and comprise an Fc region that
consists only of the CH2 and/or CH3 regions of an antibody constant
region. Preferably, the G1, G2 or G4 subclasses are used, and
particularly G1 as well as G2. These constructs are monofunctional in the
sense that the constant region itself has no particular activity, other
than to act as a structure through which dimers of the polypeptide
constructs can form. These minimal constant regions can also be altered
to provide some benefit, by incorporating the corresponding hinge regions
(SEQ ID NO:5-8) and optionally changing the cysteine residue composition.
Thus, some or all of the cysteine residues involved in bridging the two
Fc fragments or naturally used to bridge between the heavy and light
chains of a full-length antibody can be replaced or deleted. These
cysteine residues are seen in hinge sequences listed in SEQ ID NO:5-8.
First, not all of the naturally-occurring inter-hinge disulfide bonds
need to be formed for the Fc homodimerization to occur, while noting that
the stability of the Fc homodimer may depend on the number of
intermolecular disulphide bridges. Secondly and perhaps more importantly,
the presence of hinge-region cysteine residues may become problematic
when the Fc region lacks its N-terminal Fab fragment (i.e., is a headless
Fc) as in the case of some polypeptide constructs of the present
invention. This leads to untethering and exposure of these hinge-region
cysteines, and in turn that may result in complex mixtures of high-order
polymeric chains, which creates manufacturability issues in addition to
potentially diminishing the intended biological activity and efficacy.
Because it is practically impossible to predict the outcome of varying
the number of inter-hinge disulphide bridges for the "headless"
polypeptide constructs of the present invention, we generated a
systematic array of N-terminal Fc variants for all four human IgG
isotypes either by a deletion approach (in which hinge-region cysteine
residues are progressively eliminated by N-terminal truncations) or by a
mutagenesis approach (in which hinge-region cysteine residues are
progressively mutated to serine from the N-terminus of the hinge region).
Non-limiting examples of such N-terminal variants of headless Fc regions
are listed in SEQ ID NO:9-34.
[0077] In the present disclosure, an "antibody", also referred to in the
art as "immunoglobulin" (Ig), refers to a protein constructed from paired
heavy and light polypeptide chains; various Ig isotypes exist, including
IgA, IgD, IgE, IgG, and IgM. The structure of an antibody and of each of
the domains is well-established and familiar to those of skill in the
art, though is summarized herein. When an antibody is correctly folded,
each chain folds into a number of distinct globular domains joined by
more linear polypeptide sequences; the immunoglobulin light chain folds
into a variable (V.sub.L) and a constant (C.sub.L) domain, while the
heavy chain folds into a variable (V.sub.H) and three constant (C.sub.H,
C.sub.H2, C.sub.H3) domains. Once paired, interaction of the heavy and
light chain variable domains (V.sub.H and V.sub.L) and first constant
domain (C.sub.L and C.sub.H,) results in the formation of a Fab
(Fragment, antigen-binding) containing the binding region (Fv);
interaction of two heavy chains results in pairing of C.sub.H2 and
C.sub.H3 domains, leading to the formation of a Fc (Fragment,
crystallisable). Characteristics described herein for the C.sub.H2 and
C.sub.H3 domains also apply to the Fc.
[0078] While the light and heavy chain variable regions show significant
sequence diversity between antibodies, the constant regions show less
sequence diversity and are responsible for binding a number of natural
proteins to elicit important biochemical events. Specifically, and
without wishing to be limiting, the Fc fragment binds to endogenous Fc
receptors on the surface of lymphocytes.
[0079] The C.sub.H2 and C.sub.H3 domains of the first portion may be of
any isotype, including one selected from the group consisting of IgA,
IgD, IgE, and IgG. The C.sub.H2 and C.sub.H3 domains may also be from any
suitable source. For example and without wishing to be limiting, the
C.sub.H2 and C.sub.H3 domains may originate from a human, mouse and other
rodents like rats and degu, rabbit, monkey, or other mammalian source. In
one example, the C.sub.H2 and C.sub.H3 domains may be of the IgG isotype;
in another example, the C.sub.H2 and C.sub.H3 domains are from human.
[0080] In a specific, non-limiting example, the C.sub.H2 and C.sub.H3
domains of the first portion may be of an isotype or comprise a sequence
selected from the group consisting of: [0081] a human IgG1, for example
but not limited to SEQ ID NO:1
(APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK) as comprised in P01857 of
the UniProtKB/Swiss-Prot database; [0082] a human IgG2, for example but
not limited to SEQ ID NO:2
(APPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNAK
TKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK), as comprised in P01859 of
the UniProtKB/Swiss-Prot database; [0083] a human IgG3, for example but
not limited to SEQ ID NO:3
(APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNA
KTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDG
SFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK), as comprised in P01860 of
the UniProtKB/Swiss-Prot database; [0084] a human IgG4, for example but
not limited to SEQ ID NO:4
(APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA
KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK), as comprised in P01861 of
the UniProtKB/Swiss-Prot database; and [0085] a sequence substantially
identical to any of the sequences listed above.
[0086] In the protein constructs of the present invention, the first
portion may further comprise a sequence corresponding to the hinge region
at the N-terminus of the C.sub.H2 domain. For example, the first portion
may further comprise a sequence selected from the group consisting of:
TABLE-US-00001
EPKSCDKTHTCPPCP (SEQ ID NO: 5) for human IgG1;
ERKCCVECPPCP (SEQ ID NO: 6) for human IgG2;
ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPK
SCDTPPPCPRCP (SEQ ID NO: 7) for human IgG3;
ESKYGPPCPSCP (SEQ ID NO: 8) for human IgG4;
[0087] and a sequence substantially identical to any of the sequences
listed above.
[0088] Thus, the first portion of the polypeptide construct of the present
invention consists of naturally fused Fc and hinge regions for the
various IgG isoforms and in embodiments is selected from the group
consisting of SEQ ID NO:1-4 for the Fc region and SEQ ID NO:5-8 for the
hinge region.
[0089] In specific embodiments, the first portion of the polypeptide
construct of the present invention is selected from a group of sequences
displaying variation in the N-terminal sequence as exemplified by SEQ ID
NO:9-34. These differ in the number of cysteine residues retained from
the hinge region as a means to modulating the degree of Fc-region
dimerization and hence impacting on both efficacy and manufacturability.
Thus, in embodiments, the polypeptide construct comprises a variation in
the constant domain, wherein at least one cysteine residue involved in
cross-linking is deleted or substituted. Suitable substitutions include
serine or alanine, and preferably by serine. A substantially identical
sequence may comprise one or more conservative amino acid mutations that
still provide for proper folding upon secretion into the culturing
medium. It is known in the art that one or more conservative amino acid
mutations to a reference sequence may yield a mutant peptide with no
substantial change in physiological, chemical, physico-chemical or
functional properties compared to the reference sequence; in such a case,
the reference and mutant sequences would be considered "substantially
identical" polypeptides. A conservative amino acid substitution is
defined herein as the substitution of an amino acid residue for another
amino acid residue with similar chemical properties (e.g. size, charge,
or polarity). These conservative amino acid mutations may be made to the
framework regions while maintaining the overall structure of the constant
domains; thus the function of the Fc is maintained.
[0090] In a non-limiting example, a conservative mutation may be an amino
acid substitution. Such a conservative amino acid substitution may
substitute a basic, neutral, hydrophobic, or acidic amino acid for
another of the same group. By the term "basic amino acid" it is meant
hydrophilic amino acids having a side chain pK value of greater than 7,
which are typically positively charged at physiological pH. Basic amino
acids include histidine (His or H), arginine (Arg or R), and lysine (Lys
or K). By the term "neutral amino acid" (also "polar amino acid"), it is
meant hydrophilic amino acids having a side chain that is uncharged at
physiological pH, but which has at least one bond in which the pair of
electrons shared in common by two atoms is held more closely by one of
the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or
T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and
glutamine (Gln or Q). The term "hydrophobic amino acid" (also "non-polar
amino acid") is meant to include amino acids exhibiting a hydrophobicity
of greater than zero according to the normalized consensus hydrophobicity
scale of (Eisenberg et al, 1984). Hydrophobic amino acids include proline
(Pro or P), isoleucine (Ile or I), phenylalanine (Phe or F), valine (Val
or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M),
alanine (Ala or A), and glycine (Gly or G). "Acidic amino acid" refers to
hydrophilic amino acids having a side chain pK value of less than 7,
which are typically negatively charged at physiological pH. Acidic amino
acids include glutamate (Glu or E), and aspartate (Asp or D).
[0091] In another non-limiting example, a conservative mutation in the
C.sub.H2 and/or C.sub.H3 domain may be a substitution that enhances a
property selected from the group consisting of the stability, half-life,
or Fc properties of C.sub.H2 and/or C.sub.H3 domains or alter
glycosylation of the C.sub.H2 and/or C.sub.H3 domain. For example, and
without wishing to be limiting in any manner, the mutation may be an
alteration at position 228 (EU numbering, 241 according to Kabat) where
the serine is substituted by a proline (S228P), which stabilizes the
disulfide linkage within the Fc dimer. Another alteration is the mutation
at position 409 (EU numbering, 440 according to Kabat) where an arginine
is substituted to a lysine for further stabilization of the Fc homodimer
at the C.sub.H3-domain level (Yang & Ambrogelly, 2014). Yet another
alteration within the C.sub.H2 and/or C.sub.H3 domain may be a
substitution of Asn297 (EU numbering, 314 according to Kabat) by glycine
or alanine to alter glycosylation of the constant domain. In yet another
example, the C.sub.H2 and/or C.sub.H3 domain may be altered by
substitution of one or more threonine (T252L, T253S, and/or T256F; see
[U.S. 62/777,375]) to increase half-life. Particularly useful are those
alterations that enhance Fc properties while remaining silent with
respect to conformation, e.g., retaining Fc receptor binding.
[0092] In yet another non-limiting example, the conservative mutations in
the C.sub.H2 and/or C.sub.H3 domain may be a substitution that is
naturally-occurring. Such mutations may occur in nature as minor sequence
differences between species or race.
[0093] Sequence identity is used to evaluate the similarity of two
sequences; it is determined by calculating the percent of residues that
are the same when the two sequences are aligned for maximum
correspondence between residue positions. Any known method may be used to
calculate sequence identity; for example, computer software is available
to calculate sequence identity. Without wishing to be limiting, sequence
identity can be calculated by software such as NCBI BLAST2 service
maintained by the Swiss Institute of Bioinformatics (and as found at
ca.expasy.org/tools/blast/), or any other appropriate software that is
known in the art.
[0094] The substantially identical sequences of the present invention may
be at least 90% identical; in another example, the substantially
identical sequences may have an identity selected from the group
consisting of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
identical, or any percentage there between, at the amino acid level to
sequences described herein. Importantly, the substantially identical
sequences retain the activity, specificity, and functionality of the
reference sequence. In a non-limiting embodiment, the difference in
sequence identity may be due to conservative amino acid mutation(s). In a
non-limiting example, the first portion of the polypeptide construct of
the present invention may comprise a Fc comprising a sequence selected
from the group consisting of a sequence at least 95%, 98% or 99%
identical to that of the Fc described herein.
[0095] Accordingly, it will be appreciated that the first portion of a
construct will include at least an antibody region that preferably
provides for cross-linking of the polypeptide constructs, thereby to
provide a dimeric protein. This first portion comprises at least the
minimal CH2 and/or CH3 domain. That portion can be altered (i) by
substituting or deleting cysteine residues from the hinge regions (SEQ ID
NO:5-8) involved in crosslinking between the antibody heavy chains or
between the heavy and light chains in order to potentially improve
preparation homogeneity and efficacy, and/or (ii) by deleting or suitably
replacing (e.g., by mutation to alanine) the terminal lysine residue 447
(EU numbering, 478 according to Kabat) of an IgG heavy chain in order to
improve chemical stability of C-terminal fusions to enzymatic proteolysis
(e.g., by several serine proteases and typically by trypsin). These
changes have a positive impact on potency and/or manufacturability, as
revealed herein. The first portion can also be extended to become a full
Fc region, by including the CH1 domain. As a full Fc, this portion will
provide normal Fc effector functions that include involvement in immune
cell recruitment, ADCC, CDC and other antibody functions. Moreover, and
in embodiments of the present invention, the first portion can include a
complete antibody or any equivalent thereof. In certain embodiments, such
as when a construct comprises just one ectodomain that is a TGF-.beta.
receptor II ectodomain, there is the proviso that the second portion is
not an antibody that binds to an immune checkpoint protein such as PD-L1
(programmed death ligand 1) and is not an antibody that binds to an
immunomodulating agent that counteracts immune tolerance of cancer cells,
the nature and identity of which is as described in [U.S. Pat. No.
8,815,247], and is not an antibody that binds one of EGFR1, her-2, CD4,
CD6, CD20, CD25, MUC-1, IL-2, IL-6, and CTLA-4.
[0096] The second portion of the polypeptide construct of the present
invention comprises at least one and preferably two TGF-.beta.
superfamily receptor ectodomain/s (T.beta.SR-ED); for example, the second
portion may comprise one or two T.beta.SR-ED. The ectodomain of the
Transforming Growth Factor-I3 superfamily receptor (T.beta.SR) is the
N-terminal extracellular, ligand-binding portion of the receptor. Without
wishing to be limiting in any manner, the T.beta.SR ectodomain may bind a
molecule selected from the group consisting of TGF-.beta., bone
morphogenetic protein (BMP) including BMP2, BMP3, BMP4, BMPS, BMP, BMP6,
BMP7, BMP8, BMP9, BMP10, BMP11, BMP12, BMP13, BMP14, an BMP15, activin
including activins .beta.A, .beta.B and .beta.C, growth differentiation
factor (GDF-1) including GDF-3, GDF-.beta., GDF-9, and GDF-15, nodal,
inhibin-.alpha., anti-Mullerian hormone, Lefty-1, Lefty-2, arteman,
persephin, neurturin, myostatin, or other known TGF-.beta. superfamily
ligands. For example, the T.beta.R ectodomain may be selected from the
group consisting of the human TGF-.beta. receptor type II ectodomain
(TI.beta.R-II-ED), the human TGF-.beta. receptor type IIb (TI.beta.R-IIb)
ectodomain, the human activin receptor type IIa (ActR-IIa) ectodomain,
the human activin receptor type IIb (ActR-IIb) ectodomain, or the BMP
type Ia (BMPR-Ia) ectodomain.
[0097] In a preferred embodiment the ectodomain binds TGF-.beta.1 and/or
TGF-.beta.3. In another preferred embodiment, the ectodomain itself is a
human TGF-.beta. receptor type II ectodomain including particularly the
TGF-.beta. receptor type IIa (T.beta.RIIa). In one specific, non-limiting
example, the T.beta.SR-ED is the TGF-.beta. receptor type II ectodomain
(T.beta.RII-ED; SEQ ID NO:35).
[0098] In the second portion as described above, the T.beta.SR
ectodomain-based portion may further comprise natural linkers.
Appropriate, naturally-derived linkers that can be used to fuse two
ectodomains head-to-tail are known to those of skill in the art; for
example, and without wishing to be limiting, suitable natural linkers are
described in [WO2008/113185].
[0099] In this embodiment, the natural linker, if present, may be selected
from the group consisting of
TABLE-US-00002
(SEQ ID NO: 36)
IPPHVQKSVNNDMIVTDNNGAVKFP;
(SEQ ID NO: 37)
IPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFP;
(SEQ ID NO: 39)
SEEYNTSNPD;
(SEQ ID NO: 40)
SEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFP;
(SEQ ID NO: 41)
SEEYNTSNPDIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVT
DNNGAVKFP;
and
a combination thereof.
[0100] In a specific, non-limiting example, the second portion of the
polypeptide construct of the present invention may comprise the sequence
selected from the group consisting of: [0101] A single TGF-.beta. Type
II receptor ectodomain, such as:
TABLE-US-00003
[0101] (SEQ ID NO: 43, also referred to herein as T2m)
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT
SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKE
KKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD;
(SEQ ID NO: 44)
IPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQ
LCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETV
CHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSE
EYNTSNPD;
[0102] A TGF-.beta. Type II receptor ectodomain "doublet", in which a
TGF-.beta. Type II receptor ectodomain is linked with another TGF-.beta.
Type II receptor ectodomain, which ectodomains can be the same or
different TGF-.beta. superfamily receptor ectodomains, such as:
TABLE-US-00004
[0102] (SEQ ID NO: 46, also referred to herein as T22d35)
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT
SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKE
KKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTD
NNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN
DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE
CNDNIIFSEEYNTSNPD;
(SEQ ID NO: 47)
IPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQ
LCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETV
CHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSE
EYNTSNPDIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDN
NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKND
ENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDEC
NDNIIFSEEYNTSNPD;
and [0103] a sequence substantially identical thereto. "Substantially
identical" is as defined above.
[0104] In another specific, non-limiting example, the TGF-.beta. receptor
ectodomain is the bone morphogenetic protein receptor la (BMPRIa; SEQ ID
NO:69). In this embodiment, the natural linker, if present, may be
selected from the group consisting of
TABLE-US-00005
(SEQ ID NO: 70)
QNLDSMLHGTGMKSDSDQKKSENGVTLAPED;
(SEQ ID NO: 71)
PVVIGPFFDGSIR;
(SEQ ID NO: 72)
PVVIGPFFDGSIRQNLDSMLHGTGMKSDSDQKKSENGVTLAPED;
and [0105] a combination thereof.
[0106] Thus, in a specific, non-limiting example, the second portion of
the polypeptide construct of the present invention may comprise the
sequence selected from the group consisting of:
TABLE-US-00006
(SEQ ID NO: 74)
QNLDSMLHGTGMKSDSDQKKSENGVTLAPEDTLPFLKCYCSGHCPDDAINN
TCITNGHCFAIIEEDDQGETTLASGCMKYEGSDFQCKDSPKAQLRRTIECC
RTNLCNQYLQPTLPPVVIGPFFDGSIRQNLDSMLHGTGMKSDSDQKKSENG
VTLAPEDTLPFLKCYCSGHCPDDAINNTCITNGHCFAIIEEDDQGETTLAS
GCMKYEGSDFQCKDSPKAQLRRTIECCRTNLCNQYLQPTLPPVVIGPFFDG
SIR;
and [0107] a sequence substantially identical thereto. "Substantially
identical" is as defined above.
[0108] In another specific, non-limiting example, the T6SR ectodomain is
the activin receptor IIa (ActRIIA; SEQ ID NO:75). In this embodiment, the
natural linker, if present, may be selected from the group consisting of
TABLE-US-00007
(SEQ ID NO: 76)
AILGRSE;
(SEQ ID NO: 77)
EMEVTQPTSNPVTPKPPYYNI;
(SEQ ID NO: 78)
EMEVTQPTSNPVTPKPPYYNIAILGRSE;
and [0109] a combination thereof.
[0110] Thus, another specific non-limiting example of the second portion
of the polypeptide construct of the present invention comprises the
sequence selected from the group consisting of:
TABLE-US-00008
(SEQ ID NO: 80)
AILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGS
IEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEME
VTQPTSNPVTPKPPYYNIAILGRSETQECLFFNANWEKDRTNQTGVEPCYG
DKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFC
CCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPYYNI;
and [0111] a sequence substantially identical thereto. "Substantially
identical" is as defined above.
[0112] In another specific, non-limiting example, the TGF-.beta. receptor
ectodomain is the activin receptor IIb (ActRIIb; SEQ ID NO:81). In this
embodiment, the natural linker, if present, may be selected from the
group consisting of
TABLE-US-00009
(SEQ ID NO: 82)
SGRGEAET;
(SEQ ID NO: 83)
EAGGPEVTYEPPPTAPT;
(SEQ ID NO: 84)
EAGGPEVTYEPPPTAPTSGRGEAET;
and [0113] a combination thereof.
[0114] Thus, another specific non-limiting example of the second portion
of the polypeptide construct of the present invention comprises the
sequence selected from the group consisting of:
TABLE-US-00010
(SEQ ID NO: 86)
SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGT
IELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAG
GPEVTYEPPPTAPTSGRGEAETRECIYYNANWELERTNQSGLERCEGEQDK
RLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEG
NFCNERFTHLPEAGGPEVTYEPPPTAPT;
and [0115] a sequence substantially identical thereto. "Substantially
identical" is as defined above.
[0116] Thus, in various embodiments of the present invention, the present
constructs have an ectodomain comprising an amino acid sequence selected
from the group consisting of SEQ ID NO:35, SEQ ID NO:69, SEQ ID NO:75,
SEQ ID NO:81, and a sequence substantially identical thereto. In other
embodiments, the second portion comprises the entire extracellular
portion of a T.beta.SR-ED consisting of a sequence selected from the
group consisting of SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:73, SEQ ID
NO:79, SEQ ID NO:85, and a sequence substantially identical thereto.
[0117] The at least two ectodomain portion can have the same or different
ectodomains, all belonging to the superfamily. In embodiments, the
ectodomains bind the same target. In other embodiments, the ectodomains
originate from the same receptor species. In other embodiments, the
ectodomains are identical and thus are homomeric. In other embodiments
the ectodomains are different and thus are heteromeric. In these
embodiments, the ectodomain can be for instance a T.beta.RII-ED that is
type a, and another ectodomain can be a T.beta.RII-ED that is type b. A
third ectodomain could be the same as either one of these, or different
still. For example, when there is more than one ectodomain in the second
portion of the polypeptide construct of the present invention, the
ectodomains may be all the same (homomers) or all different (heteromers),
or any combination of superfamily ectodomains.
[0118] Thus, in embodiments, the second portion of the polypeptide
construct of the present invention comprises a repeat of a given
T.beta.SR-ED selected from the group consisting of SEQ ID NO:46, 47, 48,
74, 80, 86, and a sequence substantially identical thereto.
[0119] In specific embodiments, the second portion of the polypeptide
construct of the present invention comprises heteromeric repeats of two
distinct T.beta.SR-EDs genetically fused and selected from the group
consisting of SEQ ID NO:61, 62, 63, 88, and a sequence substantially
identical thereto.
[0120] In yet other embodiments, the second portion of the polypeptide
construct of the present invention comprises homo-multimeric and
hetero-multimeric repeats of one or more T.beta.SR-EDs selected for
instance from the group consisting of SEQ ID NO:49, 50, 51, 64, 65, 66,
67, 68, and a sequence substantially identical thereto.
[0121] In the protein construct of the present invention, the first and
second portions of the polypeptide construct of the present invention are
linked. By the term "linked", it is meant that the two portions are
covalently bonded. The chemical bond may be achieved by chemical
reaction, or may be the product of recombinant expression of the two
portions in a single polypeptide chain. In one specific, non-limiting
example, the C-terminus of the first portion is linked directly to the
N-terminus of the second portion, that is, no additional "linker" amino
acids are present between the two portions. In the case where no linker
is present, that is to say direct fusion of the two portions, there will
be a direct link between the N-terminus of the full ectodomain and the
C-terminus of the antibody constant regions C.sub.H2-C.sub.H3. For
example, in fusing the Fc variant SEQ ID NO:9 to the SEQ ID NO:43 via the
intrinsically disordered linker with SEQ ID NO:36, which is part of the
T.beta.RII-ED (i.e., no additional "linker" amino acids added), one
connects the glycine at the last position of SEQ ID NO:9 to the
isoleucine at the first position of SEQ ID NO:43.
[0122] A common practice when producing fusion constructs is to introduce
glycine or glycine-serine linkers (such as GGGGS, or [G.sub.4S].sub.n)
between the fused components. As taught in the above paragraph, the
polypeptide fusions of the present invention can be produced by direct
linkage without use of any additional amino-acid sequence except those
present in the Fc portion and in the receptor ectodomain portion. One
thus can refrain from utilizing foreign sequences as linkers, providing
an advantage due to their potential for undesired immunogenicity and
their added molecular weight. Entropic factors are also a potential
liability for glycine and glycine-serine linkers, which are highly
flexible and may become partially restricted upon target binding, hence
causing a loss of entropy unfavourable to binding affinity. Therefore,
only the flexible, intrinsically disordered N-terminal regions of the
T.beta.SR receptor ectodomains were employed as natural linkers in
embodiments of the present invention. However, the particular amino acid
compositions and lengths of these intrinsically disordered linkers (e.g.,
SEQ ID NO:36, 37, 70, 76, 82) precluded accurate prediction of whether
the resulting direct-fusion constructs will have the required geometry
and favourable molecular interactions for correct binding to their
intended dimeric ligands.
[0123] The first and second portions of the polypeptide construct are, in
embodiments, connected by natural intrinsically disordered polypeptide
linkers selected from the group consisting of SEQ ID NO:36, 37, 38, 53,
70, 76, 82, and a sequence substantially identical thereto.
[0124] In embodiments, when multiple T.beta.SR-ED structured regions are
present, these can be fused directly or they can be connected by natural
intrinsically disordered polypeptide linkers between the ectodomains,
such as SEQ ID NO:40, 41, 42, 55, 58, 59, 60, 72, 78, 84, 87, and a
sequence substantially identical thereto.
[0125] Non-limiting examples of full-length polypeptide constructs of the
present invention that comprise the two aforementioned portions are
selected from the group consisting of SEQ ID NO: 91-120, and a sequence
substantially identical thereto.
[0126] It is particularly important to note that, in the polypeptide
constructs of the present invention, the N-terminus of the second portion
is linked to the C-terminus of the first portion (see for example FIGS.
2A, C, D, E, F, and G).
[0127] Some of the polypeptide constructs of the present invention display
significantly greater potency of TGF-.beta. neutralization compared to
that of the TGF-.beta. superfamily receptor ectodomain alone; for
example, the polypeptide construct may be between at least 50-fold and
1.times.10.sup.6-fold more potent. For example, the polypeptide
constructs of the present invention may have a TGF-.beta. neutralization
potency selected from the group consisting of at least 50-, 75-, 100-,
150-, 200-300-, 400-, 500-, 600, 1000-1500-, 2000-, 3000-, 4000-, 5000-,
6000-, 7000-, 8000-, 9000, 10000-, 20000-, 30000-, 40000-, 50000-,
60000-, 70000-, 80000-, 90000-, 100000-, 150000-, 200000-, 250000-,
300000-, 350000-, 400000-, 450000-, 500000-, 550000-600000-, 650000-,
700000-, 750000-, 800000-, 850000-, 900000-, 950000, or 1000000-fold,
more potent than the T.beta.SR-ED alone, or any amount there between. In
one example, the potency of the construct is at least 100-fold greater
than the receptor ectodomain alone.
[0128] Additionally, when the polypeptide constructs of the present
invention include a T.beta.SR-ED that binds TGF-.beta., the polypeptide
construct may neutralize, to varying extents, all three isotypes of
TGF-.beta. (that is, TGF-.beta.1, TGF-.beta.2, and TGF-.beta.)
[0129] The polypeptide constructs of the present invention have, as
assessed in cell-based assays, TGF-.beta. neutralizing potencies that are
significantly higher (100-fold or more) than those of bivalent comparator
polypeptides, i.e. non-Fc-fused T22d35 and T2m-Fc. Within the series of
polypeptide constructs of the present invention, those that contain two
or more copies of the T.beta.RII ectodomain fused to the C-terminus of
the Fc constant region have potencies that are higher than those
constructs that contain only one copy, as assessed in cell based assays.
Additionally, within the series of polypeptide constructs of the present
invention, if the first portion within the construct is "headless", i.e.
does not contain a Fab region, the potencies of the constructs are
increased by engineering (optimizing) the number of cysteines in the
hinge region at the "revealed" N-terminus. Engineering of the cysteine
residues at the N-termini of "headless" constructs also markedly reduces
the aggregation propensity of the constructs. Lastly, within the series
of polypeptide constructs of the present invention, in vivo in tumor
models, cysteine optimized "headless" constructs exhibit higher
anti-tumor immuno-modulatory potencies than constructs in which the first
portion is a full-sized antibody.
[0130] The polypeptide construct of the present invention is expressed as
a single polypeptide chain. Once expressed, the polypeptide construct of
the present invention forms a dimer wherein the C.sub.H2 and C.sub.H3
domains of the respective polypeptide constructs interact to form a
properly assembled Fc region such as occurs when the expressed products
are secreted into the culturing medium. For example, and without wishing
to be limiting, examples of dimerized polypeptide constructs of the
present invention are shown in FIGS. 2A and C-G. In one example,
homodimers may be formed by identical polypeptide constructs.
Alternatively, heterodimers may be formed by two different polypeptide
constructs; thus, a heterodimer may be formed by two Fc region
polypeptide constructs that have been engineered to induce
heterodimerization and inhibit homodimerization.
[0131] The first portion of the polypeptide construct described above may
further comprise, at its N-terminus, any suitable antigen-binding
antibody fragment known in the art. For example, and without wishing to
be limiting in any manner, the first portion of the polypeptide construct
may comprise C.sub.H2 and C.sub.H3 domains and one selected from the
group consisting of a single-chain Fv (scFv; a molecule consisting of
V.sub.L and V.sub.H connected with a peptide linker) and a single-domain
antibody (sdAb, a fragment composed of a single V.sub.L or a single
V.sub.H; see for example FIG. 1C). In other instances, the
antigen-binding fragment may be formed by combining the polypeptide
construct with a second polypeptide chain. For example, the first portion
of the polypeptide construct may comprise C.sub.H2 and C.sub.H3 domains
along with a C.sub.H1 and V.sub.H domains, which when combined with a
second polypeptide comprising C.sub.L and V.sub.L form a full-size
antibody (i.e., Fc and Fab; see for example FIG. 1D). In another example,
the first portion of the polypeptide may comprise C.sub.H2 and C.sub.H3
domains along with V.sub.H, which when combined with a second polypeptide
comprising a V.sub.L forms an Fc fused to a Fv.
[0132] The combination of constant domains and antigen-binding fragment
may be naturally-occurring, or may be obtained by manipulation of a
naturally-occurring antibody or by using recombinant methods. The
polypeptide constructs such as those just described may require a
sequence selected from the group consisting of linker sequences,
disulfide bonds, hinge region sequences, and other type of covalent bond
to link them to the C.sub.H2 and C.sub.H3 domains; those of skill in the
art will be familiar with various suitable approaches.
[0133] In alternative constructs of the present invention, the polypeptide
construct comprises an antibody Fc fragment linked at the C-terminus of
each heavy chain to at least one TGF-.beta. superfamily receptor
ectodomain (T.beta.SR-ED), as described above and as illustrated in FIG.
2(A,D,E). The construct may further comprise an antigen-binding fragment
at the N-terminus of the Fc; the antigen-binding fragment may be selected
from the group consisting of a Fv, scFv, Fab, or sdAb, also as described
above. In the polypeptide constructs as described above, the TGF-.beta.
receptor ectodomain does not interfere in the native function or
specificity of the antigen-binding fragment.
[0134] The antigen-binding antibody fragment described above, when
present, may be directed to any suitable antigen. In certain limited
embodiments, the antigen-binding antibody or fragment does not bind to an
antigen that is PD-L1, EGFR1, her-2, CD4, CD6, CD20, CD25, MUC-1, IL-2,
IL-6, or CTLA-4.
[0135] The present constructs can further comprise antibody or antibody
fragments that target any antigen of interest. They can also comprise the
antigen itself, or any other moiety of interest that is genetically
encoded. Particular embodiments herein include the EGFR antibody
cetuximab and its active fragments, Avastin, Herceptin, Synagis, FC5, or
a poly-aspartate bone-localization motif, such a D10, or sequence
substantially identical or equivalent thereto.
[0136] The present constructs can comprise a binding protein e.g.,
antibody and binding fragments thereof, that inhibits a checkpoint
protein which may be CTLA-4, PD1, PDL1, PDL2, PDL3, PD1, B7-H3, B7-H4,
BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1,
CHK2, A2aR, CD28, CD86, or one of the B-7 family ligands or a combination
thereof.
[0137] Illustrative immune checkpoint inhibitors include Tremelimumab
(CTLA-4 blocking antibody), anti-OX40, PD-LI monoclonal Antibody
(Anti-B7-HI; MED14736), MK-3475 (PD-1 blocker), Nivolumab (anti-PDI
antibody), CT-011 (anti-PDI antibody), BY55 monoclonal antibody, AMP224
(anti-PDLI antibody), BMS-936559 (anti-PDLI antibody), MPLDL3280A
(anti-PDLI antibody), MSB0010718C (anti-PDLI antibody) and
Yervoy/ipilimumab (anti-CTLA-4 checkpoint inhibitor).
[0138] Other antibodies provided by the present constructs can include
rituximab, muromonab-CD3, abciximab, daclizumab, basiliximab,
palivizumab, infliximab, trastuzumab, gemtuzumab ozogamicin, alemtuzumab,
ibritumomab tiuxetan, adalimumab, omalizumab, tositumomab, I-131
tositumomab, efalizumab, bevacizumab, panitumumab, pertuzumab,
natalizumab, etanercept, IGN101, volociximab, Anti-CD80 mAb, Anti-CD23
mAb, CAT-3888, CDP-791, eraptuzumab, MDX-010, MDX-060, MDX-070,
matuzumab, CP-675,206, CAL, SGN-30, zanolimumab, adecatumumab,
oregovomab, EGFR-binding antibodies cetuximab, nimotuzumab, necitumumab,
panitumumab, matuzumab, and zalutumumab, as well as ABT-874, denosumab,
AM 108, AMG 714, fontolizumab, daclizumab, golimumab, CNTO 1275,
ocrelizumab, HuMax-CD20, belimumab, epratuzumab, MLN1202, visilizumab,
tocilizumab, ocrerlizumab, certolizumab, eculizumab, pexelizumab,
abciximab, ranibizimumab, mepolizumab, TNX-355, or MYO-029.
[0139] Still other antibodies that can be included in the present
constructs are rituximab, zanolimumab, hA20, AME-133, HumaLYM,
trastuzumab, pertuzumab, IMC-3G3, ch806, KSB-102, MR1-1, SC100, SC101,
SC103, alemtuzumab, muromonab-CD3, OKT4A, ibritumomab, gemtuzumab,
alefacept, abciximab, basiliximab, palivizumab, motavizumab, infliximab,
adalimumab, CDP-571, etanercept, ABX-CBL, ABX-IL8, ABX-MA1 pemtumomab,
Therex, AS1405, natalizumab, HuBC-1, natalizumab, IDEC-131, VLA-1;
CAT-152; J695, CAT-192, CAT-213, BR3-Fc, LymphoStat-B, TRAIL-R1mAb,
bevacizumab, ranibizumab, omalizumab, efalizumab, MLN-02, zanolimumab,
HuMax-IL 15, HuMax-Inflam, HuMax-Cancer, HuMax-Lymphoma, HuMax-TAC,
clenoliximab, lumiliximab, BEC2, IMC-1C11, DC101, labetuzumab,
arcitumomab, epratuzumab, tacatuzumab, MyelomaCide, LkoCide, ProstaCide,
ipilimumab, MDX-060, MDX-070, MDX-018, MDX-1106, MDX-1103, MDX-1333,
MDX-214, MDX-1100, MDX-CD4, MDX-1388, MDX-066, MDX-1307, HGS-TR2J,
FG-3019, BMS-66513, SGN-30, SGN-40, tocilizumab, CS-1008, IDM-1,
golimumab, CNTO 1275, CNTO 95, CNTO 328, mepolizumab, MOR101, MOR102,
MOR201, visilizumab, HuZAF, volocixmab, ING-1, MLN2201, daclizumab,
HCD122, CDP860, PRO542, C14, oregovomab, edrecolomab, etaracizumab,
siplizumab, lintuzumab, Hu1D10, Lym-1, efalizumab, ICM3, galiximab,
eculizumab, pexelizumab, LDP-01, huA33, WX-G250, sibrotuzumab, Chimeric
KW-2871, hu3S193, huLK26; bivatuzumab, ch14.18, 3F8, BC8, huHMFG1,
MORAb-003, MORAb-004, MORAb-009, denosumab, PRO-140, 1D09C3, huMikbeta-1,
NI-0401, NI-501, cantuzumab, HuN901, 8H9, chTNT-1/B, bavituximab, huJ591,
HeFi-1, Pentacea, abagovomab, tositumomab, 105AD7, GMA161 and GMA321.
