WO2001031346A2 - Utilisation d'un complexe de vegfr-2 et de neuropilin-1 dans l'identification de nouvelles substances actives pro-angiogeniques et anti-angiogeniques - Google Patents

Utilisation d'un complexe de vegfr-2 et de neuropilin-1 dans l'identification de nouvelles substances actives pro-angiogeniques et anti-angiogeniques Download PDF

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WO2001031346A2
WO2001031346A2 PCT/US2000/029579 US0029579W WO0131346A2 WO 2001031346 A2 WO2001031346 A2 WO 2001031346A2 US 0029579 W US0029579 W US 0029579W WO 0131346 A2 WO0131346 A2 WO 0131346A2
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WO2001031346A8 (fr
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Jan Susan Rosenbaum
George Brian Whitaker
Brian Joseph Limberg
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The Procter & Gamble Company
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Priority to AU13473/01A priority patent/AU1347301A/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • VEGF vascular endothelial growth factor
  • VEGF ⁇ 65 in the physiological angiogenesis that occurs during the female reproductive cycle, longitudinal bone growth and endochondral bone formation, and wound healing has been documented, as has involvement in pathological (dysregulated) angiogenesis such as that which occurs in tumor growth and metastases, rheumatoid arthritis, endometriosis, psoriasis, proliferative retinopathy, and atherosclerosis (N. Ferrara, J. Molec. Med. (1999), 77(7): 527-543; G. Neufeld, T. Cohen, S. Gengrinovitch, and Z. Poltorak, FASEB J. (1999), 13(1): 9-22; M.
  • the VEGF receptor system provides an attractive target for the identification of novel compounds exhibiting both pro- and anti-angiogenic activity.
  • Pro-angiogenic agents are considered useful for the treatment of cardiac ischemia and peripheral vascular disease (N. Ferrara, J. Molec. Med. (1999), 77(7): 527-543; T.D. Henry, Brit. Med. J. (1999), 318:(7197)1536-1539) in which new blood vessel formation needs to be stimulated, whereas anti-angiogenic agents are considered useful for the treatment of diseases in which dysregulated angiogenesis is a characteristic pathology.
  • the VEGF ligand family consists of VEGF and its various splice variants (discussed below), VEGF-B, VEGF-C, VEGF-D, P1GF and its splice variants (discussed below), and the viral VEGF, VEGF-E (reviewed in T. Veikkola and K. Alitalo, Semin.Cancer Biol. (1999), 9(3): 211-220).
  • the VEGF family members exert their physiological and pharmacological effects through the interaction with three different VEGF receptor tyrosine kinases, termed VEGFR- 1/FLT-l, VEGFR-2/FLK-l/KDR, and VEGFR-3/FLT-4 (for reviews, see T.
  • NP-1 Neuropilin-1
  • NP-2 Neuropilin-2
  • VEGFR-3/FLT-4 is the primary receptor for VEGF-C and VEGF-D and is considered to be responsible for the lymphatic angiogenic activities of these ligands, whereas the blood vessel angiogenic activities of these ligands is thought to be mediated in part through binding to VEGFR-2 (M.G. Achen et al., Proc Natl Acad Sci USA (1998), 95(2): 548-553; Y.H. Cao, et al., Proc.Natl.Acad.Sci. USA (1998), 95(24): 14389-14394; D.J. Dumont et al., Science (1998), 282(5390): 946-949; B. Enholm, L.
  • P1GF-1, P1GF-2, and the various forms of VEGF-B do not bind to either VEGFR-2 or VEGFR-3, are specific ligands for VEGFR-1, and do not exert appreciable mitogenic or angiogenic effects on endothelial cells (for reviews see T. Veikkola and K. Alitalo, Semin. Cancer Biol. (1999), 9(3): 21 1-220; M.G. Achen, S.A. Stacker,. InU.Exp.Pathol. (1998), 79(5): 255-265; G. Neufeld, T. Cohen, S. Gengrinovitch, and Z. Poltorak, FASEB J. (1999), 13(1): 9-22); N. Ferrara, J. Molec.
  • VEGFR-1 in angiogenesis is incompletely understood, although transgenic and knockout experiments implicate a role for this receptor in endothelial cell and monocyte chemotaxis as well as vascular organization (S. Hiratsuka, O. Minowa, J. Kuno, T. Noda, and M. Shibuya, Proc Natl Acad Sci USA (1998), 95(16): 9349-9354; G. H. Fong, L. Y. Zhang, D. M. Bryce, and J. Peng, Development (1999), 126(13): 3015-3025; G.H. Fong, J. Rossant, M.
  • VEGFR-2 in vasculogenesis and angiogenesis is established, as activation of this receptor is required for both vasculogenesis (F. Shalaby, J. Rossant, T. Yamaguchi, M. Gertsenstein, X. Wu, M. Breitman, and A.C. Schuh, Nature (1995) 376: 62-66) and angiogenesis (N. Ortega et al., Am J Pathol (1997), 151(5): 1215- 1224; B.
  • VEGF exists in multiple protein isoforms with different heparin proteoglycan and extracellular matrix binding properties. These isoforms (VEGF 1 65, VEGF 121 , VEGFus, VEGF ⁇ 8 9, VEGF 206 ) arise from alternate splicing of the VEGF gene. VEGF 16 5 is the predominant isoform and has limited heparin-binding activity, whereas the VEGF 121 isoform is freely soluble and is devoid of heparin-binding activity. Similarly, PLGF exists in three different isoforms, which also exhibit differential heparin binding ability (for reviews see G. Neufeld, T. Cohen, S. Gengrinovitch, and Z. Poltorak, FASEB J.
  • VEGF 1 65 and VEGF 121 bind to VEGFR-2 with equal affinity, their activity in biochemical assays that rely on activation of VEGFR-2 is not equivalent (B.A. Keyt et al., J. Biol. Chem. (1996), 271 : 7788-7795; S. Soker, S. Gollamudi-Payne, H. Fidder, H. Charmahelli, M. Klagsbrun, J5 o/. Chem. (1997), 272(50): 31582-31588); S. Ogawa et al., J. Biol. Chem.
  • Neuropilin- 1 (NP- 1 ) and Neuropilin-2 (NP-2) were identified as receptors that bind VEGFi 65 and PLGF-2 but do not bind either VEGF 121 or the non-heparin binding form of PLGF, PLGF-1 (S. Soker, S. Takashima, H.Q. Miao, G. Neufeld, and M. Klagsbrun, Cell (1998), 92:(6): 735-745; Migdal, et al., J Biol Chem (1998), 273(35): 22272-22278; R. Tordjman, N. Ortega, L. Coulombel, j. Plouet, P. H. Romeo, and V.
  • NP-1 and NP-2 also bind various semaphorin ligands to mediate repulsive guidance activity in certain neuronal populations (R.J. Giger, et al., Neuron (1998), 21(5): 1079-1092; T. Takahashi, F. Nakamura, Z. Jin, R.G. Kalb, S.M. Strittmatter, Nat.Neurosci. (1998), 1(6): 487- 493; F. Nakamura, M. Tanaka, T. Takahashi, R.G. Kalb, S.M. Strittmatter, Neuron (1998), 21(5): 1093-1100; D. Bagnard, M.
  • NP-1 and NP-2 are not required for semaphorin signaling (F. Nakamura, M. Tanaka, T. Takahashi, R.G. Kalb, S.M. Strittmatter, Neuron (1998), 21(5): 1093-1100) and they do not contain sequences predictive of enzymatic activity nor sequences predicted to be involved in coupling to intracellular signaling molecules, the NP-1 and NP-2 proteins appear to function as part of a signaling receptor complex for mediating semaphorin signals (T. Takahashi, A. Foumier, F. Nakamura, L-H. Wang, Y. Murakami, R.G. Kalb, H. Fujisawa, and S.M.
  • NP-1 and NP-2 are capable of stimulating signal transduction pathways, although it appears that at least some of the semaphorin-mediated signaling capability of the neuropilins requires interaction with the Plexins as a co-receptor complex (T. Takahashi, A. Fournier, F. Nakamura, L-H. Wang, Y.
  • VEGFR-2 Since binding to VEGFR-2 alone does not explain the differential activities of VEGF 121 vs. VEGFi 65 in various endothelial cell assays, and since only VEGF ⁇ 65 is capable of binding to NP-1, we postulate that the ability of VEGF ! 65 to signal through NP-1 in the presence of VEGFR- 2 may be responsible for the increased potency of VEGF 165 vs. VEGF 121 in endothelial cells co- expressing VEGFR-2 and NP-1. In order for this to be true, it is necessary to postulate the existence of a unique VEGFR-2 + NP-1 co-receptor complex to which VEGF 165 has access, but has limited availability to bind VEGF 121 .
  • VEGFR-2 + NP-1 Using a panel of polyclonal antibodies that specifically recognize either VEGFR-2 or NP-1, we demonstrate that such a receptor complex does indeed exist in the HUVEC cells in which VEGF 165 is more potent at stimulating activation of VEGFR-2 than is VEGF 12 _. Using a heterologous expression system, we further demonstrate that this VEGFR-2 + NP-1 complex has the potential to form in the absence of VEGF ligand, and that once formed, the complex binds VEGF ⁇ 65 but has a reduced ability to bind VEGF 12 ⁇ .
  • VEGFR-2 + NP-1 complex appears to be responsible for the enhanced activity of VEGF ⁇ 65 relative to VEGF 12 ⁇ .
  • agents that bind to the VEGFR-2 + NP-1 complex or stabilize the pre-existing VEGFR-2 + NP-1 complex have the potential to be superior angiogenic agents, since signaling through VEGFR-2 is enhanced in the presence of the NP-1 co-receptor.
  • agents that antagonize binding to the VEGFR-2 + NP-1 complex or agents which antagonize formation of the VEGFR-2 + NP-1 complex have the potential to be superior anti-angiogenic agents relative to agents that disrupt binding solely to VEGFR-2.
