SK15302002A3 - Treatment of non-solid mammalian tumors with vascular endothelial growth factor receptor antagonists - Google Patents

Abstract

A method of inhibiting the growth of non-solid tumor cells that are stimulated by a ligand of vascular endothelial growth factor in human patients, comprising treating the human patients with an effective amount of a vascular endothelial growth factor receptor antagonist.

Description

Technical field

The invention relates to a VEGFR receptor antagonist and its use in the manufacture of a medicament for inhibiting the growth of non-solid tumor cells that are stimulated for a mammal by a vascular endothelial growth factor receptor (VEGFR) ligand.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of death after heart attacks. Significant progress has been made in developing new therapies for the treatment of these devastating diseases. Most of this progress is due to a better understanding of cell proliferation in both normal and tumor cells.

Normal cells proliferate by highly controlled activation of growth factor receptors by their corresponding ligands. Examples of such receptors are tyrosine kinase receptors for growth factors.

Tumor cells also proliferate by activating growth factor receptors, but lose proper control of normal proliferation. Loss of control may be due to numerous factors, such as overexpression of growth factors and / or receptors, and autonomous activation of biochemical pathways regulated by growth factors.

Some examples of receptors involved in tumorigenesis are receptors for vascular endothelial growth factor (VEGFR), platelet-derived growth factor (PDGFR), insulin-like growth factor (IGFR), nerve growth factor (NGFR), and fibroblast growth factor (FGF) .

During embryonic development, hematopoietic and early endothelial cells (angioblasts) develop from a common precursor cell known as hemangioblast. Because of this common origin, several signaling pathways are common to both hematopoietic and vascular cells. One such pathway is the VEGFR signaling pathway. VEGF receptors (VEGFR) include FLT-1 sequenced by Shibuya M et al. Oncogene 5, 519524 (1990) (VEGFR-1); KDR described in PCT / US92 / 01300, registered February 20, 1992 and described by Terman et al., Oncogene 6: 1677-1683 (1991); and FLK-1 sequenced by Mathews W. et al., Proc. Natl. Acad. Sci. USA, 88: 9026-9030 (1991) (KDR and FLK-1 are collectively called VEGFR-2).

Unless otherwise stated or otherwise derived from the context, this specification follows the usual literary nomenclature of VEGF receptors. DRC refers to the human form of VEGFR-2. FLK-1 refers to the murine homolog VEGFR2. FLT-1 is different but related to the KDR / FLK-1 receptor.

VEGFR binds to several soluble factors including vascular endothelial growth factor (VEGF), which exhibits proliferative and migratory effects on the endothelium. VEGFR-2 was thought to be exclusively expressed by edothelial cells. Recently, however, VGFR has been shown to be present on a subset of multipotent hematopoietic stem cells (1). Some studies have shown that some leukemia cells also express VEGFR-2 (2).

VEGFR-2 and VEGFR-1 are two primary signaling tyrosine kinase receptors that mediate various biological effects of VEGF. Although the binding affinity of VEGFR-1 to VEGF is very high with Kd values of 10-70 pM (5), most studies have shown that VEGFR-2 is a critical receptor for the transmission of cellular signals for endothelial cell proliferation and differentiation (6). VEGFR-1 appears to be more important for vascular remodeling. The relative importance of VEGF receptors in the regulation of vasculogenesis and angiogenesis has been demonstrated in studies in which genes for VEGFR-2 and VEGFR-1 in mouse embryonic stem cells have been disrupted by homologous recombination. VEGFR-2 deficient mice had drastic defects in vasculogenesis, angiogenesis, and hematopoiesis (7). In contrast, VEGFR-1 knockout mice developed abnormal channels, suggesting the role of this receptor in regulating cell-cell or cell-matrix interactions (8).

Inhibition of angiogenesis by disruption of VRGFR-2 signaling results in growth inhibition and metastasis of solid tumors. Includ: IAD 'môriokľoháTňä ^{_ __} The antibodies XMo7Íb) ^_ ^ ňéútraližújlícä the murine VEGFR-2 inhibited tumor growth and metastasis in mouse models (9,10). In addition, glioblastoma growth was inhibited in mice dominantly negative for VEGFR-2 (11). Such inhibition of tumor growth is attributed to inhibition of angiogenesis, effectively limiting the blood supply of the tumor.

Leukemias arise from hematopoietic stem cells having self-renewal capacity, while some less aggressive leukemias, such as chronic leukemias, appear to arise from more mature, determined hematopoietic progenitor cells.

In any event, leukemia cells do not require independent blood supply. Therefore, leukemias and other non-solid tumors are not susceptible to the treatments described above.

Some studies have shown that VEGF is almost always expressed by all established leukemic cell lines as well as freshly isolated human leukemias, including the well-studied leukemic cell line HL-60 (2,3). Using RT-PCR, several studies have shown that VEGFR-2 and VEGFR-1 are only expressed by certain human leukemias (2,3). However, none of these studies demonstrated that VEGF expression is associated with any parallel expression of surface VEGFR-2 / VEGFR-1 or a functional response.

Current cancer treatment traditionally involves chemotherapy or radiation. However, this treatment is not always effective, especially for a long time.

Therefore, there is a need for new methods of treating single-cell tumors. It is an object of the present invention to provide such methods.

Description of the invention

This and other objectives, as will be apparent to those of ordinary skill in the art, have been achieved by providing a method of inhibiting the growth of non-solid tumors that are stimulated by a mammalian VEGFR ligand. The method comprises treating a mammal with an effective amount of a VEGR antagonist.

In another embodiment, the method of the present invention comprises treating VEGFR and a chemotherapeutic agent.

In yet another embodiment, the method of the present invention comprises treating human patients with a combination of an effective amount of a VEGFR antagonist and radiation.

Brief description of the figures

Figure 1. Human chloromas express FLT-1 / VEGFR-1 and KDR / VEGFR-2. Sections of human chloromas were immunohistochemically stained as described in the Methodology. A: Control IgG staining only slightly non-specific (magnification: 400x); B: Factor VIII staining, showing specific blood vessel staining (magnification: 100x); C: KDR / VEGFR-2 staining in the leukemic cell region (magnification: 400x); D: FLT-1 / VEGFR-1 expression, also detected on a leukemic cell subset (magnification: 400x). E and F: VEGF expression showing specific leukemic cell (E) and positive control, pericyt staining (white arrow, F) (magnification: 400x). These results represent detailed samples:

Figure 2A: Expression of VEGFR-2 by leukemic cell lines. The HL-60 and HEL cell lines (as well as 5 primary leukemias) expressed KDR / VEGFR-2 as detected by RT-PCR. K562 cells were KDR negative, βactin was used as an internal control.

Figure 2B: VEGF ₁₆ s induced dose-dependent VEGFR-2 phosphorylation on VEGFR-2 positive leukemia cell lines. Total cell lysates from HL-60 and HEL cells were immunoprecipitated with an antiphosphotyrosine antibody and subsequently analyzed by Western blotting using anti-VEGFR-1 (FLT-1) or VEGFR-2 (KDR) antibodies as described in the Methodology. Fibroblasts were used as negative controls and showed no signs of VEGFR-1 or VEGFR-2 phosphorylation. The results presented represent 3 separate experiments.

Figure 3. VEGF ₆ s induced proliferation of a subset of leukemias, a KDR / VEGFR-2 mediated effect. VEGF _ies induced a significant (*, p less than 0.003) increase in proliferation of all KDR / VEGFR-2 + leukemias (2 cell lines and 2 primary leukemias are shown). This effect can be blocked by neutralizing MoAbs directed against KDR / VEGFR-2 (**, p less 0.04) as described in the Methodology. The results presented represent 3 separate experiments.

Figure 4. High affinity tyrosine kinase inhibitor for KDR / VEGFR-2 blocked VEGF _165- induced leukemia cell proliferation. The tyrosine kinase inhibitor AG1433 significantly blocked leukemia cell proliferation (*, p less than 0.05), confirming that on leukemic cells mitogenic effects are mediated by KDR / VEGFR-2. These results represent 2 separate experiments.

Figure 5. VEGF ₆₅ induces MMP secretion by leukemic cells. A: Zymographic analysis of leukemic cell supernatants, with or without VEGF ₁₆₅ stimulation for 24 hours. B: Quantification of gelatinolytic activity detected in culture supernatants. Incubation of leukemia cells with VEGF _1S5 for 24 hours significantly influenced (p less than 0.05) the level of MMP released into the supernatant as detected by zymography. The results presented represent 3 separate experiments.

Figure 6. VEGF ₁₆₅ induces a dose-dependent (50-200 ng / ml) migration of leukemic cells in matrigel coated wells, a process mediated by KDR / VEGFR-2 and FLT-1 / VEGFR-1. Migration of HL-60 cells and 4 primary leukemias in matrigel coated wells is shown depending on 200 ng / ml VEGF for ₁₆ sec. This process requires MMP activity, as demonstrated by the effects of synthetic MMPi. A monoclonal antibody against KDR / VEGFR-2 partially blocked leukemic cell migration, but incubating cells with both FLT-1 / VEGFR-1 and KDR / VEGFR-2 was more effective than using either antibody alone (**, p less than 0.05) ). These results represent 3 independent experiments.

Figure 7. VEGF ₆₅ induced HL-60 cell migration is blocked by AG1433. A synthetic protein kinase inhibitor AG1433 (10 µM used) blocked VEGF ₆₅ induced HL-60 cell migration (*, p less than 0.04). These results represent 2 different experiments and each experiment was performed in a triplet.

Figure 8. Injection of HL-60 NOD-SCID cells into mice induces high concentrations of human but not mouse VEGF in mouse plasma. Mice were injected with 1x10 ⁶ HL-60 cells (iv) and plasma levels of human (A) and mouse (B) VEGF were measured by ELISA at various time intervals after injection. The results represent 2 separate experiments. B. Survival of mice (%) after injection of HL-60 cells (iv). Mice were injected with 1x10 ⁶ HL-60 cells (iv) and 3 days after injection of leukemic cells (n = 5) ip with 400 µg IMC-1C11 (neutralizing MoAb to human VEGFR-2 / KDR) or PBS / BSA ( n = 8) three times a week. One mouse was sacrificed for histological analysis. The results presented represent 2 separate experiments.

Figure 9. Histology of liver (A, B) and spleen (C, D) of mice after injection of HL-60 cells. Untreated (control) mice showed signs of liver metastatic involvement (white and red arrows, B) and spleen (D). In contrast, for mice treated with IMC-1C11, no leukemic cells were detected in the liver (A) and there were signs of apoptosis (blue arrows, C) in the spleen, but a normal histological picture.

Figure 10. In VEGFR-2 positive leukemias, VEGF can promote leukemia cell growth through paracrine (increased bone marrow and endothelial cell mass) and autocrine mechanisms. Antibodies against human VEGFR-2 (arrow) can block the autocrine loop induced by VEGF-derived leukemia.

Figure 11. Nucleotide and amino acid sequences of pIC11.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an improved method of treating non-solid tumors, particularly non-solid malignant tumors, of human patients.

Non-solid tumor cells

Non-solid tumor cells include tumors that affect hematopoietic structures, including components of the immune system. Some examples of non-solid tumor cells include leukemias, multiple myelomas and lymphomas. These tumor cells generally appear in bone marrow and peripheral circulation.

The types of non-solids that can be treated in accordance with the invention are any non-solid tumors that are stimulated by a VEGFR ligand. The VEGFR family of receptors includes, for example, VEGFR-2 (KDR, flk-1) and VEGFR-1 (flt-1). Some examples of ligands that stimulate VEGFR include VEGF.

Some examples of non-solid tumors include leukemias, multiple myelomas and lymphomas. Some examples of leukemias include acute myelocytic leukemia (AML), chronic myelocytic leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), erythrocytic leukemia or monocytic leukemia. Some examples of lymphomas include lymphomas within Hodgkin's and NonHodgkin's disease.

Non-solid tumors at concentrations or at concentrations that can express VEGFR in normal may overexpress. VEGFRs are at least 10, 100 or 1000 times higher than normal concentrations.

VEGFR antagonists

The non-solid tumors of the present invention are treated with a VEGFR antagonist. For purposes of this specification, a VEGFR antagonist is any molecule that inhibits VEGFR ligand stimulation. Such inhibition of stimulation inhibits the growth of cells that express VEGFR.

The mechanism of inhibition is not explained in the present invention. However, VEGFR tyrosine kinases are generally activated through phosphorylation reactions. Therefore, antagonists of the invention generally inhibit VEGFR phosphorylation. Therefore, the determination of phosphorylation is useful for predicting whether these antagonists will be useful in the present invention.

