U.S. patent application number 13/327624 was filed with the patent office on 2013-08-15 for monoclonal antibodies to basic fibroblast growth factor.
This patent application is currently assigned to Galaxy Biotech, LLC. The applicant listed for this patent is Kyung Jin Kim, Hangil Park, Maximiliano Vasquez, Lihong Wang. Invention is credited to Kyung Jin Kim, Hangil Park, Maximiliano Vasquez, Lihong Wang.
Application Number | 20130209482 13/327624 |
Document ID | / |
Family ID | 41398450 |
Filed Date | 2013-08-15 |
United States Patent
Application |
20130209482 |
Kind Code |
A1 |
Kim; Kyung Jin ; et
al. |
August 15, 2013 |
MONOCLONAL ANTIBODIES TO BASIC FIBROBLAST GROWTH FACTOR
Abstract
The present invention is directed toward a neutralizing
monoclonal antibody to basic fibroblast growth factor, a
pharmaceutical composition comprising same, and methods of
treatment comprising administering such a pharmaceutical
composition to a patient.
Inventors: |
Kim; Kyung Jin; (Cupertino,
CA) ; Wang; Lihong; (Palo Alto, CA) ; Park;
Hangil; (San Francisco, CA) ; Vasquez;
Maximiliano; (Palo Alto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kim; Kyung Jin
Wang; Lihong
Park; Hangil
Vasquez; Maximiliano |
Cupertino
Palo Alto
San Francisco
Palo Alto |
CA
CA
CA
CA |
US
US
US
US |
|
|
Assignee: |
Galaxy Biotech, LLC
Cupertino
CA
|
Family ID: |
41398450 |
Appl. No.: |
13/327624 |
Filed: |
December 15, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12474198 |
May 28, 2009 |
8101725 |
|
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13327624 |
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61057183 |
May 29, 2008 |
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61170561 |
Apr 17, 2009 |
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Current U.S.
Class: |
424/145.1 ;
530/387.3; 530/388.15; 530/388.24 |
Current CPC
Class: |
C07K 16/22 20130101;
C07K 2317/24 20130101; C07K 2317/76 20130101; C07K 2317/73
20130101; A61K 39/3955 20130101; A61K 39/3955 20130101; C07K
16/2863 20130101; A61K 2039/507 20130101; C07K 2319/30 20130101;
A61K 2039/505 20130101; C07K 2317/56 20130101; A61P 35/00 20180101;
A61K 2300/00 20130101 |
Class at
Publication: |
424/145.1 ;
530/388.24; 530/387.3; 530/388.15 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH OR DEVELOPMENT
[0002] The work described in this application was funded in part by
Grant 5R44 CA101283-03 from the National Institutes of Health. The
US Government has certain rights in this invention.
Claims
1. A monoclonal antibody (mAb) that binds and neutralizes human
basic fibroblast growth factor (FGF2), wherein the mAb is
genetically engineered.
2. The mAb of claim 1 which is chimeric.
3. The mAb of claim 1 which is humanized.
4. The mAb of claim 1 which is human.
5. The mAb of claim 1 which completely inhibits binding of FGF2 to
each of the FGF receptors FGFR1, FGFR2, FGFR3 and FGFR4.
6. The mAb of claim 1 which inhibits FGF2-induced proliferation of
My 1 Lu cells.
7. The mAb of claim 1 which inhibits growth of a human tumor
xenograft in a mouse.
8. The mAb of claim 1 which is a Fab or F(ab').sub.2 fragment or
single-chain antibody.
9. A pharmaceutical composition comprising a mAb of claim 1.
10. A method of treating cancer in a patient comprising
administering to the patient a pharmaceutical composition
comprising the mAb of claim 1.
11. A monoclonal antibody (mAb) that competes for binding to FGF2
with an antibody produced by hybridoma PTA-8864 and has reduced
immunogenicity.
12. The mAb of claim 11 which inhibits growth of a human tumor
xenograft in a mouse.
13. The mAb of claim 11 which is humanized or human.
14. A pharmaceutical composition comprising a mAb of claim 11.
15. A method of treating cancer in a patient comprising
administering to the patient a pharmaceutical composition
comprising the mAb of claim 11.
16. The method of claim 15 wherein said cancer is hepatocellular
carcinoma.
17. A mouse, chimeric or humanized form of a mAb produced by the
PTA-8864 hybridoma.
18. A humanized antibody comprising a humanized light chain
comprising CDRs from the sequence in FIG. 11A (GAL-F2) (SEQ ID
NO:1) and a humanized heavy chain comprising CDRs from the sequence
of FIG. 11B (GAL-F2) (SEQ ID NO:4).
19. The humanized antibody of claim 18, wherein residues H1, H27,
H30, H48, H67, H71 and H94 by Kabat numbering are occupied by the
residue occupying the corresponding position of the heavy chain
shown in FIG. 11B (GAL-F2) (SEQ ID NO:4).
20. The humanized antibody of claim 18 wherein the light chain
variable region has at least 95% sequence identity to the sequence
shown in FIG. 11A (HuGAL-F2) (SEQ ID NO:2) and the heavy chain has
at least 95% sequence identity to the sequence shown in FIG. 11B
(HuGAL-F2) (SEQ ID NO:5).
21. The humanized antibody of claim 18, comprising the three light
chain CDRs and three heavy chain CDRs shown in FIGS. 11A (HuGAL-F2)
(SEQ ID NO:2) and 11B (HuGAL-F2) (SEQ ID NO:5).
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This applications claims the benefit under 35 U.S.C.
.sctn.119(c) of U.S. Patent Application No. 61/057,183 filed May
29, 2008 and U.S. Patent Application No. 61/170,561 filed Apr. 17,
2009, which are herewith incorporated in their entirety for all
purposes.
REFERENCES TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM
LISTING SUBMITTED IN COMPUTER READABLE FORMAT
[0003] The Sequence Listing written in file
022382-000820US_SEQLIST.TXT is 12,406 bytes, and was created on May
28, 2009, for the application filed herewith, Kyung Jin Kim et al.
"MONOCLONAL ANTIBODIES TO BASIC FIBROBLAST GROWTH FACTOR." The
information contained in this file is hereby incorporated by
reference.
FIELD OF THE INVENTION
[0004] The present invention relates generally to the combination
of monoclonal antibody (mAb) and recombinant DNA technologies for
developing novel biologics, and more particularly, for example, to
the production of monoclonal antibodies that bind to and neutralize
basic Fibroblast Growth Factor.
BACKGROUND OF THE INVENTION
[0005] The prototypic Fibroblast Growth Factors (FGFs), acidic FGF
(also called FGF1) and basic FGF (also called FGF2) were first
isolated in the 1970s (FGF2 by Gospodarowicz et al., J. Biol. Chem.
250:2515, 1975). There are currently 22 known FGF family members,
which can be grouped into 7 subfamilies based upon their similarity
in activities and sequences (Ornitz et al., Genome Biol. 2: 3005.1,
2001). However, the FGF family members bind to only four tyrosine
kinase receptors (FGFR14) and their isoforms, which are expressed
in a tissue-specific manner. The FGF1 subgroup consists of FGF1 and
FGF2, which bind all four FGFRs, with FGF2 binding especially
strongly to FGFR1c (Ornitz et al., J. Biol. Chem. 271:15292,
1996).
[0006] Human FGF2 is an 18 kDa non-glycosylated polypeptide
consisting of 146 amino acids in the mature form derived from a 155
aa precursor (Ornitz et al., Genome Biol. 2:3005.1, 2001; Okada-Ban
et al., Int. J. Biochem. Cell. Biol. 32:263, 2000). This 18 kDa
form of FGF2 does not encode a signal sequence, but can be secreted
by an unconventional energy-dependent pathway independent of the
ER-Golgi complex (Mignatti et al., J. Cell Physiol. 151:81, 1992;
Florkiewicz et al., J. Cell Physiol. 162:388, 1995). The single
copy of the FGF2 gene also encodes four High Molecular Weight (HMW)
forms of the protein, in addition to the 18 kDa form, by utilizing
four alternate CUG initiation sites that provide N-terminal
extensions of various sizes, resulting in proteins of 22 kDa (196
aa), 22.5 kDa (201 aa), 24 kDa (210 aa) and 34 kDa (288 aa)
(Florkiewicz et al., Proc. Natl. Acad. Sci. USA 86:3978, 1989;
Prats et al., Proc. Natl. Acad. Sci. USA 86:1836, 1989). The HMW
forms are not secreted but are transported to the cell nucleus
where they can regulate cell growth or behavior in an intracrine
fashion (Delrieu, FEBS Lett. 468:6, 2000).
[0007] In addition to binding FGFR1-4 with high affinity, FGF2
binds to heparin sulfate proteoglycans (HSPG) with lower affinity.
Although FGF2 is secreted as a monomer, cell surface HSPG dimerizes
FGF2 in a non-covalent side-to-side configuration that is
subsequently capable of dimerizing and activating FGF receptors
(Mohammadi et al., Cytokine Growth Factor Rev 16:107, 2005). The
binding of FGF and HSPG to the extracellular domain of FGFR induces
receptor dimerization, activation and autophosphorylation.
