U.S. patent application number 09/401163 was filed with the patent office on 2002-01-24 for monoclonal antibodies specific to vegf receptors and uses thereof.
Invention is credited to GOLDSTEIN, NEIL I., ROCKWELL, PATRICIA.
Application Number | 20020009750 09/401163 |
Document ID | / |
Family ID | 46253128 |
Filed Date | 2002-01-24 |
United States Patent
Application |
20020009750 |
Kind Code |
A1 |
ROCKWELL, PATRICIA ; et
al. |
January 24, 2002 |
MONOCLONAL ANTIBODIES SPECIFIC TO VEGF RECEPTORS AND USES
THEREOF
Abstract
Monoclonal antibodies that specifically bind to an extracellular
domain of flt-1 receptor and neutralize activation of the receptor
are provided. In vitro and in vivo methods of using these
antibodies are also provided.
Inventors: |
ROCKWELL, PATRICIA; (WEST
REDDING, CT) ; GOLDSTEIN, NEIL I.; (MAPLEWOOD,
NJ) |
Correspondence
Address: |
KENYON & KENYON
ONE BROADWAY
NEW YORK
NY
10004
US
|
Family ID: |
46253128 |
Appl. No.: |
09/401163 |
Filed: |
September 22, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09401163 |
Sep 22, 1999 |
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08967113 |
Nov 10, 1997 |
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08967113 |
Nov 10, 1997 |
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08706804 |
Sep 3, 1996 |
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5861499 |
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08706804 |
Sep 3, 1996 |
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08476533 |
Jun 7, 1995 |
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08476533 |
Jun 7, 1995 |
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08326552 |
Oct 20, 1994 |
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5840301 |
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08326552 |
Oct 20, 1994 |
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08196041 |
Feb 10, 1994 |
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Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
C07K 2317/24 20130101;
C07K 2317/77 20130101; A61K 38/00 20130101; C07K 2319/00 20130101;
C07K 2317/73 20130101; C07K 16/2863 20130101; C07K 14/71
20130101 |
Class at
Publication: |
435/7.1 |
International
Class: |
A01N 001/02; G01N
033/53 |
Claims
What is claimed is:
1. Nucleic acid molecules comprising a nucleic acid sequence that
encodes an amino acid sequence wherein the amino acid sequence
consists of the variable region of a monoclonal antibody that
specifically binds to an extracellular domain of a flt-1 receptor
and neutralizes activation of the receptor.
2. The nucleic acid of claim 1, wherein the monoclonal antibody is
produced by hybridoma cell line DC101 deposited as ATCC Accession
No. HB 11534.
3. The nucleic acid of claim 1, wherein the monoclonal antibody is
produced by hybridoma cell line M25.18A1 deposited as ATCC
Accession No. HBB 12152.
4. The nucleic acid of claim 1, wherein the monoclonal antibody is
produced by hybridoma cell line M73.24 deposited as ATCC Accession
No. HB 12153.
5. Nucleic acid molecules comprising a nucleic acid sequence that
encodes an amino acid sequence wherein the amino acid sequence
consists of the hypervariable region of a monoclonal antibody that
specifically binds to an extracellular domain of a flt-1 receptor
and neu tralizes activation of the receptor.
6. The nucleic acid of claim 5, wherein the monoclonal antibody is
produced by hybridoma cell line DC101 deposited as ATCC Accession
No. HB 11534.
7. The nucleic acid of claim 5, wherein the monoclonal antibody is
produced by hybridoma cell line M25.18A1 deposited as ATCC
Accession No. HB 12152.
8. The nucleic acid of claim 5, wherein the monoclonal antibody is
produced by hybridoma cell line M73.24 deposited as ATCC Accession
No. HB 12153.
9. A method for reducing tumor growth in a mammal in need thereof
comprising treating the mammal with an effective amount of a
monoclonal antibody which specifically binds to an extracellular
domain of a flt-1 receptor and reduces tumor growth.
10. The method of claim 9, wherein the antibody is produced by a
hybridoma cell line.
11. The method of claim 10, wherein the hybridoma cell line is
deposited as ATCC Accession No. HB 11534.
12. A method for reducing tumor growth in a mammal in need thereof
comprising treating the mammal with an effective amount of a
chimeric antibody which comprises an amino acid sequence of a human
antibody constant region and an amino acid sequence of a non-human
antibody variable region, and which specifically binds to an
extracellular domain offlt-1 receptor and reduces tumor growth.
13. The method of claim 12, wherein the non-human variable region
is murne.
14. A method for reducing tumor growth in a mammal in need thereof
comprising treating the mammal with an effective amount of a
humanized antibody which comprises amino acid sequences of variable
framework and constant regions from a human antibody, and an amino
acid sequence of a non-human antibody hypervariable region, and
which specifically binds to an extracellular domain of flt-1
receptor and reduces tumor growth.
15. The method of claim 14, wherein the amino acid sequence of the
hypervariable region is murine.
16. A method for inhibiting angiogenesis in a mammal in need
thereof comprising treating the mammal with an effective amount of
a monoclonal antibody which specifically binds to an extracellular
domain of flt-1 receptor and inhibits angiogenesis.
17. The method of claim 16 wherein the antibody is produced by a
hybridoma cell line.
18. The method of claim 17, wherein the hybridoma cell line is
deposited as ATCC Accession No. HB 11534.
19. A method for inhibiting angiogenesis in a mammal in need
thereof comprising treating the mammal with an effective amount of
a chimeric antibody which comprises an amino acid sequence of a
human antibody constant region and an amino acid sequence of a
non-human antibody variable region, and which specifically binds to
an extracellular domain of flt-1 receptor and inhibits
angiogenesis.
20. A method of claim 19, wherein the non-human variable region is
murine.
21. A method for inhibiting angiogenesis in a mammal in need
thereof comprising treating the mammal with an effective amount of
a humanized antibody which comprises amino acid sequences of
variable framework and constant regions from a human antibody, and
an amino acid sequence of a non-human antibody hypervariable
region, and which specifically binds to an extracellular domain of
flt-1 receptor and inhibits angiogenesis.
22. The method of claim 21, wherein the amino acid sequence of the
hypervariable region is murine.
23. The method of claim 21, wherein the hybridoma cell line is
deposited as ATCC Accession No. HB 11534.
24. A method for reducing tumor growth in a mammal in need thereof
comprising treating the mammal with an effective amount of a single
chain antibody which specifically binds to an extracellular domain
of a flt-1 receptor and reduces tumor growth.
25. A method for inhibiting angiogenesis in a mammal in need
thereof comprising treating the mammal with an effective amount of
a single chain antibody which specifically binds to an
extracellular domain of flt-1 receptor and inhibits
angiogenesis.
26. A single chain antibody that specifically binds to an
extracellular domain of flt-1 receptor and neutralizes activation
of the receptor.
27. A single chain antibody that specifically binds to an
extracellular domain of flt-1 receptor and reduces tumor
growth.
28. A single chain antibody that specifically binds to an
extracellular domain of flt-1 receptor and inhibits
angiogenesis.
29. A process for preparing a polypeptide that comprises an amino
acid sequence that specifically binds to an extracellular domain of
flt-1 receptor and neutralizes activation of the receptor, the
process comprising: culturing cells that express a nucleic acid
molecule comprising a nucleic acid sequence that encodes an amino
acid sequence wherein the amino acid sequence consists of the
variable region of a monoclonal antibody that specifically binds to
an extracellular domain of flt-1 receptor and neutralizes
activation of the receptor; and isolating the polypeptide from the
cultured cells.
30. A process for preparing a polypeptide that comprises an amino
acid sequence that specifically binds to an extracellular domain of
flt-1 receptor and neutralizes activation of the receptor, the
process comprising: culturing cells that express a nucleic acid
molecule comprising a nucleic acid sequence that encodes an amino
acid sequence wherein the amino acid sequence consists of the
hypervariable region of a monoclonal antibody that specifically
binds to an extracellular domain of flt-1 receptor and neutralizes
activation of the receptor; and isolating the polypeptide from the
cultured cells.
31. A process for preparing chimerized monoclonal antibodies that
specifically bind to an extracellular domain of flt-1 receptor and
neutralize activation of the receptor, the process comprising:
culturing cells that express a nucleic acid molecule comprising a
nucleic acid sequence that encodes an amino acid sequence wherein
the amino acid sequence consists of: (i) a variable region of a
monoclonal antibody of a mammal other than a human wherein the
variable region specifically binds to an extracellular domain of
flt-1 receptor and neutralizes activation of the receptor, and (ii)
a constant region of a human antibody; and isolating the chimerized
monoclonal antibodies from the cultured cells.
32. A process for preparing humanized monoclonal antibodies that
specifically bind to an extracellular domain of a flt-1 receptor
and neutralize activation of the receptor, the process comprising:
culturing cells that express a nucleic acid molecule comprising a
nucleic acid sequence that encodes an amino acid sequence wherein
the amino acid sequence consists of: (i) a hypervariable region of
a monoclonal antibody of a mammal other than a human wherein the
hypervariable region specifically binds to an extracellular domain
of flt-1 receptor and neutralizes activation of the receptor, (ii)
a constant region of a human antibody, and (iii) a variable region,
other than the hypervariable region, substantially from a human
antibody; and isolating the humanized monoclonal antibodies from
the cultured cells.
33. A process for preparing a polypeptide that comprises an amino
acid sequence that specifically binds to an extracellular domain of
flt-1 receptor and inhibits tumor growth in a mammal, the process
comprising: culturing cells that express a nucleic acid molecule
comprising a nucleic acid sequence that encodes an amino acid
sequence wherein the amino acid sequence consists of the variable
region of a monoclonal antibody that specifically binds to an
extracellular domain of flt-1 receptor and inhibits tumor growth in
the mammal; and isolating the polypeptide from the cultured
cells.
34. A process for preparing a polypeptide that comprises an amino
acid sequence that specifically binds to an extracellular domain of
flt-1 receptor and inhibits tumor growth in a mammal, the process
comprising: culturing cells that express a nucleic acid molecule
comprising a nucleic acid sequence that encodes an amino acid
sequence wherein the amino acid sequence consists of the
hypervariable region of a monoclonal antibody that specifically
binds to an extracellular domain of flt-1 receptor and inhibits
tumor growth in the mammal; and isolating the polypeptide from the
cultured cells.
35. A process for preparing chimerized monoclonal antibodies that
specifically bind to an extracellular domain of flt-1 receptor and
inhibit tumor growth in a recipient mammal, the process comprising:
culturing cells that express a nucleic acid molecule comprising a
nucleic acid sequence that encodes an amino acid sequence wherein
the amino acid sequence consists of: (i) a variable region of a
monoclonal antibody of a mammal other than a human wherein the
variable region specifically binds to an extracellular domain of
flt-1 receptor and inhibits tumor growth in the recipient mammal,
and (ii) a constant region of a human antibody; and isolating the
chimerized monoclonal antibodies from the cultured cells.
36. A process for preparing humanized monoclonal antibodies that
specifically bind to an extracellular domain of flt-1 receptor and
inhibit tumor growth in a recipient mammal, the process comprising:
culturing cells that express a nucleic acid molecule comprising a
nucleic acid sequence that encodes an amino acid sequence wherein
the amino acid sequence consists of: (i) a hypervariable region of
a monoclonal antibody of a mammal other than a human wherein the
hypervariable region specifically binds to an extracellular domain
of flt-1 receptor and inhibits tumor growth in the recipient
mammal, (ii) a constant region of a human antibody, and (iii) a
variable region, other than the hypervariable region, substantially
from a human antibody; and isolating the humanized monoclonal
antibodies from the cultured cells.
37. A chimerized monoclonal antibody that specifically binds to an
extracellular domain of flt-1 receptor and neutralizes activation
of the receptor.
