WO1999030157A2 - Neuropilines et leur utilisation a des fins de diagnostic et de pronostic du cancer - Google Patents

Neuropilines et leur utilisation a des fins de diagnostic et de pronostic du cancer Download PDF

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WO1999030157A2
WO1999030157A2 PCT/US1998/026127 US9826127W WO9930157A2 WO 1999030157 A2 WO1999030157 A2 WO 1999030157A2 US 9826127 W US9826127 W US 9826127W WO 9930157 A2 WO9930157 A2 WO 9930157A2
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vegf
cells
vegfι
kdr
receptor
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PCT/US1998/026127
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WO1999030157A9 (fr
WO1999030157A3 (fr
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Michael Klagsbrun
Shay Soker
Hua-Quan Miao
Seiji Takashima
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Children's Medical Center Corporation
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Publication of WO1999030157A3 publication Critical patent/WO1999030157A3/fr
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Priority to US09/583,638 priority patent/US6635421B1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention provides methods for the diagnosis and prognosis of cancer, particularly metastatic cancer.
  • the process of tumor metastasis is a multistage event involving local invasion and destruction of intercellular matrix, intravasation into blood vessels, lymphatics or other channels of transport, survival m the circulation, extravasation out of the vessels in the secondary site and growth in the new location (Fidler, et al., Adv Cancer Res 28, 149-250 (1978), Liotta, et al., Cancer Treatment Res 40, 223-238 (1988), Nicolson, Bwchim Bwphy Ada 948, 175-224 (1988) and Zetter, N Eng J Med 322, 605-612 (1990)) Success in establishing metastatic deposits requires tumor cells to be able to accomplish these steps sequentially. Common to many steps of the metastatic process is a requirement for motihty.
  • Identifying factors that are associated with onset of tumor metastasis is extremely important.
  • those factors are an important site for identifying new compounds that can be used for treatment and as a target for treatment identifying new modes of treatment such as inhibition of metastasis is highly desirable
  • Tumor angiogenesis is essential for both primary tumor expansion and metastatic tumor spread, and angiogenesis itself requires ECM degradation (Blood et al, Bwchim Biophys Ada 1032:89-118 (1990)).
  • malignancy is a systemic disease in which interactions between the neoplastic cells and their environment play a crucial role dunng evolution of the pathological process (Fidler, I. J , Cancer Metastasis Rev 5:29-49 (1986))
  • VEGF may be a major regulator of angiogenesis (reviewed in Ferrara, et al., Endocr. Rev., 13, 18-32 (1992); Klagsbrun, et al., Curr.
  • VEGF was initially purified from the conditioned media of folliculostellate cells (Ferrara, et al., Biochem. Biophjs. Res. Commun., 161, 851-858 (1989)) and from a variety of tumor cell lines (Myoken, et al., Proc. Natl. Acad. Sci. USA, 88:5819-5823 (1991); Plouet, et al., EMBO. J, 8:3801-3806 (1991)).
  • VEGF was found to be identical to vascular permeability factor, a regulator of blood vessel permeability that was purified from the conditioned medium of U937 cells at the same time (Keck, et al., Science, 246: 1309-1312 (1989)).
  • VEGF is a specific mitogen for endothelial cells (EC) in vitro and a potent angiogenic factor in vivo.
  • EC endothelial cells
  • the expression of VEGF is up-regulated in tissue undergoing vascularization during embryogenesis and the female reproductive cycle (Brier, et al., Development, 1 14:521-532 (1992); Shweiki, et al., J. Clin. Invest, 91 :2235-2243 (1993)).
  • VEGF vascular endothelial growth factor
  • VEGF appears to play a principle role in many pathological states and processes related to neovascularization. Regulation of VEGF expression in affected tissues could therefore be key in treatment or prevention of VEGF induced neovascularization/angiogenesis .
  • VEGF exists in a number of different isoforms that are produced by alternative splicing from a single gene containing eight exons (Ferrara, et al., Endocr. Rev., 13: 18-32 (1992); Tischer, et al, J. Biol. Chem., 806: 1 1947-1 1954 (1991); Ferrara, et al, Trends Cardio Med, 3:244-250 (1993); Polterak, et al., J. Biol.
  • Human VEGF isoforms consists of monomers of 121, 145, 165, 189, and 206 amino acids, each capable of making an active homodimer (Polterak et al., J. Biol. Chem, 272:7151-7158 (1997); Houck, et al., Mo/. Endocrinol, 8: 1806-1814 (1991)).
  • the VEGF1. 21 and VEGF ⁇ 6 5 isoforms are the most abundant.
  • VEGF1.21 is the only VEGF isoforms that does not bind to heparin and is totally secreted into the culture medium.
  • VEGF ⁇ 65 is functionally different than VEGF1.
  • HSPGs heparin and cell surface heparin sulfate proteoglycans
  • VEGF receptor tyrosine kinases KDR/Flk-1 and/or Fit- 1 , are mostly expressed by EC (Terman, et al., Biochem. Biophys. Res. Commun., 187: 1579-1586 (1992); Shibuya, et al., Oncogene, 5:519-524 (1990); De Vries, et al., Science, 265:989-991 (1992); Gitay-Goran, et al, J. Biol. Chem., 287:6003-6096 (1992); Jakeman, et al., J. Clin. Invest, 89:244-253 (1992)).
  • VEGF activities such as mitogenicity, chemotaxis, and induction of morphological changes are mediated by KDR/Flk-1 but not Fit- 1 , even though both receptors undergo phosphorylation upon binding of VEGF (Millauer, et al., Cell, 72:835-846 (1993); Waltenberger, et al., J. Biol. Chem., 269:26988-26995 (1994); Seetharam, et al., Oncogene, 10: 135-147 (1995); Yoshida, et al., Growth Factors, 7: 131-138 (1996)).
  • VEGF ⁇ 65 R VEGF ⁇ 5 receptor
  • VEGF1. 65 R has a molecular mass of approximately 130 kDa, and it binds VEGF1 .65 with a Kd of about 2 X 10 "10 M, compared with approximately 5 X 10 "12 M for KDR/Flk-1.
  • Kd about 2 X 10 "10 M
  • VEGF ⁇ 65 binds to VEGF ) 65 R via its exon 7- encoded domain which is absent in VEGF1. 21 (Soker, et al, J. Biol. Chem., 271 :5761- 5767 (1996)).
  • the function of the receptor was unclear.
  • VEGF ⁇ 65 R gene SEQ ID NO: 1
  • SEQ ID NO:2 the amino acid sequence of the receptor
  • this novel VEGF receptor is structurally unrelated to Flt-1 or KDR/Flk-1 and is expressed not only by endothelial cells but by non-endothelial cells, including surprisingly tumor cells.
  • VEGF ⁇ 6> In ascertaining the function of the VEGF ⁇ 6> R we have further discovered that this receptor has been identified as a cell surface mediator of neuronal cell guidance and called neuropilin-1. Kolodkin et al., Cell 90:753-762 (1997). We refer to the receptor as VEGF ⁇ 65 R/NP-l.
  • VEGF 165 R/NP-I cDNA In addition to the expression cloning of VEGF 165 R/NP-I cDNA we isolated another human cDNA clone whose predicted amino acid sequence was 47% homologous to that of VEGF ⁇ . 5 R/NP-l and over 90% homologous to rat neuropilin-2 (NP-2) which was recently cloned (Kolodkin, et al., Cell 90, 753-762 (1997)). NP-2 binds members of the collapsin/semaphorin family selectively (Chen, et al., Neuron 19, 541-559 (1991)).
  • VEGF 165 R/NP-I and NP-2 are expressed by both endothelial and tumor cells.
  • Fig. 19 We have shown that endothelial cells expressing both KDR and VEGFK 35 R/NP-I respond with increased chemotaxis towards VEGF 165 , not VEGF 121 , when compared to endothelial cells expressing KDR alone. While not wishing to be bound by theory, we believe that VEGF ⁇ 65 R/NP-l functions in endothelial cells to mediate cell motihty as a co-receptor for KDR.
  • VEGF1. 65 stimulates 231 breast carcinoma cell motility in a dose-response manner (Fig 15 A).
  • VEGF ⁇ . 2 ! had no effect motility of these cells (Fig 15B).
  • tumor cells such as, 231 cells, do not express the VEGF receptors, KDR or Fit- 1 , while not wishing to be bound by theory, we believe that tumor cells are directly responsive to VEGF1 .65 via VEGF 165 R/NP-1.
  • VEGF 165 R/NP-1 we have also analyzed two variants of Dunning rat prostate carcinoma cells,
  • AT2.1 cells which are of low motility and low metastatic potential
  • AT3.1 cells which are highly motile, and metastatic.
  • Cross-linking and Northern blot analysis show that AT3.1 cells express abundant VEGF1 .65 R/NP-I, capable of binding VEGF ) 5 , while AT2.1 cells don't express VEGF1 .65 R/NP-I (Fig 18).
  • Immunostaining of tumor sections confirmed the expression of VEGF1 .65 R/NP-I in AT3.1, but not AT2.1 tumors (Fig 17). Additionally, immunostaining showed that in subcutaneous AT3.1 and PC3 tumors, the tumor cells expressing VEGF 165 R NP-I were found preferentially at the invading front of the tumor/dermis boundary (Fig 17).
  • the present invention provides a method of diagnosing cancer, especially prostate cancer, breast cancer, and hemangioendothelioma in a patient by measuring levels of VEGF 165 R/NP-I or NP-2 in a biological specimen obtained from the patient.
  • Levels of VEGF1. 65 R/NP-I or NP-2 in the sample greater than a base line level for that type of specimen is indicative of cancer.
  • Biological specimens include, for example, blood, tissue, serum, stool, urine, sputum, cerebrospinal fluid and supernatant from cell lysate. The determination of base lines and comparison levels is by standard modes of analysis based upon the present disclosure.
  • the present invention provides a method of prognosis in an individual having cancer, the method comprising: a. obtaining a tumor sample from said individual; b. measuring VEGF 165 R/NP-1 or NP-2 amounts to obtain an VEGF ⁇ R/NP-l level in said sample; c. correlating said VEGF 165 R NP-I levels with a baseline level; and correlating levels of VEGF 165 R/NP-I or NP-2 higher than the baseline with an indication of unfavorable prognosis and levels of VEGF 165 R/NP-I or NP-2 at the baseline or less with a favorable prognosis.