[0008] The FGFs, and in particular FGF2, have a broad spectrum of
activities on various cell types (Ornitz et al., Genome Biol.
2:3005.1, 2001). FGF2 stimulates proliferation of (i.e., is
mitogenic for) certain cells including fibroblasts and endothelial
cells and is a survival factor (anti-apoptotic) for certain cells
such as neural cells (Okada-Ban, op. cit.). It also stimulates
differentiation (morphogenesis) and migration (motility) of
endothelial cells (Dow et al., Urology 55:800, 2000). FGF2 is
involved in development, especially of the nervous system.
Importantly, FGF2 is a powerful angiogenic factor (Presta et al.,
Cytokine and Growth Factor Rev. 16:159, 2005).
[0009] FGF2 and other FGFs are believed to play a role in cancer,
both by stimulating angiogenesis and tumor cells directly (Presta
et al., op cit.) During tumor progression, cancer cells may respond
to the extracellular FGF2 secreted from the stromal cells
(paracrine), and then the tumor cells themselves may secrete FGF2
and respond to it in an autocrine manner. FGF2 or its receptor
FGFR1 has been shown to be expressed or overexpressed in most
gliomas (Takahashi et al., Proc. Natl. Acad. Sci. USA 87:5710,
1990; Morrison et al. Cancer Res. 54:2794, 1994). FGF2 is involved
in progression of prostate tumors (Dow et al., Urology 55:800,
2000) and is a key mediator of the proliferation of malignant
melanomas (Wang et al., Nature Med. 3:887, 1997). Over-expression
and/or involvement of FGF2 or FGFR1 in tumor progression has also
been reported for salivary gland tumors (Myoken et al., J. Path.
178:429, 1996), esophageal cancer (Barclay et al., Clin. Cancer
Res. 11:7683, 2005), and thyroid carcinomas (Boelaert et al., J.
Clin. Endocrin. Metabol. 88:2341, 2003).
[0010] Polyclonal antibodies (antiserum) to FGF2 have been reported
to inhibit tumor growth of a transplantable chondrosarcoma in mice
(Baird et al., J. Cell Biochem. 30:79, 1986), neutralize various
activities of FGF2 in vitro (Kurokawa et al., J. Biol. Chem.
264:7686, 1989), and to inhibit growth of U87 glioma cell
intracranial xenografts when applied locally (Stan et al., J.
Neurosurg. 82:1044, 1995). Among monoclonal antibodies, the DG2 mAb
has been reported to neutralize activities of FGF2 in vitro and in
vivo (Reilly et al., Biochem. Biophys. Res. Com. 164:736, 1989; WO
91/06668), modestly inhibit growth of rat C6 glioma xenografts in
nude mice (Gross et al., J. Nat. Cancer Inst. 85:121, 1993), and
modestly inhibit growth of rat chondrosarcomas when delivered
intralesionally but not i.p. or i.v. (Coppola et al., Anticancer
Res. 17:2033, 1997). Similarly, anti-FGF2 mAb DE6 has been reported
to inhibit growth of glioma cells in vitro (Morrison et al., J.
Neuroscience Res. 34:502, 1993). On the other hand, the anti-FGF2
mAbs bFM-1 and bFM-2 was reported to inhibit growth of endothelial
cells in vitro but not to block tumor angiogenesis in vivo
(Matsuzaki et al., Proc. Natl. Acad. Sci. USA 86:9911, 1989). The
neutralizing anti-FGF2 mAb 1E6 has been reported to inhibit growth
of RPMI4788 colon tumor xenografts (Aonuma et al., 19:4039, 1999).
The anti-FGF2 mAb 254F1 has been reported to inhibit proliferation
of human umbilical vein endothelial cells (HUVEC), while mAb FB-8
has been reported to inhibit proliferation of non-small cell lung
cancer cell lines in vitro (Kuhn et al., Lung Cancer 44:167, 2004).
The anti-FGF2 mAb 3H3 was reported to suppress growth of U87MG and
T98G glioma and HeLa cell xenografts (Takahashi et al., FEBS Let.
288:65, 1991) and growth of the K1000 FGF2-transfected 3T3 cell
line in mice (Hori et al., Cancer Res. 51:6180, 1991).
[0011] The GAL-F2 anti-FGF2 mAb described herein was discussed at
the AACR 2009 Annual Meeting in Poster #1236, which is incorporated
herein in its entirety for all purposes.
BRIEF SUMMARY OF THE INVENTION
[0012] In one embodiment, the invention provides a neutralizing mAb
to human basic fibroblast growth factor (FGF2). The mAb inhibits at
least one, and preferably several or all biological activities of
FGF2, including binding to one or more of the four FGF receptors
FGFR1-4; inducing proliferation of fibroblasts, endothelial cells,
Mv 1 Lu mink lung epithelial cells, and/or various human tumor
cells; and inducing angiogenesis. The anti-FGF2 mAb can inhibit
such an activity when used as a single agent. A preferred anti-FGF2
mAb inhibits growth of a human tumor xenograft in a mouse.
Preferably, the mAb of the invention is genetically engineered,
e.g., chimeric, humanized or human. Exemplary antibodies are GAL-F2
and its chimeric and humanized forms, and mAbs which have the same
epitope or compete for binding with GAL-F2. Cell lines producing
such antibodies are also provided. In another embodiment, a
pharmaceutical composition comprising a neutralizing anti-FGF2
antibody, e.g., chimeric or humanized GAL-F2, is provided. In a
third embodiment, the pharmaceutical composition is administered to
a patient to treat cancer or other disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1. Binding ELISA of GAL-F2 and control mouse mAb (mIgG)
to human FGF2 (hFGF2) and mouse FGF29 (mFGF2).
[0014] FIG. 2. FGFR-Fc/FGF2-Flag Binding ELISA showing inhibition
by GAL-F2 but not control mouse mAb 5G8 for each of the four FGF
receptors FGFR1-4.
[0015] FIG. 3. Competitive binding assay of biotinylated GAL F2 to
human FGF2 with various anti-FGF2 mAbs.
[0016] FIG. 4. Inhibition of FGF2-induced proliferation of BaF3
cells stably transfected with FGFR1 or FGFR2, by GAL-F2 or control
mAb 5G8. None means that no FGF2 (or mAb) was applied to the
cells.
[0017] FIG. 5. Inhibition of FGF2-induced proliferation of Mv 1 Lu
mink lung epithelial cells by GAL-F2 or control mAb 5G8. None means
that no FGF2 (or mAb) was applied to the cells.
[0018] FIG. 6. Photomicrographs of SMCC-7721 cells cloned in soft
agar in the presence of control mouse mAb (mIgG), GAL-F2 or bFM-1
anti-FGF2 mAb.
[0019] FIG. 7. Growth of RPMI 4788 human colon tumor xenografts in
mice treated with GAL-F2 (100 .mu.g twice per week) or PBS alone
starting from 5 days after tumor inoculation.
[0020] FIG. 8. Growth of RPMI 4788 human colon tumor xenografts in
mice treated with GAL-F2 or bFM-1 anti-FGF2 mAb (100 .mu.g twice
per week) or PBS alone, starting from 6 days after tumor
inoculation.
[0021] FIG. 9. Growth of SMMC-7721 human hepatoma tumor xenografts
in mice treated with PBS alone, GAL-F2 (100 .mu.g twice per week),
cisplatin (100 .mu.g once per week), or both GAL-F2 and cisplatin.
5 mice per group.
[0022] FIG. 10. Growth of HepG2 human hepatoma tumor xenografts in
mice treated with PBS alone, GAL-F2 or M225 anti-EGFR mAb (100
.mu.g twice per week), or both GAL-F2 and M225, starting from 6
days after inoculation. 6 mice per group.
[0023] FIGS. 11A and 11B. Amino acid sequences of the HuGAL-F2
light chain (A) (SEQ ID NO:2) and heavy chain (B) mature variable
regions (SEQ ID NO:5) are shown aligned with mouse GAL-F2 (SEQ ID
NOS:1 and 4) and human acceptor V regions (SEQ ID NOS:3 and 6). The
CDRs are underlined in the GAL-F2 sequences, and the amino acids
substituted with mouse L2G7 amino acids are double underlined in
the HuGAL-F2 sequences. The 1-letter amino acid code and Kabat
numbering system are used for both the light and heavy chain.
[0024] FIG. 12. Competitive binding of humanized (HuGAL-F2) and
chimeric (ChGAL-F2) mAbs and control human antibody hIgG, conducted
as described in the specification.
