U.S. patent application number 13/910792 was filed with the patent office on 2013-10-31 for anti-fgfr2 antibodies.
The applicant listed for this patent is AVEO Pharmaceuticals, Inc.. Invention is credited to Ailin Bai, Ting Chen, Jeno Gyuris, Kristan Meetze, Solly Weiler, Zhigang Weng, William M. Winston, JR..
Application Number | 20130288305 13/910792 |
Document ID | / |
Family ID | 44914959 |
Filed Date | 2013-10-31 |
United States Patent
Application |
20130288305 |
Kind Code |
A1 |
Weng; Zhigang ; et
al. |
October 31, 2013 |
ANTI-FGFR2 ANTIBODIES
Abstract
Monoclonal antibodies that bind and inhibit biological
activities of human FGFR2 are disclosed. The antibodies can be used
to treat cell proliferative diseases and disorders, including
certain forms of cancer, associated with activation or
overexpression of FGFR2.
Inventors: |
Weng; Zhigang; (Brookline,
MA) ; Winston, JR.; William M.; (Marlborough, MA)
; Bai; Ailin; (Newton, MA) ; Meetze; Kristan;
(Lexington, MA) ; Weiler; Solly; (Newton, MA)
; Chen; Ting; (Acton, MA) ; Gyuris; Jeno;
(Lincoln, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AVEO Pharmaceuticals, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
44914959 |
Appl. No.: |
13/910792 |
Filed: |
June 5, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13105521 |
May 11, 2011 |
8481688 |
|
|
13910792 |
|
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61333590 |
May 11, 2010 |
|
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Current U.S.
Class: |
435/69.6 ;
435/252.33; 435/320.1; 435/334; 536/23.53 |
Current CPC
Class: |
C07K 2317/73 20130101;
C07K 2317/92 20130101; C07K 2317/76 20130101; A61K 2039/505
20130101; A61P 35/00 20180101; C07K 16/2863 20130101; C07K 2317/24
20130101 |
Class at
Publication: |
435/69.6 ;
536/23.53; 435/320.1; 435/252.33; 435/334 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Claims
1-2. (canceled)
3. An isolated nucleic acid comprising a nucleotide sequence
encoding an immunoglobulin heavy chain variable region comprising a
CDR.sub.H1 comprising an amino acid sequence selected from the
group consisting of SEQ ID NO: 5 and SEQ ID NO: 47, a CDR.sub.H2
comprising the amino acid sequence of SEQ ID NO: 38, and a
CDR.sub.H3 comprising the amino acid sequence of SEQ ID NO: 11.
4. An isolated nucleic acid comprising a nucleotide sequence
encoding an immunoglobulin light chain variable region comprising a
CDR.sub.L1 comprising the amino acid sequence of SEQ ID NO: 41, a
CDR.sub.L2 comprising the amino acid sequence of SEQ ID NO: 42, and
a CDR.sub.L3 comprising the amino acid sequence of SEQ ID NO:
14.
5. An expression vector containing the nucleic acid of claim 3.
6. An expression vector containing the nucleic acid of claim 4.
7. The expression vector of claim 6, further comprising the nucleic
acid of claim 3.
8. A host cell comprising the expression vector of claim 5.
9. A host cell comprising the expression vector of claim 6.
10. A host cell comprising the expression vector of claim 7.
11. The host cell of claim 9, further comprising the expression
vector of claim 5.
12. A method of producing a polypeptide comprising an
immunoglobulin heavy chain variable region or an immunoglobulin
light chain variable region, the method comprising: (a) growing the
host cell of claim 8 or 9 under conditions so that the host cell
expresses the polypeptide comprising the immunoglobulin heavy chain
variable region or the immunoglobulin light chain variable region;
and (b) purifying the polypeptide comprising the immunoglobulin
heavy chain variable region or the immunoglobulin light chain
variable region.
13. A method of producing an antibody that binds human FGFR2 or an
antigen binding fragment of the antibody, the method comprising:
(a) growing the host cell of claim 10 or 11 under conditions so
that the host cell expresses a polypeptide comprising the
immunoglobulin heavy chain variable region and the immunoglobulin
light chain variable region, thereby producing the antibody or the
antigen-binding fragment of the antibody; and (b) purifying the
antibody or the antigen-binding fragment of the antibody.
14. (canceled)
15. The isolated nucleic acid of claim 3, wherein the nucleotide
sequence encodes an immunoglobulin heavy chain variable region
comprising the amino acid sequence of SEQ ID NO: 37.
16. The isolated nucleic acid of claim 4, wherein the nucleotide
sequence encodes an immunoglobulin light chain variable region
comprising the amino acid sequence of SEQ ID NO: 46.
17. An expression vector containing the nucleic acid of claim
15.
18. An expression vector containing the nucleic acid of claim
16.
19. The expression vector of claim 18, further comprising the
nucleic acid of claim 15.
20. A host cell comprising the expression vector of claim 17.
21. A host cell comprising the expression vector of claim 18.
22. A host cell comprising the expression vector of claim 19.
23. The host cell of claim 21, further comprising the expression
vector of claim 17.
24. A method of producing a polypeptide comprising an
immunoglobulin heavy chain variable region or an immunoglobulin
light chain variable region, the method comprising: (a) growing the
host cell of claim 20 or 21 under conditions so that the host cell
expresses the polypeptide comprising the immunoglobulin heavy chain
variable region of the immunoglobulin light chain variable region;
and (b) purifying the polypeptide comprising the immunoglobulin
heavy chain variable region or the immunoglobulin light chain
variable region.
25. A method of producing an antibody that binds human FGFR2 or an
antigen binding fragment of the antibody, the method comprising:
(a) growing the host cell of claim 22 or 23 under conditions so
that the host cell expresses a polypeptide comprising the
immunoglobulin heavy chain variable region and the immunoglobulin
light chain variable region, thereby producing the antibody or the
antigen-binding fragment of the antibody; and (b) purifying the
antibody or the antigen-binding fragment of the antibody.
26. (canceled)
27. The isolated nucleic acid of claim 3, wherein the nucleotide
sequence encodes an immunoglobulin heavy chain comprising the amino
acid sequence of SEQ ID NO: 56.
28. The isolated nucleic acid of claim 4, wherein the nucleotide
sequence encodes an immunoglobulin light chain comprising the amino
acid sequence of SEQ ID NO: 62.
29. An expression vector containing the nucleic acid of claim
27.
30. An expression vector containing the nucleic acid of claim
28.
31. The expression vector of claim 30, further comprising the
nucleic acid of claim 27.
32. A host cell comprising the expression vector of claim 29.
33. A host cell comprising the expression vector of claim 30.
34. A host cell comprising the expression vector of claim 31.
35. The host cell of claim 33, further comprising the expression
vector of claim 29.
36. A method of producing a polypeptide comprising an
immunoglobulin heavy chain, the method comprising: (a) growing the
host cell of claim 32 or 33 under conditions so that the host cell
expresses the polypeptide comprising the immunoglobulin heavy chain
or the immunoglobulin light chain; and (b) purifying the
polypeptide comprising the immunoglobulin heavy chain or the
immunoglobulin light chain.
37. A method of producing an antibody that binds human FGFR2 or an
antigen binding fragment of the antibody, the method comprising:
(a) growing the host cell of claim 34 of 35 under conditions so
that the host cell expresses a polypeptide comprising the
immunoglobulin heavy chain and the immunoglobulin light chain,
thereby producing the antibody or antigen-binding fragment of the
antibody; and (b) purifying the antibody or the antigen-binding
fragment of the antibody.
38-43. (canceled)
44. The isolated nucleic acid of claim 3, wherein the
immunoglobulin heavy chain variable region comprises a CDR.sub.H1
comprising the amino acid sequence of SEQ ID NO: 5, a CDR.sub.H2
comprising the amino acid sequence of SEQ ID NO: 38, and a
CDR.sub.H3 comprising the amino acid sequence of SEQ ID NO: 11.
45. The isolated nucleic acid of claim 3, wherein the
immunoglobulin heavy chain variable region comprises a CDR.sub.H1
comprising the amino acid sequence of SEQ ID NO: 47, a CDR.sub.H2
comprising the amino acid sequence of SEQ ID NO: 38, and a
CDR.sub.H3 comprising the amino acid sequence of SEQ ID NO: 11.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of and priority to U.S.
Provisional Application Ser. No. 61/333,590, filed May 11, 2010;
the content of which is incorporated herein in its entirety.
FIELD OF THE INVENTION
[0002] The field of the invention is molecular biology, immunology
and oncology. More particularly, the field is antibodies that bind
human FGFR2.
BACKGROUND
[0003] Fibroblast Growth Factor Receptor 2 (FGFR2), also known as
BEK, BFR-1, CD332, CEK3, CFD1, ECT1, F1198662, JWS, KGFR (also
known as FGFR2(IIIb)), K-SAM, TK14, and TK25, is one of four highly
conserved receptor tyrosine kinases (FGFR1, FGFR2, FGFR3 and FGFR4)
that mediate fibroblast growth factor (FGF) signaling by binding
FGFs. The FGF receptors are characterized by two or three
extracellular immunoglobulin-like domains (IgD1, IgD2 and IgD3), a
single-pass transmembrane domain, and a cytoplasmic tyrosine kinase
domain. FGF ligand binding induces FGF receptor dimerization and
tyrosine autophosphorylation, resulting in cell proliferation,
differentiation and migration (Turner et al. (2010) NATURE REVIEWS
CANCER 10:116-129; Beenken et al. (2009) NATURE REVIEWS DRUG
DISCOVERY 8:235-254; Gomez-Roman et al. (2005) CLIN. CANCER RES.
11:459-65; Chang et al. (2005) BLOOD 106:353-6; Eswarakumar et al.
(2005) CYTOKINE GROWTH FACTOR REV. 16:139-49).
[0004] Alternative splicing in the IgD3 domain yields either the Mb
or Mc isoform of FGFR1, FGFR2 and FGFR3. The FGFR4 gene is
expressed only as the Mc isoform. The different isoforms of FGF
receptors exhibit tissue-specific expression, and they respond to a
different spectrum of 18 mammalian FGFs (Beenken et al., supra).
Binding of FGFs to FGFRs in the presence of heparan sulfate
proteoglycans induces autophosphorylation of FGFRs at specific
intracellular tyrosine residues. This causes phosphorylation of
adaptor molecules, such as FGFR substrate 2 .alpha. (FRS2.alpha.),
which recruits other proteins to activate various signaling
cascades, including the mitogen-activated protein kinase (MAPK)
pathway and the phosphoinositide 3-kinase (PI3K)/Akt pathway
(Beenken et al., supra; Eswarakumar et al., supra; Turner et al.,
supra).
[0005] It has been suggested that the dysregulated FGF signaling
can directly drive the proliferation of cancer cells, promote the
survival of cancer stem cells, and support tumor angiogenesis
(Turner et al., supra). FGFR2 signaling appears to play a role in
cancer. Missense mutations in the FGFR2 gene occur in various
cancers, including endometrial cancer (Pollock et al., 2007,
ONCOGENE 26:7158-7162; Dutt et al., 2008, PROC. NATL. ACAD. SCI.
USA 105:8713-8717), ovarian cancer, breast cancer, lung cancer
(Greenman et al., 2007, Nature 446:153-158; Ding et al., 2008,
NATURE 455:1069-1075; Davies et al., 2005, CANCER RES.
65:7591-7595) and gastric cancer (Tang et al., 2001, CANCER RES.
61:3541-3543). Some of these activating mutations also have been
reported in patients with skeletal disorders (Dutt et al., supra).
Two independent genome-wide association studies have linked
specific single nucleotide polymorphisms (SNPs) in the FGFR2 gene
to increased susceptibility to breast cancer (Easton et al., 2007,
NATURE 447:1087-1093; Hunter et al., 2007, NAT. GENET. 39:870-874).
These cancer-associated SNPs appear to elevate FGFR2 gene
expression (Meyer et al., 2008, PLOS BIOL. 6:e108). The FGFR2 gene,
located at human chromosome 10q26, is amplified in a subset of
breast cancers (Adnane et al., 1991, ONCOGENE 6:659-663; Turner et
al., 2010, ONCOGENE 29:2013-2023) and gastric cancer (Hara et al.,
1998, LAB. INVEST. 78:1143-1153; Mor et al., 1993, CANCER GENET.
CYTOGENET. 65:111-114).
[0006] Naturally occurring antibodies are multimeric proteins that
contain four polypeptide chains (FIG. 1). Two of the polypeptide
chains are called immunoglobulin heavy chains (H chains), and two
of the polypeptide chains are called immunoglobulin light chains (L
chains). The immunoglobulin heavy and light chains are connected by
an interchain disulfide bond. The immunoglobulin heavy chains are
connected by interchain disulfide bonds. A light chain consists of
one variable region (V.sub.L in FIG. 1) and one constant region
(C.sub.L in FIG. 1). The heavy chain consists of one variable
region (V.sub.H in FIG. 1) and at least three constant regions
(CH.sub.1, CH.sub.2 and CH.sub.3 in FIG. 1). The variable regions
determine the specificity of the antibody. Naturally occurring
antibodies have been used as starting material for engineered
antibodies, such as chimeric antibodies and humanized
antibodies.
[0007] Each variable region contains three hypervariable regions
known as complementarity determining regions (CDRs) flanked by four
relatively conserved regions known as framework regions (FRs). The
three CDRs, referred to as CDR.sub.1, CDR.sub.2, and CDR.sub.3,
contribute to the antibody binding specificity.
[0008] Inhibitory antibodies specific against human FGFR2 have been
difficult to generate because of the high homology between mouse
and human FGFR2. In particular, the ligand binding domain of the
mouse and human FGFR2 shares approximately 98% sequence identity
(Wei et al., 2006, HYBRIDOMA 25:115-124). Thus, there is a need for
improved FGFR2 antibodies that can be used as therapeutic
agents.
SUMMARY OF THE INVENTION
[0009] The invention is based on the discovery of a family of
antibodies that specifically bind human FGFR2. The antibodies
contain FGFR2 binding sites based on the CDRs of an antibody that
specifically binds FGFR2. When used as therapeutic agents, the
antibodies are engineered, e.g., humanized, to reduce or eliminate
an immune response when administered to a human patient.
[0010] The antibodies of the invention prevent or inhibit the
activation of (i.e., neutralize) human FGFR2. The antibodies of the
invention can be used to inhibit the proliferation of tumor cells
in vitro or in vivo. When administered to a human cancer patient
(or an animal model), the antibodies inhibit or reduce tumor growth
in the human patient (or animal model).
[0011] These and other aspects and advantages of the invention are
illustrated by the following figures, detailed description and
claims. As used herein, "including" means without limitation, and
examples cited are non-limiting.
DESCRIPTION OF THE DRAWINGS
[0012] The invention can be more completely understood with
reference to the following drawings.
[0013] FIG. 1 (prior art) is a schematic representation of a
typical antibody.
[0014] FIG. 2 is a graph summarizing results from an experiment to
measure stimulation of proliferation of FGFR2-IIIb-expressing
FDCP-1 cells by FGF2 ( ), FGF7 (.gradient.), FGF9 (.quadrature.)
and FGF10 (x).
[0015] FIG. 3 is a graph summarizing results from an experiment to
measure stimulation of proliferation of FGFR2-IIIc-expressing
FDCP-1 cells by FGF2 ( ), FGF7 (.gradient.), FGF9 (.quadrature.)
and FGF10 (x).
[0016] FIG. 4 is a graph summarizing results from an experiment to
measure inhibition of proliferation of FDCP-1 cells expressing wild
type FGFR2-IIIb (.quadrature.), wild type FGFR2-IIIc (.gradient.),
or truncated FGFR2-IIIb (*), by treatment with antibody 4B9.
[0017] FIG. 5 is a graph summarizing results from an experiment to
measure inhibition of proliferation of FDCP-1 cells expressing wild
type FGFR2-IIIb (.quadrature.), FGFR2-IIIb S252W (.box-solid.), or
FGFR2-IIIb N550K (.tangle-solidup.), by treatment with antibody
4B9.
[0018] FIG. 6 is a graph summarizing results from an experiment to
measure inhibition of growth of SNU-16 xenograft tumors by
treatment with antibody 4B9 at 2 mg/kg (also referred to herein as
"mpk") (.largecircle.), 5 mpk (.DELTA.), 10 mpk (x) or 20 mpk (*),
with mIgG at 20 mpk (.diamond-solid.) serving as a negative
control.
[0019] FIG. 7 is a graph summarizing results from an experiment to
measure the effect of antibody 4B9 (.largecircle.) on the in vivo
growth of FGFR2-amplified breast cancer cell line MFM-223 (murine
IgG (.diamond-solid.)).
[0020] FIG. 8 is a schematic diagram showing the amino acid
sequences of the complete murine immunoglobulin heavy chain
variable region of 4B9 (SEQ ID NO: 2) and the complete humanized
heavy chain variable regions denoted as Hu4B9-65 (SEQ ID NO: 35)
and Hu4B9-82, -83 (SEQ ID NO: 37). The amino acid sequences for
each heavy chain variable region are aligned against one another,
and Complementary Determining Sequences (CDR) (Kabat definition),
CDR.sub.1, CDR.sub.2, and CDR.sub.3, are identified in boxes. The
unboxed sequences represent framework (FR) sequences.
[0021] FIG. 9 is a schematic diagram showing the CDR.sub.1,
CDR.sub.2, and CDR.sub.3 sequences (Kabat definition) for each of
the variable region sequences shown in FIG. 8.
[0022] FIG. 10 is a schematic diagram showing the amino acid
sequences of the complete murine immunoglobulin light chain
variable region of 4B9 (SEQ ID NO: 4) and the complete humanized
light chain variable regions denoted as Hu4B9-65 (SEQ ID NO: 40),
Hu4B9-82 (SEQ ID NO: 44), and Hu4B9-83 (SEQ ID NO: 46). The amino
acid sequences for each light chain variable region are aligned
against one another, and CDR.sub.1, CDR.sub.2, and CDR.sub.3
sequences (Kabat definition) are identified in boxes. The unboxed
sequences represent framework (FR) sequences.
[0023] FIG. 11 is a schematic diagram showing the CDR.sub.1,
CDR.sub.2, and CDR.sub.3 sequences (Kabat definition) for each of
the variable region sequences shown in FIG. 10.
[0024] FIG. 12 is a graph summarizing results from an experiment to
measure inhibition of proliferation of FDCP-1 cells expressing wild
type FGFR2-IIIb by treatment with antibody 4B9 (.quadrature.),
Hu4B9-65 (.tangle-solidup.), Hu4B9-82 () and Hu4B9-83
(.diamond-solid.).
DETAILED DESCRIPTION
[0025] The FGFR2 antibodies of the invention are based on the
antigen binding sites of a monoclonal antibody selected on the
basis of neutralizing the biological activity of human FGFR2
polypeptides. The antibodies contain immunoglobulin variable region
CDR sequences that define a binding site for human FGFR2.
[0026] Because of the neutralizing activity of these antibodies,
they are useful for inhibiting the growth and/or proliferation of
certain cancer cells and tumors. The antibodies can be engineered
to minimize or eliminate an immune response when administered to a
human patient. Various features and aspects of the invention are
discussed in more detail below.
[0027] As used herein, unless otherwise indicated, the term
"antibody" means an intact antibody (e.g., an intact monoclonal
antibody) or antigen-binding fragment of an antibody (e.g., an
antigen-binding fragment of a monoclonal antibody), including an
intact antibody or antigen-binding fragment that has been modified,
engineered or chemically conjugated. Examples of antibodies that
have been modified or engineered are chimeric antibodies, humanized
antibodies, and multispecific antibodies (e.g., bispecific
antibodies). Examples of antigen-binding fragments include Fab,
Fab', F(ab').sub.2, Fv, single chain antibodies (e.g., scFv) and
diabodies. An antibody conjugated to a toxin moiety is an example
of a chemically conjugated antibody.
Antibodies that Bind Human FGFR2
[0028] Antibodies of the invention comprise: (a) an immunoglobulin
heavy chain variable region comprising the structure
CDR.sub.H1-CDR.sub.H2-CDR.sub.H3 and (b) an immunoglobulin light
chain variable region comprising the structure
CDR.sub.L1-CDR.sub.L2-CDR.sub.L3, wherein the heavy chain variable
region and the light chain variable region together define a single
binding site for binding human FGFR2.