  • VEGF1 vascular endothelial growth factor
  • R/NP-I mRNA or protein may be measured to obtain VEGF 165 R/NP-I levels.
  • expression of VEGFi ⁇ sR/NP-1 or NP-2 in a tumor sample greater than a base line level for that particular tissue indicates a higher risk of tumor metastasis.
  • the present invention provides a method for determining the metastatic potential of a tumor by measuring the level of VEGF ⁇ 65 R/NP- 1 or NP-2 expression in the tumor. Expression of VEGFi 65 R/NP-l or NP-2 in said tumor greater than a base line level for that particular tissue indicates an increased metastatic potential.
  • changes in condition can be monitored by comparing changes in VEGF ⁇ R/NP-l or NP-2 expression levels in the tumor in that subject over time.
  • levels of VEGF ⁇ 65 R/NP-l or NP-2 can be ascertained by measuring the protein directly or indirectly by measuring transcript (mRNA) encoding VEGF ⁇ 65 R/NP-l or NP-2.
  • mRNA levels can be measured, for example, using an RNA dependent polymerase chain reaction, e.g., reverse transcriptase PCR or Northern blot analysis. DNA chip technology may also be used to measure mRNA levels.
  • Base line levels can readily be determined by measuring levels of VEGF ⁇ 5 R/NP-l or NP-2 in sample of disease free individuals.
  • the present invention also provides of a method for measuring VEGF ⁇ 5 R/NP-
  • NP-2 levels in non-neuronal tissues which comprises the steps of: a. contacting a biological specimen with an antibody or antibody fragment which selectively binds VEGF ⁇ 65 R/NP-l or NP-2, and b. detecting whether said antibody or said antibody fragment is bound by said sample and thereby measuring the levels of VEGF1. 65 R/NP-I or NP-2.
  • the receptor can serve as a target for compounds that disrupt its function.
  • compounds include, for example, VEGF antagonists, compounds that bind to NP-1 or NP-2 and antibodies that specifically binds the receptor at a region that inhibits receptor function.
  • VEGF antagonists compounds that bind to NP-1 or NP-2 and antibodies that specifically binds the receptor at a region that inhibits receptor function.
  • one can use such VEGF 165 R NP-I or NP-2 cells in an assay to discover compounds that bind to or otherwise interact with these receptors in order to discover compounds that can be used to inhibit metastasis.
  • Other aspects of the invention are disclosed infra.
  • Figure 1 shows purification of VEGF ⁇ . 5 R From 231 Cells. 12:> I-VEGF ⁇ 5 (5 ng/ml) was bound and cross-linked to receptors on 231 cells and analyzed by SDS PAGE and autoradiography (lane 1).
  • VEGF ⁇ 65 R was purified by Con A and VEGF ⁇ . 65 affinity column chromatography and analyzed by SDS-PAGE and silver stain (lane 2). Two prominent bands were detected (arrows) and N- terminally sequenced separately. Their N-terminal 18 amino acid sequences are shown to the right of the arrows.
  • the published N-terminal sequences of human and mouse neuropilin (Kawakami et al., J. Neurobiol, 29, 1-17 (1995); He and Tessier-Lavigne, Cell 90, 739-751 1997) are shown below (SEQ ID NOS: 5 and 6).
  • FIGS 2 A and 2B show isolation of VEGF ⁇ cDNA by Expression Cloning. Photomicrographs (dark field illumination) of COS 7 cells binding l25 I-VEGF ⁇ 65 . 125 I- VEGF165 was bound to transfected COS 7 cells which were then washed, fixed, and overlayed with photographic emulsion that was developed as described in the example, infra.
  • FIG. 2A shows COS 7 cells were transfected with a primary plasmid pool (#55 of the 231 cell library) representing approximately 3 x 10 3 clones and one COS 7 cell binding 12r, I-VEGF ⁇ 5 in the first round of screening is shown.
  • Figure 2 shows several COS 7 cells transfected with a single positive cDNA clone (A2) binding l23 I-VEGF ⁇ 65 after the third round of screening.
  • Figure 3 shows the Deduced Amino Acid Sequence of Human VEGF1.
  • 65 R/NP- 1 SEQ ID NO:3
  • the deduced 923 amino acid sequence of the open reading frame of VEGF ⁇ 5 R/NP-l, clone A2 (full insert size of 6.5 kb) is shown.
  • the putative signal peptide sequence (amino acids 1-21) and the putative transmembrane region (amino acids 860-883) are in boxes.
  • FIG. 4 shows the Comparison of the Deduced Amino Acid Sequence of
  • VEGF Human VEGF6 R/NP-l (SEQ ID NO 2) and NP-2 (SEQ ID NO 4)
  • the deduced open reading frame amino acid sequences of VEGF1 .63 R/NP-I and NP-2 are aligned using the DNASIS program Amino acids that are identical in both open reading frames are shaded The overall homology between the two sequences is 43%
  • Figure 5 shows a Northern Blot Analysis of VEGF ⁇ .
  • 3 R/NP-l Expression in Human EC and Tumor-Derived Cell Lines Total RNA samples prepared from HUVEC (lane 1 ) and tumor-derived breast carcinoma, prostate carcinoma and melanoma cell lines as indicated (lanes 2-8) were resolved on a 1% agarose gel and blotted onto a GeneScreen nylon membrane The membrane was probed with j2 P- labeled cDNA and exposed to X-ray film Equal RNA loading was demonstrated by ethydium bromide staining of the gel prior to blotting A major species of VEGF ⁇ . ⁇ R/NP-1 mRNA of approximately 7 kb was detected in several of the cell lines
  • Figure 6 shows a Northern Blot Analysis of VEGF1 .65 R/NP-I and KDR mRNA in Adult Human Tissues
  • a pre -made Northern blot membrane containing multiple samples of human mRNA (Clonetech) was probed with 32 P-labeled cDNA (top) as described in Fig 5, and then stripped and reprobed with P-labeled KDR cDNA (bottom)
  • Figures 7 A and 7B show a Scatchard Analysis of VEGF i65 binding to
  • Figure 7A Increasing amounts of I-VEGF ⁇ 5 (0.1-50 ng/ml) were added to subconfluent cultures of PAE cells transfected with human VEGF 165 R/NP-I cDNA (PAE/NP-1 cells) in 48 well dishes. Non-specific binding was determined by competition with a 200-fold excess of unlabeled VEGF ⁇ 65 . After binding, the cells were washed, lysed and the cell-associated radioactivity was determined using a ⁇ counter.
  • FIG 7B The binding data shown in Figure 7 A were analyzed by the method of Scatchard, and a best fit plot was obtained with the LIGAND program (Munson and Rodbard, 1980).
  • PAE/NP-1 cells express approximately 3 X 10 5 VEGF 16S binding sites per cell and bind I25 I-VEGF 165 with a Kd of 3.2 X 10" 10 M.
  • Figure 8 shows cross-linking of VEGF ⁇ 65 and VEGF.121 to PAE cells Expressing VEGF ⁇ 65 R/NP-l and/or KDR.
  • ' 2 'l-VEGF 165 (5 ng/ml) (lanes 1-6) or 12D I- VEGF 121 (10 ng/ml) (lanes 7-10) were bound to subconfluent cultures of HUVEC (lane 1), PC3 (lane 2), PAE (lanes 3 and 7), a clone of PAE cells transfected with human VEGF 165 R/NP-1 cDNA (PAE/NP-1) (lanes 4 and 8), a clone of PAE cells transfected with KDR (PAE/KDR) (lanes 5 and 9), and a clone of PAE/KDR cells transfected with human VEGF ⁇ 65 R/NP-l cDNA (PAE/KDR/NP-1) (lanes 6 and 10).
  • the binding was carried out in the presence of 1
  • each I- VEGF isoform was chemically cross-linked to the cell surface.
  • the cells were lysed and proteins were resolved by 6% SDS-PAGE.
  • the polyacrylamide gel was dried and exposed to X-ray film.
  • Solid arrows denote radiolabeled complexes containing ' ⁇ I-VEGF and KDR, open arrows denote radiolabeled complexes containing 123 I- VEGF and VEGF 165 R NP-I .
  • FIG. 9 shows cross linking of VEGF) 65 to PAE/KDR Cells Co-expressing VEGF ⁇ 5 R/NP-l Transiently.
  • PAE/KDR cells were transfected with pCPhygro or pCPhyg-NP-1 plasmids as described in "Experimental Procedures", and grown for 3 days in 6 cm dishes. ' D I-VEGF ⁇ .
  • Figure 10 shows inhibition of I25 I-VEGF 165 binding to VEGF ⁇ 65 R/NP-l
  • I-VEGF 165 (5 ng/ml) was bound to subconfluent cultures of PAE transfected with human VEGF 165 R NP-I cDNA (PAE/NP-1) (lanes 1 and 2), PAE/KDR cells (lanes 3 and 4), and PAE/KDR cells transfected with human VEGF ]65 PJNP-1 cDNA (PAE/KDR/NP-1) (lanes 5 and 16) in 35 mm dishes.
  • the binding was carried out in the presence (lanes 2, 4, and 6) or the absence (lanes 1, 3, and 5) of 25 ⁇ g/ml GST-Ex 7+8. Heparin (1 ⁇ g/ml) was added to
  • I-VEGF 165 was chemically cross- linked to the cell surface.
  • the cells were lysed and proteins were resolved by 6% SDS-PAGE as in Figure 9.
  • Solid arrows denote radiolabeled complexes containing 12:> I-VEGFi65 and KDR, open arrows denote radiolabeled complexes containing l2 7 VEGF165 and VEGF 1 65 R NP-I .
  • Figures 11A-C show a model for VEGF ⁇ 65 R/NP-l modulation of VEGF1 .65 Binding to KDR.
  • 1 1 A Cells expressing KDR alone.
  • 1 IB Cells co-expressing KDR and VEGF 165 R NP-I.
  • VEGF 165 binds to KDR via exon 4 and to VEGF ⁇ 65 R/NP-l via exon 7 (Keyt et al. J. Biol. Chem. 271,5638-5646 (1996b); Soker et al., J. Biol. Chem. 271, 5761-5767 (1996)).
  • a rectangular VEGF . 1 65 molecule represents a suboptimal conformation that doesn't bind to KDR efficiently while a rounded VEGF. ⁇ 65 molecule represents one that fits better into a binding site.
  • VEGF1 .65 binds to KDR in a sub-optimal manner.
  • VEGF1 .65 binds to KDR in a sub-optimal manner.