The growth of non-solid tumors is sufficiently inhibited in the patient to prevent tumor progression (i.e., growth, invasiveness, metastasis and / or recurrence of non-solid tumors). The VEGFR antagonist of the present invention may be cytostatic, that is, it inhibits the growth of a non-solid tumor. The ERGR antagonist is preferably cytolytic, that is, destroys the tumor.

VEGFR antagonists include biological molecules or small molecules. Biological molecules include all lipids and polymers of monosaccharides, amino acids and nucleotides having a molecular weight greater than 450. Thus, the biological molecules include, for example, oligosaccharides and oligopeptides, polypeptides, oligonucleotide peptides and polynucleotides.

polysaccharides, proteins, and

oligonucleotides

DNA and RNA.

Biological molecules further include derivatives of any of the molecules described above. Derivatives of biological molecules include, for example, lipid and glycosylation derivatives of oligopeptides, polypeptides, peptides and proteins.

Derivatives of biological molecules further include lipid derivatives of oligosaccharides and polysaccharides, for example lipopolysaccharides.

Biological molecules are the most typical antibodies or functional equivalents of antibodies. Functional equivalents of antibodies have binding characteristics comparable to antibodies and inhibit the growth of cells that express VEGFR. Such functional equivalents include, for example, chimerized, humanized and single chain antibodies, as well as fragments thereof.

The functional equivalent of the antibody is preferably a chimerized or humanized antibody. The chimerized antibody comprises a non-human antibody variable region and a human antibody constant region. The humanized antibody comprises a non-human antibody hypervariable region (CDR). The variable region other than the hypervariable region, for example, the framework variable region and the constant region of a humanized antibody, is from a human antibody.

For the purposes of this application, variable and hypervariable regions of non-human antibodies derived from antibodies produced by any non-human mammal that can produce monoclonal antibodies may be suitable. Suitable examples of non-human mammals include, for example, rabbits, rats, mice, horses, goats or primates. Mice are preferred.

Functional equivalents further include antibody fragments having binding characteristics that are the same as or comparable to the binding characteristics of the whole antibody. Suitable antibody fragments include any fragment that contains a sufficient portion of the hypervariable (i.e., determining complementarity) region for specific binding and with sufficient affinity for VEGFR to inhibit the growth of cells that express such receptors.

Such fragments may contain, for example, one or both Fab fragments or F (ab ') ₂ fragments. Preferably, antibody fragments comprise all six complementarity determining regions of the entire antibody, although functional fragments containing less than all such regions, such as three, four, or five CDRs, are also included.

Preferred fragments are single chain antibodies or Fv fragments. Single chain antibodies are polypeptides that comprise at least an antibody heavy chain variable region linked to a light chain variable region with or without an interface member. Thus, the Fv fragment comprises the entire combining site of the antibody. These chains can be produced in bacteria or in eukaryotic cells.

The antibodies and functional equivalents may be representatives of any class of immunoglobulins, such as IgG, IgM, IgA, IgD or IgE, or subclasses thereof. Preferred antibodies are antibodies of the IgG1 subclass. Functional equivalents may be equivalents by a combination of any of the above classes and subclasses.

Antibodies can be generated from the desired receptor by methods well known in the art. Receptors are either commercially available or can be isolated by well known methods. Methods for isolating and purifying KDR are known, for example, in PCT / US92 / 01300. Methods for isolating and purifying flk-1 can be found in Proc Natl Sci 88: 9026-30 (1991). Methods for isolating and purifying flt-1 can be found in Oncogene 5: 519-24.

Methods for producing monoclonal antibodies include the immunological method described by Kohler and Milstein in Nature 256, 495-497 (1975) and Campbell in Monoclonal Antibody Technology, The Production and Characterization of Rodent and Human Hybridomas in Burdon et al, Eds, Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13, Elsevier Science Publishers, Amsterdam (1985). The recombinant DNA method described by Huse et al. It is also suitable in Science 246, 1275-1281 (1989).

Briefly, in order to produce monoclonal antibodies, the host mammal is inoculated with the receptor or receptor fragment as described above and then optionally re-inoculated. For a receptor fragment to be useful, it must contain sufficient amino acid residues to define the epitope of the molecule to be detected. If the fragment is too short to be immunogenic, it can be conjugated to a carrier molecule. Some suitable carrier molecules include hemocyanin sticks and bovine serum albumin. Conjugation may be accomplished by methods known in the art. One such method combines the cysteine residue of the fragment with the cysteine residue of the carrier molecule.

Otherwise, mammalian mammals are taken away only a few days after the final inoculation. Spleen cell suspensions are fused to a tumor cell. The resulting hybridoma cells that express antibodies are isolated, cultured, and maintained in culture.

Antibodies that are substantially of human origin can be produced in transgenic mammals, especially transgenic mice that are genetically modified to express human antibodies. Methods for generating chimeric and humanized antibodies are well known in the art. Methods for generating chimeric antibodies include, for example, those described in US patents Boss (Celltech) and Cabill (Genetech). See U.S. Pat. 4816397 and 4816567, respectively. Methods of making humanized antibodies are described, for example, in US Patent Winter, no. 5,225,539th

A preferred method of humanizing antibodies is termed CDRgrafting. In this method, regions of a murine antibody that are directly involved in antigen binding, i.e., complementarity determining regions or CDRs, are grafted onto human variable regions to form reshaped human variable regions. The fully humanized variable regions are then joined to human constant regions to produce fully fully humanized antibodies.

In order to generate fully humanized antibodies that bind well to the antigen, it is advantageous to design the reshaped human variable regions with caution. The human variable regions to which the CDRs will be grafted should be carefully selected, and it is usually necessary to make several amino acid changes at critical positions within the framework regions (FRs) of the human variable regions.

For example, reshaped human variable regions may comprise up to ten amino acid changes in the FR of the selected human light chain variable regions and twelve amino acid changes in the FR of the selected human heavy chain variable regions. DNA sequences encoding these reshaped human heavy and light chain variable region genes are linked to DNA sequences encoding human heavy and light chain constant region genes, preferably γ1 and κ. The reshaped humanized antibody is then expressed in mammalian cells and its affinity for its target is compared to that of the corresponding murine antibody and the chimeric antibody.

Methods for selecting humanized antibody residues to be substituted and for substitution generation are well known in the art. See, for example, Co et al., Nature 351, 501-502 (1992); Queen et al. , Proc. Natl. Acad. Sci. 86, 100029-1003 (1989) and Rodrigues et al. , Int. J. Cancer, Supplement 7: 45-50 (1992). A method for humanizing and reshaping 225 anti-EGFR monoclonal antibodies is described by Goldstein et al. In PCT application WO 96/40210. This method can be. adapted to generate humanized and reshaped antibodies against other receptor tyrosine kinase growth factors.

Methods for generating single chain antibodies are also well known in the art. Some suitable examples include, for example, European Patent Application Wels et al. 502,812 and Int. J. Cancer 60: 137-144 (1995). Single chain antibodies can also be prepared by screening phage libraries. See below.

Other methods of generating functional equivalents described above are disclosed in PCT application WO 93/21319, European Patent Application 239 400, PCT application WO 89/09622, European Patent Application 338 745, US Patent 5 658 570, US Patent 5 693 780 and Patent Application EP 332 424.

Preferred VEGFR antibodies are chimerized, humanized, and single chain antibodies derived from a murine antibody for which the amino acid and nucleotide sequences of the hypervariable regions for the heavy and light chains are listed below. The chimerized antibody and single chain antibodies may be performed in accordance with standard methods such as those described below. The humanized antibody can be prepared in accordance with the method described in Example IV of PCT application WO 96/40210, which is hereby incorporated by reference.

The sequences of the hypervariable (CDR) regions of the light and heavy chains are reproduced below. The nucleotide sequences are listed below the amino acid sequence.

Hypervariable heavy chain (VH) regions:

CDR1

SEQ ID NO: 1

Gly Pne Asn Lys Asp Phe Tyr Met His

SEQ ID NO: 9 ggcttcaaca ttaaagactt ctatatgcac

CDR2

SEQ ID NO: 2

Asp Pro Glu Asn Gly Asp Ser Asp Tyr Ala Pro Lys Phe Gin Gly

SEQ ID NO: 10 tgattgatc ctgagaatgg tgattctgat tatgccccga agttccaggg c

CDR3

SEQ ID NO: 3

Tyr Tyr Gly Asp

SEQ ID NO: 11 actacgaagg ctac

Hypervariable light chain (VL) regions:

___ CDR1 _________._ _ .............. ..-........... - - ------------. - - ---------------- ------ - - - - - ----- .---- SEQ ID NO: 4

Ser Ala Ser Ser Ser Val Ser Tyr Met His

SEQ ID NO: 12 agtgccagct caagtgtaag ttacatgcac

CDR2

SEQ ID NO: 5

Ser Thr Ser Asn Leu Ala Ser

SEQ ID NO: 13 agcacatcca acctggcttc t

CDR3

SEQ ID NO: 6

Gin Gin Arg. Ser Ser Tyr Pro Phe Thr

SEQ ID NO: 14 cagcaaagga gtagttaccc attcacg

The entire light and heavy chain sequences are listed below. The nucleotide sequence is shown below the amino acid sequence.

Heavy chain

SEQ ID NO: 7

gin wall Lys Leu gin gin Ser Gly Ala Glu Leu wall Gly Ser Gly Ala Ser wall Lys Leu Ser Cys Thr Thr Ser Gly Phe same time Ile Lys Asp Phe Tyr Met His Trp wall Lys gin Arg for Glu gin Gly Leu Glu Trp Ile Gly Trp Ile Asp for Glu same time Gly Asp Ser Asp Tyr Ala for Lys Phe gin Gly Lys Ala Thr Met Thr Ala Asp Ser Ser Ser same time Thr Ala Tyr Leu gin Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala wall Tyr Tyr Cys same time Ala Tyr Tyr Gly Asp Tyr Glu Gly Tyr Trp Gly gin Gly Thr Thr wall Thr wall Ser Ser

SEQ ID NO: 15

caggfccaagc tgcagcagtc tggggcagag caggggcctr agtcaaattg 60 tcctgcacaa ct fc c tggc fc t caacatfcaaa tgcactgggt gaagcagagg 120 cctgaacagg gcctggagtg gattggatgg agaatggtga tfcctgattat 180 gccccgaagt tccagggcaa ggccaccatg catcctccaa cacagcctac 240 ctgcagctca gcagcctgac at c tgaggac afcfcactgtaa tgcatactat 300

cttgtggggt gacttctata attgatcctg actgcagact actgccgtct ggtgactacg aaggctactg ccgtctcctc and 351 gggccaaggg accacggtca

The light chain of SEQ ID NO: 8

Asp Ile Glu Leu Thr gin SerPro Ala and Met Met Ala Ser for Gly Glu Lys wall Thr Ile Thr Cys Ser Ala Ser Ser Ser wall Ser Tyr Met His Trp PNE gin gin Lys For Gly Thr Ser For Lys Leu Trp Tie Tyr Ser Thr Ser same time Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr White Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Arg Ser Ser Tyr for Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu White Lys

SEQ ID NO: 16 gacatcgagc tcactcagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60 ataacctgca gtgccagctc aagtgtaagt tacatgcact ggttccagca gaagccaggc 120 acttctccca aactctggat ttatagcaca cttctggagt ccctgctcgc 180 ttcagtggca gtggatctgg gacctcttac tcagccgaat ggaggctgaa 240 gatgctgcca cttattactg ccagcaaaggagtagttacc cattcacgtt cggctcgggg 300 accaagctgg aaataaaa tccaacctgg tctctcacaa

Other examples of biological molecules that are useful as antagonists include soluble receptors. (Exp. Cell Res.

241: 1,161-170; Proc. Natl. Acad. Sci. 92:23, 10457-61).

In addition to the biological molecules discussed above, small molecules may also be useful as antagonists in the present invention. Any molecule that is not a biological molecule is considered a small molecule in this specification. Some examples of small molecules include organic compounds, organometallic compounds, salts of organic and organometallic compounds, carbohydrates, amino acids, and nucleotides. Small molecules further include molecules that would otherwise be considered biological molecules unless their molecular weight is greater than 450. Thus, the small molecules may be lipids, oligosaccharides, oligopeptides and oligonucleotides and derivatives thereof having a molecular weight of 450 or less.

It is emphasized that small molecules can have any molecular weight. They are called small molecules because they typically have a molecular weight of less than 450. Small molecules include compounds found in nature as well as synthetic compounds. Preferably, small molecules inhibit the growth of non-solid tumor cells that express VEGFR tyrosine kinase.

