Novel anti-cancer agents, including, but not limited to, antibodies and
immunoconjugates, that bind to human folate receptor 1 are provided.
Methods of using the agents, antibodies, or immunoconjugates, such as
methods of inhibiting tumor growth are further provided.
| Inventors: |
AB; Olga; (Millis, MA)
; TAVARES; Daniel; (Natick, MA)
; RUI; Lingyun; (Weston, MA)
; PAYNE; Gillian; (Waban, MA)
; GOLDMAKHER; Viktor S.; (Newton, MA)
|
| Applicant: | | Name | City | State | Country | Type |
ImmunoGen, Inc. |
Waltham |
MA |
US
| | |
|---|
| Assignee: |
ImmunoGen, Inc.
Waltham
MA
|
| Family ID:
|
44507210
|
| Appl. No.:
|
16/384031
|
| Filed:
|
April 15, 2019 |
Related U.S. Patent Documents
| | | | |
|---|
|
| Application Number | Filing Date | Patent Number |
|---|
| | 15583281 | May 1, 2017 | 10301385 |
| | 16384031 | | |
| | 14819209 | Aug 5, 2015 | 9657100 |
| | 15583281 | | |
| | 13800835 | Mar 13, 2013 | 9133275 |
| | 14819209 | | |
| | 13033723 | Feb 24, 2011 | 8557966 |
| | 13800835 | | |
| | 61307797 | Feb 24, 2010 | |
| | 61346595 | May 20, 2010 | |
| | 61413172 | Nov 12, 2010 | |
|
|
| Current U.S. Class: |
1/1 |
| Current CPC Class: |
C07K 2317/14 20130101; C12N 15/63 20130101; A61K 39/39558 20130101; C07K 2317/526 20130101; C07K 2317/53 20130101; C07K 2317/54 20130101; A61K 47/6851 20170801; C07K 2317/31 20130101; C07K 2317/56 20130101; A61K 47/6849 20170801; A61K 45/06 20130101; C12N 5/16 20130101; C07K 2317/565 20130101; C12N 15/62 20130101; C07K 2317/626 20130101; A61K 47/6803 20170801; C07K 2317/622 20130101; A61K 47/6889 20170801; A61K 31/5365 20130101; C07H 21/00 20130101; A61K 2039/505 20130101; A61P 35/04 20180101; A61P 35/02 20180101; C07K 16/28 20130101; C12N 15/79 20130101; A61K 39/3955 20130101; C07K 16/30 20130101; C07K 2317/52 20130101; C07K 2317/24 20130101; C07K 2317/524 20130101; C07K 2317/55 20130101; C07K 2317/92 20130101; C07K 2317/94 20130101; C12N 15/70 20130101; C07K 2317/624 20130101; C07K 2317/71 20130101; C07K 2317/73 20130101; A61K 47/545 20170801; C07K 16/3069 20130101; C12N 2800/00 20130101; A61P 43/00 20180101; C07K 2317/732 20130101; A61P 35/00 20180101; A61K 31/537 20130101 |
| International Class: |
C07K 16/28 20060101 C07K016/28; A61K 31/537 20060101 A61K031/537; A61K 39/395 20060101 A61K039/395; A61K 45/06 20060101 A61K045/06; C07K 16/30 20060101 C07K016/30; A61K 47/68 20060101 A61K047/68; A61K 31/5365 20060101 A61K031/5365; A61K 47/54 20060101 A61K047/54 |
Claims
1-127. (canceled)
128. A method of treating cancer in a subject, comprising administering a
therapeutically effective amount of an antibody or antigen binding
fragment thereof that specifically binds human folate receptor 1 (FOLR1)
to the subject, wherein the antibody or antigen binding fragment thereof
comprises: (a) a heavy chain CDR1 comprising the amino acid sequence
SSFGMH (SEQ ID NO:72); a heavy chain CDR2 comprising the amino acid
sequence YISSGSSTIS (SEQ ID NO:73); and a heavy chain CDR3 comprising the
amino acid sequence EAYGSSMEY (SEQ ID NO:74) and (b) a light chain CDR1
comprising the amino acid sequence RASQNINNNLH (SEQ ID NO:69); a light
chain CDR2 comprising the amino acid sequence YVSQSVS (SEQ ID NO:70); and
a light chain CDR3 comprising the amino acid sequence QQSNSWPHYT (SEQ ID
NO:71).
129. The method of claim 128, wherein the heavy chain CDR2 comprises the
amino acid sequence YISSGSSTISYADSVKG (SEQ ID NO:85).
130. The method of claim 129, wherein the antibody or antigen binding
fragment thereof comprises a heavy chain variable domain comprising the
amino acid sequence of SEQ ID NO:101 and a light chain variable domain
comprising the amino acid sequence of SEQ ID NO:100.
131. The method of claim 128 wherein the antibody or antigen binding
fragment thereof is a full length antibody.
132. The method of claim 128, wherein the antibody or antigen binding
fragment thereof is an antigen binding fragment, wherein the antigen
binding fragment comprises a Fab, a Fab', a F(ab')2, a single chain Fv
(scFv), a disulfide linked Fv, an IgG-CH2, a F(ab')3, a tetrabody, a
triabody, a diabody, a (scFv)2, or a scFv-Fc.
133. The method of claim 128, wherein the antibody binds to human FOLR1
with a Kd of 1.0 nM or better.
134. The method of claim 128, wherein the antibody binds to human FOLR1
with a Kd of about 0.06 nM to about 1.0 nM.
135. The method of claim 128, wherein the cancer is selected from the
group consisting of ovarian cancer, breast cancer, uterine cancer,
pancreatic cancer, renal cancer, peritoneal cancer, and lung cancer.
136. The method of claim 128, wherein the cancer is endometrial cancer.
137. A method of treating cancer in a subject, comprising administering a
therapeutically effective amount of an immunoconjugate having the formula
(A)-(L)-(C) to the subject, wherein: (A) is the antibody or antigen
binding fragment thereof of claim 128; (L) is a linker; and (C) is a
cytotoxic agent; wherein (L) links (A) to (C).
138. The method of claim 137, wherein the linker is a cleavable linker.
139. The method of claim 137, wherein the linker is a non-cleavable
linker,
140. The method of claim 137, wherein the linker is a hydrophilic linker
or a dicarboxylic acid based linker.
141. The method of claim 140, wherein the linker is selected from the
group consisting: N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) or
N-succinimidyl 4-(2-pyridyldithio)-2-sulfopentanoate (sulfo-SPP);
N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) or N-succinimidyl
4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB); N-succinimidyl
4-(maleimidomethyl) cyclohexanecarboxylate (SMCC); N-sulfosuccinimidyl
4-(maleimidomethyl) cyclohexanecarboxylate (sulfoSMCC);
N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB); and
N-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol] ester
(NHS-PEG4-maleimide).
142. The method of claim 137, wherein the cytotoxic agent is selected
from the group consisting of a maytansinoid, benzodiazepine, taxoid,
CC-1065, duocarmycin, calicheamicin, dolastatin, auristatin, tomaymycin
and leptomycin or a prodrug of the agent.
143. The method of claim 142, wherein the maytansinoid is
N(2')-deacetyl-N(2')-(3-mercapto-1-oxopropyl)-maytansine (DM1) or
N(2')-deacetyl-N(2')-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4).
144. The method of claim 137, wherein (L) is sulfo-SPDB and (C) is DM4.
145. The method of claim 137, wherein the immunoconjugate comprises 2-6
(C) or 3-4 (C).
146. The method of claim 137, wherein the cancer is selected from the
group consisting of ovarian cancer, breast cancer, uterine cancer,
pancreatic cancer, renal cancer, peritoneal cancer, and lung cancer.
147. The method of claim 137, wherein the cancer is endometrial cancer.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser. No.
14/819,209, filed Aug. 5, 2015, now allowed, which is a divisional of
U.S. application Ser. No. 13/800,835, filed Mar. 13, 2013, now U.S. Pat.
No. 9,133,275, issued Sep. 15, 2015, which is a divisional application of
U.S. application Ser. No. 13/033,723, filed Feb. 24, 2011, now U.S. Pat.
No. 8,557,966, issued Oct. 15, 2013, which claims the priority benefit of
U.S. Provisional Application No. 61/307,797, filed Feb. 24, 2010, U.S.
Provisional Application No. 61/346,595, filed May 20, 2010, and U.S.
Provisional Application No. 61/413,172, filed Nov. 12, 2010, each of
which is hereby incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0002] The field of this invention generally relates to antibodies and
immunoconjugates that bind to human folate receptor 1, as well as to
methods of using the antibodies and immunoconjugates for the treatment of
diseases, such as cancer.
SEQUENCE LISTING
[0003] Submitted concurrently on EFS-Web as part of the originally filed
subject matter is a sequence listing of amino acid and polynucleotide
sequences described throughout the specification. The sequence listing
text file concurrently submitted with the specification is herein
incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0004] Cancer is one of the leading causes of death in the developed
world, with over one million people diagnosed with cancer and 500,000
deaths per year in the United States alone. Overall it is estimated that
more than 1 in 3 people will develop some form of cancer during their
lifetime. There are more than 200 different types of cancer, four of
which--breast, lung, colorectal, and prostate--account for over half of
all new cases (Jemal et al., 2003, Cancer J. Clin. 53:5-26).
[0005] Folate Receptor 1 (FOLR1), also known as Folate Receptor-alpha, or
Folate Binding Protein, is an N-glycosylated protein expressed on plasma
membrane of cells. FOLR1 has a high affinity for folic acid and for
several reduced folic acid derivatives. FOLR1 mediates delivery of the
physiological folate, 5-methyltetrahydrofolate, to the interior of cells.
[0006] FOLR1 is overexpressed in vast majority of ovarian cancers, as well
as in many uterine, endometrial, pancreatic, renal, lung, and breast
cancers, while the expression of FOLR1 on normal tissues is restricted to
the apical membrane of epithelial cells in the kidney proximal tubules,
alveolar pneumocytes of the lung, bladder, testes, choroid plexus, and
thyroid (Weitman S D, et al., Cancer Res 52: 3396-3401 (1992); Antony A
C, Annu Rev Nutr 16: 501-521 (1996); Kalli K R, et al. Gynecol Oncol 108:
619-626 (2008)). This expression pattern of FOLR1 makes it a desirable
target for FOLR1-directed cancer therapy.
[0007] Because ovarian cancer is typically asymptomatic until advanced
stage, it is often diagnosed at a late stage and has poor prognosis when
treated with currently available procedures, typically chemotherapeutic
drugs after surgical de-bulking (von Gruenigen V et al., Cancer 112:
2221-2227 (2008); Ayhan A et al., Am J Obstet Gynecol 196: 81 e81-86
(2007); Harry V N et al., Obstet Gynecol Surv 64: 548-560 (2009)). Thus
there is a clear unmet medical need for more effective therapeutics for
ovarian cancers.
[0008] Three anti-FOLR1 antibodies have been examined as potential
anti-cancer drugs. Murine monoclonal antibodies Mov18 and Mov19 were
isolated in the late 1980s (Miotti S et al., Int J Cancer 39: 297-303
(1987)), confirmed to target FOLR1 (Coney L R et al., Cancer Res 51:
6125-6132 (1991)), and tested in pre-clinical studies for their ability
to eradicate antigen-expressing cancer cells as conjugates with a
cytotoxic ribosome-inactivating protein (Conde F P et al., Eur J Biochem
178: 795-802 (1989)).
[0009] Mov19 was tested as a bi-specific antibody targeting cytotoxic T
cells and natural killer cells (Mezzanzanica D et al., Int J Cancer 41:
609-615 (1988); Ferrini S et al., Int J Cancer Suppl 4: 53-55 (1989);
Ferrini S et al., Int J Cancer 48: 227-233 (1991)), and as a fusion
protein of the single-chain Fv (scFv) of Mov19 with interleukin-2 in vivo
(Melani C et al., Cancer Res 58: 4146-4154 (1998)). Chimeric (murine
variable/human constant) anti-FOLR1 antibodies Mov18 and Mov19 have been
examined pre-clinically on their ability to mediate cytotoxic immune
cell-dependent killing of FOLR1-expressing tumor cells in vitro (Coney L
R et al., Cancer Res 54: 2448-2455 (1994)), and a chimeric Mov18-IgE was
tested in IgE-dependent immunotherapeutic preclinical models (Karagiannis
S N et al., J Immunol 179: 2832-2843 (2007); Gould H J et al., Eur J
Immunol 29: 3527-3537 (1999)).
[0010] Mov18 was studied in the form of conjugates with various
radionuclides in preclinical studies and then, in early 1990s, in
clinical trials (Zacchetti A et al., Nucl Med Biol 36: 759-770 (2009)),
which ended without any drug being approved for clinical use.
[0011] MORAb003, a humanized form of the murine monoclonal anti-FOLR1
antibody LK26 was evaluated pre-clinically as a non-modified antibody
(Ebel W et al., Cancer Immun 7:6 (2007)) and as a conjugate with the
.sup.111In radionuclide (Smith-Jones P M et al., Nucl Med Biol 35:
343-351 (2008)), and is currently undergoing clinical trials as a
non-modified antibody (D. K. Armstrong et al. J. Clin. Oncol. 26: 2008,
May 20 suppl; abstract 5500).
SUMMARY OF THE INVENTION
[0012] The present invention provides novel antibodies that bind to human
folate receptor 1, immunoconjugates comprising these antibodies, and
methods of their use. The present invention further provides novel
polypeptides, such as antibodies that bind human folate receptor 1,
fragments of such antibodies, and other polypeptides related to such
antibodies. Polynucleotides comprising nucleic acid sequences encoding
the polypeptides are also provided, as are vectors comprising the
polynucleotides. Cells comprising the polypeptides and/or polynucleotides
of the invention are further provided. Compositions (e.g., pharmaceutical
compositions) comprising the novel folate receptor 1 antibodies or
immunoconjugates are also provided. In addition, methods of making and
using the novel folate receptor 1 antibodies or immunoconjugates are also
provided, such as methods of using the novel folate receptor 1 antibodies
or immunoconjugates to inhibit tumor growth and/or treat cancer.
[0013] Thus, in one aspect, the invention provides a humanized antibody or
antigen binding fragment thereof that specifically binds a human folate
receptor 1, wherein the antibody comprises (a) a heavy chain CDR1
comprising GYFMN (SEQ ID NO:1); a heavy chain CDR2 comprising
RIHPYDGDTFYNQXaa.sub.1FXaa.sub.2Xaa.sub.3 (SEQ ID NO:56); and a heavy
chain CDR3 comprising YDGSRAMDY (SEQ ID NO:3); and (b) a light chain CDR1
comprising KASQSVSFAGTSLMH (SEQ ID NO:7); a light chain CDR2 comprising
RASNLEA (SEQ ID NO:8); and a light chain CDR3 comprising QQSREYPYT (SEQ
ID NO:9); wherein Xaa.sub.1 is selected from K, Q, H, and R; Xaa.sub.2 is
selected from Q, H, N, and R; and Xaa.sub.3 is selected from G, E, T, S,
A, and V. In a certain embodiment, the humanized antibody or antigen
binding fragment thereof binds a human folate receptor 1 with
substantially the same affinity as the antibody chimeric Mov19. In a
certain embodiment, the humanized antibody or antigen binding fragment
thereof comprises the heavy chain CDR2 sequence RIHPYDGDTFYNQKFQG (SEQ ID
NO:2).
[0014] In a certain embodiment, the binding affinity is measured by flow
cytometry, Biacore, or radioimmunoassay.
[0015] In another embodiment, the invention provides a humanized antibody
or antigen binding fragment thereof that specifically binds a human
folate receptor 1, wherein the antibody comprises: (a) a heavy chain CDR1
comprising GYFMN (SEQ ID NO:1), or a variant thereof comprising 1, 2, 3,
or 4 conservative amino acid substitutions; a heavy chain CDR2 comprising
RIHPYDGDTFYNQKFQG (SEQ ID NO:2), or a variant thereof comprising 1, 2, 3,
or 4 amino conservative acid substitutions; and a heavy chain CDR3
comprising YDGSRAMDY (SEQ ID NO:3), or a variant thereof comprising 1, 2,
3, or 4 conservative amino acid substitutions; and/or (b) a light chain
CDR1 comprising KASQSVSFAGTSLMH (SEQ ID NO:7), or a variant thereof
comprising 1, 2, 3, or 4 conservative amino acid substitutions; a light
chain CDR2 comprising RASNLEA (SEQ ID NO:8), or a variant thereof
comprising 1, 2, 3, or 4 conservative amino acid substitutions; and a
light chain CDR3 comprising QQSREYPYT (SEQ ID NO:9), or a variant thereof
comprising 1, 2, 3, or 4 conservative amino acid substitutions.
[0016] In a certain embodiment, the invention provides a humanized
antibody or antigen binding fragment thereof that specifically binds the
human folate receptor 1 comprising the heavy chain of SEQ ID NO:6. In
another embodiment, the humanized antibody or antigen binding fragment
thereof is encoded by the plasmid DNA deposited with the ATCC on Apr. 7,
2010 and having ATCC deposit nos. PTA-10772 and PTA-10773 or 10774.
[0017] In a certain embodiment, the invention provides a humanized
antibody or antigen binding fragment thereof that competes for binding to
FOLR1 with an antibody comprising (a) a heavy chain CDR1 comprising GYFMN
(SEQ ID NO:1); a heavy chain CDR2 comprising
RIHPYDGDTFYNQXaa.sub.1FXaa.sub.2Xaa.sub.3 (SEQ ID NO:56); and a heavy
chain CDR3 comprising YDGSRAMDY (SEQ ID NO:3); and (b) a light chain CDR1
comprising KASQSVSFAGTSLMH (SEQ ID NO:7); a light chain CDR2 comprising
RASNLEA (SEQ ID NO:8); and a light chain CDR3 comprising QQSREYPYT (SEQ
ID NO:9); wherein Xaa.sub.1 is selected from K, Q, H, and R; Xaa.sub.2 is
selected from Q, H, N, and R; and Xaa.sub.3 is selected from G, E, T, S,
A, and V. In a certain embodiment, the humanized antibody comprises the
heavy chain CDR2 sequence RIHPYDGDTFYNQKFQG (SEQ ID NO:2).
[0018] In a certain embodiment, the invention provides a polypeptide,
humanized antibody or antigen binding fragment thereof comprising a heavy
chain variable domain at least about 90% identical to SEQ ID NO:4, and a
light chain variable domain at least about 90% identical to SEQ ID NO:10
or SEQ ID NO:11. In another embodiment, the humanized antibody or antigen
binding fragment comprises a heavy chain variable domain at least about
95% identical to SEQ ID NO:4, and a light chain variable domain at least
about 95% identical to SEQ ID NO:10 or SEQ ID NO:11. In a further
embodiment, the humanized antibody comprises a heavy chain variable
domain at least about 99% identical to SEQ ID NO:4, and a light chain
variable domain at least about 99% identical to SEQ ID NO:10 or SEQ ID
NO:11. In a certain embodiment, the humanized antibody comprises the
heavy chain variable domain of SEQ ID NO:4, and the light chain variable
domain of SEQ ID NO:10 or SEQ ID NO:11. In certain embodiments, the
invention provides a polypeptide, antibody, or antigen binding fragment
at least about 90% identical to SEQ ID NOs: 88-119. In certain
embodiments, the invention provides a polypeptide, antibody, or antigen
binding fragment at least about 95% identical to SEQ ID NOs: 88-119. In
certain embodiments, the invention provides a polypeptide, antibody, or
antigen binding fragment at least about 99% identical to SEQ ID NOs:
88-119.
[0019] In a certain embodiment, the invention provides a humanized
antibody or antigen binding fragment thereof that is expressed at least
ten-fold higher than chMov19 in eukaryotic cells. In a certain
embodiment, the eukaryotic cells are HEK-293T cells.
[0020] In certain embodiments, the invention provides an antibody or
antigen binding fragment thereof that specifically binds a human folate
receptor 1, wherein the antibody comprises: (a) a heavy chain CDR1
comprising SSYGMS (SEQ ID NO:30); a heavy chain CDR2 comprising
TISSGGSYTY (SEQ ID NO:31); and/or a heavy chain CDR3 comprising
DGEGGLYAMDY (SEQ ID NO:32); and/or (b) a light chain CDR1 comprising
KASDHINNWLA (SEQ ID NO:27); a light chain CDR2 comprising GATSLET (SEQ ID
NO:28); and a light chain CDR3 comprising QQYWSTPFT (SEQ ID NO:29). In
another embodiment, the invention provides an antibody or antigen binding
fragment thereof that specifically binds a human folate receptor 1,
wherein the antibody comprises: (a) a heavy chain CDR1 comprising TNYWMQ
(SEQ ID NO:60); a heavy chain CDR2 comprising AIYPGNGDSR (SEQ ID NO:61);
and/or a heavy chain CDR3 comprising RDGNYAAY (SEQ ID NO:62); and/or (b)
a light chain CDR1 comprising RASENIYSNLA (SEQ ID NO:57); a light chain
CDR2 comprising AATNLAD (SEQ ID NO:58); and a light chain CDR3 comprising
QHFWASPYT (SEQ ID NO:59). In another embodiment, the invention provides
an antibody or antigen binding fragment thereof that specifically binds a
human folate receptor 1, wherein the antibody comprises: (a) a heavy
chain CDR1 comprising TNYWMY (SEQ ID NO:66); a heavy chain CDR2
comprising AIYPGNSDTT (SEQ ID NO:67); and/or a heavy chain CDR3
comprising RHDYGAMDY (SEQ ID NO:68); and/or (b) a light chain CDR1
comprising RASENIYTNLA (SEQ ID NO:63); a light chain CDR2 comprising
TASNLAD (SEQ ID NO:64); and a light chain CDR3 comprising QHFWVSPYT (SEQ
ID NO:65). In another embodiment, the invention provides an antibody or
antigen binding fragment thereof that specifically binds a human folate
receptor 1, wherein the antibody comprises: (a) a heavy chain CDR1
comprising SSFGMH (SEQ ID NO:72); a heavy chain CDR2 comprising
YISSGSSTIS (SEQ ID NO:73); and/or a heavy chain CDR3 comprising EAYGSSMEY
(SEQ ID NO:74); and/or (b) a light chain CDR1 comprising RASQNINNNLH (SEQ
ID NO:69); a light chain CDR2 comprising YVSQSVS (SEQ ID NO:70); and a
light chain CDR3 comprising QQSNSWPHYT (SEQ ID NO:71). In another
embodiment, the invention provides an antibody or antigen binding
fragment thereof that specifically binds a human folate receptor 1,
wherein the antibody comprises: (a) a heavy chain CDR1 comprising TSYTMH
(SEQ ID NO:78); a heavy chain CDR2 comprising YINPISGYTN (SEQ ID NO:79);
and/or a heavy chain CDR3 comprising GGAYGRKPMDY (SEQ ID NO:80); and/or
(b) a light chain CDR1 comprising KASQNVGPNVA (SEQ ID NO:75); a light
chain CDR2 comprising SASYRYS (SEQ ID NO:76); and a light chain CDR3
comprising QQYNSYPYT (SEQ ID NO:77).
[0021] In certain embodiments, the polypeptides of the invention are
full-length antibodies or antigen binding fragments. In certain
embodiments, the antibodies or antigen binding fragments are a Fab, a
Fab', a F(ab')2, a Fd, a single chain Fv or scFv, a disulfide linked Fv,
a V NAR domain, a IgNar, an intrabody, an IgG-CH2, a minibody, a F(ab')3,
a tetrabody, a triabody, a diabody, a single-domain antibody, DVD-Ig,
Fcab, mAb2, a (scFv)2, or a scFv-Fc.
[0022] In certain embodiments, an antibody or polypeptide of the invention
binds to a human folate receptor 1 with a Kd of about 1.0 to about 10 nM.
In one embodiment, the antibody or polypeptide binds to a human folate
receptor 1 with a Kd of about 1.0 nM or better. In a certain embodiment,
binding affinity is measured by flow cytometry, Biacore, or
radioimmunoassay.
[0023] The invention also provides a method of making an antibody of the
invention comprising culturing a cell expressing said antibody; and (b)
isolating the antibody from said cultured cell. In a certain embodiment,
the cell is a eukaryotic cell.
[0024] The invention also provides an immunoconjugate having the formula
(A)-(L)-(C), wherein: (A) is an antibody or antigen binding fragment or
polypeptide of the invention; (L) is a linker; and (C) is a cytotoxic
agent, wherein said linker (L) links (A) to (C).
[0025] In one embodiment, the linker is selected from the group of a
cleavable linker, a non-cleavable linker, a hydrophilic linker, and a
dicarboxylic acid based linker. In a further embodiment, the linker is
selected from the group consisting: N-succinimidyl
4-(2-pyridyldithio)pentanoate (SPP) or N-succinimidyl
4-(2-pyridyldithio)-2-sulfopentanoate (sulfo-SPP); N-succinimidyl
4-(2-pyridyldithio)butanoate (SPDB) or N-succinimidyl
4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB); N-succinimidyl
4-(maleimidomethyl) cyclohexanecarboxylate (SMCC); N-sulfosuccinimidyl
4-(maleimidomethyl) cyclohexanecarboxylate (sulfoSMCC);
N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB); and
N-succinimidyl[(N-maleimidopropionamido)-tetraethyleneglycol] ester
(NHS-PEG4-maleimide). In a certain embodiment, the linker is
N-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol] ester
(NHS-PEG4-maleimide).
[0026] In one embodiment, the immunoconjugates comprise a cytotoxic agent
selected from the group of a maytansinoid, maytansinoid analog,
benzodiazepine, taxoid, CC-1065, CC-1065 analog, duocarmycin, duocarmycin
analog, calicheamicin, dolastatin, dolastatin analog, auristatin,
tomaymycin derivative, and leptomycin derivative or a prodrug of the
agent. In a further embodiment, the cytotoxic agent is a maytansinoid. In
another embodiment, the cytotoxic agent is
N(2')-deacetyl-N(2')-(3-mercapto-1-oxopropyl)-maytansine or
N(2')-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine.
[0027] In one embodiment the invention provides an immunoconjugate
comprising: (A) a humanized antibody comprising the heavy chain variable
domain of SEQ ID NO:4, and the light chain variable domain of SEQ ID
NO:10 or SEQ ID NO:11; (L)
N-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]ester
(NHS-PEG4-maleimide); and (C)
N(2')-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine; wherein
(L) links (A) to (C).
[0028] In one embodiment the invention provides an immunoconjugate
comprising: (A) a humanized antibody comprising the heavy chain variable
domain of SEQ ID NO:4, and the light chain variable domain of SEQ ID
NO:10 or SEQ ID NO:11; (L) N-succinimidyl 4-(2-pyridyldithio)butanoate
(SPDB); and (C)
N(2)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine; wherein
(L) links (A) to (C).
[0029] In one embodiment the invention provides an immunoconjugate
comprising: (A) a humanized antibody comprising the heavy chain variable
domain of SEQ ID NO:4, and the light chain variable domain of SEQ ID
NO:10 or SEQ ID NO:11; (L) N-succinimidyl
4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB); and (C)
N(2')-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine; wherein
(L) links (A) to (C).
[0030] In one embodiment the invention provides an immunoconjugate
comprising: (A) a humanized antibody comprising the heavy chain variable
domain of SEQ ID NO:4, and the light chain variable domain of SEQ ID
NO:10 or SEQ ID NO:11; (L) N-succinimidyl
4-(2-pyridyldithio)-2-sulfopentanoate (sulfo-SPP); and (C)
N(2')-deacetyl-N(2')-(3-mercapto-1-oxopropyl)-maytansine; wherein (L)
links (A) to (C).
[0031] In one embodiment the invention provides an immunoconjugate
comprising: (A) a humanized antibody comprising the heavy chain variable
domain of SEQ ID NO:4, and the light chain variable domain of SEQ ID
NO:10 or SEQ ID NO:11; (L) N-succinimidyl 4-(2-pyridyldithio)pentanoate
(SPP); and (C) N(2)-deacetyl-N(2)-(3-mercapto-1-oxopropyl)-maytansine;
wherein (L) links (A) to (C).
[0032] The invention also provides a pharmaceutical composition comprising
an antibody, antigen binding fragment, polypeptide, or immunoconjugate of
the invention and a pharmaceutically acceptable carrier. In a certain
embodiment, the pharmaceutical composition further comprises a second
anti-cancer agent.
[0033] The invention also provides a diagnostic reagent comprising an
antibody, antigen binding fragment, polypeptide, or immunoconjugate of
the invention which is labeled. In one embodiment, the label is selected
from the group of a radiolabel, a fluorophore, a chromophore, an imaging
agent and a metal ion.
[0034] The invention also provides a kit comprising the antibody, antigen
binding fragment, polypeptide, or immunoconjugate of the invention.
[0035] The invention also provides a method of inhibiting tumor growth in
a subject, comprising administering a therapeutically effective amount of
the antibody, antigen binding fragment, polypeptide, immunoconjugate, or
pharmaceutical composition of the invention to the subject. In a certain
embodiment, the invention provides a method of inhibiting tumor growth in
a subject comprising administering a therapeutically effective amount of
an immunoconjugate having the formula (A)-(L)-(C), wherein: (A) is an
antibody or antigen binding fragment thereof that specifically binds a
human folate receptor 1; (L) is a linker; and (C) is a cytotoxin selected
from the group consisting of a maytansinoid and a maytansinoid analog;
wherein (L) links (A) to (C) and wherein the immunoconjugate reduces mean
tumor volume at least two-fold in a KB xenograft model. In a certain
embodiment, the method comprises administering an antibody or antigen
binding fragment thereof that comprises (a) a heavy chain CDR1 comprising
GYFMN (SEQ ID NO:1); a heavy chain CDR2 comprising
RIHPYDGDTFYNQXaa1FXaa2Xaa3 (SEQ ID NO:56); and a heavy chain CDR3
comprising YDGSRAMDY (SEQ ID NO:3); and (b) a light chain CDR1 comprising
KASQSVSFAGTSLMH (SEQ ID NO:7); a light chain CDR2 comprising RASNLEA (SEQ
ID NO:8); and a light chain CDR3 comprising QQSREYPYT (SEQ ID NO:9);
wherein Xaa.sub.1 is selected from K, Q, H, and R; Xaa.sub.2 is selected
from Q, H, N, and R; and Xaa.sub.3 is selected from G, E, T, S, A, and V.
In a further embodiment, the antibody comprises a heavy chain CDR2
comprising RIHPYDGDTFYNQKFQG (SEQ ID NO:2).
[0036] In a certain embodiment, the invention provides a method for
inhibiting tumor growth comprising administering an antibody or antigen
binding fragment thereof encoded by the plasmid DNA deposited with the
ATCC on Apr. 7, 2010 and having ATCC deposit nos. PTA-10772 and PTA-10773
or 10774.
[0037] In another embodiment, the method provides administering an
immunoconjugate comprising a humanized antibody comprising the heavy
chain variable domain of SEQ ID NO:4, and the light chain variable domain
of SEQ ID NO:10 or SEQ ID NO:11; (L)
N-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol] ester
(NHS-PEG4-maleimide); and (C)
N(2')-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine.
[0038] In another embodiment, the method comprises administering an
immunoconjugate which comprises (A) a humanized antibody comprising the
heavy chain variable domain of SEQ ID NO:4, and the light chain variable
domain of SEQ ID NO:10 or SEQ ID NO:11; (L) N-succinimidyl
4-(2-pyridyldithio)butanoate (SPDB); and (C)
N(2')-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine; wherein
(L) links (A) to (C).
[0039] In another embodiment, the method comprises administering an
immunoconjugate which comprises (A) a humanized antibody comprising the
heavy chain variable domain of SEQ ID NO:4, and the light chain variable
domain of SEQ ID NO:10 or SEQ ID NO:11; (L) N-succinimidyl
4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB); and (C)
N(2')-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine; wherein
(L) links (A) to (C).
[0040] In another embodiment, the method comprises administering an
immunoconjugate which comprises (A) a humanized antibody comprising the
heavy chain variable domain of SEQ ID NO:4, and the light chain variable
domain of SEQ ID NO:10 or SEQ ID NO:11; (L) N-succinimidyl
4-(2-pyridyldithio)-2-sulfopentanoate (sulfo-SPP); and (C)
N(2')-deacetyl-N(2')-(3-mercapto-1-oxopropyl)-maytansine; wherein (L)
links (A) to (C).
[0041] In another embodiment, the method comprises administering an
immunoconjugate which comprises (A) a humanized antibody comprising the
heavy chain variable domain of SEQ ID NO:4, and the light chain variable
domain of SEQ ID NO:10 or SEQ ID NO:11; (L) N-succinimidyl
4-(2-pyridyldithio)pentanoate (SPP); and (C)
N(2')-deacetyl-N(2')-(3-mercapto-1-oxopropyl)-maytansine; wherein (L)
links (A) to (C).
[0042] In another embodiment, the method comprises administering an
immunoconjugate which comprises the antibody huFR-1-21 deposited with
ATCC on Apr. 7, 2010 and having ATCC deposit nos. PTA-10775 and
PTA-10776. In a certain embodiment, the huFR1-21 antibody comprises (a) a
heavy chain CDR1 comprising SSYGMS (SEQ ID NO:30); a heavy chain CDR2
comprising TISSGGSYTY (SEQ ID NO:31); and a heavy chain CDR3 comprising
DGEGGLYAMDY (SEQ ID NO:32); and (b) a light chain CDR1 comprising
KASDHINNWLA (SEQ ID NO:27); a light chain CDR2 comprising GATSLET (SEQ ID
NO:28); and a light chain CDR3 comprising QQYWSTPFT (SEQ ID NO:29). In
certain embodiments the method comprises administering an immunoconjugate
which comprises the antibody is the huFR1-48 antibody which comprises:
(a) a heavy chain CDR1 comprising TNYWMQ (SEQ ID NO:60); a heavy chain
CDR2 comprising AIYPGNGDSR (SEQ ID NO:61); and a heavy chain CDR3
comprising RDGNYAAY (SEQ ID NO:62); and (b) a light chain CDR1 comprising
RASENIYSNLA (SEQ ID NO:57); a light chain CDR2 comprising AATNLAD (SEQ ID
NO:58); and a light chain CDR3 comprising QHFWASPYT (SEQ ID NO:59). In
certain embodiments the method comprises administering an immunoconjugate
which comprises the antibody is the huFR1-49 antibody which comprises:
(a) a heavy chain CDR1 comprising TNYWMY (SEQ ID NO:66); a heavy chain
CDR2 comprising AIYPGNSDTT (SEQ ID NO:67); and a heavy chain CDR3
comprising RHDYGAMDY (SEQ ID NO:68); and (b) a light chain CDR1
comprising RASENIYTNLA (SEQ ID NO:63); a light chain CDR2 comprising
TASNLAD (SEQ ID NO:64); and a light chain CDR3 comprising QHFWVSPYT (SEQ
ID NO:65). In certain embodiments the method comprises administering an
immunoconjugate which comprises the antibody is the huFR1-57 antibody
which comprises: (a) a heavy chain CDR1 comprising SSFGMH (SEQ ID NO:72);
a heavy chain CDR2 comprising YISSGSSTIS (SEQ ID NO:73); and a heavy
chain CDR3 comprising EAYGSSMEY (SEQ ID NO:74); and (b) a light chain
CDR1 comprising RASQNINNNLH (SEQ ID NO:69); a light chain CDR2 comprising
YVSQSVS (SEQ ID NO:70); and a light chain CDR3 comprising QQSNSWPHYT (SEQ
ID NO:71). In certain embodiments the method comprises administering an
immunoconjugate which comprises the antibody is the huFR1-65 antibody
which comprises: (a) a heavy chain CDR1 comprising TSYTMH (SEQ ID NO:78);
a heavy chain CDR2 comprising YINPISGYTN (SEQ ID NO:79); and a heavy
chain CDR3 comprising GGAYGRKPMDY (SEQ ID NO:80); and (b) a light chain
CDR1 comprising KASQNVGPNVA (SEQ ID NO:75); a light chain CDR2 comprising
SASYRYS (SEQ ID NO:76); and a light chain CDR3 comprising QQYNSYPYT (SEQ
ID NO:77).
[0043] In one embodiment, the method inhibits ovarian tumor, brain tumor,
breast tumor, uterine tumor, endometrial tumor, pancreatic tumor, renal
tumor, or lung tumor growth. In a certain embodiment, the method inhibits
ovarian tumor growth. In another embodiment, the invention inhibits lung
tumor growth. In a certain embodiment, tumor growth inhibition is used to
treat cancer. In a further embodiment, the method comprises administering
a second anti-cancer agent to the subject. In a certain embodiment, the
second anti-cancer agent is a chemotherapeutic agent.
[0044] The invention also provides an isolated cell producing the
antibody, antigen binding fragment, or polypeptide of the invention.
[0045] The invention also provides an isolated polynucleotide comprising a
sequence at least 90% identical to a sequence selected from the group
consisting of SEQ ID NOs: 5, 14, 15, 37, 38, 43, 44, 47, 48, and 120-127.
In a certain embodiment, the isolated polynucleotide is at least 95%
identical a sequence selected from the group consisting of SEQ ID NOs: 5,
14, 15, 37, 38, 43, 44, 47, 48, and 120-127. In another embodiment,
isolated polynucleotide is at least 99% identical to a sequence selected
from the group consisting of SEQ ID NOs: 5, 14, 15, 37, 38, 43, 44, 47,
48, and 120-127. The invention also provides a vector comprising any of
the polynucleotides of SEQ ID NOs: 5, 14, 15, 37, 38, 43, 44, 47, 48, and
120-127. In another embodiment, the invention provides a host cell
comprising a vector which contains a polynucleotide of SEQ ID NOs: 5, 14,
15, 37, 38, 43, 44, 47, 48, and 120-127.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0046] FIGS. 1A, 1B, 1C and 1D. Surface residues for murine (muMov19) and
humanized (huMov19) Mov19. (FIG. 1A) Murine and humanized Mov19 light
chain surface residues. The murine and humanized Mov19 light chain
variable region frame surface residues and position number (Kabat system)
are given. The human residues that are different from the original murine
sequences are underlined. *Position 74 is not a surface position, but to
remove a consensus N-linked glycosylation site in version 1.00, this
position was changed to a Threonine (the most common human residue in
this position), resulting in version 1.60. (FIG. 1B) Murine and Human
Mov19 Heavy Chain Surface Residues. The murine and humanized Mov19 heavy
chain variable region frame surface residues and position number (Kabat
system) are given. The human residues that are different from the
original murine sequences are underlined. Similar surface residues are
provided for FR1-21 (FIGS. 1C and 1D).
[0047] FIG. 2. Alignments of chimeric Mov19 and huMov19 heavy and light
chain variable domains and muFR1-21 and huFR1-21 heavy and light chain
variable domains. Alignment of resurfaced sequences for the Mov19 and
Fr1-21 variable regions with their murine counterparts. (FIG. 2, Panel A)
and (FIG. 2, Panel C) light chain variable domains; (FIG. 2, Panel B) and
(FIG. 2, Panel D) heavy chain variable domain. Dashes "-" denote identity
with the murine sequence. The CDRs (Kabat definition) are underlined.
[0048] FIG. 3. Expression of chimeric Mov19 and huMov19 in HEK cells. The
chimeric and human Mov19 expression plasmids were transiently transfected
into suspension HEK293-T cells, harvested 7 days later, and the expressed
antibody was determined by quantitative ELISA. The light chain and heavy
chain plasmids were transfected at either 3:1 or 6:1 respective molar
ratios.
[0049] FIG. 4. Binding specificity of anti-FOLR1 antibodies, as detected
by their binding to FOLR1-expressing 300-19 cells. The binding of huMov19
to 300-19-FOLR1 cells by flow cytometry. 300-19 parental cells expressing
FOLR-1. The grey solid shading represents cellular auto fluorescence; the
black dotted lines represent cells incubated with anti-human secondary
antibody conjugated with FITC, the black solid lines represent cells
incubated with the huMov-19 antibody and anti-human secondary antibody
conjugated to FITC.
[0050] FIG. 5. Binding affinities and in vitro cytotoxic activity of
anti-FOLR1 antibodies and immunoconjugates. Binding affinity of huMov19
and various murine and humanized FR-1 antibodies was measured on SKOV3
cells. In vitro cytotoxic activity of PEG4-Mal-DM4 conjugates of the
listed antibodies was also assayed.
[0051] FIG. 6. Antibody-dependent cellular cytotoxicity of
immunoconjugates. ADCC activity of huMov19, huFR1-21, and Mor003 was
assayed against Igrov1 cells. Igrov 1 were incubated at 15000 cells/well
Target:NK cell ratio of 1:4.
[0052] FIG. 7. Cytoxic activity of continuous exposure of
huFR1-21-PEG4-mal-DM4 and huMov19-PEG4-mal-DM4 on KB cells. An excess of
non-conjugated antibodies suppressed the activity of immunoconjugates
when they were co-incubated in the presence of KB cells, indicating
cytotoxic activity is antigen-dependent.
[0053] FIG. 8. In vivo efficacy of huMov19-targeted conjugates in a KB
xenograft model. FOLR1-targeting cleavable conjugate huMov19-SPDB-DM4 (B)
in comparison with non-FOLR1-targeting huC242-SPDB-DM4 (D), and
non-cleavable conjugate huMov19-PEG4-Mal-DM4 (C) in comparison with
non-targeting huC242-PEG4Mal-DM4 (E) were tested using an established
xenograft model of KB cells implanted subcutaneous into SCID mice.
Targeting of FOLR1 by huMov19 resulted in significant reduction in mean
tumor volume.
[0054] FIG. 9. In vivo efficacy of huMov19-PEG4-Mal-DM4 compared to murine
FR-1 anti-FOLR1 antibodies in a KB xenograft model. FR-1 series
antibodies, either unconjugated, or conjugated with PEG4-Mal-DM4 were
tested for their ability to reduce mean tumor volume compared to
huMov19-PEG4-Mal-DM4 in a KB xenograft tumor model. (FIG. 9, Panel A)
FR-1-9, (FIG. 9, Panel B) FR-1-13, (FIG. 9, Panel C) FR-1-22, and (FIG.
9, Panel D) FR-1-23.