  • 65 R/NP-I the binding efficiency of VEGF ⁇ 65 to KDR is enhanced. It may be that the presence of VEGF1 65 R NP-I increases the concentration of VEGF ⁇ 5 on the cell surface, thereby presenting more growth factor to KDR.
  • VEGF ⁇ 65 R/NP-l may induce a change in VEGF1.65 conformation that allows better binding to KDR, or both might occur.
  • VEGF 165 binding to VEGF ⁇ 65 R/NP-l is competitively inhibited and its binding to KDR reverts to a sub-optimal manner.
  • Figure 12 shows the human NP-2 amino acid sequence (SEQ ID NO:4).
  • Figures 13A and 13B show the human NP-2 DNA sequence (SEQ ID NO:3).
  • Figures 14A, 14B and 14C show the nucleotide (SEQ ID NO: l) and amino acid sequences (SEQ ID NO:2) of VEGF 165 R/NP-1.
  • Figures 15A and 15B show VEGF ⁇ . 65 stimulation of MDA MB 231 cell motility.
  • Figure 15 A Dose response of VEGF i65 motility activity.
  • Figure 15B Both VEGF] 65 and bFGF stimulate motility but VEGF 121 does not.
  • Figures 16A, 16B and 16C show motility and neuropilin-1 expression of Dunning rat prostate carcinoma cell lines AT3-1 (high motility, high metastatic potential) and AT2.1 (low motility, low metastatic potential) cells.
  • Figure 16A AT3.1 cells are more motile than AT2.1 cells in a Boyden chamber assay.
  • Figure 16B 125I-VEGFi 65 cross-links neuropilin-1 on AT3.1 cells but does not cross-link to AT2.1 cells.
  • Figure 16C AT3.1 but not AT2.1 cells express neuropilin-1, while both cell types express VEGF.
  • Figures 17A, 17B and 17C show immunostaining of (Figure 17A) a PC3 subcutaneous tumor in a nude mouse, ( Figure 17B) an AT3.1 tumor in a rat, ( Figure 17C) an AT2.1 tumor in rat with anti-neuropilin-1 antibodies.
  • Neuropilin lmmunostaimng is preferentially associated with PC3 and AT3 1 tumor cells at the tumor/dermis boundary Some of these cells cluster around blood vessels AT2 1 cells do not express neurop ⁇ l ⁇ n-1
  • Figures 18A and 18B show overexpression of neurop ⁇ l ⁇ n-1 in AT2 1 cells (Figure 18A) Western blot, ( Figure 18B) motihty activity Three AT2 1 clones (lanes 4,5,6) express higher amounts of neurop ⁇ l ⁇ n-1 protein and are more motile compared to parental AT2 1 cells or AT2 1 vector (AT2 1/V) controls and approach AT3 1 cell neurop ⁇ l ⁇ n-1 levels and migration activity
  • Figure 19 shows expression of NP-1, NP-2 and ⁇ -actm in cancer cell lines and endothelial cells using reverse transc ⁇ ptase PCR following primers
  • VEGF receptors VEGFR
  • neuropilms such as VEGF ⁇ . ⁇ R/NP-1 and NP-2 that are associated with metastatic potential of a malignant cell
  • Preferred ones are VEGF ⁇ ,R/NP-l and NP-2 but any neuropihn or VEGFR, where the constituents share at least about 85% homology with either of the above VEGF ⁇ R/NP-l and NP-2 can be used More preferably, such constituent shares at least 90% homology Still more preferably, each constituent shares at least 95%o homology
  • % homology can be determined by any standard algorithm used to compare homologies These include, but are not limited to BLAST 2 0 such as BLAST 2 0 4 and l 2 0 5 available from (Altschul, S F , et al Nucleic Acids Res 25 3389-3402 (1997))and DNASIS (Hitachi Software Engineering America, Ltd ) These programs should preferably be set to an automatic setting such as the standard default setting for homology compa ⁇ sons As explained by the NIH, the scoring of gapped results tends to be more biologically meaningful than ungapped results. For ease of reference, this disclosure will generally talk about VEGF i sR/NP-l and NP-2 and/or homologs thereof but all teaching are applicable to the above- described homologs.
  • a VEGFR can be used as long as it binds to a sequence having at least 90%, more preferably 95 % homology to exon 7 of VEGF ⁇ . 6 5
  • VEGF receptors and neuropi ns e.g., VEGF ⁇ 6 R/NP-l and NP-2
  • VEGF ⁇ 6 R/NP-l and NP-2 are associated with both tumor metastases and angiogenesis
  • VEGF 165 R/NP-I and NP-2 is upregulated in highly metastatic prostate cancer cell lines relative to poorly metastatic or nonmetastatic lines.
  • expression of VEGF 16 5R NP-I and NP-2 can be used to determine metastatic potential
  • the receptor and the component provide a target for treatments to inhibit the metastasis process.
  • the evaluation and comparison of levels of transcript (mRNA) or gene product, either normal or mutated, in non-neuronal tissue can be both diagnostic and prognostic of particular cancer.
  • an elevated level is indicative of a greater tendency for metastatic activity.
  • lower levels than certain baselines can also be used to indicate the metastatic potential of the tumor.
  • the present invention provides a method of diagnosing cancer, preferably prostate and breast cancer, in a patient by measuring levels of VEGF ⁇ . ⁇ R/NP-1 or NP- 2 in a biological specimen obtained from the patient.
  • Levels of VEGF ⁇ 6 R/NP-l or NP-2 in the sample greater than a base line level is indicative of cancer.
  • Baseline levels can readily be determined by measuring levels of VEGF ⁇ R NP-l or NP-2 in a sample of disease free individuals. Additionally, baselines can be obtained by measuring individuals having cancer over the course of the malignancy.
  • Biological specimens include, for example, blood, tissue, serum, stool, urine, sputum, cerebrospinal fluid and supernatant from cell lysate. Preferably, one uses tissue specimens.
  • the determination of base lines and comparison levels is by standard modes of analysis based upon the present disclosure.
  • the present invention also provides a method of prognosis in an individual having cancer, preferable prostate, breast cancer and melanoma by measuring levels of VEGF 165 R/NP-I or NP-2 in a tumor sample obtained from a patient to be tested. Expression of VEGF1. 65 R/NP-I or NP-2 in said tumor sample greater than a base line level for that particular tissue indicates a higher risk of tumor metastasis. This information can be used by the physician in determining the most effective course of treatment.
  • Changes in a patients condition can be monitored using the methods of the present invention by comparing changes in VEGF 165 R/NP-I or NP-2 expression levels in the tumor in that subject over time. For example, determining whether the level stabilizes or preferably declines.
  • the present invention further provides a method for determining the metastatic potential of a tumor by measuring the level of VEGF
  • Expression of VEGF ⁇ 65 R NP-l or NP-2 in said tumor greater than a base line level for that particular tissue indicates an increased metastatic potential.
  • Standard detection techniques well known in the art for detecting RNA, DNA, proteins and peptides can readily be applied to detect VEGF J OJ R/NP-I or NP-2 or its transcript to diagnose cancer, especially metastatic cancer or to confirm that a primary tumor has, or has not, reached a particular metastatic phase.
  • Such techniques may include detection with nucleotide probes or may comprise detection of the protein by, for example, antibodies or their equivalent.
  • the nucleotide probes hybridize to the sequence shown in SEQ ID NO: 1 for VEGF ⁇ 65 R/NP-l and SEQ ID NO:3 for NP-2.
  • a VEGFR/NP-1 homolog has at least about 85% homology to the amino acid sequence encoded by SEQ ID NO: 1, whereas a VEGFR/NP-2 homolog has at least about 85% homology to the amino acid sequence encoded by SEQ ID NO:3.
  • Types of probe include cDNA, riboprobes, synthetic oligonucleotides and genomic probes.
  • probes for in situ hybridization, and cDNA for Northern blotting, for example.
  • the most preferred probes are those which correspond to the cytoplasmic domain of the receptor. Most preferably, the probe is directed to nucleotide regions unique to the receptor. Detection of the VEGF ⁇ 5 R/NP-l or NP-2 encoding gene, per se, will be useful in screening for mutations associated with enhanced expression. Other forms of assays to detect targets more readily associated with levels of expression— transcripts and other expression products will generally be useful as well.
  • the probes may be as short as is required to differentially recognize VEGF 165 R/NP-I or NP-2 mRNA transcripts, and may be as short as, for example, 15 bases, however, probes of at least 17 bases, more preferably 18 bases and still more preferably 20 bases are preferred.
  • a probe may also be reverse-engineered by one skilled in the art from the amino acid sequence of SEQ ID NO:2 (VEGF ⁇ 65 R/NP-l) or SEQ I D NO:4 (NP-2).
  • use of such probes may be more limited than the native DNA sequence, as it will be appreciated that any one given reverse-engineered sequence will not necessarily hybridize well, or at all with any given complementary sequence reverse-engineered from the same peptide, owing to the degeneracy of the genetic code. This is a factor common in the calculations of those skilled in the art, and the degeneracy of any given sequence is frequently so broad as to yield a large number of probes for any one sequence.
  • the form of labeling of the probes may be any that is appropriate, such as the use of radioisotopes, for example, 32 P and 33 S. Labeling with radioisotopes may be achieved, whether the probe is synthesized chemically or biologically, by the use of suitably labeled bases. Other forms of labeling may include enzyme or antibody labeling such as is characteristic of ELISA.
  • RNA transcripts may be achieved by Northern blotting, for example, wherein a preparation of RNA is run on a denaturing agarose gel, and transferred to a suitable support, such as activated cellulose, nitrocellulose or glass or nylon membranes, radiolabeled cDNA or RNA is then hybridized to the preparation, washed and analyzed by autoradiography
  • In situ hybridization visualization may also be employed, wherein a radioactively labeled antisense cRNA probe is hybridized with a thin section of a biopsy sample, washed, cleaved with RNase and exposed to a sensitive emulsion for autoradiography
  • the samples may be stained with haematoxylon to demonstrate the histological composition of the sample, and dark field imaging with a suitable light filter shows up the developed emulsion
  • Non-radioactive labels such as digoxigenm may also be used.
  • Immunohistochemistry may be used to detect expression of human
  • VEGF 16 5R/NP-I or NP-2 in a biopsy sample A suitable antibody is brought into contact with, for example, a thin layer of cells, washed, and then contacted with a second, labeled antibody. Labeling may be by enzyme, such as peroxidase, avidin or by radiolabelhng. Chromogenic labels are generally preferable, as they can be detected under a microscope.
