HK1167325B - Antibodies against vascular endothelial growth factor receptor-1 - Google Patents

Description

anti-VEGF receptor-1 antibodies

The application is a divisional application of an invention patent application with the application date of 2005, 11/18 and the application number of 200580046659.X, and the same invention name and invention.

Technical Field

The present invention relates to antibodies specific for vascular endothelial growth factor receptor-1 (VEGFR-1) and methods of treating angiogenesis-related diseases and tumors with anti-VEGFR-1 antibodies.

Background

Angiogenesis, which refers to the formation of capillaries from existing blood vessels of embryonic and adult life, is considered a key factor in tumor growth, survival and metastasis. Growth factors and their receptors, including Epidermal Growth Factor (EGF), transforming growth factor-alpha (TGF-alpha), transforming growth factor-delta (TGF-delta), acidic and basic fibroblast growth factors (aFGF and bFGF), Platelet Derived Growth Factor (PDGF), and Vascular Endothelial Growth Factor (VEGF), are generally thought to play important roles in tumor angiogenesis. See klags brun and D' Amore, Annual rev. physiol., 53: 217-239(1991). Binding of these growth factors to their cell surface receptors induces receptor activation, which initiates and alters signaling pathways leading to cell proliferation and differentiation. VEGF, an endothelial cell-specific mitogen, is particularly prominent among these factors because it acts as an angiogenesis inducer by specifically promoting endothelial cell proliferation.

The biological response of VEGF is mediated through its high affinity receptor, which is selectively expressed on endothelial cells in the following processes: embryogenesis (Millauer, Cell, 72: 835-846(1993)) and tumor formation. VEGF receptors (VEGFRs) are generally class III receptor type tyrosine kinases characterized by a number, usually 5 or 7, immunoglobulin-like loops in their amino-terminal extracellular receptor ligand binding domain (Kaipain et al, J.Exp.Med., 178: 2077-2088 (1993)). The other two regions include a transmembrane region and a carboxy-terminal intracellular catalytic domain separated by a hydrophilic inteldhase sequence (also known as a kinase insert region) of variable insert length (Terman et al, Oncogene, 6: 1677-. VEGFRs include the > z, s-like tyrosine kinase receptor (flt-1) or VEGFR-1 (see sequence Shibuya et al, Oncogene, 5: 519-. The other tyrosine kinase receptor, VEGFR-3(flt-4), binds to VEGF homologues VEGF-C and VEGF-D, and is more important in lymphatic vessel development.

The importance of VEGFR-1 in regulating pathological angiogenesis has been demonstrated in vivo experimental models. Defects in the VEGFR-1 tyrosine kinase domain lead to a reduction in tumor vascularization, suggesting an important role for VEGFR-1 tyrosine kinase in pathological angiogenesis (Hiratsuka et al, cancer research, 61: 1207-1213 (2001)). The VEGFR-1 tyrosine kinase domain is also required to promote tumor pathogenesis and metastasis by inducing matrix metalloproteinase-9 (MMP-9) in endothelial cells and macrophages (Hiratsuka et al, Cancer Cell, 2: 289-300 (2002)). In addition, VEGFR-1 was shown to mediate the transfer and differentiation of BM derived progenitor cells in response to PlGF (Hattori et al, Nature Medicine, 8: 841-849 (2002)). VEGFR-1 inhibition with anti-VEGFR-1 antibodies results in a reduction in tumor angiogenesis by preventing recruitment of bone marrow derived endothelial cells and monocyte progenitor cells (myelomonocytogenic cells) from tumor angiogenesis (Lyden et al, Nature Medicine, 7: 1194-K1201 (2001)). Treatment with anti-VEGFR-1 antibodies is also effective in inhibiting pathological angiogenesis in tumors and retinal ischemia in animal models (Lutten et al, Nature Medicine, 8: 831-840 (2002)).

In addition to the role of VEGFR-1 in angiogenesis, co-expression of VEGF and its receptors is also common in hematological malignancies and in certain solid tumor cells (Bellamy, cancer research, 59: 728-. VEGF has been shown to directly induce proliferation, survival and invasion of VEGF receptor expressing leukemic cells via activation of downstream intracellular signaling pathways by ligand-stimulated autocrine loops (Dias et al, Proc. Natl. Acad. Sci. USA, 98: 10857-10862 (2001); Gerber et al, J.mol. Med., 81: 20-31 (2003)). VEGF stimulation also causes enhanced invasion of VEGFR-1 expressing breast cancer cells by inducing activation of the ERK1/2 and PI 3/Akt-kinase signaling pathways (Price et al, CellGrowth Differ., 12: 129-135 (2001)).

VEGFR-1 and its ligands have also been shown to play an important role in inflammatory diseases. VEGF-B deficiency results in a decrease in inflammation-associated vascular density and a reduction in synovial inflammation in Arthritis models (Mould et al, Arthritis Rheum., 48: 2660-2669 (2003)). PlGF also plays a crucial role in controlling skin inflammation by mediating vasodilation, inflammatory cells and monocytes/macrophages, and has also been shown to contribute to the opsonization of atherosclerosis and rheumatoid arthritis in animal models (Luttun et al, Nature Medicine, 8: 831-. Treatment with neutralizing anti-VEGFR-1 antibodies inhibits inflammatory joint damage in arthritis and reduces the growth and vulnerability of atherosclerotic plaques. The anti-inflammatory effects of anti-VEGFR-1 antibodies may be attributed to a reduction in the migration of bone marrow-derived bone marrow progenitor cells into peripheral blood in inflammatory tissues, defective activation of bone marrow cells, and impaired differentiation and infiltration of VEGFR-1 expressing leukocytes. VEGFR-1 may therefore also be a therapeutic target for the treatment of inflammation-related disorders.

There remains a need for agents that inhibit VEGF receptor activity, such as fully human monoclonal antibodies (mabs) specific for VEGFR-1. anti-VEGFR-1 antibodies may be useful as novel therapeutic antagonists for the treatment of angiogenesis-related diseases and cancers.

Brief description of the invention

In one embodiment, the present invention provides a monoclonal antibody or fragment thereof that specifically binds to VEGFR-1, the antibody or fragment thereof comprising the amino acid sequence of SEQ ID NO: 2(CDR2) and SEQ ID NO:3 light chain complementary region-3 (CDR 3).

In another embodiment, the present invention provides a monoclonal antibody or fragment thereof that specifically binds to VEGFR-1 that binds to a polypeptide comprising SEQ ID NO: 2(CDR2) and SEQ ID NO: 3(CDR3) or a fragment thereof, and has at least 70% homology to the amino acid sequence of the antibody.

In another embodiment, the present invention provides an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26 and SEQ ID NO: 27. the nucleotide sequence encodes an antibody or fragment thereof that specifically binds to VEGFR-1.

In another embodiment, the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding an antibody or fragment thereof that specifically binds to VEGFR-1, the nucleotide sequence having at least 70% homology to a nucleotide sequence selected from the group consisting of SEQ ID NO: SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26 and SEQ ID NO: 27.

in another embodiment, the invention provides a method of inhibiting angiogenesis or reducing tumor growth by administering a therapeutically effective amount of an antibody or fragment thereof that specifically binds to VEGFR-1 and comprises the amino acid sequence of SEQ ID NO: 2(CDR2) and SEQ ID NO:3 light chain complementary region-3 (CDR 3).

Brief Description of Drawings

FIG. 1 is an amino acid sequence of a light chain variable region and a heavy chain variable region of an anti-VEGFR-1 antibody according to an embodiment of the present invention.

FIG. 2 is a nucleotide sequence of a light chain variable region and a heavy chain variable region of an anti-VEGFR-1 antibody according to an embodiment of the present invention.

FIG. 3 is a graph showing the results of determining the in vitro binding activity of an anti-VEGFR-1 antibody according to an embodiment of the present invention to VEGFR-1 based on an ELISA binding assay.

FIG. 4 is a graph showing the results of an ELISA-based blocking assay for determining in vitro competitive binding of PlGF to VEGFR-1 by anti-VEGFR-1 antibodies according to embodiments of the present invention.

FIG. 5 is a graph showing the results of an ELISA-based blocking assay for determining in vitro competitive binding of an anti-VEGFR-1 antibody of an embodiment of the present invention to VEGF and VEGFR-1.

FIGS. 6A-D are graphs showing the results of specific binding of the anti-VEGFR-1 antibody 18F1 of the present invention to human VEGFR-1 (FIG. 6A), non-mouse VEGFR-1 (FIG. 6B), human VEGFR-2 (FIG. 6C), or mouse VEGFR-2 (FIG. 6D).

FIGS. 7A-E are flow cytometry analyses demonstrating the binding reactivity of anti-VEGFR-1 antibodies of embodiments of the present invention to porcine aortic endothelial cells expressing VEGFR-1.

FIGS. 8A-B are flow cytometry analyses demonstrating the binding reactivity of the anti-VEGFR-1 antibody 18F1 of the present invention to porcine endothelial cells expressing VEGFR-1 (FIG. 8A) and DU4475 human breast cancer cells (FIG. 8B).

FIG. 9 is a graph showing the results of a cell-based blocking assay to determine VEGFR-1 competitive binding of the anti-VEGFR-1 antibody 18F1 of the present invention to VEGF to endothelial cells in vitro.

FIG. 10 is a Western blot analysis demonstrating that treatment with the anti-VEGFR-1 antibody 18F1 of the present invention reduced PlGF-stimulated VEGFR-1 phosphorylation in porcine aortic endothelial VEGFR-1 expressing cells.

FIG. 11 is a Western blot analysis demonstrating that treatment with the anti-VEGFR-1 antibody 18F1 of the present invention inhibits PlGF or VEGF-stimulated VEGFR-1 phosphorylation in BT474 breast cancer cells.

FIG. 12 is a Western blot analysis demonstrating inhibition of PlGF-induced activation of ERK1/2 downstream signaling by anti-VEGFR-1 antibodies of the present invention in porcine aortic endothelial VEGFR-1 expressing cells.

FIG. 13 is a Western blot analysis demonstrating that VEGF-induced activation of ERK1/2 downstream signaling is inhibited by anti-VEGFR-1 antibodies of embodiments of the present invention in porcine aortic endothelial VEGFR-1 expressing cells.

FIGS. 14A-B are Western blot analyses demonstrating that anti-VEGFR-1 antibody 18F1 of the present invention inhibits PIGF (FIG. 14A) or VEGF (FIG. 14B) induced activation of ERK1/2 downstream signaling in porcine aortic endothelial cells expressing VEGFR-1.

FIG. 15 is a Western blot analysis demonstrating that anti-VEGFR-1 antibody 18F1 of the present invention blocks PlGF or VEGF stimulated phosphorylation of Akt in BT474 breast cancer cells.

FIG. 16 is a dose response curve showing the inhibition of VEGF-stimulated cell proliferation in a dose response manner in DU4475 breast cancer cells treated with an anti-VEGFR-1 antibody according to an embodiment of the present invention.

FIG. 17 is a dose response curve showing inhibition of PlGF-stimulated cell proliferation in a dose response manner in DU4475 breast cancer cells treated with anti-VEGFR-1 antibodies according to embodiments of the present invention.

FIGS. 18A-B are dose response curves showing inhibition of PIGF (FIG. 18A) or VEGF (FIG. 18B) stimulated cell proliferation in a dose response manner in DU4475 breast cancer cells treated with the anti-VEGFR-1 antibody 18F1 of the present invention.

FIGS. 19A and 19B are graphs plotting tumor growth of DU4475 breast tumors versus days after treatment with anti-VEGFR-1 antibodies according to embodiments of the present invention.

FIGS. 20A-C are graphs plotting tumor growth versus days for DU4475 (FIG. 20A), MDA-MB-231 (FIG. 20B), and MDA-MB-435 (FIG. 20C) breast tumors after treatment with anti-VEGFR-1 antibody 18F1 of the present invention.

FIGS. 21A-B are graphs plotting tumor growth versus days for DU4475 (FIG. 21A) and MDA-MB-231 (FIG. 21B) breast tumors after treatment with anti-human VEGFR-1 antibody 18F1 and anti-mouse VEGFR-1 antibody MF1 of the present invention.

FIG. 22 is a bar graph of colon cancer cell colony numbers after treatment with anti-human VEGFR-1 antibody 18F1 in the presence of VEGF-A and VEGF-B.

FIG. 23A is a bar graph of the number of migrating tumor cells after treatment with anti-human VEGFR-1 antibody 18F1 in the presence of VEGF-A and VEGF-B.

FIG. 23B is a micrograph of migrating cells stained after treatment with anti-human VEGFR-1 antibody 18F1 in the presence of VEGF-A and VEGF-B.