[0140] In other embodiments, the constant domain/first portion of the
constructs can comprise a polypeptide having medicinal properties, such
as agents that stimulate the immune system, in particular in relation to
the ability of the immune system to attack tumor cells. These
polypeptides can include cytokines (such as interleukin-2) or growth
factors that stimulate immune cells directly or indirectly (i.e. act by
providing gas to the immune system), as well as ectodomains or other
binding agents that neutralize ligands which inhibit immune cells, either
directly or indirectly (i.e. act by releasing a brake on the immune
system).
[0141] In other embodiments, the constant domain/first portion of the
constructs can comprise a polypeptide that does not have active medicinal
properties per se, but rather provides a localization signal. This
localization motif will serve to focus the intrinsic TGF-.beta.
neutralization activity of the second portion of the construct to a
particular region of the body. In one example, the first portion
comprised a long poly-aspartate bone-localization motif, preferably D10
or an equivalent bone-localization moiety, which acts to enhance
localisation of the construct to bone. By increasing the TGF-.beta.
neutralization activity of the construct within bone, more favourable
dosing levels and schedules may be required for the treatment of
bone-related diseases, such as osteogenesis imperfecta, relative to that
required for a similar construct without the D10 motif.
[0142] Embodiments exemplifying polypeptide constructs of the present
invention that include antigen-binding fragments at the N-terminus of the
Fc region (first portion) are selected from the group consisting of SEQ
ID NO: 121-131, and a sequence substantially identical thereto.
[0143] In other embodiments, polypeptide constructs of the present
invention that include other targeting agents, e.g., a poly-aspartate
bone-localization motif, at the N-terminus of the Fc region (first
portion), are exemplified by SEQ ID NO: 136-150.
[0144] The polypeptide construct of the present invention may also
comprise additional sequences to aid in expression, detection or
purification of a recombinant antibody or fragment thereof. Any such
sequences or tags known to those of skill in the art may be used. For
example, and without wishing to be limiting, the antibody or fragment
thereof may comprise a targeting or signal sequence (for example, but not
limited to ompA), a detection/purification tag (for example, but not
limited to c-Myc, His.sub.s, His.sub.6, or His.sub.8G), or a combination
thereof. In another example, the signal peptide may be
MVLQTQVFISLLLWISGAYG (SEQ ID NO:89) or MDVVTWRILFLVAAATGTHA (SEQ ID
NO:90). In a further example, the additional sequence may be a biotin
recognition site such as that described in [WO/1995/04069] or in
[WO/2004/076670]. As is also known to those of skill in the art, linker
sequences may be used in conjunction with the additional sequences or
tags, or may serve as a detection/purification tag.
[0145] The present invention also encompasses nucleic acid sequences
encoding the molecules as described herein. Given the degeneracy of the
genetic code, a number of nucleotide sequences would have the effect of
encoding the desired polypeptide, as would be readily understood by a
skilled artisan. The nucleic acid sequence may be codon-optimized for
expression in various micro-organisms. The present invention also
encompasses vectors comprising the nucleic acids as just described,
wherein the vectors typically comprise a promoter and signal sequence
that are operably linked to the construct-encoding polynucleotide for
driving expression thereof in the selected cellular production host. The
vectors can be the same or different provided both result in secretion of
the dimeric polypeptide construct.
[0146] Furthermore, the invention encompasses cells, also referred to
herein as transgenic cellular host, comprising the nucleic acid and/or
vector as described, encoding a first polypeptide construct. The host
cells may comprise a second nucleic acid and/or vector encoding a second
polypeptide construct different from the first polypeptide construct. The
co-expression of the first and second polypeptide constructs may lead to
the formation of heterodimers.
[0147] The present invention also encompasses a composition comprising one
or more than one polypeptide construct as described herein. The
composition may comprise a single polypeptide construct as described
above, or may be a mixture of polypeptide constructs. The composition may
also comprise one or more than one polypeptide construct of the present
invention linked to one or more than one cargo molecule. For example, and
without wishing to be limiting in any manner, the composition may
comprise one or more than one polypeptide construct of the present
invention linked to a cytotoxic drug in order to generate an
antibody-drug conjugate (ADC) in accordance with the present invention.
[0148] The composition may also comprise a pharmaceutically acceptable
diluent, excipient, or carrier. The diluent, excipient, or carrier may be
any suitable diluent, excipient, or carrier known in the art, and must be
compatible with other ingredients in the composition, with the method of
delivery of the composition, and is not deleterious to the recipient of
the composition. The composition may be in any suitable form; for
example, the composition may be provided in suspension form, powder form
(for example, but limited to lyophilised or encapsulated), capsule or
tablet form. For example, and without wishing to be limiting, when the
composition is provided in suspension form, the carrier may comprise
water, saline, a suitable buffer, or additives to improve solubility
and/or stability; reconstitution to produce the suspension is effected in
a buffer at a suitable pH to ensure the viability of the antibody or
fragment thereof. Dry powders may also include additives to improve
stability and/or carriers to increase bulk/volume; for example, and
without wishing to be limiting, the dry powder composition may comprise
sucrose or trehalose. In a specific, non-limiting example, the
composition may be so formulated as to deliver the antibody or fragment
thereof to the gastrointestinal tract of the subject. Thus, the
composition may comprise encapsulation, time-release, or other suitable
technologies for delivery of the antibody or fragment thereof. It would
be within the competency of a person of skill in the art to prepare
suitable compositions comprising the present compounds.
[0149] The constructs of the present invention may be used to treat
diseases or disorders associated with over-expression or over-activation
of ligands of the TGF-.beta. superfamily. The disease or disorder can be
selected from, but not limited to, cancer, ocular diseases, fibrotic
diseases, or genetic disorders of connective tissue.
[0150] In the field of cancer therapy, it has recently been demonstrated
that TGF-.beta. is a key factor inhibiting the antitumor response
elicited by immunotherapies, such as immune checkpoint inhibitors (ICI's)
(Hahn & Akporiaye, 2006). Specifically, therapeutic response to ICI
antibodies results primarily from the re-activation of tumor-localized
T-cells. Resistance to ICI antibodies is attributed to the presence of
immunosuppressive mechanisms that result in a dearth of T-cells in the
tumor microenvironment. Thus, it is now recognized that in order to
elicit responses in resistant patients, ICI antibodies need to be
combined with agents that can activate T-cells and induce their
recruitment into the tumor, i.e. reversing of the "non-T-cell-inflamed"
tumor phenotype. One publication noted that overcoming the
non-T-cell-inflamed tumor microenvironment is the most significant next
hurdle in immuno-oncology (Gajewski, 2015).
[0151] We have shown using a proof-of-principle TGF-.beta. trap, T22d35,
that blocking of TGF-.beta. effectively reverses the "non-T cell
inflamed" tumor phenotype (Zwaagstra et al, 2012). This positions
anti-TGF-.beta. molecules as potential synergistic combinations with
ICI's and other immunotherapeutics. In support of this, a 2014 study
(Holtzhausen et al., ASCO poster presentation) examined effects of a
TGF-.beta. blocker when combined an anti-CTLA-4 antibody in a
physiologically-relevant transgenic melanoma model. The study
demonstrated that while anti-CTLA-4 antibody monotherapy failed to
suppress melanoma progression, the combination of the TGF-.beta.
antagonist and anti-CTLA-4 antibody significantly and synergistically
suppressed both primary melanoma tumor growth as well as melanoma
metastasis. These observations correlated with significant increases in
effector T-cells in melanoma tissues.
[0152] Fibrotic diseases include those that affect any organ of the body,
including, but not limited to kidney, lung, liver, heart, skin and eye.
These diseases include, but are not limited to, chronic obstructive
pulmonary disease (COPD), glomerulonephritis, liver fibrosis,
post-infarction cardiac fibrosis, restenosis, systemic sclerosis, ocular
surgery-induced fibrosis, and scarring.
[0153] Genetic disorders of connective tissue include, but are not limited
to, Marfan syndrome (MFS) and Osteogenesis imperfecta (01).
[0154] The present invention will be further illustrated in the following
examples. However, it is to be understood that these examples are for
illustrative purposes only and should not be used to limit the scope of
the present invention in any manner.
EXAMPLE 1
Production and Purification of Fusion Molecules
[0155] Several fusion molecules comprising full-size antibody (FSA),
V.sub.HH-IgG Fc, D10-Fc or "headless" Fc C-terminally-fused to the T22d35
or T2m ectodomains were constructed (Table 1). All constructs comprising
a heavy chain included the signal sequence MDVVTWRILFLVAAATGTHA (SEQ ID
NO:89) at the N-terminus, while constructs comprising a light chain
included the signal sequence MVLQTQVFISLLLWISGAYG (SEQ ID NO:90) at the
N-terminus. The DNA coding for constructs were prepared synthetically
(Biobasic Inc. or Genescript USA Inc.). Constructs comprising FSA, D10-Fc
and "headless" Fc were cloned into the EcoR1 (5' end) and BamH1 (3' end)
sites and those comprising V.sub.HH-IgG Fc were cloned into the HindIII
(5' end) and BamH1 (3' end) sites of the pTT5 mammalian expression
plasmid vector (Durocher et al, 2002).
TABLE-US-00011
TABLE 1
FSA-, V.sub.HH-IgG Fc-, D10-Fc- and Fc-fusion constructs produced.
The letter in brackets in the construct column refers
to the type of construct as illustrated in FIG. 2.
Fusion Antibody Construct
Construct ED Scaffold source SEQ ID NO:
Cet-T2m (F) T2m FSA (hIgG1) Cetuximab 121, 123
Cet-T22d35 (D) T22d35 FSA (hIgG1) Cetuximab 121, 122
Her-T22d35 (D) T22d35 FSA (hIgG1) Herceptin 124, 125
Ava-T22d35 (D) T22d35 FSA (hIgG1) Avastin 126, 127
Syn-T22d35 (D) T22d35 FSA (hIgG1) Synagis 128, 129
FC5-Fc-T22d35 (E) T22d35 V.sub.HH-Fc FC5-Fc 130
(mIgG2)
FC5-Fc-T2m (G) T2m V.sub.HH-Fc FC5-Fc 131
(mIgG2)
D10-Fc-T2m (G) T2m Fc huIgG 136-139
(several variants with
differing D10 linkage
and IgG isotype)
Fc-T22d35 (A) T22d35 Fc huIgG 100, 105
(several variants with
differing N-termini
and IgG isotype)
Fc-T2m (C) T2m Fc huIgG 91-97
(several variants with
differing N-termini
and IgG isotype)
T2m-Fc (R&D) (B) T2m Fc huIgG1 132
T2m-Fc (B) T2m Fc huIgG2 133
[0156] The Cet-T2m and Cet-T22d35 constructs were produced by transient
co-transfection of Chinese Hamster Ovary (CHO) cells with the heavy chain
(HC)-T2m or (HC)-T22d35 construct combined with the Cetuximab light chain
(LC) construct which then assembled as the Cetuximab-T22d35 (Cet-T22d35)
or Cetuximab-T2m (Cet-T2m) fusion molecules. Briefly, CetHC-T22d35 (SEQ
ID NO:122) and CetLC (SEQ ID NO:121) plasmid DNAs (ratio=3:2) were
co-transfected into a 10 L Wavebag culture of CHO-3E7 cells in FreeStyle
F17 medium (Invitrogen) containing 4 mM glutamine and 0.1% Kolliphor
p-188 (Sigma) in a Wavebag maintained at 37.degree. C. Transfection
conditions were: DNA (50% HC+LC plasmids, 30% ssDNA, 15% AKT plasmid, 5%
GFP plasmid): PEI(polyethylenimine)pro (Polyplus) (ratio=1:2.5). At 24
hours post-transfection, 10% Tryptone N1feed (TekniScience Inc.) and 0.5
mM Vaporic acid (VPA, Sigma) were added and the temperature was shifted
to 32.degree. C. to promote the production and secretion of the fusion
proteins and maintained for 15 days post transfection after which the
cells were harvested. At final harvest the cell viability was 89.6%. The
harvest supernatant (10.8 L) was filtered (0.2 .mu.m) and loaded onto a
55 mL Protein A MabSelect Sure 55 mL column (GE Healthcare). The column
was washed with 2 column volumes of PBS and protein was eluted with 3
column volumes of 0.1 M sodium citrate pH 3.6. To maximize the yield, the
flow through was reloaded onto the Protein A column and eluted as
described above. Eluted fractions were neutralized with 1 M Tris,
evaluated by SDS-PAGE and those containing Cet-T22d35 were pooled (FIG.
3A) and subsequently loaded onto a Hi-load Superdex S200 26/60 size
exclusion chromatography (SEC) column (GE Healthcare) equilibrated in
formulation buffer (DPBS without Ca.sup.2+, without Mg.sup.2+). Protein
was eluted using 1 column volume formulation buffer, collected into
successive fractions and detected by UV absorbance at 280 nM (FIG. 3B).
The main peak SEC fractions containing Cet-T22d35 protein were then
pooled and concentrated to a concentration of 7.8 mg/mL. The final yield
was 533 mg.
[0157] Similar transfection, production and purification methods were
performed for the other FSA-trap examples listed in Table 1. In the case
of the V.sub.HH-Fc IgG-, D10 and "headless" Fc-fusion molecules the
composition of the transfection mixture was modified as follows: DNA (80%
plasmid construct, 15% AKT plasmid, 5% GFP plasmid): PElpro (ratio
1:2.5).
[0158] The integrity of the pooled Prot-A and SEC fractions of Cet-T22d35
protein was analyzed by SDS-PAGE (4-15% polyacrylamide) under reducing
and non-reducing conditions (FIG. 3C) and by UPLC-SEC (FIG. 3D). For
UPLC-SEC, 2-10 .mu.g of protein in DPBS (Hyclone, minus Ca2+, minus Mg2+)
was injected onto a Waters BEH200 SEC column (1.7 .mu.m, 4.6.times.150
mm, SN:01773430816818) and resolved under a flow rate of 0.4 mL/min for
8.5 min at room temperature, using the Waters Acquity UPLC H-Class
Bio-System. Protein peaks were detected at 280 nM (Acquity PDA detector).
Coomassie brilliant blue (CBB) staining of the gels shows the
CetHC-T22d35 (.about.110 Kd) and CetLC bands (-30 Kd) under reducing
conditions while under non-reducing conditions a 250 Kd band is detected
which represents the fully assembled and highly pure Cet-T22d35 fusion
protein. Additional UPLC-SEC analysis of the SEC purified, pooled ProtA
sample confirmed the high degree of purity (99.42%) of the Cet-T22d35
protein and the absence of aggregates. Together, these results
demonstrate the manufacturability of the Cet-T22d35 fusion protein.
[0159] Similar methods were used to analyse expression levels,
purifiability, aggregation levels, and dimeric assembly of several other
Fc-ectodomain constructs. The results from these studies are shown in
FIG. 3E to L.
[0160] FIG. 3E to 3H show the results from the analysis of the
hIgG1Fc.DELTA.K(C)-T2m construct (an example of Type C construct from
FIG. 2). FIG. 3E shows the (ProtA)-affinity column elution profile.
Fraction 12-15 were pooled, subjected to a UPLC-SEC evaluation (FIG. 3F),
further SEC purified to remove aggregates and re-evaluated by UPLC-SEC
(FIG. 3G). This confirmed the high degree of purity of the
hIgG1Fc.DELTA.K(C)-T2m construct and the absence of aggregates. SDS-PAGE
(FIG. 3H) under non-reducing (NR) and reducing (R) conditions shows bands
of expected molecular weights, demonstrating the expected assembly of
hIgG1Fc.DELTA.K(C)-T2m as a disulphide linked dimer.
[0161] FIGS. 3I and 3J compare the level of aggregation of two "headless"
Fc-T2m constructs (examples of Type C in FIG. 2). The Fc-T2m construct is
an IgG2-based construct without engineering of the hinge region, thus it
contains four cysteine residues, whereas the hIgG2Fc.DELTA.K(CC)-T2m has
been engineered by N-terminal truncation of the hinge region to have only
two cysteines in the hinge region. It can be seen that the Fc-T2m
construct contains a high level of aggregates after Protein A
purification, with a doublet peak remaining even after further SEC
purification. In contrast, hIgG2Fc.DELTA.K(CC)-T2m, which has an
engineered N-terminus, exhibited low levels of aggregates after only
Protein A purification. These results demonstrate the advantage of
carrying out N-terminal engineering of headless Fc-T2m constructs to
reduce aggregation.
[0162] FIGS. 3K and 3L compare the level of aggregation of two "headless"
Fc-T22d35 constructs (examples of Type A in FIG. 2). The Fc-T22d35
construct is without engineering of the hinge-region cysteine residues
whereas the hIgG1Fc.DELTA.K(C)-T22d35 construct has been engineered by
N-terminal truncation of the hinge to have only one cysteine in the hinge
region. It can be seen that, similarly to Fc-T2m, the Fc-T22d35 construct
contains a high level of aggregates after Protein A purification, with a
doublet peak remaining as detected by UPLC-SEC even after further SEC
purification. In contrast, hIgG1Fc.DELTA.K(C)-T22d35, which has an
engineered N-terminus, exhibited lower levels of aggregates after Protein
A purification. Further SEC purification yielded a singlet peak as
detected by UPLC-SEC, confirming the absence of aggregates. These results
demonstrate the advantage of carrying out N-terminal engineering of
headless Fc-T22d35 constructs to reduce aggregation.
EXAMPLE 2
Neutralization and Binding of TGF43 by Fusion Constructs
[0163] The TGF-.beta. neutralization potencies of purified Fc-ectodomain
fusion constructs were determined and compared to those of non-Fc-fused
T22d35. It should be noted that non-Fc-fused T2m does not neutralize any
of TGF-.beta.1, -.beta.2, or -.beta.3 (De Crescenzo et al, 2001).
[0164] TGF-.beta. neutralization potencies for TGF-.beta.1, -.beta.2 and
-.beta.3 were determined for purified fusion constructs using two
cell-based signaling assays: 1) the Mv1Lu cell luciferase reporter assay
with Mv1Lu cells having a PAI-1-luciferase reporter (as described in
(Zwaagstra et al, 2012)) and an A549 cell/IL-11 release assay adapted to
the MSD (Meso Scale Discovery) platform.
[0165] Mv1Lu cell luciferase reporter assay: Briefly, cells were seeded
onto 96-well plates (20,000 cells/well) and then treated with T2m,
T22d35, or a fusion construct+25 pM TGF-.beta. at 37.degree. C. for 16 h
in DMEM, 1% FBS, 0.1% BSA. Cells were then lysed and luciferase activity
was measured (Promega Corp.) using a Synergy 2 plate reader (BioTek
Instruments Inc.).
[0166] A549 cell IL-11 release assay: Human A549 lung cancer cells
(ATCC-CCL-185, Cedarlane Burlington ON) were seeded in 96-well plates
(5.times.103 cells/well). The following day 10 pM TGF-.beta. in complete
media in the absence or presence of a serial dilution of fusion protein
was incubated for 30 min at RT prior to adding to the cells. After 21 h
of incubation (37.degree. C., 5% CO2, humidified atmosphere) conditioned
medium was harvested and added to MSD Streptavidin Gold plates (Meso
Scale Diagnostics, Gaithersburg, Md.) that were coated with 2 .mu.g/mL
biotinylated mouse anti-human IL-11 antibody (MAB618, R&D Systems,
Minneapolis, Minn.). After 18 h (4.degree. C.) plates were washed with
PBS containing 0.02% Tween 20, a 2 .mu.g/mL SULFO-tagged goat anti-human
IL-11 antibody (AF-218-NA, R&D Systems Minneapolis, Minn.) was added and
plates were incubated for 1 h at RT. After a final wash, plates were read
in a MESO QuickPlex SQ120 machine (Meso Scale Diagnostics, Gaithersburg,
Md.). IL-11 readouts were expressed as percent IL-11 release compared to
control cells treated with TGF-.beta. alone.
[0167] In one set of experiments, using the Mv1 Lu cell reporter assay,
the neutralization potency of Cet-T2m (construct Type F in FIG. 2),
Cet-T22d35 (construct Type D in FIG. 2) and T22d35 (non-Fc-fused) were
compared. FIG. 4A shows representative TGF-.beta.1 (top panel),
TGF-.beta.3 (middle panel) and TGF-.beta.2 (bottom panel) neutralization
curves for Cet-T2m, Cet-T22d35 and T22d35 while Table 2 summarizes
TGF-.beta.1, -.beta.2 and -.beta.3 neutralization IC.sub.50 values.
Unexpectedly, the TGF-.beta.1 and TGF-.beta.3 neutralization curves for
the Cetuximab fusion constructs indicated extremely high potencies that
lie in the picomolar range (determining a single IC.sub.50 value in these
experiments is difficult due to the biphasic nature of the curves). The
observed TGF-.beta.1 IC.sub.50 value for Cet-T22d35 was in the picomolar
range. In contrast, the TGF-.beta.1 IC.sub.50 for non-Fc-fused T22d35 was
approximately 1 nM. This illustrates that there is a large increase in
T22d35 potency upon fusion to the C-terminus of the Fc region of
Cetuximab (due to the biphasic nature of the Cet-T22d35 curve, the fold
difference is difficult to determine in these experiments). The
TGF-.beta.1 IC50 value for Cet-T2m was also subpicomolar (but less potent
than Cet-T22d35), whereas unfused T2m is not able to detectably
neutralize TGF-.beta.1, even at concentrations above 500 nM (De Crescenzo
et al, 2001). This demonstrates that, similar to T22d35, a very
significant increase in T2m potency occurs upon fusion to the C-terminus
of the Fc region of Cetuximab. Both Cet-T22d35 and Cet-T2m neutralized
TGF-.beta.2 (IC.sub.50 nM range), whereas T22d35 and T2m (De Crescenzo et
al, 2001) alone did not, even at a concentration of 800 nM, again showing
the remarkable increase in neutralization potency that occurs upon fusion
of T22d35 or T2m to the C-terminus of an Fc region.
[0168] In another set of experiments using the Mv1Lu cell reporter assay,
similar extremely high potencies of TGF-.beta. neutralization were
observed for other C-terminus Fc fusion constructs (Table 2), e.g. for
constructs in which T22d35 or T2m were fused with FSAs such as Herceptin
(Her-T22d35), Avastin (Ava-T22d35) or Synagis (Syn-T22d35) [Type F and D
constructs in FIG. 2], or with the blood-brain barrier crossing Fc-fused
FC5 V.sub.HH antibody (FC5-Fc-T22d35 and FC5-Fc-T2m) [Type E and G
constructs in FIG. 2]. In addition, fusion of T22d35 or T2m to the
C-terminus of an IgG2-Fc region alone, i.e. an antibody with no Fab
region present (Fc-T22d35 and Fc-T2m) [Type A and C constructs in FIG. 2]
resulted in fusion proteins with similarly high neutralization potencies.
However, fusing T2m to the N-terminus of an IgG2-Fc (T2m-Fc) generates a
fusion protein that does not neutralize TGF-.beta.1 and -.beta.3 in the
picomolar range, but rather in the range of 1 nanomolar (0.3 to 15 nM in
Table 2) and lacks any activity towards TGF-.beta.2. These results are
similar to those obtained with commercially available N-terminally IgG1
Fc-fused TGF-.beta. Type II receptor ectodomain (T2m-Fc (R&D Systems))
(0.3 to 0.5 nM in Table 2). Together, these results thus demonstrate that
fusion of TGF-.beta. superfamily receptor ectodomains to the C-terminus
of an Fc domain in the context of full-size antibodies, a V.sub.HH-Fc, or
an Fc region alone, give rise to unexpectedly high TGF-.beta.
neutralization potencies.
TABLE-US-00012
TABLE 2
TGF-.beta. neutralization IC.sub.50 of fusion constructs. It should be
noted
that T2m does not neutralize any of TGF-.beta.1, -.beta.2, or -.beta.3 (De
Crescenzo
et al, 2001). It should also be noted that the IC.sub.50 values in the
table
below are estimates due to the biphasic nature of the curves.
TGF-.beta.1 TGF-.beta.3 TGF-.beta.2
Av IC.sub.50 (nM) Av IC.sub.50 (nM) Av IC.sub.50 (nM)
Cet-T22d35 0.000,001 0.000,002 13.6
Cet-T2m 0.000,1 0.000,0015 129.2
T22d35 1.232 0.033 No neutralization
Her-T22d35 0.000,13 0.000,05 7.89
Ava-T22d35 0.000,000,72 0.000,00038 8.60
Syn-T22d35 0.000,042 0.000,0001 35.2
FC5-Fc-T22d35 0.000,001 0.000,001 96.2
FC5-Fc-T2m 0.000,017 0.000,015 432.9
Fc-T22d35 0.001,445 0.000,026 108.4
T2m-Fc (R&D) 0.506 0.323 No neutralization
T2m-Fc 14.523 0.276 No neutralization
Fc-T2m 0.009,923 0.000,766 460.5
[0169] In order to confirm the relative potencies of Fc-ectodomain
constructs, a second cell-based assay, an A549 cell IL-11 release assay,
was used. This IL-11 release assay acts as a model of TGF-.beta.-mediated
biological responses that contribute to both tumor metastasis and
fibrosis. In the set of experiments shown in FIG. 4B, the neutralization
potencies of T22d35, Fc-T2m, Fc-T22d35, Cet-T22d35, Her-T22d35,
Ava-T22d35, Syn-T22d35 and FC5-Fc-T22d35 were compared. It can be seen
that the neutralization curves in this assay are not biphasic, making it
less challenging than the Mv1Lu assay to determine and compare IC.sub.50
values. All of the constructs in which T22d35 was fused to the C-terminus
of an Fc region exhibited IC.sub.50 values in the range of 5 pM,
corroborating the extremely high potency observed for these constructs in
the Mv1Lu assay. It can also be seen in FIG. 4B that the IC.sub.50 value
for non-Fc-fused T22d35 was .about.0.5 nM. This indicates that a
.about.100-fold increase in potency occurs upon fusion of T22d35 to the
C-terminus of an Fc region. The IC.sub.50 value for Fc-T2m was 0.05 nM.
This indicates that constructs with two ectodomains in the C-terminal
portion may be more potent than a construct with one ectodomain in the
C-terminal portion (as was observed in the Mv1 Lu assay, FIG. 4A),
however, it should be noted that Fc-T2m does not have an optimized
N-terminus.
[0170] An additional set of experiments in which the A549 cell IL-11
release assay was used to compare TGF-.beta. neutralization potencies is
shown in FIG. 4C. Here, the potencies of several "headless"-T2m
constructs were assessed along with that of non-Fc-fused T22d35. The
potency of T22d35 was determined to be .about.0.5 nM, consistent with the
data shown in FIG. 4B. This is illustrative of the robustness of the A549
cell IL-11 release assay. It can also be seen in FIG. 4C that all of the
"headless"-T2m constructs exhibited high potencies with IC.sub.50s in the
range of 5 pM (3 to 17 pM). These values are in the same range as those
of the T22d35-containing constructs shown in FIG. 4B, and are 10-fold
higher potency than that of the "headless"-T2m containing construct also
shown in FIG. 4B (Fc-T2m). Since all of the T2m-containing constructs in
FIG. 4C have engineered N-termini, whereas Fc-T2m does not, these results
indicate that engineering of the cysteine residues of the hinge region of
"headless" constructs is able to increase their potency by approximately
10-fold.
[0171] We have also compared the potencies of constructs that include
three T.beta.RII structured ectodomains with constructs carrying two
ectodomains using the A549 cell IL-11 release assay. We observed that the
triple-repeat based constructs (SEQ ID NO:111 and SEQ ID NO:116) are
potent in neutralizing TGF-.beta.1 in this assay, and typically have
improved 1050 values relative to the corresponding double-repeat based
constructs (SEQ ID NO:100 and SEQ ID NO:106, respectively). All
constructs involved in this comparative study had the same engineered
N-terminus of the Fc portion, hIgG1Fc.DELTA.K(C).
[0172] Binding to TGF-.beta.: Binding of T22d35, Cet-T22d35, and Cet-T2m
to TGF-.beta.2 was measured using a competitive SPR binding experiment.
In this assay, the molecule of interest was first allowed to bind to a
fixed amount of TGF-.beta. in solution. A 2-fold dilution series was
prepared in PBS-0.05% Tween, starting with 1000 nM T22d35 trap or 20 nM
Cet-T22d35 or Cet-T2m. Each diluted sample was pre-incubated with 1 nM
TGF-.beta.2 for 30 min at room temperature to allow binding. The mixture
was then flowed over immobilized, pan-specific anti-TGF-.beta. antibody
1D11 (2000 RU 1D11) in order to quantify the amount of ligand left
unbound (T.beta.RII ectodomain and 1D11 bind to a similar epitope on
TGF-.beta.) using a Biacore T200 instrument. The TGF-.beta.2 binding
EC.sub.50 values were determined by plotting the percent free TGF-.beta.
versus the protein concentration of the molecule of interest. Binding
curves and EC.sub.50 values are shown in FIG. 4D and Table 3. In the case
of TGF-.beta.2 binding, a 100-fold increase in binding was observed
between Cet-T22d35 and unfused T22d35 (EC.sub.50.about.1 nM versus>100
nM, respectively), indicating that C-terminal fusion of the T22d35 trap
to antibody provides a gain in affinity for the TGF-.beta.2 isoform. This
correlates with the ability of Cet-T22d35 to neutralize TGF-.beta.2 in
the 10 nM range, and the inability of unfused T22d35 to neutralize
TGF-.beta.2, as observed in the Mv1Lu-Luc cell reporter assay.
TABLE-US-00013
TABLE 3
EC.sub.50 of Cetuximab-trap binding to TGF-.beta. in solution.
EC.sub.50 is the Effective Concentration at which 50% of TGF-.beta.
is bound and is given in nM.
Trap variant EC.sub.50 for TGF-.beta.2
T22d35 >100
Cetuximab-T2m 1.17
Cetuximab-T22d35 0.50
Note:
T2m alone shows an IC50 of greater than 1000 nM (Zwaagstra et al, 2012),
and thus is not considered neutralizing.
EXAMPLE 3A
Validation of Antibody Binding
[0173] The ability of an antibody alone or in a fusion construct of
Example 1 to bind to its intended target antigen was evaluated using
surface plasmon resonance (SPR).
[0174] Direct binding of Cet-T22d35 or Cetuximab to the EGF receptor
extracellular domain (EGFR-ED) was quantified by SPR using a BIAcore T200
instrument, performed in the standard manner. Briefly, Cet-T22d35 or
Cetuximab alone were captured on the SPR CM5 chip using immobilized
anti-human IgG Fc-specific antibody (2000 RU). Variable concentrations of
EGFR-ED in PBS-0.05% Tween were then flowed over the capture surface at
100 .mu.l/min and 25.degree. C. The resulting sensorgrams (data not
shown) were analyzed using the Biacore T200 evaluation software. The
K.sub.D values of Cet-T22d35 and Cetuximab were very similar (847 and 708
pM, respectively), indicating that fusion of T22d35 to the Fc portion of
Cetuximab does not appreciably alter binding to EGFR-ED, compared to the
non-fused FSA. Similar SPR methods and analyses were performed for other
antibody-trap fusion examples, compared with their corresponding target
antigens (see Table 4).
[0175] From Table 4 it is evident for each exemplified construct that
fusion of a TGF-.beta. superfamily receptor ectodomain(s) to the
C-terminus of the Fc region of an antibody did not significantly alter
antigen-binding affinities and K.sub.D values of the antibody. This
indicates that the ectodomain(s) can be readily fused to any antibody
without compromising the ability the antibody to bind its target antigen.
TABLE-US-00014
TABLE 4
SPR determination of antigen-binding affinity of
T22d35 fusion constructs or antibodies alone.
Antigen K.sub.a (1/Ms) K.sub.d (1/s) K.sub.D (M)
Cetuximab- EGFR 1.22 .times. 10.sup.6 8.65 .times. 10.sup.-4 7.08 .times.
10.sup.-10
T22d35
Cetuximab EGFR 1.03 .times. 10.sup.6 8.45 .times. 10.sup.-4 8.47 .times.
10.sup.-10
Her-T22d35 Her2 8.30 .times. 10.sup.4 5.30 .times. 10.sup.-5 6.37 .times.
10.sup.-10
Herceptin Her2 6.88 .times. 10.sup.4 5.03 .times. 10.sup.-5 7.33 .times.
10.sup.-10
Syn-T22d35 RSV-F 3.55 .times. 10.sup.4 1.42 .times. 10.sup.-3 4.10 .times.
10.sup.-9
Synagis RSV-F 2.57 .times. 10.sup.4 1.68 .times. 10.sup.-3 6.60 .times.
10.sup.-9
NOTE:
FC-5-Fc, FC5-Fc-T2m, and FC-5-Fc-T22d35 binding affinity was assessed via
the transwell functional assay (see Example 3C).
EXAMPLE 3B
Validation of Cetuximab Function
[0176] The ability of Cetuximab to maintain its therapeutic function (i.e.
inhibition of EGF-induced EGFR phosphorylation and signaling) when fused
to either T2m or T22d35 (Example 1) was evaluated.
[0177] Phosphorylation of EGFR: The ability of Cetuximab-comprising
constructs to inhibit EGF-induced phosphorylation of EGFR in human lung
cancer A549 cells was evaluated. A549 cells were seeded in 24-well plates
(100,000 cells/well) and either mock treated (-) or pre-treated with
Cetuximab, Cet-T2m, or Cet-T22d35 (all at 10, 1 or 0.1 nM) or T22d35 (10
nM) at 37.degree. C. for 3 h, then treated with 50 ng/mL EGF at
37.degree. C. for 10 min. Whole cell lysates were prepared and resolved
by SDS-PAGE, western blotted and probed with anti-phosphoTyrosine
antibody (Clone 4G10, Millipore 05-321). As shown in FIG. 5A, Cetuximab
and Cet-T2m and Cet-T22d35 inhibited EGFR phosphorylation to similar
extents, whereas T22d35 had no effect, relative to the +EGF control.
These results thus confirm that the TGF-.beta. superfamily receptor
ectodomain moieties in Cetuximab-T2m and Cetuximab-T22d35 fusion proteins
do not interfere with the function of the Cetuximab antibody (i.e.
inhibition of EGF induced EGFR signaling).