  • Antibodies may be prepared as described below, and used in any suitable manner to detect expression of VEGF 165 R/NP-I or NP-2.
  • Antibody-based techniques include ELISA (enzyme linked immunosorbent assay) and RIA (radioimmunoassay) Any conventional procedures may be employed for such immunoassays.
  • a VEGF1. 6 5R/NP-I or NP-2 standard is labeled with a radioisotope such as 7 or " "S, or an assayable enzyme, such as horseradish peroxidase or alkaline phosphatase and, together with the unlabelled sample, is brought into contact with the corresponding antibody, whereon a second antibody is used to bind the first and radioactivity or the immobilized enzyme assayed (competitive assay), alternatively, VEGF 16 5R/NP-I or NP-2 in the sample is allowed to react with the corresponding immobilized antibody, radioisotope- or enzyme-labeled anti-receptor antibody is allowed to react with the system and radioactivity or the enzyme assayed (ELISA-sandwich assay). Other conventional methods may also be employed as suitable.
  • the above techniques may be conducted essentially as a “one-step” or “two-step” assay.
  • the “one-step” assay involves contacting antigen with immobilized antibody and, without washing, contacting the mixture with labeled antibody.
  • the “two-step” assay involves washing before contacting the mixture with labeled antibody
  • Other conventional methods may also be employed as suitable
  • Enzymatic and radio-labeling of receptor and/or the antibodies may be effected by conventional means.
  • Such means will generally include covalent linking of the enzyme to the antigen or the antibody in question, such as by glutaraldehyde, specifically so as not to adversely affect the activity of the enzyme, by which is meant that the enzyme must still be capable of interacting with its substrate, although it is not necessary for all of the enzyme to be active, provided that enough remains active to permit the assay to be effected.
  • some techniques for binding enzyme are non-specific (such as using formaldehyde), and will only yield a proportion of active enzyme.
  • Enzymes employable for labeling are not particularly limited, but may be selected from the members of the oxidase group, for example These catalyze production of hydrogen peroxide by reaction with their substrates, and glucose oxidase is often used for its good stability, ease of availability and cheapness, as well as the ready availability of its substrate (glucose)
  • Activity of the oxidase may be assayed by measuring the concentration of hydrogen peroxide formed after reaction of the enzyme-labeled antibody with the substrate under controlled conditions well-known in the art
  • VEGF1 5R/NP-I or NP-2
  • Western blotting Towbm et at , Proc Nat Acad Sci 76 4350 (1979)
  • a suitably treated sample is run on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose filter
  • Anti- receptor antibodies are then brought into contact with the support and assayed by a secondary immunological reagent, such as labeled protein A or anti-immunoglobuhn (suitable labels including n horseradish peroxidase and alkaline phosphatase)
  • Samples for diagnostic purposes may be obtained from any number of sources
  • a sample obtained direct from the tumor such as the stroma or cytosol, may be used to determine the metastatic potential of the tumor It may also be appropriate to obtain the sample from other biological specimens, such as blood or urine Such diagnosis may be of particular importance in monito ⁇ ng progress of a patient, such as after surgery to remove a tumor If a reference reading is taken after the operation, then another taken at regular intervals, any ⁇ se could be indicative of a relapse, or possibly a metastasis
  • the sample is from the tumor itself
  • the antibodies may be raised against either a peptide of the receptor or the whole molecule Such a peptide may be presented together with a earner protein, such as an KLH, to an animal system or, if it is long enough, say 25 amino acid residues, without a earner
  • a peptide may be presented together with a earner protein, such as an KLH, to an animal system or, if it is long enough, say 25 amino acid residues, without a earner
  • Polyclonal antibodies generated by the above technique may be used direct, or suitable antibody producing cells may be isolated from the animal and used to form a hybridoma by known means (Kohler and Milstein, Nature 256:795. (1975)). Selection of an appropriate hybridoma will also be apparent to those skilled in the art, and the resulting antibody may be used in a suitable assay to identify VEGF 165 R/NP-I or NP- 2.
  • This invention provides a convenient kit for measuring human VEGF ⁇ 5 R/NP-
  • This kit includes antibodies or antibody fragments which selectively bind human VEGF ⁇ 65 R/NP-l or a set of DNA oligonucleotide primers that allow synthesis of cDNA encoding the receptors.
  • the primers comprise at least 17 nucleotides and hybridizes under stringent conditions to a DNA fragment having the nucleotide sequence set forth in SEQ ID NO: 1 (VEGF ⁇ 65 R/NP-l) or SEQ ID NO:3 (NP-2).
  • stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • DNA encoding human VEGF ⁇ 65 R/NP-l or NP-2 and recombinant human VEGF ⁇ 65 R/NP-l or NP-2 may be produced according to the methods set forth in the Examples.
  • the receptors are preferably produced by recombinant methods.
  • a wide variety of molecular and biochemical methods are available for generating and expressing the polypeptides of the present invention; see e.g. the procedures disclosed in Molecular Cloning, A Laboratory Manual (2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor), Current Protocols in Molecular Biology (Eds. Aufubel, Brent, guitarist, More, Feidman, Smith and Guatemala, Greene Publ. Assoc, Wiley-Interscience, NY, N.Y. 1992) or other procedures that are otherwise known in the art.
  • the polypeptides of the invention may be obtained by chemical synthesis, expression in bacteria such as E. coli and eukaryotes such as yeast, baculovirus, or mammalian cell-based expression systems, etc., depending on the size, nature and quantity of the polypeptide.
  • isolated means that the polypeptide is removed from its original environment (e.g., the native VEGF molecule).
  • a naturally-occurring polynucleotides or polypeptides present in a living animal is not isolated, but the same polynucleotides or DNA or polypeptides, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • any suitable system can be used. The general nature of suitable vectors, expression vectors and constructions therefor will be apparent to those skilled in the art.
  • Suitable expression vectors may be based on phages or plasmids, both of which are generally host-specific, although these can often be engineered for other hosts.
  • Other suitable vectors include cosmids and retroviruses, and any other vehicles, which may or may not be specific for a given system.
  • Control sequences such as recognition, promoter, operator, inducer, terminator and other sequences essential and/or useful in the regulation of expression, will be readily apparent to those skilled in the art.
  • Conect preparation of nucleotide sequences may be confirmed, for example, by the method of Sanger et al. (Proc. Natl. Acad. Sci. USA 74:5463-7 (1977)).
  • a DNA fragment encoding the receptor or fragment thereof may readily be inserted into a suitable vector.
  • the receiving vector has suitable restriction sites for ease of insertion, but blunt-end ligation, for example, may also be used, although this may lead to uncertainty over reading frame and direction of insertion. In such an instance, it is a matter of course to test transformants for expression, 1 in 6 of which should have the correct reading frame.
  • Suitable vectors may be selected as a matter of course by those skilled in the art according to the expression system desired.
  • the desired polypeptide or protein By transforming a suitable organism or, preferably, eukaryotic cell line, such as HeLa, with the plasmid obtained, selecting the transformant with ampicillin or by other suitable means if required, and adding tryptophan or other suitable promoter-inducer (such as indoleacrylic acid) if necessary, the desired polypeptide or protein may be expressed.
  • the extent of expression may be analyzed by SDS polyacrylamide gel electrophoresis-SDS-PAGE (Lemelli, Nature 227:680-685 (1970)). Suitable methods for growing and transforming cultures etc. are usefully illustrated in, for example, Maniatis (Molecular Cloning, A Laboratory Notebook, Maniatis et al. (eds.), Cold Spring Harbor Labs, N.Y. (1989)).
  • Cultures useful for production of polypeptides or proteins may suitably be cultures of any living cells, and may vary from prokaryotic expression systems up to eukaryotic expression systems.
  • One preferred prokaryotic system is that of E. coll, owing to its ease of manipulation.
  • a higher system such as a mammalian cell line, for expression of a eukaryotic protein.
  • Currently preferred cell lines for transient expression are the HeLa and Cos cell lines.
  • Other expression systems include the Chinese Hamster Ovary (CHO) cell line and the baculovirus system.
  • streptomycetes for example, and yeasts, such as Saccharomyces spp., especially S. cerevisiae. Any system may be used as desired, generally depending on what is required by the operator. Suitable systems may also be used to amplify the genetic material, but it is generally convenient to use E. coli for this purpose when only proliferation of the DNA is required.
  • polypeptides and proteins may be isolated from the fermentation or cell culture and purified using any of a variety of conventional methods including: liquid chromatography such as normal or reversed phase, using HPLC, FPLC and the like; affinity chromatography (such as with inorganic ligands or monoclonal antibodies); size exclusion chromatography; immobilized metal chelate chromatography; gel electrophoresis; and the like.
  • liquid chromatography such as normal or reversed phase, using HPLC, FPLC and the like
  • affinity chromatography such as with inorganic ligands or monoclonal antibodies
  • size exclusion chromatography size exclusion chromatography
  • immobilized metal chelate chromatography immobilized metal chelate chromatography
  • gel electrophoresis and the like.
  • the DNA encoding N ⁇ GF 165 R/NP-1 or NP-2 can be used to treat ischemia, e.g., heart and limb.
  • the DNA can be used as an adjunct to gene therapy with VEGF (See, e.g., Isner, J., WO 97/14307).
  • VEGF See, e.g., Isner, J., WO 97/14307.
  • the DNA can be delivered to the vicinity of the area to be treated either prior to, or along with, the VEGF or DNA encoding the VEGF.
  • the present invention also provides binding assays using VEGF ⁇ 5 R/NP-l or NP-2 that permit the ready screening for compounds which affect the binding of the receptor and its ligands, e.g., VEGF1 65 .
  • these assays can be used to identify compounds that modulate, preferably inhibit metastasis. However, it is also important to know if a compound enhances metastasis so that its use can be avoided.
  • the compound of interest can be added before or after the addition of the labeled ligand, e.g., VEGF ]65 and the effect of the compound on binding or metastasis can be determined by comparing the degree of binding in that situation against a base line standard with that ligand, not in the presence of the compound.
  • the assay can be adapted depending upon precisely what is being tested.
  • the preferced technique for identifying molecules which bind to the VEGF ⁇ 5 R/NP-l receptor utilizes a receptor attached to a solid phase, such as the well of an assay plate.