  • the present invention relates to a method for determining whether a compound is capable of binding to a receptor protein complex comprising Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) and Neuroplin-1 (NP-1), which receptor protein complex is hereafter referred to as "VEGFR-2 + NP-1 protein complex” or "complex", the method comprising introducing a sample comprising the compound to the VEGFR-2 + NP-1 protein complex and allowing the compound to bind to the complex.
  • This method may utilize the full length proteins, the soluble form of either protein or a combination of full length and soluble proteins.
  • the invention further relates to a host cell co-transfected with an expression vector comprising a DNA sequence that codes for the VEGFR-2 protein and an expression vector comprising a DNA sequence that codes for the NP-1 protein.
  • the invention further relates to a method for determining whether a test compound produces a signal upon binding to a VEGFR-2 + NP-1 protein complex, the method comprising: (a) providing cells expressing a VEGFR-2 receptor protein and a NP-1 receptor protein, wherein the cells naturally express both of these receptors (e.g., HUVEC) and/or wherein the cells have been transfected with a DNA sequence coding for VEGFR-2 and/or a DNA sequence coding for NP-1 such that the cells express both receptors; (b) exposing (i) a first set of the cells to a composition containing a test compound and (ii) a second set of the cells to a composition lacking the test compound; (c) quantitatively assessing a signal derived from activation of VEGFR-2 from step (b); and (d) comparing the amount of signal from step (c) from the first set of cells to the amount of signal from step (c) for the second set of cells.
  • the invention further relates to a method for determining whether a test compound blocks a signal produced by binding of VEGF ⁇ 65 (or another heparin-binding or NP-1 binding VEGF family member) to a VEGFR-2 + NP-1 protein complex, the method comprising: (a) providing cells expressing a VEGFR-2 receptor protein and a NP-1 receptor protein, wherein the cells naturally express both of these receptors (e.g., HUVEC) and/or wherein the cells have been transfected with a DNA sequence coding for VEGFR-2 and/or a DNA sequence coding for NP-1 such that the cells express both receptors; (b) exposing (i) a first set of the cells to VEGF ⁇ 65 (or another heparin-binding VEGF family member) and a composition comprising a test compound and (ii) a second set of the cells to VEGF 165 (or another heparin-binding VEGF family member) and the composition without the test compound; (c) quantitative
  • VEGFR-2 receptor protein and a recombinant NP-1 receptor protein
  • Figure 1 shows the DNA sequence of the oligonucleotide primers used in the PCR amplification of the VEGFR-2 probe used for hybridization screening of a placenta gtlO ⁇ cDNA library to obtain a full-length VEGFR-2 cDNA.
  • the nucleotide bases adenine, thymine, cytosine, and guanine are represented by A, T, C, and G respectively.
  • the primers are derived from the sequence of the human VEGFR-2 receptor (Terman et al., Oncogene 6 (9):2 1677-1683 (1991)).
  • Figure 2 shows the construct pJFE.HFLKl, used for transient mammalian expression of human VEGFR-2.
  • SR-alpha promoter/enhancer
  • ApR ampicillin resistance marker
  • hFLKl human VEGFR-2 gene
  • BstXI restriction site
  • Xbal restriction site.
  • Figure 3 shows the DNA sequence of the oligonucleotide primers used in the PCR amplification of PGneuropilin-1.
  • the gene was isolated in two gene fragments which were ligated together forming the full-length human Neuropilin-1 coding sequence.
  • the nucleotide bases adenine, thymine, cytosine, and guanine are represented by A, T, C, and G, respectively.
  • the primers are derived from the sequence of the human Neuropilin-1 receptor (Soker, S., Takashima, S., Miao, H.Q., Neufeld, G., and Klagsbrun, M., Cell, 92: 735-745 (1998)).
  • Figure 4 shows the construct PGNP-l/pJFE14, used for transient mammalian expression of human Neuropilin-1.
  • SR-alpha promoter/enhancer
  • ApR ampicillin resistance marker
  • Neuropilin-1 human gene
  • EcoRI restriction site
  • Xbal restriction site
  • BstXI restriction site.
  • Figure 5 shows an immunoprecipitation of VEGFR-2 or Neuropilin-1 from COS-1 cells expressing VEGFR-2 only, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 only or Mock vector, and crosslinked to 261 pM [ I25 I]-VEGF ⁇ 65 .
  • Molecular weight standards are shown on the left; areas shown at the right indicate labeled protein bands migrating at the predicted molecular weight of VEGFR-2, Neuropilin-1, or potential complexes of these two receptors crosslinked to [ 125 I]-VEGFi 65 .
  • VEGFR-2 antibody R2.2C was used for the VEGFR-2 immunoprecipitates, and the Neuropilin-1 immunoprecipitations were performed with the NP1ECD4C antibody. From left to right: VEGFR-2 immunoprecipitates of COS-1 cells expressing VEGFR-2 only, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 only, and empty vector (Mock), followed by Neuropilin-1 immunoprecipitates in COS-1 cells expressing VEGFR-2 only, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 only, and empty vector (Mock).
  • Figure 6A shows an immunoprecipitation of VEGFR-2 in HUVEC cells.
  • Molecular weight standards are shown on the left; areas shown at the right indicate labeled protein bands migrating at the predicted molecular weight of VEGFR-2 or Neuropilin- 1 receptors crosslinked to 379 pM [ 125 I]-VEGF
  • unlabeled ligand was used to demonstrate the specificity of the immunoprecipitated bands. From left to right: VEGFR-2 immunoprecipitates with no competitor, 30 nM unlabeled VEGF 165 , and 100 nM VEGF 12. .
  • Figure 6B shows an immunoprecipitation of Neuropilin-1 in HUVEC cells. Molecular weight standards are shown on the left; area shown at the right indicate labeled protein bands migrating at the predicted molecular weight of the Neuropilin-1 receptor crosslinked to 379 pM [ 125 I]-VEGF ⁇ 65 . From left to right: Neuropilin-1 immunoprecipitates with no competitor, 30 nM unlabeled VEGF 165 , and 100 nM VEGF 121 .
  • FIG. 7A shows an immunoprecipitation/Western blot.
  • COS-1 cells overexpressing either VEGFR-2 alone, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 alone, or empty vector (Mock) are immunoprecipitated with the VEGFR-2 antibody (R2.2C) in the absence of ligand, in the presence (+) or absence (-) of crosslinker and, after transfer to PDVF membrane, detected using the VEGFR-2 antibody (R2.2C).
  • Lysate lanes of COS-1 cells overexpressing empty vector (Mock) or VEGFR-2 are also present to demonstrate the ability to detect the VEGFR-2 in the cell ly sates.
  • Molecular weight standards are shown on the left; the relevant area of the detected Western blot is displayed.
  • COS-1 cells overexpressing either VEGFR-2 alone, VEGFR-2 in concert with Neuropilin-1 , Neuropilin-1 alone, or empty vector (Mock) are immunoprecipitated with the Neuropilin-1 antibody (NP1ECD4C) in the absence of ligand, in the presence (+) or absence (-) crosslinker and, after transfer to PDVF membrane, detected using the VEGFR-2 antibody (R2.2C). Lysate lanes of COS-1 cells overexpressing Neuropilin-1 or Neuropilin-1 in concert with VEGFR-2 are also present to demonstrate the ability to detect VEGFR-2 in the cell lysates. Molecular weight standards are shown on the left; the relevant area of the detected Western blot is displayed.
  • the final two lanes contain 6 ⁇ g of total cell lysate from COS-1 cells expressing NP1 or VEGFR-2 in concert with Neuropilin-1 respectively.
  • Figure 7C shows an immunoprecipitation/Western blot.
  • COS-1 cells overexpressing either VEGFR-2 alone, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 alone, or empty vector (Mock) are immunoprecipitated with the VEGFR-2 antibody (R2.2C) in the absence of ligand, in the presence (+) or absence (-) of crosslinker and, after transfer to PDVF membrane, detected using the Neuropilin-1 antibody (NP1ECD1A). Lysate lanes of COS-1 cells overexpressing empty vector (Mock) or VEGFR-2 are also present to demonstrate the ability to detect the Neuropilin-1 in the cell lysates.
  • Molecular weight standards are shown on the left; the relevant area of the detected Western blot is displayed. From left to right: COS-1 cells expressing VEGFR-2, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 only or empty vector (Mock); immunoprecipitated using the VEGFR-2 antibody (R2.2C) and detected using the Neuropilin-1 antibody (NP1ECD1A). The final two lanes contain 6 ⁇ g of total cell lysate from COS-1 cells expressing empty vector (Mock) or VEGFR-2 respectively.
  • Figure 7D shows an immunoprecipitation/Western blot.
  • COS-1 cells overexpressing either VEGFR-2 alone, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 alone, or empty vector (Mock) are immunoprecipitated with the Neuropilin-1 antibody (NP1ECD4C) in the absence of ligand, in the presence (+) or absence (-) of crosslinker and, after transfer to PDVF membrane, detected using the Neuropilin-1 antibody (NP1ECD1A). Lysate lanes of COS-1 cells overexpressing Neuropilin-1 only and VEGFR-2 in concert with Neuropilin-1, are also present to demonstrate the ability to detect the Neuropilin-1 in the cell lysates.
  • NP1ECD4C Neuropilin-1 antibody
  • NP1ECD1A Neuropilin-1 antibody
  • Molecular weight standards are shown on the left; the relevant area of the detected Western blot is displayed. From left to right: COS-1 cells expressing VEGFR-2, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 only, or empty vector (Mock); immunoprecipitated using the Neuropilin-1 antibody (NP1ECD4C) and detected using the Neuropilin-1 antibody (NP1ECD1A). The final two lanes contain 6 ⁇ g of total cell lysate from COS-1 cells expressing Neuropilin-1 or VEGFR-2 in concert with Neuropilin-1, respectively.