[0055] FIG. 10. In vivo efficacy of huMov19-PEG4-Mal-DM4 and
huFR1-21-PEG4-Mal-DM4 in a KB xenograft model. 10 mg/kg single injections
of huMov19-PEG4-Mal-DM4 and huFR1-21-PEG4-Mal-DM4 on day 6 post
inoculation was performed. Both huMov19-PEG4-Mal-DM4 and
huFR1-21-PEG4-Mal-DM4 showed a significant reduction in mean tumor
volume. "Mean TV" refers to mean tumor volume.
[0056] FIG. 11. HuMov19-PEG4-mal-DM4 shows dose dependent activity in the
KB xenograft model. Dose dependent activity of the immunoconjugate was
assayed across the range of doses tested. Weekly dosing resulted in
improvement of anti-tumor activity. High drug loads only marginally
improved activity in the 10 mg/kg dose groups, with reduced activity in
the lower dose groups. 3.7 DAR refers to 3.7 drug molecules per antibody.
[0057] FIG. 12. In vivo efficacy of huMov19 conjugated with DM1 and DM4
with various linkers. huMov19 was conjugated to SMCC-DM1 at 3.9 drug
molecules per antibody (FIG. 12, Panel A), sulfo-mal-DM4 at 3.7 drug
molecules per antibody (FIG. 12, Panel B), and sulfo-mal-DM4 at 8.23 drug
molecules per antibody (FIG. 12, Panel C) and assayed for their ability
to reduce mean tumor volume at various concentrations compared to
huMov19-PEG4-mal-DM4.
[0058] FIG. 13. In vivo efficacy of huMov19 conjugated with DM1 and DM4
with various linkers. huMov19 was conjugated to SPP-DM1 at 4.3 drug
molecules per antibody; sulfo-SPDB-DM4 at 3.8 drug molecules per
antibody, SPDB-DM4 at 3.8 drug molecules per antibody, and sulfo-SPDB-DM4
at 6.8 drug molecules per antibody and assayed for their ability to
reduce mean tumor volume. Mice were treated with 5 mg/kg (FIG. 13, Panel
A) and 2.5 mg/kg (FIG. 13, Panel B) of one of the conjugates listed above
or with PBS only.
[0059] FIG. 14. In vivo efficacy of huMov19-sulfo-SPDB-DM4 in OVCAR-3
xenograft tumor model. Mice were treated with 25, 50, or 100 .mu.g/kg of
huMov19-sulfo-SPDB-DM4 or with PBS only.
[0060] FIG. 15. In vivo efficacy of huMov19-sulfo-SPDB-DM4 in IGROV-1
xenograft tumor model. Mice were treated with 25, 50, or 100 .mu.g/kg of
huMov19-sulfo-SPDB-DM4 or with PBS only
[0061] FIG. 16. In vivo efficacy of huMov19-sulfo-SPDB-DM4 in OV-90
xenograft tumor model. Mice were treated with 25, 50, or 100 .mu.g/kg of
huMov19-sulfo-SPDB-DM4 or with PBS only.
[0062] FIG. 17. Effect of cleavable and non-cleavable linkers on efficacy
of immunoconjugates in KB xenograft models.
[0063] FIG. 18. Effect of cleavabe linkers on efficacy of immunoconjugates
in (FIG. 18, Panel A) KB xenograft model (FIG. 18, Panel B) OVCAR-3
xenograft model.
[0064] FIG. 19. In vitro and in vivo efficacy of huFR1-48, huFR1-49,
huFR1-57, and huFR1-65-SMCC-DM1 in KB and xenograft tumor models. Mice
were treated with 200 .mu.g/kg single doses.
DETAILED DESCRIPTION OF THE INVENTION
[0065] The present invention provides novel agents, including, but not
limited to polypeptides such as antibodies, and immunoconjugates that
bind to human folate receptor 1 (FOLR1). Related polypeptides and
polynucleotides, compositions comprising the FOLR1-binding agents, and
methods of making the FOLR1-binding agents are also provided. Methods of
using the novel FOLR1-binding agents, such as methods of inhibiting tumor
growth and/or treating cancer, are further provided.
I. Definitions
[0066] To facilitate an understanding of the present invention, a number
of terms and phrases are defined below.
[0067] The terms "human folate receptor 1" or "FOLR1", as used herein,
refers to any native human FOLR1, unless otherwise indicated. The term
"FOLR1" encompasses "full-length," unprocessed FOLR1 as well as any form
of FOLR1 that results from processing within the cell. The term also
encompasses naturally occurring variants of FOLR1, e.g., splice variants,
allelic variants and isoforms. The FOLR1 polypeptides described herein
can be isolated from a variety of sources, such as from human tissue
types or from another source, or prepared by recombinant or synthetic
methods. Examples of FOLR1 sequences include, but are not limited to NCBI
reference numbers P15328, NP_001092242.1, AAX29268.1, AAX37119.1,
NP_057937.1, and NP_057936.1.
[0068] The term "antibody" means an immunoglobulin molecule that
recognizes and specifically binds to a target, such as a protein,
polypeptide, peptide, carbohydrate, polynucleotide, lipid, or
combinations of the foregoing through at least one antigen recognition
site within the variable region of the immunoglobulin molecule. As used
herein, the term "antibody" encompasses intact polyclonal antibodies,
intact monoclonal antibodies, antibody fragments (such as Fab, Fab',
F(ab')2, and Fv fragments), single chain Fv (scFv) mutants, multispecific
antibodies such as bispecific antibodies generated from at least two
intact antibodies, chimeric antibodies, humanized antibodies, human
antibodies, fusion proteins comprising an antigen determination portion
of an antibody, and any other modified immunoglobulin molecule comprising
an antigen recognition site so long as the antibodies exhibit the desired
biological activity. An antibody can be of any the five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)
thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on the
identity of their heavy-chain constant domains referred to as alpha,
delta, epsilon, gamma, and mu, respectively. The different classes of
immunoglobulins have different and well known subunit structures and
three-dimensional configurations. Antibodies can be naked or conjugated
to other molecules such as toxins, radioisotopes, etc.
[0069] A "blocking" antibody or an "antagonist" antibody is one which
inhibits or reduces biological activity of the antigen it binds, such as
FOLR1. In a certain embodiment blocking antibodies or antagonist
antibodies substantially or completely inhibit the biological activity of
the antigen. Desirably, the biological activity is reduced by 10%, 20%,
30%, 50%, 70%, 80%, 90%, 95%, or even 100%.
[0070] The term "anti-FOLR1 antibody" or "an antibody that binds to FOLR1"
refers to an antibody that is capable of binding FOLR1 with sufficient
affinity such that the antibody is useful as a diagnostic and/or
therapeutic agent in targeting FOLR1. The extent of binding of an
anti-FOLR1 antibody to an unrelated, non-FOLR1 protein is less than about
10% of the binding of the antibody to FOLR1 as measured, e.g., by a
radioimmunoassay (RIA). In certain embodiments, an antibody that binds to
FOLR1 has a dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100
nM, .ltoreq.10 nM, .ltoreq.1 nM, or .ltoreq.0.1 nM.
[0071] The term "antibody fragment" refers to a portion of an intact
antibody and refers to the antigenic determining variable regions of an
intact antibody. Examples of antibody fragments include, but are not
limited to Fab, Fab', F(ab')2, and Fv fragments, linear antibodies,
single chain antibodies, and multispecific antibodies formed from
antibody fragments.
[0072] A "monoclonal antibody" refers to a homogeneous antibody population
involved in the highly specific recognition and binding of a single
antigenic determinant, or epitope. This is in contrast to polyclonal
antibodies that typically include different antibodies directed against
different antigenic determinants. The term "monoclonal antibody"
encompasses both intact and full-length monoclonal antibodies as well as
antibody fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv)
mutants, fusion proteins comprising an antibody portion, and any other
modified immunoglobulin molecule comprising an antigen recognition site.
Furthermore, "monoclonal antibody" refers to such antibodies made in any
number of manners including but not limited to by hybridoma, phage
selection, recombinant expression, and transgenic animals.
[0073] The term "humanized antibody" refers to forms of non-human (e.g.
murine) antibodies that are specific immunoglobulin chains, chimeric
immunoglobulins, or fragments thereof that contain minimal non-human
(e.g., murine) sequences. Typically, humanized antibodies are human
immunoglobulins in which residues from the complementary determining
region (CDR) are replaced by residues from the CDR of a non-human species
(e.g. mouse, rat, rabbit, hamster) that have the desired specificity,
affinity, and capability (Jones et al., 1986, Nature, 321:522-525;
Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988,
Science, 239:1534-1536). In some instances, the Fv framework region (FR)
residues of a human immunoglobulin are replaced with the corresponding
residues in an antibody from a non-human species that has the desired
specificity, affinity, and capability. The humanized antibody can be
further modified by the substitution of additional residues either in the
Fv framework region and/or within the replaced non-human residues to
refine and optimize antibody specificity, affinity, and/or capability. In
general, the humanized antibody will comprise substantially all of at
least one, and typically two or three, variable domains containing all or
substantially all of the CDR regions that correspond to the non-human
immunoglobulin whereas all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody can also comprise at least a portion of an immunoglobulin
constant region or domain (Fc), typically that of a human immunoglobulin.
Examples of methods used to generate humanized antibodies are described
in U.S. Pat. No. 5,225,539 or 5,639,641.
[0074] A "variable region" of an antibody refers to the variable region of
the antibody light chain or the variable region of the antibody heavy
chain, either alone or in combination. The variable regions of the heavy
and light chain each consist of four framework regions (FR) connected by
three complementarity determining regions (CDRs) also known as
hypervariable regions. The CDRs in each chain are held together in close
proximity by the FRs and, with the CDRs from the other chain, contribute
to the formation of the antigen-binding site of antibodies. There are at
least two techniques for determining CDRs: (1) an approach based on
cross-species sequence variability (i.e., Kabat et al. Sequences of
Proteins of Immunological Interest, (5th ed., 1991, National Institutes
of Health, Bethesda Md.)); and (2) an approach based on crystallographic
studies of antigen-antibody complexes (Al-lazikani et al (1997) J. Molec.
Biol. 273:927-948)). In addition, combinations of these two approaches
are sometimes used in the art to determine CDRs.
[0075] The Kabat numbering system is generally used when referring to a
residue in the variable domain (approximately residues 1-107 of the light
chain and residues 1-113 of the heavy chain) (e.g, Kabat et al.,
Sequences of Immunological Interest. 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)).
[0076] The amino acid position numbering as in Kabat, refers to the
numbering system used for heavy chain variable domains or light chain
variable domains of the compilation of antibodies in Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public Health
Service, National Institutes of Health, Bethesda, Md. (1991). Using this
numbering system, the actual linear amino acid sequence can contain fewer
or additional amino acids corresponding to a shortening of, or insertion
into, a FR or CDR of the variable domain. For example, a heavy chain
variable domain can include a single amino acid insert (residue 52a
according to Kabat) after residue 52 of H2 and inserted residues (e.g.
residues 82a, 82b, and 82c, etc according to Kabat) after heavy chain FR
residue 82. The Kabat numbering of residues can be determined for a given
antibody by alignment at regions of homology of the sequence of the
antibody with a "standard" Kabat numbered sequence. Chothia refers
instead to the location of the structural loops (Chothia and Lesk J. Mol.
Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop when
numbered using the Kabat numbering convention varies between H32 and H34
depending on the length of the loop (this is because the Kabat numbering
scheme places the insertions at H35A and H35B; if neither 35A nor 35B is
present, the loop ends at 32; if only 35A is present, the loop ends at
33; if both 35A and 35B are present, the loop ends at 34). The AbM
hypervariable regions represent a compromise between the Kabat CDRs and
Chothia structural loops, and are used by Oxford Molecular's AbM antibody
modeling software.
TABLE-US-00001
Loop Kabat AbM Chothia
L1 L24-L34 L24-L34 L24-L34
L2 L50-L56 L50-L56 L50-L56
L3 L89-L97 L89-L97 L89-L97
H1 H31-H35B H26-H35B H26-H32 . . . 34
(Kabat Numbering)
H1 H31-H35 H26-H35 H26-H32
(Chothia Numbering)
H2 H50-H65 H50-H58 H52-H56
H3 H95-H102 H95-H102 H95-H102
[0077] The term "human antibody" means an antibody produced by a human or
an antibody having an amino acid sequence corresponding to an antibody
produced by a human made using any technique known in the art. This
definition of a human antibody includes intact or full-length antibodies,
fragments thereof, and/or antibodies comprising at least one human heavy
and/or light chain polypeptide such as, for example, an antibody
comprising murine light chain and human heavy chain polypeptides.
[0078] The term "chimeric antibodies" refers to antibodies wherein the
amino acid sequence of the immunoglobulin molecule is derived from two or
more species. Typically, the variable region of both light and heavy
chains corresponds to the variable region of antibodies derived from one
species of mammals (e.g. mouse, rat, rabbit, etc) with the desired
specificity, affinity, and capability while the constant regions are
homologous to the sequences in antibodies derived from another (usually
human) to avoid eliciting an immune response in that species.
[0079] The term "epitope" or "antigenic determinant" are used
interchangeably herein and refer to that portion of an antigen capable of
being recognized and specifically bound by a particular antibody. When
the antigen is a polypeptide, epitopes can be formed both from contiguous
amino acids and noncontiguous amino acids juxtaposed by tertiary folding
of a protein. Epitopes formed from contiguous amino acids are typically
retained upon protein denaturing, whereas epitopes formed by tertiary
folding are typically lost upon protein denaturing. An epitope typically
includes at least 3, and more usually, at least 5 or 8-10 amino acids in
a unique spatial conformation.
[0080] "Binding affinity" generally refers to the strength of the sum
total of noncovalent interactions between a single binding site of a
molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
Unless indicated otherwise, as used herein, "binding affinity" refers to
intrinsic binding affinity which reflects a 1:1 interaction between
members of a binding pair (e.g., antibody and antigen). The affinity of a
molecule X for its partner Y can generally be represented by the
dissociation constant (Kd). Affinity can be measured by common methods
known in the art, including those described herein. Low-affinity
antibodies generally bind antigen slowly and tend to dissociate readily,
whereas high-affinity antibodies generally bind antigen faster and tend
to remain bound longer. A variety of methods of measuring binding
affinity are known in the art, any of which can be used for purposes of
the present invention. Specific illustrative embodiments are described in
the following.
[0081] "Or better" when used herein to refer to binding affinity refers to
a stronger binding between a molecule and its binding partner. "Or
better" when used herein refers to a stronger binding, represented by a
smaller numerical Kd value. For example, an antibody which has an
affinity for an antigen of "0.6 nM or better", the antibody's affinity
for the antigen is <0.6 nM, i.e. 0.59 nM, 0.58 nM, 0.57 nM etc. or any
value less than 0.6 nM.
[0082] The phrase "substantially similar," or "substantially the same", as
used herein, denotes a sufficiently high degree of similarity between two
numeric values (generally one associated with an antibody of the
invention and the other associated with a reference/comparator antibody)
such that one of skill in the art would consider the difference between
the two values to be of little or no biological and/or statistical
significance within the context of the biological characteristics
measured by said values (e.g., Kd values). The difference between said
two values is less than about 50%, less than about 40%, less than about
30%, less than about 20%, or less than about 10% as a function of the
value for the reference/comparator antibody.
[0083] A polypeptide, antibody, polynucleotide, vector, cell, or
composition which is "isolated" is a polypeptide, antibody,
polynucleotide, vector, cell, or composition which is in a form not found
in nature. Isolated polypeptides, antibodies, polynucleotides, vectors,
cell or compositions include those which have been purified to a degree
that they are no longer in a form in which they are found in nature. In
some embodiments, an antibody, polynucleotide, vector, cell, or
composition which is isolated is substantially pure.
[0084] As used herein, "substantially pure" refers to material which is at
least 50% pure (i.e., free from contaminants), at least 90% pure, at
least 95% pure, at least 98% pure, or at least 99% pure.
[0085] The term "immunoconjugate" or "conjugate" as used herein refers to
a compound or a derivative thereof that is linked to a cell binding agent
(i.e., an anti-FOLR1 antibody or fragment thereof) and is defined by a
generic formula: C-L-A, wherein C=cytotoxin, L=linker, and A=cell binding
agent or anti-FOLR1 antibody or antibody fragment. Immunoconjugates can
also be defined by the generic formula in reverse order: A-L-C.
[0086] A "linker" is any chemical moiety that is capable of linking a
compound, usually a drug, such as a maytansinoid, to a cell-binding agent
such as an anti FOLR1 antibody or a fragment thereof in a stable,
covalent manner. Linkers can be susceptible to or be substantially
resistant to acid-induced cleavage, light-induced cleavage,
peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond
cleavage, at conditions under which the compound or the antibody remains
active. Suitable linkers are well known in the art and include, for
example, disulfide groups, thioether groups, acid labile groups,
photolabile groups, peptidase labile groups and esterase labile groups.
Linkers also include charged linkers, and hydrophilic forms thereof as
described herein and know in the art.
[0087] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals in which a population of cells are
characterized by unregulated cell growth. Examples of cancer include, but
are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
More particular examples of such cancers include squamous cell cancer,
small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the
lung, squamous carcinoma of the lung, cancer of the peritoneum,
hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,
glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder
cancer, hepatoma, breast cancer, colon cancer, colorectal cancer,
endometrial or uterine carcinoma, salivary gland carcinoma, kidney
cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer,
hepatic carcinoma and various types of head and neck cancers.
[0088] "Tumor" and "neoplasm" refer to any mass of tissue that result from
excessive cell growth or proliferation, either benign (noncancerous) or
malignant (cancerous) including pre-cancerous lesions.
[0089] The terms "cancer cell," "tumor cell," and grammatical equivalents
refer to the total population of cells derived from a tumor or a
pre-cancerous lesion, including both non-tumorigenic cells, which
comprise the bulk of the tumor cell population, and tumorigenic stem
cells (cancer stem cells). As used herein, the term "tumor cell" will be
modified by the term "non-tumorigenic" when referring solely to those
tumor cells lacking the capacity to renew and differentiate to
distinguish those tumor cells from cancer stem cells.
[0090] The term "subject" refers to any animal (e.g., a mammal),
including, but not limited to humans, non-human primates, rodents, and
the like, which is to be the recipient of a particular treatment.
Typically, the terms "subject" and "patient" are used interchangeably
herein in reference to a human subject.
[0091] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and consecutive
administration in any order.
[0092] The term "pharmaceutical formulation" refers to a preparation which
is in such form as to permit the biological activity of the active
ingredient to be effective, and which contains no additional components
which are unacceptably toxic to a subject to which the formulation would
be administered. Such formulation can be sterile.
[0093] An "effective amount" of an antibody as disclosed herein is an
amount sufficient to carry out a specifically stated purpose. An
"effective amount" can be determined empirically and in a routine manner,
in relation to the stated purpose.
[0094] The term "therapeutically effective amount" refers to an amount of
an antibody or other drug effective to "treat" a disease or disorder in a
subject or mammal. In the case of cancer, the therapeutically effective
amount of the drug can reduce the number of cancer cells; reduce the
tumor size; inhibit (i.e., slow to some extent and in a certain
embodiment, stop) cancer cell infiltration into peripheral organs;
inhibit (i.e., slow to some extent and in a certain embodiment, stop)
tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve
to some extent one or more of the symptoms associated with the cancer.
See the definition herein of "treating". To the extent the drug can
prevent growth and/or kill existing cancer cells, it can be cytostatic
and/or cytotoxic. A "prophylactically effective amount" refers to an
amount effective, at dosages and for periods of time necessary, to
achieve the desired prophylactic result. Typically but not necessarily,
since a prophylactic dose is used in subjects prior to or at an earlier
stage of disease, the prophylactically effective amount will be less than
the therapeutically effective amount.
[0095] The word "label" when used herein refers to a detectable compound
or composition which is conjugated directly or indirectly to the antibody
so as to generate a "labeled" antibody. The label can be detectable by
itself (e.g. radioisotope labels or fluorescent labels) or, in the case
of an enzymatic label, can catalyze chemical alteration of a substrate
compound or composition which is detectable.
[0096] A "chemotherapeutic agent" is a chemical compound useful in the
treatment of cancer, regardless of mechanism of action. Classes of
chemotherapeutic agents include, but are not limited to: alkylating
agents, antimetabolites, spindle poison plant alkaloids,
cytoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies,
photosensitizers, and kinase inhibitors. Chemotherapeutic agents include
compounds used in "targeted therapy" and conventional chemotherapy.
[0097] Terms such as "treating" or "treatment" or "to treat" or
"alleviating" or "to alleviate" refer to both 1) therapeutic measures
that cure, slow down, lessen symptoms of, and/or halt progression of a
diagnosed pathologic condition or disorder and 2) prophylactic or
preventative measures that prevent and/or slow the development of a
targeted pathologic condition or disorder. Thus, those in need of
treatment include those already with the disorder; those prone to have
the disorder; and those in whom the disorder is to be prevented. In
certain embodiments, a subject is successfully "treated" for cancer
according to the methods of the present invention if the patient shows
one or more of the following: a reduction in the number of or complete
absence of cancer cells; a reduction in the tumor size; inhibition of or
an absence of cancer cell infiltration into peripheral organs including,
for example, the spread of cancer into soft tissue and bone; inhibition
of or an absence of tumor metastasis; inhibition or an absence of tumor
growth; relief of one or more symptoms associated with the specific
cancer; reduced morbidity and mortality; improvement in quality of life;
reduction in tumorigenicity, tumorigenic frequency, or tumorigenic
capacity, of a tumor; reduction in the number or frequency of cancer stem
cells in a tumor; differentiation of tumorigenic cells to a
non-tumorigenic state; or some combination of effects.
[0098] "Polynucleotide," or "nucleic acid," as used interchangeably
herein, refer to polymers of nucleotides of any length, and include DNA
and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides,
modified nucleotides or bases, and/or their analogs, or any substrate
that can be incorporated into a polymer by DNA or RNA polymerase. A
polynucleotide can comprise modified nucleotides, such as methylated
nucleotides and their analogs. If present, modification to the nucleotide
structure can be imparted before or after assembly of the polymer. The
sequence of nucleotides can be interrupted by non-nucleotide components.
A polynucleotide can be further modified after polymerization, such as by
conjugation with a labeling component. Other types of modifications
include, for example, "caps", substitution of one or more of the
naturally occurring nucleotides with an analog, internucleotide
modifications such as, for example, those with uncharged linkages (e.g.,
methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.)
and with charged linkages (e.g., phosphorothioates, phosphorodithioates,
etc.), those containing pendant moieties, such as, for example, proteins
(e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine,
etc.), those with intercalators (e.g., acridine, psoralen, etc.), those
containing chelators (e.g., metals, radioactive metals, boron, oxidative
metals, etc.), those containing alkylators, those with modified linkages
(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms
of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily
present in the sugars can be replaced, for example, by phosphonate
groups, phosphate groups, protected by standard protecting groups, or
activated to prepare additional linkages to additional nucleotides, or
can be conjugated to solid supports. The 5' and 3' terminal OH can be
phosphorylated or substituted with amines or organic capping group
moieties of from 1 to 20 carbon atoms. Other hydroxyls can also be
derivatized to standard protecting groups. Polynucleotides can also
contain analogous forms of ribose or deoxyribose sugars that are
generally known in the art, including, for example, 2'-O-methyl-,
2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs,
.alpha.-anomeric sugars, epimeric sugars such as arabinose, xyloses or
lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic
analogs and abasic nucleoside analogs such as methyl riboside. One or
more phosphodiester linkages can be replaced by alternative linking
groups. These alternative linking groups include, but are not limited to,
embodiments wherein phosphate is replaced by P(O)S ("thioate"), P(S)S
("dithioate"), "(O)NR.sub.2 ("amidate"), P(O)R, P(O)OR', CO or CH.sub.2
("formacetal"), in which each R or R' is independently H or substituted
or unsubstituted alkyl (1-20 C) optionally containing an ether (--O--)
linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all
linkages in a polynucleotide need be identical. The preceding description
applies to all polynucleotides referred to herein, including RNA and DNA.
[0099] The term "vector" means a construct, which is capable of
delivering, and expressing, one or more gene(s) or sequence(s) of
interest in a host cell. Examples of vectors include, but are not limited
to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid
or phage vectors, DNA or RNA expression vectors associated with cationic
condensing agents, DNA or RNA expression vectors encapsulated in
liposomes, and certain eukaryotic cells, such as producer cells.
[0100] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to polymers of amino acids of any length.
The polymer can be linear or branched, it can comprise modified amino
acids, and it can be interrupted by non-amino acids. The terms also
encompass an amino acid polymer that has been modified naturally or by
intervention; for example, disulfide bond formation, glycosylation,
lipidation, acetylation, phosphorylation, or any other manipulation or
modification, such as conjugation with a labeling component. Also
included within the definition are, for example, polypeptides containing
one or more analogs of an amino acid (including, for example, unnatural
amino acids, etc.), as well as other modifications known in the art. It
is understood that, because the polypeptides of this invention are based
upon antibodies, in certain embodiments, the polypeptides can occur as
single chains or associated chains.
[0101] The terms "identical" or percent "identity" in the context of two
or more nucleic acids or polypeptides, refer to two or more sequences or
subsequences that are the same or have a specified percentage of
nucleotides or amino acid residues that are the same, when compared and
aligned (introducing gaps, if necessary) for maximum correspondence, not
considering any conservative amino acid substitutions as part of the
sequence identity. The percent identity can be measured using sequence
comparison software or algorithms or by visual inspection. Various
algorithms and software are known in the art that can be used to obtain
alignments of amino acid or nucleotide sequences. One such non-limiting
example of a sequence alignment algorithm is the algorithm described in
Karlin et al, 1990, Proc. Natl. Acad. Sci., 87:2264-2268, as modified in
Karlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-5877, and
incorporated into the NBLAST and XBLAST programs (Altschul et al., 1991,
Nucleic Acids Res., 25:3389-3402). In certain embodiments, Gapped BLAST
can be used as described in Altschul et al., 1997, Nucleic Acids Res.
25:3389-3402. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods in
Enzymology, 266:460-480), ALIGN, ALIGN-2 (Genentech, South San Francisco,
Calif.) or Megalign (DNASTAR) are additional publicly available software
programs that can be used to align sequences. In certain embodiments, the
percent identity between two nucleotide sequences is determined using the
GAP program in GCG software (e.g., using a NWSgapdna.CMP matrix and a gap
weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or
6). In certain alternative embodiments, the GAP program in the GCG
software package, which incorporates the algorithm of Needleman and
Wunsch (J. Mol. Biol. (48):444-453 (1970)) can be used to determine the
percent identity between two amino acid sequences (e.g., using either a
Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10,
8, 6, or 4 and a length weight of 1, 2, 3, 4, 5). Alternatively, in
certain embodiments, the percent identity between nucleotide or amino
acid sequences is determined using the algorithm of Myers and Miller
(CABIOS, 4:11-17 (1989)). For example, the percent identity can be
determined using the ALIGN program (version 2.0) and using a PAM120 with
residue table, a gap length penalty of 12 and a gap penalty of 4.
Appropriate parameters for maximal alignment by particular alignment
software can be determined by one skilled in the art. In certain
embodiments, the default parameters of the alignment software are used.
In certain embodiments, the percentage identity "X" of a first amino acid
sequence to a second sequence amino acid is calculated as
100.times.(Y/Z), where Y is the number of amino acid residues scored as
identical matches in the alignment of the first and second sequences (as
aligned by visual inspection or a particular sequence alignment program)
and Z is the total number of residues in the second sequence. If the
length of a first sequence is longer than the second sequence, the
percent identity of the first sequence to the second sequence will be
longer than the percent identity of the second sequence to the first
sequence.
[0102] As a non-limiting example, whether any particular polynucleotide
has a certain percentage sequence identity (e.g., is at least 80%
identical, at least 85% identical, at least 90% identical, and in some
embodiments, at least 95%, 96%, 97%, 98%, or 99% identical) to a
reference sequence can, in certain embodiments, be determined using the
Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix,
Genetics Computer Group, University Research Park, 575 Science Drive,
Madison, Wis. 53711). Bestfit uses the local homology algorithm of Smith
and Waterman, Advances in Applied Mathematics 2: 482 489 (1981), to find
the best segment of homology between two sequences. When using Bestfit or
any other sequence alignment program to determine whether a particular
sequence is, for instance, 95% identical to a reference sequence
according to the present invention, the parameters are set such that the
percentage of identity is calculated over the full length of the
reference nucleotide sequence and that gaps in homology of up to 5% of
the total number of nucleotides in the reference sequence are allowed.
[0103] In some embodiments, two nucleic acids or polypeptides of the
invention are substantially identical, meaning they have at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, and in some
embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid
residue identity, when compared and aligned for maximum correspondence,
as measured using a sequence comparison algorithm or by visual
inspection. In certain embodiments, identity exists over a region of the
sequences that is at least about 10, about 20, about 40-60 residues in
length or any integral value therebetween, or over a longer region than
60-80 residues, at least about 90-100 residues, or the sequences are
substantially identical over the full length of the sequences being
compared, such as the coding region of a nucleotide sequence for example.
[0104] A "conservative amino acid substitution" is one in which one amino
acid residue is replaced with another amino acid residue having a similar
side chain. Families of amino acid residues having similar side chains
have been defined in the art, including basic side chains (e.g., lysine,
arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., asparagine, glutamine, serine,
threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine,
alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan), beta-branched side chains (e.g., threonine, valine,
isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,
tryptophan, histidine). For example, substitution of a phenylalanine for
a tyrosine is a conservative substitution. In certain embodiments,
conservative substitutions in the sequences of the polypeptides and
antibodies of the invention do not abrogate the binding of the
polypeptide or antibody containing the amino acid sequence, to the
antigen(s), i.e., the FOLR1 to which the polypeptide or antibody binds.
Methods of identifying nucleotide and amino acid conservative
substitutions which do not eliminate antigen binding are well-known in
the art (see, e.g., Brummell et al., Biochem. 32: 1180-1 187 (1993);
Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al.
Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).
[0105] As used in the present disclosure and claims, the singular forms
"a," "an," and "the" include plural forms unless the context clearly
dictates otherwise.
[0106] It is understood that wherever embodiments are described herein
with the language "comprising," otherwise analogous embodiments described
in terms of "consisting of" and/or "consisting essentially of" are also
provided.
[0107] The term "and/or" as used in a phrase such as "A and/or B" herein
is intended to include both "A and B," "A or B," "A," and "B." Likewise,
the term "and/or" as used in a phrase such as "A, B, and/or C" is
intended to encompass each of the following embodiments: A, B, and C; A,
B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B
(alone); and C (alone).
II. FOLR1-Binding Agents
[0108] The present invention provides agents that specifically bind human
FOLR1. These agents are referred to herein as "FOLR1-binding agents." The
full-length amino acid (aa) and nucleotide (nt) sequences for FOLR1 are
known in the art and also provided herein as represented by SEQ ID NOs:25
and 26, respectively.
[0109] In certain embodiments, the FOLR1 binding agents are antibodies,
immunoconjugates or polypeptides. In some embodiments, the FOLR1 binding
agents are humanized antibodies. In certain embodiments, the FOLR-1
binding agents are humanized versions of the murine Mov19 antibody
(variable heavy and light chain shown as SEQ ID NOs: 17 and 18
respectively).
[0110] In certain embodiments, the FOLR1-binding agents have one or more
of the following effects: inhibit proliferation of tumor cells, reduce
the tumorigenicity of a tumor by reducing the frequency of cancer stem
cells in the tumor, inhibit tumor growth, increase survival, trigger cell
death of tumor cells, differentiate tumorigenic cells to a
non-tumorigenic state, or prevent metastasis of tumor cells.
[0111] In certain embodiments, immunoconjugates or other agents that
specifically bind human FOLR1 trigger cell death via a cytotoxic agent.
For example, in certain embodiments, an antibody to a human FOLR1
antibody is conjugated to a maytansinoid that is activated in tumor cells
expressing the FOLR1 by protein internalization. In certain alternative
embodiments, the agent or antibody is not conjugated.
[0112] In certain embodiments, the FOLR1-binding agents are capable of
inhibiting tumor growth. In certain embodiments, the FOLR1-binding agents
are capable of inhibiting tumor growth in vivo (e.g., in a xenograft
mouse model and/or in a human having cancer). In certain embodiments, the
FOLR1-binding agents are capable of inhibiting tumor growth in a human.
[0113] Thus, the invention provides a humanized antibody or antigen
binding fragment thereof that specifically binds a human folate receptor
1, wherein the antibody comprises: (a) a heavy chain CDR1 comprising
GYFMN (SEQ ID NO:1); a heavy chain CDR2 comprising
RIHPYDGDTFYNQXaa.sub.1FXaa.sub.2Xaa.sub.3 (SEQ ID NO:56); and a heavy
chain CDR3 comprising YDGSRAMDY (SEQ ID NO:3); and (b) a light chain CDR1
comprising KASQSVSFAGTSLMH (SEQ ID NO:7); a light chain CDR2 comprising
RASNLEA (SEQ ID NO:8); and a light chain CDR3 comprising QQSREYPYT (SEQ
ID NO:9); wherein Xaa.sub.1 is selected from K, Q, H, and R; Xaa.sub.2 is
selected from Q, H, N, and R; and Xaa.sub.3 is selected from G, E, T, S,
A, and V. In certain embodiments, the antibody is the huMov19 antibody,
which is the above-described antibody comprising the heavy chain CDR2
RIHPYDGDTFYNQKFQG (SEQ ID NO:2).
[0114] In certain embodiments, the invention provides humanized antibodies
or antigen binding fragments that specifically bind to FOLR1 that
comprise the CDRs of huMov19 with up to four (i.e. 0, 1, 2, 3, or 4)
conservative amino acid substitutions per CDR. Thus, in certain
embodiments the invention provides humanized antibodies or antigen
binding fragments that specifically binds a human folate receptor 1,
wherein the antibody comprises: (a) a heavy chain CDR1 comprising GYFMN
(SEQ ID NO:1), or a variant thereof comprising 1, 2, 3, or 4 conservative
amino acid substitutions; a heavy chain CDR2 comprising RIHPYDGDTFYNQKFQG
(SEQ ID NO:2), or a variant thereof comprising 1, 2, 3, or 4 amino
conservative acid substitutions; and a heavy chain CDR3 comprising
YDGSRAMDY (SEQ ID NO:3), or a variant thereof comprising 1, 2, 3, or 4
conservative amino acid substitutions; and/or (b) a light chain CDR1
comprising KASQSVSFAGTSLMH (SEQ ID NO:7), or a variant thereof comprising
1, 2, 3, or 4 conservative amino acid substitutions; a light chain CDR2
comprising RASNLEA (SEQ ID NO:8), or a variant thereof comprising 1, 2,
3, or 4 conservative amino acid substitutions; and a light chain CDR3
comprising QQSREYPYT (SEQ ID NO:9), or a variant thereof comprising 1, 2,
3, or 4 conservative amino acid substitutions.
[0115] The invention also provides a humanized antibody (huFR1-21) or
antigen binding fragment thereof that specifically binds a human folate
receptor 1, wherein the antibody comprises: (a) a heavy chain CDR1
comprising SSYGMS (SEQ ID NO:30); a heavy chain CDR2 comprising
TISSGGSYTY (SEQ ID NO:31); and a heavy chain CDR3 comprising DGEGGLYAMDY
(SEQ ID NO:32); and/or (b) a light chain CDR1 comprising KASDHINNWLA (SEQ
ID NO:27); a light chain CDR2 comprising GATSLET (SEQ ID NO:28); and a
light chain CDR3 comprising QQYWSTPFT (SEQ ID NO:29).
[0116] In certain embodiments, the invention provides humanized antibodies
or antigen binding fragments that specifically bind to FOLR1 that
comprise the CDRs of huFR1-21 with up to four (i.e. 0, 1, 2, 3, or 4)
conservative amino acid substitutions per CDR. Thus, in certain
embodiments the invention provides humanized antibodies or antigen
binding fragments that specifically binds a human folate receptor 1,
wherein the antibody comprises: (a) a heavy chain CDR1 comprising SSYGMS
(SEQ ID NO:30) or a variant thereof comprising 1, 2, 3, or 4 conservative
amino acid substitutions; and/or a heavy chain CDR2 comprising TISSGGSYTY
(SEQ ID NO:31) or a variant thereof comprising 1, 2, 3, or 4 conservative
amino acid substitutions; and/or and a heavy chain CDR3 comprising
DGEGGLYAMDY (SEQ ID NO:32) or a variant thereof comprising 1, 2, 3, or 4
conservative amino acid substitutions; and/or (b) a light chain CDR1
comprising KASDHINNWLA (SEQ ID NO:27) or a variant thereof comprising 1,
2, 3, or 4 conservative amino acid substitutions; and/or a light chain
CDR2 comprising GATSLET (SEQ ID NO:28) or a variant thereof comprising 1,
2, 3, or 4 conservative amino acid substitutions; and/or a light chain
CDR3 comprising QQYWSTPFT (SEQ ID NO:29) or a variant thereof comprising
1, 2, 3, or 4 conservative amino acid substitutions.
[0117] In certain embodiments, the invention provides humanized antibodies
or antigen binding fragments that specifically bind to FOLR1 that
comprise the CDRs of huFR1-48 with up to four (i.e. 0, 1, 2, 3, or 4)
conservative amino acid substitutions per CDR. Thus, in certain
embodiments the invention provides humanized antibodies or antigen
binding fragments that specifically binds a human folate receptor 1,
wherein the antibody comprises: (a) a heavy chain CDR1 comprising TNYWMQ
(SEQ ID NO:60) or a variant thereof comprising 1, 2, 3, or 4 conservative
amino acid substitutions; and/or a heavy chain CDR2 comprising IYPGNGDSR
(SEQ ID NO:61) or a variant thereof comprising 1, 2, 3, or 4 conservative
amino acid substitutions; and/or and a heavy chain CDR3 comprising
RDGNYAAY (SEQ ID NO:62) or a variant thereof comprising 1, 2, 3, or 4
conservative amino acid substitutions; and/or (b) a light chain CDR1
comprising RASENIYSNLA (SEQ ID NO:57) or a variant thereof comprising 1,
2, 3, or 4 conservative amino acid substitutions; and/or a light chain
CDR2 comprising AATNLAD (SEQ ID NO:58) or a variant thereof comprising 1,
2, 3, or 4 conservative amino acid substitutions; and/or a light chain
CDR3 comprising QHFWASPYT (SEQ ID NO:59) or a variant thereof comprising
1, 2, 3, or 4 conservative amino acid substitutions.
[0118] In certain embodiments, the invention provides humanized antibodies
or antigen binding fragments that specifically bind to FOLR1 that
comprise the CDRs of huFR1-49 with up to four (i.e. 0, 1, 2, 3, or 4)
conservative amino acid substitutions per CDR. Thus, in certain
embodiments the invention provides humanized antibodies or antigen
binding fragments that specifically binds a human folate receptor 1,
wherein the antibody comprises: (a) a heavy chain CDR1 comprising TNYWMY
(SEQ ID NO:66) or a variant thereof comprising 1, 2, 3, or 4 conservative
amino acid substitutions; and/or a heavy chain CDR2 comprising AIYPGNSDTT
(SEQ ID NO:67) or a variant thereof comprising 1, 2, 3, or 4 conservative
amino acid substitutions; and/or and a heavy chain CDR3 comprising
RHDYGAMDY (SEQ ID NO:68) or a variant thereof comprising 1, 2, 3, or 4
conservative amino acid substitutions; and/or (b) a light chain CDR1
comprising RASENIYTNLA (SEQ ID NO:63) or a variant thereof comprising 1,
2, 3, or 4 conservative amino acid substitutions; and/or a light chain
CDR2 comprising TASNLAD (SEQ ID NO:64) or a variant thereof comprising 1,
2, 3, or 4 conservative amino acid substitutions; and/or a light chain
CDR3 comprising QHFWVSPYT (SEQ ID NO:65) or a variant thereof comprising
1, 2, 3, or 4 conservative amino acid substitutions.
[0119] In certain embodiments, the invention provides humanized antibodies
or antigen binding fragments that specifically bind to FOLR1 that
comprise the CDRs of huFR1-57 with up to four (i.e. 0, 1, 2, 3, or 4)
conservative amino acid substitutions per CDR. Thus, in certain
embodiments the invention provides humanized antibodies or antigen
binding fragments that specifically binds a human folate receptor 1,
wherein the antibody comprises: (a) a heavy chain CDR1 comprising SSFGMH
(SEQ ID NO:72) or a variant thereof comprising 1, 2, 3, or 4 conservative
amino acid substitutions; and/or a heavy chain CDR2 comprising YISSGSSTIS
(SEQ ID NO:73) or a variant thereof comprising 1, 2, 3, or 4 conservative
amino acid substitutions; and/or and a heavy chain CDR3 comprising
EAYGSSMEY (SEQ ID NO:74) or a variant thereof comprising 1, 2, 3, or 4
conservative amino acid substitutions; and/or (b) a light chain CDR1
comprising RASQNINNNLH (SEQ ID NO:69) or a variant thereof comprising 1,
2, 3, or 4 conservative amino acid substitutions; and/or a light chain
CDR2 comprising YVSQSVS (SEQ ID NO:70) or a variant thereof comprising 1,
2, 3, or 4 conservative amino acid substitutions; and/or a light chain
CDR3 comprising QQSNSWPHYT (SEQ ID NO:71) or a variant thereof comprising
1, 2, 3, or 4 conservative amino acid substitutions.
[0120] In certain embodiments, the invention provides humanized antibodies
or antigen binding fragments that specifically bind to FOLR1 that
comprise the CDRs of huFR1-65 with up to four (i.e. 0, 1, 2, 3, or 4)
conservative amino acid substitutions per CDR. Thus, in certain
embodiments the invention provides humanized antibodies or antigen
binding fragments that specifically binds a human folate receptor 1,
wherein the antibody comprises: (a) a heavy chain CDR1 comprising TSYTMH
(SEQ ID NO:78) or a variant thereof comprising 1, 2, 3, or 4 conservative
amino acid substitutions; and/or a heavy chain CDR2 comprising YINPISGYTN
(SEQ ID NO:79) or a variant thereof comprising 1, 2, 3, or 4 conservative
amino acid substitutions; and/or and a heavy chain CDR3 comprising
GGAYGRKPMDY (SEQ ID NO:80) or a variant thereof comprising 1, 2, 3, or 4
conservative amino acid substitutions; and/or (b) a light chain CDR1
comprising KASQNVGPNVA (SEQ ID NO:75) or a variant thereof comprising 1,
2, 3, or 4 conservative amino acid substitutions; and/or a light chain
CDR2 comprising SASYRYS (SEQ ID NO:76) or a variant thereof comprising 1,
2, 3, or 4 conservative amino acid substitutions; and/or a light chain
CDR3 comprising QQYNSYPYT (SEQ ID NO:77) or a variant thereof comprising
1, 2, 3, or 4 conservative amino acid substitutions.
