JP2002505873A - Vascular endothelial growth factor 2 - Google Patents

Abstract

  (57) [Summary] Disclosed are human VEGF-2 polypeptides, biologically active and diagnostically or therapeutically useful fragments, analogs, or derivatives thereof, and DNA (RNA) encoding such VEGF-2 polypeptides. Is done. Also provided are procedures for producing such polypeptides by recombinant techniques, as well as antibodies and antagonists to such polypeptides. Such polypeptides and polynucleotides can be used therapeutically to stimulate wound healing and to repair vascular tissue. Also provided are methods of using the antibodies and antagonists to inhibit tumor angiogenesis and thereby tumor growth, inflammation, diabetic retinopathy, rheumatoid arthritis, and psoriasis.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION The present invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, and the use of such polynucleotides and polypeptides. For the production of polypeptides. The polypeptides of the present invention have been identified as members of the vascular endothelial growth factor family. More specifically, the polypeptide of the present invention is human vascular endothelial growth factor 2 (VEGF-2). The invention also relates to inhibiting the action of such polypeptides.

[0002] The formation of new blood vessels, or angiogenesis, is essential for embryonic development, subsequent growth, and tissue repair. Angiogenesis is also an integral part of certain pathological conditions such as neoplasms (ie, tumors and gliomas). Aberrant angiogenesis is associated with other diseases such as inflammation, rheumatoid arthritis, psoriasis, and diabetic retinopathy (Folkman, J. and Klagsbrunn, M., Sci.
ence 235: 442-447, (1987)).

[0003] Both acidic and basic fibroblast growth factor molecules are mitogens for endothelial cells and other cell types. Angiotropin
pin) and angiogenin can induce angiogenesis, but their function is unknown (Folkman, J., Cancer Medicine, Lea).
and Febiger Press (pp. 153-170) (1993)).
A highly selective mitogen for vascular endothelial cells is vascular endothelial growth factor, VEGF (Ferrara, N. et al., Endocr. Rev. 13:
19-32, (1992)), also known as vascular permeability factor (VPF).

[0004] Vascular endothelial growth factor is a secreted angiogenic mitogen whose target cell specificity appears to be restricted to vascular endothelial cells. The mouse VEGF gene is
It was characterized and its expression pattern in embryogenesis was analyzed. VEGF
Has been observed in epithelial cells adjacent to fenestrated endothelium (eg, in the choroid plexus and glomeruli). This data was consistent with the role of VEGF as a multifunctional regulator of endothelial cell proliferation and differentiation (Breier, G.
. Et al., Development, 114: 521-532 (1992)).

[0005] VEGF shares sequence homology with the human platelet-derived growth factors PDGFa and PDGFb (Leung, DW et al., Science 246: 1306).
-1309, (1989)). The degree of homology was about 21% and 23, respectively.
%. Eight cysteine residues that contribute to disulfide bond formation are strictly conserved in these proteins. These are similar, but VEGF and PD
There are specific differences from GF. While PDGF is the major growth factor for connective tissue, VEGF is highly specific for endothelial cells. Alternatively, spliced mRNAs have been identified in both VEGF, PLGF, and PDGF, and these different spliced products differ in biological activity and receptor binding specificity. VEGF and PDGF function as homodimers or heterodimers and bind to the receptor, which elicits endogenous tyrosine kinase activity after receptor dimerization.

[0006] VEGF has four different forms of 121, 165, 189, and 206 amino acids due to alternative splicing. VEGF121 and VEGF1
65 is soluble and can promote angiogenesis. On the other hand, VEGF189 and VEGF-206 are bound to heparin-containing proteoglycans on the cell surface. The temporal and spatial expression of VEGF correlated with the physiological growth of blood vessels (Gajdusek, CM, and Carbon, SJ, Ce.
11 Physiol. 139: 570-579, (1989); McNeil.
, P. L. J. et al. Cell. Biol. 109: 811-822, (1989)
)). Its high affinity binding site is located only on endothelial cells in tissue sections (Jakeman, LB et al., Clin. Invest. 89: 244-).
253, (1989)). This factor can be isolated from pituitary cells and some tumor cell lines, and has been associated with some human gliomas (Plate,
K. H. Nature 359: 845-848 (1992)). Interestingly, expression of VEGF121 or VEGF165 confers on Chinese hamster ovary cells the ability to form tumors in nude mice (Ferrara, N.
. J. et al. Clin. Invest. 91: 160-170, (1993)).
Inhibition of VEGF function by anti-VEGF monoclonal antibodies has been shown to inhibit tumor growth in immunodeficient mice (Kim, KJ, Nature).
362: 841-844 (1993)). Furthermore, a dominant negative mutant of the VEGF receptor has been shown to inhibit glioblastoma growth in mice.

[0007] Vascular permeability factor (VPF) also has a sustained microvascular hyperpermeability to plasma proteins even after cessation of injury.
rmeability), which is a characteristic feature of normal wound healing. This suggests that VPF is an important factor in wound healing. Brown, L .; F. J. et al. Exp. Med. , 176:
1375-1379 (1992).

[0008] VEGF expression is high in vascularized tissues (eg, lung, heart, placenta, and solid tumors) and correlates with both temporal and spatial angiogenesis. VEGF has also been shown to induce angiogenesis in vivo. Since angiogenesis is essential for the repair of normal tissue, especially vascular tissue, VEGF has been proposed for use in promoting vascular tissue repair (eg, in atherosclerosis).

[0009] US Pat. No. 5,073,4, issued Dec. 17, 1991 to Chen et al.
No. 92 discloses a method for synergistically enhancing endothelial cell proliferation in a suitable environment. The method involves adding VEGF, effector, and serum-derived factors to the environment. In addition, vascular endothelial cell growth factor C subunit DNA
Was prepared by the polymerase chain reaction technique. This DNA encodes a protein that can exist as either a heterodimer or a homodimer. This protein is a mammalian vascular endothelial cell mitogen and
As such, it is useful for promoting vascular development and repair, as disclosed in European Patent Application No. 9302750.2, published on Nov. 30.

SUMMARY OF THE INVENTION The polypeptides of the present invention have been putatively identified as novel vascular endothelial growth factors based on amino acid sequence homology to human VEGF.

According to one aspect of the present invention, novel mature polypeptides, and fragments, analogs thereof, that are biologically active and diagnostically or therapeutically useful
And derivatives are provided. The polypeptide of the present invention is a polypeptide of human origin.

According to another aspect of the present invention, an isolated nucleic acid molecule comprising a polynucleotide encoding a full-length or truncated VEGF-2 polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or 4, respectively, or ATCC accession number 97149 (1
Deposited on May 12, 995) or ATCC accession number 75698 (March 1994
The amino acid sequence encoded by the cDNA clone deposited in the bacterial host is provided.

[0013] The present invention also provides a biologically active and diagnostically or therapeutically useful V
It relates to fragments, analogs and derivatives of EGF-2.

According to yet another aspect of the present invention, there is provided a process for producing such a polypeptide by recombinant technology. This process involves recombining a prokaryotic and / or eukaryotic host cell containing a nucleic acid sequence encoding a polypeptide of the invention with conditions that facilitate expression of the protein and subsequent recovery of the protein. Under culturing.

According to still further aspects of the present invention, for therapeutic purposes (eg, to stimulate angiogenesis, wound healing, proliferation of damaged bone and tissue, and to promote repair of vascular tissue) ) Provides processes for utilizing such polypeptides, or polynucleotides encoding such polypeptides. In particular, provided are processes for utilizing such polypeptides or polynucleotides encoding such polypeptides for the treatment of peripheral artery disease (eg, severe limb ischemia and coronary disease).

According to yet another aspect of the present invention, there are provided antibodies against such polypeptides and processes for producing such polypeptides.

According to yet another aspect of the present invention, there are provided antagonists to such polypeptides, for example, to prevent tumor angiogenesis and thereby inhibit tumor growth, It can be used to inhibit the action of such polypeptides for the treatment of diabetic retinopathy, inflammation, rheumatoid arthritis, and psoriasis.

According to another aspect of the present invention there is provided a nucleic acid probe comprising a nucleic acid molecule of sufficient length to specifically hybridize to a nucleic acid sequence of the present invention.

According to another aspect of the present invention, there is provided a method of diagnosing a disease or susceptibility to a disease associated with a mutation in the nucleic acid sequence of the present invention and a protein encoded by such a nucleic acid sequence.

According to still further aspects of the present invention, scientific research, DNA synthesis, and DN
Processes utilizing such polypeptides, or polynucleotides encoding such polypeptides, are provided for in vitro purposes related to the production of A vectors.

[0021] These and other aspects of the invention should be apparent to those skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS The following drawings are illustrative of embodiments of the present invention and are not meant to limit the scope of the invention, which is encompassed by the claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS According to one aspect of the present invention, the deduced amino acid sequence of FIG. 1 (SEQ ID NO: 2), which was determined by sequencing the cloned cDNA An isolated nucleic acid molecule comprising a polynucleotide encoding a VEGF-2 polypeptide having the formula: A cDNA clone (this was obtained on May 12, 1995 by Am
erican Type Tissue Collection (ATCC),
10801 University Boulevard, Manassas,
(Deposited with VA 20110-2209 and given ATCC accession number 97149), yielded the nucleotide sequence shown in SEQ ID NO: 1.

According to another aspect of the present invention, a truncated VEGF-2 polypeptide having the deduced amino acid sequence of FIG. 2 (SEQ ID NO: 4), which was determined by sequencing the cloned cDNA An isolated nucleic acid molecule comprising a polynucleotide encoding A cDNA clone (this was found on March 4, 1994,
American Type Tissue Collection (ATCC)
, 10801 University Boulevard, Manassas
, VA 20110-2209, and ATCC Accession No. 75698
The nucleotide sequence shown in SEQ ID NO: 3 was obtained.

Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein are based on an automated DNA sequencer (eg, App.
lied Biosystems, Inc. The amino acid sequence of the polypeptide encoded by the DNA molecule determined by using model 373) and determined herein was predicted by translation of the determined DNA sequence above. Thus, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation will typically have at least about 90% identity to the actual sequence of the sequenced DNA molecule, more typically at least about 95% to at least about 9%.
9.9% identity. The actual sequence can be more accurately determined by other approaches, including manual DNA sequencing methods well known in the art. As also known in the art, a single insertion or deletion in a determined nucleotide sequence results in a frameshift in translation of the nucleotide sequence, as compared to the actual sequence,
As a result, the predicted amino acid sequence encoded by the determined nucleotide sequence is completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, which is the result of such insertions or deletions. Start at a position.

The polynucleotide encoding the polypeptide of the present invention can be used in an early human embryo (8-
9 weeks) can be obtained from osteoclastoma, adult heart or some breast cancer cell lines. The polynucleotide of the present invention was found in a cDNA library derived from an early 9-week-old human embryo. It is structurally related to the VEGF / PDGF family. It contains an open reading frame that encodes a protein of about 419 amino acid residues, the first approximately 23 amino acid residues being a putative leader sequence, such that the mature protein contains 396 amino acids. This protein then shows the highest amino acid sequence homology (30% identity) to human vascular endothelial growth factor, followed by PDGFa (24%) and PDGFb (22%). (See FIG. 4). It is particularly important that all eight cysteines are conserved in all four members of the family (see boxed area in FIG. 3). Furthermore, PXCVX, a feature of the PDGF / VEGF family,
XXRCXGCCN (SEQ ID NO: 8) is conserved in VEGF-2 (FIG. 3).
checking). The homology between VEGF-2, VEGF and two PDGFs is at the protein sequence level. No nucleotide sequence homology can be detected, and it is therefore difficult to isolate VEGF-2 via a simple approach (eg, low stringency hybridization).

A VEGF-2 polypeptide of the present invention is meant to include a full length polypeptide and a polynucleotide sequence encoding any leader sequence and an active fragment of the full length polypeptide. The active fragment has less than all 419 amino acids of the full length amino acid sequence shown in SEQ ID NO: 2, but still contains the eight cysteine residues shown to be conserved in FIG.
It is meant to include any portion of the full length amino acid sequence, still including two activities.

Normal tissues include at least two alternatively spliced VEGF2
mRNA sequence is present. In FIG. 7, the two bands in lane 5 show the V
4 shows the presence of alternatively spliced mRNA encoding an EGF-2 polypeptide.

The polynucleotide of the present invention may be in the form of RNA or DNA (this DN
A can include cDNA, genomic DNA, and synthetic DNA). D
The NA can be double-stranded or single-stranded, and if single-stranded, can be the coding strand or the non-coding (antisense) strand. The coding sequence encoding the mature polypeptide can be identical to the coding sequence shown in FIG. 1 or FIG. 1, 2 or a different coding sequence encoding the same mature polypeptide as the deposited cDNA.

The polynucleotide encoding the mature polypeptide of FIG. 1 or FIG. 2 or the mature polypeptide encoded by the deposited cDNA may include: the coding sequence for the mature polypeptide only; the coding sequence for the mature polypeptide. And additional coding sequences, such as leader or secretory or proprotein sequences; the coding sequence for the mature polypeptide (and optionally the additional coding sequence) and the non-coding sequences (eg, introns or coding sequence for the mature polypeptide). 5 'and / or 3' non-coding sequences).

Thus, the term “polynucleotide encoding a polypeptide” encompasses a polynucleotide that includes only the coding sequence of the polypeptide, as well as a polynucleotide that includes additional coding and / or non-coding sequences.

The present invention further provides fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of FIG. 1 or FIG. 2, or the polypeptide encoded by the cDNA of the deposited clone. It relates to a variant of the above polynucleotide. A variant of a polynucleotide can be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.

Accordingly, the present invention relates to polynucleotides encoding the same mature polypeptide as shown in FIG. 1 or FIG. 2 or the same mature polypeptide encoded by the cDNA of the deposited clone, and modifications of such polynucleotides. Includes the body. This variant encodes a fragment, derivative, or analog of the polypeptide of FIG. 1 or 2 or the polypeptide encoded by the cDNA of the deposited clone. Such nucleotide variants include deletion variants, substitution variants, and addition or insertion variants.

As indicated hereinabove, the polynucleotide may be of the type shown in FIG.
Or a coding sequence that is a naturally occurring allelic variant of the coding sequence of the deposited clone. As is known in the art, an allelic variant is another form of a polynucleotide sequence that has one or more nucleotide substitutions, deletions, or additions, which substantially alter the function of the encoded polypeptide. Do not change to

The present invention also includes polynucleotides, wherein the coding sequence for the mature polypeptide is a polynucleotide that assists in expression and secretion of the polypeptide from the host cell (eg, regulates transport of the polypeptide from the cell). A leader sequence that functions as a secretory sequence for reading) in the same reading frame. A polypeptide having a leader sequence is a preprotein and may have a leader sequence that is cleaved by a host cell to form a mature form of the polypeptide. The polynucleotide may also encode a mature protein and a proprotein that is an additional 5 'amino acid residue. A mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved, the active mature protein remains.

Thus, for example, a polynucleotide of the invention may encode a mature protein or a protein having a prosequence or a protein having both a prosequence and a presequence (leader sequence).

A polynucleotide of the invention may also have the coding sequence fused in frame with a marker sequence that allows for purification of the polypeptide of the invention. The marker sequence provides for purification of the mature polypeptide fused to the marker in the case of a bacterial host,
It may be a hexahistidine tag supplied by the pQE-9 vector. Alternatively, for example, the marker sequence can be a hemagglutinin (HA) tag when a mammalian host (eg, COS-7 cells) is used. The HA tag is consistent with an epitope derived from the influenza hemagglutinin protein (Wilson,
I. Et al., Cell, 37: 767 (1984)).

A further embodiment of the present invention is that the nucleotide sequence is at least 95% identical, and more preferably at least 96%, 97%, 98% or 9% to the following nucleotide sequences:
Includes an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence that is 9% identical: (a) a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2; (b) an amino acid sequence of SEQ ID NO: 2 (C) a nucleotide sequence encoding a polypeptide having the amino acid sequence at about position 1 to about 396 of SEQ ID NO: 2; A nucleotide sequence encoding a polypeptide having the amino acid sequence encoded by the cDNA clone contained in No. 97149; (e) a mature VEGF-2 polypeptide having an amino acid sequence encoded by the cDNA clone included in ATCC Deposit No. 97149 Nucleotides encoding Column; or (f) (a), (b), (c), (d), or a nucleotide sequence complementary to any of the nucleotide sequences of (e).

A further embodiment of the present invention is a polypeptide having a nucleotide sequence that is at least 95% identical to the following nucleotide sequences, and more preferably at least 96%, 97%, 98% or 99% identical: Isolated nucleic acid molecules comprising nucleotides: (a) a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 4;
A nucleotide sequence encoding a polypeptide lacking a terminal methionine; (c)
A nucleotide sequence encoding a polypeptide having the amino acid sequence of about 1 to about 326 of SEQ ID NO: 4; (d) the cDN contained in ATCC Deposit No. 75698
A nucleotide sequence encoding the polypeptide having the amino acid sequence encoded by the A clone; (e) a nucleotide sequence encoding the mature VEGF-2 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 75698. Or (f) (a), (b), (c), (d)
Or a nucleotide sequence complementary to any of the nucleotide sequences of (e).

A polypeptide having a nucleotide sequence that is at least 95% “identical” to a reference nucleotide sequence encoding a VEGF-2 polypeptide is a reference nucleotide sequence whose polynucleotide sequence encodes a VEGF-2 polypeptide. It is intended that the nucleotide sequence of this polynucleotide be identical to the reference sequence, except that it may contain up to 5 point mutations for every 100 nucleotides of the polynucleotide. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or replaced with another nucleotide, Alternatively, as many as 5% of all nucleotides in the reference sequence can be inserted into the reference sequence. These mutations in the reference sequence correspond to the 5N-terminal position or 3
At the N-terminal position or somewhere between these terminal positions, either individually dispersed between nucleotides in the reference sequence or dispersed in one or more contiguous groups in the reference sequence. Can occur.

As a practical matter, any particular nucleic acid molecule may be, for example, SEQ ID NO: 1 or 3
Or at least 95%, 96%, 97%, 98%, and 99% identical to the nucleotide sequence of the deposited cDNA clone, using a known computer program (eg, Bestfit program (eg, Wisconsin Sequence Analysis Package
, Version 8 for Unix, Genetics Compute
r Grupup, University Research Park, 57
5 Science Drive, Madison, WI 53371)). Bestfit finds Smith and Waterman (Adv) to find the best segment of homology between two sequences.
ances in Applied Matrices 2: 482-4
89 (1981)). To determine whether a particular sequence is 95% identical to a reference sequence according to the present invention, for example, Be
When using stfit or any other sequence alignment program, of course, the percent identity is calculated over the entire length of the reference nucleotide sequence, and gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are tolerated. Set the parameters as follows.

As described in detail below, the polypeptides of the present invention can be used to raise polyclonal and monoclonal antibodies, which detect VEGF-2 protein expression as described below. In agonists and antagonists that can enhance or inhibit the function of VEGF-2 protein. Further, such polypeptides can be used in a yeast two-hybrid system that “captures” a VEGF-2 protein binding protein that is also a candidate agonist and antagonist according to the invention. The yeast two-hybrid system is described in Fields and Song, Nature.
340: 245-246 (1989).

In another aspect, the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention. For the selection of peptides or polypeptides bearing an antigenic epitope (ie, including the region of the protein molecule to which the antibody can bind), relatively short synthetic peptides that mimic a portion of the protein sequence were partially mimicked It is well known in the art that antisera reactive with proteins can be routinely raised. See, for example, Sutcliffe, J. et al. G. FIG. , Shi
nnick, T .; M. Green, N .; And Learner, R .; A. (1
983) Antibodies that react with prede
Terminated sites on proteins. Science 2
19: 660-666. Peptides that can elicit protein-reactive sera are often represented in the primary sequence of a protein, can be characterized by a series of simple chemical rules, and can be characterized by immunodominant regions (ie, immunogenic epitopes) or amino acids of intact proteins. It is not restricted to either the terminal or the carboxy terminal. Extremely hydrophobic peptides and their six or fewer residues are generally ineffective at inducing antibodies that bind to the mimicked protein; longer soluble peptides, especially those containing proline residues Peptides are usually effective. Sutcliffe et al., Supra at 661. For example, 18 out of 20 peptides designed according to these guidelines, including 8 to 39 residues covering 75% of the sequence of the influenza virus hemagglutinin HA1 polypeptide chain, are HA1 protein or intact. Antibodies reactive with the virus were induced; and 12/12 peptide from MuLV polymerase and 18/18 from rabies glycoprotein induced antibodies that precipitated the respective proteins.

Accordingly, the antigenic epitope-bearing peptide and the antigenic epitope-bearing polypeptide of the present invention are useful for raising antibodies (including monoclonal antibodies) that specifically bind to the polypeptide of the present invention. Thus, a high proportion of hybridomas, obtained by fusion of spleen cells from a donor immunized with an antigenic epitope-bearing peptide, generally secretes antibodies that react with the native protein. S
utcliffe et al., supra, 663. Antibodies elicited by antigenic epitope-bearing peptides or polypeptides are useful for detecting mimicked proteins, and antibodies to different peptides follow the fate of various regions of the protein precursor. And undergoes post-translational processing. Peptides and anti-peptide antibodies can be used in various qualitative or quantitative assays for the mimicked protein, for example, in competitive assays. This is because shorter peptides (eg, about 9 amino acids) have been shown in immunoprecipitation assays to be able to bind and even substitute larger peptides. See, eg, Wilson et al., Cell 37: 767.
778 (1984) 777. The anti-peptide antibodies of the present invention are also useful for the purification of mimicked proteins, for example, by adsorption chromatography using methods well known in the art.

[0045] The antigenic epitope-bearing peptide and the antigenic epitope-bearing polypeptide of the present invention designed according to the above guidelines are contained in the amino acid sequence of the polypeptide of the present invention, preferably at least 7, more preferably at least 7, 9
And most preferably between about 15 and about 30 amino acids. However, the invention comprises about 30, 40, 50, 60, 70, 80, 90, 100, or 150 amino acids, or up to any length of the amino acid sequence of the polypeptide of the invention and in its entirety. Peptides or polypeptides comprising a larger portion of the amino acid sequence of the polypeptide of the invention are also considered epitope-bearing peptides or polypeptides of the invention, and also for inducing antibodies that react with the mimicked protein. Useful for Preferably, the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in an aqueous solvent (ie, the sequence comprises relatively hydrophilic residues and a highly hydrophobic sequence). Is preferably avoided); and the sequence comprising the proline residue is
Particularly preferred.

[0046] Non-limiting examples of antigenic polypeptides or peptides that can be used to raise VEGF-2 specific antibodies include: about leu-3 of SEQ ID NO: 2.
7 to about glu-45, from about Tyr-58 to about Gly-66 of SEQ ID NO: 2, from about Gln-73 to about Glu-81 of SEQ ID NO: 2, about As of SEQ ID NO: 2
From p-100 to about Cys-108, from about Gly-140 to about L of SEQ ID NO: 2
eu-148, from about Pro-168 to about Val-176 of SEQ ID NO: 2,
From about His-183 to about Lys-191 of SEQ ID NO: 2, about Il of SEQ ID NO: 2
e-201 to about Thr-209, from about Ala-216 to about T
yr-224, from Asp-244 of SEQ ID NO: 2 to about His-254, from about Gly-258 to about Glu-266 of SEQ ID NO: 2, about Cys of SEQ ID NO: 2
-272 to about Ser-280, from about Pro-283 to about Se of SEQ ID NO: 2
r-291, from about Cys-296 to about Gln-304 of SEQ ID NO: 2, from about Ala-307 to about Cys-316 of SEQ ID NO: 2, about Val of SEQ ID NO: 2
From -319 to Cys-335, from about Cys-339 to about Leu of SEQ ID NO: 2
From about Cys-360 to about Glu-373 of SEQ ID NO: 2, from about Tyr-378 to about Val-386 of SEQ ID NO: 2, and about S of SEQ ID NO: 2.
A polypeptide comprising the amino acid residues er-388 to about Ser-396.
These polypeptide fragments have been determined to have an antigenic epitope of the VEGF-2 protein by analysis of the Jameson-Wolf antigenic index.

The epitope-bearing peptides and polypeptides of the present invention can be produced by any conventional means for making peptides or polypeptides, including recombinant means using the nucleic acid molecules of the present invention. For example, a short epitope-bearing amino acid sequence
It can be fused to a larger polypeptide that acts as a carrier during recombinant production and purification, as well as during immunization to produce anti-peptide antibodies. Epitope-bearing peptides can also be synthesized using known methods of chemical synthesis. For example, Houghten has 248 different 13 residues representing a large number of peptides (eg, a single amino acid variant of a segment of the HA1 polypeptide prepared and characterized (by ELISA-type binding studies) in less than 4 weeks). 10 to 2 peptides
(0 mg). Houghten, R .; A
. (1985) General method for the rapid
solid-phase synthesis of large number
rs of peptides: specificity of antiges
n-antibody interaction at the level
of individual amino acids. Proc. Natl
. Acad. Sci. ScL USA 82: 5131-5135. This “simultaneous multiple peptide synthesis (Simultaneous Multiple Peptide S)
synthesis (SMPS) "process is described in Houghten et al. (1986).
) Are further described in U.S. Pat. In this procedure, the individual resins for solid phase synthesis of various peptides are placed in separate solvent permeable packets, allowing optimal use of many of the same iterative steps involved in solid phase methods. I have. By a completely manual procedure, it is possible to simultaneously carry out more than 500 to 1000 syntheses. Houghten et al., Supra, p. 5134.