[0025] FIG. 13. Amino acid sequences of the entire mature HuGAL-F2
antibody light chain (A) (SEQ ID NO:7) and heavy chain (B) (SEQ ID
NO:8). The first amino acid on each line is numbered; the numbering
is sequential. In the light chain, the first amino acid of the
C.kappa. region is underlined, and in the heavy chain, the first
amino acids of the CH1, hinge, CH2 and CH3 regions are
underlined.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The invention provides neutralizing anti-FGF2 monoclonal
antibodies, pharmaceutical compositions comprising them, and
methods of using them for the treatment of disease.
1. Antibodies
[0027] Antibodies are very large, complex molecules (molecular
weight of .about.150,000 or about 1320 amino acids) with intricate
internal structure. A natural antibody molecule contains two
identical pairs of polypeptide chains, each pair having one light
chain and one heavy chain. Each light chain and heavy chain in turn
consists of two regions: a variable ("V") region involved in
binding the target antigen, and a constant ("C") region that
interacts with other components of the immune system. The light and
heavy chain variable regions come together in 3-dimensional space
to form a variable region that binds the antigen (for example, a
receptor on the surface of a cell). Within each light or heavy
chain variable region, there are three short segments (averaging 10
amino acids in length) called the complementarity determining
regions ("CDRs"). The six CDRs in an antibody variable domain
(three from the light chain and three from the heavy chain) fold up
together in 3-D space to form the actual antibody binding site
which locks onto the target antigen. The position and length of the
CDRs have been precisely defined by Kabat, E. et al., Sequences of
Proteins of Immunological Interest, U.S. Department of Health and
Human Services, 1983, 1987. The part of a variable region not
contained in the CDRs is called the framework, which forms the
environment for the CDRs. Chothia et al., J. Mol. Biol. 196:901,
1987, have defined the related concept of hypervariable regions or
loops determined by structure.
[0028] A humanized antibody is a genetically engineered antibody in
which the CDRs from a mouse antibody ("donor antibody", which can
also be rat, hamster or other non-human species) are grafted onto a
human antibody ("acceptor antibody"). The sequence of the acceptor
antibody can be, for example, a mature human antibody sequence, a
consensus sequence of human antibody sequences or a germline
sequence. Thus, a humanized antibody is an antibody having CDRs
from a donor antibody and variable region framework and constant
regions from a human antibody. In addition, in order to retain high
binding affinity, at least one of two additional structural
elements can be employed. See, U.S. Pat. Nos. 5,530,101 and
5,585,089, incorporated herein by reference, which provide detailed
instructions for construction of humanized antibodies. A humanized
antibody typically has a humanized heavy chain and a humanized
light chain In general neither the heavy chain variable region
framework of a humanized heavy chain nor the light chain variable
region framework of a humanized light chain includes more than ten
or twelve substitutions resulting in residues not present in the
acceptor human heavy or light chain variable region framework
(including human consensus variable region frameworks and composite
human variable region frameworks).
[0029] Although humanized antibodies often incorporate all six
intact CDRs (as defined by Kabat) from a mouse antibody, they can
also be made with less than the complete CDRs from a mouse antibody
(e.g., Pascalis et al., J. Immunol. 169:3076, 2002). Numerous
antibodies have been described in the scientific literature in
which one or two CDRs can be dispensed with for binding. Padlan et
al., FASEB Journal 9: 133-139 (1995); Vajdos et al., Journal of
Molecular Biology, 320: 415-428 (2002); Iwahashi et al., Mol.
Immunol. 36:1079-1091, (1999); Tamura et al, Journal of Immunology,
164:1432-1441 (2000). Similarly, it may be necessary to incorporate
only part of the CDRs, namely the subset of CDR residues required
for binding, termed the SDRs, into the humanized antibody.
[0030] CDR residues not contacting antigen and not in the SDRs can
be identified based on previous studies (for example residues
H60-H65 in CDRH2 are often not required), from regions of Kabat
CDRs lying outside Chothia hypervariable loops, by molecular
modeling and/or empirically, or as described in Gonzales et al.,
Mol. Immunol. 41: 863, 2004. If a CDR or residue(s) thereof is
omitted, it is usually substituted with an amino acid occupying the
corresponding position in the human acceptor sequence supplying the
variable region framework sequences. The number of such
substitutions to include reflects a balance of competing
considerations. Such substitutions are potentially advantageous in
decreasing the number of mouse amino acids in a humanized antibody
and consequently decreasing potential immunogenicity. However,
substitutions can also cause changes of affinity, and significant
reductions in affinity are preferably avoided. Positions for
substitution within CDRs and amino acids to substitute can also be
selected empirically.
[0031] In the first structural element referred to above for
retaining high binding affinity in a humanized antibody, the
framework of the heavy chain variable region of the humanized
antibody is chosen to have maximal sequence identity (between 65%
and 95%) with the framework of the heavy chain variable region of
the donor antibody, by suitably selecting the acceptor antibody
from among the many known human antibodies. In the second
structural element, in constructing the humanized antibody,
selected amino acids in the framework of the human acceptor
antibody (outside the CDRs) are replaced with corresponding amino
acids from the donor antibody, in accordance with specified rules.
Specifically, the amino acids to be replaced in the framework are
chosen on the basis of their ability to interact with the CDRs. For
example, the replaced amino acids can be adjacent to a CDR in the
donor antibody sequence or within 4-6 angstroms of a CDR in the
humanized antibody as measured in 3-dimensional space.
[0032] A chimeric antibody is an antibody in which the variable
region of a mouse (or other rodent) antibody is combined with the
constant region of a human antibody; their construction by means of
genetic engineering is well-known. Such antibodies retain the
binding specificity of the mouse antibody, while being about
two-thirds human. The proportion of nonhuman sequence present in
mouse, chimeric and humanized antibodies suggests that the
immunogenicity of chimeric antibodies is intermediate between mouse
and humanized antibodies. Other types of genetically engineered
antibodies that may have reduced immunogenicity relative to mouse
antibodies include human antibodies made using phage display
methods (Dower et al., WO91/17271; McCafferty et al., WO92/001047;
Winter, WO92/20791; and Winter, FEBS Lett. 23:92, 1998, each of
which is incorporated herein by reference) or using transgenic
animals (Lonberg et al., WO93/12227; Kucherlapati WO91/10741, each
of which is incorporated herein by reference).
[0033] As used herein, the term "human-like" antibody refers to a
mAb in which a substantial portion of the amino acid sequence of
one or both chains (e.g., about 50% or more) originates from human
immunoglobulin genes. Hence, human-like antibodies encompass but
are not limited to chimeric, humanized and human antibodies. As
used herein, a mAb with "reduced immunogenicity" is one expected to
have significantly less immunogenicity than a mouse antibody when
administered to human patients. Such antibodies encompass chimeric,
humanized and human mAbs as well as mAbs made by replacing specific
amino acids in mouse antibodies that may contribute to B- or T-cell
epitopes, for example exposed residues (Padlan, Mol. Immunol.
28:489, 1991). As used herein, a "genetically engineered" mAb is
one for which the genes have been constructed or put in an
unnatural environment (e.g., human genes in a mouse or on a
bacteriophage) with the help of recombinant DNA techniques, and
would therefore, e.g., not encompass a mouse mAb made with
conventional hybridoma technology.
[0034] The epitope of a mAb is the region of its antigen to which
the mAb binds. Two antibodies bind to the same or overlapping
epitope if each competitively inhibits (blocks) binding of the
other to the antigen. That is, a 1.times., 5.times., 10.times.,
20.times. or 100.times. excess of one antibody inhibits binding of
the other by at least 50% but preferably 75%, 90% or even 99% as
measured in a competitive binding assay (see, e.g., Junghans et
al., Cancer Res. 50:1495, 1990). Alternatively, two antibodies have
the same epitope if they bind the same region of the antigen or if
essentially all amino acid mutations in the antigen that reduce or
eliminate binding of one antibody reduce or eliminate binding of
the other. Two antibodies have overlapping epitopes if some amino
acid mutations that reduce or eliminate binding of one antibody
reduce or eliminate binding of the other.
2. Neutralizing Anti-FGF2 Antibodies
[0035] A monoclonal antibody (mAb) that binds FGF2 (i.e., an
anti-FGF2 mAb) is said to neutralize FGF2, or be neutralizing, if
the binding partially or completely inhibits one or more biological
activities of FGF2 (i.e., when the mAb is used as a single agent).
Among the biological properties of FGF2 that a neutralizing
antibody may inhibit are the ability of FGF2 to bind to one or more
of the four FGF receptors, to stimulate proliferation of (i.e., be
mitogenic for) certain cells including fibroblasts, endothelial
cells, My 1 Lu mink lung epithelial cells, and various human tumor
cells; to stimulate differentiation and migration of cells such as
endothelial cells, or to stimulate angiogenesis, for example as
measured by stimulation of human vascular endothelial cell (HUVEC)
proliferation or tube formation or by induction of blood vessels
when applied to the chick embryo chorioallantoic membrane
(CAM).