  • Figure 8A shows a whole cell binding competition of 287 pM [125 vEGF ⁇ 65 with either
  • VEGF, 2 i (-•-) or VEGF, 65 (- ⁇ -) in COS-1 cells overexpressing VEGFR-2 The Y axis is in total DPM, the X axis is in log units of the molar concentration of competing unlabeled ligand used.
  • IC 50 3.19 x 10 " " M vs. 6.85 x 10 " " M for VEGF, 2 ⁇ and VEGF, 65 , repectively).
  • Figure 8B shows whole cell binding competition of 320 pM [12 vEGF ⁇ 65 with either VEGF, 2 i (-•-) or VEGF I65 (- ⁇ -) in HUVEC cells.
  • VEGF 121 and VEGF )65 to compete for [125i]VEGF
  • Figure 9 A shows an immunoprecipitation of VEGFR-2 or Neuropilin-1 from COS-1 cells over-expressing VEGFR-2 and crosslinked to 420 pM [ 125 I]VEGF ⁇ 65 .
  • Molecular weight standards are shown on the left; area shown at the right indicate labeled protein bands migrating at the predicted molecular weight of VEGFR-2 crosslinked to [ l25 I]-VEGF ⁇ 65 .
  • the VEGFR-2 antibody R2.2C was used for the VEGFR-2 immunoprecipitates, and the Neuropilin-1 immunoprecipitations were performed with the NP1ECD4C antibody. In this experiment binding was performed in the presence of unlabeled ligand to demonstrate the binding specificity of the immunoprecipitated bands.
  • VEGFR-2 immunoprecipitates from cells incubated in the absence of competitor or in the presence of either 30 nM unlabeled VEGF 165 , or 100 nM VEGF 121 .
  • Neuropilin-1 immunoprecipitates from cells incubated in the absence of competitor or in the presence of either 30 nM unlabeled VEGF ⁇ 65 , or 100 nM VEGF, 2 ..
  • Figure 9B shows an immunoprecipitation of VEGFR-2 or Neuropilin-1 from COS-1 cells over-expressing VEGFR-2 in concert with Neuropilin-1, and crosslinked to 420 pM [ 125 I]VEGFi 65 .
  • Molecular weight standards are shown on the left; areas shown at the right indicate labeled protein bands migrating at the predicted molecular weight of VEGFR-2, Neuropilin-1, or potential complexes involving these two receptors, crosslinked to [ 125 I]VEGF ⁇ 65 .
  • the VEGFR-2 antibody R2.2C was used for the VEGFR-2 immuno-precipitates, and the Neuropilin-1 immunoprecipitations were performed with the NP1ECD4C antibody.
  • binding was performed in the presence of unlabeled ligand to demonstrate the binding specificity of the immunoprecipitated bands.
  • VEGFR-2 immunoprecipitates from cells incubated in the absence of competitors, or in the presence of either 30 nM unlabeled VEGF ⁇ 65 , or 100 nM VEGF .
  • Neuropilin- 1 immunoprecipitates from cells incubated in the absence of competitors, or in the presence of either 30 nM unlabeled VEGF, 65 , or 100 nM VEGF .
  • Figure 9C shows an immunoprecipitation of VEGFR-2 or Neuropilin-1 from COS-1 cells over-expressing Neuropilin-1, and crosslinked to 420 pM [ 125 I]VEGF ⁇ 65 .
  • Molecular weight standards are shown on the left; areas shown at the right indicate labeled protein bands migrating at the predicted molecular weight of VEGFR-2, or complexes involving this receptor, crosslinked to [ 125 I]VEGF ⁇ 65 .
  • the VEGFR-2 antibody R2.2C was used for the VEGFR-2 immunoprecipitates, and the Neuropilin-1 immunoprecipitations were performed with the NP1ECD4C antibody.
  • binding was performed in the presence of unlabeled ligand to demonstrate the binding specificity of the immunoprecipitated bands.
  • VEGFR-2 from cells incubated in the absence of competitors, or in the presence of either 30 nM unlabeled VEGF
  • Neuropilin-1 immunoprecipitates from cells incubated in the absence of competitors, or in the presence of either 30 nM unlabeled VEGF, 65 , or 100 nM VEGF, 2 ⁇ .
  • Figure 10A shows an anti-phosphotyrosine Western blot performed in HUVEC cells.
  • the upper panel shows the anti-phosphotyrosine Western blot of VEGFR-2 immunoprecipitates (detection via the 4G10 anti-phosphotyrosine antibody), and the lower panel shows the same blot stripped and re-probed with the VEGFR-2 antibody (R2.2C) for purpose of normalization.
  • VEGF 165 From left to right, the lanes are treated with: no VEGF 165 , 0.1 pM VEGF ⁇ 65 , 1 pM VEGF ⁇ 65 , 3 pM VEGF 165 , 10 pM VEGF 165 , 30 pM VEGF 165 , 100 pM VEGF 165 , 300 pM VEGF ⁇ 65 , 1,000 pM VEGF 165 , or 10,000 pM VEGF i65 .
  • Only the most mature form of VEGFR-2 top band in lower panel
  • Figure 10B shows an anti-phosphotyrosine Western blot performed in HUVEC cells.
  • the upper panel shows the anti-phosphotyrosine Western blot of VEGFR-2 immunoprecipitates (detection via the 4G10 anti-phosphotyrosine antibody), and the lower shows the same blot stripped and re-probed with the VEGFR-2 antibody (R2.2C) for purpose of normalization.
  • the lanes are treated with: no VEGFm, 100 pM VEGF 12 ⁇ , 300 pM VEGF , 1,000 pM VEGFm, 3,000 pM VEGF 121 , 10,000 pM VEGF 12 ⁇ , 30,000 pM VEGFm, 100,000 pM VEGFm, 300,000 pM VEGF 121 , or 1,000,000 pM VEGF, 21 .
  • Only the most mature form of VEGFR-2 (top band in lower panel) is phosphorylated on tyrosine in response to ligand.
  • ATCC American Type Culture Collection, Rockville, Maryland.
  • biologically active means that a particular molecule shares sufficient amino acid sequence similarity with the embodiments of the present invention disclosed herein to be capable of binding detectable quantities of VEGF ⁇ 65 or another heparin-binding VEGF or NP- 1 -binding family member, or transmitting a VEGF ⁇ 65 stimulus to a cell, e.g., as a component of a hybrid receptor construct.
  • a biologically active VEGFR-2 + NP-1 receptor protein complex within the scope of the present invention means the receptor protein complex is capable of binding and can be immunoprecipitated with antibodies generated against either VEGFR-2 or NP-1.
  • host cell means a cell comprising a recombinant expression vector described herein. Host cells may be stably transfected or transiently transfected within a recombinant expression plasmid or infected by a recombinant virus vector.
  • the host cells include prokaryotic cells, such as Escherichia coli, fungal systems such as Saccharomyces cerevisiae, permanent cell lines derived from insects such as Sf-9 and Sf-21, and permanent mammalian cell lines such as Chinese hamster ovary (CHO), SV40-transformed African green monkey kidney cells (COS), Balb/c3T3 A31 cells, or any other cell line known to those skilled in the art.
  • prokaryotic cells such as Escherichia coli
  • fungal systems such as Saccharomyces cerevisiae
  • permanent cell lines derived from insects such as Sf-9 and Sf-21
  • permanent mammalian cell lines such as Chinese hamster ovary (CHO),
  • isolated in reference to the receptor protein of the present invention or DNA sequences encoding said protein, means that the protein or DNA sequence is removed from the complex cellular milieu in which it naturally occurs, and said protein is expressible from said DNA sequence in a cell that does not naturally express it when operably linked to the appropriate regulatory sequences.
  • NP-1 means a protein having the amino acid sequence SEQ ID NO. 4, as well as proteins having amino acid sequences substantially similar to SEQ ID NO. 4 and which are biologically active in that they are capable of binding a VEGF family member (including, but not limited to VEGF ⁇ 65 and P1GF-2), as well as various semaphorin family members, or crossreacting with antibodies raised against NP-1 protein, or peptides derived from the protein sequence of NP-1 protein.
  • VEGF family member including, but not limited to VEGF ⁇ 65 and P1GF-2
  • semaphorin family members include antibodies raised against NP-1 protein, or peptides derived from the protein sequence of NP-1 protein.
  • the term NP-1 includes truncated and/or mutated proteins wherein regions of the receptor molecule not required for VEGF binding, signaling, or complex formation with VEGFR-2 have been deleted or modified.
  • operably linked refers to a condition in which portions of a linear DNA sequence are capable of influencing the activity of other portions of the same linear DNA sequence.
  • DNA for a signal peptide secretory leader
  • a promoter is operably linked to a coding sequence if it controls the transcription of the sequence
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.
  • operably linked means contiguous and, in the case of secretory leaders, contiguous in reading frame.
  • recombinant expression vector refers to a DNA construct used to express DNA which encodes a desired protein (for example, VEGFR-2 or NP-1) and which includes a transcriptional subunit comprising an assembly of 1) genetic elements having a regulatory role in gene expression, for example, promoters and enhancers, 2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and 3) appropriate transcription and translation initiation and termination sequences.
  • recombinant expression vectors of the present invention can be constructed. Possible vectors for use in the present invention include, but are not limited to: for mammalian cells, pJT4 (as described in World Patent Publication No. 96/14579, published by J.
  • soluble receptor refers to an amino acid sequence corresponding to the extracellular region of VEGFR-2, or a portion thereof, which is capable of binding VEGF ]65 or another VEGF family member (including, but not limited to, VEGF 165 , VEGFm, VEGF 206 , VEGF 189 , VEGF 145 , VEGF-C, VEGF-D and the viral VEGF ligands).