[0029] As disclosed herein, an antibody may comprise: (a) an
immunoglobulin heavy chain variable region comprising the structure
CDR.sub.H1-CDR.sub.H2-CDR.sub.H3 and (b) immunoglobulin light chain
variable region, wherein the heavy chain variable region and the
light chain variable region together define a single binding site
for binding human FGFR.sub.2. A CDR.sub.H1 comprises an amino acid
sequence selected from the group consisting of SEQ ID NO: 5 (4B9;
Hu4B9-65; Hu4B9-82, -83), SEQ ID NO: 7 (4B9; Hu4B9-65), and SEQ ID
NO: 47 (Hu4B9-82, -83); a CDR.sub.H2 comprises an amino acid
sequence selected from the group consisting of SEQ ID NO: 6 (4B9;
Hu4B9-65) and SEQ ID NO: 38 (Hu4B9-82, -83); and a CDR.sub.H3
comprises an amino acid sequence selected from the group consisting
of amino acid sequence FDY (4B9; Hu4B9-65; Hu4B9-82, -83) and SEQ
ID NO: 11 (4B9; Hu4B9-65; Hu4B9-82, -83). Throughout the
specification a particular SEQ ID NO. is followed in parentheses by
the antibody that was the origin of that sequence. For example,
"SEQ ID NO: 47 (Hu4B9-82, -83)" means that SEQ ID NO: 47 comes from
the humanized antibody 4B9 denoted Hu4B9-82, -83.
[0030] In some embodiments, the heavy chain variable region
comprises a CDR.sub.H1 comprising the amino acid sequence of SEQ ID
NO: 5 or SEQ ID NO: 7 (4B9; Hu4B9-65; Hu4B9-82, -83), a CDR.sub.H2
comprising the amino acid sequence of SEQ ID NO: 6 (4B9; Hu4B9-65),
and a CDR.sub.H3 comprising the amino acid sequence of SEQ ID NO:
11 (4B9; Hu4B9-65; Hu4B9-82, -83).
[0031] In some embodiments, the heavy chain variable region
comprises a CDR.sub.H1 comprising the amino acid sequence of SEQ ID
NO: 5 (4B9; Hu4B9-65; Hu4B9-82, -83) or SEQ ID NO: 47 (Hu4B9-82,
-83), a CDR.sub.H2 comprising the amino acid sequence of SEQ ID NO:
38 (Hu4B9-82, -83), and a CDR.sub.H3 comprising the amino acid
sequence of SEQ ID NO: 11 (4B9; Hu4B9-65; Hu4B9-82, -83).
[0032] Preferably, the CDR.sub.H1, CDR.sub.H2, and CDR.sub.H3
sequences are interposed between human or humanized immunoglobulin
FRs. The antibody can be an intact antibody or an antigen-binding
antibody fragment.
[0033] In other embodiments, the antibody comprises (a) an
immunoglobulin light chain variable region comprising the structure
CDR.sub.L1-CDR.sub.L2-CDR.sub.L3, and (b) an immunoglobulin heavy
chain variable region, wherein the IgG light chain variable region
and the IgG heavy chain variable region together define a single
binding site for binding human FGFR.sub.2. A CDR.sub.L1 comprises
an amino acid sequence selected from the group consisting of SEQ ID
NO: 12 (4B9) and SEQ ID NO: 41 (Hu4B9-65; Hu4B9-82; Hu4B9-83); a
CDR.sub.L2 comprises an amino acid sequence selected from the group
consisting of SEQ ID NO: 13 (4B9) and SEQ ID NO: 42 (Hu4B9-65;
Hu4B9-82; Hu4B9-83); and a CDR.sub.L3 comprises an amino acid
sequence of SEQ ID NO: 14 (4B9; Hu4B9-65; Hu4B9-82; Hu4B9-83).
[0034] In some embodiments, the light chain variable region
comprises a CDR.sub.L1 comprising the amino acid sequence of SEQ ID
NO: 12 (4B9); a CDR.sub.L2 comprising the amino acid sequence of
SEQ ID NO: 13 (4B9); and a CDR.sub.L3 comprising the amino acid
sequence of SEQ ID NO: 14 (4B9; Hu4B9-65; Hu4B9-82; Hu4B9-83).
[0035] In some embodiments, the light chain variable region
comprises a CDR.sub.L1 comprising the amino acid sequence of SEQ ID
NO: 41 (Hu4B9-65; Hu4B9-82; Hu4B9-83); a CDR.sub.L2 comprising the
amino acid sequence of SEQ ID NO: 42 (Hu4B9-65; Hu4B9-82;
Hu4B9-83); and a CDR.sub.L3 comprising the amino acid sequence of
SEQ ID NO: 14 (4B9; Hu4B9-65; Hu4B9-82; Hu4B9-83).
[0036] Preferably, the CDR.sub.L1, CDR.sub.L2, and CDR.sub.L3
sequences are interposed between human or humanized immunoglobulin
FRs. The antibody can be an intact antibody or an antigen-binding
antibody fragment.
[0037] In some embodiments, the antibody comprises: (a) an
immunoglobulin heavy chain variable region comprising the structure
CDR.sub.H1-CDR.sub.H2-CDR.sub.H3 and (b) an immunoglobulin light
chain variable region comprising the structure
CDR.sub.L1-CDR.sub.L2-CDR.sub.L3, wherein the heavy chain variable
region and the light chain variable region together define a single
binding site for binding human FGFR.sub.2. The CDR.sub.H1 is an
amino acid sequence selected from the group consisting of SEQ ID
NO: 5 or SEQ ID NO: 7 (4B9; Hu4B9-65; Hu4B9-82, -83); the
CDR.sub.H2 is an amino acid sequence selected from the group
consisting of SEQ ID NO: 6 (4B9; Hu4B9-65) and SEQ ID NO: 38
(Hu4B9-82, -83); and the CDR.sub.H3 is an amino acid sequence
selected from the group consisting of amino acid sequence FDY and
SEQ ID NO: 11 (4B9; Hu4B9-65; Hu4B9-82, -83). The CDR.sub.L1 is an
amino acid sequence selected from the group consisting of SEQ ID
NO: 12 (4B9) and SEQ ID NO: 41 (Hu4B9-65; Hu4B9-82; Hu4B9-83); the
CDR.sub.L2 is an amino acid sequence selected from the group
consisting of SEQ ID NO: 13 (4B9) and SEQ ID NO: 42 (Hu4B9-65;
Hu4B9-82; Hu4B9-83); and the CDR.sub.L3 comprises the amino acid
sequence of SEQ ID NO: 14 (4B9; Hu4B9-65; Hu4B9-82; Hu4B9-83).
[0038] In another embodiment, the antibody comprises an
immunoglobulin heavy chain variable region selected from the group
consisting of SEQ ID NO: 2 (4B9), SEQ ID NO: 35 (Hu4B9-65), and SEQ
ID NO: 37 (Hu4B9-82, -83), and an immunoglobulin light chain
variable region selected from the group consisting of SEQ ID NO: 4
(4B9), SEQ ID NO: 40 (Hu4B9-65), SEQ ID NO: 44 (Hu4B9-82) and SEQ
ID NO: 46 (Hu4B9-83).
[0039] In some embodiments, the antibody comprises an
immunoglobulin heavy chain variable region comprising the amino
acid sequence of SEQ ID NO: 2 (4B9), and an immunoglobulin light
chain variable region comprising the amino acid sequence of SEQ ID
NO: 4 (4B9).
[0040] In some embodiments, the antibody comprises an
immunoglobulin heavy chain variable region comprising the amino
acid sequence of SEQ ID NO: 35 (Hu4B9-65), and an immunoglobulin
light chain variable region comprising the amino acid sequence of
SEQ ID NO: 40 (Hu4B9-65).
[0041] In some embodiments, the antibody comprises an
immunoglobulin heavy chain variable region comprising the amino
acid sequence of SEQ ID NO: 37 (Hu4B9-82, -83), and an
immunoglobulin light chain variable region comprising the amino
acid sequence of SEQ ID NO: 44 (Hu4B9-82).
[0042] In some embodiments, the antibody comprises an
immunoglobulin heavy chain variable region comprising the amino
acid sequence of SEQ ID NO: 37 (Hu4B9-82, -83), and an
immunoglobulin light chain variable region comprising the amino
acid sequence of SEQ ID NO: 46 (Hu4B9-83).
[0043] In other embodiments, the antibody comprises (i) an
immunoglobulin heavy chain selected from the group consisting of
SEQ ID NO: 21 (4B9), SEQ ID NO: 54 (Hu4B9-65), and SEQ ID NO: 56
(Hu4B9-82, -83), and (ii) an immunoglobulin light chain selected
from the group consisting of SEQ ID NO: 23 (4B9), SEQ ID NO: 58
(Hu4B9-65), SEQ ID NO: 60 (Hu4B9-82) and SEQ ID NO: 62
(Hu4B9-83).
[0044] In certain embodiments, the antibody comprises (i) an
immunoglobulin heavy chain comprising the amino acid sequence of
SEQ ID NO: 21 (4B9), and (ii) an immunoglobulin light chain
comprising the amino acid sequence of SEQ ID NO: 23 (4B9).
[0045] In certain embodiments, the antibody comprises (i) an
immunoglobulin heavy chain comprising the amino acid sequence of
SEQ ID NO: 54 (Hu4B9-65), and (ii) an immunoglobulin light chain
comprising the amino acid sequence of SEQ ID NO: 58 (Hu4B9-65).
[0046] In certain embodiments, the antibody comprises (i) an
immunoglobulin heavy chain comprising the amino acid sequence of
SEQ ID NO: 56 (Hu4B9-82, -83), and (ii) an immunoglobulin light
chain comprising the amino acid sequence of SEQ ID NO: 60
(Hu4B9-82).
[0047] In certain embodiments, the antibody comprises (i) an
immunoglobulin heavy chain comprising the amino acid sequence of
SEQ ID NO: 56 (Hu4B9-82, -83), and (ii) an immunoglobulin light
chain comprising the amino acid sequence of SEQ ID NO: 62
(Hu4B9-83).
[0048] In other embodiments, an isolated antibody that binds human
FGFR2 comprises an immunoglobulin heavy chain variable region
comprising an amino acid sequence that is at least 70%, 75%, 80%,
85%, 90%, 95%, 98%, or 99% identical to the entire variable region
or the framework region sequence of SEQ ID NO: 2 (4B9), SEQ ID NO:
35 (Hu4B9-65), and SEQ ID NO: 37 (Hu4B9-82, -83).
[0049] In other embodiments, an isolated antibody that binds human
FGFR2 comprises an immunoglobulin light chain variable region
comprising an amino acid sequence that is at least 70%, 75%, 80%,
85%, 90%, 95%, 98%, or 99% identical to the entire variable region
or the framework region sequence of SEQ ID NO: 4 (4B9), SEQ ID NO:
40 (Hu4B9-65), SEQ ID NO: 44 (Hu4B9-82) and SEQ ID NO: 46
(Hu4B9-83).
[0050] Homology or identity may be determined in various ways that
are within the skill in the art, for instance, using publicly
available computer software such as BLAST, BLAST-2, ALIGN or
Megalign (DNASTAR) software. BLAST (Basic Local Alignment Search
Tool) analysis using the algorithm employed by the programs blastp,
blastn, blastx, tblastn and tblastx (Karlin et al., (1990) PROC.
NATL. ACAD. SCI. USA 87, 2264-2268; Altschul, (1993) J. MOL. EVOL.
36, 290-300; Altschul et al., (1997) NUCLEIC ACIDS RES. 25,
3389-3402, incorporated by reference) are tailored for sequence
similarity searching. The approach used by the BLAST program is to
first consider similar segments between a query sequence and a
database sequence, then to evaluate the statistical significance of
all matches that are identified and finally to summarize only those
matches which satisfy a preselected threshold of significance. For
a discussion of basic issues in similarity searching of sequence
databases see Altschul et al., (1994) NATURE GENETICS 6, 119-129
which is fully incorporated by reference. Those skilled in the art
can determine appropriate parameters for measuring alignment,
including any algorithms needed to achieve maximal alignment over
the full length of the sequences being compared. The search
parameters for histogram, descriptions, alignments, expect (i.e.,
the statistical significance threshold for reporting matches
against database sequences), cutoff, matrix and filter are at the
default settings. The default scoring matrix used by blastp,
blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et
al., (1992) PROC. NATL. ACAD. SCI. USA 89, 10915-10919, fully
incorporated by reference). Four blastn parameters may be adjusted
as follows: Q=10 (gap creation penalty); R=10 (gap extension
penalty); wink=1 (generates word hits at every wink.sup.th position
along the query); and gapw=16 (sets the window width within which
gapped alignments are generated). The equivalent Blastp parameter
settings may be Q=9; R=2; wink=1; and gapw=32. Searches may also be
conducted using the NCBI (National Center for Biotechnology
Information) BLAST Advanced Option parameter (e.g.: -G, Cost to
open gap [Integer]: default=5 for nucleotides/11 for proteins; -E,
Cost to extend gap [Integer]: default=2 for nucleotides/1 for
proteins; -q, Penalty for nucleotide mismatch [Integer]:
default=-3; -r, reward for nucleotide match [Integer]: default=1;
-e, expect value [Real]: default=10; -W, wordsize [Integer]:
default=11 for nucleotides/28 for megablast/3 for proteins; -y,
Dropoff (X) for blast extensions in bits: default=20 for blastn/7
for others; -X, X dropoff value for gapped alignment (in bits):
default=15 for all programs, not applicable to blastn; and -Z,
final X dropoff value for gapped alignment (in bits): 50 for
blastn, 25 for others). ClustalW for pairwise protein alignments
may also be used (default parameters may include, e.g., Blosum62
matrix and Gap Opening Penalty=10 and Gape Extenstion Penalty=0.1).
A Bestfit comparison between sequences, available in the GCG
package version 10.0, uses DNA parameters GAP=50 (gap creation
penalty) and LEN=3 (gap extension penalty) and the equivalent
settings in protein comparisons are GAP=8 and LEN=2.
[0051] In each of the foregoing embodiments, it is contemplated
herein that immunoglobulin heavy chain variable region sequences
and/or light chain variable region sequences that together bind
human FGFR2 may contain amino acid alterations (e.g., at least 1,
2, 3, 4, 5, or 10 amino acid substitutions, deletions, or
additions) in the framework regions of the heavy and/or light chain
variable regions.
[0052] In some embodiments, an isolated antibody binds human FGFR2
with a K.sub.D of 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 950 .mu.M, 900
.mu.M, 850 .mu.M, 800 .mu.M, 750 .mu.M, 700 .mu.M, 650 .mu.M, 600
.mu.M, 550 .mu.M, 500 .mu.M, 450 .mu.M, 400 .mu.M, 350 .mu.M, 300
.mu.M, 250 .mu.M, 200 .mu.M, 150 .mu.M, 100 .mu.M, 50 .mu.M or
lower. Unless otherwise specified, K.sub.D values are determined by
surface plasmon resonance methods under the conditions described,
for example, in Examples 5 and 9.
Production of Antibodies
[0053] Methods for producing antibodies of the invention are known
in the art. For example, DNA molecules encoding light chain
variable regions and heavy chain variable regions can be chemically
synthesized using the sequence information provided herein.
Synthetic DNA molecules can be ligated to other appropriate
nucleotide sequences, including, e.g., constant region coding
sequences, and expression control sequences, to produce
conventional gene expression constructs encoding the desired
antibody. Production of defined gene constructs is within routine
skill in the art. Alternatively, the sequences provided herein can
be cloned out of hybridomas by conventional hybridization
techniques or polymerase chain reaction (PCR) techniques, using
synthetic nucleic acid probes whose sequences are based on sequence
information provided herein, or prior art sequence information
regarding genes encoding the heavy and light chains of murine
antibodies in hybridoma cells.
[0054] Nucleic acids encoding desired antibodies can be
incorporated (ligated) into expression vectors, which can be
introduced into host cells through conventional transfection or
transformation techniques. Exemplary host cells are E. coli cells,
Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney
(BHK) cells, monkey kidney cells (COS), human hepatocellular
carcinoma cells (e.g., Hep G2), and myeloma cells that do not
otherwise produce IgG protein. Transformed host cells can be grown
under conditions that permit the host cells to express the genes
that encode the immunoglobulin light or heavy chain variable
regions.
[0055] Specific expression and purification conditions will vary
depending upon the expression system employed. For example, if a
gene is to be expressed in E. coli, it is first cloned into an
expression vector by positioning the engineered gene downstream
from a suitable bacterial promoter, e.g., Trp or Tac, and a
prokaryotic signal sequence. The expressed secreted protein
accumulates in refractile or inclusion bodies, and can be harvested
after disruption of the cells by French press or sonication. The
refractile bodies then are solubilized, and the proteins refolded
and cleaved by methods known in the art.
[0056] If the engineered gene is to be expressed in eukaryotic host
cells, e.g., CHO cells, it is first inserted into an expression
vector containing a suitable eukaryotic promoter, a secretion
signal, IgG enhancers, and various introns. This expression vector
optionally contains sequences encoding all or part of a constant
region, enabling an entire, or a part of, a heavy or light chain to
be expressed. The gene construct can be introduced into eukaryotic
host cells using convention techniques. The host cells express
V.sub.L or V.sub.H fragments, V.sub.L-V.sub.H heterodimers,
V.sub.H-V.sub.L or V.sub.L-V.sub.H single chain polypeptides,
complete heavy or light immunoglobulin chains, or portions thereof,
each of which may be attached to a moiety having another function
(e.g., cytotoxicity). In some embodiments, a host cell is
transfected with a single vector expressing a polypeptide
expressing an entire, or part of, a heavy chain (e.g., a heavy
chain variable region) or a light chain (e.g., a light chain
variable region). In other embodiments, a host cell is transfected
with a single vector encoding (a) a polypeptide comprising a heavy
chain variable region and a polypeptide comprising a light chain
variable region, or (b) an entire immunoglobulin heavy chain and an
entire immunoglobulin light chain. In still other embodiments, a
host cell is co-transfected with more than one expression vector
(e.g., one expression vector expressing a polypeptide comprising an
entire, or part of, a heavy chain or heavy chain variable region,
and another expression vector expressing a polypeptide comprising
an entire, or part of, a light chain or light chain variable
region).
[0057] A polypeptide comprising an immunoglobulin heavy chain
variable region or a light chain variable region can be produced by
growing a host cell transfected with an expression vector encoding
such variable region, under conditions that permit expression of
the polypeptide. Following expression, the polypeptide can be
harvested and purified using techniques well known in the art,
e.g., affinity tags such as glutathione-S-transferase (GST) and
histidine tags.
[0058] A monoclonal antibody that binds human FGFR2, or an
antigen-binding fragment of the antibody, can be produced by
growing a host cell transfected with: (a) an expression vector that
encodes a complete or partial immunoglobulin heavy chain, and a
separate expression vector that encodes a complete or partial light
chain; or (b) a single expression vector that encodes both chains
(e.g., complete or partial heavy and light chains) under conditions
that permit expression of both chains. The intact antibody (or the
antigen-binding fragment of the antibody) can be harvested and
purified using techniques well known in the art, e.g., Protein A,
Protein G, affinity tags such as glutathione-S-transferase (GST)
and histidine tags. It is within ordinary skill in the art to
express the heavy chain and the light chain from a single
expression vector or from two separate expression vectors.
Modifications to the Antibodies
[0059] Methods for reducing or eliminating the antigenicity of
antibodies and antibody fragments are known in the art. When the
antibodies are to be administered to a human, the antibodies
preferably are "humanized" to reduce or eliminate antigenicity in
humans. Preferably, the humanized antibodies have the same, or
substantially the same, affinity for the antigen as the
non-humanized mouse antibody from which it was derived.
[0060] In one humanization approach, chimeric proteins are created
in which mouse immunoglobulin constant regions are replaced with
human immunoglobulin constant regions. See, e.g., Morrison et al.,
1984, PROC. NAT. ACAD. SCI. 81:6851-6855, Neuberger et al., 1984,
NATURE 312:604-608; U.S. Pat. Nos. 6,893,625 (Robinson); 5,500,362
(Robinson); and 4,816,567 (Cabilly).
[0061] In an approach known as CDR grafting, the CDRs of the light
and heavy chain variable regions are grafted into frameworks from
another species. For example, murine CDRs can be grafted into human
FRs. In some embodiments of the invention, the CDRs of the light
and heavy chain variable regions of an anti-FGFR2 antibody are
grafted into human FRs or consensus human FRs. To create consensus
human FRs, FRs from several human heavy chain or light chain amino
acid sequences are aligned to identify a consensus amino acid
sequence. CDR grafting is described in U.S. Pat. Nos. 7,022,500
(Queen); 6,982,321 (Winter); 6,180,370 (Queen); 6,054,297 (Carter);
5,693,762 (Queen); 5,859,205 (Adair); 5,693,761 (Queen); 5,565,332
(Hoogenboom); 5,585,089 (Queen); 5,530,101 (Queen); Jones et al.