[0048] The epitope-bearing peptides and polypeptides of the present invention are used to induce antibodies according to methods well known in the art. For example, Sutcliffe et al.
See, Wilson et al., Supra; Chow, M .; Proc. Natl. Aca
d. Sci. USA 82: 910-914; and Bittle, F .; J. J. et al. Gen. Virol. 66: 2347-2354 (1985). In general, animals can be immunized with free peptide; however, anti-peptide antibody titers can be used to conjugate the peptide to a macromolecular carrier, such as keyhole limpet hemocyanin (KLH) or tetanus toxin. Can be boosted. For example, a peptide containing cysteine is m-maleimidobenzoyl-
Linkers such as N-hydroxysuccinimide ester (MBS) can be attached to the carrier, while other peptides can be attached to the carrier using a more common binder such as glutaraldehyde. Animals such as rabbits, rats, and mice can be either free peptide or peptide bound to the carrier, for example, by intraperitoneal and / or intradermal injection of an emulsion containing about 100 mg of the peptide or carrier protein and Freund's adjuvant. Immunized with For example, some booster injections provide useful titers of anti-peptide antibodies that can be detected at about two-week intervals, for example, by an ELISA assay, using free peptide adsorbed to a solid surface. May be needed for The titer of the anti-peptide antibody in the serum from the immunized animal can be determined, for example, by adsorbing to the peptide on a solid phase and eluting the selected antibody, according to methods well known in the art. Can be increased.

[0049] The immunogenic epitope-bearing peptides of the invention, ie, those portions of the protein that elicit an antibody response when the entire protein is an immunogen, are identified according to methods known in the art. For example, Geysen et al., Supra, disclose a procedure for rapid, simultaneous synthesis of hundreds of sufficiently pure peptides on a solid support to react in an enzyme-linked immunosorbent assay. I do. The interaction of the synthesized peptides with the antibody is then easily detected without removing them from the support. In this way, peptides having an immunogenic epitope of the desired protein can be routinely identified by those skilled in the art. For example, an immunologically important epitope in the coat protein of foot-and-mouth disease virus is a 7 amino acid amino acid sequence synthesized by Geysen et al. By synthesis of an overlapping set of all 208 possible hexapeptides covering the entire 213 amino acid sequence of the protein. Positioned in resolution. The complete substitution set of peptides, in which all 20 amino acids were successively substituted at all positions within the epitope, was then synthesized and the specific amino acids conferring specificity for reaction with the antibody were determined. Thus, peptide analogs of the epitope-bearing peptides of the invention can be routinely made by this method. Geysen (19
US Pat. No. 4,708,781 to No. 87) further describes this method of identifying a peptide bearing an immunogenic epitope of a desired protein.

Still further, US Pat. No. 5,194,392 to Geysen (199
0) detects or detects the sequence of a monomer (amino acid or other compound) that is the geometric equivalent (ie, Amimotope) of an epitope complementary to a particular paratope (antigen binding site) of the antibody of interest. The general method of determination is described. More generally, U.S. Pat. No. 4,433,030 to Geysen
No. 92 (1989) describes a method for detecting or determining the sequence of a monomer that is the geometric equivalent of a ligand that is complementary to the ligand binding site of the particular receptor of interest. Similarly, peralkylated oligopeptide mixtures (Peral
Ho about killed Oligopeptide Mixtures
Ughten, R.A. A. U.S. Pat. No. 5,480,971 (1996).
) Is used to determine the sequence of linear C ₁ -C ₇ alkyl peralkylated oligopeptides and sets and libraries of such peptides, and the peralkylated oligopeptides that bind preferentially to the acceptor molecule of interest. Disclosed are methods for using such sets and libraries of oligopeptides. Thus, non-peptide analogs of the epitope-bearing peptides of the present invention can also be routinely made by these methods.

As will be appreciated by those of skill in the art, the VEGF-2 polypeptides of the invention described above and epitope-bearing fragments thereof can be combined with a portion of an immunoglobulin (IgG) constant domain to yield a chimeric polypeptide. . These fusion proteins facilitate purification and exhibit an increased half-life in vivo. this is,
For example, a chimeric protein consisting of the first two domains of the human CD4 polypeptide and various domains of the constant region of the heavy or light chain of a mammalian immunoglobulin has been shown (EPA 394,827; Traunecker et al., N.
Nature 331: 84-86 (1988)).

In accordance with the present invention, novel variants of VEGF-2 are also described. These are V
It can be produced by deleting or substituting one or more amino acids of EGF-2. Natural variants are called allelic variants. An allelic variant can be silent (no change in the encoded polypeptide) or have an altered amino acid sequence.

In an attempt to improve or alter the characteristics of native VEGF-2, protein engineering can be utilized. Recombinant DNA techniques known to those of skill in the art can be used to make new polypeptides. Muteins and deletions can, for example, exhibit enhanced activity or increased stability. In addition, they can be purified in higher yields and exhibit better solubility, at least under certain purification and storage conditions. Shown below are examples of mutations that can be constructed.

(Amino and carboxyl terminal deletions) In addition, VEGF-2, when run on an SDS-PAGE gel, appears to be proteolytically cleaved upon expression, resulting in a polypeptide fragment of the following size: Some (approximate sizes) (see, for example, FIGS. 6-8): 80, 59, 45, 43, 41, 40, 39, 38, 37, 36, 31, 2
9, 21, and 15 kDa. These polypeptide fragments are the result of proteolytic cleavage at both the N-terminal and C-terminal portions of the protein. These proteolytically generated fragments, especially the 21 kDa fragment, appear to be active.

In addition, protein engineering can be utilized to improve or alter one or more characteristics of native VEGF-2. Deletion of the carboxy-terminal amino acid can enhance the activity of the protein. One example is interferon gamma, which shows up to 10-fold higher activity due to the deletion of 10 amino acid residues from the carboxy terminus of the protein (Dobeli et al., J. of Biotechnology 7: 199-21).
6 (1988)). Accordingly, one aspect of the invention is a VEGF that exhibits enhanced stability (eg, when exposed to typical pH, heat, or other storage conditions) to a native VEGF-2 polypeptide. -2 polypeptide analogs and nucleotide sequences encoding such analogs.

Particularly preferred VEGF-2 polypeptides are shown below (numbering begins with the first amino acid (Met) in the protein (FIG. 1 (SEQ ID NO: 18)):

[0057]

Embedded image Preferred embodiments include the following deletion mutants:

[0058]

Embedded image The invention also includes deletion mutants having amino acid deletions from both the NB and C termini. Such variants include all combinations of the above N-terminal deletion mutants and C-terminal deletion mutants. These combinations can be made using recombinant techniques known to those skilled in the art.

In particular, an N-terminal deletion of a VEGF-2 polypeptide may be described by the general formula m-396, where m is an integer from -23 to 388, and m is identified in SEQ ID NO: 2. Corresponding to the position of the amino acid residue. Preferably, the N-terminal deletion is in the conserved boxed region (PXCVXXXRCXGCCN) of FIG. 3 (SEQ ID NO: 8).
And comprises a polypeptide comprising the amino acid sequence of the following residues: The N-terminal deletion of the polypeptide of the invention as set forth in SEQ ID NO: 1 comprises a polypeptide comprising the amino acid sequence of the following residues: SEQ ID NO: 2

[0060]

Embedded image One preferred embodiment includes amino acids S-205 to S-396 of SEQ ID NO: 2. Polynucleotides encoding these polypeptides are also preferred.

Further, the C-terminal deletion of a VEGF-2 polypeptide can also be described by the general formula 23-n, where n is an integer from -15 to 395, and n is identified in SEQ ID NO: 2. Corresponding to the position of the amino acid residue to be determined. Preferably, the C-terminal deletion retains the conserved boxed region of FIG. 3 (PXCVXXXRCXGCCN) (SEQ ID NO: 8) and comprises a polypeptide comprising the amino acid sequence of the following residues: The C-terminal deletion of the polypeptide of the invention shown in SEQ ID NO: 2 includes a polypeptide comprising the amino acid sequence of the following residues:

[0062]

Embedded image Polynucleotides encoding these polypeptides are also preferred.

In addition, the present invention also provides polypeptides having one or more amino acid deletions from both the amino and carboxyl termini, which generally comprise SEQ ID NO: 2
Has the residues mn, where n and m are integers as described above.

Similarly, C-terminal deletions of the VEGF-2 polypeptides of the present invention set forth in SEQ ID NO: 2 are also preferred, which include polypeptides comprising the amino acid sequence of the following residues of SEQ ID NO: 2:

[0065]

Embedded image Particularly preferred are polypeptide fragments comprising amino acid residues F-9 to R-203 of SEQ ID NO: 2, as well as polynucleotides encoding this polypeptide.
Preferably, this F-9 to R-203 of the polypeptide of SEQ ID NO: 2 binds to S-205 to S-396 of the polypeptide of SEQ ID NO: 2. The linkage can be by disulfide, covalent or non-covalent interactions, by linkage via a linker (eg, a serine, glycine, proline linkage), or by an antibody.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 1 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Thus, preferably, from the present invention, the nucleotide sequence described by the general formula ab (where a is any integer between 1-1660 of SEQ ID NO: 1 and b is 15-16
An integer of 74, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 1, and wherein b is a + 14 or greater, excluding one or more polynucleotides You.

Thus, in one aspect, the present invention provides N-terminal deletion mutants.
Such variants include the amino acid sequence shown in FIG. 1 (SEQ ID NO: 18) (FIG.
SEQ ID NO: 18) excluding at least the first 24 N-terminal amino acid residues (i.e., deletion of at least Met (1) -Glu (24)), but at least the first 115 or less N-terminal amino acid residues ) Are included. Or
The first 24 N-terminal amino acid residues (i.e., at least Met (1)-Glu (24) deletion in FIG. 1 (SEQ ID NO: 18), but no more than the first 103 N-terminal amino acid residues And the like.

In another aspect, the present invention provides a C-terminal deletion mutant. Such mutants include the amino acid sequence shown in FIG. 1 (SEQ ID NO: 18) (without deletion of at least the last C-terminal amino acid residue (Ser (419)), The amino acid residue (ie, amino acid residue Va of FIG. 1 (SEQ ID NO: 18)
l (199) -Ser (419) deletion). Alternatively, the deletion comprises at least the last C-terminal amino acid residue of FIG. 1 (SEQ ID NO: 18), but no more than the last 216 C-terminal amino acid residues. Alternatively, the deletion comprises at least the last C-terminal amino acid residue of FIG.
No more than 04 C-terminal amino acid residues. Alternatively, the deletion is shown in FIG. 1 (SEQ ID NO: 1
8) containing at least the last C-terminal amino acid residue, but not more than the last 192
Terminal amino acid residue. Alternatively, the deletion comprises at least the last C-terminal amino acid residue of FIG. 1 (SEQ ID NO: 18), but no more than the last 156 C-terminal amino acid residues. Alternatively, the deletion comprises at least the last C-terminal amino acid residue of FIG. 1 (SEQ ID NO: 18), but no more than the last 108 C-terminal amino acid residues. Alternatively, the deletion comprises at least the last C-terminal amino acid residue of FIG. 1 (SEQ ID NO: 18), but no more than the last 52 C-terminal amino acid residues.

In yet another aspect, the present invention also includes deletion mutants having amino acid deletions from both N-terminal and C-terminal residues. Such variants include all combinations of the above N-terminal deletion mutants and C-terminal deletion mutants.

The term “gene” refers to a segment of DNA involved in producing a polypeptide chain; the regions before and after the coding region (leaders and trailers), and the individual coding segments (exons). Including intervening sequences (introns) between them.

The invention further relates to fragments of the isolated nucleic acid molecules described herein. The deposited cDNA or a fragment of the isolated nucleic acid molecule having the nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, at least 15 nt, and more preferably at least about 20 nt;
Still more preferably, at least about 30 nt, and even more preferably,
Fragments of at least about 40 nt in length are contemplated, and are useful as diagnostic probes and primers as discussed herein. Of course,

[0072]

(Equation 1) Large nucleotide length fragments may also be deposited according to the present invention.
Useful as well as fragments corresponding to most (if not all) of the nucleotide sequence of NA or the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. For example, a fragment comprising at least 20 nt in length is intended to include a nucleotide sequence of the deposited cDNA or a fragment comprising 20 or more contiguous bases from the nucleotide sequence shown in SEQ ID NO: 1 or 3.

In addition, representative examples of VEGF-2 polynucleotide fragments include, for example,

[0074]

(Equation 2) To the end of SEQ ID NO: 1 or the cDN contained in the deposited clone
Fragments with A are included. In this context, “about” includes the particularly recited range, and a few nucleotides (5, 4, 5) at either or both termini.
, 3, 2, or 1) larger or smaller ranges. Preferably,
These fragments encode a polypeptide having biological activity.

The fragments of the full-length gene of the present invention can be used to isolate full-length cDNAs and other cDNs with high sequence similarity or similar biological activity to the gene.
It can be used as a hybridization probe on a cDNA library to isolate A. Probes of this type preferably have at least 3
It has zero bases and can contain, for example, 50 or more bases. Probes can also be used to identify cDNA clones corresponding to the full-length transcript and genomic clones containing the complete gene containing regulatory and promoter regions, exons, and introns. An example of a screen involves isolating the coding region of a gene by using a known DNA sequence to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to the sequence of the gene of the invention can be used to determine a library of human cDNA, genomic DNA, or mRNA to determine which library member probes will hybridize. Used to screen.

VEGF-2 “polynucleotide” also refers to a polynucleotide that is capable of hybridizing under stringent hybridization conditions to the sequence contained in SEQ ID NO: 1, or, for example, ATCC accession numbers 97149 or 75698,
And the cDNA clone contained in the complement thereof. “Stringent hybridization conditions” are 50% formamide at 42 ° C., 5 × SSC (
Overnight incubation in a solution containing 750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA Followed by filter washing at about 65 ° C. in 0.1 × SSC.

[0077] Nucleic acid molecules that hybridize to VEGF-2 polynucleotides under low stringency hybridization conditions are also contemplated. Changes in hybridization stringency and signal detection are mainly due to formamide concentration (lower percentages of formamide result in lower stringency)
Achieved by manipulating the salt conditions or temperature. For example, lower stringency conditions include 6 × SSPE (20 × SSPE = 3M NaCl;
0.2 M NaH ₂ PO ₄ ; 0.02 M EDTA, pH 7.4), 0.5% SD
S, 30% formamide, 100 μg / ml salmon sperm blocking DNA in solution overnight at 37 ° C .; followed by 1 × SSP at 50 ° C.
E, includes washing with 0.1% SDS. In addition, to achieve even lower stringency, washing that follows stringent hybridization can be performed at higher salt concentrations (eg, 5 × SSC).

Modification of the above conditions can be achieved by including and / or replacing alternative blocking reagents used to reduce background in hybridization experiments. Typical blocking reagents include De.
Includes nhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercial proprietary formulations. Including certain blocking reagents may require changing the above hybridization conditions due to compatibility issues.

Of course, a polynucleotide that hybridizes only to a poly A + sequence (eg, any 3 ′ terminal poly A + region of the cDNA shown in the sequence listing) or a complementary stretch of T (or U) residues is: It is not included in the definition of "polynucleotide" because it hybridizes to any nucleic acid molecule containing a poly (A) stretch or its complement (eg, any real double-stranded cDNA clone).

A polynucleotide that hybridizes to a “portion” of a polynucleotide is at least about 15 nucleotides (nt) of the reference polynucleotide, more preferably at least about 20 nt, even more preferably at least about 30 nt, and even more preferably Polynucleotides (either DNA or RNA) that hybridize to at least about 30-70 nt are contemplated. As discussed above and below in more detail, they are useful as diagnostic probes and primers.

A polynucleotide portion “at least about 20 nt in length” intends, for example, 20 or more contiguous nucleotides derived from the nucleotide sequence of a reference polynucleotide (eg, as deposited cDNA or SEQ ID NO: 1). Nucleotide sequence as shown). Of course, a polynucleotide that hybridizes only to the polyA sequence (eg, the 3N-terminal polyA + region of the VEGF-2 cDNA shown in SEQ ID NO: 1 or 3), or the complementary stretch of T (or U) residues, Any nucleic acid molecule comprising a poly (A) stretch or a complement thereof (eg,
Since it hybridizes to any double-stranded cDNA clone), it is not included in the polynucleotide of the present invention used to hybridize to a part of the nucleic acid of the present invention.

The present application relates to a nucleic acid sequence as set forth in SEQ ID NO: 1 or 3 or a deposited c
A nucleic acid molecule that is at least 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of DNA. This is because certain nucleic acid molecules are VEGF-
Even if they do not encode a polypeptide with two activities, the skilled person still knows how to use this nucleic acid molecule (eg as a hybridization probe or a polymerase chain reaction (PCR) primer).
. Uses of the nucleic acid molecules of the invention that do not encode a polypeptide having VEGF-2 activity specifically include: (1) isolation of the VEGF-2 gene or allelic variants thereof in a cDNA library; (2) ) In situ hybridization to metaphase chromosomal spreads (eg, "FISH") to define the exact chromosomal location of the VEGF-2 gene (Verma et al., Human Chromosome).
mosomes: A Manual of Basic Technologies
. Pergamon Press, New York (1988)); and Northern blot analysis to detect VEGF-2 mRNA expression in specific tissues.

However, the nucleic acid sequence shown in SEQ ID NO: 1 or 3 or the deposited cDNA
Preferred are nucleic acid molecules having a sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of (which actually encodes a polypeptide having VEGF-2 protein activity) . By "polypeptide having VEGF-2 activity" is intended a polypeptide that exhibits VEGF-2 activity in a particular biological assay. For example, VEGF-2 protein activity can be measured using, for example, mitogen assays and endothelial cell migration assays.
See, for example, Olofsson et al., Proc. Natl. Acad. Sci. USA
93: 2576-2581 (1996) and Joukov et al., EMBO J.
. 5: 290-298 (1996).

Of course, due to the degeneracy of the genetic code, those skilled in the art will recognize that the nucleic acid sequence of the deposited cDNA or the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, at least 90%, It is immediately recognized that many nucleic acid molecules with 96%, 97%, 98% or 99% sequence identity encode polypeptides having "VEGF-2 protein activity". In fact, this is obvious to one skilled in the art without performing the above-described comparative assays, since all of these degenerate variants of the nucleic acid sequence encode the same polypeptide. For such nucleic acid molecules that are degenerate variants, those of skill in the art will further recognize that a reasonable number also encodes a polypeptide having VEGF-2 protein activity. This is because one of skill in the art is well aware of amino acid substitutions that are either not likely or do not significantly affect protein function (eg, aliphatic amino acids and secondary amino acids).
With an aliphatic amino acid).

For example, guidance on how to make phenotypically silent amino acid substitutions can be found in Bowie, J. et al. U. Et al., "Deciphering the M
message in Protein Sequences: Toleranc
e to Amino Acid Substitutions ", Science
ce 247: 1306-1310 (1990), where we show that the protein is surprisingly tolerant of amino acid substitutions.

Thus, the present invention relates to the polypeptides of SEQ ID NO: 2 or 4, and fragments thereof (these fragments are at least 30 base pairs, preferably 50
At least 70% identity, preferably at least 90% and more preferably at least 95% to the polynucleotide encoding
Polynucleotides having 96%, 97%, or 98% identity, as well as polypeptides encoded by such polynucleotides.

“Identity” per sequence (se) has an art-recognized meaning and can be calculated using published techniques (eg, COMPUTATINAL).
MOLECULAR BIOLOGY, Lesk, A. M. Et al., Oxford
University Press, New York, (1988); BI.
OCMPUTING: INFORMATICS AND GENOME PRO
JECTS, Smith, D.A. W. Hen, Academic Press, Ne
w York, (1993); COMPUTER ANALYSIS OF S
EQUEENCE DATA, PART I, Griffin, A. M. And G
riffin, H .; G. FIG. Hen, Human Press, New Jersey,
(1994); SEQUENCE ANALYSIS IN MOLECULA
R BIOLOGY, von Heinje, G .; , Academic Pre
ss, (1987); and SEQUENCE ANALYSIS PRIM
ER, Gribskov, M.E. And Devereux, J. et al. Hen, M Stoc
See kman Press, New York, (1991). 2.) While there are many methods for determining the identity between two polynucleotide sequences or two polypeptide sequences, the term "identity" is well known to those skilled in the art.
(Carillo, H. and Lipton, D., SIAM J. Appli
ed Math. 48: 1073 (1988). ) Methods commonly used to determine identity or similarity between two sequences include the following,
Without limitation, these methods are described in “Guide to Huge Com”.
puters, "Martin J. Bishop, Academic Pr
ess, San Diego, (1994), and Carillo, H .; And Lipton, D .; , SIAM J. et al. Applied Math. 48:
1073 (1988). Methods for aligning polynucleotides or polypeptides are assembled in computer programs, including: the GCG program package (Devereux, J. et al.
Et al., Nucleic Acid Research 12 (1): 387 (198
4)), BLASTP, BLASTN, FASTA (Atschul, SF
J. et al. Molec. Biol. 215: 403 (1990), Bestfit
Program (Wisconsin Sequence Analysis Pa)
Cage, Version 8 for Unix, Genetics Compute
r Group, University Research Park, 575
Science Drive, Madison, WI 53711 (Smith
Advance, using the local homology algorithm of Waterman and Waterman
s in Applied Mathematicals 2: 482-489 (1
981)). At least, for example, 9
A polynucleotide having a 5% "identical" nucleotide sequence is one in which the polynucleotide sequence has 10% of the reference nucleotide sequence encoding the VEGF-2 peptide.
It is intended that the nucleotide sequence of the polynucleotide be identical to the reference sequence, except that it may contain up to 5 point mutations per 0 nucleotide. That is, to obtain a polynucleotide having a nucleic acid sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or replaced with another nucleotide, or Up to 5% of the total nucleotides within can be inserted into the reference sequence. The query array is
It can be the entire sequence of SEQ ID NO: 1, the ORF (open reading frame), or any particular fragment described herein.

As a practical matter, any particular nucleic acid molecule or polypeptide will have at least 90%, 95%, 96%, 97%, 98%,
Or whether they are 99% identical can be determined conventionally using known computer programs. A preferred method for determining the best overall match (also referred to as an overall sequence alignment) between a query sequence (sequence of the invention) and a subject sequence is described in Brutlag et al. (Comp. App.Biosci
. (1990) 6: 237-245) can be determined using the FASTDB computer program. In sequence alignment, the query and subject sequences are both DNA sequences. RNA sequence is U to T
Can be compared by converting The result of this global sequence alignment is expressed as percent identity (%). The preferred parameters used in FASTDB alignment of DNA sequences to calculate percent identity are: Matrix =
Unitary, k-tuple = 4, Mismatch Penalty = 1
, Joining Penalty = 30, Randomization Gr
up Length = 0, Cutoff Score = 1, Gap Pena
lty = 5, Gap Size Penalty 0.05, Window S
size = 500 or the length of the nucleotide sequence of interest (whichever is shorter).

If the subject sequence is shorter than the query sequence due to a 5 ′ or 3 ′ deletion (not due to an internal deletion), a manual correction must be made to the result. This is because the FASTDB program does not consider 5 'and 3' truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5 'or 3' end, the percent identity to the query sequence is the query sequence 5 'and 3' of the subject sequence not matched / aligned as a percentage of the total bases of the query sequence. Is calculated by calculating the number of bases. Whether nucleotides are matched / aligned is determined by the result of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity and calculated by the FASTDB program described above using the specified parameters, to arrive at a final percent identity score. This corrected score is used for the purpose of the present invention. Only those bases outside of the 5 'and 3' bases of the subject sequence that are not matched / aligned with the query sequence, as indicated by FASTDB alignment, are calculated for the purpose of manually adjusting the percent identity score.

For example, a 90 base subject sequence is aligned with a 100 base query sequence to determine percent identity. The deletion occurs at the 5 'end of the subject sequence, so the FASTDB alignment does not show a match / alignment of the first 10 bases at the 5' end. 1
Zero unpaired bases represent 10% of the sequence (number of bases at the unmatched 5 'and 3' ends / total number of bases in the query sequence), so 10% is FASTD
It is subtracted from the percent identity score calculated by the B program.
If the remaining 90 bases are a perfect match, the final percent identity is 90%. In another example, a 90 base subject sequence is compared to a 100 base query sequence. In this case, the deletion is an internal deletion, such that there is no base at 5 'or 3' of the subject sequence that does not match / align with the query sequence. In this case, FASTDB
The percent identity calculated by is not manually corrected. Again, only the 5 'or 3' bases of the subject sequence that do not match / align with the query sequence are manually corrected. No other manual corrections are made for the purposes of the present invention.