FIG. 24A is a bar graph of the number of tumor cells that migrated through MATRIGELTM layer after treatment with anti-human VEGFR-1 antibody 18F1 in the presence of VEGF-A or VEGF-B.

FIG. 24B is a micrograph of migrating cells stained after treatment with anti-human VEGFR-1 antibody 18F1 in the presence of VEGF-A and VEGF-B.

FIG. 25 is a graph plotting tumor growth versus days for DU4475 (FIG. 25A) and MDA-MB-435 (FIG. 25B) breast tumors after treatment with anti-VEGFR-1 antibodies 18F1, 6F9, and 15F 11.

FIG. 26 is a graph plotting growth of HT-29 (FIG. 26A), DLD-1 (FIG. 26B) and GEO (FIG. 26C) colon cancer cells versus days after treatment with the prescribed dose of anti-human VEGFR-1 antibody 18F 1.

FIG. 27 is a photomicrograph of MDS-MB-231 xenograft tumors after treatment with anti-human VEGFR-1 antibody 18F 1.

FIG. 28 is a graph plotting tumor growth versus days after treatment with the prescribed dose of anti-human anti-VEGFR-1 antibody 18F1, anti-mouse anti-VEGFR-1 antibody MF1, or both, in MDA-MB-231 (FIG. 28A) and DU4475 (FIG. 28B) xenografts.

FIG. 29 is a graph plotting tumor growth versus days after treatment with anti-human anti-VEGFR-1 antibody 18F1 and anti-mouse anti-VEGFR-1 antibody MF1 in MDS-MB-231 xenografts in combination with cyclophosphamide.

FIGS. 30A and 30B are graphs plotting tumor growth versus days after treatment with anti-human anti-VEGFR-1 antibody 18F1 and anti-mouse anti-VEGFR-1 antibody MF1 in combination with 5-FU/LV or doxorubicin in MDA-MB-231 xenografts.

FIG. 31 is a bar graph plotting total tumor cell number versus concentration of varying amounts of 18F1 antibody after treatment with desferrioxamine in the presence of VEGF-A (FIG. 31A) or PlGF (FIG. 31B).

FIGS. 32A, 32B and 32C are graphs showing the specificity of anti-human anti-VEGFR-1 antibody 18F1 and anti-mouse anti-VEGFR-1 antibody MF 1.

Detailed Description

In one embodiment, the present invention provides monoclonal antibodies and fragments thereof that specifically bind to VEGFR-1 (the antibodies and fragments thereof are also referred to herein as "anti-VEGFR-1 antibodies," unless otherwise specified). The anti-VEGFR-1 antibodies of the present invention comprise SEQ ID NO: 2(CDR2) and SEQ ID NO:3 light chain complementary region-3 (CDR 3). Alternatively, it is preferred that the anti-VEGFR-1 antibody of the present invention comprises light chain complementary region-1 (CDR1) having the sequence: RASQSX₁SSSYLA in which X₁Is V or G (SEQ ID NO:1 or 4). Alternatively, it is preferred that the anti-VEGFR-1 antibody of the present invention comprises a peptide having the sequenceHeavy chain CDR1 of column: GFX₂FSSYGMH where X₂Is T or A (SEQ ID NO: 5 or 11). Alternatively, it is preferred that the anti-VEGFR-1 antibodies of the present invention comprise a heavy chain CDR2 having the sequence: VIWX₃DGSNKYYADSVX₄G, wherein X₃Is Y or F, X₄Is K or R (SEQ ID NO: 6, 9 or 12). Alternatively, it is also preferred that the anti-VEGFR-1 antibodies of the present invention comprise a heavy chain CDR3 having the sequence: DHX₅GSGX₆HX₇YX₈YYGX₉DV, wherein X₅Is F or Y; x₆Is A or V; x₇Y, S or H; x₈Is Y or F; x₉M or L (SEQ ID NO: 7, 8, 10, 13). The CDR amino acid sequences of preferred anti-VEGFR-1 antibodies (also known as clones "6F 9", "13G 12", "15F 11" and "18F 1" (or "MC-18F 1")) are shown in Table 1 below.

In another embodiment, an anti-VEGFR-1 antibody of the present invention has the amino acid sequence of SEQ ID NO: 14. 15 or 16 light chain variable region (V)_L) And/or SEQ ID NO: 17. 18, 19 or 20 (V)_H). The amino acid sequences of the light and heavy chain variable regions of the preferred anti-VEGFR-1 antibodies of the present invention are shown in Table 2 below.

In a preferred embodiment, the anti-VEGFR-1 antibodies of the present invention are human antibodies.

The anti-VEGFR-1 antibodies of the present invention include whole antibodies and antibody fragments that specifically bind to VEGFR-1. Non-limiting examples of types of antibodies of the invention include naturally occurring antibodies; a single chain antibody; multivalent single chain antibodies such as diabodies (diabodies) and triabodies (tribodies); monovalent fragments such as Fab (antigen binding fragment), bivalent fragmentsSegment for example (FaV)₂(ii) a Fv (variable) fragments or derivatives thereof such as single chain Fv (scFv) fragments and single domain antibodies (single domain antibodies) that specifically bind to VEGFR-1.

Naturally occurring antibodies typically have two identical heavy chains and two identical light chains, each light chain being covalently linked to a heavy chain by an interchain disulfide bond, with multiple disulfide bonds further linking the two heavy chains to each other. Each chain can be folded into domains of similar size (110-125 amino acids) and structure, but with different functions. The light chain may comprise one V_LZone and a constant region (C)_L). The heavy chain may also comprise a V_HThe domains and/or the constant domains (C) comprising 3 or 4 according to the antibody class or isotype_H1、C_H2、C_H3 and C_H4). The human isotypes are IgA, IgD, IgE, IgG and IgM, and the subclasses or subtypes are subdivided for IgA and IgG (IgA and IgG)_1-2And IgG_1-4)。

Single chain antibodies lack some or all of the constant regions of the intact antibody from which they are derived. The peptide linker used to generate the single chain antibody may be a flexible peptide of choice to ensure formation of the appropriate three-dimensional folded V_LRegion and V_HAnd (4) a zone. In general, V_LOr V_HThe carboxy terminus of the sequence may be joined to the complementary V through such a peptide linker_HOr V_LThe amino terminus of the sequences are covalently linked. The linker is typically 10-50 amino acid residues, preferably 10-30 amino acid residues, more preferably 12-30 amino acid residues, most preferably 15-25 amino acid residues. Examples of such a linker peptide include (Gly-Gly-Gly-Gly-Ser)₃(SEQ ID NO：28)。

A plurality of single chain antibodies, each single chain having a V covalently linked by a first peptide linker_HA region and a V_LThe region, which may be covalently linked to at least one or more peptide linkers to form a multivalent single chain antibody, may be monospecific or multispecific. Each chain of a multivalent single chain antibody comprises a variable light chain fragment and a variable heavy chain fragment, and is linked to at least one other chain by a peptide linker.

Two single chain antibodies can combine to form a diabody, also known as a trivalent dimer. Diabodies have two chains and two binding sites and can be monospecific or bispecific. Each chain of a diabody comprises V_HZone and V connected thereto_LAnd (4) a zone. The domains are joined with linkers short enough to prevent pairing between the domains of the same strand, thereby facilitating pairing of complementary domains on different strands to recreate the two antigen binding sites.

The three single-chain antibodies can be combined to form a three-chain antibody, also known as a trivalent trimer. Will V_LRegion or V_HAmino terminus of domain and V_LRegion or V_HThe carboxy-terminal end of the region is fused directly, that is, without any linking sequences, to construct a three-chain antibody. Tri-chain antibodies have three Fv heads, the polypeptides of which are arranged in a circular, head-to-tail fashion. One possible three-chain antibody construct is planar, with the three binding sites lying at 120 ° angles to each other. The three-chain antibody may be monospecific, bispecific or trispecific.

Fab fragment means the fragment consisting of V_LC_LVHCH₁A region comprising an antibody fragment. The fragment produced by papain digestion is called "Fab" and does not retain the heavy chain hinge region. The fragment produced by digestion with pepsin is known as "(Fab')₂", in which case the interchain disulfide bond is intact, also known as Fab', in which case the disulfide bond is not retained. Bivalent (Fab)₂The affinity of the fragment for the antigen is higher than that of a monovalent Fab fragment.

The Fv fragment consists of_LRegion and V_HThe antibody portion of the domain constitutes the antigen binding site. scFv comprising V on one polypeptide chain_LRegion and V_HAntibody fragments of regions in which the N-terminus of one region and the C-terminus of the other region are joined together by a flexible linker such that the two fragments associate to form a functional antigen binding site (see, e.g., U.S. Pat. No. 4,946,778 (Ladner et al), WO 88/09344(Huston et al), the contents of both of which are incorporated herein by reference). By leadingWith WO 92/01047(McCafferty et al) incorporated herein, the display of scFv fragments on the surface of a soluble recombinant genetic display package (e.g.phage) is described.

Single domain antibodies have a single variable region (single variable domain) that is capable of efficiently binding to an antigen. Examples of antibodies in which binding affinity and specificity are primarily due to an overall variable region are known in the art, see, e.g., Jeffrey, p.d., et al, proc.natl.acad.sci.usa 90: 10310-4(1993), the contents of which are incorporated herein by reference, and discloses anti-digoxin antibodies that bind to digoxin primarily through the heavy chain of the antibody. Thus, it was determined that the single antibody domain binds well to the VEGF receptor. It should be understood that in order to contain V_HRegion and V_LAntibodies to regions single domain antibodies are prepared, and certain amino acid substitutions outside the CDR regions may be required to enhance binding, expression, or solubility. For example, it may be desirable to modify amino acid residues that would otherwise be buried in V_H-V_LAnd (6) an interface.

The individual domains of the anti-VEGFR-1 antibodies of the present invention may be the entire antibody heavy or light chain variable region, or it may be a functional equivalent or mutant or derivative of a naturally occurring domain, or a synthetic domain, for example constructed in vitro using techniques such as those described in WO 93/11236(Griffiths et al). For example, it is possible to associate a domain with a corresponding antibody variable region lacking at least one amino acid. An important characteristic feature is the ability of each domain to associate with a complementary domain to form an antigen binding site. Thus, the term "variable heavy/light chain fragment" should not be construed as excluding variant fragments that do not materially contribute to VEGFR-1 binding specificity.

As used herein, an "anti-VEGFR-1 antibody" includes various modifications of the anti-VEGFR-1 antibodies of the present invention that retain the VEGFR-1 specificity. These modifications include, but are not limited to, conjugation to effector molecules such as chemotherapeutic drugs (e.g., cisplatin, taxol, doxorubicin) or cytotoxins (e.g., proteinaceous or non-proteinaceous organic chemotherapeutic drugs). Modifications also include, but are not limited to, conjugation to a detectable reporter moiety. Modifications that extend the half-life of the antibody (e.g., pegylation) are also included.

Protein and non-protein drugs can be conjugated to antibodies by methods known in the art. Conjugation methods include direct bonding, linker bonding through covalent linkage, and specific binding of partner components (e.g., avidin-biotin). For example, these methods include the method described in Greenfield et al, Cancer Research 50, 6600-.

The anti-VEGFR-1 antibodies of the present invention also include antibodies having improved binding characteristics by direct mutation, affinity maturation, phage display, or chain shuffling. Antigen binding sites with desired properties can be screened, modified and improved affinity and specificity can be obtained by mutating any CDR of an antibody of the invention (see, e.g., Yang et al, J.mol.biol., 254: 392-403(1995), the contents of which are incorporated herein by reference). The CDRs can be mutated in various ways known to those skilled in the art. For example, one approach is to arbitrarily arrange individual residues or combinations of residues such that all 20 amino acids are at a particular position on a set of non-identical antigen binding sites. Or by error-prone PCR, mutations are induced over the CDR residues (see, e.g., Hawkins et al, J.mol.biol., 226: 889-896(1992), the contents of which are incorporated herein by reference). For example, phage display vectors containing heavy and light chain variable region genes can be propagated in E.coli (E.coli) mutants (see, e.g., Low et al, J.mol.biol., 250: 359-368(1996), the contents of which are incorporated herein by reference).

anti-VEGFR-1 antibodies also include functional equivalents comprising polypeptides having an amino acid sequence substantially identical to the amino acid sequence of the variable or hypervariable region of an antibody of the invention. "substantially identical" amino acid sequences includes amino acid sequences that are at least 70%, preferably at least 80%, and more preferably at least 90% identical to one another when the amino acids of the two sequences are optimally aligned and compared to determine the exact alignment of amino acids between the two sequences. "substantially identical" amino acid sequences also include amino acid sequences that are at least 70%, preferably at least 80%, more preferably at least 90% homologous to one another as determined by the FASTA search method according to Pearson and Lipman, Proc.Natl.Acad.Sci.USA 85, 2444-8 (1988).