[0178] Inhibition of EGFR signaling: Inhibition of autocrine EGFR
signaling results in varying degrees of cytotoxicity in EGFR-expressing
cells treated with Cetuximab. Cytotoxicity of Cet-T22d35 was compared to
Cetuximab and T22d35 in human breast cancer cells (MDA-MB-468) and
immortalized keratinocyte cells (HaCaT). These cells exhibited
significant Cetuximab cytotoxicity, due to their intrinsic dependence on
the EGF signaling pathway for basal growth. The cells were seeded onto
96-well plates (MDA-MB-468, 2300 cells/well; HaCaT, 1500 cells/well) and
then treated with different doses of inhibitor at 37.degree. C. for 5
days. Cell viability was measured using sulforhodamine reagent to
determine the percentage of viable cells relative to mock-treated
controls. Results are shown in FIG. 5B and Table 5. The IC.sub.50 values
for Cet-T22d35 and Cetuximab were similar in both cell lines (0.2-1.4 nM
range), while T22d35 resulted in no cytotoxicity. These results further
confirm that the TGF-.beta. superfamily receptor ectodomain moiety in
Cetuximab-T22d35 does not interfere with the function of the Cetuximab
antibody.
TABLE-US-00015
TABLE 5
Cetuximab-T22d35 cytotoxicity in MDA-MB-468 and HaCat cells.
The cytotoxic potency IC.sub.50 values are given in nM.
MDA-MB-468 IC.sub.50 HaCaT IC.sub.50
Cetuximab 0.50 0.33
Cetuximab-T22d35 1.42 0.22
T22d35 0 0
EXAMPLE 3C
Validation of FC5 Function
[0179] The ability of FC5 V.sub.HH to maintain its function (i.e.
transmigrate the blood-brain barrier) when fused with the T2m or T22d35
moieties (see description in Example 2 and activities in Table 2) was
evaluated.
[0180] Briefly, SV40-immortalized Adult Rat Brain Endothelial Cells
(Sv-ARBEC) were used to generate an in vitro blood-brain barrier (BBB)
model as described (Garberg et al, 2005; Haqqani et al, 2013). Sv-ARBEC
cells (80,000 cells/membrane) were seeded on 0.1 mg/mL rat tail collagen
type I-coated tissue culture inserts (pore size-1 .mu.m; surface area 0.9
cm.sup.2, Falcon) in 1 ml of growth medium. The bottom chamber of the
insert assembly contained 2 ml of growth medium supplemented with the
immortalized neonatal rat astrocytes-conditioned medium in a 1:1 (v/v)
ratio. Equimolar amounts (5.6 .mu.M) of positive (FC5-Fc) control;
negative controls (A20.1); and T22d35, T2m, FC5-Fc-T22d35 or FC5-Fc-T2m
were tested for their ability to cross the Sv-ARBEC cell monolayer.
Following exposure of equimolar amounts of the proteins to the luminal
side of the BBB, samples were taken after 15, 30, and 60 min from the
abluminal side. The protein content of each sample was then quantified by
mass spectrometry (multiple reaction monitoring-isotype labeled internal
standards; MRM-ILIS) as described by (Haqqani et al, 2013) (see method
description below).
[0181] Quantified values can be directly plotted or the P.sub.app
(apparent permeability coefficient) values can be determined using the
following formula
Papp = d Q r / d t A .times. C 0 ##EQU00001##
[0182] The P.sub.app value is commonly used to determine the specific
permeability of a molecule, and is a measure of transport across the
brain endothelial monolayer. [Qr/dt=cumulative amount in the receiver
(bottom) compartment versus time; A=area of the cell monolayer;
C0=initial concentration of the dosing solution (top chamber)].
[0183] FIG. 6 shows the results of the experiment. The P.sub.app value of
FC5-Fc-T22d35 was similar to the control FC5-Fc, indicating it was
transported efficiently and that the fused T22d35 did not interfere with
transport. The P.sub.app value for FC5-Fc-T2m was approximately 50% less,
compared to FC5-Fc-T22d35 and FC5-Fc, indicating somewhat reduced
permeability. Nevertheless, the level of transport of FC5-Fc-T2m was
about 4-fold greater than the negative controls (T2m, T22d35, and
antibody A20.1).
EXAMPLE 4
Inhibition of Epithelial to Mesenchymal Transition
[0184] Treatment of A549 cells with EGF plus TGF-.beta. results in a
strong epithelial to mesenchymal transition (EMT). The EMT is
phenotypically characterized by changes in cell morphology (tight
cellular junctions with "cobble-stone" appearance converts to elongated
cells, see FIG. 7A) and changes in the adherin junction proteins
E-cadherin and N-cadherin. The ability of the fusion constructs to block
EMT was assessed in A549 cells by western blotting (E-cadherin) and flow
cytometry (E-cadherin and N-cadherin).
[0185] Briefly, for the western blot analysis, A549 cells were seeded in
24-well plates (8000 cells/well) and then treated with EGF (50
ng/mL)+TGF-.beta.1 (50 pM) at 37.degree. C. for 3 days in the presence of
Cet-T22d35, Cetuximab, or T22d35 (0, 0.05, 0.5, 5, 50, or 500 nM). Whole
cell lysates were prepared and resolved by SDS-PAGE. The proteins were
transferred to nitrocellulose and then probed with an E-cadherin antibody
(BD Transduction laboratories Biosciences) (FIG. 7B). The E-Cadherin
positive bands in the Western blot were quantified by densitometer
detection and ImageJ analysis (FIG. 7C). EGF+TGF-.beta. treatment
resulted in an EMT, as indicated by the disappearance of E-cadherin
(compare non-treated and EGF+TGF-.beta. lanes in the absence of
inhibitors). Cet-T22d35 blocked the EMT (E-cadherin disappearance) in a
dose-dependent manner whereas 500 nM Cetuximab or T22d35 treatments only
modestly blocked the EMT (E-cadherin levels .about.20-25% of the
non-treated control level).
[0186] The ability of Cet-T22d35, Cetuximab and T22d35 to block the
EGF+TGF-.beta. EMT response was further examined by flow cytometry using
A549 cells treated with Cet-T22d35 or Cetuximab, (all at 50, 5, 0.5 nM)
or the Cetuximab+T22d35 combination (50 nM+100 nM, 5 nM+10 nM, or 0.5
nM+1 nM, respectively) and evaluating the EMT associated changes in
E-cadherin and N-cadherin cell surface expression levels (FIG. 7D). In
this experiment the molar amounts of the molecules of interest used in
`T22d35 alone` and `Cetuximab+T22d35` groups were two-fold higher than
for Cet-T22d35 in order to correspond with a 2:1 trap/antibody ratio in
the Cet-T22d35 fusion molecule. A549 cells were seeded in 6-well plates
(30,000 cells/well) and pre-treated with the inhibitors at 37.degree. C.
for 1 h, followed by added treatment with EGF (10 ng/mL)+TGF-.beta.1 (10
pM) and incubation at 37.degree. C. for 3 days. Cells were then
dissociated from the plate using 1 mL Dissociation Buffer (Sigma)/well,
centrifuged at 2000 rpm for 2 min and re-suspended in 100 .mu.l RPMI-5
media at 4.degree. C. AlexaFluor488-E-cadherin (Santa Cruz, SC21791) and
AlexaFluor647-N-cadherin (BD Biosciences, 563434) antibodies (1/25 v/v
dilutions) were added and samples were incubated at 4.degree. C. for 1 h.
Cells were then centrifuged, washed once in RPMI-5, and re-suspended in
400 .mu.l RPMI-5 containing 15 .mu.g/mL propidium iodide (Life
Technologies) at 4.degree. C. Mean fluorescent intensities (MFI) were
measured by flow cytometry (BD LS RII flow cytometer, BD Biosciences) to
quantify E-cadherin and N-cadherin levels. The results show that
Cet-T22d35 was more effective in preventing down-regulation of E-cadherin
(FIG. 7D, top panel) and up-regulation of N-cadherin (FIG. 7D, bottom
panel), as a measure of blocking EMT, compared to Cetuximab, T22d34 or
the Cetuximab+T22d35 combination at each respective dose, and is most
notable at the lowest dose used (0.5 nM).
EXAMPLE 5
Pharmacokinetic (PK) Studies on Constructs with and Without a Lysine
Residue at the Fusion Site Between the C-Terminus of the Fc Region and
the N-Terminus of the Ectodomain
[0187] PK studies were carried out in normal, healthy mice to determine
whether fusion of T22d35 to an antibody increased its half-life in vivo,
and whether removal of a lysine at the fusion site within constructs
reduced the amount of cleavage occurring in vivo.
[0188] Results from Cet-T22d35 (construct Type D in FIG. 2--containing a
lysine at the fusion site): A single bolus of Cet-T22d35 protein (10
mgs/Kg) formulated in DPBS was intravenously injected (IV) into the tail
vein of normal Balb/c mice and serum samples were collected from the
submandibular vein at selected time points (0.5, 1, 2, 4, 8, 14, 24, 48,
96 h). Blood samples were centrifuged at 2000 g at 4.degree. C. for 10
min and the serum supernatant was removed and stored frozen at
-80.degree. C., prior to analyses. The samples were thawed at 4.degree.
C. and analyzed via mass spectrometry (multiple reaction
monitoring-isotype labeled internal standards; MRM-LIS) in order to
measure the levels of both the Cetuximab and T22d35 trap moieties.
Briefly, 20 .mu.l of sample was thawed and treated with mild detergents
(0.1% RapiGest SF, Waters; 5.5 mM TCEP) at 95.degree. C. for 10 min. The
sample was cooled to room temperature and Iodoacetamide (IAA) in 50 mM
Ammonium Bicarbonate was added to a final concentration of 10 mM IAA,
followed by incubation for 40 min in the dark. DTT (10 mM final) was then
added and the sample was incubated at room temperature for 15 min,
followed by trypsin digestion (Sigma, 0.8 mg/mL final) at 37.degree. C.
for 18 h. A mixture of 5 .mu.M each of isotope-labeled trap and cetuximab
peptides (formulated in 30% acetonitrile, 0.1% formic acid) were added to
final concentrations of 1 .mu.M, as internal standards for
quantification. The isotope-labeled peptides were
13C/15N--(H2N-LPYHDFILEDAASPK-OH; SEQ ID NO:134) and
13C/15N--(H2N-ALPAPIEK-OH; SEQ ID NO:135) for T22d35 and Cetuximab,
respectively (NewEngland Peptide). Trifluoroacetic acid was then added
(0.5% final), followed by incubation at 37.degree. C. for 30 min. The
samples were centrifuged at 13000 rpm for 20 min and the supernatant was
used analyzed via MRM-ILIS using an Agilent 1260 HPLC system coupled with
Agilent QQQ6410B at 55.degree. C. The PK profiles seen in FIG. 8A show
that the levels of the Cetuximab and T22d35 moieties diverge at early
time points (<10 h) after injection, indicating different kinetics and
suggesting possible cleavage of T22d35 from the Cet-T22d35 protein in
vivo. Nevertheless, analysis of these curves using a two compartmental
model (Phoenix WinNonlin SoftwareVersion 6.3) indicated that the average
terminal half-life (T1/2 .beta.) of the T22d35 component was 45.8 h. This
represents a 7.6-fold increase compared to the previously determined
half-life for unfused T22d35 (T1/2 .beta..about.6 h). As well, the PK
profile shows that the Cetuximab moiety was maintained in the blood, with
a T1/2 .beta.=262.5 h, indicating that the Cetuximab moiety has a long
circulating half-life.
[0189] Results from several constructs with lysine deleted at the fusion
site:
[0190] The same methods that were used in the PK study of Cet-T22d35 (FIG.
8A) were applied to assess the PK of Cet.DELTA.K-T2m (Type F in FIG. 2)
as well as several "headless" T2m constructs (Type C in FIG. 2;
hIgG1Fc.DELTA.K(SS)-T2m, hIgG1Fc.DELTA.K(.DELTA.C)-T2m, and
hIgG2Fc.DELTA.K(SS)-T2m), all of which have the lysine deleted at the
fusion site. The data shown in FIG. 8B indicate that no detectable
cleavage of these constructs is occurring in vivo since the levels of the
Fc moieties (closed symbols) and ectodomain moieties (open symbols) do
not diverge over time. Additionally, all of the constructs exhibit
similar long circulating half-lives with T1/2 .beta.s of approximately
100 h. This represents an improvement to the half-life of the ectodomain
moiety of the construct which has a lysine at the fusion site, presented
in FIG. 8A (45.8 h).
EXAMPLE 6
Efficacy Studies Comparing the Effect of "Headless" Fc-T2m and FSA-T2M
Constructs on Tumor Growth (A) and T-Cell Function (B and C) in an
Immune-Competent Syngeneic Triple Negative Breast Cancer (4T1) Model
[0191] A FSA-T2m construct (Cet-T2m--Type F in FIG. 2) and three headless
constructs, all with engineered N-termini, (hIgG1Fc.DELTA.K(CC)-T2m,
hIgG1Fc.DELTA.K(C)-T2m, and hIgG2Fc.DELTA.K(CC)-T2m--Type C in FIG. 2)
were evaluated for their ability to inhibit tumor growth and to affect
T-cell function in a syngeneic tumor model derived from 4T1 triple
negative breast cancer cells. The results presented in FIG. 9 show the
effect on tumor growth (A) and T-cell function (B, C). The effects of
these Fc-fused ectodomain constructs were compared to those of a
pan-specific neutralizing anti-TGF-.beta. antibody, 1D11 and a
non-Fc-fused ectodomain construct, T22d35.
[0192] The protocols used for these syngeneic mouse model studies are
described in (Zheng et al, 2013). Briefly, female BALB/c (H-2Kd) mice 6
weeks of age were purchased from The Jackson Laboratories and kept in
filter-top cages. The 4T1 breast cancer cells and B16F10 cells were
purchased from American type culture collection and cultured in RPMI-1640
supplemented with 2 mmol/L L-glutamine, 100 U/mL penicillin, 100
microgm/L streptomycin, 50 micromol/L 2-mercaptoethanol, and 10% fetal
calf serum. Mice were inoculated subcutaneously in the left flank with
100 uL sterile saline containing 5.times.10.sup.4 4T1 cells. Tumors were
grown to .about.100 mm.sup.3, as measured by caliper, then mice were
randomized and divided into the six treatment groups (8 animals/group)
(Day 0). Treatments commenced on Day 1 and continued for 15 days with the
animals being dosed at 5 mg/kg twice per week such that they received a
total of 4 doses. Tumor growth was monitored by caliper measurements
3.times. per week. Animals were euthanized by exsanguination under
anaesthesia on Day 15; T cells were isolated from draining lymph nodes
and assessed for their capacity to kill mouse 4T1 and B16F10 tumour cells
ex vivo. The capacity of T cells from mice treated with or without test
agents to lyse target 4T1 tumor cells was measured using a CytoTox 96
nonradioactive cytotoxicity assay kit (Promega) according to the
manufacturer's instructions. Briefly, naive target 4T1 cells or melanoma
cell line B16F10 cells are plated and incubated for 4 hr with CD8+
effector T cells isolated from 4T1 tumor-bearing mice using CD8 magnetic
MicroBeads (BD Bioscience). The isolated CD8+ cells are confirmed, by
flow cytometry, to be over 85% CD8+. A range of ratios of effectors to
target cells is tested (100:1, 50:1, 25:1). Lactate dehydrogenase (LDH)
release in response to effector T cells is measured in the buffer bathing
target cells. Target cells incubated in the absence of effector cells are
used as a comparator to control for spontaneous LDH release. Released LDH
in culture supernatants is detected after a 30-min incubation using a
coupled enzymatic assay. The intensity of the color formed is
proportional to the number of lysed cells. Cytotoxic activity of CTL is
calculated using the following formula:
Cytotoxic activity %=[(absorbance)-(spontaneous effector cell LDH
release)-(spontaneous target cell LDH release)]/[(maximal LDH
release)--(spontaneous target cell LDH release)].times.100
[0193] The results presented in FIG. 9A show the effect of the Fc-fused
ectodomain constructs listed above on 4T1 tumor growth (the pan-specific
neutralizing anti-TGF-.beta. antibody, 1 D11, and a non-Fc-fused
ectodomain construct, T22d35, being tested as comparators). As can be
seen in FIG. 9A, all of the Fc-fused ectodomain constructs reduced tumor
growth relative to the saline treatment when tested for significance by
t-test. The 1 D11 and T22d35 comparator treatments were observed to be
less effective relative to the Fc-fused ectodomain treatments, and not
significantly different from the saline control. These results
demonstrate that constructs with an ectodomain fused to the C-terminus of
an Fc region have significant anti-tumor potency, with the efficacy being
higher than the 1 D11 and T22d35 comparators. There was no significant
difference between the FSA-T2m construct (Cet-T2m) and the constructs
that have no Fab, i.e. are "headless" (hIgG1Fc.DELTA.K(CC)-T2m,
hIgG1Fc.DELTA.K(C)-T2m, and hIgG2Fc.DELTA.K(CC)-T2m) with respect to
their effect on tumor growth. The high anti-tumor potency of these
Fc-ectodomain fusions relative to comparators likely results from high
potency neutralization of TGF-.beta. combined with a favourable
circulating half-life.
[0194] To investigate whether these Fc-ectodomain fusions exhibit an
immuno-modulatory effect in vivo on cytotoxic T lymphocyte cells (CTLs)
present in tumor draining lymph nodes, lymph nodes were removed from mice
treated with or without test agents; T-cells were then isolated and
tested for their capacity to lyse target 4T1 tumor cells (and B16F10
cells as a test of tumor specificity) using the methods described above.
[0195] As shown in FIG. 9B, treatment of the animals with Fc-ectodomain
fusions significantly increased the ability of draining lymph node
T-cells to lyse target 4T1 tumor cells ex vivo. It can be seen that this
immuno-stimulatory effect is specific to 4T1 cells since the T-cells were
not able to effectively lyse B16F10 melanoma cells (FGURE 9C; maximal
lysis of .about.15% for B16F10 cells and .about.80% for 4T1 cells). When
administered at the same 5 mg/kg dose as the Fc-ectodomain fusions, the
non-Fc-fused comparator molecule, T22d35, had no effect above saline.
This is consistent with its lack of effect on tumor volume. Although the
1 D11 antibody had no statistically significant effect on tumor volume
(FIG. 9A), it did increase the ability of lymph node T-cells to lyse 4T1
tumor cells.
[0196] Interestingly, with respect to the Fc-ectodomain fusions of this
invention, the most potent constructs were the hIgG1Fc.DELTA.K(CC)-T2m
and hIgG2Fc.DELTA.K(CC)-T2m constructs; both of these constructs
containing two cysteines in the engineered hinge region. These constructs
were more potent than the construct with one cysteine in the engineered
hinge region, hIgG1Fc.DELTA. K(C)-T2m, as well as being more potent than
the full-size antibody-T2m construct, Cet-T2m. The difference in potency
between constructs with one versus two hinge region cysteines may result
from the construct with one cysteine having a lower relative stability.
The lower potency of the full size antibody construct relative to the
headless constructs with two hinge region cysteines may result from a
difference in molecular weight, with the smaller constructs being able to
penetrate the tumor microenvironment more effectively.
EXAMPLE 7
In Vitro and In Vivo Studies Illustrating Enhanced Bone Localization of
Constructs Containing a Deca-Aspartate Motif for Bone Targeting at the
N-Terminus of the Fc Region
[0197] In vivo studies were carried out to investigate whether the
addition of a 10 amino acid long poly-aspartate bone-localization motif
(D10) to the N-terminus of the Fc region of constructs will promote their
localization to bone. Optical imaging of D10-hIgG1Fc-T2m fusions: Upon
arrival, male Balb/c mice were housed 3 mice/cage. On the day of the
experiment, animals were shaved dorsal and ventral and treated with the
hair removal cream, NAIR.RTM.. Mice were injected with a single
intravenous bolus of 10 mg/kg of two CF770 labeled constructs with a
deca-aspartate motif for bone targeting (D10) at their N-termini
(D10-hIgG1Fc.DELTA.K(CC)-T2m, D10-GSL-hIgG1Fc.DELTA.K(CC)-T2m) or with a
control construct without the D10 motif (hIgG1Fc.DELTA.K(CC)-T2m) and
whole body bio-distribution followed using both in vivo and ex-vivo near
infrared imaging. Imaging was conducted with a small-animal time-domain
eXplore Optix pre-clinical imager MX3 (Advanced Research Technologies,
ART) at various time points (prescan, 5 mins, 3 hr, 6 hr, 24 h, 48 h, 72
h, 96 h and 120 h).
[0198] The small animal time-domain eXplore Optix preclinical imagers
consists of a 785-nm pulsed laser diode with a repetition frequency of 80
MHz and a time resolution of 12.5 ps light pulse was used for excitation.
The fluorescence emission beyond 813 nm was collected by a highly
sensitive time-correlated single photon counting system and detected
through a fast photomultiplier tube. The data were recorded as temporal
point-spread functions (TPSF) and the images were presented as
fluorescence intensity maps using ART Optix Optiview analysis software
3.02.
[0199] For in vivo optical imaging, mice were first anesthetized using
isofluorane (1.5-2%), positioned on the animal stage within a chamber
which allows for gaseous anesthesia and maintenance of animal temperature
at 36.degree. C. The scanning of the mouse at each time point lasted up
to 20 mins using a 2.5 mm step size and the mouse is placed back in its
home cage between imaging time points.
[0200] At the end of the imaging protocol (120 hrs) animals were
sacrificed by intracardiac perfusion using heparnized saline with deep
anesthesia. The organs (brain, heart, lungs, liver, kidney, spleen and
right and left leg bones) were imaged ex-vivo using a 1.0 mm step size
using the eXplore Optix pre-clinical imager MX3.
[0201] Data analysis was done using eXplore Optix Optiview analysis
software 3.02 (Advanced Research Technologies, Montreal, QC) to estimate
the fluorescence total and average fluorescence intensity in region of
interest containing the ex-vivo organs.
[0202] The results shown in FIGS. 10A and B demonstrate that the fusion of
the deca-aspartate D10 motif on the N-termini of the fusion constructs
had no impact on their ability to neutralize TGF-.beta., i.e. the
IC.sub.50 of the construct lacking the D10 motif
(hIgG1Fc.DELTA.K(CC)-T2m) was 3 nM, which is the same as the value
determined in FIG. 4C, while the IC.sub.50s of the D10 containing
constructs, D10-hIgG1Fc.DELTA.K(CC)-T2m and
D10-GSL-hIgG1Fc.DELTA.K(CC)-T2m, were very similar at 4-5 nM. The results
in FIGS. 10A and B also indicate that labeling with the CF770 dye reduced
the ability of the constructs to neutralize TGF-.beta. by approximately
4-fold. Since dye conjugation occurs at lysine residues, and since it is
know that lysines are at the binding interface between the Type II
ectodomain and TGF-.beta., it is not entirely surprising that labeling
reduced neutralization potency. In any case, since this is a comparative
study of differences in in vivo localization promoted by the D10 peptide,
it was felt that partially active constructs would be informative.
[0203] The results shown in FIGS. 10C and D demonstrate that the addition
of the D10 peptide to the N-termini of the constructs greatly enhanced
bone localization. Images taken 120 h post-injection of the CF770 labeled
fusions show a clear accumulation of the D10-fusions in the vertebrae.
Further ex vivo imaging of the brain, heart, lungs, liver, kidneys,
spleen, and the left and right legs 120 h post-injection confirmed the
specific accumulation of the D10-fusions in the bones. The fluorescent
signals observed in the kidneys and liver were similar for all fusions
indicating that accumulation in these organs was not affected by the
presence of the D10 sequence. These results indicate that the TGF-.beta.
neutralization activity of constructs may be increased within bone
through the addition of the D10 peptide. This could result in more
favourable dosing levels and schedules for the treatment of bone-related
diseases, such as osteogenesis imperfecta, relative to that required for
a similar construct without the D10 motif.
[0204] The embodiments and examples described herein are illustrative and
are not meant to limit the scope of the invention as claimed. Variations
of the foregoing embodiments, including alternatives, modifications and
equivalents, are intended by the inventors to be encompassed by the
claims. Furthermore, the discussed combination of features might not be
necessary for the inventive solution.
TABLE-US-00016
LISTING OF SEQUENCES
SEQ
ID
NO: Sequence Description
1 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG Human IgG1 Fc
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI region
EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN
HYTQKSLSLSPGK
2 APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGV Human IgG2 Fc
EVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE region
KTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH
YTQKSLSLSPGK
3 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDG Human IgG3 Fc
VEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI region
EKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES
SGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHN
RFTQKSLSLSPGK
4 APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG Human IgG4 Fc
VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI region
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN
HYTQKSLSLSLGK
5 EPKSCDKTHTCPPCP Human IgG1
hinge region
6 ERKCCVECPPCP Human IgG2
hinge region
7 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKS Human IgG3
CDTPPPCPRCP hinge region
8 ESKYGPPCPSCP Human IgG4
hinge region
9 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
hIgG1Fc.DELTA.K-.DELTA.C
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI Fc variant
EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN
HYTQKSLSLSPG
10 PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW hIgG1Fc.DELTA.K-C
Fc
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL variant
PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPG
11 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP hIgG1Fc.DELTA.K-CC
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK Fc variant
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPG
12 EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV hIgG1Fc.DELTA.K-S
Fc
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK variant
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPG
13 EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV hIgG1.DELTA.K-SS
Fc
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK variant
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPG
14 EPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
hIgG1Fc.DELTA.K-SSS
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK Fc variant
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPG
15 APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGV
hIgG2Fc.DELTA.K-.DELTA.C
EVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE Fc variant
KTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH
YTQKSLSLSPG
16 PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWY hIgG2Fc.DELTA.K-C
Fc
VDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLP variant
APIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVE
WESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
LHNHYTQKSLSLSPG
17 VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF hIgG2Fc.DELTA.K-CC
NWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNK Fc variant
GLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI
SVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPG
18 ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED hIgG2Fc-CCCC
PEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKC Fc variant
KVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF
YPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGK
19 ERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED hIgG2Fc.DELTA.K-SS
PEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKC Fc variant
KVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF
YPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPG
20 ERKSSVESPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
hIgG2Fc.DELTA.K-SSS
PEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKC Fc variant
KVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF
YPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPG
21 ERKSSVESPPSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
hIgG2Fc.DELTA.K-SSSS
PEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKC Fc variant
KVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF
YPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPG
22 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDG
hIgG3Fc.DELTA.K-.DELTA.C
VEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI Fc variant
EKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES
SGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHN
RFTQKSLSLSPG
23 PRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKW hIgG3Fc.DELTA.K-C
Fc
YVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKAL variant
PAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV
EWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHE
ALHNRFTQKSLSLSPG
24 DTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP hIgG3Fc.DELTA.K-CC
EVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCK Fc variant
VSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFS
CSVMHEALHNRFTQKSLSLSPG
25 EPKSSDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV hIgG3Fc.DELTA.K-S
Fc
SHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGK variant
EYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQ
GNIFSCSVMHEALHNRFTQKSLSLSPG
26 EPKSSDTPPPSPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV hIgG3Fc.DELTA.K-SS
SHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGK Fc variant
EYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQ
GNIFSCSVMHEALHNRFTQKSLSLSPG
27 EPKSSDTPPPSPRSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
hIgG3Fc.DELTA.K-SSS
SHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGK Fc variant
EYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQ
GNIFSCSVMHEALHNRFTQKSLSLSPG
28 APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG hIgG4Fc.DELTA.K-AC
VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI Fc variant
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN
HYTQKSLSLSLG
29 PSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW hIgG4Fc.DELTA.K-C
Fc
YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL variant
PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE
ALHNHYTQKSLSLSLG
30 ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE hIgG4Fc.DELTA.K-CC
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK Fc variant
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV
FSCSVMHEALHNHYTQKSLSLSLG
31 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE
hIgG4Fc.DELTA.K-CC-
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK 228P Fc variant
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV
FSCSVMHEALHNHYTQKSLSLSLG
32 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE
hIgG4Fc.DELTA.K-CC-
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK 228P-409K Fc
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG variant
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNV
FSCSVMHEALHNHYTQKSLSLSLG
33 ESKYGPPSPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE hIgG4Fc.DELTA.K-S
Fc
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK variant
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV
FSCSVMHEALHNHYTQKSLSLSLG
34 ESKYGPPSPSSPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE hIgG4Fc.DELTA.K-SS
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK Fc variant
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV
FSCSVMHEALHNHYTQKSLSLSLG
35 QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLET T.beta.RII-ED
VCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF structured domain
36 IPPHVQKSVNNDMIVTDNNGAVKFP T.beta.RII-ED N-term
unstructured
region and natural
linker
37 IPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFP T.beta.RIIb-ED
N-term
unstructured
region and natural
linker
38 IPPHVQKSDVEMEAQKDEIIAPSANRTAHPLRHINNDMIVTDNNGAVKFP T.beta.RIIb-ED Cys-
mutated N-term
unstructured
region and natural
linker
39 SEEYNTSNPD T.beta.RII-ED C-term
unstructured
region and natural
linker
40 SEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFP T.beta.RII-ED natural
linker
41 SEEYNTSNPDIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVT T.beta.RIIb-ED
natural
DNNGAVKFP linker
42 SEEYNTSNPDIPPHVQKSDVEMEAQKDEIIAPSANRTAHPLRHINNDMIVT T.beta.RIIb-ED
Cys-
DNNGAVKFP mutated linker
43 IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT T.beta.RII-ED
SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKE monomer, also
KKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD termed T2 or T2m
44 IPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQ T.beta.bRIIb-ED
LCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETV monomer, also
CHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSE termed T2b
EYNTSNPD
45 IPPHVQKSDVEMEAQKDEIIAPSANRTAHPLRHINNDMIVTDNNGAVKFPQ T.beta.RIIb-ED
LCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETV monomer Cys-
CHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSE mutated in the
EYNTSNPD linker region, also
termed T2b.sup.AA
46 IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT T.beta.RII-ED
dimer,
SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKE also termed T2-
KKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTD T2 or T22d35
NNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN
DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE
CNDNIIFSEEYNTSNPD
47 IPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQ T.beta.RIIb-ED
dimer,
LCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETV also termed T2-
CHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSE T2b
EYNTSNPDIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDN
NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKND
ENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDEC
NDNIIFSEEYNTSNPD
48 IPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQ T.beta.RIIb-ED
dimer
LCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETV Cys-mutated in
CHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSE the linker region,
EYNTSNPDIPPHVQKSDVEMEAQKDEIIAPSANRTAHPLRHINNDMIVTDN also termed T2-
NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKND T2b.sup.AA
ENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDEC
NDNIIFSEEYNTSNPD
49 IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT T.beta.RII-ED
trimer,
SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKE also termed T2-
KKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTD T2-T2
NNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN
DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE
CNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVR
FSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY
HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
50 IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT T.beta.RIIb-ED
trimer,
SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKE also termed T2-
KKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSDVEMEAQKD T2b-T2b
EIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKS
CMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAA
SPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSD
VEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRF
STCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYH
DFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
51 IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT T.beta.RIIb-ED
trimer
SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKE Cys-mutated in
KKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSDVEMEAQKD the linker regions,
EIIAPSANRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKS also termed T2-
CMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAA T2b.sup.AA-T2b.sup.AA
SPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSD
VEMEAQKDEIIAPSANRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRF
STCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYH
DFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
52 ALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRP T.beta.RI-ED
FVCAPSSKTGSVTTTYCCNQDHCNKIEL structured domain
53 AALLPGAT T.beta.RI-ED N-term
unstructured
region and natural
linker
54 PTTVKSSPGLGPVE T.beta.RI-ED C-term
unstructured
region and natural
linker
55 PTTVKSSPGLGPVEAALLPGAT T.beta.RI-ED natural
linker
56 AALLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEI T.beta.RI-ED
DLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVE monomer, also
termed T1 or T1m
57 AALLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEI T.beta.RI-ED
dimer,
DLIPRDRPFVCAPSSKTGSVTITYCCNQDHCNKIELPTTVKSSPGLGPVEA also termed T1-
ALLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEID T1
LIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVE
58 PTTVKSSPGLGPVEIPPHVQKSVNNDMIVTDNNGAVKFP T.beta.RI-T.beta.RII-ED
natural linker
59 PTTVKSSPGLGPVEIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINND
T.beta.RI-T.beta.RIIb-ED
MIVTDNNGAVKFP natural linker
60 SEEYNTSNPDAALLPGAT T.beta.RII-T.beta.RI-ED
natural linker
61 AALLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEI
T.beta.RI-T.beta.RII-ED
DLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVEI dimer T1-T2
PPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITS
ICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEK
KKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
62 AALLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEI
T.beta.RI-T.beta.RII-ED
DLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVEI dimer T1-T2b
PPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQL
CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVC
HDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE
YNTSNPD
63 IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT
T.beta.RI-T.beta.RII-ED
SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKE dimer T2-T1
KKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDAALLPGATALQCFCHLC
TKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKT
GSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVE
64 AALLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEI
T.beta.RI-T.beta.RII-ED
DLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVEI trimer T1-T2-T2
PPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITS
ICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEK
KKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDN
NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKND
ENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDEC
NDNIIFSEEYNTSNPD
65 AALLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEI
T.beta.RI-T.beta.RII-ED
DLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVEI trimer T1-T2-T2b
PPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITS
ICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEK
KKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSDVEMEAQKDE
IICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSC
MSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAAS
PKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
66 AALLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEI
T.beta.RI-T.beta.RII-ED
DLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVEI trimer T1-T2-
PPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQL T2b.sup.AA
CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVC
HDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE
YNTSNPDIPPHVQKSDVEMEAQKDEIIAPSANRTAHPLRHINNDMIVTDNN
GAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDE
NITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECN
DNIIFSEEYNTSNPD
67 IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT
T.beta.RI-T.beta.RII-ED
SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKE trimer T2-T2-T1
KKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTD
NNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN
DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE
CNDNIIFSEEYNTSNPDAALLPGATALQCFCHLCTKDNFTCVTDGLCFVSV
TETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKI
ELPTTVKSSPGLGPVE
68 IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT
T.beta.RI-T.beta.RII-ED
SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKE timer T2-T2b.sup.AA-
KKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSDVEMEAQKD T1
EIIAPSANRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKS
CMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAA
SPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDAALLPGATA
LQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPF
VCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVE
69 TLPFLKCYCSGHCPDDAINNTCITNGHCFAIIEEDDQGETTLASGCMKYEG BMPRIa-ED
SDFQCKDSPKAQLRRTIECCRTNLCNQYLQPTLP structured domain
70 QNLDSMLHGTGMKSDSDQKKSENGVTLAPED BMPRIa-ED N-
term unstructured
region and natural
linker
71 PVVIGPFFDGSIR BMPRIa-ED C-
term unstructured
region and natural
linker
72 PVVIGPFFDGSIRQNLDSMLHGTGMKSDSDQKKSENGVTLAPED BMPRIa-ED
natural linker
73 QNLDSMLHGTGMKSDSDQKKSENGVTLAPEDTLPFLKCYCSGHCPDDAINN BMPRIa-ED
TCITNGHCFAIIEEDDQGETTLASGCMKYEGSDFQCKDSPKAQLRRTIECC monomer
RTNLCNQYLQPTLPPVVIGPFFDGSIR
74 QNLDSMLHGTGMKSDSDQKKSENGVTLAPEDTLPFLKCYCSGHCPDDAINN BMPRIa-ED
TCITNGHCFAIIEEDDQGETTLASGCMKYEGSDFQCKDSPKAQLRRTIECC dimer
RTNLCNQYLQPTLPPVVIGPFFDGSIRQNLDSMLHGTGMKSDSDQKKSENG
VTLAPEDTLPFLKCYCSGHCPDDAINNTCITNGHCFAIIEEDDQGETTLAS
GCMKYEGSDFQCKDSPKAQLRRTIECCRTNLCNQYLQPTLPPVVIGPFFDG
SIR
75 TQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQG ActRIIa-ED
CWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFP structured domain
76 AILGRSE ActRIIa-ED N-
term unstructured
region and natural
linker
77 EMEVTQPTSNPVTPKPPYYNI ActRIIa-ED C-
term unstructured
region and natural
linker
78 EMEVTQPTSNPVTPKPPYYNIAILGRSE ActRIIa-ED
natural linker
79 AILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGS ActRIIa-ED
IEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEME monomer
VTQPTSNPVTPKPPYYNI
80 AILGRSETQECLFFNANwEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGS ActRIIa-ED dimer
IEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEME
VTQPTSNPVTPKPPYYNIAILGRSETQECLFFNANWEKDRTNQTGVEPCYG
DKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFC
CCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPYYNI
81 RECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGC ActRIIb-ED
WLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLP structured domain
82 SGRGEAET ActRIIb-ED N-
term unstructured
region and natural
linker
83 EAGGPEVTYEPPPTAPT ActRIIb-ED C-
term unstructured
region and natural
linker
84 EAGGPEVTYEPPPTAPTSGRGEAET ActRIIb-ED
natural linker
85 SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGT ActRIIb-ED
IELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAG monomer
GPEVTYEPPPTAPT
86 SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDRRLHCYASWRNSSGT ActRIIb-ED dimer
IELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAG
GPEVTYEPPPTAPTSGRGEAETRECIYYNANWELERTNQSGLERCEGEQDK
RLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEG
NFCNERFTHLPEAGGPEVTYEPPPTAPT
87 EMEVTQPTSNPVTPKPPYYNIQNLDSMLHGTGMKSDSDQKKSENGVTLAPE ActRIIa-BMPRIa-
D ED natural linker
88 AILGRSETQECLFFNANwEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGS ActRIla-BMPRIa-
IEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEME ED dimer
VTQPTSNPVTPKPPYYNIQNLDSMLHGTGMKSDSDQKKSENGVTLAPEDTL
PFLKCYCSGHCPDDAINNTCITNGHCFAIIEEDDQGETTLASGCMKYEGSD
FQCKDSPKAQLRRTIECCRTNLCNQYLQPTLPPVVIGPFFDGSIR
89 MDWTWRILFLVAAATGTHA Signal peptide
90 MVLQTQVFISLLLWISGAYG Signal peptide
91 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
hIgG1Fc.DELTA.K-.DELTA.C-
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI T2m fusion
EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN
HYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCD
NQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFIL
EDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
92 PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW hIgG1Fc.DELTA.K-C-
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL T2m fusion
PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRF
STCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYH
DFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
93 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP
hIgG1Fc.DELTA.K-CC-
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK T2m fusion
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVKFPQLCK
FCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHD
PKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYN
TSNPD
94 APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGV
hIgG2Fc.DELTA.K-.DELTA.C-
EVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE T2m fusion
KTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH
YTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDN
QKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILE
DAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
95 PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWY hIgG2Fc.DELTA.K-C-
VDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLP T2m fusion
APIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVE
WESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
LHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFS
TCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHD
FILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
96 APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGV
hIgG2Fc.DELTA.K-CC-
EVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE T2m fusion
KTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH
YTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDN
QKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILE
DAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
97 MDWTWRILFLVAAATGTHAERKCCVECPPCPAPPVAGPSVFLFPPKPKDTL hIgG2Fc-CCCC-
MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV T2 fusion, also
VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP termed Fc-T2m
SREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIPPHVQKS
VNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQE
VCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF
FMCSCSSDECNDNIIFSEEYNTSNPD
98 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE
hIgG4Fc.DELTA.K-CC-
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK 228P-T2m fusion
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV
FSCSVMHEALHNHYTQKSLSLSLGIPPHVQKSVNNDMIVTDNNGAVKFPQL
CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVC
HDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE
YNTSNPD
99 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE
hIgG4Fc.DELTA.K-CC-