(1986) NATURE 321: 522-525; Riechmann et al. (1988) NATURE 332:
323-327; Verhoeyen et al. (1988) SCIENCE 239: 1534-1536; and Winter
(1998) FEBS LETT 430: 92-94.
[0062] In an approach called "SUPERHUMANIZATION.TM.," human CDR
sequences are chosen from human germline genes, based on the
structural similarity of the human CDRs to those of the mouse
antibody to be humanized. See, e.g., U.S. Pat. No. 6,881,557
(Foote); and Tan et al., 2002, J. IMMUNOL 169:1119-1125.
[0063] Other methods to reduce immunogenicity include "reshaping,"
"hyperchimerization," and "veneering/resurfacing." See, e.g.,
Vaswami et al., 1998, ANNALS OF ALLERGY, ASTHMA, & IMMUNOL.
81:105; Roguska et al., 1996, PROT. ENGINEER 9:895-904; and U.S.
Pat. No. 6,072,035 (Hardman). In the veneering/resurfacing
approach, the surface accessible amino acid residues in the murine
antibody are replaced by amino acid residues more frequently found
at the same positions in a human antibody. This type of antibody
resurfacing is described, e.g., in U.S. Pat. No. 5,639,641
(Pedersen).
[0064] Another approach for converting a mouse antibody into a form
suitable for medical use in humans is known as ACTIVMAB.TM.
technology (Vaccinex, Inc., Rochester, N.Y.), which involves a
vaccinia virus-based vector to express antibodies in mammalian
cells. High levels of combinatorial diversity of IgG heavy and
light chains are said to be produced. See, e.g., U.S. Pat. Nos.
6,706,477 (Zauderer); 6,800,442 (Zauderer); and 6,872,518
(Zauderer).
[0065] Another approach for converting a mouse antibody into a form
suitable for use in humans is technology practiced commercially by
KaloBios Pharmaceuticals, Inc. (Palo Alto, Calif.). This technology
involves the use of a proprietary human "acceptor" library to
produce an "epitope focused" library for antibody selection.
[0066] Another approach for modifying a mouse antibody into a form
suitable for medical use in humans is HUMAN ENGINEERING.TM.
technology, which is practiced commercially by XOMA (US) LLC. See,
e.g., PCT Publication No. WO 93/11794 and U.S. Pat. Nos. 5,766,886;
5,770,196; 5,821,123; and 5,869,619.
[0067] Any suitable approach, including any of the above
approaches, can be used to reduce or eliminate human immunogenicity
of an antibody disclosed herein.
[0068] If the antibody is for use as a therapeutic agent, it can be
conjugated to an effector moiety such as a small molecule toxin or
a radionuclide using standard in vitro conjugation chemistries. If
the effector moiety is a polypeptide, the antibody can be
chemically conjugated to the effector or joined to the effector as
a fusion protein. Construction of fusion proteins is within
ordinary skill in the art.
Use of Antibodies
[0069] Antibodies disclosed herein can be used to treat various
forms of cancer, e.g., breast, ovarian, prostate, cervical,
colorectal, lung, pancreatic, gastric, and head and neck cancers.
The cancer cells are exposed to a therapeutically effective amount
of the antibody so as to inhibit or reduce proliferation of the
cancer cells. In some embodiments, the antibodies inhibit cancer
cell proliferation by at least 40%, 50%, 60%, 70%, 80%, 90%, 95%,
98%, 99%, or 100%.
[0070] In some embodiments, the disclosed antibodies can be used in
a method to inhibit tumor growth in a human patient. The method
comprises administering to the patient a therapeutically effective
amount of the antibody. Cancers associated with FGFR2
overexpression and/or activation include breast cancer, ovarian
cancer, prostate cancer, cervical cancer, lung cancer, some forms
of brain cancer, melanomas, and gastrointestinal cancers (e.g.,
colorectal, pancreatic, gastric, head and neck).
[0071] As used herein, "treating" a disease means: (a) reducing
symptoms of the disease; (b) inhibiting progression of the disease;
(c) causing regression of the disease; or (d) curing the
disease.
[0072] Generally, a therapeutically effective amount of active
component is in the range of 0.1 mg/kg to 100 mg/kg, e.g., 1 mg/kg
to 100 mg/kg, 1 mg/kg to 10 mg/kg. The amount administered will
depend on variables such as the type and extent of disease or
indication to be treated, the overall health of the patient, the in
vivo potency of the antibody, the pharmaceutical formulation, and
the route of administration. The initial dosage can be increased
beyond the upper level in order to rapidly achieve the desired
blood-level or tissue level. Alternatively, the initial dosage can
be smaller than the optimum, and the daily dosage may be
progressively increased during the course of treatment. Human
dosage can be optimized, e.g., in a conventional Phase I dose
escalation study designed to run from 0.5 mg/kg to 20 mg/kg. Dosing
frequency can vary, depending on factors such as route of
administration, dosage amount and the disease being treated.
Exemplary dosing frequencies are once per day, once per week and
once every two weeks. A preferred route of administration is
parenteral, e.g., intravenous infusion. Formulation of monoclonal
antibody-based drugs is within ordinary skill in the art. In some
embodiments of the invention a monoclonal antibody is lyophilized
and reconstituted in buffered saline at the time of
administration.
[0073] For therapeutic use, an antibody preferably is combined with
a pharmaceutically acceptable carrier. As used herein,
"pharmaceutically acceptable carrier" means buffers, carriers, and
excipients suitable for use in contact with the tissues of human
beings and animals without excessive toxicity, irritation, allergic
response, or other problem or complication, commensurate with a
reasonable benefit/risk ratio. The carrier(s) should be
"acceptable" in the sense of being compatible with the other
ingredients of the formulations and not deleterious to the
recipient. Pharmaceutically acceptable carriers include buffers,
solvents, dispersion media, coatings, isotonic and absorption
delaying agents, and the like, that are compatible with
pharmaceutical administration. The use of such media and agents for
pharmaceutically active substances is known in the art.
[0074] Pharmaceutical compositions containing antibodies of the
invention can be presented in a dosage unit form and can be
prepared by any suitable method. A pharmaceutical composition
should be formulated to be compatible with its intended route of
administration. Examples of routes of administration are
intravenous (IV), intradermal, inhalation, transdermal, topical,
transmucosal, and rectal administration. A preferred route of
administration for monoclonal antibodies is IV infusion. Useful
formulations can be prepared by methods well known in the
pharmaceutical art. For example, see Remington's Pharmaceutical
Sciences, 18th ed. (Mack Publishing Company, 1990). Formulation
components suitable for parenteral administration include a sterile
diluent such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as EDTA; buffers such as acetates,
citrates or phosphates; and agents for the adjustment of tonicity
such as sodium chloride or dextrose.
[0075] For intravenous administration, suitable carriers include
physiological saline, bacteriostatic water, Cremophor EL.TM. (BASF,
Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier
should be stable under the conditions of manufacture and storage,
and should be preserved against microorganisms. The carrier can be
a solvent or dispersion medium containing, for example, water,
ethanol, polyol (for example, glycerol, propylene glycol, and
liquid polyetheylene glycol), and suitable mixtures thereof.
[0076] Pharmaceutical formulations preferably are sterile.
Sterilization can be accomplished, for example, by filtration
through sterile filtration membranes. Where the composition is
lyophilized, filter sterilization can be conducted prior to or
following lyophilization and reconstitution.
EXAMPLES
[0077] The following Examples are merely illustrative and are not
intended to limit the scope or content of the invention in any
way.
Example 1
Cell Lines and Reagents
[0078] KATO III, HEC-1-A, AN3 CA, SNU-16, and human lung cancer
cell lines were acquired from the American Type Culture Collection
(Rockville, Md.). FDCP-1 and Ba/F3, MFM-223, MFE-296, MFE-280,
MFE-319 and ESS-1 cells were obtained from the German Collection of
Microorganisms and Cell Cultures. All human cell lines were
cultured according to the instructions specified by the suppliers,
at 37.degree. C., in an atmosphere containing 5% CO.sub.2. All FGFs
were purchased from R&D Systems, Inc. (Minneapolis, Minn.).
[0079] To establish cell-based assays to screen for functional
FGFR2 antibodies, we first engineered Ba/F3 and FDCP-1 cells to
express wild type FGFR2 and cancer-associated mutants or variants
of FGFR2. FGFR-driven FDCP cells and Ba/F3 cells were obtained by
the following methods. FDCP-1 cells were transfected by
electroporation with plasmids encoding the Mb, Mc isoform or
C-terminally truncated variant of human FGFR2 as well as
cancer-associated FGFR2-IIIb S252W, or FGFR2-IIIb N550K mutants.
Following selection with G418 (600 .mu.g/ml), single clones were
isolated and tested for their FGF1-dependent proliferation in the
absence of IL3 by the MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
assay (Sigma-Aldrich, St. Louis, Mo.). MTT reagent (10 .mu.l) was
added to the cells and the reaction was stopped with 100 .mu.l of
10% SDS with 2N HCL after four hours. The plates were analyzed the
following day. The clones that exhibited robust FGF-1-dependent
proliferation in the absence of IL3 were used for subsequent
studies. To generate retroviruses expressing FGFR2, cDNAs encoding
various human FGFR2 variants were each inserted into a retroviral
vector. Retroviruses were produced by transfecting Phoenix cells
using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.).
Supernatants containing the retroviruses were used to infect Ba/F3
cells by centrifugation at 2500 rpm for 90 minutes, in the presence
of 8 .mu.g/ml of polybrene (Sigma-Aldrich). Individual clones were
isolated by limiting dilution, and cell surface receptor expression
was verified by flow cytometry.
[0080] Cancer cell lines with FGFR amplification were identified as
follows. The CGP copy number database at the Wellcome Trust Sanger
Institute (www.sanger.ac.uk) was queried for FGFR2 amplification
(gene copy number>7). The copy number of the cell lines with
potential FGFR2 amplification was analyzed by quantitative PCR
(qPCR) using FGFR2 specific primers (5'-ACTTGGGCTGGAGTGATTTG-3'
(SEQ ID NO: 24) and 5'-AATCCCATCTGCACACTTCC-3' (SEQ ID NO: 25)) and
reference gene (transketolase) primers (5'-CAAAAACATGGCTGAGCAGA-3'
(SEQ ID NO: 26) and 5'-GAAACAGGCCCCACTTTGTA-3' (SEQ ID NO: 27)).
The FGFR2 gene copy number was calculated essentially as described
in Toyokawa et al., 2009, ONCOL. REP. 21:875-880.
[0081] FGFR gene expression analysis was performed as follows.
Total RNA was isolated by the RNeasy.TM. mini kit (Qiagen,
Valencia, Calif.). Quantitative RT-PCR (qRT-PCR) was performed
using a QuantiTect.TM. SYBR Green RT-PCR kit (Qiagen), with primers
specific for FGFR2, FGFR2-IIIb, FGFR2-IIIc, and HPRT. The
expression levels were normalized to HPRT.
[0082] Previous studies have demonstrated that ectopic expression
of FGFRs in murine pro-B Ba/F3 or bone marrow FDCP-1 cells confers
FGF1-dependent proliferation in the absence of IL-3 (Tannheimer et
al., 2000, BREAST CANCER RES. 2:311-320; Ornitz et al., 1996, J.
BIOL. CHEM. 271:15292-15297). As expected, there was no noticeable
proliferation of FDCP-1 cells stably expressing wild-type FGFR2 in
the absence of IL-3 and FGF1. It was known that FGF1, 3, 7, 10 and
22 transduce signals through FGFR2-IIIb, and that FGFR2-IIIc
responds to a broader panel of ligands including FGF1, 2, 4, 6, 9,
16, 17, 18 and 20 (Tannheimer et al., supra; Ornitz et al., supra;
Zhang et al., 2006, J. BIOL. CHEM. 281:15964-15700). The
proliferation of FDCP-1 cells expressing the Mb isoform of FGFR2
was stimulated by FGF7 and FGF10, but not by FGF2 and FGF9 (FIG.
2). The proliferation of cells expressing the Mc isoform was
enhanced by FGF2 and FGF9 specifically (FIG. 3).
Example 2
Production of Anti-FGFR2Monoclonal Antibodies
[0083] Mice were immunized with a 1:1 mixture of human
FGFR21gD2-IgD3 (Mb) and human FGFR2 IgD2-IgD3 (Mc) fused with a
human Fc moiety at their C-termini. Mouse immunizations and cell
fusions were performed by a commercial vendor (Precision Antibody,
Columbia, Md.).
[0084] In a primary screen, hybridoma supernatants were screened to
detect binding to human FGFR2 IgD2-IgD3, using an ELISA format.
Antibodies that passed the primary screen were subjected to a
secondary screen, which was a cell-based proliferation assay
described in Example 3 (below).
[0085] The primary screen was performed using the supernatants of
the murine hybridoma clones yielded from the splenic fusion of the
mice immunized with the extracellular domain of human FGFR2. Assay
plates were coated with 100 ng/well of recombinant soluble FGFR2
extracellular domain and then blocked with 5% milk in PBS for one
hour at room temperature. Then 50 .mu.l of hybridoma supernatant
was added to each well to allow antibody binding for one hour at
room temperature. Plates were washed three times with wash buffer
(PBS with 0.1% Tween 20) followed by incubation with a
HRP-conjugated goat anti-mouse IgG heavy and light chain secondary
antibody. The assay was developed using TMB (tetramethylbenzene) as
a substrate, and absorbance was read at 620 nm.
Example 3
Identification of FGFR2Antagonist Antibodies
[0086] To screen for FGFR2 antagonist antibodies, hybridoma
supernatants containing FGFR2 antibodies were added to FDCP cells
ectopically expressing one of the following five forms of FGFR2:
(1) wild type FGFR2-IIIb; (2) wild type FGFR2-IIIc; (3)
FGFR2-III(b) S252W; (4) FGFR2-III(b) N550K; and (5) FGFR2-III(b)
with C-terminal truncation. The supernatants were added to the
FGFR2-expressing cells at a 1:1 ratio (volume) in a flat-bottomed
96-well plate (70,000 cells/well) with heparin (5 .mu.g/ml).+-.FGF1
(8 ng/ml). After incubation at 37.degree. C. for 2 days, MTT assays
were conducted as described above.
[0087] The supernatant of clone 4B9 demonstrated potent and
selective inhibition of the FDCP-1 proliferation driven by the
IIIb-isoform of FGFR2. Antibody 4B9 (also referred to as antibody
GP369), produced by clone 4B9, was purified by conventional
techniques for further characterization. Surface plasmon resonance
analysis indicated that antibody 4B9 exhibited strong affinity
towards human FGFR2-IIIb and showed no detectable binding to the
human FGFR2-IIIc. No binding of antibody 4B9 to human FGFR1-IIIc or
FGFR3-IIIb was detected.
Example 4
Sequence Analysis
[0088] The light chain isotype and heavy chain isotype of antibody
4B9 in Example 1 was determined using the IsoStrip.TM. Mouse
Monoclonal Antibody Isotyping Kit according to the manufacturer's
instructions (Roche Applied Science, Indianapolis, Ind.). The
antibody was determined to be Kappa light chain and IgG1 heavy
chain.
[0089] The heavy and light chain variable regions of antibody 4B9
were sequenced using 5' RACE (Rapid Amplification of cDNA Ends).
Total RNA was extracted from the 4B9 monoclonal hybridoma cell line
using the RNeasy.TM. Miniprep kit according to the vendor's
instructions (Qiagen, Valencia, Calif.). Full-length first strand
cDNA containing 5' ends was generated using SMARTer.TM. RACE cDNA
Amplification Kit (Clontech, Palo Alto, Calif.) according to the
manufacturer's instructions using random primers for 5' RACE.
[0090] The variable regions of the kappa and heavy IgG1 chains were
amplified by PCR, using KOD Hot Start.TM. Polymerase (EMD
Chemicals, Gibbstown, N.J.) according to the manufacturer's
instructions. For amplification of 5' cDNA ends in conjunction with
the SMARTer.TM. RACE cDNA Amplification Kit, the Universal Primer
Mix A primer (Clontech), a mix of
5'CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT 3' (SEQ ID NO: 28)
and 5' CTAATACGACTCACTATAGGGC 3' (SEQ ID NO: 29), was used as a 5'
primer. The heavy chain variable region was amplified using the
above 5' primers and a 3' IgG 1 constant region specific primer, 5'
TATGCAAGGCTTACAACCACA 3' (SEQ ID NO: 30). The kappa chain variable
region was amplified with the above 5' primers and a 3' kappa
constant region specific primer, CGACTGAGGCACCTCCAGATGTT 3' (SEQ ID
NO: 31).
[0091] Individual PCR products were isolated by agarose gel
electrophoresis and purified using the Qiaquick.TM. Gel
Purification kit according to the manufacturer's instructions
(Qiagen). The PCR products were subsequently cloned into the
pCR4Blunt plasmid using the Zero Blunt TOPO.RTM. PCR Cloning Kit
according to the manufacturer's instructions (Invitrogen) and
transformed into DH5-.alpha. bacteria (Invitrogen) through standard
molecular biology techniques. Plasmid DNA isolated from transformed
bacterial clones was sequenced using M13 Forward (5'
GTAAAACGACGGCCAGT 3') (SEQ ID NO: 32) and M13 Reverse primers (5'
CAGGAAACAGCTATGACC 3') (SEQ ID NO: 33) by Beckman Genomics
(Danvers, Mass.), using standard dideoxy DNA sequencing methods to
identify the sequence of the variable region sequences. The
sequences were analyzed using Vector NTI software (Invitrogen) and
the IMGT/V-Quest web server to identify and confirm variable region
sequences.
[0092] The nucleic acid sequences encoding and the protein
sequences defining variable regions of antibody 4B9 are summarized
below (amino terminal signal peptide sequences are not shown). CDR
sequences (Kabat definition) are shown in bold/underlined in the
amino acid sequences.
[0093] Nucleic Acid Sequence Encoding the Heavy Chain Variable
Region of Antibody 4B9 (SEQ ID NO: 1)
TABLE-US-00001 1 gaggttcagc tccagcagtc tgggactgtg ctggcaaggc
ctggggcttc agtgaagatg 61 tcctgcaaga cttctggcta cacatttacc
agctactgga tgcactgggt aaaacagagg 121 cctggacagg gtctggaatg
gataggggct atttatcctg gaaatagtga tactgactac 181 agccagaagt
tcaagggcaa ggccacactg actgcagtca catccgccac cactgcctac 241
atggaactca gcagcctgac aaatgaggac tctgcggtct attactgttc aaagtttgac
301 tactggggcc aaggcaccac tctcacagtc tcctca
[0094] Protein Sequence Defining the Heavy Chain Variable Region of
Antibody 4B9 (SEQ ID NO: 2)
TABLE-US-00002 1 evqlqqsgtv larpgasvkm scktsgytft sywmhwvkqr
pgqglewiga iypgnsdtdy 61 sqkfkgkatl tavtsattay melssltned
savyycskfd ywgqgttltv ss
[0095] Nucleic Acid Sequence Encoding the Kappa Chain Variable
Region of Antibody 4B9 (SEQ ID NO: 3)
TABLE-US-00003 1 caaattgttc tcacccagtc tccagcactc atgtctgcat
ctccagggga gaaggtcacc 61 atgacctgca gtgccagctc aagtgtaaat
tacatgtact ggtaccagca gaagccaaga 121 tcctccccca aaccctggat
ttatctcaca tccaacctgg cttctggagt ccctgctcgc 181 ttcagtggca
gggggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa 241
gatgctgcca cttattactg ccagcagtgg agtagtaacc cgtacacgtt cggagggggg
301 accaagctgg aaataaaa
[0096] Protein Sequence Defining the Kappa Chain Variable Region of
Antibody 4B9 (SEQ ID NO: 4)
TABLE-US-00004 1 qivltqspal msaspgekvt mtcsasssvn ymywyqqkpr
sspkpwiylt snlasgvpar 61 fsgrgsgtsy sltissmeae daatyycqqw
ssnpytfggg tkleik
[0097] Table 1 is a concordance chart showing the SEQ ID NO. of
each sequence discussed in this Example.