Some examples of small molecules that are useful as VEGFR molecules include the quinazolines, quinolines, and cinolines described by Hennequin et al. J. Med. Chem. 42, 5369-5389 (1999). See also Annie et al. , Journal of Aquired Immune Deficiency Syndromes and Human Retrovirology 17, A41 (1998).

Administration of VEGFR antagonists

The present invention includes administering to a human patient an effective amount of a VEGFR antagonist. Administration of the VEGFR antagonist may be accomplished by a variety of routes including systemic administration by parenteral and enteral routes. The VEGFR antagonist of the present invention can be easily administered intravenously (e.g., by intravenous injection), which is the preferred route of administration. Intravenous administration can be accomplished by contacting VEGFR antagonists with a suitable pharmaceutical carrier (vehicle) or excipient by a person skilled in the art.

The VEGFR antagonists of the present invention significantly inhibit the growth of non-solid tumor cells when administered to a human patient in an effective amount. As used herein, an effective amount is an amount effective to achieve the specified result of inhibiting the growth of non-solid tumors.

Optimal dosages of VEGFR antagonists can be determined by the physician depending on a variety of parameters including age, sex, weight, severity of the disease to be treated, the type of antagonist to be administered, and the route of administration. Generally, a serum concentration of the antagonist that permits saturation of the target receptor is desirable. For example, for polypeptides and antibodies, a concentration of about 0.1 nM is normally sufficient. For example, an antibody dose of 100 mg / m ² provides a serum concentration of about 20 nM for eight days.

As a guide, doses of 10-300 mg / m ^{2 of} antibody may be administered weekly. Equivalent doses of antibody fractions should be used at more frequent intervals to maintain serum levels at a concentration that allows receptor saturation.

Combination therapy

In one preferred embodiment, the non-solid tumor may be treated with an effective amount of a combination of a VEGFR antagonist as described above and chemotherapeutic agents, radiation, or combinations thereof.

Examples of chemotherapeutic agents include alkylating agents, for example, nitrogen mustard, ethyleneimine compounds, and alkyl sulfonates; antimetabolites such as folic acid or pyrimidine antagonists; mitosis inhibitors such as vinea alkaloids and podophyllotoxin derivatives; cytotoxic antibiotics; compounds that damage or interfere with DNA expression.

Specific examples of chemotherapeutic agents or chemotherapy include cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine, (nitrogen mustard), streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (cytriorcinin, prounamycin, munacarin, prounamycin), daunorcinin, prounamycin, daunorcinin, etoposide, methotrexate, 5-fluorouracil, vinblastine, vineristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere), aldesleukin, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludicabamine, pludicabine, pludicabine, pludicabine, pludicabine, pludicabine , leuprolide, megestrol, mercaptopurine, mitotane, pegaspargase, pentostatin, pipobroman, testolactone, thioguanine, tamoxifen, teniposide, tiotepa, uracil mustrad, vinorelbine, chlorambucil, taxol and combinations thereof.

Administration of chemotherapeutic agents may be accomplished by a variety of routes including systemic administration by parenteral and eneteral routes as described above.

In yet another embodiment, the non-solid tumor can be treated with an effective amount of a VEGFR antagonist in combination with radiation. The source may be either external or internal to the patient to be treated. When the source is external, the treatment is known as external beam radiation therapy (EBRT). When the radiation source is internal, the treatment is called brachytherapy (BT).

The irradiation is applied in accordance with well known standard techniques with standard equipment made for this purpose, such as AECL Theratron and Varian Clinac. The dose of radiation depends on a number of factors, as is well known in the art. Such factors include the type of organ to be treated, healthy organs in the radiation pathway that may be inadvertently or undesirably affected, the patient's tolerance to radiation therapy, and the area of the body to be treated. The dose will typically be between 1 and 100 Gy and more particularly between 2 and 80 Gy. Some doses that have been described include 35 Gy per spinal cord, 15 Gy per kidney, 20 Gy per liver and 65-80 Gy per prostate. However, it should be emphasized that the invention is not limited to any particular dose. The dose will be determined by the physician in accordance with the particular factors in the situation, including those mentioned above.

The distance between the source of external radiation and the point of entry into the patient may be any distance that represents an acceptable balance between killing target cells and minimizing side effects. The external radiation source is typically between 70 and 100 cm from the point of entry into the patient.

Brachytherapy is generally accomplished by placing the radiation source in the patient. The radiation source is typically approximately 0-3 cm from the tissue to be treated. Known techniques include interstitial, intercavitary and superficial brachytherapy. The radioactive grains may be implanted permanently or temporarily. Some typical radioactive atoms that are used in permanent implants include iodine 125 and radon. Some typical radioactive atoms that are used as temporary implants include radium, cesium 137 and iridium 192. Some other radioactive atoms that are used in brachytherapy include americium 241 and gold 198.

The radiation doses for brachytherapy may be the same as those mentioned above for traditional external beam therapy. In addition to the factors mentioned above for determining the dose for radiation therapy by external beam, the nature of the radioactive atom is also taken into account for determining the dose of brachytherapy.

In a preferred embodiment, there is synergy in the treatment of non-solid tumors of human patients with a VEGFR antagonist and chemotherapeutic agents or radiation, or combinations thereof. In other words, inhibition of tumor growth by a VEGFR antagonist is enhanced when combined with chemotherapeutic agents or radiation or combinations thereof. Synergy can be demonstrated, for example, by greater inhibition of non-solid tumor growth by combination therapy than might be expected for treatment with either a VEGFR antagonist, a chemotherapeutic agent, or radiation alone. Synergy is preferably demonstrated by remission of a cancer, where remission is not expected from treatment with a VEGFR antagonist, a chemotherapeutic agent alone, or radiation alone.

The VEGFR antagonist is administered before, during, or after initiation of chemotherapy or radiation therapy, as well as a combination of such administration, i.e. before and during, before and after, during and after, or before, during and after initiation of chemotherapy treatment, and / or radiation therapy. For example, when the VEGFR antagonist is an antibody, it is typically administered between day 1 and day 30, preferably between day 3 and day 20, more preferably between day 5 and day 12, before starting treatment with radiation and / or chemotherapeutic agents.

The examples below demonstrate that certain subgroups of leukemias not only produce VEGF but also express functional VEGFR-2 in vivo and in vitro, resulting in the formation of an autocrine loop that enhances the proliferation and migration of leukemic cells. Approximately 50% of the investigated freshly isolated acute myeloblastic leukemias (AML) express mRNA and protein for VEGFR-2. VEGFigs induced VEGFR-2 phosphorylation and increased leukemia cell proliferation. VEGF ₆₅ also induced expression of metalloproteinase-9 (MMP-9) by leukemic cells and promoted their migration across the reconstituted basement membrane.

The neutralizing MoAb to VEGFR-2 and the specific inhibitor of VEGFR-2 synthesis blocked leukemia cell proliferation of human leukemia leukemia cells resulted in significant VEGF ₁₆ mediated VEGF-induced migration. Xenotransplantation of immunocompromised NOD-SCID mice increased human but not murine VEGF in plasma and death of inoculated mice for two weeks. Injection of a human specific neutralizing monoclonal antibody that selectively blocks signaling by human

VEGF-2, inhibited the proliferation of xenotransplanted human leukemias and increased mouse survival during the experiment.

All chemicals were obtained from Sigma unless otherwise noted.

DETAILED DESCRIPTION OF THE INVENTION

Example 1. Inhibition of VEGFR

Sampling of primary AML and isolation of mononuclear cells by Ficoll gradient

Peripheral blood samples from patients with leukemia (diagnosis of acute leukemia) were collected by phlebotomy, diluted half with Hank buffer (Gibco BRL) and overlaid with 5 ml Lymhoprep (Ficoll, Accurate Chemical and Scientific Corporation, NY). Each sample was centrifuged at 4000 rpm for 30 minutes and the interphase mononuclear cells were collected in a fresh tube and washed twice with Hank buffer for 5 minutes at 2500 rpm. The resulting cell pellet was finally resuspended in RPMI / 10% FCS.

Cell culture

The three AML cell lines used in this study, HL-60 (promyelomonocytic), HEL (megakaryocytic) and K562 (erythroid) were cultured in RPMI with 10% FCS, penicillin (100 U / ml), streptomycin (100 µg / ml) and fungizone (0.25 µg / ml). Before incubation with VEGF and / or antibodies / protein kinase inhibitor, cells were cultured for 16 hours to overnight only in RPMI alone.

After collection and isolation of peripheral blood mononuclear cells, primary leukemic cells were cultured overnight in RPMI / 10% FCS to eliminate possible contamination with monocytes / macrophages that adhere more readily to tissue culture vessels. The cells remaining in suspension are mainly leukemic blasts. These leukemic cells were subsequently transferred to another vessel and before adding VEGF and / or antiVEGFR antibodies, the cells were cultured without serum for 16 hours to overnight in RPMI alone.

For proliferation experiments, cells were cultured in 6-well plates (Corning), at a cell density of 1x10 ⁵ cells / well in serum-free RPMI. Cells were treated (10-50 ng / ml VEGF) or untreated (medium alone) and cultured in the presence or absence of 500 ng to 1 µg / ml immunoneutralizing MoAb to KDR / VEGFR-2, IMC-1C11 (12), or MoAb to FLT-1 / VEGFR-1, clone 6.12 (both from ImClone Systems Incorporated). A synthetic protein kinase inhibitor with high affinity for VEGFR-2 (AG1433, IC ₅₀ ) was also used to confirm the mitogenic effects of VEGF on leukemia cell lines that were mediated by VEGFR-2.

9.3 µM, Calbiochem, La Jolla, CA) at a concentration of 10 µM as recommended by the manufacturers. After 24, 48 and 96 hours, live cells (determined by trypan blue exclusion) were counted in a triplet using a haemocytometer. Each experiment was performed in a triplet and experiments with leukemia cell lines were repeated three times.

RNA extraction, cDNA synthesis and RT-PCR

Total RNA was extracted using TRI reagent following manufacturer's instructions. The cDNA was then synthesized from total RNA using a Ready-To-go Kit (Amersham Pharmacia Biotech, Piscataway, NJ) and PCR was performed using a PCR thermocycler (MWG Biotech, High Point, NC). The PCR program used to amplify KDR, FLT-1 and β-actin consisted of a pre-cycle of 5 minutes at 94 ° C, 45 seconds at 60 ° C and 45 seconds at 72 ° C. After the initial cycle, the reaction was continued for 35 cycles of 1 minute at 94 ° C, 45 seconds at 65 ° C and 2 minutes at 72 ° C and the reaction was terminated for 7 minutes at 72 ° C. The primer sequences were as follows: β-actin forward primer: tcatgtttgagaccttcaa (SEQ ID NO: 17); β-actin reverse primer: gtctttgcggatgtccacg (SEQ ID NO: 18) (β-actin PCR product: 513 bp); VEGFR-2 forward primer: gtgaccaacatggagtcgtg (SEQ ID NO:

19); VEGFR-2 reverse primer: ccagagattccatgccactt (SEQ ID NO:

20) (VEGFR-2 PCR product: 660 bp); VEGFR-1 forward primer: atttgtgattttggccttgc (SEQ ID NO: 21); VEGFR-1 reverse primer: caggctcatgaacttgaaagc (SEQ ID NO: 22) (VEGFR-1 PCR product: 550 bp). Endothelial cell cDNA was used as a positive control for all 3 sets of primers.

Protein extraction and western blotting

Phosphorylated VEGF receptors (VEGFR-1 and VEGFR-2) were detected by Western blotting after incubation of cells with 20 ng / ml VEGF ₁₆₅ (RD Systems, MN) for 10 minutes at 37 ° C. After this brief stimulation, total protein extracts from primary leukemic cells and cell lines were obtained by lysing the cells in cold RIPA buffer (50 mM Tris, 5 mM EDTA, 1% Triton X-114, 0.4% sodium cacodylate, and 150 mM NaCl). presence of protease inhibitors (1 mg / ml aprotinin, 10 mg / ml leupeptin, 1 mM β-glycerol phosphate, 1 mM sodium orthovanadate and 1 mM PMSF). Embryonic fibroblasts (MRC5 cell line) were used as a negative control. After centrifugation, which was used to remove cell debris, supernatants (minimum total protein 500 ng) were immunoprecipitated overnight at 4 ° C with protein-G agarose beads and antiphosphotyrosine antibody (Santa Cruz Biotechnology, CA) to precipitate phosphorylated proteins . These proteins were resuspended in sample buffer and subjected to SDS-PAGE-acrylamide gel electrophoresis (7.5% gels) under reducing conditions (in the presence of β-mercaptoethanol). Proteins were blotted onto nitrocellulose membrane according to conventional protocols. The blots were finally blocked with 1% BSA / PBS-1% Tween-20 for 1 hour at RT (RT) followed by incubation with primary and secondary antibodies. Rabbit polyclonal anti-VEGFR-2 antibody (Santa Cruz Biotechnology, CA) and goat monoclonal antiVEGFR-1 antibody (RD Systems, MN) were used at a concentration of 1 µg / ml, and a secondary anti-rabbit IgG-HRP antibody (for VEGFR- 2) or anti-rabbit IgG-HRP antibody (for VEGFR-1) was used at a concentration of 1: 6000. ECL chemiluminescence detection system and ECL film (Amersham Pharmacia Biotech, Piscataway, NJ) were used to visualize the presence of proteins on nitrocellulose blots.