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK 228P-409K-T2m
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG fusion
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNV
FSCSVMHEALHNHYTQKSLSLSLGIPPHVQKSVNNDMIVTDNNGAVKFPQL
CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVC
HDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE
YNTSNPD
100 PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW hIgG1Fc.DELTA.K-C-
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL T22d35 fusion
PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRF
STCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYH
DFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDI
PPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITS
ICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEK
KKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
101 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP
hIgG1Fc.DELTA.K-CC-
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK T22d35 fusion
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVKFPQLCK
FCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHD
PKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYN
TSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMS
NCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPK
CIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
102 VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF
hIgG2Fc.DELTA.K-CC-
NWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNK T22d35 fusion
GLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI
SVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDV
RFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLP
YHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNP
DIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI
TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMK
EKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
103 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE
hIgG4Fc.DELTA.K-CC-
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK 228P-T22d35
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG fusion
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV
FSCSVMHEALHNHYTQKSLSLSLGIPPHVQKSVNNDMIVTDNNGAVKFPQL
CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVC
HDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE
YNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSC
MSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAAS
PKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
104 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE
hIgG4Fc.DELTA.K-CC-
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK 228P-409K-
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG T22d35 fusion
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNV
FSCSVMHEALHNHYTQKSLSLSLGIPPHVQKSVNNDMIVTDNNGAVKFPQL
CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVC
HDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE
YNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSC
MSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAAS
PKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
105 MDWTWRILFLVAAATGTHAERKCCVECPPCPAPPVAGPSVFLFPPKPKDTL hIgG2Fc-CCCC-
MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV T22d35 fusion,
VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP also termed Fc-
SREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSF T22d35
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIPPHVQKS
VNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQE
VCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF
FMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFP
QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLET
VCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFS
EEYNTSNPD
106 PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
hIgG1Fc.DELTA.K-C-T2-
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL T2b.sup.AA fusion
PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRF
STCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYH
DFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDI
PPHVQKSDVEMEAQKDEIIAPSANRTAHPLRHINNDMIVTDNNGAVKFPQL
CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVC
HDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE
YNTSNPD
107 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP
hIgG1Fc.DELTA.K-CC-
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK T2-T2b.sup.AA fusion
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVKFPQLCK
FCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHD
PKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYN
TSNPDIPPHVQKSDVEMEAQKDEIIAPSANRTAHPLRHINNDMIVTDNNGA
VKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENI
TLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDN
IIFSEEYNTSNPD
108 VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF
hIgG2Fc.DELTA.K-CC-
NWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNK T2-T2b.sup.AA fusion
GLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI
SVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDV
RFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLP
YHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNP
DIPPHVQKSDVEMEAQKDEIIAPSANRTAHPLRHINNDMIVTDNNGAVKFP
QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLET
VCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFS
EEYNTSNPD
109 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE
hIgG4Fc.DELTA.K-CC-
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK 228P-T2-T2b.sup.AA
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG fusion
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV
FSCSVMHEALHNHYTQKSLSLSLGIPPHVQKSVNNDMIVTDNNGAVKFPQL
CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVC
HDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE
YNTSNPDIPPHVQKSDVEMEAQKDEIIAPSANRTAHPLRHINNDMIVTDNN
GAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDE
NITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECN
DNIIFSEEYNTSNPD
110 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE
hIgG4Fc.DELTA.K-CC-
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK 228P-409K-T2-
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG T2b.sup.AA fusion
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNV
FSCSVMHEALHNHYTQKSLSLSLGIPPHVQKSVNNDMIVTDNNGAVKFPQL
CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVC
HDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE
YNTSNPDIPPHVQKSDVEMEAQKDEIIAPSANRTAHPLRHINNDMIVTDNN
GAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDE
NITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECN
DNIIFSEEYNTSNPD
111 PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
hIgG1Fc.DELTA.K-C-T2-
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL T2-T2 fusion
PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRF
STCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYH
DFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDI
PPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITS
ICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEK
KKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDN
NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKND
ENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDEC
NDNIIFSEEYNTSNPD
112 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP
hIgG1Fc.DELTA.K-CC-
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK T2-T2-T2 fusion
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVKFPQLCK
FCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHD
PKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYN
TSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMS
NCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPK
CIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNND
MIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVA
VWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCS
CSSDECNDNIIFSEEYNTSNPD
113 VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF
hIgG2Fc.DELTA.K-CC-
NWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNK T2-T2-T2 fusion
GLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI
SVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDV
RFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLP
YHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNP
DIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI
TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMK
EKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVT
DNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK
NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD
ECNDNIIFSEEYNTSNPD
114 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE
hIgG4Fc.DELTA.K-CC-
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK 228P-T2-T2-T2
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG fusion
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV
FSCSVMHEALHNHYTQKSLSLSLGIPPHVQKSVNNDMIVTDNNGAVKFPQL
CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVC
HDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE
YNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSC
MSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAAS
PKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVN
NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVC
VAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFM
CSCSSDECNDNIIFSEEYNTSNPD
115 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE
hIgG4Fc.DELTA.K-CC-
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK 228P-409K-T2-
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG T2-T2 fusion
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNV
FSCSVMHEALHNHYTQKSLSLSLGIPPHVQKSVNNDMIVTDNNGAVKFPQL
CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVC
HDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE
YNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSC
MSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAAS
PKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVN
NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVC
VAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFM
CSCSSDECNDNIIFSEEYNTSNPD
116 PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
hIgG1Fc.DELTA.K-C-T2-
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL T2b.sup.AA-T2b.sup.AA
PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV fusion
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRF
STCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYH
DFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDI
PPHVQKSDVEMEAQKDEIIAPSANRTAHPLRHINNDMIVTDNNGAVKFPQL
CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVC
HDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE
YNTSNPDIPPHVQKSDVEMEAQKDEIIAPSANRTAHPLRHINNDMIVTDNN
GAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDE
NITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECN
DNIIFSEEYNTSNPD
117 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP
hIgG1Fc.DELTA.K-CC-
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
T2-T2b.sup.AA-T2b.sup.AA
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY fusion
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVKFPQLCK
FCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHD
PKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYN
TSNPDIPPHVQKSDVEMEAQKDEIIAPSANRTAHPLRHINNDMIVTDNNGA
VKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENI
TLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDN
IIFSEEYNTSNPDIPPHVQKSDVEMEAQKDEIIAPSANRTAHPLRHINNDM
IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV
WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSC
SSDECNDNIIFSEEYNTSNPD
118 VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF
hIgG2Fc.DELTA.K-CC-
NWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNK
T2-T2b.sup.AA-T2b.sup.AA
GLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI fusion
SVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDV
RFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLP
YHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNP
DIPPHVQKSDVEMEAQKDEIIAPSANRTAHPLRHINNDMIVTDNNGAVKFP
QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLET
VCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFS
EEYNTSNPDIPPHVQKSDVEMEAQKDEIIAPSANRTAHPLRHINNDMIVTD
NNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN
DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE
CNDNIIFSEEYNTSNPD
119 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE
hIgG4Fc.DELTA.K-CC-
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK 228P-T2-T2b.sup.AA-
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG T2b.sup.AA fusion
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV
FSCSVMHEALHNHYTQKSLSLSLGIPPHVQKSVNNDMIVTDNNGAVKFPQL
CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVC
HDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE
YNTSNPDIPPHVQKSDVEMEAQKDEIIAPSANRTAHPLRHINNDMIVTDNN
GAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDE
NITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECN
DNIIFSEEYNTSNPDIPPHVQKSDVEMEAQKDEIIAPSANRTAHPLRHINN
DMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCV
AVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMC
SCSSDECNDNIIFSEEYNTSNPD
120 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE
hIgG4Fc.DELTA.K-CC-
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK 228P-409K-T2-
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG T2b.sup.AA-T2b.sup.AA
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNV fusion
FSCSVMHEALHNHYTQKSLSLSLGIPPHVQKSVNNDMIVTDNNGAVKFPQL
CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVC
HDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE
YNTSNPDIPPHVQKSDVEMEAQKDEIIAPSANRTAHPLRHINNDMIVTDNN
GAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDE
NITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECN
DNIIFSEEYNTSNPDIPPHVQKSDVEMEAQKDEIIAPSANRTAHPLRHINN
DMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCV
AVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMC
SCSSDECNDNIIFSEEYNTSNPD
121 DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYA Cetuximab LC
SESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGT
KLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA
LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
VTKSFNRGEC
122 QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVI Cetuximab HC-
WSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYY T22d35
DYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIPPHVQKSVN
NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVC
VAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFM
CSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQL
CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVC
HDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE
YNTSNPD
123 QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVI Cetuximab HC-
WSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYY T2m
DYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIPPHVQKSVN
NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVC
VAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFM
CSCSSDECNDNIIFSEEYNTSNPD
124 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSA Herceptin LC
SFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGT
KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA
LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
VTKSFNRGEC
125 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARI Herceptin HC-
YPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGD T22d35
GFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM
ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIPPHVQKSV
NNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEV
CVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFF
MCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQ
LCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETV
CHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSE
EYNTSNPD
126 DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKCLIYFT Avastin LC
SSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGT
KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA
LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
VTKSFNRGEC
127 EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWI Avastin HC-
NTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHY T22d35
YGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIPPHVQ
KSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKP
QEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGE
TFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVK
FPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITL
ETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNII
FSEEYNTSNPD
128 DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLLIYDTS Synagis LC
KLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTK
LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV
TKSFNRGEC
129 QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLA Synagis HC-
DIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTATYYCARSMI T22d35
TNWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSAAAAGGTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM
ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGIPPHVQKSVN
NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVC
VAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFM
CSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQL
CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVC
HDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE
YNTSNPD
130 EVQLQASGGGLVQAGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSRI FC5-Fc-T22d35
TWGGDNTFYSNSVKGRFTISRDNAKNTVYLQMNSLKPEDTADYYCAAGSTS
TATPLRVDYWGKGTQVTVSSASEPRGPTIKPCPPCKCPAPNLLGGPSVFIF
PPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHRE
DYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRA
PQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEP
VLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGT
GIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI
TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMK
EKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVT
DNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK
NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD
ECNDNIIFSEEYNTSNPD
131 EVQLQASGGGLVQAGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSRI FC5-Fc-T2m
TWGGDNTFYSNSVKGRFTISRDNAKNTVYLQMNSLKPEDTADYYCAAGSTS
TATPLRVDYWGKGTQVTVSSASEPRGPTIKPCPPCKCPAPNLLGGPSVFIF
PPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHRE
DYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRA
PQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEP
VLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGT
GIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI
TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMK
EKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
132 MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLC T2-hIgG1Fc
KFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCH fusion from R&D,
DPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEY also termed T2m-
NTSNPDMDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV Fc (R&D)
TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
133 MDWTWRILFLVAAATGTHAIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCD T2-hIgG2Fc-
VRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKL CCCC fusion,
PYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSN also termed T2m-
PDERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH Fc
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEY
KCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPG
134 LPYHDFILEDAASPK Isotope-labelled
peptide for
T22d35
135 ALPAPIEK Isotope-labelled
peptide for
Cetuximab
136 DDDDDDDDDDDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC
D10-hIgG1Fc.DELTA.K-
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD CC-T2m fusion
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNN
GAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDE
NITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECN
DNIIFSEEYNTSNPD
137 DDDDDDDDDDGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP D10-G.sub.3S-
EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV hIgG1Fc.DELTA.K-CC-
LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT T2m fusion
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGIPPHVQKSVNNDMIV
TDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWR
KNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSS
DECNDNIIFSEEYNTSNPD
138 DDDDDDDDDDPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
D10-hIgG1Fc.DELTA.K-
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE C-T2m fusion
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVKFP
QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLET
VCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFS
EEYNTSNPD
139 DDDDDDDDDDGGGSGGGSPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV
D10-(G.sub.3S).sub.2-
TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH hIgG1Fc.DELTA.K-C-
QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN T2m fusion
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTD
NNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN
DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE
CNDNIIFSEEYNTSNPD
140 DDDDDDDDDDVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVD
D10-hIgG2Fc.DELTA.K-
VSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNG CC-T2m fusion
KEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVK
FPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITL
ETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNII
FSEEYNTSNPD
141 DDDDDDDDDDESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEV
D10-hIgG4Fc.DELTA.K-
TCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH CC-228P-T2m
QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN fusion
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV
DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGIPPHVQKSVNNDMIVTD
NNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN
DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE
CNDNIIFSEEYNTSNPD
142 DDDDDDDDDDESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEV
D10-hIgG4Fc.DELTA.K-
TCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH CC-228P-409K-
QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN T2m fusion
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGIPPHVQKSVNNDMIVTD
NNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN
DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE
CNDNIIFSEEYNTSNPD
143 DDDDDDDDDDDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC
D10-hIgG1Fc.DELTA.K-
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD CC-T22d35 fusion
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNN
GAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDE
NITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECN
DNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFS
TCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHD
FILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
144 DDDDDDDDDDVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVD
D10-hIgG2Fc.DELTA.K-
VSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNG CC-T22d35 fusion
KEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVK
FPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITL
ETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNII
FSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDN
QKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILE
DAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
145 DDDDDDDDDDESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEV
D10-hIgG4Fc.DELTA.K-
TCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH CC-228P-T22d35
QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN fusion
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV
DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGIPPHVQKSVNNDMIVTD
NNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN
DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE
CNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVR
FSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY
HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
146 DDDDDDDDDDESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEV
D10-hIgG4Fc.DELTA.K-
TCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKpREEQFNSTYRVVSVLTVLH CC-228P-409K-
QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN T22d35 fusion
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGIPPHVQKSVNNDMIVTD
NNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN
DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE
CNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVR
FSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY
HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
147 DDDDDDDDDDDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC
D10-hIgG1Fc.DELTA.K-
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD CC-T2-T2-T2
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV fusion
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNN
GAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDE
NITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECN
DNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFS
TCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHD
FILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIP
PHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSI
CEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKK
KPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
148 DDDDDDDDDDVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVD
D10-hIgG2Fc.DELTA.K-
VSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNG CC-T2-T2-T2
KEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTC fusion
LVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVK
FPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITL
ETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNII
FSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDN
QKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILE
DAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQ
KSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKP
QEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGE
TFFMCSCSSDECNDNIIFSEEYNTSNPD
149 DDDDDDDDDDESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEV
D10-hIgG4Fc.DELTA.K-
TCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH CC-228P-T2-T2-
QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN T2 fusion
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV
DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGIPPHVQKSVNNDMIVTD
NNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN
DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE
CNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVR
FSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY
HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT
SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKE
KKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
150 DDDDDDDDDDESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEV
D10-hIgG4Fc.DELTA.K-
TCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH CC-228P-409K-
QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN T2-T2-T2 fusion
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGIPPHVQKSVNNDMIVTD
NNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN
DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE
CNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVR
FSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY
HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT
SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKE
KKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
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[0235] US2015/0225483
Sequence CWU
1
1
1501217PRTArtificial SequenceHuman IgG1 Fc region 1Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5
10 15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val 20 25 30Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35
40 45Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu 50 55
60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65
70 75 80Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85
90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln 100 105
110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
115 120 125Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135
140Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn145 150 155 160Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185
190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln 195 200 205Lys Ser Leu Ser
Leu Ser Pro Gly Lys 210 2152216PRTArtificial
SequenceHuman IgG2 Fc region 2Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro1 5 10
15Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
20 25 30Val Asp Val Ser His Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val 35 40
45Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln 50 55 60Phe Asn Ser Thr Phe Arg
Val Val Ser Val Leu Thr Val Val His Gln65 70
75 80Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly 85 90
95Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro
100 105 110Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 115 120
125Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser 130 135 140Asp Ile Ser Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr145 150
155 160Lys Thr Thr Pro Pro Met Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr 165 170
175Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
180 185 190Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys 195
200 205Ser Leu Ser Leu Ser Pro Gly Lys 210
2153217PRTArtificial SequenceHuman IgG3 Fc region 3Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5
10 15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val 20 25
30Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr
35 40 45Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu 50 55
60Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His65
70 75 80Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85
90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Thr Lys Gly Gln 100 105
110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135
140Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn
Asn145 150 155 160Tyr Asn
Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Ile 180 185
190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe
Thr Gln 195 200 205Lys Ser Leu Ser
Leu Ser Pro Gly Lys 210 2154217PRTArtificial
SequenceHuman IgG4 Fc region 4Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys1 5 10
15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30Val Val Asp Val Ser Gln Glu
Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40
45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu 50 55 60Gln Phe Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His65 70
75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90
95Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 115 120
125Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro 130 135 140Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn145 150
155 160Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu 165 170
175Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
180 185 190Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln 195
200 205Lys Ser Leu Ser Leu Ser Leu Gly Lys 210
215515PRTArtificial SequenceHuman IgG1 hinge region 5Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro1 5
10 15612PRTArtificial SequenceHuman IgG2 hinge
region 6Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro1 5
10762PRTArtificial SequenceHuman IgG3 hinge region 7Glu
Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys1
5 10 15Pro Glu Pro Lys Ser Cys Asp
Thr Pro Pro Pro Cys Pro Arg Cys Pro 20 25
30Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys
Pro Glu 35 40 45Pro Lys Ser Cys
Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 50 55
60812PRTArtificial SequenceHuman IgG4 hinge region 8Glu Ser Lys
Tyr Gly Pro Pro Cys Pro Ser Cys Pro1 5
109216PRTArtificial SequencehIgG1FcdeltaK-deltaC Fc variant 9Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5
10 15Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val 20 25
30Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55
60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65
70 75 80Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85
90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln 100 105
110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
115 120 125Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135
140Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn145 150 155 160Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185
190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln 195 200 205Lys Ser Leu Ser
Leu Ser Pro Gly 210 21510220PRTArtificial
SequencehIgG1FcdeltaK-C Fc variant 10Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu1 5 10
15Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu 20 25 30Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 35
40 45Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys 50 55 60Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu65 70
75 80Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys 85 90
95Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys 100 105 110Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 115
120 125Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys 130 135 140Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln145
150 155 160Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly 165
170 175Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln 180 185 190Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 195
200 205His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly 210 215
22011226PRTArtificial SequencehIgG1FcdeltaK-CC Fc variant 11Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5
10 15Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 20 25
30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65
70 75 80Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile 100 105
110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135
140Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu145 150 155 160Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 195 200 205His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220Pro Gly22512231PRTArtificial
SequencehIgG1FcdeltaK-S Fc variant 12Glu Pro Lys Ser Ser Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala1 5 10
15Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro 20 25 30Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35
40 45Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val 50 55 60Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln65 70
75 80Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln 85 90
95Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala 100 105 110Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115
120 125Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr 130 135 140Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser145
150 155 160Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165
170 175Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr 180 185 190Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195
200 205Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys 210 215
220Ser Leu Ser Leu Ser Pro Gly225 23013231PRTArtificial
SequencehIgG1deltaK-SS Fc variant 13Glu Pro Lys Ser Ser Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala1 5 10
15Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro 20 25 30Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35
40 45Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val 50 55 60Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln65 70
75 80Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln 85 90
95Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala 100 105 110Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115
120 125Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr 130 135 140Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser145
150 155 160Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165
170 175Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr 180 185 190Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195
200 205Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys 210 215
220Ser Leu Ser Leu Ser Pro Gly225 23014231PRTArtificial
SequencehIgG1FcdeltaK-SSS Fc variant 14Glu Pro Lys Ser Ser Asp Lys Thr
His Thr Ser Pro Pro Ser Pro Ala1 5 10
15Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro 20 25 30Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35
40 45Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val 50 55 60Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln65
70 75 80Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln 85 90
95Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala 100 105 110Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115
120 125Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr 130 135 140Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser145
150 155 160Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165
170 175Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr 180 185 190Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195
200 205Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys 210 215
220Ser Leu Ser Leu Ser Pro Gly225 23015215PRTArtificial
SequencehIgG2FcdeltaK-deltaC Fc variant 15Ala Pro Pro Val Ala Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro1 5 10
15Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val 20 25 30Val Asp Val
Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 35
40 45Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln 50 55 60Phe Asn
Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln65
70 75 80Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Gly 85 90
95Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys
Gly Gln Pro 100 105 110Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 115
120 125Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser 130 135 140Asp
Ile Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr145
150 155 160Lys Thr Thr Pro Pro Met
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 165
170 175Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe 180 185 190Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 195
200 205Ser Leu Ser Leu Ser Pro Gly 210
21516219PRTArtificial SequencehIgG2FcdeltaK-C Fc variant
16Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe1
5 10 15Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val 20 25
30Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Gln Phe 35 40 45Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 50
55 60Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val
Ser Val Leu Thr65 70 75
80Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
85 90 95Ser Asn Lys Gly Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr 100
105 110Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg 115 120 125Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 130
135 140Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu
Ser Asn Gly Gln Pro145 150 155
160Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser
165 170 175Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 180
185 190Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His 195 200 205Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 210
21517222PRTArtificial SequencehIgG2FcdeltaK-CC Fc variant 17Val Glu Cys
Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val1 5
10 15Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr 20 25
30Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
35 40 45Val Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 50 55
60Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser65
70 75 80Val Leu Thr Val
Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 85
90 95Cys Lys Val Ser Asn Lys Gly Leu Pro Ala
Pro Ile Glu Lys Thr Ile 100 105
110Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
115 120 125Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu 130 135
140Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Ser
Asn145 150 155 160Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser
165 170 175Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg 180 185
190Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 195 200 205His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 210 215
22018228PRTArtificial SequencehIgG2Fc-CCCC Fc variant 18Glu Arg
Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val1 5
10 15Ala Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu 20 25
30Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser 35 40 45His Glu Asp Pro Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55
60Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser Thr65 70 75 80Phe
Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn
85 90 95Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ala Pro 100 105
110Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu
Pro Gln 115 120 125Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 130
135 140Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ser Val145 150 155
160Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
165 170 175Pro Met Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180
185 190Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val 195 200 205Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210
215 220Ser Pro Gly Lys22519227PRTArtificial
SequencehIgG2FcdeltaK-SS Fc variant 19Glu Arg Lys Ser Ser Val Glu Cys Pro
Pro Cys Pro Ala Pro Pro Val1 5 10
15Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu 20 25 30Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35
40 45His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu 50 55 60Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr65 70
75 80Phe Arg Val Val Ser Val Leu Thr
Val Val His Gln Asp Trp Leu Asn 85 90
95Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ala Pro 100 105 110Ile Glu Lys
Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln 115
120 125Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val 130 135 140Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val145
150 155 160Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165
170 175Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr 180 185 190Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 195
200 205Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu 210 215
220Ser Pro Gly22520227PRTArtificial SequencehIgG2FcdeltaK-SSS Fc variant
20Glu Arg Lys Ser Ser Val Glu Ser Pro Pro Cys Pro Ala Pro Pro Val1
5 10 15Ala Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25
30Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser 35 40 45His Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 50
55 60Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr65 70 75
80Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn
85 90 95Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ala Pro 100
105 110Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro
Arg Glu Pro Gln 115 120 125Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 130
135 140Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ser Val145 150 155
160Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
165 170 175Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180
185 190Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val 195 200 205Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210
215 220Ser Pro Gly22521227PRTArtificial
SequencehIgG2FcdeltaK-SSSS Fc variant 21Glu Arg Lys Ser Ser Val Glu Ser
Pro Pro Ser Pro Ala Pro Pro Val1 5 10
15Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu 20 25 30Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35
40 45His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu 50 55 60Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr65
70 75 80Phe Arg Val Val Ser Val Leu
Thr Val Val His Gln Asp Trp Leu Asn 85 90
95Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ala Pro 100 105 110Ile Glu
Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln 115
120 125Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val 130 135 140Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val145
150 155 160Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165
170 175Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr 180 185 190Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 195
200 205Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu 210 215
220Ser Pro Gly22522216PRTArtificial SequencehIgG3FcdeltaK-deltaC Fc
variant 22Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys1 5 10 15Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20
25 30Val Val Asp Val Ser His Glu Asp Pro Glu
Val Gln Phe Lys Trp Tyr 35 40
45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50
55 60Gln Tyr Asn Ser Thr Phe Arg Val Val
Ser Val Leu Thr Val Leu His65 70 75
80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys 85 90 95Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 100
105 110Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met 115 120
125Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140Ser Asp Ile Ala Val Glu Trp
Glu Ser Ser Gly Gln Pro Glu Asn Asn145 150
155 160Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly
Ser Phe Phe Leu 165 170
175Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile
180 185 190Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn Arg Phe Thr Gln 195 200
205Lys Ser Leu Ser Leu Ser Pro Gly 210
21523220PRTArtificial SequencehIgG3FcdeltaK-C Fc variant 23Pro Arg Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu1 5
10 15Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu 20 25
30Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln
35 40 45Phe Lys Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys 50 55
60Pro Arg Glu Glu Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu65
70 75 80Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 85