TABLE-US-00005 TABLE 1 SEQ. ID NO. Antibody 4B9 Nucleic Acid or
Protein 1 Heavy Chain Variable Region--nucleic acid 2 Heavy Chain
Variable Region--protein 3 Light (kappa) Chain Variable
Region--nucleic acid 4 Light (kappa) Chain Variable Region--protein
5 Heavy Chain CDR.sub.1 (Kabat definition) 6 Heavy Chain CDR.sub.2
(Kabat definition) 11 Heavy Chain CDR.sub.3 (IGMT definition) 12
Light (kappa) Chain CDR.sub.1 (Kabat definition) 13 Light (kappa)
Chain CDR.sub.2 (Kabat definition) 14 Light (kappa) Chain CDR.sub.3
(Kabat definition)
[0098] Mouse monoclonal antibody heavy chain CDR sequences (Kabat,
Chothia, and IMGT definitions) are shown in Table 2.
TABLE-US-00006 TABLE 2 CDR1 CDR2 CDR3 Kabat 4B9 SYWMH
AIYPGNSDTDYSQKFKG FDY (SEQ ID (SEQ ID NO: 6) NO: 5) Chothia 4B9
GYTFTSY YPGNSD FDY (SEQ ID (SEQ ID NO: 8) NO: 7) IMGT 4B9 GYTFTSYW
IYPGNSDT SKFDY (SEQ ID (SEQ ID NO: 10) (SEQ ID NO: 11) NO: 9)
[0099] Mouse monoclonal antibody Kappa light chain CDR sequences
(Kabat, Chothia, and IMGT definitions) are shown in Table 3.
TABLE-US-00007 TABLE 3 CDR1 CDR2 CDR3 Kabat/Chothia 4B9 SASSSVNYMY
LTSNLAS QQWSSNPYT (SEQ ID (SEQ ID (SEQ ID NO: 14) NO: 12) NO: 13)
IMGT 4B9 SSVNY LTS QQWSSNPYT (SEQ ID (SEQ ID NO: 14) NO: 15)
[0100] To create the complete heavy or kappa chain antibody
sequences, each variable sequence above is combined with its
respective constant region. For example, a complete heavy chain
comprises the heavy variable sequence followed by the murine IgG1
heavy chain constant sequence and the complete kappa chain
comprises a kappa variable sequence followed by the murine kappa
light chain constant sequence.
[0101] Nucleic Acid Sequence Encoding the Murine IgG1 Heavy Chain
Constant Region (SEQ ID NO: 16)
TABLE-US-00008 1 gccaaaacga cacccccatc tgtctatcca ctggcccctg
gatctgctgc ccaaactaac 61 tccatggtga ccctgggatg cctggtcaag
ggctatttcc ctgagccagt gacagtgacc 121 tggaactctg gatccctgtc
cagcggtgtg cacaccttcc cagctgtcct gcagtctgac 181 ctctacactc
tgagcagctc agtgactgtc ccctccagca cctggcccag ccagaccgtc 241
acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg
301 gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt
cttcatcttc 361 cccccaaagc ccaaggatgt gctcaccatt actctgactc
ctaaggtcac gtgtgttgtg 421 gtagacatca gcaaggatga tcccgaggtc
cagttcagct ggtttgtaga tgatgtggag 481 gtgcacacag ctcagacgca
accccgggag gagcagttca acagcacttt ccgctcagtc 541 agtgaacttc
ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc 601
aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg
661 aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa
ggataaagtc 721 agtctgacct gcatgataac agacttcttc cctgaagaca
ttactgtgga gtggcagtgg 781 aatgggcagc cagcggagaa ctacaagaac
actcagccca tcatggacac agatggctct 841 tacttcgtct acagcaagct
caatgtgcag aagagcaact gggaggcagg aaatactttc 901 acctgctctg
tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac 961
tctcctggta aa
[0102] Protein Sequence Defining the Murine IgG1 Heavy Chain
Constant Region (SEQ ID NO: 17)
TABLE-US-00009 1 akttppsvyp lapgsaaqtn smvtlgclvk gyfpepvtvt
wnsgslssgv htfpavlqsd 61 lytlsssvtv psstwpsqtv tcnvahpass
tkvdkkivpr dcgckpcict vpevssvfif 121 ppkpkdvlti tltpkvtcvv
vdiskddpev qfswfvddve vhtaqtqpre eqfnstfrsv 181 selpimhqdw
lngkefkcrv nsaafpapie ktisktkgrp kapqvytipp pkeqmakdkv 241
sltcmitdff peditvewqw ngqpaenykn tqpimdtdgs yfvysklnvq ksnweagntf
301 tcsvlheglh nhhtekslsh spgk
[0103] Nucleic Acid Sequence Encoding the Murine Kappa Light Chain
Constant Region (SEQ ID NO: 18)
TABLE-US-00010 1 cgggctgatg ctgcaccaac tgtatccatc ttcccaccat
ccagtgagca gttaacatct 61 ggaggtgcct cagtcgtgtg cttcttgaac
aacttctacc ccagagacat caatgtcaag 121 tggaagattg atggcagtga
acgacaaaat ggtgtcctga acagttggac tgatcaggac 181 agcaaagaca
gcacctacag catgagcagc accctcacat tgaccaagga cgagtatgaa 241
cgacataaca gctatacctg tgaggccact cacaagacat caacttcacc cattgtcaag
301 agcttcaaca ggaatgagtg t
[0104] Protein Sequence Defining the Murine Kappa Light Chain
Constant Region (SEQ ID NO: 19)
TABLE-US-00011 1 radaaptvsi fppsseqlts ggasvvcfln nfyprdinvk
wkidgserqn gvlnswtdqd 61 skdstysmss tltltkdeye rhnsytceat
hktstspivk sfnrnec
[0105] The following sequences represent the actual or contemplated
full length heavy and light chain sequences (i.e., containing both
the variable and constant regions sequences) for each antibody
described in this Example. Signal sequences for proper secretion of
the antibodies are also included at the 5' end of the DNA sequences
or the amino terminal end of the protein sequences. The variable
region sequences can be ligated to other constant region sequences,
to produce active full length IgG heavy and light chains.
[0106] Nucleic Acid Sequence Encoding the Full Length Heavy Chain
Sequence (Heavy Chain Variable Region and IgG1 Constant Region) of
4B9 (SEQ ID NO: 20)
TABLE-US-00012 1 atggaatgta actggatact tccttttatt ctgtcggtaa
cttcaggggt ctactcagag 61 gttcagctcc agcagtctgg gactgtgctg
gcaaggcctg gggcttcagt gaagatgtcc 121 tgcaagactt ctggctacac
atttaccagc tactggatgc actgggtaaa acagaggcct 181 ggacagggtc
tggaatggat aggggctatt tatcctggaa atagtgatac tgactacagc 241
cagaagttca agggcaaggc cacactgact gcagtcacat ccgccaccac tgcctacatg
301 gaactcagca gcctgacaaa tgaggactct gcggtctatt actgttcaaa
gtttgactac 361 tggggccaag gcaccactct cacagtctcc tcagccaaaa
cgacaccccc atctgtctat 421 ccactggccc ctggatctgc tgcccaaact
aactccatgg tgaccctggg atgcctggtc 481 aagggctatt tccctgagcc
agtgacagtg acctggaact ctggatccct gtccagcggt 541 gtgcacacct
tcccagctgt cctgcagtct gacctctaca ctctgagcag ctcagtgact 601
gtcccctcca gcacctggcc cagccagacc gtcacctgca acgttgccca cccggccagc
661 agcaccaagg tggacaagaa aattgtgccc agggattgtg gttgtaagcc
ttgcatatgt 721 acagtcccag aagtatcatc tgtcttcatc ttccccccaa
agcccaagga tgtgctcacc 781 attactctga ctcctaaggt cacgtgtgtt
gtggtagaca tcagcaagga tgatcccgag 841 gtccagttca gctggtttgt
agatgatgtg gaggtgcaca cagctcagac gcaaccccgg 901 gaggagcagt
tcaacagcac tttccgctca gtcagtgaac ttcccatcat gcaccaggac 961
tggctcaatg gcaaggagtt caaatgcagg gtcaacagtg cagctttccc tgcccccatc
1021 gagaaaacca tctccaaaac caaaggcaga ccgaaggctc cacaggtgta
caccattcca 1081 cctcccaagg agcagatggc caaggataaa gtcagtctga
cctgcatgat aacagacttc 1141 ttccctgaag acattactgt ggagtggcag
tggaatgggc agccagcgga gaactacaag 1201 aacactcagc ccatcatgga
cacagatggc tcttacttcg tctacagcaa gctcaatgtg 1261 cagaagagca
actgggaggc aggaaatact ttcacctgct ctgtgttaca tgagggcctg 1321
cacaaccacc atactgagaa gagcctctcc cactctcctg gtaaa
[0107] Protein Sequence Defining the Full Length Heavy Chain
Sequence (Heavy Chain Variable Region and IgG1 Constant Region) of
4B9 (SEQ ID NO: 21)
TABLE-US-00013 1 mecnwilpfi lsvtsgvyse vqlqqsgtvl arpgasvkms
cktsgytfts ywmhwvkqrp 61 gqglewigai ypgnsdtdys qkfkgkatlt
avtsattaym elssltneds avyycskfdy 121 wgqgttltvs sakttppsvy
plapgsaaqt nsmvtlgclv kgyfpepvtv twnsgslssg 181 vhtfpavlqs
dlytlsssvt vpsstwpsqt vtcnvahpas stkvdkkivp rdcgckpcic 241
tvpevssvfi fppkpkdvlt itltpkvtcv vvdiskddpe vqfswfvddv evhtaqtqpr
301 eeqfnstfrs vselpimhqd wlngkefkcr vnsaafpapi ektisktkgr
pkapqvytip 361 ppkeqmakdk vsltcmitdf fpeditvewq wngqpaenyk
ntqpimdtdg syfvysklnv 421 qksnweagnt ftcsvlhegl hnhhteksls
hspgk
[0108] Nucleic Acid Sequence Encoding the Full Length Light Chain
Sequence (Kappa Chain Variable Region and Constant Region) of 4B9
(SEQ ID NO: 22)
TABLE-US-00014 1 atggattttc aagtgcagat tttcagcttc ctgctaatga
gtgcctcagt cataatgtcc 61 aggggacaaa ttgttctcac ccagtctcca
gcactcatgt ctgcatctcc aggggagaag 121 gtcaccatga cctgcagtgc
cagctcaagt gtaaattaca tgtactggta ccagcagaag 181 ccaagatcct
cccccaaacc ctggatttat ctcacatcca acctggcttc tggagtccct 241
gctcgcttca gtggcagggg gtctgggacc tcttactctc tcacaatcag cagcatggag
301 gctgaagatg ctgccactta ttactgccag cagtggagta gtaacccgta
cacgttcgga 361 ggggggacca agctggaaat aaaacgggct gatgctgcac
caactgtatc catcttccca 421 ccatccagtg agcagttaac atctggaggt
gcctcagtcg tgtgcttctt gaacaacttc 481 taccccagag acatcaatgt
caagtggaag attgatggca gtgaacgaca aaatggtgtc 541 ctgaacagtt
ggactgatca ggacagcaaa gacagcacct acagcatgag cagcaccctc 601
acattgacca aggacgagta tgaacgacat aacagctata cctgtgaggc cactcacaag
661 acatcaactt cacccattgt caagagcttc aacaggaatg agtgt
[0109] Protein Sequence Defining the Full Length Light Chain
Sequence (Kappa Chain Variable Region and Constant Region) of 4B9
(SEQ ID NO: 23)
TABLE-US-00015 1 mdfqvqifsf llmsasvims rgqivltqsp almsaspgek
vtmtcsasss vnymywyqqk 61 prsspkpwiy ltsnlasgvp arfsgrgsgt
sysltissme aedaatyycq qwssnpytfg 121 ggtkleikra daaptvsifp
psseqltsgg asvvcflnnf yprdinvkwk idgserqngv 181 lnswtdqdsk
dstysmsstl tltkdeyerh nsytceathk tstspivksf nrnec
[0110] Table 4 shows the correspondence between the full length
sequences of the antibodies discussed in this Example with those
presented in the Sequence Listing.
TABLE-US-00016 TABLE 4 SEQ ID NO. Antibody 4B9 Nucleic Acid or
Protein 20 Heavy Variable + IgG1 Constant--nucleic acid 21 Heavy
Variable + IgG1 Constant--protein 22 Kappa Variable +
Constant--nucleic acid 23 Kappa Variable + Constant--protein
Example 5
Binding Affinities
[0111] The binding affinities and binding kinetics of monoclonal
antibody 4B9 were measured with respect to the following proteins
(R&D Systems, Inc., Minneapolis, Minn.): recombinant human
FGFR1 beta (IIIb)/Fc Chimera (rhFGFR1.beta.-IIIc-Fc), recombinant
human FGFR2 beta (IIIb)/Fc Chimera (rhFGFR2.beta.-IIIb-Fc),
recombinant human FGFR2 beta (IIIc)/Fc Chimera
(rhFGFR2.beta.-IIIc-Fc), recombinant human FGFR3 beta (IIIb)/Fc
Chimera (rhFGFR3.beta.-IIIb-Fc), and a version of recombinant human
FGFR2 beta (IIIb)/Fc (in which the Fc region was removed
enzymatically). Binding affinities and binding kinetics were
measured by surface plasmon resonance using a Biacore T100
instrument (GE Healthcare, Piscataway, N.J.).
[0112] Rabbit anti-mouse IgGs (GE Healthcare) were immobilized on
carboxymethylated dextran CM4 sensor chips (GE Healthcare) by amine
coupling, using a standard coupling protocol, according to the
vendor's instructions (GE Healthcare). The analyses were performed
at 25.degree. C. and 37.degree. C., using PBS containing 0.05%
surfactant P20 (GE Healthcare) as running buffer.
[0113] The antibodies were captured in individual flow cells at a
flow rate of 10 .mu.l/min. Injection time was varied for each
antibody to yield an Rmax between 30 and 60 RU. Buffer and FGFR
proteins diluted in running buffer were injected sequentially over
a reference surface (no antibody captured) and the active surface
(antibody to be tested) for 240 seconds at 60 .mu.l/min. The
dissociation phase was monitored for up to 900 seconds. The surface
was then regenerated with two 60-second injections of 10 mM
Glycine-HCl (pH 1.7), at a flow rate of 60 .mu.l/minute. The FGFR
protein concentration range tested was 50 to 3.125 nM (two-fold
dilutions).
[0114] Kinetic parameters were determined using the kinetic
function of the BlAevalutation software (GE Healthcare) with double
reference subtraction. Kinetic parameters for each antibody,
k.sub.a (association rate constant), k.sub.d (dissociation rate
constant) and K.sub.D (equilibrium dissociation constant) were
determined. Kinetic values of the monoclonal antibodies on FGFR
proteins at 25.degree. C. and 37.degree. C. are summarized in Table
5.
TABLE-US-00017 TABLE 5 Anti- Temp body Target (.degree. C.) k.sub.a
(M.sup.-1 s.sup.-1) k.sub.d (s.sup.-1) K.sub.D (M) 4B9
rhFGFR1.beta.-IIIb-Fc 25 no binding no binding no binding 4B9
rhFGFR2.beta.-IIIb-Fc 25 9.4E+04 4.6E-05 6.1E-10 4B9
rhFGFR2.beta.-IIIb-Fc 37 3.44E+04 3.16E-05 2.96E-09 4B9
rhFGFR2.beta.-IIIb- 25 5.5E+04 8.1E-05 4.2E-09 cleaved 4B9
rhFGFR2.beta.-IIIb- 37 2.54E+05 2.23E-04 1.20E-09 cleaved 4B9
rhFGFR2.beta.-IIIc-Fc 25 no binding no binding no binding 4B9
rhFGFR3.beta.-IIIb-Fc 25 no binding no binding no binding
[0115] The results in Table 5 demonstrate that antibody 4B9 binds
rhFGFR2.beta.-IIIb with a K.sub.D of about 5 nM, 4 nM, 3 nM, 2 nM,
1 nM, 750 pM, 650 pM, 610 pM or less. The results also demonstrate
that antibody 4B9 does not bind rhFGFR1.beta.-IIIb,
rhFGFR2.beta.-IIIc, and rhFGFR3.beta.-IIIb.
Example 6
Anti-Proliferative Activity
[0116] To assess the potency of antibody 4B9 quantitatively, we
carried out dose-response studies, using FDCP-1 cells expressing
FGFR2-IIIb or FGFR2-IIIc. FDCP-1 cells expressing FGFR2-IIIb or
FGFR2-IIIc were seeded in a 96-well plate in the absence of IL3.
Varied amounts of FGFs and heparin were added. MTT assays were
carried out after 2-3 days. Varied amounts of antibody
4B9-containing supernatants were added to FDCP-1 cells expressing
FGFR2-IIIb, FGFR2-IIIc, or C-terminally truncated FGFR2-IIIb, in
the presence of FGF1 and heparin. MTT assays were carried out after
2 days. Varied amounts of purified antibody 4B9 were added to
FDCP-1 cells expressing FGFR2-IIIb S252W or FGFR2-IIIb N550K in the
presence of FGF1 and heparin. MTT assays were carried out after 2
days.
[0117] Antibody 4B9 potently inhibited FGF1-induced proliferation
of FDCP-1 cells driven by FGFR2-IIIb, in a dose-dependent manner,
while 4B9 had no significant effect on the FGF1-induced
proliferation of FDCP cells expressing the FGFR2-IIIc (FIG. 4).
C-terminally truncated FGFR2-IIIb, which causes constitutive
phosphorylation of FRS2 adaptor molecule and activation of
downstream signaling, is found in gastric and breast cancer cell
lines (Itoh et al., 1994, CANCER RES. 54:3237-3241; Moffa et al.,
2004, MOL. CANCER RES. 2:643-652). Antibody 4B9 potently inhibited
the proliferation of FDCP-1 cells driven by the C-terminally
truncated FGFR2-IIIb (FIG. 4).
[0118] FGFR2 mutations have been reported in approximately 12% of
endometrial tumor sample (Pollock et al., supra; Dutt et al.,
supra). Somatic activating mutations in FGFR2 cluster within the
linker region between IgD2 and IgD3, the extracellular
juxtamembrane domain, or the kinase domain. Two of the most common
mutations in endometrial tumors are the S252W mutation (which
alters ligand specificity and increases affinity of ligand binding)
and the N550K mutation in the kinase domain (which enhances kinase
activity). Purified antibody 4B9 potently inhibited cell
proliferation driven by the wild type FGFR2-IIIb, as well as
FGFR2-IIIb S252W and FGFR2-IIIb N550K, with IC.sub.50 values of 0.3
nM, 3.0 nM and 8.1 nM, respectively (FIG. 5).
Example 7
Inhibition of FGFR2-Activated Signaling Pathways
[0119] We investigated the effect of antibody 4B9 on
FGFR2-activated signaling pathways. To examine the effect of
antibody 4B9 on tyrosine phosphorylation of FGFR2, SNU-16 cells
were treated with antibodies at a dose of 5 .mu.g/ml for 1 hour at
37.degree. C., followed by stimulation with heparin alone (20
.mu.g/ml) or heparin-plus-FGF7 (30 ng/ml) for 15 minutes. The cells
were lysed in NP-40 lysis buffer containing 1% NP-40, 20 mM
Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 2 mM EDTA and
supplemented with protease inhibitors (Roche Applied Science) and
Halt phosphatase inhibitors (Thermo Scientific).
[0120] The lysates were analyzed by Western blot with anti-FGFR
(Y653/Y654) (R&D Systems, Inc., Minneapolis, Minn.), anti-FGFR2
(sc-122) (Santa Cruz Biotechnology, Santa Cruz, Calif.),
anti-phospho-ERK1/2 and anti-ERK1/2 (Cell Signaling Technology,
Danvers, Mass.), anti-.beta.-tubulin, clone AA2 (Millipore
Corporation; Billerica, Mass.) antibodies. The immunoblots were
detected by chemiluminescent substrate (ECL Plus.TM., Amersham
Pharmacia Biotech, Piscataway, N.J.). Human Phospho-RTK and MAPK
kinase arrays (R&D systems) were carried out according to
manufacturer's instructions (R&D systems). For phospho-RTK
arrays, the cells were lysed in NP-40 lysis buffer. The arrays were
blocked in Array Buffer 1 at room temperature for one hour prior to
the addition of cell lysates diluted in Array Buffer 1 and were
then incubated at 4.degree. C. overnight. The arrays were
visualized by chemiluminescence. For phospho-MAPK arrays, the cells
were lysed in Lysis Buffer 6. The diluted cell lysates were added
to arrays. After incubation at 4.degree. C. overnight, the arrays
were mixed with anti-phospho-MAPK antibody for two hours at room
temperature and visualized as described above.