A polypeptide having an amino acid sequence that is at least 95% “identical” to the query amino acid sequence of the present invention, for example, is that the amino acid sequence of the subject polypeptide is It is intended to be identical to the query sequence except that it can include up to 5 amino acid changes per 100 amino acids. In other words, at least 95%
To obtain a polypeptide having the same amino acid sequence, up to 5% of the amino acid residues in the subject sequence can be inserted, deleted, (indels) or replaced with another amino acid. These changes in the reference sequence may occur at the amino or carboxy terminal site of the reference amino acid sequence, or may occur anywhere between those terminal sites, individually between residues in the reference sequence, or One of
Interspersed with any of one or more adjacent groups.

As a practical matter, any particular polypeptide will be at least 90%, 95% less than the amino acid sequence shown in Table 1 or the amino acid sequence encoded by the deposited DNA clone. Whether it is%, 96%, 97%, 98%, or 99% identical can be determined conventionally using known computer programs. A preferred method for determining the best overall match (also referred to as an overall sequence alignment) between a query sequence (a sequence of the invention) and a subject sequence is described in Brutlag et al. (Comp. App. Biosci. 1990)
6: 237-245) can be determined using the FASTDB computer program based on the algorithm. In a sequence alignment, the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The results of the overall sequence alignment described above are expressed as percent identity. Preferred parameters used for the FASTDB amino acid alignment are: Matri
x = PAM 0, k-tuple = 2, Mismatch Penalty = 1
, Joining Penalty = 20, Randomization Gr
up Length = 0, Cutoff Score = 1, Window S
size = sequence length, Gap Penalty = 5, Gap Size P
energy = 0.05, Window Size = 500 or the length of the amino acid sequence of interest (whichever is shorter).

If the subject sequence is shorter than the query sequence due to N-terminal or C-terminal deletions (not due to internal deletions), manual corrections must be made to the results. This is because the FASTDB program does not consider N- and C-terminal truncations of the subject sequence when calculating the overall percent identity. For subject sequences that are truncated at the N-terminus and C-terminus, for the query sequence, the percent identity is the percent of total bases in the query sequence, the N-terminus and C- It is corrected by calculating the number of residues in the query sequence that are terminal. Whether residues are matched / aligned is determined by the result of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity and calculated by the FASTDB program described above using the specified parameters to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only the N- and C-terminal residues of the subject sequence that are not matched / aligned with the query sequence are considered for the purpose of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence,
And therefore the FASTDB alignment does not show a match / alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of unmatched N- and C-terminal residues / total number of residues in the query sequence) resulting in FAS
10% is subtracted from the percent identity score calculated by the TDB program. If the remaining 90 residues match perfectly, the final percent identity is 90%. In another example, a 90 residue subject sequence is compared to a 100 residue query sequence. In this case, the deletion is an internal deletion, so that there are no N- or C-terminal residues of the subject sequence that do not match / align with the query sequence.
In this case, the percent identity calculated by FASTDB is not manually corrected. Again, only residue positions outside the N- and C-termini of the subject sequence that do not match / align with the query sequence, as shown in the FASTDB alignment, are manually corrected. No other manual corrections are made for the purposes of the present invention.

VEGF-2 Polypeptides The present invention further provides polypeptides having the deduced amino acid sequence of FIG. 1 or FIG. 2, or having the amino acid sequence encoded by the deposited cDNA, and such polypeptides. It relates to fragments, analogs and derivatives of the peptides.

The terms “fragment”, “derivative”, and “analog” when referring to the polypeptide of FIG. 1 or FIG. 2 or the polypeptide encoded by the deposited cDNA, are conserved of the VEGF protein shown in FIG. Motifs and polypeptides that retain essentially the same biological function or activity.

In the present invention, “polypeptide fragment” refers to the short amino acid sequence contained in SEQ ID NO: 2 or the short amino acid sequence encoded by the cDNA contained in the deposited clone. Protein fragments are "free"
-Standing) or may be contained within a larger polypeptide of a portion or region formed by fragments, most preferably one contiguous region. Representative examples of polypeptide fragments of the present invention include, for example, approximate amino acid numbers 1-20, 21-40, 41-60, 61-80,
81-100, 102-120, 121-140, 141-160, 161-1
80,181-200,201-220,221-240,241-260,2
Fragments from 61 to 280 or 281 to the end of the coding region. In addition, polypeptide fragments can be about 20, 30, 40, 50,
60, 70, 80, 90, 100, 110, 120, 130, 140, or 1
It can be 50 amino acids. In this context, “about” means at either or both termini, the specifically recited range, only some (5, 4, 3, 2, 2, or 1) amino acids, greater or less than Including small areas.

[0098] Preferred polypeptide fragments include the secreted VEGF-2 protein as well as the mature form. More preferred polypeptide fragments include the secreted VEGF-2 protein or the mature form having a contiguous series of deleted residues from the amino terminus or the carboxy terminus, or both. For example,
Any number of amino acids (ranging from 1 to 60) can be deleted from the amino terminus of either the secreted VEGF-2 polypeptide or the mature form. Similarly, any number of amino acids (ranging from 1 to 30) can be deleted from the carboxy terminus of the secreted VEGF-2 protein or the mature form. Further, any combination of the above amino terminal and carboxy terminal deletions is preferred. Similarly, these VEGF
Polynucleotide fragments encoding the -2 polypeptide fragment are also preferred.

Structural or functional domains (eg, α-helix and α-helix forming regions, β-sheet and β-sheet forming regions, turns and turn-forming regions, coils and coil-forming regions, hydrophilic regions, hydrophobic regions, α-amphiphiles Also preferred are polypeptide and polynucleotide fragments of VEGF-2, characterized by a fragment comprising an avidity region, a beta amphipathic region, a variable region, a surface-forming region, a substrate binding region, and a high antigenic index region. . Polypeptide fragments of SEQ ID NO: 2 that fall within the conserved domains are specifically contemplated by the present invention (see Table 2). In addition, polynucleotide fragments encoding these domains are also contemplated.

[0100] Another preferred fragment is a biologically active VEGF-2 fragment. Biologically active fragments are those that exhibit activity similar to, but not necessarily identical to, that of the VEGF-2 polypeptide.
The biological activity of the fragment may include an improved desired activity, or a reduced unwanted activity.

The polypeptide of the present invention can be a recombinant polypeptide, a natural peptide, or a synthetic polypeptide, and is preferably a recombinant polypeptide.

[0102] It is recognized in the art that some amino acid sequences of VEGF-2 polypeptides can be changed without significant effect on protein structure or function.
If such differences in sequence are taken into account, it must be remembered that there are important regions on the protein that determine activity.

Accordingly, the present invention further provides a method for demonstrating substantial VEGF-2 polypeptide activity,
Or a variant of the VEGF-2 polypeptide comprising a region of the VEGF-2 protein (eg, the protein portion discussed below). Such variants include deletions, insertions, inversions, repeats, and type substitutions. As indicated above, guidance on amino acid changes that are phenotypically silent can be found in Bowie.
, J. et al. U. Et al., "Deciphering the Message in P
protein Sequences: Tolerance to Amino
Acid Substitutions, "Science 247: 1306.
-1310 (1990).

Thus, a fragment, derivative, or analog of the polypeptide of FIG. 1 or 2, or the polypeptide encoded by the deposited cDNA, is: (I)
There, one or more amino acid residues are replaced with conserved or non-conserved amino acid residues (preferably conserved amino acid residues), and such substituted amino acid residues are the amino acid residues encoded by this genetic code. Or (ii) wherein one or more amino acid residues contain a substituent; or (iii) a compound wherein the mature polypeptide increases the half-life of the polypeptide ( (Iv) where an additional amino acid is fused to the mature polypeptide (eg, a leader or secretory sequence or a mature polypeptide sequence or (A sequence used for purification of the proprotein sequence); or (v) It includes Suyori fewer amino acid residues, and retains the conserved motif and yet still may be those which retain the characteristic activity of the VEGF family of polypeptides. Such fragments, derivatives and analogs are deemed to be within the skill of the art in light of the teachings herein.

Of particular interest is the replacement of a charged amino acid with another charged amino acid and a neutral or negatively charged amino acid. The latter result reduces the positive charge on the protein and improves the properties of the VEGF-2 protein. Prevention of agglomeration is highly preferred. Aggregation of proteins not only results in reduced activity when preparing pharmaceutical formulations, but can also have problems. Because they can be immunogenic. (Pinkard et al., Clin. Exp. Immunol. 2
: 331-340 (1967); Robbins et al., Diabetes 36:
838-845 (1987); Cleland et al., Crit. Rev .. Ther
apeutic Drug Carrier Systems 10: 307-
377 (1993)).

[0106] Amino acid substitutions can also alter the selectivity of binding to cell surface receptors.
Ostade et al., Nature 361: 266-268 (1993) describe that certain mutations result in the selective binding of TNF-a to only one of the two known TNF receptor types. Thus, VEGF-2 of the present invention may include one or more amino acid substitutions, deletions or additions, either from natural variants or man-made operations.

As indicated, the changes are preferably of a mild nature so that conservative amino acid substitutions do not have a significant effect on protein folding or activity (see Table 1).

[0108]

[Table 1] Of course, the number of amino acid substitutions made by one of skill in the art will depend on many factors, including the factors described above. Generally, the number of substitutions for any given VEGF-2 polypeptide is not more than 50, 40, 30, 25, 20, 15, 10, 5, or 3.

The amino acids in the VEGF-2 protein of the invention that are essential for function are those such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081-1085 (1989)). It can be identified by methods known in the art. The latter procedure introduces a single alanine mutation at every residue in the molecule. The resulting mutant molecules are then tested for biological activity (eg, receptor binding activity or in vitro) or for proliferative activity in vitro. Sites critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance, or photoaffinity labeling (Smith et al., J. Mol. Biol. 224: 899-904 (
1992) and de Vos et al., Science 255: 306-312 (
1992)).

The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and are preferably purified to homogeneity.

The term “isolated” means that the material has been removed from its original environment (eg, the natural environment if it occurs naturally). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is the same polynucleotide or polypeptide that has not been isolated but has been separated from some or all of the coexisting materials in the natural system. DNA or polypeptide has been isolated. Such a polynucleotide can be part of a vector,
And / or such a polynucleotide or polypeptide may be part of a composition, and is still isolated in that such a vector or composition is not part of its natural environment .

In certain embodiments, the polynucleotides of the present invention comprise 300 kb, 200 kb
, 100 kb, 50 kb, 15 kb, 10 kb, or less than 7.5 kb in length. In a further embodiment, the polynucleotide of the present invention comprises at least 15 contiguous nucleotides of the VEGF-2 coding sequence, but does not contain any VEGF-
It does not include all or some of the two introns. In another embodiment, the nucleic acid comprising the VEGF-2 coding sequence is a genomic flanking gene (ie, VE in this genome).
It does not contain the coding sequence 5 'or 3' to the GF-2 gene).

The polypeptides of the present invention may comprise at least 7 polypeptides of SEQ ID NOs: 2 and 4 (especially mature polypeptides) and at least 7 polypeptides of SEQ ID NOs: 2 and 4.
0% similarity (preferably at least 70% identity), and more preferably at least 90% similarity (more preferably at least 95% identity) to the polypeptides of SEQ ID NOs: 2 and 4, and even more preferably SEQ ID NOs: 2 and 4
And polypeptides that have at least 95% similarity (even more preferably at least 90% identity) to a polypeptide of the invention, and also include portions of such polypeptides, such portions of the polypeptides. Generally comprises at least 30 amino acids, and more preferably at least 50 amino acids.

As is known in the art, “similarity” between two polypeptides refers to the amino acid sequence of one polypeptide and conserved amino acid substitutions thereof relative to the sequence of a second polypeptide. Determined by comparison.

A fragment or portion of a polypeptide of the present invention may be used to produce the corresponding full-length polypeptide by peptide synthesis; therefore, this fragment may be used as an intermediate to produce a full-length polypeptide. Can be Fragments or portions of the polynucleotides of the invention can be used to synthesize full length polynucleotides of the invention.

A polypeptide of the invention is a polypeptide encoded by a deposited cDNA, including a leader; a mature polypeptide (ie, a mature protein) encoded by a deposited cDNA without a leader; SEQ ID NO: About-in 2
A polypeptide comprising 23 to about 396 amino acids;
A polypeptide comprising about 396 amino acids; a polypeptide comprising about 1 to about 396 amino acids in SEQ ID NO: 2; and at least 95% identical to the above polypeptide, and more preferably at least 96%, 97%, 98 % Or 99% identical polypeptides, and also includes portions of such polypeptides that include at least 30 amino acids, and more preferably at least 50 amino acids.

Fusion Proteins Any VEGF-2 polypeptide can be used to produce a fusion protein. For example, a VEGF-2 polypeptide can be used as an antigenic tag when fused to a second protein. Antibodies raised against the VEGF-2 polypeptide can be used to indirectly detect this second protein by binding to VEGF-2. In addition, VEGF-2 polypeptides can be used as targeting molecules, once fused to other proteins, since secreted proteins target cell locations based on transport signals.

Examples of domains that can be fused to a VEGF-2 polypeptide include not only a heterologous signal sequence, but also other heterologous functional regions. The fusion need not be direct, but can occur via a linker sequence.

[0119] In addition, fusion proteins can also be engineered to improve VEGF-2 polypeptide characteristics. For example, additional amino acids, especially regions of charged amino acids,
VEGF-2 polypeptides may be added at the N-terminus to improve stability and persistence during purification or subsequent handling and storage from host cells. Also, a peptide moiety can be added to the VEGF-2 polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the VEGF-2 polypeptide. The addition of peptide moieties to facilitate handling of the polypeptide is well known in the art and is a conventional technique.

In addition, VEGF-2 polypeptides (including fragments, and particularly epitopes) can be combined with portions of the constant domain of an immunoglobulin (IgG), resulting in a chimeric polypeptide. These fusion proteins facilitate purification and show an increased half-life in vivo. One reported example is human CD
A chimeric protein consisting of the first two domains of the four polypeptides and various domains of the constant regions of the heavy or light chains of a mammalian immunoglobulin has been described (EPA 394,827; Traunecker et al., Nature 3).
31: 84-86 (1988)). Fusion proteins with disulfide-linked dimeric structures (due to IgG) may also be more efficient at binding and neutralizing other molecules than monomeric secreted proteins or protein fragments alone (Funtoulakis et al., J. Biochem. 270: 395).
8-3964 (1995)).

Similarly, EP-A-O 464 533 (Canadian Corresponding Patent No. 2045869)
Discloses fusion proteins comprising various parts of the constant region of an immunoglobulin molecule together with another human protein or part thereof. In many cases, the Fc portion of the fusion protein is beneficial in therapy and diagnosis, and thus may, for example, result in improved pharmacokinetic properties (EP-A 0232 262). Alternatively, it may be desirable to delete the Fc portion after the fusion protein has been expressed, detected, and purified. For example, if the fusion protein is used as an antigen for immunization, the Fc portion can interfere with therapy and diagnosis. For example, in drug discovery, human proteins such as hIL-5 have been fused with an Fc portion for the purpose of high-throughput screening assays to identify antagonists of hIL-5 (D. Bennett et al., J. Molecular Reco
8: 52-58 (1995); Johanson et al.
Biol. Chem. 270: 9449-9471 (1995)).
.

In addition, VEGF-2 polypeptides have a marker sequence (eg, VEGF-2).
Peptide that facilitates purification of In a preferred embodiment, the marker amino acid sequence is, inter alia, a hexahistidine peptide (eg, pQE
Vector (QIAGEN, Inc., 9259 Eton Avenue, Ch.
atsworth, CA, 91311), many of which are commercially available. Gentz et al., Proc. Natl. Acad. S
ci. USA 86: 821-824 (1989), for example, hexahistidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the “HA” tag, corresponds to an epitope from the influenza hemagglutinin protein (Wilson et al., Cell 37:
767 (1984)).

Accordingly, any of these above fusions can be engineered using a VEGF-2 polynucleotide or VEGF-2 polypeptide.

VEGF-2 Biological Activity VEGF-2 polynucleotides and VEGF-2 polypeptides can be used in assays to test for one or more biological activities. If VEGF-2 polynucleotides and VEGF-2 polypeptides show activity in a particular assay, it is possible that VEGF-2 may be involved in diseases associated with its biological activity. Thus, VEGF-2 can be used to treat this related disease.

(Immune activity) VEGF-2 polypeptide or VEGF-2 polynucleotide activates or inhibits the proliferation, differentiation, or recruitment (chemotaxis) of immune cells, thereby arresting or impairing the immune system. It may be useful for treating. The immune cells produce a process called hematopoiesis, a process that produces myeloid cells (platelets, red blood cells, neutrophils, and macrophages) and lymphoid cells (B and T lymphocytes) from pluripotent stem cells. Occurs through. The etiology of these immunodeficiencies or disorders can be hereditary, somatic (eg, cancer or some autoimmune disorders), acquired (eg, due to chemotherapy or toxins), or infectious. further,
VEGF-2 polynucleotides or VEGF-2 polypeptides can be used as markers or detectors for certain immune system diseases or disorders.

A VEGF-2 polynucleotide or VEGF-2 polypeptide may be useful in treating or detecting a hematopoietic cell deficiency or disorder. In an effort to treat disorders associated with the reduction of certain (or many) types of hematopoietic cells,
VEGF-2 polypeptides or polynucleotides can be used to increase the differentiation and proliferation of hematopoietic cells, including pluripotent stem cells. Examples of immunodeficiency syndromes include, but are not limited to: blood protein disorders (eg, agammaglobulinemia, hypogammaglobulinemia), telangiectasia ataxia, unclassified immunodeficiency, Di George syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia,
Phagocytic bactericidal dysfunction, severe combined immunodeficiency (SCID), Viscott-Aldrich disorder, anemia, thrombocytopenia, or hemoglobinuria.

In addition, VEGF-2 polypeptides or VEGF-2 polynucleotides can also be used to modulate hemostatic activity (stop bleeding) or thrombolytic activity (clot formation). For example, by increasing haemostatic or thrombolytic activity, VEGF-2 polynucleotides or VEGF-2 polypeptides may cause blood coagulation disorders (eg, afibrinogenemia, factor deficiency), blood platelet disorders (
For example, thrombocytopenia), or can be used to treat wounds resulting from trauma, surgery or other causes. Alternatively, coagulation can be inhibited or lysed using a VEGF-2 polynucleotide or VEGF-2 polypeptide, which can reduce hemostatic or thrombolytic activity. These molecules are used in heart attacks (infarctions), strokes,
Or it may be important in treating scarring.

A VEGF-2 polynucleotide or VEGF-2 polypeptide may also be useful in treating or detecting an autoimmune disorder. Many autoimmune disorders result from improper recognition of self as a foreign substance by immune cells. This inappropriate recognition results in an immune response that leads to destruction of the host tissue. Therefore, administration of VEGF-2 polypeptides or VEGF-2 polynucleotides that can inhibit the immune response, particularly T cell proliferation, differentiation, or chemotaxis, is an effective treatment in preventing autoimmune disorders. obtain.

Examples of autoimmune disorders that can be treated or detected by VEGF-2 include, but are not limited to, Addison's disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, Allergic encephalomyelitis, glomerulonephritis,
Goodpasture's syndrome, Graves' disease, multiple sclerosis, myasthenia gravis, neuritis, conjunctivitis, bullous pemphigoid, pemphigus, polyendocrine disease, purpura, Reiter's disease, stiff man syndrome, autoimmune thyroid Inflammation, systemic lupus erythematosus, autoimmune pulmonary inflammation, Guillain-Barre syndrome, insulin-dependent diabetes mellitus, and autoimmune inflammatory eye disease.

[0130] Similarly, allergic reactions and conditions, such as, for example, asthma (particularly allergic asthma) or other respiratory problems, may also include VEGF-2 polypeptide or VEGF.
-2 polynucleotide. In addition, VEGF-2 can be used to treat anaphylaxis, hypersensitivity to antigenic molecules, or blood group incompatibility.

[0131] VEGF-2 polynucleotides or VEGF-2 polypeptides can also be used to treat and / or prevent organ rejection or graft versus host disease (GVHD). Organ rejection occurs when host immune cells destroy the transplanted tissue via an immune response. Similarly, the immune response also involves GVHD,
In this case, the foreign transplanted immune cells destroy the host tissue. Immune response, especially T
VEGF-2 polypeptide or V that inhibits cell growth, differentiation, or chemotaxis
Administration of an EGF-2 polynucleotide may be an effective treatment in preventing organ rejection or GVHD. Similarly, a VEGF-2 polypeptide or VEGF-2
Polynucleotides can also be used to regulate inflammation. For example, VEG
An F-2 polypeptide or VEGF-2 polynucleotide can inhibit the growth and differentiation of cells involved in the inflammatory response. These molecules can be used to treat inflammatory conditions (both chronic and acute conditions), including: infection-related inflammation such as septic shock, sepsis, or systemic inflammatory response syndrome (S
IRS)), ischemic reperfusion injury, endotoxin death, arthritis, complement-mediated hyperacute rejection,
Nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or conditions resulting from overproduction of cytokines (eg, TNF or IL-1).

Hyperproliferative Disorders VEGF-2 polypeptides or VEGF-2 polynucleotides can be used to treat or detect hyperproliferative disorders, including neoplasms. A VEGF-2 antagonist polypeptide or VEGF-2 antagonist polynucleotide can inhibit the growth of a disorder by direct or indirect interaction. Or VE
GF-2 antagonist polypeptides or VEGF-2 antagonist polynucleotides can proliferate other cells that can inhibit hyperproliferative disorders.

For example, by increasing the immune response, in particular by increasing the antigenic quality of the hyperproliferative disorder, or by expanding, differentiating or recruiting T cells, The disorder can be treated. This immune response can be raised either by enhancing an existing immune response or by initiating a new immune response. Alternatively, reducing the immune response can also be a method of treating a hyperproliferative disorder (eg, a chemotherapeutic agent).

Examples of hyperproliferative disorders that can be treated or detected by a VEGF-2 antagonist polynucleotide or VEGF-2 antagonist polypeptide include, but are not limited to, neoplasms located at: abdomen, bone, chest , Digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal gland, parathyroid, pituitary, testis, ovary, thymus, thyroid), eyes, head and neck, nerves (central and peripheral), lymphatic system, pelvis , Skin, soft tissue, spleen, rib cage, and genitourinary.

Similarly, other proliferative disorders can also be treated or detected by a VEGF-2 antagonist polynucleotide or VEGF-2 antagonist polypeptide. Examples of such hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemia, purpura, sarcoidosis, Sesary syndrome, Waldenstrom gamma globulin Located in the blood systems, Gaucher disease, histiocytosis, and the organ systems listed above,
Any other hyperproliferative disease other than neoplasm.

Infectious Disease VEGF-2 polypeptides or VEGF-2 polynucleotides can be used to treat or detect infectious agents. For example, an infectious disease can be treated by increasing the immune response, particularly by increasing the proliferation and differentiation of B and / or T cells. The immune response can be increased either by enhancing an existing immune response or by initiating a new immune response. Alternatively, a VEGF-2 polypeptide or VEGF-2 polynucleotide may also directly inhibit an infectious agent without necessarily eliciting an immune response.

A virus is an example of an infectious agent that can cause a disease or condition that can be treated or detected by a VEGF-2 polynucleotide or VEGF-2 polypeptide. Examples of viruses include, but are not limited to, the following DNAviridae and RNAviridae: arboviridae, adenoviridae,
Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae,
Caliciviridae, Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (eg, cytomegalovirus, herpes simplex, herpes zoster), mononegavirus. (Eg, paramyxoviridae, measles virus, rhabdoviridae), orthomyxoviridae (eg, influenza), papovaviridae, parvoviridae, picornaviridae, poxviridae (eg, pox or variola), Reoviridae (for example, rotavirus), Retroviridae (HTLV-I, HTLV-II, lentivirus), and Togaviridae (for example, genus Rubivirus). Viruses that fall into these families can cause a variety of diseases or conditions, including but not limited to: arthritis, bronchiolitis, encephalitis, ocular infections (eg, conjunctivitis, keratitis), Chronic fatigue syndrome, hepatitis (types A, B, C,
Type E, chronic activity, delta), meningitis, opportunistic infections (eg, AIDS), pneumonia, Burkitt's lymphoma, chickenpox, hemorrhagic fever, measles, mumps, parainfluenza, rabies, cold, Polio, leukemia, rubella, sexually transmitted diseases, skin diseases (eg, capos, warts), and viremia. VEGF-2 polypeptide or VEG
Any of these conditions or diseases can be treated or detected using the F-2 polynucleotide.