38. A cell line producing the antibody of claim 37.
39. A composition comprising the antibody of claim 37 and a
pharmaceutically acceptable carrier.
40. The composition of claim 39 further comprising a
chemotherapeutic agent and a pharmaceutically acceptable
carrier.
41. A chimerized mono clonal antibody that specifically binds to an
extracellular domain of flt-1 receptor and reduces tumor
growth.
42. A cell line producing the antibody of claim 41.
43. A composition comprising the antibody of claim 41 and a
pharmaceutically acceptable carrier.
44. The composition of claim 43 fuirther comprising a
chemotherapeutic agent and a pharmaceutically acceptable
carrier.
45. A humanized monoclonal antibody that specifically binds to an
extracellular domain of flt-1 receptor and neutralizes activation
of the receptor.
46. A cell line producing the antibody of claim 45.
47. A composition comprising the antibody of claim 45 and a
pharmaceutically acceptable carrier.
48. The composition of claim 47 further comprising a
chemotherapeutic agent and a pharmaceutically acceptable
carrier.
49. A humanized monoclonal antibody that specifically binds to an
extracellular domain of flt-1 receptor and reduces tumor
growth.
50. A cell line producing the antibody of claim 49.
51. A composition comprising the antibody of claim 49 and a
pharmaceutically acceptable carrier.
52. The composition of claim 51 further comprising a
chemotherapeutic agent and a pharmaceutically acceptable carrier.
Description
[0001] This application is a continuation of Ser. No. 08/967,113
filed on Nov. 10, 1997, pending; which is a continuation-in-part of
U.S. Pat. No. 5,861,499 filed Sep. 3, 1996, which is a
continuation-in-part of Ser. No. 08/476,533 filed Jun. 7, 1995,
abandoned; which is a continuation of U.S. Pat. No. 5,840,301 filed
Oct. 20, 1994; which is a continuation-in-part of Ser. No.
08/196,041 filed Feb. 10, 1994, abandoned. The entire disclosure of
the aforementioned prior applications are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] Angiogenesis is the process of developing new blood Docket
Number:______ vessels that involves the proliferation, migration
and tissue infiltration of capillary endothelial cells from
pre-existing blood vessels. Angiogenesis is important in normal
physiological processes including embryonic development, follicular
growth, and wound healing as well as in pathological conditions
involving tumor growth and nonneoplastic diseases involving
abnormal neovascularization, including neovascular glaucoma
(Folkman, J. and Klagsbrun, M. Science 235:442-447 (1987)).
[0003] The vascular endothelium is usually quiescent and its
activation is tightly regulated during angiogenesis. Several
factors have been implicated as possible regulators of angiogenesis
in vivo. These include transforming growth factor (TGFb), acidic
and basic fibroblast growth factor (aFGF and bFGF), platelet
derived growth factor (PDGF), and vascular endothelial growth
factor (VEGF) (Klagsbrun, M. and D'Amore, P. (1991) Annual Rev.
Physiol. 53: 217-239). VEGF, an endothelial cellspecific mitogen,
is distinct among these factors in that it acts as an angiogenesis
inducer by specifically promoting the proliferation of endothelial
cells. resulting from alternative splicing of mRNA. These include
two membrane bound forms (VEGF.sub.206 and VEGF.sub.189) and two
soluble forms (VEGF.sub.165 and VEGF.sub.121). In all human tissues
except placenta, VEGF.sub.165 is the most abundant isoform.
[0004] VEGF is expressed in embryonic tissues (Breier et al.,
Development (Camb.) 114:521 (1992)), macrophages, proliferating
epidermal keratinocytes during wound healing (Brown et al., J. Exp.
Med., 176:1375 (1992)), and may be responsible for tissue edema
associated with inflammation (Ferrara et al., Endocr. Rev. 13:18
(1992)). In situ hybridization studies have demonstrated high VEGF
expression in a number of human tumor lines including glioblastoma
multiforme, hemangioblastoma, central nervous system neoplasms and
AIDS-associated Kaposi's sarcoma (Plate, K. et al. (1992) Nature
359: 845-848; Plate, K. et al. (1993) Cancer Res. 53: 5822-5827;
Berkman, R. et al. (1993) J. Clin. Invest. 91: 153-159; Nakamura,
S. et al. (1992) AIDS Weekly, 13 (1)). High levels of VEGF were
also observed in hypoxia induced angiogenesis (Shweiki, D. et al.
(1992) Nature 359: 843-845).
[0005] The biological response of VEGF is mediated through its high
affinity VEGF receptors which are selectively expressed on
endothelial cells during embryogenesis (Millauer, B., et al. (1993)
Cell 72: 835-846) and during tumor formation. VEGF receptors
typically are class III receptor-type tyrosine kinases
characterized by having several, typically 5 or 7,
immunoglobulin-like loops in their amino-terminal extracellular
receptor ligand-binding domains (Kaipainen et al., J. Exp. Med.
178:2077-2088 (1993)). The other two regions include a
transmembrane region and a carboxy-terminal intracellular catalytic
domain interrupted by an insertion of hydrophilic interkinase
sequences of variable lengths, called the kinase insert domain
(Terman et al., Oncogene 6:1677-1683 (1991). VEGF receptors include
FLT-1, sequenced by Shibuya M. et al., Oncogene 5, 519-524 (1990);
KDR, described in PCT/US92/01300, filed Feb. 20, 1992, and in
Terman et al., Oncogene 6:1677-1683 (1991); and FLK-1, sequenced by
Matthews W. et al. Proc. Natl. Acad. Sci. USA, 88:9026-9030
(1991).
[0006] High levels of FLK-1 are expressed by endothelial cells that
infiltrate gliomas (Plate, K. et al., (1992) Nature 359: 845-848).
FLK-1 levels are specifically upregulated by, VEGF produced by
human glioblastomas (Plate, K. et al. (1993) Cancer Res. 53:
5822-5827). The finding of high levels of FLK-1 expression in
glioblastoma associated endothelial cells (GAEC) indicates that
receptor activity is probably induced during tumor formation since
FLK-1 transcripts are barely detectable in normal brain endothelial
cells. This upregulation is confined to the vascular endothelial
cells in close proximity to the tumor. Blocking VEGF activity with
neutralizing anti-VEGF monoclonal antibodies (mAbs) resulted in an
inhibition of the growth of human tumor xenografts in nude mice
(Kim, K. et al. (1993) Nature 362: 841-844), indicating a direct
role for VEGF in tumor-related angiogenesis.
[0007] Although the VEGF ligand is upregulated in tumor cells, and
its receptors are upregulated in tumor infiltrated vascular
endothelial cells, the expression of the VEGF ligand and its
receptors is low in normal cells that are not associated with
angiogenesis. Therefore, such normal cells would not be affected by
blocking the interaction between VEGF and its receptors to inhibit
angiogenesis, and therefore tumor growth. Blocking this VEGF-VEGF
receptor interaction by using a monoclonal antibody to the VEGF
receptor has not been demonstrated prior to the subject
invention.
[0008] One advantage of blocking the VEGF receptor as opposed to
blocking the VEGF ligand to inhibit angiogenesis, and thereby to
inhibit pathological conditions such as tumor growth, is that fewer
antibodies may be needed to achieve such inhibition. Furthermore,
receptor expression levels may be more constant than those of the
environmentally induced ligand. Another advantage of blocking the
VEGF receptor is that more efficient inhibition may be achieved
when combined with blocking of the VEGF ligand.
[0009] The object of this invention is to provide monoclonal
antibodies that neutralizes the interaction between VEGF and its
receptor by binding to a VEGF receptor and thereby preventing VEGF
phosphorylation of the receptor. A further object of this invention
is to provide methods to inhibit angiogenesis and thereby to
inhibit tumor growth in mammals using such monoclonal
antibodies.
SUMMARY OF THE INVENTION
[0010] The present invention provides a monoclonal antibody which
specifically binds to an extracellular domain of a VEGF receptor
and neutralizes activation of the receptor.
[0011] The invention also provides hybridoma cell lines as well as
the monoclonal antibodies produced therefrom: DC101 (IgG1k)
deposited as ATCC Accession No. ATCC HB 11534: Mab25 (IgG1)
deposited as HB12152; and Mab 73 (IgG1) deposited as HB-12153.
[0012] Further, the invention provides a method of neutralizing
VEGF activation of a VEGF receptor in endothelial cells comprising
contacting the cells with the monoclonal antibody of the
invention.
[0013] The invention also provides a method of inhibiting
angiogenesis in a mammal comprising administering an effective
amount of any one of the antibodies of the invention to the mammal.
In addition, the invention provides a method of inhibiting tumor
growth in a mammal comprising administering an effective amount of
any one of the antibodies of the invention to the mammal.
[0014] The invention also provides a pharmaceutical composition
comprising any one of the antibodies of the invention and a
pharmaceutically acceptable carrier.
DESCRIPTION OF THE FIGURES
[0015] FIG. 1: Western Blot of FLK-1/SEAPS immunoprecipitation with
monoclonal antibody DC101 demonstrating that DC101
immunoprecipitates murine FLK-1:SEAPS but not SEAPS alone.
[0016] FIGS. 2A and 2B: Competitive inhibition assay indicating the
effect of anti-FLK-1 monoclonal antibody DC101 on VEGF.sub.165
induced phosphorylation of the FLK-1/fms receptor in transfected
3T3 cells.
[0017] FIG. 2B: Sensitivity of VEGF induced phosphorylation of the
FLK-1/fms receptor to inhibition by monoclonal antibody DC101. C441
cells were assayed at maximal stimulatory concentrations of
VEGF.sub.165 (40 ng/ml) combined with varying levels of the
antibody.
[0018] FIGS. 3A and 3B: Titration of VEGF-induced phosphorylation
of the FLK-1/fins receptor in the presence of mAb DC101. C441 cells
were stimulated with the concentrations of VEGF indicated in the
presence (Lanes 1 to 4) or absence (Lanes 5 to 8) of 5 ug/ml of MAb
DC101. Unstimulated cells assayed in the presence of antibody (Lane
9) serves as the control.
[0019] FIG. 3B: Densitometry scans of the level of phosphorylated
receptor in each lane in FIG. 3A relative to each VEGF
concentration is plotted to show the extent of Mab inhibition at
excess ligand concentrations. Cell lysates were prepared for
detection by anti-phosphotyrosine as described in the Examples
below.
[0020] FIG. 4: Inhibition of VEGF-FLK-1/fins activation by prebound
mAb DC101. C441 cells were stimulated with the concentrations of
VEGF indicated in the absence (Lanes 3 and 4) and presence (Lanes 5
and 6) of DC101. Unstimulated cells (Lanes 1 and 2) serve as
controls. MAb was assayed using two sets of conditions. For P,
cells were prebound with Mab followed by stimulation with VEGF for
15 minutes at room temperature. For C, MAb and ligand were added
simultaneously and assayed as above.
[0021] FIG. 5: VEGF-induced phosphorylation ofthe FLK-1/fins
receptor by treatments with varying concentrations of monoclonal
antibody DC101 and conditioned media from glioblastoma cells (GB
CM).
[0022] FIG. 6: FACS analysis of anti-FLK-1 mAb binding to FLK-1/fms
transfected 3T3 Cells (C441). Transfected FLK-1/fms 3T3 cells were
incubated on ice for 60 minutes with 10 ug/ml of the anti-FLK-1 MAb
DC101 or the isotype matched irrelevant anti-FLK-1 MAb 23H7. Cells
were washed and reincubated with 5 ug of goat anti-mouse IgG
conjugated to FITC, washed, and analyzed by flow cytometry to
determine antibody binding. Data shows the level of fluorescence
for DC101 to C441 cells relative to that detected with the
irrelevant MAb 23H7.