  • Soluble receptors include truncated and/or mutated proteins wherein regions of the receptor molecule not required for VEGF binding or complex formation with NP-1 have been deleted or modified.
  • Examples of such soluble receptors for VEGFR-2 include, but are not limited to, polypeptides having the amino acid sequences substantially similar to SEQ ID NO:6 (i.e, amino acid residues 1-760 depicted in SEQ ID NO. 2), especially those encoding amino acid number 124-320 corresponding to the Ig domains 2 and 3 of human VEGFR-2; or polypeptides encoded by nucleic acid residues substantially similar to SEQ ID NO. 5 (i.e., nucleic acid residues 71-2350 depicted in SEQ ID NO. 1), especially base pairs 442-1030 corresponding to the Ig domains 2 and 3 of human VEGFR-2.
  • polypeptides having the amino acid sequences substantially similar to SEQ ID NO:6 i.e, amino acid residues 1-760 depicted in SEQ ID NO. 2
  • polypeptides encoded by nucleic acid residues substantially similar to SEQ ID NO. 5 i.e., nucleic acid residues 71-2350 depicted in SEQ ID NO
  • soluble receptor refers to an amino acid sequence corresponding to the extracellular region of NP-1, or a portion thereof, which is capable of binding VEGF 165 or other heparin-binding VEGF family members.
  • Soluble receptors include truncated and/or mutated proteins wherein regions of the receptor molecule not required for VEGF binding or complex formation with VEGFR-2 have been deleted or modified. Examples of such soluble receptors for NP-1 include, but are not limited to, polypeptides having the amino acid sequences substantially similar to SEQ ID NO. 8 (i.e., amino acid residues 1-856 depicted in SEQ ID NO.
  • nucleic acid residues substantially similar to SEQ ID NO. 7 i.e., nucleic acid residues 1-2568 depicted in SEQ ID NO. 3
  • substantially similar when used to define either amino acid or nucleic acid sequences, means that a particular subject sequence, for example, a sequence altered by mutagenesis, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which is to retain biological activity of the protein.
  • nucleic acid sequences and analogs are "substantially similar” to the specific DNA sequence disclosed herein if the DNA sequences, as a result of degeneracy in the genetic code, encode an amino acid sequence substantially similar to the reference amino acid sequence.
  • substantially similar means a receptor protein that will react with antibodies generated against the protein or peptides derived from the protein sequence.
  • VEGFR-2 means a protein having the amino acid sequence SEQ ID NO. 2, as well as proteins having amino acid sequences substantially similar to SEQ ID NO. 2 and which are biologically active in that they are capable of binding a VEGF family member (including, but not limited to VEGF 165 , VEGFm, VEGF 206 , VEGF 189 , VEGF ⁇ 45 , VEGF-C, VEGF- D and the viral VEGF ligands), or transducing a biological signal initiated by the VEGF family member binding to a cell expressing VEGFR-2, or crossreacting with antibodies raised against VEGFR-2 protein, or peptides derived from the protein sequence of VEGFR-2 protein.
  • VEGFR-2 includes truncated and/or mutated proteins wherein regions of the receptor molecule not required for VEGF binding, signaling, or complex formation with NP-1 have been deleted or modified.
  • VEGFR-2 and NP-1 receptor proteins For purposes of illustrating the methods and expression systems of the present invention, the following non-limiting examples are discussed in detail. While these examples describe the use of human VEGFR-2 and human NP-1 receptor proteins, the skilled artisan will recognize that sequences from other species are readily obtainable and may be used in place of either or both of the human receptor proteins. Examples of such known sequences include, but are not limited: VEGFR-2 from mouse (GENBANK #X70842), rat (GENBANK #U93306) and quail (GENBANK #X83288); NP-1 from mouse (GENBANK #D50086), rat (GENBANK #AF010296) and chicken (GENBANK #D45416). The skilled artisan will also recognize that VEGFR-2 and NP-1 receptor proteins from other species are obtainable using well known methods. The following abbreviations are used in the Examples:
  • VEGF vascular endothelial growth factor
  • VEGFR-2 VEGF Receptor-2
  • FLK-1 Fetal Liver Kinase- 1
  • KDR Kinase Domain Receptor
  • VEGFR-1 VEGF Receptor 1 FLT-1: Ems-like Tyrosine Kinase- 1
  • BSA Bovine Serum Albumin
  • DPM Disintegrations Per Minute
  • ⁇ CL Enhanced Chemiluminescence HUVEC: Human Umbilical
  • NP-1 Neuropilin-1
  • NP-2 Neuropilin-2
  • PBS Phosphate Buffered Saline
  • TBS Tris Buffered Saline
  • DTT Dithiothreitol DSG: Disuccinimidyl Glutarate P1GF: Placental Growth Factor
  • primers shown in Figure 1 are designed from the GENBANK Accession #X61656 (Terman et al., Oncogene 6 (9):2 1677-1683 (1991)) to generate a hybridization probe by PCR corresponding to the first 347 nucleotides.
  • PCR is performed in a 100 ⁇ l reaction using 1 ⁇ l of human placenta cDNA library (see below) as template, 0.25 ⁇ M primers, 25 ⁇ M dNTPs (dTTP, dGTP, and dATP) (Perkin Elmer Cetus, Foster City, CA), 100 ⁇ Ci P-32 dCTP (Cat# BLU513, Dupont-NEN, Boston, MA), IX polymerase buffer, and 5 U polymerase (TaKaRa Shuzo Panver, Kyoto, Japan).
  • the temperature cycle is carried out as follows for 12 cycles: melting, 95°C for 30 sec; annealing, 50°C for 30 sec; extension, 70°C for 30 sec. After the 12 th cycle, the reaction is held at 70°C for an additional 30 seconds to complete extension.
  • the probe was purified by a Nick Column (Cat# 17-0855-01, Pharmacia-Biotech, Upsala, Sweden) according to the manufacturer's instructions. The probe generated is used to screen a gtlO ⁇ cDNA library (Cat.#HL5014A, Clontech, Palo Alto, CA) and obtain a human VEGFR-2 cDNA.
  • the placenta cDNA library is screened according to the Clontech Lambda Library Protocol Handbook (Clontech, Palo Alto, CA).
  • the primary screen is performed on Optitran membranes (Cat# 68350, Schleicher & Schuell, Keene, NH) at a density of 2.5 x 10 5 plaques/filter.
  • the secondary screen is performed on Optitran membranes (Cat#68320, Schleicher & Schuell, Keene, NH) at a density of 1.0 x 10 4 or 1.0 x 10 5 plaques/filter.
  • Each round of hybridization screening is carried out overnight at 65°C in Phosphate Buffer (0.25M Na2P04, 7% sodium dodecyl sulfate, 1% BSA, 1 mM EDTA, 40 ⁇ g/ml ssDNA) using 1 x 10 6 dpm/ml of radio-labeled probe.
  • the membranes are washed in 2X SSC, 6 times for 10 minutes at room temperature, followed by a 65°C wash in 2X SSC for 15 minutes.
  • LambaFlkl#12 is digested with the restriction enzymes BamHI and Fspl (Stratagene, La Jolla, CA) and subcloned into a pBluescript KS vector (Stratagene, La Jolla, CA) previously digested with BamHI and EcorV (Stratagene, La Jolla, CA).
  • the resulting construct is digested with Hindlll (Stratagene, La Jolla, CA) to excise LambaFlkl#12 and then treated with pfu DNA polymerase (Cat# 600140 Stratagene, La Jolla, CA) according to the manufacturer's instructions, the 2.4kb gene fragment is then digested with BamHI and recovered by gel purification. Additionally, LambdaFlkl#l is digested with Xbal and BamHI (Stratagene, La Jolla, CA ) and the resulting 2.3 kb fragment is recovered by gel purification.
  • the two gene fragments are then ligated into pJFE14 mammalian expression vector previously digested with Xbal and Smal (Stratagene, La Jolla, CA).
  • the pJFE14 expression vector is derived from the pCDL-SR ⁇ 296 vector described by Takebe et al. (Y. Takebe et al., MCB 8: 466-472 (1988)).
  • the resulting mammalian construct containing VEGFR-2 is designated pJFE.HFLKl ( Figure 2).
  • the coding region for hVEGFR-2 is identical to the GENBANK sequence X61656.
  • the VEGFR-2 sequences contained within the pJFE14 expression vector contains 4226 nucleotides encompassing the entire coding region plus 86 nucleotides of 3' untranslated DNA sequence (SEQ ID NO. 1).
  • the cDNA clone described herein differs from GENBANK X61656 as follows: 70 nucleotides of 5' untranslated sequence; and 6 additional nucleotides at the 5' end coding for the first two amino acids (Met, Gin) of human VEGFR-2, as described by GENBANK AF035121 (L.Y. Lin et al., unpublished).
  • primers shown in Figure 3 are designed from the AFO 16050 GENBANK sequence, incorporating the indicated restriction sites utilized for subcloning (described below).
  • the template cDNA is generated from human heart polyA+ RNA (Cat.# 6533-1, Clontech, Palo Alto, CA) by reverse transcription.
  • the reverse transcription carried out in a 20 ⁇ l volume, contains approximately 1 ⁇ g-500 ng human heart polyA+ RNA, 10 ⁇ M random hexamers (Perkin Elmer Cetus, Foster City, CA), 1 mM dNTPs, 0.02 mM DTT, 10 U RNase inhibitors (Boehringer Mannheim Biochemical, Indianapolis, IN), 400 U Superscript II MMLV Reverse transcriptase (Gibco-BRL, Grand Island, NY) at 37°C for 1 hour. The reverse transcriptase is heat killed at 72°C for 10 min and each PCR is performed in a 100 ⁇ l volume.
  • Primers BJL-265 and BJL-238R are used to generate the 910 bp 5' region of NP- 1 while primers BJL-259 and BJL-258 are used to generate the 1862 bp 3' region of NP-1.