Gelatinolytic zymography

The supernatants of the leukemia cell lines and primary cell cultures were withdrawn after overnight incubation in serum free medium with or without VEGF ₆ 5, and the metalloproteinase activity was measured by gelatinolytic zymography, as previously described (13). Briefly, cell culture supernatants were incubated with gelatin-agarose beads to concentrate gelatinases, and subjected to SDS-PAGE acrylamide electrophoresis containing 1% gelatin. The gels were then incubated in 2.5% Triton X-100 for 1 hour at RT, washed in distilled water (DW) and placed in low salt buffer and collagenase buffer (50 nM Tris, pH 7.6, 0.2 M). NaCl, 5 mM CaCl ₂ and 0.2% v / v Brij-35) at 37 ° C for 18 hours. Beads with gelatinolytic activity were visualized after gel staining with 10 ml of 0.2% Coomasie blue solution and 190 ml of staining solution (distilled water, methanol and glacial acetic acid, 6: 3: 1) for 30-60 minutes at RT. . For each experiment was 1 x 10 ^s cells located in each well, and experiments were performed in triplicate. Adobe Photoshop 4.0 and Umax Astra scanner were used to scan the gels and the intensity of the gelatinolytic strips was evaluated using NIH Image 1.58.

Migration experiments

Freshly isolated leukemic cells and cell lines were resuspended in serum free RPMI and a sample of 10 ⁶ cells / ml was prepared. A modified version of the well migration technique described above (14) was used. Briefly, LC aliquots (100 µl), coated with 25 µg of Matrigel without growth factor (Beckton and Dickinson, San Jose, CA) were added to the 8 µm pore well inserts and placed in wells of a 24-well plate. The lower compartment contained serum free RPMI with or without 200 ng / ml VEGF ₁₆ s (RD Systems, MN). For the purpose of blocking migration, each condition was prepared in a separate aliquot and incubated with 1 µg / ml IMC-1C11, anti-VEGFR-1 (clone 6.12) MoAb neutralizing, 10 µM AG1433 (Calbiochem, CA) or broad MMP inhibitor. range, 5,10 - phenanthroline (1 µM). The antibodies and tyrosine kinase inhibitor used in these studies have been shown to block VEGF-induced endothelial cell proliferation and receptor phosphorylation (12,15,16).

Migration was performed at 37 ° C and 5% CO 2 for 14-18 hours. The migrated cells were harvested from the lower compartment, centrifuged at 8000 rpm and counted on a hemocytometer. Only living cells were used for quantification as determined by trypan blue exclusion. The experiments were performed in a triplet and the results are shown as the number of cells migrated in response to VEGF.

Immunohistochemical detection of KDR / VEGFR-2, FLT-1 / VEGFR-1 and VEGF for ectopically implanted leukemias (chloromas).

Chlorine paraffin sections were immunohistochemically stained for VEGFR-1 and VEGFR-2 using conventional protocols. The protocols used were: murine MoAb to VEGFR-2, 6.64 clone (ImClone Systems Incorporated) used at a concentration of 300 ng / ml; rabbit polyclonal antibody to VEGFR-1 (RD Systems) used at a concentration of 200 ng / ml; vWF polyclonal antibody used at 200 ng / ml; VEGF polyclonal antibody (BioGenex, Ab No. 360 p) used at a concentration of 200 ng / ml. Peroxidase-labeled secondary antibodies (against mouse and rabbit immunoglobulins) were used at a 1/6000 dilution. Sections prefabricated with Hematoxylin / Eosin and observed under a light microscope.

In vivo experiments with HL-60 cells

Non-obese diabetic immunocompromised mice (NON-SCID) paired by age and sex were used in all experiments. HL-60 cells (1x10 ⁶ / mouse) were injected intravenously (iv) into 10 NON-SCID mice, and 3 days after injection, the mice were divided into 2 groups of 5 mice each. One group was treated intraperitoneally with 400 µg IMC-1C11 (12) three times a week while the control group was injected with PBS / 1%

BSA (antibody dilution control) for the duration of the experiment.

Quantification of VEGF levels in mouse plasma

Two ELISA kits (both from RD Systems) specific for human and murine VEGF, respectively, were used to determine plasma VEGF concentrations in mice injected with HL-60 cells. Plasma samples were collected at various time points after injection of leukemic cells and samples were used without further dilution. Each sample was analyzed in a triplet and determinations were performed in two separate experiments. Both analyzes had a sensitivity limit of 7.5 pg / ml and were performed according to the manufacturer's instructions.

human chloromas express VEGF, VEGFR-2 and VEGFR-1 human leukemias are not only localized to bone marrow and peripheral circulation, but can also metastasize to tissue and form solids referred to as chloromas.

Immunohistochemical staining of human chloromas with VEGFR-1 and VEGFR-2 specific antibiotics revealed that in addition to staining the endothelial lining of blood vessels, these receptors were also expressed on a subset of leukemic cells (Fig. IC and D). Staining localized to the cell membrane and positive cells appeared scattered in sections (Fig. IC and D). Overall, the areas in the sections analyzed were more VEGFR-2 positive than VEGFR-1 positive. These receptors were primarily detected in different tumor regions, suggesting that staining for VEGFR-1 and VEGFR-2 may identify different cell populations. Furthermore, leukemia cells were stained for VRGF in the sections analyzed (FIG.

1, E and F), suggesting that autocrine loop between VEGF and its receptors may be involved in the formation of chloromas.

Primary leukemias and leukemic cell lines produce VEGF and express VEGFR-2.

For the production of VEGF and VEGFR-2 expression, three leukemic cell lines and 10 primary acute leukemias (isolated from peripheral blood samples) were analyzed by ELISA and RT-PCR.

Of the analyzed leukemic cell lines, HL-60 and HEL cells expressed VEGFR-2 at the mRNA level, while K532 cells were negative (Fig. 2A). RT-PCR analysis of primary leukemia samples showed that 5 out of 10 primary AML samples (50% total) expressed VEGFR-2. VEGFR-1 was also detected by RT-PCR on VEGFR-2 positive leukemias. Furthermore, all VEGFR-2 positive leukemia cell lines produced VEGF in vitro. Thus, the presence of functional receptors on VEGF-producing leukemic cells can form an autocrine loop promoting cell growth and survival.

VEGF165 induces phosphorylation of VEGFR-1 and VEGFR-2 on leukemic cells.

5 ₁₆ VEGF induced a dose dependent increase in phosphorylation of VEGFR-1 and VEGFR-2 in leukemia cell lines and primary leukemias, but not fibroblasts or VEGFR negative leukemia cells (FIG. 2B). In the absence of VEGF, the leukemia cells have basal phosphorylation of VEGFR-1 and VRGFR-2 (Fig. 2B), which may be due to VEGF production and expression of its receptors by the same cells.

The observation that VEGF ₁₆ s induced phosphorylation of its receptors on the leukemia subset suggests that it can also induce phenotypic changes on these cells. Therefore, the effect of VEGFies was further investigated by observing phenotypic changes such as increased proliferation, matrix metalloproteinase (MMP) formation and migration across the reconstituted basement membrane. Anti-VEGFR MoAb and the specific protein kinase inhibitor AG1433 were used to investigate whether VEGF _{16 was} inducing its effects on leukemic cells via VEGFR-2.

VEGF induces leukemia cell proliferation, an effect mediated by VEGFR-2.

VEGF ₁₆ induced in a dose-dependent manner an increase in the proliferation of VEGFR-2 positive leukemias and leukemic cell lines (Fig. 3). This effect could be blocked by incubating the cells with IMC-1C11, used at a concentration of 1 µg / ml (Fig. 3, *, p less than 0.05). Mitogenic effects of VEGF ₁₆ of the leukemia cells seem to be mediated primarily through its receptor, as the MoAb neutralizing VEGFR-1 had no effect on the proliferation of leukemia cells induced by VEGF ₆ 5 · Importantly, the leukemia cells, which did not respond to VEGF _165, as for example, the K562 cell line and primary samples 6, 7, 8, 9 and 10, incubation with IMC-1C11 had no effect on VEGF-induced cell proliferation (Fig. 3).

Experiments using the protein kinase inhibitor AG1433 confirmed that VEGF ₆₅ induced leukemia cell proliferation mainly via VEGFR-2 (Fig. 4). AG1433 significantly blocked the growth of VEGF _165- induced leukemia cells in 72 hours. (**, p less than 0.05, Fig. 4). As with neutralizing MoAbs, incubation of K562 cells, VEGFR-2 negative leukemias or fibroblasts (not shown) with AG1433 in the presence or absence of VEGF ₁₆ with no effect on VEGF-induced cell proliferation (Fig. 4).

These results demonstrated that VRGF1 ₆₅ induced cell proliferation on a subset of cell lines and primary leukemias. This effect can be blocked by MoAb to VEGFR-2 and synthetic protein kinase inhibitors with high affinity for this receptor. This suggests that on leukemic cells, as demonstrated on endothelial cells, VEGF ₁₆₅ transmits its mitogenic effects via VEGFR-2.

VEGF induces MMP secretion / production by leukemic cells.

VEGFies induces the production of smooth muscle MMPs, an effect that is usually correlated with obtaining a more invasive phenotype (17). We have investigated whether VEGF ₆ s can induce a similar effect on leukemic cells.

Without stimulation under serum-free conditions, MMP levels released into the supernatant by each sample were variable (Fig. 5). It is suggested that the production of MMPs by leukemic cells may identify a leukemic subtype or subpopulation with a more invasive phenotype. The primary leukemias and / or cell lines examined were found to show that myelomonocyte subtypes (shown in Figure 5: HL-60 cells, samples 2 and 3) have consistently higher basal production and MMP release. For MMP-producing leukemias, incubation with VEGF ₁₆₅ increased significantly MMP-9 secretion by leukemic cells (Figs. 5A and B) for an 18-hour period, as determined by densitometry. In primary leukemias, MMP-9 was the major MMP released into cell supernatants where MMP-2 was not present in most cases (Fig. 5). TIMP-1 levels produced by leukemic cells showed only slight deviations after stimulation with VEGF ₆₅ as demonstrated by Western blot.

VEGF induces migration of leukemic cells by matrigel coated wells, an effect mediated by VEGFR-1 and VEGFR-2. We investigated whether the VEGF-induced increase of ₁₆ 5 MMP leukemia cells reflects the acquisition of invasive phenotype. This has been demonstrated using a migration system in which the well inserts are coated with a thin layer of matrigel, serving as a model of invasion through the basement membrane. VEGFiss induced the migration of HL-60 leukemia cells and VEGFR-2 + primary leukemias in a dose-dependent manner (Fig. 6). This process requires the generation and activation of MMPs because VEGF-induced cell migration was blocked using a synthetic MMP inhibitor, 5,10-phenanthroline (Fig. 6) and recombinant human TIMP-1.

The remaining cell lines and VEGFR-2 negative primary leukemias did not migrate depending on VEGF, not even the bare (uncoated) wells. This correlated with the decreased ability of these cells to secrete MMPs, but may also be due to a decreased level of VEGFR expression on these cells, particularly VEGFR-1.