Similarly, bacterial or fungal factors that can cause a disease or condition and that can be treated or detected by a VEGF-2 polynucleotide or VEGF-2 polypeptide include the following Gram-negative and Gram-positive bacteria families and Including but not limited to fungi: Actinomycetales (eg, Co
rynebacterium, Mycobacterium, Norcardi
a), Aspergillosis, Bacillaceae (for example, Ant
hrax, Clostridium), Bacteroidaceae, Bla
stomycosis, Bordetella, Borrelia, Bruce
llosis, Candidiasis, Campylobacter, Coc
Cidioidomycosis, Cryptococcosis, Derma
tocycoses, Enterobacteriaceae (Klebsie
lla, Salmonella, Serratia, Yersinia), Er
ysipelothrix, Helicobacter, Legionello
sis, Leptospirosis, Listeria, Mycoplasm
atales, Neisseriaceae (eg, Acinetobacter)
er, Gonorrhea, Menigococcal), Pasteurel
racea infections (eg, Actinobacillus, Heamorph)
ilus, Pasteurella), Pseudomonas, Ricket
tsiaaceae, Chlamydiaceae, Syphilis, and S
taphyococcal. These bacterial or fungal families can cause diseases or conditions including, but not limited to: bacteremia, endocarditis, ocular infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic. Infectious diseases (eg, AIDS
Infections associated with), periungitis, prosthesis-related infections, Reiter's disease, respiratory tract infections (eg, pertussis or pyometra), sepsis, Lyme disease, cat scratching disease, dysentery, paratyphoid fever, food poisoning, typhoid fever, pneumonia, Gonorrhea, meningitis, chlamydia, syphilis,
Diphtheria, leprosy, paratuberculosis, tuberculosis, lupus, botulism, gangrene, tetanus,
Impetigo, rheumatic fever, scarlet fever, sexually transmitted diseases, skin diseases (eg, cellulitis, dermatomycoses), toxicemia, urinary tract infections, wound infections. VE
A GF-2 polypeptide or VEGF-2 polynucleotide can be used to treat or detect any of these conditions or diseases.

In addition, parasite agents that cause a disease or condition that can be treated or detected by a VEGF-2 polynucleotide or VEGF-2 polypeptide include, but are not limited to, the following families: amoeba, babesia,
Coccidium, Cryptosporidium, dinuclear amoeba, quarantine, ectoparasites, Giardia flagellates, helminths, Leishmania, Theileria, Toxoplasma, Trypanosoma, and Trichomonas. These parasites can cause a variety of diseases or conditions, including but not limited to: scabies, tsutsugamushi disease, ocular infections, bowel disease (eg, dysentery, giardiasis), liver disease, lung Diseases, opportunistic infections (eg, AIDS-related), malaria, pregnancy complications, and toxoplasma. VEGF-2 polypeptides or polynucleotides can be used to treat or detect any of these conditions or diseases.

Preferably, treatment with the VEGF-2 polypeptide or VEGF-2 polynucleotide comprises administering to the patient an effective amount of the VEGF-2 polypeptide, or removing cells from the patient, and removing the VEGF-2 polynucleotide. To the cells and return the engineered cells to the patient (ex vivo treatment). In addition, VEGF-2 polypeptides or VEGF-2 polynucleotides can be used as an antigen in a vaccine to elicit an immune response against an infectious disease.

Regeneration VEGF-2 polynucleotides or VEGF-2 polypeptides can be used to differentiate, proliferate, and attract cells, which leads to tissue regeneration. (See Science 276: 59-87 (1997)). Tissue regeneration includes birth defects, trauma (wounds, burns, incisions, or ulcers), aging, diseases (eg, osteoporosis, osteoarthritis, periodontal disease, liver failure), surgery (cosmetic formation) (Including surgery), fibrosis, reperfusion injury, or systemic cytokine damage can be used to repair, replace, or protect tissue damaged.

Tissues that can be regenerated using the present invention include: Organs (eg,
Pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth muscle, skeletal muscle or myocardium), blood vessels (including vascular endothelium), lymph tissue (including lymphatic endothelium), nervous tissue, hematopoietic tissue, and skeleton ( Bone, cartilage, tendons, and ligaments) tissue. Preferably, regeneration occurs without scarring or with reduced scarring. Regeneration may also include angiogenesis.

Further, a VEGF-2 polynucleotide or VEGF-2 polypeptide can be
It can increase the regeneration of tissue that is difficult to heal. For example, increased tendon / ligament regeneration
Faster recovery time after injury. VEGF-2 polynucleotide or VE of the invention
GF-2 polypeptides can also be used prophylactically in efforts to avoid damage. Specific diseases that can be treated include tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. Further examples of non-healing wound tissue regeneration include pressure ulcers, ulcers associated with vascular vascular insufficiency, surgical and traumatic wounds.

Similarly, neural and brain tissue can also be regenerated by VEGF-2 polynucleotides or polypeptides to proliferate and differentiate neural cells. Diseases that can be treated using the present methods include central and peripheral nervous system disorders, neurological or mechanical disorders and traumatic disorders such as spinal cord disorders, head trauma, cerebrovascular disorders, and stroke ( stokes)). In particular, diseases associated with peripheral nerve injury, peripheral neuropathy (eg, resulting from chemotherapy or other medical therapy), regional neuropathy, and central nervous system diseases (eg, Alzheimer's disease,
Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome) all use these VEGF-2 polynucleotides or VEs.
It can be treated using a GF-2 polypeptide.

Chemotaxis A VEGF-2 polynucleotide or VEGF-2 polypeptide may have chemotactic activity. Chemotactic molecules convert cells (eg, monocytes, fibroblasts, neutrophils, T cells, mast cells, eosinophils, epithelial cells and / or endothelial cells) into specific parts of the body (eg, inflammatory cells). Site, site of infection, or site of hyperproliferation). The migrated cells can then repel and / or heal certain traumas or abnormalities.

[0146] VEGF-2 polynucleotides or VEGF-2 polypeptides can increase chemotactic activity of certain cells. These chemotactic molecules are then used to treat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells targeted to specific locations in the body. Can be used. For example, chemotactic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the site of the injury. As a chemotactic molecule, VEGF-2 can also attract fibroblasts. Fibroblasts can be used to treat wounds.

It is also contemplated that a VEGF-2 polynucleotide or VEGF-2 polypeptide may inhibit chemotactic activity. These molecules can also be used to treat disorders. Accordingly, VEGF-2 polynucleotides or VEGF-2 polypeptides can be used as inhibitors of chemotaxis.

(Binding Activity) VEGF-2 polypeptides can be used to screen for VEGF-2 binding molecules or for VEGF-2 binding molecules.
The binding of VEGF-2 and its molecule, the activity of VEGF-2 or the bound molecule,
It can be activated (agonist), increased, inhibited (antagonist) or decreased. Examples of such molecules include antibodies, oligonucleotides, proteins (eg,
Receptors) or small molecules.

Preferably, the molecule is closely related to a natural ligand of VEGF-2 (eg, a fragment of that ligand) or a natural substrate, ligand, structural or functional mimetic. (Coligan et al., Current Prot.
ocols in Immunology 1 (2): see Chapter 5 (1991)). Similarly, this molecule is a natural receptor (to which VEGF-2 binds)
That is, it may be closely related to Flt-4) or at least to fragments of the receptor that can bind to VEGF-2 (eg, the active site). In each case, the molecule can be rationally designed using known techniques. Preferably, screening for these molecules involves generating appropriate cells that express VEGF-2, either as a secreted protein or on the cell membrane. Preferred cells are mammals, yeast, Drosophila, or E. coli.
E. coli-derived cells. The cell expressing VEGF-2 (or the cell membrane containing the expressed polypeptide) is then preferably used to observe binding, stimulation or inhibition of the activity of VEGF-2 or any of the molecules. , Is contacted with a test compound that may contain this molecule.

This assay can simply test the binding of the candidate compound to VEGF-2. Here, binding is detected by a label or in an assay involving competition with a labeled competitor. In addition, the assay indicates that the candidate compound is VEGF-2
Can produce a signal generated by binding to

Alternatively, the assay may be performed using a cell-free preparation, a polypeptide / molecule immobilized on a solid support, a chemical library, or a mixture of natural products. The assay also comprises mixing the candidate compound with a solution comprising VEGF-2, measuring VEGF-2 / molecular activity or VEGF-2 binding molecule,
And comparing the VEGF-2 / molecular activity or VEGF-2 binding molecule to a standard can be included simply.

Preferably, an ELISA assay may use a monoclonal or polyclonal antibody to measure VEGF-2 levels or activity in a sample (eg, a biological sample). The antibody can measure VEGF-2 levels or VEGF-2 activity, either by binding directly or indirectly to VEGF-2, or by competing with VEGF-2 for a substrate.

All of these above assays can be used as diagnostic or prophylactic markers. The molecule discovered using these assays is the VEGF-2
/ Can be used to treat a disease by activating or inhibiting a molecule, or to cause a particular outcome in a patient (eg, vascular growth).
In addition, this assay provides for substances that can inhibit or enhance VEGF-2 production.
It can be found in properly engineered cells or tissues.

Accordingly, the invention includes a method of identifying a compound that binds to VEGF-2. The method comprises the steps of: (a) incubating a candidate binding compound with VEGF-2; and (b) determining whether binding has occurred. Further, the invention includes a method of identifying an agonist / antagonist comprising the steps of: (a) incubating a candidate compound with VEGF-2; (b) assaying for biological activity; (B) VEGF-2
Determining whether the biological activity of has been altered.

Other Activities VEGF-2 polypeptides or VEGF-2 polynucleotides may also increase or decrease the differentiation or proliferation of embryonic stem cells in addition to hematopoietic lineages, as discussed above. .

VEGF-2 polypeptides or VEGF-2 polynucleotides may also have mammalian characteristics such as length, weight, hair color, eye color, skin, percentage of adipose tissue,
It can be used to control pigmentation, size, and shape (eg, cosmetic surgery). Similarly, VEGF-2 polypeptides or VEGF-2 polynucleotides can be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and energy storage.

[0157] VEGF-2 polypeptides or VEGF-2 polynucleotides can also be used to alter the mental or physical state of a mammal by affecting: biorhythms, caricadic rhythms, depression (depressive disorder) Propensity for violence, tolerance to pain, fertility (preferably by activin-like or inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive abilities.

[0158] VEGF-2 polypeptides or VEGF-2 polynucleotides may also be used as food additives or food preservatives, for example, holding capacity, fat content, lipid proteins, carbohydrates, vitamins, minerals, cofactors, or other nutritional factors. It can be used to increase or decrease components.

Vectors, Host Cells, and Protein Production The present invention also provides recombinant vectors comprising the isolated nucleic acid molecules of the present invention, and host cells comprising the recombinant vectors, and such vectors and hosts. The present invention relates to methods of making cells, as well as methods of using them for the production of VEGF-2 polypeptides or peptides by recombinant techniques.

A host cell is genetically engineered (transduced, transformed, or transfected) with a vector of the invention, which can be, for example, a cloning vector or an expression vector. ) Is done. The vector can be in the form of, for example, a plasmid, a virus particle, a phage, and the like. The engineered host cells can activate the promoter, select for transformants, or use the VEGF-
The two genes can be cultured in conventional nutrient media suitably modified to amplify. Culture conditions (eg, temperature, pH, etc.) are those previously used for the host cell selected for expression, and will be apparent to those skilled in the art.

[0161] The polynucleotides of the present invention can be used to produce polypeptides by recombinant techniques. Thus, for example, a polynucleotide sequence can be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, non-chromosomal, and synthetic DNA sequences.
Such vectors include, for example, derivatives of SV40; bacterial plasmids; phage DNA; yeast plasmids; vectors derived from a combination of plasmid DNA and phage DNA; viruses such as vaccinia, adenovirus, fowlpox virus, and pseudorabies. Contains DNA. However, any other plasmid or vector can be used as long as it is replicable and viable in the host.

The appropriate DNA sequence can be inserted into the vector by various procedures. In general,
The DNA sequence is inserted into an appropriate restriction endonuclease site by procedures known in the art. Such and other procedures are deemed to be within the purview of those skilled in the art.

[0163] The DNA sequence in the expression vector is operably linked to an appropriate expression control sequence (promoter) to direct the synthesis of mRNA. Representative examples of such promoters may include: LTR or SV40 promoter, E. coli. co
li. lac or trp promoters, phage lambda P _L promoters, and other promoters known to control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. Vectors may also include appropriate sequences for amplifying expression.

Further, the expression vector preferably contains at least one selectable marker gene that provides a phenotypic trait for the selection of transformed host cells. Such markers include dihydrofolate reductase (DHFR) or neomycin resistance for eukaryotic cell culture, and E. coli. For culture in E. coli and other bacteria, tetracycline resistance or ampicillin resistance may be mentioned.

A vector containing a suitable DNA sequence as described herein above and a suitable promoter or control sequence can be used to transform a suitable host and express the protein in that host. Representative examples of suitable hosts include, but are not limited to, bacterial cells (eg, E. coli, Salmonella typhimurium and Streptomyces); fungal cells (eg, yeast); insect cells (eg, , Drosophila S2 and Spodoptera Sf9); animal cells (eg, CHO, COS, and B)
owes melanoma); and plant cells. The selection of an appropriate host is considered to be within the skill of the art in light of the teachings herein.

More particularly, the present invention also includes a recombinant construct comprising one or more sequences broadly described above. These constructs include a vector (eg, a plasmid vector or a viral vector) into which the sequence of the present invention has been inserted in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises a regulatory sequence operably linked to the sequence (eg, including a promoter). Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided as examples. Bacterial: pQE70, pQE60, pQE-9 (available from Qiagen); pB
S vector, Phasescript vector, Bluescript vector, pNH8A, pNH16a, pNH18A, pNH46A (Stratage
and ptrc99a, pKK223-3, pKK233
-3, pDR540, pRIT5 (available from Pharmacia). Among preferred eukaryotic vectors are: pWLNEO, pSV2CAT,
pOG44, pXT1, pSG (available from Stratagene); and pSVK3, pBPV, pMSG, pSVL (available from Pharmacia). Other suitable vectors will be readily apparent to one of skill in the art.

In addition to the use of expression vectors in the practice of the present invention, the present invention further includes novel expression vectors that include an operator and a promoter element operably linked to a nucleotide sequence encoding a protein of interest. One example of such a vector is pHE4a, described in detail below.

As summarized in FIGS. 28 and 29, the pHE4a vector (SEQ ID NO: 16)
Examples of the components of (1) include the following: 1) a neomycin phosphotransferase gene as a selection marker; E. coli origin of replication, 3) T5 phage promoter sequence, 4) two lac operator sequences, 5) Shine-Dalgarno sequence, 6) lactose operon repressor gene (lacIq), and 7) multicloning site linker region. The origin of replication (oriC) is p
It is derived from UC19 (LTI, Gaithersburg, MD). Promoter and operator sequences were made synthetically. Synthetic generation of nucleic acid sequences is well known in the art. CLONTECH 95/96 Catalog, 215
216, CLONTECH. 1020 East Meadow Circ
le, Palo Alto, CA 94303. The pHE4a vector contains 199
Deposited with the ATCC on February 25, 2008 and given accession number 209645.

The nucleotide sequence encoding VEGF-2 (SEQ ID NO: 1) restricted the vector to pHE4a with NdeI and either XbaI, BamHI, XhoI, or Asp718, and the larger fragment (multi- The cloning site region is about 310 nucleotides).
Operably linked to a promoter and operator. The nucleotide sequence encoding VEGF-2 with the appropriate restriction sites (SEQ ID NO: 1) can be obtained, for example, according to the PCR protocol described in Example 1 using the restriction sites for NdeI (
5 ′ primer), and XbaI, BamHI, XhoI, or As
PCR with restriction site (as 3 'primer) for any of p718
Created using primers. This PCR insert is gel purified and restricted with compatible enzymes. The insert and the vector are ligated according to standard protocols.

As mentioned above, the pHE4a vector contains the lacIq gene. LacIq
Is an allele of the lacI gene that confers tight regulation of the lac operator. Amann, E .; Et al., Gene 69: 301-315 (1988); S
star, M .; Gene 51: 255-267 (1987). The lacIq gene encodes a repressor protein that binds to the lac operator sequence and blocks transcription of downstream (ie, 3 ′) sequences. However, the lacIq gene product dissociates from the lac operator in the presence of either lactose or certain lactose analogs (eg, isopropyl BD-thiogalactopyranoside (IPTG). It is not produced in measurable amounts in uninduced host cells containing the vector, but does not
Induction of these host cells by the addition of an agent such as TG results in expression of the VEGF-2 coding sequence.

The promoter / operator sequence of the pHE4a vector (SEQ ID NO: 17)
It contains a T5 phage promoter and two lac operator sequences. One operator is located 5 'to the transcription start site and the other is located 3' to the same site. These operators, when present in combination with the lacIq gene product, confer robust repression of downstream sequences in the absence of a lac operon inducer (eg, IPTG). Expression of an operably linked sequence located downstream from the lac operator can be induced by the addition of a lac operon inducer, such as IPTG. Binding of the lac inducer to the lacIq protein results in their release from the lac operator sequence and initiation of transcription of the operably linked sequence. The lac operon regulation of gene expression is described in Devlin, T.
. , Textbook of Biochemistry with Clin
ical Correlations, 4th edition (1997), pp. 802-807.

The pHE4 series of vectors contains all p
Contains the components of the HE4a vector. Features of the pHE4a vector include an optimized synthetic T5 phage promoter, a lac operator, and a Shine-Dalgarno sequence. In addition, these sequences are also optimally spaced so that expression of the inserted gene can be tightly regulated and high levels of expression upon induction.

Known bacterial promoters suitable for use for the production of the proteins of the invention include the following: E. coli lacI promoter and lac
Z promoter, T3 promoter and T7 promoter, gpt promoter, λ PR promoter and PL promoter, and trp promoter. Suitable eukaryotic promoters include: the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoter of the retroviral LTR (eg, the promoter of the Rous sarcoma virus (RSV), and Metallothionein promoter (eg, mouse metallothionein I promoter).

The pHE4a vector also contains a Shine-Dalgarno sequence 5 ′ to the AUG start codon. A Shine-Dalgarno sequence is generally a short sequence located about 10 nucleotides upstream (ie, 5 ′) from the AUG start codon. These sequences essentially direct the prokaryotic ribosome to the AUG start codon.

Accordingly, the present invention also relates to expression vectors useful for producing the proteins of the present invention. This aspect of the invention is illustrated by the pHE4a vector (SEQ ID NO: 16).

The promoter region can be selected from any desired gene using a CAT (chloramphenicol transferase) vector, or other vector with a selectable marker. Two suitable vectors are pKK232-8 and pC232
M7. Particularly famous bacterial promoters include the lacI promoter, la
cZ promoter, T3 promoter, T7 promoter, gpt promoter, lambda P _R promoter, P _L promoter and trp promoter. Eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, LTR promoter from retrovirus, and mouse metallothionein I promoter. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell (eg, a mammalian cell) or a lower eukaryotic cell (eg, a yeast cell), or the host cell can be a prokaryotic cell (eg, a yeast cell).
For example, bacterial cells). Introduction of the construct into host cells can be achieved by calcium phosphate transfection, DEAE-dextran mediated transfection, electroporation, transduction, infection, or other methods (Davis, L. et al., Basic Methods in Molecular).
ar Biology (1986)).

The construct in a host cell can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the present invention can be produced synthetically by a conventional peptide synthesizer.

[0179] The mature protein can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be used to produce such proteins using RNA from the DNA constructs of the present invention. Suitable cloning and expression vectors for use in prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cl.
oning: A Laboratory Manual, 2nd edition, Cold S
spring Harbor Ladatory, Cold Spring
Harbor, N .; Y. (1989), the disclosure of which is incorporated herein by reference.

[0180] Transcription of a DNA encoding a polypeptide of the invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancer is D
The cis-acting element of NA, usually about 10 to about 300 bp, which acts on the promoter to increase its transcription. As an example, the replication origin (bp
100-270), the SV40 enhancer on the late side, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

In general, recombinant expression vectors contain an origin of replication and a selectable marker that allows for the transformation of host cells, such as the ampicillin resistance gene of E. coli and the S. coli.
cerevisiae TRP1 gene) and promoters from highly expressed genes that direct transcription of downstream structural sequences. Such promoters include, inter alia, glycolytic enzymes (eg, 3-phosphoglycerate kinase (PGK)), α-factor,
It can be derived from an operon encoding acid phosphatase or a heat shock protein or the like. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably a leader sequence capable of directing secretion of the translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein containing an N-terminal identifying peptide that confers the desired characteristics (eg, stabilization or simplified purification of the expressed recombinant product).

[0182] Useful expression vectors for bacterial use include the structure D encoding the desired protein.
The NA sequence is constructed by inserting in a read phase operable with a functional promoter, along with the appropriate translation initiation and termination signals. The vector contains one or more phenotypic selectable markers, and an origin of replication, to ensure maintenance of the vector and, if desired, provide for amplification in the host. Prokaryotic hosts suitable for transformation include E. coli. E. coli, Bacillus subtil
is, Salmonella typhimurium, and Pseudom
genus onas, Streptomyces, and Staphylococcus
Although encompassing various species within the genus s, other species may also be used for selection.

As a representative but non-limiting example, expression vectors useful for bacterial use include selectable markers derived from commercially available plasmids containing the genetic elements of the well-known cloning vector pBR322 (ATCC 37017) and bacterial Origin of replication. Such a commercially available vector is, for example, pKK223-3 (
Pharmacia Fine Chemicals, Uppsala, Swe
den) and GEM1 (Promega Biotec, Madison, W.)
I, USA). These pBR322 "backbone" portions are combined with an appropriate promoter and the structural sequence to be expressed.

Following transformation of the appropriate host strain and growth of the host strain to an appropriate cell density, the selected promoter is derepressed by appropriate means (eg, temperature shift or chemical induction) and Is cultured for an additional period.

The cells are typically recovered by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification.

The microbial cells used in protein expression can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents, and such methods include: It is well known to those skilled in the art.

[0187] Various mammalian cell culture systems can also be used to express the recombinant protein. Examples of mammalian expression systems are described in Gluzman, Cell 23: 175 (
1981) and other cell lines capable of expressing compatible vectors (e.g., C127, 3T3, CHO, HeLa).
, And BHK cell lines). The mammalian expression vector contains an origin of replication, an appropriate promoter and enhancer, and any necessary ribosome binding, polyadenylation, splice donor and splice acceptor sites, transcription termination sequences, and 5 'flanking non-transcribed sequences. Including. DNA sequences derived from the SV40 viral genome, such as the SV40 origin, early promoter, enhancer, splice and polyadenylation sites, can be used to provide the necessary non-transcribed genetic elements.

In addition to including host cells containing the vector constructs discussed herein, the present invention also provides for deleting or replacing endogenous genetic material (eg, VEGF-2 sequences). And / or comprises genetic material (eg, a heterologous promoter) operably linked to a VEGF-2 sequence of the invention and that activates, alters, and / or amplifies an endogenous VEGF-2 polynucleotide. It includes engineered primary, secondary, and immortalized host cells of vertebrate origin, especially mammalian origin. For example, heterologous regulatory regions and endogenous polynucleotide sequences (eg, encoding VEGF-2) can be operably linked via homologous recombination using techniques known in the art (eg, 1997). June 24
U.S. Patent No. 5,641,670, issued on Sep. 26, 1996; International Publication No. WO 96/29411; published on Aug. 4, 1994; 12650; Koller et al., Proc. Natl
. Acad. Sci. USA 86: 8932-8935 (1989); and Zijlstra et al., Nature 342: 435-438 (1989), each of which disclosures are incorporated by reference in their entirety).

The host cell can be a higher eukaryotic cell, such as a mammalian cell (eg, a human-derived cell), or a lower eukaryotic cell (eg, a yeast cell), or the host cell can be a prokaryote. It can be a cell (eg, a bacterial cell). A host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired. Expression from a particular promoter can be increased in the presence of a particular inducer; thus, expression of a generally engineered polypeptide can be controlled. In addition, different host cells have characteristics and specific mechanisms during the translational and post-translational processing and modification (eg, glycosylation, phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modification and processing of the expressed protein.

[0190] Polypeptides can be recovered and purified from recombinant cell culture by previously used methods. These methods include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. I do. Preferably, low concentrations (about 0.1-5 mM) of calcium ions are present during purification (Price et al., J. Biol.
Chem. 244: 917 (1969)). If necessary, a protein refolding step can be used to bring the mature protein into full conformation. Finally, high performance liquid chromatography (HPLC) can be used for the final purification step.

The polypeptides of the present invention can be purified from nature or the products of chemical synthesis procedures, or can be prokaryotic or eukaryotic hosts (eg, bacterial cells, yeast cells, higher plants in culture). Cells, insect cells, and mammalian cells)
From recombinant techniques. Depending on the host used in the recombinant production procedure, the polypeptides of the present invention may or may not be glycosylated with mammalian or other eukaryotic carbohydrates. The polypeptides of the present invention may also include an initiating methionine amino acid residue.