[0023] FIG. 7: Saturation binding of mAb DC101 to the FLK-1/fms
receptor on the transfected 3T3 cell line C441. Confluent C441
cells were incubated in 24 well plates with increasing
concentrations of MAb DC101 (50 ng/ml to 2 ug/ml) for two hours at
4.degree. C. Cells were washed and incubated with 5 ug anti-rat
IgG-biotin conjugate. To detect binding, cells were washed,
incubated with a 1:1000 dilution of streptavidin-HRP, washed and
incubated in a colormetric detection system (TMB). Data represents
the absorbance at 540 nm versus increasing concentrations of MAb
DC101. The binding of the secondary antibody to cells alone was
subtracted from each determination to adjust for non-specific
binding. Data represents the average of three independent
experiments.
[0024] FIG. 8: Immunoprecipitation of phosphorylated FLK-1/fms from
VEGF stimulated FLK-1/fms transfected 3T3 cells. Cells were
stimulated with VEGF as described in the Experimental Procedures
and lysates were immunoprecipitated with irrelevant or relevant
antibodies as follows: 1. rat anti-FLK2 IgG2a (Mab 2A13); 2. rat
anti-FLK-1 IgGI (Mab DC101); 3. rat anti-FLK2 IgGI (Mab 23H7); 4.
rabbit antifms polyclonal antibody. Immunoprecipitated protein was
subjected to SDS PAGE followed by Western blotting. The
immunoprecipitation of VEGF activated receptor was detected by
probing the blots with an anti-phosphotyrosine antibody.
[0025] FIG. 9: Sensitivity of VEGF-induced phosphorylation ofthe
FLK-1/fms receptor to inhibition by mAb DC101. Prebound and
competitive assays were performed with 40 ng/ml of VEGF at the
antibody concentrations indicated. Cell lysates were prepared for
receptor detection with anti-phophotyrosine as described in the
Examples below.
[0026] FIG. 10: Effect of mAb DC101 on CSF-1 induced
phosphorylation of the FMS receptor. In (B), the fms/FLK-2
transfected 3T3 cell line, 10A2, was stimulated with optimal
stimulatory levels of CSF-1 in the absence (Lanes 3 and 4) and
presence (Lanes 5 and 6) of 5 ug/ml of MAb DC 101. Unstimulated
cells assayed in the absence (Lane 1) or presence (Lane 2) of
antibody serve as controls. Cell lysates were prepared for
detection by anti-phosphotyrosine as described in the Examples
below.
[0027] FIG. 11: Specificity of mAb DC101 neutralization of the
activated FLK-1/fms receptor. C441 cells were stimulated with 20 or
40 ng/ml of VEGF in the presence of DC101 (IgGI) or the irrelevant
anti-FLK-2 rat monoclonal antibodies 2A13 (IgG2a) or 23H7 (IgGI).
Assays were performed with each antibody in the absence of VEGF
(Lanes 1 to 3) and in the presence of VEGF under competitive (lanes
4 to 8) or prebound (lanes 9 to 11) conditions. Cell lysates were
prepared for detection by anti-phosphotyrosine as described in the
Examples below. Blots were stripped and reprobed to detect the
FLK-1/fms receptor using a rabbit polyclonal antibody to the
C-terminal region of the fms receptor.
[0028] FIG. 12: Immunoprecipitation of phosphorylated receptor
bands from VEGF stimulated HUVEC cells. HUVEC cells were grown to
subconfluency in endothelial growth medium (EGM) for three days
without a change of medium. Receptor forms were immunoprecipated by
MAb DC101 from lysates of unstimulated cells (Lane 1), VEGF
stimulated cells (lane 2), and cells stimulated with VEGF in the
presence of 1 ug/ml heparin (Lane 3). Phosphorylation assays,
immunoprecipitations, and detection of the phosphorylated receptor
forms were performed as described in the Experimental
Procedures.
[0029] FIG. 13: Effect of mAb DC101 on the proliferation of HUVEC
cells in response to VEGF. Cells were grown for 48 hours as
described in the legend to FIG. 6. Cells were then subjected to the
following assay conditions: no addition to medium (untreated); a
change of fresh endothelial growth medium (complete medium); the
addition of 10 ng/ml of VEGF in the absence or presence of 1 ug/ml
heparin; and VEGF and VEGF-heparin treated cells assayed in the
presence of 1 ug/ml of DC101. Cells were assayed for proliferation
by colormetric detection at 550 nm using a cell proliferation assay
kit (Promega).
[0030] FIGS. 14A and 14B FIG. 14A: Reduction in tumor growth of
individual animals with DC101 (rat anti-flik-1 monoclonal
antibody). FIG. 14B: Reduction in tumor growth in individual
animals with the control 2A13 group (rat anti-flk-2 monoclonal
antibody).
[0031] FIG. 15: Athymic nude mice were injected subcutaneously with
human glioblastoma cell line GBM-18 and divided into three groups:
a PBS control, an irrelevant rat IgGI control 23H7, and DC101.
Treatments were administered simultaneously with tumor xenografts
and continued for four weeks.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The present invention provides monoclonal antibodies that
bind specifically to an extracellular domain of a VEGF receptor. An
extracellular domain of a VEGF receptor is herein defined as a
ligand-binding domain on extracellular region of the receptor,
normally found at the amino-terminal end of the protein, typical of
class III tyrosine kinase receptors.
[0033] Some examples of VEGF receptors include the protein tyrosine
kinase receptors referred to in the literature as FLT-1, KDR and
FLK-1. Unless otherwise stated or clearly inferred otherwise by
context, this specification will follow the customary literature
nomenclature of VEGF receptors. KDR will be referred to as the
human form of a VEGF receptor having MW 180 kd. FLK-1 will be
referred to as the murine homolog of KDR. FLT-1 will be referred to
as a form of VEGF receptor different from, but related to, the
KDR/FLK-1 receptor.
[0034] Other VEGF receptors include those that can be cross-link
labeled with VEGF, or that can be co-immunoprecipitated with KDR
(MW 180 KD). Some known forms of these VEGF receptors have
molecular weights of approximately 170 KD, 150 KD, 130-135 KD,
120-125 KD and 85 KD. See, for example, Quinn et al. Proc. Nat'l.
Acad. Sci 90, 7533-7537 (1993). Scher et al. J. Biol. Chem. 271,
5761-5767 (1996).
[0035] Equivalent receptors having substantially the same amino
acid sequence, as defined above, occur in mammals, ie human, mouse.
The binding of an antibody to one VEGF receptor does not
necessarily imply binding to another VEGF receptor, and binding to
a VEGF receptor in one mammal does not necessarily imply binding to
the equivalent receptor in another mammal.
[0036] The VEGF receptor is usually bound to a cell, such as an
endothelial cell. The VEGF receptor may also be bound to a
non-endothelial cell, such as a tumor cell. Alternatively, the VEGF
receptor may be free from the cell, preferably in soluble form The
antibodies of the invention neutralize VEGF receptors. In this
specification, neutralizing a receptor means inactivating the
intrinsic kinase activity of the receptor to transduce a signal. A
reliable assay for VEGF receptor neutralization is the inhibition
of receptor phosphorylation.
[0037] The present invention is not limited by any particular
mechanism of VEGF receptor neutralization. At the time of filing
this application, the mechanism of VEGF receptor neutralization by
antibodies is not well understood, and the mechanism followed by
one antibody is not necessarilly the same as that followed by
another antibody. Some possible mechanisms include preventing
binding of the VEGF ligand to the extracellular binding domain of
the VEGF receptor, and preventing dimerization or oligomerization
of receptors. Other mechanisms cannot, however, be ruled out.
[0038] UTILITY
[0039] A. Neutralizing VEGF activation of VEGF receptors:
[0040] Neutralization of VEGF activation of a VEGF receptor in a
sample of endothelial or non-endothelial cells, such as tumor
cells, may be performed in vitro or in vivo. Neutralizing VEGF
activation of a VEGF receptor in a sample of VEGFreceptor
expressing cells comprises contacting the cells with an antibody of
the invention. In vitro, the cells are contacted with the antibody
before, simultaneously with, or after, adding VEGF to the cell
sample.
[0041] In vivo, an antibody of the invention is contacted with a
VEGF receptor by administration to a mammal. Methods of
administration to a mammal include, for example, oral, intravenous,
intraperitoneal, subcutaneous, or intramuscular administration.
[0042] This in vivo neutralization method is useflil for inhibiting
angiogenesis in a mammal. Angiogenesis inhibition is a useful
therapeutic method, such as for preventing or inhibiting
angiogenesis associated with pathological conditions such as tumor
growth. Accordingly, the antibodies of the invention are
anti-angiogenic and anti-tumor immunotherapeutic agents.
[0043] VEGF receptors are found on some non-endothelial cells, such
as tumor cells, indicating the unexpected presence of an autocrine
and/or paracrine loop in these cells. The antibodies of this
invention are useful in neutralizing activity of VEGF receptors on
such cells, thereby blocking the autocrine and/or paracrine loop,
and inhibiting tumor growth.
[0044] The methods of inhibiting angiogenesis and of inhibiting
pathological conditions such as tumor growth in a mammal comprises
administering an effective amount of any one of the invention's
antibodies, including any of the functional equivalents thereof,
systemically to a mammal, or directly to a tumor within the mammal.
The mammal is preferably human.
[0045] This method is effective for treating subjects with tumors
and neoplasms, including malignant tumors and neoplasms, such as
blastomas, carcinomas or sarcomas, and especially highly vascular
tumors and neoplasms. Some examples of tumors that can be treated
with the antibodies and fragments of the invention include
epidermoid tumors, squamous tumors, such as head and neck tumors,
colorectal tumors, prostate tumors, breast tumors, lung tumors,
including small cell and non15 small cell lung tumors, pancreatic
tumors, thyroid tumors, ovarian tumors, and liver tumors.
[0046] For example, antibodies of the invention are effective in
treating vascularized skin cancers, including squamous cell
carcinoma, basal cell carcinoma, and skin cancers that can be
treated by suppressing the growth of malignant keratinocytes, such
as human malignant keratinocytes. Other cancers that can be treated
by the antibodies described in this application include Kaposi's
sarcoma, CNS neoplasms (neuroblastomas, capillary
hemangioblastomas, meningiomas and cerebral metastases), melanoma,
gastrointestinal and renal carcinomas and sarcomas,
rhabdomyosarcoma, glioblastoma, preferably glioblastoma multiforme,
and leiomyosarcoma.
[0047] Experimental results described later demonstrate that
antibodies of the invention specifically block VEGF induced
phosphorylation of a mouse extracellular FLK-1/intracellular fms
chimeric receptor expressed in transfected 3T3 cells. The
antibodies had no effect on a fully stimulated chimeric
extracellular fms/intracellular FLK2 receptor by CSF-1. In vivo
studies also described below show that the antibodies were able to
significantly inhibit tumor growth in nude mice.
[0048] A cocktail of monoclonal antibodies of the invention
provides an especially efficient treatment for inhibiting the
growth of tumor cells. The cocktail may include as few as 2, 3 or 4
antibodies, and as many as 6, 8 or 10 antibodies.
[0049] The combined treatment of one or more of the antibodies of
the invention with anti-VEGF antibodies provides a more efficient
treatment for inhibiting the growth of tumor cells than the use of
the antibody or antibodies alone. Anti-VEGF antibodies have been
described by Kim et al in Nature 362, 841-844 (1993).
[0050] Furthermore, the combined treatment of one or more of the
antibodies of the invention with an anti-neoplastic or
chemotherapeutic drug such as, for example, doxorubicin, cisplatin
or taxol provides an even more efficient treatment for inhibiting
the growth of tumor cells than the use of the antibody by itself In
one embodiment, the pharmaceutical composition comprises the
antibody and the anti-neoplastic or chemotherapeutic drug as
separate molecules. In another embodiment, the pharmaceutical
composition comprises the antibody attached, such as, for example,
by conjugation, to an chemotherapeutic drug.