As mentioned above, the mitogenic effects of VEGF ₁₆ s on leukemic cells were mediated via VEGFR-2. However, in the migration system used in our studies, incubation of HL-60 cells with IMC-1C11 (at a concentration of 1 µg / ml) could only partially (40%) block VEGF _55- induced matigel migration (Fig. 6). On the other hand MoAb to VEGFR-1 used in the same concentration was significantly blocked 60-70% the migration of HL-60 cells (FIG. 6 *, p < 0.05), indicating that these cells can induce VEGF activation 5 ₆ MMPs and cell migration by interaction with both receptors. To confirm this possibility, incubation of HL-60 cells with a combination of VEGFR-1 and VEGFR-2 MoAbs resulted in a more effective blockade of VEGF165-induced migration (70-80%) than either antibody alone (Fig. 6). Incubation of HL-60 cells with VEGFR-2 specific tyrosine kinase inhibitor AG1433, however, significantly blocked VEGF- ₆₅ induced migration (70%) (Fig. 7, *, p less than 0.05). Taken together, these results suggest that on HL-60 cells, the interaction of VEGF ₆ s with VEGFR-1 and VEGFR-2 may be required to induce cell migration and invasion, but the exact contribution of each receptor in this process requires further investigation.

In primary leukemias, both anti-VEGFR-1 and VEGFR-2 MoAbs showed overall comparable migration-blocking effects (Fig. 6). However, as for HL-60 cells, incubation of primary leukemias with a combination of anti-VEGFR-1 and VEGFR-2 MoAbs was more effective than either antibody alone, blocking the migration induced by VEGF ₆₅ by 80% (Fig. 6). Finally, leukemic cell migration in response to VEGF ₆₅ could not be blocked by anti-MMP-2 antibodies in any of the cells tested, but was significantly reduced when cells were incubated with MMP-2 neutralizing antibodies, suggesting that MMP-9 may be the major a proteinase that plays a role in the migration process.

As mentioned above, the determination of migration used in our studies requires secretion and activation of active MMP, as well as cellular chemotaxis in response to VEGF3.65. Furthermore, studies with antibodies and protein kinase inhibitors indicate that signaling induced by VEGF ₆ s via VEGFR-1 and VEGFR-2 may be required for cells to migrate and invade through the basement membrane. This is not surprising as VEGFR-1 has been shown to mediate monocyte migration (18) as well as smooth muscle MMP production (17).

Taken together, these results suggest that VEGF ₁₆ s induces a more invasive phenotype on the leukemic cell subset, as determined by increased proliferation, MMP formation and migration through VEGFR-2, although signaling via VEGFR-1 may also be needed especially for MMP migration and production .

HL-60 cells release VEGF in vivo

It is now well established that HL-60 cells release VEGF in picogram amounts in vitro. Using NOD-SCID mice, we examined whether these cells also release VEGF in vivo. As shown in Figure 8A, plasma levels of human VEGF increased significantly following leukemic cell injection, which correlated with an increase in circulating leukemic cells and a corresponding decrease in mouse survival (Fig. 8B). Importantly, the plasma levels of mouse VEGF remained very low (at or below the detection level, Fig. 8A) throughout the experiment. These results support the autocrine role of VEGF in the growth and survival of leukemia cells.

Anti-human VEGFR-2 / KDR moAb (IMC-1C11) blocks leukemia growth in vivo.

In the absence of any treatment, injected 1 x 10 ⁶ HL-60 (iv) cells survived for only 14-18 days (n = 8), emphasizing the aggressive nature of these leukemic cells. Using IMC1C11, we investigated whether VEGF derived from leukemic cells promoted leukemic cell growth by autocrine interaction with VEGFR-2. As shown in Figure 8B, mice treated with IMC-1C11 survived during the experiment (more than 80 days, Fig. 8B, p less than 0.005). Importantly, it has recently been confirmed that the antibody used in these studies is human specific

VEGFR-2 (KDR) and does not cross-react with mouse flk-1 (Lu et al, published). Also, as mentioned above, there was an increase in human VEGF plasma levels for control (untreated) mice, which correlated with decreased overall survival of the mice (Fig. 8A and B).

These in vivo results confirm the role of VEGF and VEGFR-2 in the regulation of leukemia cell growth and suggest that antibodies to VEGF receptors may represent a new therapeutic approach for the treatment of a subset of leukemias.

MoAb against human VEGFR-2 / KDR blocks the formation of liver and spleen metastases.

Histological analysis of the liver and spleen of mice injected with HL-60 cells and treated with PBS 14 days after initiation of treatment revealed the presence of leukemia infiltrates (chloromas) in both organs (Figs. 9B and D). Liver sections had several regions invaded by leukemia cells showing signs of active cell proliferation (Fig. 9B). Furthermore, the spleen red pulp region of these mice was largely replaced by leukemic cells (Fig. 9D). On the other hand, mice treated with neutralizing MoAb IMC-1C11 had normal liver and spleen histology (Fig. 9A and C). There was no evidence of liver leukemia infiltrates in treated mice (Fig. 9A) and the spleen appeared normal with signs of apoptosis (Fig. 9C). These results indicate that moAbs to human VEGFR-2 block the proliferation and invasiveness of HL-60 cells for inoculated mice.

Interpretation of the Embodiment of the Invention 1

To define the role of the VEGF / VEGFR-2 autocrine loop in the regulation of leukemia cells in vivo, we used a well established xenotransplant model where the human leukemia cell line HL-60 is transplanted into immunodeficient mice (19-21). Injection of 1 million HL-60 NON-SCID cells into mice results in healing and leukemic chlorine formation in the bone marrow, spleen, liver and peripheral circulation. We demonstrate that HL-60 cells produce human VEGF, which can be detected in the plasma of mice with xenografted HL-60 cells at high concentrations as determined by human specific ELISA (Figure 8A). Increasing levels of human VEGF were detected in plasma samples and increased over time as HL-60 cells proliferated in NON-SCID mice. In contrast, the plasma levels of VEGF produced were insignificant and did not increase during the proliferation of HL-60 cells (Figure 8A).

In the absence of any intervention, NON-SCID mice xenografted with HL-60 cells undergo metastatic chlorine proliferation within 14 days of inoculation. However, injection of MoAb specific for the VEGF binding domain of human VEGFR-2 resulted in inhibition of HL-60 human leukemia proliferation in NON-SCID mice and their long-term survival during the experimental period (more than 80 days, Figure 8B). Histological examination of the liver and spleen has shown that treatment of MoAb against VEGFR-2 effectively inhibits the proliferation of leukemia infiltrates and chloromas in the liver and spleen (Figure 9). These data strongly suggest that endogenously produced human VEGF by HL-60 cells promotes the growth of these leukemic cells through interaction with human VEGFR-2 forming an autocrine loop and confirming in vitro data. Taken together, these data illustrate the plasticity of VEGFR signaling in vascular and hematopoietic lineages and their potential to support; proliferation of certain subgroups of VEGFR-2 + acute leukemias.

In humans, acute leukemias are classified according to the type of progenitor cell and the state of differentiation of the stem cell from which they originate (M1 to M7). Most leukemias are caused by malignant transformation of myeloid progenitors (M1, M2, M3, M4, M5), which are mostly located in bone marrow and peripheral circulation. However, leukemic cells occasionally acquire the capacity to invade various tissues creating niches to form large tumor masses called chloromas. Depending on our in vivo results, most of the chloroforms tested to date express VEGFR-2 and VEGFR-1. Given that chlorine formation, like vessel formation, requires sequential invasion, proliferation, and stabilization of leukemic cells, it is not surprising that leukemic cells with a chlorine-forming potential also express VEGFR. For example, HL60 cells represent a single leukemia model with the capacity to metastasize and form solid, vascularized tumors when implanted subcutaneously. Therefore, chlorine-forming leukemias can utilize VEGFR to increase regulation of MMP expression and facilitate tissue invasion. Expression of VEGFR-2 may subsequently promote the proliferation of leukemia cells. In the absence of anti-regulatory factors, leukemias can continue to proliferate.

Based on our in vivo results, HL-60 cell growth was inhibited, suggesting that in addition to paracrine loops, autocrine loops generated by VEGF / VEGFR-2 mediate leukemia cell proliferation (Figure 10).

Example II. Single chain antibody production

Cell lines and proteins

Primary cultured HUVEC cells were maintained in media

EBM at 37 ° C, 5% CO 2. Cells were used for all analyzes between passages 2-5. The VRGF ₁₆₅ protein was expressed in baculovirus and purified. The cDNA encoding the extracellular domain of KDR was isolated by RT-PCR from human fetal kidney mRNA and subcloned into the Bgl II and BspEI sites of the AP-Tag vector. In this plasmid, the cDNA for the KDR extracellular domain is fused to the cDNA for human placental AP. The plasmid was electroporated into NIH 3T3 cells along with the neomycin expression vector pSV-Neo and stable cell clones were selected by G418. The soluble KDR-ΡΡ fusion protein was purified from cell culture supernatant by affinity chromatography using immobilized anti-AP monoclonal antibodies.

Mouse immunization and construction of a single chain antibody phage library

Female BALB / C mice received two intraperitoneal (ip) injections of 10 µg KDR-ΡΡ in 200 µl RIBI adjuvant system over a two month period followed by a single ip injection without RIBI adjuvant. Mice were also injected subcutaneously (sc) with 10 µg KDR-Ρ-in 200 µl RIBI at the time of first immunization. Three days prior to euthanasia, mice were challenged with challenge i.p. injection of KDR-ΑΡ. Donor spleens were removed and cells were isolated. RNA was extracted and mRNA was purified from total RNA of splenocytes. The scFv phage library was constructed using mRNA that was exposed on the surface of filamentous phage M13.

When exposed to scFvs on the surface of filamentous phage, the V _H and V _L domains of the antibody are linked together by 15 amino acids by a long binding element (GGGGS) ³ (SEQ ID NO 23) and fused to the non-terminal phage protein III. A 15 amino acid long E tag, followed by an amber codon (TAG), was inserted between the C-terminal portion. V _L and protein III for detection and other analytical purposes. The amber codon located between the E tag protein III allows the construct to create a format with exposure to scFv on the surface after transformation in a suppressor host (such as TGI cells) and scFv in a soluble form upon transformation into a non-suppressor host (such as HB2151 cells).

The assembled scFv DNA was ligated into the pCANTAB 5E vector. Transformed TGI cells were plated on 2YTAG plates and incubated. Colonies were scraped into 10 ml of 2YT medium, mixed with 5 ml of 50% glycerol and stored at -70 ° C as a library sample.

bio-panning

The library sample was cultured to log phase, rescued with M13K07 helper phage, and amplified overnight in 2YTAK medium (2YT containing 100 µg / ml ampicillin and 50 µg / ml kanamycin) at 30 ° C. The phage preparation was precipitated in 4% PEG / 0.5 M NaCl, resuspended in 3% fat-free milk / PBS containing 500 µg / ml AP protein and incubated at 37 ° C for 1 hour to capture the phage with anti-AP scFv for blocking other non-specific binding.

Maxisorp Star tubes coated with KDR-AP (10 µg / ml) (Nunc Denmark) were first blocked with 3% milk / PBS at 37 ° C for 1 hour and then incubated with the phage preparation at room temperature for 1 hour. The tubes were washed 10 times with PBST followed by 10 times with PBS (ΡΒΞ containing 0.1% Tween 20). Bound phage were eluted at room temperature for 10 minutes with 1 ml freshly prepared 100 mM triethylamine solution. The eluted phage were incubated with 10 ml with medium log phase TGI cells at 37 ° C for 30 minutes stationary and 30 minutes with shaking. The infected TG1 cells were then plated on 2YTAG plates and incubated overnight at 30 ° C.

99% (185/186) of the clones examined after the 3rd round of panning were found to specifically bind DRC. However, only 15 (8%) of these binding agents could block KDR binding to immobilized VEGF. DNA BstN I fingerprinting of these 15 clones showed the presence of 2 different cleaved forms, while 21 randomly removed non-VEGF blocking clones resulted in 4 different forms. All digested forms were also evident in the clones identified after 2. round panning. Representative clones of each digested form were harvested from clones obtained after round 2 of panning and subjected to DNA sequencing. Of the 15 sequenced clones, 2 unique VEGF blocking clones and 3 non-VEGF blocking clones were identified. One scFv, p2A7, which neither bound KDR nor blocked VEGF binding to KDR was selected as a negative control for all studies.

ELISA phages

Individual TG1 clones were cultured at 37 ° C in 96-well plates and rescued with M13K07 helper phage as described above. The amplified phage preparation was blocked with 1/6 volume of 18% milk / PBS at room temperature for 1 hour and added to 96-well Maxisorp (Nunc) plates coated with KDR-ΑΡ or AP (1 µg / ml x 100 µl). After incubation at room temperature, the plates were washed 3 times with PBST and incubated with rabbit anti-M13 phage Ab-HRP conjugate. Plates were washed 5 times, peroxidase substrate was added, optical density was read at 450 nm using a microplate reader, and csFv antibodies were identified and sequenced.