In addition, polypeptides of the invention can be chemically synthesized using techniques known in the art (eg, Creighton, 1983, Proteins:
Structures and Molecular Principles,
W. H. Freeman & Co. , N .; Y. , And Hunkapille
r, M. Et al., 1984, Nature 310: 105-111).
. For example, a peptide corresponding to a fragment of a VEGF-2 polypeptide of the present invention can be synthesized by use of a peptide synthesizer. Further, if desired, non-classical amino acids or chemical amino acid analogs can be introduced as a substitution or addition to the VEGF-2 polynucleotide sequence. Non-classical amino acids include, but are not limited to, the D isomer of a common amino acid, 2,
4-diaminobutyric acid, a-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine , Norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid,
t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoroamino acid, designed
Amino acids (eg, b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids), and generally amino acid analogs. Further, the amino acid can be D (dextrorotary) or L (levorotary).

The present invention provides for differentially modifying (eg, glycosylation,
Acetylated, phosphorylated, amidated, derivatized with known protecting / blocking groups, proteolytic cleavage, binding to antibody molecules or other cellular ligands, etc.). Any of a number of chemical modifications include, but below may be performed by known techniques including but not limited to, cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, specific chemical cleavage by NaBH _4; acetylation , Formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin;

Additional post-translational modifications encompassed by the present invention include, for example, N-linked or O-linked carbohydrate chains, N-terminal or C-terminal processing, attachment of a chemical moiety to the amino acid backbone, N-linked Chemical modification of carbohydrate or O-linked carbohydrate chains, and addition or deletion of N-terminal methionine residues as a result of prokaryotic host cell expression. Polypeptides can also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label, to allow detection and isolation of the protein.

[0195] The present invention also provides chemically modified derivatives of VEGF-2. This may provide additional benefits such as increased solubility, stability, and circulation time of the polypeptide, or reduced immunogenicity (US Pat. No. 4,179,33).
No. 7). The chemical moiety for derivatization may be selected from water-soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol and the like. The polypeptide may be modified at random positions in the molecule, or may be modified at predetermined positions in the molecule, and may have one, two, three or more attached May contain chemical moieties.

The polymer can be of any molecular weight and can be branched or unbranched. For polyethylene glycol, for ease of operation and manufacture, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about" refers to the definition of some molecules in a preparation of polyethylene glycol). Larger than the molecular weight, and some are smaller). Other sizes include the desired therapeutic profile (eg, desired sustained release duration, effect (if any) on biological activity), ease of manipulation, degree or deficiency of antigenicity, And other known effects of polyethylene glycol on therapeutic proteins or analogs).

A polyethylene glycol molecule (or other chemical moiety) should be attached to a protein in view of its effect on the functional or antigenic domain of the protein. There are many attachment methods available to those skilled in the art (eg, EP 0 401 384 (PEG G-CSF), which is incorporated herein by reference).
To Malik et al., Exp. Hematol. 20: 1028-
1035 (1992) (tresyl chloride)
PEG-PEGylation of GM-CSF using). For example, polyethylene glycol can be covalently linked through an amino acid residue via a reactive group (eg, a free amino or carboxyl group). A reactive group is a group to which an activated polyethylene glycol molecule can be bound. Amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residue: amino acid residues having a free carboxyl group include aspartic acid residues, glutamic acid residues, and C-terminal Amino acid residues may be mentioned. Sulfhydryl groups can also be used as reactive groups for attaching polyethylene glycol molecules. Preferred for therapeutic purposes is N
Attachment at an amino group, such as attachment at a terminal or lysine group.

Proteins that are chemically modified at the N-terminus may be particularly desirable. Using polyethylene glycol as an illustration of the composition of the present invention, the ratio of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mixture from various polyethylene glycol molecules (depending on molecular weight, branching, etc.), the PEG to be performed The method of obtaining the protein with the selected N-terminus PEGylated can be selected for the type of the reaction. Obtain a preparation pegylated at the N-terminus (ie, if necessary, separate this part from other monopegylated parts)
The method is obtained by purifying N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins that are chemically modified in the N-terminal modification include different types of major amino groups (available for derivatization of a particular protein).
It can be achieved by reductive alkylation, exploiting the differential reactivity of lysine (N-terminal). Under appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a polymer containing carbonyl groups is achieved.

The VEGF-2 polypeptides of the present invention can be in monomers or multimers (ie, dimers, trimers, tetramers, and higher multimers). Accordingly, the present invention relates to monomers and multimers of VEGF-2 polypeptides of the present invention, their preparation, and compositions (preferably pharmaceutical compositions) containing them. In certain embodiments, a polypeptide of the invention is a monomer, dimer, trimer, or tetramer. In a further embodiment, the multimer of the invention is at least a dimer, at least a trimer, or at least a tetramer.

The multimers encompassed by the present invention may be homomeric or heteromeric. As used herein, the term homomer refers to a multimer comprising only a VEGF-2 polypeptide of the present invention (as described herein, a VEGF-2 fragment, variant, Splice variants, and fusion proteins).
These homomers have VEGF-2 with the same or different amino acid sequence.
It can include a polypeptide. In certain embodiments, a homomer of the invention is a multimer comprising only a VEGF-2 polypeptide having the same amino acid sequence. In another particular embodiment, a homomer of the invention is a multimer comprising a VEGF-2 polypeptide having a different amino acid sequence. In certain embodiments, the multimers of the invention are homodimers (eg, including VEGF-2 polypeptides having the same or different amino acid sequences), or homotrimers (eg, the same and And / or VEGF-2 polypeptides having different amino acid sequences). In a further embodiment, the multimer of a homomer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer comprising one or more heterologous polypeptides (ie, polypeptides of different proteins) in addition to a VEGF-2 polypeptide of the present invention. Say. In certain embodiments, a multimer of the invention is a heterodimer, heterotrimer, or heterotetramer. In a further embodiment, the multimer of a homomer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.

[0202] Multimers of the invention can be the result of hydrophobic, hydrophilic, ionic, and / or covalent associations and / or can be indirectly linked, for example, by liposome formation. Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when the polypeptides of the invention contact one another in solution. In another embodiment, a heteromultimer of the invention, eg, a heterotrimer or heterotetramer, is a polypeptide of the invention wherein the polypeptide of the invention is an antibody against the polypeptide of the invention (solution of the fusion protein of the invention). (Including antibodies to the heterologous polypeptide sequence). In other embodiments, the multimers of the invention are formed by covalent association with VEGF-2 polypeptides of the invention and / or covalent associations between VEGF-2 polypeptides. Such covalent associations include (eg, those contained in the polypeptides set forth in SEQ ID NO: 2 or encoded by the deposited clone).
It may include one or more amino acid residues contained in a polypeptide sequence. In one example, the covalent association is a bridge between cysteine residues located within the interacting polypeptide sequence in the native (ie, naturally occurring) polypeptide. In another example, the covalent association is a result of chemical or recombinant manipulation. Alternatively, such a covalent association may involve one or more amino acid residues contained in a heterologous polypeptide sequence in a VEGF-2 fusion protein. In one example, the covalent association is between heterologous sequences contained in a fusion protein of the invention (see, eg, US Pat. No. 5,478,925). In certain instances, the covalent association is:
Between the heterologous sequences contained in the VEGF-2-Fc fusion protein of the invention (as described herein). In another specific example, the covalent association of the fusion proteins of the invention is such that another TNF family ligand / receptor member capable of forming covalently associated multimers, such as oseteoprotegerin (o
setoprotegerin) (for example, International Publication No. WO98 / 4930)
See FIG. This content lies between the heterologous polypeptide sequences from ())), which is hereby incorporated by reference in its entirety.

The multimers of the present invention can be produced using chemical techniques known in the art. For example, a polypeptide that is desired to be included in a multimer of the invention can be chemically cross-linked using linker molecule and linker molecule length optimization techniques known in the art (eg, US Pat. No. 5,478,925 (this is, in its entirety,
See herein incorporated by reference). Further, the multimers of the present invention may be used in the art to form one or more intermolecular bridges between cysteine residues located within the sequence of the polypeptide desired to be included in the multimer. It can be produced using known techniques (see, for example, US Pat. No. 5,478,925, which is incorporated herein by reference in its entirety). Further, the polypeptide of the present invention may have a C-terminal or N-terminal relative to the polypeptide.
It can be modified conventionally by adding cysteine or biotin, and techniques known in the art can be applied to produce multimers comprising one or more of these modified polypeptides (eg, US See US Patent No. 5,478,925, which is hereby incorporated by reference in its entirety. further,
Techniques known in the art can be applied to generate liposomes containing the polypeptide component desired to be included in the multimers of the invention (see, for example, US Pat. No. 5,47,47).
No. 8,925, which is incorporated herein by reference in its entirety).

Alternatively, the multimers of the present invention can be produced using genetic engineering techniques known in the art. In one embodiment, the polypeptide comprised in the multimer of the invention is recombinantly produced using fusion protein techniques described herein or otherwise known in the art. (Eg, US Pat. No. 5,478,
No. 925, which is hereby incorporated by reference in its entirety). In certain embodiments, the polynucleotide encoding the homodimer of the invention comprises a polynucleotide sequence encoding a polypeptide of the invention replaced by a sequence encoding a linker polypeptide, and then further from the native C-terminus. Produced by ligation to a synthetic polynucleotide encoding the translation product of the polypeptide in the opposite direction of the N-terminus (without the leader sequence) (see, eg, US Pat. No. 5,478,925, which is incorporated in its entirety). Is incorporated herein by reference)). In another embodiment, a recombinant technique described herein or otherwise known in the art is applied to comprise a transmembrane domain (or hydrophobic or signal peptide) and Produce recombinant polypeptides of the invention that can be incorporated into liposomes by construction techniques (eg, US Pat. No. 5,478,925, which is hereby incorporated by reference in its entirety). checking).

Therapeutic Use VEGF-2 polypeptides of the present invention are potent mitogens for vascular and lymphatic endothelial cells. As shown in FIGS. 12 and 13, the VEGF-2 polypeptide of SEQ ID NO: 2 (with the first 46 amino acids subtracted)
Are powerful mitogens for vascular endothelial cells and stimulate their growth and proliferation. The results of Northern blot analysis performed on the VEGF-2 nucleic acid sequence encoding this polypeptide (where 20 mg of RNA from several human tissues was probed with ³² P-VEGF-2) indicate that the protein was It illustrates that it is actively expressed in heart and lung. This is further evidence of mitogenic activity.

Thus, VEGF-2 or a biologically active portion thereof can be used to treat vascular trauma by promoting angiogenesis. To stimulate the growth of transplanted tissue, for example, when performing coronary artery bypass surgery. VE
GF-2 or a biologically active portion thereof also promotes wound healing and revascularizes damaged tissue, especially during ischemia and when new capillary angiogenesis is desired. Or to stimulate collateral blood flow. V
EGF-2 or a biologically active portion thereof can be used to treat full-thickness wounds such as skin ulcers (
(Including ulcers, venous ulcers, and diabetic ulcers). Further, VEGF-2 or a biologically active portion thereof may be used in skin grafts or flaps to repair such burns and damages. Can be used to treat injury. VEGF-2 or a biologically active portion thereof can also be used for use in plastic surgery (eg, for repairing a tear, burn or other trauma). In addition, VEGF-2 can be used to promote healing of wounds and injuries to the eye and to treat eye diseases.

In these same manners, VEGF-2 or a biologically active portion thereof can also be used to induce the growth of damaged bone, periodontal tissue, or ligament tissue. VEGF-2 or a biologically active portion thereof can also be used to regenerate tooth support tissue, including, for example, cementum and periodontal ligament damaged by periodontal disease or trauma.

Since angiogenesis is important in keeping the wound clean and uninfected, VEGF-2 or a biologically active portion thereof may be involved in surgery and incision and amputation. Can be used after repair. VEGF-2 or a biologically active portion thereof can also be used for the treatment of abdominal injuries at high risk of infection.

[0209] VEGF-2 or a biologically active portion thereof can be used to promote endothelialization in vascular transplant surgery. For vascular grafts using either graft or synthetic materials, VEGF
-2 or a biologically active portion thereof can be applied to the surface of the graft or its junction to promote vascular endothelial cell growth. VEGF-2 or a biologically active portion thereof can also be used to repair myocardial tissue damage resulting from myocardial infarction. VEGF-2 or a biologically active portion thereof can also be used to repair the cardiovascular system after ischemia. VEGF-2 or a biologically active portion thereof can also be used to treat coronary artery disease and damaged vascular tissue that is a consequence of peripheral and CNS vascular disease.

VEGF-2, or a biologically active portion thereof, may also be used to reduce the rejection of the graft material and to stimulate angiogenesis of the graft material, either by means of an artificial prosthesis or a natural prosthesis within the body. Can be used to coat organs.

VEGF-2 or a biologically active portion thereof may also be used to repair vascular tissue injuries resulting from trauma (eg, occurring during atherosclerosis and balloon angioplasty where vascular tissue is damaged) Post-operative repair).

[0212] VEGF-2 or a biologically active portion thereof can also be used for the treatment of peripheral artery disease. Thus, in a further aspect, there is provided a process of utilizing a VEGF-2 polypeptide to treat peripheral artery disease. Preferably, VEGF-2 polypeptide is administered to an individual for the purpose of reducing or treating peripheral arterial disease. Suitable doses, formulations, and routes of administration are described below.

VEGF-2 or a biologically active portion thereof also promotes endothelial function of lymphoid tissues and lymphatic vessels, such as to treat lymphatic loss, lymphatic obstruction, and lymphangioma Can be used to VEGF-2 can also be used to stimulate lymphocyte production.

[0214] VEGF-2 or a biologically active portion thereof can also be used to treat hemangiomas in neonates. Thus, in a further aspect, there is provided a process of utilizing a VEGF-2 polypeptide to treat hemangiomas in a neonate. Preferably, VEGF-2 polypeptide is administered to an individual for the purpose of reducing or treating hemangiomas in a newborn. Suitable doses, formulations, and routes of administration are described below.

[0215] VEGF-2 or a biologically active portion thereof can also be used to prevent or treat abnormal retinal development in premature neonates. Thus, in a further aspect, there is provided a process of utilizing a VEGF-2 polypeptide to treat abnormal retinal development in a premature newborn. Preferably, VEGF-2 polypeptide is administered to an individual for the purpose of reducing or treating abnormal retinal development in a premature newborn. Suitable doses, formulations, and routes of administration are described below.

[0216] VEGF-2 or a biologically active portion thereof can be used to treat primary (idiopathic) lymphedema, including Milroy's disease and lymphedema precox. Thus, in a further aspect, there is provided a process of utilizing a VEGF-2 polypeptide to treat primary (idiopathic) lymphedema, including Milroy's disease and lymphedema precox. Preferably, the VEGF-2 polypeptide is administered to an individual for the purpose of reducing or treating primary (idiopathic) lymphedema, including Milroy's disease and lymphedema precox. VEGF-2 or a biologically active portion thereof can also be used to treat edema as well as to affect blood pressure in animals. Suitable doses, formulations, and routes of administration are described below.

VEGF-2 or a biologically active portion thereof may also be derived from (I) removal of lymph nodes and lymph vessels, (ii) radiation therapy and surgery in the treatment of cancer, and (iii) trauma and infection. Secondary (occlusive) lifespan, including those that occur (se
condary lifetime). Therefore,
In further aspects, treating (I) removal of lymph nodes and lymph vessels, (ii) radiation therapy and surgery in the treatment of cancer, and (iii) secondary (obstructive) lifespan, including those resulting from trauma and infection. In order to do so, a process utilizing a VEGF-2 polypeptide is provided. Preferably, the VEGF-2 polypeptide is secondary (including those resulting from (I) removal of lymph nodes and lymph vessels, (ii) radiation therapy and surgery in the treatment of cancer, and (iii) trauma and infection. (Occlusive) administered to an individual for the purpose of life. Suitable doses, formulations, and routes of administration are described below.

[0219] VEGF-2 or a biologically active portion thereof can also be used to treat Kaposi's sarcoma. Thus, in a further aspect, there is provided a process utilizing a VEGF-2 polypeptide to treat Kaposi's sarcoma. Preferably, VEGF-2 polypeptide is administered to an individual for the purpose of reducing or treating Kaposi's sarcoma. Suitable doses, formulations, and routes of administration are described below.

[0219] VEGF-2 antagonists can be used to treat cancer by inhibiting angiogenesis, which is required to support cancer and tumor growth.

Cardiovascular Disorders We have shown that VEGF-2 stimulates vascular endothelial cell growth, stimulates endothelial cell migration, stimulates angiogenesis in CAM assays, spontaneous Reduced blood pressure in ischemic hypertensive rats and increased blood flow to ischemic limbs in rabbits. Accordingly, VEGF-2 polypeptides or polynucleotides encoding VEGF-2 can be used to treat cardiovascular disorders including peripheral arterial disease (eg, limb ischemia).

[0221] Cardiovascular disorders include arterio-arterial fistulas, arteriovenous fistulas, cerebral arteriovenous malformations, congenital heart defects, lung obstruction, and cardiovascular abnormalities such as Scimitar syndrome. Congenital heart defects include aortic constriction, triatrial heart, coronary artery anomaly, crossed heart, right thoracic heart, arterial tract, Ebstein malformation, Eisenmenger complex, left ventricular dysgenesis syndrome, left thoracic heart, tetralogy of Fallot Disease, transposition of the great arteries, right ventricular bilateral great vessels, tricuspid regurgitation, remnant ductus arteriosus, and cardiac septum defect (
For example, aorto-pulmonary septal defect, endocardial bed defect, Lutanbache syndrome, trilogy of Fallot, ventricular septal defect).

[0222] Cardiovascular disorders also include arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), cardiac aneurysms, cardiac arrest, congestion Congenital heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, cardiac hypertrophy,
Left ventricular hypertrophy, right ventricular hypertrophy, post-fracture heart laceration, ventricular septum rupture, heart valve disease, myocardial disease, myocardial ischemia, endocardial effusion, pericarditis (including contractile and tuberculous), airway Includes heart diseases such as membrane disease, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, scimitar syndrome, cardiovascular syphilis, and cardiovascular tuberculosis .

Arrhythmias include sinus arrhythmias, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes syndrome, leg block, sinoatrial block, long QT syndrome, accessory contraction,
Examples include Lone-Gannong-Levine syndrome, Mahem-type abnormal premature excitement syndrome, Wolf-Parkinson-White syndrome, sinus dysfunction syndrome, tachycardia, and ventricular fibrillation. Tachycardia includes paroxysmal tachycardia, supraventricular tachycardia, accelerated ventricular specific rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial Nodal reentry tachycardia, sinus tachycardia, torsades de pointes, and ventricular tachycardia.

Heart valve diseases include aortic valve insufficiency, aortic stenosis, cardiac murmur, aortic valve prolapse, mitral valve prolapse, tricuspid prolapse, mitral valve insufficiency, mitral stenosis, pulmonary atresia Pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid regurgitation, tricuspid insufficiency, and tricuspid stenosis.

Examples of cardiomyopathy include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, hypoaortic stenosis, pulmonary subvalvular stenosis, restricted cardiomyopathy, Chagas cardiomyopathy, endocardium These include fibroelastosis, endocardial myocardial fibrosis, Keynes syndrome, myocardial reperfusion injury, and myocarditis.

Myocardial ischemia includes angina, coronary artery aneurysm, coronary atherosclerosis, coronary thrombosis,
Coronary vasospasm, myocardial infarction and myocardial stun
ning).

Cardiovascular diseases also include aneurysms, angiodysplasia, hemangiomatosis, bacterial hemangioma symptoms, Hippel-Lindau disease, Kripel-Tornone-Weber syndrome, Sturgi-Weber syndrome, vasomotor edema, Aortic disease, Takayasu arteritis, aorticitis, Leish syndrome, arterial occlusive disease, arteritis, arteritis
itis), polyarteritis nodosa, cerebrovascular disorder, diabetic vascular disorder, diabetic retinopathy, embolism, thrombosis, erythromelalgia, hemorrhoids, hepatic venous occlusion (veno-occl)
use) disease, hypertension, hypotension, ischemia, peripheral vascular disease, phlebitis, pulmonary vein obstruction disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, scimitar syndrome,
Superior vena cava syndrome, telangiectasia, telangiectasia ataxia (atasia tel)
angiectasia), hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcers, vasculitis, and vascular disease such as venous insufficiency.

Aneurysms include dissecting aneurysms, pseudoaneurysms, infectious aneurysms, ruptured aortic aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, cardiac aneurysms, and iliac aneurysms Is mentioned.

Arterial occlusive diseases include arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasia, mesenteric vascular obstruction, Moyamoya disease, renal artery obstruction, retinal artery obstruction, and thrombotic vasculitis Is mentioned.

Cerebrovascular disorders include carotid artery disease, amyloid angiopathy of the brain, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral vein malformation, cerebral artery disease, cerebral embolism and cerebral thrombosis, Arterial thrombosis, sinus thrombosis, Wallenberg syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subarachnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient)
, Subclavian steal syndrome, periventricular leukomalacia
leukomalacia), vascular headache, cluster headache, migraine, and spinal basilar insufficiency.

Embolisms include air embolism, amniotic fluid embolism, cholesterol embolism, toe cyanosis syndrome, fat embolism, pulmonary embolism, and thromboembolism
oembolism). Thrombosis includes coronary artery thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg syndrome, and thrombophlebitis.

Examples of ischemia include cerebral ischemia, ischemic colitis, partition syndrome, anterior partition syndrome (
anterior compartment syndrome), myocardial ischemia,
Reperfusion injury, and peripheral limb ischemia. As vasculitis, aoritis, arteritis, Behcet's syndrome, Churg-Strauss syndrome, mucocutaneous lymph node syndrome, obstructive thrombotic vasculitis, irritable vasculitis, Schönlein-Henoch purpura, allergic cutaneous vasculitis, And Wegener's granulomatosis.

[0233] VEGF-2 polypeptides or polynucleotides are particularly effective for the treatment of severe limb ischemia, and coronary artery disease. As shown in Example 18,
Administration of VEGF-2 polynucleotides and polypeptides to hind limbs of experimentally induced ischemic rabbits restored blood pressure ratios, blood flow, angiographic scores, and capillary density.

The VEGF-2 polypeptide can be prepared by any method known in the art (direct needle injection at the site of delivery, intravenous injection, topical administration, catheter infusion, biolistic injector, particle accelerator, gel foam sponge accumulation (Gelfoam sponge depo
t), other commercial depots, osmotic pumps, oral or suppository (suppos)
itorial) solid pharmaceutical formulation, decanted during surgery (dec)
ant) application or topical application, including, but not limited to, aerosol delivery). Such methods are known in the art. VEGF-2 polypeptides, as described in detail below,
It can be administered as part of a pharmaceutical composition. Methods of delivering VEGF-2 polynucleotides are described in more detail below.

(Gene Therapy Method) Another aspect of the present invention relates to a gene therapy method for treating disorders, diseases and conditions. Gene therapy methods involve introducing nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an animal to achieve expression of a VEGF-2 polypeptide of the invention. The method requires a VEGF-2 polypeptide-encoding polynucleotide operably linked to a promoter and any other genetic elements required for expression of the polypeptide by the target tissue. . Such gene therapy and delivery techniques are known in the art. See, for example, WO 90/11092, which is incorporated herein by reference.

Thus, for example, cells from a patient can be engineered with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a VEGF-2 polynucleotide ex vivo, and then the engineered cells Is provided to the patient to be treated with the polypeptide. Such methods are well-known in the art. See, for example, Belldegrun, A .; J. et al. Natl. Cancer
Inst. 85: 207-216 (1993); Ferrantini, M .;
Et al., Cancer Research 53: 1107-1112 (1993).
Ferrantini, M .; J. et al. Immunology 153: 460
4-4615 (1994); Kaido, T .; Et al., Int. J. Cancer
60: 221-229 (1995); Ogura, H .; Et al., Cancer Re
search 50: 5102-5106 (1990); Santodonat.
o, L. Et al., Human Gene Therapy 7: 1-10 (1996).
); Santodonato, L .; Et al., Gene Therapy 4: 124.
6-1255 (1997); and Zhang, J. et al. -F. Et al., Cancer
See Gene Therapy 3: 31-38 (1996), which are incorporated herein by reference. In one embodiment, the engineered cells are arterial cells. The cells of the artery can be reintroduced into the patient by direct injection into the artery, the tissue around the artery, or by catheter infusion.

As discussed in more detail below, VEGF-2 polynucleotide constructs can be used to deliver any injectable substance to the cells of an animal, including tissues (heart, muscle, skin, lung, liver, etc.). Etc.) into the interstitial space). VE
The GF-2 polynucleotide construct can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

In one embodiment, a VEGF-2 polynucleotide is delivered as a naked polynucleotide. The term "naked" polynucleotide, DNA or RNA
Is any delivery vehicle that acts to assist, promote, or enhance entry into a cell, such as a viral sequence formulation, a virus particle formulation, a liposome formulation, a lipofection or precipitating agent, etc.
Refers to a sequence that does not contain However, VEGF-2 polynucleotides can also be delivered in liposome formulations, and lipofection formulations and the like can be prepared by methods well known to those skilled in the art. Such a method is described, for example, in US Pat.
Nos. 93,972, 5,589,466, and 5,580,859, which are incorporated herein by reference.