[0051] Preventing or inhibiting angiogenesis is also useful to
treat non-neoplastic pathologic conditions charaqcterized by
excessive angiogenesis, such as neovascular glaucoma, proliferative
retinopathy including proliferative diabetic retinopathy, macular
degeneration, hemangiomas, angiofibromas, and psoriasis.
[0052] B. Using the Antibodies of the Invention to Isolate and
Purify the VEGF Receptor
[0053] The antibodies of the present invention may be used to
isolate and purify the VEGF receptor using conventional methods
such as affinity chromatography (Dean, P.D. G. et al., Affinity
Chromatography: A Practical Approach, IRL Press, Arlington, Va.
(1985)). Other methods well known in the art include magnetic
separation with antibody-coated magnetic beads, "panning" with an
antibody attached to a solid matrix, and flow cytometry.
[0054] The source of VEGF receptor is typically vascular cells, and
especially vascular endothelial cells, that express the VEGF
receptor. Suitable sources of vascular endothelial cells are blood
vessels, such as umbilical cord blood cells, especially, human
umbilical cord vascular endothelial cells (HUVEC).
[0055] The VEGF receptors may be used as starting material to
produce other materials, such as antigens for making additional
monoclonal and polyclonal antibodies that recognize and bind to the
VEGF receptor or other antigens on the surface of VEGF-expressing
cells.
[0056] C. Using the Antibodies of the Invention to Isolate and
Purify FLK-1 Positive Tumor Cells
[0057] The antibodies of the present invention may be used to
isolate and purify FLK-1 positive tumor cells, i.e., tumor cells
expressing the FLK-1 receptor, using conventional methods such as
affinity chromatography (Dean, P. D. G. et al., Affinity
Chromatography:A Practical Approach, IRL Press, Arlington, Va.
(1985)). Other methods well known in the art include magnetic
separation with antibody-coated magnetic beads, cytotoxic agents,
such as complement, conjugated to the antibody, "panning" with an
antibody attached to a solid matrix, and flow cytometry.
[0058] D. Monitoring Levels of VEGF and VEGF Receptors In Vitro or
In Vivo
[0059] The antibodies of the invention may be used to monitor
levels of VEGF or VEGF receptors in vitro or in vivo in biological
samples using standard assays and methods known in the art. Some
examples of biological samples include bodily fluids, such as
blood. Standard assays involve, for example, labeling the
antibodies and conducting standard immunoassays, such as
radioimmunoassays, as is well know in the art.
[0060] PREPARATION OF ANTIBODIES
[0061] The monoclonal antibodies of the invention that specifically
bind to the VEGF receptor may be produced by methods known in the
art. These methods include the immunological method described by
Kohler and Milstein in Nature 256, 495-497 (1975) and Campbell in
"Monoclonal Antibody Technology, The Production and
Characterization of Rodent and Human Hybridomas" in Burdon et al.,
Eds., Laboratory Techniques in Biochemistry and Molecular Biology,
Volume 13, Elsevier Science Publishers, Amsterdam (1985); as well
as by the recombinant DNA method described by Huse et al in Science
246, 1275-1281 (1989).
[0062] The antibodies of the invention may be prepared by
immunizing a mammal with a soluble VEGF receptor. The soluble
receptors may be used by themselves as immunogens, or may be
attached to a carrier protein or to other objects, such as beads,
i.e. sepharose beads. After the mammal has produced antibodies, a
mixture of antibody-producing cells, such as the splenocytes, is
isolated. Monoclonal antibodies may be produced by isolating
individual antibody-producing cells from the mixture and making the
cells immortal by, for example, fusing them with tumor cells, such
as myeloma cells. The resulting hybridomas are preserved in
culture, and express monoclonal antibodies, which are harvested
from the culture medium.
[0063] The antibodies may also be prepared from VEGF receptors
bound to the surface of cells that express the VEGF receptor. The
cell to which the VEGF receptors are bound may be a cell that
naturally expresses the receptor, such as a vascular endothelial
cell. Alternatively, the cell to which the receptor is bound may be
a cell into which the DNA encoding the receptor has been
transfected, such as 3T3 cells.
[0064] The antibody may be prepared in any mammal, including mice,
rats, rabbits, goats and humans. The antibody may be a member of
one of the following immunoglobulin classes: IgG, IgM, IgA, IgD, or
IgE, and the subclasses thereof, and preferably is an IgG1
antibody.
[0065] In one embodiment the antibody is a monoclonal antibody
directed to an epitope of a VEGF receptor present on the surface of
a cell. In another embodiment the monoclonal antibody is a rat IgG1
monoclonal antibody, specific for the murine VEGF receptor FLK-1,
and produced by hybridoma DC101. Hybridoma cell line DC101 was
deposited Jan. 26, 1994 with the American Type Culture Collection,
designated ATCC HB 11534. In a preferred embodiment, the monoclonal
antibody is directed to an epitope of a human FLT-1 receptor or to
a human KDR receptor.
[0066] Functional Equivalents of Antibodies
[0067] The invention also includes functional equivalents of the
antibodies described in this specification. Functional equivalents
have binding characteristics comparable to those of the antibodies,
and include, for example, chimerized, humanized and single chain
antibodies as well as fragments thereof Methods of producing such
functional equivalents are disclosed in PCT Application WO
93/21319, European Patent Application No. 239,400; PCT Application
WO 89/09622; European Patent Application 338,745; and European
Patent Application EP 332,424.
[0068] Functional equivalents include polypeptides with amino acid
sequences substantially the same as the amino acid sequence of the
variable or hypervariable regions of the antibodies of the
invention. "Substantially the same" amino acid sequence is defined
herein as a sequence with at least 70%, preferably at least about
80%, and more preferably at least about 90% homology to another
amino acid sequence, as determined by the FASTA search method in
accordance with Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85,
2444-2448 (1988).
[0069] Chimerized antibodies preferably have constant regions
derived substantially or exclusively from human antibody constant
regions and variable regions derived substantially or exclusively
from the sequence of the variable region from a mammal other than a
human. Humanized antibodies preferably have constant regions and
variable regions other than the complement determining regions
(CDRs) derived substantially or exclusively from the corresponding
human antibody regions and CDRs derived substantially or
exclusively from a mammal other than a human.
[0070] Suitable mammals other than a human include any mammal from
which monoclonal antibodies may be made. Suitable examples of
mammals other than a human include, for example a rabbit, rat,
mouse, horse, goat, or primate. Mice are preferred.
[0071] Functional equivalents also include single-chain antibody
fragments, also known as single-chain antibodies (scFvs).
Single-chain antibody fragments are recombinant polypeptides which
typically may bind with antigens or receptors; these fragments
contain at least one fragment of an antibody variable heavy-chain
amino acid sequence (V.sub.H) tethered to at least one fragment of
an antibody variable light-chain sequence (V.sub.L) with or without
one or more interconnecting linkers. Such a linker may be a short,
flexible peptide selected to assure that the proper
three-dimensional folding of the (V.sub.L) and (V.sub.H) domains
may occur once they are linked so as maintain the target molecule
binding-specificity of the whole antibody from which the
single-chain antibody fragment is derived. Generally, the carboxyl
terminus of the (V.sub.L) or (V.sub.H) sequence may be covalently
linked by such a peptide linker to the amino acid terminus of a
complementary (V.sub.L) and (V.sub.H) sequence. Single-chain
antibody fragments may be generated by molecular cloning, antibody
phage display library or similar techniques. These proteins may be
produced either in eukaryotic cells or prokaryotic cells, including
bacteria.
[0072] Single-chain antibody fragments contain amino acid sequences
having at least one of the variable or complentarity determining
regions (CDR's) of the whole antibodies described in this
specification, but are lacking some or all of the constant domains
of those antibodies. These constant domains are not necessary for
antigen binding, but constitute a major portion of the structure of
whole antibodies. Singlechain antibody fragments may therefore
overcome some of the problems associated with the use of antibodies
containing a part or all of a constant domain. For example,
single-chain antibody fragments tend to be free of undesired
interactions between biological molecules and the heavy-chain
constant region, or other unwanted biological activity.
Additionally, single-chain antibody fragments are considerably
smaller than whole antibodies and may therefore have greater
capillary permeability than whole antibodies, allowing single-chain
antibody fragments to localize and bind to target antigen-binding
sites more efficiently. Also, antibody fragments can be produced on
a relatively large scale in prokaryotic cells, thus facilitating
their production. Furthermore, the relatively small size of
single-chain antibody fragments makes them less likely to provoke
an immune response in a recipient than whole antibodies.
[0073] Functional equivalents further include fragments of
antibodies that have the same, or binding characteristics
comparable to, those of the whole antibody. Such fragments may
contain one or both Fab fragments or the F(ab').sub.2 fragment.
Preferably the antibody fragments contain all six complement
determining regions of the whole antibody, although fragments
containing fewer than all of such regions, such as three, four or
five CDRs, are also functional.
[0074] Further, the functional equivalents may be or may combine
members of any one of the following immunoglobulin classes: IgG,
IgM, IgA, IgD, or IgE, and the subclasses thereof.
[0075] Preparation of VEGF Receptor Immunogens
[0076] The VEGF receptor may be used as an immunogen to raise an
antibody of the invention. The receptor peptide may be obtained
from natural sources, such as from cells that express VEGF
receptors, i.e. vascular endothelial cells. Alternatively,
synthetic VEGF receptor peptides may be prepared using commercially
available machines and the VEGF receptor amino acid sequence
provided by, for example, Shibuya M. et al., Oncogene 5, 519-524
(1990) for FLT-1; PCT/US92/01300 and Terman et al., Oncogene
6:1677-1683 (1991) for KDR; and Matthews W. et al. Proc. Natl.
Acad. Sci. USA, 88:9026-9030 (1991) for FLK-1.
[0077] As a further alternative, DNA encoding a VEGF receptor, such
as a cDNA or a fragment thereof, may be cloned and expressed and
the resulting polypeptide recovered and used as an immunogen to
raise an antibody of the invention. In order to prepare the VEGF
receptors against which the antibodies are made, nucleic acid
molecules that encode the VEGF receptors of the invention, or
portions thereof, especially the extracellular portions thereof,
may be inserted into known vectors for expression in host cells
using standard recombinant DNA techniques, such as those described
below. Suitable sources of such nucleic acid molecules include
cells that express VEGF receptors, i.e. vascular endothelial
cells.
[0078] Preparation of Equivalents
[0079] Equivalents of antibodies are prepared by methods known in
the art. For example, fragments of antibodies may be prepared
enzymatically from whole antibodies.
[0080] Preferably, equivalents of antibodies are prepared from DNA
encoding such equivalents. DNA encoding fragments of antibodies may
be prepared by deleting all but the desired portion of the DNA that
encodes the full length antibody.
[0081] DNA encoding chimerized antibodies may be prepared by
recombining DNA substantially or exclusively encoding human
constant regions and DNA encoding variable regions derived
substantially or exclusively from the sequence of the variable
region of a mammal other than a human. DNA encoding humanized
antibodies may be prepared by recombining DNA encoding constant
regions and variable regions other than the complementarity
determining regions (CDRs) derived substantially or exclusively
from the corresponding human antibody regions and DNA encoding CDRs
derived substantially or exclusively from a mammal other than a
human.
[0082] Suitable sources of DNA molecules that encode fragments of
antibodies include cells, such as hybridomas, that express the full
length antibody. The fragments may be used by themselves as
antibody equivalents, or may be recombined into equivalents, as
described above.
[0083] The DNA deletions and recombinations described in this
section may be carried out by known methods, such as those
described in the published patent applications listed above in the
section entitled "Functional Equivalents of Antibodies" and/or
other standard recombinant DNA techniques, such as those described
below.