  • the temperature cycle is carried out as follows for 35 cycles: melting, 95°C for 1 min; annealing, 53°C for 1 min; extension, 72°C for 3 min. After the 35 th cycle, the reaction is held at 72°C min for an additional 10 minutes to complete extension.
  • the respective PCR reactions are purified using Strataprep PCR purification columns (Stratagene, La Jolla, CA) according to the manufacturer's instructions, then eluted with 50 ⁇ l water.
  • the 910 bp and 1862 bp PCR products are subcloned, respectively, into the PCR-ScriptTM Amp vector (Stratagene, La Jolla, CA) using the PCR-ScriptTM Amp Electroporation-Competent Cell Cloning kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Positive clones are identified by DNA sequencing analysis.
  • NP-1 coding sequence is constructed and subcloned into the pJFE14 mammalian expression vector (discussed above).
  • the NP-1 cDNA fragments are excised from the pCRScript vectors by restriction digestion.
  • the plasmid construct for the 5' region of NP-1 is digested with Xbal and Pstl restriction enzymes (Boehringer Mannheim Biochemical, Indianapolis, IN) while the plasmid construct containing the 1862 bp 3' region of NP-1 is digested with Pstl and EcoRI.
  • QIAEX II Qiagen, Chatsworth, CA; a kit for gel purification of DNA fragments, including activated silica spheres and buffers
  • the resulting DNA is recovered by centrifugation at 14,000xg for 20 min, rinsed briefly with 75% cold ethanol to remove salts, and air-dried 20 min.
  • the DNA is resuspended in 10 ⁇ l water then 5 ⁇ l is transformed into XL-1 Blue E.coli (Stratagene, La Jolla, CA) by electroporation using the GenePulserTM (Bio-Rad, Hercules, CA).
  • the resulting cDNA from this clone is designated PGNP-l/pJFE14 ( Figure 4).
  • DNA sequence analysis is performed on the PGNP-1 cDNA (SEQ ID NO. 3). The DNA sequence is identical to the GENBANK NP-1 sequence AF016050 (S.
  • VEGFR-2 and Neuropilin- 1 Transient expression of the receptors in mammalian cells for the binding and immunoprecipitation studies (Examples 3-5), is carried out in COS-1 cells (ATCC CRL 1650) using Lipofectamine 2000 (LF 2000, Gibco-BRL, Grand Island, NY) and the expression plasmids described above (Example 1).
  • COS-1 cells are grown to approximately 70%-90% confluency in DME high glucose media (Gibco-BRL) supplemented with 10% fetal bovine serum (HyClone, Logan, Utah), nonessential amino acids, and glutamine in T-175 flasks (Corning, San Diego, CA).
  • Solution A contains 625 ⁇ l of OptiMEM (Gibco-BRL) and the cDNAs of interest: for VEGFR-2, 5 ⁇ g of pJFE.HFLK-1, for Neuropilin-1, 1.5 ⁇ g of PGNP-1 PJFE14, pre-incubated at room temperature for 10 minutes.
  • Solution B contains 37.5 ⁇ l of LF2000 reagent in 625 ⁇ l OptiMEM.
  • the empty expression vector pJFE14 is substituted for the corresponding receptor cDNA, such that all transfections contain the same amount of total cDNA.
  • the transfected cells are split into 12 well plates 24 hr post transfection for whole cell binding (Example 5) or 100 mm plates for immunoprecipitation/Western Blot (Examples 4 and 6), affinity labeling/immunoprecipitation (Examples 3 and 5), and Western blot experiments (Examples 4 and 6). The cells are suitable for binding analysis 36 to 72 hours after transfection.
  • VEGF 165 Recombinant human VEGF 165 is purchased from R&D Systems, Inc. (Minneapolis, MN) [ 125 I]VEGFi65 is prepared using Chloromine T (Sigma, St. Louis MO), using a modification of methods previously described (K. Pajusola et al., Oncogene, 9: 3545-3555 (1994), B. A. Keyt et al., The Journal of Biological Chemistry 271(13): 7778-7795 (1996)). Lyophilized VEGF, 65 (5 ⁇ g) is taken up in 90 ⁇ l of DPBS (Gibco-BRL) which is then divided equally into two 1.7 ml pre- lubricated conical tubes (VWR, St. Louis MO).
  • DPBS Gibco-BRL
  • the mixture is applied to a PD-10 gel filtration column (Pharmacia, Piscataway, NJ) previously equilibrated in PBS containing 0.5% BSA (Sigma, St. Louis MO) and 0.01% Tween 20 (Sigma, St. Louis MO).
  • the resulting labeled material is >95% precipitable by trichloroacetic acid, indicating that all of the isolated [ 125 I] is protein associated, and has a typical specific activity of 4000 to 12000 Ci/mmol.
  • the Neuropilin-1 receptor has previously been demonstrated to bind VEGF ⁇ 65 (S. Soker, et al., Cell 92(6):735-745, (1998)).
  • COS-1 cells are co-transfected with the cDNA for VEGFR-2 and Neuropilin-1 and plated at a density of 3 x 10 6 cells/dish into 100 mm dishes (Corning, San Diego, CA), as described in Examples 1-2.
  • the receptors are crosslinked to [ 125 I]VEGFi 65 , then subjected to immunoprecipitation with antibodies specific for either receptor using a modification of the method previously described (B. B.
  • the cells are washed three times at 4°C with 5 ml of binding buffer having the same composition as described above, except that no BSA, heparin, or protease inhibitors are added.
  • To each plate is then added 4 ml of fresh BSA-free binding buffer, followed by freshly prepared DSG (Pierce, Rockford, IL) to a final concentration of 186 ⁇ M. After swirling gently to mix the DSG, the plates are incubated for exactly 15 minutes at 4°C with gentle shaking. The media is then aspirated and the cells washed with 15 ml ice cold PBS (Gibco-BRL).
  • the supernatants are transferred to fresh 1.5 ml conical tubes to which 10 ⁇ g of the antibody of choice (see below), 10 ⁇ l of 10% SDS (Gibco-BRL), and 100 ⁇ l of a 50:50 slurry of Fast Flow protein G beads (Pharmacia, Piscataway, NJ) is added.
  • the samples are boiled for 2 min and centrifuged (13,0000 x g, 5 min). 20 ⁇ l of the supernatants are loaded onto 6% SDS-polyacrylamide gels (Novex, San Diego, CA) and subjected to (SDS- PAGE) electrophoresis.
  • the gels are treated to prevent cracking in gel drying buffer (10% methanol, 40% acetic acid, 50% H 2 O) for 15 minutes, and subsequently dried. Radiolabeled bands that were immunoprecipitated by the antibody of choice are visualized and quantitated on a Storm System (Molecular Dynamics, Sunnyvale, CA) .
  • VEGFR-2 rabbit polyclonal antibody used to precipitate VEGFR-2 is produced by Quality Control Biochemicals, Inc. (Hopkinton, MA).
  • the R2.2C antibody is raised against the peptide sequence Ac-SKRKSRPVSVKTFEDIPLEEPC-amide found in the carboxy- terminus of human VEGFR-2 (identical to AA# 1225-1245, SWISSPROT # P35968, except that a C-terminal Cys is added for conjugation). This sequence is conserved in human, mouse, and rat, and this antibody has demonstrated reactivity with human, mouse, rat, canine and bovine KDR/VEGFR-2/FLK-l (data not shown).
  • R2.2C does not cross-react with the homologous VEGFR-1 receptor protein (data not shown), nor does it cross-react with Neuropilin-1 ( Figure 5).
  • the Neuropilin-1 polyclonal antibody (NP1ECD4C) used to precipitate Neuropilin-1 is also produced by Quality Control Biochemicals (Hopkinton, MA).
  • the NP1ECD4C antibody is raised against the peptide sequence AcDLDKKNPEIKIDETGST-C-amide in the extracellular juxtamembrane region of human Neuropilin-1 (identical to AA# 814-830, GENBANK . # AF016050, AF018956 (S.
  • NP-1 antibody is the anti-neuropilin- 1 polyclonal antibody C-19 from Santa Cruz Biotechnology, Inc., Santa Cruz, California (cat. No. sc-7239).
  • NP1ECD4C precipitates a band of the predicted size (-148 kDa) for the VEGF 165 :Neuropilin-l complex (S. Soker et al., Cell (1998), 92: 735-745) as well as a higher molecular weight band that may represent a Neuropilin-1 homodimeric complex.
  • the R2.2C antibody does not immunoprecipitate Neuropilin-1 in these cells, indicating that it does not cross-react with Neuropilin-1.
  • the R2.2C antibody precipitates an intense band the correct size for VEGFR-2 (-230 kDa) as well as a smaller band that corresponds to the molecular weight anticipated for VEGF bound to Neuropilin-1 (-148 kDa). Because the R2.2C antibody does not cross-react with Neuropilin-1, this indicates that VEGFR-2 and Neuropilin-1 are forming a complex in the presence of VEGF ⁇ 65 .
  • the Neuropilin-1 antibody NP1ECD4C precipitates the same bands observed in the COS cells overexpressing only NP-1.
  • the NP1ECD4C antibody does not precipitate a band the size of VEGFR-2 (-230 kDa) which might suggest that Neuropilin-1 is expressed to a much greater extent then is VEGFR-2 at the ratios of cDNA used for this experiment; such that the majority of Neuropilin- 1 that is immunoprecipitated is not in a complex with VEGFR-2.
  • the ability of the R2.2C antibody to co-immunoprecipitate Neuropilin-1 indicates that the majority of VEGFR-2 exists in a complex with Neuropilin-1 in the presence of VEGF ⁇ 65 ligand, under the conditions of this experiment.