  • the binding of the candidate molecules, which are optionally labeled (e.g., radiolabeled), to the immobilized receptor can be measured.
  • competition for binding of a known, labeled receptor ligand, such as I- 12 VEGF1. 65 can be measured.
  • the VEGF1 .65 R/NP-I receptor can be exposed to a receptor ligand, e.g., VEGF ⁇ 5; followed by the putative antagonist, or the ligand and antagonist can be added to the VEGF 165 R/NP-I receptor simultaneously, and the ability of the antagonist to block receptor activation can be evaluated.
  • VEGF antagonist activity may also be determined by inhibition of binding of labeled VEGF1. 65 to VEGF 165 R as disclosed in the Examples.
  • the ability of discovered antagonists to influence angiogenesis or metastasis can also be determined using a number of know in vivo and in vitro assays. Such assays are disclosed in Jain et al., Nature Medicine 3, 1203-1208(1997), and the examples.
  • the present invention further provides use of the VEGF1.
  • 65 R/NP-I for intracellular or extracellular targets to affect binding.
  • Intracellular targeting can be accomplished through the use of intracellularly expressed antibodies refened to as intrabodies.
  • Extracellular targeting can be accomplished through the use of receptor specific antibodies.
  • the soluble form of the receptor can be used as a receptor decoy to inhibit binding. These methods can be used to inhibit metastasis in malignant cells as we have found that the presence of these receptors is positively correlated with metastasis.
  • One prefened approach is the use of antibodies that specifically block VEGF binding to the receptor.
  • an antibody to the VEGF binding site can be prepared by standard means. For example, one can use single chain antibodies to target these binding sites.
  • One type of decoy can be obtained by deleting or otherwise changing the transmembrane portion of the molecule.
  • the cytoplasmic tail can also be deleted in one embodiment, or retained in another.
  • isolated antibodies or antibody fragments which selectively binds the receptor.
  • the antibody fragments include, for example, Fab, Fab', F(ab')2 or Fv fragments.
  • the antibody may be a single chain antibody, a humanized antibody or a chimeric antibody.
  • Antibodies, or their equivalents, or other receptor antagonists may also be used in accordance with the present invention for the treatment or prophylaxis of cancers.
  • Administration of a suitable dose of the antibody or the antagonist may serve to block the receptor and this may provide a crucial time window in which to treat the malignant growth.
  • Prophylaxis may be appropriate even at very early stages of the disease, as it is not known what specific event actually triggers metastasis in any given case.
  • administration of the antibodies, their equivalents, intrabodies, decoys or antagonists which interfere with receptor activity may be effected as soon as cancer is diagnosed, and treatment continued for as long as is necessary, preferably until the threat of the disease has been removed.
  • Such treatment may also be used prophylactically in individuals at high risk for development of certain cancers, e.g., prostate or breast.
  • a method of treatment involves attachment of a suitable toxin to the antibodies which then target the area of the tumor.
  • Such toxins are well known in the art, and may comprise toxic radioisotopes, heavy metals, enzymes and complement activators, as well as such natural toxins as ricin which are capable of acting at the level of only one or two molecules per cell. It may also be possible to use such a technique to deliver localized doses of suitable physiologically active compounds, which may be used, for example, to treat cancers.
  • antibodies for use in accordance with the present invention may be monoclonal or polyclonal as appropriate.
  • Antibody equivalents of these may comprise: the Fab' fragments of the antibodies, such as Fab, Fab', F(ab')2 and Fv; idiotopes; or the results of allotope grafting (where the recognition region of an animal antibody is grafted into the appropriate region of a human antibody to avoid an immune response in the patient), for example.
  • Single chain antibodies may also be used.
  • Other suitable modifications and/or agents will be apparent to those skilled in the art.
  • Chimeric and humanized antibodies are also within the scope of the invention. It is expected that chimeric and humanized antibodies would be less immunogenic in a human subject than the corresponding non-chimeric antibody.
  • a variety of approaches for making chimeric antibodies, comprising for example a non-human variable region and a human constant region, have been described. See, for example, Morrison et al., Proc. Natl. Acad. Sci. U.S.A. 81,6851 (1985); Takeda, et al., Nature 314,452(1985), Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al, U.S. Pat. No. 4,816,397; Tanaguchi et al..
  • a chimeric antibody can be further "humanized" such that parts of the variable regions, especially the conserved framework regions of the antigen-binding domain, are of human origin and only the hypervariable regions are of non-human origin.
  • Such altered immunoglobulin molecules may be made by any of several techniques known in the art, (e.g., Teng et al., Proc. Natl. Acad Sci. U.S.A., 80, 7308-7312 (1983); Kozbor et al., Immunology Today, 4, 7279 (1983); Olsson et al., Meth.
  • Humanized antibodies can be commercially produced by, for example, Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain.
  • the antibody can be administered by a number of methods.
  • One prefereed method is set forth by Marasco and Haseltine in PCT WO94/02610, which is incorporated herein by reference.
  • This method discloses the intracellular delivery of a gene encoding the antibody.
  • the antibody would preferably contain a nuclear localization sequence.
  • the present invention provides for the administration of, for example, antibodies to a patient, then this may be by any suitable route. If the tumor is still thought to be, or diagnosed as, localized, then an appropriate method of administration may be by injection direct to the site. Administration may also be by injection, including subcutaneous, intramuscular, intravenous and intradermal injections.
  • Formulations may be any that are appropriate to the route of administration, and will be apparent to those skilled in the art.
  • the formulations may contain a suitable earner, such as saline, and may also comprise bulking agents, other medicinal preparations, adjuvants and any other suitable pharmaceutical ingredients. Catheters are one preferred mode of administration.
  • VEGF 165 R/NP-I expression may also be inhibited in vivo by the use of antisense technology.
  • Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.
  • An antisense nucleic acid molecule which is complementary to a nucleic acid molecule encoding receptor can be designed based upon the isolated nucleic acid molecules encoding the receptor provided by the invention.
  • An antisense nucleic acid molecule can comprise a nucleotide sequence which is complementary to a coding strand of a nucleic acid, e.g.
  • an antisense sequence complementary to a sequence of an mRNA can be complementary to a sequence in the coding region of the mRNA or can be complementary to a 5' or 3' untranslated region of the mRNA.
  • an antisense nucleic acid can be complementary m sequence to a regulatory region of the gene encoding the mRNA, for instance a transcription initiation sequence or regulatory element.
  • an antisense nucleic acid complementary to a region preceding or spanning the initiation codon or in the 3' untranslated region of an mRNA is used.
  • An antisense nucleic acid can be designed based upon the nucleotide sequence shown in SEQ ID NO: l (VEGF ⁇ 6 R/NP-l) or SEQ ID NO:3/NP-2).
  • a nucleic acid is designed which has a sequence complementary to a sequence of the coding or untranslated region of the shown nucleic acid
  • an antisense nucleic acid can be designed based upon sequences of a VEGF ⁇ 6 R gene, which can be identified by screening a genomic DNA library with an isolated nucleic acid of the invention
  • the sequence of an important regulatory element can be determined by standard techniques and a sequence which is antisense to the regulatory element can be designed.
  • the antisense nucleic acids and oligonucleotides of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • the antisense nucleic acid or oligonucleotide can be chemically synthesized using naturally occumng nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids e.g. phosphorothioate derivatives and acndine substituted nucleotides can be used.
  • the antisense nucleic acids and oligonucleotides can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (l e. nucleic acid transcribed from the inserted nucleic acid will be of an antisense onentation to a target nucleic acid of interest)
  • the antisense expression vector is introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol 1 (1)1986
  • pharmaceutically acceptable salts include mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • antibodies or nucleic acids of the invention are administered orally, topically, or by parenteral means, including subcutaneous and intramuscular injection, implantation of sustained release depots, intravenous injection, intranasal administration, and the like. Accordingly, antibodies or nucleic acids of the invention may be administered as a pharmaceutical composition comprising the antibody or nucleic acid of the invention in combination with a pharmaceutically acceptable carrier. Such compositions may be aqueous solutions, emulsions, creams, ointments, suspensions, gels, liposomal suspensions, and the like.
  • Suitable earners include water, saline, Ringer's solution, dextrose solution, and solutions of ethanol, glucose, sucrose, dextran, mannose, mannitol, sorbitol, polyethylene glycol (PEG), phosphate, acetate, gelatin, collagen, Carbopol Registered TM , vegetable oils, and the like.
  • Cream or ointment bases useful in formulation include lanolin, Silvadene Registered TM (Marion), Aquaphor Registered TM (Duke).
  • Topical formulations include aerosols, bandages, and other wound dressings.
  • Other devices include indwelling catheters and devices such as the Alzet Registered TM minipump.
  • Ophthalmic preparations may be formulated using commercially available vehicles such as Sorbi-care Registered TM (Allergan), Neodecadron Registered TM (Merck, Sharp & Dohme), Lacrilube Registered TM , and the like, or may employ topical preparations such as that described in U.S. Pat. No.
  • VEGF antagonist in solid form, especially as a lyophilized powder.
  • Lyophilized formulations typically contain stabilizing and bulking agents, for example human serum albumin, sucrose, mannitol, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co.).
  • antibody or nucleic acid required to treat any particular disorder will of course vary depending upon the nature and severity of the disorder, the age and condition of the subject, and other factors readily determined by one of ordinary skill in the art.
  • VEGF vascular endothelial growth factor
  • GST VEGF exons 7+8 fusion protein was prepared in E.Coli and purified as previously described (Soker et al., J. Biol. Chem., 271, 5761-5767 (1996)). Heparin, hygromycin B and protease inhibitors were purchased from Sigma (St. Louis, MO). 12:> I-Sodium, 32 P-dCTP, and GeneScreen-Plus hybridization transfer membrane were purchased from DuPont NEN (Boston, MA).
  • DSS Disuccinimidyl suberate
  • IODO-BEADS were purchased from Pierce Chemical Co. (Rockford, IL).
  • Con A Sepharose was purchased from Pharmacia LKB Biotechnology Inc. (Piscataway, NJ).
  • RNAzol-B was purchased from TEL-TEST Inc. (Friendswood, TX).
  • Silver Stain kit and Trans-Blot PVDF membranes were purchased from Bio-Rad Laboratories (Hercules, CA). Multiple tissue northern blot membranes were purchased from Clontech (Palo Alto, CA).