[0084] Standard Recombinant DNA Techniques
[0085] Standard recombinant DNA techniques are described in
Sambrook et al., "Molecular Cloning," Second Edition, Cold Spring
Harbor Laboratory Press (1987) and by Ausubel et al. (Eds) "Current
Protocols in Molecular Biology," Green Publishing Associates/
Wiley-Interscience, New York (1990).
[0086] Briefly, a suitable source of cells containing nucleic acid
molecules that express the desired DNA, such as an antibody,
antibody equivalent or VEGF receptor, is selected. See above.
[0087] Total RNA is prepared by standard procedures from a suitable
source. The total RNA is used to direct cDNA synthesis. Standard
methods for isolating RNA and synthesizing cDNA are provided in
standard manuals of molecular biology such as, for example, those
described above.
[0088] The cDNA may be amplified by known methods. For example, the
cDNA may be used as a template for amplification by polymerase
chain reaction (PCR); see Saiki et al., Science, 239, 487 (1988) or
Mullis et al., U.S. Pat. No. 4,683,195. The sequences of the
oligonucleotide primers for the PCR amplification are derived from
the known sequence to be amplified. The oligonucleotides are
synthesized by methods known in the art. Suitable methods include
those described by Caruthers in Science 230, 281-285 (1985).
[0089] A mixture of upstream and downstream oligonucleotides are
used in the PCR amplification. The conditions are optimized for
each particular primer pair according to standard procedures. The
PCR product is analyzed, for example, by electrophoresis for cDNA
having the correct size, corresponding to the sequence between the
primers.
[0090] Alternatively, the coding region may be amplified in two or
more overlapping fragments. The overlapping fragments are designed
to include a restriction site permitting the assembly of the intact
cDNA from the fragments.
[0091] In order to isolate the entire protein-coding regions for
the VEGF receptors, for example, the upstream PCR oligonucleotide
primer is complementary to the sequence at the 5' end, preferably
encompassing the ATG start codon and at least 5-10 nucleotides
upstream of the start codon. The downstream PCR oligonucleotide
primer is complementary to the sequence at the 3' end of the
desired DNA sequence. The desired DNA sequence preferably encodes
the entire extracellular portion of the VEGF receptor, and
optionally encodes all or part of the transmembrane region, and/or
all or part of the intracellular region, including the stop
codon.
[0092] The DNA to be amplified, such as that encoding antibodies,
antibody equivalents, or VEGF receptors, may also be replicated in
a wide variety of cloning vectors in a wide variety of host cells.
The host cell may be prokaryotic or eukaryotic.
[0093] The vector into which the DNA is spliced may comprise
segments of chromosomal, non-chromosomal and synthetic DNA
sequences. Some suitable prokaryotic cloning vectors include
plasmids from E. coli, such as colE1, pCR1, pBR322, pMB9, pUC,
pKSM, and RP4. Prokaryotic vectors also include derivatives of
phage DNA such as M13 and other filamentous single-stranded DNA
phages.
[0094] A preferred vector for cloning nucleic acid encoding the
VEGF receptor is the Baculovirus vector.
[0095] The vector containing the DNA to be expressed is transfected
into a suitable host cell. The host cell is maintained in an
appropriate culture medium, and subjected to conditions under which
the cells and the vector replicate. The vector may be recovered
from the cell. The DNA to be expressed may be recovered from the
vector.
[0096] Expression and Isolation of Antibodies, Antibody
Equivalents. or VEGF Receptors
[0097] The DNA to be expressed, such as that encoding antibodies,
antibody equivalents, or VEGF receptors, may be inserted into a
suitable expression vector and expressed in a suitable prokaryotic
or eucaryotic host cell.
[0098] For example, the DNA inserted into a host cell may encode
the entire extracellular portion of the VEGF receptor, or a soluble
fragment of the extracellular portion of the VEGF receptor. The
extracellular portion of the VEGF receptor encoded by the DNA is
optionally attached at either, or both, the 5' end or the 3' end to
additional amino acid sequences. The additional amino acid
sequences may be attached to the VEGF receptor extracellular region
in nature, such as the leader sequence, the transmembrane region
and/or the intracellular region of the VEGF receptor. The
additional amino acid sequences may also be sequences not attached
to the VEGF receptor in nature. Preferably, such additional amino
acid sequences serve a particular purpose, such as to improve
expression levels, secretion, solubility, or immunogenicity.
[0099] Vectors for expressing proteins in bacteria, especially E.
coli, are known. Such vectors include the PATH vectors described by
Dieckmann and Tzagoloff in J. Biol. Chem. 260, 1513-1520 (1985).
These vectors contain DNA sequences that encode anthranilate
synthetase (TrpE) followed by a polylinker at the carboxy terminus.
Other expression vector systems are based on beta-galactosidase
(pEX); lambda P.sub.L; maltose binding protein (pMAL); and
glutathione S-transferase (PGST) -see Gene 67, 31 (1988) and
Peptide Research 3, 167 (1990).
[0100] Vectors useful in yeast are available. A suitable example is
the 2.mu. plasmid.
[0101] Suitable vectors for expression in mammalian cells are also
known. Such vectors include well-known derivatives of SV-40,
adenovirus, retrovirus-derived DNA sequences and shuttle vectors
derived from combination of fulnctional mammalian vectors, such as
those described above, and functional plasmids and phage DNA.
[0102] Further eukaryotic expression vectors are known in the art
(e.g., P. J. Southern and P. Berg, J. Mol. Appl. Genet. 1, 327-341
(1982); S. Subramani et al, Mol. Cell. Biol. 1, 854-864 (1981); R.
J. Kaufmann and P. A. Sharp, "Amplification And Expression Of
Sequences Cotransfected with A Modular Dihydrofolate Reductase
Complementary DNA Gene," J. Mol. Biol. 159, 601-621 (1982); R. J.
Kaufrnann and P. A. Sharp, Mol. Cell. Biol. 159, 601-664 (1982); S.
I. Scahill et al, "Expression And Characterization Of The Product
Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary
Cells," Proc. Natl. Acad. Sci. USA 80 4654-4659 (1983); G. Urlaub
and L. A. Chasin, Proc. Natl. Acad. Sci. USA 77, 4216-4220,
(1980).
[0103] The expression vectors useful in the present invention
contain at least one expression control sequence that is
operatively linked to the DNA sequence or fragment to be expressed.
The control sequence is inserted in the vector in order to control
and to regulate the expression of the cloned DNA sequence. Examples
of useful expression control sequences are the lac system, the XM
system, the tac system, the trc system, major operator and promoter
regions of phage lambda, the control region of fd coat protein, the
glycolytic promoters of yeast, e.g., the promoter for
3-phosphoglycerate kinase, the promoters of yeast acid phosphatase,
e.g., Pho5, the promoters of the yeast alpha-mating factors, and
promoters derived from polyoma, adenovirus, retrovirus, and simian
virus, e.g., the early and late promoters or SV40, and other
sequences known to control the expression of genes of prokaryotic
or eukaryotic cells and their viruses or combinations thereof.
[0104] Vectors containing the control signals and DNA to be
expressed, such as that encoding antibodies, antibody equivalents,
or VEGF receptors, are inserted into a host cell for expression.
Some useful expression host cells include well-known prokaryotic
and eukaryotic cells. Some suitable prokaryotic hosts include, for
example, E. coli, such as E. coli SG-936, E. coli HB 101, E. coli
W3110, E. coli X1776, E. coli X2282, E. Coli DHI, and E. coli MRC1,
Pseudomonas, Bacillus, such as Bacillus subtilis, and Streptomyces.
Suitable eukaryotic cells include yeast and other fungi, insect,
animal cells, such as COS cells and CHO cells, human cells and
plant cells in tissue culture.
[0105] Following expression in a host cell maintained in a suitable
medium, the polypeptide or peptide to be expressed, such as that
encoding antibodies, antibody equivalents, or VEGF receptors, may
be isolated from the medium, and purified by methods known in the
art. If the, the polypeptide or peptide is not secreted into the
culture medium, the host cells are lysed prior to isolation and
purification.
EXAMPLES
[0106] The Examples which follow are set forth to aid in
understanding the invention but are not intended to, and should not
be construed to, limit its scope in any way. The Examples do not
include detailed descriptions of conventional methods, such as
those employed in the construction of vectors and plasmids, the
insertion of genes encoding polypeptides into such vectors and
plasmids, or the introduction of plasmids into host cells. Such
methods are well known to those of ordinary skill in the art and
are described in numerous publications including Sambrook, J.,
Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A
Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory
Press.
Example I
CELL LINES AND MEDIA
[0107] NIH 3T3 cells were obtained from the American Type Culture
Collection (Rockville Md.). The C441 cell line was constructed by
transfecting 3T3 cells with the chimeric receptor mouse FLK1/human
frs. 10A2 is a 3T3 transfectant containing the chimeric receptor
human fms/mouse FLK2, the isolation and characterization of which
has been described (Dosil, M. et al., Mol. Cell. Biol. 13:6572-6585
(1993)). Cells were routinely maintained in Dulbecco's modified
Eagle's medium (DME) supplemented with 10% calf serum (CS), 1 mM
L-glutamine, antibiotics, and 600 ug/ml G418 (Geneticin; Sigma, St
Louis Mo.).
[0108] A glioblastoma cell line, GBM-18, was maintained in DME
supplemented with 5% calf serum, ImmM L-glutamine, and
antibiotics.
[0109] A stable 3T3 line secreting the soluble chimeric protein,
mouse FLKI:SEAPs (secretory alkaline phosphastase), was generated
and maintained in DMEM and 10% calf serum. Conditioned media was
collected. Soluble FLK-1 :SEAP is isolated from the conditioned
media.
Example II
Isolation of Monoclonal Antibodies
Example II-1
Rat Anti Mouse FLK-1 Monoclonal Antibody DC101 (IgG1)
[0110] Lewis rats (Charles River Labs) were hyperimmunized with an
immune complex consisting of the mouse FLK-1: SEAPs soluble
receptor, a rabbit anti-alkaline phosphatase polyclonal antibody
and Protein-G sepharose beads. The animals received 7
intraperitoneal injections of this complex spread over three months
(at days 0, 14, 21, 28, 49, 63, 77). At various times, the animals
were bled from the tail vein and immune sera screened by ELISA for
high titer binding to mFLK-1:SEAPs. Five days after the final
injection, rats were sacrificed and the spleens aseptically
removed. Splenocytes were washed, counted, and fused at a 2:1 ratio
with the murine myeloma cell line NS1. Hybridomas were selected in
HAT medium and colonies screened by ELISA for specific binding to
mFLK-1:SEAPs but not the SEAPs protein. A number of positive
hybridomas were expanded and cloned three times by limiting
dilution. One subclone, designated DC101, was further
characterized.
Example II-2
Mouse Anti Mouse FLK-1 Monoclonal Antibodies Mab 25 and Mab 73
[0111] Murine anti-FLK-1 monoclonal antibodies (Mabs) were produced
using a similar protocol as that employed for DC101. Briefly, mice
were injected with a complex of FLK-1/SEAP soluble receptor bound
to either an anti-SEAP-Protein/A Sepharose complex or wheat germ
agglutinin Sepharose from conditioned medium of transfected NIH 3T3
cell. Mice were hyperimmunized at periodic intervals over a 6 month
period. Immune splenocytes were pooled and fused with the murine
myeloma cell line, NSI. Hybidomas were selected in HAT medium and
following incubation, colonies were screened for mouse Mab
produciton. Unlike the protocol employed for DC101, positive
supernatants were initially screened for binding to the FLK-1/fms
receptor captured from C441 cell lysates on ELISA plates coated
witha peptide generated polyclonal antibody against the C-terminal
region of fms. Reactive Mabs were then assayed by ELISA for binding
to intact C441 cells and to purified FLK-1/SEAP versus SEAP alone.