[0036] A neutralizing mAb of the invention at a concentration of,
e.g., 0.01, 0.1, 0.5, 1, 2, 5, 10, 20 or 50 .mu.g/ml inhibits a
biological function of FGF2 by about at least 50% but preferably
75%, more preferably by 90% or 95% or even 99%, and most preferably
approximately 100% (essentially completely) as assayed by methods
described under Examples or known in the art. Typically, the extent
of inhibition is measured when the amount of FGF2 used is just
sufficient to fully stimulate the biological activity, or is 1, 2,
or 5 ng/ml or 0.01, 0.02, 0.05, 0.1, 0.5, 1, 3 or 10 .mu.g/ml.
Preferably, the mAb is neutralizing, i.e., inhibits the biological
activity, when used as a single agent, but optionally 2 mAbs can be
used together to give inhibition. Most preferably, the mAb
neutralizes not just one but two, three or several of the
biological activities listed above; for purposes herein, an
anti-FGF2 mAb that used as a single agent neutralizes all the
biological activities of FGF2 is called "fully neutralizing", and
such mAbs are most preferable. MAbs of the invention are preferably
specific for FGF2, that is they do not bind, or only bind to a much
lesser extent (e.g., at least 10-fold less), proteins that are
related to FGF2 such as the other FGFs, e.g., FGF1, and vascular
endothelial growth factor (VEGF). Some mAbs of the invention bind
both human FGF2 and mouse FGF2, or bind human FGF2 and one, two or
more or all of mouse, rat, rabbit, chicken, dog and/or monkey
(e.g., cynomolgus monkey) FGF2. Other mAbs are specific for human
FGF2. MAbs of the invention typically have a binding affinity (Ka)
for FGF2 of at least 107 M.sup.-1 but preferably 10.sup.8 M.sup.-1
or higher, and most preferably 10.sup.9 M.sup.-1 or higher or even
10.sup.10 M.sup.-1 or higher.
[0037] MAbs of the invention include anti-FGF2 antibodies in their
natural tetrameric form (2 light chains and 2 heavy chains) and may
be of any of the known isotypcs IgG, IgA, IgM, IgD and IgE and
their subtypes, i.e., human IgG1, IgG2, IgG3, IgG4 and mouse IgG1,
IgG2a, IgG2b, and IgG3. The mAbs of the invention are also meant to
include fragments of antibodies such as Fv, Fab and F(ab')2;
bifunctional hybrid antibodies (e.g., Lanzavecchia et al., Eur. J.
Immunol. 17:105, 1987), single-chain antibodies (Huston et al.,
Proc. Natl. Acad. Sci. USA 85:5879, 1988; Bird et al., Science
242:423, 1988); single-arm antibodies (Nguyen et al., Cancer Gene
Ther. 10:840, 2003); and antibodies with altered constant regions
(e.g., U.S. Pat. No. 5,624,821). The mAbs may be of animal (e.g.,
mouse, rat, hamster or chicken) origin, or they may be genetically
engineered. Rodent mAbs are made by standard methods well-known in
the art, comprising multiple immunization with FGF2 in appropriate
adjuvant i.p., i.v., or into the footpad, followed by extraction of
spleen or lymph node cells and fusion with a suitable immortalized
cell line, and then selection for hybridomas that produce antibody
binding to FGF2, e.g., see under Examples. Chimeric and humanized
mAbs, made by art-known methods mentioned supra, are preferred
embodiments of the invention. Human antibodies made, e.g., by phage
display or transgenic mice methods are also preferred (see e.g.,
Dower et al., McCafferty et al., Winter, Lonberg et al.,
Kucherlapati, supra).
[0038] The neutralizing anti-FGF2 mAb GAL-F2 described infra is an
example of the invention. Once a single, archetypal anti-human-FGF2
mAb, for example GAL-F2, has been isolated that has the desired
properties described herein of neutralizing FGF2, it is
straightforward to generate other mAbs with similar properties, by
using art-known methods. For example, mice may be immunized with
FGF2 as described above, hybridomas produced, and the resulting
mAbs screened for the ability to compete with the archetypal mAb
for binding to FGF2. Mice can also be immunized with a smaller
fragment of FGF2 containing the epitope to which GAL-F2 binds. The
epitope can be localized by, e.g., screening for binding to a
series of overlapping peptides spanning FGF2. Alternatively, the
method of Jespers et al., Biotechnology 12:899, 1994, which is
incorporated herein by reference, may be used to guide the
selection of mAbs having the same epitope and therefore similar
properties to the archetypal mAb, e.g., GAL-F2. Using phage
display, first the heavy chain of the archetypal antibody is paired
with a repertoire of (preferably human) light chains to select an
FGF2-binding mAb, and then the new light chain is paired with a
repertoire of (preferably human) heavy chains to select a
(preferably human) FGF2-binding mAb having the same epitope as the
archetypal mAb. Alternatively variants of GAL-F2 can be obtained by
mutagenesis of cDNA encoding the heavy and light chains of GAL-F2
obtained from the hybridoma.
[0039] Neutralizing mAbs with the same or overlapping epitope as
GAL-F2, e.g., that compete for binding to FGF2 with GAL-F2, provide
other examples. A chimeric or humanized form of GAL-F2 is an
especially preferred embodiment. MAbs that are 90%, 95% or 99%
identical to GAL-F2 in amino acid sequence of the heavy and/or
light chain variable regions and maintain its functional
properties, and/or which differ from it by a small number of
functionally inconsequential amino acid substitutions (e.g.,
conservative substitutions), deletions, or insertions are also
included in the invention. MAbs having at least one and preferably
all six CDR(s) that are 90%, 95% or 99% or 100% identical to
corresponding CDRs of GAL-F2 are also included. Here, as elsewhere
in this application, percentage sequence identities are determined
with antibody sequences maximally aligned by the Kabat numbering
convention. After alignment, if a subject antibody region (e.g.,
the entire mature variable region of a heavy or light chain) is
being compared with the same region of a reference antibody, the
percentage sequence identity between the subject and reference
antibody regions is the number of positions occupied by the same
amino acid in both the subject and reference antibody region
divided by the total number of aligned positions of the two
regions, with gaps not counted, multiplied by 100 to convert to
percentage.
[0040] Native mAbs of the invention may be produced from their
hybridomas. Genetically engineered mAbs, e.g., chimeric or
humanized mAbs, may be expressed by a variety of art-known methods.
For example, genes encoding their light and heavy chain V regions
may be synthesized from overlapping oligonucleotides and inserted
together with available C regions into expression vectors (e.g.,
commercially available from Invitrogen) that provide the necessary
regulatory regions, e.g., promoters, enhancers, poly A sites, etc.
Use of the CMV promoter-enhancer is preferred. The expression
vectors may then be transfected using various well-known methods
such as lipofection or electroporation into a variety of mammalian
cell lines such as CHO or non-producing myclomas including Sp2/0
and NSO, and cells expressing the antibodies selected by
appropriate antibiotic selection. See, e.g., U.S. Pat. No.
5,530,101. Larger amounts of antibody may be produced by growing
the cells in commercially available bioreactors.
[0041] Once expressed, the mAbs or other antibodies of the
invention may be purified according to standard procedures of the
art such as microfiltration, ultrafiltration, protein A or G
affinity chromatography, size exclusion chromatography, anion
exchange chromatography, cation exchange chromatography and/or
other forms of affinity chromatography based on organic dyes or the
like. Substantially pure antibodies of at least about 90 or 95%
homogeneity are preferred, and 98% or 99% or more homogeneity most
preferred, for pharmaceutical uses.
3. Treatment Methods
[0042] The invention provides methods of treatment in which the mAb
of the invention (e.g., anti-FGF2) is administered to patients
having a disease (therapeutic treatment) or at risk of occurrence
or recurrence of a disease (prophylactic treatment). The term
"patient" includes human patients; veterinary patients, such as
cats, dogs and horses; farm animals, such as cattle, sheep, and
pigs; and laboratory animals used for testing purposes, such as
mice and rats. The methods are particularly amenable to treatment
of human patients. The mAb used in methods of treating human
patients binds to the human FGF2 protein, the sequence of which is
provided by, e.g., Ornitz et al., Genome Biol. 2: 3005.1, 2001 or
Okada-Ban et al., Int. J. Biochem. Cell. Biol. 32:263, 2000, also
Locus P09038 of Swiss-Prot database. A mAb to a human protein can
also be used in other species in which the species homolog has
antigenic crossreactivity with the human protein. In species
lacking such crossreactivity, an antibody is used with appropriate
specificity for the species homolog present in that species.
However, in xenograft experiments in laboratory animals, a mAb with
specificity for the human protein expressed by the xenograft is
generally used.
[0043] In a preferred embodiment, the present invention provides a
pharmaceutical formulation comprising the antibodies described
herein. Pharmaceutical formulations of the antibodies contain the
mAb in a physiologically acceptable carrier, optionally with
excipients or stabilizers, in the form of lyophilized or aqueous
solutions. Acceptable carriers, excipients or stabilizers are
nontoxic to recipients at the dosages and concentrations employed,
and include buffers such as phosphate, citrate, or acetate at a pH
typically of 5.0 to 8.0, most often 6.0 to 7.0; salts such as
sodium chloride, potassium chloride, etc. to make isotonic;
antioxidants, preservatives, low molecular weight polypeptides,
proteins, hydrophilic polymers such as polysorbate 80, amino acids,
carbohydrates, chelating agents, sugars, and other standard
ingredients known to those skilled in the art (Remington's
Pharmaceutical Science 16th edition, Osol, A. Ed. 1980). The mAb is
typically present at a concentration of 1-100 mg/ml, e.g., 10
mg/ml.