[0121] FGF7 induced tyrosine phosphorylation of FGFR2 and
subsequent activation of extracellular signal-regulated kinase 1
and 2 (ERK1/2) in Ba/F3 cells overexpressing FGFR2, and in
FGFR2-amplified SNU-16 cells. Antibody 4B9 effectively suppressed
the ligand-induced tyrosine phosphorylation of FGFR2 and activation
of ERK1/2 in these cells. In addition, antibody 4B9 downregulated
the FGFR2 protein level in SNU-16 cells. A slight decrease in the
FGFR2 protein level was observed as early as two hours after
exposure to the antibody. A dramatic reduction in the protein level
was seen at the six-hour time point.
[0122] We investigated activation of downstream signaling pathways
in these cell lines, using a phospho-MAPK array, which measures
phosphorylation of ERKs, c-Jun NH.sub.2-Terminal Kinases (JNKs),
p38 MAPKs, AKTs, and their downstream effector molecules. We found
little phosphorylation of ERK1/2 in the absence of ligand
stimulation. Stimulation of SNU-16 cells with FGF7 significantly
increased the phosphorylation of ERK1/2. We observed an increase in
the phosphorylation of mitogen- and stress-activated kinase 2
(MSK2), p38.alpha. MAPK, 90-kD ribosomal protein kinase 1 (RSK1),
Akt1, and p70S6 kinase (p70S6K). Antibody 4B9 effectively blocked
the phosphorylation of all the downstream signaling proteins
activated by FGF7.
Example 8
Inhibition of Tumor Xenograft Growth
[0123] To assess the activity of antibody 4B9 in vivo, we tested
the effect of antibody 4B9 on the growth of human cancer xenografts
harboring amplification of the FGFR2 gene. Out of the four
FGFR2-amplified cell lines that were tested, only SNU-16 and
MFM-223 yielded tumors in mice. Therefore, we tested the efficacy
of antibody 4B9 against SNU-16 and MFM-223 xenograft tumors.
[0124] All mice were treated in accordance with the OLAW Public
Health Service Policy on Human Care and Use of Laboratory Animals
and the ILAR Guide for the Care and Use of Laboratory Animals. All
in vivo studies were conducted following the protocols approved by
the AVEO Institutional Animal Care and Use Committee. For the
SNU-16 in vivo studies, 10 week old female C.B-17 SCID mice
(Taconic, Germantown, N.Y.) were inoculated subcutaneously into the
right flank with 5.times.10.sup.6 cells in 1:1 RPMI 1640
(Invitrogen, Carlsbad, Calif.)/Matrigel (BD Biosciences, San Jose
Calif.). Tumor measurements were taken twice weekly, using vernier
calipers. Tumor volume was calculated using the formula:
V=0.5.times.width.times.width.times.length. When tumors approached
a volume of 200 mm.sup.3, mice were randomized into five groups of
ten animals. The next day, mice were treated with 20 mg/kg mIgG
(BioXCell; West Lebanon, N.H.), 2 mg/kg 4B9, 5 mg/kg 4B9, 10 mg/kg
4B9, or 20 mg/kg 4B9 by intraperitoneal injection. Mice were dosed
twice weekly for the duration of the study. Seventy-two hours after
the final dose tumor volumes were measured again for calculation of
tumor growth inhibition. All statistical analysis was done using
GraphPad PRISM.RTM. Version 4.00. Final tumor volumes were analyzed
using with a one-way analysis of variance and Tukey multiple
comparison test.
[0125] SNU-16 xenograft tumors were treated with a control murine
IgG at 20 mg/kg or antibody 4B9 at 2, 5, 10 or 20 mg/kg. As shown
in FIG. 6, each 4B9 treatment group showed significant tumor growth
inhibition, as compared to mIgG treated controls (70, 72, 77, and
82%, respectively p<0.001) at day 43, which was the last day for
the control group to remain in the study. All treatments were
well-tolerated with no significant body weight loss. The tumor
lysates were also analyzed. Concomitant with inhibition of tyrosine
phosphorylation of FGFR2, antibody 4B9 downregulated the total
amount of FGFR2 protein in tumors. No significant difference in the
total ERK1/2 or phospho-ERK1/2 was detected in the tumors samples
treated with control IgG or 4B9 from tumors collected at the end of
study. In contrast to the phospho-receptor tyrosine kinase (RTK)
profile of SNU-16 cells in vitro, RTK array analysis of SNU-16
xenografts revealed that FGFR2 was the predominant RTK that was
tyrosine phosphorylated in vivo, and 4B9 significantly inhibited
FGFR2 tyrosine phosphorylation in two of the 4B9-treated SNU-16
tumors tested. In vitro, the proliferation of SNU-16 cells was not
sensitive to the treatment of 4B9. Tyrosine phosphorylation of
FGFR2 in SNU-16 cells in vivo suggests that the dependence of
SNU-16 xenografts on activated FGFR2 signaling in vivo explains
their sensitivity to treatment with antibody 4B9.
[0126] The effect of antibody 4B9 was also investigated on the in
vivo growth of FGFR2-amplified breast cancer cell line MFM-223. For
these studies, 5-week old female NCr nude mice (Taconic;
Germantown, N.Y.) were implanted subcutaneously on the left flank
with 0.72 mg 90-day release 17-.beta. estradiol pellets (Innovative
Research; Sarasota, Fla.) and inoculated subcutaneously into the
right flank with 10.times.10.sup.6 MFM-223 cells in 1:1 EMEM (ATCC;
Manassas, Va.)/Matrigel. When tumors approached a volume of 200
mm.sup.3, mice were randomized into two groups of ten animals and
treated IP with 20 mg/kg mIgG (BioXCell; West Lebanon, N.H.) or 20
mg/kg 4B9. Mice were dosed twice weekly for the duration of the
study. All statistical analysis was done using GraphPad PRISM.RTM.
Version 4.00. Since there were only two groups in this study final
tumor volumes and weights (Day 27, 48 hours after final dose) were
analyzed with an unpaired two tailed t-test.
[0127] On day 25, in the MFM-223 xenografts, there was greater than
66% inhibition of tumor volumes (p=0.0015; FIG. 7) and final tumor
weights (p=0.0188) in 4B9 treated mice, as compared to mIgG-treated
controls. All treatments were well-tolerated, with no significant
body weight loss. Similar to what was observed in SNU-16
xenografts, 4B9 strongly down-regulated the total FGFR2 protein in
tumors, concomitant with inhibition of tyrosine phosphorylation of
FGFR2. No significant difference in the total or phosphor-ERK1/2
was detected in the tumors samples either treated with the control
IgG or 4B9 from tumors collected at the end of study.
Example 9
Humanization of Anti-FGFR2Antibodies
[0128] A. Construction of Humanized FGFR2Antibodies
[0129] This Example describes the humanization of the murine
antibody designated 4B9, and the characterization of the resulting
humanized antibodies. The humanized anti-FGFR2Mb antibodies were
designed using methods well-known in the art. The designed amino
acid sequences were converted to codon-optimized DNA sequences and
synthesized by DNA2.0, Inc. to include (in the following order): 5'
HindIII restriction site, Kozak consensus sequence, amino terminal
signal sequence, humanized variable region, human IgG 1 or Kappa
constant region, stop codon, and a 3' EcoRI restriction site.
[0130] The humanized heavy chains were subcloned into pEE6.4
(Lonza, Basel, Switzerland) via HindIII and EcoRI sites using
In-Fusion.TM. PCR cloning (Clontech, Mountain View, Calif.). The
humanized Kappa light chains were subcloned into pEE14.4 (Lonza)
via HindIII and EcoRI sites using In-Fusion.TM. PCR cloning.
[0131] Humanized antibody chains were transiently transfected into
293T cells to produce antibody. Antibody was purified for
subsequent in vitro analysis. Binding of the humanized antibodies
to human FGFR2Mb was measured as described below. The results are
summarized in Tables 12 and 13.
[0132] Each of the possible combinations of the humanized
immunoglobulin heavy chain and immunoglobulin light chain variable
regions are set forth below in Table 6.
TABLE-US-00018 TABLE 6 Light Chain Variable Region Heavy Chain
Variable Region Hu4B9-65 Kappa (SEQ ID NO: 40) Hu4B9-65 Heavy (SEQ
ID NO: 35) Hu4B9-65 Kappa (SEQ ID NO: 40) Hu4B9-82, -83 Heavy (SEQ
ID NO: 37) Hu4B9-82 Kappa (SEQ ID NO: 44) Hu4B9-65 Heavy (SEQ ID
NO: 35) Hu4B9-82 Kappa (SEQ ID NO: 44) Hu4B9-82, -83 Heavy (SEQ ID
NO: 37) Hu4B9-83 Kappa (SEQ ID NO: 46) Hu4B9-65 Heavy (SEQ ID NO:
35) Hu4B9-83 Kappa (SEQ ID NO: 46) Hu4B9-82, -83 Heavy (SEQ ID NO:
37)
[0133] The nucleic acid sequences encoding and the protein
sequences defining variable regions of the humanized 4B9 antibodies
are summarized below (amino terminal signal peptide sequences are
not shown). CDR sequences (Kabat definition) are shown in bold and
are underlined in the amino acid sequences.
[0134] Nucleic Acid Sequence Encoding the Hu4B9-65 Heavy Chain
Variable Region (SEQ ID NO: 34)
TABLE-US-00019 1 caagtgcagc tcgtccaatc gggagccgaa gtgaagaagc
ctggttcctc ggtaaaagta 61 agctgtaagg cgtccggtta cacgtttacc
tcatattgga tgcactgggt cagacaggca 121 cccggacagg gactcgagtg
gatgggagcg atctacccgg gcaattcgga cactgattac 181 agccagaaat
tcaaggggag ggtcacgatc acggcagatg agagcacatc aacagcctat 241
atggagctgt cgtcgcttcg gagcgaggac acggcggtct actactgctc caaattcgac
301 tattgggggc aggggacctt ggtgaccgtg tcatcc
[0135] Protein Sequence Defining the Hu4B9-65 Heavy Chain Variable
Region (SEQ ID NO: 35)
TABLE-US-00020 1 qvqlvqsgae vkkpgssvkv sckasgytft sywmhwvrqa
pgqglewmga iypgnsdtdy 61 sqkfkgrvti tadeststay melsslrsed
tavyycskfd ywgqgtlvtv ss
[0136] Nucleic Acid Sequence Encoding the Hu4B9-82, -83 Heavy Chain
Variable Region (SEQ ID NO: 36)
TABLE-US-00021 1 caagtgcagc tcgtccaatc gggagccgaa gtgaagaagc
ctggttcctc ggtaaaagta 61 agctgtaagg cgtccggtta cacgttttcc
tcatattgga tgcactgggt cagacaggca 121 cccggacagg gactcgagtg
gatgggagcg atctacccgg gcaattcgga cactgattac 181 agccagaaat
tccaggggag ggtcacgatc acggcagatg agagcacatc aacagcctat 241
atggagctgt cgtcgcttcg gagcgaggac acggcggtct actactgctc caaattcgac
301 tattgggggc aggggacctt ggtgaccgtg tcatcc
[0137] Protein Sequence Defining the Hu4B9-82, -83 Heavy Chain
Variable Region (SEQ ID NO: 37)
TABLE-US-00022 1 qvqlvqsgae vkkpgssvkv sckasgytfs sywmhwvrqa
pgqglewmga iypgnsdtdy 61 sqkfqgrvti tadeststay melsslrsed
tavyycskfd ywgqgtlvtv ss
[0138] Nucleic Acid Sequence Encoding the Hu4B9-65 Kappa Chain
Variable Region (SEQ ID NO: 39)
TABLE-US-00023 1 gaaattgtgc tgacccagag cccggcgacc ctgagcctga
gcccgggcga acgcgcgacc 61 ctgagctgcc gcgcgagcag cagcgtgaac
tatatgtatt ggtatcagca gaaaccgggc 121 caggcgccgc gcccgtggat
ttatctgacc agcaaccgcg cgaccggcgt gccggcgcgc 181 tttagcggca
gcggcagcgg caccgattat accctgacca ttagcagcct ggaaccggaa 241
gattttgcgg tgtattattg ccagcagtgg agcagcaacc cgtatacctt tggccagggc
301 accaaactgg aaattaaa
[0139] Protein Sequence Defining the Hu4B9-65 Kappa Chain Variable
Region (SEQ ID NO: 40)
TABLE-US-00024 1 eivltqspat lslspgerat lscrasssvn ymywyqqkpg
qaprpwiylt snratgvpar 61 fsgsgsgtdy tltisslepe dfavyycqqw
ssnpytfgqg tkleik
[0140] Nucleic Acid Sequence Encoding the Hu4B9-82 Kappa Chain
Variable Region (SEQ ID NO: 43)
TABLE-US-00025 1 gaaatcgtac ttactcagag ccctgccaca ttgtcattgt
cacccgggga acgcgccaca 61 ctgtcgtgcc gggcttcatc gagcgtgaac
tacatgtatt ggtatcaaca gaaaccaggc 121 caagcaccgc gaccttggat
ctacttgacg agcaatcgag ccacgggtat ccccgcgagg 181 ttctccggtt
cggggtcggg aactgattac acactgacaa tttcctcgct ggagcccgag 241
gacttcgcgg tgtactattg tcagcagtgg tcatccaacc cgtacacgtt tggacagggg
301 acgaagctcg agatcaag
[0141] Protein Sequence Defining the Hu4B9-82 Kappa Chain Variable
Region (SEQ ID NO: 44)
TABLE-US-00026 1 eivltqspat lslspgerat lscrasssvn ymywyqqkpg
qaprpwiylt snratgipar 61 fsgsgsgtdy tltisslepe dfavyycqqw
ssnpytfgqg tkleik
[0142] Nucleic Acid Sequence Encoding the Hu4B9-83 Kappa Chain
Variable Region (SEQ ID NO: 45)
TABLE-US-00027 1 gaaatcgtac ttactcagag ccctgccaca ttgtcattgt
cacccgggga acgcgccaca 61 ctgtcgtgcc gggcttcatc gagcgtgaac
tacatgtatt ggtatcaaca gaaaccaggc 121 caagcaccgc gaccttggat
ctacttgacg agcaatcgag ccacgggtat ccccgcgagg 181 ttctccggtt
cggggtcggg aactgatttc acactgacaa tttcctcgct ggagcccgag 241
gacttcgcgg tgtactattg tcagcagtgg tcatccaacc cgtacacgtt tggacagggg
301 acgaagctcg agatcaag
[0143] Protein Sequence Defining the Hu4B9-83 Kappa Chain Variable
Region (SEQ ID NO: 46)
TABLE-US-00028 1 eivltqspat lslspgerat lscrasssvn ymywyqqkpg
qaprpwiylt snratgipar 61 fsgsgsgtdf tltisslepe dfavyycqqw
ssnpytfgqg tkleik
[0144] The amino acid sequences defining the immunoglobulin heavy
chain variable regions for the antibodies produced in Example 9 are
aligned in FIG. 8. Amino terminal signal peptide sequences (for
proper expression/secretion) are not shown. CDR.sub.1, CDR.sub.2,
and CDR.sub.3 (Kabat definition) are identified by boxes (See FIG.
9).
[0145] The amino acid sequences defining the immunoglobulin light
chain variable regions for the antibodies in Example 9 are aligned
in FIG. 10. Amino terminal signal peptide sequences (for proper
expression/secretion) are not shown. CDR.sub.1, CDR.sub.2 and
CDR.sub.3 (Kabat definition) are identified by boxes (See FIG.
11).
[0146] Table 7 is a concordance chart showing the SEQ ID NO. of
each sequence discussed in this Example.
TABLE-US-00029 TABLE 7 SEQ. ID NO. Nucleic Acid or Protein 34
Hu4B9-65 Heavy Chain Variable Region--nucleic acid 35 Hu4B9-65
Heavy Chain Variable Region--protein 5 Hu4B9-65 Heavy Chain
CDR.sub.1 (Kabat definition) 6 Hu4B9-65 Heavy Chain CDR.sub.2
(Kabat definition) 11 Hu4B9-65 Heavy Chain CDR.sub.3 (IGMT
definition) 36 Hu4B9-82, -83 Heavy Chain Variable Region--nucleic
acid 37 Hu4B9-82, -83 Heavy Chain Variable Region--protein 5
Hu4B9-82, -83 Heavy Chain CDR.sub.1 (Kabat definition) 38 Hu4B9-82,
-83 Heavy Chain CDR.sub.2 (Kabat definition) 11 Hu4B9-82, -83 Heavy
Chain CDR.sub.3 (IGMT definition) 39 Hu4B9-65 Light (kappa) Chain
Variable Region--nucleic acid 40 Hu4B9-65 Light (kappa) Chain
Variable Region--protein 41 Hu4B9-65 Light (kappa) Chain CDR.sub.1
(Kabat definition) 42 Hu4B9-65 Light (kappa) Chain CDR.sub.2 (Kabat
definition) 14 Hu4B9-65 Light (kappa) Chain CDR.sub.3 (Kabat
definition) 43 Hu4B9-82 Light (kappa) Chain Variable
Region--nucleic acid 44 Hu4B9-82 Light (kappa) Chain Variable
Region--protein 41 Hu4B9-82 Light (kappa) Chain CDR.sub.1 (Kabat
definition) 42 Hu4B9-82 Light (kappa) Chain CDR.sub.2 (Kabat
definition) 14 Hu4B9-82 Light (kappa) Chain CDR.sub.3 (Kabat
definition) 45 Hu4B9-83 Light (kappa) Chain Variable
Region--nucleic acid 46 Hu4B9-83 Light (kappa) Chain Variable
Region--protein 41 Hu4B9-83 Light (kappa) Chain CDR.sub.1 (Kabat
definition) 42 Hu4B9-83 Light (kappa) Chain CDR.sub.2 (Kabat
definition) 14 Hu4B9-83 Light (kappa) Chain CDR.sub.3 (Kabat
definition)
[0147] Murine and humanized monoclonal antibody heavy chain CDR
sequences (Kabat, Chothia, and IMGT definitions) are shown in Table
8.
TABLE-US-00030 TABLE 8 CDR1 CDR2 CDR3 Kabat 4B9 SYWMH AIYPGNSDTDYSQ
FDY (SEQ ID KFKG NO: 5) (SEQ ID NO: 6) Hu4B9-65 SYWMH AIYPGNSDTDYSQ
FDY (SEQ ID KFKG NO: 5) (SEQ ID NO: 6) Hu4B9-82, -83 SYWMH
AIYPGNSDTDYSQ FDY (SEQ ID KFQG NO: 5) (SEQ ID NO: 38) CHOTHIA 4B9
GYTFTSY YPGNSD FDY (SEQ ID (SEQ ID NO: 8) NO: 7) Hu4B9-65 GYTFTSY
YPGNSD FDY (SEQ ID (SEQ ID NO: 8) NO: 7) Hu4B9-82, -83 GYTFSSY
YPGNSD FDY (SEQ ID (SEQ ID NO: 8) NO: 47) IMGT 4B9 GYTFTSYW
IYPGNSDT SKFDY (SEQ ID (SEQ ID NO: 10) (SEQ ID NO: 9) NO: 11)
Hu4B9-65 GYTFTSYW IYPGNSDT SKFDY (SEQ ID (SEQ ID NO: 10) (SEQ ID
NO: 9) NO: 11) Hu4B9-82, -83 GYTFSSYW IYPGNSDT SKFDY (SEQ ID (SEQ
ID NO: 10) (SEQ ID NO: 48) NO: 11)
[0148] Murine and humanized monoclonal antibody Kappa light chain
CDR sequences (Kabat, Chothia, and IMGT definitions) are shown in
Table 9.