[0239] The VEGF-2 polynucleotide vector construct used in the gene therapy method is preferably a construct that does not integrate into the host genome and does not contain sequences enabling replication. Suitable vectors include pWLNEO, pSV2C
AT, pOG44, pXT1 and pSG (available from Stratagene); pSVK3, pBPV, pMSG and pSVL (available from Pharmacia); and pEF / V5, pcDNA3.1 and pRc / CMV2
(Available from Invitrogen). Other suitable vectors are
It will be readily apparent to one skilled in the art.

[0240] Any strong promoter known to those of skill in the art can be used to drive expression of VEGF-2 DNA. Suitable promoters include adenovirus promoters (eg, adenovirus major late promoter); or heterologous promoters (eg, cytomegalovirus (CMV) promoter); RS virus (RSV) promoter; inducible promoters (eg, MMT promoter, Heat shock promoter; albumin promoter; ApoAI promoter; human globin promoter; viral thymidine kinase promoter (eg, herpes simplex thymidine kinase promoter); retroviral LTR; b-actin promoter; and human growth hormone promoter. The promoter can also be a native promoter for VEGF-2.

[0241] Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transient nature of polynucleotide synthesis in cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for up to six months.

[0242] VEGF-2 polynucleotide constructs can be used in tissues (muscle tissue, skin tissue, brain tissue, lung tissue, liver tissue, spleen tissue, bone marrow tissue, thymus tissue, heart tissue, lymph tissue, blood tissue, bone tissue) in animals. Tissue, cartilage tissue, pancreas tissue, kidney tissue, gallbladder tissue, stomach tissue,
(Including intestinal, testicular, ovarian, uterine, rectal, nervous, ocular, glandular, and connective tissues). The interstitial space of the tissue may be the intercellular fluid mucopolysaccharide matrix between the reticulum fibers of the organ tissue, the elastic fibers in the vessel or chamber walls, the collagen fibers of the fibrous tissue, or the connective tissue covering the muscle cells or Includes the same matrix within the bone cavity. This is likewise the space occupied by the circulating plasma and the lymph of the lymph channels. Delivery of muscle tissue to the interstitial space is preferred for reasons discussed below. They can be conveniently delivered by injection into the tissue containing these cells. They are preferably delivered to and expressed in differentiated, persistent, non-dividing cells, but the delivery and expression is dependent on undifferentiated or less fully differentiated cells (eg, blood fibroblasts or skin (Fibroblast stem cells). Muscle cells in vivo are particularly suitable for their ability to take up and express polynucleotides.

For naked nucleic acid sequence injection, the effective dosage of DNA or RNA is about 0.05
mg / kg to about 50 mg / kg body weight. Preferably, the dosage will be from about 0.005 mg / kg to about 20 mg / kg, and more preferably about 0.
05 mg / kg to about 5 mg / kg. Of course, as those skilled in the art understand,
This dosage will vary according to the tissue site of the injection. Appropriate and effective dosages of nucleic acid sequences can be readily determined by one skilled in the art, and will depend on the condition being treated and the route of administration.

The preferred route of administration is by parenteral route of injection into the interstitial space of the tissue. However, other parenteral routes, such as inhalation of an aerosol formulation for delivery to the lung or bronchial tissue, especially to the throat or nasal mucosa, may also be used. In addition, naked VEGF-2 DNA constructs can be delivered to arteries during angioplasty with the catheter used in this procedure.

Naked polynucleotides can be prepared by any method known in the art, including direct needle injection at the site of delivery, intravenous injection, topical administration, catheter infusion, and so-called “
Gene guns). These delivery methods are known in the art.

As evidenced by Example 18, the naked VEGF-2 nucleic acid sequence was
In vivo administration can result in successful expression of VEGF-2 polypeptide in the rabbit femoral artery.

The construct can also be delivered in a delivery vehicle such as a viral sequence, viral particle, liposome formulation, lipofectin, precipitant, and the like. Such delivery methods are known in the art.

In certain embodiments, a VEGF-2 polynucleotide construct is complexed in a liposome preparation. A liposome preparation for use in the present invention comprises
Includes cationic (positively charged), anionic (negatively charged), and neutral preparations. However, cationic liposomes are particularly preferred because strong charged complexes can be formed between the cationic liposomes and the polyanionic nucleic acids. Cationic liposomes have been shown to mediate the intracellular delivery of the following in a functional form: Plasmid DNA (Felgner et al., Proc. Nat.
l. Acad. Sci. USA (1987) 84: 7413-7416, which is incorporated herein by reference); mRNA (Malone et al., Proc.
Natl. Acad. Sci. USA (1989) 86: 6077-6081;
Which is incorporated herein by reference); and purified transcription factors (De
bs et al. Biol. Chem. (1990) 265: 10189-1019.
2, which is incorporated herein by reference).

[0249] Cationic liposomes are readily available. For example, N [1-2,3-
Dioleyloxy) propyl] -N, N, N-triethylammonium (DOT
MA) Liposomes are particularly useful, and GIBCO BRL, Grand
Island, N.M. Y. (Felgner et al., Proc. Natl. Acad. Sci. USA (198).
7) 84: 7413-7416, which is also incorporated herein by reference). Other commercially available liposomes are transfectase (tran)
surface) (DDAB / DOPE) and DOTAP / DOPE (Bo
ehringer).

Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. For example, for a description of the synthesis of DOTAP (1,2-bis (oleoyloxy) -3- (trimethylammonio) propane) liposomes, PCT Publication No. WO 90/11092, which is incorporated herein by reference. See). The preparation of DOTMA liposomes is described in the literature and is described, for example, in Felgner et al., Proc. Natl. Aca
d. Sci. USA 84: 7413-7417, which is incorporated herein by reference. Similar methods can be used to prepare liposomes from other cationic lipid substances.

Similarly, anionic liposomes and neutral liposomes are, for example, Avanti
It may be readily available from Polar Lipids (Birmingham, Ala.) Or may be readily prepared using readily available materials. Such substances include, inter alia, phosphatidylcholine, cholesterol,
Phosphatidylethanolamine, dioleoylphosphatidylcholine (DOP
C), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylethanolamine (DOPE). These substances are also DO
It can be mixed with TMA and DOTAP starting material in an appropriate ratio. Methods for making liposomes using these materials are well known in the art.

For example, commercially available dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations with or without the addition of cholesterol. Without it, conventional liposomes can be made. Thus, for example, DOPG / DOPC vesicles can be treated under a stream of nitrogen gas into sonicated vials.
Can be prepared by drying 50 mg each of DOPG and DOPC.
The sample is placed under a vacuum pump overnight and hydrated with deionized water the next day. The sample was then placed in a capped vial for 2 hours in an inverted cup (
Heat System with inverted cup (tub type) probe
Sonication using a ms model 350 sonicator at maximum setting while rotating the tub at 15 EC. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles, or through the nucleopore membrane to produce discrete sized unilamellar vesicles. Can be prepared by extrusion. Other methods are known and available to those skilled in the art.

The liposomes can comprise multilamellar vesicles (MLV), small unilamellar vesicles (SUV), or large unilamellar vesicles (LUV), with SUV being preferred. Various liposomes-
A nucleic acid complex is prepared using methods well known in the art. For example, St
Raubinger et al., Methods of Immunology (198
3) See 101: 512-527, which is incorporated herein by reference. For example, MLVs containing nucleic acids can be prepared by depositing a thin film of phospholipid on a glass tube wall and subsequently hydrating with a solution of the substance to be encapsulated. SUVs are prepared by prolonged sonication of MLVs to generate a homogeneous population of unilamellar liposomes. The substance to be captured is added to a pre-formed suspension of MLV and then sonicated. If liposomes containing cationic lipids are used, the dried lipid film is applied to a suitable solution (eg, sterile water or an isotonic buffer such as 10 mM Tris / NaCl).
The liposomes are resuspended in, sonicated and then preformed liposomes are mixed directly with the DNA. Liposomes and DNA form very stable complexes due to the binding of positively charged liposomes to cationic DNA. SUV finds use with small nucleic acid fragments. LUVs are prepared by a number of methods well known in the art. Commonly used methods include Ca ²⁺ -EDTA chelation (Papahadjopoulos et al., Biochim. Biophys).
. Acta (1975) 394: 483; Wilson et al., Cell (1979).
) 17:17); Ether injection (Deamer, D. and Bangham, A.).
. Biochim. Biophys. Acta (1976) 443: 629;
Ostro et al., Biochem. Biophys. Res. Commun. (1
977) 76: 836; Fraley et al., Proc. Natl. Acad. Sc
i. USA (1979) 76: 3348): Surfactant dialysis (Enoch, H .;
And Strittmatter, P .; Proc. Natl. Acad. Sc
i. USA (1979) 76: 145); and reverse phase evaporation (REV).
) (Fraley et al., J. Biol. Chem. (1980) 255: 1043).
1; Szoka, F .; And Papahadjopoulos, D.A. , Proc
. Natl. Acad. Sci. USA (1978) 75: 145; Schae.
fer-Ridder et al., Science (1982) 215: 166), which are incorporated herein by reference.

Generally, the ratio of DNA to liposomes is from about 10: 1 to about 1:10. Preferably, this ratio is from about 5: 1 to about 1: 5. More preferably, this ratio is about 3
: 1 to about 1: 3. Even more preferably, this ratio is about 1: 1.

US Pat. No. 5,676,954, which is incorporated herein by reference, reports the injection of genetic material, complexed with a cationic liposome carrier, into mice. I have. U.S. Pat. Nos. 4,897,355 and 4,946,78
No. 7, No. 5,049,386, No. 5,459,127, No. 5,589
, 466, 5,693,622, 5,580,859, 5,
No. 703,055, and International Publication No. WO 94/9469, which are incorporated herein by reference, provide cationic lipids for use in transfecting DNA into cells and mammals. I do. U.S. Pat. No. 5,589,
Nos. 466, 5,693,622, 5,580,859 and 5,7
No. 03,055, and International Publication No. WO 94/9469, which are incorporated herein by reference, provide methods for delivering DNA-cationic lipid complexes to mammals.

In certain embodiments, retroviral particles comprising RNA comprising a sequence encoding VEGF-2 are used to manipulate ex vivo or in vivo. Retroviruses from which retroviral plasmid vectors can be derived include Moloney murine leukemia virus, spleen necrosis virus, Rous sarcoma virus, Harvey sarcoma virus, chicken leukemia virus, gibbon monkey leukemia virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and Breast cancer virus, but is not limited thereto.

The retroviral vector is used to transduce the packaging cell line to form a producer cell line. Examples of packaging cells that can be transfected include PE501, PA317, R-2, R-AM, PA12, T19-1.
4X, VT-19-17-H2, RCRE, RCRIP, GP + E-86, GP
+ EnvAm12, and a DAN cell line (Miller, Human Gene Therapy, 1: 5-, which is incorporated herein by reference in its entirety).
14 (1990)). This vector can transduce the packaging cells via any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO ₄ precipitation. In another, the retrovirus plasmid vector can be encapsulated in liposomes or conjugated to a lipid and then administered to a host.

The producer cell line produces infectious retroviral vector particles that contain the polynucleotide encoding VEGF-2. Such retroviral vector particles can then be used to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells are VEGF-
Express 2.

[0259] In certain embodiments, the cell is a VE contained in an adenovirus vector.
Engineered ex vivo or in vivo with a GF-2 polynucleotide. The adenovirus can be engineered so that it encodes and expresses VEGF-2;
And at the same time it is inactivated for its ability to replicate in the normal lytic virus life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thereby reducing concerns about insertional mutagenesis. further,
Adenovirus has been used for many years as a live intestinal vaccine with an excellent safety profile (Schwartz, AR et al. (1974) Am. Rev.
Respir. Dis. 109: 233-238). Finally, adenovirus-mediated gene transfer has been demonstrated in a number of examples, including the transfer of alpha-1-antitrypsin and CFTR to the cotton rat lung (Rosenfeld, MA et al. 1991) Science 252: 43.
1-434; Rosenfeld et al. (1992) Cell 68: 143-15.
5). In addition, extensive research into attempts to establish adenovirus as a causative factor in human cancer has been uniformly negative (Green, M. et al. (19).
79) Proc. Natl. Acad. Sci. ScL USA 76: 6606).

Suitable adenoviral vectors useful in the present invention are described, for example, below, and they are incorporated herein by reference: Koza
rsky and Wilson, Curr. Opin. Genet. Devel.
3: 499-503 (1993); Rosenfeld et al., Cell 68: 1.
43-155 (1992); Engelhardt et al., Human Genet.
. Ther. 4: 759-769 (1993); Yang et al., Nature.
Genet. 7: 362-369 (1994); Wilson et al., Nature.
365: 691-692 (1993); and U.S. Patent No. 5,652,224.
issue. For example, the adenovirus vector Ad2 is useful and can be propagated in human 293 cells. These cells contain the E1 region of the adenovirus,
It then constitutively expresses E1a and E1b, which complement the defective adenovirus by providing the product of the gene deleted from the vector. In addition to Ad2, other types of adenovirus (eg, Ad3, Ad5, and Ad7) are also useful in the present invention.

Preferably, the adenovirus used in the present invention is replication deficient. Replication deficient adenoviruses require the help of a helper virus and / or a packaging cell line to form infectious particles. The resulting virus can infect cells and express a polynucleotide of interest operably linked to a promoter (eg, a HARP promoter of the present invention), but cannot replicate in most cells. A replication-defective adenovirus may have one or more of all or a portion of the following genes deleted: E1a, E1b, E3, E4, E2.
a, or L1 to L5.

In certain embodiments, cells are engineered ex vivo or in vivo with adeno-associated virus (AAV). AAV is a naturally occurring defective virus that requires a helper virus to produce infectious particles (Muzycz
ka, N .; Curr. Topics in Microbiol. Immun
ol. 158: 97 (1992)). AAV is also one of several viruses that can integrate its DNA into non-dividing cells. Vectors containing as few as 300 base pairs of AAV can be packaged, but the space for exogenous DNA is limited to about 4.5 kb. Methods for producing and using such AAV are known in the art. For example, US Pat. No. 5,139,9
No. 41, 5,173,414, 5,354,678, 5,43
See 6,146, 5,474,935, 5,478,745, and 5,589,377.

For example, suitable AAV vectors for use in the present invention include DNA replication,
Includes all sequences necessary for encapsidation and host cell integration. V
The EGF-2 polynucleotide construct was prepared as described in Sambrook et al., Moleculara.
r Cloning: A Laboratory Manual, Cold S
Insert into the AAV vector using standard cloning methods such as those found in the Spring Harbor Press (1989). Then
The recombinant AAV vector is transfected into packaging cells infected with the helper virus using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, and the like. Suitable helper viruses include adenovirus, cytomegalovirus, vaccinia virus, or herpes virus. Once the packaging cells are transfected and infected, they produce infectious AAV virions containing the VEGF-2 polynucleotide construct. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells contain the VEGF-2 polynucleotide construct integrated into its genome and express VEGF-2.

Another method of gene therapy involves operably linking a heterologous control region and an endogenous polynucleotide sequence (eg, encoding VEGF-2) via homologous recombination (see, eg, US Pat. No. 5,641,670 (June 1997
International Publication No. WO 96/29411 (published September 26, 1996); International Publication Number WO 94/12650 (published August 4, 1994); Kol
ler et al., Proc. Natl. Acad. Sci. USA 86: 8932-
8935 (1989); and Zijlstra et al., Nature 342: 4.
35-438 (1989)). The method involves the activation of a gene that is present in the target cell, but is not normally expressed in the cell, or is expressed at a level lower than desired.

A polynucleotide construct comprising a promoter with a target sequence flanking the promoter is made using standard techniques known in the art. Suitable promoters are described herein. The targeting sequence is sufficiently complementary to endogenous to allow homologous recombination between the promoter-targeting sequence and the endogenous sequence. The target sequence is sufficiently close to the 5 ′ end of the desired endogenous polynucleotide sequence of VEFG-2 that the promoter is operably linked to the endogenous sequence during homologous recombination.

[0266] Promoters and targeting sequences can be amplified using PCR. Preferably,
The promoter to be amplified contains different restriction sites on the 5 'and 3' ends. Preferably, the 3 'end of the first targeting sequence contains the same restriction enzyme site as the 5' end of the promoter to be amplified, and the 5 'end of the second targeting sequence has the 3' end of the promoter to be amplified. 'Includes the same restriction sites as the ends. The amplified promoter and targeting sequence are digested and ligated together.

The promoter-targeting sequence construct can be used as a naked polynucleotide or with a transfection facilitating agent such as liposomes, viral sequences, viral particles, whole viruses, lipofections, sedimentation agents, etc. (described in more detail above). Deliver to cells either in combination. The P promoter-targeting sequence can be delivered by any method, including direct needle injection, intravenous injection, local administration, catheter injection, particle accelerator, and the like. This method is described in more detail below.

[0268] The promoter-targeting sequence construct is taken up by the cell. Homologous recombination between the construct and the endogenous sequence occurs, resulting in the endogenous VEGF-2 sequence being placed under the control of the promoter. This promoter is then converted to the endogenous VE.
Drives expression of the GF-2 sequence.

The polynucleotide encoding VEGF-2 may be derived from other blood vessels (anion).
genic) protein can be administered together with other polynucleotides. Angiogenic proteins include, but are not limited to: acidic and basic fibroblast growth factors, VEGF-1, epidermal growth factors α and β, platelet-derived endothelial growth factor, Platelet-derived growth factor, tumor necrosis factor α, hepatocyte growth factor, insulin-like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte / macrophage colony stimulating factor,
And nitric oxide synthase.

Preferably, the polynucleotide encoding VEGF-2 contains a secretory signal sequence that facilitates secretion of the protein. Typically, this signal sequence is located in the 5 'end of the coding region or at the 5' end of the coding region of the polynucleotide to be expressed. The signal sequence can be homologous or heterologous to the polynucleotide of interest, and can be homologous or heterologous to the cell to be transfected. Further, the signal sequence can be chemically synthesized using methods known in the art.

[0271] Any mode of administration of any of the above-described polynucleotide constructs can be used, so long as the mode results in expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injections, systemic injections, catheter infusions, biolistic injectors, particle accelerators (or "gene guns"), gel foam sponge accumulations, other commercially available storage materials, osmotic pumps (e.g., Alza minipumps), oral or suppository solid (tablets or pills) pharmaceutical formulations, and decantation or topical application during surgery. For example, direct injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen, or direct injection of protein-coated plasmid into porcine vein results in gene expression of foreign genes in rat liver (Kaneda et al., Science 243: 375 ( 1989)).

The preferred method of topical administration is by direct injection. Preferably, the recombinant molecule of the invention complexed with a delivery vehicle is administered by direct injection into the area of the artery or is administered locally therein. Administering the composition locally into the area of the artery refers to injecting the composition several centimeters, and preferably several millimeters, into the artery.

Another method of topical administration is to contact the polynucleotide construct of the present invention in or around a surgical wound. For example, a patient can undergo surgery and the polynucleotide construct can be coated on the surface of the tissue inside the wound, or the construct can be injected into a region of tissue inside the wound.

[0274] Therapeutic compositions useful for systemic administration comprise a recombinant molecule of the invention conjugated to a targeted delivery vehicle of the invention. Suitable delivery vehicles for use in systemic administration include liposomes containing ligands for targeting the vehicle to a particular site.

Preferred methods of systemic administration include intravenous injection, aerosol, oral and transdermal (topical)
Including delivery. Intravenous injections can be performed using methods standard in the art. Aerosol delivery may also be performed using methods standard in the art (eg, Stribling et al., Proc. Natl. Acad. Sci. USA).
189: 11277-11281, 1992, which is incorporated herein by reference). Oral delivery can be performed by conjugating the polynucleotide construct of the present invention to a carrier that can withstand degradation by digestive enzymes in the intestine of the animal. Examples of such carriers include plastic capsules or tablets, such as those known in the art. Topical delivery involves the transfer of a polynucleotide construct of the present invention to a lipophilic reagent (eg, DMS) that can pass through the skin.
O).

Determining the effective amount of a substance to be delivered can depend on a number of factors. Such factors include, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition requiring treatment and its severity, and the route of administration. The frequency of treatment depends on a number of factors. Such factors include, for example, the amount of polynucleotide construct administered per dose, and the health and medical history of the subject. The exact amount, frequency of administration, and timing of administration are determined by your physician or veterinarian.

The therapeutic compositions of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits, sheep, cows, horses, and pigs. Humans are particularly preferred.

VEGF-2 nucleic acid sequences and VEGF-2 polypeptides may also be useful in scientific studies,
It can be used for in vitro purposes related to DNA synthesis and production of DNA vectors, and for the production of diagnostics and therapeutics for treating human diseases. For example, VEGF-2 can be used for the in vitro culture of vascular endothelial cells, where VEGF-2 is added to the conditioned medium at a concentration of 10 pg / ml to 10 ng / ml.

[0279] Fragments of the full-length VEGF-2 gene can be used as hybridization probes for a cDNA library to isolate other genes with high sequence similarity or similar biological activity to this gene. Probes of this type generally have at least 50 base pairs, but they can have many more bases. This probe can also be used to identify cDNA clones corresponding to the full-length transcript, and genomic clone (s) containing the complete VEGF-2 gene, including regulatory and promoter regions, exons, and introns. . An example of a screen includes isolating the coding region of the VEGF-2 gene by using a known DNA sequence to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to the sequence of the gene of the invention screens a library of human cDNA, genomic DNA, or mRNA and determines to which member of the library the probe hybridizes. Used to

[0280] The present invention provides methods for the identification of the VEGF-2 receptor. The gene encoding this receptor can be prepared by a number of methods known to those skilled in the art, such as ligand panning and FACS sorting (Coligan et al., Current P
rotocols in Immun. , 1 (2), Chapter 5, (1991)))
Can be identified by Preferably, expression cloning is used, in which polyadenylated RNA is prepared from cells responsive to VEGF-2, and a cDNA library made from this RNA is divided into pools, and COS cells or VEGF- Used to transfect other cells that do not respond to 2. Transfected cells growing on glass slides were labeled VE
Expose to GF-2. VEGF-2 can be labeled by a variety of means, including iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified and subpools are prepared and re-transfected using an iterative subpooling and rescreening process, ultimately resulting in a single clone encoding the putative receptor.

As an alternative approach for receptor identification, labeled VEGF-2 is
It may be optical affinity coupled to a cell membrane expressing the receptor molecule or an extract preparation. The crosslinked material is separated by PAGE and exposed to X-ray film. The labeled complex containing VEGF-2 is then excised, broken down into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from the microsequencing was used to design a set of degenerate oligonucleotide probes,
It is used to screen DNA libraries to identify genes encoding putative receptors.

(VEGF-2 Agonist and Antagonist) The present invention also relates to a compound screening method for identifying a compound that is a VEGF-2 agonist or antagonist. One example of such a method utilizes the ability of VEGF-2 to significantly stimulate the proliferation of human endothelial cells in the presence of Comitogen Con A. Endothelial cells were obtained and 96-well flat bottom culture plates (Costar, Cambridge, Calif.) In reaction mixtures supplemented with Con-A (Calbiochem, La Jolla, Calif.).
MA). Add Con-A, the polypeptide of the invention, and the compound to be screened. After incubation at 37 ° C., ³ enough time to take in the [H], ³ [H] thymidine 1μCi intermittently added to the culture (5Ci / mmol;; 1C i = 37BGq NEN) and glass, Fiber Filter (Cambridge Technology, Watertown)
, MA). The average ³ [H] thymidine incorporation (cpm) of triplicate cultures was determined using a liquid scintillation counter (Beckman Instrument).
nts, Irvine, CA). Significant ³ [H] thymidine incorporation compared to a control assay without compound indicates stimulation of endothelial cell proliferation.

To assay for antagonists, the assays described above were performed, and the ability of the compound to inhibit ³ [H] thymidine incorporation in the presence of VEGF-2 indicates that the compound is an antagonist to VEGF-2. Show. Alternatively, VEGF-2 antagonists can be obtained under conditions appropriate for competitive inhibition assays.
It can be detected by combining GF-2 and a potential antagonist with the VEGF-2 receptor bound to the membrane or a recombinant receptor. VEG
F-2 can be labeled, for example, by radioactivity, such that the number of VEGF-2 molecules bound to the receptor can determine the efficacy of a potential antagonist.

Alternatively, the response of a known second messenger system following the interaction of VEGF-2 with the receptor is measured in the presence or absence of the compound and compared. Such second messenger systems include, but are not limited to, cAMP guanylate cyclase, ion channels, or phosphoinositide hydrolysis. In another method, a mammalian cell or membrane preparation expressing the VEGF-2 receptor is labeled with VEGF-II in the presence of the compound.
2 The ability of the compound to enhance or block this interaction can then be measured.