[0044] In another preferred embodiment, the invention provides a
method of treating a patient with a disease using an anti-FGF2 mAb
in a pharmaceutical formulation. The mAb prepared in a
pharmaceutical formulation can be administered to a patient by any
suitable route, especially parentally by intravenous infusion or
bolus injection, intramuscularly or subcutaneously. Intravenous
infusion can be given over as little as 15 minutes, but more often
for 30 minutes, or over 1, 2 or even 3 hours. The mAb can also be
injected directly into the site of disease (e.g., a tumor), or
encapsulated into carrying agents such as liposomes. The dose given
is sufficient at least partially to alleviate the condition being
treated ("therapeutically effective dose") and is optionally 0.1 to
5 mg/kg body weight, for example 1, 2, 3 or 4 mg/kg, but may be as
high as 10 mg/kg or even 15, 20 or 30 mg/kg. A fixed unit dose may
also be given, for example, 100, 200, 500, 1000 or 2000 mg, or the
dose may be based on the patient's surface area, e.g., 1000
mg/m.sup.2. Usually between 1 and 8 doses, (e.g., 1, 2, 3, 4, 5, 6,
7 or 8) are administered to treat cancer, but 10, 20 or more doses
may be given. The mAb can be administered daily, biweekly, weekly,
every other week, monthly or at some other interval, depending,
e.g. on the half-life of the mAb, for 1 week, 2 weeks, 4 weeks, 8
weeks, 3-6 months or longer or until the disease progresses.
Repeated courses of treatment are also possible, as is chronic
administration.
[0045] A combination of a dose, frequency of administration and
route of administration effective to at least partially alleviate a
disease present in a patient being treated is referred to as
therapeutically effective regime. A combination of a dose,
frequency of administration and route of administration effective
to inhibit or delay onset of a disease in a patient is referred to
as a prophylactically effective regime.
[0046] Diseases especially susceptible to treatment with the
anti-FGF2 mAbs of this invention include solid tumors believed to
require angiogenesis, or to be associated with elevated levels of
FGF2, or to be associated with expression of FGF2. Such tumors, for
which treatment with the anti-FGF2 mAb is appropriate, include for
example ovarian cancer, breast cancer, lung cancer (small cell or
non-small cell), colon cancer, prostate cancer, cervical cancer,
endometrial cancer, pancreatic cancer, gastric cancer,
hepatocellular carcinoma or hepatoma (liver cancer), head-and-neck
tumors, melanoma, sarcomas, carcinomas, and brain tumors (e.g.,
gliomas such as glioblastomas). Hematologic malignancies such as
leukemias and lymphomas and multiple myeloma can also be
susceptible to such treatment. Other diseases associated with
angiogenesis for which treatment with the anti-FGF mAbs of the
invention are suitable include age-related macular degeneration
(AMD), diabetic retinopathy, neovascular glaucoma and other
diseases of the eye; psoriasis and other diseases of the skin; and
rheumatoid arthritis.
[0047] In a preferred embodiment, the anti-FGF2 mAb is administered
in combination with (i.e., together with, that is, before, during
or after) other therapy. For example, to treat cancer, the
anti-FGF2 mAb may be administered together with any one or more of
the known chemotherapeutic drugs, for example alkylating agents
such as carmustine, chlorambucil, cisplatin, carboplatin,
oxaliplatin, procarbazine, and cyclophosphamide; antimetabolites
such as fluorouracil, floxuridine, fludarabine, gemcitabine,
methotrexate and hydroxyurea; natural products including plant
alkaloids and antibiotics such as bleomycin, doxorubicin,
daunorubicin, idarubicin, etoposide, mitomycin, mitoxantrone,
vinblastine, vincristine, and Taxol (paclitaxel) or related
compounds such as Taxotere.RTM.; the topoisomerase 1 inhibitor
irinotecan; agents specifically approved for brain tumors including
temozolomide and Gliadel.RTM. wafer containing carmustine; and
inhibitors of tyrosine kinases such as Gleevec.RTM. (imatinib
mesylate), Sutent.RTM. (sunitinib malate), Nexavar.RTM.
(sorafenib), Tarceva.RTM. (erlotinib) and Iressa.RTM. (gefitinib);
inhibitors of angiogenesis; and all approved and experimental
anti-cancer agents listed in WO 2005/017107 A2 (which is herein
incorporated by reference). The anti-FGF2 mAb may be used in
combination with 1, 2, 3 or more of these other agents used in a
standard chemotherapeutic regimen. Normally, the other agents are
those already known to be effective for the particular type of
cancer being treated. The anti-FGF2 mAb is especially useful in
overcoming resistance to chemotherapeutic drugs and thereby
increasing their effectiveness (see Song et al. Proc. Natl. Acad.
Sci. USA 97:8658, 2000).
[0048] Other agents with which the anti-FGF2 mAb can be
administered to treat cancer include biologics such as monoclonal
antibodies, including Herceptin.TM. against the HER2 antigen;
Avastin.RTM. against VEGF; or antibodies to the Epidermal Growth
Factor (EGF) receptor such as Erbitux.RTM. (cetuximab) and
Vectibix.RTM. (panitumumab). Antibodies against Hepatocyte Growth
Factor (HGF) are especially preferred for use with the anti-FGF2
mAb, including mAb L2G7 (Kim et al., Clin Cancer Res 12:1292, 2006
and U.S. Pat. No. 7,220,410) and particularly its chimeric and
humanized forms such as HuL2G7 (WO 07115049 A2); the human anti-HGF
mAbs described in WO 2005/017107 A2, particularly 2.12.1; and the
HGF binding proteins described in WO 07143090 A2 or WO 07143098 A2;
and other neutralizing anti-HGF mAbs that compete for binding with
any of the aforementioned mAbs. A mAb that binds the cMet receptor
of HGF is also preferred, for example the anti-cMet mAb OA-5D5
(Martens et al., Clin. Cancer Res. 12:6144, 2006) that has been
genetically engineered to have only one "arm", i.e. binding domain.
Moreover, the anti FGF2 mAb can be used together with any form of
surgery and/or radiation therapy including external beam radiation,
intensity modulated radiation therapy (IMRT) and any form of
radiosurgery such as, e.g. Gamma Knife.
[0049] Treatment (e.g., standard chemotherapy) including the
anti-FGF2 mAb antibody may alleviate a disease by increasing the
median progression-free survival or overall survival time of
patients with cancer by at least 30% or 40% but preferably 50%, 60%
to 70% or even 100% or longer, compared to the same treatment
(e.g., chemotherapy) but without the anti-FGF2 mAb. In addition or
alternatively, treatment (e.g., standard chemotherapy) including
the anti-FGF2 mAb may increase the complete response rate, partial
response rate, or objective response rate (complete+partial) of
patients with these tumors (e.g., ovarian, breast, lung, colon and
glioblastomas especially when relapsed or refractory) by at least
30% or 40% but preferably 50%, 60% to 70% or even 100% compared to
the same treatment (e.g., chemotherapy) but without the anti-FGF2
mAb.
[0050] Typically, in a clinical trial (e.g., a phase II, phase
II/III or phase III trial), the aforementioned increases in median
progression-free survival and/or response rate of the patients
treated with chemotherapy plus the anti-FGF2 mAb, relative to the
control group of patients receiving chemotherapy alone (or plus
placebo), are statistically significant, for example at the p=0.05
or 0.01 or even 0.001 level. The complete and partial response
rates are determined by objective criteria commonly used in
clinical trials for cancer, e.g., as listed or accepted by the
National Cancer Institute and/or Food and Drug Administration.
4. Other Methods
[0051] The anti-FGF2 mAbs of the invention also find use in
diagnostic, prognostic and laboratory methods. They may be used to
measure the level of FGF2 in a tumor or in the circulation of a
patient with a tumor, to determine if the level is measurable or
even elevated, and therefore to follow and guide treatment of the
tumor, since tumors associated with measurable or elevated levels
of FGF2 will be most susceptable to treatment with the anti-FGF2
mAb. For example, a tumor associated with high levels of FGF2 would
be especially susceptible to treatment with an anti-FGF2 mAb. In
particular embodiments, the mAbs can be used in an ELISA or
radioimmunoassay to measure the level of FGF2, e.g., in a tumor
biopsy specimen or in serum or in media supernatant of
FGF2-secreting cells in cell culture. The use of two anti-FGF2 mAbs
binding to different epitopes (i.e., not competing for binding)
will be especially useful in developing a sensitive "sandwich"
ELISA to detect FGF2. For various assays, the mAb may be labeled
with fluorescent molecules, spin-labeled molecules, enzymes or
radioisotopes, and may be provided in the form of kit with all the
necessary reagents to perform the assay for FGF2. In other uses,
the anti-FGF2 mAbs will be used to purify FGF2, e.g., by affinity
chromatography.