  • PolyATract mRNA isolation kits were purchased from Promega (Madison, WI) RediPnme DNA labeling kits and molecular weight markers were purchased from Amersham (Arlington Heights, IL) Plasmids pcDNA3 1 was purchased from Invitrogen (Carlsbad, CA), and pCPhygro, containing the CMV promoter and encoding hygromycin B phosphorylase, was kindly provided by Dr Urban Deutsch (Max Plank Institute, Bad Nauheim, Germany) Restriction endonucleases and Ligase were purchased from New England Biolabs, Inc (Beverly, MA) NT-B2 photographic emulsion and x-ray film were purchased from the Eastman Kodak company (Roley NY)
  • Human umbilical vein EC (HUVEC) were obtained from Amencan Type Culture Collection (ATCC) (Rockville, MD), and grown on gelatin coated dishes in M-199 medium containing 20%> fetal calf serum (FCS) and a mixture of glutamine, penicillin and streptomycin (GPS) Basic FGF (2 ng/ml) was added to the culture medium every other day
  • Parental porcine aortic endothelial (PAE) cells and PAE cells expressing KDR (PAE/KDR) (Waltenberger et al , J Biol Chem 269, 26988-26995 (1994)) were kindly provided by Dr Lena Claesson-Welsh and were grown in F12 medium containing 10% FCS and GPS MDA-MB-231 cells and MDA-MB-453 cells were obtained from ATCC, and grown in DMEM containing 10% FCS and GPS
  • the human melanoma cell lines, RU-mel, EP-mel and WK-mel were kmdly
  • MDA-MB-231 cells grown in 150 cm dishes were washed with PBS containing 5 mM EDTA, scraped and centrifuged for 5 mm at 500g
  • the cell pellet was lysed with 150 ml of 20 mM HEPES, pH 8 0, 0 5% t ⁇ ton X- 100 and protease inhibitors including 1 mM AEBSF, 5 ⁇ g/ml leupeptin and 5 ⁇ g/ml aprotinin for 30 mm on ice, and the lysate was centrifuged at 30,000 x g for 30 min.
  • MnCb and CaCb were added to the supernatant to obtain a final concentration of ImM each.
  • the lysate was absorbed onto a Con A Sepharose column (7 ml) and bound proteins were eluted with 15 ml 20 mM HEPES, pH 8.0, 0.2 M NaCl, 0.1% triton X-100 and 1 M methyl- ⁇ -D-mannopyranoside at 0.2 ml/min. The elution was repeated twice more at 30 minute intervals.
  • the Con A column eluates were pooled and incubated for 12 h at 4° C with 0.5 ml of VEGF ⁇ 5 - Sepharose beads, containing about 150 ⁇ g VEGF ⁇ . 65 , prepared as described previously (Wilchek and Miron, Biochem. Int. 4, 629-635. (1982)).
  • the VEGF] 65 -Sepharose beads were washed with 50 ml of 20 mM HEPES, pH 8.0, 0.2 M NaCl and 0.1% triton X-100 and then with 25 ml of 20 mM HEPES, pH 8.0.
  • the beads were boiled in SDS-PAGE buffer and bound proteins were separated by 6% SDS-PAGE. Proteins were transferred to a TransBlot PVDF membrane using a semi-dry electric blotter (Hoeffer Scientific), and the PVDF membrane was stained with 0.1 % Coomassie Brilliant Blue in 40% methanol.
  • the two prominent proteins in a 130-140 kDa doublet were cut out separately and N-terminally sequenced using an Applied Biosystems model 477 A microsequenator as a service provided by Dr. William Lane of the Harvard Microchemistry facility (Cambridge, MA).
  • cDNA Complementary DNA
  • Double-stranded cDNA was ligated to EcoRl adaptors, and size-fractionated on a 5- 20%) potassium acetate gradient .
  • DNA fragments larger than 2kb were ligated to an eukaryotic expression plasmid, pcDNA3.1.
  • the plasmid library was transfected into ⁇
  • E.coh to yield a primary library of approximately 1 x 10 individual clones.
  • a portion of the transformed bacteria was divided into 240 pools, each representing approximately 3 x 10 individual clones.
  • DNA prepared from each pool was used to transfect COS-7 cells seeded in 12 well dishes, using the Lipofectin reagent according to the manufacturer's instructions. Three days after transfection, the cells were incubated on ice for 2 h with I23 I-VEGF I 65 (10 ng/ml) in the presence of 1 ⁇ g/ml heparin, washed and fixed with 4%> paraformaldehyde in PBS.
  • OVEGF 165 binding to individual cells was detected by overlaying the monolayers with photographic emulsion, NT-B2, and developing the emulsion after two days as described (Gearing et al.,1989) Seven positive DNA pools were identified and DNA from one of the positive pools was used to transform E Coh .
  • the E coh were sub-divided into 50 separate pools and plated onto 50 LB ampicillin dishes, with each pool representing approximately 100 clones. DNA made from these pools was transfected into COS-7 cells which were screened for l2: T-NEGF ⁇ 5 binding as descnbed above.
  • RNA was prepared from cells in culture using RNAzol according to the manufacturer's instructions. Samples of 20 ⁇ g RNA were separated on a 1% formaldehide-agarose gel, and transferred to a GeneScreen-Plus membrane. The membrane was hybridized with a P labeled fragment of human NEGF ⁇ . 5 R/NP-l cDNA, corresponding to nucleotides 63-454 in the ORF, at 63°C for 18 h. The membrane was washed and exposed to an x-ray film for 18 h. A commercially- obtained multiple human adult tissue mRNA blot (Clonetech, 2 ⁇ g/lane) was probed for human NP-1 in a similar manner.
  • the multiple tissue blot was stnpped by boiling in the presence of 0.5% SDS and re-probed with a P labeled fragment of KDR cDNA corresponding to nucleotides 2841-3251 of the ORF (Terman et al., Oncogene 6, 1677-1683 (1991)).
  • PAE/KDR Parental PAE cells and PAE cells expressing KDR (PAE/KDR) (Waltenberger et al., 1994) were obtained from Dr. Lena Claesson-Welsh. Human NP-1 cDNA was digested with Xhol and Xbal restriction enzymes and subcloned into the corresponding sites of pCPhygro, to yield pCPhyg-NP-1. PAE and PAE/KDR cells were grown in 6 cm dishes and transfected with 5 ⁇ g of pCPhyg-NP-1 using Lipofectamine, according to the manufacturer's instructions. Cells were allowed to grow for an additional 48 h and the medium was replaced with fresh medium containing 200 ⁇ g/ml hygromycin B.
  • Radio-iodination of VEGF, binding and cross-linking experiments The radio-iodination of VEGFj 5 and VEGF1. 21 using IODO-BEADS was carried out as previously described (Soker et al., J. Biol. Chem. 272, 31582-31588 (1997)). The specific activity ranged from 40,000-100,000 cpm/ng protein. Binding and cross-linking experiments using 5 I-VEGF ⁇ 65 and 5 I-NEGF ⁇ 2 ⁇ were performed as previously described (Gitay-Goren et al, J. Biol. Chem. 267, 6093-6098 (1992); Soker et al., J. Biol. Chem. 271, 5761-5767 (1996)).
  • VEGF binding was quantitated by measuring the cell-associated radioactivity in a ⁇ -counter (Beckman, Gamma 5500). The counts represent the average of three wells. All experiments were repeated at least three times and similar results were obtained. The results of the binding experiments were analyzed by the method of Scatchard using the LIGAND program (Munson and Rodbard, 1980). 12: T-VEGFi65 and I2:, I-VEGFi2i cross linked complexes were resolved by 6% SDS/PAGE and the gels were exposed to an X-Ray film. X-ray films were subsequently scanned by using an IS- 1000 digital imaging system (Alpha Innotech Corporation) Purification of VEGF ⁇ 65 R
  • Cross-linking of 12r, I-VEGF ⁇ 65 to cell surface receptors of 231 cells results in formation of a 165-175 kDa labeled complex (Soker et al., J. Biol. Chem. 271, 5761- 5767 (1996)). These cells have about 1-2 x 10 3 VEGF1 .65 binding sites/cell. In contrast to VEGF1. 65 , VEGF 1 21 does not bind to the 231 cells and does not form a ligand-receptor complex (Soker et al., J. Biol. Chem. 271, 5761-5767 (1996)).
  • VEGF1 .65 R is a glycoprotein and accordingly, a 231 cell lysate prepared from approximately 5 x 10 8 cells was absorbed onto a Con A Sepharose column. Bound proteins, eluted from the Con A column, were incubated with VEGFi 65 -Sepharose and the VEGF 165 - affinity purified proteins were analyzed by SDS-PAGE and silver staining ( Figure 9, lane 2). A prominent doublet with a molecular mass of about 130-135 kDa was detected. This size is consistent with the formation of a 165-175 kDa complex of 40-45 kDa
  • VEGF1 65 bound to receptors approximately 130-135 kDa in size (Figure 9, lane 1).
  • the two bands were excised separately and N-terminal amino acid sequencing was carried out (Figure 1, right). Both the upper and lower bands had similar N-terminal amino acid sequences which showed high degrees of sequence homology to the predicted amino acid sequences in the N-terminal regions of mouse (Kawakami et al, J. Neurobiol, 29, 1-17 (1995)) and human neuroplilin-1 (NP-1) (He and Tessier- Lavigne, Cell 90739-151 (1997)).
  • VEGF 165 R was cloned by expression cloning (Aruffo and Seed, Proc. Natl. Acad. Sci. USA 84, 8573-8577 (1987a); Aruffo and Seed, EMBO J., 6, 3313-3316 (1987b); Gearing et al, EMBO J. 8,3667-3676 (1989)).
  • expression cloning 231 cell mRNA was used to prepare a cDNA library of approximately 10 7 clones in a eukaryotic expression plasmid. E. coli transformed with the plasmid library were divided into pools.
  • VEGF ⁇ 65 R After we had cloned human VEGF ⁇ 65 R, two groups reported the cloning of rat and human receptors for semaphorin III and identified them to be NP- 1 (He and Tessier-Lavigne, Cell 90, 739-751 (1997); Kolodkin et al., Cell 90, 753-762 (1997)).
  • the 231 cell-derived VEGF ⁇ 65 R cDNA sequence is virtually identical (see figure legend 3 for exceptions) to the human NP- 1 sequence (He and Tessier-Lavigne, Cell 90, 739-751 (1997)).