90 95Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys 100 105
110Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
115 120 125Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys 130 135
140Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly
Gln145 150 155 160Pro Glu
Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly
165 170 175Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln 180 185
190Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn 195 200 205Arg Phe Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 210 215
22024226PRTArtificial SequencehIgG3FcdeltaK-CC Fc variant 24Asp Thr Pro
Pro Pro Cys Pro Arg Cys Pro Ala Pro Glu Leu Leu Gly1 5
10 15Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 20 25
30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45Glu Asp Pro Glu Val Gln Phe
Lys Trp Tyr Val Asp Gly Val Glu Val 50 55
60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Phe65
70 75 80Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile 100 105
110Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser 130 135
140Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu145 150 155 160Trp Glu
Ser Ser Gly Gln Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro
165 170 175Met Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe Ser Cys Ser
Val Met 195 200 205His Glu Ala Leu
His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220Pro Gly22525231PRTArtificial
SequencehIgG3FcdeltaK-S Fc variant 25Glu Pro Lys Ser Ser Asp Thr Pro Pro
Pro Cys Pro Arg Cys Pro Ala1 5 10
15Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro 20 25 30Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35
40 45Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe
Lys Trp Tyr Val 50 55 60Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln65 70
75 80Tyr Asn Ser Thr Phe Arg Val Val
Ser Val Leu Thr Val Leu His Gln 85 90
95Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala 100 105 110Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro 115
120 125Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met Thr 130 135 140Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser145
150 155 160Asp Ile Ala Val Glu Trp Glu
Ser Ser Gly Gln Pro Glu Asn Asn Tyr 165
170 175Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr 180 185 190Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe 195
200 205Ser Cys Ser Val Met His Glu Ala Leu
His Asn Arg Phe Thr Gln Lys 210 215
220Ser Leu Ser Leu Ser Pro Gly225 23026231PRTArtificial
SequencehIgG3FcdeltaK-SS Fc variant 26Glu Pro Lys Ser Ser Asp Thr Pro Pro
Pro Ser Pro Arg Cys Pro Ala1 5 10
15Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro 20 25 30Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35
40 45Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe
Lys Trp Tyr Val 50 55 60Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln65 70
75 80Tyr Asn Ser Thr Phe Arg Val Val
Ser Val Leu Thr Val Leu His Gln 85 90
95Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala 100 105 110Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro 115
120 125Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met Thr 130 135 140Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser145
150 155 160Asp Ile Ala Val Glu Trp Glu
Ser Ser Gly Gln Pro Glu Asn Asn Tyr 165
170 175Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr 180 185 190Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe 195
200 205Ser Cys Ser Val Met His Glu Ala Leu
His Asn Arg Phe Thr Gln Lys 210 215
220Ser Leu Ser Leu Ser Pro Gly225 23027231PRTArtificial
SequencehIgG3FcdeltaK-SSS Fc variant 27Glu Pro Lys Ser Ser Asp Thr Pro
Pro Pro Ser Pro Arg Ser Pro Ala1 5 10
15Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro 20 25 30Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35
40 45Val Asp Val Ser His Glu Asp Pro Glu Val Gln
Phe Lys Trp Tyr Val 50 55 60Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln65
70 75 80Tyr Asn Ser Thr Phe Arg Val
Val Ser Val Leu Thr Val Leu His Gln 85 90
95Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala 100 105 110Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro 115
120 125Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr 130 135 140Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser145
150 155 160Asp Ile Ala Val Glu Trp
Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr 165
170 175Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr 180 185 190Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe 195
200 205Ser Cys Ser Val Met His Glu Ala Leu
His Asn Arg Phe Thr Gln Lys 210 215
220Ser Leu Ser Leu Ser Pro Gly225 23028216PRTArtificial
SequencehIgG4FcdeltaK-deltaC Fc variant 28Ala Pro Glu Phe Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys1 5 10
15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val 20 25 30Val Val Asp
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35
40 45Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu 50 55 60Gln Phe
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65
70 75 80Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90
95Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln 100 105 110Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 115
120 125Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro 130 135 140Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn145
150 155 160Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165
170 175Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val 180 185 190Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195
200 205Lys Ser Leu Ser Leu Ser Leu Gly
210 21529220PRTArtificial SequencehIgG4FcdeltaK-C Fc
variant 29Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe
Leu1 5 10 15Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 20
25 30Val Thr Cys Val Val Val Asp Val Ser Gln
Glu Asp Pro Glu Val Gln 35 40
45Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 50
55 60Pro Arg Glu Glu Gln Phe Asn Ser Thr
Tyr Arg Val Val Ser Val Leu65 70 75
80Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys 85 90 95Val Ser
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 100
105 110Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser 115 120
125Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
130 135 140Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln145 150
155 160Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly 165 170
175Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
180 185 190Glu Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn 195 200
205His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 210
215 22030228PRTArtificial
SequencehIgG4FcdeltaK-CC Fc variant 30Glu Ser Lys Tyr Gly Pro Pro Cys Pro
Ser Cys Pro Ala Pro Glu Phe1 5 10
15Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr 20 25 30Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35
40 45Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val Asp Gly Val 50 55 60Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser65 70
75 80Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu 85 90
95Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ser 100 105 110Ser Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115
120 125Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys Asn Gln 130 135 140Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala145
150 155 160Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165
170 175Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Arg Leu 180 185 190Thr
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195
200 205Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser 210 215
220Leu Ser Leu Gly22531228PRTArtificial SequencehIgG4FcdeltaK-CC-228P Fc
variant 31Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
Phe1 5 10 15Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20
25 30Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val 35 40
45Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50
55 60Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser65 70 75
80Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu 85 90 95Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100
105 110Ser Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro 115 120
125Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala145 150
155 160Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr 165 170
175Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
180 185 190Thr Val Asp Lys Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200
205Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser 210 215 220Leu Ser Leu
Gly22532228PRTArtificial SequencehIgG4FcdeltaK-CC-228P-409K Fc variant
32Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe1
5 10 15Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25
30Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val 35 40 45Ser Gln Glu
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50
55 60Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser65 70 75
80Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
85 90 95Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100
105 110Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro 115 120 125Gln Val
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130
135 140Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala145 150 155
160Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 180
185 190Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser Cys Ser 195 200 205Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210
215 220Leu Ser Leu Gly22533228PRTArtificial
SequencehIgG4FcdeltaK-S Fc variant 33Glu Ser Lys Tyr Gly Pro Pro Ser Pro
Ser Cys Pro Ala Pro Glu Phe1 5 10
15Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr 20 25 30Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35
40 45Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val Asp Gly Val 50 55 60Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser65 70
75 80Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu 85 90
95Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ser 100 105 110Ser Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115
120 125Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys Asn Gln 130 135 140Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala145
150 155 160Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165
170 175Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Arg Leu 180 185 190Thr
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195
200 205Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser 210 215
220Leu Ser Leu Gly22534228PRTArtificial SequencehIgG4FcdeltaK-SS Fc
variant 34Glu Ser Lys Tyr Gly Pro Pro Ser Pro Ser Ser Pro Ala Pro Glu
Phe1 5 10 15Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20
25 30Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val 35 40
45Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50
55 60Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser65 70 75
80Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu 85 90 95Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100
105 110Ser Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro 115 120
125Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala145 150
155 160Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr 165 170
175Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
180 185 190Thr Val Asp Lys Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200
205Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser 210 215 220Leu Ser Leu
Gly22535101PRTArtificial SequenceTbetaRII-ED structured domain 35Gln Leu
Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln1 5
10 15Lys Ser Cys Met Ser Asn Cys Ser
Ile Thr Ser Ile Cys Glu Lys Pro 20 25
30Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile
Thr 35 40 45Leu Glu Thr Val Cys
His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile 50 55
60Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys
Lys Lys65 70 75 80Pro
Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn
85 90 95Asp Asn Ile Ile Phe
1003625PRTArtificial SequenceTbetaRII-ED N-term unstructured region and
natural linker 36Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met
Ile Val Thr1 5 10 15Asp
Asn Asn Gly Ala Val Lys Phe Pro 20
253750PRTArtificial SequenceTbetaRIIb-ED N-term unstructured region and
natural linker 37Ile Pro Pro His Val Gln Lys Ser Asp Val Glu Met Glu
Ala Gln Lys1 5 10 15Asp
Glu Ile Ile Cys Pro Ser Cys Asn Arg Thr Ala His Pro Leu Arg 20
25 30His Ile Asn Asn Asp Met Ile Val
Thr Asp Asn Asn Gly Ala Val Lys 35 40
45Phe Pro 503850PRTArtificial SequenceTbetaRIIb-ED Cys-mutated
N-term unstructured region and natural linker 38Ile Pro Pro His Val
Gln Lys Ser Asp Val Glu Met Glu Ala Gln Lys1 5
10 15Asp Glu Ile Ile Ala Pro Ser Ala Asn Arg Thr
Ala His Pro Leu Arg 20 25
30His Ile Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys
35 40 45Phe Pro 503910PRTArtificial
SequenceTbetaRII-ED C-term unstructured region and natural linker
39Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp1 5
104035PRTArtificial SequenceTbetaRII-ED natural linker 40Ser Glu Glu
Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro His Val Gln1 5
10 15Lys Ser Val Asn Asn Asp Met Ile Val
Thr Asp Asn Asn Gly Ala Val 20 25
30Lys Phe Pro 354160PRTArtificial SequenceTbetaRIIb-ED
natural linker 41Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro His
Val Gln1 5 10 15Lys Ser
Asp Val Glu Met Glu Ala Gln Lys Asp Glu Ile Ile Cys Pro 20
25 30Ser Cys Asn Arg Thr Ala His Pro Leu
Arg His Ile Asn Asn Asp Met 35 40
45Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro 50
55 604260PRTArtificial SequenceTbetaRIIb-ED Cys-mutated
linker 42Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro His Val Gln1
5 10 15Lys Ser Asp Val
Glu Met Glu Ala Gln Lys Asp Glu Ile Ile Ala Pro 20
25 30Ser Ala Asn Arg Thr Ala His Pro Leu Arg His
Ile Asn Asn Asp Met 35 40 45Ile
Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro 50 55
6043136PRTArtificial SequenceTbetaRII-ED monomer, also
termed T2 or T2m 43Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met
Ile Val Thr1 5 10 15Asp
Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20
25 30Val Arg Phe Ser Thr Cys Asp Asn
Gln Lys Ser Cys Met Ser Asn Cys 35 40
45Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val
50 55 60Trp Arg Lys Asn Asp Glu Asn Ile
Thr Leu Glu Thr Val Cys His Asp65 70 75
80Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala
Ala Ser Pro 85 90 95Lys
Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met
100 105 110Cys Ser Cys Ser Ser Asp Glu
Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120
125Glu Tyr Asn Thr Ser Asn Pro Asp 130
13544161PRTArtificial SequenceTbetabRIIb-ED monomer, also termed T2b
44Ile Pro Pro His Val Gln Lys Ser Asp Val Glu Met Glu Ala Gln Lys1
5 10 15Asp Glu Ile Ile Cys Pro
Ser Cys Asn Arg Thr Ala His Pro Leu Arg 20 25
30His Ile Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly
Ala Val Lys 35 40 45Phe Pro Gln
Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp 50
55 60Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr
Ser Ile Cys Glu65 70 75
80Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn
85 90 95Ile Thr Leu Glu Thr Val
Cys His Asp Pro Lys Leu Pro Tyr His Asp 100
105 110Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile
Met Lys Glu Lys 115 120 125Lys Lys
Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu 130
135 140Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr
Asn Thr Ser Asn Pro145 150 155
160Asp45161PRTArtificial SequenceTbetaRIIb-ED monomer Cys-mutated in
the linker region, also termed T2bAA 45Ile Pro Pro His Val Gln Lys
Ser Asp Val Glu Met Glu Ala Gln Lys1 5 10
15Asp Glu Ile Ile Ala Pro Ser Ala Asn Arg Thr Ala His
Pro Leu Arg 20 25 30His Ile
Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys 35
40 45Phe Pro Gln Leu Cys Lys Phe Cys Asp Val
Arg Phe Ser Thr Cys Asp 50 55 60Asn
Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu65
70 75 80Lys Pro Gln Glu Val Cys
Val Ala Val Trp Arg Lys Asn Asp Glu Asn 85
90 95Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu
Pro Tyr His Asp 100 105 110Phe
Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys 115
120 125Lys Lys Pro Gly Glu Thr Phe Phe Met
Cys Ser Cys Ser Ser Asp Glu 130 135
140Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro145
150 155
160Asp46272PRTArtificial SequenceTbetaRII-ED dimer, also termed T2-T2 or
T22d35 46Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr1
5 10 15Asp Asn Asn Gly
Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20
25 30Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser
Cys Met Ser Asn Cys 35 40 45Ser
Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50
55 60Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu
Glu Thr Val Cys His Asp65 70 75
80Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser
Pro 85 90 95Lys Cys Ile
Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100
105 110Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp
Asn Ile Ile Phe Ser Glu 115 120
125Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys Ser 130
135 140Val Asn Asn Asp Met Ile Val Thr
Asp Asn Asn Gly Ala Val Lys Phe145 150
155 160Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser
Thr Cys Asp Asn 165 170
175Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys
180 185 190Pro Gln Glu Val Cys Val
Ala Val Trp Arg Lys Asn Asp Glu Asn Ile 195 200
205Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His
Asp Phe 210 215 220Ile Leu Glu Asp Ala
Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys225 230
235 240Lys Pro Gly Glu Thr Phe Phe Met Cys Ser
Cys Ser Ser Asp Glu Cys 245 250
255Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp
260 265 27047322PRTArtificial
SequenceTbetaRIIb-ED dimer, also termed T2-T2b 47Ile Pro Pro His Val Gln
Lys Ser Asp Val Glu Met Glu Ala Gln Lys1 5
10 15Asp Glu Ile Ile Cys Pro Ser Cys Asn Arg Thr Ala
His Pro Leu Arg 20 25 30His
Ile Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys 35
40 45Phe Pro Gln Leu Cys Lys Phe Cys Asp
Val Arg Phe Ser Thr Cys Asp 50 55
60Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu65
70 75 80Lys Pro Gln Glu Val
Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn 85
90 95Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys
Leu Pro Tyr His Asp 100 105
110Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys
115 120 125Lys Lys Pro Gly Glu Thr Phe
Phe Met Cys Ser Cys Ser Ser Asp Glu 130 135
140Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn
Pro145 150 155 160Asp Ile
Pro Pro His Val Gln Lys Ser Asp Val Glu Met Glu Ala Gln
165 170 175Lys Asp Glu Ile Ile Cys Pro
Ser Cys Asn Arg Thr Ala His Pro Leu 180 185
190Arg His Ile Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly
Ala Val 195 200 205Lys Phe Pro Gln
Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys 210
215 220Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile
Thr Ser Ile Cys225 230 235
240Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu
245 250 255Asn Ile Thr Leu Glu
Thr Val Cys His Asp Pro Lys Leu Pro Tyr His 260
265 270Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys
Ile Met Lys Glu 275 280 285Lys Lys
Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp 290
295 300Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu
Tyr Asn Thr Ser Asn305 310 315
320Pro Asp48322PRTArtificial SequenceTbetaRIIb-ED dimer Cys-mutated
in the linker region, also termed T2-T2bAA 48Ile Pro Pro His Val Gln
Lys Ser Asp Val Glu Met Glu Ala Gln Lys1 5
10 15Asp Glu Ile Ile Cys Pro Ser Cys Asn Arg Thr Ala
His Pro Leu Arg 20 25 30His
Ile Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys 35
40 45Phe Pro Gln Leu Cys Lys Phe Cys Asp
Val Arg Phe Ser Thr Cys Asp 50 55
60Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu65
70 75 80Lys Pro Gln Glu Val
Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn 85
90 95Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys
Leu Pro Tyr His Asp 100 105
110Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys
115 120 125Lys Lys Pro Gly Glu Thr Phe
Phe Met Cys Ser Cys Ser Ser Asp Glu 130 135
140Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn
Pro145 150 155 160Asp Ile
Pro Pro His Val Gln Lys Ser Asp Val Glu Met Glu Ala Gln
165 170 175Lys Asp Glu Ile Ile Ala Pro
Ser Ala Asn Arg Thr Ala His Pro Leu 180 185
190Arg His Ile Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly
Ala Val 195 200 205Lys Phe Pro Gln
Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys 210
215 220Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile
Thr Ser Ile Cys225 230 235
240Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu
245 250 255Asn Ile Thr Leu Glu
Thr Val Cys His Asp Pro Lys Leu Pro Tyr His 260
265 270Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys
Ile Met Lys Glu 275 280 285Lys Lys
Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp 290
295 300Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu
Tyr Asn Thr Ser Asn305 310 315
320Pro Asp49408PRTArtificial SequenceTbetaRII-ED trimer, also termed
T2-T2-T2 49Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val
Thr1 5 10 15Asp Asn Asn
Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20
25 30Val Arg Phe Ser Thr Cys Asp Asn Gln Lys
Ser Cys Met Ser Asn Cys 35 40
45Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50
55 60Trp Arg Lys Asn Asp Glu Asn Ile Thr
Leu Glu Thr Val Cys His Asp65 70 75
80Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala
Ser Pro 85 90 95Lys Cys
Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100
105 110Cys Ser Cys Ser Ser Asp Glu Cys Asn
Asp Asn Ile Ile Phe Ser Glu 115 120
125Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys Ser
130 135 140Val Asn Asn Asp Met Ile Val
Thr Asp Asn Asn Gly Ala Val Lys Phe145 150
155 160Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser
Thr Cys Asp Asn 165 170
175Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys
180 185 190Pro Gln Glu Val Cys Val
Ala Val Trp Arg Lys Asn Asp Glu Asn Ile 195 200
205Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His
Asp Phe 210 215 220Ile Leu Glu Asp Ala
Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys225 230
235 240Lys Pro Gly Glu Thr Phe Phe Met Cys Ser
Cys Ser Ser Asp Glu Cys 245 250
255Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp
260 265 270Ile Pro Pro His Val
Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 275
280 285Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys
Lys Phe Cys Asp 290 295 300Val Arg Phe
Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys305
310 315 320Ser Ile Thr Ser Ile Cys Glu
Lys Pro Gln Glu Val Cys Val Ala Val 325
330 335Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr
Val Cys His Asp 340 345 350Pro
Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 355
360 365Lys Cys Ile Met Lys Glu Lys Lys Lys
Pro Gly Glu Thr Phe Phe Met 370 375
380Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu385
390 395 400Glu Tyr Asn Thr
Ser Asn Pro Asp 40550458PRTArtificial SequenceTbetaRIIb-ED
trimer, also termed T2-T2b-T2b 50Ile Pro Pro His Val Gln Lys Ser Val Asn
Asn Asp Met Ile Val Thr1 5 10
15Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp
20 25 30Val Arg Phe Ser Thr Cys
Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40
45Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val
Ala Val 50 55 60Trp Arg Lys Asn Asp
Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp65 70
75 80Pro Lys Leu Pro Tyr His Asp Phe Ile Leu
Glu Asp Ala Ala Ser Pro 85 90
95Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met
100 105 110Cys Ser Cys Ser Ser
Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115
120 125Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro His
Val Gln Lys Ser 130 135 140Asp Val Glu
Met Glu Ala Gln Lys Asp Glu Ile Ile Cys Pro Ser Cys145
150 155 160Asn Arg Thr Ala His Pro Leu
Arg His Ile Asn Asn Asp Met Ile Val 165
170 175Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu
Cys Lys Phe Cys 180 185 190Asp
Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn 195
200 205Cys Ser Ile Thr Ser Ile Cys Glu Lys
Pro Gln Glu Val Cys Val Ala 210 215
220Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His225
230 235 240Asp Pro Lys Leu
Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser 245
250 255Pro Lys Cys Ile Met Lys Glu Lys Lys Lys
Pro Gly Glu Thr Phe Phe 260 265
270Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser
275 280 285Glu Glu Tyr Asn Thr Ser Asn
Pro Asp Ile Pro Pro His Val Gln Lys 290 295
300Ser Asp Val Glu Met Glu Ala Gln Lys Asp Glu Ile Ile Cys Pro
Ser305 310 315 320Cys Asn
Arg Thr Ala His Pro Leu Arg His Ile Asn Asn Asp Met Ile
325 330 335Val Thr Asp Asn Asn Gly Ala
Val Lys Phe Pro Gln Leu Cys Lys Phe 340 345
350Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys
Met Ser 355 360 365Asn Cys Ser Ile
Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val 370
375 380Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu
Glu Thr Val Cys385 390 395
400His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala
405 410 415Ser Pro Lys Cys Ile
Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe 420
425 430Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp
Asn Ile Ile Phe 435 440 445Ser Glu
Glu Tyr Asn Thr Ser Asn Pro Asp 450
45551458PRTArtificial SequenceTbetaRIIb-ED trimer Cys-mutated in the
linker regions, also termed T2-T2bAA-T2bAA 51Ile Pro Pro His Val Gln
Lys Ser Val Asn Asn Asp Met Ile Val Thr1 5
10 15Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys
Lys Phe Cys Asp 20 25 30Val
Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35
40 45Ser Ile Thr Ser Ile Cys Glu Lys Pro
Gln Glu Val Cys Val Ala Val 50 55
60Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp65
70 75 80Pro Lys Leu Pro Tyr
His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85
90 95Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly
Glu Thr Phe Phe Met 100 105
110Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu
115 120 125Glu Tyr Asn Thr Ser Asn Pro
Asp Ile Pro Pro His Val Gln Lys Ser 130 135
140Asp Val Glu Met Glu Ala Gln Lys Asp Glu Ile Ile Ala Pro Ser
Ala145 150 155 160Asn Arg
Thr Ala His Pro Leu Arg His Ile Asn Asn Asp Met Ile Val
165 170 175Thr Asp Asn Asn Gly Ala Val
Lys Phe Pro Gln Leu Cys Lys Phe Cys 180 185
190Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met
Ser Asn 195 200 205Cys Ser Ile Thr
Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala 210
215 220Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu
Thr Val Cys His225 230 235
240Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser
245 250 255Pro Lys Cys Ile Met
Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe 260
265 270Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn
Ile Ile Phe Ser 275 280 285Glu Glu
Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys 290
295 300Ser Asp Val Glu Met Glu Ala Gln Lys Asp Glu
Ile Ile Ala Pro Ser305 310 315
320Ala Asn Arg Thr Ala His Pro Leu Arg His Ile Asn Asn Asp Met Ile
325 330 335Val Thr Asp Asn
Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe 340
345 350Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln
Lys Ser Cys Met Ser 355 360 365Asn
Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val 370
375 380Ala Val Trp Arg Lys Asn Asp Glu Asn Ile
Thr Leu Glu Thr Val Cys385 390 395
400His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala
Ala 405 410 415Ser Pro Lys
Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe 420
425 430Phe Met Cys Ser Cys Ser Ser Asp Glu Cys
Asn Asp Asn Ile Ile Phe 435 440
445Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 450
4555279PRTArtificial SequenceTbetaRI-ED structured domain 52Ala Leu Gln
Cys Phe Cys His Leu Cys Thr Lys Asp Asn Phe Thr Cys1 5
10 15Val Thr Asp Gly Leu Cys Phe Val Ser
Val Thr Glu Thr Thr Asp Lys 20 25
30Val Ile His Asn Ser Met Cys Ile Ala Glu Ile Asp Leu Ile Pro Arg
35 40 45Asp Arg Pro Phe Val Cys Ala
Pro Ser Ser Lys Thr Gly Ser Val Thr 50 55
60Thr Thr Tyr Cys Cys Asn Gln Asp His Cys Asn Lys Ile Glu Leu65
70 75538PRTArtificial SequenceTbetaRI-ED
N-term unstructured region and natural linker 53Ala Ala Leu Leu Pro
Gly Ala Thr1 55414PRTArtificial SequenceTbetaRI-ED C-term
unstructured region and natural linker 54Pro Thr Thr Val Lys Ser Ser
Pro Gly Leu Gly Pro Val Glu1 5
105522PRTArtificial SequenceTbetaRI-ED natural linker 55Pro Thr Thr Val
Lys Ser Ser Pro Gly Leu Gly Pro Val Glu Ala Ala1 5
10 15Leu Leu Pro Gly Ala Thr
2056101PRTArtificial SequenceTbetaRI-ED monomer, also termed T1 or T1m
56Ala Ala Leu Leu Pro Gly Ala Thr Ala Leu Gln Cys Phe Cys His Leu1
5 10 15Cys Thr Lys Asp Asn Phe
Thr Cys Val Thr Asp Gly Leu Cys Phe Val 20 25
30Ser Val Thr Glu Thr Thr Asp Lys Val Ile His Asn Ser
Met Cys Ile 35 40 45Ala Glu Ile
Asp Leu Ile Pro Arg Asp Arg Pro Phe Val Cys Ala Pro 50
55 60Ser Ser Lys Thr Gly Ser Val Thr Thr Thr Tyr Cys
Cys Asn Gln Asp65 70 75
80His Cys Asn Lys Ile Glu Leu Pro Thr Thr Val Lys Ser Ser Pro Gly
85 90 95Leu Gly Pro Val Glu
10057202PRTArtificial SequenceTbetaRI-ED dimer, also termed T1-T1
57Ala Ala Leu Leu Pro Gly Ala Thr Ala Leu Gln Cys Phe Cys His Leu1
5 10 15Cys Thr Lys Asp Asn Phe
Thr Cys Val Thr Asp Gly Leu Cys Phe Val 20 25
30Ser Val Thr Glu Thr Thr Asp Lys Val Ile His Asn Ser
Met Cys Ile 35 40 45Ala Glu Ile
Asp Leu Ile Pro Arg Asp Arg Pro Phe Val Cys Ala Pro 50
55 60Ser Ser Lys Thr Gly Ser Val Thr Thr Thr Tyr Cys
Cys Asn Gln Asp65 70 75
80His Cys Asn Lys Ile Glu Leu Pro Thr Thr Val Lys Ser Ser Pro Gly
85 90 95Leu Gly Pro Val Glu Ala
Ala Leu Leu Pro Gly Ala Thr Ala Leu Gln 100
105 110Cys Phe Cys His Leu Cys Thr Lys Asp Asn Phe Thr
Cys Val Thr Asp 115 120 125Gly Leu
Cys Phe Val Ser Val Thr Glu Thr Thr Asp Lys Val Ile His 130
135 140Asn Ser Met Cys Ile Ala Glu Ile Asp Leu Ile
Pro Arg Asp Arg Pro145 150 155
160Phe Val Cys Ala Pro Ser Ser Lys Thr Gly Ser Val Thr Thr Thr Tyr
165 170 175Cys Cys Asn Gln
Asp His Cys Asn Lys Ile Glu Leu Pro Thr Thr Val 180
185 190Lys Ser Ser Pro Gly Leu Gly Pro Val Glu
195 2005839PRTArtificial SequenceTbetaRI-TbetaRII-ED
natural linker 58Pro Thr Thr Val Lys Ser Ser Pro Gly Leu Gly Pro Val Glu
Ile Pro1 5 10 15Pro His
Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn 20
25 30Asn Gly Ala Val Lys Phe Pro
355964PRTArtificial SequenceTbetaRI-TbetaRIIb-ED natural linker 59Pro Thr
Thr Val Lys Ser Ser Pro Gly Leu Gly Pro Val Glu Ile Pro1 5
10 15Pro His Val Gln Lys Ser Asp Val
Glu Met Glu Ala Gln Lys Asp Glu 20 25
30Ile Ile Cys Pro Ser Cys Asn Arg Thr Ala His Pro Leu Arg His
Ile 35 40 45Asn Asn Asp Met Ile
Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro 50 55
606018PRTArtificial SequenceTbetaRII-TbetaRI-ED natural
linker 60Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ala Ala Leu Leu Pro Gly1
5 10 15Ala
Thr61237PRTArtificial SequenceTbetaRI-TbetaRII-ED dimer T1-T2 61Ala Ala
Leu Leu Pro Gly Ala Thr Ala Leu Gln Cys Phe Cys His Leu1 5
10 15Cys Thr Lys Asp Asn Phe Thr Cys
Val Thr Asp Gly Leu Cys Phe Val 20 25
30Ser Val Thr Glu Thr Thr Asp Lys Val Ile His Asn Ser Met Cys
Ile 35 40 45Ala Glu Ile Asp Leu
Ile Pro Arg Asp Arg Pro Phe Val Cys Ala Pro 50 55
60Ser Ser Lys Thr Gly Ser Val Thr Thr Thr Tyr Cys Cys Asn
Gln Asp65 70 75 80His
Cys Asn Lys Ile Glu Leu Pro Thr Thr Val Lys Ser Ser Pro Gly
85 90 95Leu Gly Pro Val Glu Ile Pro
Pro His Val Gln Lys Ser Val Asn Asn 100 105
110Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro
Gln Leu 115 120 125Cys Lys Phe Cys
Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser 130
135 140Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu
Lys Pro Gln Glu145 150 155
160Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu
165 170 175Thr Val Cys His Asp
Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu 180
185 190Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys
Lys Lys Pro Gly 195 200 205Glu Thr
Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn 210
215 220Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn
Pro Asp225 230 23562262PRTArtificial
SequenceTbetaRI-TbetaRII-ED dimer T1-T2b 62Ala Ala Leu Leu Pro Gly Ala
Thr Ala Leu Gln Cys Phe Cys His Leu1 5 10
15Cys Thr Lys Asp Asn Phe Thr Cys Val Thr Asp Gly Leu
Cys Phe Val 20 25 30Ser Val
Thr Glu Thr Thr Asp Lys Val Ile His Asn Ser Met Cys Ile 35
40 45Ala Glu Ile Asp Leu Ile Pro Arg Asp Arg
Pro Phe Val Cys Ala Pro 50 55 60Ser
Ser Lys Thr Gly Ser Val Thr Thr Thr Tyr Cys Cys Asn Gln Asp65
70 75 80His Cys Asn Lys Ile Glu
Leu Pro Thr Thr Val Lys Ser Ser Pro Gly 85
90 95Leu Gly Pro Val Glu Ile Pro Pro His Val Gln Lys
Ser Asp Val Glu 100 105 110Met
Glu Ala Gln Lys Asp Glu Ile Ile Cys Pro Ser Cys Asn Arg Thr 115
120 125Ala His Pro Leu Arg His Ile Asn Asn
Asp Met Ile Val Thr Asp Asn 130 135
140Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg145
150 155 160Phe Ser Thr Cys
Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile 165
170 175Thr Ser Ile Cys Glu Lys Pro Gln Glu Val
Cys Val Ala Val Trp Arg 180 185
190Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys
195 200 205Leu Pro Tyr His Asp Phe Ile
Leu Glu Asp Ala Ala Ser Pro Lys Cys 210 215
220Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys
Ser225 230 235 240Cys Ser
Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr
245 250 255Asn Thr Ser Asn Pro Asp
26063237PRTArtificial SequenceTbetaRI-TbetaRII-ED dimer T2-T1 63Ile
Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr1
5 10 15Asp Asn Asn Gly Ala Val Lys
Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25
30Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser
Asn Cys 35 40 45Ser Ile Thr Ser
Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55
60Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val
Cys His Asp65 70 75
80Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro
85 90 95Lys Cys Ile Met Lys Glu
Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100
105 110Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile
Ile Phe Ser Glu 115 120 125Glu Tyr
Asn Thr Ser Asn Pro Asp Ala Ala Leu Leu Pro Gly Ala Thr 130
135 140Ala Leu Gln Cys Phe Cys His Leu Cys Thr Lys
Asp Asn Phe Thr Cys145 150 155
160Val Thr Asp Gly Leu Cys Phe Val Ser Val Thr Glu Thr Thr Asp Lys
165 170 175Val Ile His Asn
Ser Met Cys Ile Ala Glu Ile Asp Leu Ile Pro Arg 180
185 190Asp Arg Pro Phe Val Cys Ala Pro Ser Ser Lys
Thr Gly Ser Val Thr 195 200 205Thr
Thr Tyr Cys Cys Asn Gln Asp His Cys Asn Lys Ile Glu Leu Pro 210
215 220Thr Thr Val Lys Ser Ser Pro Gly Leu Gly
Pro Val Glu225 230 23564373PRTArtificial
SequenceTbetaRI-TbetaRII-ED trimer T1-T2-T2 64Ala Ala Leu Leu Pro Gly Ala
Thr Ala Leu Gln Cys Phe Cys His Leu1 5 10
15Cys Thr Lys Asp Asn Phe Thr Cys Val Thr Asp Gly Leu
Cys Phe Val 20 25 30Ser Val
Thr Glu Thr Thr Asp Lys Val Ile His Asn Ser Met Cys Ile 35
40 45Ala Glu Ile Asp Leu Ile Pro Arg Asp Arg
Pro Phe Val Cys Ala Pro 50 55 60Ser
Ser Lys Thr Gly Ser Val Thr Thr Thr Tyr Cys Cys Asn Gln Asp65
70 75 80His Cys Asn Lys Ile Glu
Leu Pro Thr Thr Val Lys Ser Ser Pro Gly 85
90 95Leu Gly Pro Val Glu Ile Pro Pro His Val Gln Lys
Ser Val Asn Asn 100 105 110Asp
Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu 115
120 125Cys Lys Phe Cys Asp Val Arg Phe Ser
Thr Cys Asp Asn Gln Lys Ser 130 135
140Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu145
150 155 160Val Cys Val Ala
Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu 165
170 175Thr Val Cys His Asp Pro Lys Leu Pro Tyr
His Asp Phe Ile Leu Glu 180 185
190Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly
195 200 205Glu Thr Phe Phe Met Cys Ser
Cys Ser Ser Asp Glu Cys Asn Asp Asn 210 215
220Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro
Pro225 230 235 240His Val
Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn
245 250 255Gly Ala Val Lys Phe Pro Gln
Leu Cys Lys Phe Cys Asp Val Arg Phe 260 265
270Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser
Ile Thr 275 280 285Ser Ile Cys Glu
Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys 290
295 300Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His
Asp Pro Lys Leu305 310 315
320Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile
325 330 335Met Lys Glu Lys Lys
Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys 340
345 350Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser
Glu Glu Tyr Asn 355 360 365Thr Ser
Asn Pro Asp 37065398PRTArtificial SequenceTbetaRI-TbetaRII-ED trimer
T1-T2-T2b 65Ala Ala Leu Leu Pro Gly Ala Thr Ala Leu Gln Cys Phe Cys His
Leu1 5 10 15Cys Thr Lys
Asp Asn Phe Thr Cys Val Thr Asp Gly Leu Cys Phe Val 20
25 30Ser Val Thr Glu Thr Thr Asp Lys Val Ile
His Asn Ser Met Cys Ile 35 40
45Ala Glu Ile Asp Leu Ile Pro Arg Asp Arg Pro Phe Val Cys Ala Pro 50
55 60Ser Ser Lys Thr Gly Ser Val Thr Thr
Thr Tyr Cys Cys Asn Gln Asp65 70 75
80His Cys Asn Lys Ile Glu Leu Pro Thr Thr Val Lys Ser Ser
Pro Gly 85 90 95Leu Gly
Pro Val Glu Ile Pro Pro His Val Gln Lys Ser Val Asn Asn 100
105 110Asp Met Ile Val Thr Asp Asn Asn Gly
Ala Val Lys Phe Pro Gln Leu 115 120
125Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser
130 135 140Cys Met Ser Asn Cys Ser Ile
Thr Ser Ile Cys Glu Lys Pro Gln Glu145 150
155 160Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn
Ile Thr Leu Glu 165 170
175Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu
180 185 190Asp Ala Ala Ser Pro Lys
Cys Ile Met Lys Glu Lys Lys Lys Pro Gly 195 200
205Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn
Asp Asn 210 215 220Ile Ile Phe Ser Glu
Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro225 230
235 240His Val Gln Lys Ser Asp Val Glu Met Glu
Ala Gln Lys Asp Glu Ile 245 250
255Ile Cys Pro Ser Cys Asn Arg Thr Ala His Pro Leu Arg His Ile Asn
260 265 270Asn Asp Met Ile Val
Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln 275
280 285Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys
Asp Asn Gln Lys 290 295 300Ser Cys Met
Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln305
310 315 320Glu Val Cys Val Ala Val Trp
Arg Lys Asn Asp Glu Asn Ile Thr Leu 325
330 335Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His
Asp Phe Ile Leu 340 345 350Glu
Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro 355
360 365Gly Glu Thr Phe Phe Met Cys Ser Cys
Ser Ser Asp Glu Cys Asn Asp 370 375
380Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp385
390 39566423PRTArtificial SequenceTbetaRI-TbetaRII-ED
trimer T1-T2-T2bAA 66Ala Ala Leu Leu Pro Gly Ala Thr Ala Leu Gln Cys Phe
Cys His Leu1 5 10 15Cys
Thr Lys Asp Asn Phe Thr Cys Val Thr Asp Gly Leu Cys Phe Val 20
25 30Ser Val Thr Glu Thr Thr Asp Lys
Val Ile His Asn Ser Met Cys Ile 35 40
45Ala Glu Ile Asp Leu Ile Pro Arg Asp Arg Pro Phe Val Cys Ala Pro
50 55 60Ser Ser Lys Thr Gly Ser Val Thr
Thr Thr Tyr Cys Cys Asn Gln Asp65 70 75
80His Cys Asn Lys Ile Glu Leu Pro Thr Thr Val Lys Ser
Ser Pro Gly 85 90 95Leu
Gly Pro Val Glu Ile Pro Pro His Val Gln Lys Ser Asp Val Glu
100 105 110Met Glu Ala Gln Lys Asp Glu
Ile Ile Cys Pro Ser Cys Asn Arg Thr 115 120
125Ala His Pro Leu Arg His Ile Asn Asn Asp Met Ile Val Thr Asp
Asn 130 135 140Asn Gly Ala Val Lys Phe
Pro Gln Leu Cys Lys Phe Cys Asp Val Arg145 150
155 160Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met
Ser Asn Cys Ser Ile 165 170
175Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg
180 185 190Lys Asn Asp Glu Asn Ile
Thr Leu Glu Thr Val Cys His Asp Pro Lys 195 200
205Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro
Lys Cys 210 215 220Ile Met Lys Glu Lys
Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser225 230
235 240Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile
Ile Phe Ser Glu Glu Tyr 245 250
255Asn Thr Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys Ser Asp Val
260 265 270Glu Met Glu Ala Gln
Lys Asp Glu Ile Ile Ala Pro Ser Ala Asn Arg 275
280 285Thr Ala His Pro Leu Arg His Ile Asn Asn Asp Met
Ile Val Thr Asp 290 295 300Asn Asn Gly
Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val305
310 315 320Arg Phe Ser Thr Cys Asp Asn
Gln Lys Ser Cys Met Ser Asn Cys Ser 325
330 335Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys
Val Ala Val Trp 340 345 350Arg
Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro 355
360 365Lys Leu Pro Tyr His Asp Phe Ile Leu
Glu Asp Ala Ala Ser Pro Lys 370 375
380Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys385
390 395 400Ser Cys Ser Ser
Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu 405
410 415Tyr Asn Thr Ser Asn Pro Asp
42067373PRTArtificial SequenceTbetaRI-TbetaRII-ED trimer T2-T2-T1 67Ile
Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr1
5 10 15Asp Asn Asn Gly Ala Val Lys
Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25
30Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser
Asn Cys 35 40 45Ser Ile Thr Ser
Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55
60Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val
Cys His Asp65 70 75
80Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro
85 90 95Lys Cys Ile Met Lys Glu
Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100
105 110Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile
Ile Phe Ser Glu 115 120 125Glu Tyr
Asn Thr Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys Ser 130
135 140Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn
Gly Ala Val Lys Phe145 150 155
160Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn
165 170 175Gln Lys Ser Cys
Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys 180
185 190Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys
Asn Asp Glu Asn Ile 195 200 205Thr
Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe 210
215 220Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys
Ile Met Lys Glu Lys Lys225 230 235
240Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu
Cys 245 250 255Asn Asp Asn
Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 260
265 270Ala Ala Leu Leu Pro Gly Ala Thr Ala Leu
Gln Cys Phe Cys His Leu 275 280
285Cys Thr Lys Asp Asn Phe Thr Cys Val Thr Asp Gly Leu Cys Phe Val 290
295 300Ser Val Thr Glu Thr Thr Asp Lys
Val Ile His Asn Ser Met Cys Ile305 310
315 320Ala Glu Ile Asp Leu Ile Pro Arg Asp Arg Pro Phe
Val Cys Ala Pro 325 330
335Ser Ser Lys Thr Gly Ser Val Thr Thr Thr Tyr Cys Cys Asn Gln Asp
340 345 350His Cys Asn Lys Ile Glu
Leu Pro Thr Thr Val Lys Ser Ser Pro Gly 355 360
365Leu Gly Pro Val Glu 37068398PRTArtificial
SequenceTbetaRI-TbetaRII-ED trimer T2-T2bAA-T1 68Ile Pro Pro His Val Gln
Lys Ser Val Asn Asn Asp Met Ile Val Thr1 5
10 15Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys
Lys Phe Cys Asp 20 25 30Val
Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35
40 45Ser Ile Thr Ser Ile Cys Glu Lys Pro
Gln Glu Val Cys Val Ala Val 50 55
60Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp65
70 75 80Pro Lys Leu Pro Tyr
His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85
90 95Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly
Glu Thr Phe Phe Met 100 105
110Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu
115 120 125Glu Tyr Asn Thr Ser Asn Pro
Asp Ile Pro Pro His Val Gln Lys Ser 130 135
140Asp Val Glu Met Glu Ala Gln Lys Asp Glu Ile Ile Ala Pro Ser
Ala145 150 155 160Asn Arg
Thr Ala His Pro Leu Arg His Ile Asn Asn Asp Met Ile Val
165 170 175Thr Asp Asn Asn Gly Ala Val
Lys Phe Pro Gln Leu Cys Lys Phe Cys 180 185
190Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met
Ser Asn 195 200 205Cys Ser Ile Thr
Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala 210
215 220Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu
Thr Val Cys His225 230 235
240Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser
245 250 255Pro Lys Cys Ile Met
Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe 260
265 270Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn
Ile Ile Phe Ser 275 280 285Glu Glu
Tyr Asn Thr Ser Asn Pro Asp Ala Ala Leu Leu Pro Gly Ala 290
295 300Thr Ala Leu Gln Cys Phe Cys His Leu Cys Thr
Lys Asp Asn Phe Thr305 310 315
320Cys Val Thr Asp Gly Leu Cys Phe Val Ser Val Thr Glu Thr Thr Asp
325 330 335Lys Val Ile His
Asn Ser Met Cys Ile Ala Glu Ile Asp Leu Ile Pro 340
345 350Arg Asp Arg Pro Phe Val Cys Ala Pro Ser Ser
Lys Thr Gly Ser Val 355 360 365Thr
Thr Thr Tyr Cys Cys Asn Gln Asp His Cys Asn Lys Ile Glu Leu 370
375 380Pro Thr Thr Val Lys Ser Ser Pro Gly Leu
Gly Pro Val Glu385 390
3956985PRTArtificial SequenceBMPRIa-ED structured domain 69Thr Leu Pro
Phe Leu Lys Cys Tyr Cys Ser Gly His Cys Pro Asp Asp1 5
10 15Ala Ile Asn Asn Thr Cys Ile Thr Asn
Gly His Cys Phe Ala Ile Ile 20 25
30Glu Glu Asp Asp Gln Gly Glu Thr Thr Leu Ala Ser Gly Cys Met Lys
35 40 45Tyr Glu Gly Ser Asp Phe Gln
Cys Lys Asp Ser Pro Lys Ala Gln Leu 50 55
60Arg Arg Thr Ile Glu Cys Cys Arg Thr Asn Leu Cys Asn Gln Tyr Leu65
70 75 80Gln Pro Thr Leu
Pro 857031PRTArtificial SequenceBMPRIa-ED N-term
unstructured region and natural linker 70Gln Asn Leu Asp Ser Met Leu
His Gly Thr Gly Met Lys Ser Asp Ser1 5 10
15Asp Gln Lys Lys Ser Glu Asn Gly Val Thr Leu Ala Pro
Glu Asp 20 25
307113PRTArtificial SequenceBMPRIa-ED C-term unstructured region and
natural linker 71Pro Val Val Ile Gly Pro Phe Phe Asp Gly Ser Ile Arg1
5 107244PRTArtificial SequenceBMPRIa-ED
natural linker 72Pro Val Val Ile Gly Pro Phe Phe Asp Gly Ser Ile Arg Gln
Asn Leu1 5 10 15Asp Ser
Met Leu His Gly Thr Gly Met Lys Ser Asp Ser Asp Gln Lys 20
25 30Lys Ser Glu Asn Gly Val Thr Leu Ala
Pro Glu Asp 35 4073129PRTArtificial
SequenceBMPRIa-ED monomer 73Gln Asn Leu Asp Ser Met Leu His Gly Thr Gly
Met Lys Ser Asp Ser1 5 10
15Asp Gln Lys Lys Ser Glu Asn Gly Val Thr Leu Ala Pro Glu Asp Thr
20 25 30Leu Pro Phe Leu Lys Cys Tyr
Cys Ser Gly His Cys Pro Asp Asp Ala 35 40
45Ile Asn Asn Thr Cys Ile Thr Asn Gly His Cys Phe Ala Ile Ile
Glu 50 55 60Glu Asp Asp Gln Gly Glu
Thr Thr Leu Ala Ser Gly Cys Met Lys Tyr65 70
75 80Glu Gly Ser Asp Phe Gln Cys Lys Asp Ser Pro
Lys Ala Gln Leu Arg 85 90
95Arg Thr Ile Glu Cys Cys Arg Thr Asn Leu Cys Asn Gln Tyr Leu Gln
100 105 110Pro Thr Leu Pro Pro Val
Val Ile Gly Pro Phe Phe Asp Gly Ser Ile 115 120
125Arg74258PRTArtificial SequenceBMPRIa-ED dimer 74Gln Asn
Leu Asp Ser Met Leu His Gly Thr Gly Met Lys Ser Asp Ser1 5
10 15Asp Gln Lys Lys Ser Glu Asn Gly
Val Thr Leu Ala Pro Glu Asp Thr 20 25
30Leu Pro Phe Leu Lys Cys Tyr Cys Ser Gly His Cys Pro Asp Asp
Ala 35 40 45Ile Asn Asn Thr Cys
Ile Thr Asn Gly His Cys Phe Ala Ile Ile Glu 50 55