[0121] Polypeptides comprising one of the individual light chains or heavy
chains described herein, as well as polypeptides (e.g., antibodies)
comprising both a light chain and a heavy chain are also provided. The
polypeptides of SEQ ID NOs: 4 and 6 comprise the variable domain of the
heavy chain of huMov19, and the heavy chain of huMov19, respectively. The
polypeptides of SEQ ID NOs:10-13 comprise the variable domain light chain
version 1.00, the variable domain light chain version 1.60, the light
chain version 1.00, and the light chain version 1.60 of huMov19,
respectively. The polypeptides of SEQ ID NOs: 42 and 46 comprise the
variable domain of the heavy chain of huFR1-21, and the heavy chain of
huFR1-21, respectively. The polypeptides of SEQ ID NOs:41 and 45 comprise
the variable domain light chain and light chain of huFR1-21,
respectively. The polypeptides of SEQ ID NOs: 97 and 113 comprise the
variable domain of the heavy chain of huFR1-48, and the heavy chain of
huFR1-48, respectively. The polypeptides of SEQ ID NOs:96 and 112
comprise the variable domain light chain and light chain of huFR1-48,
respectively. The polypeptides of SEQ ID NOs: 99 and 115 comprise the
variable domain of the heavy chain of huFR1-49, and the heavy chain of
huFR1-49, respectively. The polypeptides of SEQ ID NOs:98 and 114
comprise the variable domain light chain and light chain of huFR1-49,
respectively. The polypeptides of SEQ ID NOs: 101 and 117 comprise the
variable domain of the heavy chain of huFR1-57, and the heavy chain of
huFR1-57, respectively. The polypeptides of SEQ ID NOs:100 and 116
comprise the variable domain light chain and light chain of huFR1-57,
respectively. The polypeptides of SEQ ID NOs:103 and 119 comprise the
variable domain of the heavy chain of huFR1-65, and the heavy chain of
huFR1-65, respectively. The polypeptides of SEQ ID NOs:102 and 118
comprise the variable domain light chain and light chain of huFR1-65,
respectively.
[0122] Also provided are polypeptides that comprise: (a) a polypeptide
having at least about 90% sequence identity to SEQ ID NO:4 or 6; and/or
(b) a polypeptide having at least about 90% sequence identity to SEQ ID
NOs:10-13. Also provided are polypeptides that comprise: (a) a
polypeptide having about 90% sequence identity to SEQ ID NO: 42 or 46;
and/or (b) a polypeptide having at least about 90% sequence identity to
SEQ ID NOs: 41 and 45. Also provided are polypeptides that comprise: (a)
a polypeptide having at least about 90% sequence identity to SEQ ID NO:97
or 113; and/or (b) a polypeptide having at least about 90% sequence
identity to SEQ ID NOs:96 or 112. Also provided are polypeptides that
comprise: (a) a polypeptide having at least about 90% sequence identity
to SEQ ID NO:99 or 115; and/or (b) a polypeptide having at least about
90% sequence identity to SEQ ID NOs:98 or 114. Also provided are
polypeptides that comprise: (a) a polypeptide having at least about 90%
sequence identity to SEQ ID NO:101 or 117; and/or (b) a polypeptide
having at least about 90% sequence identity to SEQ ID NOs:100 or 116.
Also provided are polypeptides that comprise: (a) a polypeptide having at
least about 90% sequence identity to SEQ ID NO:103 or 119; and/or (b) a
polypeptide having at least about 90% sequence identity to SEQ ID NOs:102
or 118. In certain embodiments, the polypeptide comprises a polypeptide
having at least about 95%, at least about 96%, at least about 97%, at
least about 98%, or at least about 99% sequence identity to SEQ ID NOs:4,
6, 10-13, 41, 42, 45 or 46. Thus, in certain embodiments, the polypeptide
comprises (a) a polypeptide having at least about 95% sequence identity
to SEQ ID NO:4 or 6, and/or (b) a polypeptide having at least about 95%
sequence identity to SEQ ID NOs:10-13. In certain embodiments, the
polypeptide comprises (a) a polypeptide having at least about 95%
sequence identity to SEQ ID NO:42 or 46, and/or (b) a polypeptide having
at least about 95% sequence identity to SEQ ID NOs:41 or 45. Also
provided are polypeptides that comprise: (a) a polypeptide having at
least about 95% sequence identity to SEQ ID NO:97 or 113; and/or (b) a
polypeptide having at least about 95% sequence identity to SEQ ID NOs:96
or 112. Also provided are polypeptides that comprise: (a) a polypeptide
having at least about 95% sequence identity to SEQ ID NO:99 or 115;
and/or (b) a polypeptide having at least about 95% sequence identity to
SEQ ID NOs:98 or 114. Also provided are polypeptides that comprise: (a) a
polypeptide having at least about 95% sequence identity to SEQ ID NO:101
or 117; and/or (b) a polypeptide having at least about 95% sequence
identity to SEQ ID NOs:100 or 116. Also provided are polypeptides that
comprise: (a) a polypeptide having at least about 95% sequence identity
to SEQ ID NO:103 or 119; and/or (b) a polypeptide having at least about
95% sequence identity to SEQ ID NOs:102 or 118. In certain embodiments,
the polypeptide comprises (a) a polypeptide having the amino acid
sequence of SEQ ID NO: 4; and/or (b) a polypeptide having the amino acid
sequence of SEQ ID NO:10 or SEQ ID NO:11. In certain embodiments, the
polypeptide comprises (a) a polypeptide having the amino acid sequence of
SEQ ID NO:45; and/or (b) a polypeptide having the amino acid sequence of
SEQ ID NO:46. In certain embodiments, the polypeptide comprises (a) a
polypeptide having the amino acid sequence of SEQ ID NO: 6; and/or (b) a
polypeptide having the amino acid sequence of SEQ ID NO:12 or SEQ ID
NO:13. In certain embodiments, the polypeptide is an antibody and/or the
polypeptide specifically binds human folate receptor 1. In certain
embodiments, the polypeptide is a humanized antibody that specifically
binds human folate receptor 1. For example, the invention provides an
antibody or humanized antibody that specifically binds a human FOLR1 that
comprises (a) a polypeptide having the amino acid sequence of SEQ ID NO:
4; and (b) a polypeptide having the amino acid sequence of SEQ ID NO:10
or SEQ ID NO:11. In certain embodiments the polypeptide comprising SEQ ID
NO:4 is a heavy chain variable region. In certain embodiments, the
polypeptide comprising SEQ ID NO:10 or 11 is a light chain variable
region. The invention also provides an antibody or humanized antibody
that specifically binds a human FOLR1 that comprises (a) a polypeptide
having the amino acid sequence of SEQ ID NO: 6; and (b) a polypeptide
having the amino acid sequence of SEQ ID NO:12 or SEQ ID NO:13. The
invention also provides and antibody or humanized antibody that
specifically binds a human FOLR1 that comprises (a) a polypeptide having
the amino acid sequence of SEQ ID NO:45; and (b) a polypeptide having the
amino acid sequence of SEQ ID NO:46. The invention also provides and
antibody or humanized antibody that specifically binds a human FOLR1 that
comprises (a) a polypeptide having the amino acid sequence of SEQ ID
NO:112; and (b) a polypeptide having the amino acid sequence of SEQ ID
NO:113. The invention also provides and antibody or humanized antibody
that specifically binds a human FOLR1 that comprises (a) a polypeptide
having the amino acid sequence of SEQ ID NO:114; and (b) a polypeptide
having the amino acid sequence of SEQ ID NO:115. The invention also
provides and antibody or humanized antibody that specifically binds a
human FOLR1 that comprises (a) a polypeptide having the amino acid
sequence of SEQ ID NO:116; and (b) a polypeptide having the amino acid
sequence of SEQ ID NO:117. The invention also provides and antibody or
humanized antibody that specifically binds a human FOLR1 that comprises
(a) a polypeptide having the amino acid sequence of SEQ ID NO:118; and
(b) a polypeptide having the amino acid sequence of SEQ ID NO:119. In
certain embodiments, the polypeptide having a certain percentage of
sequence identity to SEQ ID NOs: 4, 6, 10-13, 41, 42, 45, 46, 96-103 and
112-119 differs from SEQ ID NO: 4, 6, 10-13, 41, 42, 45, 46, 96-103 and
112-119 by conservative amino acid substitutions only.
[0123] In certain embodiments, the FOLR1-binding agent comprises, consists
essentially of, or consists of an anti-FOLR1 antibody selected from the
group consisting of huMov19, FR-1-21, FR1-48, FR1-49, FR1-57, and FR1-65
antibodies.
[0124] In certain embodiments, the huMov19 antibody is encoded by the
plasmids deposited with the American Type Culture Collection (ATCC) on
Apr. 7, 2010 and having ATCC deposit nos. PTA-10772 and PTA-10773 or
10774.
[0125] In certain embodiments, the FR-1-21 antibody is encoded by the
plasmids deposited with the ATCC on Apr. 7, 2010, and assigned deposit
designation numbers PTA-10775 and 10776.
[0126] In certain embodiments, the humanized antibodies bind FOLR1 with
substantially the same affinity as the antibody chimeric Mov19. The
affinity or avidity of an antibody for an antigen can be determined
experimentally using any suitable method well known in the art, e.g. flow
cytometry, enzyme-linked immunoabsorbent assay (ELISA), or
radioimmunoassay (MA), or kinetics (e.g., BIACORE.TM. analysis). Direct
binding assays as well as competitive binding assay formats can be
readily employed. (See, for example, Berzofsky, et al., "Antibody-Antigen
Interactions," In Fundamental Immunology, Paul, W. E., Ed., Raven Press:
New York, N.Y. (1984); Kuby, Janis Immunology, W. H. Freeman and Company:
New York, N.Y. (1992); and methods described herein. The measured
affinity of a particular antibody-antigen interaction can vary if
measured under different conditions (e.g., salt concentration, pH,
temperature). Thus, measurements of affinity and other antigen-binding
parameters (e.g., KD or Kd, K.sub.on, K.sub.off) are made with
standardized solutions of antibody and antigen, and a standardized
buffer, as known in the art and such as the buffer described herein.
[0127] In one aspect, binding assays can be performed using flow cytometry
on cells expressing the FOLR1 antigen on the surface. For example,
FOLR1-positive cells such as SKOV3 were incubated with varying
concentrations of anti-FOLR1 antibodies using 1.times.105 cells per
sample in 100 .mu.L FACS buffer (RPMI-1640 medium supplemented with 2%
normal goat serum). Then, the cells were pelleted, washed, and incubated
for 1 h with 100 .mu.L of FITC-conjugated goat-anti-mouse or
goat-anti-human IgG-antibody (such as is obtainable from, for example
Jackson Laboratory, 6 .mu.g/mL in FACS buffer). The cells were pelleted
again, washed with FACS buffer and resuspended in 200 .mu.L of PBS
containing 1% formaldehyde. Samples were acquired, for example, using a
FACSCalibur flow cytometer with the HTS multiwell sampler and analyzed
using CellQuest Pro (all from BD Biosciences, San Diego, US). For each
sample the mean fluorescence intensity for FL1 (MFI) was exported and
plotted against the antibody concentration in a semi-log plot to generate
a binding curve. A sigmoidal dose-response curve is fitted for binding
curves and EC50 values are calculated using programs such as GraphPad
Prism v4 with default parameters (GraphPad software, San Diego, Calif.).
EC50 values can be used as a measure for the apparent dissociation
constant "Kd" or "KD" for each antibody.
[0128] Monoclonal antibodies can be prepared using hybridoma methods, such
as those described by Kohler and Milstein (1975) Nature 256:495. Using
the hybridoma method, a mouse, hamster, or other appropriate host animal,
is immunized as described above to elicit the production by lymphocytes
of antibodies that will specifically bind to an immunizing antigen.
Lymphocytes can also be immunized in vitro. Following immunization, the
lymphocytes are isolated and fused with a suitable myeloma cell line
using, for example, polyethylene glycol, to form hybridoma cells that can
then be selected away from unfused lymphocytes and myeloma cells.
Hybridomas that produce monoclonal antibodies directed specifically
against a chosen antigen as determined by immunoprecipitation,
immunoblotting, or by an in vitro binding assay (e.g. radioimmunoassay
(MA); enzyme-linked immunosorbent assay (ELISA)) can then be propagated
either in vitro culture using standard methods (Goding, Monoclonal
Antibodies: Principles and Practice, Academic Press, 1986) or in vivo as
ascites tumors in an animal. The monoclonal antibodies can then be
purified from the culture medium or ascites fluid as described for
polyclonal antibodies above.
[0129] Alternatively monoclonal antibodies can also be made using
recombinant DNA methods as described in U.S. Pat. No. 4,816,567. The
polynucleotides encoding a monoclonal antibody are isolated from mature
B-cells or hybridoma cell, such as by RT-PCR using oligonucleotide
primers that specifically amplify the genes encoding the heavy and light
chains of the antibody, and their sequence is determined using
conventional procedures. The isolated polynucleotides encoding the heavy
and light chains are then cloned into suitable expression vectors, which
when transfected into host cells such as E. coli cells, simian COS cells,
Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise
produce immunoglobulin protein, monoclonal antibodies are generated by
the host cells. Also, recombinant monoclonal antibodies or fragments
thereof of the desired species can be isolated from phage display
libraries expressing CDRs of the desired species as described (McCafferty
et al., 1990, Nature, 348:552-554; Clackson et al., 1991, Nature,
352:624-628; and Marks et al., 1991, J. Mol. Biol., 222:581-597).
[0130] The polynucleotide(s) encoding a monoclonal antibody can further be
modified in a number of different manners using recombinant DNA
technology to generate alternative antibodies. In some embodiments, the
constant domains of the light and heavy chains of, for example, a mouse
monoclonal antibody can be substituted 1) for those regions of, for
example, a human antibody to generate a chimeric antibody or 2) for a
non-immunoglobulin polypeptide to generate a fusion antibody. In some
embodiments, the constant regions are truncated or removed to generate
the desired antibody fragment of a monoclonal antibody. Site-directed or
high-density mutagenesis of the variable region can be used to optimize
specificity, affinity, etc. of a monoclonal antibody.
[0131] In some embodiments, the monoclonal antibody against the human
FOLR1 is a humanized antibody. In certain embodiments, such antibodies
are used therapeutically to reduce antigenicity and HAMA (human
anti-mouse antibody) responses when administered to a human subject.
[0132] Methods for engineering, humanizing or resurfacing non-human or
human antibodies can also be used and are well known in the art. A
humanized, resurfaced or similarly engineered antibody can have one or
more amino acid residues from a source that is non-human, e.g., but not
limited to, mouse, rat, rabbit, non-human primate or other mammal. These
non-human amino acid residues are replaced by residues that are often
referred to as "import" residues, which are typically taken from an
"import" variable, constant or other domain of a known human sequence.
[0133] Such imported sequences can be used to reduce immunogenicity or
reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity,
specificity, half-life, or any other suitable characteristic, as known in
the art. In general, the CDR residues are directly and most substantially
involved in influencing FOLR1 binding. Accordingly, part or all of the
non-human or human CDR sequences are maintained while the non-human
sequences of the variable and constant regions can be replaced with human
or other amino acids.
[0134] Antibodies can also optionally be humanized, resurfaced, engineered
or human antibodies engineered with retention of high affinity for the
antigen FOLR1 and other favorable biological properties. To achieve this
goal, humanized (or human) or engineered anti-FOLR1 antibodies and
resurfaced antibodies can be optionally prepared by a process of analysis
of the parental sequences and various conceptual humanized and engineered
products using three-dimensional models of the parental, engineered, and
humanized sequences. Three-dimensional immunoglobulin models are commonly
available and are familiar to those skilled in the art. Computer programs
are available which illustrate and display probable three-dimensional
conformational structures of selected candidate immunoglobulin sequences.
Inspection of these displays permits analysis of the likely role of the
residues in the functioning of the candidate immunoglobulin sequence,
i.e., the analysis of residues that influence the ability of the
candidate immunoglobulin to bind its antigen, such as FOLR1. In this way,
framework (FR) residues can be selected and combined from the consensus
and import sequences so that the desired antibody characteristic, such as
increased affinity for the target antigen(s), is achieved.
[0135] Humanization, resurfacing or engineering of antibodies of the
present invention can be performed using any known method, such as but
not limited to those described in, Winter (Jones et al., Nature 321:522
(1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al.,
Science 239:1534 (1988)), Sims et al., J. Immunol. 151: 2296 (1993);
Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc.
Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol.
151:2623 (1993), U.S. Pat. Nos. 5,639,641, 5,723,323; 5,976,862;
5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886;
5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089;
5,225,539; 4,816,567; PCT/US98/16280; US96/18978; US91/09630; US91/05939;
US94/01234; GB89/01334; GB91/01134; GB92/01755; WO90/14443; WO90/14424;
WO90/14430; EP 229246; U.S. Pat. Nos. 7,557,189; 7,538,195; and
7,342,110, each of which is entirely incorporated herein by reference,
including the references cited therein.
[0136] In certain alternative embodiments, the antibody to FOLR1 is a
human antibody. Human antibodies can be directly prepared using various
techniques known in the art. Immortalized human B lymphocytes immunized
in vitro or isolated from an immunized individual that produce an
antibody directed against a target antigen can be generated (See, e.g.,
Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.
77 (1985); Boemer et al., 1991, J. Immunol., 147 (1):86-95; and U.S. Pat.
No. 5,750,373). Also, the human antibody can be selected from a phage
library, where that phage library expresses human antibodies, as
described, for example, in Vaughan et al., 1996, Nat. Biotech.,
14:309-314, Sheets et al., 1998, Proc. Nat'l. Acad. Sci., 95:6157-6162,
Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381, and Marks et al.,
1991, J. Mol. Biol., 222:581). Techniques for the generation and use of
antibody phage libraries are also described in U.S. Pat. Nos. 5,969,108,
6,172,197, 5,885,793, 6,521,404; 6,544,731; 6,555,313; 6,582,915;
6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe et
al., 2007, J. Mol. Bio., doi:10.1016/j.jmb.2007.12.018 (each of which is
incorporated by reference in its entirety). Affinity maturation
strategies and chain shuffling strategies (Marks et al., 1992,
Bio/Technology 10:779-783, incorporated by reference in its entirety) are
known in the art and can be employed to generate high affinity human
antibodies.
[0137] Humanized antibodies can also be made in transgenic mice containing
human immunoglobulin loci that are capable upon immunization of producing
the full repertoire of human antibodies in the absence of endogenous
immunoglobulin production. This approach is described in U.S. Pat. Nos.
5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.
[0138] This invention also encompasses bispecific antibodies that
specifically recognize a human folate receptor 1. Bispecific antibodies
are antibodies that are capable of specifically recognizing and binding
at least two different epitopes. The different epitopes can either be
within the same molecule (e.g. the same human folate receptor 1) or on
different molecules such that both, for example, the antibodies can
specifically recognize and bind a human folate receptor 1 as well as, for
example, 1) an effector molecule on a leukocyte such as a T-cell receptor
(e.g. CD3) or Fc receptor (e.g. CD64, CD32, or CD16) or 2) a cytotoxic
agent as described in detail below.
[0139] Exemplary bispecific antibodies can bind to two different epitopes,
at least one of which originates in a polypeptide of the invention.
Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be
combined with an arm which binds to a triggering molecule on a leukocyte
such as a T cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc
receptors for IgG so as to focus cellular defense mechanisms to the cell
expressing the particular antigen. Bispecific antibodies can also be used
to direct cytotoxic agents to cells which express a particular antigen.
These antibodies possess an antigen-binding arm and an arm which binds a
cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA,
or TETA. Techniques for making bispecific antibodies are common in the
art (Millstein et al., 1983, Nature 305:537-539; Brennan et al., 1985,
Science 229:81; Suresh et al, 1986, Methods in Enzymol. 121:120;
Traunecker et al., 1991, EMBO J. 10:3655-3659; Shalaby et al., 1992, J.
Exp. Med. 175:217-225; Kostelny et al., 1992, J. Immunol. 148:1547-1553;
Gruber et al., 1994, J. Immunol. 152:5368; and U.S. Pat. No. 5,731,168).
Antibodies with more than two valencies are also contemplated. For
example, trispecific antibodies can be prepared (Tutt et al., J. Immunol.
147:60 (1991)). Thus, in certain embodiments the antibodies to FOLR1 are
multispecific.
[0140] In certain embodiments are provided an antibody fragment to, for
example, increase tumor penetration. Various techniques are known for the
production of antibody fragments. Traditionally, these fragments are
derived via proteolytic digestion of intact antibodies (for example
Morimoto et al., 1993, Journal of Biochemical and Biophysical Methods
24:107-117; Brennan et al., 1985, Science, 229:81). In certain
embodiments, antibody fragments are produced recombinantly. Fab, Fv, and
scFv antibody fragments can all be expressed in and secreted from E. coli
or other host cells, thus allowing the production of large amounts of
these fragments. Such antibody fragments can also be isolated from the
antibody phage libraries discussed above. The antibody fragment can also
be linear antibodies as described in U.S. Pat. No. 5,641,870, for
example, and can be monospecific or bispecific. Other techniques for the
production of antibody fragments will be apparent to the skilled
practitioner.
[0141] According to the present invention, techniques can be adapted for
the production of single-chain antibodies specific to human folate
receptor 1 (see U.S. Pat. No. 4,946,778). In addition, methods can be
adapted for the construction of Fab expression libraries (Huse, et al.,
Science 246:1275-1281 (1989)) to allow rapid and effective identification
of monoclonal Fab fragments with the desired specificity for a folate 1
receptor, or derivatives, fragments, analogs or homologs thereof.
Antibody fragments can be produced by techniques in the art including,
but not limited to: (a) a F(ab')2 fragment produced by pepsin digestion
of an antibody molecule; (b) a Fab fragment generated by reducing the
disulfide bridges of an F(ab')2 fragment, (c) a Fab fragment generated by
the treatment of the antibody molecule with papain and a reducing agent,
and (d) Fv fragments.
[0142] It can further be desirable, especially in the case of antibody
fragments, to modify an antibody in order to increase its serum
half-life. This can be achieved, for example, by incorporation of a
salvage receptor binding epitope into the antibody fragment by mutation
of the appropriate region in the antibody fragment or by incorporating
the epitope into a peptide tag that is then fused to the antibody
fragment at either end or in the middle (e.g., by DNA or peptide
synthesis).
[0143] Heteroconjugate antibodies are also within the scope of the present
invention. Heteroconjugate antibodies are composed of two covalently
joined antibodies. Such antibodies have, for example, been proposed to
target immune cells to unwanted cells (U.S. Pat. No. 4,676,980). It is
contemplated that the antibodies can be prepared in vitro using known
methods in synthetic protein chemistry, including those involving
crosslinking agents. For example, immunotoxins can be constructed using a
disulfide exchange reaction or by forming a thioether bond. Examples of
suitable reagents for this purpose include iminothiolate and
methyl-4-mercaptobutyrimidate.
[0144] For the purposes of the present invention, it should be appreciated
that modified antibodies can comprise any type of variable region that
provides for the association of the antibody with the polypeptides of a
human FOLR1. In this regard, the variable region can comprise or be
derived from any type of mammal that can be induced to mount a humoral
response and generate immunoglobulins against the desired tumor
associated antigen. As such, the variable region of the modified
antibodies can be, for example, of human, murine, non-human primate (e.g.
cynomolgus monkeys, macaques, etc.) or lupine origin. In some embodiments
both the variable and constant regions of the modified immunoglobulins
are human. In other embodiments the variable regions of compatible
antibodies (usually derived from a non-human source) can be engineered or
specifically tailored to improve the binding properties or reduce the
immunogenicity of the molecule. In this respect, variable regions useful
in the present invention can be humanized or otherwise altered through
the inclusion of imported amino acid sequences.
[0145] In certain embodiments, the variable domains in both the heavy and
light chains are altered by at least partial replacement of one or more
CDRs and, if necessary, by partial framework region replacement and
sequence changing. Although the CDRs can be derived from an antibody of
the same class or even subclass as the antibody from which the framework
regions are derived, it is envisaged that the CDRs will be derived from
an antibody of different class and in certain embodiments from an
antibody from a different species. It may not be necessary to replace all
of the CDRs with the complete CDRs from the donor variable region to
transfer the antigen binding capacity of one variable domain to another.
Rather, it may only be necessary to transfer those residues that are
necessary to maintain the activity of the antigen binding site. Given the
explanations set forth in U.S. Pat. Nos. 5,585,089, 5,693,761 and
5,693,762, it will be well within the competence of those skilled in the
art, either by carrying out routine experimentation or by trial and error
testing to obtain a functional antibody with reduced immunogenicity.
[0146] Alterations to the variable region notwithstanding, those skilled
in the art will appreciate that the modified antibodies of this invention
will comprise antibodies (e.g., full-length antibodies or immunoreactive
fragments thereof) in which at least a fraction of one or more of the
constant region domains has been deleted or otherwise altered so as to
provide desired biochemical characteristics such as increased tumor
localization or reduced serum half-life when compared with an antibody of
approximately the same immunogenicity comprising a native or unaltered
constant region. In some embodiments, the constant region of the modified
antibodies will comprise a human constant region. Modifications to the
constant region compatible with this invention comprise additions,
deletions or substitutions of one or more amino acids in one or more
domains. That is, the modified antibodies disclosed herein can comprise
alterations or modifications to one or more of the three heavy chain
constant domains (CH1, CH2 or CH3) and/or to the light chain constant
domain (CL). In some embodiments, modified constant regions wherein one
or more domains are partially or entirely deleted are contemplated. In
some embodiments, the modified antibodies will comprise domain deleted
constructs or variants wherein the entire CH2 domain has been removed
(.DELTA.CH2 constructs). In some embodiments, the omitted constant region
domain will be replaced by a short amino acid spacer (e.g. 10 residues)
that provides some of the molecular flexibility typically imparted by the
absent constant region.
[0147] Besides their configuration, it is known in the art that the
constant region mediates several effector functions. For example, binding
of the C1 component of complement to antibodies activates the complement
system. Activation of complement is important in the opsonisation and
lysis of cell pathogens. The activation of complement also stimulates the
inflammatory response and can also be involved in autoimmune
hypersensitivity. Further, antibodies bind to cells via the Fc region,
with a Fc receptor site on the antibody Fc region binding to a Fc
receptor (FcR) on a cell. There are a number of Fc receptors which are
specific for different classes of antibody, including IgG (gamma
receptors), IgE (eta receptors), IgA (alpha receptors) and IgM (mu
receptors). Binding of antibody to Fc receptors on cell surfaces triggers
a number of important and diverse biological responses including
engulfment and destruction of antibody-coated particles, clearance of
immune complexes, lysis of antibody-coated target cells by killer cells
(called antibody-dependent cell-mediated cytotoxicity, or ADCC), release
of inflammatory mediators, placental transfer and control of
immunoglobulin production.
[0148] In certain embodiments, the FOLR1-binding antibodies provide for
altered effector functions that, in turn, affect the biological profile
of the administered antibody. For example, the deletion or inactivation
(through point mutations or other means) of a constant region domain can
reduce Fc receptor binding of the circulating modified antibody thereby
increasing tumor localization. In other cases it may be that constant
region modifications, consistent with this invention, moderate complement
binding and thus reduce the serum half life and nonspecific association
of a conjugated cytotoxin. Yet other modifications of the constant region
can be used to eliminate disulfide linkages or oligosaccharide moieties
that allow for enhanced localization due to increased antigen specificity
or antibody flexibility. Similarly, modifications to the constant region
in accordance with this invention can easily be made using well known
biochemical or molecular engineering techniques well within the purview
of the skilled artisan.
[0149] In certain embodiments, a FOLR1-binding agent that is an antibody
does not have one or more effector functions. For instance, in some
embodiments, the antibody has no antibody-dependent cellular cytoxicity
(ADCC) activity and/or no complement-dependent cytoxicity (CDC) activity.
In certain embodiments, the antibody does not bind to an Fc receptor
and/or complement factors. In certain embodiments, the antibody has no
effector function.
[0150] It will be noted that in certain embodiments, the modified
antibodies can be engineered to fuse the CH3 domain directly to the hinge
region of the respective modified antibodies. In other constructs it may
be desirable to provide a peptide spacer between the hinge region and the
modified CH2 and/or CH3 domains. For example, compatible constructs could
be expressed wherein the CH2 domain has been deleted and the remaining
CH3 domain (modified or unmodified) is joined to the hinge region with a
5-20 amino acid spacer. Such a spacer can be added, for instance, to
ensure that the regulatory elements of the constant domain remain free
and accessible or that the hinge region remains flexible. However, it
should be noted that amino acid spacers can, in some cases, prove to be
immunogenic and elicit an unwanted immune response against the construct.
Accordingly, in certain embodiments, any spacer added to the construct
will be relatively non-immunogenic, or even omitted altogether, so as to
maintain the desired biochemical qualities of the modified antibodies.
[0151] Besides the deletion of whole constant region domains, it will be
appreciated that the antibodies of the present invention can be provided
by the partial deletion or substitution of a few or even a single amino
acid. For example, the mutation of a single amino acid in selected areas
of the CH2 domain may be enough to substantially reduce Fc binding and
thereby increase tumor localization. Similarly, it may be desirable to
simply delete that part of one or more constant region domains that
control the effector function (e.g. complement C1Q binding) to be
modulated. Such partial deletions of the constant regions can improve
selected characteristics of the antibody (serum half-life) while leaving
other desirable functions associated with the subject constant region
domain intact. Moreover, as alluded to above, the constant regions of the
disclosed antibodies can be modified through the mutation or substitution
of one or more amino acids that enhances the profile of the resulting
construct. In this respect it may be possible to disrupt the activity
provided by a conserved binding site (e.g. Fc binding) while
substantially maintaining the configuration and immunogenic profile of
the modified antibody. Certain embodiments can comprise the addition of
one or more amino acids to the constant region to enhance desirable
characteristics such as decreasing or increasing effector function or
provide for more cytotoxin or carbohydrate attachment. In such
embodiments it can be desirable to insert or replicate specific sequences
derived from selected constant region domains.
[0152] The present invention further embraces variants and equivalents
which are substantially homologous to the chimeric, humanized and human
antibodies, or antibody fragments thereof, set forth herein. These can
contain, for example, conservative substitution mutations, i.e. the
substitution of one or more amino acids by similar amino acids. For
example, conservative substitution refers to the substitution of an amino
acid with another within the same general class such as, for example, one
acidic amino acid with another acidic amino acid, one basic amino acid
with another basic amino acid or one neutral amino acid by another
neutral amino acid. What is intended by a conservative amino acid
substitution is well known in the art.
[0153] The polypeptides of the present invention can be recombinant
polypeptides, natural polypeptides, or synthetic polypeptides comprising
an antibody, or fragment thereof, against a human FOLR1. It will be
recognized in the art that some amino acid sequences of the invention can
be varied without significant effect of the structure or function of the
protein. Thus, the invention further includes variations of the
polypeptides which show substantial activity or which include regions of
an antibody, or fragment thereof, against a human folate receptor
protein. Such mutants include deletions, insertions, inversions, repeats,
and type substitutions.
[0154] The polypeptides and analogs can be further modified to contain
additional chemical moieties not normally part of the protein. Those
derivatized moieties can improve the solubility, the biological half life
or absorption of the protein. The moieties can also reduce or eliminate
any desirable side effects of the proteins and the like. An overview for
those moieties can be found in REMINGTON'S PHARMACEUTICAL SCIENCES, 20th
ed., Mack Publishing Co., Easton, Pa. (2000).
[0155] The isolated polypeptides described herein can be produced by any
suitable method known in the art. Such methods range from direct protein
synthetic methods to constructing a DNA sequence encoding isolated
polypeptide sequences and expressing those sequences in a suitable
transformed host. In some embodiments, a DNA sequence is constructed
using recombinant technology by isolating or synthesizing a DNA sequence
encoding a wild-type protein of interest. Optionally, the sequence can be
mutagenized by site-specific mutagenesis to provide functional analogs
thereof. See, e.g. Zoeller et al., Proc. Nat'l. Acad. Sci. USA
81:5662-5066 (1984) and U.S. Pat. No. 4,588,585.
[0156] In some embodiments a DNA sequence encoding a polypeptide of
interest would be constructed by chemical synthesis using an
oligonucleotide synthesizer. Such oligonucleotides can be designed based
on the amino acid sequence of the desired polypeptide and selecting those
codons that are favored in the host cell in which the recombinant
polypeptide of interest will be produced. Standard methods can be applied
to synthesize an isolated polynucleotide sequence encoding an isolated
polypeptide of interest. For example, a complete amino acid sequence can
be used to construct a back-translated gene. Further, a DNA oligomer
containing a nucleotide sequence coding for the particular isolated
polypeptide can be synthesized. For example, several small
oligonucleotides coding for portions of the desired polypeptide can be
synthesized and then ligated. The individual oligonucleotides typically
contain 5' or 3' overhangs for complementary assembly.
[0157] Once assembled (by synthesis, site-directed mutagenesis or another
method), the polynucleotide sequences encoding a particular isolated
polypeptide of interest will be inserted into an expression vector and
operatively linked to an expression control sequence appropriate for
expression of the protein in a desired host. Proper assembly can be
confirmed by nucleotide sequencing, restriction mapping, and expression
of a biologically active polypeptide in a suitable host. As is well known
in the art, in order to obtain high expression levels of a transfected
gene in a host, the gene must be operatively linked to transcriptional
and translational expression control sequences that are functional in the
chosen expression host.
[0158] In certain embodiments, recombinant expression vectors are used to
amplify and express DNA encoding antibodies, or fragments thereof,
against human FOLR1. Recombinant expression vectors are replicable DNA
constructs which have synthetic or cDNA-derived DNA fragments encoding a
polypeptide chain of an anti-FOLR1 antibody, or fragment thereof,
operatively linked to suitable transcriptional or translational
regulatory elements derived from mammalian, microbial, viral or insect
genes. A transcriptional unit generally comprises an assembly of (1) a
genetic element or elements having a regulatory role in gene expression,
for example, transcriptional promoters or enhancers, (2) a structural or
coding sequence which is transcribed into mRNA and translated into
protein, and (3) appropriate transcription and translation initiation and
termination sequences, as described in detail below. Such regulatory
elements can include an operator sequence to control transcription. The
ability to replicate in a host, usually conferred by an origin of
replication, and a selection gene to facilitate recognition of
transformants can additionally be incorporated. DNA regions are
operatively linked when they are functionally related to each other. For
example, DNA for a signal peptide (secretory leader) is operatively
linked to DNA for a polypeptide if it is expressed as a precursor which
participates in the secretion of the polypeptide; a promoter is
operatively linked to a coding sequence if it controls the transcription
of the sequence; or a ribosome binding site is operatively linked to a
coding sequence if it is positioned so as to permit translation.
Structural elements intended for use in yeast expression systems include
a leader sequence enabling extracellular secretion of translated protein
by a host cell. Alternatively, where recombinant protein is expressed
without a leader or transport sequence, it can include an N-terminal
methionine residue. This residue can optionally be subsequently cleaved
from the expressed recombinant protein to provide a final product.
[0159] The choice of expression control sequence and expression vector
will depend upon the choice of host. A wide variety of expression
host/vector combinations can be employed. Useful expression vectors for
eukaryotic hosts, include, for example, vectors comprising expression
control sequences from SV40, bovine papilloma virus, adenovims and
cytomegalovirus. Useful expression vectors for bacterial hosts include
known bacterial plasmids, such as plasmids from Esherichia coli,
including pCR 1, pBR322, pMB9 and their derivatives, wider host range
plasmids, such as M13 and filamentous single-stranded DNA phages.
[0160] Suitable host cells for expression of a FOLR1-binding polypeptide
or antibody (or a FOLR1 protein to use as an antigen) include
prokaryotes, yeast, insect or higher eukaryotic cells under the control
of appropriate promoters. Prokaryotes include gram negative or gram
positive organisms, for example E. coli or bacilli. Higher eukaryotic
cells include established cell lines of mammalian origin as described
below. Cell-free translation systems could also be employed. Appropriate
cloning and expression vectors for use with bacterial, fungal, yeast, and
mammalian cellular hosts are described by Pouwels et al. (Cloning
Vectors: A Laboratory Manual, Elsevier, N.Y., 1985), the relevant
disclosure of which is hereby incorporated by reference. Additional
information regarding methods of protein production, including antibody
production, can be found, e.g., in U.S. Patent Publication No.
2008/0187954, U.S. Pat. Nos. 6,413,746 and 6,660,501, and International
Patent Publication No. WO 04009823, each of which is hereby incorporated
by reference herein in its entirety.
[0161] Various mammalian or insect cell culture systems are also
advantageously employed to express recombinant protein. Expression of
recombinant proteins in mammalian cells can be performed because such
proteins are generally correctly folded, appropriately modified and
completely functional. Examples of suitable mammalian host cell lines
include HEK-293 and HEK-293T, the COS-7 lines of monkey kidney cells,
described by Gluzman (Cell 23:175, 1981), and other cell lines including,
for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and
BHK cell lines. Mammalian expression vectors can comprise nontranscribed
elements such as an origin of replication, a suitable promoter and
enhancer linked to the gene to be expressed, and other 5' or 3' flanking
nontranscribed sequences, and 5' or 3' nontranslated sequences, such as
necessary ribosome binding sites, a polyadenylation site, splice donor
and acceptor sites, and transcriptional termination sequences.
Baculovirus systems for production of heterologous proteins in insect
cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988).
[0162] The proteins produced by a transformed host can be purified
according to any suitable method. Such standard methods include
chromatography (e.g., ion exchange, affinity and sizing column
chromatography), centrifugation, differential solubility, or by any other
standard technique for protein purification. Affinity tags such as
hexahistidine, maltose binding domain, influenza coat sequence and
glutathione-S-transferase can be attached to the protein to allow easy
purification by passage over an appropriate affinity column. Isolated
proteins can also be physically characterized using such techniques as
proteolysis, nuclear magnetic resonance and x-ray crystallography.
[0163] For example, supernatants from systems which secrete recombinant
protein into culture media can be first concentrated using a commercially
available protein concentration filter, for example, an Amicon or
Millipore Pellicon ultrafiltration unit. Following the concentration
step, the concentrate can be applied to a suitable purification matrix.
Alternatively, an anion exchange resin can be employed, for example, a
matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The
matrices can be acrylamide, agarose, dextran, cellulose or other types
commonly employed in protein purification. Alternatively, a cation
exchange step can be employed. Suitable cation exchangers include various
insoluble matrices comprising sulfopropyl or carboxymethyl groups.
Finally, one or more reversed-phase high performance liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g.,
silica gel having pendant methyl or other aliphatic groups, can be
employed to further purify a FOLR1-binding agent. Some or all of the
foregoing purification steps, in various combinations, can also be
employed to provide a homogeneous recombinant protein.
[0164] Recombinant protein produced in bacterial culture can be isolated,
for example, by initial extraction from cell pellets, followed by one or
more concentration, salting-out, aqueous ion exchange or size exclusion
chromatography steps. High performance liquid chromatography (HPLC) can
be employed for final purification steps. Microbial cells employed in
expression of a recombinant protein can be disrupted by any convenient
method, including freeze-thaw cycling, sonication, mechanical disruption,
or use of cell lysing agents.
[0165] Methods known in the art for purifying antibodies and other
proteins also include, for example, those described in U.S. Patent
Publication No. 2008/0312425, 2008/0177048, and 2009/0187005, each of
which is hereby incorporated by reference herein in its entirety.
[0166] In certain embodiments, the FOLR1-binding agent is a polypeptide
that is not an antibody. A variety of methods for identifying and
producing non-antibody polypeptides that bind with high affinity to a
protein target are known in the art. See, e.g., Skerra, Curr. Opin.
Biotechnol., 18:295-304 (2007), Hosse et al., Protein Science, 15:14-27
(2006), Gill et al., Curr. Opin. Biotechnol., 17:653-658 (2006), Nygren,
FEBS J., 275:2668-76 (2008), and Skerra, FEBS J., 275:2677-83 (2008),
each of which is incorporated by reference herein in its entirety. In
certain embodiments, phage display technology has been used to
identify/produce the FOLR1-binding polypeptide. In certain embodiments,
the polypeptide comprises a protein scaffold of a type selected from the
group consisting of protein A, a lipocalin, a fribronectin domain, an
ankyrin consensus repeat domain, and thioredoxin.
[0167] In some embodiments, the agent is a non-protein molecule. In
certain embodiments, the agent is a small molecule. Combinatorial
chemistry libraries and techniques useful in the identification of
non-protein FOLR1-binding agents are known to those skilled in the art.
See, e.g., Kennedy et al., J. Comb. Chem, 10:345-354 (2008), Dolle et al,
J. Comb. Chem., 9:855-902 (2007), and Bhattacharyya, Curr. Med. Chem.,
8:1383-404 (2001), each of which is incorporated by reference herein in
its entirety. In certain further embodiments, the agent is a
carbohydrate, a glycosaminoglycan, a glycoprotein, or a proteoglycan.
[0168] In certain embodiments, the agent is a nucleic acid aptamer.
Aptamers are polynucleotide molecules that have been selected (e.g., from
random or mutagenized pools) on the basis of their ability to bind to
another molecule. In some embodiments, the aptamer comprises a DNA
polynucleotide. In certain alternative embodiments, the aptamer comprises
an RNA polynucleotide. In certain embodiments, the aptamer comprises one
or more modified nucleic acid residues. Methods of generating and
screening nucleic acid aptamers for binding to proteins are well known in
the art. See, e.g., U.S. Pat. Nos. 5,270,163, 5,683,867, 5,763,595,
6,344,321, 7,368,236, 5,582,981, 5,756,291, 5,840,867, 7,312,325,
7,329,742, International Patent Publication No. WO 02/077262,
International Patent Publication No. WO 03/070984, U.S. Patent
Application Publication No. 2005/0239134, U.S. Patent Application
Publication No. 2005/0124565, and U.S. Patent Application Publication No.
2008/0227735, each of which is incorporated by reference herein in its
entirety.