  • the human VEGF 165 R NP-I cDNA sequence predicts an open reading frame (ORF) of 923 amino acids with two hydrophobic regions representing putative signal peptide and transmembrane domains ( Figure 3). Overall, the sequence predicts ectodomain, transmembrane and cytoplasmic domains consistent with the structure of a cell surface receptor.
  • the N-terminal sequence obtained via protein purification as shown in Figure 1 is downstream of a 21 amino acid putative hydrophobic signal peptide domain, thereby indicating directly where the signal peptide domain is cleaved and removed.
  • the short cytoplasmic tail of 40 amino acids is consistent with results demonstrating that soluble VEGF1.
  • VEGF ⁇ 65 R/NP-l Expression of VEGF ⁇ 65 R/NP-l in adult cell lines and tissues
  • NP-1 gene expression has been limited so far to the nervous system of the developing embryo (Takagi et al., Dev. Biol. 122, 90-100 (1987); Kawakami et al., J. Neurobiol. 29, 1-17 (1995); Takagi et al, Dev. Biol. 170, 207-222 (1995)).
  • Cell surface VEGF 165 R NP-I is associated with non-neuronal adult cell types such as EC and a variety of tumor-derived cells (Soker et al., J. Biol. Chem. 271, 5761-5767 (1996)).
  • VEGF ⁇ 65 R NP-l mRNA levels were highest in 231 and PC3 cells.
  • VEGF ⁇ 65 R/NP-l mRNA was detected to a lesser degree in HUVEC, LNCaP, EP-mel and RU-mel cells. There was little if any expression in MDA-MB-453 and WK-mel cells.
  • the VEGF ⁇ 65 R/NP-l gene expression patterns were consistent with our previous results showing that HUVEC, 231, PC3, LNCaP, EP-mel and RU-mel cells bind 125 I- VEGF 165 to cell surface VEGF 165 R NP-1 but that MDA-MB-453 and WK-mel cells do not (Soker et al, J. Biol. Chem. Ill, 5761-5767 (1996)).
  • VEGF ⁇ 5 R/NP-l gene expression was analyzed also by Northern blot in a variety of adult tissues in comparison to KDR gene expression (Figure 6).
  • VEGF ⁇ 5 R/NP-l mRNA levels were relatively highly in adult heart and placenta and relatively moderate in lung, liver, skeletal muscle, kidney and pancreas.
  • a relatively low level of VEGF 165 R/NP-I mRNA was detected in adult brain.
  • previous analysis of NP-1 gene expression in mouse and chicken brain suggested that this gene was expressed primarily during embryonic development and was greatly diminished after birth (Kawakami et al., J. Neurobiol. 29, 1-17 (1995); Takagi et al., Dev. Biol. 170, 207-222 (1995)).
  • VEGFi 6 sR/NP-l is expressed widely m adult non- neuronal tissue, including tissues in which angiogenesis occurs such as heart and placenta
  • porcine aortic endothelial (PAE) cells were transfected with the cDNA of VEGF .
  • ⁇ 6 R/NP-l The PAE cells were chosen for these expression studies because they express neither KDR, Flt-1 (Waltenberger et al , J Biol Chem 269, 26988-26995 (1994)) nor VEGF , 65 R Stable cell lines synthesizing VEGF , 6 S R/NP- 1 (PAE/NP- 1 ) were established and OVEGF ⁇ 6 5 binding experiments were carried out (Fig 7) 12 ⁇ I- VEGFi 6 5 binding to PAE/NP-1 cells increased in a dose-dependent manner and reached saturation at approximately 30 ng/ml demonstrating that VEGF ⁇ 6 R NP-l is a specific VEGFi receptor ( Figure 7A) Scatchard analysis of VEGF] ⁇ binding revealed a single class of VEGF ⁇ .65 binding sites with
  • VEGFie but not VEGF,2i, binds to VEGF, 6 5R/NP-1 on HUVEC and 231 cells (Gitay-Goren et al., J Biol Chem 111, 5519-5523 (1992).
  • PAE cells that were previously transfected with KDR cDNA to produce stable clones of PAE/KDR cells (Waltenberger et al , J Biol Chem 269, 26988-26995 (1994)), were transfected with VEGF, 6 5R NP-1 cDNA and stable clones expressing both receptors (PAE/KDR/NP-1) were obtained These cells bound 125 I-VEGFi 6 s to KDR ( Figure 8, lane 6, upper complex) and to VEGF 16 R/NP-l ( Figure 8, lane 6, lower complex) to yield a cross-lmking profile similar to HUVEC ( Figure 8, lane 1) On the other hand, the PAE/KDR/NP-1 cells bound 12 'l-VEGF ⁇ 2 ⁇ to form a complex only with KDR ( Figure 8, lanes 9 and 10), consistent with the inability of VEGF 12 , to bind
  • a GST- VEGF Exon 7+8 fusion protein inhibits VEGF 16 , binding to VEGF 16 ,R/NP-1 and KDR
  • Neuropilin-1 is an isoform-specific VEGF ⁇ 65 receptor
  • VEGF ⁇ 65 receptor Recently, we described a novel 130-135 kDa VEGF cell surface receptor that binds VEGF1 65 but not VEGF121 and that we named, accordingly, NEGF ⁇ 6 R (Soker et al., J. Biol. Chem. 271, 5761-5767 (1996)).
  • NEGF ⁇ 6 R Soker et al., J. Biol. Chem. 271, 5761-5767 (1996).
  • NP- 1 human neuropilin-1
  • VEGF 165 R is identical to NP-1 and that NP-1 serves as a receptor for VEGF ⁇ 65 is as follows: i) purification of VEGF, 65 R protein from human MDA-MB-231 (231) cells using VEGF affinity, yielded a 130-140 kDa doublet upon SDS-PAGE and silver stain. N-terminal sequencing of both proteins yielded the same N-terminal sequence of 18 amino acids that demonstrated a high degree of homology to mouse NP-1 (Kawakami et al., J. Neurobiol.
  • VEGF ⁇ 65 R After we purified VEGF ⁇ 65 R from human 231 cells, the cloning of human NP-1 was reported (He and Tessier-Lavigne, Cell 90, 739-751 (1997)) and the N-terminal sequence of human VEGF 165 R was found to be identical to a sequence in the N-terminal region of human NP-1; iii) Expression cloning using a 231 cell cDNA library resulted in isolation of several cDNA clones and their sequences were identical to the human NP-1 cDNA sequence (He and Tessier- Lavigne, Cell 90, 739-751 (1997)).
  • VEGF 165 Cells that bound VEGF 165 to VEGF1 65 synthesized relatively abundant NP- 1 mRNA while cells that showed very little if any VEGF ] 6 5 binding, did not synthesize much if any NP-1 mRNA; v) when NP-1 was expressed in PAE cells, the transfected , but not the parental cells, were able to bind VEGF ⁇ 65 but not VEGF 12 1, consistent with the isoform specificity of binding previously shown for HUVEC and 231 cells (Soker et al, J. Biol. Chem. 271, 5761-5767 (1996)).
  • the K d of 12" I -VEGF 165 binding of to PAE expressing NP-1 was about 3 x 10 "10 M, consistent with previous K d binding values of 2-2.8 x 10 "10 M for 231 cells and HUVEC (Soker et al., J. Biol. Chem. 271, 5761-5767 (1996)); and vi)
  • the binding of VEGF, 65 to cells expressing NP-1 post-transfection was more efficient in the presence of heparin as was the binding of this ligand to HUVEC and 231 cells (Gitay-Goren et al., J. Biol. Chem. 267, 6093-6098 (1992); Soker et al., J. Biol. Chem.
  • VEGF ⁇ 65 R is identical to NP-1 but that it is a functional receptor that binds VEGF1 65 in an isoform-specific manner. Accordingly, we have named this VEGF receptor VEGF 165 R NP-I.
  • VEGF 165 R NP-I cDNA In addition to the expression cloning of VEGF 165 R NP-I cDNA, another human cDNA clone was isolated whose predicted amino acid sequence was 47% homologous to that of VEGF 165 R/NP-I and over 90% homologous to rat neuropilin-2 (NP-2) which was recently cloned (Kolodkin et al., Cell 90, 753-762 (1997)). NP-2 binds members of the collapsin/semaphorin family selectively (Chen et al., Neuron 19, 547-559 (1997)).
  • NP-1 serves as a receptor for NEGF ⁇ 65
  • NP-1 had previously been shown to be associated solely with the nervous system during embryonic development (Kawakami et al., J. Neurobiol. 29, 1-17 (1995); Takagi et al., Dev. Biol. 170, 207-222 (1995)) and more recently as a receptor for members of the collapsin/semaphorin family (He and Tessier-Lavigne, Cell 90739- 751 (1997); Kolodkin et al., Cell 90, 753-762 (1997)).
  • NP-1 is a 130-140 kDa transmembrane glycoprotein first identified in the developing Xenopus optic system (Takagi et al., Dev. Biol. 122, 90-100 (1987); Takagi et al., Neuron 7, 295-307 (1991)).
  • NP-1 expression in the nervous system is highly regulated spatially and temporally during development and in particular is associated with those developmental stages when axons are actively growing to form neuronal connections. (Fujisawa et al., Dev. Neurosci. 17, 343-349 (1995); Kawakami et al., J. Neurobiol 29, 1-17 (1995); Takagi et al., Dev. Biol.
  • NP-1 protein is associated with neuronal axons but not the stomata (Kawakami et al., J. Neurobiol 29, 1-17 (1995)). Functionally, neuropilin has been shown to promote neurite outgrowth of optic nerve fibers in vitro (Hirata et al., Neurosci. Res. 17, 159- 169 (1993)) and to promote cell adhesiveness (Tagaki et al., Dev. Biol. 170, 207-222 (1995)). Targeted disruption of NP-1 results in severe abnormalities in the trajectory of efferent fibers of the peripheral nervous system (Kitsukawa et al., Neuron 19, 995- 1005 (1997)).
  • NP-1 is a neuronal cell recognition molecule that plays a role in axon growth and guidance (Kawakami et al., J. Neurobiol. 29, 1-17 (1995); He and Tessier-Lavigne, Cell 90, 739-751 (1997); Kitsukawa et al., Neuron 19, 995-1005 1997; Kolodkin et al, Cell 90, 753-762 (1997)).