The supernatants from hybridomas showing binding to C441 and
reactivity with FLK-1/SEAP but not SEAP were expanded, grown in
ascites, and purified (EZ-PREP, Pharmacia). Purified Mabs were
subjected to assays on C441 cells to determine their cell surface
binding by FACS and their ability to inhibit VEGF induced
activation of FLK-1/frns in phosphorylation assays. The results of
these studies led to the cloning of Mabs 25 and 73 (isotype IgG1)
for further characterization based on their capabilities to bind
specifically to FLK-1 and block receptor activation at levels
comparable to that observed for DC101.
Example III
ASSAYS
Example III-1
ELISA Methods
[0112] Antibodies were screened by a solid state ELISA in which the
binding characteristics of the various mAbs to FLK-1:SEAP and SEAP
protein were compared. Microtiter plates were coated with 50-100
ng/well of either FLK-1 :SEAP or AP in pH9.6 carbonate buffer
overnight at 4.degree. C. Plates were blocked with phosphate
buffered saline supplemented with 10% new born calf serum (NB 10)
for one hour at 37.degree. C. Hybridoma supernatants or purified
antibodies were added to the plates for two hours at 37.degree. C.
followed by goat anti-rat IgG conjugated to horse radish peroxidase
(Tago) added for an additional hour at 37.degree. C. After
extensive washing, TMB (Kirkegaard and Perry, Gaithersburg Md.)
plus hydrogen peroxide was added as the chromogen and the plates
read at 450 nm in an ELISA reader.
Example III-2
Isotyping
[0113] Isotyping of the various monoclonal antibodies was done as
previously described (Songsakphisarn, R. and Goldstein, N. I.,
Hybridoma 12: 343-348, 1993) using rat isotype specific reagents
(Zymed Labs, South San Francisco Calif.).
Example III-3
Phosphorylation, Immunoprecipitation and Immunoblot Assays
[0114] The phosphorylation assays and Western blot analysis with
C441 and 10A2 cells were performed as previously described (Tessler
et al., 1994) with some modifications. Briefly, cells were grown to
90% confluency in DME-10% CS and then serum starved in DME-0.5% CS
for 24 hours prior to experimentation. HUVEC cells were grown to
subconfluence in EGM basal media. For neutralization assays, cells
were stimulated with various concentrations of the appropriate
ligand under serum free conditions (DME-0.1% BSA) in the presence
and absence of mAb DC101 for 15 minutes at room temperature. The
ligands, VEGF and CSF-1, were assayed at concentrations of 10-80
ng/ml and 20-40 ng/ml, respectively. Monoclonal antibodies were
assayed at concentrations ranging from 0.5 ug/ml to 10 ug/ml. To
evaluate the effects of mAb DC101 on the VEGF induced activation of
the FLK-1-fms receptor, antibody was either added simultaneously
(competitive inhibition) or prebound to cells for 15 minutes at
room temperature prior to the addition of ligand. Cells incubated
in serum free medium in the absence and presence of DC101 served as
controls for receptor autophosphorylation in the absence of ligand
and the presence of antibody, respectively. A control cell line
expressing the fms/FLK2 chimeric receptor (10A2) was starved and
stimulated with 20 and 40 ng/ml CSF-1 and assayed in the presence
and absence of 5 ug/ml DC101.
[0115] Following stimulation, monolayers were washed with ice cold
PBS containing 1 mM sodium orthovanadate. Cells were then lysed in
lysis buffer (20 mM Tris-HCI, pH 7.4, 1% Triton X-100, 137 mM NaCl,
10% glycerol, 10 mM EDTA, 2 mM sodium orthovanadate, 100 mM NaF,
100 mM sodium pyrophosphate, 5 mM Pefabloc (Boehringer Mannheim
Biochemicals, Indianapolis Ind.), 100 ug aprotinin and 100 ug/ml
leupeptin) and centrifuged at 14000.times.g for 10 minutes. Protein
was immunoprecipitated from cleared lysates of transfected cells
using polyclonal antibodies generated to peptides corresponding to
the C-terminal region of the human fms receptor (Tessler et al., J.
Biol. Chem. 269, 12456-12461, 1994) or the murine FLK-2 interkinase
domain (Small et al., Proc. Natl. Acad. Sci. USA, 91, 459-463,
1994) coupled to Protein A Sepharose beads. Where indicated,
immunoprecipitations with DC101 or irrelevant rat IgG were
performed with 10 ug of antibody coupled to Protein G beads. The
beads were then washed once with 0.2% Triton X-100, 10 mM Tris-HCI
pH8.0, 150 mM NaCl, 2 mM EDTA (Buffer A), twice with Buffer A
containing 500 mM NaCl and twice with Tris-HCI, pH 8.0. Drained
beads were mixed with 30 ul in 2.times.SDS loading buffer and
subjected to SDS PAGE in 4-12% gradient gels (Novex, San Diego
Calif.). After electrophoresis, proteins were blotted to
nitrocellulose filters for analysis. Filters were blocked overnight
in blocking buffer (50 mM Tris-HCI, pH7.4, 150 mM NaCl (TBS)
containing 5% bovine serum albumin and 10% nonfat dried milk
(Biorad, Calif.). To detect phosphorylated receptor, blots were
probed with a monoclonal antibody directed to phosphotyrosine (UBI,
Lake Placid, N.Y.) in blocking buffer for 1 hour at room
temperature. Blots were then washed extensively with 0.5.times.TBS
containing 0.1% Tween-20 (TBS-T) and incubated with goat anti-mouse
Ig conjugated to horseradish peroxidase (Amersham). Blots were
washed with TBS and incubated for 1 minute with a chemiluminescence
reagent (ECL, Amersham). Anti-phosphotyrosine reacting with
phosphorylated proteins was detected by exposure to a high
performance luminescence detection film (Hyperfilm-ECL, Amersham)
for 0.5 to 10 minutes.
[0116] To detect FLK-1/fms in C441 cells receptor levels, blots
were stripped according to manufacturer's protocols (Amersham) and
reprobed with the anti-fns rabbit polyclonal antibody.
Example III-4
Flow Cytometer Binding Assays
[0117] C441 cells were grown to near confluency in 10 cm plates.
Cells were removed with a non-enzymatic dissociation buffer
(Sigma), washed in cold serum free medium and resuspended in Hanks
balanced salt solution supplemented with 1% BSA (BBSS51 BSA) at a
concentration of 1 million cells per tube. mAb DC101 or an isotype
matched irrelevant antibody anti FLK-2 23H7 was added at 10 ug per
tube for 60 minutes on ice. After washing, 5 ul of goat anti-mouse
IgG conjugated to FITC (TAGO) was added for an additional 30
minutes on ice. Cells were washed three times, resuspended in Imid
of BBSS-BSA, and analyzed on a Coulter Epics Elite Cytometer.
Non-specific binding of the fluorescent secondary antibody was
determined from samples lacking the primary antibody.
Example III-5
Binding Assays to Intact Cells
[0118] Assays with C441 cells were performed with cells grown to
confluency in 24 well dishes. HUVEC cells were grown to confluency
in 6 well dishes. Monolayers were incubated at 4.degree. C. for 2
hours with various amounts of mAb DC101 in binding buffer (DMEM, 50
Mm HEPES pH 7.0, 0.5% bovine serum albumin). Cells were then washed
with cold phosphate buffered saline (PBS) and incubated with a
secondary anti-rat IgG antibody conjugated with biotin at a final
concentration of 2.5 ug/ml. After 1 hour at 4.degree. C. cells were
washed and incubated with a streptavidin-horse radish peroxidase
complex for 30 minutes at 4.degree. C. Following washing,
cell-bound antibody was determined by measuring the absorbance at
540 nm obtained with a colormetric detection system (TMB,
Kirkegaard and Perry). The OD 540 nm of the secondary antibody
alone served as the control for non-specific binding.
Example III-6
Cell Proliferation Assays
[0119] Mitogenic assays were performed using the Cell Titer 96 Non
Radioactive Cell Proliferation Assay Kit (Promega Corp., Madison,
Wis.). In this assay proliferation is measured color metrically as
the value obtained from the reduction of a tetrazolium salt by
viable cells to a formazan product. Briefly, HUVEC cells were grown
in 24 well gelatin-coated plates in EGM basal media at 1000
cells/well. After a 48-hour incubation various components were
added to the wells. VEGF was added at 10 ng/ml to the media in the
presence and absence of 1 ug/ml of mAb DC101. Where indicated,
heparin (Sigma) was added to a final concentration of 1 ug/ml.
Cells were then incubated for an additional 3 days. To measure cell
growth, a 20 ul aliquot of tetrazolum dye was added to each well
and cells were incubated for 3 hrs at 37.degree. C. Cells were
solubilized and the absorbance (OD570) of the formazan product was
measured as a quantitation of proliferation.
Example IV
IN VITRO ACTIVITY ASSAYS
Example IV-1
Murine Anti-FLK-1 Mabs 25 and 73 Elicit a Specific Neutralization
of VEGF Induced Activation of the FLK-1/fms Receptor
[0120] Assays were performed with immunoprecipitated FLK/fms and
PDGF receptors from equal concentrations of the FLK-1/fms
transfected 3T3 cell line, C441 whereas the human EGFR was
immunoprecipitated from the tumor cell line, KB. Cells were
stimulated with RPMI-0.5% BSA containing 20 ng/ml VEGF (FLK-1/fms),
DMEM-10% calf serum (PDGFR), or 10 ng/ml EGF (EGFR), in the
presence and absence of 10 ug/ml of the murine anti-FLK-1 Mabs, 25
and 73. Following stimulation, cells were washed with PBS-lmM
sodium orthovanadate and lysed. FLK-1/fms and PDGFR were
immunoprecipitated from lysates with peptide generated polyclonal
antibodies against the C-terminal region of the c-frns (IM 133) and
the PDGF (UBI) receptors, respectively. EGFR was immunoprecipitated
with a Mab (C225) raised against the N-terminal region of the human
receptor. Immunoprecipitated lystates were subjected to SDS
polyacrylamide electrophoresis followed by western blotting. Blots
were probed with an anti-PTyr Mab (UBI) to detect receptor
activation. Receptor neutralization of stimulated cells was
assessed relative to an irrelevant Mab and the unstimulated
control.
Example IV-2
Detection of the FLK-1/fms Receptor by Western Blotting Using Mab
25 and Mab 73 as Probes
[0121] Receptor was detected by the murine anti-FLK-1 Mabs on
western blots of the FLK1/fms receptor immunoprecipiated by a
peptide generated polyclonal antibody against the C-terminal region
of the c-fms receptor from lysated prepared from equal
concentrations of transfected 3T3 cell line C441. Following
analysis by SDS gel electrophoresis and western blotting, the blot
was divided into four parts and each section was probed with 50
ug/ml of the anti-FLK-1 Mabs 25 and 73. Blots were then stripped
and reprobed with the anti-fms polyclonal antibody to verify that
the bands deted by each Mab represented the FLK-1/fms receptor.