As previously described, the anti-VEGFR-1 antibodies of the present invention specifically bind to VEGFR-1. These antibodies may be monospecific or bispecific, so long as the antigen binding site is specific for VEGFR-1. Antibody specificity, which refers to the selective recognition of a particular epitope by an antibody, the antibody specificity of an antibody for VEGFR-1 can be determined by affinity and/or avidity. Affinity, using the dissociation equilibrium constant (K) of antigen and antibody_d) This is a measure of the strength of binding between an antigenic determinant (epitope) and the binding site of an antibody. Avidity is a measure of the strength of binding between an antibody and its antigen. General K of antibody_dIs 10^-5-10^-11Liter/mole. It is generally considered that any less than 10^-4Liter/mole of K_dBoth indicate non-specific binding. K_dThe smaller the value, the stronger the binding strength between the antigenic determinant and the antibody binding site.

The anti-VEGFR-1 antibodies of the present invention specifically bind to the extracellular domain of VEGFR-1, preferably by preventing binding of the ligand of VEGFR-1 to the receptor, thereby neutralizing VEGFR-1 activation. In these preferred embodiments, the antibodies bind VEGFR-1 at least as strongly as the native ligands of VEGFR-1 (including VEGF-A, VEGF-B and PlGF).

Neutralizing activation of VEGFR-1 includes reducing, inhibiting, inactivating, and/or blocking one or more activities associated with signal transduction. These activities include receptor dimerization, VEGFR-1 autophosphorylation, activation of the internal cytoplasmic tyrosine kinase domain of VEGFR-1, and initiation of multiple signal transduction and transactivation pathways involved in regulating DNA synthesis (gene activation) and cell cycle progression or division. One approach to VEGFR-1 neutralization is to inhibit VEGFR-1 tyrosine kinase activity. Inhibition of tyrosine kinases can be measured by well known methods, such as phosphorylation assays that measure the level of autophosphorylation of recombinant kinase receptors and/or phosphorylation of natural or synthetic substrates. Phosphorylation can be detected in an ELISA assay or Western blot using, for example, an antibody specific for phosphotyrosine. Some assays for tyrosine kinase activity are described in Panek et al, j. 1433-44(1997) and Batley et al, Life Sci, 62: 143-50(1998), the contents of both of which are incorporated by reference.

Alternatively, assays for protein expression may be used to determine whether the antibody neutralizes VEGFR-1 activation, wherein the protein to be detected is modulated by VEGFR-1 tyrosine kinase activity. These methods include Immunohistochemistry (IHC) to detect protein expression, Fluorescence In Situ Hybridization (FISH) to detect gene amplification, competitive radioligand binding assays, solid matrix blotting techniques such as northern blotting and southern blotting, reverse transcriptase-polymerase chain reaction (RT-PCR), and ELISA. See, e.g., Grandis et al, Cancer, 78: 1284-92 (1996); shimizu et al, Japan j. cancer res, 85: 567-71 (1994); sauter et al, am.j.path, 148: 1047-53 (1996); collins, Glia, 15: 289-96 (1995); radinsky et al, clin. cancer res, 1: 19-31 (1995); petrides et al, Cancer res, 50: 3934-39 (1990); hoffmann et al, Anticancer Res, 17: 4419-26 (1997); wikstrand et al, Cancer res, 55: 3140-48(1995), the contents of all of which are incorporated by reference.

VEGFR-1 neutralization can be detected using in vivo assays. Cell lines stimulated with receptor ligands can observe inhibition of receptor tyrosine kinases, for example, by mitogenic assays, in the presence or absence of inhibitors. For example, HUVEC cells (ATCC) stimulated with VEGF-A or VEGF-B can be used to analyze VEGFR-1 inhibition. Another method involves testing for inhibition of growth of VEGF-expressing tumor cells by injection into mice using, for example, human tumor cells. See, for example, U.S. patent No. 6,365,157 (Rockwell et al), the contents of which are incorporated herein by reference.

Of course, the present invention is not limited by any particular mechanism for VEGFR-1 neutralization. For example, an anti-VEGFR-1 antibody of the present invention may bind externally to VEGFR-1, block ligand binding to VEGFR-1, and block subsequent signal transduction mediated through receptor-associated tyrosine kinases, preventing phosphorylation of VEGFR-1 and other downstream proteins in the signal transduction cascade. Receptor-antibody complexes can also be internalized and degraded, resulting in receptor cell surface down-regulation. Matrix metalloproteinases, whose function is tumor cell invasion and metastasis, can also be down-regulated by the anti-VEGFR-1 antibodies of the present invention.

The human anti-VEGFR-1 antibody may be derived from a naturally occurring antibody, or from a Fab or scFv phage display library constructed from, for example, human heavy and light chain variable region genes, into which the CDR sequences of the anti-VEGFR-1 antibody of the present invention may be inserted.

Human anti-VEGFR-1 antibodies can be produced by methods well known to those skilled in the art. These methods include methods of using transgenic mouse hybridomas as well as methods of using recombinant DNA. Methods for hybridomas are described in Kohler and Milstein, Nature, 256: 495-497(1975) and Campbell, Monoclonal Antibody Technology, The production and Characterization of Rodent and Human Hybridomas (Monoclonal Antibody Technology: production and Characterization of Rodent and Human Hybridomas); burdon et al, eds, laboratory techniques in Biochemistry and Molecular Biology, Vol.13, Elsevier Science Publishers, Amsterdam (1985), the contents of all of which are incorporated herein by reference; methods for recombinant DNA are described in Huse et al, Science, 246, 1275-1281(1989), the contents of which are incorporated herein by reference.

Antibody fragments can be prepared by cleaving an intact antibody, or by expressing DNA encoding the fragment. These can be obtained by methods described in Lamoyi et al, j.immunol.methods, 56: 235-243(1983) andparham, j.immunol.131: 2895-2902(1983), the contents of both of which are incorporated herein by reference. These fragments may contain one or two Fab fragments or F (ab')₂And (3) fragment. These fragments may also contain antibody single chain fragment variable regions, i.e., scFv, diabody or other antibody fragments. PCT application WO93/21319, European patent application No. 239,400, PCT application WO 89/09622, European patent application No. 338,745, and European patent application EP 332,424, the contents of all of which are incorporated herein by reference, disclose methods for preparing these antibodies.

In another embodiment, the present invention provides polynucleotides encoding the anti-VEGFR-1 antibodies of the present invention. These polynucleotides encode SEQ ID NO:2, light chain CDR2, SEQ id no:3 and preferably one or more other CDRs listed in table 1. Table 3 shows the nucleic acid sequences of preferred anti-VEGFR-1 antibodies.

DNA encoding human antibodies can be prepared by recombining DNAs encoding human constant regions and variable regions, and DNAs encoding CDRs derived from human (SEQ ID NOS: 1-4 light chain variable region CDRs and SEQ ID NOS: 5-13 heavy chain variable region CDRs), in addition to DNAs substantially or completely derived from the corresponding human antibody regions CDRs.

The polynucleotides encoding the anti-VEGFR-1 antibodies of the present invention include polynucleotides having a nucleic acid sequence that is substantially identical to the nucleic acid sequence of the polynucleotides of the present invention. A nucleic acid sequence that is "substantially identical" is defined herein as one that is at least 70%, preferably at least 80%, more preferably at least 90% identical to another nucleic acid sequence when the two sequences are optimally aligned (with appropriate nucleotide insertions or deletions) and compared to determine the exact alignment of the nucleotides between the two sequences.

Suitable sources of DNA encoding antibody fragments include any cell, such as hybridoma cells and spleen cells expressing full-length antibodies. The fragments themselves may be used as antibody equivalents or may be recombined into equivalents as described above. The DNA deletion and recombination described in this section can be carried out by known methods, for example, the method of the invention entitled "functional equivalents of antibodies" described in the above-listed published patent section and/or other standard recombinant DNA techniques (e.g., the methods described below). As is known in the art, another source of DNA is single chain antibodies generated from phage display libraries.

In addition, the present invention provides an expression vector comprising the polynucleotide sequence described above operably linked to an expression sequence, a promoter sequence, and an enhancer sequence. Various expression vectors have been developed for the efficient synthesis of antibody polypeptides in prokaryotic (e.g., bacterial) and eukaryotic systems, including but not limited to yeast and mammalian cell culture systems. The vectors of the invention may comprise chromosomal segments, non-chromosomal segments and synthetic DNA sequences.

Any suitable expression vector may be used. For example, prokaryotic cloning vectors include plasmids from E.coli, such as colEl, pCRl, pBR322, pMB9, pUC, pKSM and RP 4. Prokaryotic vectors also include derivatives of phage DNA, such as M13 and other filamentous single stranded DNA phages. An example of a vector for yeast is a 2 μ plasmid. Suitable vectors for expression in mammalian cells include the well-known SV-40 derivatives, adenovirus-derived DNA sequences, retrovirus-derived DNA sequences, and shuttle vectors derived from functional mammalian vectors (e.g., the vectors described above) in combination with functional plasmid and phage DNA.

Additional eukaryotic Expression vectors are known In The art (e.g., PJ. southern And P.berg, J.mol. appl. Gene., 1: 327. 341 (1982); Subramann et al, mol. cell. biol., 1: 854. 864 (1981); Kaufinann And Sharp, "Amplification And Expression Of sequences transfected with a Modular Dihydrofolate reductase complementary DNA Gene", 159: 601. cell. biol. 621 (1982); Kaufinann And Sharp, mol. cell. biol., 159: 601. alpha. 664 (1982); Scahian et al, "Expression Of expressed And modified genes Of sequences Of genes Of The sequence Of interest", Na. cell. Biol. 54. 1982.; Kaufinan And Sharp, mol. cell. biol. 159: 601. 664. (1982); And Nature DNA Of The Human immune Cells, 4254, Nature DNA, USA, 19820, USA, And Na. interferon, USA).

The expression vectors used in the present invention contain at least one expression control sequence operably linked to the DNA sequence or fragment to be expressed. To control and regulate the expression of the cloned DNA sequences, control sequences are inserted into the vector. Examples of useful expression control sequences are the lac system, trp system, tac system, trc system, the major operator and promoter regions of bacteriophage lambda, the fd coat protein control regions, yeast glycolytic promoters such as the 3-phosphoglycerate kinase promoter, yeast acid phosphatase promoters such as Pho5, the promoters of yeast alpha-mating factor, promoters obtained from polyoma, adenovirus, retrovirus and simian viruses such as the early and late promoters or SV40, and other sequences known to control the expression of prokaryotic or eukaryotic cells and their viral or viral combination genes.

The present invention also provides a recombinant host cell containing the aforementioned expression vector. The anti-VEGFR-1 antibodies of the present invention may be expressed in cell lines other than hybridomas. Nucleic acids comprising sequences encoding the polypeptides of the invention may be used to transform suitable mammalian host cells.

Particularly preferred cell lines are selected for high expression levels, constitutive expression of the protein of interest and minimal contamination of the host protein. Available mammalian cell lines for use as expression hosts are well known in the art and include a number of immortalized cell lines such as, but not limited to, Chinese Hamster Ovary (CHO) cells, Baby Hamster Kidney (BHK) cells, and many other cell lines. Suitable other eukaryotic cells include yeast and other fungi. Useful prokaryotic hosts include, for example, Escherichia coli (E.coli) (e.g., Escherichia coli SG-936, Escherichia coli HB 101, Escherichia coli W3110, Escherichia coli X1776, Escherichia coli X2282, Escherichia coli DHI, and Escherichia coli MRC1), Pseudomonas (Pseudomonas), Bacillus (Bacillus) such as Bacillus subtilis, and Streptomyces (Streptomyces).

These recombinant host cells can be used to produce antibodies by culturing the cells under conditions that allow for expression of the antibody and purifying the antibody from the host cells and the medium surrounding the host cells. Secretion of expressed antibody from recombinant host cells is facilitated by insertion of a signal or secretory leader peptide coding sequence at the 5' end of the gene encoding the target for the antibody (see Shokri et al, (2003) appl. Microbiol. Biotechnol., 60 (6): 654-, (654-), (Nielsen et al, prot. Eng., 10: 1-6 (1997); von Heinje et al, Nucl. acids sRs., 14: 4683-) (1986), the contents of all of which are incorporated herein by reference). These secretory leader peptide elements may be obtained from prokaryotic or eukaryotic sequences. Thus, a secretory leader, i.e., an amino acid linked to the N-terminus of the polypeptide to direct transfer and secretion of the polypeptide from the cytosol of the host cell into the culture medium, is suitably used.

The anti-VEGFR-1 antibodies of the present invention may be fused to other amino acid residues. For example, these amino acid residues may be peptide tags to facilitate separation. Other amino acid residues useful for the antibody to home to a specific organ or tissue are also included.