  • VEGF 165 R/NP-I is also expressed in adult tissues, in contrast to the earlier studies that have shown that NP-1 expression in Xenopus, chicken and mouse is limited to the developmental and early post-natal stages (Fujisawa et al., Dev. Neurosci. 17, 343-349 (1995); Kawakami et al., J. Neurobiol. 29, 1-17 (1995); Takagi et al., E»ev. Biol. 70, 207-222 (1995)).
  • NP-1 is expressed in the developing nervous system starting in the dorsal root ganglia at day 9 and ceases at day 15 (Kawakami et al., J. Neurobiol.
  • VEGF1 .65 R/NP-I mRNA transcripts in heart, placenta, lung, liver, skeletal muscle, kidney and pancreas. Interestingly, there is very little relative expression in adult brain, consistent with the mouse nervous system expression studies (Kawakami et al., J. Neurobiol. 29, 1-17 (1995)).
  • VEGF1. 65 R/NP-I is also expressed in a number of cultured non-neuronal cell lines including EC and a variety of tumor- derived cells. A possible function of VEGF ⁇ 65 R/NP-l in these cells is to mediate angiogenesis as will be discussed below.
  • NP-1 has been identified as a receptor for the collapsin/semaphorin family by expression cloning of a cDNA library obtained from rat E14 spinal cord and dorsal root ganglion (DRG) tissue (He and Tessier-Lavigne, Cell 90, 739-751 (1997); Kolodkin et al., Cell 90, 753-762 (1997)).
  • DRG dorsal root ganglion
  • collapsin/semaphorins comprise a large family of transmembrane and secreted glycoproteins that function in repulsive growth cone and axon guidance (Kolodkin et al., Cell 75, 1389-1399 (1993)).
  • the repulsive effect of sema III for DRG cells was blocked by anti-NP-1 antibodies (He and Tessier- Lavigne, Cell 90, 739-751 (1997); Kolodkin et al., Cell 90, 753-762 (1997)).
  • VEGF ⁇ 5 R/NP-l has five discrete domains in its ectodomain, and it has been suggested that this diversity of protein modules in NP-1 is consistent with the possibility of multiple binding ligands for NP-1 (Takagi et al., Neuron 7, 295-307 (1991); Feiner et al, Neuron 19539-545 (1997); He and Tessier-Lavigne, Cell 90 739-751 (1997). Preliminary analysis does not indicate any large degree of sequence homology between sema III and VEGF exon 7 which is responsible for VEGF binding to VEGF1.
  • VEGF 165 displays any neuronal guidance activity and whether sema III has any EC growth factor activity. These possibilities have not been examined yet. However, it may be that VEGF requires two receptors, KDR and NP-1 for optimal EC growth factor activity (Soker et al, J. Biol. Chem.
  • VEGF ⁇ 65 R NP-l modulates the binding of VEGF ⁇ 65 to KDR, a high affinity RTK that is an important regulator of angiogenesis as evidenced by KDR knock out experiments in mice (Shalaby et al., Nature 376, 62-66 (1995).
  • the affinity of KDR for VEGF 165 is about 50 times greater than for VEGF . 1 65 R/NP-I (Gitay-Goren et al., J. Biol. Chem. 287, 6003-6096 (1992); Waltenberger et al, J. Biol. Chem. 269, 26988- 26995 (1994)).
  • VEGF ⁇ 65 R/NP-l and KDR When VEGF ⁇ 65 R/NP-l and KDR are co-expressed, the binding of 125 I- VEGF1 65 to KDR is enhanced by about 4-fold compared to cells expressing KDR alone.
  • the enhanced binding can be demonstrated in stable clones co-expressing VEGF 165 R NP-I and KDR (PAE/KDR/NP-1 cells), and also in PAE/KDR cells transfected transiently with VEGF ⁇ 65 R/NP-l cDNA where clonal selection does not take place.
  • VEGF 165 R/NP-I with its relatively high receptor/cell number, about 0.2-2 x 10 s (Gitay-Goren et al, J. Biol. Chem. 287, 6003-6096 (1992); Soker et al., J. Biol. Chem. 271, 5761-5767 (1996)), appears to serve to concentrate VEGF165 on the cell surface, thereby providing greater access of VEGF ]65 to KDR.
  • VEGF 165 undergoes a conformational change that enhances its binding to KDR.
  • the end result would be elevated KDR signaling and increased VEGF activity.
  • KDR signaling and increased VEGF activity.
  • VEGF mitogenicity for PAE/KDR/NP-1 cells compared to PAE/KDR cells.
  • VEGF, 6 . which binds to both KDR and VEGF 16 ,R/NP-1, is a better mitogen for HUVEC than is VEGF 1 21, which binds only to KDR (Keyt et al , J Biol Chem 271, 5638-5646 (1996b), Soker et al , J Biol Chem 272,
  • VEGF1. 21 mitogemc activity The concept that dual receptors regulate growth factor binding and activity has been previously demonstrated for TGF- ⁇ , bFGF and NGF (Lopez-Casillas et al , Cell 67, 785-795 (1991), Yayon et al , Cell 64, 841-848 (1991 , Barbacid, Curr Opin Cell Biol 7, 148-155 (1995))
  • Another connection between VEGFi 6 ⁇ R NP-l and angiogenesis comes from studies in which NP-1 was overexpressed ectopically in transgenic mice (Kitsuskawa et al , Develop 121, 4309-4318 (1995))
  • NP-1 overexpression resulted in embryonic lethality and the mice died in utero no later than on embryonic day 15 5 and those that survived the best had lower levels of NP-1 expression
  • VEGF 165 R/NP -1 is associated with tumor-derived cells
  • the greatest degree of VEGF 165 R/NP-I expression that we have detected so far occurs in tumor-derived cells such as 231 breast carcinoma cells and PC3 prostate carcinoma cells, far more than occurs in HUVEC.
  • the tumor cells express abundant levels of VEGF ⁇ R/NP-l mRNA and about 200,000 VEGF, 65 receptors/cell (Soker et al., J. Biol. Chem. 271, 5761-5767 (1996)). On the other hand, these tumor cells do not express KDR or Flt-1 so that VEGF1.
  • 65 R/NP-I is the only VEGF receptor associated with these cells. The tumor cells are therefore useful for testing whether VEGF1.
  • R/NP-I is a functional receptor for VEGF . 1 65 in the absence of a KDR background.
  • VEGF1 .65 R/NP-I mediates a VEGF 165 signal in tumor-derived cells as measured by receptor tyrosine phopshorylation.
  • VEGF 165 might have an effect on tumor cells by inducing some, as yet undetermined activity such as enhanced survival, differentiation, or motility.
  • a recent report has demonstrated that glioma cells express a 190 kDa protein that binds VEGF i65 but not VEGF 121 efficiently (Omura et al, J. Biol. Chem. 272, 23317-23322 (1997)). No stimulation of tyrosine phosphorylation could be demonstrated upon binding of VEGF 165 to this receptor.
  • Whether the 190 kDa isoform-specific receptor is related to VEGF 165 R NP-I is not known presently.
  • VEGF1 65 R NP-I may have a storage and sequestration function for VEGF ⁇ 65 .
  • VEGF ⁇ 65 is produced by a tumor cell and binds to VEGF 165 R/NP-I on that cell via the exon 7-encoded domain (Soker et al., J. Biol. Chem. 271, 5761-5767 (1996)). The stored VEGF ⁇ 65 could be then released to stimulate tumor angiogenesis in a paracrine manner.
  • VEGF 165 R NP-I may mediate a juxtacrine effect in which VEGF] 65 is bound to VEGF ⁇ 65 R/NP-l on a tumor cell via the exon 7-encoded domain and is also bound to KDR on a neighboring EC via the exon 4-encoded domain (Keyt et al., J. Biol. Chem. 271, 5638-5646 (1996b)).
  • VEGF] 65 is bound to VEGF ⁇ 65 R/NP-l on a tumor cell via the exon 7-encoded domain and is also bound to KDR on a neighboring EC via the exon 4-encoded domain
  • VEGF ⁇ sR vascular endothelial growth factor receptor
  • NP-1 a cell surface protein previously identified as playing a role in embryonic development of the nervous system and as being a receptor for the collapsins/semaphonns
  • binding to VEGF 16 5R/NP-I enhances the binding of VEGF 1 65 to KDR on EC and tumor cells

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Abstract

L'invention porte sur des récepteurs du facteur de croissance de l'endothélium vasculaire (VEGFR), et des neuropilines telles que les VEGF165R/NP-1 et NP-2 qui sont associées au potentiel métastatique des cellules malignes, et sur leur utilisation pour diagnostiquer ou pronostiquer le cancer. Les neuropilines VEGF165R/NP-1 et NP-2 sont préférées, mais toute neuropiline ou tout VEGFR dont les constituants présentent 85 % d'homologie avec lesdites VEGF165R/NP-1 et NP-2, ou mieux 90 %, ou mieux encore 95 %, peut être utilisé.
PCT/US1998/026127 1997-12-09 1998-12-09 Neuropilines et leur utilisation a des fins de diagnostic et de pronostic du cancer WO1999030157A2 (fr)

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US8637262B2 (en) 1995-08-01 2014-01-28 Vegenics Pty Limited Vascular endothelial growth factor C (VEGF-C) protein and gene, mutants thereof, and uses thereof
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US7309604B2 (en) 2000-02-25 2007-12-18 Licentia, Ltd. Materials and methods involving hybrid vascular endothelial growth factor DNAs and proteins
US7566566B2 (en) 2000-02-25 2009-07-28 Vengenics Limited Materials and methods involving hybrid vascular endothelial growth factor DNAs and proteins
US7902149B2 (en) 2000-02-25 2011-03-08 Vegenics Pty Limited Materials and methods involving hybrid vascular endothelial growth factor DNAs and proteins
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WO2007022287A2 (fr) 2005-08-15 2007-02-22 Vegenics Limited Vegf-a modifié aux propriétés angiogéniques améliorées
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US11807687B2 (en) 2019-10-03 2023-11-07 Atyr Pharma, Inc. Therapeutic compositions comprising anti-NRP2 antibodies

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AU1810899A (en) 1999-06-28
WO1999030157A9 (fr) 1999-10-21
JP2001526032A (ja) 2001-12-18
EP1037994A1 (fr) 2000-09-27
AU1906099A (en) 1999-06-28
JP2009148293A (ja) 2009-07-09
CA2313390A1 (fr) 1999-06-17
WO1999029861A1 (fr) 1999-06-17
JP4312955B2 (ja) 2009-08-12
WO1999030157A3 (fr) 1999-07-22

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