  • the NP1ECD4C antibody is unable to co-immunoprecipitate VEGFR-2 in the HUVEC cells; indicating that the majority of NP-1 may not exist in complex with VEGFR-2 in these endothelial cells. Nevertheless, the ability of an antibody that is specific for VEGFR-2 (R2.2C) to co-immunoprecipitate NP-1 in the presence of VEGF ⁇ 65 ligand suggests that the majority of the VEGFR-2 that is present in HUVECs exists in a complex with NP-1 upon ligand addition.
  • VEGF 12 ⁇ is unable to compete for [ ,25 I]VEGF ⁇ 65 binding at either VEGFR-2 or Neuropilin-1 (Figs. 6A and 6B). Since VEGFm exhibits equal affinity at VEGFR-2 as does VEGF 165 (B.A. Keyt et al., J. Biol. Chem. (1996), 271: 7788-7795), these data suggest that VEGF 121 has only limited access to VEGFR-2 when it is in a complex with NP-1 in these endothelial cells.
  • COS-1 cells are co-transfected with the cDNA for VEGFR-2 and Neuropilin-1 and plated in 100 mM dishes as described in Example 2. 48-68 hours after transfection, the cells are subjected to the binding and affinity labeling protocol described in Example 3, except that [ 125 I]VEGF
  • the supernatants are transferred to fresh 1.5 ml conical tubes to which 10 ⁇ g of the antibody of choice (see below), and 100 ⁇ l of a 50:50 slurry of Fast Flow protein G beads (Pharmacia, Piscataway, NJ) is added.
  • the samples are immunoprecipitated at 4°C with neuration overnight, and subjected to SDS-PAGE as described in Example 3.
  • cell lysates are prepared and analyzed by SDS-PAGE. In this procedure, the cells are seeded 24 hours post transfection at 3 x 10 6 cells into 100 mm plates.
  • the cells are scraped on ice using a standard cell scraper and transferred to 1.5 ml conical tubes where the cells are collected by centrifugation (13,000xg, 5 min., 4°C). After centrifugation the cells are re-suspended in 80 ⁇ l of RIPA buffer (described above) per plate, and run through a 23 gauge needle to help solubilize the lysates.
  • the lysates are vortexed for 30 minutes at 4°C prior to another centrifugation (13,000xg, 5 min., 4°C).
  • the cleared supernatants are then transferred to fresh tubes where the total protein concentration is determined using the Pierce BCA assay system (Pierce, Rockford, IL).
  • Six micrograms of total protein is then diluted to 20 ⁇ l with water and 5X loading dye (4 ⁇ l, Five Prime Three Prime, Boulder, CO, Cat # 2- 910675).
  • lysates or immunoprecipitated protein is prepared (described above), 20 ⁇ l of the supernatants are loaded onto 8% SDS-polyacrylamide gels (Novex, San Diego, CA) and subjected to (SDS-PAGE) electrophoresis. The gels are then transferred to PDVF membranes (Owl Scientific, Woburn, MA) using standard Western blot transfer techniques. Post transfer the membranes are blocked using Western blocking buffer (5% BSA (Cat# 05479, Pharmacia, Piscataway, NJ), 0.1% Tween 20, TBS (Sigma, St. Louis, MO)) for 30 minutes.
  • Western blocking buffer 5% BSA (Cat# 05479, Pharmacia, Piscataway, NJ), 0.1% Tween 20, TBS (Sigma, St. Louis, MO)
  • the membranes are then washed three times for 5 minutes (recovering and storing the blocking buffer used for re- blocking the following day) in TBST-0.1% (0.1% Tween 20, TBS) and placed at 4°C overnight without agitation. The following morning the membranes are re-blocked for 2 hours in the same blocking buffer (saved from the previous night). Post blocking, the membranes are probed with the R2.2C antibody (1 :5,000, described in Example 3) for detection of VEGFR-2 or NP1ECD1A antibody for detection of NP-1 at (1 :1,000; see below) in primary probe buffer (Western blocking buffer diluted 1:1 with TBST-0.1%) for two hours at room temperature with agitation.
  • the NP1ECD1A antibody is created using the same methodology as described for NP1ECD4C in Example 3, but is generated against the peptide sequence Ac-TEKPTVIDSTIQSEFPTC-amide (identical to AA# 629-645, GENBANK # AF016050, AF018956 (S. Soker et al., Cell (1998), 92: 735-745, Z. He and M. Tessier-Lavigne, Cell (1997), 90: 739-757, except that a C-terminal Cys is added for conjugation) located in the B2-MAM domain linker region of NP-1.
  • the membranes are then washed five times for six minutes in TBST-0.1% at room temperature with agitation prior to the addition of the HRP-labeled Goat anti-Rabbit secondary antibody at 1 :40,000 (Pierce, Rockford, IL).
  • the secondary antibody is incubated on the membranes for one hour.
  • Post secondary incubation the membranes are washed at room temperature with agitation, successively as follows: 3 X 10 minutes with TBST-0.1%, 2 X 10 minutes with TBST-0.3%, and 3 X 5 minutes with TBS.
  • the membranes are incubated in the ECL solution for 1 minute according to the manufacturer's instructions prior to exposure to Hyperfim (Amersham, Piscataway, NJ).
  • Figure 7 demonstrates the R2.2C antibody clearly immunoprecipitates and detects a band of the correct size for VEGFR-2 (-230 kDa) in COS-1 cells either expressing VEGFR-2 alone or in concert with Neuropilin- 1 , as well as detecting a low level expression of VEGFR-2 in empty vector- (mock-) or NP-1 -transfected COS-1 cells (Figure 7A).
  • Figure 7B demonstrates the ability of VEGFR-2 (-230 kDa Band) to be precipitated by the NP1ECD4C antibody, but only when both VEGFR-2 and Neuropilin-1 are co-expressed.
  • Figures 7C and 7D The reciprocal immunoprecipitation is illustrated in Figures 7C and 7D.
  • Figure 7C demonstrates that the R2.2 antibody can only precipitate Neuropilin-1 (-130 kDa band) when VEGFR-2 and Neuropilin-1 are co-expressed in COS-1 cells.
  • the lack of cross-reactivity of the VEGFR-2 antibody with NP-1 is illustrated by the failure of the R2.2C antibody to immunoprecipitate NP-1 in cells only expressing NP-1 ( Figure 7C), further illustrating that the NP-1 that is immunoprecipitated by the R2.2C antibody in cells co-expressing VEGFR-2 + NP-1 must be in a complex with VEGFR-2.
  • NP-1 expression is demonstrated both in cells expressing NP-1 alone, and in cells co- expressing VEGFR-2 + NP-1 ( Figure 7D).
  • the NP1ECD1A Western antibody appears to preferentially recognize Neuropilin-1 when it is co-expressed with VEGFR-2 vs. when it is expressed alone ( Figure 7D). This conclusion is supported by the data in cell lysates where the NP1ECD1A antibody preferentially recognizes the Neuropilin-1 in the VEGFR-2 + Neuropilin-1 co-transfected cell lysate vs. lysate from cells only overexpressing Neuropilin-1 ( Figure 7D).
  • Neuropilin-1 may be more easily detected by this antibody when Neuropilin-1 is in a complex with VEGFR-2, and further supports the conclusion of the existence of the VEGFR-2 + NP-1 complex in this system.
  • the preferential recognition of the complex by the NP1ECD1A antibody suggests that the B2-MAM-domain linker region epitope recognized by this antibody may undergo a conformational change when VEGFR-2 is in proximity to NP-1 that is maintained even under the SDS-PAGE conditions. This is also consistent with the proposed role of the B domains in binding of VEGF ⁇ 65 to NP-1 (R. J. Giger, et al..
  • NP-1 is expressed more efficiently when it is co-expressed with VEGFR-2, since levels of NP-1 are undetectable by the NP1EDC1A antibody in 6 ⁇ g of total protein lysates from cells solely over expressing NP-1.
  • Figure 7D Such apparent differences in NP-1 expression are not apparent when NP-1 is assessed by affinity labeling with VEGF 165 followed by immunoprecipitation by the NP1ECD4C antibody ( Figure 5), again suggesting that the Western antibody is particularly sensitive to detection of differences of NP-1 expression in the presence or absence of VEGFR-2.
  • FIG. 7 illustrates the ability of the VEGFR-2 + NP-1 complex to form in the absence of ligand. Detection of the co-receptor complex is not an artifact created by use of the crosslinking agent (see Example 3 for methods) as it is readily detectable even in the absence of crosslinker ( Figures 7A through 7D). This suggests that the VEGFR-2 + Neuropilin-1 receptor complex is ligand-independent and may exist as a pre-existing heterodimer on the cell surface under these assay conditions.
  • Example 5 Characterization of the VEGF Isoform Binding Profile of the VEGFR-2 + NP-1 Complex
  • the first is the affinity labeling/immunoprecipitation technique described in Example 3.
  • the second is referred to as whole cell binding competition analysis.
  • COS-1 cells are transfected with cDNA for VEGFR-2 or Neuropilin-1 by the methods described in Example 2, or endogenous HUVEC cells are utilized.
  • VEGF 165 is added to the binding buffer at a final concentration of 10 to 50 nM. The cells are incubated for 4 hr at 4°C with gentle shaking.
  • the buffer is aspirated, and the cells are rinsed 3 times with 1 ml BSA- free binding buffer (Example 3).
  • the solubilized cells are then transferred to fresh tubes and counted in a Packard Model 5005 COBRA Gamma Counter (Packard Instruments, Meriden, CT).
  • the binding curves are analyzed using the Prizm program (GraphPad, San Diego, CA).
  • Figure 8 demonstrates the results of the whole cell binding assay in either COS-1 cells overexpressing VEGFR-2 alone (Figure 8A) or in HUVECs ( Figure 8B).