TABLE-US-00031 TABLE 9 CDR1 CDR2 CDR3 Kabat/Chothia 4B9 SASSSVNYMY
LTSNLAS QQWSSNPYT (SEQ ID NO: 12) (SEQ ID (SEQ ID NO: 13) NO: 14)
Hu4B9- RASSSVNYMY LTSNRAT QQWSSNPYT 65 (SEQ ID NO: 41) (SEQ ID (SEQ
ID NO: 42) NO: 14) Hu4B9- RASSSVNYMY LTSNRAT QQWSSNPYT 82 (SEQ ID
NO: 41) (SEQ ID (SEQ ID NO: 42) NO: 14) Hu4B9- RASSSVNYMY LTSNRAT
QQWSSNPYT 83 (SEQ ID NO: 41) (SEQ ID (SEQ ID NO: 42) NO: 14) IGMT
4B9 SSVNY QQWSSNPYT (SEQ ID NO: 15) LTS (SEQ ID NO: 14) Hu4B9-
SSVNY QQWSSNPYT 65 (SEQ ID NO: 15) LTS (SEQ ID NO: 14) Hu4B9- SSVNY
QQWSSNPYT 82 (SEQ ID NO: 15) LTS (SEQ ID NO: 14) Hu4B9- SSVNY
QQWSSNPYT 83 (SEQ ID NO: 15) LTS (SEQ ID NO: 14)
[0149] To create the complete humanized heavy or kappa chain
antibody sequences, each variable sequence above is combined with
its respective human constant region. For example, a complete heavy
chain comprises a heavy variable sequence followed by a human IgG1
heavy chain constant sequence. A complete kappa chain comprises a
kappa variable sequence followed by the human kappa light chain
constant sequence.
[0150] Nucleic Acid Sequence Encoding the Human IgG1 Heavy Chain
Constant Region (SEQ ID NO: 49)
TABLE-US-00032 1 gcctcaacaa aaggaccaag tgtgttccca ctcgccccta
gcagcaagag tacatccggg 61 ggcactgcag cactcggctg cctcgtcaag
gattattttc cagagccagt aaccgtgagc 121 tggaacagtg gagcactcac
ttctggtgtc catacttttc ctgctgtcct gcaaagctct 181 ggcctgtact
cactcagctc cgtcgtgacc gtgccatctt catctctggg cactcagacc 241
tacatctgta atgtaaacca caagcctagc aatactaagg tcgataagcg ggtggaaccc
301 aagagctgcg acaagactca cacttgtccc ccatgccctg cccctgaact
tctgggcggt 361 cccagcgtct ttttgttccc accaaagcct aaagatactc
tgatgataag tagaacaccc 421 gaggtgacat gtgttgttgt agacgtttcc
cacgaggacc cagaggttaa gttcaactgg 481 tacgttgatg gagtcgaagt
acataatgct aagaccaagc ctagagagga gcagtataat 541 agtacatacc
gtgtagtcag tgttctcaca gtgctgcacc aagactggct caacggcaaa 601
gaatacaaat gcaaagtgtc caacaaagca ctcccagccc ctatcgagaa gactattagt
661 aaggcaaagg ggcagcctcg tgaaccacag gtgtacactc tgccacccag
tagagaggaa 721 atgacaaaga accaagtctc attgacctgc ctggtgaaag
gcttctaccc cagcgacatc 781 gccgttgagt gggagagtaa cggtcagcct
gagaacaatt acaagacaac ccccccagtg 841 ctggatagtg acgggtcttt
ctttctgtac agtaagctga ctgtggacaa gtcccgctgg 901 cagcagggta
acgtcttcag ctgttccgtg atgcacgagg cattgcacaa ccactacacc 961
cagaagtcac tgagcctgag cccagggaag
[0151] Protein Sequence Defining the Human IgG1 Heavy Chain
Constant Region (SEQ ID NO: 50)
TABLE-US-00033 1 astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs
wnsgaltsgv htfpavlqss 61 glyslssvvt vpssslgtqt yicnvnhkps
ntkvdkrvep kscdkthtcp pcpapellgg 121 psvflfppkp kdtlmisrtp
evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn 181 styrvvsvlt
vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree 241
mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw
301 qqgnvfscsv mhealhnhyt qkslslspgk
[0152] Nucleic Acid Sequence Encoding the Human Kappa Light Chain
Constant Region (SEQ ID NO: 51)
TABLE-US-00034 1 cgcacagttg ctgcccccag cgtgttcatt ttcccaccta
gcgatgagca gctgaaaagc 61 ggtactgcct ctgtcgtatg cttgctcaac
aacttttacc cacgtgaggc taaggtgcag 121 tggaaagtgg ataatgcact
tcaatctgga aacagtcaag agtccgtgac agaacaggac 181 agcaaagact
caacttattc actctcttcc accctgactc tgtccaaggc agactatgaa 241
aaacacaagg tatacgcctg cgaggttaca caccagggtt tgtctagtcc tgtcaccaag
301 tccttcaata ggggcgaatg t
[0153] Protein Sequence Defining the Human Kappa Light Chain
Constant Region (SEQ ID NO: 52)
TABLE-US-00035 1 rtvaapsvfi fppsdeqlks gtasvvclln nfypreakvq
wkvdnalqsg nsqesvteqd 61 skdstyslss tltlskadye khkvyacevt
hqglsspvtk sfnrgec
[0154] The following sequences represent the actual or contemplated
full length heavy and light chain sequences (i.e., containing both
the variable and constant regions sequences) for each antibody
described in this Example. Signal sequences for proper secretion of
the antibodies are also included at the 5' end of the DNA sequences
or the amino terminal end of the protein sequences. It is also
contemplated herein that the variable region sequences can be
ligated to other constant region sequences to produce active full
length IgG heavy and light chains.
[0155] Nucleic Acid Sequence Encoding the Full Length Humanized
Hu4B9-65 Heavy Chain (Humanized Heavy Chain Variable Region and
Human IgG1 Constant Region) (SEQ ID NO: 53)
TABLE-US-00036 1 atggacatga gagttcctgc tcagctgctc gggttgctgt
tgctttggct ccggggtgct 61 aggtgccaag tgcagctcgt ccaatcggga
gccgaagtga agaagcctgg ttcctcggta 121 aaagtaagct gtaaggcgtc
cggttacacg tttacctcat attggatgca ctgggtcaga 181 caggcacccg
gacagggact cgagtggatg ggagcgatct acccgggcaa ttcggacact 241
gattacagcc agaaattcaa ggggagggtc acgatcacgg cagatgagag cacatcaaca
301 gcctatatgg agctgtcgtc gcttcggagc gaggacacgg cggtctacta
ctgctccaaa 361 ttcgactatt gggggcaggg gaccttggtg accgtgtcat
ccgcctcaac aaaaggacca 421 agtgtgttcc cactcgcccc tagcagcaag
agtacatccg ggggcactgc agcactcggc 481 tgcctcgtca aggattattt
tccagagcca gtaaccgtga gctggaacag tggagcactc 541 acttctggtg
tccatacttt tcctgctgtc ctgcaaagct ctggcctgta ctcactcagc 601
tccgtcgtga ccgtgccatc ttcatctctg ggcactcaga cctacatctg taatgtaaac
661 cacaagccta gcaatactaa ggtcgataag cgggtggaac ccaagagctg
cgacaagact 721 cacacttgtc ccccatgccc tgcccctgaa cttctgggcg
gtcccagcgt ctttttgttc 781 ccaccaaagc ctaaagatac tctgatgata
agtagaacac ccgaggtgac atgtgttgtt 841 gtagacgttt cccacgagga
cccagaggtt aagttcaact ggtacgttga tggagtcgaa 901 gtacataatg
ctaagaccaa gcctagagag gagcagtata atagtacata ccgtgtagtc 961
agtgttctca cagtgctgca ccaagactgg ctcaacggca aagaatacaa atgcaaagtg
1021 tccaacaaag cactcccagc ccctatcgag aagactatta gtaaggcaaa
ggggcagcct 1081 cgtgaaccac aggtgtacac tctgccaccc agtagagagg
aaatgacaaa gaaccaagtc 1141 tcattgacct gcctggtgaa aggcttctac
cccagcgaca tcgccgttga gtgggagagt 1201 aacggtcagc ctgagaacaa
ttacaagaca acccccccag tgctggatag tgacgggtct 1261 ttctttctgt
acagtaagct gactgtggac aagtcccgct ggcagcaggg taacgtcttc 1321
agctgttccg tgatgcacga ggcattgcac aaccactaca cccagaagtc actgagcctg
1381 agcccaggga ag
[0156] Protein Sequence Defining the Full Length Humanized Hu4B9-65
Heavy Chain (Humanized Heavy Chain Variable Region and Human IgG1
Constant Region) (SEQ ID NO: 54)
TABLE-US-00037 1 mdmrvpaqll gllllwlrga rcqvqlvqsg aevkkpgssv
kvsckasgyt ftsywmhwvr 61 qapgqglewm gaiypgnsdt dysqkfkgrv
titadestst aymelsslrs edtavyycsk 121 fdywgqgtlv tvssastkgp
svfplapssk stsggtaalg clvkdyfpep vtvswnsgal 181 tsgvhtfpav
lqssglysls svvtvpsssl gtqtyicnvn hkpsntkvdk rvepkscdkt 241
htcppcpape llggpsvflf ppkpkdtlmi srtpevtcvv vdvshedpev kfnwyvdgve
301 vhnaktkpre eqynstyrvv svltvlhqdw lngkeykckv snkalpapie
ktiskakgqp 361 repqvytlpp sreemtknqv sltclvkgfy psdiavewes
ngqpennykt tppvldsdgs 421 fflyskltvd ksrwqqgnvf scsvmhealh
nhytqkslsl spgk
[0157] Nucleic Acid Sequence Encoding the Full Length Humanized
Hu4B9-82, -83 Heavy Chain (Humanized Heavy Chain Variable Region
and Human IgG1 Constant Region) (SEQ ID NO: 55)
TABLE-US-00038 1 atggacatga gagttcctgc tcagctgctc gggttgctgt
tgctttggct ccggggtgct 61 aggtgccaag tgcagctcgt ccaatcggga
gccgaagtga agaagcctgg ttcctcggta 121 aaagtaagct gtaaggcgtc
cggttacacg ttttcctcat attggatgca ctgggtcaga 181 caggcacccg
gacagggact cgagtggatg ggagcgatct acccgggcaa ttcggacact 241
gattacagcc agaaattcca ggggagggtc acgatcacgg cagatgagag cacatcaaca
301 gcctatatgg agctgtcgtc gcttcggagc gaggacacgg cggtctacta
ctgctccaaa 361 ttcgactatt gggggcaggg gaccttggtg accgtgtcat
ccgcctcaac aaaaggacca 421 agtgtgttcc cactcgcccc tagcagcaag
agtacatccg ggggcactgc agcactcggc 481 tgcctcgtca aggattattt
tccagagcca gtaaccgtga gctggaacag tggagcactc 541 acttctggtg
tccatacttt tcctgctgtc ctgcaaagct ctggcctgta ctcactcagc 601
tccgtcgtga ccgtgccatc ttcatctctg ggcactcaga cctacatctg taatgtaaac
661 cacaagccta gcaatactaa ggtcgataag cgggtggaac ccaagagctg
cgacaagact 721 cacacttgtc ccccatgccc tgcccctgaa cttctgggcg
gtcccagcgt ctttttgttc 781 ccaccaaagc ctaaagatac tctgatgata
agtagaacac ccgaggtgac atgtgttgtt 841 gtagacgttt cccacgagga
cccagaggtt aagttcaact ggtacgttga tggagtcgaa 901 gtacataatg
ctaagaccaa gcctagagag gagcagtata atagtacata ccgtgtagtc 961
agtgttctca cagtgctgca ccaagactgg ctcaacggca aagaatacaa atgcaaagtg
1021 tccaacaaag cactcccagc ccctatcgag aagactatta gtaaggcaaa
ggggcagcct 1081 cgtgaaccac aggtgtacac tctgccaccc agtagagagg
aaatgacaaa gaaccaagtc 1141 tcattgacct gcctggtgaa aggcttctac
cccagcgaca tcgccgttga gtgggagagt 1201 aacggtcagc ctgagaacaa
ttacaagaca acccccccag tgctggatag tgacgggtct 1261 ttctttctgt
acagtaagct gactgtggac aagtcccgct ggcagcaggg taacgtcttc 1321
agctgttccg tgatgcacga ggcattgcac aaccactaca cccagaagtc actgagcctg
1381 agcccaggga ag
[0158] Protein Sequence Defining the Full Length Humanized
Hu4B9-82, -83 Heavy Chain (Humanized Heavy Chain Variable Region
and Human IgG1 Constant Region) (SEQ ID NO: 56)
TABLE-US-00039 1 mdmrvpaqll gllllwlrga rcqvqlvqsg aevkkpgssv
kvsckasgyt fssywmhwvr 61 qapgqglewm gaiypgnsdt dysqkfqgrv
titadestst aymelsslrs edtavyycsk 121 fdywgqgtlv tvssastkgp
svfplapssk stsggtaalg clvkdyfpep vtvswnsgal 181 tsgvhtfpav
lqssglysls svvtvpsssl gtqtyicnvn hkpsntkvdk rvepkscdkt 241
htcppcpape llggpsvflf ppkpkdtlmi srtpevtcvv vdvshedpev kfnwyvdgve
301 vhnaktkpre eqynstyrvv svltvlhqdw lngkeykckv snkalpapie
ktiskakgqp 361 repqvytlpp sreemtknqv sltclvkgfy psdiavewes
ngqpennykt tppvldsdgs 421 fflyskltvd ksrwqqgnvf scsvmhealh
nhytqkslsl spgk
[0159] Nucleic Acid Sequence Encoding the Full Length Humanized
Hu4B9-65 Light Chain (Humanized Kappa Chain Variable Region and
Human Constant Region) (SEQ ID NO: 57)
TABLE-US-00040 1 atggacatga gggtgcccgc tcaactgctg gggctgctgc
tgctgtggct gagaggagct 61 cgttgcgaaa ttgtgctgac ccagagcccg
gcgaccctga gcctgagccc gggcgaacgc 121 gcgaccctga gctgccgcgc
gagcagcagc gtgaactata tgtattggta tcagcagaaa 181 ccgggccagg
cgccgcgccc gtggatttat ctgaccagca accgcgcgac cggcgtgccg 241
gcgcgcttta gcggcagcgg cagcggcacc gattataccc tgaccattag cagcctggaa
301 ccggaagatt ttgcggtgta ttattgccag cagtggagca gcaacccgta
tacctttggc 361 cagggcacca aactggaaat taaacgcaca gttgctgccc
ccagcgtgtt cattttccca 421 cctagcgatg agcagctgaa aagcggtact
gcctctgtcg tatgcttgct caacaacttt 481 tacccacgtg aggctaaggt
gcagtggaaa gtggataatg cacttcaatc tggaaacagt 541 caagagtccg
tgacagaaca ggacagcaaa gactcaactt attcactctc ttccaccctg 601
actctgtcca aggcagacta tgaaaaacac aaggtatacg cctgcgaggt tacacaccag
661 ggtttgtcta gtcctgtcac caagtccttc aataggggcg aatgt
[0160] Protein Sequence Defining the Full Length Humanized Hu4B9-65
Light Chain (Humanized Kappa Chain Variable Region and Human
Constant Region) (SEQ ID NO: 58)
TABLE-US-00041 1 mdmrvpaqll gllllwlrga rceivltqsp atlslspger
atlscrasss vnymywyqqk 61 pgqaprpwiy ltsnratgvp arfsgsgsgt
dytltissle pedfavyycq qwssnpytfg 121 qgtkleikrt vaapsvfifp
psdeqlksgt asvvcllnnf ypreakvqwk vdnalqsgns 181 qesvteqdsk
dstyslsstl tlskadyekh kvyacevthq glsspvtksf nrgec
[0161] Nucleic Acid Sequence Encoding the Full Length Humanized
Hu4B9-82 Light Chain (Humanized Kappa Chain Variable Region and
Human Constant Region) (SEQ ID NO: 59)
TABLE-US-00042 1 atggacatga gggtgcccgc tcaactgctg gggctgctgc
tgctgtggct gagaggagct 61 cgttgcgaaa tcgtacttac tcagagccct
gccacattgt cattgtcacc cggggaacgc 121 gccacactgt cgtgccgggc
ttcatcgagc gtgaactaca tgtattggta tcaacagaaa 181 ccaggccaag
caccgcgacc ttggatctac ttgacgagca atcgagccac gggtatcccc 241
gcgaggttct ccggttcggg gtcgggaact gattacacac tgacaatttc ctcgctggag
301 cccgaggact tcgcggtgta ctattgtcag cagtggtcat ccaacccgta
cacgtttgga 361 caggggacga agctcgagat caagcgcaca gttgctgccc
ccagcgtgtt cattttccca 421 cctagcgatg agcagctgaa aagcggtact
gcctctgtcg tatgcttgct caacaacttt 481 tacccacgtg aggctaaggt
gcagtggaaa gtggataatg cacttcaatc tggaaacagt 541 caagagtccg
tgacagaaca ggacagcaaa gactcaactt attcactctc ttccaccctg 601
actctgtcca aggcagacta tgaaaaacac aaggtatacg cctgcgaggt tacacaccag
661 ggtttgtcta gtcctgtcac caagtccttc aataggggcg aatgt
[0162] Protein Sequence Defining the Full Length Humanized Hu4B9-82
Light Chain (Humanized Kappa Chain Variable Region and Human
Constant Region) (SEQ ID NO: 60)
TABLE-US-00043 1 mdmrvpaqll gllllwlrga rceivltqsp atlslspger
atlscrasss vnymywyqqk 61 pgqaprpwiy ltsnratgip arfsgsgsgt
dytltissle pedfavyycq qwssnpytfg 121 qgtkleikrt vaapsvfifp
psdeqlksgt asvvcllnnf ypreakvqwk vdnalqsgns 181 qesvteqdsk
dstyslsstl tlskadyekh kvyacevthq glsspvtksf nrgec
[0163] Nucleic Acid Sequence Encoding the Full Length Humanized
Hu4B9-83 Light Chain (Humanized Kappa Chain Variable Region and
Human Constant Region) (SEQ ID NO: 61)
TABLE-US-00044 1 atggacatga gggtgcccgc tcaactgctg gggctgctgc
tgctgtggct gagaggagct 61 cgttgcgaaa tcgtacttac tcagagccct
gccacattgt cattgtcacc cggggaacgc 121 gccacactgt cgtgccgggc
ttcatcgagc gtgaactaca tgtattggta tcaacagaaa 181 ccaggccaag
caccgcgacc ttggatctac ttgacgagca atcgagccac gggtatcccc 241
gcgaggttct ccggttcggg gtcgggaact gatttcacac tgacaatttc ctcgctggag
301 cccgaggact tcgcggtgta ctattgtcag cagtggtcat ccaacccgta
cacgtttgga 361 caggggacga agctcgagat caagcgcaca gttgctgccc
ccagcgtgtt cattttccca 421 cctagcgatg agcagctgaa aagcggtact
gcctctgtcg tatgcttgct caacaacttt 481 tacccacgtg aggctaaggt
gcagtggaaa gtggataatg cacttcaatc tggaaacagt 541 caagagtccg
tgacagaaca ggacagcaaa gactcaactt attcactctc ttccaccctg 601
actctgtcca aggcagacta tgaaaaacac aaggtatacg cctgcgaggt tacacaccag
661 ggtttgtcta gtcctgtcac caagtccttc aataggggcg aatgt
[0164] Protein Sequence Defining the Full Length Humanized Hu4B9-83
Light Chain (Humanized Kappa Chain Variable Region and Human
Constant Region) (SEQ ID NO: 62)
TABLE-US-00045 1 mdmrvpaqll gllllwlrga rceivltqsp atlslspger
atlscrasss vnymywyqqk 61 pgqaprpwiy ltsnratgip arfsgsgsgt
dftltissle pedfavyycq qwssnpytfg 121 qgtkleikrt vaapsvfifp
psdeqlksgt asvvcllnnf ypreakvqwk vdnalqsgns 181 qesvteqdsk
dstyslsstl tlskadyekh kvyacevthq glsspvtksf nrgec
[0165] For convenience, Table 10 provides a concordance chart
showing the SEQ ID NO. of each sequence discussed in this
Example.