Example IV-3
Detection of Activated KDR from VEGF Stimulated HUVEC and OVCAR-3
Cells by Immunoprecipiation with Anti-FLK-1 Mabs
[0122] Proteins were immunoprecipitated by different antibodies
from a lysate of freshly isolated HUVEC. Prior to lysis, cells were
stimulated with 20 ng/ml VEGF for 10 minutes at room temperature in
RPMI-0.5% BSA and washed with PBS containing 1 mM sodium
orthovanadate. Individual immunoprecipitations were performed with
equal volumes of lysate and then subjected to SDS polyacrylamide
electrophoresis followed by western blotting. The blot was probed
initially with an anti-PTyr Mab (UBI) and then sequentially
stripped and reprobed with a peptide generated polyclonal antibody
against the interkinase of FLK-1/KDR (IM 142), followed by a
polyclonal antibody against the C-terminal region of FLT-1 (Santa
Cruz Biotechnology, Inc). The immunoprecipitations were performed
with an irrelevant rat Mab, 23H7, an irrelevant mouse Mab, DAB 8,
versus the anti-FLK-1 Mabs, DC101, 73, 25 and an anti-FLK-1/KDR
polyclonal antibody, IM 142. In some cases blots were stripped and
reprobed with the anti-FLK-1 Mabs 73 and 25 to detect cross
reactive bands.
[0123] A similar protocol was employed to detect KDR receptor
form(s) in the ovarian carcinoma cell line OVCAR-3.
Example V
ACTIVITY OF ANTIBODIES
Example V-1
ELISA and Immunoprecipitation with DC101
[0124] Rat IgG1 monoclonal antibody DC101 was found to be specific
for the murine tyrosine kinase receptor FLK-1. ELISA data showed
that the antibody bound to purified FLK1:SEAP but not alkaline
phosphatase or other receptor tyrosine kinases such as FLK2. As
seen in FIG. 1, DC101 immunoprecipitates murine FLK-1 :SEAPS but
not SEAPS alone.
Example V-2
INHIBITION OF FLK-1 RECEPTOR PHOSPHORYLATION WITH DC101
[0125] Experiments were then performed to determine whether DC101
could neutralize phosphorylation of FLK1 in C441 cells by its
cognate ligand, VEGF.sub.165. In these studies, monoclonal antibody
and VEGF were added simultaneously to monolayers for 15 minutes at
room temperature. These conditions were designed to determine the
competitive effects (competitive inhibition) of the antibody on
receptor/ligand binding. The results of these assays, shown in FIG.
2A, indicate that VEGF.sub.165 induced phosphorylation of the
FLK1/fms receptor was markedly reduced when cells were assayed in
the presence of DC101. In addition, these data suggest that the Mab
competes with VEGF.sub.165 to prevent a full activation of receptor
by ligand. To determine the sensitivity of the VEGF-FLK1
interaction to inhibition by DC101, C441 cells were assayed at
maximal stimulatory concentrations of VBGF.sub.165 (40 ng/ml)
combined with varying levels of the antibody. The results of these
Mab titrations are shown in FIG. 2B. A marked decrease in the
phosphorylation of FLK1 by VEGF.sub.165 was observed when DC101 was
included at concentrations greater than 0.5 ug/ml. These data show
that relatively low concentrations of antibody (<1 ug/mI) are
sufficient to inhibit receptor activation by ligand. At 5 ug/ml the
antibody is able to neutralize VEGF.sub.165 stimulation of FLK1 in
the presence of excess ligand at 80 ng/ml (FIGS. 3A and 3B). As a
control, the effect of DC101 was tested on the fully stimulated
fms/FLK2 receptor (10A2 cell line) using CSF-1. Under these
conditions, DC101 showed no effect on receptor activation.
Example V-3
Inhibition Studies with DC 101
[0126] The extent and specificity of Mab inhibition was further
assessed by studies in which DC101 was preincubated with cells
before the addition of ligand to allow maximal interaction of
antibody with receptor. In these experiments, monolayers were
incubated with 5 ug/ml of DC101, a rat anti-FLK2 Mab (2A13)
prepared by conventional techniques (ImClone, N.Y.), and control
rat IgG1 (Zymed Labs) for 15 minutes at room temperature prior to
the addition of 40 ng/ml of VEGF.sub.165 for an additional 15
minutes. For comparison, assays were run in which DC101 and
VEGF.sub.165 were added simultaneously (competitive inhibition).
The results of these studies (FIG. 4) show that preincubation of
the anti-FLK-1 monoclonal antibody with FLK1/fms transfected cells
completely abrogates receptor activation by VEGF.sub.165. Similar
results were observed using VEGF.sub.121 for stimulation. While
phosphorylation of FLK1 by VEGF is not affected by the addition of
irrelevant isotype matched rat antibodies, the reactivity of the
same blot probed with the anti-fms polyclonal antibody shows an
equal level of receptor protein per lane. These data indicate that
the inhibition of phosphorylation observed with DC101 was due to
the blockage of receptor activation rather than a lack of receptor
protein in the test samples.
Example V-4
Binding of DC101 to C441 Cells by FACS Analysis
[0127] The mAb was assayed by FACS analysis for binding to 3T3
cells transfected with the FLK-1/frns receptor (C441 cells). The
results, shown in FIG. 6, demonstrate that the chimeric FLK-1/fms
expressed on the surface of C441 cells is specifically recognized
by mAb DC101 and not by an antibody of the same isotype raised
against the related tyrosine kinase receptor, FLK-2. The efficacy
of the mAb-receptor interaction at the cell surface was determined
from assays in which varying levels of mAb binding was measured on
intact C441 cells. These results, shown in FIG. 7, indicate that
mAb binds to the FLK-1/fms receptor with a relative apparent
affinity of approximately 500 ng/ml. These results indicate that
the mAb has a strong affinity for cell surface expressed FLK-1.
Example V-5
Reactivity of DC101 by Immunoprecipitation
[0128] The extent of DC101 reactivity with the FLK-1/fms receptor
was further assessed by determining the capacity of the antibody to
immunoprecipitate the receptor following activation by VEGF. FIG. 8
shows an immunoprecipitation by mAb DC101 of the phosphorylated
FLK-1/fms receptor from VEGF stimulated C441 cells. The results
show that the DC101 monoclonal and anti-fms polyclonal antibodies
display similar levels of receptor interaction while rat anti FLK-2
antibodies 2H37 (IgG1) and 2A13 (IgG2a) show no reactivity.
Experiments were then performed to determine whether mAb DC101
could neutralize the VEGF induced phosphorylation of FLK-1/fms at
maximal stimulatory concentrations of ligand (40 ng/ml). In these
studies, monoclonal antibody was added to monolayers either
simultaneously with ligand or prior to ligand stimulation and
assayed for 15 minutes at room temperature. These conditions were
studied to determine both the competitive effects (competitive
inhibition) of the antibody on receptor/ligand binding as well as
the efficacy of prebound antibody to prevent receptor activation.
The results of these assays, shown in FIG. 4, indicate that
phosphorylation of the FLK-1/fms is reduced by the simultaneous
addition of mAb with VEGF and markedly inhibited by antibody
prebound to the receptor. A densitometry scan of these data
revealed that nAb DC101 interacts with FLK-1/fms to inhibit
phosphorylation to a level that is 6% (lane 5, B) and 40% (lane 6,C
) of the fully stimulated receptor control (lane 4). From these
data we infer that mAb DC101 strongly competes with the
ligand-receptor interaction to neutralize FLK-1 receptor
activation. To determine the sensitivity of the VEGF-FLK-1
interaction to inhibition by mAb DC101, C441 cells were assayed
with maximal VEGF levels in the presence of increasing
concentrations of antibody. Assays were performed with the mAb
under competitive and prebinding conditions. The results of these
mAb titrations are shown in FIG. 9. A marked decrease in the
phosphorylation of FLK-1 is observed when mAb DC101 competes with
VEGF antibody at concentrations greater than 0.5 ug/ml. These data
also show that relatively low concentrations of prebound antibody
(<1 ug/ml) are sufficient to completely inhibit receptor
activation by ligand.
Example V-6
Activity of DC101 by Phosphorylation Assay
[0129] To further evaluate the antagonistic behavior of mAb DC101
on receptor activation, phosphorylation assays were performed in
which a fixed amount of antibody (5 ug/ml) was added to C441 cells
stimulated with increasing amounts of ligand (FIG. 3A). The level
of phosphorylation induced by each ligand concentration in the
presence and absence of mAb DC101 was also quantitated by
densitometry readings. The plot of these data given in FIG. 3B
indicates that the antibody was able to partially neutralize
receptor phosphorylation even in the presence of excess amounts of
VEGF. To evaluate the specificity of mAb DC101 on receptor
activation, the antibody was tested for its ability to
competitively inhibit CSF-1 induced activation of the fms/FLK-2
receptor in the 3T3 transfected cell line, 10A2. In these
experiments 5 ug/ml of mAb DC101 was tested together with CSF-1
concentrations (20-40 ng/ml) that are known to result in full
activation of the receptor. These results, which are shown in FIG.
10, indicate that mAb DC101 has no effect on the CSF-1 mediated
phosphorylation of the fms/FLK-2 receptor.
Example V-7
DC101 Inhibition by Pre-Incubation Studies
[0130] The extent and specificity of antibody inhibition was
further assessed by studies in which DC101 or an irrelevant
antibodies were preincubated with cells before the addition of
ligand to assure maximal interaction of antibody with receptor. In
these experiments, monolayers were preincubated with either 5 ug/ml
of DC 101, a rat anti-FLK2 mAb (2A13) or a control rat IgG1 (Zymed
Labs) prior to the addition of 40 ng/ml of VEGF. For comparison,
competitive assays were run in which antibodies and VEGF were added
simultaneously. The results of these studies show that only the
preincubation of the anti-FLK-1 monoclonal antibody with FLK1/fms
transfected cells completely abrogates receptor activation by VEGF
while phosphorylation of FLK1 by VEGF is not affected by the
addition of irrelevant isotype matched rat antibodies. The
reactivity of the same blot probed with the anti-fms polyclonal
(FIG. 11) shows an equal level of receptor protein per lane. These
data indicate that the lack of phosphorylation observed with mAb
DC101 treated cells was due to the blockage of a VEGF-induced
phosphorylation of equal amounts of expressed receptor.
Example V-8
Interaction of Antibodies with Homologous Receptor Forms
[0131] Experiments were then conducted to determine whether the
anti FLK-1 monoclonal antibodies interact with homologous receptor
forms on human endothelial cells. A titration of increasing
concentrations of DC101 on cloned HUVEC cells (ATCC) indicated that
the antibody displayed a complex binding behavior. The data
represent differential antibody interactions with VEGF receptors
reported to occur on endothelial cells (Vaisman et al., J. Biol.
Chem. 265, 19461-19466, 1990). The specificity of DC101 interaction
with VEGF stimulated HUVEC cells was then addressed using
phosphorylation assays under similar conditions as those reported
for FIG. 8. In these studies DC101 immunoprecipitates protein bands
from HUVEC cells that have molecular weights similar to those
reported for cross linked VEGF-receptor bands when the ligand
component is subtracted (FIG. 12). These bands display an increased
phosphorylation when cells are stimulated by VEGF (compare lanes 1
and 2 in FIG. 12). In addition, the VEGF induced phosphorylation of
the receptor bands is potentiated by the inclusion of 1 ug/ml
heparin in the assay (lane 3 in FIG. 12). These findings are
consistent with previous reports of increased VEGF binding to
endothelial cells in the presence of low concentrations of heparin
(Gitay-Goren et al., J. Biol. Chem. 267, 6093-6098.1992).
[0132] It is difficult to ascertain which immunoprecipitated
protein interacts with DC101 to generate the complex of
phosphorylated bands observed in FIG. 12 given the various receptor
forms shown to bind VEGF on HUVEC and the possibility of their
association upon stimulation. Cell surface expressed receptor forms
with molecular weights of approximately 180 (KDR), 155, 130-135,
120-125 and 85 have been reported to bind VEGF on HUVEC. Such
findings address the possibility that several different receptor
forms may heterodimerize upon ligand stimulation in a manner
similar to that reported for KDR-FLT-1. However, with the exception
of KDR, the exact nature and role of these receptor forms have yet
to be defined. Consequently, antibody reactivity may result from
interaction(s) with one of several VEGF receptors independent of
KDR.