  • VEGF, 65 and VEGFm exhibit equivalent binding affinity at VEGFR-2, as indicated by the similar IC 50 values (6.85 x 10 " 1 1 M and 3.19 x 10 "U M for VEGF 1 and VEGF 121 , respectively) in COS-1 cells overexpressing VEGFR-2.
  • VEGF 1 65 and VEGF 121 are not identical in HUVEC cells ( Figure 8B), despite the finding that HUVECs contain a substantial amount of immunoprecipitatable VEGFR-2 that is competent to both bind VEGF 165 and be activated by it ( Figures 6 and 10).
  • NP-1 the binding sites to which VEGFm do not have access may be explained as being NP-1, wherein NP-1 appears to represent the majority of the binding sites observable in HUVEC at the whole cell level.
  • NP-1 Since the expression of NP-1 appears to be in excess of that of VEGFR-2 (defining VEGFR-2 as accessible to both VEGF 165 and VEGFm), and since VEGFR-2 can form a complex with NP-1 in the absence of ligand (Example 4), the inability of VEGFm to compete for a substantial portion of the [ 125 I]VEGFi 65 binding sites at either the whole cell level ( Figure 8B), or in the affinity labeling/immunoprecipitation assay ( Figure 6), can be explained as either an inaccessibility to NP-1 alone, or to VEGFR-2 when it is in a complex with NP-1.
  • Figure 9 illustrates that, while VEGFm is able to completely compete with [ 125 I]VEGF 165 binding to VEGFR-2 when it is expressed alone in COS-1 cells (Figure 9A), it does not compete with [ I25 I]VEGF ⁇ 65 binding to NP-1 when it is expressed alone ( Figure 9C) or in combination with VEGFR-2 ( Figure 9B).
  • VEGFm only partially competes for binding at VEGFR-2 in COS cells co- expressing VEGFR-2 + NP-1 (R2.2C IP, Figure 9B) indicating that VEGFm has only limited access to VEGFR-2 when it is in a complex with NP- 1.
  • HUVEC cells BioWhittaker, Walkersville, MD
  • EGM media BioWhittaker, Walkersville, MD
  • the cells are left undisturbed.
  • the day of stimulation the growth media is removed, the cells are rinsed, and returned to the 37°C incubator in serum-free DMEM (Gibco-BRL).
  • the ligands for stimulation are pre-diluted in 1.5 ml of stimulation buffer (25 mM HEPES, Gibco-BRL, DMEM, Gibco-BRL, 0.2% BSA, Sigma, St. Louis, MO, 1 ⁇ g/ml Heparin cat# H7399, Sigma, St. Louis, MO).
  • the media is quickly removed, the stimulation buffer including the ligand is applied, and the cells are returned to the incubator. After exactly 5 minutes at 37°C the cells are placed on ice and the stimulation buffer is removed. A secondary aspiration is performed to ensure complete removal of the stimulation media.
  • the lysates are then transferred to 1.5 ml conical tubes on ice and further solubilized by 3 passes through a 23 gauge needle.
  • the lysates are then centrifuged (13,000 x g, 5 minutes, 4°C) to clear the supernatant and the supernatants are transferred to fresh 1.5 ml conical tubes.
  • Example 4 10 ⁇ g of the R2.2C antibody, and 100 ⁇ l of a 50:50 slurry of the protein G beads (described in Example 4) is then added to each sample. The samples are then placed at 4°C with neuration overnight, or for up to 60-72 hours. Following the overnight incubation the beads are pelleted at 13,000 x g for 5 minutes at 4°C and the supernatants aspirated. Three successive washes of the beads are performed using the RIPA buffer (described above) with the protease inhibitor cocktail (described in Example 3).
  • the samples are boiled for 2 min and centrifuged (13,0000 x g, 5 min). 20 ⁇ l of the supernatants are loaded onto 6% SDS-polyacrylamide gels (Novex, San Diego, CA) and subjected to (SDS-PAGE) electrophoresis; followed by the Western blotting procedure and blocked as described in Example 4.
  • the blocking buffer contains a form of BSA (FLUKA, obtained from Pharmacia, Piscataway, NJ, Cat # 05479), with low endogenous peroxidase activity to avoid high background with the ECL detection.
  • BSA FLUKA
  • Post blocking the membranes are probed for anti-phosphotyrosine using the 4G10 antibody (1:5,000, Upstate Biotechnology Inc., Lake Placid, NY) in primary probe buffer (Western blocking buffer diluted 1 :1 with TBST-0.1%) for two hours at room temperature with agitation.
  • the membranes Prior to the addition of the HRP-labeled Goat anti-Mouse secondary antibody at 1 :40,000 (Pierce, Rockford, IL), the membranes are washed five times for six minutes in TBST-0.1% at room temperature with agitation. The secondary antibody is incubated on the membranes for one hour, after which they are washed at room temperature with agitation, successively as follows: 3 X 10 minutes with TBST-0.1%, 2 X 10 minutes with TBST-0.3%, and 3 X 5 minutes with TBS. Following the final wash step the membranes are incubated in the ECL solution for 1 minute according to the manufacturer's instructions, prior to exposure to Hyperf ⁇ m (Amersham, Piscataway, NJ).
  • Re-probing with the VEGFR-2 antibody allows the phosphotyrosine signal to be normalized to the amount of VEGFR-2 present in the immunoprecipitate for quantitation of the response. Quantitation of the signal is achieved using Image Quant software (Molecular Dynamics, Sunnyvale, CA) after scanning the film on a Scanmaster 2500 (Howtek, Hudson, NH). The results of the anti-phosphotyrosine assay are illustrated in Figure 10. In HUVEC cells the activation of VEGFR-2 autophosphorylation by VEGF ⁇ 6 s is readily apparent at a concentration of 100 pM, and is maximal at 300 pM (EC 50 -1.25 x 10 "10 M; Figure 10A).
  • VEGF ⁇ 65 and VEGFm have similar binding affinity to VEGFR-2 (Example 5 and B.A. Keyt et al, (1996), J. Biol. Chem. 271 : 7788-7795), and the response that is measured here is a direct activation of VEGFR-2, rather than a more downstream event that may be subject to post-receptor regulation.
  • VEGFR-2 we postulate that this difference in potency in the ability to activate VEGFR-2 results from an inability of VEGFm to bind to the VEGFR-2 + NP-1 complex, and that the presence of NP-1 in the VEGFR-2 complex augments signaling through VEGFR-2.
  • VEGF 16 s and VEGFm may have similar binding affinity at VEGFR-2
  • the ability of VEGF ⁇ 6 s to bind to the VEGFR-2 + NP-1 complex allows VEGF to signal more efficiently through VEGFR-2, thereby inducing receptor autophosphorylation at lower concentrations than that observed with VEGF 121 .
  • Example 7 Use of the VEGFR-2 + NP-1 Complex in a Receptor Binding Assay for the Identification of VEGF Receptor Agonists and Antagonists
  • Identification of ligands that interact with the VEGFR-2 + NP-1 complex can be achieved through the use of assays that are designed to measure the interaction of the ligands with this VEGF receptor complex.
  • a receptor binding assay that uses the VEGFR-2 + NP-1 complex and is adapted to handle large numbers of samples is carried out as follows:
  • the whole cell binding assay is carried out as described in Example 5, except that either single or increasing concentrations of test compounds are used in place of VEGFm (Example 5, Figure 8).
  • binding can be tested either at NP-1 alone, VEGFR-2 alone, or at the VEGFR-2 + NP-1 complex in COS-1 cells overexpressing these receptors, or in alternate cell lines engineered to stably overexpress VEGFR-2 + NP-1, using methods readily available to those skilled in the art.
  • the binding data is analyzed using the Prizm program (GraphPad, San Diego, CA) as described in Example 5.
  • test compounds which interact with the VEGFR-2 + NP-1 receptor complex are observed to compete for binding to the receptor complex with the [ IjVEGFi ⁇ s tracer, such that less [ I]VEGF 16 s tracer is bound in the presence of the test compound in comparison to the binding observed when
  • the binding assay can be carried out using soluble receptor proteins in an ELISA-based capture assay format, similar to that described in the scientific literature for soluble VEGFR-2 (B.A. Keyt et al, (1996), J. Biol. Chem. 271: 7788-7795; G. Fuh, B. Li, C. Crowley, B. Cunningham, J.A. Wells, (1998), J. Biol. Chem. 273: (18):11197-11204) or for soluble NP-1 (L.M. Wise, et al, (1999), Proc. Nat. Acad. Sci.
  • the soluble VEGFR-2 receptor would include the extracellular domains present in SEQ ID NO. 5 and SEQ ID NO. 6, or portions thereof, especially those portions including Ig domains 2 and 3, encompassing amino acids 124 to 320, as Ig domains 2 and 3 have been determined to be necessary for high affinity VEGF )65 binding to VEGFR-2 (G. Fuh, B. Li, C. Crowley, B. Cunningham, J.A. Wells, (1998), J Biol Chem 273: (18):11197-11204; A.
  • the soluble NP-1 receptor would include the extracellular domains present in SEQ ID NO. 7 and SEQ ID NO. 8, or portions thereof, especially those portions including the B domains (also known as the coagulation factor domains), encompassing amino acids 274 to 647, as the B domains have been determined to be necessary for VEGF 165 binding to NP-1 (RJ. Giger, et al, (1998), Neuron 21 : (5):1079-1092); see also the PCT publication Number WO 99/29858).
  • B domains also known as the coagulation factor domains
  • VEGF, 65 is either radiolabeled (e.g. as [ l25 I]VEGF, 65 ) or is labeled with a tracer than can then be detected by a fluorometer (e.g., europium-labeled VEGF, 6 s).
  • test compounds which interact with the VEGFR-2 + NP- 1 receptor complex are observed to compete for binding to the receptor complex with the labeled VEGF, 65 tracer, such that less VEGF, 65 tracer is bound in the presence of the test compound in comparison to the binding observed when the tracer is incubated in the absence of the novel compound.