TABLE-US-00046 TABLE 10 SEQ ID NO. Nucleic Acid or Protein 49 Human
IgG1 constant--nucleic acid 50 Human IgG1 constant--protein 51
Human Kappa constant--nucleic acid 52 Human Kappa constant--protein
53 Humanized Hu4B9-65 Heavy Human Variable + Human IgG1
constant--nucleic acid 54 Humanized Hu4B9-65 Heavy Human Variable +
Human IgG1 constant--protein 55 Humanized Hu4B9-82, -83 Heavy Human
Variable + Human IgG1 constant--nucleic acid 56 Humanized
Hu4B9-82,-83 Heavy Human Variable + Human IgG1 constant--protein 57
Humanized Hu4B9-65 Human Variable + Human Kappa constant--nucleic
acid 58 Humanized Hu4B9-65 Human Variable + Human Kappa
constant--protein 59 Humanized Hu4B9-82 Human Variable + Human
Kappa constant--nucleic acid 60 Humanized Hu4B9-82 Human Variable +
Human Kappa constant--protein 61 Humanized Hu4B9-83 Human Variable
+ Human Kappa constant--nucleic acid 62 Humanized Hu4B9-83 Human
Variable + Human Kappa constant--protein
[0166] Table 11 below shows antibodies containing each of the
possible combinations of the full-length humanized immunoglobulin
heavy and light chains.
TABLE-US-00047 TABLE 11 Antibody Name Light Chain Heavy Chain
Hu4B9-65 Hu4B9-65 Kappa Hu4B9-65 Heavy (SEQ ID NO: 58) (SEQ ID NO:
54) Hu4B9-84 Hu4B9-65 Kappa Hu4B9-82, -83 Heavy (SEQ ID NO: 58)
(SEQ ID NO: 56) Hu4B9-85 Hu4B9-82 Kappa Hu4B9-65 Heavy (SEQ ID NO:
60) (SEQ ID NO: 54) Hu4B9-82 Hu4B9-82 Kappa Hu4B9-82, -83 Heavy
(SEQ ID NO: 60) (SEQ ID NO: 56) Hu4B9-86 Hu4B9-83 Kappa Hu4B9-65
Heavy (SEQ ID NO: 62) (SEQ ID NO: 54) Hu4B9-83 Hu4B9-83 Kappa
Hu4B9-82, -83 Heavy (SEQ ID NO: 62) (SEQ ID NO: 56)
[0167] Three of the possible antibody constructs containing the
full length immunoglobulin heavy and light chains containing
humanized variable regions are designated below: [0168]
Hu4B9-65=Humanized Hu4B9-65 Heavy Chain Variable Region and Human
IgG1 Constant Region (SEQ ID NO: 54) plus Hu4B9-65 Light Chain
Variable Region and Human Kappa Constant Region (SEQ ID NO: 58)
[0169] Hu4B9-82=Humanized Hu4B9-82, -83 Heavy Chain Variable Region
and Human IgG1 Constant Region (SEQ ID NO: 56) plus Hu4B9-82 Light
Chain Variable Region and Human Kappa Constant Region (SEQ ID NO:
60) [0170] Hu4B9-83=Humanized Hu4B9-82, -83 Heavy Chain Variable
Region and Human IgG1 Constant Region (SEQ ID NO: 56) plus Hu4B9-83
Light Chain Variable Region and Human Kappa Constant Region (SEQ ID
NO: 62)
[0171] B. Binding Affinities of Humanized Anti-FGFR2Monoclonal
Antibodies
[0172] The binding affinities and kinetics of interaction of
monoclonal antibodies produced in Example 9 against monomeric
recombinant human FGFR2 beta Mb (rhFGFR2.beta.-IIIb-cleaved) were
measured by surface plasmon resonance using a Biacore T100 (Biacore
(GE Healthcare), Piscataway, N.J.) instrument.
[0173] Goat anti-human IgG Fc (Jackson ImmunoResearch, Catalog No.
109-005-098) was immobilized on carboxymethylated dextran CM4
sensor chips (Biacore) by amine coupling (Biacore) using a standard
coupling protocol according to the vendor's instructions. The
analyses were performed at 25.degree. C. and 37.degree. C. using
PBS (Invitrogen) containing 0.05% surfactant P20 (Biacore) as
running buffer.
[0174] Purified antibodies were captured in individual flow cells
at a flow rate of 10 .mu.l/minute. Injection time was varied for
each antibody to yield an R.sub.max between 30 and 90 RU. Buffer or
rhFGFR2.beta.-IIIb-cleaved diluted in running buffer was injected
sequentially over a reference surface (no antibody captured) and
the active surface (antibody to be tested) for 240 seconds at 60
.mu.l/minute. The dissociation phase was monitored for up to 900
seconds. The surface was then regenerated with two 60 second
injections of glycine pH 2.25 (made from glycine pH 2.0 (Biacore)
and pH 2.5 (Biacore)) at 30 .mu.l/minute. Experiments were
conducted using concentrations of rhFGFR2.beta.-IIIb-cleaved
between 20 and 1.25 nM (a two-fold serial dilution).
[0175] Kinetic parameters were determined using the kinetic
function of the BlAevaluation software (Biacore) with double
reference subtraction. Kinetic parameters for each antibody,
k.sub.a (association rate constant), k.sub.d (dissociation rate
constant) and K.sub.D (equilibrium dissociation constant) were
determined. The kinetic values of certain purified monoclonal
antibodies (i.e., Hu4B9-65, Hu4B9-82, and Hu4B9-83) on
rhFGFR2.beta.-IIIb-cleaved at 25.degree. C. are summarized in Table
12.
TABLE-US-00048 TABLE 12 ka KD Antibody (1/Ms) kd (1/s) (M) n
hu4B9-65 2.4E+05 6.5E-05 2.6E-10 4 hu4B9-82 1.9E+05 9.4E-05 4.9E-10
2 hu4B9-83 2.6E+05 8.9E-05 3.5E-10 3
[0176] The results in Table 12 demonstrate the purified antibodies
have affinities ranging from about 260 pM to about 490 pM when
tested at 25.degree. C.
[0177] The kinetic values of certain purified monoclonal antibodies
(i.e., Hu4B9-65, Hu4B9-82, and Hu4B9-83) on
rhFGFR2.beta.-IIIb-cleaved at 37.degree. C. are summarized in Table
13.
TABLE-US-00049 TABLE 13 ka KD Antibody (1/Ms) kd (1/s) (M) n
hu4B9-65 3.7E+05 2.8E-04 8.9E-10 7 hu4B9-82 4.0E+05 3.6E-04 9.3E-10
3 hu4B9-83 3.2E+05 2.9E-04 9.2E-10 3
[0178] The results in Table 13 demonstrate the purified antibodies
have affinities ranging from about 890 pM to about 930 pM when
tested at 37.degree. C.
Example 10
Anti-Proliferative Activity of Humanized Anti-FGFR2Monoclonal
Antibodies
[0179] The potency of humanized anti-FGFR2 antibodies was assessed
in a cell-based proliferation assay. FDCP-1 cells expressing
FGFR2-IIIb were seeded in a 96-well plate in IL-3 free medium
containing 8 ng/ml of FGF1 and 5 .mu.g/ml of heparin. Serial
dilutions of the antibodies were prepared and added to the plate.
After two days of incubation, cell proliferation was examined by a
MTT assay as described above in Example 1.
[0180] As shown in FIG. 12, humanized antibodies (Hu4B9-65,
Hu4B9-82, and Hu4B9-83) demonstrated dose-dependent inhibition of
FGF1-induced FDCP-FGFR2-IIIb cell proliferation. The average IC50s
of the 4B9, Hu4B9-65, Hu4B9-82 and Hu4B9-83 from three independent
experiments are 1.4, 4.9, 5.7 and 4.7 nM, respectively.
INCORPORATION BY REFERENCE
[0181] The entire disclosure of each of the patent documents and
scientific articles referred to herein is incorporated by reference
for all purposes.
EQUIVALENTS
[0182] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting on the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes that come within the meaning and the range of equivalency
of the claims are intended to be embraced therein.
Sequence CWU 1
1
621336DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 1gaggttcagc tccagcagtc tgggactgtg
ctggcaaggc ctggggcttc agtgaagatg 60tcctgcaaga cttctggcta cacatttacc
agctactgga tgcactgggt aaaacagagg 120cctggacagg gtctggaatg
gataggggct atttatcctg gaaatagtga tactgactac 180agccagaagt
tcaagggcaa ggccacactg actgcagtca catccgccac cactgcctac
240atggaactca gcagcctgac aaatgaggac tctgcggtct attactgttc
aaagtttgac 300tactggggcc aaggcaccac tctcacagtc tcctca
3362112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 2Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu
Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Thr Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln
Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro
Gly Asn Ser Asp Thr Asp Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Lys
Ala Thr Leu Thr Ala Val Thr Ser Ala Thr Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys 85 90
95 Ser Lys Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
100 105 110 3318DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 3caaattgttc tcacccagtc tccagcactc
atgtctgcat ctccagggga gaaggtcacc 60atgacctgca gtgccagctc aagtgtaaat
tacatgtact ggtaccagca gaagccaaga 120tcctccccca aaccctggat
ttatctcaca tccaacctgg cttctggagt ccctgctcgc 180ttcagtggca
gggggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa
240gatgctgcca cttattactg ccagcagtgg agtagtaacc cgtacacgtt
cggagggggg 300accaagctgg aaataaaa 3184106PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
4Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly 1
5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr
Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro
Trp Ile Tyr 35 40 45 Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala
Arg Phe Ser Gly Arg 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr
Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys
Gln Gln Trp Ser Ser Asn Pro Tyr Thr 85 90 95 Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys 100 105 55PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 5Ser Tyr Trp Met His 1 5
617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Ala Ile Tyr Pro Gly Asn Ser Asp Thr Asp Tyr Ser
Gln Lys Phe Lys 1 5 10 15 Gly 77PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 7Gly Tyr Thr Phe Thr Ser
Tyr 1 5 86PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 8Tyr Pro Gly Asn Ser Asp 1 5 98PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 9Gly
Tyr Thr Phe Thr Ser Tyr Trp 1 5 108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 10Ile
Tyr Pro Gly Asn Ser Asp Thr 1 5 115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 11Ser
Lys Phe Asp Tyr 1 5 1210PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 12Ser Ala Ser Ser Ser Val Asn
Tyr Met Tyr 1 5 10 137PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 13Leu Thr Ser Asn Leu Ala Ser
1 5 149PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 14Gln Gln Trp Ser Ser Asn Pro Tyr Thr 1 5
155PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 15Ser Ser Val Asn Tyr 1 5 16972DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
16gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac
60tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc
120tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct
gcagtctgac 180ctctacactc tgagcagctc agtgactgtc ccctccagca
cctggcccag ccagaccgtc 240acctgcaacg ttgcccaccc ggccagcagc
accaaggtgg acaagaaaat tgtgcccagg 300gattgtggtt gtaagccttg
catatgtaca gtcccagaag tatcatctgt cttcatcttc 360cccccaaagc
ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg
420gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga
tgatgtggag 480gtgcacacag ctcagacgca accccgggag gagcagttca
acagcacttt ccgctcagtc 540agtgaacttc ccatcatgca ccaggactgg
ctcaatggca aggagttcaa atgcagggtc 600aacagtgcag ctttccctgc
ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg 660aaggctccac
aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc
720agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga
gtggcagtgg 780aatgggcagc cagcggagaa ctacaagaac actcagccca
tcatggacac agatggctct 840tacttcgtct acagcaagct caatgtgcag
aagagcaact gggaggcagg aaatactttc 900acctgctctg tgttacatga
gggcctgcac aaccaccata ctgagaagag cctctcccac 960tctcctggta aa
97217324PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 17Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu
Ala Pro Gly Ser Ala 1 5 10 15 Ala Gln Thr Asn Ser Met Val Thr Leu
Gly Cys Leu Val Lys Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val
Thr Trp Asn Ser Gly Ser Leu Ser Ser 35 40 45 Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu 50 55 60 Ser Ser Ser
Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val 65 70 75 80 Thr
Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 85 90
95 Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro
100 105 110 Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp
Val Leu 115 120 125 Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val
Val Asp Ile Ser 130 135 140 Lys Asp Asp Pro Glu Val Gln Phe Ser Trp
Phe Val Asp Asp Val Glu 145 150 155 160 Val His Thr Ala Gln Thr Gln
Pro Arg Glu Glu Gln Phe Asn Ser Thr 165 170 175 Phe Arg Ser Val Ser
Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn 180 185 190 Gly Lys Glu
Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro 195 200 205 Ile
Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln 210 215
220 Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val
225 230 235 240 Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp
Ile Thr Val 245 250 255 Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn
Tyr Lys Asn Thr Gln 260 265 270 Pro Ile Met Asp Thr Asp Gly Ser Tyr
Phe Val Tyr Ser Lys Leu Asn 275 280 285 Val Gln Lys Ser Asn Trp Glu
Ala Gly Asn Thr Phe Thr Cys Ser Val 290 295 300 Leu His Glu Gly Leu
His Asn His His Thr Glu Lys Ser Leu Ser His 305 310 315 320 Ser Pro
Gly Lys 18321DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 18cgggctgatg ctgcaccaac
tgtatccatc ttcccaccat ccagtgagca gttaacatct 60ggaggtgcct cagtcgtgtg
cttcttgaac aacttctacc ccagagacat caatgtcaag 120tggaagattg
atggcagtga acgacaaaat ggtgtcctga acagttggac tgatcaggac
180agcaaagaca gcacctacag catgagcagc accctcacat tgaccaagga
cgagtatgaa 240cgacataaca gctatacctg tgaggccact cacaagacat
caacttcacc cattgtcaag 300agcttcaaca ggaatgagtg t
32119107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 19Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe
Pro Pro Ser Ser Glu 1 5 10 15 Gln Leu Thr Ser Gly Gly Ala Ser Val
Val Cys Phe Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Asp Ile Asn Val
Lys Trp Lys Ile Asp Gly Ser Glu Arg 35 40 45 Gln Asn Gly Val Leu
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser
Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 65 70 75 80 Arg
His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 85 90
95 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 100 105
201365DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 20atggaatgta actggatact tccttttatt
ctgtcggtaa cttcaggggt ctactcagag 60gttcagctcc agcagtctgg gactgtgctg
gcaaggcctg gggcttcagt gaagatgtcc 120tgcaagactt ctggctacac
atttaccagc tactggatgc actgggtaaa acagaggcct 180ggacagggtc
tggaatggat aggggctatt tatcctggaa atagtgatac tgactacagc
240cagaagttca agggcaaggc cacactgact gcagtcacat ccgccaccac
tgcctacatg 300gaactcagca gcctgacaaa tgaggactct gcggtctatt
actgttcaaa gtttgactac 360tggggccaag gcaccactct cacagtctcc
tcagccaaaa cgacaccccc atctgtctat 420ccactggccc ctggatctgc
tgcccaaact aactccatgg tgaccctggg atgcctggtc 480aagggctatt
tccctgagcc agtgacagtg acctggaact ctggatccct gtccagcggt
540gtgcacacct tcccagctgt cctgcagtct gacctctaca ctctgagcag
ctcagtgact 600gtcccctcca gcacctggcc cagccagacc gtcacctgca
acgttgccca cccggccagc 660agcaccaagg tggacaagaa aattgtgccc
agggattgtg gttgtaagcc ttgcatatgt 720acagtcccag aagtatcatc
tgtcttcatc ttccccccaa agcccaagga tgtgctcacc 780attactctga
ctcctaaggt cacgtgtgtt gtggtagaca tcagcaagga tgatcccgag
840gtccagttca gctggtttgt agatgatgtg gaggtgcaca cagctcagac
gcaaccccgg 900gaggagcagt tcaacagcac tttccgctca gtcagtgaac
ttcccatcat gcaccaggac 960tggctcaatg gcaaggagtt caaatgcagg
gtcaacagtg cagctttccc tgcccccatc 1020gagaaaacca tctccaaaac
caaaggcaga ccgaaggctc cacaggtgta caccattcca 1080cctcccaagg
agcagatggc caaggataaa gtcagtctga cctgcatgat aacagacttc
1140ttccctgaag acattactgt ggagtggcag tggaatgggc agccagcgga
gaactacaag 1200aacactcagc ccatcatgga cacagatggc tcttacttcg
tctacagcaa gctcaatgtg 1260cagaagagca actgggaggc aggaaatact
ttcacctgct ctgtgttaca tgagggcctg 1320cacaaccacc atactgagaa
gagcctctcc cactctcctg gtaaa 136521455PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
21Met Glu Cys Asn Trp Ile Leu Pro Phe Ile Leu Ser Val Thr Ser Gly 1
5 10 15 Val Tyr Ser Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala
Arg 20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly
Tyr Thr Phe 35 40 45 Thr Ser Tyr Trp Met His Trp Val Lys Gln Arg
Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Ala Ile Tyr Pro Gly
Asn Ser Asp Thr Asp Tyr Ser 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala
Thr Leu Thr Ala Val Thr Ser Ala Thr 85 90 95 Thr Ala Tyr Met Glu
Leu Ser Ser Leu Thr Asn Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys
Ser Lys Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 115 120 125 Val
Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro 130 135
140 Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val
145 150 155 160 Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn
Ser Gly Ser 165 170 175 Leu Ser Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Asp Leu 180 185 190 Tyr Thr Leu Ser Ser Ser Val Thr Val
Pro Ser Ser Thr Trp Pro Ser 195 200 205 Gln Thr Val Thr Cys Asn Val
Ala His Pro Ala Ser Ser Thr Lys Val 210 215 220 Asp Lys Lys Ile Val
Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys 225 230 235 240 Thr Val
Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys 245 250 255
Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val 260
265 270 Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val
Asp 275 280 285 Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu
Glu Gln Phe 290 295 300 Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro
Ile Met His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Phe Lys
Cys Arg Val Asn Ser Ala Ala Phe 325 330 335 Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys 340 345 350 Ala Pro Gln Val
Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys 355 360 365 Asp Lys
Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp 370 375 380
Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys 385
390 395 400 Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val
Tyr Ser 405 410 415 Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly
Asn Thr Phe Thr 420 425 430 Cys Ser Val Leu His Glu Gly Leu His Asn
His His Thr Glu Lys Ser 435 440 445 Leu Ser His Ser Pro Gly Lys 450
455 22705DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 22atggattttc aagtgcagat tttcagcttc
ctgctaatga gtgcctcagt cataatgtcc 60aggggacaaa ttgttctcac ccagtctcca
gcactcatgt ctgcatctcc aggggagaag 120gtcaccatga cctgcagtgc
cagctcaagt gtaaattaca tgtactggta ccagcagaag 180ccaagatcct
cccccaaacc ctggatttat ctcacatcca acctggcttc tggagtccct
240gctcgcttca gtggcagggg gtctgggacc tcttactctc tcacaatcag
cagcatggag 300gctgaagatg ctgccactta ttactgccag cagtggagta
gtaacccgta cacgttcgga 360ggggggacca agctggaaat aaaacgggct
gatgctgcac caactgtatc catcttccca 420ccatccagtg agcagttaac
atctggaggt gcctcagtcg tgtgcttctt gaacaacttc 480taccccagag
acatcaatgt caagtggaag attgatggca gtgaacgaca aaatggtgtc
540ctgaacagtt ggactgatca ggacagcaaa gacagcacct acagcatgag
cagcaccctc 600acattgacca aggacgagta tgaacgacat aacagctata
cctgtgaggc cactcacaag 660acatcaactt cacccattgt caagagcttc
aacaggaatg agtgt 70523235PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 23Met Asp Phe Gln Val Gln
Ile Phe Ser Phe Leu Leu Met Ser Ala Ser 1 5 10 15 Val Ile Met Ser
Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Leu 20 25 30 Met Ser
Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser 35 40 45
Ser Ser Val Asn Tyr Met Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser 50
55 60 Pro Lys Pro Trp Ile Tyr Leu Thr Ser Asn Leu Ala Ser Gly Val
Pro 65 70 75 80 Ala Arg Phe Ser Gly Arg Gly Ser Gly Thr Ser Tyr Ser
Leu Thr Ile 85 90 95 Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr
Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Tyr Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys 115 120 125 Arg Ala Asp Ala Ala Pro Thr
Val Ser Ile Phe Pro Pro Ser Ser Glu 130 135 140 Gln Leu Thr Ser Gly
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 145 150 155 160 Tyr Pro
Arg Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 165 170 175
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 180
185 190 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr
Glu 195
200 205 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr
Ser 210 215 220 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230
235 2420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 24acttgggctg gagtgatttg 202520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
25aatcccatct gcacacttcc 202620DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 26caaaaacatg gctgagcaga
202720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 27gaaacaggcc ccactttgta 202845DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
28ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagt
452922DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 29ctaatacgac tcactatagg gc 223021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
30tatgcaaggc ttacaaccac a 213123DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 31cgactgaggc acctccagat gtt