In another embodiment, the invention provides a method of treating a medical condition by administering to a mammal in need thereof a therapeutically effective amount of an anti-VEGFR-1 antibody of the invention. A therapeutically effective amount refers to an amount effective to produce the desired therapeutic effect (e.g., inhibition of tyrosine kinase activity).

In a preferred embodiment, the present invention provides a method of reducing tumor growth or inhibiting angiogenesis by administering to a mammal in need thereof a therapeutically effective amount of an anti-VEGFR-1 antibody of the present invention. While not intending to be bound by a particular mechanism, diseases that can be treated by the methods of the invention include, for example, diseases in which tumor growth or pathological angiogenesis is stimulated by the paracrine and/or autocrine loops of VEGFR.

For slowing tumor growth, these tumors include primary and metastatic tumors as well as refractory tumors. Refractory tumors include tumors that are non-responsive or resistant to other forms of treatment, such as treatment with chemotherapeutic drugs only, antibodies only, radiopharmaceuticals only, or combinations thereof. Refractory tumors also include tumors that appear to be inhibited after treatment with these drugs, but recur again up to 5 years, sometimes up to 10 years or more, after treatment is discontinued.

The anti-VEGFR-1 antibodies of the present invention are useful for treating VEGFR-1 expressing tumors. These tumors have unique sensitivity to VEGF present in their environment and can further produce and be stimulated by VEGF in an autocrine stimulatory loop. Thus, the method is effective for treating solid tumors or non-solid tumors that do not form vessels or that do not substantially form vessels.

Thus, examples of solid tumors that may be treated include breast cancer, lung cancer, colorectal cancer, pancreatic cancer, glioma, and lymphoma. Some examples of such tumors include epidermoid tumors, squamous tumors such as head and neck tumors, colorectal tumors, prostate tumors, breast tumors, lung tumors including small cell and non-small cell lung tumors, pancreatic tumors, thyroid tumors, ovarian tumors, and liver tumors. Other examples include Kaposi's sarcoma, CNS tumors (CNS neoplams), neuroblastoma, capillary hemangioblastoma, meningioma and brain metastases, melanoma, gastrointestinal cancer, gastrointestinal sarcoma, renal cancer, renal sarcoma, rhabdomyosarcoma, glioblastoma (preferably glioblastoma multiforme), and leiomyosarcoma. Examples of vascularized skin cancers for which the anti-VEGFR-1 antibodies of the present invention are effective include squamous cell carcinoma, basal cell carcinoma, and skin cancers that can be treated by inhibiting the growth of malignant keratinocytes (e.g., human malignant keratinocytes).

Examples of non-solid tumors include leukemia, multiple myeloma, and lymphoma. Some examples of leukemias include Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), Acute Lymphocytic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), erythrocytic leukemia, or monocytic leukemia. Some examples of lymphomas include Hodgkin's lymphoma and non-Hodgkin's lymphoma.

For inhibition of angiogenesis, the anti-VEGFR-1 antibodies of the present invention are useful for treating patients with vascularized tumors or vascularized neoplasms or angiogenic diseases characterized by excessive angiogenesis. These tumors and neoplasms include, for example, malignant tumors and neoplasms such as blastoma, carcinoma or sarcoma, and highly vascularized tumors and neoplasms. Cancers that may be treated by the methods of the present invention include, for example, cancers of the brain, genitourinary tract, lymphatic system, stomach, kidney, colon, larynx and lung and bone. Non-limiting examples also include epidermoid tumors, squamous tumors such as head and neck tumors, colorectal tumors, prostate tumors, breast tumors, lung tumors (including lung adenocarcinoma and small cell and non-small cell lung tumors), pancreatic tumors, thyroid tumors, ovarian tumors, and liver tumors. The method is also useful for treating vascularized skin cancers, including squamous cell carcinoma, basal cell carcinoma, and skin cancers that can be treated by inhibiting the growth of malignant keratinocytes (e.g., human malignant keratinocytes). Other cancers that may be treated include kaposi's sarcoma, CNS tumors (neuroblastoma, capillary hemangioblastoma, meningioma and brain metastases), melanoma, gastrointestinal cancer, gastrointestinal sarcoma, renal carcinoma, renal sarcoma, rhabdomyosarcoma, glioblastoma (including glioblastoma multiforme), and leiomyosarcoma.

Non-limiting examples of pathological angiogenic diseases characterized by excessive angiogenesis (involving, for example, inflammation) and/or vascularization include atherosclerosis, Rheumatoid Arthritis (RA), neovascular glaucoma, proliferative retinopathies including proliferative diabetic retinopathy, macular degeneration, hemangiomas, angiofibromas, and psoriasis. Other non-limiting examples of non-neoplastic angiogenic diseases are retinopathy of prematurity (retrolental fibroplasia), corneal graft rejection, insulin dependent diabetes mellitus, multiple sclerosis, myasthenia gravis, Crohn's disease, autoimmune nephritis, primary biliary cirrhosis, psoriasis, acute pancreatitis, allograft rejection, allergic inflammation, contact dermatitis and delayed hypersensitivity reactions, inflammatory bowel disease, septic shock, osteoporosis, osteoarthritis, neuronal inflammation-induced cognitive deficits, Osier-Weber syndrome, restenosis and fungal infections, parasitic infections and viral infections, including cytomegalovirus infections.

Those skilled in the art are knowledgeable in the art and techniques for identifying medical conditions treatable by the anti-VEGFR-1 antibodies of the present invention. For example, human subjects suffering from clinically significant neoplasia or angiogenic disease or at risk of developing clinically significant symptoms are suitable for administration of the VEGF receptor antibodies of the invention. For example, by applying clinical trials, physical examinations, and medical/family history, clinicians in the art can readily determine whether an individual is eligible for such treatment.

The anti-VEGFR-1 antibodies of the present invention may be administered to a patient suffering from a tumor or angiogenesis-related pathological condition for therapeutic treatment in an amount sufficient to prevent, inhibit or slow the progression of the tumor or pathological condition. Progression includes, for example, growth, invasion, metastasis and/or recurrence of a tumor or pathological condition. The effective amount used will depend on the severity of the disease and the general state of the patient's own immune system. The dosage regimen will vary with the disease state and condition of the patient and will generally range from a single bolus dose or continuous infusion to multiple administrations per day (e.g. every 4-6 hours), or as determined by the treating physician and the patient. It is noted, however, that the present invention is not limited to any particular dosage.

In another embodiment, the invention provides methods of treating medical conditions by administering an anti-VEGFR-1 antibody of the invention in combination with one or more additional agents. For example, one embodiment of the present invention provides methods of treating a medical condition by administering an anti-VEGFR-1 antibody of the present invention in conjunction with an anti-neoplastic agent or an anti-angiogenic agent. The anti-VEGFR-1 antibody may be chemically linked or biosynthetically linked to one or more antineoplastic or anti-angiogenic agents.

Any suitable antineoplastic agent may be used, such as a chemotherapeutic agent or a radiopharmaceutical. Examples of chemotherapeutic drugs include, but are not limited to, cisplatin, doxorubicin, cyclophosphamide, paclitaxel, irinotecan (CPT-I1), topotecan, or combinations thereof. When the antineoplastic drug is a radiopharmaceutical, the radiation source may be external (external beam radiation therapy-EBRT) or internal (brachytherapy-BT) to be administered to the patient undergoing treatment.

In addition, the anti-VEGFR-1 antibodies of the present invention may be administered with antibodies that neutralize other receptors involved in tumor growth or angiogenesis. An example of such a receptor is VEGFR-2/KDR. In one embodiment, the anti-VEGR-I antibodies of the invention are used in combination with receptor antagonists that specifically bind VEGFR-2. Particularly preferred are antigen binding proteins that bind to the extracellular domain of VEGFR-2 and block binding of VEGFR-2 to any one of its ligands, such as VEGF-A, VEGF-C, VEGF-D or VEGF-E.

Another example of such a receptor is EGFR. In one embodiment of the invention, an anti-VEGFR-1 antibody is used in combination with an EGFR antagonist. The EGFR antagonist can be an antibody that binds to EGFR or an EGFR ligand and inhibits binding of EGFR to its ligand. Ligands for EGFR include, for example, EGF, TGF-ce amphiregulin, heparin-binding EGF (HB-EGF), and betamullulin. It is generally believed that EGF and TGF- α are the major endogenous ligands responsible for EGFR-mediated stimulation, although TGF- α has been shown to be more effective in promoting angiogenesis. It will be appreciated that EGFR antagonists may bind externally to the extracellular portion of EGFR, which may or may not inhibit ligand binding, or internally to the tyrosine kinase domain. Examples of EGFR antagonists that bind to EGFR include, but are not limited to, biomolecules such as EGFR-specific antibodies (and functional equivalents thereof) and small molecules such as synthetic kinase inhibitors that act directly on the EGFR cytoplasmic domain.

Other examples of growth factor receptors involved in tumorigenesis are platelet-derived growth factor receptor (PDGFR), insulin-like growth factor receptor (IGFR), Nerve Growth Factor Receptor (NGFR) and Fibroblast Growth Factor Receptor (FGFR).

In another alternative embodiment, the present invention provides methods of treating medical conditions by administering an anti-VEGFR-1 antibody of the present invention in combination with one or more suitable adjuvants, such as cytokines (e.g., IL-10 and IL-13) or other immune stimulators. See, e.g., Larrive et al, supra.

In combination therapy, the anti-VEGFR-1 antibody can be administered before, during, or after the initiation of treatment with another drug, as well as any combination thereof, that is, before and during, before and after, during, or after the initiation of antineoplastic drug treatment. For example, the anti-VEGFR-1 antibody of the present invention may be administered between 1 day and the first 30 days, preferably between the first 3 days and the first 20 days, more preferably between the first 5 days and the first 12 days before radiation treatment is initiated. However, the present invention is not limited to any particular dosing regimen. The dosage of the other drug administered depends on a variety of factors including, for example, the type of drug, the type and severity of the medical condition to be treated, and the route of administration. However, the present invention is not limited to any particular dosage.

Any suitable method or route may be used for administration of the anti-VEGFR-1 antibodies of the present invention, optionally in conjunction with anti-neoplastic agents and/or other receptor antagonists. For example, routes of administration include oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration. However, it should be emphasized that the present invention is not limited to any particular method or route of administration.

Notably, the anti-VEGFR-1 antibodies of the present invention can be administered as a conjugate that specifically binds to the receptor and releases a toxic lethal load upon internalization of the ligand-toxin.

It will be appreciated that when used in mammals for prophylactic or therapeutic purposes, the anti-VEGFR-1 antibodies of the present invention will be administered in a composition that further comprises a pharmaceutically acceptable carrier. For example, suitable pharmaceutically acceptable carriers include one or more of the following: water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. The pharmaceutically acceptable carrier may also contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers to prolong shelf life or to enhance the effectiveness of the binding protein. Injectable compositions, well known in the art, may also be prepared to provide immediate, sustained or delayed release of the active ingredient after administration to the mammal.

Although the human antibodies of the invention are particularly useful for administration to humans, they may also be administered to other mammals. The term "mammal" as used herein includes, but is not limited to, humans, laboratory animals, domestic pets, and farm animals.

The invention may also include kits for inhibiting tumor growth and/or angiogenesis comprising a therapeutically effective amount of an anti-VEGFR-1 antibody of the invention. The kit may also contain any suitable antagonist, such as an additional antagonist of a growth factor receptor involved in tumorigenesis or angiogenesis (e.g., VEGFR-2/FKDR, EGFR, PDGFR, IGFR, NGFR, FGFR, etc., as described above). Alternatively, the kit of the invention may comprise an anti-tumour agent, or the kit of the invention may comprise any suitable antagonist and may additionally comprise an anti-tumour agent. Examples of suitable antineoplastic agents have been described in the present disclosure. The kit of the invention may additionally comprise an adjuvant, examples of which have been described above.

In another embodiment, the invention provides methods of in vivo or in vitro research or diagnosis using the anti-VEGFR-1 antibodies of the invention. In these methods, the anti-VEGFR-1 antibody can be linked to a target or reporter moiety.

Examples

The following examples do not include detailed descriptions of conventional methods such as methods for vector and plasmid construction, insertion of genes encoding polypeptides into these vectors and plasmids, or introduction of plasmids into host cells. These 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 manitis, T. (1989), Molecular Cloning: a Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, the contents of which are incorporated herein by reference.