EXAMPLES
Example 1
Reagents and Assays
[0052] Preparation of GST-FGF2, FGF2-Fc and FGF2-Flag. The cDNA
sequence for human FGF2 (the precursor form with 155 amino acids;
Sommer et al., 1987) was synthesized (GenScript, Inc), PCR
amplified and cloned into a derivative of the pDisplay vector
(Invitrogen). These plasmids were transformed into E. coli
BL21(DE3) cells and FGF2 expression was induced using 1 mM IPTG.
The level of FGF2 expression was determined using an FGF2 specific
ELISA Kit (R&D Systems), and FGF2 was purified using
heparin-Sepharose CL-6B beads (Amersham Biosciences) as described
(Wiedlocha et al., Mol. Cell. Biol. 16:270, 1996). Fusion proteins
of FGF2 with respectively glutamine synthetase (GST-FGF2), Flag
peptide (FGF2-Flag), and human IgG1 Fc domain (amino acids 216 to
446; FGF2-Fc), which were used in the FGF2/FGFR binding assays as
well as for immunization, were produced similarly from the
appropriate genetic constructs using standard molecular biology
techniques. GST-FGF2 was purified using an anti-GST column, FGF2-Fc
was purified using a protein A/G column, and FGF-Flag was used in
culture supernatant after determination of the FGF2-Flag
concentration. In addition, purified human FGF2 (QED Bioscience
Inc.) and murine FGF2 (ProSpec-Tany Technogene Ltd.) were
purchased.
[0053] Preparation of FGFR-Fc proteins. The extracellular domain
(ECD) of human FGFR1 alpha (IIIc) (designated as FGFR1c) and human
FGFR2c alpha (IIIc) (designated as FGFR2c) were expressed as
immunoadhesin molecules. DNA fragments encoding the entire ECD of
FGFR1c and FGFR2c were fused to human Fc via a polypeptide linker.
These FGFR-Fc molecules were expressed by transfecting human 293F
cells and selecting stable 293 transfectants in the presence of
G418 (1 mg/ml) in 293 expression medium (Invitrogen). The FGFR-Fc
secreted from 293F transfected cells was purified using a protein
A/G column. In addition, human FGFR3c-Fc and human FGFR4-Fc fusion
were obtained from R & D Systems.
[0054] Synthesis of FGF2 fragments. Peptides consisting of amino
acid residues 29-44 (Peptide #1), 101-118 (Peptide #3) and 137-155
(peptide #4) of FGF2 were synthesized by SynBioSci. Extra cysteine
residues were added to the c-terminus of Peptide #1 and #3 and the
N-terminus of peptide #4. These peptide fragments were then
conjugated to keyhole limpet hemocyanin (KLH).
[0055] FGF2 binding ELISA. ELISA wells were coated with 50 .mu.g/ml
of heparin (Sigma) overnight at 4.degree. C. and then incubated
with 0.3-1 .mu.g/ml of either human or mouse FGF2 for 1 hr at room
temperature (RT) so that the heparin could capture the FGF2. After
blocking with 2% BSA for 1 hr at RT, hybridoma culture supernatants
or purified mAbs were added for 1 hr at RT. The bound anti-FGF2
antibodies were detected by the addition of HRP-goat anti-mouse IgG
Fc for 1 hr at RT, followed by washing, addition of TMB substrate
(Sigma) and reading at 450 nm.
[0056] FGFR-Fc/FGF2-Flag Binding ELISA. The blocking activity of
anti-FGF2 mAbs was determined in the FGFR-Fc/FGF2-Flag binding
ELISA. ELISA wells were coated with 2 .mu.g/ml of goat antibody
specific for human IgG-Fc overnight at 4 C. After blocking with 2%
BSA for 1 hr at RT, the wells were incubated with 0.5 .mu.g/ml of
FGFR1c-Fc, FGFR2c-Fc, FGFR3c-Fc or FGFR4-Fc for 1 hr. After
washing, wells were incubated with FGF2-Flag (0.2 .mu.g/ml) in the
presence of various concentrations of mAbs for 1 hr. The bound
FGF2-Flag was detected by the addition of HRP-anti-Flag M2 antibody
(Sigma).
Example 2
Generation of Monoclonal Antibodies
[0057] Balb/c mice (5-6 week old female) were immunized by
injection in their rear footpads 14 times at 1 week intervals with
GST-FGF2, FGF2-Fc and/or KLH conjugated FGF2 synthetic peptides,
resuspended in MPL/TDM (Sigma-Aldrich), as described in the table
below. Three days after the final injection, popliteal lymphoid
cells were fused with P3/X63-Ag8U1 mouse myeloma cells using
standard fusion methods with 35% polyethylene glycol as described
(Chuntharapai et al., Methods Enzymol 288:15, 1997). Ten days after
the fusion, hybridoma culture supernatants were screened for their
ability to bind to FGF2 using the FGF2 binding ELISA described
above. Selected mAbs were then screened for their blocking
activities in the FGFR1-Fc/FGF-Flag binding ELISA. Selected
hybridomas were then cloned twice using the limiting dilution
technique as described (Harlow et al., 1988).
TABLE-US-00001 TABLE Immunization protocol Injection No. Antigens
(in MPL/TDM) per footpad 1 GST-FGF2 10 .mu.g 2 GST-FGF2 5 .mu.g 3
GST-FGF2 2.5 .mu.g 4-7 GST-FGF2 5 .mu.g 8-9 GST-FGF2 10 .mu.g 10
GST-FGF2 (5 .mu.g) + FGF2-Fc (5 .mu.g) 11 FGF2-Fc (2 .mu.g) +
Peptide #1 & # 3 (2 .mu.g) 12 FGF2-Fc (2 .mu.g) + Peptide #1,
#3 & #4 (3 .mu.g) 13 FGF2-Fc (2 .mu.g) + Peptide #1, #3 &
#4 (3 .mu.g) 14 FGF2-Fc 2 .mu.g
[0058] FIG. 1 shows that the mAb GAL-F2 generated in this way binds
to both human and mouse FGF2, approximately equally well, whereas a
negative control mouse IgG mAb does not bind to FGF2. FIG. 2 shows
that GAL-F2 but not the control mAb 5G8 is able to inhibit binding
of human FGF2 to each of the four FGF receptors FGFR1-4. In another
experiment using the FGFR-Fc/FGF2-Flag Binding ELISA described
above, GAL-F2 (at a concentration of 10 .mu.g/ml) completely
inhibited the binding of FGF2 to each of the four FGFRs--FGFR 1,
FGFR2, FGFR3 and FGFR4--that is, reduced the signal to background
level within experimental error, while the anti-FGF2 mAbs bFM-1 and
3H3 reduced binding substantially but not completely.
Example 3
Epitope of GAL-F2
[0059] Several commercially available anti-FGF2 mAbs that have been
reported to have certain neutralizing activities--3H3 (Calbiochem),
mAb bFM-1 (Millipore) and mAb FB-8 (Abcam)--were purchased. A
competitive binding ELISA was performed to determine if GAL-F2 has
the same epitope as any of these mAbs. ELISA plates were coated
with 50 .mu.l/well of heparin (50 .mu.g/ml) overnight, decanted and
incubated with 50 .mu.l/well of FGF2 for 1 hr. After blocking with
2% BSA, wells were incubated with 0.5 .mu.g/ml of biotinylated
GAL-F2 mAb in the presence of various concentrations of each
anti-FGF2 mAb to be tested. The level of biotinylated GAL-F2
binding to the FGF2 in each well was detected by the addition of
HRP-streptavidin followed by TMB substrate. FIG. 3 shows that none
of the mAbs 3H3, bFM-1 and FB-8 nor a negative control mouse mAb
(mIgG) competed for binding to FGF2 with GAL-F2, while of course
GAL F2 competed with itself. Hence no previous anti-FGF2 mAb tested
has the same or overlapping epitope on FGF2 as GAL-F2.
Example 4
Inhibition of FGF2-Induced Proliferation by GAL-F2
[0060] BaF3 cells (DSMZ, Germany) are maintained in RPMI 1640
medium (GIBCO) supplemented with 10% FCS, 10% WEHI-3 conditioned
medium and antibiotics. Stable FGFR1c or FGFR2c expressing Ba/F3
cells were generated by electroporation of linearized FGFR1c and
FGFR2c expression vector DNAs as described (Ornitz et al., 1996).