III. Immunoconjugates
[0169] The present invention is also directed to conjugates (also referred
to herein as immunoconjugates), comprising the anti-FOLR1 antibodies,
antibody fragments, functional equivalents, improved antibodies and their
aspects as disclosed herein, linked or conjugated to a cytotoxin (drug)
or prodrug. Thus, in a certain embodiment, the invention provides an
immunoconjugate comprising a humanized antibody or antigen binding
fragment thereof that specifically binds a human folate receptor 1,
wherein the antibody comprises: (a) a heavy chain CDR1 comprising GYFMN
(SEQ ID NO:1); a heavy chain CDR2 comprising
RIHPYDGDTFYNQXaa.sub.1FXaa.sub.2Xaa.sub.3 (SEQ ID NO:56); and a heavy
chain CDR3 comprising YDGSRAMDY (SEQ ID NO:3); and (b) a light chain CDR1
comprising KASQSVSFAGTSLMH (SEQ ID NO:7); a light chain CDR2 comprising
RASNLEA (SEQ ID NO:8); and a light chain CDR3 comprising QQSREYPYT (SEQ
ID NO:9); wherein Xaa.sub.1 is selected from K, Q, H, and R; Xaa.sub.2 is
selected from Q, H, N, and R; and Xaa.sub.3 is selected from G, E, T, S,
A, and V. In certain embodiments, the antibody is the huMov19 antibody,
which is the above-described antibody comprising the heavy chain CDR2
RIHPYDGDTFYNQKFQG (SEQ ID NO:2). In other embodiments, the antibody is
FR1-21 and comprises (a) a heavy chain CDR1 comprising SSYGMS (SEQ ID
NO:30); a heavy chain CDR2 comprising TISSGGSYTY (SEQ ID NO:31); and/or a
heavy chain CDR3 comprising DGEGGLYAMDY (SEQ ID NO:32); and (b) a light
chain CDR1 comprising KASDHINNWLA (SEQ ID NO:27); a light chain CDR2
comprising GATSLET (SEQ ID NO:28); and a light chain CDR3 comprising
QQYWSTPFT (SEQ ID NO:29). In other embodiments, the antibody is FR1-48
and comprises: (a) a heavy chain CDR1 comprising TNYWMQ (SEQ ID NO:60); a
heavy chain CDR2 comprising AIYPGNGDSR (SEQ ID NO:61); and/or a heavy
chain CDR3 comprising RDGNYAAY (SEQ ID NO:62); and/or (b) a light chain
CDR1 comprising RASENIYSNLA (SEQ ID NO:57); a light chain CDR2 comprising
AATNLAD (SEQ ID NO:58); and a light chain CDR3 comprising QHFWASPYT (SEQ
ID NO:59). In other embodiments, the antibody is FR1-49 and comprises:
(a) a heavy chain CDR1 comprising TNYWMY (SEQ ID NO:66); a heavy chain
CDR2 comprising AIYPGNSDTT (SEQ ID NO:67); and/or a heavy chain CDR3
comprising RHDYGAMDY (SEQ ID NO:68); and/or (b) a light chain CDR1
comprising RASENIYTNLA (SEQ ID NO:63); a light chain CDR2 comprising
TASNLAD (SEQ ID NO:64); and a light chain CDR3 comprising QHFWVSPYT (SEQ
ID NO:65). In other embodiments, the antibody is FR1-57 and comprises:
(a) a heavy chain CDR1 comprising SSFGMH (SEQ ID NO:72); a heavy chain
CDR2 comprising YISSGSSTIS (SEQ ID NO:73); and/or a heavy chain CDR3
comprising EAYGSSMEY (SEQ ID NO:74); and/or (b) a light chain CDR1
comprising RASQNINNNLH (SEQ ID NO:69); a light chain CDR2 comprising
YVSQSVS (SEQ ID NO:70); and a light chain CDR3 comprising QQSNSWPHYT (SEQ
ID NO:71). In yet another embodiment, the antibody is FR1-65 and
comprises: (a) a heavy chain CDR1 comprising TSYTMH (SEQ ID NO:78); a
heavy chain CDR2 comprising YINPISGYTN (SEQ ID NO:79); and/or a heavy
chain CDR3 comprising GGAYGRKPMDY (SEQ ID NO:80); and/or (b) a light
chain CDR1 comprising KASQNVGPNVA (SEQ ID NO:75); a light chain CDR2
comprising SASYRYS (SEQ ID NO:76); and a light chain CDR3 comprising
QQYNSYPYT (SEQ ID NO:77).
[0170] Suitable drugs or prodrugs are known in the art. In certain
embodiments, drugs or prodrugs are cytotoxic agents. The cytotoxic agent
used in the cytotoxic conjugate of the present invention can be any
compound that results in the death of a cell, or induces cell death, or
in some manner decreases cell viability, and includes, for example,
maytansinoids and maytansinoid analogs, benzodiazepines, taxoids, CC-1065
and CC-1065 analogs, duocarmycins and duocarmycin analogs, enediynes,
such as calicheamicins, dolastatin and dolastatin analogs including
auristatins, tomaymycin derivaties, leptomycin derivaties, methotrexate,
cisplatin, carboplatin, daunorubicin, doxorubicin, vincristine,
vinblastine, melphalan, mitomycin C, chlorambucil and morpholino
doxorubicin. In certain embodiments, the cytotoxic agents are
maytansinoids and maytansinoids analogs.
[0171] Such conjugates can be prepared by using a linking group in order
to link a drug or prodrug to the antibody or functional equivalent.
Suitable linking groups are well known in the art and include, for
example, disulfide groups, thioether groups, acid labile groups,
photolabile groups, peptidase labile groups and esterase labile groups.
[0172] The drug or prodrug can, for example, be linked to the anti-FOLR1
antibody or fragment thereof through a disulfide bond. The linker
molecule or crosslinking agent comprises a reactive chemical group that
can react with the anti-FOLR1 antibody or fragment thereof. In certain
embodiments, reactive chemical groups for reaction with the cell-binding
agent are N-succinimidyl esters and N-sulfosuccinimidyl esters.
Additionally the linker molecule comprises a reactive chemical group, in
certain embodiments a dithiopyridyl group that can react with the drug to
form a disulfide bond. In certain embodiments, linker molecules include,
for example, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) (see,
e.g., Carlsson et al., Biochem. J., 173: 723-737 (1978)), N-succinimidyl
4-(2-pyridyldithio)butanoate (SPDB) (see, e.g., U.S. Pat. No. 4,563,304),
N-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB) (see US
Publication No. 20090274713), N-succinimidyl 4-(2-pyridyldithio)
pentanoate (SPP) (see, e.g., CAS Registry number 341498-08-6),
2-iminothiolane, or acetylsuccinic anhydride. For example, the antibody
or cell binding agent can be modified with crosslinking reagents and the
antibody or cell binding agent containing free or protected thiol groups
thus derived is then reacted with a disulfide- or thiol-containing
maytansinoid to produce conjugates. The conjugates can be purified by
chromatography, including but not limited to HPLC, size-exclusion,
adsorption, ion exchange and affinity capture, dialysis or tangential
flow filtration. In certain embodiments, the anti-FOLR1 antibody is
linked to the cytoxin via a SPDB or sulfo-SPDB linker. In a certain
embodiment, the huMov19 antibody is linked to a cytotoxin via a SPDB or
sulfo-SPDB linker.
[0173] In another aspect of the present invention, the anti-FOLR1 antibody
is linked to cytotoxic drugs via disulfide bonds and a polyethylene
glycol spacer in enhancing the potency, solubility or the efficacy of the
immunoconjugate. Such cleavable hydrophilic linkers are described in
WO2009/0134976. The additional benefit of this linker design is the
desired high monomer ratio and the minimal aggregation of the
antibody-drug conjugate. Specifically contemplated in this aspect are
conjugates of cell-binding agents and drugs linked via disulfide group
(--S--S--) bearing polyethylene glycol spacers
((CH.sub.2CH.sub.2O).sub.n=1-14) with a narrow range of drug load of 2-8
are described that show relatively high potent biological activity toward
cancer cells and have the desired biochemical properties of high
conjugation yield and high monomer ratio with minimal protein
aggregation.
[0174] Specifically contemplated in this aspect is an anti-FOLR1 antibody
drug conjugate of formula (I) or a conjugate of formula (I'):
A-[X.sub.1--(--CH.sub.2--CH.sub.2O--).sub.n--Y--C].sub.m (I)
[C--Y--(--CH.sub.2--CH.sub.2O).sub.nX.sub.l].sub.m-A (I')
[0175] wherein: [0176] A represents an anti-FOLR1 antibody or fragment;
[0177] C represents a cytotoxin or drug; [0178] X represents an
aliphatic, an aromatic or a heterocyclic unit attached to the
cell-binding agent via a thioether bond, an amide bond, a carbamate bond,
or an ether bond; [0179] Y represents an aliphatic, an aromatic or a
heterocyclic unit attached to the drug via a disulfide bond; [0180] l is
0 or 1; [0181] m is an integer from 2 to 8; and [0182] n is an integer
from 1 to 24. [0183] In certain embodiments, m is an integer from 2 to 6.
[0184] In certain embodiments, m is an integer from 3 to 5.
[0185] Also, In certain embodiments, n is an integer form 2 to 8.
Alternatively, as disclosed in, for example, U.S. Pat. Nos. 6,441,163 and
7,368,565, the drug can be first modified to introduce a reactive ester
suitable to react with a cell-binding agent. Reaction of these drugs
containing an activated linker moiety with a cell-binding agent provides
another method of producing a cell-binding agent drug conjugate.
Maytansinoids can also be linked to anti-FOLR1 antibody or fragment using
PEG linking groups, as set forth for example in U.S. Pat. No. 6,716,821.
These PEG non-cleavable linking groups are soluble both in water and in
non-aqueous solvents, and can be used to join one or more cytotoxic
agents to a cell binding agent. Exemplary PEG linking groups include
heterobifunctional PEG linkers that react with cytotoxic agents and cell
binding agents at opposite ends of the linkers through a functional
sulfhydryl or disulfide group at one end, and an active ester at the
other end. As a general example of the synthesis of a cytotoxic conjugate
using a PEG linking group, reference is again made to U.S. Pat. No.
6,716,821 which is incorporated entirely by reference herein. Synthesis
begins with the reaction of one or more cytotoxic agents bearing a
reactive PEG moiety with a cell-binding agent, resulting in displacement
of the terminal active ester of each reactive PEG moiety by an amino acid
residue of the cell binding agent, to yield a cytotoxic conjugate
comprising one or more cytotoxic agents covalently bonded to a cell
binding agent through a PEG linking group. Alternatively, the cell
binding can be modified with the bifunctional PEG crosslinker to
introduce a reactive disulfide moiety (such as a pyridyldisulfide), which
can then be treated with a thiol-containing maytansinoid to provide a
conjugate. In another method, the cell binding can be modified with the
bifunctional PEG crosslinker to introduce a thiol moiety which can then
can be treated with a reactive disulfide-containing maytansinoid (such as
a pyridyldisulfide), to provide a conjugate.
[0186] Antibody-maytansinoid conjugates with non-cleavable links can also
be prepared. Such crosslinkers are described in the art (see
ThermoScientific Pierce Crosslinking Technical Handbook and US Patent
Application Publication No. 2005/0169933) and include but are not limited
to, N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC),
N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproa-
te), which is a "long chain" analog of SMCC (LC-SMCC),
.kappa.-maleimidoundecanoic acid N-succinimidyl ester (KMUA),
.beta.-maleimidopropanoic acid N-succinimidyl ester (BMPS),
.gamma.-maleimidobutyric acid N-succinimidyl ester (GMBS),
.epsilon.-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS),
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),
N-(.alpha.-maleimidoacetoxy)-succinimide ester (AMAS),
succinimidyl-6-(.beta.-maleimidopropionamido)hexanoate (SMPH),
N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), and
N-(p-maleimidophenyl)isocyanate (PMPI),
N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl
iodoacetate (SIA), N-succinimidyl bromoacetate (SBA), and N-succinimidyl
3-(bromoacetamido)propionate (SBAP). In certain embodiments, the antibody
is modified with crosslinking reagents such as succinimidyl
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), sulfo-SMCC,
maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS or
succinimidyl-iodoacetate, as described in the literature, to introduce
1-10 reactive groups (Yoshitake et al, Eur. J. Biochem., 101:395-399
(1979); Hashida et al, J. Applied Biochem., 56-63 (1984); and Liu et al,
Biochem., 18:690-697 (1979)). The modified antibody is then reacted with
the thiol-containing maytansinoid derivative to produce a conjugate. The
conjugate can be purified by gel filtration through a Sephadex G25 column
or by dialysis or tangential flow filtration. The modified antibodies are
treated with the thiol-containing maytansinoid (1 to 2 molar
equivalent/maleimido group) and antibody-maytansinoid conjugates are
purified by gel filtration through a Sephadex G-25 column, chromatography
on a ceramic hydroxyapatite column, dialysis or tangential flow
filtration or a combination of methods thereof. Typically, an average of
1-10 maytansinoids per antibody are linked. One method is to modify
antibodies with succinimidyl
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) to introduce
maleimido groups followed by reaction of the modified antibody with a
thiol-containing maytansinoid to give a thioether-linked conjugate. Again
conjugates with 1 to 10 drug molecules per antibody molecule result.
Maytansinoid conjugates of antibodies, antibody fragments, protein
hormones, protein growth factors and other proteins are made in the same
way.
[0187] In another aspect of the invention, the FOLR1 antibody (e.g.
huMov19, FR1-21, FR1-48, FR1-49, FR1-57, or FR1-65) is linked to the drug
via a non-cleavable bond through the intermediacy of a PEG spacer.
Suitable crosslinking reagents comprising hydrophilic PEG chains that
form linkers between a drug and the anti-FOLR1 antibody or fragment are
also well known in the art, or are commercially available (for example
from Quanta Biodesign, Powell, Ohio). Suitable PEG-containing
crosslinkers can also be synthesized from commercially available PEGs
themselves using standard synthetic chemistry techniques known to one
skilled in the art. The drugs can be reacted with bifunctional
PEG-containing cross linkers to give compounds of the following formula,
Z--X.sub.l--(--CH.sub.2--CH.sub.2--O--).sub.n--Y.sub.p-D, by methods
described in detail in US Patent Publication 20090274713 and in
WO2009/0134976, which can then react with the cell binding agent to
provide a conjugate. Alternatively, the cell binding can be modified with
the bifunctional PEG crosslinker to introduce a thiol-reactive group
(such as a maleimide or haloacetamide) which can then be treated with a
thiol-containing maytansinoid to provide a conjugate. In another method,
the cell binding can be modified with the bifunctional PEG crosslinker to
introduce a thiol moiety which can then be treated with a thiol-reactive
maytansinoid (such as a maytansinoid bearing a maleimide or
haloacetamide), to provide a conjugate.
[0188] Accordingly, another aspect of the present invention is an
anti-FOLR1 antibody drug conjugate of formula (II) or of formula (II'):
A-[X.sub.1--(--CH.sub.2--CH.sub.2--O--).sub.n--Y.sub.p--C].sub.m (II)
[C--Y.sub.p--(--CH.sub.2--CH.sub.2--O--).sub.n--X.sub.l].sub.m-A (II')
[0189] wherein, A represents an anti-FOLR1 antibody or fragment; [0190]
C represents a cytotoxin or drug; [0191] X represents an aliphatic, an
aromatic or a heterocyclic unit bonded to the cell-binding agent via a
thioether bond, an amide bond, a carbamate bond, or an ether bond; [0192]
Y represents an aliphatic, an aromatic, or a heterocyclic unit bonded to
the drug via a covalent bond selected from the group consisting of a
thioether bond, an amide bond, a carbamate bond, an ether bond, an amine
bond, a carbon-carbon bond and a hydrazone bond; [0193] l is 0 or 1;
[0194] p is 0 or 1; [0195] m is an integer from 2 to 15; and [0196] n is
an integer from 1 to 2000. [0197] In a certain embodiment, m is an
integer from 2 to 8; and [0198] n is an integer from 1 to 24. [0199] In a
certain embodiment, m is an integer from 2 to 6. [0200] In a certain
embodiment, n is an integer from 2 to 8.
[0201] In a certain embodiment, m is an integer from 3 to 5. In a certain
embodiment, the antibody is huMov19. In another embodiment, the antibody
is FR-1-21. In another embodiment, the antibody is FR-1-48. In another
embodiment, the antibody is FR-1-49. In another embodiment, the antibody
is FR-1-57. In another embodiment, the antibody is FR-1-65.
Examples of suitable PEG-containing linkers include linkers having an
N-succinimidyl ester or N-sulfosuccinimidyl ester moiety for reaction
with the anti-FOLR1 antibody or fragment thereof, as well as a maleimido-
or haloacetyl-based moiety for reaction with the compound. A PEG spacer
can be incorporated into any crosslinker known in the art by the methods
described herein.
[0202] Many of the linkers disclosed herein are described in detail in
U.S. Patent Publication Nos. 20050169933 and 20090274713, and in
WO2009/0134976; the contents of which are entirely incorporated herein by
reference.
[0203] The present invention includes aspects wherein about 2 to about 8
drug molecules ("drug load"), for example, maytansinoid, are linked to an
anti-FOLR1 antibody or fragment thereof, the anti-tumor effect of the
conjugate is much more efficacious as compared to a drug load of a lesser
or higher number of drugs linked to the same cell binding agent. "Drug
load", as used herein, refers to the number of drug molecules (e.g., a
maytansinoid) that can be attached to a cell binding agent (e.g., an
anti-FOLR1 antibody or fragment thereof). In one aspect the number of
drug molecules that can be attached to a cell binding agent can average
from about 2 to about 8 (e.g., 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,
2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,
4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4,
5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,
6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1). In
certain embodiments, the drug is
N.sup.2'-deacetyl-N.sup.2'-(3-mercapto-1-oxopropyl)-maytansine (DM1) or
N.sup.2'-deacetyl-N.sup.2'-(4-mercapto-4-methyl-1-oxopentyl) maytansine
(DM4). Thus, in a certain embodiment, the antibody huMov19 is conjugated
to DM1 or DM4. In another embodiment, the antibody FR-1-21 is conjugated
to DM1 or DM4. In another embodiment, the antibody FR-1-48 is conjugated
to DM1 or DM4. In another embodiment, the antibody FR-1-49 is conjugated
to DM1 or DM4. In another embodiment, the antibody FR-1-57 is conjugated
to DM1 or DM4. In another embodiment, the antibody FR-1-65 is conjugated
to DM1 or DM4.
[0204] Thus, in one aspect, an immunoconjugate comprises 1 maytansinoid
per antibody. In another aspect, an immunoconjugate comprises 2
maytansinoids per antibody. In another aspect, an immunoconjugate
comprises 3 maytansinoids per antibody. In another aspect, an
immunoconjugate comprises 4 maytansinoids per antibody. In another
aspect, an immunoconjugate comprises 5 maytansinoids per antibody. In
another aspect, an immunoconjugate comprises 6 maytansinoids per
antibody. In another aspect, an immunoconjugate comprises 7 maytansinoids
per antibody. In another aspect, an immunoconjugate comprises 8
maytansinoids per antibody.
[0205] In one aspect, an immunoconjugate comprises about 1 to about 8
maytansinoids per antibody. In another aspect, an immunoconjugate
comprises about 2 to about 7 maytansinoids per antibody. In another
aspect, an immunoconjugate comprises about 2 to about 6 maytansinoids per
antibody. In another aspect, an immunoconjugate comprises about 2 to
about 5 maytansinoids per antibody. In another aspect, an immunoconjugate
comprises about 3 to about 5 maytansinoids per antibody. In another
aspect, an immunoconjugate comprises about 3 to about 4 maytansinoids per
antibody.
[0206] In one aspect, a composition comprising immunoconjugates has an
average of about 2 to about 8 (e.g., 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,
2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,
4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3,
5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7,
6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1)
drug molecules (e.g., maytansinoids) attached per antibody. In one
aspect, a composition comprising immunoconjugates has an average of about
1 to about 8 drug molecules (e.g., maytansinoids) per antibody. In one
aspect, a composition comprising immunoconjugates has an average of about
2 to about 7 drug molecules (e.g., maytansinoids) per antibody. In one
aspect, a composition comprising immunoconjugates has an average of about
2 to about 6 drug molecules (e.g., maytansinoids) per antibody. In one
aspect, a composition comprising immunoconjugates has an average of about
2 to about 5 drug molecules (e.g., maytansinoids) per antibody. In one
aspect, a composition comprising immunoconjugates has an average of about
3 to about 5 drug molecules (e.g., maytansinoids) per antibody. In one
aspect, a composition comprising immunoconjugates has an average of about
3 to about 4 drug molecules (e.g., maytansinoids) per antibody. In one
aspect, a composition comprising immunoconjugates has an average of about
3.5 to about 4 drug molecules (e.g., maytansinoids) per antibody.
[0207] In one aspect, a composition comprising immunoconjugates has an
average of about 2.+-.0.5, about 2.5.+-.0.5, about 3.+-.0.5, about
3.5.+-.0.5, about 4.+-.0.5, about 4.5.+-.0.5, about 5.+-.0.5, about
5.5.+-.0.5, about 6.+-.0.5, about 6.5.+-.0.5, about 7.+-.0.5, about
7.5.+-.0.5, or about 8.+-.0.5 drug molecules (e.g., maytansinoids)
attached per antibody. In one aspect, a composition comprising
immunoconjugates has an average of about 3.5.+-.0.5 drug molecules (e.g.,
maytansinoids) per antibody.
[0208] The anti-FOLR1 antibody or fragment thereof can be modified by
reacting a bifunctional crosslinking reagent with the anti-FOLR1 antibody
or fragment thereof, thereby resulting in the covalent attachment of a
linker molecule to the anti-FOLR1 antibody or fragment thereof. As used
herein, a "bifunctional crosslinking reagent" is any chemical moiety that
covalently links a cell-binding agent to a drug, such as the drugs
described herein. In another method, a portion of the linking moiety is
provided by the drug. In this respect, the drug comprises a linking
moiety that is part of a larger linker molecule that is used to join the
cell-binding agent to the drug. For example, to form the maytansinoid
DM1, the side chain at the C-3 hydroxyl group of maytansine is modified
to have a free sulfhydryl group (SH). This thiolated form of maytansine
can react with a modified cell-binding agent to form a conjugate.
Therefore, the final linker is assembled from two components, one of
which is provided by the crosslinking reagent, while the other is
provided by the side chain from DM1.
[0209] The drug molecules can also be linked to the antibody molecules
through an intermediary carrier molecule such as serum albumin.
[0210] As used herein, the expression "linked to a cell-binding agent" or
"linked to an anti-FOLR1 antibody or fragment" refers to the conjugate
molecule comprising at least one drug derivative bound to a cell-binding
agent anti-FOLR1 antibody or fragment via a suitable linking group, or a
precursor thereof. In certain embodiments, the linking group is SMCC.
[0211] In certain embodiments, cytotoxic agents useful in the present
invention are maytansinoids and maytansinoid analogs. Examples of
suitable maytansinoids include esters of maytansinol and maytansinol
analogs. Included are any drugs that inhibit microtubule formation and
that are highly toxic to mammalian cells, as are maytansinol and
maytansinol analogs.
[0212] Examples of suitable maytansinol esters include those having a
modified aromatic ring and those having modifications at other positions.
Such suitable maytansinoids are disclosed in U.S. Pat. Nos. 4,424,219;
4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598;
4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533;
5,208,020; 5,416,064; 5,475,092; 5,585,499; 5,846,545; 6,333,410;
7,276,497 and 7,473,796.
[0213] In a certain embodiment, the immunoconjugates of the invention
utilize the thiol-containing maytansinoid (DM1), formally termed
N.sup.2'-deacetyl-N.sup.2'-(3-mercapto-1-oxopropyl)-maytansine, as the
cytotoxic agent. DM1 is represented by the following structural formula
(III):
##STR00001##
[0214] In another embodiment, the conjugates of the present invention
utilize the thiol-containing maytansinoid
N.sup.2'-deacetyl-N.sup.2'(4-methyl-4-mercapto-1-oxopentyl)-maytansine
(e.g., DM4) as the cytotoxic agent. DM4 is represented by the following
structural formula (IV):
##STR00002##
[0215] Another maytansinoid comprising a side chain that contains a
sterically hindered thiol bond is
N.sup.2'-deacetyl-N-.sup.2'(4-mercapto-1-oxopentyl)-maytansine (termed
DM3), represented by the following structural formula (V):
##STR00003##
[0216] Each of the maytansinoids taught in U.S. Pat. Nos. 5,208,020 and
7,276,497, can also be used in the conjugate of the present invention. In
this regard, the entire disclosure of U.S. Pat. Nos. 5,208,020 and
7,276,697 is incorporated herein by reference.
[0217] Many positions on maytansinoids can serve as the position to
chemically link the linking moiety. For example, the C-3 position having
a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15
position modified with hydroxy and the C-20 position having a hydroxy
group are all expected to be useful. In certain embodiments, the C-3
position is utilized. In certain embodiments, the C-3 position of
maytansinol is utilized.
[0218] Structural representations of certain conjugates are shown below:
##STR00004## ##STR00005## ##STR00006##
In a certain embodiment, the antibody is huMov19. In another embodiment,
the antibody is FR1-21.
[0219] Several descriptions for producing such antibody-maytansinoid
conjugates are provided in U.S. Pat. Nos. 6,333,410, 6,441,163,
6,716,821, and 7,368,565, each of which is incorporated herein in its
entirety.
[0220] In general, a solution of an antibody in aqueous buffer can be
incubated with a molar excess of maytansinoids having a disulfide moiety
that bears a reactive group. The reaction mixture can be quenched by
addition of excess amine (such as ethanolamine, taurine, etc.). The
maytansinoid-antibody conjugate can then be purified by gel filtration.
The number of maytansinoid molecules bound per antibody molecule can be
determined by measuring spectrophotometrically the ratio of the
absorbance at 252 nm and 280 nm. An average of 1-10 maytansinoid
molecules/antibody molecule is used and an average of 2-5 is also used in
certain embodiments. The average number of maytansinoid
molecules/antibody can be, for example, about 1-10, 2-5, 3-4, 3.5-4 or
3.5. In one aspect, the average number of maytansinoid molecules/antibody
is about 3.5.+-.0.5. In one aspect, the average number of maytansinoid
molecules/antibody is about 3.5-4.
[0221] Conjugates of antibodies with maytansinoid drugs can be evaluated
for their ability to suppress proliferation of various unwanted cell
lines in vitro. For example, cell lines such as the human KB cell line,
can easily be used for the assessment of cytotoxicity of these compounds.
Cells to be evaluated can be exposed to the compounds for 4 to 5 days and
the surviving fractions of cells measured in direct assays by known
methods. IC.sub.50 values can then be calculated from the results of the
assays.
[0222] Benzodiazepine compounds described, for example, in U.S. Patent
Application Publication No. 2010/0203007 (e.g., indolinobenzodiazepines
or oxazolidinobenzodiazepines), derivatives thereof, intermediates
thereof, may also be used to prepare anti-FOLR1 antibody fragment or
conjugates.
[0223] Useful benzodiazepines include compounds of formula (XIV), (XV) and
(XVI), in which the dimer compounds optionally bear a linking group that
allows for linkage to cell binding agents.
##STR00007##
wherein the double line between N and C represents a single bond or a
double bond, provided that when it is a double bond X is absent and Y is
H, and when it is a single bond, X is H or an amine protecting moiety
that converts the compound into a prodrug; Y is selected from --OR, an
ester represented by --OCOR', a carbonate represented by --OCOOR', a
carbamate represented by --OCONR'R'', an amine or a hydroxyl amine
represented by NR'R'', amide represented by --NRCOR', a peptide
represented by NRCOP, wherein P is an amino acid or a polypeptide
containing between 2 to 20 amino acid units, a thioether represented by
SR', a sulfoxide represented by SOR', a sulfone represented by
--SO.sub.2R', a sulfite --SO.sub.3, a bisulfite --OSO.sub.3, a halogen,
cyano, an azido, or a thiol, wherein R, R' and R'' are same or different
and are selected from H, substituted or unsubstituted linear, branched or
cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a
polyethylene glycol unit (--OCH.sub.2CH.sub.2)n, wherein n is an integer
from 1 to 2000, aryl having from 6 to 10 carbon atoms, heterocyclic ring
having from 3 to 10 carbon atoms wherein the substituent is selected from
halogen, OR.sub.7, NR.sub.8R.sub.9, NO.sub.2, NRCOR', SR.sub.10,a
sulfoxide represented by SOR', a sulfone represented by --SO.sub.2R', a
sulfite --SO.sub.3, a bisulfite --OSO.sub.3, a sulfonamide represented by
SO.sub.2NRR', cyano, an azido, --COR.sub.11, OCOR.sub.11 or
OCONR.sub.11R.sub.12, wherein the definitions of R.sub.7, R.sub.8,
R.sub.9, R.sub.10, R.sub.11 and R.sub.12 are as given above, optionally
R'' is OH; W is C.dbd.O, C.dbd.S, CH.sub.2, BH, SO or SO.sub.2; R.sub.1,
R.sub.2, R.sub.3, R.sub.4, R.sub.1', R.sub.2', R.sub.3' and R.sub.4' are
each independently selected from H, substituted or unsubstituted linear,
branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon
atoms, a polyethylene glycol unit (--OCH.sub.2CH.sub.2)n, wherein n is an
integer from 1 to 2000, or a substituent selected from a halogen,
guanidinium [--NH(C.dbd.NH)NH.sub.2], OR.sub.7, NR.sub.8R.sub.9,
NO.sub.2, NRCOR', SR.sub.10,a sulfoxide represented by SOR', a sulfone
represented by --SO.sub.2R % a sulfite --SO.sub.3, a bisulfite
--OSO.sub.3, a sulfonamide represented by SO.sub.2NRR', cyano, an azido,
OCOR.sub.11 or OCONR.sub.11R.sub.12 wherein R.sub.7, R.sub.8, R.sub.9,
R.sub.10, R.sub.11 and R.sub.12 are each independently selected from H,
linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10
carbon atoms, a polyethylene glycol unit (--OCH.sub.2CH.sub.2).sub.n--,
wherein n is an integer from 1 to 2000, aryl having from 6 to 10 carbon
atoms, heterocyclic ring having from 3 to 10 carbon atoms, optionally
R.sub.10 is SR.sub.13 or COR.sub.13, wherein R.sub.13 is selected from
linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10
carbon atoms, a polyethylene glycol unit (--OCH.sub.2CH.sub.2)--, wherein
n is an integer from 1 to 2000, aryl having from 6 to 10 carbon atoms,
heterocyclic ring having from 3 to 10 carbon atoms, optionally R.sub.11
is OR.sub.14, wherein R.sub.14 has the same definition as R, optionally,
any one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.1', R.sub.2',
R.sub.3', or R.sub.4' is a linking group that enables linkage to a cell
binding agent via a covalent bond or is selected from a polypyrrolo,
poly-indolyl, poly-imidazolyl, polypyrollo-imidazolyl,
poly-pyrollo-indolyl or polyimidazolo-indolyl unit optionally bearing a
linking group that enables linkage to a cell binding agent; Z is selected
from (CH.sub.2).sub.n, wherein n is 1, 2 or 3, CR.sub.15R.sub.16,
NR.sub.17, O or S, wherein R.sub.15, R.sub.16 and R.sub.17 are each
independently selected from H, linear, branched or cyclic alkyl having
from 1 to 10 carbon atoms, a polyethylene glycol unit
(--OCH.sub.2CH.sub.2)--, wherein n is an integer from 1 to 2000; R.sub.6
is OR, SR or NRR', wherein R and R' have the same definition as given
above; X' is selected from CH.sub.2, NR, CO, BH, SO or SO.sub.2 wherein R
has the same definition as given above; Y' is O, CH.sub.2, NR or S,
wherein R has the same definition as given above; Z' is CH.sub.2 or
(CH.sub.2).sub.n, wherein n is 2, 3 or 4, provided that X', Y' and Z' are
not all CH.sub.2 at the same time; A and A' are the same or different and
are selected from O, --CRR'O, S, --CRR'S, --NR.sub.15 or CRR'NHR.sub.15,
wherein R and R' have the same definition as given above and wherein
R.sub.15 has the same definition as given above for R; D and D' are same
or different and independently selected from linear, branched or cyclic
alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, optionally
substituted with any one of halogen, OR.sub.7, NR.sub.8R.sub.9, NO.sub.2,
NRCOR', SR.sub.10,a sulfoxide represented by SOR', a sulfone represented
by --SO.sub.2R', a sulfite --SO.sub.3, a bisulfite --OSO.sub.3, a
sulfonamide represented by SO.sub.2NRR', cyano, an azido, --COR.sub.11,
OCOR.sub.11 or OCONR.sub.11R.sub.12, wherein the definitions of R.sub.7,
R.sub.8, R.sub.9, R.sub.10, R.sub.11 and R.sub.12 are as given above, a
polyethylene glycol unit (--OCH.sub.2CH.sub.2).sub.n, wherein n is an
integer from 1 to 2000; L is an optional phenyl group or a heterocycle
ring having from 3 to 10 carbon atoms that is optionally substituted,
wherein the substituent is a linking group that enables linkage to a cell
binding agent via a covalent bond, or is selected from linear, branched
or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms,
optionally substituted with any one of halogen, OR.sub.7,
NR.sub.8R.sub.9, NO.sub.2, NRCOR', SR.sub.10, a sulfoxide represented by
SOR', a sulfone represented by --SO.sub.2R', a sulfite --SO.sub.3, a
bisulfite --OSO.sub.3, a sulfonamide represented by SO.sub.2NRR', cyano,
an azido, --COR.sub.11, OCOR.sub.11 or OCONR.sub.11R.sub.12, wherein the
definitions of R.sub.7, R.sub.8, R.sub.9, R.sub.10, R.sub.11 and R.sub.12
are as given above, a polyethylene glycol unit (--OCH.sub.2CH.sub.2)n,
wherein n is an integer from 1 to 2000; optionally, L itself is a linking
group that enables linkage to a cell binding agent via a covalent bond;
or their pharmaceutically acceptable solvates, salts, hydrates or
hydrated salts, their optical isomers, racemates, diastereomers,
enantiomers or the polymorphic crystalline structures of these compounds;
provided that the compound has no more than one linking group that
enables linkage to a cell binding agent via a covalent bond.
[0224] In one aspect, the double line between N and C represents a single
bond or a double bond, provided that when it is a double bond X is absent
and Y is H, and when it is a single bond, X is H or an amine protecting
group that converts the compound into a prodrug;
Y is selected from --OR, NR'R'', a sulfite --SO.sub.3, or a bisulfite
--OSO.sub.3, wherein R is selected from H, linear, branched or cyclic
alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a
polyethylene glycol unit (--OCH.sub.2CH.sub.2).sub.n, wherein n is an
integer from 1 to 2000, aryl having from 6 to 10 carbon atoms,
heterocyclic ring having from 3 to 10 carbon atoms; W is C.dbd.O,
CH.sub.2 or SO.sub.2; R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.1'.
R.sub.2'. R.sub.3' and R.sub.4' are each independently selected from H,
NO.sub.2 or a linking group that enables linkage to a cell binding agent
via a covalent bond; R.sub.6 is OR.sub.18, wherein R.sub.18 has the same
definition as R; Z is selected from (CH.sub.2).sub.n, wherein n is 1, 2
or 3, CR.sub.15R.sub.16, NR.sub.17, O or S, wherein R.sub.15, R.sub.16
and R.sub.17 are each independently selected from H, linear, branched or
cyclic alkyl having from 1 to 10 carbon atoms, a polyethylene glycol unit
(--OCH.sub.2CH.sub.2).sub.n, wherein n is an integer from 1 to 2000; X'
is selected from CH.sub.2, or C.dbd.O; Y' is O, NR, or S, wherein R is
defined as above; Z' is CH.sub.2 or (CH.sub.2).sub.2; A and A' are each
O; D and D' are same or different and independently selected from linear,
branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon
atoms; L is an optional phenyl group or a heterocycle ring having from 3
to 10 carbon atoms that is optionally substituted, wherein the
substituent is a linking group that enables linkage to a cell binding
agent via a covalent bond, or is selected from linear, branched or cyclic
alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, optionally
substituted with any one of halogen, OR.sub.7, NR.sub.8R.sub.9, NO.sub.2,
NRCOR', SR.sub.10, a sulfoxide represented by SOR', a sulfone represented
by --SO.sub.2R', a sulfite --SO.sub.3, a bisulfate --OSO.sub.3, a
sulfonamide represented by SO.sub.2NRR', cyano, an azido, --COR.sub.11,
OCOR.sub.11 or OCONR.sub.11R.sub.12, a polyethylene glycol unit
(--OCH.sub.2CH.sub.2)n, wherein n is an integer from 1 to 2000;
optionally, L itself is a linking group that enables linkage to a cell
binding agent via a covalent bond; or their pharmaceutically acceptable
solvates, salts, hydrates or hydrated salts, their optical isomers,
racemates, diastereomers, enantiomers or the polymorphic crystalline
structures of these compounds.
[0225] In another aspect the compound is represented by formula (XVII):
##STR00008##
wherein the double line between N and C represents a single bond or a
double bond, provided that when it is a double bond X is absent and Y is
H, and when it is a single bond, X is H or an amine protecting group that
converts the compound into a prodrug, and Y is selected from OH, an ether
represented by --OR, a sulfite --SO.sub.3, or a bisulfite --OSO.sub.3,
wherein R is selected from linear, branched or cyclic alkyl, alkenyl or
alkynyl bearing from 1 to 10 carbon atoms one of R2, R3 is a linking
group that enables linkage to a cell binding agent via a covalent bond
and the other is H, one of L', L'' or L''' is a linking group that
enables linkage to a cell binding agent, while the others are H; L' can
be the linking group and G is CH or N. Other examples are described in
U.S. Patent Application No. 61/150,201, the entire content of which is
incorporated herein by reference. Thus, in a certain embodiment, the
antibody huMov19 is conjugated to a benzodiazepine having a structure
shown in XIX-XXII above. In another embodiment, the antibody FR-1-21 is
conjugated to a benzodiazepine having a structure shown in XIX-XXII
above.
IV. Polynucleotides
[0226] In certain embodiments, the invention encompasses polynucleotides
comprising polynucleotides that encode a polypeptide that specifically
binds a human FOLR1 receptor or a fragment of such a polypeptide. For
example, the invention provides a polynucleotide comprising a nucleic
acid sequence that encodes an antibody to a human FOLR1 or encodes a
fragment of such an antibody. The polynucleotides of the invention can be
in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA,
and synthetic DNA; and can be double-stranded or single-stranded, and if
single stranded can be the coding strand or non-coding (anti-sense)
strand.
[0227] In certain embodiments, the polynucleotides are isolated. In
certain embodiments, the polynucleotides are substantially pure.
[0228] The invention provides a polynucleotide comprising a polynucleotide
encoding a polypeptide comprising a sequence selected from the group
consisting of SEQ ID NOs:4, 10, 11, 41, 42, and 88-103. Also provided is
a polynucleotide encoding a polypeptide having at least about 95%, at
least about 96%, at least about 97%, at least about 98%, or at least
about 99% sequence identity to SEQ ID NOs: 4, 10, 11, 41, 42, and 88-103.
[0229] The polynucleotides SEQ ID NOs: 5, 14, and 15 comprise the coding
sequence for huMov19 variable domain heavy chain, variable domain light
chain version 1.00, and variable domain light chain version 1.60,
respectively.
[0230] The invention further provides a polynucleotide comprising a
sequence selected from the group consisting of SEQ ID NOs:5, 14, 15, 37,
38, 43, 44, 47, 48, and 120-127. Also provided is a polynucleotide having
at least about 95%, at least about 96%, at least about 97%, at least
about 98%, or at least about 99% sequence identity to SEQ ID NOs: 5, 14,
15, 37, 38, 43, 44, 47, 48, and 120-127. Thus, in certain embodiments,
the polynucleotide comprises (a) a polynucleotide having at least about
95% sequence identity to SEQ ID NO:5, and/or (b) a polynucleotide having
at least about 95% sequence identity to SEQ ID NO:14 or 15. In certain
embodiments, the polynucleotide comprises (a) a polynucleotide having the
amino acid sequence of SEQ ID NO: 5; and/or (b) a polynucleotide having
the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:15.
[0231] In certain embodiments the polynucleotides comprise the coding
sequence for the mature polypeptide fused in the same reading frame to a
polynucleotide which aids, for example, in expression and secretion of a
polypeptide from a host cell (e.g. a leader sequence which functions as a
secretory sequence for controlling transport of a polypeptide from the
cell). The polypeptide having a leader sequence is a preprotein and can
have the leader sequence cleaved by the host cell to form the mature form
of the polypeptide. The polynucleotides can also encode for a proprotein
which is the mature protein plus additional 5' amino acid residues. A
mature protein having a prosequence is a proprotein and is an inactive
form of the protein. Once the prosequence is cleaved an active mature
protein remains.
[0232] In certain embodiments the polynucleotides comprise the coding
sequence for the mature polypeptide fused in the same reading frame to a
marker sequence that allows, for example, for purification of the encoded
polypeptide. For example, the marker sequence can be a hexa-histidine tag
supplied by a pQE-9 vector to provide for purification of the mature
polypeptide fused to the marker in the case of a bacterial host, or the
marker sequence can be a hemagglutinin (HA) tag derived from the
influenza hemagglutinin protein when a mammalian host (e.g. COS-7 cells)
is used.
[0233] The present invention further relates to variants of the
hereinabove described polynucleotides encoding, for example, fragments,
analogs, and derivatives.
[0234] The polynucleotide variants can contain alterations in the coding
regions, non-coding regions, or both. In some embodiments the
polynucleotide variants contain alterations which produce silent
substitutions, additions, or deletions, but do not alter the properties
or activities of the encoded polypeptide. In some embodiments, nucleotide
variants are produced by silent substitutions due to the degeneracy of
the genetic code. Polynucleotide variants can be produced for a variety
of reasons, e.g., to optimize codon expression for a particular host
(change codons in the human mRNA to those preferred by a bacterial host
such as E. coli).
[0235] Vectors and cells comprising the polynucleotides described herein
are also provided.