Material

AU reagents and chemicals were purchased from Sigma (st. louis, MO) unless otherwise indicated. Human VEGF165 and soluble recombinant human VEGFR-1 alkaline phosphatase (rhuVEGFR-1AP) proteins were expressed in stably transfected cells and purified from the cell culture supernatant according to methods known to those skilled in the art (Tessler, J.biol.chem., 269: 12456-12461(1994), the contents of which are incorporated herein by reference). PlGF and soluble recombinant VEGFR-1Fc (rhuVEGFR-1Fc) proteins were purchased from R & D Systems Inc. Minneapolis, MN. Cell culture dishes and assay plates were purchased from BD Biosciences, Bedford, MA.

Cell lines

Human breast cancer cell lines DU4475, MDA-MB-231, MDA-MB-435 and mouse myeloma cell lines P3-X63-Ag8.653 and NSO were obtained from the American Type Culture Collection (American Type Tissue Culture Collection, Manassas, Va.). The P3-X63-Ag8.653Bc1/2 transfected cell line was prepared autonomously as described previously (Ray S, Diam)ond B.Proc.Natl.Acad.Sci.USA 91: 5548-51, 1994). Tumor cells were maintained in RPMI1640 medium (Invitrogen/life technologies, inc., Rockville, MD) with 10% FCS (Hyclone, Logan, UT). Porcine aortic endothelial VEGFR-1 expressing cell lines were cultured in F12 medium (Invitrogen/Life Technologies, inc., Rockville, MD) containing 10% FCS (Hyclone, Logan, UT), supplied by doctor l. All cells were humidified at 37 ℃ and 5% CO₂Storing under atmosphere.

Example 1:production of anti-VEGFR-1 antibodies

Human anti-VEGFR-1 monoclonal Antibodies (also referred to herein as "anti-VEGFR-1 Antibodies") were generated using KM transgenic mice that produce human immunoglobulin gamma heavy and kappa light chains (Metarex, San Jose, Calif.) by standard hybridoma techniques (master eds. Harlow and Lane, Antibodies: antibody Manual (antibody laboratory Manual), Cold Spring Harbor, 211-213(1998), the contents of which are incorporated herein by reference). KM mice were immunized subcutaneously (s.c.) with complete Freund's adjuvant containing VEGFR-1 crystalline fragment (Fc). Animals were boosted intraperitoneally (i.p.) three times with incomplete freund's adjuvant containing the same VEGFR-1 protein prior to fusion. The animals were allowed to rest for one month and then a final boost was performed intraperitoneally with Phosphate Buffered Saline (PBS) containing 25 micrograms of VEGFR-1 protein. Four days later, splenocytes were harvested from immunized mice and plasmacytoma cells were transfected with polyethylene glycol (PEG, molecular weight 1450KD) and P3-X63-Ag8.653Bcl-2 for fusion. After fusion, the cells were resuspended in HAT (hypoxanthine, aminopterin, thymidine) medium supplemented with 10% Fetal Bovine Serum (FBS) and added to a 96-well plate at a density of 200 μ l/well to establish hybridoma cells. On day 6 after the fusion, 100. mu.l of the original medium was aspirated, and 100. mu.l of fresh medium was added.

Example 2A:the anti-VEGFR-1 antibodies from example 1 bind to VEGFR-1 and inhibition of VEGFR-1 binding to its ligand

a.VEGFR-1 binding and blocking assays

10-12 days after fusion, hybridomas were screened for antibody production and for specific binding activity of the antibodies in the culture supernatants to rhuVEGFR-1 protein in ELISA-based binding and blocking assays. Positive hybridomas were subcloned three times by establishing limiting dilution cultures of monoclonal hybridomas.

Specifically, hybridoma supernatant and purified antibody were diluted with PBS (ELISA buffer) containing 5% FBS and 0.05% Tween 20(Tween 20) and incubated for 30 minutes in rhu VEGFR-1AP or AP coated 96-well microtiter plates. Each plate was washed with ELISA buffer and incubated with goat anti-mouse IgG-horseradish peroxidase (HRP) conjugate (biosource international, Camarillo, CA) for 30 minutes. Color was developed with TMB (3, 3 ', 5, 5 ' -tetramethylbenzidine) substrate (Kierkegaard and Perry Lab, inc., Gaithersburg, MD) according to the manufacturer's instructions. The absorbance was read at 450 nanometers (nm) and the antibody binding activity was quantitatively determined. To identify hybridomas that produce anti-VEGFR-1 antibodies, hybridoma supernatants were preincubated for 1 hour with VEGFR-1 AP. The mixture was incubated with ELISA buffer for 1 hour in VEGF or PlGF coated 96 well microtiter plates. Color was developed with PNPP (p-nitrophenyl phosphate) substrate for AP according to the manufacturer's instructions. Absorbance was read at 405nm and binding of VEGFR-1 to VEGF or PlGF was quantitated. Optical Density (OD) values were read on a microtiter plate reader (Molecular Devices corp., Sunnyvale, CA). Antibodies were analyzed for ED50 and IC50 using GraphPad Prism 3 Software (GraphPad Software, inc., san diego, CA).

FIG. 3 shows the binding activity of purified antibodies produced by hybridomas designated "6F 9", "13G 12", "15F 11" and "18F 1". These antibodies showed binding activity in ELISA-based binding assays with ED50 of 0.1-0.3 nM. FIGS. 4 and 5 show that clones 6F9, 13G12, 15F11, and 18F1 effectively blocked PlGF binding to VEGFR-1 (IC50 at 0.4-0.8nM) and VEGF binding to VEGFR-1 (IC50 at 0.7-0.8nM), respectively. Table 4 summarizes the binding and blocking characteristics of the antibodies.

b.Determination of affinity of anti-VEGFR-1 antibody

The affinities of anti-VEGFR-1 antibody clones 6F9, 13G12, 15F11, 18F1 were determined by surface plasmon resonance (plasmon resonance) using BIAcore 2000(Pharmacia, Piscataway, NJ) according to the manufacturer's protocol. The recombinant extracellular domain of VEGFR-1 was immobilized at low density on the sensor surface and the antibody was subjected to kinetic analysis. Determined by BIAevaluation 2.1 software supplied by the manufacturer (K)_{Association of}) And dissociation (koff) rate.

anti-VEGFR-1 antibody clones 6F9, 13G12, F11 and 18F1 showed high affinity, K_DThe values were 69pM, 121pM, 70pM and 54pM, respectively. Table 5 summarizes the antibody kinetics.

c.Evaluation of specificity of anti-VEGFR-1 antibody

To determine the specificity of the anti-VEGFR-1 monoclonal antibody for human VEGFR-1, the purified antibody 18F1 was tested in an ELISA-based assay. Mu.g/ml recombinant human VEGFR-1Fc, mouse VEGFR-2Fc, or human VEGFR-2 alkaline phosphatase was coated in a 96-well microtiter plate with PBS overnight at 4 ℃. After washing, receptor-coated plates were blocked with PBS containing 5% Milk powder (Dry Milk) and 0.05% tween 20. Serially diluted anti-human VEGFR-1 primary antibody 18F1, anti-mouse VEGFR-1 primary antibody MF1, anti-human VEGFR-2 primary antibody ICl1, or anti-mouse VEGFR-2 primary antibody DClOl were incubated in receptor coated plates for 30 minutes. After washing, the secondary anti-primary HRP conjugate antibody was incubated in the plate for 30 minutes. And washing the plate, adding a substrate TMB (3, 3 ', 5, 5' -tetramethyl benzidine), incubating and developing color. The absorbance OD was read at 450nm to quantitatively determine the binding activity of the antibody. Data were analyzed using GraphPad Prism Software.

FIGS. 6A-D show the specificity of anti-human VEGFR-1 monoclonal antibody 18F1 (FIG. 6A), which is non-cross-reactive with mouse VEGFR-1 (FIG. 6B), human VEGFR-2 (FIG. 6C), and mouse VEGFR-2 (FIG. 6D). The results show that anti-human VEGFR-1 antibody 18F1 has strict binding specificity to its corresponding receptor.

d.Western blot

Confluent porcine aortic endothelial VEGFR-1 expressing (PAE-VEGFR-1) cells and BT474 human breast cancer cells were cultured in serum-depleted F12 medium for 48 hours. The cells were then preincubated for 1 hour with anti-VEGFR-1 antibody clone 18F1 at a concentration ranging from 0.1 to 30 μ g/ml, and stimulated with VEGF or PlGF for 5 minutes at 37 ℃. The cells were then rinsed in ice cold PBS and lysed in lysis buffer (50mM HEPES, 150mM NaCl, 1% TritonX-100 and 10% glycerol containing 1mM phenylmethanesulfonyl fluoride, 10. mu.g/ml aprotinin, 10. mu.g/ml leupeptin and 1mM sodium vanadate). Cell lysates were subjected to SDS-PAGE and transferred to Immobilon membranes (Millipore Corp. Billerica, Mass.). After transfer, the blot was incubated with blocking solution, probed with an anti-phosphotyrosine antibody (PY20, Santa Cruz biotechnology, Santa Cruz, CA), and then washed. The protein content was observed with horseradish peroxidase-conjugated secondary antibodies followed by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, N.J.). anti-VEGFR-1 specific antibodies (OncogeneResearch Products, San Diego, Calif.) were used for double blotting (re-blot) of VEGFR-1.

All anti-VEGFR-1 antibodies recognized the VEGFR-1 recombinant protein as a 180KD molecule.

Example 2B:anti-human anti-VEGFR-1 antibodies are specific for human VEGFR-1

Human VEGFR-1-Fc, mouse VEGFR-1-AP (hnclone systems) or huVEGFR-2-AP (Imclone systems) (100 ng/well) were coated onto 96-well plates and blocked with 5% milk/PBS. Binding of 18F1 and other anti-human VEGFR-1 antibodies or rat anti-mouse VEGFR-1 antibody MF1(Imclone Systems, ref.18) to VEGFR-1 or VEGFR-2 of the binding plate was evaluated as described above for screening of hybridoma supernatants except that for 18F1, the bound antibodies were detected with goat anti-human kappa-HRP antibody (BioSource International, Camarillo, Calif.) and for MF1, the bound antibodies were detected with anti-human VEGFR-2 antibody ICl1 or goat anti-rat IgG-HRSP antibody (BioSource International).

8F1 showed specific reactivity with human VEGFR-1 (FIG. 32A), but no cross-reactivity with mouse VEGFR-1 (FIG. 32B) and human VEGFR-2 (FIG. 32C). The anti-mouse VEGFR-1 blocking antibody MF1 also demonstrated interspecies specificity for binding to mouse (FIG. 32B) but not to human VEGFR-1 (FIG. 32A).

Example 3:anti-VEGFR-1 antibodies and natural anti-VEGFR-1 antibodies in VEGFR-1 expressing cells VEGFR-1 binding

a.Flow cytometry analysis

Harvest from a confluent culture equant 10⁶The PAE-VEGFR-1 cells of (1), together with anti-VEGFR-1 antibody clones 6F9, 13G12, F11 and 18F1, were incubated on ice for 1 hour in PBS (staining buffer) containing 1% Bovine Serum Albumin (BSA) and 0.02% sodium azide. Harvest from a confluent culture equant 10⁶DU4475 human breast cancer cells were incubated with anti-VEGFR-1 antibody clone 18F1 for 1 hour on ice in PBS (staining buffer) containing 1% Bovine Serum Albumin (BSA) and 0.02% sodium azide. A matching IgG isotype (Jackson ImmunoResearch, West Grove, PA) was used as a negative control. Cells were washed twice with running buffer and then incubated with Fluorescein Isothiocyanate (FITC) -labeled goat anti-human IgG antibody (BioSource International, Camarillo, CA) for 30 minutes on ice in staining buffer. Cells were washed as described above and analyzed in an Epics XL flow cytometer (Beckman-Coulter, Hialeah, FL). Dead cells and debris were removed from the analysis based on forward and side light scattering. Mean fluorescence intensity units (MFRJ) were calculated as mean logarithmic fluorescence multiplied by the percentage of positive population.

FIG. 7 shows the binding reactivity of clones 6F9, 13G12, 15F11 and 18F1 to PAE-VEGFR-1 expressing cells. FIGS. 8A and 8B show the binding reactivity of clone 18F1 to PAE-VEGFR-1 expressing cells and DU4475 human breast cancer, respectively. These results indicate that human anti-VEGFR-1 antibodies bind to native VEGFR-1 expressed on the cell surface.

b.Surface VEGFR-1 blocking assay

Binding of 125I-VEGF to VEGFR-1 was performed on the cell surface using PAE-VEGFR-1 expressing cells. Cells were grown in uncoated plastic cell culture plates and reduced non-specific binding was found, but without affecting¹²⁵Specific binding of I-VEGF. Confluent cells were cultured in Dulbecco's Modified Eagle Medium (DMEM)/F-12 medium (Invitrogen, Carlsbad, Calif.) without serum and without growth supplements for 24 hours. Cells were rinsed once with ice-cold DMEM/F-12 medium containing 0.025M HEPES and 1mg/ml Bovine Serum Albumin (BSA). Serial dilutions of anti-VEGFR-1 antibody 18F1 or cold VEGF were added to each well of the plate at a molar concentration 200-fold greater than the labeled VEGF and incubated for 1 hour at 4 ℃. After washing, 2ng/ml of a solution is added¹²⁵I-VEGF, incubated for 2 hours at 4 ℃ in a bench top shaker. Cells were incubated with 1mg/ml BSA and 0.25mM CaCl₂Washed three times with 1% Triton X-100, 1mg/ml BSA and 0.16% NaN₃Incubate in the presence for 5 minutes to remove bound VEGF. The soluble content of each well was counted using a gamma counter. In at least three independent experiments, experiments were performed in triplicate and data was analyzed using Prism GraphPad software 3.03.