[0133] DC101 does not react with human KDR in an ELISA format nor
bind to freshly isolated HUVEC by FACS analysis. These results
suggest that a direct interaction of DC101 with human KDR is highly
unlikely.
[0134] Unlike DC 101, Mab 25 and Mab 73 both react with human KDR
in an ELISA format and bind to freshly isolated HUVEC by FACS
analysis.
Example V-9
Mitogenic Assays of HUVEC.
[0135] An inhibitory effect of DC101 on endothelial cells was
observed when the antibody was tested in mitogenic assays of HUVEC
cells (ATCC) stimulated with VEGF in the presence and absence of
antibody (FIG. 12). These results show that a marked increase in
cell proliferation by VEGF is reduced approximately 35% by DC101.
Heparin shows no differential effect on cell growth under the
growth conditions employed in these assays.
[0136] Since DC101 can exert effects on VEGF induced proliferation
and receptor phosphorylation of HUVEC it is conceivable that these
results are due to a Mab interaction with an undefined receptor
form which is poorly accessible at the cell surface, but which
plays some role, albeit minor, in HUVEC growth. Also, the
immunoprecipitation of phosphorylated bands of the correct
molecular weight by DC101 from VEGF stimulated HUVEC also supports
the notion that DC101 may interact with an undefined FLK-1 like
protein that associates with an activated receptor complex.
Example V-10
Binding of Mab 25 and Mab 73 to C441 Cells and HUVEC
[0137] Mabs 25 and 73 bind to C441 and HUVEC by FACS analysis and
show internalization in both cell lines. Results from western blots
show that both anti-FLK-1 Mabs can detect the band(s) for the
FLK/fms receptor in immunoprecipitates by an anti-fms polyclonal
antibody from C441 cells. (See example IV-2 above for protocol.)
These antibodies elicit a specific neutralization of VEGF induced
activation of the FLK-1/fms receptor and have no effect on the
phosphorylation of the mouse PDGF receptor by PDGF or the human EGF
receptor by EGF. (See example IV-1 above for protocol.) They have
the capacity to inhibit VEGF stimulated HUVEC in proliferation
assays to 50% whereas DCIO1 affects growth to a far lesser
extent.
Example V-11
Immunoprecipitation of KDR with Mab25 and Mab73
[0138] KDR represents one of the phosphoproteins immunoprecipitated
by the Mab25 and Mab 73 from activated HuvEC. KDR was detected in
western blot and immunoprecipitation analyses using an
anti-FLK-1/KDR polyclonal antibody (IM142) from VEGF-stimulated
early passage HUVEC. Conversely, bands immunoprecipitated by these
antibodies from VEGF-stimulated HUVEC are cross reactive with IM142
but not an anti-FLT-1 polyclonal antibody. These findings infer
that the Mabs may affect the activity of KDR in KUVEC based on
experimental evidence implicating KDR as the VEGF receptor
responsible for the proliferative response in activated endothelial
cells. (See example IV-3 above for protocol.)
Example VI
Presence of VEGF Receptor Forms on Non-Endothelial (Tumor)
Cells
[0139] Several tumor lines were screened for protein reactivity
with DC101 by immunoprecipitation and detection with
antiphosphotyrosine. Immunoblots from the cell lines 8161
(melanoma) and A431 (epidermoid carcinoma) yielded phosphorylated
bands with molecular weights of approximately 170 and 120 kD. These
results indicate that a human VEGF receptor form is expressed in
non-endothelial cells, such as tumor cells.
[0140] Similar experiments have shown that a KDR like receptor is
expressed in an ovarian carcinoma cell line, OVCAR-3. These cells
also appear to secrete VEGF. Phosphorylated bands are
immunoprecipitated by an anti-KDR polyclonal antibody from
VEGF-stimulated OVCAR-3 cells that are reactive with anti-FLK-1
Mabs by western blotting. Also, bands immunoprecipitated by the
murine Mabs from these cells show cross reactivity with the same
polyclonal antibody. Furthermore certain murine anti-FLK-1 Mabs
elicit an inhibitory effect on these cells in proliferation assays.
These results demonstrate nonendothelial expression (i.e. on tumor
cells) of human VEGF receptor forms. The data from the
phosphorylation and proliferation assays also suggest that VEGF can
modulate receptor activity in an autocrine and paracrine manner
during tumorigenesis. (See Example IV-3 above for protocol.)
Example VII
IN VIVO STUDIES USING DC101
Example VII-1
Inhibition in vivo of Angiogenesis by dc101
[0141] In vivo studies were designed to determine if an anti-FLK1
monoclonal antibody would block the growth of VEGF-expressing tumor
cells. In these experiments, a human glioblastoma multiform cell
line was used that has high levels of VEGF message and secretes
about 5 ng/ml of VEGF growth factor after a 24 hour conditioning in
serum free medium (FIG. 5).
[0142] On day zero, athymic nude mice (nu/nu; Charles River Labs)
were injected in the flank with 1-2 million glioblastoma cells.
Beginning on the same day, animals received intraperitoneal
injections of either DC101 and control antibodies (100 ug/animal).
The mice received subsequent antibody treatments on days 3, 5, 7,
10, 12, 14, 17, 19, and 21. Animals received injections of 100 ug
of either DC101 or a control rat antibody to the murine FLK2 (2A13)
receptor on days 0, 3, 5, 7, 10, 12, 14, 17, 19, and 21 for a total
inoculation of 1 mg/animal. Tumors began to appear by day 5 and
followed for 50 days. Tumor size was measured daily with a caliper
and tumor volume calculated by the following formula:
p/6.times.larger diameter.times.(smaller diameter).sup.2 (Baselga
J. Natl. Cancer Inst. 85: 1327-1333). Measurements were taken at
least three times per week and tumor volume calculated as described
above. One tumor bearing animal in the DC101 group died early in
the study and was not used to determine statistical significance
between the groups.
[0143] FIGS. 14A and 14B show a comparison between the DC101 and
the control 2A13 group of reduction in tumor growth over 38 days in
individual animals. Although all animals developed tumors of
varying sizes and number during the course of the study, DC101
-treated mice showed an overall delay in tumor progression. One
mouse in the DC101 group remained tumor free until day 49 when a
small growth was observed.
[0144] Even then, tumor growth was markedly suppressed. Statistical
analysis of the data was done to assess differences in tumor size
between the two groups. Data was subjected to a standard analysis
of covariance where tumor size was regressed on time with treatment
as a covariate. The results showed that reduction in tumor size
over time for the DC101 group was significantly different
(p<0.0001) from that seen for 2A13 injected mice.
[0145] FIG. 15 shows the therapeutic efficacy of DC101 in athymic
nude mice transplanted with the human glioblastoma tumor cell line
GBM-18, which secretes VEGF. Nude mice were injected subcutaneously
with GBM-18 cells and divided into three groups of treatment: a PBS
control, an irrelevant rat IgG1 control, and DC101. Treatments were
administered simultaneously with tumor xenografts and continued for
four weeks. The results showed that GBM-18 tumor growth in DC101
treated nude mice was significantly reduced relative to controls.
This experiment indicates that DC101 suppresses tumor growth by
blocking VEGF activation of FLK-1 on tumor associated vascular
endothelial cells, and that DC101 has therapeutic value as an
anti-angiogenic reagent against vascularized tumors secreting
VEGF.
[0146] Monoclonal antibodies to FLK-1 receptor tyrosine kinase
inhibit tumor invasion by abrogating angiogenesis. Invasive growth
and angiogenesis are essential characteristics of malignant tumors.
Both phenomena proved to be suitable to discriminate benign from
malignant keratinocytes in a surface transplantation assay. After
transplantation of a cell monolayer attached to a collagen gel onto
the back muscle of nude mice, all tumor cells initially formed
organized squamous epithelia, but only malignant keratinocytes grew
invasively within 2-3 weeks. Both benign and malignant cells
induced angiogenesis. Angiogenic response to malignant cells,
however, occurred earlier, is much stronger, and capillary growth
directed toward malignant epithelia. Moreover, in transplants of
benign tumor cells, capillaries regressed after 2-3 weeks, whereas
malignant keratinocytes maintain the level of ongoing angiogenesis.
The vascular endothelial growth factor (VEGF) and its cognate
receptor play a pivotal role in tumor angiogenesis. The
administration of DC101 disrupted ongoing angiogenesis leading to
inhibition of tumor invasion. The antibody prevented maturation and
further expansion of newly formed vascular network, but did not
significantly interfere with initial angiogenesis induction. These
results provide evidence that tumor invasion requires precedent
angiogenesis, and that the VEGF receptors are crucial in
maintaining angiogenesis in this model system.
Example VII-2
Effect of Different Concentrations of DC101 on Established
Glioblastoma (gbm-18) Tumors
[0147] Athymic mice (nu/nu) were inoculated subcutaneously with
GBM-18 (human glioblastoma multiformae). Antibody therapy was
initiated when the tumors reached an average volume of 100-200
mm.sup.3. Treatment consisted of six injections (twice weekly for 3
weeks) of the following: (i) DC-101 at 200, 400 or 800
ug/injection; (ii) an irrelevant isotype matched rat IgG (400
ug/injection); or, (iii) PBS. Tumor volumes were measured with a
caliper. Tumor inhibition in the DC-101 groups was found to be
significant (*) vs. the PBS and irrelevant monoclonal antibody
groups.
[0148] Another experiment demonstrates the effects of the rat
anti-FLK-1 monoclonal antibody DC101 on the growth of GBM-18 tumors
in nude mice. Animals (nu/nu; Charles River Labs; ten animals per
group) were injected subcutaneously with GBM-18 cells (human
glioblastoma [100]; 1 million per animal) on day 0. Treatments with
PBS or DC101 (200 .mu.g per injection) were begun on day 7 and
continued twice weekly for 3 weeks (6.times.). Graphs show a plot
of the mean tumor volumes and regressed data for each group over
time with their respective tumor growth rates (slopes given as
.gamma.; solid lines) and 99% confidence limits (dotted lines). The
slope of the line for animals treated with DC101 was significantly
different from that of PBS (p.ltoreq.0.01). It is important to note
that an irrelevant rat IgG1 monoclonal antibody (anti-mouse IgA;
Pharmigen) had no effect on the growth of GBM-18 xenografts and
gave results similar to that observed with PBS (data not
shown).
[0149] SUPPLEMENTAL ENABLEMENT
[0150] The invention as claimed is enabled in accordance with the
above specification and readily available references and starting
materials. Nevertheless, Applicants have deposited with the
American Type Culture Collection, 12301 Parklawn Drive, Rockville,
Md., 20852 USA (ATCC) the hybridoma cell lines that produce the
monoclonal antibodies listed below:
[0151] Hybridoma cell line DC101 producing rat anti-mouse FLK-1
monoclonal antibody deposited on Jan. 26, 1994 (ATCC Accession
Number BB 11534).
[0152] Hybridoma cell line M25.18A1 producing mouse anti-mouse
FLK-1 monoclonal antibody Mab 25 deposited on Jul. 19, 1996 (ATCC
Accession Number HB 12152).
[0153] Hybridoma cell line M73.24 producing mouse anti-mouse FLK-1
monoclonal antibody Mab 73 deposited on Jul. 19, 1996 (ATCC
Accession Number HB 12153).
[0154] These deposits were made under the provisions of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure and the
regulations thereunder (Budapest Treaty). This assures maintenance
of a viable culture for 30 years from date of deposit. The organism
will be made available by ATCC under the terms of the Budapest
Treaty, and subject to an agreement between Applicants and ATCC
which assures unrestricted availability upon issuance of the
pertinent U.S. patent. Availability of the deposited strains is not
to be construed as a license to practice the invention in
contravention of the rights granted under the authority of any
government in accordance with its patent laws.
* * * * *