[0285] Potential VEGF-2 antagonists include antibodies, or in some cases, oligonucleotides, which bind to the polypeptide and
-2 function is effectively deleted. Alternatively, a potential antagonist is VEG
It may be a closely related protein that binds to the F-2 receptor, but they are an inactive form of this polypeptide, thereby preventing the action of VEGF-2. Examples of these antagonists include negative dominant variants of the VEGF-2 polypeptide, for example, one chain of the heterodimeric form of VEGF-2 may be dominant and will not retain biological activity As described above. Examples of negative dominant variants include truncated versions of dimeric VEGF-2. It can interact with another dimer to form wild-type VEGF-2, but the resulting homodimer is inactive and cannot exhibit characteristic VEGF activity.

[0286] Another potential VEGF-2 antagonist is an antisense construct prepared using antisense technology. Antisense technology can be used to control gene expression via triple helix formation or antisense DNA or RNA. Both of these methods are based on the binding of a polynucleotide to DNA or RNA. For example, the 5 'coding portion of the polynucleotide sequence encodes a mature polypeptide of the invention and has an antisense RN of about 10-40 base pairs in length.
Used to design A oligonucleotides. DNA oligonucleotides are designed to be complementary to the region of the gene involved in transcription (triple helix-
Lee et al., Nucl. Acids Res. , 6: 3073 (1979); Co.
oney et al., Science, 241: 456 (1988); and Derva
n et al., Science, 251: 1360 (1991)), thereby preventing VEGF-2 transcription and production. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and the V of the mRNA molecule
Block translation into EGF-2 polypeptide (antisense-Okano,
J. Neurochem. , 56: 560 (1991); oligodeoxynucleotides as antisense inhibitors of gene expression, CRC Press,
Boca Raton, FL (1988)). The oligonucleotides described above can also be delivered to cells so that antisense RNA or DNA can be expressed in vivo to inhibit VEGF-2 production.

[0287] Potential VEGF-2 antagonists also bind to and occupy the active site of the polypeptide, thereby rendering the catalytic site inaccessible to the substrate, such that normal biological activity is reduced. Contains small molecules that are hindered. Examples of small molecules are
Includes, but is not limited to, small peptides or peptide-like molecules.

[0288] Antagonists can be used to limit the angiogenesis required for solid tumor metastasis. The identification of VEGF-2 can be used for the generation of specific inhibitors of vascular endothelial growth factor. Since angiogenesis and neovascularization are essential steps in the growth of solid tumors, inhibition of the angiogenic activity of vascular endothelial growth factor is very important to prevent further growth, retardation, or even suppression of solid tumors. Useful. VEGF
-2 expression levels can be found to be extremely low in normal tissues, including the breast, but moderately expressed in at least two breast cancer cell lines derived from malignant tumors. Therefore, VEGF-2 is thought to be involved in tumor angiogenesis and proliferation.

Glioma is also one type of neoplasm that can be treated with an antagonist of the present invention.

[0290] Antagonists can also be used to treat chronic inflammation caused by increased vascular permeability. In addition to these disorders, antagonists can also be used to treat retinopathy, rheumatoid arthritis, and psoriasis associated with diabetes.

The antagonist may be used in a composition with a pharmaceutically acceptable carrier (eg, as described herein below).

Pharmaceutical Compositions VEGF-2 polypeptides, polynucleotides, and agonists and antagonists can be used in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the polypeptide or agonist or antagonist, and a pharmaceutically acceptable carrier or excipient. Such carriers include saline, buffered saline, dextrose, water, glycerol,
Including but not limited to ethanol, and combinations thereof. The formulation should suit the mode of administration.

The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical composition of the present invention. Such containers may be accompanied by a notice in the form specified by the governmental agency that controls the manufacture, use, or sale of the drug or biological product, the notice comprising the manufacture, use, or use for administration to humans. , Or an authorization by the agency of sale. In addition, the pharmaceutical compositions can be used in combination with other therapeutic compounds.

The pharmaceutical compositions can be administered in a convenient manner, such as by the topical, intravenous, intraperitoneal, intramuscular, intratumoral, subcutaneous, intranasal, or intradermal route. Pharmaceutical compositions are administered in an amount effective to treat and / or prevent the particular indication. Generally, the pharmaceutical compositions will be administered in an amount of at least about 10 μg / kg body weight, and most often they will be administered in an amount not to exceed about 8 mg / kg body weight per day.
In most cases, the dosage is about 10 μm / day, taking into account the route of administration, symptoms, etc.
g / kg body weight to about 1 mg / kg body weight.

[0295] VEGF-2 polypeptides, and agonists or antagonists that are polypeptides, can also be used according to the present invention by expression of such polypeptides in vivo, as described above, which is often referred to as "gene therapy". Will be

Thus, for example, cells, such as bone marrow cells, can be engineered ex vivo with a polynucleotide (DNA or RNA) encoding a polypeptide, and then
The engineered cells are provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, cells can be manipulated by procedures known in the art by use of retroviral particles containing RNA encoding a polypeptide of the invention.

Similarly, cells can be engineered in vivo for expression of the polypeptide in vivo, for example, by procedures known in the art. As is known in the art, producer cells for producing retroviral particles containing RNA encoding a polypeptide of the invention can be used to engineer cells in vivo, and for expression of a polypeptide in vivo. May be administered to the patient. These and other methods for administering the polypeptides of the present invention by such methods should be apparent to those skilled in the art from the teachings of the present invention. For example, an expression vehicle for manipulating cells can be other than a retroviral particle (eg, an adenovirus). This can be used to manipulate cells in vivo after combining with a suitable delivery vehicle.

The retroviruses from which the retroviral plasmid vectors described herein above may be derived include Moloney murine leukemia virus, spleen necrosis virus, retroviruses (eg, Rous sarcoma virus, Harvey sarcoma virus, chicken leukemia virus, gibbon Leukemia virus, human immunodeficiency virus), adenovirus, myeloproliferative sarcoma virus, and mammary adenocarcinoma virus. In one embodiment, the retroviral plasmid vector is derived from Moloney murine leukemia virus.

[0299] The vector contains one or more promoters. Suitable promoters that can be used are the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter (Miller et al., Biotechniques).
, 7: 980-990 (1989)), or any other promoter (including, for example, cellular promoters such as eukaryotic cellular promoters (including histone, pol III, and β-actin promoters). But not limited to). Other viral promoters that can be used are the adenovirus promoter, thymidine kinase (TK
) Promoters and B19 parvovirus promoters. The selection of an appropriate promoter will be apparent to the skilled artisan from the teachings contained herein.

The nucleic acid sequence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters that can be used include adenovirus promoters, such as the adenovirus major late promoter; or cytomegalovirus (CM
V) Heterologous promoters such as promoter; RS virus (RSV) promoter; inducible promoters such as MMT promoter, metallothionein promoter; heat shock promoter; albumin promoter; ApoAI promoter; human globin promoter; Viral thymidine kinase promoter; retroviral LTR (
Β-actin promoter; and human growth hormone promoter, including but not limited to the modified retroviral LTR herein above. The promoter can also be a native promoter that controls the gene encoding the polypeptide.

[0301] Retroviral plasmid vectors are used to transduce packaging cell lines to form producer cell lines. Examples of packaging cells that can be transfected are PE501, PA317, ψ-2, ψ-AM, PA
12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP
+ E-86, GP + envAm12, and a DAN cell line (Miller, Human Gene Thera, incorporated herein by reference in its entirety).
py 1: 5-14 (1990)). The vector can transduce the packaging cells via any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO ₄ precipitation. In another, the retroviral plasmid vector can be encapsulated in liposomes or conjugated to a lipid and then administered to a host.

[0302] The producer cell line produces infectious retroviral vector particles that contain the nucleic acid sequence encoding the polypeptide. Such retroviral vector particles can then be used to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cell will express the nucleic acid sequence encoding the polypeptide. Eukaryotic cells that can be transduced include, but are not limited to, embryonic stem cells, embryonic cancer cells, and hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells. Not limited.

Diagnostic Assays The present invention also relates to the use of the VEGF-2 gene as part of a diagnostic assay for detecting a disease or susceptibility to a disease associated with the presence of a mutation in a VEGF-2 nucleic acid sequence. .

An individual having a mutation in the VEGF-2 gene can be detected at the DNA level by various techniques. Nucleic acids for diagnosis can be obtained from patient cells such as blood, urine, saliva, tissue biopsies, and autopsy material. Genomic DNA can be used directly for detection or PCR (Saiki et al., Nature, 32) prior to analysis.
4: 163-166 (1986)). RNA or cDNA can also be used for the same purpose. As an example, VEGF-2
PCR primers complementary to the nucleic acid encoding the can be used to identify and analyze VEGF-2 mutations. For example, deletions and insertions can be detected by a change in the size of the amplification product in comparison to the normal genotype. Point mutations can be identified by hybridizing the amplified DNA with radiolabeled VEGF-2 RNA or with a radiolabeled VEGF-2 antisense DNA sequence. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.

Genetic testing based on DNA sequence differences can be performed with or without denaturing agents.
This can be achieved by detecting a change in electrophoretic mobility of the DNA fragment in the gel. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels, with the mobility of the different DNA fragments lagging at different positions in the gel according to their specific or partial melting temperatures (eg, Myers et al.,
Science, 230: 1242 (1985)).

Sequence changes at specific positions may also be due to nuclease protection assays, such as RNase protection, and S1 protection, or chemical cleavage methods (eg, Cotton et al., PNAS, USA, 85: 4397-440).
1 (1985)).

Thus, the detection of specific DNA sequences can be accomplished by hybridization, RNase protection, chemical cleavage, direct DNA sequencing, or the use of restriction enzymes (eg, restriction fragment length polymorphism (RFLP)) and genomic It can be achieved by methods such as Southern blots of DNA.

In addition to more traditional gel electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

[0309] The present invention also relates to the use of a variety of tissues, as overexpression of VEGF-2 protein compared to normal control tissue samples can detect the presence of, or susceptibility to, disease (eg, abnormal cell differentiation). A diagnostic assay for detecting altered levels of VEGF-2 protein in E. coli. V in host-derived sample
Assays used to detect levels of EGF-2 protein are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, ELISA assays, and "sandwich" assays. ELISA assay (Coligan et al., Current Protocol)
ls in Immunology, 1 (2), Chapter 6, (1991)) involves first preparing an antibody specific to the VEGF-2 antigen, preferably a monoclonal antibody. In addition, reporter antibodies are prepared against the monoclonal antibodies. The reporter antibody is bound to a detectable reagent, such as radioactivity, fluorescence, or, in this example, horseradish peroxidase enzyme. The sample is removed from the host and incubated on a solid support (eg, a polystyrene dish) that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin. Next, the monoclonal antibody is incubated in the dish, during which time the monoclonal antibody binds to any VEGF-2 protein bound to the polystyrene dish. All unbound monoclonal antibodies are removed by washing with buffer. A reporter antibody conjugated to horseradish peroxidase is placed in a dish and VE
The reporter antibody is bound to any monoclonal antibody bound to GF-2.
The unbound reporter antibody is then washed away. The amount of color development within a given time is then a measure of the amount of VEGF-2 protein present in a given volume of a patient sample when a peroxidase substrate is added to the dish and compared to a standard curve. .

[0310] A competition assay may be used, wherein an antibody specific for VEGF-2 is bound to a solid support. The polypeptide of the invention is then labeled, e.g., by radioactivity, and a sample from the host is passed through a solid support, and
The amount of label detected by liquid scintillation chromatography can be correlated to the amount of VEGF-2 in the sample.

[0311] The "sandwich" assay is similar to the ELISA assay. In a "sandwich" assay, VEGF-2 is passed through a solid support and bound to an antibody attached to the solid support. Then, the second antibody is allowed to bind to VEGF-2. A third antibody, labeled and specific for the second antibody, can then be passed through the solid support and allowed to bind to the second antibody, and the amount can be quantified.

Chromosome Identification The sequences of the present invention are also valuable for chromosome identification. This sequence can specifically target a particular location on an individual human chromosome and hybridize at that location. In addition, it is now necessary to identify specific sites on the chromosome. Currently, there are few chromosome marking reagents based on actual sequence data (repetitive polymorphisms) available for marking chromosomal locations. The mapping of DNA to chromosomes according to the present invention comprises:
This is an important first step in correlating these sequences with genes for disease.

[0313] Briefly, the sequence was derived from cDNA by PCR primers (preferably 15-
25 bp) can be mapped to the chromosome. Computer analysis of the cDNA is used to rapidly select primers that do not span more than one exon in the genomic DNA and thus complicate the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to this primer will yield an amplified fragment.

[0314] PCR mapping of somatic cell hybrids is a rapid procedure for assigning specific DNA to specific chromosomes. Using the present invention with the same oligonucleotide primers, in a similar fashion, using a panel of fragments from a particular chromosome, or using a pool of large genomic clones to sublocate (su
blocking can be achieved. Other mapping strategies that can also be used to map to that chromosome include in situ hybridization, prescreening on labeled chromosomes selected by flow cytometry, and hybridization to construct chromosome-specific cDNA libraries. By preselection.

[0315] Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosome spread can be used to provide a precise chromosomal location in one step. This technique can be used with probes from cDNAs as short as 50 or 60 base pairs; for a review of this technique, see Verma et al.,
Human Chromosomes: a Manual of Basic
Techniques, Pergamon Press, New York (1
988).

Once a sequence has been mapped to a precise chromosomal location, the physical location of the sequence on the chromosome can be correlated with genetic map data. Such data, for example,
V. McKusick, Mendelian Inheritance i
n Man (Johns Hopkins University)
Available online from Welch Medical Library))). The relationship between the disease and the gene mapped to the same chromosomal region is then identified through linkage analysis (simultaneous inheritance of physically adjacent genes).

Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If the mutation is observed in some or all affected individuals but not in normal individuals, then the mutation appears to be a causative agent of the disease.

At the current resolution of physical and genetic mapping techniques, a cDNA that is correctly located in a chromosomal region associated with a disease is one of between 50 and 500 potential causative genes. possible. (This assumes 1 megabase mapping resolution and 1 gene per 20 kb.) Comparisons between affected and unaffected individuals generally show structural changes in chromosomes (eg, as seen from chromosome spread) Or deletions or translocations that are detectable using PCR based on the cDNA sequence). Ultimately, complete sequencing of genes from several individuals is required to confirm the presence of the mutation and to distinguish mutations from polymorphisms.

Antisense The present invention further provides VEGF-2 in vivo by use of antisense technology.
Related to inhibiting. Antisense technology can be used to control gene expression through triple helix formation or antisense DNA or RNA. Both of these methods are based on the binding of a polynucleotide to DNA or RNA. For example, the 5 'coding portion of a mature polynucleotide sequence encodes a mature polypeptide of the invention and is used to design an antisense RNA oligonucleotide 10-40 base pairs in length. DNA oligonucleotides are designed to be complementary to the region of the gene involved in transcription (triple helix-Lee et al., Nucl.
Acids Res. , 6: 3073 (1979); Cooney et al., Sci.
ence, 241: 456 (1988); and Dervan et al., Science.
e, 251: 1360 (1991)), thereby preventing VEGF2 transcription and production. Antisense RNA oligonucleotides hybridize to mRNA in vivo and block translation of the mRNA molecule into VEGF2 (antisense-Okano, J. Neurochem., 56: 5).
60 (1991); oligodeoxynucleotides as antisense inhibitors of gene expression, CRC Press, Boca Raton, FL (198
8)).

Alternatively, the above-described oligonucleotides can be delivered to cells by procedures in the art, so that antisense RNA or DNA can be expressed in vivo and inhibit the production of VEGF2 in the manner described above.

Thus, antisense constructs against VEGF-2 may inhibit the angiogenic activity of VEGF-2 and may prevent further growth or even loss of solid tumors. Angiogenesis and neovascularization are essential steps in the growth of solid tumors. These antisense constructs can also be used to treat rheumatoid arthritis, psoriasis, diabetic retinopathy and Kaposi's sarcoma, all characterized by abnormal angiogenesis.

(Epitope-Carrying Portion) In another aspect, the present invention provides peptides and polypeptides containing the epitope-bearing portion of the polypeptide of the present invention. These epitopes are immunogenic or antigenic epitopes of the polypeptide of the invention. An "immunogenic epitope" is defined as a portion of a protein that elicits an antibody response in vivo when the entire polypeptide of the invention, or a fragment thereof, is an immunogen. On the other hand, regions of the polypeptide to which the antibody can bind are defined as "antigenic determinants" or "antigenic epitopes". The number of in vivo immunogenic epitopes of a protein is generally less than the number of antigenic epitopes. See, for example, Geysen et al., (1983) Proc. Natl. Acad. Sci. USA 81: 399
See 8-4002. However, by using methods such as phage display, antibodies, whether or not they are immunogenic epitopes,
It can be generated for any antigenic epitope. For example, Petersen G
. (1995) Mol. Gen. Genet. 249: 425-431. Thus, the present invention includes both immunogenic and antigenic epitopes.

The immunogenic epitope was determined by Jameson-Wolf analysis,
It is specifically pointed out that it contains predicted important amino acid residues. Thus, additional flanking residues, either at the N-terminus, the C-terminus, or both the N-terminus and the C-terminus, can be added to these sequences to produce an epitope-bearing polypeptide of the invention. Thus, an immunogenic epitope can include additional N-terminal or C-terminal amino acid residues. This additional flanking amino acid residue can be a contiguous flanking N-terminal and / or C-terminal sequence from a polypeptide of the invention, a heterologous polypeptide sequence, or a contiguous flanking amino acid residue from a polypeptide of the invention. A polypeptide of the invention comprising an immunogenic or antigenic epitope, which may include both ranking and heterologous polypeptide sequences,
It is at least 7 amino acid residues long. "At least" means that the polypeptide of the invention comprising an immunogenic or antigenic epitope can be 7 amino acid residues in length, or 7 amino acids and the amino acid residues of a full-length polypeptide of the invention. Means that it can be any integer between the numbers. Preferred polypeptides comprising an immunogenic or antigenic epitope are at least 10, 15, 20
, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80
, 85, 90, 95 or 100 amino acid residues in length. However, it is pointed out that each and every integer between 7 and the number of amino acid residues of the full length polypeptide is included in the present invention.

[0324] Immune and antigenic epitope-bearing fragments can be identified by any of the number of contiguous amino acid residues as described above, or the N-terminal position and C-terminal of these fragments in the amino acid sequence of SEQ ID NO: 2. It can be further specified by location. For example, a fragment of at least 7 or at least 15 contiguous amino acid residues in length may occupy the amino acid sequence of SEQ ID NO: 2,
Any combination of terminal and C-terminal positions is included in the present invention. Again, the "length of at least seven consecutive amino acid residues" refers to the length of seven amino acid residues, or any integer between 7 amino acids and the number of amino acid residues of the full-length polypeptide of the invention. means. In particular, each and every integer between 7 and the number of amino acid residues of the full length polypeptide is included in the invention.

The immunogenic epitope-bearing polypeptides and antigenic epitope-bearing polypeptides of the invention are useful, for example, in generating antibodies that specifically bind to a polypeptide of the invention, and polypeptides of the invention. Useful in immunoassays that detect This antibody is useful, for example, in affinity purification of the polypeptide of the present invention. The antibodies can also be routinely used for various qualitative and quantitative immunoassays, especially for polypeptides of the invention using methods known in the art. For example, Harlow et al., Antibodies: A
Laboratory Manual, (Cold Spring Harbo
r Laboratory Press; 2nd ed., 1988).

The epitope-bearing polypeptides of the present invention can be produced by any conventional method for producing polypeptides, including synthetic and recombinant methods known in the art. For example, epitope-bearing peptides can be synthesized using known methods of chemical synthesis. For example, Houghten describes a simple method for the synthesis of a large number of peptides (eg, 10-20 mg in 248 individuals) and a different 13 residue peptide representing a segment of a single amino acid variant of the HA1 polypeptide. All of which have been prepared and characterized in less than 4 weeks (by ELISA-type binding studies) (Houghten, RAP).
rc. Natl. Acad. Sci. USA 82: 5131-5135 (1
985)). This “simultaneous multiple peptide synthesis (SMPS)” process is described in Houg
US Patent No. 4,631,21 to Hten and co-workers et al. (1986).
No. 1 is further described. In this procedure, the individual resins for the solid phase synthesis of the various peptides are included in separate solvent permeation packets, which allows for optimal use of many of the same iterative steps involved in the solid phase method. To A complete manual procedure can perform over 500-1000 or more syntheses simultaneously (Houghten
(1985) Proc. Natl. Acad. Sci. 82: 5131-51
35, 5134).

[0327] The epitope-bearing polypeptides of the present invention can be used to raise antibodies according to methods well known in the art, including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. Induce. For example, Su
tcliffe et al., supra; Wilson et al., supra, and Bittle et al. (19
85) J. Gen. Virol. 66: 2347-2354. If in vivo immunization is used, animals can be immunized with free peptide; however, the titer of the anti-peptide antibody depends on the level of the peptide and the carrier of the macromolecule (eg, keyhole limpet haem). It can be boosted by conjugation with cyanine (KLH) or diphtheria toxoid). For example, a peptide containing a cysteine residue can be attached to a carrier using a linker (eg, -maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides have more common binders. (Eg, glutaraldehyde) .Animals such as rabbits, rats and mice may be, for example, about 100 μg
Are immunized with either the free epitope or the peptide bound to the carrier by intraperitoneal and / or intradermal injection of an emulsion containing the peptide or carrier protein and Freund's adjuvant. Some booster injections are necessary to provide useful titers of the anti-peptide antibody, for example, at intervals of about two weeks, for example, which can be detected by an ELISA assay using free peptide adsorbed to a solid surface. possible. The titer of the anti-peptide antibody in the serum from the immunized animal is increased by selection of the anti-peptide antibody, for example, by adsorption to the peptide on a solid support and elution of the selected antibody according to methods well known in the art. obtain.

As the skilled artisan will appreciate, and as discussed above, polypeptides of the invention that include an immunogenic or antigenic epitope can be fused to a heterologous polypeptide sequence. For example, a polypeptide of the invention may comprise an immunoglobulin (Ig
A, IgE, IgG, IgM), or portions thereof (CH1
, CH2, CH3, any combination thereof, including both the entire domain and a portion thereof), resulting in a chimeric polypeptide. These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, for example, for chimeric proteins consisting of the first two domains of the human CD4 polypeptide and various domains of the heavy or light chain constant regions of mammalian immunoglobulins. For example, ERA 0,394,82
7; See Traunecker et al. (1988) Nature 331: 84-86. Fusion proteins with disulfide-bridged dimeric structures due to IgG moieties may also be more effective at binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. For example,
Toulakis et al. Biochem. 270: 3958-39
See 64. Nucleic acids encoding the above-mentioned epitopes can also be recombined with the gene of interest as epitope tags to aid in the detection and purification of the expressed polypeptide.

(Antibodies) The present invention further relates to antibodies and T cell antigen receptors (TCRs) that specifically bind the polypeptides of the present invention. The antibody of the present invention comprises an IgG (IgG1,
G2, IgG3, and IgG4), IgA (IgA1, and IgA2
), IgD, IgE, or IgM, and IgY. As used herein, the term "antibody" (Ab) is meant to include whole antibodies, including whole single-chain antibodies, and to include antigen-binding fragments thereof. Most preferably, the antibody comprises Fab, Fab ', and F (ab') 2, Fd, single chain Fvs (scFv), single chain antibody, disulfide-linked Fvs (sdFv) and any of the _VL or _VH domains. Human antigen-binding antibody fragments of the invention, including, but not limited to, fragments. Antibodies can be from any animal origin, including birds and mammals. Preferably, the antibody is human, mouse, rabbit, goat, guinea pig, camel, horse or chicken.

[0330] Antigen binding antibody fragments (including single chain antibodies) may comprise only the variable region, or may be comprised in combination with all or part of the following: hinge region, CH1
, CH2 and CH3 domains. The invention also includes the variable region and any combination of hinge regions, CH1, CH2, and CH3 domains. The present invention further includes chimeric, humanized, and human monoclonal and polyclonal antibodies that specifically bind the polypeptides of the present invention. The present invention further includes antibodies that are anti-idiotypic to the antibodies of the present invention.

The antibodies of the present invention can be monospecific, bispecific, trivalent or more polyspecific. Multispecific antibodies can be specific for different epitopes of a polypeptide of the invention, or can comprise a polypeptide of the invention as well as a heterologous composition (
(For example, a heterologous polypeptide or a solid support).
For example, WO93 / 17715; WO92 / 08802; WO91 / 00360.
WO 92/05793; Tutt, A .; (1991) J. Am. Immunol.
147: 60-69; U.S. Patent Nos. 5,573,920 and 4,474,89.
No. 3, 5,601, 819, 4,714,681, 4,925,648;
Kostelny, S.M. A. (1992) J. et al. Immunol. 148: 15
See 47-1553.