FIG. 9 shows that the anti-VEGFR-1 antibody 18F1 has a very strong blocking activity, 18F1 significantly prevents native VEGFR-1 from occurring on porcine aortic endothelial cells¹²⁵I-VEGF binds.

Example 4:anti-VEGFR-1 antibodies inhibit VEGFR-1 in response to VEGF and PlGF Autophosphorylation and activation of MAPK and Akt

a.VEGFR-1 phosphorylation assay

The autophosphorylation of VEGFR-1 is induced by its ligands, resulting in the activation of typical MAPK, extracellular signal-regulated protein kinase 1/2(ERK1/2) and PBK/Atk downstream signaling pathways that mediate cellular biological responses such as proliferation, motility, survival and differentiation. Cells transfected with PAE-VEGFR-1 and BT474 breast cancer cells were assayed for the ability of anti-VEGFR-1 antibodies to inhibit VEGFR-1 phosphorylation and activation of signals downstream of ERK1/2 and Akt kinases.

Density of 5X 10⁵PAE-VEGFR-1 and BT474 cells/well were seeded into 100mm²Or 150mm²Cultured in serum-free medium for 18-48 hours on plate. After medium change, cells were treated with anti-VEGFR-1 antibody clones 6F9, 15F11, and 18F1 or isotype controls for 1 hour at 37 ℃ and then incubated with 50ng/ml VEGF or 100ng/ml PlGF for 10 minutes. After treatment, lysis buffer [20mM HEPES (pH 7.4), 10mM MgCl₂、2mM MnCl₂0.05% Triton X-100 and 1mM DTT]Total cellular protein extracts were isolated and immunoprecipitated with anti-VEGFR-1 antibody (C-17, Santa Cruz Biotechnology, Santa Cruz, Calif.). Western blot of phosphorylated VEGFR-1 was detected with an anti-phospho-kinase antibody (PY-20, Santa Cruz Biotechnology, Santa Cruz, Calif.). Proteins were detected using an electrochemiluminescence system (ECL) (Amersham Pharmacia Biotech, Piscataway, N.J.) and quantified by densitometry using NIH Image (National Institute of Mental Health, Bethesda, Md.).

b.In vitro kinase assay

To evaluate MAPK and Akt phosphorylation, densities were 5X 10⁵BT474 cells per well were seeded into 12-well plates and treated for 18 hours under serum-free conditions. Cells were treated with anti-VEGFR-1 antibody clone 18F1 or isotype control for 1 hour at 37 deg.C and then incubated with 50ng/ml VEGF or 100ng/ml PlGF for 5-10 minutes. Cells were lysed, proteins were isolated, and electroblotted. The membrane was incubated with an anti-phosphorylated p44/p42MAP kinase (Thr202/Tyr204, Santa Cruz Biotechnology, Santa Cruz, CA) antibody or an anti-phosphorylated Akt (Ser473, Cell Signaling Technology, Beverly, MA) antibody at a concentration of 1. mu.g/ml, followed by incubation with a second IgG-HRP (1: 5000). To ensure the same amount of loaded sample, the membrane was peeled off and probed again with anti-p 44/p42 antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) or anti-Akt antibody (Cell Signaling Technology, Beverly, Mass.).

c.Results

As shown in FIGS. 10-14, significant phosphorylation of VEGFR-1 and activation of ERK1/2 and Akt signaling was induced by VEGF and PlGF stimulation in PAE-VEGFR-1 transfected cells and BT474 breast cancer cells, suggesting intrinsic activity of VEGFR-1 and receptor-associated downstream kinase signaling pathways in both breast and endothelial cells. As shown in FIGS. 10 and 11, respectively, treatment with anti-VEGFR-1 antibody 18F1 significantly reduced PlGF or VEGF-stimulated VEGFR-1 phosphorylation in PAE-VEGFR-1 transfected cells and BT474 breast cancer cells compared to untreated controls. As shown in fig. 12 and 13, respectively, treatment with anti-VEGFR-1 antibodies 15F11 and 6F9 also significantly inhibited PlGF and VEGF induced activation of ERK1/2 downstream signaling by PlGF and VEGF in PAE-VEGFR-1 transfected cells. In breast cancer, activation of Akt protein kinases is an important intracellular signaling activity that mediates cell survival. As shown in FIGS. 14A and 14B, respectively, in PAE-VEGFR-1 transfected cells, treatment with anti-VEGFR-1 antibody 18F1 significantly inhibited PlGF or VEGF-induced activation of ERK1/2 downstream signaling by PlGF and VEGF.

As shown in figure 15, anti-VEGFR-1 antibody 18F1 significantly blocked PlGF-stimulated Akt phosphorylation in BT474 breast cancer cells. These results demonstrate that treatment with an anti-VEGFR-1 antibody effectively inhibits the activation of VEGFR-1 and downstream signaling kinase pathways in both breast cancer cells and endothelial cells.

Example 5:anti-VEGFR-1 antibodies block growth of breast tumor cells in vitro

Tumor hypoxia is associated with accelerated malignant progression, enhanced invasiveness and enhanced resistance to chemotherapeutic drugs. Hypoxic tumor cells undergo a biological response that activates survival and proliferation signaling pathways by upregulating expression of various genes, including VEGFR-1 (Harris AL. nat Revcancer. 2: 38-47, 2002).

Cell growth assay

Density of 5X 10³DU4475 cancer cells/well were seeded in 96-well plates and treated for 18 hours in serum-free conditions and in some cases for 5 hours more with 100nM deferoxamine. The inhibitory effect of anti-VEGFR-1 antibodies on tumor cell growth was determined by incubating the cells with anti-VEGFR-1 antibody clones 6F9, 13G12, 15F11 and 18F1 at doses of 3, 10 and 30 μ G/ml each in the presence of 50ng/ml VEGF or 200ng/ml PlGF for 48 hours. Viable cells were then counted in a Coulter cell counter (Coulter Electronics Ltd. Luton, Beds, England) in triplicate. Each experiment was performed in triplicate.

The growth rate of DU4475 tumor cells pretreated with the hypoxia-mimetic agent deferoxamine increased approximately 2-fold in response to stimulation with VEGF or PlGF. Treatment with anti-VEGFR-1 antibody was effective in reducing proliferation of VEGF and PlGF stimulated DU4475 breast cancer cells in a dose response manner, as shown in fig. 16 and 17, respectively. Fig. 18A and 18B are each plotted as the antibody concentration of antibody clone 18F1 against the cell count of VEGF and PlGF stimulated proliferation of DU4475 breast cancer cells. Table 6 summarizes the inhibition of PlGF-induced growth of DU4475 cells in vitro as indicated by anti-VEGFR-1 antibodies represented by IC50 values.

Example 6A:anti-VEGFR-1 antibodies inhibit growth of breast tumor xenograftsTreatment of human breast cancer xenografts

The anti-tumor efficacy of human anti-VEGFR-1 antibodies was tested in a human xenograft breast tumor model.

In the left flank of athymic nude mice (Charles River Laboratories, Wilmington, Mass.), mixed in Matrigel (Matrigel) (collagen research biochemicals, Bedford, Mass.) was injected subcutaneously2×10⁶DU4475 cells or 5X 10⁶MDA-MB-231 cells and MDA-MB-435 cells. In the DU4475 and MDA-MB-231 models, tumors grew to approximately 200mm³At size, mice were randomly grouped into groups of 12-16 animals each. The anti-VEGFR-1 antibody clone 6F9, 15F11, or 18F1 was administered to the animals intraperitoneally at a dose of 0.5mg (MDA-MB-231) or 1mg (DU4475) three times per week. In the MDA-MB-435 model, tumor cells were transplanted into the subcutaneous sites of mammary fat pads in mice. The tumor grows up to about 200mm³In size, mice were randomized into groups of 15 animals each, and 0.5 mg/dose of 18F1 antibody was administered intraperitoneally three times a week. The mice in the control group were given an equivalent amount of saline solution. Treatment of the animals was continued during the experiment. Tumors were measured twice weekly using calipers. Applying the formula [ pi/6 (w1 xw 2 xw 2)]Tumor volume was calculated, where "w 1" represents the tumor maximum diameter and "w 2" represents the tumor minimum diameter.

As shown in FIGS. 19A and 19B, the systemic administration of anti-VEGFR-1 antibodies 6F9, 15F11, 13G12, and 18F1 at a dose of 1 mg/dose three times per week resulted in a statistically significant inhibition of the growth of DU4475 xenograft tumors (p < 0.05). Systemic administration of anti-VEGFR-1 antibody 18F1 resulted in statistically significant inhibition of tumor growth in DU4475, MDA-MB-231, and MDA-MB-435 xenografts three times per week (ANOVA p < 0.05), as shown in FIGS. 20A, 20B, and 20C, respectively, at doses of 0.5 mg/dose or 1 mg/dose. As shown in FIGS. 21A and 21B, twice weekly treatment with anti-human VEGFR-1 antibody clone 18F1, which inhibits cancer cell growth, and anti-mouse VEGFR-1 clone MF1, which inhibits tumor angiogenesis, inhibited tumor growth more strongly (P < 0.05) than treatment with one antibody alone in the DU4475 and MDA-MB-231 xenograft models. These results indicate that VEGFR-1 is blocked by anti-VEGFR-1 antibodies in the xenograft model to directly promote the growth of cancer cells and to regulate the in vivo function of tumor angiogenesis, effectively inhibiting the growth of VEGFR-1 positive breast tumors.

Example 6B:anti-human anti-VEGFR-1 antibody blocks growth of breast cancer cells in vitro

DU4475 cancer cells(2×10⁴/well) were seeded into 24-well plates and treated for 18 hours under serum-free conditions. Then, it was treated with the hypoxia-mimetic drug desferrioxamine (Sigma) for another 6 hours. Serial dilutions of anti-human VEGFR-1 antibody 18F1 were added to the plates in triplicate at 50ng/ml VEGF-A (R)&D Systems) or 200ng/ml PlGF for 48 hours. The total number of cells (cells in both fixed and suspended) was determined for each well using a Coulter cell counter (Coulter Electronics Ltd., England).

IMC-18F1 treatment significantly blocked VEGF-A and PlGF stimulated proliferation of DU4475 breast cancer cells (see FIG. 31A and FIG. 31B, respectively, estimated IC 50: 30-50 nM). Isotype control antibodies had no effect on cell proliferation. Thus, 18F1 inhibited VEGFR-1 ligand-induced promotion of tumor cell proliferation/survival.

Example 7:anti-VEGFR-1 antibodies inhibit VEGF-A and VEGF-B stimulated colon Colony formation of cancer cells

1ml of DMEM medium containing 10% FBS and 1% agarose (Cambrex Corporation, EastRutherford, NJ) was added to each well of the 6-well plate. HT-29 human colon cancer cells in serum-free medium were treated with 66nM 18F1 or control IgG for 1 hour, followed by 10ng/ml VEGF-A or 50ng/ml VEGF-B for 4 hours. Treated cells were mixed with 1ml 10% FBS DMEM containing 0.5% agarose and appropriate antibodies and/or ligands. 1ml of this suspension (containing 250 cells) was seeded on top of a 1% agarose bottom layer for each well. After 2 days, medium containing additional antibody and/or ligand was added to each well to keep the agarose hydrated. Cells were grown at 37 ℃ for 14 days. Colonies larger than 50 μm in diameter were then counted using a dissecting microscope. Statistical analysis was performed using InStat Statistical software (V2.03, GraphPadSoftware, San Diego, Calif.).

The number and size of colonies in each well of cells treated with VEGF-A or VEGF-B was significantly increased compared to untreated cells with complete medium alone. As shown in fig. 22, treatment with 18F1 completely inhibited ligand-induced colony formation (p < 0.03) compared to basal activity in the absence of ligand stimulation (fig. 22). Thus, 18F1 has the ability to inhibit tumor cell survival and growth for both adherent and nonadherent cells.