Stable transfected cells are selected using G418 (600 .mu.g/ml) for
10 days. The stable Ba/F3 transfectants expressing FGFR1c or FGFR2c
were shown to proliferate in response to FGF2 in the absence of
WEHI-3 medium. To determine the blocking (neutralizing) activity of
GAL-F2 mAb, the FGFR1c- or FGFR2c-expressing BaF3 cells (10,000
cells/well) were washed and resuspended in RPMI with 10% FCS plus 2
.mu.g/ml of heparin and 20 ng/ml of FGF-2 in the presence of
various concentrations of GAL-F2. After incubation for 36-48 hrs at
37.degree. C., 5% CO.sub.2, the level of proliferation was
determined by the addition of WST-1 (Roche Applied Science) for 2
hrs. FIG. 4 shows that GAL F2 inhibited FGF2-induced proliferation,
almost completely at 10 .mu.g/ml mAb.
[0061] The blocking activities GAL-F2 on normal epithelial cells
were determined using Mv 1 Lu mink lung epithelial cells (CCL-64
from ATCC). Mv 1 Lu cells (2.times.103 cells/100 .mu.l/well) grown
in DMEM containing 10% FCS were resuspended in serum-free DMEM and
stimulated with 1-2 ng/ml of FGF2, plus 1 ng/ml of TGF-.beta. to
inhibit background proliferation, in the presence of various
concentrations of mAb for 24 hrs. The level of cell proliferation
was determined by the addition of WST-1 (Roche Applied Science) for
24-48 hr. FIG. 5 shows that GAL-F2 inhibited FGF2-induced
proliferation of the Mv 1 Lu cells, almost completely at 1 .mu.g/ml
mAb. In a similar experiment, GAL-F2 inhibited proliferation of
HUVEC induced by 10 ng/ml FGF2, by about 75% at a concentration of
0.1 .mu.g/ml (i.e., at an approximately equimolar ratio to FGF2)
and completely at a concentration of 1.0 .mu.g/ml. This was
somewhat better than the extent of inhibition by the bFM-1
anti-FGF2 mAb and substantially better than by the 3H3 anti-FGF2
mAb, and suggests that GAL-F2 inhibits angiogenesis, since
proliferation of endothelial cells is an essential step in
angiogenesis.
Example 5
Clonogenic Assay
[0062] The anti-tumor activity of GAL-F2 was investigated in vitro
by its effect on the colony formation of human tumor cells in soft
agar. The assay was performed as follows: E-well plates was coated
with 1.5 ml/well of 0.6% agar in DMEM containing 10% FCS and then
layered with 1.5 ml of 0.3% agar in DMEM with 10% FCS. The top agar
layer was mixed with 2.times.10.sup.4 SMMC-7721 human hepatoma
tumor cells plus 10 .mu.g/ml of mAb. Plates were incubated at
37.degree. C. in a humidified incubator for 10 to 14 days and then
stained with 0.005% crystal violet for 1 hr and examined by
photomicroscopy. FIG. 6 shows that compared to cells in the
presence of irrelevant control mouse IgG mAb, cells in the presence
of GAL-F2 formed many fewer colonies, while the bFM-1 anti-FGF2 mAb
did not reduce the number of colonies. Hence, GAL-F2 inhibited
colony formation in soft agar of human tumor cells.
Example 6
Angiogenesis Assay
[0063] The anti-angiogenic activity of GAL-F2 was determined in
BALB/c mice injected in the back with 0.4 ml of matrigel (BD
Biosciences) with or without 30 ng of FGF2 and/or 3 .mu.g GAL-F2.
Matrigel plugs were harvested for photography on day 6. FGF2
stimulated the formation of blood vessels in this assay, while
GAL-F2 at least partially inhibited this stimulation.
Example 7
Xenograft Models
[0064] Xenograft experiments are carried out as described
previously (Kim et al., Nature 362:841, 1993). Human tumor cells
typically grown in complete DMEM medium are harvested in HBSS.
Female athymic nude mice or NIH-III Xid/Beige/nude mice (4-6 wks
old) are injected subcutaneously with 2-10.times.10.sup.6 cells in
0.1 ml of HBSS in the dorsal areas. When the tumor size reaches
50-100 mm.sup.3, the mice are grouped randomly and 5 mg/kg (100
.mu.g total) of mAbs are administered i.p. twice per week in a
volume of 0.1 ml. Tumor sizes are determined twice a week by
measuring in two dimensions [length (a) and width (b)]. Tumor
volume is calculated according to V=ab2/2 and expressed as mean
tumor volume.+-.SEM. The number of mice in each treatment group is
typically 5-7 mice. Statistical analysis can be performed, e.g.,
using Student's t test.
[0065] FIG. 7 shows that GAL-F2 strongly inhibited the growth of
RPMI 4788 colon tumor xenografts, and FIG. 8 shows that the bFM-1
anti-FGF2 mAb inhibited xenograft growth to a lesser extent than
GAL-F2. FIG. 9 shows that GAL-F2 inhibited SMMC-7721 hepatoma tumor
xenografts in Xid/Beige/nude mice, while the chemotherapeutic drug
cisplatin at 5 mg/kg once per week inhibited xenograft growth to a
lesser extent. The combination of GAL-F2 and cisplatin inhibited
more strongly than either agent alone, showing an additive or
synergistic effect of these agents. FIG. 10 shows that GAL-F2
inhibited HepG2 hepatoma tumor xenografts in nude mice more
strongly than the anti-EGF receptor mAb M225 from which
Erbitux.RTM. was derived, but the combination of GAL-F2 and M225
inhibited somewhat more strongly than either agent, again showing
an additive or synergistic effect of these agents. However, GAL F2
was not able to inhibit the growth of all xenografts tested,
possibly because they are not dependent on FGF2 for their
growth.
Example 8
Humanization of GAL-F2
[0066] Cloning of the light and heavy chain variable regions of the
GAL-F2 mAb, construction and expression of a chimeric mAb, and
design, construction, expression and purification of a humanized
GAL-F2 mAb were all performed using standard methods of molecular
biology, e.g. as described in U.S. patent application Ser. No.
11/731,774 for the L2G7 mAb, which is herein incorporated by
reference for all purposes. The amino acid sequences of the
(mature) light and heavy chain variable (V) regions of GAL-F2 are
shown respectively in FIGS. 11A and 11B, top lines labeled GAL-F2.
More specifically, to design a humanized GAL-F2 mAb, the methods of
Queen et al., U.S. Pat. Nos. 5,530,101 and 5,585,089 were generally
followed. The human V.kappa. sequence ABA70776 and VH sequence
AAL04519, as shown respectively in FIGS. 11A and 11B, bottom lines,
were respectively chosen to serve as acceptor sequences for the
GAL-F2 VL and VH sequences because they have particularly high
framework homology (i.e., sequence identity) to them. A
computer-generated molecular model of the GAL-F2 variable domain
was used to locate the amino acids in the GAL-F2 framework that are
close enough to the CDRs to potentially interact with them. To
design the humanized GAL-F2 light and heavy chain variable regions,
the CDRs from the mouse GAL-F2 mAb were first conceptually grafted
into the acceptor framework regions. At framework positions where
the computer model suggested significant contact with the CDRs,
which may be needed to maintain the CDR conformation, the amino
acids from the mouse antibody were substituted for the human
framework amino acids. For the humanized GAL-F2 mAb designated
HuGAL-F2, this was done at residues 1, 27 and 30 (residues 27 and
30 being within Chothia hypervariable loop H1), 48, 67, 71 and 94
of the heavy chain and at no residues in the light chain, using
Kabat numbering. The light and heavy chain V region sequences of
HuGAL-F2 are shown in FIGS. 11A and 11B respectively, middle lines
labeled HuGAL-F2, where they are aligned against the respective
GAL-F2 donor and human acceptor V regions--the CDRs (as defined by
Kabat) are underlined and the substituted amino acids listed above
are double-underlined.
[0067] The invention provides not only a humanized GAL-F2 mAb
HuGAL-F2 including the light and heavy chain V regions shown in
FIG. 11, but also variant humanized GAL-F2 mAbs whose light and
heavy chain variable regions differ from the sequences of HuGAL-F2
by a small number (e.g., typically no more than 1, 2, 3, 5 or 10)
of replacements, deletions or insertions, usually in the framework
but possibly in the CDRs. In particular, only a subset of the
substitutions described above can be made in the acceptor
frameworks, or additional substitution(s) can be made, e.g., the
mouse GAL-F2 VH amino acid 69L may replace the acceptor amino acid
691, or the mouse amino acids may replace the respective amino
acids in the humanized light chain at any or all of the positions
1, 3, 60 and/or 67 by Kabat numbering. On the other hand, the VH
amino acid 1E (Glu) may instead be Q (Gln). Indeed, many of the
framework residues not in contact with the CDRs in HuGAL-F2 can
accommodate substitutions of amino acids from the corresponding
positions of GAL-F2 or other mouse or human antibodies, and even
many potential CDR-contact residues are also amenable to
substitution or even amino acids within the CDRs may be altered.
One example of a CDR substitution is to substitute a residue in a
CDR with the residue occupying the corresponding position of the
human acceptor sequence used to supply variable region
frameworks.