V. Methods of Use and Pharmaceutical Compositions
[0236] The FOLR1-binding agents (including antibodies, immunoconjugates,
and polypeptides) of the invention are useful in a variety of
applications including, but not limited to, therapeutic treatment
methods, such as the treatment of cancer. In certain embodiments, the
agents are useful for inhibiting tumor growth, inducing differentiation,
reducing tumor volume, and/or reducing the tumorigenicity of a tumor. The
methods of use may be in vitro, ex vivo, or in vivo methods. In certain
embodiments, the FOLR1-binding agent or antibody or immunoconjugate, or
polypeptide is an antagonist of the human FOLR1 to which it binds.
[0237] In one aspect, anti-FOLR1 antibodies and immunoconjugates of the
invention are useful for detecting the presence of FOLR1 in a biological
sample. The term "detecting" as used herein encompasses quantitative or
qualitative detection. In certain embodiments, a biological sample
comprises a cell or tissue. In certain embodiments, such tissues include
normal and/or cancerous tissues that express FOLR1 at higher levels
relative to other tissues. In certain embodiments, FOLR1 overexpression
detects the presence of ovarian cancer, lung cancer, brain cancer, breast
cancer, uterine cancer, renal cancer or pancreatic cancer.
[0238] In one aspect, the invention provides a method of detecting the
presence of FOLR1 in a biological sample. In certain embodiments, the
method comprises contacting the biological sample with an anti-FOLR1
antibody under conditions permissive for binding of the anti-FOLR1
antibody to FOLR1, and detecting whether a complex is formed between the
anti-FOLR1 antibody and FOLR1.
[0239] In one aspect, the invention provides a method of diagnosing a
disorder associated with increased expression of FOLR1. In certain
embodiments, the method comprises contacting a test cell with an
anti-FOLR1 antibody; determining the level of expression (either
quantitatively or qualitatively) of FOLR1 by the test cell by detecting
binding of the anti-FOLR1 antibody to FOLR1; and comparing the level of
expression of FOLR1 by the test cell with the level of expression of
FOLR1 by a control cell (e.g., a normal cell of the same tissue origin as
the test cell or a cell that expresses FOLR1 at levels comparable to such
a normal cell), wherein a higher level of expression of FOLR1 by the test
cell as compared to the control cell indicates the presence of a disorder
associated with increased expression of FOLR1. In certain embodiments,
the test cell is obtained from an individual suspected of having a
disorder associated with increased expression of FOLR1. In certain
embodiments, the disorder is a cell proliferative disorder, such as a
cancer or a tumor.
[0240] In certain embodiments, a method of diagnosis or detection, such as
those described above, comprises detecting binding of an anti-FOLR1
antibody to FOLR1 expressed on the surface of a cell or in a membrane
preparation obtained from a cell expressing FOLR1 on its surface. In
certain embodiments, the method comprises contacting a cell with an
anti-FOLR1 antibody under conditions permissive for binding of the
anti-FOLR1 antibody to FOLR1, and detecting whether a complex is formed
between the anti-FOLR1 antibody and FOLR1 on the cell surface. An
exemplary assay for detecting binding of an anti-FOLR1 antibody to FOLR1
expressed on the surface of a cell is a "FACS" assay.
[0241] Certain other methods can be used to detect binding of anti-FOLR1
antibodies to FOLR1. Such methods include, but are not limited to,
antigen-binding assays that are well known in the art, such as western
blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),
"sandwich" immunoassays, immunoprecipitation assays, fluorescent
immunoassays, protein A immunoassays, and immunohistochemistry (IHC).
[0242] In certain embodiments, anti-FOLR1 antibodies are labeled. Labels
include, but are not limited to, labels or moieties that are detected
directly (such as fluorescent, chromophoric, electron-dense,
chemiluminescent, and radioactive labels), as well as moieties, such as
enzymes or ligands, that are detected indirectly, e.g., through an
enzymatic reaction or molecular interaction.
[0243] In certain embodiments, anti-FOLR1 antibodies are immobilized on an
insoluble matrix. Immobilization entails separating the anti-FOLR1
antibody from any FOLR1 that remains free in solution. This
conventionally is accomplished by either insolubilizing the anti-FOLR1
antibody before the assay procedure, as by adsorption to a
water-insoluble matrix or surface (Bennich et al., U.S. Pat. No.
3,720,760), or by covalent coupling (for example, using glutaraldehyde
cross-linking), or by insolubilizing the anti-FOLR1 antibody after
formation of a complex between the anti-FOLR1 antibody and FOLR1, e.g.,
by immunoprecipitation.
[0244] Any of the above embodiments of diagnosis or detection may be
carried out using an immunoconjugate of the invention in place of or in
addition to an anti-FOLR1 antibody.
[0245] In certain embodiments, the disease treated with the FOLR1-binding
agent or antagonist (e.g., a huMov19 antibody or immunoconjugate) is a
cancer. In certain embodiments, the cancer is characterized by tumors
expressing folate receptor 1 to which the FOLR1-binding agent (e.g.,
antibody) binds.
[0246] The present invention provides for methods of treating cancer
comprising administering a therapeutically effective amount of a
FOLR1-binding agent to a subject (e.g., a subject in need of treatment).
In certain embodiments, the cancer is a cancer selected from the group
consisting of colorectal cancer, pancreatic cancer, lung cancer, ovarian
cancer, liver cancer, breast cancer, brain cancer, kidney cancer,
prostate cancer, gastrointestinal cancer, melanoma, cervical cancer,
bladder cancer, glioblastoma, and head and neck cancer. In certain
embodiments, the cancer is ovarian cancer. In certain embodiments, the
cancer is lung cancer. In certain embodiments, the subject is a human.
[0247] The present invention further provides methods for inhibiting tumor
growth using the antibodies or other agents described herein. In certain
embodiments, the method of inhibiting the tumor growth comprises
contacting the cell with a FOLR1-binding agent (e.g., antibody) in vitro.
For example, an immortalized cell line or a cancer cell line that
expresses FOLR1 is cultured in medium to which is added the antibody or
other agent to inhibit tumor growth. In some embodiments, tumor cells are
isolated from a patient sample such as, for example, a tissue biopsy,
pleural effusion, or blood sample and cultured in medium to which is
added an FOLR1-binding agent to inhibit tumor growth.
[0248] In some embodiments, the method of inhibiting tumor growth
comprises contacting the tumor or tumor cells with the FOLR1-binding
agent (e.g., antibody) in vivo. In certain embodiments, contacting a
tumor or tumor cell with a FOLR1-binding agent is undertaken in an animal
model. For example, FOLR1-binding agents can be administered to
xenografts expressing one or more FOLR1s that have been grown in
immunocompromised mice (e.g. NOD/SCID mice) to inhibit tumor growth. In
some embodiments, cancer stem cells are isolated from a patient sample
such as, for example, a tissue biopsy, pleural effusion, or blood sample
and injected into immunocompromised mice that are then administered a
FOLR1-binding agent to inhibit tumor cell growth. In some embodiments,
the FOLR1-binding agent is administered at the same time or shortly after
introduction of tumorigenic cells into the animal to prevent tumor
growth. In some embodiments, the FOLR1-binding agent is administered as a
therapeutic after the tumorigenic cells have grown to a specified size.
[0249] In certain embodiments, the method of inhibiting tumor growth
comprises administering to a subject a therapeutically effective amount
of a FOLR1-binding agent. In certain embodiments, the subject is a human.
In certain embodiments, the subject has a tumor or has had a tumor
removed.
[0250] In certain embodiments, the tumor expresses the folate receptor to
which the FOLR1-binding agent or antibody binds. In certain embodiments,
the tumor overexpresses the human FOLR1.
[0251] In certain embodiments, the tumor is a tumor selected from the
group consisting of brain tumor, colorectal tumor, pancreatic tumor, lung
tumor, ovarian tumor, liver tumor, breast tumor, kidney tumor, prostate
tumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor,
glioblastoma, and head and neck tumor. In certain embodiments, the tumor
is an ovarian tumor.
[0252] In addition, the invention provides a method of reducing the
tumorigenicity of a tumor in a subject, comprising administering a
therapeutically effective amount of a FOLR1-binding agent to the subject.
In certain embodiments, the tumor comprises cancer stem cells. In certain
embodiments, the frequency of cancer stem cells in the tumor is reduced
by administration of the agent.
[0253] Thus, in certain embodiments the inventions provides methods of
treating cancer using huMov19 antibody and immunoconjugates. In certain
embodiments, the huMov19 immunoconjugate is huMov19-SPDB-DM4;
huMov19-sulfo-SPP-DM1; huMov19-SPP-DM1; or huMov19-PEG4-Mal-DM4.
[0254] The invention further provides methods of differentiating
tumorigenic cells into non-tumorigenic cells comprising contacting the
tumorigenic cells with a FOLR1-binding agent (for example, by
administering the FOLR1-binding agent to a subject that has a tumor
comprising the tumorigenic cells or that has had such a tumor removed. In
certain embodiments, the tumorigenic cells are ovarian tumor cells.
[0255] The present invention further provides methods of reducing
myofibroblast activation in the stroma of a solid tumor, comprising
contacting the stroma with an effective amount of the FOLR1-binding
agent, polypeptide or antibody.
[0256] The present invention further provides pharmaceutical compositions
comprising one or more of the FOLR1-binding agents described herein. In
certain embodiments, the pharmaceutical compositions further comprise a
pharmaceutically acceptable vehicle. These pharmaceutical compositions
find use in inhibiting tumor growth and treating cancer in human
patients.
[0257] In certain embodiments, formulations are prepared for storage and
use by combining a purified antibody or agent of the present invention
with a pharmaceutically acceptable vehicle (e.g. carrier, excipient)
(Remington, The Science and Practice of Pharmacy 20th Edition Mack
Publishing, 2000). Suitable pharmaceutically acceptable vehicles include,
but are not limited to, nontoxic buffers such as phosphate, citrate, and
other organic acids; salts such as sodium chloride; antioxidants
including ascorbic acid and methionine; preservatives (e.g.
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl
alcohol; alkyl parabens, such as methyl or propyl paraben; catechol;
resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight
polypeptides (e.g. less than about 10 amino acid residues); proteins such
as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such
as polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; carbohydrates such as
monosaccharides, disaccharides, glucose, mannose, or dextrins; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or
sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g.
Zn-protein complexes); and non-ionic surfactants such as TWEEN or
polyethylene glycol (PEG).
[0258] The pharmaceutical compositions of the present invention can be
administered in any number of ways for either local or systemic
treatment. Administration can be topical (such as to mucous membranes
including vaginal and rectal delivery) such as transdermal patches,
ointments, lotions, creams, gels, drops, suppositories, sprays, liquids
and powders; pulmonary (e.g., by inhalation or insufflation of powders or
aerosols, including by nebulizer; intratracheal, intranasal, epidermal
and transdermal); oral; or parenteral including intravenous,
intraarterial, subcutaneous, intraperitoneal or intramuscular injection
or infusion; or intracranial (e.g., intrathecal or intraventricular)
administration.
[0259] An antibody or immunoconjugate of the invention can be combined in
a pharmaceutical combination formulation, or dosing regimen as
combination therapy, with a second compound having anti-cancer
properties. The second compound of the pharmaceutical combination
formulation or dosing regimen preferably has complementary activities to
the ADC of the combination such that they do not adversely affect each
other. Pharmaceutical compositions comprising the FOLR1-binding agent and
the second anti-cancer agent are also provided.
[0260] For the treatment of the disease, the appropriate dosage of an
antibody or agent of the present invention depends on the type of disease
to be treated, the severity and course of the disease, the responsiveness
of the disease, whether the antibody or agent is administered for
therapeutic or preventative purposes, previous therapy, patient's
clinical history, and so on all at the discretion of the treating
physician. The antibody or agent can be administered one time or over a
series of treatments lasting from several days to several months, or
until a cure is effected or a diminution of the disease state is achieved
(e.g. reduction in tumor size). Optimal dosing schedules can be
calculated from measurements of drug accumulation in the body of the
patient and will vary depending on the relative potency of an individual
antibody or agent. The administering physician can easily determine
optimum dosages, dosing methodologies and repetition rates. In certain
embodiments, dosage is from 0.01 .mu.g to 100 mg per kg of body weight,
and can be given once or more daily, weekly, monthly or yearly. In
certain embodiments, the antibody or other FOLR1-binding agent is given
once every two weeks or once every three weeks. In certain embodiments,
the dosage of the antibody or other FOLR1-binding agent is from about 0.1
mg to about 20 mg per kg of body weight. The treating physician can
estimate repetition rates for dosing based on measured residence times
and concentrations of the drug in bodily fluids or tissues.
[0261] The combination therapy can provide "synergy" and prove
"synergistic", i.e. the effect achieved when the active ingredients used
together is greater than the sum of the effects that results from using
the compounds separately. A synergistic effect can be attained when the
active ingredients are: (1) co-formulated and administered or delivered
simultaneously in a combined, unit dosage formulation; (2) delivered by
alternation or in parallel as separate formulations; or (3) by some other
regimen. When delivered in alternation therapy, a synergistic effect can
be attained when the compounds are administered or delivered
sequentially, e.g. by different injections in separate syringes. In
general, during alternation therapy, an effective dosage of each active
ingredient is administered sequentially, i.e. serially, whereas in
combination therapy, effective dosages of two or more active ingredients
are administered together.
VI. Kits Comprising FOLR1-Binding Agents
[0262] The present invention provides kits that comprise the antibodies,
immunoconjugates or other agents described herein and that can be used to
perform the methods described herein. In certain embodiments, a kit
comprises at least one purified antibody against human folate receptor 1
in one or more containers. In some embodiments, the kits contain all of
the components necessary and/or sufficient to perform a detection assay,
including all controls, directions for performing assays, and any
necessary software for analysis and presentation of results. One skilled
in the art will readily recognize that the disclosed antibodies,
immunoconjugates or other agents of the present invention can be readily
incorporated into one of the established kit formats which are well known
in the art.
[0263] Further provided are kits comprising a FOLR1-binding agent (e.g., a
FOLR1-binding antibody), as well as a second anti-cancer agent. In
certain embodiments, the second anti-cancer agent is a chemotherapeutic
agent (e.g., gemcitabine or irinotecan).
[0264] Embodiments of the present disclosure can be further defined by
reference to the following non-limiting examples, which describe in
detail preparation of certain antibodies of the present disclosure and
methods for using antibodies of the present disclosure. It will be
apparent to those skilled in the art that many modifications, both to
materials and methods, can be practiced without departing from the scope
of the present disclosure.
EXAMPLES
[0265] It is understood that the examples and embodiments described herein
are for illustrative purposes only and that various modifications or
changes in light thereof will be suggested to persons skilled in the art
and are to be included within the spirit and purview of this application.
Example 1
Chimerization of Murine Monoclonal Antibody Mov19
[0266] The variable region amino acid sequences for Mov19 were obtained
from the NCBI database (accessions CAA68253 for the light chain (SEQ ID
NO:24) and CAA68252 for the heavy chain (SEQ ID NO:23)) and then
codon-optimized and synthesized by Blue Heron Biotechnology. The light
chain variable region was cloned into the EcoRI and BsiWI sites of the
pAbKZeo plasmid and the heavy chain variable region was cloned into the
HindIII and Apa1 sites of the pAbG1Neo plasmid.
Example 2
Humanization of Murine Monoclonal Antibodies Mov19 and FR1-21
[0267] The Mov19 antibody was humanized following framework resurfacing
methods previously described (Roguska M. et. al, Proc. Natl. Acad. Sci.
USA 1994 February; 91:969-973) and (Roguska et al., Protein Eng.
9(10):895-904 (1996)). Briefly, the average solvent accessibility for
each variable region framework residue was calculated using closely
related solved antibody structures from the PDB database, and positions
with greater than a 30% average accessibility were marked as surface
residues (Pedersen J. T. et. Al, J. Mol. Biol. 1994; 235: 959-973). The
human surface replacement sequence was selected by aligning the surface
positions of murine antibody sequences with the corresponding positions
of the human antibody germline sequences in the Kabat database (Johnson,
G. and Wu, T. T. (2001) Nucleic Acids Research, 29: 205-206). The most
homologous human light chain variable region surface (clone DPK19, IMGT
locus IGKV2D-30*01 for Mov19 and IMGT locus IGKV1/OR2-0*01 for FR1-21)
and the most homologous human heavy chain variable region surface (clone
8M27, IMGT locus IGHV1-69*08 for Mov19 and IMGT locus IGHV5-51*02 for
FR1-21) was selected to replace the murine Mov19 framework surface
positions, leaving the 6 CDRs (Table 1) unaltered. The murine and human
Mov19 and FR1-21 surface positions and residues are given in FIGS. 1A-D.
TABLE-US-00002
TABLE 1A
Mov19 CDRs FR1-21 CDRs
Light Chain Light Chain
CDR1: KASQSVSFAGTSLMH CDR1: KASDHINNWLA
(SEQ ID NO: 7) (SEQ ID NO: 27)
CDR2: RASNLEA CDR2: GATSLET
(SEQ ID NO: 8) (SEQ ID NO: 28)
CDR3: QQSREYPYT CDR3: QQYWSTPFT
(SEQ ID NO: 9) (SEQ ID NO: 29)
Heavy Chain Heavy Chain
CDR1: GYFMN CDR1SSYGMS
(SEQ ID NO: 1) (SEQ ID NO: 30)
CDR2 (AbM): RIHPYDGDTF CDR2 (AbM): TISSGGS
(SEQ ID NO: 131) YTY (SEQ ID NO: 31)
CDR3: YDGSRAMDY CDR3: DGEGGLYAMDY
(SEQ ID NO: 3) (SEQ ID NO: 32)
Kabat Defined Mov19 Kabat Defined
HC CDR2 FR1-21 HC CDR2
Murine Murine
HC CDR2: RIHPYDGDTFYNQN HC CDR2: TISSGGSYTYY
FKD (SEQ ID NO: 128) PDGVKG (SEQ ID NO: 33)
Human Human
HC CDR2: RIHPYDGDTFYNQK HC CDR2:TISSGGSYTY
FQG(SEQ ID NO: 129) ID NO: 34)
The Mov19 and FR1-21 light and heavy chain CDRs as defined for resurfacing
are provided.
The Kabat definition for heavy chain CDR2 is also given for both the
murine and human antibodies.
[0268] None of the residue changes raised concerns for impacting the
interactions of either the Mov19 or FR1-21 CDRs with their target
epitopes on folate receptor 1, so no surface back mutations were
considered for the humanized sequences of either antibody. The resurfaced
Mov19 sequence did however introduce a consensus N-linked glycosylation
site at the light chain N74 (light chain version 1.00), so a second
humanized light chain version was made to remove this site. A review of
the Kabat human light chain sequence database revealed that threonine is
the most common residue found at light chain position 74 so the humanized
Mov19 light chain version 1.60 was built with a threonine at position 74.
Position 74 is not a surface residue so this residue substitution has no
impact on the humanization by resurfacing. Alignments of the variable
region sequences of murine and humanized Mov19, and FR1-21 are given in
FIG. 2.
[0269] The variable region sequences for humanized Mov19 and FR1-21 were
codon-optimized and synthesized by Blue Heron Biotechnology. The
sequences are flanked by restriction enzyme sites to facilitate cloning
in-frame with the respective constant sequences in single chain mammalian
expression plasmids. The light chain variable region was cloned into the
EcoRI and BsiWI sites of the pAbKZeo plasmid. The resulting plasmid DNAs
encoding huMov19 light chain were deposited with the ATCC as ATCC Deposit
Nos. PTA-10773 and PTA-10774 and the resulting plasmid DNA encoding
huFR1-21 light chain was deposited as ATCC Deposit No. PTA-10776. The
heavy chain variable region was cloned into the HindIII and Apa1 sites of
the pAbG1Neo plasmid. The resulting plasmid DNA encoding huMov19 heavy
chain was deposited with the ATCC as ATCC Deposit No. PTA-10772 and the
resulting plasmid DNA encoding huFR1-21 heavy chain was deposited as ATCC
Deposit No. PTA-10775. These plasmids, were then transfected as described
in example 3 to produce huMov19. The plasmid encoding either huMov19
light chain (i.e., that deposited as ATCC Deposit No. PTA-10773 or
PTA-10774) can be paired with the plasmid encoding huMov19 heavy chain to
create a huMov19 antibody according to the methods provided herein and as
are well-known by one of ordinary skill in the art.
Example 3
Recombinant Antibody Expression
[0270] The chimeric and humanized antibody constructs were transiently
produced in either adherent HEK-293T cells using a standard calcium
phosphate procedure (BD Biosciences, CalPhos Mammalian Transfection Kit,
Cat #631312) or in suspension adapted HEK-293T cells using a modified PEI
procedure [Durocher Y, Perret S, Kamen A High-level and high-throughput
recombinant protein production by transient transfection of
suspension-growing human 293-EBNA1 cells. Nucleic Acids Res. 2002 Jan.
15; 30(2):E9] in spinner flasks. The PEI transient transfections were
performed as previously described (Durocher, Y. et al., Nucleic Acids
Res. 30(2):E9 (2002)), except the HEK-293T cells were grown in Freestyle
293 (Invitrogen) and the culture volume was left undiluted after the
addition of the PEI-DNA complexes. Both the adherent and suspension
transient transfections were incubated for a week and then the cleared
supernatant was purified by a Protein A column followed by a CM column
ion exchange chromatography as described below. As shown in FIG. 3,
expression of huMov19 was at least 10-fold higher than expression of
chimeric Mov19 in transfected cells.
Example 4
Antibody Purification
[0271] Antibodies were purified from cleared cell culture supernatants
using standard methods, such as, for example Protein A or G
chromatography (HiTrap Protein A or G HP, 1 mL, Amersham Biosciences).
Briefly, supernatant was prepared for chromatography by the addition of
1/10 volume of 1 M Tris/HCl buffer, pH 8.0. The pH-adjusted supernatant
was filtered through a 0.22 .mu.m filter membrane and loaded onto column
equilibrated with binding buffer (PBS, pH 7.3). The column was washed
with binding buffer until a stable baseline was obtained with no
absorbance at 280 nm. Antibody was eluted with 0.1 M acetic acid buffer
containing 0.15 M NaCl, pH 2.8, using a flow rate of 0.5 mL/min.
Fractions of approximately 0.25 mL were collected and neutralized by the
addition of 1/10 volume of 1M Tris/HCl, pH 8.0. The peak fraction(s) was
dialyzed overnight twice against 1.times.PBS and sterilized by filtering
through a 0.2 .mu.m filter membrane. Purified antibody was quantified by
absorbance at A.sub.280.
[0272] Protein A purified fractions were further purified using ion
exchange chromatography (IEX) with carboxymethyl (CM) chromatography.
Briefly, samples from protein A purification were buffer exchanged into
the start buffer (10 mM potassium phosphate, 10 mM sodium chloride, pH
7.5) and filtered through 0.22 .mu.m filer. The prepared sample was then
loaded onto a CM fast flow resin (GE lifesciences) that was equilibrated
with the start buffer at a flow rate of 120 cm/hr. Column size was chosen
to have sufficient capacity to bind all the antibody in the sample. The
column was then washed with binding buffer until a stable baseline was
obtained with no absorbance at 280 nm. Antibody was eluted by initiating
a gradient from 10 mM to 500 mM sodium chloride in 20 column volume (CV).
Fractions with the UV reading above 50 mAu of the major peak were
collected. The purity (the percentage of monomer and soluble high
molecular weight aggregates) was assessed with size exclusion
chromatography (SEC) on a TSK gel G3000SWXL, 7.8.times.300 mm with a SWXL
guard column, 6.0.times.40 mm (Tosoh Bioscience, Montgomeryville, Pa.)
using an Agilent HPLC 1100 system (Agilent, Santa Clara, Calif.).
Fractions with desired purity (>95%) were pooled, buffer exchanged to
PBS (pH 7.4) using TFF system, and sterilized by filtering through a 0.2
.mu.m filter membrane. Purified antibody was further tested for its
purity by SEC and the IgG concentration was determined by absorbance
measurement at 280 nm using an extinction coefficient of 1.47. Dilution
was made if necessary. Alternatively, ceramic hydroxyapatite (CHT) can be
used to polish both murine and humanized antibodies with improved
selectivity. Type II CHT resin with 40 .mu.m particle size (Bio-Rad
Laboratories) was applied to the polishing of antibodies with similar
protocol as IEX chromatography. The start buffer for CHT was 20 mM sodium
phosphate, pH 7.0 and antibody was eluted with a gradient of 20-160 mM
sodium phosphate over 20 CV.
Example 5
Development of Murine Anti-FOLR1 Antibodies
[0273] There were two different immunization/screening series. First
series has led to generation of FR1-21 clone, second series has resulted
in generation of FR1-48, FR1-49, FR1-57 and FR1-65 clones. In the first
series mice were subcutaneously immunized with approximately
5.times.10.sup.6 FOLR1-expressing KB cells (American Tissue Culture
Collection, ATCC CCL-17). In the second series 300-19 cells expressing
human FOLR1 on their surface were used to immunize mice. To make these
cells, the human FOLR1 amino acid sequence was obtained from the NCBI
website (accession NP_057937), then it was codon optimized and
synthesized by Blue Heron biotechnologies, flanked by EcoRI and Xba1
restriction sites to facilitate cloning into the pSRa mammalian
expression vector. 300-19 cells, a pre-B cell line derived from a Balb/c
mouse (Reth et al., Nature, 317:353-355 (1985)), were transfected with
the pSRa-FolR1 expression plasmid to stably express high levels of human
FOLR1 on the cell surface. Standard immunization protocols known to those
of skill, for example, such as those used at ImmunoGen, Inc were applied
for both series. Immunized mice were boosted with antigen three days
before being sacrificed for hybridoma generation. Spleens from mice was
collected according to standard animal protocols, such as, for example
grinding tissue between two sterile, frosted microscopic slides to obtain
a single cell suspension in RPMI-1640 medium. The spleen cells were
centrifuged, pelleted, washed, and fused with a murine myeloma, such as,
for example P3X63Ag8.653 cells (Kearney et al., J. Immunol.,
123:1548-1550 (1979)) using polyethylene glycol-1500 (Roche 783 641). The
fused cells were resuspended in RPMI-1640 selection medium containing
hypoxanthine-aminopterin-thymidine (HAT) (Sigma H-0262) and selected for
growth in 96-well flat-bottomed culture plates (Corning-Costar 3596, 0.2
ml of cell suspension per well) at 37.degree. C. with 5% CO.sub.2. After
5 days of incubation, 0.1 ml of culture supernatant were removed from
each well and replaced with 0.1 ml of RPMI-1640 medium containing
hypoxanthine-thymidine (HT) supplement (Sigma H-0137). Incubation at
37.degree. C. with 5% CO.sub.2 was continued until hydridoma clones were
ready for antibody screening. Other techniques of immunization and
hybridoma production can also be used, including those described in
Langone et al. (Eds., "Immunochemical Techniques, Part I", Methods in
Enzymology, Academic Press, volume 121, Florida) and Harlow et al.
("Antibodies: A Laboratory Manual"; Cold Spring Harbor Laboratory Press,
New York (1988)).
TABLE-US-00003
TABLE 1B
FR1-48 CDRs FR1-49 CDRs FR1-57 CDRs FR1-65 CDRs
Light Chain Light Chain Light Chain Light Chain
CDR1-0RASENIYSNLA CDR1-RASENIYTNLA CDR1- CDR1-KASQNVGPNVA
(SEQ ID NO: 57) (SEQ ID NO: 63) RASQNINNNLH (SEQ ID NO: 75)
(SEQ ID NO: 69)
CDR2-AATNLAD(SEQ CDR2-TASNLAD (SEQ CDR2-YVSQSVS CDR2-SASYRYS
ID NO: 58) ID NO: 64) (SEQ ID NO: 70) (SEQ ID NO: 76)
CDR3-QHFWASPYT CDR3-QHFWVSPYT CDR3-QQSNSWPHYT CDR3-QQYNSYPYT
(SEQ ID NO: 59) (SEQ ID NO: 65) (SEQ ID NO: 71) (SEQ ID NO: 77)
Heavy Chain Heavy Chain Heavy Chain Heavy Chain
CDR1-TNYWMQ (SEQ CDR1-TNYWMY (SEQ CDR1-SSFGMH CDR1-TSYTMH
ID NO: 60) ID NO: 66) (SEQ ID NO: 72) (SEQ ID NO: 78)
CDR2-AIYPGNGDSR CDR2-AIYPGNSDTT CDR2-YISSGSSTIS CDR2-YINPISGYTN
(SEQ ID NO: 61) (SEQ ID NO: 67) (SEQ ID NO: 73) (SEQ ID NO: 79)
CDR3-RDGNYAAY CDR3-RHDYGAMDY CDR3-EAYGSSMEY CDR3-GGAYGRKPMDY
(SEQ ID NO: 62) (SEQ ID NO: 68) (SEQ ID NO: 74) (SEQ ID NO: 80)
Kabat HC CDR2 Kabat HC CDR2 Kabat HC CDR2 Kabat HC CDR2
Murine Murine Murine Murine
AIYPGNGDSRYTQKFKG AIYPGNSDTTYNLKFKG YISSGSSTISYADT YINPISGYTNYNQK
(SEQ ID NO: 81) (SEQ ID NO: 130) VKG FKD
(SEQ ID NO: 84) (SEQ ID NO: 86)
Human Human Human Human
AIYPGNGDSRYTQKFQG AIYPGNSDTTYNQKFQG YISSGSSTISYADS YINPISGYTNYNQK
(SEQ ID NO: 82) (SEQ ID NO: 83) VKG FQG
(SEQ ID NO: 85) (SEQ ID NO: 87)
The FR1-48, 49, 57, and 65 light and heavy chain CDRs are provided.
The Kabat definition for heavy chain CDR2 isalso given for both the murine
and human antibodies.
Example 6
Hybridoma Screening and Selection
[0274] FOLR1-300-19 cells transfected with human FOLR1 and KB cells were
used in the first and second series of screenings correspondently.
Culture supernatants from the hybridoma were screened by flow cytometry
for secretion of mouse monoclonal antibodies that bind to FOLR1 positive
cells, such as FOLR1-expressing 300-19 cells or KB cells, but not to the
FOLR1 negative cells, such as non-transfected 300-19 cells. 0.1 ml of
hybridoma supernatants was incubated for 3 h with either FOLR1-positive
cells or the non-transfected 300-19 cells (1.times.10.sup.5 cells per
sample) in 0.1 ml FACS buffer (RPMI-1640 medium supplemented with 2%
normal goat serum). Then, the cells were centrifuged, pelleted, washed,
and incubated for 1 hour with 0.1 ml of PE-conjugated goat anti mouse
IgG-antibody (such as obtainable from, for example Jackson Laboratory, 6
.mu.g/mL in FACS buffer). The cells were centrifuged, pelleted again,
washed with FACS buffer and resuspended in 0.2 ml of PBS containing 1%
formaldehyde. Cell-associated fluorescence was measured using a
FACSCalibur flow cytometer with the HTS multiwell sampler or a FACS array
flow cytometer and analyzed using CellQuest Pro (all from BD Biosciences,
San Diego, US). Positive hybridoma clones were subcloned by limiting
dilution. One subclone from each hybridoma, which showed the same
reactivity against FOLR1 as the parental cells by flow cytometry, was
chosen for subsequent analysis. Stable subclones were cultured and the
isotype of each secreted anti-FOLR1 antibody was identified using
commercial isotyping reagents (Roche 1493027). Murine antibodies were
protein A purified from cleared hybridoma media as described above. These
antibodies were designated FR-1 antibodies.
Example 7
Murine Monoclonal Antibody Purification
[0275] Antibodies were purified from hybridoma subclone supernatants using
standard methods, such as, for example Protein A or G chromatography
(HiTrap Protein A or G HP, 1 mL, Amersham Biosciences). Briefly,
supernatant was prepared for chromatography by the addition of 1/10
volume of 1 M Tris/HCl buffer, pH 8.0. The pH-adjusted supernatant was
filtered through a 0.22 .mu.m filter membrane and loaded onto column
equilibrated with binding buffer (PBS, pH 7.3). The column was washed
with binding buffer until a stable baseline was obtained with no
absorbance at 280 nm. Antibody was eluted with 0.1 M acetic acid buffer
containing 0.15 M NaCl, pH 2.8, using a flow rate of 0.5 mL/min.
Fractions of approximately 0.25 mL were collected and neutralized by the
addition of 1/10 volume of 1M Tris/HCl, pH 8.0. The peak fraction(s) was
dialyzed overnight twice against 1x PBS and sterilized by filtering
through a 0.2 .mu.m filter membrane. Purified antibody was quantified by
absorbance at A.sub.280.
Example 8
Binding Characterization by Flow Cytometry
[0276] Binding specificity was tested by flow cytometry using purified
antibodies. FACS histograms demonstrating the binding of anti-FOLR1 to
FOLR1-expressing 300-19 cells and the absence of binding to the parental
300-19 cells are shown in FIG. 4. Each antibody was incubated for 3 hours
with either FOLR1-expressing 300-19 cells or the non-transfected 300-19
cells (1.times.10.sup.5 cells per sample) in 0.1 ml FACS buffer
(RPMI-1640 medium supplemented with 2% normal goat serum). Then, the
cells were pelleted, washed, and incubated for 1 hour with 0.1 ml of
FITC-conjugated goat anti-mouse IgG-antibody (such as is obtainable from,
for example Jackson Laboratory, 6 .mu.g/mL in FACS buffer). The cells
were pelleted again, washed with FACS buffer and resuspended in 200 .mu.L
of PBS containing 1% formaldehyde. Samples were acquired using a
FACSCalibur flow cytometer with the HTS multiwell sampler or a FACS array
flow cytometer and analyzed using CellQuest Pro (all from BD Biosciences,
San Diego, US). The FACS histograms of anti-FOLR1 antibodies showed a
fluorescence shift, while parental 300-19 cells did not. Also, no
significant fluorescence shift was detected when either of the cell lines
was incubated only with FITC conjugated goat anti-human IgG-antibody
alone.
Example 9
Cloning and Sequencing of the VL and VH Regions of muFR1-21
[0277] Total cellular RNA was prepared from 5.times.10.sup.6 hybridoma
cells using an RNeasy kit (QIAgen) according to the manufacturer's
protocol. cDNA was subsequently synthesized from total RNA using the
SuperScript II cDNA synthesis kit (Invitrogen). The procedure for the
first round degenerate PCR reaction on the cDNA derived from hybridoma
cells was based on methods described in Wang et al. ((2000) J Immunol
Methods. January 13; 233(1-2):167-77) and Co et al. ((1992) J Immunol.
February 15; 148(4):1149-54). VH sequences were amplified by PCR using
the following degenerate primers: EcoMH1 CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC
(SEQ ID NO:50) EcoMH2 CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG (SEQ ID NO:51)
and BamIgG1 GGAGGATCCATAGACAGATGGGGGTGTCGTTTTGGC (SEQ ID NO:52). VL
sequences were amplified by PCR using the following degenerate primers:
SacIMK GGAGCTCGAYATTGTGMTSACMCARWCTMCA (SEQ ID NO:53) and HindKL
TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC (SEQ ID NO:54). (Mixed
bases are defined as follows: N=G+A+T+C, S=G+C, Y=C+T, M=A+C, R=A+G,
W=A+T).
[0278] The PCR reaction mixtures were then run on a 1% low melt agarose
gel, the 300 to 400 bp bands were excised, purified using Zymo DNA mini
columns, and sent to Agencourt Biosciences for sequencing. The respective
5' and 3' PCR primers were used as sequencing primers to generate the
variable region cDNAs from both directions. The amino acid sequences of
VH and VL regions were obtained by translating the DNA sequencing results
with VectorNTI software.
[0279] To identify 5'end primer sequencing artifacts in the preliminary
variable region cDNA sequences, the NCBI IgBlast site was utilized to
search for the murine germline sequences from which the antibody
sequences were derived. The cleaned up variable region sequences were
then combined with the NCBI reference sequences for the specific antibody
constant regions to assemble expected full length murine antibody
sequences. The molecular weight of the expected murine Fr1-21 light and
heavy chains were then calculated and compared with the mass measured by
liquid chromatography/mass spectrophotometric analysis (LC/MS). The
murine FR1-21 heavy chain matched the measured mass, but the light chain
required a follow up sequencing effort to determine the 5' end sequence.
The CD37-1LClead1 PCR primer ttttgaattcgccaccatgaagtttccttctcaacttct (SEQ
ID NO:55) was designed to anneal to the germline linked leader sequence
of the murine antibody so that this new PCR reaction would yield a
complete variable region cDNA sequence, unaltered by the primers. The PCR
reactions, band purifications, and sequencing were performed as described
above and the new complete sequence encoded a light chain that matched
the Fr1-21 light chain mass measured by LC/MS.
Example 10
Expression of Reference Antibodies
[0280] The Morphotech anti-FOLR1 antibody, MorAb-003 (Farletuzumab), amino
acid sequence was obtained from the World Health Organization (WHO)
International Nonproprietary Names for Pharmaceutical Substances (INN)
list and was codon-optimized and synthesized by Blue Heron Biotechnology.
The light chain variable region sequence is flanked by EcoRI and BsiWI
restriction enzyme sites and the heavy chain variable region sequence
flanked by HindIII and Apa1 restriction enzyme sites for cloning in-frame
with the respective constant sequences in single chain mammalian
expression plasmids. Cloning, expression and purification was carried out
as described for humanized Mov19 and Fr1-21 above.
Example 11
ADCC Activity of huMov19
[0281] A lactate dehydrogenase (LDH) release assay was used to measure
antibody-dependent cell mediated cytotoxicity (ADCC) of tumor cells lines
using freshly isolated human natural killer (NK) cells as effector cells
(e.g., Shields, J. Biol. Chem., 276(9):6591-6604 (2001)). NK cells were
first isolated from human blood from a normal donor (Research Blood
Components, Inc., Brighton, Mass.) using a modified protocol for the NK
Isolation Kit II (Miltenyi Biotech, 130-091-152). Blood was diluted
2-fold with 1x PBS. 25 mL of diluted blood was carefully layered over 25
mL of Ficoll Paque in a 50 mL conical tube and centrifuged at 400 g for
45 min at RT. The peripheral blood mononuclear cells (PBMC) were
collected from the interface, transferred into a new conical 50 mL tube,
and washed once with 1x PBS. The PBMC were resuspended in 2 mL of
NK-isolation buffer (1.times.PBS, 0.5% BSA, 2 mM EDTA), and then 500
.mu.L of Biotin-Antibody Cocktail were added to the cell suspension. The
Biotin-Antibody Cocktail contains biotinylated antibodies that bind to
the lymphocytes, except for NK cells, resulting in a negative selection
of NK cells. The mixture was incubated at 4.degree. C. for 10 minutes,
and then 1.5 mL of NK-isolation buffer and 1 mL of Anti-Biotin Micro
Beads were added. The cell antibody mixture was incubated for another 15
minutes at 4.degree. C. Next, cells were washed once with 50 mL of
NK-isolation buffer and resuspended in 3 mL of NK-isolation buffer. Then,
a MACS LS column was mounted on the autoMACS separator (Miltenyi Biotech)
and pre-washed with 3 mL of NK-isolation Buffer. The cell suspension was
automatically applied onto the column, washed and the effluent fraction
with unlabeled NK cells was collected into a new 50 mL conical tube. The
resulting NK cells were plated into 30 mL of complete RPMI media
(RPMI-1640 supplemented with 5% fetal bovine serum, 1%
penicillin-streptomycin, 1 mM HEPES, 1 mM Sodium Pyruvate, 1%
100.times.MEM non-essential Amino Acid Solution) overnight. The
subsequent assay and all dilutions were carried out in RHBP medium (RPMI
1640 medium supplemented with 20 mM HEPES, pH 7.4, 0.1% BSA and 1%
penicillin streptomycin). Various concentrations of antibodies in RHBP
medium were aliquoted in duplicate at 50 .mu.L/well into a round bottom
96-well plate. The target cells were resuspended at 10.sup.6 cells/mL in
RHBP medium and added at 100 .mu.L/well to each well containing antibody
dilutions. The plate containing target cells and antibody dilutions was
incubated for 30 minutes at 37.degree. C. NK cells were then added to the
wells containing the target cells at 50 .mu.L/well. The typical ratio was
about 1 target cell to 3-4 NK cells. At least the following controls were
set up for each experiment: NK cells alone, target cells alone
(spontaneous LDH release), target cells with NK cells (antibody
independent LDH release), target cells with 10% TritonX-100 (maximum LDH
release). The mixtures were incubated at 37.degree. C. for 4 hours to
allow for cell lysis. Plates were centrifuged for 10 minutes at 1200 rpm,
and 100 .mu.L of the supernatant was carefully transferred to a new flat
bottom 96-well plate. LDH reaction mixture (100 .mu.L/well) from the
Cytotoxicity Detection Kit (Roche 1 644 793) was added to each well and
incubated at room temperature for 5 to 30 min. The optical density of
samples was measured at 490 nm (OD.sub.490). The percent specific lysis
of each sample was determined using the following formula: percent
specific lysis=(sample value-spontaneous release)/(maximum
release-spontaneous release)*100.
[0282] Incubation with huMov19 lead to good ADCC activity against IGROV-1
cells in the presence of human NK effector cells. ADCC activity on
IGROV-1 cells was compared for huMov19, huFR-1-21, Mor003, and chTK1
(isotype control) (FIG. 6). Treatment with 0.9 ng/ml huMov19 resulted in
approximately 30% IGROV-1 cell lysis, similar to activity that was
observed with the other anti-FOLR1 antibodies. ADCC activity by huMov19
had an EC.sub.50 of 0.20 ng/mL, huFr-1-21 had an EC.sub.50 of 0.11 ng/mL,
Mor003 of 0.16 ng/mL and chTK1 did not show any activity against IGROV-1
cells.
Example 12
Preparation of Anti-FOLR1 Immunoconjugates
[0283] Preparation of huMOV19v1.6-Sulfo-SPDB-DM4
[0284] The exemplary 2-sulfo-SPDB linker was dissolved in DMA. The
huMOV19v1.6 antibody was incubated at 8 mg/mL with a 12 fold molar excess
of 2-sulfo-SPDB linker for approximately 2 hours at 25.degree. C. at pH
7.5. The reaction mixture was purified using a SEPHADEX.TM. G25F column
equilibrated with 50 mM potassium phosphate buffer containing 50 mM NaCl,
2 mM EDTA, pH 6.5. The maytansinoid DM4 was dissolved in
dimethylacetamide (DMA, final concentration is 5%) and a 1.7 fold molar
excess compared to the linker was added drop wise to the sulfo-SPDB
modified antibody. The reaction mixture was adjusted to pH 7.5 with 1 m
HEPES buffer. After overnight incubation at room temperature, the
conjugated antibody was purified by chromatography on SEPHADEX.TM. G25F
equilibrated with 10 mM histidine, 250 mM glycine, 1% sucrose, pH 5.5 The
number of DM4 molecules linked per antibody molecule was determined using
the previously reported extinction coefficients for antibody and
maytansinoid (Widdison, W C, et al. J Med Chem, 49:4392-4408 (2006)). The
percentage of total free maytansinoid species were determined as
described above. Conjugates with 3.5-4 DM4 molecules per huMov19v1.6
antibody were obtained with <1% present as unconjugated maytansinoid.