Example 8:anti-VEGFR-1 antibodies inhibit VEGF-A and VEGF-B induced colon Migration and invasion of cancer cells

HT-29 cells (2.5X 10)⁴) Or SW480 cells (1.5X 10)⁴) In a medium containing 1% FBS, with 24-well MATRIGEL^TM(Becton Dickinson Labware, Bedford, Mass.) the anti-VEGFR-1 antibody 18F1(66nM) on the upper surface of the membrane plug (pore size 8.0 μm) coated (HT-29) or uncoated (SW480) was incubated together. Plug insertion into a 10ng/ml VEGF-A (R) containing solution&D Systems) or 50ng/ml VEGF-B (R)&D Systems) for 48 hours in the lower chamber (lower chambers). Cells remaining in the upper chamber of the plug were removed with a cotton swab. Cells that migrated into the interior of the plug were stained with Diff-Quik (Harleco, Gibbstown, NJ) and counted at 100X magnification for 10 random fields. Statistical analysis was performed using InStat Statistical Software (V2.03, GraphPad Software, San Diego, Calif.).

As shown in FIGS. 23A and 23B, VEGF-A or VEGF-B induced HT-29 cell migration through the uncoated membrane towards the ligand. As shown in fig. 24A and 24B, these ligands also induced invasion of SW480 cells through matrigel coat. Compared to the basal activity in the absence of ligand stimulation, 18F1 completely blocked VEGFR-1 ligand-induced migration and invasion (p < 0.05, FIGS. 23 and 24). Thus, in addition to the negative effects on tumor cell proliferation and survival, 18F1 also provides a means to inhibit tumor cell invasion and subsequent metastasis.

Example 9:treatment of human with anti-VEGFR-1 specific antibodies to inhibit VEGFR-1 expression In vivo growth of xenograft tumors

Female athymic nu/nu mice, 6-8 weeks old, containing human tumor cell lines subcutaneously injected on dorsal surface with MATRIGEL^TM(BD Biosciences) 1: 1 diluted suspension in a volume of 0.4 ml. Cell lines used in xenograft models (cell doses are indicated in parentheses (10)⁶Cell/mouse)) are: human colon cancer cell line DLD-1(5) GEO (5) and HT-29 (5); human breast cancer cell lines DU4475(2), MDA-MB-231(5), MDA-MB-435(5) and BT474 (5). When the tumor grows to about 200-300mm³At the time, mice were randomly divided into treatment groups by tumor size. Tumor growth was evaluated approximately twice a week for tumor volume in pi/6 x (length x width)²) And calculating, wherein the length is the longest diameter, and the width is the diameter perpendicular to the length. Tumor size was measured with calipers. T/C% was calculated from 100 × (final treated tumor volume/initial treated tumor volume)/(final control tumor volume/initial control tumor volume).

18F1 was diluted with 0.9% USP saline (Braun) or Phosphate Buffered Saline (PBS) and given to each mouse intraperitoneally in a volume of 0.5 ml. Treatment effects on tumor growth were analyzed using repeated measures analysis of variance (RM ANOVA), with p < 0.05 considered significant.

As shown in FIG. 25, intraperitoneal administration of 18F1 significantly (p < 0.05) inhibited the growth of DU4475 (FIG. 25A), MDA-MB-231, and MDA-MB-435 (FIG. 25B) xenograft tumors. As shown in FIG. 26, a significant antitumor effect of 18F1 monotherapy against HT-29 (FIG. 26A), DLD-1 (FIG. 26B) and GEO (FIG. 26C) colon cancer xenografts was also observed. These results indicate that blocking human VEGFR-1 effectively inhibits the growth of xenograft tumors established with human tumor cell lines expressing VEGFR-1.

Example 10:in vivo signaling inhibition of proliferation and survival pathways by treatment with anti-human VEGFR-1 Induction of tumor cell apoptosis

Paraffin-embedded MDA-MB-231 xenografts were evaluated for markers of tumor cell proliferation, survival and apoptosis by immunohistochemistry. Proliferation and survival markers include Ki-67 (rabbit pAb; Lab Vision Corporation, Fremont, Calif.), phosphate-specific p44/42MAPK (Thr202/Tyr204) (rabbit pAb; Cell Signaling Technology), and phosphate-specific Akt (Ser473) (rabbit pAb; Cell Signaling Technology). The EnVision + System was used with 3, 3' -Diaminobenzidine (DAB) as the chromogen for rabbit antibodies (DAKO Cytomation, Carpenteria, CA) according to kit instructions. Through Mayer hematoxylinAfter a brief counterstaining, all sections were dehydrated, cleared, and coverslipped with permanent mounting medium. Apoptosis of tumor cells by TUNEL assay according to kit instructionsPeroxidase in situ apoptosis detection kit (Chemicon, Temecula, Calif.) was evaluated. Stained sections were coverslipped with Gelmount (Biomeda, Foster City, Calif.). Positive immunostaining and TUNEL positive immunofluorescence were analyzed and photographed using an Axioskop light microscope equipped with an Axiocam digital camera (Carl Zeiss, Germany).

As shown in FIG. 27, after 14 days of treatment with 20mg/kg (about 0.5 mg/dose in female nu/nu athymic mice) of 18F1, 2 times/week, the proliferative cell marker (Ki-67) was significantly reduced (study No. 3067-04). In addition, 18F1 treatment resulted in a significant reduction in MAPK activation at this time point (fig. 27). Increased apoptosis (FIG. 27) and significant reduction in Akt phosphorylation as measured by TUNEL positive events were also detected in MDA-MB-231 xenograft tumors 1 week after treatment with 18F1(0.5 mg/dose, M-W-F).

Example 11:blocking of human and murine VEGFR-1 in vivo results in xenografts against human breast cancer Better antitumor activity of the product

18F1 was used in combination with the anti-mouse VEGFR-1 antibody MF 1. 18F1 was diluted with 0.9% USP saline (Braun) or Phosphate Buffered Saline (PBS) and given intraperitoneally to each mouse in a volume of 0.5 ml. Treatment effects on tumor growth were analyzed using repeated measures analysis of variance (RM ANOVA), with p < 0.05 considered significant. As shown in FIG. 28, 18F1 inhibited tumors expressing human VEGFR-1 and MF1 inhibited tumors expressing endogenous mouse VEGFR-1 in MDA-MB-231 (FIG. 28A) and DU4475 (FIG. 28B) xenograft models, resulting in significant tumor growth inhibition (p < 0.05). MF1 has been shown to inhibit tumor growth by reducing tumor angiogenesis. 18F1 in combination with MF1 resulted in more significant tumor growth inhibition (p < 0.05) than monotherapy. The combination treatment of 18F1+ MF1 was not associated with weight loss. These data support dual inhibition of tumor angiogenesis and tumor cell proliferation and survival in patients treated with 18F 1.

Example 12:combination of anti-VEGFR-1 antibodies with chemotherapy

In the MDA-MB-231 model, 18F1+ MF1 was combined with cytotoxic therapy (5-fluorouracil, leucovorin and paclitaxel). 18F1 was diluted with 0.9% USP saline (Braun) or Phosphate Buffered Saline (PBS). Antibody treatment was given at a constant dose per mouse, giving a volume of 0.5ml per mouse. The dose of antibody and cytotoxic therapy administered is proportional to body weight and is 10 μ l volume/gram body weight. 5-Fluorouracil and folinic acid (5-FU/LV) were diluted separately with USP saline and administered separately. Paclitaxel was prepared either with 5% benzyl alcohol (Sigma), 5% Cremophor EL (Sigma) and 90% USP saline, or with 5% ethanol (Sigma), 5% polyoxyethylene castor oil and 90% USP saline. Cyclophosphamide and doxorubicin were dissolved in USP saline for administration. AU treatment was given intraperitoneally. Treatment effects on tumor growth were analyzed using repeated measures analysis of variance (RM ANOVA), with p < 0.05 considered significant.

As shown in figure 29, the antitumor effect was significantly increased in the MDA-MB-231 model in the treatment of 18F1+ ME1 added to the active dose of cyclophosphamide. As shown in FIG. 30, 18F1+ MF1 increased the anti-tumor effect of 5-FU/LV and doxorubicin chemotherapy when both were administered at specific dose levels.

In the DU4475 xenograft model, there was a trend towards an increase in activity (lower T/C%) when IMC-18F1+ MF1 was used in combination with 5-FU/LV, doxorubicin and paclitaxel, although the effect did not reach statistical significance compared to IMC-18F1+ MF1 alone, or compared to cytotoxic drug monotherapy. In MDA-MB-231, the same is true for doxorubicin, although in combination with 5-FU/LV and paclitaxel, there is no tendency for activity enhancement. The lack of additive effect may be due to the minimal effect of 5-FU/LV and paclitaxel as monotherapy at the selected dose level. In the MDA-MB-435 model (T/C% ═ 51), the activity was also increased in combination with cyclophosphamide compared to IMC-18F1+ MF1 alone (T/C% ═ 60) or cyclophosphamide monotherapy (T/C% ═ 65), although these differences were not statistically significant. The same is true for doxorubicin and paclitaxel in the same study. Similar to the data for MDA-MB-231 and MDA-MB-435 described above, in the DU4475 xenograft model, BVIC-18F1, MF1, and cyclophosphamide in combination showed enhanced antitumor activity compared to antibody or cytotoxic treatment alone, although this trend was not statistically significant.

Statistical analysis

Tumor volumes and in vitro tumor cell growth were analyzed using Student's test and the SigmaStat statistical analysis package (version 2.03; Jandel Scientific, San Rafael, Calif.). Variance p < 0.05 was considered statistically significant.

Example 13:cloning and sequencing of anti-VEGFR-1 antibody VH/VL regions

Poly (A +) mRNA was isolated from hybridoma cells producing clones 6F9, 13G12, 15F11, and 18F1 obtained from VEGFR-1 immunized KM mice using the Fast-Track kit (Invitrogen, Carlsbad, Calif.). Thereafter, random primed cDNA was generated by Polymerase Chain Reaction (PCR) using the Clontech kit. Primers (Forward: 5 '-ATGGAGTTTGGGCTGAGCTG and reverse: 3' -TGCCAGGGGGAAGACCGATGG) and (Forward: 5 '-ATG GAA ACC CCAGCG CAG CTT CTC and reverse: 3' -CGGGAAGATGAAGACAGATG) were used to bind to the heavy chain variable region and the kappa light chain variable region, respectively. The sequences of human immunoglobulin-derived heavy and kappa chain transcripts from hybridomas were obtained by direct sequencing of PCR products generated from poly (A +) RNA using the primers described above. The PCR products were cloned into pCR2.1 using the TA cloning kit (Invitrogen, Carlsbad, Calif.) and both strands were sequenced using the Prism dye-terminator sequencing kit and ABI 3730 sequencer (GENEWIZ, North Brunswick, N.J.). All sequences were analyzed by alignment with the Kataman antibody sequence program using DNASTAR software.

Table 2 above shows the amino acid sequences of the light and heavy chain variable regions of anti-VEGFR-1 antibody clones 6F9, 13G12, 15F11 and 18F 1. The underlined sections represent the sequences of the CDR1, CDR2 and CDR3 regions. Table 3 above shows the nucleotide sequences of cDNAs encoding the heavy and light chain variable regions of clones 6F9, 13G12, 15F11 and 18F 1.

Example 14:engineering and expression of human IgG1 anti-VEGFR-1 antibody

DNA sequences encoding the heavy and light chain variable regions of anti-VEGFR-1 antibody clones 6F9, 13G12, 15F11 and 18F1 were cloned into expression vectors by PCR for propagation. In the vector pEE Lou.1 (Lonza biologices pic, Slough, Berkshire, UK), the heavy chain variable region is fused in-frame to the human immunoglobulin heavy chain gamma constant region. The entire human light chain cDNA was directly cloned into the vector pEE12.1(Lonza biologices PLC, Slough, Berkshire, UK). The engineered immunoglobulin expression vectors are stably transfected into NSO myeloma cells by electroporation and selected in glutamine synthetase selective medium. Stable clones were screened for antibody expression by anti-Fc and VEGFR-1 specific binding ELISA. Positive clones were expanded in serum-free medium and cultured in spinner flasks (spinner flash) or bioreactors for a period of two weeks to produce antibodies. Full-length IgG1 antibody was purified by protein a affinity chromatography (Poros a, perstraction Biosystems inc., Foster City, CA) and eluted into a neutral buffered saline solution.

The above specification and examples are illustrative of the invention only and are not limiting thereof. Each of the disclosed aspects and embodiments of the invention may be considered independently or in combination with other aspects, embodiments and variations of the invention. Additionally, unless otherwise indicated, none of the steps in the methods of the present invention are limited by any particular order of practice. Modifications of the disclosed embodiments, which include the spirit and substance of the invention, are possible to those skilled in the art and are within the scope of the invention. In addition, all cited references are incorporated by reference in their entirety.