Preparation of soluble scFv

Phages of individual clones were used to infect the non-suppressor E.coli HB2151 host and the infectious agent was selected on 2YTAG-N plates. ScFv expression in HB2 151 cells was induced by culturing the cells in 2YTA medium containing 1 mM isopropyl-1-thio-β-D-galktopyranoside at 30 ° C. Periplasmic cell extract was prepared by resuspending the cell pellet in 25 mM Tris (pH 7.5) containing 20% (w / v) sucrose, 200 mM NaCl, 1 mM EDTA and 0.1 mM PMSF followed by incubation at 4 ° C. with gentle stirring for 1 hour. After centrifugation at 15,000 rpm, the soluble scFv was purified from the supernatant by affinity chromatography using the RPAS Purification Module (Pharmacia Biotech).

Example III. Analyzes

Quantitative DRC binding analysis

Two assays were used to investigate the quantitative binding of purified soluble scFv to KDR.

Four different clones, including two VEGF blocking clones, pIC11 and plF12, one non-blocking clone, the dominant p2A6 clone, and the non-p2A7 clone were expressed in shake flasks using non-suppressor E. coli HB2151 host cells and the heavy chain Seq. Figure 11. Soluble scFv was purified from E. coli anti-E tag periplasmic extracts by affinity chromatography. The yield of purified scFv of these clones was in the range of 100-400 µg / liter culture.

In direct binding analysis, various amounts of soluble scFv were added to KDR-coated 96-well Maxisorp microtiter plates and incubated at room temperature for 1 hour, after which the plates were washed 3 times with PBST. Plates were then incubated at room temperature for 1 hour with 100 µl of mouse anti E tag antibody followed by incubation with 100 µl of rabbit anti-mouse antibody-HRP conjugate. Plates were washed and developed according to the procedure described above for ELISA phage.

In a further assay, i.e. a competitive VEGF blocking assay, various amounts of scFv were mixed with a fixed amount of KDR-AP (50 ng) and incubated at room temperature for 1 hour. The mixture was then transferred to 96-well plates coated with VEGF ₁₆₅ (200 ng / well) and incubated at room temperature for an additional 2 hours, after which the plates were washed 5 times and substrate for AP was added to quantify bound KDR-AP molecules. The IC ₅₀ value, i.e. the concentration of scFv required for 50% inhibition of KDR binding to VEGF, was then calculated.

The clone pIL11 and pIL12 but not p2A6 also block binding to immobilized VEGF. The plC11 clone, the dominant clone after each panning round, showed the highest KDR binding capacity and the highest potential to block VEGF binding to the DRC (Table 1). The antibody concentrations of the clC11 clone required for 50% maximal KDR binding and for 50% inhibition of KDR binding to VEGF were 0.2 nM and 3 nM, respectively (see Table 1). FACS analysis showed that the clC11, plF12 and p2A6 clones were also able to bind to the expressed cell surface receptor on HUVEC cells.

BIAcore analysis of soluble scFv

The scFv binding kinetics to KDR were measured using a BIAcore biosensor (Pharmacia Biosensor). The KDR-AP fusion protein was immobilized on a sensor chip and soluble scFvs were injected at concentrations ranging from 62.56 nM to 1000 nM. At each concentration, sensograms were obtained and evaluated using the BIA Evaluation 2.0 program to determine the rate constant k on and k off. The Kd value was calculated from the constant koff / kon ratio.

Table 1 shows the results of surface plasmon resonance on a BIAcore device. VEGFs block scFvs, pIC11 and plF12, bound to immobilized KDR with approximately 6 times weaker affinity (K d, 11.2 nM) than the best binding pC11 binding mainly due to much higher dissociation. As expected, p2A7 clone did not bind to immobilized KDR on a BIAcore.

Phosphorylation analysis

Phosphorylation analyzes were performed by early passage of HUVEC cells followed by the protocol described above. Briefly, HUVEC cells were incubated in EBM-2 basal copper even without serum supplemented with 0.5% bovine serum albumin at room temperature for 10 minutes in the presence or absence of scFv antibodies at a concentration of 5 µg / ml followed by stimulation with 20 ng / ml VEGFi ₆₅ at room temperature for a further 15 minutes. Cells were lysed and KDR receptor was immunoprecipitated from cell lysate using Protein A Sepharose beads bound to rabbit anti-KDR polyclonal antibody (ImClone Systems Incorporated). The beads were washed and mixed with SDS sample buffer and the supernatant was subjected to Western blot analysis. To detect KDR phosphorylation, blots were exposed to anti-phosphotyrosine Mab, 4G10. To determine MAP kinase activity, cell lysates were resolved by SDS-PAGE followed by Western blot analysis using a phospho-specific anti-MAP kinase antibody. All signals were detected using ECL.

The results showed that VEGF blocking scFv pIC1 but not non-blocking scFv p-2A6 were able to inhibit VEGF-stimulated KDR receptor phosphorylation. Furthermore, pIC11 also effectively inhibited VEGF-stimulated activation of p44 / p42 MAP kinases. In contrast, neither pIC11 nor IMC-2A6 inhibited VEGF-stimulated activation of p44 / p42 MAP kinases.

Anti-mitogenic analysis

HUVEC cells (5 x 10 3 cells / well) were plated in 96-well tissue culture plates (Wallach, Inc., Gaithersburg, MD) in 200 µl EBM-2 medium without VEGF, bFGF or EGF and incubated at 37 ° C for 72 hours . Different amounts of antibodies were added to the wells in duplicate and preincubated at 37 ° C for 1 hour, after which VEGFies were added to a final concentration of 16 ng / ml. After 18 hours of incubation, 0.25 µCi (3H) -TdR (Amersham) was added to each well and incubated for an additional 4 hours. Cells were placed on ice, washed twice with serum-containing medium followed by incubation for 10 minutes at 4 ° C with 10% TCA. The cells were then washed once with water and solubilized in 25 µl of 2% SDS. Scintillation fluid (150 µl / well) was added and radioactivity incorporated into DNA was determined on a scintillation counter (Wallach, Model 1450 Microbeta Scintillation Counter).

The ability of scFv antibodies to block VEGF-stimulated mitogenic activity on HUVEC cells is shown in FIG. 3. The VEGF blocking scFv p1C11 strongly inhibited DNA-induced DNA synthesis in HUVEC cells with an EC ₅₀ value, i.e., the concentration of antibodies that inhibited 50% of VEGF-stimulated HUVEC mitogenesis of the cells by approximately 5 nM. Non-blocking scFv p2A6 showed no inhibitory effect on mitogenic VEGF activity. Neither pIC11 nor p2A6 inhibited bFGF-induced HUVEC DNA synthesis,

Example IV. Generation of chimeric antibodies

Cell lines and proteins

Primary cultured human umbilical vein endothelial cells (HUVEC) were maintained in EBM-2 medium at 37 ° C, 5% CO _2. Cells between passages 2-5 were used for all assays. VEGF ₁₆₅ and KDR-alkaline phosphatase fusion proteins (KDR-AP) were expressed in baculovirus and NIH 3T3 cells, respectively, and purified according to the procedures described above. Anti-KDR scFv and scFv p2A6 antibodies that bind to KDR but do not block KDR-VEGF interaction were isolated from a phage library constructed from a mouse immunized with KDR as described above. C225 is a chimeric IgG1 antibody directed against the epidermal growth factor (EGF) receptor.

Cloning of scFv pIC11 variable domains

The variable domains of the light (V _L ) and heavy (V _H ) chains of pIC11 were cloned from the scFv expression vector by PCR using primers 1 and 2, and primers 3 and 4, respectively. The leader peptide sequence for protein secretion in mammalian cells was then added to 5 'ends of V _L and V _H by PCR using primers 5 and 2 and primers 5 and 4, respectively.

Primer 1: 5 CRA GTA GCA ACT

GAG CTC3

Primer 2: 5 TCG ATC TAG AAG

BamHI

Primer 3: 5 CTA GTA GCA ACT GCA GTA GTA GTA CAT TCA CAG GTC

AAG CTG3

Primer 4: 5TCG AAG GAT CCA ACC TGA GGA GAC GGT3

BamHI

Primer 5: 5 GGT CTIA AAG CTT ATG

CTT TTT CTA GTA GCA ACT3 '

Construction of expression vectors for chimeric pIC11 IgG.

Separate vectors have been constructed to express chimeric IgG light and heavy chains. The cloned V _L gene was digested with Hind III and Bam HI and ligated into the pKN100 vector containing the human κ light chain constant region (C _L ) to create an expression vector for the chimeric pIC11 light chain, IMC-1C11-L. The cloned V _H gene was digested with Hind III and BamHI and ligated into the pGID105 vector containing the human IgG1 (γ) heavy chain (C _H ) constant region to create an expression vector for the chimeric pIC11 heavy chain, IMC-1C11-H. Both constructs were screened by restriction enzyme digestion and verified by dideoxynucleotide sequencing.

As seen in Figure 4, both the V _H and V _L domains are fused precisely at their 5 'ends to a segment of the gene encoding the leader peptide sequence as indicated. The V _H and V _L domains are ligated with Hind III / Bam HI sites into the expression vector pG1D105, which contains the human IgG1 (γ) heavy chain (C _H ) constant region, and pKN100, which contains the cDNA version of the human κ light chain (C) _L ). In both cases, expression is under the control of the HCMV 1 promoter and is terminated by an artificial termination sequence. The light and heavy chain regions of the complementary determining regions (CDRs) defined according to the hypervariable sequence definitions of Kabat et al. Are underlined and designated CDR-H1 to H3 and CDR-L1 to L3.

IgG expression and purification.

COS cells were transfected with the same amount of plasmids IMC-1C11-L and IMC 1C11-H for transient expression of IgG. Subconfluent COS cells grown in DMEM / 10% FCS in 150 mm culture flasks were washed once with 20 ml DMEM containing 40 mM Tris (pH 7.4), followed by incubation at 37 ° C for 4.5 hours with 4 ml DMEM / DEAE-Dextran / DNA mixture (DMEM containing 40 nM Tris, 0.4 mg / ml DEAE-Dextran (Sigma) and 20 µg plasmid IMC-1C11-L and IMC-1C11-H). Cells were incubated at 37 ° C for 1 hour with 4 ml DMEM / 2% FCS containing 100 nM chloroquine (Sigma) followed by incubation with 1.5 ml 20% glycerol / PBS at room temperature for 1 minute. Cells were washed twice with DMEM / 5% FCS and incubated in 20 ml of the same medium overnight. Cell culture medium was changed to serum-free DMEM / HEPES after cells were washed twice with pure DMEM. Cell culture supernatant was withdrawn 48 hours and 120 hours after transfection. chimeric

IgG was purified from the pooled supernatant by affinity chromatography using the protocol described by the protein G column manufacturer according to (Pharmacia Biotech). Fractions containing IgG were pooled, buffer exchanged into PBS, and terminated using a Centricon 10 concentration apparatus (Amicon Corp., Beverly, MA). IgG purity was analyzed by SDS-PAGE. The concentration of purified antibody was determined by ELISA analysis using a specific anti-human γ chain antibody as scavenger. The standard curve was calibrated using clinical grade human C225 antibody.

Following Protein G affinity purification, one band of approximately 150 kD protein was detected on SDSPAGE. Western blot analysis using HRP-conjugated specific human IgG1 Fc fragment confirmed the presence of human IgG Fc fragment in the purified protein.

ELISA results demonstrate that IMC-1C11 binds more effectively to immobilized KDR than maternal scFv.

Example V. Determination and Analysis

FACS analysis.

Early passaged HUVEC cells were cultured overnight in growth factor-free EBM-2 to induce KDR expression. Cells were harvested and washed three times with PBS, incubated with IMC-pIC11 IgG (5µg / ml) for 1 hour at 4 ° C followed by incubation with FITC-labeled rabbit anti-human Fc fragment (Capper, Organon Teknika Corp., West Chester, PA) for an additional 60 minutes. Cells were washed and analyzed by flow cytometer (Model EPICS ^IR , Coulter

Corp., Edison, N.J.). As previously evident for maternal scFv pIC11, IMC-1C11 specifically binds to KDR expressed on timely passaged HUVEC cells.