  • a decrease in binding of the VEGF, 65 tracer by > 30% at the highest concentration of the test compound that is studied demonstrates that the test compound binds to the VEGFR-2 + NP-1 receptor complex.
  • VEGFR-2 + NP-1 Use of VEGFR-2 + NP-1 in a Signaling Assay for the Identification of VEGF Receptor Agonists and Antagonists
  • Identification of ligands that signal upon interaction with the VEGFR-2 + NP-1 receptor complex can be achieved through the use of assays that are designed to measure the activation of the receptor protein kinase domain after binding of the ligand to the receptor complex.
  • One assay that is used is similar to that described for measuring the relative potency of VEGF ,65 vs. VEGF ,2, alone (Example 6), except that in this case test compounds would be added in the absence of VEGF, 65 or VEGFm, at either a single concentration or at multiple concentrations.
  • a test compound that measurably increases the phosphotyrosine content of the immunoprecipitated VEGFR-2 above that observed in the absence of compound is considered an agonist of either VEGFR-2 or of the VEGFR-2 + NP-1 receptor complex.
  • a compound that is an antagonist at the VEGFR-2 + NP-1 complex can be detected using an anti-phosphotyrosine Western blot assay similar to that previously described in Example 6 ( Figure 10), with the following modifications: (1) In one assay format, increasing concentrations of either VEGF, 6 s or VEGF, 2 , are used alone, or in the presence of a single concentration of the test compound. A compound is determined to be an antagonist of the VEGFR-2 + NP- 1 complex if the concentration-response curve is shifted to the right for VEGF, 65 , but the concentration- response curve for VEGFm is not shifted substantially to the right.
  • a “substantial" shift is one that results in an increase in the EC 5 o of VEGF, 65 or VEGFm by a factor > 5.
  • antagonistic activity can be detected using a single concentration of either VEGF, 65 or VEGF 12 , (e.g., a concentration that is > EC 5 0 for increasing phosphotyrosine content of the immunoprecipitated VEGFR-2 receptor), in the presence of increasing concentrations of the test compound.
  • the response of VEGF,65 should be decreased by at least 30%, whereas a substantial decrease in the VEGFm response would not be obtained.
  • the anti-phosphotyrosine assay described above can be performed in HUVECs or any other cell line determined to express VEGFR-2 + NP-1, in which VEGF, 65 is a more potent agent than is VEGFm in signaling assays that are dependent on activation of VEGFR-2.
  • the assay can also be performed in cells engineered to overexpress VEGFR-2 + NP-1, using methodology common to those skilled in the art.
  • a stable cell line (generated from Balb/c 3T3 clone A31 ; ATCC # CCL-163) exhibiting these characteristics, named D7R2/NP1#4, has been deposited with the ATCC on September 21, 2000, and assigned ATCC Designation No. .
  • the human NP-1 gene (SEQ ID NO. 3) was incorporated into the vector pBluescript II SK+, which was deposited with the ATCC on October 20, 1999, and assigned ATCC Designation No. PTA-858.
  • the vector is commercially available from Stratagene (La Jolla, CA). The gene was sub-cloned into the EcoRI and Xbal restriction sites of the vector.
  • AAA TCT CTA ATC TCT CCT GTG GAT TCC TAC CAG TAC GGC ACC ACT CAA TTT AGA GAT TAG AGA GGA CAC CTA AGG ATG GTC ATG CCG TGG TGA GTT Lys Ser Leu He Ser Pro Val Asp Ser Tyr Gin Tyr Gly Thr Thr Gln>
  • 2690 2700 2710 2720 2730 CTG GAG AAC TAT AAC TTT GAA CTT GTG GAT GGT GTG AAG TTG
  • GCC ATT CCT CCC CCG CAT CAC ATC CAC TGG TAT TGG CAG TTG GAG GAA CGG TAA GGA GGG GGC GTA GTG TAG GTG ACC ATA ACC GTC AAC CTC CTT Ala He Pro Pro Pro His His He His Trp Tyr Trp Gin Leu Glu Glu>
  • GTC TGC CTT GCT CAA GAC AGG AAG ACC AAG AAA AGA CAT TGC GTG GTC CAG ACG GAA CGA GTT CTG TCC TTC TGG TTC TTT TCT GTA ACG CAC CAG Val Cys Leu Ala Gin Asp Arg Lys Thr Lys Lys Arg His Cys Val Val>
  • TCA TCC AAC CAA GGG GAC AGA AAC TGG ATG CCT GAA AAC ATC 1485 AGT AGG TTG GTT CCC CTG TCT TTG ACC TAC GGA CTT TTG TAG
  • SEQUENCE ID NO:8 (i) SEQUENCE CHARACTERISTICS: 1665 (A) LENGTH: 856 amino acids

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Abstract

L'invention concerne une technique permettant de déterminer si un composé peut se lier à un complexe protéine de récepteur comprenant les récepteurs VEGFR-2 (récepteur du facteur de croissance vasculaire endothéliale) et Neuropilin-1 (NP-1), laquelle technique consiste à introduire un échantillon contenant le composé dans le complexe protéine VEGFR-2+NP-1 et à laisser que le composé se lie au complexe. Cette technique peut utiliser les protéines pleine longueur, la forme soluble d'une des protéines ou une combinaison des protéines pleine longueur et solubles. L'invention concerne également une cellule hôte co-transfectée au moyen d'un vecteur d'expression contenant une séquence d'ADN codant pour la protéine VEGFR-2 et un vecteur d'expression contenant une séquence d'ADN codant pour la protéine NP-1. L'invention concerne en outre un complexe formé par l'interaction d'une protéine réceptrice de VEGFR-2 recombinant et d'une protéine réceptrice de NP-1 recombinant. L'invention concerne enfin une technique permettant de déterminer si un composé test produit un signal à la suite de sa liaison avec un complexe protéine VEGFR-2+NP-1, à savoir s'il s'agit d'un antagoniste dudit complexe, ainsi qu'une technique servant à déterminer si un composé test bloque un signal lorsque VEGF165 se lie audit complexe, c'est à dire s'il s'agit d'un antagoniste du complexe.
PCT/US2000/029579 1999-10-28 2000-10-26 Utilisation d'un complexe de vegfr-2 et de neuropilin-1 dans l'identification de nouvelles substances actives pro-angiogeniques et anti-angiogeniques WO2001031346A2 (fr)

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EP00975419A EP1281088A2 (fr) 1999-10-28 2000-10-26 Utilisation d'un complexe de vegfr-2 et de neuropilin-1 dans l'identification de nouvelles substances actives pro-angiogeniques et anti-angiogeniques
CA002406927A CA2406927A1 (fr) 1999-10-28 2000-10-26 Utilisation d'un complexe de vegfr-2 et de neuropilin-1 dans l'identification de nouvelles substances actives pro-angiogeniques et anti-angiogeniques
AU13473/01A AU1347301A (en) 1999-10-28 2000-10-26 The use of a complex of vegfr-2 and neuropilin-1 for identification of novel pro- and anti-angiogenic actives

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003029814A3 (fr) * 2001-10-01 2003-12-31 Ludwig Inst Cancer Res Matieres a base de neuropiline/vegf c/vegfr 3 et procedes correspondants
US8846386B2 (en) * 2007-12-18 2014-09-30 University Of Kentucky Research Foundation sVEGFR-2 and its role in lymphangiogenesis modulation

Citations (1)

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WO1997034920A1 (fr) * 1996-03-21 1997-09-25 Sugen, Inc. Dosages pour inhibiteurs du recepteur tyrosine kinase kdr/flk-1

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WO1997034920A1 (fr) * 1996-03-21 1997-09-25 Sugen, Inc. Dosages pour inhibiteurs du recepteur tyrosine kinase kdr/flk-1

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GIGER ROMAN J ET AL: "Neuropilin-2 is a receptor for semaphorin IV: Insight into the structural basis of receptor function and specificity." NEURON, vol. 21, no. 5, November 1998 (1998-11), pages 1079-1092, XP001001417 ISSN: 0896-6273 cited in the application *
KENDALL R L ET AL: "IDENTIFICATION OF A NATURAL SOLUBLE FORM OF THE VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR, FLT-1, AND ITS HETERODIMERIZATION WITH KDR" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS,US,ACADEMIC PRESS INC. ORLANDO, FL, vol. 226, no. 2, 13 September 1996 (1996-09-13), pages 324-328, XP000611908 ISSN: 0006-291X *
KEYT B A ET AL: "THE CARBOXYL-TERMINAL DOMAIN (111-165) OF VASCULAR ENDOTHELIAL GROWTH FACTOR IS CRITICAL FOR ITS MITOGENIC POTENCY" JOURNAL OF BIOLOGICAL CHEMISTRY,US,AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, vol. 271, no. 13, 29 March 1996 (1996-03-29), pages 7788-7795, XP000611911 ISSN: 0021-9258 cited in the application *
POLTORAK ZOYA ET AL: "The VEGF splice variants: Properties, receptors, and usage for the treatment of ischemic diseases." HERZ, vol. 25, no. 2, March 2000 (2000-03), pages 126-129, XP001001418 ISSN: 0340-9937 *
SHINKAI AKEO ET AL: "Mapping of the sites involved in ligand association and dissociation at the extracellular domain of the kinase insert domain-containing receptor for vascular endothelial growth factor." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 47, 20 November 1998 (1998-11-20), pages 31283-31288, XP002166839 ISSN: 0021-9258 cited in the application *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003029814A3 (fr) * 2001-10-01 2003-12-31 Ludwig Inst Cancer Res Matieres a base de neuropiline/vegf c/vegfr 3 et procedes correspondants
US8846386B2 (en) * 2007-12-18 2014-09-30 University Of Kentucky Research Foundation sVEGFR-2 and its role in lymphangiogenesis modulation

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