233217DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 32gtaaaacgac ggccagt 173318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
33caggaaacag ctatgacc 1834336DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 34caagtgcagc
tcgtccaatc gggagccgaa gtgaagaagc ctggttcctc ggtaaaagta 60agctgtaagg
cgtccggtta cacgtttacc tcatattgga tgcactgggt cagacaggca
120cccggacagg gactcgagtg gatgggagcg atctacccgg gcaattcgga
cactgattac 180agccagaaat tcaaggggag ggtcacgatc acggcagatg
agagcacatc aacagcctat 240atggagctgt cgtcgcttcg gagcgaggac
acggcggtct actactgctc caaattcgac 300tattgggggc aggggacctt
ggtgaccgtg tcatcc 33635112PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 35Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Ala Ile Tyr Pro Gly Asn Ser Asp Thr Asp Tyr Ser Gln Lys Phe 50
55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ser Lys Phe Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 100 105 110 36336DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
36caagtgcagc tcgtccaatc gggagccgaa gtgaagaagc ctggttcctc ggtaaaagta
60agctgtaagg cgtccggtta cacgttttcc tcatattgga tgcactgggt cagacaggca
120cccggacagg gactcgagtg gatgggagcg atctacccgg gcaattcgga
cactgattac 180agccagaaat tccaggggag ggtcacgatc acggcagatg
agagcacatc aacagcctat 240atggagctgt cgtcgcttcg gagcgaggac
acggcggtct actactgctc caaattcgac 300tattgggggc aggggacctt
ggtgaccgtg tcatcc 33637112PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 37Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr 20 25 30 Trp Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Ala Ile Tyr Pro Gly Asn Ser Asp Thr Asp Tyr Ser Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ser Lys Phe Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 100 105 110 3817PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 38Ala
Ile Tyr Pro Gly Asn Ser Asp Thr Asp Tyr Ser Gln Lys Phe Gln 1 5 10
15 Gly 39318DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 39gaaattgtgc tgacccagag
cccggcgacc ctgagcctga gcccgggcga acgcgcgacc 60ctgagctgcc gcgcgagcag
cagcgtgaac tatatgtatt ggtatcagca gaaaccgggc 120caggcgccgc
gcccgtggat ttatctgacc agcaaccgcg cgaccggcgt gccggcgcgc
180tttagcggca gcggcagcgg caccgattat accctgacca ttagcagcct
ggaaccggaa 240gattttgcgg tgtattattg ccagcagtgg agcagcaacc
cgtatacctt tggccagggc 300accaaactgg aaattaaa 31840106PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
40Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1
5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr
Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro
Trp Ile Tyr 35 40 45 Leu Thr Ser Asn Arg Ala Thr Gly Val Pro Ala
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr
Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Trp Ser Ser Asn Pro Tyr Thr 85 90 95 Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys 100 105 4110PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 41Arg Ala Ser Ser Ser Val
Asn Tyr Met Tyr 1 5 10 427PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 42Leu Thr Ser Asn Arg Ala Thr
1 5 43318DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 43gaaatcgtac ttactcagag ccctgccaca
ttgtcattgt cacccgggga acgcgccaca 60ctgtcgtgcc gggcttcatc gagcgtgaac
tacatgtatt ggtatcaaca gaaaccaggc 120caagcaccgc gaccttggat
ctacttgacg agcaatcgag ccacgggtat ccccgcgagg 180ttctccggtt
cggggtcggg aactgattac acactgacaa tttcctcgct ggagcccgag
240gacttcgcgg tgtactattg tcagcagtgg tcatccaacc cgtacacgtt
tggacagggg 300acgaagctcg agatcaag 31844106PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
44Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1
5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr
Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro
Trp Ile Tyr 35 40 45 Leu Thr Ser Asn Arg Ala Thr Gly Ile Pro Ala
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr
Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Trp Ser Ser Asn Pro Tyr Thr 85 90 95 Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys 100 105 45318DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 45gaaatcgtac
ttactcagag ccctgccaca ttgtcattgt cacccgggga acgcgccaca 60ctgtcgtgcc
gggcttcatc gagcgtgaac tacatgtatt ggtatcaaca gaaaccaggc
120caagcaccgc gaccttggat ctacttgacg agcaatcgag ccacgggtat
ccccgcgagg 180ttctccggtt cggggtcggg aactgatttc acactgacaa
tttcctcgct ggagcccgag 240gacttcgcgg tgtactattg tcagcagtgg
tcatccaacc cgtacacgtt tggacagggg 300acgaagctcg agatcaag
31846106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 46Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Ser Ser Val Asn Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Pro Trp Ile Tyr 35 40 45 Leu Thr Ser Asn Arg
Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp
Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Tyr Thr 85 90
95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 477PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 47Gly
Tyr Thr Phe Ser Ser Tyr 1 5 488PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 48Gly Tyr Thr Phe Ser Ser Tyr
Trp 1 5 49990DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 49gcctcaacaa aaggaccaag
tgtgttccca ctcgccccta gcagcaagag tacatccggg 60ggcactgcag cactcggctg
cctcgtcaag gattattttc cagagccagt aaccgtgagc 120tggaacagtg
gagcactcac ttctggtgtc catacttttc ctgctgtcct gcaaagctct
180ggcctgtact cactcagctc cgtcgtgacc gtgccatctt catctctggg
cactcagacc 240tacatctgta atgtaaacca caagcctagc aatactaagg
tcgataagcg ggtggaaccc 300aagagctgcg acaagactca cacttgtccc
ccatgccctg cccctgaact tctgggcggt 360cccagcgtct ttttgttccc
accaaagcct aaagatactc tgatgataag tagaacaccc 420gaggtgacat
gtgttgttgt agacgtttcc cacgaggacc cagaggttaa gttcaactgg
480tacgttgatg gagtcgaagt acataatgct aagaccaagc ctagagagga
gcagtataat 540agtacatacc gtgtagtcag tgttctcaca gtgctgcacc
aagactggct caacggcaaa 600gaatacaaat gcaaagtgtc caacaaagca
ctcccagccc ctatcgagaa gactattagt 660aaggcaaagg ggcagcctcg
tgaaccacag gtgtacactc tgccacccag tagagaggaa 720atgacaaaga
accaagtctc attgacctgc ctggtgaaag gcttctaccc cagcgacatc
780gccgttgagt gggagagtaa cggtcagcct gagaacaatt acaagacaac
ccccccagtg 840ctggatagtg acgggtcttt ctttctgtac agtaagctga
ctgtggacaa gtcccgctgg 900cagcagggta acgtcttcag ctgttccgtg
atgcacgagg cattgcacaa ccactacacc 960cagaagtcac tgagcctgag
cccagggaag 99050330PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 50Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65
70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
51321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 51cgcacagttg ctgcccccag cgtgttcatt
ttcccaccta gcgatgagca gctgaaaagc 60ggtactgcct ctgtcgtatg cttgctcaac
aacttttacc cacgtgaggc taaggtgcag 120tggaaagtgg ataatgcact
tcaatctgga aacagtcaag agtccgtgac agaacaggac 180agcaaagact
caacttattc actctcttcc accctgactc tgtccaaggc agactatgaa
240aaacacaagg tatacgcctg cgaggttaca caccagggtt tgtctagtcc
tgtcaccaag 300tccttcaata ggggcgaatg t 32152107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
52Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1
5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser
Phe Asn Arg Gly Glu Cys 100 105 531392DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
53atggacatga gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct
60aggtgccaag tgcagctcgt ccaatcggga gccgaagtga agaagcctgg ttcctcggta
120aaagtaagct gtaaggcgtc cggttacacg tttacctcat attggatgca
ctgggtcaga 180caggcacccg gacagggact cgagtggatg ggagcgatct
acccgggcaa ttcggacact 240gattacagcc agaaattcaa ggggagggtc
acgatcacgg cagatgagag cacatcaaca 300gcctatatgg agctgtcgtc
gcttcggagc gaggacacgg cggtctacta ctgctccaaa 360ttcgactatt
gggggcaggg gaccttggtg accgtgtcat ccgcctcaac aaaaggacca
420agtgtgttcc cactcgcccc tagcagcaag agtacatccg ggggcactgc
agcactcggc 480tgcctcgtca aggattattt tccagagcca gtaaccgtga
gctggaacag tggagcactc 540acttctggtg tccatacttt tcctgctgtc
ctgcaaagct ctggcctgta ctcactcagc 600tccgtcgtga ccgtgccatc
ttcatctctg ggcactcaga cctacatctg taatgtaaac 660cacaagccta
gcaatactaa ggtcgataag cgggtggaac ccaagagctg cgacaagact
720cacacttgtc ccccatgccc tgcccctgaa cttctgggcg gtcccagcgt
ctttttgttc 780ccaccaaagc ctaaagatac tctgatgata agtagaacac
ccgaggtgac atgtgttgtt 840gtagacgttt cccacgagga cccagaggtt
aagttcaact ggtacgttga tggagtcgaa 900gtacataatg ctaagaccaa
gcctagagag gagcagtata atagtacata ccgtgtagtc 960agtgttctca
cagtgctgca ccaagactgg ctcaacggca aagaatacaa atgcaaagtg
1020tccaacaaag cactcccagc ccctatcgag aagactatta gtaaggcaaa
ggggcagcct 1080cgtgaaccac aggtgtacac tctgccaccc agtagagagg
aaatgacaaa gaaccaagtc 1140tcattgacct gcctggtgaa aggcttctac
cccagcgaca tcgccgttga gtgggagagt 1200aacggtcagc ctgagaacaa
ttacaagaca acccccccag tgctggatag tgacgggtct 1260ttctttctgt
acagtaagct gactgtggac aagtcccgct ggcagcaggg taacgtcttc
1320agctgttccg tgatgcacga ggcattgcac aaccactaca cccagaagtc
actgagcctg 1380agcccaggga ag 139254464PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
54Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1
5 10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala
Glu 20 25 30 Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys
Ala Ser Gly 35 40 45 Tyr Thr Phe Thr Ser Tyr Trp Met His Trp Val
Arg Gln Ala Pro Gly 50 55 60 Gln Gly Leu Glu Trp Met Gly Ala Ile
Tyr Pro Gly Asn Ser Asp Thr 65
70 75 80 Asp Tyr Ser Gln Lys Phe Lys Gly Arg Val Thr Ile Thr Ala
Asp Glu 85 90 95 Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ser Lys Phe Asp
Tyr Trp Gly Gln Gly Thr 115 120 125 Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro 130 135 140 Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 145 150 155 160 Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 165 170 175 Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 180 185
190 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
195 200 205 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 210 215 220 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser
Cys Asp Lys Thr 225 230 235 240 His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser 245 250 255 Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg 260 265 270 Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 275 280 285 Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 290 295 300 Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 305 310
315 320 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr 325 330 335 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr 340 345 350 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu 355 360 365 Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys 370 375 380 Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser 385 390 395 400 Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 405 410 415 Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 420 425 430
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 435
440 445 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 450 455 460 551392DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 55atggacatga
gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct 60aggtgccaag
tgcagctcgt ccaatcggga gccgaagtga agaagcctgg ttcctcggta
120aaagtaagct gtaaggcgtc cggttacacg ttttcctcat attggatgca
ctgggtcaga 180caggcacccg gacagggact cgagtggatg ggagcgatct
acccgggcaa ttcggacact 240gattacagcc agaaattcca ggggagggtc
acgatcacgg cagatgagag cacatcaaca 300gcctatatgg agctgtcgtc
gcttcggagc gaggacacgg cggtctacta ctgctccaaa 360ttcgactatt
gggggcaggg gaccttggtg accgtgtcat ccgcctcaac aaaaggacca
420agtgtgttcc cactcgcccc tagcagcaag agtacatccg ggggcactgc
agcactcggc 480tgcctcgtca aggattattt tccagagcca gtaaccgtga
gctggaacag tggagcactc 540acttctggtg tccatacttt tcctgctgtc
ctgcaaagct ctggcctgta ctcactcagc 600tccgtcgtga ccgtgccatc
ttcatctctg ggcactcaga cctacatctg taatgtaaac 660cacaagccta
gcaatactaa ggtcgataag cgggtggaac ccaagagctg cgacaagact
720cacacttgtc ccccatgccc tgcccctgaa cttctgggcg gtcccagcgt
ctttttgttc 780ccaccaaagc ctaaagatac tctgatgata agtagaacac
ccgaggtgac atgtgttgtt 840gtagacgttt cccacgagga cccagaggtt
aagttcaact ggtacgttga tggagtcgaa 900gtacataatg ctaagaccaa
gcctagagag gagcagtata atagtacata ccgtgtagtc 960agtgttctca
cagtgctgca ccaagactgg ctcaacggca aagaatacaa atgcaaagtg
1020tccaacaaag cactcccagc ccctatcgag aagactatta gtaaggcaaa
ggggcagcct 1080cgtgaaccac aggtgtacac tctgccaccc agtagagagg
aaatgacaaa gaaccaagtc 1140tcattgacct gcctggtgaa aggcttctac
cccagcgaca tcgccgttga gtgggagagt 1200aacggtcagc ctgagaacaa
ttacaagaca acccccccag tgctggatag tgacgggtct 1260ttctttctgt
acagtaagct gactgtggac aagtcccgct ggcagcaggg taacgtcttc
1320agctgttccg tgatgcacga ggcattgcac aaccactaca cccagaagtc
actgagcctg 1380agcccaggga ag 139256464PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
56Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1
5 10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala
Glu 20 25 30 Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys
Ala Ser Gly 35 40 45 Tyr Thr Phe Ser Ser Tyr Trp Met His Trp Val
Arg Gln Ala Pro Gly 50 55 60 Gln Gly Leu Glu Trp Met Gly Ala Ile
Tyr Pro Gly Asn Ser Asp Thr 65 70 75 80 Asp Tyr Ser Gln Lys Phe Gln
Gly Arg Val Thr Ile Thr Ala Asp Glu 85 90 95 Ser Thr Ser Thr Ala
Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 100 105 110 Thr Ala Val
Tyr Tyr Cys Ser Lys Phe Asp Tyr Trp Gly Gln Gly Thr 115 120 125 Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 130 135
140 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
145 150 155 160 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp Asn 165 170 175 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln 180 185 190 Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser 195 200 205 Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro Ser 210 215 220 Asn Thr Lys Val Asp
Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr 225 230 235 240 His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 245 250 255
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 260
265 270 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro 275 280 285 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala 290 295 300 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val 305 310 315 320 Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr 325 330 335 Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 340 345 350 Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 355 360 365 Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 370 375 380
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 385
390 395 400 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp 405 410 415 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser 420 425 430 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala 435 440 445 Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460 57705DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
57atggacatga gggtgcccgc tcaactgctg gggctgctgc tgctgtggct gagaggagct
60cgttgcgaaa ttgtgctgac ccagagcccg gcgaccctga gcctgagccc gggcgaacgc
120gcgaccctga gctgccgcgc gagcagcagc gtgaactata tgtattggta
tcagcagaaa 180ccgggccagg cgccgcgccc gtggatttat ctgaccagca
accgcgcgac cggcgtgccg 240gcgcgcttta gcggcagcgg cagcggcacc
gattataccc tgaccattag cagcctggaa 300ccggaagatt ttgcggtgta
ttattgccag cagtggagca gcaacccgta tacctttggc 360cagggcacca
aactggaaat taaacgcaca gttgctgccc ccagcgtgtt cattttccca
420cctagcgatg agcagctgaa aagcggtact gcctctgtcg tatgcttgct
caacaacttt 480tacccacgtg aggctaaggt gcagtggaaa gtggataatg
cacttcaatc tggaaacagt 540caagagtccg tgacagaaca ggacagcaaa
gactcaactt attcactctc ttccaccctg 600actctgtcca aggcagacta
tgaaaaacac aaggtatacg cctgcgaggt tacacaccag 660ggtttgtcta
gtcctgtcac caagtccttc aataggggcg aatgt 70558235PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
58Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1
5 10 15 Leu Arg Gly Ala Arg Cys Glu Ile Val Leu Thr Gln Ser Pro Ala
Thr 20 25 30 Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser 35 40 45 Ser Ser Val Asn Tyr Met Tyr Trp Tyr Gln Gln
Lys Pro Gly Gln Ala 50 55 60 Pro Arg Pro Trp Ile Tyr Leu Thr Ser
Asn Arg Ala Thr Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Tyr Thr Leu Thr Ile 85 90 95 Ser Ser Leu Glu Pro
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn
Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 115 120 125 Arg
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135
140 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
145 150 155 160 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln 165 170 175 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu 195 200 205 Lys His Lys Val Tyr Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser 210 215 220 Pro Val Thr Lys Ser
Phe Asn Arg Gly Glu Cys 225 230 235 59705DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
59atggacatga gggtgcccgc tcaactgctg gggctgctgc tgctgtggct gagaggagct
60cgttgcgaaa tcgtacttac tcagagccct gccacattgt cattgtcacc cggggaacgc
120gccacactgt cgtgccgggc ttcatcgagc gtgaactaca tgtattggta
tcaacagaaa 180ccaggccaag caccgcgacc ttggatctac ttgacgagca
atcgagccac gggtatcccc 240gcgaggttct ccggttcggg gtcgggaact
gattacacac tgacaatttc ctcgctggag 300cccgaggact tcgcggtgta
ctattgtcag cagtggtcat ccaacccgta cacgtttgga 360caggggacga
agctcgagat caagcgcaca gttgctgccc ccagcgtgtt cattttccca
420cctagcgatg agcagctgaa aagcggtact gcctctgtcg tatgcttgct
caacaacttt 480tacccacgtg aggctaaggt gcagtggaaa gtggataatg
cacttcaatc tggaaacagt 540caagagtccg tgacagaaca ggacagcaaa
gactcaactt attcactctc ttccaccctg 600actctgtcca aggcagacta
tgaaaaacac aaggtatacg cctgcgaggt tacacaccag 660ggtttgtcta
gtcctgtcac caagtccttc aataggggcg aatgt 70560235PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
60Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1
5 10 15 Leu Arg Gly Ala Arg Cys Glu Ile Val Leu Thr Gln Ser Pro Ala
Thr 20 25 30 Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser 35 40 45 Ser Ser Val Asn Tyr Met Tyr Trp Tyr Gln Gln
Lys Pro Gly Gln Ala 50 55 60 Pro Arg Pro Trp Ile Tyr Leu Thr Ser
Asn Arg Ala Thr Gly Ile Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Tyr Thr Leu Thr Ile 85 90 95 Ser Ser Leu Glu Pro
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn
Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 115 120 125 Arg
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135
140 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
145 150 155 160 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln 165 170 175 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu 195 200 205 Lys His Lys Val Tyr Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser 210 215 220 Pro Val Thr Lys Ser
Phe Asn Arg Gly Glu Cys 225 230 235 61705DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
61atggacatga gggtgcccgc tcaactgctg gggctgctgc tgctgtggct gagaggagct
60cgttgcgaaa tcgtacttac tcagagccct gccacattgt cattgtcacc cggggaacgc
120gccacactgt cgtgccgggc ttcatcgagc gtgaactaca tgtattggta
tcaacagaaa 180ccaggccaag caccgcgacc ttggatctac ttgacgagca
atcgagccac gggtatcccc 240gcgaggttct ccggttcggg gtcgggaact
gatttcacac tgacaatttc ctcgctggag 300cccgaggact tcgcggtgta
ctattgtcag cagtggtcat ccaacccgta cacgtttgga 360caggggacga
agctcgagat caagcgcaca gttgctgccc ccagcgtgtt cattttccca
420cctagcgatg agcagctgaa aagcggtact gcctctgtcg tatgcttgct
caacaacttt 480tacccacgtg aggctaaggt gcagtggaaa gtggataatg
cacttcaatc tggaaacagt 540caagagtccg tgacagaaca ggacagcaaa
gactcaactt attcactctc ttccaccctg 600actctgtcca aggcagacta
tgaaaaacac aaggtatacg cctgcgaggt tacacaccag 660ggtttgtcta
gtcctgtcac caagtccttc aataggggcg aatgt 70562235PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
62Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1
5 10 15 Leu Arg Gly Ala Arg Cys Glu Ile Val Leu Thr Gln Ser Pro Ala
Thr 20 25 30 Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser 35 40 45 Ser Ser Val Asn Tyr Met Tyr Trp Tyr Gln Gln
Lys Pro Gly Gln Ala 50 55 60 Pro Arg Pro Trp Ile Tyr Leu Thr Ser
Asn Arg Ala Thr Gly Ile Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95 Ser Ser Leu Glu Pro
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn
Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 115 120 125 Arg
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135
140 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
145 150 155 160 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln 165 170 175 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu 195 200 205 Lys His Lys Val Tyr Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser 210 215 220 Pro Val Thr Lys Ser
Phe Asn Arg Gly Glu Cys 225 230 235
* * * * *