Preparation of huMOV19v1.6-SPP-DM1
[0285] The exemplary N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP)
linker was dissolved in ethanol. The huMOV19v1.6 antibody was incubated
at 8 mg/mL with a 6.5 to 6-fold molar excess of SPP linker for
approximately 2 hours at room temperature in 50 mM potassium phosphate
buffer (pH 6.5) containing 50 mM NaCl, 2 mM EDTA, and 5% ethanol. The SPP
modified antibody was diluted 2-fold in PBS, pH 6.5 and modified with a
1.5 fold molar excess of the maytansinoid DM1 by the addition of a
concentrated solution (15-30 mM) of DM1 in dimethylacetamide (DMA). The
concentration of DMA was adjusted to 5% and after overnight incubation at
room temperature, the conjugated antibody was purified by chromatography
on SEPHADEX.TM. G25F equilibrated 10 mM, 250 mM glycine, 1% sucrose pH
5.5. The number of DM1 molecules linked per antibody molecule was
determined using the previously reported extinction coefficients for
antibody and DM1 (Liu et al., Proc. Natl. Acad. Sci. USA, 93, 8618-8623
(1996)). The percentage of free maytansinoid present after the
conjugation reaction was determined by injecting 20-50 .mu.g conjugate
onto a HiSep.TM. column equilibrated in 25% acetonitrile in 100 mM
ammonium acetate buffer, pH 7.0, and eluting in acetonitrile. The peak
area of total free maytansinoid species (eluted in the gradient and
identified by comparison of elution time with known standards) was
measured using an absorbance detector set to a wavelength of 252 nm and
compared with the peak area related to bound maytansinoid (eluted in the
conjugate peak in the column flow-through fractions) to calculate the
percentage of total free maytansinoid species. Conjugates with 3.5-4 DM1
molecules per huMOV19v1.6 were obtained with <1% present as
unconjugated maytansinoid.
Preparation of huMOV19v1.6 SPDB-DM4
[0286] The exemplary N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB)
linker was dissolved in ethanol. The huMOV19v1.6 antibody was incubated
at 8 mg/mL with a 5.5-5 fold molar excess of SPDB linker for
approximately 2 hours at room temperature in 50 mM potassium phosphate
buffer (pH 6.5) containing 50 mM NaCl, 2 mM EDTA, and 3% ethanol. The
SPDB modified antibody was diluted 2-fold in PBS, pH 6.5 and modified
with a 1.5 fold molar excess of the maytansinoid DM4 by the addition of a
concentrated solution (15-30 mM) of DM4 in dimethylacetamide (DMA). After
overnight incubation at room temperature, the conjugated antibody was
purified by chromatography on SEPHADEX.TM. G25F equilibrated with 10 mM
histidine, 250 mM glycine, 1% sucrose pH 5.5. The number of DM4 molecules
linked per antibody molecule was determined using the previously reported
extinction coefficients for antibody and maytansinoid (Widdison, W C, et
al. J Med Chem, 49:4392-4408 (2006)). The percentage of total free
maytansinoid species were determined as described above. Conjugates with
3.5-4 DM4 molecules per huMOV19v1.6antibody were obtained with <1%
present as unconjugated maytansinoid.
Preparation of huMOV19v1.0-3-Sulfo-Mal-DM4
[0287] The NHS-3-sulfo-mal linker and DM4 were dissolved separately in
DMA. The linker and DM4 thiol were mixed together in a solution of DMA
containing 40% 200 mM succinate buffer, 2 mM EDTA, pH5.0 to give a molar
ratio of DM4 to linker of 1.6:1 and a final concentration of DM4 equal to
10 mM. The mixture was reacted for 2 hours at 25 C. Without purification,
the reaction mixture was added so that an equivalent of 9.6 molar excess
of linker to antibody was added to a solution of huMOV19v1.0 antibody in
phosphate buffer (pH7.5) under final conjugation conditions of 4 mg/mL
antibody, 90% phosphate buffer/10% DMA pH7.5 (v/v). After an overnight
incubation at room temperature, the conjugation mixture was purified by
chromatography on SEPHADEX G25 equilibrated in PBS pH7.5. The
huMOV19v1.0-3-sulfo-mal-DM4 was then dialyzed into a buffer containing
9.55 mM Phosphate, 139.6 mM NaCl, pH6.5. The number of DM4 molecules
linked per antibody molecule was determined using the previously reported
extinction coefficients for antibody and maytansinoid (Widdison, W C, et
al. J Med Chem, 49:4392-4408 (2006)). The percentage of total free
maytansinoid species was determined as described above. Conjugates with
3.5-4 DM4 molecules per huMOV19v1.0 antibody were obtained with <1%
present as unconjugated maytansinoid.
Preparation of huMOV19v1.0-SMCC-DM1
[0288] The NHS-sulfo-SMCC linker and DM1 were dissolved separately in DMA.
The linker and DM1 thiol were mixed together in a solution of DMA
containing 40% 200 mM succinate buffer, 2 mM EDTA, pH5.0 to give a molar
ratio of DM1 to linker of 1.2:1 and a final concentration of DM1 equal to
3.75 mM. The mixture was reacted for 75 minutes at 20.degree. C. Without
purification, the reaction mixture was added so that an equivalent of 6.4
molar excess of linker to antibody was added to a solution of huMOV19v1.0
antibody in phosphate buffer (pH7.5) under final conjugation conditions
of 4 mg/mL antibody, 88% 50 mM Potassium Phosphate, 50 mM NaCl, 2 mM
EDTA, pH 7.5/12% DMA pH7.5 (v/v). After 2 hour incubation at 20.degree.
C., the conjugation mixture was purified by chromatography on SEPHADEX
G25 equilibrated in PBS pH7.5. The huMOV19v1.0-SMCC-DM1 was then dialyzed
into a buffer containing 250 mM Glycine, 10 mM Histidine pH5.5. The
number of DM1 molecules linked per antibody molecule was determined using
the previously reported extinction coefficients for antibody and
maytansinoid (Widdison, W C, et al. J Med Chem, 49:4392-4408 (2006)). The
percentage of total free maytansinoid species was determined as described
above. Conjugates with 3.5-4 DM1 molecules per huMOV19v1.0 antibody were
obtained with <2.8% present as unconjugated maytansinoid.
Preparation of huMOV19v1.0-PEG4-mal-DM1
[0289] The NHS-PEG4-mal-DM1 1 step reagent was dissolved in DMA. The
huMov19v1.0 antibody was incubated at 5 mg/mL with a 5.7 fold molar
excess of NHS-PEG4-mal-DM1 overnight at 25.degree. C. in 50 mM KPi, 50 mM
NaCl, 2 mM EDTA, pH 7.5 and 10% DMA by volume. The reaction mixture was
purified by SEPHADEX G25 column equilibrated in PBS pH7.5. The
huMOV19v1.0-PEG4-mal-DM1 was dialyzed into buffer containing 250 mM
Glycine, 10 mM Histidine pH5.5. The number of DM1 molecules linked per
antibody molecule was determined using the previously reported extinction
coefficients for antibody and maytansinoid (Widdison, W C, et al. J Med
Chem, 49:4392-4408 (2006)). The percentage of total free maytansinoid
species was determined as described above. Conjugates with 3.5-4 DM1
molecules per huMOV19v1.0 antibody were obtained with <1.1% present as
unconjugated maytansinoid.
Example 13
Binding Affinity of Antibodies and Conjugates
[0290] Binding affinities of anti-FOLR1 antibodies and of their SPDB-DM4,
PEG4Mal-DM4, SMCC-DM1, or anti-FOLR1-sulfo-SPDB-DM4 conjugates were
assayed by Flow Cytometry. FOLR1-expressing SKOV3 cells were incubated
with varying concentrations of anti-FOLR1 antibodies or their conjugates
and processed as described above for flow cytometry analysis. Data
analysis was performed using CellQuest Pro (BD Biosciences, San Diego,
US) and for each sample the mean fluorescence intensity for FL1 (MFI) was
exported and plotted against the antibody concentration in a semi-log
plot. A dose-response curve was generated by non-linear regression and
the value for the apparent equilibrium dissociation constant (K.sub.d) of
the test-samples for the binding to SKOV3 cells was calculated using
GraphPad Prism v4 (GraphPad software, San Diego, Calif.) and presented in
FIG. 5. The results demonstrate that conjugation to either DM1 or DM4
through either of the linkers used, did not notably alter the affinity of
either of the antibodies (e.g., huMov19).
Example 14
In Vitro Cytotoxicity Assays
[0291] The ability of exemplary muFR1-9, muFR1-13, muFR1-22, muFR1-23,
huFR1-23, muFR1-21, and huFR1-21 conjugates to inhibit cell growth was
measured using in vitro cytotoxicity assays by the method described in
Kovtun Y V et al. (Cancer Res 66: 3214-3221 (2006)). A PEG4-mal-DM4
conjugate in various concentrations was added to FOLR1-expressing KB
cells in a 96 well plate at 1,000 cells per well in 100 .mu.L in complete
RPMI medium (RPMI-1640, 10% fetal bovine serum, 2 mM glutamine, 1%
gentamycin, all reagents from Invitrogen). Antibodies and conjugates were
diluted into complete RPMI medium using 3-fold dilution series and 100
.mu.L were added per well. The final concentration typically ranged from
3.times.10.sup.-8 M to 4.6.times.10.sup.-12 M. Control wells containing
cells and the medium but lacking the conjugates, and wells containing
medium only were included in each assay plate. The plates were incubated
from four to six days at 37.degree. C. in a humidified atmosphere
containing 5% CO.sub.2. WST-8 reagent, 10% v/v (Dojindo Molecular
Technologies, Gaithersburg, Md., US) was then added to the wells and the
plates were incubated at 37.degree. C. for 2-6 h. WST-8 is reduced by
dehydrogenases in living cells to an orange (maximum formazan product
that is soluble in tissue culture medium. The amount of formazan produced
is directly proportional to the number of living cells. Plates were
analyzed by measuring the absorbance at 450 nm (A.sub.450) and at and 650
nm (A.sub.650) in a multiwell plate reader. First, the background of
cells' opalescence (A.sub.650) was subtracted from A.sub.650. The
resulting A*.sub.450 was then used to determine the surviving fraction of
cells. Background A*.sub.450 absorbance was that of wells with medium and
WST-8 only. The surviving fraction was calculated as follows: Percent
viability=100.times.(A*.sub.450 treated sample-A*.sub.450
background)/(A*.sub.450 untreated sample-A*.sub.450 background). The
surviving fraction values were plotted against antibody or conjugate
concentration in a semi-log plot for each treatment. From these data
IC.sub.50 values were then determined using GraphPad Prism v4 (GraphPad
software, San Diego, Calif.) and presented in FIG. 5. The results shown
in FIG. 5 demonstrate that all conjugates are similarly active in their
cytotoxic potency against FOLR1-expressing KB cells. To further verify
the specificity of the anti-FOLR1-maytansinoid conjugates towards FOLR1,
their activities were evaluated in the presence of an excess of
non-conjugated antibodies against KB cells. Addition of an excess of
competing non-conjugated antibody to the conjugates suppressed their
cytotoxicity, as seen in FIG. 7. These data indicate that the conjugates
kill KB cells in an antigen-dependent manner. Additional data
demonstrated that huMov19-SPDB-DM4 induced cell cycle arrest in the G2/M
phase in KB cells in in vitro assays.
Example 15
In Vivo Efficacy of huMov19-PEG4Mal-DM4 and huMov19-SPDB-DM4 Conjugates in
Comparison with Similar Non-Targeting Conjugates in a KB Xenograft Model
[0292] FOLR1-targeting cleavable conjugate huMov19-SPDB-DM4 in comparison
with non-targeting huC242-SPDB-DM4, and non-cleavable conjugate
huMov19-PEG4-Mal-DM4 in comparison with non-targeting huC242-PEG4Mal-DM4
were tested using an established xenograft model of KB cells implanted
subcutaneous into SCID mice. Mice were randomized by body weight into
treatment groups and treated either singly (SPDB conjugates) on day 3
post cell inoculation, or three times weekly on days 3, 10, and 17 post
cell inoculation with 5 and 10 mg/kg of a conjugate, respectively. The
median tumor volume of the different treatment groups is plotted in FIG.
8. The treatments with either huMov19-SPDB-DM4, or huMov19-PEG4Mal-DM4
resulted in a decrease in median tumor volume as compared to the PBS
control, while the treatments with either of the respective non-targeting
conjugate did not produce any significant effect.
Example 16
In Vivo Efficacy of Anti-FOLR1-PEG4Mal-DM4 Conjugates in a KB Xenograft
Model
[0293] PEG4Mal-DM4 conjugates of the exemplary anti-FOLR1 antibodies
huMov19, muFR-1-9, muFR-1-13, muFR-1-22, muFR-1-23, and huFR-1-21 were
tested using an established xenograft model of KB cells implanted
subcutaneous into SCID mice. Mice were randomized by body weight into
treatment groups and treated once on day 3 post cell inoculation with 10
mg/kg of one of the conjugates listed above or with PBS only.
HuMov19-PEG4Mal-DM4 was shown above to be similar to PEG4Mal-DM4
conjugates of muFR-1-9, muFR-1-13, muFR-1-22, muFR-1-23, and huFR-1-21 in
its cytotoxic potency in vitro. HuMov19-PEG4Mal-DM4 and
huFR-1-21-PEG4Mal-DM4 were significantly more potent in vivo than any of
the other conjugates, resulting in a more pronounced decrease in median
tumor volume (FIGS. 9 and 10). The potency was also demonstrated to be
dose-dependent (FIG. 11) and choice of linker played a role as well
(FIGS. 12 and 13).
Example 17
In Vivo Efficacy of Anti-FOLR1-Sulfo-SPDB-DM4 Conjugates in a Xenograft
Models
[0294] Anti-FOLR1 huMov19-sulfo-SPDB-DM4 conjugates were tested in three
ovarian serous adenocarcinoma xenografts: OVCAR-3, IGROV-1, and OV-90.
Each of these xenograft tumors showed FOLR1 expression levels comparable
to patient tumors when measured using a calibrated immunohistochemical
(IHC) staining method on formalin-fixed paraffin-embedded sections. Mice
bearing established subcutaneous xenograft tumors (approximately 100
mm.sup.3) were treated with a single intravenous injection of
huMov19-sulfo-SPDB-DM4 conjugate at 1.2, 2.5, and 5.0 mg/kg (based on
antibody concentration; FIGS. 14-16 show the concentration of the
maytansinoid conjugate in .mu.g/kg). The conjugate was active in all
three models evaluated. In OVCAR-3 xenografts, the minimally efficacious
dose (MED) was 1.2 mg/kg (FIG. 14). The higher dose levels were highly
active, resulting in complete regressions (CR) in 4/6 and 2/6 mice in the
2.5 and 5.0 mg/kg treatment groups, respectively. Treatment with the
conjugate resulted in strong anti-tumor activity in both IGROV-1 and
OV-90 xenograft models, with a MED of 2.5 mg/kg, single injection (FIGS.
15 and 16). These data demonstrate the strong anti-tumor activity of
huMov19-sulfo-SPDB-DM4 conjugates against ovarian xenograft tumors with
FOLR1 expression levels comparable to patient tumors.
Example 18
Effect of Linkers on Immunoconjugate Efficacy
[0295] The anti-FOLR1 antibody huMov19 was linked to DM1 or DM4 via the
disulfide-containing cleavable linkers SPP, SPDB, or sulfo-SPDB, or via
the non-cleavable linker SMCC. The in vitro cytotoxic activities of these
conjugates on KB, IGROV-1 and JEG-3 cell lines was examined. FACS
analysis indicated that the KB (cervical) cells had >2,000,000
antibody binding sites per cell. The IGROV-1 (ovarian) cells had 260,000
antibody binding sites per cell, and the JEG-3 (choriocarcinoma) cells
had 40,000 antibody binding sites per cell. The results of the in vitro
cytotoxicity are summarized in Table 2 below. The cleavable conjugates
displayed markedly greater in vitro activities compared with those of the
SMCC-conjugate.
TABLE-US-00004
TABLE 2
Effect of immunoconjugate linkers on cytotoxicity in vitro.
IC.sub.50, nM (n = 3), Ab-based
Cells SPP-DM1 SPDB-DM4 Sulfo-SPDM-DM4 SMCC-DM1
KB 0.1 0.1 0.1 0.1
Igrov 1 0.1 0.1 0.3 1.0
Jeg3 0.2 0.2 3.0 20
[0296] The in vivo activities of the conjugates in FOLR1-positive KB- and
OVCAR-3-tumor models were also tested. The results shown in FIG. 17
demonstrate that cleavable SPDB-DM4 and sulfo-SPDB-DM4 conjugates are
more patent than non-cleavable SMCC-DM1 conjugates in vivo. In addition,
among the cleavable conjugates, the SPP-DM1 conjugate was less active
than either the SPDB-DM4 or sulfo-SPDB-DM4 conjugates in both xenograft
models (FIG. 18). The two latter conjugates were similarly active against
KB tumors, whereas the sulfo-SPDB-DM4 conjugate was more active against
the OVCAR-3 model. The data obtained using the OVCAR-3 model is
summarized in Table 3 below.
TABLE-US-00005
TABLE 3
Effect of immunoconjugate linkers on tumor size in OVCAR-3
xenograft model.
Tumor over Partial Complete
Conjugate control (%) Response Response Response
SPP-DM1 54 0/6 0/6 Inactive
SPDB-DM4 9 6/6 1/6 Highly active
Sulfo-DPDB- 0 6/6 4/6 Highly active
DM4
[0297] These data demonstrate that immunoconjugates containing a cleavable
linker show increased efficacy both in vitro and in vivo, and anti-FOLR1
immunoconjugates containing sulfo-SPDB are highly active in tumor models.
Example 19
In Vitro and In Vivo Efficacy of huFR1 Antibody SMCC-DM1 Conjugate
[0298] Anti-FOLR1 huFR1-48, huFR1-49, huFR1-57, and huFR1-65 were
conjugated with SMCC linker and DM1 and the effects on KB cells, and in
vivo using the above-described xenograft models were analyzed as
described above. While each of the antibodies showed similar efficacy in
the KB cell model, the huFR1-48, huFR1-49, huFR1-57, and huFR1-65
immunoconjugates showed variable, but significant, in vivo efficacy at a
200 .mu.g/kg dose in a xenograft model system (Table 4 and FIG. 19).
TABLE-US-00006
TABLE 4
In vitro and in vivo efficacy of huFR1 antibody SMCC-DM1 conjugate
huAb-smcc-DM1
Apparent affinity activity on KB in huAb-smcc-DM1
Clone # (nM) vitro (nM) activity in vivo
huFR1-48 0.13 0.05 +
huFR1-49 0.08 0.10 +
huFR1-57 0.14 0.10 +
huFR1-65 0.15 0.10 +
huMov19 0.06 0.10 ++
[0299] All publications, patents, patent applications, internet sites, and
accession numbers/database sequences (including both polynucleotide and
polypeptide sequences) cited herein are hereby incorporated by reference
in their entirety for all purposes to the same extent as if each
individual publication, patent, patent application, internet site, or
accession number/database sequence were specifically and individually
indicated to be so incorporated by reference.
TABLE-US-00007
SEQUENCES
SEQ ID NO: 1-huMov19 vHC CDR1
GYFMN
SEQ ID NO: 2-huMov19 vHC CDR2
RIHPYDGDTFYNQKFQG
SEQ ID NO: 3-huMov19 vHC CDR3
YDGSRAMDY
SEQ ID NO: 4-huMov19 vHC
QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFYNQ
KFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVTVSS
SEQ ID NO: 5-huMov19 vHC nucleic acid sequence
aagcttgccaccatgggttggtcatgcatcatcctcttcttggttgcaactgctaccggagtgcacagtcaggt-
acagctcgtgcagtccggcgccga
ggtggtgaagcctggtgccagcgtgaagatctcctgtaaagccagtggatacacattcaccggttattttatga-
attgggtgaaacagagcccaggcc
aatccctcgaatggatagggcgaatccacccatatgacggggacaccttttacaaccagaaattccaggggaaa-
gccactctgacagtggacaaga
gttccaacactgcacacatggagcttctctccctgaccagcgaagacttcgctgtttattactgtacccgttat-
gatggttcccgtgcaatggactactgg
ggccaagggaccactgtcaccgtaagttccgccagcaccaagggccc
SEQ ID NO: 6-huMov19 HC amino acid sequence
QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFYNQ
KFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVTVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY
TQKSLSLSPGK
SEQ ID NO: 7-huMov19 vLC CDR1
KASQSVSFAGTSLMH
SEQ ID NO: 8-huMov19 vLC CDR2
RASNLEA
SEQ ID NO: 9-huMov19 vLC CDR3
QQSREYPYT
SEQ ID NO: 10-huMov19 vLCv1.00
DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPD
RFSGSGSKTDFTLNISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKR
SEQ ID NO: 11-huMov19 vLCv1.60
DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPD
RFSGSGSKTDFTLTISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKR
SEQ ID NO: 12-huMov19 LCv1.00
DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPD
RFSGSGSKTDFTLNISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQL
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE
KHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 13-huMov19 LCv1.60
DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPD
RFSGSGSKTDFTLTISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQL
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE
KHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 14-huMov19 LCv1.00 nucleic acid
gaattcgccaccatgggctggagctgcattatcctttttctggtagccacagctacaggcgtgcatagcgatat-
cgtgctgacacaatcccccctctctc
tggccgtgtcactcggacagcccgctatcatcagctgcaaagccagccagtctgtcagatcgctggaacaagtc-
ttatgcattggtatcatcagaag
cctggccagcaacccaggctgctgatctatcgagcctcaaacttggaagcaggagtgccagaccggttttctgg-
gtccgggagtaaaaccgatttta
cacttaatatctcacctgtcgaggccgaggacgccgccacctactactgtcagcagagccgagagtaccataca-
cttttggcggtgggactaaactg
gaaataaaacgtacg
SEQ ID NO: 15-huMov19 LCv1.60 nucleic acid
gaattcgccaccatgggctggtcttgtatcatcctgtttctggtggccaccgcaaccggtgttcactccgacat-
tgtgctgacacagtcccccctttcact
ggctgtatccctcggccagcccgctatcatcagctgcaaggctagccagagcgtgagtMgccggcacttcactt-
atgcattggtaccatcagaaac
caggccagcaacctaggctgctgatttatcgggctagcaacctggaggccggcgtgcccgaccgctttagcggg-
agcggctccaagactgacttc
actctgaccatctcccccgtagaagcagaagatgctgcaacctactactgtcagcagtctcgcgagtatcctta-
tacattcggaggcggaactaaact
ggagattaaacgtacg
SEQ ID NO: 16-muMov19 vHC CDR2
RIHPYDGDTFYNQNFKD
SEQ ID NO: 17-muMov19 vHC_CAA68252
QVQLQQSGAELVKPGASVKISCKASGYSFTGYFMNWVKQSHGKSLEWIGRIHPYDGDTFYNQ
NFKDKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVTVS
SEQ ID NO: 18-muMov19 vLC_CAA68253
DIELTQSPASLAVSLGQRAIISCKASQSVSFAGTSLMHWYHQKPGQQPKLLTYRASNLEAGVPT
RFSGSGSKTDFTLNIHPVEEEDAATYYCQQSREYPYTFGGGTKL
SEQ ID NO: 19-chMov19 HC
QVQLQQSGAELVKPGASVKISCKASGYSFTGYFMNWVKQSHGKSLEWIGRIHPYDGDTFYNQ
NFKDKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVTVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY
TQKSLSLSPGK
SEQ ID NO: 20-chMov19 LC
DIELTQSPASLAVSLGQRAIISCKASQSVSFAGTSLMHWYHQKPGQQPKLLIYRASNLEAGVPT
RFSGSGSKTDFTLNIHPVEEEDAATYYCQQSREYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQL
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE
KHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 21-chMov19 HC nucleic acid
aagcttgccaccatgggttggtatgtattatcctctttctcgtcgcaaccgcaacaggcgtccattcacaagtc-
caactgcagcaatccggcgccgaa
ctcgttaaacctggagcatctgttaaaatctcatgtaaagcatcaggatactcatttactggctattttatgaa-
ctgggtcaaacaatcacacggaaaatc
acttgaatggatcggacgtattcacccctatgatggcgatacttMacaaccagaacttcaaagacaaagctaca-
ctcaccgttgacaaatcatctaac
accgctcacatggaactcctttcactcacatctgaagacttcgctgtttattactgtactagatacgatggatc-
aagagctatggattattggggacaagg
aacaacagtcacagtctcatctgcatcaactaagggccca
SEQ ID NO: 22-chMov19 LC nucleic acid
gaattcgccaccatgggttggtcttgtattatcctattctcgtcgcaaccgcaacaggcgtccattcagatatc-
gaactcacacaatcaccagcttccct
cgcagtctctctcggtcaacgcgcaatcatctcttgtaaagcctcccaatcagtctcattcgccggcacgtccc-
tcatgcattggtaccatcaaaaaccc
ggtcagcaacccaaactccttatctatagagcaagcaacctcgaagcaggcgttcccaccagatttagcggatc-
aggaagtaaaaccgatttcacac
tcaacattcatccagtcgaagaagaagatgcagctacttattattgccaacagtctagagaatatccatacaca-
ttcggagggggtaccaaacttgaaa
ttaaacgtacg
SEQ ID NO: 23-muMov19 vHC_CAA68252
QVQLQQSGAELVKPGASVKISCKASGYSFTGYFMNWVKQSHGKSLEWIGRIHPYDGDTFYNQ
NFKDKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVTVS
SEQ ID NO: 24-muMov19 vLC_CAA68253
DIELTQSPASLAVSLGQRAIISCKASQSVSFAGTSLMHWYHQKPGQQPKLLIYRASNLEAGVPT
RFSGSGSKTDFTLNIHPVEEEDAATYYCQQSREYPYTFGGGTKL
SEQ ID NO: 25-human folate receptor 1
MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPW
RKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVD
QSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHF
YEPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGPWAA
WPFLLSLALMLLWLLS
SEQ ID NO: 26-human folate receptor 1 nucleic acid sequence
Atggctcagcggatgacaacacagctgctgctccttctagtgtgggtggctgtagtaggggaggctcagacaag-
gattgcatgggccaggactga
gcttctcaatgtctgcatgaacgccaagcaccacaaggaaaagccaggccccgaggacaagttgcatgagcagt-
gtcgaccctggaggaagaatg
cctgctgttctaccaacaccagccaggaagcccataaggatgMcctacctatatagattcaactggaaccactg-
tggagagatggcacctgcctgc
aaacggcatttcatccaggacacctgcctctacgagtgctcccccaacttggggccctggatccagcaggtgga-
tcagagctggcgcaaagagcg
ggtactgaacgtgcccctgtgcaaagaggactgtgagcaatggtgggaagattgtcgcacctcctacacctgca-
agagcaactggcacaagggct
ggaactggacttcagggtttaacaagtgcgcagtgggagctgcctgccaacctttccatttctacttccccaca-
cccactgttctgtgcaatgaaatctg
gactcactcctacaaggtcagcaactacagccgagggagtggccgctgcatccagatgtggttcgacccagccc-
agggcaaccccaatgaggag
gtggcgaggttctatgctgcagccatgagtggggctgggccctgggcagcctggcctttcctgcttagcctggc-
cctaatgctgctgtggctgctcag
c
SEQ ID NO: 27-FR1-21 vLC CDR1
KASDHINNWLA
SEQ ID NO: 28-FR1-21 vLC CDR2
GATSLET
SEQ ID NO: 29-FR1-21 vLC CDR3
QQYWSTPFT
SEQ ID NO: 30-FR1-21 vHC CDR1
SSYGMS
SEQ ID NO: 31-FR1-21 vHC CDR2
TISSGGSYTY
SEQ ID NO: 32-FR1-21 vHC CDR3
DGEGGLYAMDY
SEQ ID NO: 33-FR1-21 Kabat murine CDR-H2
TISSGGSYTYYPDGVKG
SEQ ID NO: 34-FR1-21 Kabat human CDR-H2
TISSGGSYTYYSPGFQG
SEQ ID NO: 35-muFR1-21 vLC
DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSG
SGSGKDYTLSISSLQTEDVATYYCQQYWSTPFTFGSGTKLEIKR
SEQ ID NO: 36-muFR1-21 vHC
EVKLVESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLECVATISSGGSYTYYPD
GVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARDGEGGLYAMM(WGQGTSVTVSS
SEQ ID NO: 37-muFR1-21 vLC DNA sequence
gacatccagatgacacaatcttcatcctacttgtctgtatctctaggaggcagagtcaccattacttgcaaggc-
aagtgaccacataaataattggttag
cctggtatcagcagaaaccaggaaatgctcctaggctcttaatatctggtgcaaccagtttggaaactggggtt-
ccttcaagattcagtggcagtggat
ctggaaaggattacactctcagcatttccagtatcagactgaagatgttgctacttattactgtcaacagtatt-
ggagtactccattcacgttcggctcgg
ggacaaagttggaaataaaacg
SEQ ID NO: 38-muFR1-21HCvarPat
gaagtgaagctggtggagtctgggggagacttagtgaagcctggagggtccctgaaactctcctgtgcagcctc-
tggattcactttcagtagctatgg
catgtcttgggttcgccagactccagacaagaggttggagtgtgtcgcaaccattagtagtggtggtagttaca-
cctactatccagacggtgtgaagg
ggcgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgagcagtctgaagtctgaggac-
acagccatgtattactgtgcaag
ggacggcgaggggggcctctatgctatggactactggggtcaaggaacctcagtcaccgtctcctca
SEQ ID NO: 39-muER1-21 LC
DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSG
SGSGKDYTLSISSLQTEDVATYYCQQYWSTPFTEGSGTKLEIKRADAAPTVSIFPPSSEQLTSGG
ASVVCELNNEYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNS
YTCEATHKTSTSPIVKSFNRNEC
SEQ ID NO: 40-muER1-21 HC
EVKLVESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLECVATISSGGSYTYYPD
GVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARDGEGGLYAMDYWGQGTSVTVSSAK
TTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSS
SVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVEIF
PPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWEVNNVEVHTAQTQTHREDYNSTIRVVSTL
PIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVV
GFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYEIYSKLNMKTSKWEKTDSFSCNVRHEGL
KNYYLKKTISRSPGK
SEQ ID NO: 41-huER1-21 vLC
DIQMTQSSSSLSVSVGGRVTITCKASDHINNWLAWYQQKPGKAPKLLISGATSLETGVPSRFSG
SGSGKDYTLSISSLQPEDVATYYCQQYWSTPFTFGQGTKLEIKR
SEQ ID NO: 42-huER1-21 vHC
EVQLVESGGDVVKPGGSLKLSCAASGFTFSSYGMSWVRQTPGKGLECVATISSGGSYTYYSP
GEQGRFTISRDKSKNTLYLQMSSLKAEDTAMYYCARDGEGGLYAMDYWGQ GTSVTVSS
SEQ ID NO: 43-huER1-21VH_co
aagcttgccaccatgggatggtcatgcatcattctttttctcgtcgccactgccacaggtgtgcattccgaggt-
gcaacttgtagaatctggcggggatg
ttgtgaagcctggaggtagtctcaagttgtcctgtgctgcatctgggtttaccttctcttcctacggaatgagc-
tgggtgagacagactcctggcaaggg
gctggagtgcgttgccaccattagtagtggaggttcttacacctactattcacctggtMcagggacgctttaca-
atctcccgcgataagtctaagaaca
ccctttacctccagatgagtagccttaaggctgaggacacagccatgtattattgcgctcgcgatggggaggga-
gggattacgctatggactactgg
ggccagggtaccagcgtgaccgtttcctctgctagtaccaagggccc
SEQ ID NO: 44-huER21VL_co
gaattcgccaccatgggatggtcatgtatcattctgttcttggtagcaacagcaactggcgtccattctgacat-
ccagatgacccaatcctccagcagct
tgtcagtatccgttgggggccgcgttactattacctgtaaggcctccgaccatataaataactggcttgcatgg-
tatcaacagaagcctgggaaggca
cctaaactgcttatctctggggccacaagcctggagaccggcgtgccttccaggttctctggaagtggatctgg-
caaggactataccttgagcattagt
agccttcaacctgaggacgtcgccacctactattgtcagcagtattggtctacaccattaccMggacagggcac-
taaattggagataaaacgtacg
SEQ ID NO: 45-huER1-21 LC
DIQMTQSSSSLSVSVGGRVTITCKASDHINNWLAWYQQKPGKAPKLLISGATSLETGVPSRFSG
SGSGKDYTLSISSLQPEDVATYYCQQYWSTPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGT
ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
YACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 46-huFR1-21 HC
EVQLVESGGDVVKPGGSLKLSCAASGFTFSSYGMSWVRQTPGKGLECVATISSGGSYTYYSP
GFQGRFTISRDKSKNTLYLQMSSLKAEDTAMYYCARDGEGGLYAMDYWGQGTSVTVSSAST
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICNVNHKSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN
HYTQKSLSLSPG
SEQ ID NO: 47-huFR1-21LC DNA sequence
gacatccagatgacccaatcctccagcagcttgtcagtatccgttgggggccgcgttactattacctgtaaggc-
ctccgaccatataaataactggctt
gcatggtatcaacagaagcctgggaaggcacctaaactgcttatctctggggccacaagcctggagaccggcgt-
gccttccaggttctctggaagtg
gatctggcaaggactataccttgagcattagtagccttcaacctgaggacgtcgccacctactattgtcagcag-
tattggtctacaccctttacctttgga
cagggcactaaattggagataaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagca-
gttgaaatctggaactgcctctgttg
tgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggt-
aactcccaggagagtgtcacaga
gcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacaca-
aagtctacgcctgcgaagt
cacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt
SEQ ID NO: 48-huFR1-21HC DNA sequence
gaggtgcaacttgtagaatctggcggggatgttgtgaagcctggaggtagtctcaagttgtcctgtgctgcatc-
tgggtttaccttctcttcctacggaat
gagctgggtgagacagactcctggcaaggggctggagtgcgttgccaccattagtagtggaggttcttacacct-
actattcacctggttttcagggac
gctttacaatctcccgcgataagtctaagaacaccctttacctccagatgagtagccttaaggctgaggacaca-
gccatgtattattgcgctcgcgatg
gggagggagggctttacgctatggactactggggccagggtaccagcgtgaccgtttcctctgctagtaccaag-
ggcccatcagttttccccttggct
ccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgt-
gacagtgagctggaatagcggagc
attgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaa-
ccgtcccttctagcagcttgggaaccc
agacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgt-
gataagacacatacatgccctcc
ttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatga-
tctctcgtactccagaggtcacctgtg
ttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaat-
gccaagaccaagcccagggagg
agcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagag-
tacaagtgcaaggtgtccaacaag
gctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacatt-
gcccccatctagagacgagctg
accaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtc-
taacggacagccagaaaacaacta
caagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtcta-
gatggcagcaaggaaacgtifict
cctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccaggg
SEQ ID NO: 49-huFo1R1 DNA sequence EcoRI to Xba1
gaattcgccaccatggcacagcgcatgaccactcagctcctgcttctgttggtttgggtggcagtcgtgggaga-
ggcccagaccaggattgcttggg
cacgcacagagctgcttaatgtttgcatgaacgcaaagcaccataaagagaaacccggtcccgaggataagttg-
cacgaacagtgccgcccttgga
gaaagaatgcatgctgtagcacgaacacctctcaggaggcgcataaagacgtaagctatttgtatagatttaac-
tggaaccattgcggtgaaatggca
cctgcctgtaaacggcactttatccaggatacttgcttgtacgagtgtagcccgaatctcgggccctggattca-
gcaagttgatcagagttggcgcaaa
gagagggtgctgaacgttccgctttgcaaggaggactgcgagcaatggtgggaagactgtagaaccagctacac-
ctgtaagtctaactggcacaaa
ggatggaactggacatccgggtttaacaaatgcgctgtcggcgctgcctgccagccatttcatttctactttcc-
aactcccactgtcctgtgtaacgaga
tttggacgcattcatataaagtcagcaactacagccggggctccggccgctgcattcagatgtggttcgaccct-
gcacagggcaaccctaacgagga
ggtcgcacgcttctacgctgcagcaatgtctggagccggtccttgggctgcttggccatttctccttagcctcg-
ccctcatgcttctctggctgttgtcat
aatctaga
SEQ ID NO: 50-Primer EcoMH1 CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC
SEQ ID NO: 51-Primer EcoMH2 CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG
SEQ ID NO: 52-Primer BamIgG1 GGAGGATCCATAGACAGATGGGGGTGTCGTTTTGGC
SEQ ID NO: 53-SacIMK GGAGCTCGAYATTGTGMTSACMCARWCTMCA
SEQ ID NO: 54-HindKL TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC
Mixed bases are defined as follows:
N = G + A + T + C, S = G + C, Y = C + T, M = A + C, R = A + G, W = A + T
SEQ ID NO: 55-cd37-1LClead
ttttgaattcgccaccatgaagtttccttctcaacttct
SEQ ID NO: 56-human and chimeric Mov19 vHC CDR2 composite
RIHPYDGDTFYNQXaa.sub.1FXaa.sub.2Xaa.sub.3
Xaa.sub.1 = Q, H, K, or R
Xaa.sub.2 = R, Q, H, or N
Xaa.sub.3 = E, T, S, G, A, or V
SEQ ID NO: 57-FR1-48vL CDR1
RASENIYSNLA
SEQ ID NO: 58-FR1-48vL CDR2
AATNLAD
SEQ ID NO: 59-FR1-48vL CDR3
QHFWASPYT
SEQ ID NO: 60-FR1-48vH CDR1
TNYWMQ
SEQ ID NO: 61-FR1-48vH CDR2
AIYPGNGDSR
SEQ ID NO: 62-FR1-48vH CDR3
RDGNYAAY
SEQ ID NO: 63-FR1-49vL CDR1
RASENIYTNLA
SEQ ID NO: 64-FR1-49vL CDR2
TASNLAD
SEQ ID NO: 65-FR1-49vL CDR3
QHFWVSPYT
SEQ ID NO: 66-FR1-49vH CDR1
TNYWMY
SEQ ID NO: 67-FR1-49vH CDR2
AIYPGNSDTT
SEQ ID NO: 68-FR1-49vH CDR3
RHDYGAMDY
SEQ ID NO: 69-FR1-57vL CDR1
RASQNINNNLH
SEQ ID NO: 70-FR1-57vL CDR2
YVSQSVS
SEQ ID NO: 71-FR1-57vL CDR3
QQSNSWPHYT
SEQ ID NO: 72-FR1-57vH CDR1
SSFGMH
SEQ ID NO: 73-FR1-57vH CDR2
YISSGSSTIS
SEQ ID NO: 74-FR1-57vH CDR3
EAYGSSMEY
SEQ ID NO: 75-FR1-65vL CDR1
KASQNVGPNVA
SEQ ID NO: 76-FR1-65vL CDR2
SASYRYS
SEQ ID NO: 77-FR1-65vL CDR3
QQYNSYPYT
SEQ ID NO: 78-FR1-65vH CDR1
TSYTMH
SEQ ID NO: 79-FR1-65vH CDR2
YINPISGYTN
SEQ ID NO: 80-FR1-65vH CDR3
GGAYGRKPMDY
SEQ ID NO: 81-muFR1-48 Kabat defined HC CDR2
AIYPGNGDSRYTQKFKG
SEQ ID NO: 82-huFR1-48 Kabat defined HC CDR2
AIYPGNGDSRYTQKFQG
SEQ ID NO: 130-muFR1-49 Kabat defined HC CDR2
AIYPGNSDTTYNLKFKG
SEQ ID NO: 83-huFR1-49 Kabat defined HC CDR2
AIYPGNSDTTYNQKFQG
SEQ ID NO: 84-muFR1-57 Kabat defined HC CDR2
YISSGSSTISYADTVKG
SEQ ID NO: 85-huFR1-57 Kabat defined HC CDR2
YISSGSSTISYADSVKG
SEQ ID NO: 86-muFR1-65 Kabat defined HC CDR2
YINPISGYTNYNQKFKD
SEQ ID NO: 87-huFR1-65 Kabat defined HC CDR2
YINPISGYTNYNQKFQG
SEQ ID NO: 88-muFR1-48vL
DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYAATNLADGVPSRFS
GSESGTQYSLKINSLQSEDFGSYYCQHFWASPYTFGGGTKLEIKR
SEQ ID NO: 89-muFR1-48vH
QVQLQQSGAELARPGASVKLSCRASGYTFTNYWMQWIKQRPGQGLEWIGAIYPGNGDSRYT
QKFKGKATLTADKSSSTAYMQVSSLTSEDSAVYYCARRDGNYAAYWGQGTLVTVSA
SEQ ID NO: 90-muFR1-49vL
DIQMTQSPASLSVSVGETVTITCRASENIYTNLAWYQQKQGKSPQLLVYTASNLADGVPSRFS
GSGSGTQYSLKINSLQSEDFGTYYCQHFWVSPYTFGGGTKLEIKR
SEQ ID NO: 91-muFR1-49vH
EVQLQQSGTVLARPGASVKMSCKASGYKFTNYWMYWIKQRPGQGLELIGAIYPGNSDTTYN
LKFKGKAKLTAVTSANTVYMEVSSLTNEDSAVYYCTKRHDYGAMDYWGQGTSVTVSS
SEQ ID NO: 92-muFR1-57vL
DIVLTQSPATLSVTPGDSVSLSCRASQNINNNLHWYQQKSHESPRLLIKYVSQSVSGIPSRFSGS
GSGTDFTLSINSVETEDFGMYFCQQSNSWPHYTFGGGTKLEIKR
SEQ ID NO: 93-muFR1-57vH
DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSSTISYADT
VKGRFTISRDNSKKTLLLQMTSLRSEDTAMYYCAREAYGSSMEYWGQGTSVTVSS
SEQ ID NO: 94-muFR1-65vL
DIVIVITQSQKFMSTSVGDRVSVTCKASQNVGPNVAWYQQKPGQSPKALIYSASYRYSEVPDRF
TGSGSGTDFTLTISNMQSADLAEYFCQQYNSYPYTFGGGTKLEIKR
SEQ ID NO: 95-muFR1-65vH
QVQLQQSGAELARPGASVKMSCKASGYTFTSYTMHWVKQRPGQGLAWIGYINPISGYTNYN
QKFKDKATLTADKSSSTAYMQLNSLTSEDSAVYYCASGGAYGRKPMDYWGQGTSVTVSS
SEQ ID NO: 96-huFR1-48vL
DIQMTQSPSSLSVSVGERVTITCRASENIYSNLAWYQQKPGKSPKLLVYAATNLADGVPSRFS
GSESGTDYSLKINSLQPEDFGSYYCQHFWASPYTFGQGTKLEIKR
SEQ ID NO: 97-huFR1-48vH
QVQLVQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQRPGQGLEWIGAIYPGNGDSRYT
QKFQGKATLTADKSSSTAYMQVSSLTSEDSAVYYCARRDGNYAAYWGQGTLVTVSA
SEQ ID NO: 98-huFR1-49vL
DIQMTQSPSSLSVSVGERVTITCRASENIYTNLAWYQQKPGKSPKLLVYTASNLADGVPSRFSG
SGSGTDYSLKINSLQPEDFGTYYCQHFWVSPYTFGQGTKLEIKR
SEQ ID NO: 99-huFR1-49vH
QVQLQQSGAVVAKPGASVKMSCKASGYTFTNYWMYWIKQRPGQGLELIGAIYPGNSDTTYN
QKFQGKATLTAVTSANTVYMEVSSLTSEDSAVYYCTKRHDYGAMDYWGQGTSVTVSS
SEQ ID NO: 100-huFR1-57vL
EIVLTQSPATLSVTPGDRVSLSCRASQNINNNLHWYQQKPGQSPRLLIKYVSQSVSGIPDRFSGS
GSGTDFTLSISSVEPEDFGMYFCQQSNSWPHYTFGQGTKLEIKR
SEQ ID NO: 101-huFR1-57vH
EVQLVESGGGLVQPGGSRRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSSTISYADS
VKGRFTISRDNSKKTLLLQMTSLRAEDTAMYYCAREAYGSSMEYWGQGTLVTVSS
SEQ ID NO: 102-huFR1-65vL
EIVIVITQSPATMSTSPGDRVSVTCKASQNVGPNVAWYQQKPGQSPRALIYSASYRYSGVPARF
TGSGSGTDFTLTISNMQSEDLAEYFCQQYNSYPYTFGQGTKLEIKR
SEQ ID NO: 103-huFR1-65vH
QVQLVQSGAEVAKPGASVKMSCKASGYTFTSYTMHWVKQRPGQGLAWIGYINPISGYTNYN
QKFQGKATLTADKSSSTAYMQLNSLTSEDSAVYYCASGGAYGRKPMDYWGQGTSVTVSS
SEQ ID NO: 104-muFR1-48LC
DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYAATNLADGVPSRFS
GSESGTQYSLKINSLQSEDEGSYYCQHFWASPYTEGGGTKLEIKRADAAPTVSIFPPSSEQLTSG
GASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHN
SYTCEATHKTSTSPIVKSFNRNEC
SEQ ID NO: 105-muFR1-48HC
QVQLQQSGAELARPGASVKLSCRASGYTFTNYWMQWIKQRPGQGLEWIGAIYPGNGDSRYT
QKFKGKATLTADKSSSTAYMQVSSLTSEDSAVYYCARRDGNYAAYWGQGTLVTVSAAKTTP
PSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSV
TVPSSMRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTP
KVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTERSVSELPIMHQDWLNGKEF
KCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQW
NGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSP
GK
SEQ ID NO: 106-muFR1-49LC
DIQMTQSPASLSVSVGETVTITCRASENIYTNLAWYQQKQGKSPQLLVYTASNLADGVPSRFS
GSGSGTQYSLKINSLQSEDFGTYYCQHFWVSPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTS
GGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERH
NSYTCEATHKTSTSPIVKSFN
RNEC
SEQ ID NO: 107-muFR1-49HC
EVQLQQSGTVLARPGASVKMSCKASGYKFTNYWMYWIKQRPGQGLELIGAIYPGNSDTTYN
LKFKGKAKLTAVTSANTVYMEVSSLTNEDSAVYYCTKRHDYGAMDYWGQGTSVTVSSAKT
TAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSS
VTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDV
LMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDW
MSGKEEKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDEMPE
DIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHEI
TTKSFSRTPGK
SEQ ID NO: 108-muFR1-57LC
DIVLTQSPATLSVTPGDSVSLSCRASQNINNNLHWYQQKSHESPRLLIKYVSQSVSGIPSRFSGS
GSGTDFTLSINSVETEDFGMYFCQQSNSWPHYTFGGGTKLEIKRADAAPTVSTEPPSSEQLTSG
GASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHN
SYTCEATHKTSTSPIVKSF
NRNEC
SEQ ID NO: 109-muFR1-57HC
DVQLVESGGGLVQPGGSRKLSCAASGETESSEGMHWVRQAPEKGLEWVAYISSGSSTISYADT
VKGRETISRDNSKKTLLLQMTSLRSEDTAMYYCAREAYGSSMEYWGQGTSVTVSSAKTTAPS
VYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTV
TSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIEPPKIKDVLMI
SLSPIVTCVVVDVSEDDPDVQISWEVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMS
GKEEKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDEMPEDIY
VEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTT
KSFSRTPGK
SEQ ID NO: 110-muER1-65LC
DIVIVITQSQKEMSTSVGDRVSVTCKASQNVGPNVAWYQQKPGQSPKALIYSASYRYSEVPDRE
TGSGSGTDETLTISNMQSADLAEYECQQYNSYPYTEGGGTKLEIKRADAAPTVSIEPPSSEQLTS
GGASVVCELNNEYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERH
NSYTCEATHKTSTSPIVKSFNRNEC
SEQ ID NO: 111-muER1-65HC
QVQLQQSGAELARPGASVKMSCKASGYTFTSYTMHWVKQRPGQGLAWIGYINPISGYTNYN
QKFKDKATLTADKSSSTAYMQLNSLTSEDSAVYYCASGGAYGRKPMDYWGQGTSVTVSSAK
TTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLS
SSVTVPSSMRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIEPPKPKDVLTI
TLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLN
GKEEKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDEEPEDITVE
WQWNGQPAENYKNTQPIMNTNGSYEVYSKLNVQKSNWEAGNTETCSVLHEGLHNHHTEKS
LSHSPGK
SEQ ID NO: 112-huER1-48LC
DIQMTQSPSSLSVSVGERVTITCRASENIYSNLAWYQQKPGKSPKLLVYAATNLADGVPSRFS
GSESGTDYSLKINSLQPEDEGSYYCQHFWASPYTEGQGTKLEIKRTVAAPSVFIEPPSDEQLKSG
TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK
VYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 113-huER1-48HC
QVQLVQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQRPGQGLEWIGAIYPGNGDSRYT
QKFQGKATLTADKSSSTAYMQVSSLTSEDSAVYYCARRDGNYAAYWGQGTLVTVSAASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVELEPPKPKDT
LMISRTPEVTCVVVDVSHEDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVESCSVMHEALHNHY
TQKSLSLSPG
SEQ ID NO: 114-huER1-49LC
DIQMTQSPSSLSVSVGERVTITCRASENIYTNLAWYQQKPGKSPKLLVYTASNLADGVPSRESG
SGSGTDYSLKINSLQPEDEGTYYCQHFWVSPYTEGQGTKLEIKRTVAAPSVFIEPPSDEQLKSG
TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK
VYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 115-huFR1-49HC
QVQLQQSGAVVAKPGASVKMSCKASGYTFTNYWMYWIKQRPGQGLELIGAIYPGNSDTTYN
QKFQGKATLTAVTSANTVYMEVSSLTSEDSAVYYCTKRHDYGAMDYWGQGTSVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH
YTQKSLSLSPG
SEQ ID NO: 116-huFR1-57LC
EIVLTQSPATLSVTPGDRVSLSCRASQNINNNLHWYQQKPGQSPRLLIKYVSQSVSGIPDRFSGS
GSGTDFTLSISSVEPEDFGMYFCQQSNSWPHYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGT
ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
YACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 117-huFR1-57HC
EVQLVESGGGLVQPGGSRRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSSTISYADS
VKGRFTISRDNSKKTLLLQMTSLRAEDTAMYYCAREAYGSSMEYWGQGTLVTVSSASTKGPS
VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT
VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
SEQ ID NO: 118-huFR1-65LC
EIVMTQSPATMSTSPGDRVSVTCKASQNVGPNVAWYQQKPGQSPRALIYSASYRYSGVPARF
TGSGSGTDFTLTISNMQSEDLAEYFCQQYNSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS
GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
KVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 119-huFR1-65HC
QVQLVQSGAEVAKPGASVKMSCKASGYTFTSYTMHWVKQRPGQGLAWIGYINPISGYTNYN
QKFQGKATLTADKSSSTAYMQLNSLTSEDSAVYYCASGGAYGRKPMDYWGQGTSVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV
LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPG
SEQ ID NO: 120-huFR1-48_VL
gaattcgccaccatgggatggagttgtatcatcctgtttcttgtggctacagccacaggggtacactccgatat-
tcaaatgacacagtccccttcatccct
gtccgtcagtgtgggggaaagggttaccatcacctgccgtgcatcagagaacatctattccaacctcgcctggt-
accaacagaaacctggcaagtcc
cctaagctgttggtctacgccgctacaaacctcgccgatggggtgccttcccgtttcagtgggtcagagtcagg-
caccgactattctctgaagatcaac
tccctccagcctgaggatttcggctcctattactgtcagcacttctgggctagtccatatactttcggccaggg-
aaccaaacttgaaattaaacgtacg
SEQ ID NO: 121-huFR1-48_VH
aagcttgccaccatggggtggagctgcatcatcctttttctggtggccactgccaccggcgtgcactctcaggt-
ccaacttgtgcagagcggagccg
aggtggccaaacccggagctagtgttaagctctcatgtaaagcatctggctacacctttactaactactggatg-
cagtggatcaagcaacggccagg
ccagggcctggagtggattggtgctatttatcccggaaacggggatagcaggtacactcagaaatttcagggaa-
aggctacccttaccgccgataag
agttcttccacagcatatatgcaagtctcctctctgacctcagaggatagtgctgtctattactgcgctcgccg-
ggatggcaactatgcagcctattggg
gtcaaggcacccttgtgactgtatccgcagcaagcaccaagggccc
SEQ ID NO: 122-huFR1-49_VL
gaattcgccaccatgggttggtcatgcattatcctgtttctggtcgcaacagcaacaggtgtgcacagtgacat-
tcagatgacccaaagcccctccagt
ctgagcgtttccgtgggggaacgtgtcactatcacatgcagagcttccgagaatatttacactaacctcgcatg-
gtaccagcagaaacccgggaagt
ctccaaaacttctcgtatatacagccagcaacttggcagatggggtgcccagccggtttagcggatctggttca-
ggcaccgactattctttgaaaattaa
ttccctgcagcctgaggattttggtacctactattgccagcatttttgggtatcaccatacacttttggacagg-
gaacaaagctggagatcaagcgtacg
SEQ ID NO: 123-huFR1-49_VH
aagcttgccaccatgggctggtatgtattattctttttatgtggccacagccacaggagtccattcacaggtac-
agctccaacagtctggcgcagttgt
cgccaagcccggcgcctctgtgaagatgagttgcaaggcctctggctacaccttcactaattattggatgtact-
ggatcaaacaacgccccggccag
ggtctggaactcattggagccatctacccaggcaactccgacacaacatacaatcagaagtttcagggcaaagc-
aaccctgaccgctgtaacctcag
ctaataccgtgtacatggaggtaagtagcttgactagtgaagattccgcagtatactattgcaccaagcgccat-
gattacggcgccatggattactggg
gccaaggtaccagtgtgaccgtgtcttccgcttccaccaagggccc
SEQ ID NO: 124-huFR1-57_VL
gaattcgccaccatgggctggtcatgcattattttgttcctggtcgccaccgcaaccggcgttcattccgaaat-
tgttcttactcagagccctgcaacctt
gagtgtgacacccggcgatcgggtctcactgagttgcagagcttcccagaatatcaacaataatctgcactggt-
atcagcagaagcctggccagtct
cctcgcttgctgattaagtatgtctcacagagcgtgtcaggtatccctgaccgtttctccgggtcaggttcagg-
caccgacttcacactgtccatttctag
cgtggagcctgaggatttcggaatgtacttttgccagcagagcaatagctggcctcactacacctttggccaag-
ggaccaagctggagatcaagcgt
acg
SEQ ID NO: 125-huFR1-57_VH
aagcttgccaccatgggctggagctgtatcatcttgttccttgtggccacagctactggcgtgcactccgaggt-
gcagctggtcgaatccggcggag
gcctggtgcagcctggggggagtagacggctgtcctgcgctgcctctgggtttactttctcaagtttcggtatg-
cactgggtgcgtcaggcccccggg
aagggcctggaatgggttgcttatatatcatctggcagctccaccatttcttatgctgattccgttaagggacg-
cttcaccatttccagagacaacagtaa
gaaaacccttctgctgcagatgacctctctccgcgccgaagacaccgcaatgtattattgtgctagagaggcct-
acggcagtagtatggaatactggg
ggcaggggaccctggtgaccgtgtcttccgcatctactaagggccc
SEQ ID NO: 126-huFR1-65_VL
gaattcgccaccatgggctggtcttgcattattctgttcctggttgcaacagccactggcgtccattccgaaat-
cgtgatgacccaatctcccgccacca
tgtctacctctcccggggaccgggtgtctgtgacctgcaaggcctctcagaatgttggcccaaacgtggcatgg-
tatcaacagaaaccagggcagtc
acccagagccctgatttactccgcttcttacagatattcaggagttcccgcccggttcacaggtagtgggtccg-
gcactgactttaccttgaccatttcca
acatgcaatccgaggacctggccgaatacttctgtcagcagtacaattcatatccctatacattcggccagggg-
accaagctggaaataaagcgtac
g
SEQ ID NO: 127-huFR1-65_VH
Aagcttgccaccatgggctggtcatgcataatcctgttcctggtcgcaaccgctacaggtgtacactcccaggt-
gcagttggtgcagagcggggcc
gaagttgctaagcccggtgcaagtgtaaaaatgtcctgcaaagctagcgggtacacattcacatcctatactat-
gcattgggtaaaacagcgcccagg
acaggggctcgcctggataggctatattaacccaatatcaggatacacaaactacaatcagaaatttcagggaa-
aggcaaccctgaccgccgacaa
gtcctcttctaccgcatatatgcagctcaactccctgaccagtgaagatagcgcagtgtattactgtgcctccg-
gcggtgcttatggccggaaacccat
ggattactggggacaaggcacctccgtcacagtgagtagcgcctcaaccaagggccc
SEQ ID NO: 128-Kabat Defined Mov19 HC CDR2 Murine
RIHPYDGDTFYNQNFKD
SEQ ID NO: 129-Kabat Defined Mov19 HC CDR2 Human
RIHPYDGDTFYNQKFQG
Sequence CWU
1
1
13115PRTArtificial sequencehuMov19 vHC CDR1 1Gly Tyr Phe Met Asn1
5217PRTArtificial sequencehuMov19 vHC CDR2 2Arg Ile His Pro Tyr Asp
Gly Asp Thr Phe Tyr Asn Gln Lys Phe Gln1 5
10 15Gly39PRTArtificial sequencehuMov19 vHC CDR3 3Tyr
Asp Gly Ser Arg Ala Met Asp Tyr1 54118PRTArtificial
sequencehuMov19 vHC 4Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys
Pro Gly Ala1 5 10 15Ser
Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20
25 30Phe Met Asn Trp Val Lys Gln Ser
Pro Gly Gln Ser Leu Glu Trp Ile 35 40
45Gly Arg Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gln Lys Phe
50 55 60Gln Gly Lys Ala Thr Leu Thr Val
Asp Lys Ser Ser Asn Thr Ala His65 70 75
80Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Phe Ala Val
Tyr Tyr Cys 85 90 95Thr
Arg Tyr Asp Gly Ser Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110Thr Val Thr Val Ser Ser
1155440DNAArtificial sequencehuMov19 vHC nucleic acid sequence
5aagcttgcca ccatgggttg gtcatgcatc atcctcttct tggttgcaac tgctaccgga
60gtgcacagtc aggtacagct cgtgcagtcc ggcgccgagg tggtgaagcc tggtgccagc
120gtgaagatct cctgtaaagc cagtggatac acattcaccg gttattttat gaattgggtg
180aaacagagcc caggccaatc cctcgaatgg atagggcgaa tccacccata tgacggggac
240accttttaca accagaaatt ccaggggaaa gccactctga cagtggacaa gagttccaac
300actgcacaca tggagcttct ctccctgacc agcgaagact tcgctgttta ttactgtacc
360cgttatgatg gttcccgtgc aatggactac tggggccaag ggaccactgt caccgtaagt
420tccgccagca ccaagggccc
4406448PRTArtificial sequencehuMov19 HC amino acid sequence 6Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala1 5
10 15Ser Val Lys Ile Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Gly Tyr 20 25
30Phe Met Asn Trp Val Lys Gln Ser Pro Gly Gln Ser Leu Glu Trp Ile
35 40 45Gly Arg Ile His Pro Tyr Asp
Gly Asp Thr Phe Tyr Asn Gln Lys Phe 50 55
60Gln Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala His65
70 75 80Met Glu Leu Leu
Ser Leu Thr Ser Glu Asp Phe Ala Val Tyr Tyr Cys 85
90 95Thr Arg Tyr Asp Gly Ser Arg Ala Met Asp
Tyr Trp Gly Gln Gly Thr 100 105
110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135
140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn145 150 155 160Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185
190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 275 280 285Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340
345 350Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys 355 360 365Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370
375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp385 390 395
400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser 405 410 415Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420
425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 435 440
445715PRTArtificial sequencehuMov19 vLC CDR1 7Lys Ala Ser Gln Ser Val Ser
Phe Ala Gly Thr Ser Leu Met His1 5 10
1587PRTArtificial sequencehuMov19 vLC CDR2 8Arg Ala Ser Asn
Leu Glu Ala1 599PRTArtificial sequencehuMov19 vLC CDR3 9Gln
Gln Ser Arg Glu Tyr Pro Tyr Thr1 510112PRTArtificial
sequencehuMov19 vLCv1.00 10Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu
Ala Val Ser Leu Gly1 5 10
15Gln Pro Ala Ile Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Phe Ala
20 25 30Gly Thr Ser Leu Met His Trp
Tyr His Gln Lys Pro Gly Gln Gln Pro 35 40
45Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro
Asp 50 55 60Arg Phe Ser Gly Ser Gly
Ser Lys Thr Asp Phe Thr Leu Asn Ile Ser65 70
75 80Pro Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr
Cys Gln Gln Ser Arg 85 90
95Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 11011112PRTArtificial
sequencehuMov19 vLCv1.60 11Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu
Ala Val Ser Leu Gly1 5 10
15Gln Pro Ala Ile Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Phe Ala
20 25 30Gly Thr Ser Leu Met His Trp
Tyr His Gln Lys Pro Gly Gln Gln Pro 35 40
45Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro
Asp 50 55 60Arg Phe Ser Gly Ser Gly
Ser Lys Thr Asp Phe Thr Leu Thr Ile Ser65 70
75 80Pro Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr
Cys Gln Gln Ser Arg 85 90
95Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 11012218PRTArtificial
sequencehuMov19 LCv1.00 12Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Ala
Val Ser Leu Gly1 5 10
15Gln Pro Ala Ile Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Phe Ala
20 25 30Gly Thr Ser Leu Met His Trp
Tyr His Gln Lys Pro Gly Gln Gln Pro 35 40
45Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro
Asp 50 55 60Arg Phe Ser Gly Ser Gly
Ser Lys Thr Asp Phe Thr Leu Asn Ile Ser65 70
75 80Pro Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr
Cys Gln Gln Ser Arg 85 90
95Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120
125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr 130 135 140Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser145 150
155 160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr 165 170
175Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195
200 205Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
21513218PRTArtificial sequencehuMov19 LCv1.60 13Asp Ile Val
Leu Thr Gln Ser Pro Leu Ser Leu Ala Val Ser Leu Gly1 5
10 15Gln Pro Ala Ile Ile Ser Cys Lys Ala
Ser Gln Ser Val Ser Phe Ala 20 25
30Gly Thr Ser Leu Met His Trp Tyr His Gln Lys Pro Gly Gln Gln Pro
35 40 45Arg Leu Leu Ile Tyr Arg Ala
Ser Asn Leu Glu Ala Gly Val Pro Asp 50 55
60Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Thr Ile Ser65
70 75 80Pro Val Glu Ala
Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Arg 85
90 95Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys Arg 100 105
110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135
140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser145 150 155 160Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185
190His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro 195 200 205Val Thr Lys Ser
Phe Asn Arg Gly Glu Cys 210 21514408DNAArtificial
sequencehuMov19 LCv1.00 nucleic acid 14gaattcgcca ccatgggctg gagctgcatt
atcctttttc tggtagccac agctacaggc 60gtgcatagcg atatcgtgct gacacaatcc
cccctctctc tggccgtgtc actcggacag 120cccgctatca tcagctgcaa agccagccag
tctgtcagct tcgctggaac aagtcttatg 180cattggtatc atcagaagcc tggccagcaa
cccaggctgc tgatctatcg agcctcaaac 240ttggaagcag gagtgccaga ccggttttct
gggtccggga gtaaaaccga ttttacactt 300aatatctcac ctgtcgaggc cgaggacgcc
gccacctact actgtcagca gagccgagag 360tacccttaca cttttggcgg tgggactaaa
ctggaaataa aacgtacg 40815408DNAArtificial sequencehuMov19
LCv1.60 nucleic acid 15gaattcgcca ccatgggctg gtcttgtatc atcctgtttc
tggtggccac cgcaaccggt 60gttcactccg acattgtgct gacacagtcc cccctttcac
tggctgtatc cctcggccag 120cccgctatca tcagctgcaa ggctagccag agcgtgagtt
ttgccggcac ttcacttatg 180cattggtacc atcagaaacc aggccagcaa cctaggctgc
tgatttatcg ggctagcaac 240ctggaggccg gcgtgcccga ccgctttagc gggagcggct
ccaagactga cttcactctg 300accatctccc ccgtagaagc agaagatgct gcaacctact
actgtcagca gtctcgcgag 360tatccttata cattcggagg cggaactaaa ctggagatta
aacgtacg 4081617PRTArtificial sequencemuMov19 vHC CDR2
16Arg Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gln Asn Phe Lys1
5 10 15Asp17117PRTArtificial
sequencemuMov19 vHC_CAA68252 17Gln Val Gln Leu Gln Gln Ser Gly Ala Glu
Leu Val Lys Pro Gly Ala1 5 10
15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30Phe Met Asn Trp Val Lys
Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40
45Gly Arg Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gln
Asn Phe 50 55 60Lys Asp Lys Ala Thr
Leu Thr Val Asp Lys Ser Ser Asn Thr Ala His65 70
75 80Met Glu Leu Leu Ser Leu Thr Ser Glu Asp
Phe Ala Val Tyr Tyr Cys 85 90
95Thr Arg Tyr Asp Gly Ser Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110Thr Val Thr Val Ser
11518108PRTArtificial sequencemuMov19 vLC_CAA68253 18Asp Ile Glu Leu
Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5
10 15Gln Arg Ala Ile Ile Ser Cys Lys Ala Ser
Gln Ser Val Ser Phe Ala 20 25
30Gly Thr Ser Leu Met His Trp Tyr His Gln Lys Pro Gly Gln Gln Pro
35 40 45Lys Leu Leu Ile Tyr Arg Ala Ser
Asn Leu Glu Ala Gly Val Pro Thr 50 55
60Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Asn Ile His65
70 75 80Pro Val Glu Glu Glu
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Arg 85
90 95Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys
Leu 100 10519448PRTArtificial sequencechMov19
HC 19Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala1
5 10 15Ser Val Lys Ile Ser
Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20
25 30Phe Met Asn Trp Val Lys Gln Ser His Gly Lys Ser
Leu Glu Trp Ile 35 40 45Gly Arg
Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gln Asn Phe 50
55 60Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser
Ser Asn Thr Ala His65 70 75
80Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Phe Ala Val Tyr Tyr Cys
85 90 95Thr Arg Tyr Asp Gly
Ser Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr 100
105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130
135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn145 150 155
160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180
185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser 195 200 205Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg 245 250 255Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala 275 280
285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr305 310
315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 325 330
335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360
365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser 370 375 380Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390
395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser 405 410
415Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435
440 44520218PRTArtificial sequencechMov19 LC 20Asp Ile
Glu Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5
10 15Gln Arg Ala Ile Ile Ser Cys Lys
Ala Ser Gln Ser Val Ser Phe Ala 20 25
30Gly Thr Ser Leu Met His Trp Tyr His Gln Lys Pro Gly Gln Gln
Pro 35 40 45Lys Leu Leu Ile Tyr
Arg Ala Ser Asn Leu Glu Ala Gly Val Pro Thr 50 55
60Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Asn
Ile His65 70 75 80Pro
Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Arg
85 90 95Glu Tyr Pro Tyr Thr Phe Gly
Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105
110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln 115 120 125Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130
135 140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser145 150 155
160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180
185 190His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro 195 200 205Val Thr
Lys Ser Phe Asn Arg Gly Glu Cys 210
21521441DNAArtificial sequencechMov19 HC nucleic acid 21aagcttgcca
ccatgggttg gtcttgtatt atcctctttc tcgtcgcaac cgcaacaggc 60gtccattcac
aagtccaact gcagcaatcc ggcgccgaac tcgttaaacc tggagcatct 120gttaaaatct
catgtaaagc atcaggatac tcatttactg gctattttat gaactgggtc 180aaacaatcac
acggaaaatc acttgaatgg atcggacgta ttcaccccta tgatggcgat 240actttttaca
accagaactt caaagacaaa gctacactca ccgttgacaa atcatctaac 300accgctcaca
tggaactcct ttcactcaca tctgaagact tcgctgttta ttactgtact 360agatacgatg
gatcaagagc tatggattat tggggacaag gaacaacagt cacagtctca 420tctgcatcaa
ctaagggccc a
44122408DNAArtificial sequencechMov19 LC nucleic acid 22gaattcgcca
ccatgggttg gtcttgtatt atcctctttc tcgtcgcaac cgcaacaggc 60gtccattcag
atatcgaact cacacaatca ccagcttccc tcgcagtctc tctcggtcaa 120cgcgcaatca
tctcttgtaa agcctcccaa tcagtctcat tcgccggcac gtccctcatg 180cattggtacc
atcaaaaacc cggtcagcaa cccaaactcc ttatctatag agcaagcaac 240ctcgaagcag
gcgttcccac cagatttagc ggatcaggaa gtaaaaccga tttcacactc 300aacattcatc
cagtcgaaga agaagatgca gctacttatt attgccaaca gtctagagaa 360tatccataca
cattcggagg gggtaccaaa cttgaaatta aacgtacg
40823117PRTArtificial sequencemuMov19 vHC_CAA68252 23Gln Val Gln Leu Gln
Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala1 5
10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr
Ser Phe Thr Gly Tyr 20 25
30Phe Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45Gly Arg Ile His Pro Tyr Asp Gly
Asp Thr Phe Tyr Asn Gln Asn Phe 50 55
60Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala His65
70 75 80Met Glu Leu Leu Ser
Leu Thr Ser Glu Asp Phe Ala Val Tyr Tyr Cys 85
90 95Thr Arg Tyr Asp Gly Ser Arg Ala Met Asp Tyr
Trp Gly Gln Gly Thr 100 105
110Thr Val Thr Val Ser 11524108PRTArtificial sequencemuMov19
vLC_CAA68253 24Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser
Leu Gly1 5 10 15Gln Arg
Ala Ile Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Phe Ala 20
25 30Gly Thr Ser Leu Met His Trp Tyr His
Gln Lys Pro Gly Gln Gln Pro 35 40
45Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro Thr 50
55 60Arg Phe Ser Gly Ser Gly Ser Lys Thr
Asp Phe Thr Leu Asn Ile His65 70 75
80Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln
Ser Arg 85 90 95Glu Tyr
Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu 100
10525257PRTHomo sapiensMISC_FEATURE(1)..(257) 25Met Ala Gln Arg Met Thr
Thr Gln Leu Leu Leu Leu Leu Val Trp Val1 5
10 15Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp
Ala Arg Thr Glu 20 25 30Leu
Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly 35
40 45Pro Glu Asp Lys Leu His Glu Gln Cys
Arg Pro Trp Arg Lys Asn Ala 50 55
60Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr65
70 75 80Leu Tyr Arg Phe Asn
Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys 85
90 95Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr
Glu Cys Ser Pro Asn 100 105
110Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg
115 120 125Val Leu Asn Val Pro Leu Cys
Lys Glu Asp Cys Glu Gln Trp Trp Glu 130 135
140Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly
Trp145 150 155 160Asn Trp
Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln
165 170 175Pro Phe His Phe Tyr Phe Pro
Thr Pro Thr Val Leu Cys Asn Glu Ile 180 185
190Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser
Gly Arg 195 200 205Cys Ile Gln Met
Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu 210
215 220Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala
Gly Pro Trp Ala225 230 235
240Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu
245 250 255Ser26771DNAHomo
sapiensmisc_feature(1)..(771) 26atggctcagc ggatgacaac acagctgctg
ctccttctag tgtgggtggc tgtagtaggg 60gaggctcaga caaggattgc atgggccagg
actgagcttc tcaatgtctg catgaacgcc 120aagcaccaca aggaaaagcc aggccccgag
gacaagttgc atgagcagtg tcgaccctgg 180aggaagaatg cctgctgttc taccaacacc
agccaggaag cccataagga tgtttcctac 240ctatatagat tcaactggaa ccactgtgga
gagatggcac ctgcctgcaa acggcatttc 300atccaggaca cctgcctcta cgagtgctcc
cccaacttgg ggccctggat ccagcaggtg 360gatcagagct ggcgcaaaga gcgggtactg
aacgtgcccc tgtgcaaaga ggactgtgag 420caatggtggg aagattgtcg cacctcctac
acctgcaaga gcaactggca caagggctgg 480aactggactt cagggtttaa caagtgcgca
gtgggagctg cctgccaacc tttccatttc 540tacttcccca cacccactgt tctgtgcaat
gaaatctgga ctcactccta caaggtcagc 600aactacagcc gagggagtgg ccgctgcatc
cagatgtggt tcgacccagc ccagggcaac 660cccaatgagg aggtggcgag gttctatgct
gcagccatga gtggggctgg gccctgggca 720gcctggcctt tcctgcttag cctggcccta
atgctgctgt ggctgctcag c 7712711PRTArtificial sequenceFR1-21
vLC CDR1 27Lys Ala Ser Asp His Ile Asn Asn Trp Leu Ala1 5
10287PRTArtificial sequenceFR1-21 vLC CDR2 28Gly Ala Thr
Ser Leu Glu Thr1 5299PRTArtificial sequenceFR1-21 vLC CDR3
29Gln Gln Tyr Trp Ser Thr Pro Phe Thr1 5306PRTArtificial
sequenceFR1-21 vHC CDR1 30Ser Ser Tyr Gly Met Ser1
53110PRTArtificial sequenceFR1-21 vHC CDR2 31Thr Ile Ser Ser Gly Gly Ser
Tyr Thr Tyr1 5 103211PRTArtificial
sequenceFR1-21 vHC CDR3 32Asp Gly Glu Gly Gly Leu Tyr Ala Met Asp Tyr1
5 103317PRTArtificial sequenceFR1-21 Kabat
murine CDR-H2 33Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Gly
Val Lys1 5 10
15Gly3417PRTArtificial sequenceFR1-21 Kabat human CDR-H2 34Thr Ile Ser
Ser Gly Gly Ser Tyr Thr Tyr Tyr Ser Pro Gly Phe Gln1 5
10 15Gly35108PRTArtificial sequencemuFR1-21
vLC 35Asp Ile Gln Met Thr Gln Ser Ser Ser Tyr Leu Ser Val Ser Leu Gly1
5 10 15Gly Arg Val Thr Ile
Thr Cys Lys Ala Ser Asp His Ile Asn Asn Trp 20
25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro
Arg Leu Leu Ile 35 40 45Ser Gly
Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50
55 60Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile
Ser Ser Leu Gln Thr65 70 75
80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Phe
85 90 95Thr Phe Gly Ser Gly
Thr Lys Leu Glu Ile Lys Arg 100
10536120PRTArtificial sequencemuFR1-21 vHC 36Glu Val Lys Leu Val Glu Ser
Gly Gly Asp Leu Val Lys Pro Gly Gly1 5 10
15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Ser Tyr 20 25 30Gly Met
Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Cys Val 35
40 45Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr
Tyr Tyr Pro Asp Gly Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Ser Ser Leu
Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85
90 95Ala Arg Asp Gly Glu Gly Gly Leu Tyr Ala Met Asp
Tyr Trp Gly Gln 100 105 110Gly
Thr Ser Val Thr Val Ser Ser 115
12037323DNAArtificial sequencemuFR1-21 vLC DNA sequence 37gacatccaga
tgacacaatc ttcatcctac ttgtctgtat ctctaggagg cagagtcacc 60attacttgca
aggcaagtga ccacataaat aattggttag cctggtatca gcagaaacca 120ggaaatgctc
ctaggctctt aatatctggt gcaaccagtt tggaaactgg ggttccttca 180agattcagtg
gcagtggatc tggaaaggat tacactctca gcatttccag tcttcagact 240gaagatgttg
ctacttatta ctgtcaacag tattggagta ctccattcac gttcggctcg 300gggacaaagt
tggaaataaa acg
32338360DNAArtificial sequencemuFR1-21HCvarPat 38gaagtgaagc tggtggagtc
tgggggagac ttagtgaagc ctggagggtc cctgaaactc 60tcctgtgcag cctctggatt
cactttcagt agctatggca tgtcttgggt tcgccagact 120ccagacaaga ggttggagtg
tgtcgcaacc attagtagtg gtggtagtta cacctactat 180ccagacggtg tgaaggggcg
attcaccatc tccagagaca atgccaagaa caccctgtac 240ctgcaaatga gcagtctgaa
gtctgaggac acagccatgt attactgtgc aagggacggc 300gaggggggcc tctatgctat
ggactactgg ggtcaaggaa cctcagtcac cgtctcctca 36039214PRTArtificial
sequencemuFR1-21 LC 39Asp Ile Gln Met Thr Gln Ser Ser Ser Tyr Leu Ser Val
Ser Leu Gly1 5 10 15Gly
Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Ile Asn Asn Trp 20
25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Asn Ala Pro Arg Leu Leu Ile 35 40
45Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Lys Asp Tyr Thr
Leu Ser Ile Ser Ser Leu Gln Thr65 70 75
80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser
Thr Pro Phe 85 90 95Thr
Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala
100 105 110Pro Thr Val Ser Ile Phe Pro
Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120
125Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp
Ile 130 135 140Asn Val Lys Trp Lys Ile
Asp Gly Ser Glu Arg Gln Asn Gly Val Leu145 150
155 160Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Met Ser 165 170
175Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
180 185 190Thr Cys Glu Ala Thr His
Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200
205Phe Asn Arg Asn Glu Cys 21040456PRTArtificial
sequencemuFR1-21 HC 40Glu Val Lys Leu Val Glu Ser Gly Gly Asp Leu Val Lys
Pro Gly Gly1 5 10 15Ser
Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20
25 30Gly Met Ser Trp Val Arg Gln Thr
Pro Asp Lys Arg Leu Glu Cys Val 35 40
45Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Gly Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met
Tyr Tyr Cys 85 90 95Ala
Arg Asp Gly Glu Gly Gly Leu Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110Gly Thr Ser Val Thr Val Ser
Ser Ala Lys Thr Thr Pro Pro Ser Val 115 120
125Tyr Pro Leu Ala Pro Gly Cys Gly Asp Thr Thr Gly Ser Ser Val
Thr 130 135 140Leu Gly Cys Leu Val Lys
Gly Tyr Phe Pro Glu Ser Val Thr Val Thr145 150
155 160Trp Asn Ser Gly Ser Leu Ser Ser Ser Val His
Thr Phe Pro Ala Leu 165 170
175Leu Gln Ser Gly Leu Tyr Thr Met Ser Ser Ser Val Thr Val Pro Ser
180 185 190Ser Thr Trp Pro Ser Gln
Thr Val Thr Cys Ser Val Ala His Pro Ala 195 200
205Ser Ser Thr Thr Val Asp Lys Lys Leu Glu Pro Ser Gly Pro
Ile Ser 210 215 220Thr Ile Asn Pro Cys
Pro Pro Cys Lys Glu Cys His Lys Cys Pro Ala225 230
235 240Pro Asn Leu Glu Gly Gly Pro Ser Val Phe
Ile Phe Pro Pro Asn Ile 245 250
255Lys Asp Val Leu Met Ile Ser Leu Thr Pro Lys Val Thr Cys Val Val
260 265 270Val Asp Val Ser Glu
Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val 275
280 285Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr
His Arg Glu Asp 290 295 300Tyr Asn Ser
Thr Ile Arg Val Val Ser Thr Leu Pro Ile Gln His Gln305
310 315 320Asp Trp Met Ser Gly Lys Glu
Phe Lys Cys Lys Val Asn Asn Lys Asp 325
330 335Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ile
Lys Gly Leu Val 340 345 350Arg
Ala Pro Gln Val Tyr Ile Leu Pro Pro Pro Ala Glu Gln Leu Ser 355
360 365Arg Lys Asp Val Ser Leu Thr Cys Leu
Val Val Gly Phe Asn Pro Gly 370 375
380Asp Ile Ser Val Glu Trp Thr Ser Asn Gly His Thr Glu Glu Asn Tyr385
390 395 400Lys Asp Thr Ala
Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Ile Tyr 405
410 415Ser Lys Leu Asn Met Lys Thr Ser Lys Trp
Glu Lys Thr Asp Ser Phe 420 425
430Ser Cys Asn Val Arg His Glu Gly Leu Lys Asn Tyr Tyr Leu Lys Lys
435 440 445Thr Ile Ser Arg Ser Pro Gly
Lys 450 45541108PRTArtificial sequencehuFR1-21 vLC
41Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Leu Ser Val Ser Val Gly1
5 10 15Gly Arg Val Thr Ile Thr
Cys Lys Ala Ser Asp His Ile Asn Asn Trp 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45Ser Gly Ala
Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50
55 60Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Ser
Ser Leu Gln Pro65 70 75
80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Phe
85 90 95Thr Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys Arg 100
10542120PRTArtificial sequencehuFR1-21 vHC 42Glu Val Gln Leu Val Glu Ser
Gly Gly Asp Val Val Lys Pro Gly Gly1 5 10
15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Ser Tyr 20 25 30Gly Met
Ser Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Cys Val 35
40 45Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr
Tyr Tyr Ser Pro Gly Phe 50 55 60Gln
Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Ser Ser Leu
Lys Ala Glu Asp Thr Ala Met Tyr Tyr Cys 85
90 95Ala Arg Asp Gly Glu Gly Gly Leu Tyr Ala Met Asp
Tyr Trp Gly Gln 100 105 110Gly
Thr Ser Val Thr Val Ser Ser 115
12043446DNAArtificial sequencehuFR1-21VH_co 43aagcttgcca ccatgggatg
gtcatgcatc attctttttc tcgtcgccac tgccacaggt 60gtgcattccg aggtgcaact
tgtagaatct ggcggggatg ttgtgaagcc tggaggtagt 120ctcaagttgt cctgtgctgc
atctgggttt accttctctt cctacggaat gagctgggtg 180agacagactc ctggcaaggg
gctggagtgc gttgccacca ttagtagtgg aggttcttac 240acctactatt cacctggttt
tcagggacgc tttacaatct cccgcgataa gtctaagaac 300accctttacc tccagatgag
tagccttaag gctgaggaca cagccatgta ttattgcgct 360cgcgatgggg agggagggct
ttacgctatg gactactggg gccagggtac cagcgtgacc 420gtttcctctg ctagtaccaa
gggccc 44644396DNAArtificial
sequencehuFR21VL_co 44gaattcgcca ccatgggatg gtcatgtatc attctgttct
tggtagcaac agcaactggc 60gtccattctg acatccagat gacccaatcc tccagcagct
tgtcagtatc cgttgggggc 120cgcgttacta ttacctgtaa ggcctccgac catataaata
actggcttgc atggtatcaa 180cagaagcctg ggaaggcacc taaactgctt atctctgggg
ccacaagcct ggagaccggc 240gtgccttcca ggttctctgg aagtggatct ggcaaggact
ataccttgag cattagtagc 300cttcaacctg aggacgtcgc cacctactat tgtcagcagt
attggtctac accctttacc 360tttggacagg gcactaaatt ggagataaaa cgtacg
39645214PRTArtificial sequencehuFR1-21 LC 45Asp
Ile Gln Met Thr Gln Ser Ser Ser Ser Leu Ser Val Ser Val Gly1
5 10 15Gly Arg Val Thr Ile Thr Cys
Lys Ala Ser Asp His Ile Asn Asn Trp 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Ser Gly Ala Thr
Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Ser Ser
Leu Gln Pro65 70 75
80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Phe
85 90 95Thr Phe Gly Gln Gly Thr
Lys Leu 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 21046449PRTArtificial sequencehuFR1-21 HC 46Glu
Val Gln Leu Val Glu Ser Gly Gly Asp Val Val Lys Pro Gly Gly1
5 10 15Ser Leu Lys Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Gly Met Ser Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu
Cys Val 35 40 45Ala Thr Ile Ser
Ser Gly Gly Ser Tyr Thr Tyr Tyr Ser Pro Gly Phe 50 55
60Gln Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn
Thr Leu Tyr65 70 75
80Leu Gln Met Ser Ser Leu Lys Ala Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95Ala Arg Asp Gly Glu Gly
Gly Leu Tyr Ala Met Asp Tyr Trp Gly Gln 100
105 110Gly Thr Ser 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 445Gly47642DNAArtificial sequencehuFR1-21LC DNA
sequence 47gacatccaga tgacccaatc ctccagcagc ttgtcagtat ccgttggggg
ccgcgttact 60attacctgta aggcctccga ccatataaat aactggcttg catggtatca
acagaagcct 120gggaaggcac ctaaactgct tatctctggg gccacaagcc tggagaccgg
cgtgccttcc 180aggttctctg gaagtggatc tggcaaggac tataccttga gcattagtag
ccttcaacct 240gaggacgtcg ccacctacta ttgtcagcag tattggtcta caccctttac
ctttggacag 300ggcactaaat tggagataaa acgtacggtg gctgcaccat ctgtcttcat
cttcccgcca 360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa
taacttctat 420cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg
taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag
caccctgacg 540ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac
ccatcagggc 600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt
642481347DNAArtificial sequencehuFR1-21HC DNA sequence
48gaggtgcaac ttgtagaatc tggcggggat gttgtgaagc ctggaggtag tctcaagttg
60tcctgtgctg catctgggtt taccttctct tcctacggaa tgagctgggt gagacagact
120cctggcaagg ggctggagtg cgttgccacc attagtagtg gaggttctta cacctactat
180tcacctggtt ttcagggacg ctttacaatc tcccgcgata agtctaagaa caccctttac
240ctccagatga gtagccttaa ggctgaggac acagccatgt attattgcgc tcgcgatggg
300gagggagggc tttacgctat ggactactgg ggccagggta ccagcgtgac cgtttcctct
360gctagtacca agggcccatc agttttcccc ttggctccaa gttctaaatc cacaagcggt
420ggaacagctg cactgggatg cctcgttaaa gattatttcc ctgagcctgt gacagtgagc
480tggaatagcg gagcattgac ttcaggtgtg cacacttttc ccgctgtgtt gcagtcctcc
540ggtctgtact cactgtccag tgtcgtaacc gtcccttcta gcagcttggg aacccagacc
600tacatctgta acgtcaacca taaaccatcc aacacaaagg tggataagaa ggttgaacca
660aagagctgtg ataagacaca tacatgccct ccttgtcctg caccagagct cctcggaggt
720ccatctgtgt tcctgtttcc ccccaaaccc aaggacactc ttatgatctc tcgtactcca
780gaggtcacct gtgttgttgt cgacgtgagc catgaagatc ccgaggttaa attcaactgg
840tacgtggatg gagtcgaggt tcacaatgcc aagaccaagc ccagggagga gcaatataat
900tctacatatc gggtagtgag cgttctgacc gtgctccacc aagattggct caatggaaaa
960gagtacaagt gcaaggtgtc caacaaggct cttcccgctc ccattgagaa aactatctcc
1020aaagccaagg ggcagccacg ggaaccccag gtgtatacat tgcccccatc tagagacgag
1080ctgaccaaga accaggtgag tctcacttgt ctggtcaagg ggttttaccc ttctgacatt
1140gctgtagagt gggagtctaa cggacagcca gaaaacaact acaagacaac tcccccagtg
1200ctggacagcg acgggagctt cttcctctac tccaagttga ctgtagacaa gtctagatgg
1260cagcaaggaa acgttttctc ctgctcagta atgcatgagg ctctgcacaa tcactatacc
1320cagaaatcac tgtcccttag cccaggg
134749792DNAArtificial sequencehuFolR1 DNA sequence EcoRI to Xba1
49gaattcgcca ccatggcaca gcgcatgacc actcagctcc tgcttctgtt ggtttgggtg
60gcagtcgtgg gagaggccca gaccaggatt gcttgggcac gcacagagct gcttaatgtt
120tgcatgaacg caaagcacca taaagagaaa cccggtcccg aggataagtt gcacgaacag
180tgccgccctt ggagaaagaa tgcatgctgt agcacgaaca cctctcagga ggcgcataaa
240gacgtaagct atttgtatag atttaactgg aaccattgcg gtgaaatggc acctgcctgt
300aaacggcact ttatccagga tacttgcttg tacgagtgta gcccgaatct cgggccctgg
360attcagcaag ttgatcagag ttggcgcaaa gagagggtgc tgaacgttcc gctttgcaag
420gaggactgcg agcaatggtg ggaagactgt agaaccagct acacctgtaa gtctaactgg
480cacaaaggat ggaactggac atccgggttt aacaaatgcg ctgtcggcgc tgcctgccag
540ccatttcatt tctactttcc aactcccact gtcctgtgta acgagatttg gacgcattca
600tataaagtca gcaactacag ccggggctcc ggccgctgca ttcagatgtg gttcgaccct
660gcacagggca accctaacga ggaggtcgca cgcttctacg ctgcagcaat gtctggagcc
720ggtccttggg ctgcttggcc atttctcctt agcctcgccc tcatgcttct ctggctgttg
780tcataatcta ga
7925032DNAArtificial sequencePrimer EcoMH1misc_feature(13)..(13)s is g or
cmisc_feature(15)..(15)r is a or gmisc_feature(18)..(18)n is g, a, t or
cmisc_feature(19)..(19)m is a or cmisc_feature(25)..(25)s is g or
cmisc_feature(28)..(28)s is g or c 50cttccggaat tcsargtnma gctgsagsag tc
325135DNAArtificial sequencePrimer
EcoMH2misc_feature(13)..(13)s is g or cmisc_feature(15)..(15)r is a or
gmisc_feature(18)..(18)n is g, a, t or cmisc_feature(19)..(19)m is a or
cmisc_feature(25)..(25)s is g or cmisc_feature(28)..(28)s is g or
cmisc_feature(33)..(33)w is a or t 51cttccggaat tcsargtnma gctgsagsag
tcwgg 355236DNAArtificial sequencePrimer
BamIgG1 52ggaggatcca tagacagatg ggggtgtcgt tttggc
365331DNAArtificial sequenceSacIMKmisc_feature(10)..(10)y is c or
tmisc_feature(17)..(17)m is a or cmisc_feature(19)..(19)s is g or
cmisc_feature(22)..(22)m is a or cmisc_feature(25)..(25)r is a or
gmisc_feature(26)..(26)w is a or tmisc_feature(29)..(29)m is a or c
53ggagctcgay attgtgmtsa cmcarwctmc a
315446DNAArtificial sequenceHindKL 54tatagagctc aagcttggat ggtgggaaga
tggatacagt tggtgc 465539DNAArtificial
sequencecd37-1LClead 55ttttgaattc gccaccatga agtttccttc tcaacttct
395617PRTArtificial sequencehuman and chimeric Mov19
vHC CDR2 compositeMISC_FEATURE(14)..(14)Xaa can be Gln, His, Lys, or
ArgMISC_FEATURE(16)..(16)Xaa can be Arg, Gln, His, or
AsnMISC_FEATURE(17)..(17)Xaa can be Glu, Thr, Ser, Gly, Ala, or Val 56Arg
Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gln Xaa Phe Xaa1
5 10 15Xaa5711PRTArtificial
sequenceFR1-48vL CDR1 57Arg Ala Ser Glu Asn Ile Tyr Ser Asn Leu Ala1
5 10587PRTArtificial sequenceFR1-48vL CDR2
58Ala Ala Thr Asn Leu Ala Asp1 5599PRTArtificial
sequenceFR1-48vL CDR3 59Gln His Phe Trp Ala Ser Pro Tyr Thr1
5606PRTArtificial sequenceFR1-48vH CDR1 60Thr Asn Tyr Trp Met Gln1