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Bohlen，Peter

Hicklin，Daniel J.

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Ala Val Ile Trp Phe Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp His Tyr Gly Ser Gly Ala His Ser Tyr Tyr Tyr Tyr Gly

100 105 110

Leu Asp Val Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120 125

<210>20

<211>126

<212>PRT

<213> Intelligent people

<400>20

Gln Ala Gln Val Val Glu Ser Gly Gly Gly Val Val Gln Ser Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser SerTyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp His Tyr Gly Ser Gly Val His His Tyr Phe Tyr Tyr Gly

100 105 110

Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210>21

<211>360

<212>DNA

<213> Intelligent people

<400>21

gaaattgtgt tgacgcagtc tccaggcacc ctgtccttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtggtagc agcagctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcaccgct cactttcggc 300

ggagggacca aggtggagat caaacgaact gtggctgcac catctgtctt catcttcccg 360

<210>22

<211>360

<212>DNA

<213> Intelligent people

<400>22

gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcacctct caccttcggc 300

caagggacac gactggagat taaacgaact gtggctgcac catctgtctt catcttcccg 360

<210>23

<211>360

<212>DNA

<213> Intelligent people

<400>23

gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcaccgct cactttcggc 300

ggagggacca aggtggagat caaacgaact gtggctgcac catctgtctt catctttccg 360

<210>24

<211>378

<212>DNA

<213> Intelligent people

<400>24

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cgtctggatt caccttcagt agttatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacggtgtat 240

ctgcaaatga acagcctgag agccgaggac acggctgtgt atcactgtac gagagatcac 300

tttggttcgg gggctcacta ctactactac tacggtatgg acgtctgggg ccaagggacc 360

acggtcaccg tctcctca 378

<210>25

<211>378

<212>DNA

<213> Intelligent people

<400>25

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagatcac 300

tatggttcgg gggctcacta ctactactac tacggtatgg acgtctgggg ccaagggacc 360

acggtcaccg tctcctca 378

<210>26

<211>378

<212>DNA

<213> Intelligent people

<400>26

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatggtttg atggaagtaa taaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagatcac 300

tatggttcgg gggctcactc ctactactac tacggtttgg acgtttgggg ccaagggacc 360

tcggtcaccg tctcctca 378

<210>27

<211>378

<212>DNA

<213> Intelligent people

<400>27

caggcgcagg tggtggagtc tgggggaggc gtggtccagt ctgggaggtc cctgagactc 60

tcctgtgcag cgtctggatt cgccttcagt agctacggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaatactat 180

gcagactccg tgaggggccg attcaccatc tccagagaca attccgagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac accgctgtgt attactgtgc cagagatcac 300

tatggttcgg gggtgcacca ctatttctac tacggtctgg acgtctgggg ccaagggacc 360

acggtcaccg tctcctca 378

<210>28

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic linker peptides

<400>28

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210>29

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic primers

<400>29

atggagtttg ggctgagctg 20

<210>30

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic primers

<400>30

tgccaggggg aagaccgatg g 21

<210>31

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic primers

<400>31

atggaaaccc cagcgcagct tctc 24

<210>32

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic primers

<400>32

cgggaagatg aagacagatg 20

<210>33

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221>MOD_RES

<222>(6)..(6)

<223> Val or Gly

<400>33

Arg Ala Ser Gln Ser Xaa Ser Ser Ser Tyr Leu Ala

1 5 10

<210>34

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221>MOD_RES

<222>(3)..(3)

<223> Thr or Ala

<400>34

Gly Phe Xaa Phe Ser Ser Tyr Gly Met His

1 5 10

<210>35

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221>MOD RES

<222>(4)..(4)

<223> Tyr or Phe

<220>

<221>MOD_RES

<222>(16)..(16)

<223> Lys or Arg

<400>35

Val Ile Trp Xaa Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Xaa

1 5 10 15

Gly

<210>36

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221>MOD_RES

<222>(3)..(3)

<223> Phe or Tyr

<220>

<221>MOD_RES

<222>(7)..(7)

<223> Ala or Val

<220>

<221>MOD_RES

<222>(9)..(9)

<223> Tyr, Ser, or His

<220>

<221>MOD_RES

<222>(11)..(11)

<223> Tyr or Phe

<220>

<221>MOD_RES

<222>(15)..(15)

<223> Met or Leu

<400>36

Asp His Xaa Gly Ser Gly Xaa His Xaa Tyr Xaa Tyr Tyr Gly Xaa Asp

1 5 10 15

Val

<210>37

<211>140

<212>PRT

<213> Intelligent people

<400>37

Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Glu Ser Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser

20 25 30

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

35 40 45

Gly Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro

65 70 75 80

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

85 90 95

Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr

100 105 110

Gly Ser Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

115 120 125

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

130 135 140

<210>38

<211>145

<212>PRT

<213> Intelligent people

<400>38

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly

1 5 10 15

Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln

20 25 30

Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

85 90 95

Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr His Cys Thr Arg Asp His Phe Gly Ser Gly Ala His Tyr Tyr Tyr

115 120 125

Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

130 135 140

Ser

145

<210>39

<211>140

<212>PRT

<213> Intelligent people

<400>39

Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Glu Ser Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser

20 25 30

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

35 40 45

Gly Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro

65 70 75 80

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

85 90 95

Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr

100 105 110

Gly Ser Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

115 120 125

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

130 135 140

<210>40

<211>145

<212>PRT

<213> Intelligent people

<400>40

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly

1 5 10 15

Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln

20 25 30

Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Asp His Tyr Gly Ser Gly Ala His Tyr Tyr Tyr

115 120 125

Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

130 135 140

Ser

145

<210>41

<211>140

<212>PRT

<213> Intelligent people

<400>41

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser

20 25 30

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

35 40 45

Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro

65 70 75 80

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

85 90 95

Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr

100 105 110

Gly Ser Ser Pro Leu Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

115 120 125

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

130 135 140

<210>42

<211>145

<212>PRT

<213> Intelligent people

<400>42

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly

1 5 10 15

Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln

20 25 30

Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Val Ile Trp Phe Asp Gly Ser Asn Lys Tyr Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Asp His Tyr Gly Ser Gly Ala His Ser Tyr Tyr

115 120 125

Tyr Tyr Gly Leu Asp Val Trp Gly Gln Gly Thr Ser Val Thr Val Ser

130 135 140

Ser

145

<210>43

<211>140

<212>PRT

<213> Intelligent people

<400>43

Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser

20 25 30

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

35 40 45

Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro

65 70 75 80

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

85 90 95

Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr

100 105 110

Gly Ser Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

115 120 125

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

130 135 140

<210>44

<211>145

<212>PRT

<213> Intelligent people

<400>44

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly

1 5 10 15

Val Gln Cys Gln Ala Gln Val Val Glu Ser Gly Gly Gly Val Val Gln

20 25 30

Ser Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe

35 40 45

Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala

65 70 75 80

Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Asp His Tyr Gly Ser Gly Val His His Tyr Phe

115 120 125

Tyr Tyr Gly Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

130 135 140

Ser

145

<210>45

<211>420

<212>DNA

<213> Intelligent people

<400>45

atggaaaccc cagcgcagct tctcttcctc ctgctactct ggctcccaga aagcaccgga 60

gaaattgtgt tgacgcagtc tccaggcacc ctgtccttgt ctccagggga aagagccacc 120

ctctcctgca gggccagtca gagtggtagc agcagctact tagcctggta ccagcagaaa 180

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 240

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 300

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcaccgct cactttcggc 360

ggagggacca aggtggagat caaacgaact gtggctgcac catctgtctt catcttcccg 420

<210>46

<211>435

<212>DNA

<213> Intelligent people

<400>46

atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt ccagtgtcag 60

gtgcagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct gagactctcc 120

tgtgcagcgt ctggattcac cttcagtagt tatggcatgc actgggtccg ccaggctcca 180

ggcaaggggc tggagtgggt ggcagttata tggtatgatg gaagtaataa atactatgca 240

gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac ggtgtatctg 300

caaatgaaca gcctgagagc cgaggacacg gctgtgtatc actgtacgag agatcacttt 360

ggttcggggg ctcactacta ctactactac ggtatggacg tctggggcca agggaccacg 420

gtcaccgtct cctca 435

<210>47

<211>420

<212>DNA

<213> Intelligent people

<400>47

atggaaaccc cagcgcagct tctcttcctc ctgctactct ggctcccaga aagcaccgga 60

gaaattgtgt tgacgcagtc tccaggcacc ctgtccttgt ctccagggga aagagccacc 120

ctctcctgca gggccagtca gagtggtagc agcagctact tagcctggta ccagcagaaa 180

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 240

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 300

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcaccgct cactttcggc 360

ggagggacca aggtggagat caaacgaact gtggctgcac catctgtctt catcttcccg 420

<210>48

<211>435

<212>DNA

<213> Intelligent people

<400>48

atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt ccagtgtcag 60

gtgcagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct gagactctcc 120

tgtgcagcgt ctggattcac cttcagtagc tatggcatgc actgggtccg ccaggctcca 180

ggcaaggggc tggagtgggt ggcagttata tggtatgatg gaagtaataa atactatgca 240

gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctg 300

caaatgaaca gcctgagagc cgaggacacg gctgtgtatt actgtgcgag agatcactat 360

ggttcggggg ctcactacta ctactactacggtatggacg tctggggcca agggaccacg 420

gtcaccgtct cctca 435

<210>49

<211>420

<212>DNA

<213> Intelligent people

<400>49

atggaagccc cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60

gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 120

ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 180

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 240

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 300

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcacctct caccttcggc 360

caagggacac gactggagat taaacgaact gtggctgcac catctgtctt catcttcccg 420

<210>50

<211>435

<212>DNA

<213> Intelligent people

<400>50

atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt ccagtgtcag 60

gtgcagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct gagactctcc 120

tgtgcagcgt ctggattcac cttcagtagc tatggcatgc actgggtccg ccaggctcca 180

ggcaaggggc tggagtgggt ggcagttata tggtttgatg gaagtaataa atactatgca 240

gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctg 300

caaatgaaca gcctgagagc cgaggacacg gctgtgtatt actgtgcgag agatcactat 360

ggttcggggg ctcactccta ctactactac ggtttggacg tttggggcca agggacctcg 420

gtcaccgtct cctca 435

<210>51

<211>420

<212>DNA

<213> Intelligent people

<400>51

atggaaaccc cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60

gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 120

ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 180

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 240

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 300

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcaccgct cactttcggc 360

ggagggacca aggtggagat caaacgaact gtggctgcac catctgtctt catctttccg 420

<210>52

<211>435

<212>DNA

<213> Intelligent people

<400>52

atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt ccagtgtcag 60

gcgcaggtgg tggagtctgg gggaggcgtg gtccagtctg ggaggtccct gagactctcc 120

tgtgcagcgt ctggattcgc cttcagtagc tacggcatgc actgggtccg ccaggctcca 180

ggcaaggggc tggagtgggt ggcagttata tggtatgatg gaagtaataa atactatgca 240

gactccgtga ggggccgatt caccatctcc agagacaatt ccgagaacac gctgtatctg 300

caaatgaaca gcctgagagc cgaggacacc gctgtatatt actgtgccag agatcactat 360

ggttcggggg tgcaccacta tttctactac ggtctggacg tctggggcca agggaccacg 420

gtcaccgtct cctca 435

<210>53

<211>20

<212>DNA

<213> Intelligent people

<400>53

atggagtttg ggctgagctg 20

<210>54

<211>21

<212>DNA

<213> Intelligent people

<400>54

tgccaggggg aagaccgatg g 21

<210>55

<211>24

<212>DNA

<213> Intelligent people

<400>55

atggaaaccc cagcgcagct tctc 24

<210>56

<211>20

<212>DNA

<213> Intelligent people

<400>56

cgggaagatg aagacagatg 20

Claims (2)

1. Use of an isolated human monoclonal antibody in combination with a chemotherapeutic agent for the manufacture of a medicament for reducing breast tumor growth in a mammal, wherein said antibody specifically binds to VEGFR-1 and comprises:

(i) LCDR1 with sequence SEQIDNO 4;

(ii) LCDR2 with sequence SEQ ID NO. 2;

(iii) LCDR3 with sequence SEQIDNO 3;

(iv) HCDR1 with sequence SEQ ID NO. 11;

(v) HCDR2 having the sequence of SEQ ID NO. 12; and

(vi) HCDR3 with the sequence of SEQ ID NO. 13,

wherein the chemotherapeutic agent is 5-fluorouracil and folinic acid; or the chemotherapeutic drug is paclitaxel, doxorubicin or cyclophosphamide.

2. The use of claim 1, wherein said chemotherapeutic agent and said antibody are administered simultaneously, sequentially or separately.