Quantitative DRC binding study

Various amounts of antibodies were added to 96-well Maxi-sorp KDR-coated microtiter plates (Nunc. Denmark) and incubated at room temperature for 1 hour, after which the plates were washed 3 times with PBS containing 0.1% Tween-20. Plates were then incubated at room temperature for 1 hour with 100 µl mouse anti E tag antibody-HRP conjugate (Pharmacia Biotech) for scFv, or rabbit conjugate rabbit anti-human IgG-HRP Fc fragment (Cappel, Organon Teknika Corp.) for chimeric IgG. Plates were washed 5 times, TMB peroxidase substrate (KPL, Gaithersburg, MD) was added and OD read at 450 nm using a microplate reader (Molecular Device, Sunnyvale, CA). IMC-1C11 binds more effectively to immobilized KDR than the parent scFv.

Biacore analysis.

The binding kinetics of anti-KDR antibodies were measured using a BIAcore biosensor (Pharmacia Biosensor). The KDR-ΡΡ fusion protein was immobilized on a sensor chip and antibodies or VEGF were injected at concentrations ranging from 25 nM to 200 nM. The sensorgrams were obtained at each concentration and were evaluated using the BIA Evaluation 2.0 program to determine the rate constants k on and k off. The Kd value was calculated as the ratio of the rate constants koff / kon.

BIAcore analysis revealed that IMC-1C11 binds to KDR with higher affinity than the parent scFv (Table 2). The Kd of IMC1C11 is 0.82 nM compared to 2.1 nM for scFv. The increased affinity of IMC-1C11 is mainly due to the lower dissociation rate (k off) of bivalent chimeric IgG. It is important to note that the affinity of IMC-1C11 for binding to KDR is similar to that of natural VEGF ligand for KDR binding, which is 0.93 nM as determined in our BIAcore analysis (Table 2).

Competitive VEGF Binding Analysis.

In the first analysis, different amounts of antibody were mixed with a mixed amount of KDR-Ρ (50ng) and incubated at room temperature for 1 hour. The mixtures were then transferred to 96-well microtiter plates coated with VEGF ₁₆ 5 (200 ng / well) and incubated at room temperature for an additional 2 hours, after which the plates were washed 5 times and substrate for AP (p-nitrophenyl phosphate, Sigma) was added. quantification of KDR-ΡΡ molecules. The EC ₅₀ value, i.e. the concentration of antibody required for 50% inhibition of KDR binding to VEGF, was then calculated.

MC-1C11 also blocks the KDR receptor from binding with immobilized VEGF in a dose-dependent manner. The chimeric antibody more effectively blocks the interaction of VEGF-KDR with an IC ₅₀ value (i.e., the concentration of antibody required to inhibit 50% of KDR binding to VEGF) by 0.8 nM compared to 2.0 nM for scFv. The control scFv also binds KDR but does not block VEGF-KDR.

In a second assay, different amounts of IMC-1C11 antibody or unlabeled VEGF ₁₆₅ protein mixed with a fixed amount of 1251-labeled VEGFiss were added to 96-well microtiter plates coated with KDR receptor. Plates were incubated at room temperature for 2 hours, washed 5 times, and the amount of radionuclide labeled VEGF ₁₆₅ that bound to the immobilized KDR receptor was calculated. The concentrations of IMC-1C11 and unlabeled VEGF ₁₆₅ required to block 50% of the binding of radionuclide labeled VEGF to the immobilized KDR receptor were determined.

IMC-1C11 effectively competes with VEGF designated 1251 for binding to the immobilized KDR receptor in a dose-dependent manner. As expected, C225, a chimeric antibody directed against the EGF receptor, does not bind to the KDR receptor or block the VEGF-KDR interaction.

Phosphorylation analysis.

HUVEC subconfluent cells were cultured in ABM-2 without growth factor for 24 to 48 hours prior to the experiment. After pretreatment with 50 nM sodium orthovanadate for 30 minutes, cells were incubated in the presence or absence of antibodies for 30 minutes followed by stimulation with 20 ng / ml VEGF ₁₆ 5 or 10 ng / ml FGF at room temperature for an additional 15 minutes. Cells were then lysed in lysis buffer (50 nM Tris, 150 mM, 1% NP-40, 2 mM EDTA, 0.25% sodium deoxycholate, 1 mM PMSF, 1 µg / ml leupeptin, 1 µg / ml pepstatin, 10 µg / ml aprotinin, pH 7.5) and cell lysate were used for both KDR and MAP kinase phosphorylation analysis. The KDR receptor was immunoprecipitated from cell lysates by Protein A Sepharose beads (Santa Cruz Biotechnology, Inc., CA) bound to the anti-KDR antibody, Mab 4.13 (ImClone Systems). Proteins were resolved on SDS-PAGE and subjected to Western blot analysis. To detect KDR phosphorylation, blots were exposed to antiphosphotyrosine Mab, PY20 (ICN Biomedicals, Inc. Aurora, OH). For analysis of MAP kinase activity, cell lysates were resolved on SDS-PAGE followed by Western blot analysis using a phospho-specific anti-MAP kinase antibody (New England BioLabs, Beverly, MA). All signals were detected using ECL (Amersham, Arlington Heights, IL). In both assays, the blots were re-exposed to a polyclonal anti-KDR antibody (ImClone Systems) to ensure that equal amounts of protein were loaded into each trace of SDS-PGA gels.

IMC-1C11 effectively inhibits VEGF KDR receptor-stimulated phosphorylation and activation of p44 / p42 MAP kinases. In contrast, C225 shows no inhibition of VEGF-stimulated KDR receptor and MAP kinase activation. Neither IMC-1C11 nor C225 alone had any effect on KDR receptor activity and p44 / p42 MAP kinases. As was previously seen for scFv pIC11, IMC-1C11 does not inhibit FGF-stimulated activation of p44 / p42 MAP kinases. Furthermore, neither the scFv p2A6 nor the chimeric IgG form of p2A6 (c-p2A6) inhibits VEGF-stimulated KDR receptor and MAP kinase activation.

Antimitogenic analysis

The effect of anti-KDR antibodies on mitogenesis of human VEGF-stimulated endothelial cells was determined by ( ³ H) -TdR DNA incorporation analysis using HUVEC cells. HUVEC cells (5x10 ³ cells / well) were plated in 96-well tissue culture plates in 200 µl of EBM2 medium without VEGF, bFGF or EGF and incubated at 37 ° C for 72 hours. Different amounts of antibodies were added to the wells in duplicate and preincubated at 37 ° C for 1 hour, after which VEGF ₁₆₅ was added to a final concentration of 16 ng / ml). After 18 hours of incubation, 0.5 µCi ( ³ H) -TdR was added to each well and incubated for an additional 4 hours. The radioactivity incorporated into the DNA was determined by scintillation counting.

IMC-1C11 and scFv pIC11 effectively inhibit VEGF-stimulated HUVEC cell mitogenesis. IMC-1C11 is a potent inhibitor of HUVEC-induced VEGF-induced mitogenesis than maternal scFv. The antibody concentrations required for 50% inhibition of HUVEC-induced EGF mitogenesis are 0.8 nM for IMC-1C11 and 6 nM for scFv, respectively. As expected, scFv p2A6 showed no inhibitory effect on the proliferation of VEGF-stimulated endothelial cells.

References

1. Ziegler, B.L., Valtieri, M., Porada, G.A., De Maria,

R., Muller, R., Masella, B., Gabbianelli, M., Casella, L., Pelosi, E., Bock, T., Zanjani, ED, and Peschle, C. (1999) Science 285 (5433), 1553-8

2. Fiedier, W., Graeven, U., Ergun, S., Verago, S., Kilic, N.,

Stockschlader, M., and Hossfeld, D. K. (1997) Blood 89 (6), 18705

3. Bellamy, W.T., Richter, L., Frutiger, Y., and Grogan, T. M. (1999) Cancer Res 59 (3), 728-33.

4. Katoh, O., Tauchi, H., Kawaishi, K., Kimura, A., and Satow, Y. (1995) Cancer Res 55 (23), 5687-92.

5. Klagsbrun, M., and D'Amore, P. A. (1996) Cytokine Growth Factor Rev 7 (3), 259-70.

Ortega, N., Jonca, F., Vincent, S., Favard, C., Ruchoux, M.

M., and Plouet, J. (1991) Am J Pathol 151 (5), 1215-24

7. Shalaby, F., Rossant, J., Yamaguchi, T. P., Gertsenstein, M., Wu, X. F., Breitman, M. L., and Schuh, A. C. (1995) Nature 376 (6535), 62-6.

8. Fong, G.H., Rossant, J., Gertsenstein, M., and Breitman, M. L. (1995) Nature 376 (6535), 66-70.

9. Skobe, M., Rockwell, P., Goldstein, K, Vosseler, S., and Fusenig, N.E. (1997) Nat Med 3 (11), 1222-7.

10. Prewett, M., Huber, J., Li, Y., Santiago, A., O'Connor,

W., King, K., Overholser, J. Hooper, A. Pytowski, B., Witte, L., Bohlen, P., and Hicklin, D . J. (1999) Cancer Res 59 (21), 5209 -18 11th Millauer, Shawver, L. K., Plate, K.H., Risau, W., et al

Ullrich, A. (1994) Nature 367 (6463), 576-9

12. Zim, Z., Lu, D., Kotanides, H., Santiago, A., Jimenez, X.,

Simcox, T., Hicklin, D.J., Bohlen, P., and Witte, L. (1999)

Cancer Lett 136 (2), 203-13

13. Leber, T. M., and Balkwill, F. R. (1997) Anal Biochem 249 (1), 24-8.

14. Hamada, T., Mohle, R., Hesselgesser, J., Hoxie, J., Nachman, R. L., Moore, M.A.S, and Rafii, S. (1998) J Exp Med 188, 539548

Strawn, LM, McMahon, G., App, H., Schreck, R., Kuchler, W.R, Longhi, MP, Hui, TH, Tang, C, Levitzki, A., Gazit, A, Chen, I., Keri, G., Orfi, L., Risau, W., Flamme, I., Ullrich, A., Kirta KP, and Shawyer. L. K. (1996) Cancer Res 56 (15), 3540-5

16. Zhu, Z., Rockwell, P., Lu, D., Kotanides, H., Pytowski, B., Hicklin, DJ, Bohlen, P., and Witte, L. (1998) Cancer Res 58 (15) , 3209-14

17. Wang, H., and Keiser, J.A. (1998) Circ Res 83 (8), 832-40

18. Barleon, B., Sozzani, S., Zhou, D., Weich, H.A., Mantovani. A., and Marme, D. (1996) Blood 87 (8), 3336-43

19. Clutterbuck, R. D., Millar, B. C., Powles, R. L., Newman,

A., Catovsky, D., Jarman, M., and Millar, J. L. (1998) Br J

Haematol 102 (2): 522-7

20. Terpstra, W., Prins, A., Visser, T., Wognum, B., Wagemaker, G., Lowenberg, B., and Wielenga, J. (1995) Leukemia 9 (9), 1573-7.

21. Machado, E.A., Gerard, D.A., Lozzio, C. B., Lozzio, B.

B., Mitchell, J. R., and Golde, D. W. (1984) Blood 63 (5), 1015-22

22. Soker, S., Takashima, S., Miao, H. Q., Neufeld, G., and Klagsbrun, M. (1998) Cell 92 (6), 735-45.

23. Salven, P., Manpaa, H., Orpana, A., Aliialo, K., and Joensuu, H. (1997) Clin Cancer Res 3 (5), 647-51.

24. Obermair, A., Tempfer, C., Hefler, L., Preyer, O., Kaider, A. Zeillinger, R., Leodolter, S., and Kainz, C. (1998) Br J Cancer 77 (11). ), 1870-4

25. Kranz, A., Mattfeldt, T., and Waltenberger, J. (1999) Int J Cancer 84 (3), 293-8.

26. Abu-Jawdeh, G.M., Faíx, JD, Niloff, J, Tognazzi, K., Manseau, E., Dvorak, H.F., and Brown, L.F. (1996) Lab Invest

74 (6): 1105-15 27. Ferrer, F. A., Miller, L.J., Lindquist, R. ₃ Kowalczyk, P., Laudone, V.P. Albertsen, P.C. , and Kreutzer, D.L. (1999) Urology 54 (2), 567-72 28. Folkman, J . (1997) Exs 79, 1-8 29. Perez-Atayde, A.R., Sallan, S.E., Tedrow, U., Cormors,

S., Allred, E., and Folkman, J. (1997) Am J Pathol 150 (3), 81521

30. Fielder, W., Graeveo, U., Ergun, S., Verago, S., Kilic, N., Stockschlader, M., and Hossfeld, D. K. (1997) Leukemia (8), 1234-7.