56110PRTArtificial sequenceFR1-48vH CDR2 61Ala Ile Tyr Pro Gly
Asn Gly Asp Ser Arg1 5 10628PRTArtificial
sequenceFR1-48vH CDR3 62Arg Asp Gly Asn Tyr Ala Ala Tyr1
56311PRTArtificial sequenceFR1-49vL CDR1 63Arg Ala Ser Glu Asn Ile Tyr
Thr Asn Leu Ala1 5 10647PRTArtificial
sequenceFR1-49vL CDR2 64Thr Ala Ser Asn Leu Ala Asp1
5659PRTArtificial sequenceFR1-49vL CDR3 65Gln His Phe Trp Val Ser Pro Tyr
Thr1 5666PRTArtificial sequenceFR1-49vH CDR1 66Thr Asn Tyr
Trp Met Tyr1 56710PRTArtificial sequenceFR1-49vH CDR2 67Ala
Ile Tyr Pro Gly Asn Ser Asp Thr Thr1 5
10689PRTArtificial sequenceFR1-49vH CDR3 68Arg His Asp Tyr Gly Ala Met
Asp Tyr1 56911PRTArtificial sequenceFR1-57vL CDR1 69Arg Ala
Ser Gln Asn Ile Asn Asn Asn Leu His1 5
10707PRTArtificial sequenceFR1-57vL CDR2 70Tyr Val Ser Gln Ser Val Ser1
57110PRTArtificial sequenceFR1-57vL CDR3 71Gln Gln Ser Asn
Ser Trp Pro His Tyr Thr1 5
10726PRTArtificial sequenceFR1-57vH CDR1 72Ser Ser Phe Gly Met His1
57310PRTArtificial sequenceFR1-57vH CDR2 73Tyr Ile Ser Ser Gly
Ser Ser Thr Ile Ser1 5 10749PRTArtificial
sequenceFR1-57vH CDR3 74Glu Ala Tyr Gly Ser Ser Met Glu Tyr1
57511PRTArtificial sequenceFR1-65vL CDR1 75Lys Ala Ser Gln Asn Val Gly
Pro Asn Val Ala1 5 10767PRTArtificial
sequenceFR1-65vL CDR2 76Ser Ala Ser Tyr Arg Tyr Ser1
5779PRTArtificial sequenceFR1-65vL CDR3 77Gln Gln Tyr Asn Ser Tyr Pro Tyr
Thr1 5786PRTArtificial sequenceFR1-65vH CDR1 78Thr Ser Tyr
Thr Met His1 57910PRTArtificial sequenceFR1-65vH CDR2 79Tyr
Ile Asn Pro Ile Ser Gly Tyr Thr Asn1 5
108011PRTArtificial sequenceFR1-65vH CDR3 80Gly Gly Ala Tyr Gly Arg Lys
Pro Met Asp Tyr1 5 108117PRTArtificial
sequencemuFR1-48 Kabat defined HC CDR2 81Ala Ile Tyr Pro Gly Asn Gly Asp
Ser Arg Tyr Thr Gln Lys Phe Lys1 5 10
15Gly8217PRTArtificial sequencehuFR1-48 Kabat defined HC
CDR2 82Ala Ile Tyr Pro Gly Asn Gly Asp Ser Arg Tyr Thr Gln Lys Phe Gln1
5 10
15Gly8317PRTArtificial sequencehuFR1-49 Kabat defined HC CDR2 83Ala Ile
Tyr Pro Gly Asn Ser Asp Thr Thr Tyr Asn Gln Lys Phe Gln1 5
10 15Gly8417PRTArtificial
sequencemuFR1-57 Kabat defined HC CDR2 84Tyr Ile Ser Ser Gly Ser Ser Thr
Ile Ser Tyr Ala Asp Thr Val Lys1 5 10
15Gly8517PRTArtificial sequencehuFR1-57 Kabat defined HC
CDR2 85Tyr Ile Ser Ser Gly Ser Ser Thr Ile Ser Tyr Ala Asp Ser Val Lys1
5 10
15Gly8617PRTArtificial sequencemuFR1-65 Kabat defined HC CDR2 86Tyr Ile
Asn Pro Ile Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys1 5
10 15Asp8717PRTArtificial
sequencehuFR1-65 Kabat defined HC CDR2 87Tyr Ile Asn Pro Ile Ser Gly Tyr
Thr Asn Tyr Asn Gln Lys Phe Gln1 5 10
15Gly88108PRTArtificial sequencemuFR1-48vL 88Asp Ile Gln Met
Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly1 5
10 15Glu Thr Val Thr Ile Thr Cys Arg Ala Ser
Glu Asn Ile Tyr Ser Asn 20 25
30Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
35 40 45Tyr Ala Ala Thr Asn Leu Ala Asp
Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Glu Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser65
70 75 80Glu Asp Phe Gly Ser
Tyr Tyr Cys Gln His Phe Trp Ala Ser Pro Tyr 85
90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
Arg 100 10589117PRTArtificial
sequencemuFR1-48vH 89Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg
Pro Gly Ala1 5 10 15Ser
Val Lys Leu Ser Cys Arg Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20
25 30Trp Met Gln Trp Ile Lys Gln Arg
Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45Gly Ala Ile Tyr Pro Gly Asn Gly Asp Ser Arg Tyr Thr Gln Lys Phe
50 55 60Lys Gly Lys Ala Thr Leu Thr Ala
Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75
80Met Gln Val Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95Ala
Arg Arg Asp Gly Asn Tyr Ala Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110Val Thr Val Ser Ala
11590108PRTArtificial sequencemuFR1-49vL 90Asp Ile Gln Met Thr Gln Ser
Pro Ala Ser Leu Ser Val Ser Val Gly1 5 10
15Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile
Tyr Thr Asn 20 25 30Leu Ala
Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35
40 45Tyr Thr Ala Ser Asn Leu Ala Asp Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser65
70 75 80Glu Asp Phe Gly Thr Tyr
Tyr Cys Gln His Phe Trp Val Ser Pro Tyr 85
90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 10591118PRTArtificial sequencemuFR1-49vH
91Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Ala1
5 10 15Ser Val Lys Met Ser Cys
Lys Ala Ser Gly Tyr Lys Phe Thr Asn Tyr 20 25
30Trp Met Tyr Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu
Glu Leu Ile 35 40 45Gly Ala Ile
Tyr Pro Gly Asn Ser Asp Thr Thr Tyr Asn Leu Lys Phe 50
55 60Lys Gly Lys Ala Lys Leu Thr Ala Val Thr Ser Ala
Asn Thr Val Tyr65 70 75
80Met Glu Val Ser Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95Thr Lys Arg His Asp Tyr
Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr 100
105 110Ser Val Thr Val Ser Ser
11592109PRTArtificial sequencemuFR1-57vL 92Asp Ile Val Leu Thr Gln Ser
Pro Ala Thr Leu Ser Val Thr Pro Gly1 5 10
15Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Asn Ile
Asn Asn Asn 20 25 30Leu His
Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35
40 45Lys Tyr Val Ser Gln Ser Val 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 Thr65
70 75 80Glu Asp Phe Gly Met Tyr
Phe Cys Gln Gln Ser Asn Ser Trp Pro His 85
90 95Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
Arg 100 10593118PRTArtificial
sequencemuFR1-57vH 93Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser
Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20
25 30Gly Met His Trp Val Arg Gln Ala
Pro Glu Lys Gly Leu Glu Trp Val 35 40
45Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Ser Tyr Ala Asp Thr Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Lys Thr Leu Leu65 70 75
80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met
Tyr Tyr Cys 85 90 95Ala
Arg Glu Ala Tyr Gly Ser Ser Met Glu Tyr Trp Gly Gln Gly Thr
100 105 110Ser Val Thr Val Ser Ser
11594108PRTArtificial sequencemuFR1-65vL 94Asp Ile Val Met Thr Gln Ser
Gln Lys Phe Met Ser Thr Ser Val Gly1 5 10
15Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val
Gly Pro Asn 20 25 30Val Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile 35
40 45Tyr Ser Ala Ser Tyr Arg Tyr Ser Glu Val
Pro Asp Arg Phe Thr Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Met Gln Ser65
70 75 80Ala Asp Leu Ala Glu Tyr
Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr 85
90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 10595120PRTArtificial sequencemuFR1-65vH
95Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala1
5 10 15Ser Val Lys Met Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25
30Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
Ala Trp Ile 35 40 45Gly Tyr Ile
Asn Pro Ile Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50
55 60Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
Ser Thr Ala Tyr65 70 75
80Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95Ala Ser Gly Gly Ala Tyr
Gly Arg Lys Pro Met Asp Tyr Trp Gly Gln 100
105 110Gly Thr Ser Val Thr Val Ser Ser 115
12096108PRTArtificial sequencehuFR1-48vL 96Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Val Ser Val Gly1 5
10 15Glu Arg Val Thr Ile Thr Cys Arg Ala Ser Glu
Asn Ile Tyr Ser Asn 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val
35 40 45Tyr Ala Ala Thr Asn Leu Ala Asp
Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Glu Ser Gly Thr Asp Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro65
70 75 80Glu Asp Phe Gly Ser
Tyr Tyr Cys Gln His Phe Trp Ala Ser Pro Tyr 85
90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
Arg 100 10597117PRTArtificial
sequencehuFR1-48vH 97Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Ala Lys
Pro Gly Ala1 5 10 15Ser
Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20
25 30Trp Met Gln Trp Ile Lys Gln Arg
Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45Gly Ala Ile Tyr Pro Gly Asn Gly Asp Ser Arg Tyr Thr Gln Lys Phe
50 55 60Gln Gly Lys Ala Thr Leu Thr Ala
Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75
80Met Gln Val Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95Ala
Arg Arg Asp Gly Asn Tyr Ala Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110Val Thr Val Ser Ala
11598108PRTArtificial sequencehuFR1-49vL 98Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Val Ser Val Gly1 5 10
15Glu Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile
Tyr Thr Asn 20 25 30Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val 35
40 45Tyr Thr Ala Ser Asn Leu Ala Asp Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro65
70 75 80Glu Asp Phe Gly Thr Tyr
Tyr Cys Gln His Phe Trp Val Ser Pro Tyr 85
90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 10599118PRTArtificial sequencehuFR1-49vH
99Gln Val Gln Leu Gln Gln Ser Gly Ala Val Val Ala Lys Pro Gly Ala1
5 10 15Ser Val Lys Met Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25
30Trp Met Tyr Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu
Glu Leu Ile 35 40 45Gly Ala Ile
Tyr Pro Gly Asn Ser Asp Thr Thr Tyr Asn Gln Lys Phe 50
55 60Gln Gly Lys Ala Thr Leu Thr Ala Val Thr Ser Ala
Asn Thr Val Tyr65 70 75
80Met Glu Val Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95Thr Lys Arg His Asp Tyr
Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr 100
105 110Ser Val Thr Val Ser Ser
115100109PRTArtificial sequencehuFR1-57vL 100Glu Ile Val Leu Thr Gln Ser
Pro Ala Thr Leu Ser Val Thr Pro Gly1 5 10
15Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Asn Ile
Asn Asn Asn 20 25 30Leu His
Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile 35
40 45Lys Tyr Val Ser Gln Ser Val Ser Gly Ile
Pro Asp Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser Ser Val Glu Pro65
70 75 80Glu Asp Phe Gly Met Tyr
Phe Cys Gln Gln Ser Asn Ser Trp Pro His 85
90 95Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
Arg 100 105101118PRTArtificial
sequencehuFR1-57vH 101Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser
Arg Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20
25 30Gly Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Ser Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Lys Thr Leu Leu65 70 75
80Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Met
Tyr Tyr Cys 85 90 95Ala
Arg Glu Ala Tyr Gly Ser Ser Met Glu Tyr Trp Gly Gln Gly Thr
100 105 110Leu Val Thr Val Ser Ser
115102108PRTArtificial sequencehuFR1-65vL 102Glu Ile Val Met Thr Gln Ser
Pro Ala Thr Met Ser Thr Ser Pro Gly1 5 10
15Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val
Gly Pro Asn 20 25 30Val Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Ala Leu Ile 35
40 45Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val
Pro Ala Arg Phe Thr Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Met Gln Ser65
70 75 80Glu Asp Leu Ala Glu Tyr
Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr 85
90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105103120PRTArtificial sequencehuFR1-65vH
103Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Ala Lys Pro Gly Ala1
5 10 15Ser Val Lys Met Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25
30Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
Ala Trp Ile 35 40 45Gly Tyr Ile
Asn Pro Ile Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50
55 60Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
Ser Thr Ala Tyr65 70 75
80Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95Ala Ser Gly Gly Ala Tyr
Gly Arg Lys Pro Met Asp Tyr Trp Gly Gln 100
105 110Gly Thr Ser Val Thr Val Ser Ser 115
120104214PRTArtificial sequencemuFR1-48LC 104Asp Ile Gln Met Thr
Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly1 5
10 15Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu
Asn Ile Tyr Ser Asn 20 25
30Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
35 40 45Tyr Ala Ala Thr Asn Leu Ala Asp
Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Glu Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser65
70 75 80Glu Asp Phe Gly Ser
Tyr Tyr Cys Gln His Phe Trp Ala Ser Pro Tyr 85
90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
Arg Ala Asp Ala Ala 100 105
110Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
115 120 125Gly Ala Ser Val Val Cys Phe
Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135
140Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val
Leu145 150 155 160Asn Ser
Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175Ser Thr Leu Thr Leu Thr Lys
Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185
190Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val
Lys Ser 195 200 205Phe Asn Arg Asn
Glu Cys 210105441PRTArtificial sequencemuFR1-48HC 105Gln Val Gln Leu
Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala1 5
10 15Ser Val Lys Leu Ser Cys Arg Ala Ser Gly
Tyr Thr Phe Thr Asn Tyr 20 25
30Trp Met Gln Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45Gly Ala Ile Tyr Pro Gly Asn Gly
Asp Ser Arg Tyr Thr Gln Lys Phe 50 55
60Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65
70 75 80Met Gln Val Ser Ser
Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85
90 95Ala Arg Arg Asp Gly Asn Tyr Ala Ala Tyr Trp
Gly Gln Gly Thr Leu 100 105
110Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu
115 120 125Ala Pro Gly Ser Ala Ala Gln
Thr Asn Ser Met Val Thr Leu Gly Cys 130 135
140Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn
Ser145 150 155 160Gly Ser
Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Glu Ser
165 170 175Asp Leu Tyr Thr Leu Ser Ser
Ser Val Thr Val Pro Ser Ser Met Arg 180 185
190Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser
Ser Thr 195 200 205Lys Val Asp Lys
Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys 210
215 220Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile
Phe Pro Pro Lys225 230 235
240Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val
245 250 255Val Val Asp Ile Ser
Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe 260
265 270Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln
Pro Arg Glu Glu 275 280 285Gln Phe
Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His 290
295 300Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys
Arg Val Asn Ser Ala305 310 315
320Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg
325 330 335Pro Lys Ala Pro
Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met 340
345 350Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile
Thr Asp Phe Phe Pro 355 360 365Glu
Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn 370
375 380Tyr Lys Asn Thr Gln Pro Ile Met Asn Thr
Asn Gly Ser Tyr Phe Val385 390 395
400Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn
Thr 405 410 415Phe Thr Cys
Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu 420
425 430Lys Ser Leu Ser His Ser Pro Gly Lys
435 440106214PRTArtificial sequencemuFR1-49LC 106Asp Ile
Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly1 5
10 15Glu Thr Val Thr Ile Thr Cys Arg
Ala Ser Glu Asn Ile Tyr Thr Asn 20 25
30Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu
Val 35 40 45Tyr Thr Ala Ser Asn
Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu
Gln Ser65 70 75 80Glu
Asp Phe Gly Thr Tyr Tyr Cys Gln His Phe Trp Val Ser Pro Tyr
85 90 95Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105
110Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr
Ser Gly 115 120 125Gly Ala Ser Val
Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130
135 140Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln
Asn Gly Val Leu145 150 155
160Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175Ser Thr Leu Thr Leu
Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180
185 190Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro
Ile Val Lys Ser 195 200 205Phe Asn
Arg Asn Glu Cys 210107448PRTArtificial sequencemuFR1-49HC 107Glu Val
Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Ala1 5
10 15Ser Val Lys Met Ser Cys Lys Ala
Ser Gly Tyr Lys Phe Thr Asn Tyr 20 25
30Trp Met Tyr Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Leu
Ile 35 40 45Gly Ala Ile Tyr Pro
Gly Asn Ser Asp Thr Thr Tyr Asn Leu Lys Phe 50 55
60Lys Gly Lys Ala Lys Leu Thr Ala Val Thr Ser Ala Asn Thr
Val Tyr65 70 75 80Met
Glu Val Ser Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95Thr Lys Arg His Asp Tyr Gly
Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105
110Ser Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val
Tyr Pro 115 120 125Leu Ala Pro Val
Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 130
135 140Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr
Leu Thr Trp Asn145 150 155
160Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Asp Leu Tyr Thr
Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr 180
185 190Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His
Pro Ala Ser Ser 195 200 205Thr Lys
Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro 210
215 220Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu
Leu Gly Gly Pro Ser225 230 235
240Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu
245 250 255Ser Pro Ile Val
Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro 260
265 270Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val
Glu Val His Thr Ala 275 280 285Gln
Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val 290
295 300Ser Ala Leu Pro Ile Gln His Gln Asp Trp
Met Ser Gly Lys Glu Phe305 310 315
320Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg
Thr 325 330 335Ile Ser Lys
Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu 340
345 350Pro Pro Pro Glu Glu Glu Met Thr Lys Lys
Gln Val Thr Leu Thr Cys 355 360
365Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn 370
375 380Asn Gly Lys Thr Glu Leu Asn Tyr
Lys Asn Thr Glu Pro Val Leu Asp385 390
395 400Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg
Val Glu Lys Lys 405 410
415Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly
420 425 430Leu His Asn His His Thr
Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 435 440
445108215PRTArtificial sequencemuFR1-57LC 108Asp Ile Val Leu
Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly1 5
10 15Asp Ser Val Ser Leu Ser Cys Arg Ala Ser
Gln Asn Ile Asn Asn Asn 20 25
30Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile
35 40 45Lys Tyr Val Ser Gln Ser Val 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 Thr65
70 75 80Glu Asp Phe Gly Met
Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro His 85
90 95Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys Arg Ala Asp Ala 100 105
110Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser
115 120 125Gly Gly Ala Ser Val Val Cys
Phe Leu Asn Asn Phe Tyr Pro Lys Asp 130 135
140Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly
Val145 150 155 160Leu Asn
Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met
165 170 175Ser Ser Thr Leu Thr Leu Thr
Lys Asp Glu Tyr Glu Arg His Asn Ser 180 185
190Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile
Val Lys 195 200 205Ser Phe Asn Arg
Asn Glu Cys 210 215109448PRTArtificial
sequencemuFR1-57HC 109Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser
Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20
25 30Gly Met His Trp Val Arg Gln Ala
Pro Glu Lys Gly Leu Glu Trp Val 35 40
45Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Ser Tyr Ala Asp Thr Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Lys Thr Leu Leu65 70 75
80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met
Tyr Tyr Cys 85 90 95Ala
Arg Glu Ala Tyr Gly Ser Ser Met Glu Tyr Trp Gly Gln Gly Thr
100 105 110Ser Val Thr Val Ser Ser Ala
Lys Thr Thr Ala Pro Ser Val Tyr Pro 115 120
125Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu
Gly 130 135 140Cys Leu Val Lys Gly Tyr
Phe Pro Glu Pro Val Thr Leu Thr Trp Asn145 150
155 160Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln 165 170
175Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr
180 185 190Trp Pro Ser Gln Ser Ile
Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200
205Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile
Lys Pro 210 215 220Cys Pro Pro Cys Lys
Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser225 230
235 240Val Phe Ile Phe Pro Pro Lys Ile Lys Asp
Val Leu Met Ile Ser Leu 245 250
255Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro
260 265 270Asp Val Gln Ile Ser
Trp Phe Val Asn Asn Val Glu Val His Thr Ala 275
280 285Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr
Leu Arg Val Val 290 295 300Ser Ala Leu
Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe305
310 315 320Lys Cys Lys Val Asn Asn Lys
Asp Leu Pro Ala Pro Ile Glu Arg Thr 325
330 335Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln
Val Tyr Val Leu 340 345 350Pro
Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys 355
360 365Met Val Thr Asp Phe Met Pro Glu Asp
Ile Tyr Val Glu Trp Thr Asn 370 375
380Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp385
390 395 400Ser Asp Gly Ser
Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys 405
410 415Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys
Ser Val Val His Glu Gly 420 425
430Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys
435 440 445110214PRTArtificial
sequencemuFR1-65LC 110Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr
Ser Val Gly1 5 10 15Asp
Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Pro Asn 20
25 30Val Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ser Pro Lys Ala Leu Ile 35 40
45Tyr Ser Ala Ser Tyr Arg Tyr Ser Glu Val Pro Asp Arg Phe Thr Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Asn Met Gln Ser65 70 75
80Ala Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser
Tyr Pro Tyr 85 90 95Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala
100 105 110Pro Thr Val Ser Ile Phe Pro
Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120
125Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp
Ile 130 135 140Asn Val Lys Trp Lys Ile
Asp Gly Ser Glu Arg Gln Asn Gly Val Leu145 150
155 160Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Met Ser 165 170
175Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
180 185 190Thr Cys Glu Ala Thr His
Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200
205Phe Asn Arg Asn Glu Cys 210111444PRTArtificial
sequencemuFR1-65HC 111Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg
Pro Gly Ala1 5 10 15Ser
Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20
25 30Thr Met His Trp Val Lys Gln Arg
Pro Gly Gln Gly Leu Ala Trp Ile 35 40
45Gly Tyr Ile Asn Pro Ile Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60Lys Asp Lys Ala Thr Leu Thr Ala
Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75
80Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95Ala
Ser Gly Gly Ala Tyr Gly Arg Lys Pro Met Asp Tyr Trp Gly Gln
100 105 110Gly Thr Ser Val Thr Val Ser
Ser Ala Lys Thr Thr Pro Pro Ser Val 115 120
125Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val
Thr 130 135 140Leu Gly Cys Leu Val Lys
Gly Tyr Phe Pro Glu Pro Val Thr Val Thr145 150
155 160Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His
Thr Phe Pro Ala Val 165 170
175Leu Glu Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser
180 185 190Ser Met Arg Pro Ser Glu
Thr Val Thr Cys Asn Val Ala His Pro Ala 195 200
205Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys
Gly Cys 210 215 220Lys Pro Cys Ile Cys
Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe225 230
235 240Pro Pro Lys Pro Lys Asp Val Leu Thr Ile
Thr Leu Thr Pro Lys Val 245 250
255Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe
260 265 270Ser Trp Phe Val Asp
Asp Val Glu Val His Thr Ala Gln Thr Gln Pro 275
280 285Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val
Ser Glu Leu Pro 290 295 300Ile Met His
Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val305
310 315 320Asn Ser Ala Ala Phe Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Thr 325
330 335Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile
Pro Pro Pro Lys 340 345 350Glu
Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp 355
360 365Phe Phe Pro Glu Asp Ile Thr Val Glu
Trp Gln Trp Asn Gly Gln Pro 370 375
380Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asn Thr Asn Gly Ser385
390 395 400Tyr Phe Val Tyr
Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala 405
410 415Gly Asn Thr Phe Thr Cys Ser Val Leu His
Glu Gly Leu His Asn His 420 425
430His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435
440112214PRTArtificial sequencehuFR1-48LC 112Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Val Ser Val Gly1 5
10 15Glu Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn
Ile Tyr Ser Asn 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val 35
40 45Tyr Ala Ala Thr Asn Leu Ala Asp Gly
Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Glu Ser Gly Thr Asp Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro65
70 75 80Glu Asp Phe Gly Ser
Tyr Tyr Cys Gln His Phe Trp Ala Ser Pro Tyr 85
90 95Thr Phe Gly Gln Gly Thr Lys Leu 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 210113446PRTArtificial sequencehuFR1-48HC 113Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Ala Lys Pro Gly Ala1 5
10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asn Tyr 20 25
30Trp Met Gln Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45Gly Ala Ile Tyr Pro Gly Asn Gly
Asp Ser Arg Tyr Thr Gln Lys Phe 50 55
60Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65
70 75 80Met Gln Val Ser Ser
Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85
90 95Ala Arg Arg Asp Gly Asn Tyr Ala Ala Tyr Trp
Gly Gln Gly Thr Leu 100 105
110Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135
140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser145 150 155 160Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser 180 185
190Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn 195 200 205Thr Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210
215 220Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val225 230 235
240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu 260
265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys 275 280 285Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290
295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys305 310 315
320Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340
345 350Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu 355 360 365Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370
375 380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser385 390 395
400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg 405 410 415Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420
425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly 435 440
445114214PRTArtificial sequencehuFR1-49LC 114Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Val Ser Val Gly1 5 10
15Glu Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile
Tyr Thr Asn 20 25 30Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val 35
40 45Tyr Thr Ala Ser Asn Leu Ala Asp Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro65
70 75 80Glu Asp Phe Gly Thr Tyr
Tyr Cys Gln His Phe Trp Val Ser Pro Tyr 85
90 95Thr Phe Gly Gln Gly Thr Lys Leu 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
210115447PRTArtificial sequencehuFR1-49HC 115Gln Val Gln Leu Gln Gln Ser
Gly Ala Val Val Ala Lys Pro Gly Ala1 5 10
15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Asn Tyr 20 25 30Trp Met
Tyr Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Leu Ile 35
40 45Gly Ala Ile Tyr Pro Gly Asn Ser Asp Thr
Thr Tyr Asn Gln Lys Phe 50 55 60Gln
Gly Lys Ala Thr Leu Thr Ala Val Thr Ser Ala Asn Thr Val Tyr65
70 75 80Met Glu Val Ser Ser Leu
Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85
90 95Thr Lys Arg His Asp Tyr Gly Ala Met Asp Tyr Trp
Gly Gln Gly Thr 100 105 110Ser
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115
120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly 130 135
140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145
150 155 160Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165
170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser Ser 180 185
190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215
220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser225 230 235 240Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro 260 265
270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala 275 280 285Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340
345 350Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys 355 360 365Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370
375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp385 390 395
400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420
425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly 435 440
445116215PRTArtificial sequencehuFR1-57LC 116Glu Ile Val Leu Thr Gln Ser
Pro Ala Thr Leu Ser Val Thr Pro Gly1 5 10
15Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Asn Ile
Asn Asn Asn 20 25 30Leu His
Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile 35
40 45Lys Tyr Val Ser Gln Ser Val Ser Gly Ile
Pro Asp Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser Ser Val Glu Pro65
70 75 80Glu Asp Phe Gly Met Tyr
Phe Cys Gln Gln Ser Asn Ser Trp Pro His 85
90 95Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
Arg Thr Val Ala 100 105 110Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115
120 125Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu 130 135
140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser145
150 155 160Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165
170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val 180 185
190Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205Ser Phe Asn Arg Gly Glu Cys
210 215117447PRTArtificial sequencehuFR1-57HC 117Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Arg Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Phe 20 25
30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Tyr Ile Ser Ser
Gly Ser Ser Thr Ile Ser Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr
Leu Leu65 70 75 80Leu
Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95Ala Arg Glu Ala Tyr Gly Ser
Ser Met Glu Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro 115 120 125Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130
135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn145 150 155
160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180
185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser 195 200 205Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala 275 280 285Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr 325 330 335Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340
345 350Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr Cys 355 360
365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370
375 380Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp385 390
395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser 405 410
415Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445118214PRTArtificial sequencehuFR1-65LC 118Glu Ile Val Met Thr
Gln Ser Pro Ala Thr Met Ser Thr Ser Pro Gly1 5
10 15Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln
Asn Val Gly Pro Asn 20 25
30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Ala Leu Ile
35 40 45Tyr Ser Ala Ser Tyr Arg Tyr Ser
Gly Val Pro Ala Arg Phe Thr Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Met Gln Ser65
70 75 80Glu Asp Leu Ala Glu
Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr 85
90 95Thr Phe Gly Gln Gly Thr Lys Leu 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 210119449PRTArtificial sequencehuFR1-65HC 119Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Ala Lys Pro Gly Ala1 5
10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Ser Tyr 20 25
30Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Ala Trp Ile
35 40 45Gly Tyr Ile Asn Pro Ile Ser Gly
Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55
60Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65
70 75 80Met Gln Leu Asn Ser
Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85
90 95Ala Ser Gly Gly Ala Tyr Gly Arg Lys Pro Met
Asp Tyr Trp Gly Gln 100 105
110Gly Thr Ser 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
445Gly120396DNAArtificial sequencehuFR1-48_VL 120gaattcgcca ccatgggatg
gagttgtatc atcctgtttc ttgtggctac agccacaggg 60gtacactccg atattcaaat
gacacagtcc ccttcatccc tgtccgtcag tgtgggggaa 120agggttacca tcacctgccg
tgcatcagag aacatctatt ccaacctcgc ctggtaccaa 180cagaaacctg gcaagtcccc
taagctgttg gtctacgccg ctacaaacct cgccgatggg 240gtgccttccc gtttcagtgg
gtcagagtca ggcaccgact attctctgaa gatcaactcc 300ctccagcctg aggatttcgg
ctcctattac tgtcagcact tctgggctag tccatatact 360ttcggccagg gaaccaaact
tgaaattaaa cgtacg 396121437DNAArtificial
sequencehuFR1-48_VH 121aagcttgcca ccatggggtg gagctgcatc atcctttttc
tggtggccac tgccaccggc 60gtgcactctc aggtccaact tgtgcagagc ggagccgagg
tggccaaacc cggagctagt 120gttaagctct catgtaaagc atctggctac acctttacta
actactggat gcagtggatc 180aagcaacggc caggccaggg cctggagtgg attggtgcta
tttatcccgg aaacggggat 240agcaggtaca ctcagaaatt tcagggaaag gctaccctta
ccgccgataa gagttcttcc 300acagcatata tgcaagtctc ctctctgacc tcagaggata
gtgctgtcta ttactgcgct 360cgccgggatg gcaactatgc agcctattgg ggtcaaggca
cccttgtgac tgtatccgca 420gcaagcacca agggccc
437122396DNAArtificial sequencehuFR1-49_VL
122gaattcgcca ccatgggttg gtcatgcatt atcctgtttc tggtcgcaac agcaacaggt
60gtgcacagtg acattcagat gacccaaagc ccctccagtc tgagcgtttc cgtgggggaa
120cgtgtcacta tcacatgcag agcttccgag aatatttaca ctaacctcgc atggtaccag
180cagaaacccg ggaagtctcc aaaacttctc gtatatacag ccagcaactt ggcagatggg
240gtgcccagcc ggtttagcgg atctggttca ggcaccgact attctttgaa aattaattcc
300ctgcagcctg aggattttgg tacctactat tgccagcatt tttgggtatc accatacact
360tttggacagg gaacaaagct ggagatcaag cgtacg
396123440DNAArtificial sequencehuFR1-49_VH 123aagcttgcca ccatgggctg
gtcttgtatt attctttttc ttgtggccac agccacagga 60gtccattcac aggtacagct
ccaacagtct ggcgcagttg tcgccaagcc cggcgcctct 120gtgaagatga gttgcaaggc
ctctggctac accttcacta attattggat gtactggatc 180aaacaacgcc ccggccaggg
tctggaactc attggagcca tctacccagg caactccgac 240acaacataca atcagaagtt
tcagggcaaa gcaaccctga ccgctgtaac ctcagctaat 300accgtgtaca tggaggtaag
tagcttgact agtgaagatt ccgcagtata ctattgcacc 360aagcgccatg attacggcgc
catggattac tggggccaag gtaccagtgt gaccgtgtct 420tccgcttcca ccaagggccc
440124399DNAArtificial
sequencehuFR1-57_VL 124gaattcgcca ccatgggctg gtcatgcatt attttgttcc
tggtcgccac cgcaaccggc 60gttcattccg aaattgttct tactcagagc cctgcaacct
tgagtgtgac acccggcgat 120cgggtctcac tgagttgcag agcttcccag aatatcaaca
ataatctgca ctggtatcag 180cagaagcctg gccagtctcc tcgcttgctg attaagtatg
tctcacagag cgtgtcaggt 240atccctgacc gtttctccgg gtcaggttca ggcaccgact
tcacactgtc catttctagc 300gtggagcctg aggatttcgg aatgtacttt tgccagcaga
gcaatagctg gcctcactac 360acctttggcc aagggaccaa gctggagatc aagcgtacg
399125440DNAArtificial sequencehuFR1-57_VH
125aagcttgcca ccatgggctg gagctgtatc atcttgttcc ttgtggccac agctactggc
60gtgcactccg aggtgcagct ggtcgaatcc ggcggaggcc tggtgcagcc tggggggagt
120agacggctgt cctgcgctgc ctctgggttt actttctcaa gtttcggtat gcactgggtg
180cgtcaggccc ccgggaaggg cctggaatgg gttgcttata tatcatctgg cagctccacc
240atttcttatg ctgattccgt taagggacgc ttcaccattt ccagagacaa cagtaagaaa
300acccttctgc tgcagatgac ctctctccgc gccgaagaca ccgcaatgta ttattgtgct
360agagaggcct acggcagtag tatggaatac tgggggcagg ggaccctggt gaccgtgtct
420tccgcatcta ctaagggccc
440126396DNAArtificial sequencehuFR1-65_VL 126gaattcgcca ccatgggctg
gtcttgcatt attctgttcc tggttgcaac agccactggc 60gtccattccg aaatcgtgat
gacccaatct cccgccacca tgtctacctc tcccggggac 120cgggtgtctg tgacctgcaa
ggcctctcag aatgttggcc caaacgtggc atggtatcaa 180cagaaaccag ggcagtcacc
cagagccctg atttactccg cttcttacag atattcagga 240gttcccgccc ggttcacagg
tagtgggtcc ggcactgact ttaccttgac catttccaac 300atgcaatccg aggacctggc
cgaatacttc tgtcagcagt acaattcata tccctataca 360ttcggccagg ggaccaagct
ggaaataaag cgtacg 396127446DNAArtificial
sequencehuFR1-65_VH 127aagcttgcca ccatgggctg gtcatgcata atcctgttcc
tggtcgcaac cgctacaggt 60gtacactccc aggtgcagtt ggtgcagagc ggggccgaag
ttgctaagcc cggtgcaagt 120gtaaaaatgt cctgcaaagc tagcgggtac acattcacat
cctatactat gcattgggta 180aaacagcgcc caggacaggg gctcgcctgg ataggctata
ttaacccaat atcaggatac 240acaaactaca atcagaaatt tcagggaaag gcaaccctga
ccgccgacaa gtcctcttct 300accgcatata tgcagctcaa ctccctgacc agtgaagata
gcgcagtgta ttactgtgcc 360tccggcggtg cttatggccg gaaacccatg gattactggg
gacaaggcac ctccgtcaca 420gtgagtagcg cctcaaccaa gggccc
44612817PRTArtificial sequenceKabat Defined Mov19
HC CDR2 Murine 128Arg Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gln Asn
Phe Lys1 5 10
15Asp12917PRTArtificial sequenceKabat Defined Mov19 HC CDR2 Human 129Arg
Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gln Lys Phe Gln1
5 10 15Gly13017PRTArtificial
sequencemuFR1-49 Kabat defined HC CDR2 130Ala Ile Tyr Pro Gly Asn Ser Asp
Thr Thr Tyr Asn Leu Lys Phe Lys1 5 10
15Gly13110PRTArtificial SequencehuMov19 vHC CDR2 131Arg Ile
His Pro Tyr Asp Gly Asp Thr Phe1 5 10
* * * * *
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