60Glu Asp Asp Gln Gly Glu Thr Thr Leu Ala Ser Gly Cys Met
Lys Tyr65 70 75 80Glu
Gly Ser Asp Phe Gln Cys Lys Asp Ser Pro Lys Ala Gln Leu Arg
85 90 95Arg Thr Ile Glu Cys Cys Arg
Thr Asn Leu Cys Asn Gln Tyr Leu Gln 100 105
110Pro Thr Leu Pro Pro Val Val Ile Gly Pro Phe Phe Asp Gly
Ser Ile 115 120 125Arg Gln Asn Leu
Asp Ser Met Leu His Gly Thr Gly Met Lys Ser Asp 130
135 140Ser Asp Gln Lys Lys Ser Glu Asn Gly Val Thr Leu
Ala Pro Glu Asp145 150 155
160Thr Leu Pro Phe Leu Lys Cys Tyr Cys Ser Gly His Cys Pro Asp Asp
165 170 175Ala Ile Asn Asn Thr
Cys Ile Thr Asn Gly His Cys Phe Ala Ile Ile 180
185 190Glu Glu Asp Asp Gln Gly Glu Thr Thr Leu Ala Ser
Gly Cys Met Lys 195 200 205Tyr Glu
Gly Ser Asp Phe Gln Cys Lys Asp Ser Pro Lys Ala Gln Leu 210
215 220Arg Arg Thr Ile Glu Cys Cys Arg Thr Asn Leu
Cys Asn Gln Tyr Leu225 230 235
240Gln Pro Thr Leu Pro Pro Val Val Ile Gly Pro Phe Phe Asp Gly Ser
245 250 255Ile
Arg7592PRTArtificial SequenceActRIIa-ED structured domain 75Thr Gln Glu
Cys Leu Phe Phe Asn Ala Asn Trp Glu Lys Asp Arg Thr1 5
10 15Asn Gln Thr Gly Val Glu Pro Cys Tyr
Gly Asp Lys Asp Lys Arg Arg 20 25
30His Cys Phe Ala Thr Trp Lys Asn Ile Ser Gly Ser Ile Glu Ile Val
35 40 45Lys Gln Gly Cys Trp Leu Asp
Asp Ile Asn Cys Tyr Asp Arg Thr Asp 50 55
60Cys Val Glu Lys Lys Asp Ser Pro Glu Val Tyr Phe Cys Cys Cys Glu65
70 75 80Gly Asn Met Cys
Asn Glu Lys Phe Ser Tyr Phe Pro 85
90767PRTArtificial SequenceActRIIa-ED N-term unstructured region and
natural linker 76Ala Ile Leu Gly Arg Ser Glu1
57721PRTArtificial SequenceActRIIa-ED C-term unstructured region and
natural linker 77Glu Met Glu Val Thr Gln Pro Thr Ser Asn Pro Val Thr Pro
Lys Pro1 5 10 15Pro Tyr
Tyr Asn Ile 207828PRTArtificial SequenceActRIIa-ED natural
linker 78Glu Met Glu Val Thr Gln Pro Thr Ser Asn Pro Val Thr Pro Lys Pro1
5 10 15Pro Tyr Tyr Asn
Ile Ala Ile Leu Gly Arg Ser Glu 20
2579120PRTArtificial SequenceActRIIa-ED monomer 79Ala Ile Leu Gly Arg Ser
Glu Thr Gln Glu Cys Leu Phe Phe Asn Ala1 5
10 15Asn Trp Glu Lys Asp Arg Thr Asn Gln Thr Gly Val
Glu Pro Cys Tyr 20 25 30Gly
Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp Lys Asn Ile 35
40 45Ser Gly Ser Ile Glu Ile Val Lys Gln
Gly Cys Trp Leu Asp Asp Ile 50 55
60Asn Cys Tyr Asp Arg Thr Asp Cys Val Glu Lys Lys Asp Ser Pro Glu65
70 75 80Val Tyr Phe Cys Cys
Cys Glu Gly Asn Met Cys Asn Glu Lys Phe Ser 85
90 95Tyr Phe Pro Glu Met Glu Val Thr Gln Pro Thr
Ser Asn Pro Val Thr 100 105
110Pro Lys Pro Pro Tyr Tyr Asn Ile 115
12080240PRTArtificial SequenceActRIIa-ED dimer 80Ala Ile Leu Gly Arg Ser
Glu Thr Gln Glu Cys Leu Phe Phe Asn Ala1 5
10 15Asn Trp Glu Lys Asp Arg Thr Asn Gln Thr Gly Val
Glu Pro Cys Tyr 20 25 30Gly
Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp Lys Asn Ile 35
40 45Ser Gly Ser Ile Glu Ile Val Lys Gln
Gly Cys Trp Leu Asp Asp Ile 50 55
60Asn Cys Tyr Asp Arg Thr Asp Cys Val Glu Lys Lys Asp Ser Pro Glu65
70 75 80Val Tyr Phe Cys Cys
Cys Glu Gly Asn Met Cys Asn Glu Lys Phe Ser 85
90 95Tyr Phe Pro Glu Met Glu Val Thr Gln Pro Thr
Ser Asn Pro Val Thr 100 105
110Pro Lys Pro Pro Tyr Tyr Asn Ile Ala Ile Leu Gly Arg Ser Glu Thr
115 120 125Gln Glu Cys Leu Phe Phe Asn
Ala Asn Trp Glu Lys Asp Arg Thr Asn 130 135
140Gln Thr Gly Val Glu Pro Cys Tyr Gly Asp Lys Asp Lys Arg Arg
His145 150 155 160Cys Phe
Ala Thr Trp Lys Asn Ile Ser Gly Ser Ile Glu Ile Val Lys
165 170 175Gln Gly Cys Trp Leu Asp Asp
Ile Asn Cys Tyr Asp Arg Thr Asp Cys 180 185
190Val Glu Lys Lys Asp Ser Pro Glu Val Tyr Phe Cys Cys Cys
Glu Gly 195 200 205Asn Met Cys Asn
Glu Lys Phe Ser Tyr Phe Pro Glu Met Glu Val Thr 210
215 220Gln Pro Thr Ser Asn Pro Val Thr Pro Lys Pro Pro
Tyr Tyr Asn Ile225 230 235
2408191PRTArtificial SequenceActRIIb-ED structured domain 81Arg Glu Cys
Ile Tyr Tyr Asn Ala Asn Trp Glu Leu Glu Arg Thr Asn1 5
10 15Gln Ser Gly Leu Glu Arg Cys Glu Gly
Glu Gln Asp Lys Arg Leu His 20 25
30Cys Tyr Ala Ser Trp Arg Asn Ser Ser Gly Thr Ile Glu Leu Val Lys
35 40 45Lys Gly Cys Trp Leu Asp Asp
Phe Asn Cys Tyr Asp Arg Gln Glu Cys 50 55
60Val Ala Thr Glu Glu Asn Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly65
70 75 80Asn Phe Cys Asn
Glu Arg Phe Thr His Leu Pro 85
90828PRTArtificial SequenceActRIIb-ED N-term unstructured region and
natural linker 82Ser Gly Arg Gly Glu Ala Glu Thr1
58317PRTArtificial SequenceActRIIb-ED C-term unstructured region and
natural linker 83Glu Ala Gly Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro Thr
Ala Pro1 5 10
15Thr8425PRTArtificial SequenceActRIIb-ED natural linker 84Glu Ala Gly
Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro Thr Ala Pro1 5
10 15Thr Ser Gly Arg Gly Glu Ala Glu Thr
20 2585116PRTArtificial SequenceActRIIb-ED
monomer 85Ser Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr Asn
Ala1 5 10 15Asn Trp Glu
Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu 20
25 30Gly Glu Gln Asp Lys Arg Leu His Cys Tyr
Ala Ser Trp Arg Asn Ser 35 40
45Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp Asp Phe 50
55 60Asn Cys Tyr Asp Arg Gln Glu Cys Val
Ala Thr Glu Glu Asn Pro Gln65 70 75
80Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg
Phe Thr 85 90 95His Leu
Pro Glu Ala Gly Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro 100
105 110Thr Ala Pro Thr
11586232PRTArtificial SequenceActRIIb-ED dimer 86Ser Gly Arg Gly Glu Ala
Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala1 5
10 15Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu
Glu Arg Cys Glu 20 25 30Gly
Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg Asn Ser 35
40 45Ser Gly Thr Ile Glu Leu Val Lys Lys
Gly Cys Trp Leu Asp Asp Phe 50 55
60Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln65
70 75 80Val Tyr Phe Cys Cys
Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr 85
90 95His Leu Pro Glu Ala Gly Gly Pro Glu Val Thr
Tyr Glu Pro Pro Pro 100 105
110Thr Ala Pro Thr Ser Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile
115 120 125Tyr Tyr Asn Ala Asn Trp Glu
Leu Glu Arg Thr Asn Gln Ser Gly Leu 130 135
140Glu Arg Cys Glu Gly Glu Gln Asp Lys Arg Leu His Cys Tyr Ala
Ser145 150 155 160Trp Arg
Asn Ser Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp
165 170 175Leu Asp Asp Phe Asn Cys Tyr
Asp Arg Gln Glu Cys Val Ala Thr Glu 180 185
190Glu Asn Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe
Cys Asn 195 200 205Glu Arg Phe Thr
His Leu Pro Glu Ala Gly Gly Pro Glu Val Thr Tyr 210
215 220Glu Pro Pro Pro Thr Ala Pro Thr225
2308752PRTArtificial SequenceActRIIa-BMPRIa-ED natural linker 87Glu Met
Glu Val Thr Gln Pro Thr Ser Asn Pro Val Thr Pro Lys Pro1 5
10 15Pro Tyr Tyr Asn Ile Gln Asn Leu
Asp Ser Met Leu His Gly Thr Gly 20 25
30Met Lys Ser Asp Ser Asp Gln Lys Lys Ser Glu Asn Gly Val Thr
Leu 35 40 45Ala Pro Glu Asp
5088249PRTArtificial SequenceActRIIa-BMPRIa-ED dimer 88Ala Ile Leu Gly
Arg Ser Glu Thr Gln Glu Cys Leu Phe Phe Asn Ala1 5
10 15Asn Trp Glu Lys Asp Arg Thr Asn Gln Thr
Gly Val Glu Pro Cys Tyr 20 25
30Gly Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp Lys Asn Ile
35 40 45Ser Gly Ser Ile Glu Ile Val Lys
Gln Gly Cys Trp Leu Asp Asp Ile 50 55
60Asn Cys Tyr Asp Arg Thr Asp Cys Val Glu Lys Lys Asp Ser Pro Glu65
70 75 80Val Tyr Phe Cys Cys
Cys Glu Gly Asn Met Cys Asn Glu Lys Phe Ser 85
90 95Tyr Phe Pro Glu Met Glu Val Thr Gln Pro Thr
Ser Asn Pro Val Thr 100 105
110Pro Lys Pro Pro Tyr Tyr Asn Ile Gln Asn Leu Asp Ser Met Leu His
115 120 125Gly Thr Gly Met Lys Ser Asp
Ser Asp Gln Lys Lys Ser Glu Asn Gly 130 135
140Val Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe Leu Lys Cys Tyr
Cys145 150 155 160Ser Gly
His Cys Pro Asp Asp Ala Ile Asn Asn Thr Cys Ile Thr Asn
165 170 175Gly His Cys Phe Ala Ile Ile
Glu Glu Asp Asp Gln Gly Glu Thr Thr 180 185
190Leu Ala Ser Gly Cys Met Lys Tyr Glu Gly Ser Asp Phe Gln
Cys Lys 195 200 205Asp Ser Pro Lys
Ala Gln Leu Arg Arg Thr Ile Glu Cys Cys Arg Thr 210
215 220Asn Leu Cys Asn Gln Tyr Leu Gln Pro Thr Leu Pro
Pro Val Val Ile225 230 235
240Gly Pro Phe Phe Asp Gly Ser Ile Arg
2458919PRTArtificial SequenceSignal peptide 89Met Asp Trp Thr Trp Arg Ile
Leu Phe Leu Val Ala Ala Ala Thr Gly1 5 10
15Thr His Ala9020PRTArtificial SequenceSignal peptide
90Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser1
5 10 15Gly Ala Tyr Gly
2091352PRTArtificial SequencehIgG1FcdeltaK-deltaC-T2m fusion 91Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5
10 15Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val 20 25
30Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr 35 40 45Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55
60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His65 70 75 80Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105
110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu 115 120 125Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130
135 140Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn145 150 155
160Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180
185 190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln 195 200 205Lys Ser
Leu Ser Leu Ser Pro Gly Ile Pro Pro His Val Gln Lys Ser 210
215 220Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn
Gly Ala Val Lys Phe225 230 235
240Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn
245 250 255Gln Lys Ser Cys
Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys 260
265 270Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys
Asn Asp Glu Asn Ile 275 280 285Thr
Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe 290
295 300Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys
Ile Met Lys Glu Lys Lys305 310 315
320Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu
Cys 325 330 335Asn Asp Asn
Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 340
345 35092356PRTArtificial
SequencehIgG1FcdeltaK-C-T2m fusion 92Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu1 5 10
15Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu 20 25 30Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 35
40 45Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys 50 55 60Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu65 70
75 80Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys 85 90
95Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys 100 105 110Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 115
120 125Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys 130 135 140Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln145
150 155 160Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly 165
170 175Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln 180 185 190Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 195
200 205His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Ile Pro Pro His 210 215
220Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly225
230 235 240Ala Val Lys Phe
Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser 245
250 255Thr Cys Asp Asn Gln Lys Ser Cys Met Ser
Asn Cys Ser Ile Thr Ser 260 265
270Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn
275 280 285Asp Glu Asn Ile Thr Leu Glu
Thr Val Cys His Asp Pro Lys Leu Pro 290 295
300Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile
Met305 310 315 320Lys Glu
Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser
325 330 335Ser Asp Glu Cys Asn Asp Asn
Ile Ile Phe Ser Glu Glu Tyr Asn Thr 340 345
350Ser Asn Pro Asp 35593362PRTArtificial
SequencehIgG1FcdeltaK-CC-T2m fusion 93Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly1 5 10
15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 20 25 30Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70
75 80Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90
95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile 100 105 110Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115
120 125Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145
150 155 160Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser 210 215
220Pro Gly Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile225
230 235 240Val Thr Asp Asn
Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe 245
250 255Cys Asp Val Arg Phe Ser Thr Cys Asp Asn
Gln Lys Ser Cys Met Ser 260 265
270Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val
275 280 285Ala Val Trp Arg Lys Asn Asp
Glu Asn Ile Thr Leu Glu Thr Val Cys 290 295
300His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala
Ala305 310 315 320Ser Pro
Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe
325 330 335Phe Met Cys Ser Cys Ser Ser
Asp Glu Cys Asn Asp Asn Ile Ile Phe 340 345
350Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 355
36094351PRTArtificial SequencehIgG2FcdeltaK-deltaC-T2m fusion
94Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro1
5 10 15Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val 20 25
30Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn
Trp Tyr Val 35 40 45Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 50
55 60Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr
Val Val His Gln65 70 75
80Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
85 90 95Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro 100
105 110Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr 115 120 125Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 130
135 140Asp Ile Ser Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr145 150 155
160Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
165 170 175Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 180
185 190Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys 195 200 205Ser
Leu Ser Leu Ser Pro Gly Ile Pro Pro His Val Gln Lys Ser Val 210
215 220Asn Asn Asp Met Ile Val Thr Asp Asn Asn
Gly Ala Val Lys Phe Pro225 230 235
240Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn
Gln 245 250 255Lys Ser Cys
Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro 260
265 270Gln Glu Val Cys Val Ala Val Trp Arg Lys
Asn Asp Glu Asn Ile Thr 275 280
285Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile 290
295 300Leu Glu Asp Ala Ala Ser Pro Lys
Cys Ile Met Lys Glu Lys Lys Lys305 310
315 320Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser
Asp Glu Cys Asn 325 330
335Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp
340 345 35095355PRTArtificial
SequencehIgG2FcdeltaK-C-T2m fusion 95Pro Pro Cys Pro Ala Pro Pro Val Ala
Gly Pro Ser Val Phe Leu Phe1 5 10
15Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val 20 25 30Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe 35
40 45Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro 50 55 60Arg Glu Glu
Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr65 70
75 80Val Val His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val 85 90
95Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Thr 100 105 110Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 115
120 125Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly 130 135 140Phe Tyr
Pro Ser Asp Ile Ser Val Glu Trp Glu Ser Asn Gly Gln Pro145
150 155 160Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Met Leu Asp Ser Asp Gly Ser 165
170 175Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln 180 185 190Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 195
200 205Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Ile Pro Pro His Val 210 215
220Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala225
230 235 240Val Lys Phe Pro
Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr 245
250 255Cys Asp Asn Gln Lys Ser Cys Met Ser Asn
Cys Ser Ile Thr Ser Ile 260 265
270Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp
275 280 285Glu Asn Ile Thr Leu Glu Thr
Val Cys His Asp Pro Lys Leu Pro Tyr 290 295
300His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met
Lys305 310 315 320Glu Lys
Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser
325 330 335Asp Glu Cys Asn Asp Asn Ile
Ile Phe Ser Glu Glu Tyr Asn Thr Ser 340 345
350Asn Pro Asp 35596351PRTArtificial
SequencehIgG2FcdeltaK-CC-T2m fusion 96Ala Pro Pro Val Ala Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro1 5 10
15Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val 20 25 30Val Asp Val Ser
His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 35
40 45Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln 50 55 60Phe Asn Ser
Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln65 70
75 80Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly 85 90
95Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
Gln Pro 100 105 110Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 115
120 125Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser 130 135 140Asp Ile
Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr145
150 155 160Lys Thr Thr Pro Pro Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr 165
170 175Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe 180 185 190Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 195
200 205Ser Leu Ser Leu Ser Pro Gly Ile Pro
Pro His Val Gln Lys Ser Val 210 215
220Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro225
230 235 240Gln Leu Cys Lys
Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln 245
250 255Lys Ser Cys Met Ser Asn Cys Ser Ile Thr
Ser Ile Cys Glu Lys Pro 260 265
270Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr
275 280 285Leu Glu Thr Val Cys His Asp
Pro Lys Leu Pro Tyr His Asp Phe Ile 290 295
300Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys
Lys305 310 315 320Pro Gly
Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn
325 330 335Asp Asn Ile Ile Phe Ser Glu
Glu Tyr Asn Thr Ser Asn Pro Asp 340 345
35097383PRTArtificial SequencehIgG2Fc-CCCC-T2 fusion, also
termed Fc-T2m 97Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala
Thr Gly1 5 10 15Thr His
Ala Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala 20
25 30Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys 35 40
45Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50
55 60Asp Val Ser His Glu Asp Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp65 70 75
80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe 85 90 95Asn Ser
Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp 100
105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu 115 120
125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg
130 135 140Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys145 150
155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp 165 170
175Ile Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
180 185 190Thr Thr Pro Pro Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200
205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser 210 215 220Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser225 230
235 240Leu Ser Leu Ser Pro Gly Lys Ile Pro Pro
His Val Gln Lys Ser Val 245 250
255Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro
260 265 270Gln Leu Cys Lys Phe
Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln 275
280 285Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile
Cys Glu Lys Pro 290 295 300Gln Glu Val
Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr305
310 315 320Leu Glu Thr Val Cys His Asp
Pro Lys Leu Pro Tyr His Asp Phe Ile 325
330 335Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys
Glu Lys Lys Lys 340 345 350Pro
Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn 355
360 365Asp Asn Ile Ile Phe Ser Glu Glu Tyr
Asn Thr Ser Asn Pro Asp 370 375
38098364PRTArtificial SequencehIgG4Fc K-CC-228P-T2m fusion 98Glu Ser Lys
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe1 5
10 15Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr 20 25
30Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
35 40 45Ser Gln Glu Asp Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55
60Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser65
70 75 80Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85
90 95Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro Ser 100 105
110Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125Gln Val Tyr Thr Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135
140Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala145 150 155 160Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185
190Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
Cys Ser 195 200 205Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210
215 220Leu Ser Leu Gly Ile Pro Pro His Val Gln Lys Ser
Val Asn Asn Asp225 230 235
240Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys
245 250 255Lys Phe Cys Asp Val
Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys 260
265 270Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys
Pro Gln Glu Val 275 280 285Cys Val
Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr 290
295 300Val Cys His Asp Pro Lys Leu Pro Tyr His Asp
Phe Ile Leu Glu Asp305 310 315
320Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu
325 330 335Thr Phe Phe Met
Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile 340
345 350Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro
Asp 355 36099364PRTArtificial
SequencehIgG4FcdeltaK-CC-228P-409K-T2m fusion 99Glu Ser Lys Tyr Gly Pro
Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe1 5
10 15Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr 20 25 30Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35
40 45Ser Gln Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr Val Asp Gly Val 50 55
60Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser65
70 75 80Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85
90 95Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro Ser 100 105
110Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125Gln Val Tyr Thr Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135
140Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala145 150 155 160Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu 180 185
190Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
Cys Ser 195 200 205Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210
215 220Leu Ser Leu Gly Ile Pro Pro His Val Gln Lys Ser
Val Asn Asn Asp225 230 235
240Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys
245 250 255Lys Phe Cys Asp Val
Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys 260
265 270Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys
Pro Gln Glu Val 275 280 285Cys Val
Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr 290
295 300Val Cys His Asp Pro Lys Leu Pro Tyr His Asp
Phe Ile Leu Glu Asp305 310 315
320Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu
325 330 335Thr Phe Phe Met
Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile 340
345 350Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro
Asp 355 360100492PRTArtificial
SequencehIgG1FcdeltaK-C-T22d35 fusion 100Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu1 5 10
15Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu 20 25 30Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 35
40 45Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys 50 55 60Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu65
70 75 80Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys 85 90
95Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys 100 105 110Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 115
120 125Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys 130 135 140Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln145
150 155 160Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 165
170 175Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln 180 185 190Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 195
200 205His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Ile Pro Pro His 210 215
220Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly225
230 235 240Ala Val Lys Phe
Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser 245
250 255Thr Cys Asp Asn Gln Lys Ser Cys Met Ser
Asn Cys Ser Ile Thr Ser 260 265
270Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn
275 280 285Asp Glu Asn Ile Thr Leu Glu
Thr Val Cys His Asp Pro Lys Leu Pro 290 295
300Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile
Met305 310 315 320Lys Glu
Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser
325 330 335Ser Asp Glu Cys Asn Asp Asn
Ile Ile Phe Ser Glu Glu Tyr Asn Thr 340 345
350Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys Ser Val Asn
Asn Asp 355 360 365Met Ile Val Thr
Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys 370
375 380Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn
Gln Lys Ser Cys385 390 395
400Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val
405 410 415Cys Val Ala Val Trp
Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr 420
425 430Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe
Ile Leu Glu Asp 435 440 445Ala Ala
Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu 450
455 460Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu
Cys Asn Asp Asn Ile465 470 475
480Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp
485 490101498PRTArtificial
SequencehIgG1FcdeltaK-CC-T22d35 fusion 101Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly1 5 10
15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 50 55 60His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65
70 75 80Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly 85 90
95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 100 105 110Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115
120 125Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser 130 135 140Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145
150 155 160Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser 210 215
220Pro Gly Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile225
230 235 240Val Thr Asp Asn
Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe 245
250 255Cys Asp Val Arg Phe Ser Thr Cys Asp Asn
Gln Lys Ser Cys Met Ser 260 265
270Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val
275 280 285Ala Val Trp Arg Lys Asn Asp
Glu Asn Ile Thr Leu Glu Thr Val Cys 290 295
300His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala
Ala305 310 315 320Ser Pro
Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe
325 330 335Phe Met Cys Ser Cys Ser Ser
Asp Glu Cys Asn Asp Asn Ile Ile Phe 340 345
350Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro His
Val Gln 355 360 365Lys Ser Val Asn
Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val 370
375 380Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg
Phe Ser Thr Cys385 390 395
400Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys
405 410 415Glu Lys Pro Gln Glu
Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu 420
425 430Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys
Leu Pro Tyr His 435 440 445Asp Phe
Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu 450
455 460Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys
Ser Cys Ser Ser Asp465 470 475
480Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn
485 490 495Pro
Asp102494PRTArtificial SequencehIgG2FcdeltaK-CC-T22d35 fusion 102Val Glu
Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val1 5
10 15Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr 20 25
30Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu 35 40 45Val Gln Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 50 55
60Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val
Val Ser65 70 75 80Val
Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
85 90 95Cys Lys Val Ser Asn Lys Gly
Leu Pro Ala Pro Ile Glu Lys Thr Ile 100 105
110Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 115 120 125Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 130
135 140Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu
Trp Glu Ser Asn145 150 155
160Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser
165 170 175Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 180
185 190Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu 195 200 205His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ile Pro 210
215 220Pro His Val Gln Lys Ser Val Asn Asn Asp Met
Ile Val Thr Asp Asn225 230 235
240Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg
245 250 255Phe Ser Thr Cys
Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile 260
265 270Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys
Val Ala Val Trp Arg 275 280 285Lys
Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys 290
295 300Leu Pro Tyr His Asp Phe Ile Leu Glu Asp
Ala Ala Ser Pro Lys Cys305 310 315
320Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys
Ser 325 330 335Cys Ser Ser
Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr 340
345 350Asn Thr Ser Asn Pro Asp Ile Pro Pro His
Val Gln Lys Ser Val Asn 355 360
365Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln 370
375 380Leu Cys Lys Phe Cys Asp Val Arg
Phe Ser Thr Cys Asp Asn Gln Lys385 390
395 400Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys
Glu Lys Pro Gln 405 410
415Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu
420 425 430Glu Thr Val Cys His Asp
Pro Lys Leu Pro Tyr His Asp Phe Ile Leu 435 440
445Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys
Lys Pro 450 455 460Gly Glu Thr Phe Phe
Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp465 470
475 480Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr
Ser Asn Pro Asp 485 490103500PRTArtificial
SequencehIgG4FcdeltaK-CC-228P-T22d35 fusion 103Glu Ser Lys Tyr Gly Pro
Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe1 5
10 15Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr 20 25 30Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35
40 45Ser Gln Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr Val Asp Gly Val 50 55
60Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser65
70 75 80Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85
90 95Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro Ser 100 105
110Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125Gln Val Tyr Thr Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135
140Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala145 150 155 160Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185
190Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
Cys Ser 195 200 205Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210
215 220Leu Ser Leu Gly Ile Pro Pro His Val Gln Lys Ser
Val Asn Asn Asp225 230 235
240Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys
245 250 255Lys Phe Cys Asp Val
Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys 260
265 270Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys
Pro Gln Glu Val 275 280 285Cys Val
Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr 290
295 300Val Cys His Asp Pro Lys Leu Pro Tyr His Asp
Phe Ile Leu Glu Asp305 310 315
320Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu
325 330 335Thr Phe Phe Met
Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile 340
345 350Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro
Asp Ile Pro Pro His 355 360 365Val
Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly 370
375 380Ala Val Lys Phe Pro Gln Leu Cys Lys Phe
Cys Asp Val Arg Phe Ser385 390 395
400Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr
Ser 405 410 415Ile Cys Glu
Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn 420
425 430Asp Glu Asn Ile Thr Leu Glu Thr Val Cys
His Asp Pro Lys Leu Pro 435 440
445Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met 450
455 460Lys Glu Lys Lys Lys Pro Gly Glu
Thr Phe Phe Met Cys Ser Cys Ser465 470
475 480Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu
Glu Tyr Asn Thr 485 490
495Ser Asn Pro Asp 500104500PRTArtificial
SequencehIgG4FcdeltaK-CC-228P-409K-T22d35 fusion 104Glu Ser Lys Tyr Gly
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe1 5
10 15Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr 20 25
30Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
35 40 45Ser Gln Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val 50 55
60Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser65
70 75 80Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85
90 95Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro Ser 100 105
110Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125Gln Val Tyr Thr Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135
140Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala145 150 155 160Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu 180 185
190Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
Cys Ser 195 200 205Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210
215 220Leu Ser Leu Gly Ile Pro Pro His Val Gln Lys Ser
Val Asn Asn Asp225 230 235
240Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys
245 250 255Lys Phe Cys Asp Val
Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys 260
265 270Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys
Pro Gln Glu Val 275 280 285Cys Val
Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr 290
295 300Val Cys His Asp Pro Lys Leu Pro Tyr His Asp
Phe Ile Leu Glu Asp305 310 315
320Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu
325 330 335Thr Phe Phe Met
Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile 340
345 350Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro
Asp Ile Pro Pro His 355 360 365Val
Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly 370
375 380Ala Val Lys Phe Pro Gln Leu Cys Lys Phe
Cys Asp Val Arg Phe Ser385 390 395
400Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr
Ser 405 410 415Ile Cys Glu
Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn 420
425 430Asp Glu Asn Ile Thr Leu Glu Thr Val Cys
His Asp Pro Lys Leu Pro 435 440
445Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met 450
455 460Lys Glu Lys Lys Lys Pro Gly Glu
Thr Phe Phe Met Cys Ser Cys Ser465 470
475 480Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu
Glu Tyr Asn Thr 485 490
495Ser Asn Pro Asp 500105519PRTArtificial
SequencehIgG2Fc-CCCC-T22d35 fusion, also termed Fc- T22d35 105Met
Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly1
5 10 15Thr His Ala Glu Arg Lys Cys
Cys Val Glu Cys Pro Pro Cys Pro Ala 20 25
30Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 35 40 45Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55
60Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr Val Asp65 70 75
80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
85 90 95Asn Ser Thr Phe Arg Val
Val Ser Val Leu Thr Val Val His Gln Asp 100
105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu 115 120 125Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg 130
135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys145 150 155
160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
165 170 175Ile Ser Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180
185 190Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser 195 200 205Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210
215 220Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser225 230 235
240Leu Ser Leu Ser Pro Gly Lys Ile Pro Pro His Val Gln Lys Ser
Val 245 250 255Asn Asn Asp
Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro 260
265 270Gln Leu Cys Lys Phe Cys Asp Val Arg Phe
Ser Thr Cys Asp Asn Gln 275 280
285Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro 290
295 300Gln Glu Val Cys Val Ala Val Trp
Arg Lys Asn Asp Glu Asn Ile Thr305 310
315 320Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr
His Asp Phe Ile 325 330
335Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys
340 345 350Pro Gly Glu Thr Phe Phe
Met Cys Ser Cys Ser Ser Asp Glu Cys Asn 355 360
365Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro
Asp Ile 370 375 380Pro Pro His Val Gln
Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp385 390
395 400Asn Asn Gly Ala Val Lys Phe Pro Gln Leu
Cys Lys Phe Cys Asp Val 405 410
415Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser
420 425 430Ile Thr Ser Ile Cys
Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp 435
440 445Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val
Cys His Asp Pro 450 455 460Lys Leu Pro
Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys465
470 475 480Cys Ile Met Lys Glu Lys Lys
Lys Pro Gly Glu Thr Phe Phe Met Cys 485
490 495Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile
Phe Ser Glu Glu 500 505 510Tyr
Asn Thr Ser Asn Pro Asp 515106517PRTArtificial
SequencehIgG1FcdeltaK-C-T2-T2bAA fusion 106Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu1 5 10
15Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu 20 25 30Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 35
40 45Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys 50 55 60Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu65
70 75 80Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 85
90 95Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys 100 105 110Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 115
120 125Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys 130 135
140Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln145
150 155 160Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 165
170 175Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln 180 185
190Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
195 200 205His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Ile Pro Pro His 210 215
220Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn
Gly225 230 235 240Ala Val
Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser
245 250 255Thr Cys Asp Asn Gln Lys Ser
Cys Met Ser Asn Cys Ser Ile Thr Ser 260 265
270Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg
Lys Asn 275 280 285Asp Glu Asn Ile
Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro 290
295 300Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro
Lys Cys Ile Met305 310 315
320Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser
325 330 335Ser Asp Glu Cys Asn
Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr 340
345 350Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys Ser
Asp Val Glu Met 355 360 365Glu Ala
Gln Lys Asp Glu Ile Ile Ala Pro Ser Ala Asn Arg Thr Ala 370
375 380His Pro Leu Arg His Ile Asn Asn Asp Met Ile
Val Thr Asp Asn Asn385 390 395
400Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe
405 410 415Ser Thr Cys Asp
Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr 420
425 430Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val
Ala Val Trp Arg Lys 435 440 445Asn
Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu 450
455 460Pro Tyr His Asp Phe Ile Leu Glu Asp Ala
Ala Ser Pro Lys Cys Ile465 470 475
480Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser
Cys 485 490 495Ser Ser Asp
Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn 500
505 510Thr Ser Asn Pro Asp
515107523PRTArtificial SequencehIgG1FcdeltaK-CC-T2-T2bAA fusion 107Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1
5 10 15Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25
30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr65 70 75
80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100
105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val 115 120 125Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu145 150 155
160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180
185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met 195 200 205His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220Pro Gly Ile Pro Pro His Val Gln Lys Ser
Val Asn Asn Asp Met Ile225 230 235
240Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys
Phe 245 250 255Cys Asp Val
Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser 260
265 270Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys
Pro Gln Glu Val Cys Val 275 280
285Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys 290
295 300His Asp Pro Lys Leu Pro Tyr His
Asp Phe Ile Leu Glu Asp Ala Ala305 310
315 320Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro
Gly Glu Thr Phe 325 330
335Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe
340 345 350Ser Glu Glu Tyr Asn Thr
Ser Asn Pro Asp Ile Pro Pro His Val Gln 355 360
365Lys Ser Asp Val Glu Met Glu Ala Gln Lys Asp Glu Ile Ile
Ala Pro 370 375 380Ser Ala Asn Arg Thr
Ala His Pro Leu Arg His Ile Asn Asn Asp Met385 390
395 400Ile Val Thr Asp Asn Asn Gly Ala Val Lys
Phe Pro Gln Leu Cys Lys 405 410
415Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met
420 425 430Ser Asn Cys Ser Ile
Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys 435
440 445Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr
Leu Glu Thr Val 450 455 460Cys His Asp
Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala465
470 475 480Ala Ser Pro Lys Cys Ile Met
Lys Glu Lys Lys Lys Pro Gly Glu Thr 485
490 495Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn
Asp Asn Ile Ile 500 505 510Phe
Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 515
520108519PRTArtificial SequencehIgG2FcdeltaK-CC-T2-T2bAA fusion 108Val
Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val1
5 10 15Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr 20 25
30Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu 35 40 45Val Gln Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 50 55
60Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg
Val Val Ser65 70 75
80Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
85 90 95Cys Lys Val Ser Asn Lys
Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile 100
105 110Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro 115 120 125Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 130
135 140Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val
Glu Trp Glu Ser Asn145 150 155
160Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser
165 170 175Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 180
185 190Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu 195 200 205His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ile Pro 210
215 220Pro His Val Gln Lys Ser Val Asn Asn Asp
Met Ile Val Thr Asp Asn225 230 235
240Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val
Arg 245 250 255Phe Ser Thr
Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile 260
265 270Thr Ser Ile Cys Glu Lys Pro Gln Glu Val
Cys Val Ala Val Trp Arg 275 280
285Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys 290
295 300Leu Pro Tyr His Asp Phe Ile Leu
Glu Asp Ala Ala Ser Pro Lys Cys305 310
315 320Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe
Phe Met Cys Ser 325 330
335Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr
340 345 350Asn Thr Ser Asn Pro Asp
Ile Pro Pro His Val Gln Lys Ser Asp Val 355 360
365Glu Met Glu Ala Gln Lys Asp Glu Ile Ile Ala Pro Ser Ala
Asn Arg 370 375 380Thr Ala His Pro Leu
Arg His Ile Asn Asn Asp Met Ile Val Thr Asp385 390
395 400Asn Asn Gly Ala Val Lys Phe Pro Gln Leu
Cys Lys Phe Cys Asp Val 405 410
415Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser
420 425 430Ile Thr Ser Ile Cys
Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp 435
440 445Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val
Cys His Asp Pro 450 455 460Lys Leu Pro
Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys465
470 475 480Cys Ile Met Lys Glu Lys Lys
Lys Pro Gly Glu Thr Phe Phe Met Cys 485
490 495Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile
Phe Ser Glu Glu 500 505 510Tyr
Asn Thr Ser Asn Pro Asp 515109525PRTArtificial
SequencehIgG4FcdeltaK-CC-228P-T2-T2bAA fusion 109Glu Ser Lys Tyr Gly Pro
Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe1 5
10 15Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr 20 25 30Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35
40 45Ser Gln Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr Val Asp Gly Val 50 55
60Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser65
70 75 80Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85
90 95Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro Ser 100 105
110Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125Gln Val Tyr Thr Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135
140Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala145 150 155 160Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185
190Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
Cys Ser 195 200 205Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210
215 220Leu Ser Leu Gly Ile Pro Pro His Val Gln Lys Ser
Val Asn Asn Asp225 230 235
240Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys
245 250 255Lys Phe Cys Asp Val
Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys 260
265 270Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys
Pro Gln Glu Val 275 280 285Cys Val
Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr 290
295 300Val Cys His Asp Pro Lys Leu Pro Tyr His Asp
Phe Ile Leu Glu Asp305 310 315
320Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu
325 330 335Thr Phe Phe Met
Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile 340
345 350Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro
Asp Ile Pro Pro His 355 360 365Val
Gln Lys Ser Asp Val Glu Met Glu Ala Gln Lys Asp Glu Ile Ile 370
375 380Ala Pro Ser Ala Asn Arg Thr Ala His Pro
Leu Arg His Ile Asn Asn385 390 395
400Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln
Leu 405 410 415Cys Lys Phe
Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser 420
425 430Cys Met Ser Asn Cys Ser Ile Thr Ser Ile
Cys Glu Lys Pro Gln Glu 435 440
445Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu 450
455 460Thr Val Cys His Asp Pro Lys Leu
Pro Tyr His Asp Phe Ile Leu Glu465 470
475 480Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys
Lys Lys Pro Gly 485 490
495Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn
500 505 510Ile Ile Phe Ser Glu Glu
Tyr Asn Thr Ser Asn Pro Asp 515 520
525110525PRTArtificial SequencehIgG4FcdeltaK-CC-228P-409K-T2-T2bAA
fusion 110Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
Phe1 5 10 15Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20
25 30Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val 35 40
45Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50
55 60Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser65 70 75
80Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu 85 90 95Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100
105 110Ser Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro 115 120
125Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala145 150
155 160Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr 165 170
175Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
180 185 190Thr Val Asp Lys Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200
205Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser 210 215 220Leu Ser Leu Gly Ile
Pro Pro His Val Gln Lys Ser Val Asn Asn Asp225 230
235 240Met Ile Val Thr Asp Asn Asn Gly Ala Val
Lys Phe Pro Gln Leu Cys 245 250
255Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys
260 265 270Met Ser Asn Cys Ser
Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val 275
280 285Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile
Thr Leu Glu Thr 290 295 300Val Cys His
Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp305
310 315 320Ala Ala Ser Pro Lys Cys Ile
Met Lys Glu Lys Lys Lys Pro Gly Glu 325
330 335Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys
Asn Asp Asn Ile 340 345 350Ile
Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro His 355
360 365Val Gln Lys Ser Asp Val Glu Met Glu
Ala Gln Lys Asp Glu Ile Ile 370 375
380Ala Pro Ser Ala Asn Arg Thr Ala His Pro Leu Arg His Ile Asn Asn385
390 395 400Asp Met Ile Val
Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu 405
410 415Cys Lys Phe Cys Asp Val Arg Phe Ser Thr
Cys Asp Asn Gln Lys Ser 420 425
430Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu
435 440 445Val Cys Val Ala Val Trp Arg
Lys Asn Asp Glu Asn Ile Thr Leu Glu 450 455
460Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu
Glu465 470 475 480Asp Ala
Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly
485 490 495Glu Thr Phe Phe Met Cys Ser
Cys Ser Ser Asp Glu Cys Asn Asp Asn 500 505
510Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp
515 520 525111628PRTArtificial
SequencehIgG1FcdeltaK-C-T2-T2-T2 fusion 111Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu1 5 10
15Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu 20 25 30Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 35
40 45Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys 50 55 60Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu65
70 75 80Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 85
90 95Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys 100 105 110Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 115
120 125Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys 130 135
140Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln145
150 155 160Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 165
170 175Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln 180 185
190Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
195 200 205His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Ile Pro Pro His 210 215
220Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn
Gly225 230 235 240Ala Val
Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser
245 250 255Thr Cys Asp Asn Gln Lys Ser
Cys Met Ser Asn Cys Ser Ile Thr Ser 260 265
270Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg
Lys Asn 275 280 285Asp Glu Asn Ile
Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro 290
295 300Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro
Lys Cys Ile Met305 310 315
320Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser
325 330 335Ser Asp Glu Cys Asn
Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr 340
345 350Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys Ser
Val Asn Asn Asp 355 360 365Met Ile
Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys 370
375 380Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp
Asn Gln Lys Ser Cys385 390 395
400Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val
405 410 415Cys Val Ala Val
Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr 420
425 430Val Cys His Asp Pro Lys Leu Pro Tyr His Asp
Phe Ile Leu Glu Asp 435 440 445Ala
Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu 450
455 460Thr Phe Phe Met Cys Ser Cys Ser Ser Asp
Glu Cys Asn Asp Asn Ile465 470 475
480Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro
His 485 490 495Val Gln Lys
Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly 500
505 510Ala Val Lys Phe Pro Gln Leu Cys Lys Phe
Cys Asp Val Arg Phe Ser 515 520
525Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser 530
535 540Ile Cys Glu Lys Pro Gln Glu Val
Cys Val Ala Val Trp Arg Lys Asn545 550
555 560Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp
Pro Lys Leu Pro 565 570
575Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met
580 585 590Lys Glu Lys Lys Lys Pro
Gly Glu Thr Phe Phe Met Cys Ser Cys Ser 595 600
605Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr
Asn Thr 610 615 620Ser Asn Pro
Asp625112634PRTArtificial SequencehIgG1FcdeltaK-CC-T2-T2-T2 fusion 112Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1
5 10 15Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25
30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr65 70 75
80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100
105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val 115 120 125Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130
135 140Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu145 150 155
160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180
185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met 195 200 205His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220Pro Gly Ile Pro Pro His Val Gln Lys Ser
Val Asn Asn Asp Met Ile225 230 235
240Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys
Phe 245 250 255Cys Asp Val
Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser 260
265 270Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys
Pro Gln Glu Val Cys Val 275 280
285Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys 290
295 300His Asp Pro Lys Leu Pro Tyr His
Asp Phe Ile Leu Glu Asp Ala Ala305 310
315 320Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro
Gly Glu Thr Phe 325 330
335Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe
340 345 350Ser Glu Glu Tyr Asn Thr
Ser Asn Pro Asp Ile Pro Pro His Val Gln 355 360
365Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly
Ala Val 370 375 380Lys Phe Pro Gln Leu
Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys385 390
395 400Asp Asn Gln Lys Ser Cys Met Ser Asn Cys
Ser Ile Thr Ser Ile Cys 405 410
415Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu
420 425 430Asn Ile Thr Leu Glu
Thr Val Cys His Asp Pro Lys Leu Pro Tyr His 435
440 445Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys
Ile Met Lys Glu 450 455 460Lys Lys Lys
Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp465
470 475 480Glu Cys Asn Asp Asn Ile Ile
Phe Ser Glu Glu Tyr Asn Thr Ser Asn 485
490 495Pro Asp Ile Pro Pro His Val Gln Lys Ser Val Asn
Asn Asp Met Ile 500 505 510Val
Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe 515
520 525Cys Asp Val Arg Phe Ser Thr Cys Asp
Asn Gln Lys Ser Cys Met Ser 530 535
540Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val545
550 555 560Ala Val Trp Arg
Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys 565
570 575His Asp Pro Lys Leu Pro Tyr His Asp Phe
Ile Leu Glu Asp Ala Ala 580 585
590Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe
595 600 605Phe Met Cys Ser Cys Ser Ser
Asp Glu Cys Asn Asp Asn Ile Ile Phe 610 615
620Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp625
630113630PRTArtificial SequencehIgG2FcdeltaK-CC-T2-T2-T2 fusion 113Val
Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val1
5 10 15Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr 20 25
30Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu 35 40 45Val Gln Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 50 55
60Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg
Val Val Ser65 70 75
80Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
85 90 95Cys Lys Val Ser Asn Lys
Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile 100
105 110Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro 115 120 125Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 130
135 140Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val
Glu Trp Glu Ser Asn145 150 155
160Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser
165 170 175Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 180
185 190Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu 195 200 205His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ile Pro 210
215 220Pro His Val Gln Lys Ser Val Asn Asn Asp
Met Ile Val Thr Asp Asn225 230 235
240Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val
Arg 245 250 255Phe Ser Thr
Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile 260
265 270Thr Ser Ile Cys Glu Lys Pro Gln Glu Val
Cys Val Ala Val Trp Arg 275 280
285Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys 290
295 300Leu Pro Tyr His Asp Phe Ile Leu
Glu Asp Ala Ala Ser Pro Lys Cys305 310
315 320Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe
Phe Met Cys Ser 325 330
335Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr
340 345 350Asn Thr Ser Asn Pro Asp
Ile Pro Pro His Val Gln Lys Ser Val Asn 355 360
365Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe
Pro Gln 370 375 380Leu Cys Lys Phe Cys
Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys385 390
395 400Ser Cys Met Ser Asn Cys Ser Ile Thr Ser
Ile Cys Glu Lys Pro Gln 405 410
415Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu
420 425 430Glu Thr Val Cys His
Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu 435
440 445Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu
Lys Lys Lys Pro 450 455 460Gly Glu Thr
Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp465
470 475 480Asn Ile Ile Phe Ser Glu Glu
Tyr Asn Thr Ser Asn Pro Asp Ile Pro 485
490 495Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile
Val Thr Asp Asn 500 505 510Asn
Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg 515
520 525Phe Ser Thr Cys Asp Asn Gln Lys Ser
Cys Met Ser Asn Cys Ser Ile 530 535
540Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg545
550 555 560Lys Asn Asp Glu
Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys 565
570 575Leu Pro Tyr His Asp Phe Ile Leu Glu Asp
Ala Ala Ser Pro Lys Cys 580 585
590Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser
595 600 605Cys Ser Ser Asp Glu Cys Asn
Asp Asn Ile Ile Phe Ser Glu Glu Tyr 610 615
620Asn Thr Ser Asn Pro Asp625 630114636PRTArtificial
SequencehIgG4FcdeltaK-CC-228P-T2-T2-T2 fusion 114Glu Ser Lys Tyr Gly Pro
Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe1 5
10 15Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr 20 25 30Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35
40 45Ser Gln Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr Val Asp Gly Val 50 55
60Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser65
70 75 80Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85
90 95Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro Ser 100 105
110Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125Gln Val Tyr Thr Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135
140Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala145 150 155 160Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185
190Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
Cys Ser 195 200 205Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210
215 220Leu Ser Leu Gly Ile Pro Pro His Val Gln Lys Ser
Val Asn Asn Asp225 230 235
240Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys
245 250 255Lys Phe Cys Asp Val
Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys 260
265 270Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys
Pro Gln Glu Val 275 280 285Cys Val
Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr 290
295 300Val Cys His Asp Pro Lys Leu Pro Tyr His Asp
Phe Ile Leu Glu Asp305 310 315
320Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu
325 330 335Thr Phe Phe Met
Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile 340
345 350Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro
Asp Ile Pro Pro His 355 360 365Val
Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly 370
375 380Ala Val Lys Phe Pro Gln Leu Cys Lys Phe
Cys Asp Val Arg Phe Ser385 390 395
400Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr
Ser 405 410 415Ile Cys Glu
Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn 420
425 430Asp Glu Asn Ile Thr Leu Glu Thr Val Cys
His Asp Pro Lys Leu Pro 435 440
445Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met 450
455 460Lys Glu Lys Lys Lys Pro Gly Glu
Thr Phe Phe Met Cys Ser Cys Ser465 470
475 480Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu
Glu Tyr Asn Thr 485 490
495Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp
500 505 510Met Ile Val Thr Asp Asn
Asn Gly Ala Val Lys Phe Pro Gln Leu Cys 515 520
525Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys
Ser Cys 530 535 540Met Ser Asn Cys Ser
Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val545 550
555 560Cys Val Ala Val Trp Arg Lys Asn Asp Glu
Asn Ile Thr Leu Glu Thr 565 570
575Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp
580 585 590Ala Ala Ser Pro Lys
Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu 595
600 605Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys
Asn Asp Asn Ile 610 615 620Ile Phe Ser
Glu Glu Tyr Asn Thr Ser Asn Pro Asp625 630
635115636PRTArtificial SequencehIgG4FcdeltaK-CC-228P-409K-T2-T2-T2
fusion 115Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
Phe1 5 10 15Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20
25 30Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val 35 40
45Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50
55 60Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser65 70 75
80Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu 85 90 95Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100
105 110Ser Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro 115 120
125Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala145 150
155 160Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr 165 170
175Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
180 185 190Thr Val Asp Lys Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200
205Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser 210 215 220Leu Ser Leu Gly Ile
Pro Pro His Val Gln Lys Ser Val Asn Asn Asp225 230
235 240Met Ile Val Thr Asp Asn Asn Gly Ala Val
Lys Phe Pro Gln Leu Cys 245 250
255Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys
260 265 270Met Ser Asn Cys Ser
Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val 275
280 285Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile
Thr Leu Glu Thr 290 295 300Val Cys His
Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp305
310 315 320Ala Ala Ser Pro Lys Cys Ile
Met Lys Glu Lys Lys Lys Pro Gly Glu 325
330 335Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys
Asn Asp Asn Ile 340 345 350Ile
Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro His 355
360 365Val Gln Lys Ser Val Asn Asn Asp Met
Ile Val Thr Asp Asn Asn Gly 370 375
380Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser385
390 395 400Thr Cys Asp Asn
Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser 405
410 415Ile Cys Glu Lys Pro Gln Glu Val Cys Val
Ala Val Trp Arg Lys Asn 420 425
430Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro
435 440 445Tyr His Asp Phe Ile Leu Glu
Asp Ala Ala Ser Pro Lys Cys Ile Met 450 455
460Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys
Ser465 470 475 480Ser Asp
Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr
485 490 495Ser Asn Pro Asp Ile Pro Pro
His Val Gln Lys Ser Val Asn Asn Asp 500 505
510Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln
Leu Cys 515 520 525Lys Phe Cys Asp
Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys 530
535 540Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys
Pro Gln Glu Val545 550 555
560Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr
565 570 575Val Cys His Asp Pro
Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp 580
585 590Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys
Lys Pro Gly Glu 595 600 605Thr Phe
Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile 610
615 620Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro
Asp625 630 635116678PRTArtificial
SequencehIgG1FcdeltaK-C-T2-T2bAA-T2bAA fusion 116Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu1 5
10 15Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu 20 25 30Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 35
40 45Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys 50 55
60Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu65
70 75 80Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 85
90 95Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys 100 105
110Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
115 120 125Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys 130 135
140Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln145 150 155 160Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
165 170 175Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln 180 185
190Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn 195 200 205His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Ile Pro Pro His 210
215 220Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr
Asp Asn Asn Gly225 230 235
240Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser
245 250 255Thr Cys Asp Asn Gln
Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser 260
265 270Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val
Trp Arg Lys Asn 275 280 285Asp Glu
Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro 290
295 300Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser
Pro Lys Cys Ile Met305 310 315
320Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser
325 330 335Ser Asp Glu Cys
Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr 340
345 350Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys
Ser Asp Val Glu Met 355 360 365Glu
Ala Gln Lys Asp Glu Ile Ile Ala Pro Ser Ala Asn Arg Thr Ala 370
375 380His Pro Leu Arg His Ile Asn Asn Asp Met
Ile Val Thr Asp Asn Asn385 390 395
400Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg
Phe 405 410 415Ser Thr Cys
Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr 420
425 430Ser Ile Cys Glu Lys Pro Gln Glu Val Cys
Val Ala Val Trp Arg Lys 435 440
445Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu 450
455 460Pro Tyr His Asp Phe Ile Leu Glu
Asp Ala Ala Ser Pro Lys Cys Ile465 470
475 480Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe
Met Cys Ser Cys 485 490
495Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn
500 505 510Thr Ser Asn Pro Asp Ile
Pro Pro His Val Gln Lys Ser Asp Val Glu 515 520
525Met Glu Ala Gln Lys Asp Glu Ile Ile Ala Pro Ser Ala Asn
Arg Thr 530 535 540Ala His Pro Leu Arg
His Ile Asn Asn Asp Met Ile Val Thr Asp Asn545 550
555 560Asn Gly Ala Val Lys Phe Pro Gln Leu Cys
Lys Phe Cys Asp Val Arg 565 570
575Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile
580 585 590Thr Ser Ile Cys Glu
Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg 595
600 605Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys
His Asp Pro Lys 610 615 620Leu Pro Tyr
His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys625
630 635 640Ile Met Lys Glu Lys Lys Lys
Pro Gly Glu Thr Phe Phe Met Cys Ser 645
650 655Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe
Ser Glu Glu Tyr 660 665 670Asn
Thr Ser Asn Pro Asp 675117684PRTArtificial
SequencehIgG1FcdeltaK-CC-T2-T2bAA-T2bAA fusion 117Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5
10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met 20 25 30Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 50 55
60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65
70 75 80Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile 100 105
110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135
140Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu145 150 155 160Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 195 200 205His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220Pro Gly Ile Pro Pro His Val Gln Lys Ser Val Asn
Asn Asp Met Ile225 230 235
240Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe
245 250 255Cys Asp Val Arg Phe
Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser 260
265 270Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln
Glu Val Cys Val 275 280 285Ala Val
Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys 290
295 300His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile
Leu Glu Asp Ala Ala305 310 315
320Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe
325 330 335Phe Met Cys Ser
Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe 340
345 350Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile
Pro Pro His Val Gln 355 360 365Lys
Ser Asp Val Glu Met Glu Ala Gln Lys Asp Glu Ile Ile Ala Pro 370
375 380Ser Ala Asn Arg Thr Ala His Pro Leu Arg
His Ile Asn Asn Asp Met385 390 395
400Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys
Lys 405 410 415Phe Cys Asp
Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met 420
425 430Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu
Lys Pro Gln Glu Val Cys 435 440
445Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val 450
455 460Cys His Asp Pro Lys Leu Pro Tyr
His Asp Phe Ile Leu Glu Asp Ala465 470
475 480Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys
Pro Gly Glu Thr 485 490
495Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile
500 505 510Phe Ser Glu Glu Tyr Asn
Thr Ser Asn Pro Asp Ile Pro Pro His Val 515 520
525Gln Lys Ser Asp Val Glu Met Glu Ala Gln Lys Asp Glu Ile
Ile Ala 530 535 540Pro Ser Ala Asn Arg
Thr Ala His Pro Leu Arg His Ile Asn Asn Asp545 550
555 560Met Ile Val Thr Asp Asn Asn Gly Ala Val
Lys Phe Pro Gln Leu Cys 565 570
575Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys
580 585 590Met Ser Asn Cys Ser
Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val 595
600 605Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile
Thr Leu Glu Thr 610 615 620Val Cys His
Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp625
630 635 640Ala Ala Ser Pro Lys Cys Ile
Met Lys Glu Lys Lys Lys Pro Gly Glu 645
650 655Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys
Asn Asp Asn Ile 660 665 670Ile
Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 675
680118680PRTArtificial SequencehIgG2FcdeltaK-CC-T2-T2bAA-T2bAA fusion
118Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val1
5 10 15Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 20 25
30Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu 35 40 45Val Gln Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 50
55 60Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
Arg Val Val Ser65 70 75
80Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
85 90 95Cys Lys Val Ser Asn Lys
Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile 100
105 110Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro 115 120 125Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 130
135 140Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val
Glu Trp Glu Ser Asn145 150 155
160Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser
165 170 175Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 180
185 190Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu 195 200 205His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ile Pro 210
215 220Pro His Val Gln Lys Ser Val Asn Asn Asp
Met Ile Val Thr Asp Asn225 230 235
240Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val
Arg 245 250 255Phe Ser Thr
Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile 260
265 270Thr Ser Ile Cys Glu Lys Pro Gln Glu Val
Cys Val Ala Val Trp Arg 275 280
285Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys 290
295 300Leu Pro Tyr His Asp Phe Ile Leu
Glu Asp Ala Ala Ser Pro Lys Cys305 310
315 320Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe
Phe Met Cys Ser 325 330
335Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr
340 345 350Asn Thr Ser Asn Pro Asp
Ile Pro Pro His Val Gln Lys Ser Asp Val 355 360
365Glu Met Glu Ala Gln Lys Asp Glu Ile Ile Ala Pro Ser Ala
Asn Arg 370 375 380Thr Ala His Pro Leu
Arg His Ile Asn Asn Asp Met Ile Val Thr Asp385 390
395 400Asn Asn Gly Ala Val Lys Phe Pro Gln Leu
Cys Lys Phe Cys Asp Val 405 410
415Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser
420 425 430Ile Thr Ser Ile Cys
Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp 435
440 445Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val
Cys His Asp Pro 450 455 460Lys Leu Pro
Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys465
470 475 480Cys Ile Met Lys Glu Lys Lys
Lys Pro Gly Glu Thr Phe Phe Met Cys 485
490 495Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile
Phe Ser Glu Glu 500 505 510Tyr
Asn Thr Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys Ser Asp 515
520 525Val Glu Met Glu Ala Gln Lys Asp Glu
Ile Ile Ala Pro Ser Ala Asn 530 535
540Arg Thr Ala His Pro Leu Arg His Ile Asn Asn Asp Met Ile Val Thr545
550 555 560Asp Asn Asn Gly
Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 565
570 575Val Arg Phe Ser Thr Cys Asp Asn Gln Lys
Ser Cys Met Ser Asn Cys 580 585
590Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val
595 600 605Trp Arg Lys Asn Asp Glu Asn
Ile Thr Leu Glu Thr Val Cys His Asp 610 615
620Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser
Pro625 630 635 640Lys Cys
Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met
645 650 655Cys Ser Cys Ser Ser Asp Glu
Cys Asn Asp Asn Ile Ile Phe Ser Glu 660 665
670Glu Tyr Asn Thr Ser Asn Pro Asp 675
680119686PRTArtificial SequencehIgG4FcdeltaK-CC-228P-T2-T2bAA-T2bAA
fusion 119Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
Phe1 5 10 15Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20
25 30Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val 35 40
45Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50
55 60Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser65 70 75
80Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu 85 90 95Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100
105 110Ser Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro 115 120
125Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala145 150
155 160Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr 165 170
175Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
180 185 190Thr Val Asp Lys Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200
205Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser 210 215 220Leu Ser Leu Gly Ile
Pro Pro His Val Gln Lys Ser Val Asn Asn Asp225 230
235 240Met Ile Val Thr Asp Asn Asn Gly Ala Val
Lys Phe Pro Gln Leu Cys 245 250
255Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys
260 265 270Met Ser Asn Cys Ser
Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val 275
280 285Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile
Thr Leu Glu Thr 290 295 300Val Cys His
Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp305
310 315 320Ala Ala Ser Pro Lys Cys Ile
Met Lys Glu Lys Lys Lys Pro Gly Glu 325
330 335Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys
Asn Asp Asn Ile 340 345 350Ile
Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro His 355
360 365Val Gln Lys Ser Asp Val Glu Met Glu
Ala Gln Lys Asp Glu Ile Ile 370 375
380Ala Pro Ser Ala Asn Arg Thr Ala His Pro Leu Arg His Ile Asn Asn385
390 395 400Asp Met Ile Val
Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu 405
410 415Cys Lys Phe Cys Asp Val Arg Phe Ser Thr
Cys Asp Asn Gln Lys Ser 420 425
430Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu
435 440 445Val Cys Val Ala Val Trp Arg
Lys Asn Asp Glu Asn Ile Thr Leu Glu 450 455
460Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu
Glu465 470 475 480Asp Ala
Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly
485 490 495Glu Thr Phe Phe Met Cys Ser
Cys Ser Ser Asp Glu Cys Asn Asp Asn 500 505
510Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile
Pro Pro 515 520 525His Val Gln Lys
Ser Asp Val Glu Met Glu Ala Gln Lys Asp Glu Ile 530
535 540Ile Ala Pro Ser Ala Asn Arg Thr Ala His Pro Leu
Arg His Ile Asn545 550 555
560Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln
565 570 575Leu Cys Lys Phe Cys
Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys 580
585 590Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys
Glu Lys Pro Gln 595 600 605Glu Val
Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu 610
615 620Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr
His Asp Phe Ile Leu625 630 635
640Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro
645 650 655Gly Glu Thr Phe
Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp 660
665 670Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser
Asn Pro Asp 675 680
685120686PRTArtificial SequencehIgG4FcdeltaK-CC-228P-409K-T2-T2bAA-T2bAA
fusion 120Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
Phe1 5 10 15Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20
25 30Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val 35 40
45Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50
55 60Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser65 70 75
80Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu 85 90 95Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100
105 110Ser Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro 115 120
125Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala145 150
155 160Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr 165 170
175Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
180 185 190Thr Val Asp Lys Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200
205Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser 210 215 220Leu Ser Leu Gly Ile
Pro Pro His Val Gln Lys Ser Val Asn Asn Asp225 230
235 240Met Ile Val Thr Asp Asn Asn Gly Ala Val
Lys Phe Pro Gln Leu Cys 245 250
255Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys
260 265 270Met Ser Asn Cys Ser
Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val 275
280 285Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile
Thr Leu Glu Thr 290 295 300Val Cys His
Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp305
310 315 320Ala Ala Ser Pro Lys Cys Ile
Met Lys Glu Lys Lys Lys Pro Gly Glu 325
330 335Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys
Asn Asp Asn Ile 340 345 350Ile
Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro His 355
360 365Val Gln Lys Ser Asp Val Glu Met Glu
Ala Gln Lys Asp Glu Ile Ile 370 375
380Ala Pro Ser Ala Asn Arg Thr Ala His Pro Leu Arg His Ile Asn Asn385
390 395 400Asp Met Ile Val
Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu 405
410 415Cys Lys Phe Cys Asp Val Arg Phe Ser Thr
Cys Asp Asn Gln Lys Ser 420 425
430Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu
435 440 445Val Cys Val Ala Val Trp Arg
Lys Asn Asp Glu Asn Ile Thr Leu Glu 450 455
460Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu
Glu465 470 475 480Asp Ala
Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly
485 490 495Glu Thr Phe Phe Met Cys Ser
Cys Ser Ser Asp Glu Cys Asn Asp Asn 500 505
510Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile
Pro Pro 515 520 525His Val Gln Lys
Ser Asp Val Glu Met Glu Ala Gln Lys Asp Glu Ile 530
535 540Ile Ala Pro Ser Ala Asn Arg Thr Ala His Pro Leu
Arg His Ile Asn545 550 555
560Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln
565 570 575Leu Cys Lys Phe Cys
Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys 580
585 590Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys
Glu Lys Pro Gln 595 600 605Glu Val
Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu 610
615 620Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr
His Asp Phe Ile Leu625 630 635
640Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro
645 650 655Gly Glu Thr Phe
Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp 660
665 670Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser
Asn Pro Asp 675 680
685121214PRTArtificial SequenceCetuximab LC 121Asp Ile Leu Leu Thr Gln
Ser Pro Val Ile Leu Ser Val Ser Pro Gly1 5
10 15Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser
Ile Gly Thr Asn 20 25 30Ile
His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35
40 45Lys Tyr Ala Ser Glu Ser Ile Ser Gly
Ile Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser65
70 75 80Glu Asp Ile Ala Asp
Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr 85
90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln145 150 155 160Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly
Glu Cys 210122721PRTArtificial SequenceCetuximab HC-T22d35 122Gln Val
Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln1 5
10 15Ser Leu Ser Ile Thr Cys Thr Val
Ser Gly Phe Ser Leu Thr Asn Tyr 20 25
30Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp
Leu 35 40 45Gly Val Ile Trp Ser
Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55
60Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val
Phe Phe65 70 75 80Lys
Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95Arg Ala Leu Thr Tyr Tyr Asp
Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105
110Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser
Val Phe 115 120 125Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130
135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp145 150 155
160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180
185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro 195 200 205Ser Asn
Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210
215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro225 230 235
240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260
265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn 275 280 285Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290
295 300Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu305 310 315
320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys 325 330 335Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340
345 350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser Leu Thr 355 360
365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370
375 380Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu385 390
395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys 405 410
415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445Lys Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met
Ile Val 450 455 460Thr Asp Asn Asn Gly
Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys465 470
475 480Asp Val Arg Phe Ser Thr Cys Asp Asn Gln
Lys Ser Cys Met Ser Asn 485 490
495Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala
500 505 510Val Trp Arg Lys Asn
Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His 515
520 525Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu
Asp Ala Ala Ser 530 535 540Pro Lys Cys
Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe545
550 555 560Met Cys Ser Cys Ser Ser Asp
Glu Cys Asn Asp Asn Ile Ile Phe Ser 565
570 575Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro
His Val Gln Lys 580 585 590Ser
Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys 595
600 605Phe Pro Gln Leu Cys Lys Phe Cys Asp
Val Arg Phe Ser Thr Cys Asp 610 615
620Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu625
630 635 640Lys Pro Gln Glu
Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn 645
650 655Ile Thr Leu Glu Thr Val Cys His Asp Pro
Lys Leu Pro Tyr His Asp 660 665
670Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys
675 680 685Lys Lys Pro Gly Glu Thr Phe
Phe Met Cys Ser Cys Ser Ser Asp Glu 690 695
700Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn
Pro705 710 715
720Asp123585PRTArtificial SequenceCetuximab HC-T2m 123Gln Val Gln Leu Lys
Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln1 5
10 15Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe
Ser Leu Thr Asn Tyr 20 25
30Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45Gly Val Ile Trp Ser Gly Gly Asn
Thr Asp Tyr Asn Thr Pro Phe Thr 50 55
60Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe65
70 75 80Lys Met Asn Ser Leu
Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 85
90 95Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala
Tyr Trp Gly Gln Gly 100 105
110Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp145 150 155 160Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser 180 185
190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205Ser Asn Thr Lys
Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210
215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro225 230 235
240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp 260
265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290
295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu305 310 315
320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340
345 350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr 355 360 365Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370
375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu385 390 395
400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys 405 410 415Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420
425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 435 440
445Lys Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val 450
455 460Thr Asp Asn Asn Gly Ala Val Lys
Phe Pro Gln Leu Cys Lys Phe Cys465 470
475 480Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser
Cys Met Ser Asn 485 490
495Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala
500 505 510Val Trp Arg Lys Asn Asp
Glu Asn Ile Thr Leu Glu Thr Val Cys His 515 520
525Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala
Ala Ser 530 535 540Pro Lys Cys Ile Met
Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe545 550
555 560Met Cys Ser Cys Ser Ser Asp Glu Cys Asn
Asp Asn Ile Ile Phe Ser 565 570
575Glu Glu Tyr Asn Thr Ser Asn Pro Asp 580
585124214PRTArtificial SequenceHerceptin LC 124Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp
Val Asn Thr Ala 20 25 30Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85
90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln145 150 155 160Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly
Glu Cys 210125722PRTArtificial SequenceHerceptin HC-T22d35 125Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Asn Ile Lys Asp Thr 20 25
30Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Arg Ile Tyr Pro
Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr
Ala Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ser Arg Trp Gly Gly Asp Gly
Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val 115 120 125Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130
135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser145 150 155
160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180
185 190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys 195 200 205Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210
215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly225 230 235
240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260
265 270Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His 275 280 285Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290
295 300Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys305 310 315
320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu 325 330 335Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340
345 350Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser Leu 355 360
365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370
375 380Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val385 390
395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp 405 410
415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445Gly Lys Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp
Met Ile 450 455 460Val Thr Asp Asn Asn
Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe465 470
475 480Cys Asp Val Arg Phe Ser Thr Cys Asp Asn
Gln Lys Ser Cys Met Ser 485 490
495Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val
500 505 510Ala Val Trp Arg Lys
Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys 515
520 525His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu
Glu Asp Ala Ala 530 535 540Ser Pro Lys
Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe545
550 555 560Phe Met Cys Ser Cys Ser Ser
Asp Glu Cys Asn Asp Asn Ile Ile Phe 565
570 575Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro
Pro His Val Gln 580 585 590Lys
Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val 595
600 605Lys Phe Pro Gln Leu Cys Lys Phe Cys
Asp Val Arg Phe Ser Thr Cys 610 615
620Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys625
630 635 640Glu Lys Pro Gln
Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu 645
650 655Asn Ile Thr Leu Glu Thr Val Cys His Asp
Pro Lys Leu Pro Tyr His 660 665
670Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu
675 680 685Lys Lys Lys Pro Gly Glu Thr
Phe Phe Met Cys Ser Cys Ser Ser Asp 690 695
700Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser
Asn705 710 715 720Pro
Asp126214PRTArtificial SequenceAvastin LC 126Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile
Ser Asn Tyr 20 25 30Leu Asn
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35
40 45Tyr Phe Thr Ser Ser Leu His Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85
90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
Thr Val Ala Ala 100 105 110Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115
120 125Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro Arg Glu Ala 130 135
140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145
150 155 160Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165
170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 180 185
190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205Phe Asn Arg Gly Glu Cys
210127725PRTArtificial SequenceAvastin HC-T22d35 127Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr
Thr Phe Thr Asn Tyr 20 25
30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Gly Trp Ile Asn Thr Tyr Thr Gly
Glu Pro Thr Tyr Ala Ala Asp Phe 50 55
60Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His
Trp Tyr Phe Asp Val 100 105
110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135
140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val145 150 155 160Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185
190Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val 195 200 205Asn His Lys Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210
215 220Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu225 230 235
240Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
245 250 255Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260
265 270Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val 275 280 285Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290
295 300Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu305 310 315
320Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
325 330 335Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340
345 350Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln 355 360 365Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370
375 380Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr385 390 395
400Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu 405 410 415Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420
425 430Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser 435 440
445Leu Ser Pro Gly Lys Ile Pro Pro His Val Gln Lys Ser Val Asn Asn 450
455 460Asp Met Ile Val Thr Asp Asn Asn
Gly Ala Val Lys Phe Pro Gln Leu465 470
475 480Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp
Asn Gln Lys Ser 485 490
495Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu
500 505 510Val Cys Val Ala Val Trp
Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu 515 520
525Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile
Leu Glu 530 535 540Asp Ala Ala Ser Pro
Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly545 550
555 560Glu Thr Phe Phe Met Cys Ser Cys Ser Ser
Asp Glu Cys Asn Asp Asn 565 570
575Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro
580 585 590His Val Gln Lys Ser
Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn 595
600 605Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys
Asp Val Arg Phe 610 615 620Ser Thr Cys
Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr625
630 635 640Ser Ile Cys Glu Lys Pro Gln
Glu Val Cys Val Ala Val Trp Arg Lys 645
650 655Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His
Asp Pro Lys Leu 660 665 670Pro
Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile 675
680 685Met Lys Glu Lys Lys Lys Pro Gly Glu
Thr Phe Phe Met Cys Ser Cys 690 695
700Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn705
710 715 720Thr Ser Asn Pro
Asp 725128213PRTArtificial SequenceSynagis LC 128Asp Ile
Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Lys
Cys Gln Leu Ser Val Gly Tyr Met 20 25
30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
Tyr 35 40 45Asp Thr Ser Lys Leu
Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55
60Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro Asp65 70 75 80Asp
Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr
85 90 95Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105
110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
Gly Thr 115 120 125Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130
135 140Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser Gln Glu145 150 155
160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180
185 190Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser Phe 195 200 205Asn Arg
Gly Glu Cys 210129721PRTArtificial SequenceSynagis HC-T22d35 129Gln
Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1
5 10 15Thr Leu Thr Leu Thr Cys Thr
Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25
30Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala
Leu Glu 35 40 45Trp Leu Ala Asp
Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser 50 55
60Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys
Asn Gln Val65 70 75
80Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr
85 90 95Cys Ala Arg Ser Met Ile
Thr Asn Trp Tyr Phe Asp Val Trp Gly Ala 100
105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val 115 120 125Phe Pro
Leu Ala Pro Ser Ser Ala Ala Ala Ala Gly Gly Thr Ala Ala 130
135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser145 150 155
160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180
185 190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys 195 200 205Pro
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 210
215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly225 230 235
240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile 245 250 255Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260
265 270Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His 275 280
285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290
295 300Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys305 310
315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu 325 330
335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360
365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp 370 375 380Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390
395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp 405 410
415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435
440 445Gly Ile Pro Pro His Val Gln Lys Ser Val Asn Asn
Asp Met Ile Val 450 455 460Thr Asp Asn
Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys465
470 475 480Asp Val Arg Phe Ser Thr Cys
Asp Asn Gln Lys Ser Cys Met Ser Asn 485
490 495Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu
Val Cys Val Ala 500 505 510Val
Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His 515
520 525Asp Pro Lys Leu Pro Tyr His Asp Phe
Ile Leu Glu Asp Ala Ala Ser 530 535
540Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe545
550 555 560Met Cys Ser Cys
Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser 565
570 575Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile
Pro Pro His Val Gln Lys 580 585
590Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys
595 600 605Phe Pro Gln Leu Cys Lys Phe
Cys Asp Val Arg Phe Ser Thr Cys Asp 610 615
620Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys
Glu625 630 635 640Lys Pro
Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn
645 650 655Ile Thr Leu Glu Thr Val Cys
His Asp Pro Lys Leu Pro Tyr His Asp 660 665
670Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys
Glu Lys 675 680 685Lys Lys Pro Gly
Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu 690
695 700Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn
Thr Ser Asn Pro705 710 715
720Asp130630PRTArtificial SequenceFC5-Fc-T22d35 130Glu Val Gln Leu Gln
Ala Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Lys Ile Thr His Tyr 20 25
30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45Ser Arg Ile Thr Trp Gly Gly Asp
Asn Thr Phe Tyr Ser Asn Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Asp Tyr Tyr Cys 85
90 95Ala Ala Gly Ser Thr Ser Thr Ala Thr Pro Leu
Arg Val Asp Tyr Trp 100 105
110Gly Lys Gly Thr Gln Val Thr Val Ser Ser Ala Ser Glu Pro Arg Gly
115 120 125Pro Thr Ile Lys Pro Cys Pro
Pro Cys Lys Cys Pro Ala Pro Asn Leu 130 135
140Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp
Val145 150 155 160Leu Met
Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val
165 170 175Ser Glu Asp Asp Pro Asp Val
Gln Ile Ser Trp Phe Val Asn Asn Val 180 185
190Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr
Asn Ser 195 200 205Thr Leu Arg Val
Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met 210
215 220Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys
Asp Leu Pro Ala225 230 235
240Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro
245 250 255Gln Val Tyr Val Leu
Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln 260
265 270Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro
Glu Asp Ile Tyr 275 280 285Val Glu
Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr 290
295 300Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe
Met Tyr Ser Lys Leu305 310 315
320Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser
325 330 335Val Val His Glu
Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser 340
345 350Arg Thr Pro Gly Thr Gly Ile Pro Pro His Val
Gln Lys Ser Val Asn 355 360 365Asn
Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln 370
375 380Leu Cys Lys Phe Cys Asp Val Arg Phe Ser
Thr Cys Asp Asn Gln Lys385 390 395
400Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro
Gln 405 410 415Glu Val Cys
Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu 420
425 430Glu Thr Val Cys His Asp Pro Lys Leu Pro
Tyr His Asp Phe Ile Leu 435 440
445Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro 450
455 460Gly Glu Thr Phe Phe Met Cys Ser
Cys Ser Ser Asp Glu Cys Asn Asp465 470
475 480Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn
Pro Asp Ile Pro 485 490
495Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn
500 505 510Asn Gly Ala Val Lys Phe
Pro Gln Leu Cys Lys Phe Cys Asp Val Arg 515 520
525Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys
Ser Ile 530 535 540Thr Ser Ile Cys Glu
Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg545 550
555 560Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr
Val Cys His Asp Pro Lys 565 570
575Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys
580 585 590Ile Met Lys Glu Lys
Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser 595
600 605Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe
Ser Glu Glu Tyr 610 615 620Asn Thr Ser
Asn Pro Asp625 630131494PRTArtificial SequenceFC5-Fc-T2m
131Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Lys Ile Thr His Tyr 20 25
30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg
Glu Phe Val 35 40 45Ser Arg Ile
Thr Trp Gly Gly Asp Asn Thr Phe Tyr Ser Asn Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Val Tyr65 70 75
80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Asp Tyr Tyr Cys
85 90 95Ala Ala Gly Ser Thr Ser
Thr Ala Thr Pro Leu Arg Val Asp Tyr Trp 100
105 110Gly Lys Gly Thr Gln Val Thr Val Ser Ser Ala Ser
Glu Pro Arg Gly 115 120 125Pro Thr
Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu 130
135 140Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro
Lys Ile Lys Asp Val145 150 155
160Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val
165 170 175Ser Glu Asp Asp
Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val 180
185 190Glu Val His Thr Ala Gln Thr Gln Thr His Arg
Glu Asp Tyr Asn Ser 195 200 205Thr
Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met 210
215 220Ser Gly Lys Glu Phe Lys Cys Lys Val Asn
Asn Lys Asp Leu Pro Ala225 230 235
240Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala
Pro 245 250 255Gln Val Tyr
Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln 260
265 270Val Thr Leu Thr Cys Met Val Thr Asp Phe
Met Pro Glu Asp Ile Tyr 275 280
285Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr 290
295 300Glu Pro Val Leu Asp Ser Asp Gly
Ser Tyr Phe Met Tyr Ser Lys Leu305 310
315 320Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser
Tyr Ser Cys Ser 325 330
335Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser
340 345 350Arg Thr Pro Gly Thr Gly
Ile Pro Pro His Val Gln Lys Ser Val Asn 355 360
365Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe
Pro Gln 370 375 380Leu Cys Lys Phe Cys
Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys385 390
395 400Ser Cys Met Ser Asn Cys Ser Ile Thr Ser
Ile Cys Glu Lys Pro Gln 405 410
415Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu
420 425 430Glu Thr Val Cys His
Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu 435
440 445Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu
Lys Lys Lys Pro 450 455 460Gly Glu Thr
Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp465
470 475 480Asn Ile Ile Phe Ser Glu Glu
Tyr Asn Thr Ser Asn Pro Asp 485
490132392PRTArtificial SequenceT2-hIgG1Fc fusion from R&D, also termed
T2m-Fc (R&D) 132Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His
Ile Val Leu1 5 10 15Trp
Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Val 20
25 30Asn Asn Asp Met Ile Val Thr Asp
Asn Asn Gly Ala Val Lys Phe Pro 35 40
45Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln
50 55 60Lys Ser Cys Met Ser Asn Cys Ser
Ile Thr Ser Ile Cys Glu Lys Pro65 70 75
80Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu
Asn Ile Thr 85 90 95Leu
Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile
100 105 110Leu Glu Asp Ala Ala Ser Pro
Lys Cys Ile Met Lys Glu Lys Lys Lys 115 120
125Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys
Asn 130 135 140Asp Asn Ile Ile Phe Ser
Glu Glu Tyr Asn Thr Ser Asn Pro Asp Met145 150
155 160Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala 165 170
175Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
180 185 190Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val 195 200
205Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val 210 215 220Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln225 230
235 240Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln 245 250
255Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
260 265 270Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 275
280 285Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr 290 295 300Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser305
310 315 320Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr 325
330 335Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr 340 345 350Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 355
360 365Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys 370 375
380Ser Leu Ser Leu Ser Pro Gly Lys385
390133382PRTArtificial SequenceT2-hIgG2Fc-CCCC fusion, also termed T2m-Fc
133Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly1
5 10 15Thr His Ala Ile Pro Pro
His Val Gln Lys Ser Val Asn Asn Asp Met 20 25
30Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln
Leu Cys Lys 35 40 45Phe Cys Asp
Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met 50
55 60Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro
Gln Glu Val Cys65 70 75
80Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val
85 90 95Cys His Asp Pro Lys Leu
Pro Tyr His Asp Phe Ile Leu Glu Asp Ala 100
105 110Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys
Pro Gly Glu Thr 115 120 125Phe Phe
Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile 130
135 140Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp
Glu Arg Lys Cys Cys145 150 155
160Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val
165 170 175Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 180
185 190Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu 195 200 205Val
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 210
215 220Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
Thr Phe Arg Val Val Ser225 230 235
240Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys 245 250 255Cys Lys Val
Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile 260
265 270Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro 275 280
285Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 290
295 300Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ser Val Glu Trp Glu Ser Asn305 310
315 320Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Met Leu Asp Ser 325 330
335Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
340 345 350Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu 355 360
365His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
370 375 38013415PRTArtificial
SequenceIsotope-labelled peptide for T22d35 134Leu Pro Tyr His Asp Phe
Ile Leu Glu Asp Ala Ala Ser Pro Lys1 5 10
151358PRTArtificial SequenceIsotope-labelled peptide for
Cetuximab 135Ala Leu Pro Ala Pro Ile Glu Lys1
5136372PRTArtificial SequenceD10-hIgG1FcdeltaK-CC-T2m fusion 136Asp Asp
Asp Asp Asp Asp Asp Asp Asp Asp Asp Lys Thr His Thr Cys1 5
10 15Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu 20 25
30Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu 35 40 45Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys 50 55
60Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys65 70 75 80Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
85 90 95Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys 100 105
110Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys 115 120 125Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 130
135 140Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys145 150 155
160Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
165 170 175Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 180
185 190Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln 195 200 205Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 210
215 220His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Ile Pro Pro His225 230 235
240Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly
245 250 255Ala Val Lys Phe
Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser 260
265 270Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn
Cys Ser Ile Thr Ser 275 280 285Ile
Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn 290
295 300Asp Glu Asn Ile Thr Leu Glu Thr Val Cys
His Asp Pro Lys Leu Pro305 310 315
320Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile
Met 325 330 335Lys Glu Lys
Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser 340
345 350Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe
Ser Glu Glu Tyr Asn Thr 355 360
365Ser Asn Pro Asp 370137376PRTArtificial
SequenceD10-G3S-hIgG1FcdeltaK-CC-T2m fusion 137Asp Asp Asp Asp Asp Asp
Asp Asp Asp Asp Gly Gly Gly Ser Asp Lys1 5
10 15Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro 20 25 30Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 35
40 45Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp 50 55
60Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn65
70 75 80Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 85
90 95Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 100 105
110Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
115 120 125Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr 130 135
140Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr145 150 155 160Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
165 170 175Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu 180 185
190Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys 195 200 205Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 210
215 220Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly225 230 235
240Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr
245 250 255Asp Asn Asn Gly Ala
Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 260
265 270Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys
Met Ser Asn Cys 275 280 285Ser Ile
Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 290
295 300Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu
Thr Val Cys His Asp305 310 315
320Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro
325 330 335Lys Cys Ile Met
Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 340
345 350Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn
Ile Ile Phe Ser Glu 355 360 365Glu
Tyr Asn Thr Ser Asn Pro Asp 370 375138366PRTArtificial
SequenceD10-hIgG1FcdeltaK-C-T2m fusion 138Asp Asp Asp Asp Asp Asp Asp Asp
Asp Asp Pro Pro Cys Pro Ala Pro1 5 10
15Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 20 25 30Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 35
40 45Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp 50 55 60Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr65
70 75 80Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp 85 90
95Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu 100 105 110Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 115
120 125Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys 130 135 140Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp145
150 155 160Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 165
170 175Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser 180 185 190Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 195
200 205Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser 210 215
220Leu Ser Leu Ser Pro Gly Ile Pro Pro His Val Gln Lys Ser Val Asn225
230 235 240Asn Asp Met Ile
Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln 245
250 255Leu Cys Lys Phe Cys Asp Val Arg Phe Ser
Thr Cys Asp Asn Gln Lys 260 265
270Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln
275 280 285Glu Val Cys Val Ala Val Trp
Arg Lys Asn Asp Glu Asn Ile Thr Leu 290 295
300Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile
Leu305 310 315 320Glu Asp
Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro
325 330 335Gly Glu Thr Phe Phe Met Cys
Ser Cys Ser Ser Asp Glu Cys Asn Asp 340 345
350Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp
355 360 365139374PRTArtificial
SequenceD10-(G3S)2-hIgG1FcdeltaK-C-T2m fusion 139Asp Asp Asp Asp Asp Asp
Asp Asp Asp Asp Gly Gly Gly Ser Gly Gly1 5
10 15Gly Ser Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val 20 25 30Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 35
40 45Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu 50 55
60Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys65
70 75 80Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 85
90 95Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys 100 105
110Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
115 120 125Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro 130 135
140Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu145 150 155 160Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
165 170 175Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser 180 185
190Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg 195 200 205Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 210
215 220His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Ile Pro225 230 235
240Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn
245 250 255Asn Gly Ala Val Lys
Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg 260
265 270Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser
Asn Cys Ser Ile 275 280 285Thr Ser
Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg 290
295 300Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val
Cys His Asp Pro Lys305 310 315
320Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys
325 330 335Ile Met Lys Glu
Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser 340
345 350Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile
Phe Ser Glu Glu Tyr 355 360 365Asn
Thr Ser Asn Pro Asp 370140368PRTArtificial
SequenceD10-hIgG2FcdeltaK-CC-T2m fusion 140Asp Asp Asp Asp Asp Asp Asp
Asp Asp Asp Val Glu Cys Pro Pro Cys1 5 10
15Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys 20 25 30Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 35
40 45Val Val Asp Val Ser His Glu Asp Pro Glu
Val Gln Phe Asn Trp Tyr 50 55 60Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu65
70 75 80Gln Phe Asn Ser Thr Phe
Arg Val Val Ser Val Leu Thr Val Val His 85
90 95Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys 100 105 110Gly
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 115
120 125Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met 130 135
140Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro145
150 155 160Ser Asp Ile Ser
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 165
170 175Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser
Asp Gly Ser Phe Phe Leu 180 185
190Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln 210 215
220Lys Ser Leu Ser Leu Ser Pro Gly Ile Pro Pro His Val Gln Lys
Ser225 230 235 240Val Asn
Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe
245 250 255Pro Gln Leu Cys Lys Phe Cys
Asp Val Arg Phe Ser Thr Cys Asp Asn 260 265
270Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys
Glu Lys 275 280 285Pro Gln Glu Val
Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile 290
295 300Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro
Tyr His Asp Phe305 310 315
320Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys
325 330 335Lys Pro Gly Glu Thr
Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys 340
345 350Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr
Ser Asn Pro Asp 355 360
365141374PRTArtificial SequenceD10-hIgG4FcdeltaK-CC-228P-T2m fusion
141Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Glu Ser Lys Tyr Gly Pro1
5 10 15Pro Cys Pro Pro Cys Pro
Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 20 25
30Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr 35 40 45Pro Glu Val
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 50
55 60Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys65 70 75
80Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
85 90 95Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 100
105 110Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile 115 120 125Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 130
135 140Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu145 150 155
160Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
165 170 175Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 180
185 190Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
Val Asp Lys Ser Arg 195 200 205Trp
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 210
215 220His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Leu Gly Ile Pro225 230 235
240Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp
Asn 245 250 255Asn Gly Ala
Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg 260
265 270Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys
Met Ser Asn Cys Ser Ile 275 280
285Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg 290
295 300Lys Asn Asp Glu Asn Ile Thr Leu
Glu Thr Val Cys His Asp Pro Lys305 310
315 320Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala
Ser Pro Lys Cys 325 330
335Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser
340 345 350Cys Ser Ser Asp Glu Cys
Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr 355 360
365Asn Thr Ser Asn Pro Asp 370142374PRTArtificial
SequenceD10-hIgG4FcdeltaK-CC-228P-409K-T2m fusion 142Asp Asp Asp Asp Asp
Asp Asp Asp Asp Asp Glu Ser Lys Tyr Gly Pro1 5
10 15Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu
Gly Gly Pro Ser Val 20 25
30Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
35 40 45Pro Glu Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro Glu 50 55
60Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys65
70 75 80Thr Lys Pro Arg Glu
Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 85
90 95Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys 100 105
110Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
115 120 125Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro 130 135
140Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu145 150 155 160Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
165 170 175Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser 180 185
190Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg 195 200 205Trp Gln Glu Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 210
215 220His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Leu Gly Ile Pro225 230 235
240Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn
245 250 255Asn Gly Ala Val Lys
Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg 260
265 270Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser
Asn Cys Ser Ile 275 280 285Thr Ser
Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg 290
295 300Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val
Cys His Asp Pro Lys305 310 315
320Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys
325 330 335Ile Met Lys Glu
Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser 340
345 350Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile
Phe Ser Glu Glu Tyr 355 360 365Asn
Thr Ser Asn Pro Asp 370143508PRTArtificial
SequenceD10-hIgG1FcdeltaK-CC-T22d35 fusion 143Asp Asp Asp Asp Asp Asp Asp
Asp Asp Asp Asp Lys Thr His Thr Cys1 5 10
15Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu 20 25 30Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 35
40 45Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys 50 55 60Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys65
70 75 80Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu 85
90 95Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys 100 105 110Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 115
120 125Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser 130 135
140Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys145
150 155 160Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 165
170 175Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly 180 185
190Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
195 200 205Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn 210 215
220His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ile Pro Pro
His225 230 235 240Val Gln
Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly
245 250 255Ala Val Lys Phe Pro Gln Leu
Cys Lys Phe Cys Asp Val Arg Phe Ser 260 265
270Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile
Thr Ser 275 280 285Ile Cys Glu Lys
Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn 290
295 300Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp
Pro Lys Leu Pro305 310 315
320Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met
325 330 335Lys Glu Lys Lys Lys
Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser 340
345 350Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu
Glu Tyr Asn Thr 355 360 365Ser Asn
Pro Asp Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp 370
375 380Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys
Phe Pro Gln Leu Cys385 390 395
400Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys
405 410 415Met Ser Asn Cys
Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val 420
425 430Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn
Ile Thr Leu Glu Thr 435 440 445Val
Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp 450
455 460Ala Ala Ser Pro Lys Cys Ile Met Lys Glu
Lys Lys Lys Pro Gly Glu465 470 475
480Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn
Ile 485 490 495Ile Phe Ser
Glu Glu Tyr Asn Thr Ser Asn Pro Asp 500
505144504PRTArtificial SequenceD10-hIgG2FcdeltaK-CC-T22d35 fusion 144Asp
Asp Asp Asp Asp Asp Asp Asp Asp Asp Val Glu Cys Pro Pro Cys1
5 10 15Pro Ala Pro Pro Val Ala Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys 20 25
30Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val 35 40 45Val Val Asp Val
Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 50 55
60Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu65 70 75
80Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His
85 90 95Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 100
105 110Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Thr Lys Gly Gln 115 120 125Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 130
135 140Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro145 150 155
160Ser Asp Ile Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175Tyr Lys Thr Thr
Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 180
185 190Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val 195 200 205Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 210
215 220Lys Ser Leu Ser Leu Ser Pro Gly Ile Pro
Pro His Val Gln Lys Ser225 230 235
240Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys
Phe 245 250 255Pro Gln Leu
Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn 260
265 270Gln Lys Ser Cys Met Ser Asn Cys Ser Ile
Thr Ser Ile Cys Glu Lys 275 280
285Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile 290
295 300Thr Leu Glu Thr Val Cys His Asp
Pro Lys Leu Pro Tyr His Asp Phe305 310
315 320Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met
Lys Glu Lys Lys 325 330
335Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys
340 345 350Asn Asp Asn Ile Ile Phe
Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 355 360
365Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile
Val Thr 370 375 380Asp Asn Asn Gly Ala
Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp385 390
395 400Val Arg Phe Ser Thr Cys Asp Asn Gln Lys
Ser Cys Met Ser Asn Cys 405 410
415Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val
420 425 430Trp Arg Lys Asn Asp
Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 435
440 445Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp
Ala Ala Ser Pro 450 455 460Lys Cys Ile
Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met465
470 475 480Cys Ser Cys Ser Ser Asp Glu
Cys Asn Asp Asn Ile Ile Phe Ser Glu 485
490 495Glu Tyr Asn Thr Ser Asn Pro Asp
500145510PRTArtificial SequenceD10-hIgG4FcdeltaK-CC-228P-T22d35 fusion
145Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Glu Ser Lys Tyr Gly Pro1
5 10 15Pro Cys Pro Pro Cys Pro
Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 20 25
30Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr 35 40 45Pro Glu Val
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 50
55 60Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys65 70 75
80Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
85 90 95Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 100
105 110Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile 115 120 125Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 130
135 140Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu145 150 155
160Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
165 170 175Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 180
185 190Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
Val Asp Lys Ser Arg 195 200 205Trp
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 210
215 220His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Leu Gly Ile Pro225 230 235
240Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp
Asn 245 250 255Asn Gly Ala
Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg 260
265 270Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys
Met Ser Asn Cys Ser Ile 275 280
285Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg 290
295 300Lys Asn Asp Glu Asn Ile Thr Leu
Glu Thr Val Cys His Asp Pro Lys305 310
315 320Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala
Ser Pro Lys Cys 325 330
335Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser
340 345 350Cys Ser Ser Asp Glu Cys
Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr 355 360
365Asn Thr Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys Ser
Val Asn 370 375 380Asn Asp Met Ile Val
Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln385 390
395 400Leu Cys Lys Phe Cys Asp Val Arg Phe Ser
Thr Cys Asp Asn Gln Lys 405 410
415Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln
420 425 430Glu Val Cys Val Ala
Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu 435
440 445Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His
Asp Phe Ile Leu 450 455 460Glu Asp Ala
Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro465
470 475 480Gly Glu Thr Phe Phe Met Cys
Ser Cys Ser Ser Asp Glu Cys Asn Asp 485
490 495Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn
Pro Asp 500 505
510146510PRTArtificial SequenceD10-hIgG4FcdeltaK-CC-228P-409K-T22d35
fusion 146Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Glu Ser Lys Tyr Gly
Pro1 5 10 15Pro Cys Pro
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 20
25 30Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr 35 40
45Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 50
55 60Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys65 70 75
80Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
Val Ser 85 90 95Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 100
105 110Cys Lys Val Ser Asn Lys Gly Leu Pro
Ser Ser Ile Glu Lys Thr Ile 115 120
125Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
130 135 140Pro Ser Gln Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu145 150
155 160Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn 165 170
175Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
180 185 190Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 195 200
205Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 210 215 220His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Leu Gly Ile Pro225 230
235 240Pro His Val Gln Lys Ser Val Asn Asn Asp
Met Ile Val Thr Asp Asn 245 250
255Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg
260 265 270Phe Ser Thr Cys Asp
Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile 275
280 285Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val
Ala Val Trp Arg 290 295 300Lys Asn Asp
Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys305
310 315 320Leu Pro Tyr His Asp Phe Ile
Leu Glu Asp Ala Ala Ser Pro Lys Cys 325
330 335Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe
Phe Met Cys Ser 340 345 350Cys
Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr 355
360 365Asn Thr Ser Asn Pro Asp Ile Pro Pro
His Val Gln Lys Ser Val Asn 370 375
380Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln385
390 395 400Leu Cys Lys Phe
Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys 405
410 415Ser Cys Met Ser Asn Cys Ser Ile Thr Ser
Ile Cys Glu Lys Pro Gln 420 425
430Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu
435 440 445Glu Thr Val Cys His Asp Pro
Lys Leu Pro Tyr His Asp Phe Ile Leu 450 455
460Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys
Pro465 470 475 480Gly Glu
Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp
485 490 495Asn Ile Ile Phe Ser Glu Glu
Tyr Asn Thr Ser Asn Pro Asp 500 505
510147644PRTArtificial SequenceD10-hIgG1FcdeltaK-CC-T2-T2-T2 fusion
147Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Lys Thr His Thr Cys1
5 10 15Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 20 25
30Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu 35 40 45Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 50
55 60Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys65 70 75
80Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
85 90 95Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 100
105 110Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys 115 120 125Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 130
135 140Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys145 150 155
160Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
165 170 175Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 180
185 190Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln 195 200 205Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 210
215 220His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Ile Pro Pro His225 230 235
240Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn
Gly 245 250 255Ala Val Lys
Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser 260
265 270Thr Cys Asp Asn Gln Lys Ser Cys Met Ser
Asn Cys Ser Ile Thr Ser 275 280
285Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn 290
295 300Asp Glu Asn Ile Thr Leu Glu Thr
Val Cys His Asp Pro Lys Leu Pro305 310
315 320Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro
Lys Cys Ile Met 325 330
335Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser
340 345 350Ser Asp Glu Cys Asn Asp
Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr 355 360
365Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys Ser Val Asn
Asn Asp 370 375 380Met Ile Val Thr Asp
Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys385 390
395 400Lys Phe Cys Asp Val Arg Phe Ser Thr Cys
Asp Asn Gln Lys Ser Cys 405 410
415Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val
420 425 430Cys Val Ala Val Trp
Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr 435
440 445Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe
Ile Leu Glu Asp 450 455 460Ala Ala Ser
Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu465
470 475 480Thr Phe Phe Met Cys Ser Cys
Ser Ser Asp Glu Cys Asn Asp Asn Ile 485
490 495Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp
Ile Pro Pro His 500 505 510Val
Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly 515
520 525Ala Val Lys Phe Pro Gln Leu Cys Lys
Phe Cys Asp Val Arg Phe Ser 530 535
540Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser545
550 555 560Ile Cys Glu Lys
Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn 565
570 575Asp Glu Asn Ile Thr Leu Glu Thr Val Cys
His Asp Pro Lys Leu Pro 580 585
590Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met
595 600 605Lys Glu Lys Lys Lys Pro Gly
Glu Thr Phe Phe Met Cys Ser Cys Ser 610 615
620Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn
Thr625 630 635 640Ser Asn
Pro Asp148640PRTArtificial SequenceD10-hIgG2FcdeltaK-CC-T2-T2-T2 fusion
148Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Val Glu Cys Pro Pro Cys1
5 10 15Pro Ala Pro Pro Val Ala
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 20 25
30Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val 35 40 45Val Val Asp
Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 50
55 60Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu65 70 75
80Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His
85 90 95Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 100
105 110Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Thr Lys Gly Gln 115 120 125Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 130
135 140Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro145 150 155
160Ser Asp Ile Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175Tyr Lys Thr Thr
Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 180
185 190Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val 195 200 205Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 210
215 220Lys Ser Leu Ser Leu Ser Pro Gly Ile Pro
Pro His Val Gln Lys Ser225 230 235
240Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys
Phe 245 250 255Pro Gln Leu
Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn 260
265 270Gln Lys Ser Cys Met Ser Asn Cys Ser Ile
Thr Ser Ile Cys Glu Lys 275 280
285Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile 290
295 300Thr Leu Glu Thr Val Cys His Asp
Pro Lys Leu Pro Tyr His Asp Phe305 310
315 320Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met
Lys Glu Lys Lys 325 330
335Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys
340 345 350Asn Asp Asn Ile Ile Phe
Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 355 360
365Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile
Val Thr 370 375 380Asp Asn Asn Gly Ala
Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp385 390
395 400Val Arg Phe Ser Thr Cys Asp Asn Gln Lys
Ser Cys Met Ser Asn Cys 405 410
415Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val
420 425 430Trp Arg Lys Asn Asp
Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 435
440 445Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp
Ala Ala Ser Pro 450 455 460Lys Cys Ile
Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met465
470 475 480Cys Ser Cys Ser Ser Asp Glu
Cys Asn Asp Asn Ile Ile Phe Ser Glu 485
490 495Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro His
Val Gln Lys Ser 500 505 510Val
Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe 515
520 525Pro Gln Leu Cys Lys Phe Cys Asp Val
Arg Phe Ser Thr Cys Asp Asn 530 535
540Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys545
550 555 560Pro Gln Glu Val
Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile 565
570 575Thr Leu Glu Thr Val Cys His Asp Pro Lys
Leu Pro Tyr His Asp Phe 580 585
590Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys
595 600 605Lys Pro Gly Glu Thr Phe Phe
Met Cys Ser Cys Ser Ser Asp Glu Cys 610 615
620Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro
Asp625 630 635
640149646PRTArtificial SequenceD10-hIgG4FcdeltaK-CC-228P-T2-T2-T2 fusion
149Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Glu Ser Lys Tyr Gly Pro1
5 10 15Pro Cys Pro Pro Cys Pro
Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 20 25
30Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr 35 40 45Pro Glu Val
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 50
55 60Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys65 70 75
80Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
85 90 95Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 100
105 110Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile 115 120 125Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 130
135 140Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu145 150 155
160Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
165 170 175Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 180
185 190Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
Val Asp Lys Ser Arg 195 200 205Trp
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 210
215 220His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Leu Gly Ile Pro225 230 235
240Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp
Asn 245 250 255Asn Gly Ala
Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg 260
265 270Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys
Met Ser Asn Cys Ser Ile 275 280
285Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg 290
295 300Lys Asn Asp Glu Asn Ile Thr Leu
Glu Thr Val Cys His Asp Pro Lys305 310
315 320Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala
Ser Pro Lys Cys 325 330
335Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser
340 345 350Cys Ser Ser Asp Glu Cys
Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr 355 360
365Asn Thr Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys Ser
Val Asn 370 375 380Asn Asp Met Ile Val
Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln385 390
395 400Leu Cys Lys Phe Cys Asp Val Arg Phe Ser
Thr Cys Asp Asn Gln Lys 405 410
415Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln
420 425 430Glu Val Cys Val Ala
Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu 435
440 445Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His
Asp Phe Ile Leu 450 455 460Glu Asp Ala
Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro465
470 475 480Gly Glu Thr Phe Phe Met Cys
Ser Cys Ser Ser Asp Glu Cys Asn Asp 485
490 495Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn
Pro Asp Ile Pro 500 505 510Pro
His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn 515
520 525Asn Gly Ala Val Lys Phe Pro Gln Leu
Cys Lys Phe Cys Asp Val Arg 530 535
540Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile545
550 555 560Thr Ser Ile Cys
Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg 565
570 575Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr
Val Cys His Asp Pro Lys 580 585
590Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys
595 600 605Ile Met Lys Glu Lys Lys Lys
Pro Gly Glu Thr Phe Phe Met Cys Ser 610 615
620Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu
Tyr625 630 635 640Asn Thr
Ser Asn Pro Asp 645150649PRTArtificial
SequenceD10-hIgG4FcdeltaK-CC-228P-409K-T2-T2-T2 fusion 150Asp Asp Asp Asp
Asp Asp Asp Asp Asp Asp Glu Ser Lys Tyr Gly Pro1 5
10 15Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
Leu Gly Gly Pro Ser Val 20 25
30Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
35 40 45Pro Glu Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro Glu 50 55
60Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys65
70 75 80Thr Lys Pro Arg Glu
Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 85
90 95Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys 100 105
110Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Ser Glu Gln Glu
115 120 125Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr 130 135
140Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
Leu145 150 155 160Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
165 170 175Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val 180 185
190Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp 195 200 205Lys Ser Arg Trp
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 210
215 220Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Leu225 230 235
240Gly Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val
245 250 255Thr Asp Asn Asn Gly
Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys 260
265 270Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser
Cys Met Ser Asn 275 280 285Cys Ser
Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala 290
295 300Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu
Glu Thr Val Cys His305 310 315
320Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser
325 330 335Pro Lys Cys Ile
Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe 340
345 350Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp
Asn Ile Ile Phe Ser 355 360 365Glu
Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys 370
375 380Ser Val Asn Asn Asp Met Ile Val Thr Asp
Asn Asn Gly Ala Val Lys385 390 395
400Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys
Asp 405 410 415Asn Gln Lys
Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu 420
425 430Lys Pro Gln Glu Val Cys Val Ala Val Trp
Arg Lys Asn Asp Glu Asn 435 440
445Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp 450
455 460Phe Ile Leu Glu Asp Ala Ala Ser
Pro Lys Cys Ile Met Lys Glu Lys465 470
475 480Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys
Ser Ser Asp Glu 485 490
495Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro
500 505 510Asp Ile Pro Pro His Val
Gln Lys Ser Val Asn Asn Asp Met Ile Val 515 520
525Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys
Phe Cys 530 535 540Asp Val Arg Phe Ser
Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn545 550
555 560Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro
Gln Glu Val Cys Val Ala 565 570
575Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His
580 585 590Asp Pro Lys Leu Pro
Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser 595
600 605Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly
Glu Thr Phe Phe 610 615 620Met Cys Ser
Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser625
630 635 640Glu Glu Tyr Asn Thr Ser Asn
Pro Asp 645
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