[0068] Most often the replacements made in the variant humanized
GAL-F2 sequences are conservative with respect to the replaced
HuGAL-F2 amino acids. Amino acids can be grouped as follows for
determining conservative substitutions, i.e., substitutions within
a group: Group I (hydrophobic sidechains): met, ala, val, leu, ile;
Group II (neutral hydrophilic side chains): cys, ser, thr; Group
III (acidic side chains): asp, glu; Group IV (basic side chains):
asn, gln, his, lys, arg; Group V (residues influencing chain
orientation): gly, pro; and Group VI (aromatic side chains): trp,
tyr, phe.
[0069] Preferably, replacements in HuGAL-F2 (whether or not
conservative) have no substantial effect on the binding affinity or
potency of the humanized mAb, that is, its ability to neutralize
the biological activities of FGF2 (e.g., the potency in some or all
of the assays described herein of the variant humanized GAL-F2 mAb
is essentially the same, i.e., within experimental error, as that
of HuGAL-F2). Preferably the mature variant light and heavy chain V
region sequences are at least 90%, more preferably at least 95%,
and most preferably at least 98% identical to the respective
HuGAL-F2 mature light and heavy chain V regions. Alternatively,
other human antibody variable regions with high sequence identity
to those of GAL-F2 are also suitable to provide the humanized
antibody framework, especially kappa V regions from human subgroup
I and heavy chain V regions from human subgroup I, or consensus
sequences of these subgroups.
[0070] In other humanized antibodies, at least 1, 2, 3, 4, 5, 6 or
all seven of the positions of acceptor to donor substitutions
mentioned in connection with the exemplified antibody (i.e., H1,
H27, H30, H48, H67, H71 and 94) are preferably occupied by the
residue occupying the corresponding position of the mouse donor
antibody heavy chain. If the heavy chain acceptor sequence is other
than AAL04519 an acceptor to donor substitution may or may not be
required for the specified occupancy of a particular variable
framework region position depending whether the residue occupying
the specified position is already the same between the acceptor and
donor.
[0071] The exemplary mAb HuGAL-F2 discussed here has human .kappa.
and .gamma.1 constant regions, e.g., as presented in U.S. patent
application Ser. No. 11/731,774, and is therefore an IgG1. The
complete sequences of the (mature) light and heavy chains of
HuGAL-F2 are shown in FIG. 13. Thus, an exemplary humanized
antibody comprises a light chain of SEQ ID NO:7 and a heavy chain
of SEQ ID NO:8. While these sequences are respectively of the Km(3)
and G1m(3) allotypes, it is understood that IgG1 mAbs of any (IgG1,
.kappa.) allotype are encompassed by the designation HuGAL-F2. It
will also be understood that when HuGAL-F2 is manufactured by
conventional procedures, one to several amino acids at the amino or
carboxy terminus of the light and/or heavy chain, such as the
C-terminal lysine of the heavy chain, may be missing or derivatized
in a proportion or all of the molecules, and such a composition
will still be encompassed by the designation HuGAL-F2 and
considered a humanized GAL-F2 mAb. Humanized mAbs of other isotypes
(e.g., IgG2, IgG3 and IgG4) can be made by combining the HuGAL-F2
variable regions with the appropriate human constant regions.
Replacements can be made in the HuGAL-F2 constant regions to reduce
or increase effector function such as complement-mediated
cytotoxicity or ADCC (see, e.g., Winter et al., U.S. Pat. No.
5,624,821; Tso et al., U.S. Pat. No. 5,834,597; and Lazar et al.,
Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to prolong half-life
in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213,
2004). Specifically but without limitation, HuGAL-F2 having
mutations in the IgG constant region to a Gln at position 250
and/or a Leu at position 428 are embodiments of the present
invention.
[0072] To compare the binding affinity of HuGAL-F2 with that of the
mouse-human chimeric mAb ChGAL-F2, a competitive binding experiment
was performed using standard ELISA technology. Specifically, human
FGF2 was immobilized on a heparin-coated ELISA plate. The wells
were incubated with biotinylated GAL-F2 mAb (0.5 .mu.g/ml) in the
presence of increasing concentrations of unlabeled ChGAL-F2,
HuGAL-F2 or control human antibody hIgG. The level of biotinylated
GAL-F2 bound was determined by the addition of HRP-streptavidin and
substrate. As shown in FIG. 12, HuGAL-F2 and ChGAL-F2 competed
approximately equally well, with HuGAL-F2 possibly slightly better,
indicating that the binding affinity for FGF2 of HuGAL-F2 is at
least as high as ChGAL-F2 and therefore as the original mouse
GAL-F2 mAb. From the concentration of HuGAL-F2 required to inhibit
binding of the labeled mAb by 50%, one may estimate that the
binding affinity Ka of HuGAL-F2 for FGF2 is at least approximately
10.sup.9 M.sup.-1. HuGAL-F2 may also be tested in any of the
biological assays for FGF2 activity described herein, and will
inhibit FGF2 activity comparably to the GAL-F2 mAb.
[0073] Although the invention has been described with reference to
the presently preferred embodiments, it should be understood that
various modifications can be made without departing from the
invention. Unless otherwise apparent from the context any step,
element, embodiment, feature or aspect of the invention can be used
with any other. All publications, patents and patent applications
including accession numbers and the like cited are herein
incorporated by reference in their entirety for all purposes to the
same extent as if each individual publication, patent and patent
application was specifically and individually indicated to be
incorporated by reference in its entirety for all purposes. If more
than one sequence is associated with an accession number at
different times, the sequence associated with the accession number
as of May 29, 2008 is intended. In the event of any discrepancy
between corresponding sequences in the figures and sequences
listing, the figures control.
[0074] The hybridoma producing the monoclonal antibody GAL-F2, ATCC
Number PTA-8864, has been deposited at the American Type Culture
Collection, P.O. Box 1549 Manassas, Va. 20108, on Jan. 8, 2008
under the Budapest Treaty. This deposit will be maintained at an
authorized depository and replaced in the event of mutation,
nonviability or destruction for a period of at least five years
after the most recent request for release of a sample was received
by the depository, for a period of at least thirty years after the
date of the deposit, or during the enforceable life of the related
patent, whichever period is longest. All restrictions on the
availability to the public of these cell lines will be irrevocably
removed upon the issuance of a patent from the application.
Sequence CWU 1
1
81107PRTMus musculusGAL-F2 1Ser Ile Val Met Thr Gln Thr Pro Lys Phe
Leu Leu Val Ser Ala Gly1 5 10 15 Asp Arg Val Thr Met Thr Cys Lys
Ala Ser Gln Ser Val Ser Ser Asp 20 25 30 Val Gly Trp Tyr Gln Gln
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Gly Ser
Asn Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly
Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala65 70 75 80
Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Tyr Ser Pro Trp 85
90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
2107PRTArtificial SequenceHuGAL-F2 2Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Lys Ala Ser Gln Ser Val Ser Ser Asp 20 25 30 Val Gly Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser
Gly Ser Asn Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Tyr Ser
Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 3107PRTHomo sapiensimmunoglobulin kappa light chain variable
region 3Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Trp 85 90 95 Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 105 4121PRTMus musculusGAL-F2
4Glu Val His Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5
10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn
Tyr 20 25 30 Val Ile Asn Trp Val Lys Gln Lys Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Tyr Asn Asp Pro Tyr Asn Asp Val Ser Lys
Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp
Lys Ser Ser Ser Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Thr
Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Gly Gly
Gly Lys Tyr Val Tyr Ala Met Asp Ser Trp Gly 100 105 110 Gln Gly Thr
Ser Val Thr Val Ser Ser 115 120 5121PRTArtificial SequenceHuGAL-F2
5Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5
10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn
Tyr 20 25 30 Val Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Tyr Asn Asp Pro Tyr Asn Asp Val Ser Lys
Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Ile Thr Ser Asp
Lys Ser Thr Ser Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Gly Gly
Gly Lys Tyr Val Tyr Ala Met Asp Ser Trp Gly 100 105 110 Gln Gly Thr
Thr Val Thr Val Ser Ser 115 120 6122PRTHomo
sapiensanti-pneumococcal capsular polysaccharide immunoglobulin
heavy chain variable region 6Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile
Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln
Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Val Gly Gln Leu Gly Tyr Tyr Tyr Tyr Gly Met Asp
Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
7214PRTArtificial SequenceHuGAL-F2 antibody light chain 7Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Ser Asp 20
25 30 Val Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr Ser Gly Ser Asn Arg Tyr Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Asp Tyr Tyr Ser Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150
155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
8451PRTArtificial SequenceHuGAL-F2 antibody heavy chain 8Glu Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20
25 30 Val Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45 Gly Tyr Asn Asp Pro Tyr Asn Asp Val Ser Lys Tyr Asn
Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Ile Thr Ser Asp Lys Ser
Thr Ser Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Gly Gly Gly Lys
Tyr Val Tyr Ala Met Asp Ser Trp Gly 100 105 110 Gln Gly Thr Thr Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150
155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys
Arg Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230 235 240 Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275
280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395
400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450
* * * * *