Table 1: KDR binding analysis of scFv antibodies against KDR

klone scFv KDR binding ¹ (ED _S0 , nM) VEGF Block ² (XC ₅₀ , nM) Coupling kinetics ³ Kon (10 ⁵ Μ ^_1s ' ¹ ) Kof f (10 ^ ^{- -1} ) K D (10 ^-9 M) PlCll Yes (0,3) Yes (3,0) 1.1 2.3 2.1 P1F12 Yes (1,0) Yes (15) 0.24 1.4 5.9 P2A6 Yes (5,0) No (over 300) 4.1 46.1 11.2 P2A7 No (NA) No (over 300) ON THE ON THE ON THE

1. Determined by direct binding ELISA, the numbers in parentheses represent the concentrations of scFv that give 50% maximal binding;

2. Determined by competitive VEGF blocking ELISA, the numbers in parentheses represent the concentrations of scFv required to inhibit 50% of KDR binding to immobilized VEGF;

3. Determined by BIAcore analysis

NA = not applicable

Table 2. Receptor binding kinetics of pIC11 scFv and IMC-1C11

DRC *

antibody Kon (ío ^^ s' ¹ ) K off (10 ^-4 s ^_1) Kd (10 ' ^9m ) PlC11 scFv 1.11 2.27 2.1 IMC-1C11 0.63 0.52 0.82 VEGF 1.87 1.81 0.93

* All rates were determined by surface plasmon resonance using the BIAcore system and are the average of at least three separate determinations.

Claims (1)

PATENT A VEGFR receptor antagonist for use in inhibiting the growth of non-solid tumor cells that are stimulated in a mammal by a vascular endothelial growth factor receptor (VEGFR) ligand. 2. Antagonist receptor VEGFR by claim 1 where VEGFR is a DRC. 3. Antagonist receptor VEGFR by claim 1 where VEGFR is a flk -1. 4. Antagonist receptor VEGFR by claim 1 where VEGFR is flt-1. 5. antagonist receptor VEGFR by claim 1 where mammals is human. 6. VEGFR antagonist the antagonist is a biological molecule. by claim 1 where 7th antagonist receptor VEGFR by claim 6. where the biological molecule is a monoclonal antibody specific for VEGFR or a fragment containing its hypervariable region. The VEGFR antagonist of claim 7, wherein the antibody comprises at least one variable heavy chain fragment comprising: CDRH1 having the amino acid sequence shown in SEQ ID NO 1; ami nokys e1i new sequence said in SEQ ID CDRH2 maj úci NO 2 i a NO CDRH3 3; maj úci amino acid sequence said in SEQ ID and at least one variable fragment light chain containing NO CDRLl 4; having amino acid sequence said in SEQ ID NO CDRL2 5; and having amino acid sequence said in SEQ ID NO CDRL3 6th having amino acid sequence said in SEQ ID The VEGFR antagonist of claim 7, wherein the antibody comprises at least one variable heavy chain fragment having the amino acid sequence set forth in SEQ ID NO 7; and at least one light chain variable fragment having the amino acid sequence set forth in SEQ ID NO 8. The VEGFR antagonist of claim 7, wherein the monoclonal antibody is chimerized or humanized. The VEGFR antagonist of claim 1, wherein the antagonist is a small molecule. The VEGFR antagonist of claim 1, wherein the medicament is intended to be administered in combination with treatment of non-solid tumor cells by radiation, chemotherapy, or combinations thereof. The VEGFR antagonist of claim 1, wherein the tumor cells are cells of hematopoietic structures. 14. tumor antagonist the cells are cell receptor y bone VEGFR marrow. according to claim 13 where 15 Dec antagonist receptor VEGFR by claim 14 where tumor the cells are affected by leukemia. 16th antagonist receptor VEGFR by claim 15 where leukemia is acute myelocytic leukemia (AML), chronic myelocytic leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), erythrocytic leukemia or monocytic leukemia. The VEGFR antagonist of claim 14, wherein the tumor cells are multiple myleoma cells. The VEGFR antagonist of claim 13, wherein the tumor cells are lymphoid cells. The VEGFR antagonist of claim 18, wherein the lymphoid cells are related to Hodgkin's disease or non-Hodgkin's disease. A VEGFR receptor antagonist for use in conjunction with radiation for inhibiting the growth of non-solid tumor cells that are stimulated to a mammalian vascular endothelial growth factor receptor (VEGFR) ligand. The VEGFR antagonist of claim 20, wherein the mammal is a human. The VEGFR antagonist of claim 20, wherein the antagonist is to be administered prior to radiation. The VEGFR antagonist of claim 20, wherein the antagonist is to be administered during radiation. The VEGFR antagonist of claim 20, the antagonist to be administered after radiation. The VEGFR antagonist of claim 20, the antagonist to be administered before and during radiation. The VEGFR antagonist of claim 20, wherein the antagonist is to be administered during and after radiation. The VEGFR antagonist of claim 20, the antagonist to be administered before and after radiation. The VEGFR antagonist of claim 20, the antagonist to be administered before, during and after radiation. The VEGFR antagonist of claim 20, wherein the radiation source is located outside of the mammal. The VEGFR antagonist of claim 20, wherein the radiation source is located internally to the mammal. The VEGFR antagonist of claim 20, wherein the antagonist is a monoclonal antibody. The VEGFR antagonist of claim 20, tumors affect hematopoietic structures. where where where where where where where where where where A VEGFR receptor antagonist for use with a chemotherapeutic agent to inhibit the growth of non-solid tumor cells that are stimulated in a mammal by a ligand for vascular endothelial growth factor receptor (VEGFR). The VEGFR antagonist of claim 33, wherein the antagonist is to be administered prior to treatment with a chemotherapeutic agent. The VEGFR antagonist of claim 33, wherein the antagonist is to be administered during treatment with a chemotherapeutic agent. The VEGFR antagonist of claim 33, wherein the antagonist is to be administered after treatment with a chemotherapeutic agent. 37. The VEGFR antagonist of the antagonist is to be administered prior to and with the chemotherapeutic agent. of claim 33 wherein during treatment The VEGFR antagonist of claim 10, wherein the antagonist is to be administered during and after the chemotherapeutic agent. 33 where treatment The VEGFR antagonist of claim 33, wherein the antagonist is to be administered before and after treatment with the chemotherapeutic agent. The VEGFR antagonist of claim 33, wherein the antagonist is to be administered before, during, and after treatment with the chemotherapeutic agent. 41. The VEGFR antagonist of claim 33, wherein the chemotherapeutic agent is selected from the group consisting of cisplatin, dacarbazine, dactinomycin, mechlorethamine, streptozocin, cyclophosphamide, carmustine, lomustine, doxorubicin, daunorubicin, procarbazine, cytomethine, mitomycin, mitomycin, mitomycin, mitomycin. , vinblastine, vincristine, bleomycin, paclitaxel, docetaxel, aldesleukin, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon alpha, leuprolide, megestrol, penthamine, mercaptopine, mercaptopine, mercaptopine plicamycin, streptozocin, tamoxifen, teniposide, testolactone, thioguanine, tiotepa, uracil mustard, vinorelbine, chlorambucil, taxol and combinations thereof. The VEGFR antagonist of claim 33, wherein the chemotherapeutic agent is selected from the group consisting of cisplatin, doxorubicin, taxol, and combinations thereof. The VEGFR antagonist of claim 33, wherein the tumor cells are cells of hematopoietic structures. The VEGFR antagonist of claim 33, wherein the antagonist is a monoclonal antibody. Use of a VEGFR antagonist for the manufacture of a medicament for inhibiting the growth of non-solid tumor cells that are stimulated in a mammal by a vascular endothelial growth factor receptor (VEGFR) ligand. The use of claim 45, wherein the VEGFR is KDR. The use of claim 45, wherein the VEGFR is flk-1. Use according to claim 45, Use according to claim 45, Use according to claim biological molecule. wherein the VEGFR is flt-1. wherein the mammal is a human. 45, wherein the antagonist is The use of claim 50, wherein the biological molecule is a monoclonal antibody specific for VEGFR or a fragment that comprises a hypervariable region thereof. The use of claim 51, wherein the antibody comprises at least one variable heavy chain fragment comprising: NO CDRH1 1; having amino acid sequence said in SEQ ID NO CDRH2 2 a having amino acid sequence said in SEQ ID NO CDRH3 3; maj úci amino acid sequence said in SEQ ID and at least one variable fragment light chain containing : NO CDRL1 4; having amino acid sequence said in SEQ ID NO CDRL2 5; and having amino acid sequence said in SEQ ID NO CDRL3 6th having amino acid sequence said in SEQ ID 53. The use according to at least one variable amino acid sequence of at least one variable amino acid sequence of claim 51, a fragment referred to in the fragment set forth in wherein the heavy chain antibody SEQ ID NO of light SEQ ID NO 7; and strings 8. contain owners have owners The use of claim 51, wherein the monoclonal antibody is chimerized or humanized. The use of claim 45, wherein the antagonist is a small molecule. The use of claim 45, wherein the medicament is intended to be administered in combination with the treatment of non-solid tumor cells by radiation, chemotherapy, or combinations thereof. 57th Use as claimed 45. where tumor cells They are cell hematopoietic structures. 58th . Use according to claim 57, where tumor cells are cells bone marrow. 59th Use as claimed 58. where tumor cells They are affected by leukemia. The use of claim 59, wherein the leukemia is acute myelocytic leukemia (AML), chronic myelocytic leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), erythrocytic leukemia or monocytic leukemia. The use of claim 58, wherein the tumor cells are multiple myleoma cells. The use of claim 58, wherein the tumor cells are lymphoid cells. The use of claim 62, wherein the lymphoid cells are related to Hodgkin's disease or non-Hodgkin's disease. 64. The use of a VEGFR antagonist for the manufacture of a medicament for use in conjunction with radiation to inhibit the growth of non-solid tumor cells that are stimulated to a mammalian vascular endothelial growth factor receptor (VEGFR) ligand. 65. The use of claim 64, wherein the mammal is a human. 66. served Use according to claim 64, before irradiation. wherein the antagonist is to be 67. served Use according to claim 64, during irradiation. wherein the antagonist is to be 68. served Use according to claim 64, after irradiation. wherein the antagonist is to be 69. served Use according to claim 64, before and during irradiation. wherein the antagonist is to be 70. served Use according to claim 64, during and after irradiation. wherein the antagonist is to be 71st served Use according to claim 64, before and after radiation. wherein the antagonist is to be 72. served Use according to claim 64, before, during and after radiation. wherein the antagonist is to be 73. The use of claim 64, wherein the radiation source is located outside of the mammal. The use of claim 64, wherein the radiation source is located internally to the mammal. The use of claim 64, wherein the antagonist is a monoclonal antibody. Use according to claim 64, wherein the tumors affect hematopoietic structures. 77. The use of a VEGFR antagonist for the manufacture of a medicament for use with a chemotherapeutic agent to inhibit the growth of non-solid tumor cells that are stimulated in a mammal by a vascular endothelial growth factor receptor (VEGFR) ligand. 78. The use of claim 77, wherein the antagonist is to be administered prior to treatment with a chemotherapeutic agent. The use of claim 77, wherein the antagonist is to be administered during treatment with a chemotherapeutic agent. The use of claim 77, wherein the antagonist is to be administered after treatment with a chemotherapeutic agent. The use of claim 77, wherein the antagonist is to be administered before and during treatment with a chemotherapeutic agent. The use of claim 77, wherein the antagonist is to be administered during and after treatment with the chemotherapeutic agent. 83. The use of claim 77, wherein the antagonist is to be administered before and after treatment with the chemotherapeutic agent. The use of claim 77, wherein the antagonist is to be administered before, during and after treatment with the chemotherapeutic agent. The use according to claim 77, wherein the chemotherapeutic agent is selected from the group consisting of cisplatin, dacarbazine, dactinomycin, mechloretamine, streptozocin, cyclophosphamide, carmustine, lomustine, doxorubicin, daunorubicin, procarbazine, mitomycin, cytarabine, etarpine, etarpine, etarpine, etopotin, etopotin, , bleomycin, paclitaxel, docetaxel, aldesleukin, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, ifosfamide, interferon alpha, leuprolide, mercaptopurine, plicamycin, miticane, pipotepin, pegasparosinic, pegasparosing testolactone, thioguanine, tiotepa, uracil mustard, vinorelbine, chlorambucil, taxol and combinations thereof. hydroxyureu, megestrol, The use of claim 77, wherein the chemotherapeutic agent is selected from the group consisting of cisplatin, doxorubicin, taxol, and combinations thereof. The use of claim 77, wherein the tumor cells are cells of hematopoietic structures. The use of claim 77, wherein the antagonist is a monoclonal antibody.