An antibody of the invention can be described or specified in terms of an epitope or portion of a polypeptide of the invention, which is recognized or specifically bound by an antibody. The epitope or polypeptide moiety may be selected as described herein (using
For example, by N- and C-terminal portions, by the size of adjacent amino acid residues), or as listed in the tables and figures. Antibodies that specifically bind to any epitope or polypeptide of the present invention may also be excluded. Accordingly, the present invention includes and allows for the elimination of antibodies that specifically bind to a polypeptide of the present invention.

The antibodies of the present invention may also be described or specified for their cross-reactivity.
obtain. Any other analog, ortholog or homolog of a polypeptide of the invention
Antibodies that do not bind to Less than 95%, 90 relative to the polypeptide of the invention
%, Less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, 60%
%, 55% and 50% identity (methods and methods known in the art)
Polypeptide having (as calculated using the method described in the specification)
Antibodies that do not bind the peptide are also included in the invention. Further, the present invention provides a
Under gentle hybridization conditions (as described herein),
Encoded by a polynucleotide that hybridizes to a polypeptide of the invention
Antibodies that bind only the polypeptide are included. The antibodies of the present invention also exhibit their binding affinity.
Sex may be described or specified. Preferred binding affinity is 5 × 10 ^-6 M, 10^-6M, 5 × 10^-7M, 10^-7M, 5 × 10^-8M, 10^-8M, 5 × 10 ^-9 M, 10^-9M, 5 × 10^-TenM, 10^-TenM, 5 × 10^-11M, 10^-11M, 5x
10^-12M, 10^-12M, 5 × 10^-13M, 10^-13M, 5 × 10^-14M, 10^-14M
, 5 × 10^-15M, and 10^-15Dissociation constants less than M, ie, affinity for Kd
Can be

The antibodies of the invention include, but are not limited to, methods known in the art for purifying, detecting and targeting polypeptides of the invention, including both in vitro and in vivo diagnostic and therapeutic methods. Has unlimited uses. For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of a polypeptide of the invention in biological samples. For example, Harl
ow et al., ANTIBODIES: A LABORATORY MANUAL, (
Cold Spring Harbor Laboratory Press,
Second Edition, 1988), which is incorporated by reference in its entirety.

The antibodies of the present invention can be used either alone or in combination with other compositions. The antibody can further be recombinantly fused at the N-terminus or C-terminus to a heterologous polypeptide, or chemically conjugated (covalently and non-covalently conjugated) to the polypeptide or other composition. Including). For example, the antibodies of the present invention may be useful as labels and effector molecules (in effector molecules) in detection assays.
(Eg, a heterologous polypeptide, drug or toxin). For example, WO92 / 08495; WO91 / 14438; WO89 / 1
U.S. Pat. No. 5,314,995; and EP 0396387.
See issue. The antibodies of the present invention can be prepared by any suitable method known in the art. For example, the polypeptide of the present invention or an antigenic fragment thereof
It can be administered to animals to induce the production of serum containing polyclonal antibodies. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma technology and recombinant technology. For example, Harlow et al., A
NTIBODIES: A LABORATORY MANUAL (Cold S
spring Harbor Laboratory Press, 2nd edition, 19
88); Hammerling et al., MONOCLONAL ANTIBODIE.
S AND T-CELL HYBRIDOMAS 563-681 (Else
viar, N .; Y. , 1981) (these references are incorporated by reference in their entirety). Fab and F (ab ') 2 fragments can be made by proteolytic cleavage, using enzymes such as papain (for making Fab fragments) or pepsin (for making F (ab') 2 fragments).

Alternatively, the antibodies of the present invention can be made by the application of recombinant DNA technology using methods known in the art or by synthetic chemistry. For example, the antibody of the present invention
It can be prepared using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences that encode them. Phage with the desired binding properties are selected directly by using an antigen (typically an antigen bound or captured to a solid surface or beads) to provide a repertoire or combinatorial antibody library (eg, , Human or mouse). The phage used in these methods is typically
Filamentous phage comprising fd and M13 having the antibody domain of a Fab, Fv or disulfide stabilized Fv recombinantly fused to either the phage gene III protein or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the invention include those disclosed below: Brinkman U.S. Pat. (1995) J. Am. Im
munol. Methods 182: 41-50; Ames, R .; S. (1
995) J. Immunol. Methods 184: 177-186; Ke
tleborough, C .; A. (1994) Eur. J. Immunol
. 24: 952-958; Persic, L .; (1997) Gene 187.
9-18; Burton, D .; R. Et al. (1994) Advances in
Immunology 57: 191-280; PCT / GB91 / 01134
WO 90/02809; WO 91/10737; WO 92/01047
WO 92/18619; WO 93/11236; WO 95/15982
WO 95/20401; and U.S. Patent Nos. 5,698,426;
No. 5,223,409, No. 5,403,484, No. 5,580,717,
No. 5,427,908, No. 5,750,753, No. 5,821,04
No. 7, No. 5,571,698, No. 5,427,908, No. 5,516
Nos., 637, 5,780,225, 5,658,727 and 5,733,743 (these references are incorporated by reference in their entirety).

As described in the above references, after phage selection, antibodies encoding regions from the phage can be used to generate whole human antibodies or antibodies, including any other desired antigen-binding fragments. It can be isolated and used, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria. For example, techniques for making Fab, Fab 'and F (ab') 2 fragments recombinantly can also be used using methods known in the art. This method is, for example, the method disclosed below: WO 92/22324
Mullinax, R .; L. (1992) BioTechniques 1
2 (6): 864-869; and Sawai, H .; (1995) AJRI
34: 26-34; and Better, M .; Et al. (1998) Science
240: 1041-1043 (these references are incorporated by reference in their entirety).

Examples of techniques that can be used to make single chain Fvs and antibodies include US Pat. Nos. 4,946,778 and 5,258,498; Huston et al. (1991) Methods in Enzymology. 203: 46-8
8; Shu, L .; (1993) PNAS 90: 7995-7999; and Skerra, A. et al. (1988) Science 240: 1038-104.
0. For some uses, including in vivo use of antibodies in humans and in vitro detection assays, the use of chimeric, humanized or human antibodies may be preferred. A method for producing a chimeric antibody comprises:
It is known in the art. For example, Morrison, Science 2
29: 1202 (1985); Oi et al., BioTechniques 4:21.
4 (1986); Gillies, S .; D. (1989) J. Am. Immunol
. Methods 125: 191-202; and US Pat. No. 5,807,7.
See No. 15. Antibodies can be humanized using various techniques, including:
DR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101 and 5,585,089), veneering or resurfacing.
) (EP 0 592 106; EP 0 519 596; Pa)
dlan E. A. (1991) Molecular Immunology
28 (4/5): 489-498; Studnicka G. et al. M. (1994) Protein Engineering 7 (6): 805-814; R
oguska M .; A. (1994) PNAS 91: 969-973) and chain shuffling (U.S. Pat. No. 5,565,332). Human antibodies can be produced by various methods known in the art, including the phage display method described above. Also, U.S. Pat.
Nos. 4,887, 4,716,111, 5,545,806 and 5,814,318; and WO 98/46645 (these references are incorporated by reference in their entirety. )checking.

In addition, the present invention includes antibodies recombinantly fused or chemically conjugated (including both covalent and non-covalent bonds) to a polypeptide of the present invention. This antibody may be specific for an antigen other than the polypeptide of the invention. For example, an antibody targets a polypeptide of the invention to a particular cell type, either in vitro or in vivo, by fusing or binding the polypeptide of the invention to an antibody specific for a particular cell surface receptor. Can be used for Antibodies fused or conjugated to a polypeptide of the invention can also be used in in vitro immunoassays and purification methods using methods known in the art. For example,
Harbor et al., Supra, and WO 93/21232; EP 0 439.
No. 095; Naramura, M .; (1994) Immunol. Lett
. 39: 91-99; U.S. Patent No. 5,474,981;
O. (1992) PNAS 89: 1428-1432; Fell, H .; P.
(1991) J. Am. Immunol. 146: 2446-2452, which are incorporated by reference in their entirety.

The present invention further includes compositions containing a polypeptide of the present invention fused or conjugated to an antibody domain other than the variable region. For example, a polypeptide of the invention can be fused or conjugated to the Fc region of an antibody or a portion thereof. The antibody portion fused to the polypeptide of the present invention may comprise the hinge region, CH1 domain, CH2 domain and CH3 domain or any combination of all or part of those domains. The polypeptides of the present invention can be fused or conjugated to an antibody moiety described above to extend the in vivo half-life of the polypeptide, or for use in immunoassays using methods known in the art. The polypeptide can also be fused or conjugated to the antibody portion described above to form a multimer. For example, an Fc portion fused to a polypeptide of the invention can form a dimer through disulfide bonds between the Fc portions. The more multivalent multimeric forms are IgA and I
It can be made by fusing a polypeptide to a portion of gM. Methods for fusing or binding a polypeptide of the invention to an antibody moiety are known in the art. For example, U.S. Patent Nos. 5,336,603 and 5,622,929
No. 5,359,046, No. 5,349,053, No. 5,447,
No. 851, No. 5,112,946; European Patent No. 0 307 434, European Patent No. 0 367 166; WO 96/04388, WO 91/0657.
0; Ashkenazi, A .; (1991) PNAS 88: 10535-1.
0539; Zheng, X .; X. (1995) J. Am. Immunol. 154:
5590-5600; and Vil, H .; (1992) PNAS 89:11.
337-11341 (these references are incorporated by reference in their entirety)
checking ...

The present invention further relates to antibodies that act as agonists or antagonists of the polypeptides of the present invention. For example, the invention includes antibodies that disrupt the receptor / ligand interaction with a polypeptide of the invention, either partially or completely. Both receptor-specific and ligand-specific antibodies are included. Receptor-specific antibodies that do not prevent ligand binding but do prevent receptor activation are included. Receptor activation (ie, signal transduction) can be determined by the techniques described herein, or other techniques known in the art. Also included are receptor-specific antibodies that prevent both ligand binding and receptor activation. Also included are neutralizing antibodies that bind to the ligand and prevent binding of the ligand to the receptor, and antibodies that bind to the ligand, thereby preventing activation of the receptor but not preventing the ligand from binding to the receptor. . Furthermore, antibodies that activate the receptor are included. These antibodies may act as agonists for any or all of the biological activities affected by ligand-mediated receptor activation. The antibody can be specified as an agonist or antagonist for a biological activity, including the specific activities disclosed herein. The antibody agonist can be made using methods known in the art. For example, WO 96/40281; U.S. Patent No. 5,811,097; D
eng, B .; (1998) Blood 92 (6): 1981-1988; C
hen, Z .; (1998) Cancer Res. 58 (16): 3668-
3678; Harrop, J. M .; A. (1998) J. Am. Immunol. 161
(4): 1786-1794; Zhu, Z .; Et al. (1998) Cancer Re.
s. 58 (15): 3209-3214; Yoon, D .; Y. Et al. (1998) J
. Immunol. 160 (7): 3170-3179; Prat, M .; (1
998). Cell. Sci. 111 (Pt2): 237-247; Pita
rd, V .; (1997) J. Am. Immunol. Methods 205 (2)
177-190; Liauard, J .; Et al. (1997) Cytokinde
9 (4): 233-241; Carlson, N .; G. FIG. (1997) J. Am. B
iol. Chem. 272 (17): 11295-11301; Taryman
, R .; E. FIG. (1995) Neuron 14 (4): 755-762; Mul
ler, Y .; A. (1998) Structure 6 (9): 1153-1.
167; Bartunek, P .; (1996) Cytokine 8 (1):
14-20 (these references are incorporated by reference in their entirety).

The antibodies can further be used in immunoassays to detect the presence of a tumor in a particular individual. An enzyme immunoassay can be performed from a blood sample of an individual. Elevated levels of VEGF-2 can be considered a diagnosis of cancer.

Wild-type VEGF- to form a dimer that does not activate endothelial cell growth
VEGF, which inactivates endogenous VEGF-2.
A shortened version of -2 can also be produced. Alternatively, the mutated forms of VEGF-2 form dimers themselves and occupy the ligand binding domain of the appropriate tyrosine kinase receptor on the target cell surface, but do not activate cell growth.

Alternatively, antagonists to a polypeptide of the invention that bind to a receptor to which the polypeptide of the invention binds normally may be used. The antagonists can be closely related proteins, so that they recognize and bind to the receptor site of the native protein, but they are inactive forms of the native protein, thereby VE because the site is occupied
Prevents the action of GF-2. In these methods, the action of VEGF-2 is prevented and the antagonist / inhibitor is occupied by occupying the receptor site of the tumor recognized by VEGF-2, or by inactivating VEGF-2 itself. It can be used therapeutically as an antitumor drug. This antagonist / inhibitor can also be used to prevent inflammation due to the increased vascular permeability effect of VEGF-2. The antagonist / inhibitor may also be used for the treatment of solid tumor growth, diabetic retinopathy, psoriasis and rheumatoid arthritis.

The antagonist / inhibitor can be used, for example, in combination with a pharmaceutically acceptable carrier as described herein above.

The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts are by weight unless otherwise specified.

To facilitate understanding of the following examples, methods and / or terms that appear at a particular frequency will be described.

"Plasmids" are designated by a lower case p preceded and / or followed by capital letters and / or numbers. The starting plasmids herein are either commercially available and publicly available without limitation, or can be constructed from available plasmids according to published procedures. In addition, plasmids equivalent to the described plasmids are known in the art and will be apparent to those skilled in the art.

"Digestion" of DNA is a restriction enzyme that acts only at certain sequences in the DNA.
Is catalytically cleaved. The various restriction enzymes used herein are commercially available, and their reaction conditions, cofactors, and other requirements used were known to those skilled in the art. For analytical purposes, typically 1 mg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 Fl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5-50 mg of DNA is digested with 20-250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37EC are usually used, but this can be varied according to the supplier's instructions. After digestion, the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.

The size separation of the cleaved fragments is described in Goeddel, D .; Et al., Nucl
eic Acids Res. 8: 4057 (1980) 8%
This is performed using a polyacrylamide gel.

“Oligonucleotide” refers to either a single-stranded polydeoxynucleotide or two complementary polydeoxynucleotide chains, which can be chemically synthesized. Such synthetic oligonucleotides do not have a 5 'phosphate and therefore will not ligate to another oligonucleotide without the addition of phosphate and ATP in the presence of a kinase. Synthetic oligonucleotides ligate to fragments that have not been dephosphorylated.

"Ligation" refers to the process of forming a phosphodiester bond between two double-stranded nucleic acid fragments (Sambrook et al., Molecular Cloni).
ng: A Laboratory Manual, 2nd edition, Cold Spri
ng Harbor Laboratory, Cold Spring Har
bor, N .; Y. (1989), 146). Unless otherwise provided, ligation can be accomplished using known buffers and conditions, using approximately equimolar amounts of 10 units of T4 DNA ligase per 0.5 μg of DNA fragment to be ligated (“ligase”). .

Unless otherwise stated, transformation was performed according to Graham, F. et al. And Van der
Eb, A .; , Virology, 52: 456-457 (1973).

EXAMPLES Example 1 VEGF-2 Expression Pattern in Human Tissues and Breast Cancer Cell Lines Northern blot analysis was performed to determine the expression of VEGF-2 in human tissues and breast cancer cell lines in human tissues. The levels were tested. Total cell RNA samples were prepared using RNAz
ol ^™ B system (Biotecx Laboratories, Inc.). Approximately 1 isolated from each breast cancer tissue and specialized cell line
0 mg of total RNA was separated on a 1% agarose gel and blotted onto nylon filters (Sambrook et al., Molecular Clonin).
g: A Laboratory Manual, Second Edition
, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.M. Y. (1989)). Labeling reaction is 50 ng
Was performed according to the Stratagene Prime-It kit. The labeled DNA was prepared using 5 Prime @ 3 Prime, Inc (B
(Selector-G.Oulder, CO) using a Select-G-50 column. The filters were then hybridized overnight at 65 ° C. with 1,000,000 cpm / ml of the radiolabeled full length VEGF-2 gene in 0.5 M NaPO ₄ and 7% SDS. After two washes with 0.5 × SSC, 0.1% SDS at room temperature and two washes at 60 ° C., the filters were then exposed to an intensifying screen at −70 ° C. overnight. . A 1.6 Kd message was observed in two breast cancer cell lines. FIG. 5, lane 4, shows a highly neoplastic cell line,
Estrogen-independent for growth.

Also, 10 mg of total RNA from 10 adult human tissues was separated on an agarose gel and blotted on nylon filters. The filters were then hybridized overnight at 65 ° C. with a radiolabeled VEGF-2 probe in 0.5 M NaPO ₄ , pH 7.2; 1% BSA. 6 in 0.2 × SSC
After washing at 5 ° C., the filters were washed with an intensifying screen at −70 ° C. for 24 hours.
Exposure to film for hours. See FIG.

Example 2 Expression of Truncated Form of VEGF-2 (SEQ ID NO: 4) by In Vitro Transcription and Translation VEGF-2 cDNA is transcribed and translated in vitro, and truncated forms of VEGF-2 and The size of the translatable polypeptide encoded by the partial VEGF-2 cDNA was determined. The two inserts of VEGF-2 in the pBluescript SK vector were amplified by PCR using the following three pairs of primers: 1) M13 reverse and forward primers; 2) M13 reverse and VEGF primer F4; And 3) M13 reverse primer and VEGF primer F5. The sequences of these primers are as follows.

M13-2 Reverse Primer: 5′-ATGCTTCCGGCTCGTATG
-3 '(SEQ ID NO: 9). This sequence is the VE in the pBluescript vector.
It is located upstream of the 5 'end of the GF-2 cDNA insert and is in the antisense orientation with the cDNA. The T3 promoter sequence is composed of this primer and VEGF
-2 cDNA. M13-2 forward primer: 5'GGGT
TTTCCCAGTCACGAC-3 ′ (SEQ ID NO: 10). This sequence is pBl
Located downstream of the 3 'end of the VEGF-2 cDNA insert in the uiscript vector and in antisense orientation with the cDNA insert. VEGF primer F4: 5'-CCACATGGTTCAGGGAAAGACA-3 '(SEQ ID NO: 11). This sequence is included in the VEGF-2 cDNA in the antisense orientation,
Located at bp 1259-1239, which is about 169 bp away from the 3 'end of the stop codon of the cDNA and about 266 bp before the last nucleotide of the cDNA.

The PCR reaction using all three pairs of primers generates an amplification product that contains the T3 promoter sequence before the cDNA insert. The first and third pairs of primers
A PCR product encoding the VEGF-2 polypeptide set forth in SEQ ID NO: 4 is generated. The second pair of primers generates a PCR product lacking the 36 amino acid coding sequence at the C-terminus of the VEGF-2 polypeptide.

About 0.5 mg from the first pair of primers, about 1 mg from the second pair of primers
About 1 mg of PCR product from the third pair of primers was used for in vitro transcription / translation. The in vitro transcription / translation reaction is performed in a volume of 25 Fl using _TN TJ Coupled Reticulocyte Lysates
Promega, CAT # L4950). In particular, the reaction is, T _N T rabbit reticulocyte lysate 12.5Fl, T _N T reaction buffer 2FL,
1 Fl of T3 polymerase, 1 Fl of a 1 mM mixture of amino acids (without methionine), 4 Fl of ³⁵ S-methionine (> 1000 Ci / mmol, 10 mCi / m
l) 40 U / μl of 1 Fl; RNasin ribonuclease inhibitor, 0
. Contains 5 or 1 mg of PCR product. And H ₂ O was added nuclease free, combined capacity 25 fL. The reaction was incubated at 30 ° C. for 2 hours.
5 μl of the reaction product was analyzed on a 4-20% gradient SDS-PAGE gel. 2
After fixation with 5% isopropanol and 10% acetic acid, the gel was dried and
Exposure to X-ray film overnight at 0 ° C.

As shown in FIG. 7, a PCR product containing a truncated VEGF-2 cDNA (ie, as shown in SEQ ID NO: 3) and a 3 ′ untranslated region (3′-UTR)
CDNAs lacking 266 bp in) produce translation products of the same length and their molecular weight is estimated to be 38-40 dk (lanes 1 and 3). All 3 '
A cDNA lacking UTR and lacking the sequence encoding the C-terminal 36 amino acids,
It was translated into a polypeptide with an estimated molecular weight of 36-38 kd (lane 2).

Example 3 Cloning and Expression of VEGF-2 Using Baculovirus Expression System DNA encoding VEGF-2 protein without the N-terminal 46 amino acids
The sequence (see ATCC No. 97149) was added to the 5 'and 3'
Amplify using PCR oligonucleotide primers corresponding to the sequence.

The 5 ′ primer has the sequence TGT AAT ACG ACT CAC TAT
AG G GAT CC C GCC ATG GAG GCC ACG GCT
One that has TAT GC (SEQ ID NO: 12) and is complementary to the BamH1 restriction enzyme site (underlined) and the 5 ′ sequence of VEGF-2 (nucleotides 150-166).
7 nucleotide sequence.

The 3 ′ primer has the sequence GATC TCT AGA TTA GCT CAT
It has TTG TGG TCT (SEQ ID NO: 13) and contains 18 nucleotides complementary to the cleavage site for the restriction enzyme XbaI and the 3 ′ sequence of VEGF-2 (including the stop codon and the 15 nucleotide sequence before the stop codon).

The amplified sequence was prepared using a commercially available kit (“Geneclean” BIO 101).
Inc. , La Jolla, CA) using a 1% agarose gel. This fragment was then converted to the endonucleases BamH1 and X
digested with baI and then purified again on a 1% agarose gel. This fragment was transformed with the pAcGP67A baculovirus transfer vector (Pharminge
n) was ligated at the BamH1 and XbaI sites. VEGF-
The 2 cDNA was cloned in frame with the signal sequence of the baculovirus gp67 gene and located at the 3 'end of the signal sequence of the vector. This is designated as pAcGP67A-VEGF-2.

PRG1 for expressing VEGF-2 with the signal sequence of the gp67 gene
For cloning into a vector, VEGF-2 having the signal sequence and some upstream sequences was ligated with XhoI and XbaI restriction enzymes at the Xho and XbaI restriction endonuclease sites located upstream of the VEGF-2 cDNA. , PAcGP67A-VEGF-2 plasmid. This fragment was separated from the rest of the vector on a 1% agarose gel and purified using the "Geneclean" kit. This is F
No. 2.

The PRG1 vector (a modification of the pVL941 vector) was transformed into a baculovirus expression system (for review: Summers, MD and Smith, GE,
"A Manual of Methods for Baculovirus
Vectors and Insect Cell Culture Pro
cedures "Texas Agricultural Expertimen
tal Station Bulletin No. 1555 (1987)) for expression of VEGF-2 protein. This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the restriction endonucleases BamHI, Smal, Xbal, BglII and Asp7.
Includes recognition sites for 18. The site for the restriction endonuclease Xho1 is located upstream of the BamH1 site. The sequence between Xho1 and BamH1 is the same as the sequence of the PAcGp67A (type-static) vector. The simian virus (SV) 40 polyadenylation site is used for efficient polyadenylation. For easy selection of recombinant viruses, E. coli The β-galactosidase gene from E. coli is inserted in the same direction as the polyhedrin promoter, followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequence is flanked on both sides by viral sequences for cell-mediated homologous recombination of co-transfected wild-type viral DNA. Many other baculovirus vectors are
It can be used in place of pRG1 (eg, pAc373, pVL941 and pAcIM1 (Luckow, VA and Summers, MD, Vi.
rology 170: 31-39 (1989)).

The plasmid was digested with restriction enzymes XboI and XbaI and then dephosphorylated using bovine intestinal phosphatase by procedures known in the art. The DN
A is then replaced with a commercially available kit ("Geneclean" BIO 101 Inc.
, La Jolla, Ca) using a 1% agarose gel. This vector DNA is called V2.

The fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNA ligase. Then, E. coli HB101 cells, and VE
Bacteria containing the plasmid with the GF-2 gene (pBac gp67-VEGF-2) were identified using the enzymes BamH1 and XbaI. The sequence of the cloned fragment was confirmed by DNA sequencing.

The 5 mg of plasmid pBac gp67-VEGF-2 was ligated with the lipofectin method (Felgner et al., Proc. Natl. Acad. Sci. USA 84:
7413-7417 (1987)) using 1.0 mg of commercially available linearized baculovirus DNA ("BaculoGoldJ baculovirus D").
NA ", Pharmingen, San Diego, CA. ) And co-transfected.

1 mg of BaculoGoldJ viral DNA and 5 mg of plasmid p
Bac gp67-VEGF-2 was added to 50 ml of serum-free Grace's medium (Life
Technologies Inc. , Gaithersburg, MD) in a sterile well of a microtiter plate. Thereafter, 10 ml of Lipofectin and 90 ml of Grace's medium were added, mixed and incubated for 15 minutes at room temperature. This transfection mixture is then
S seeded in 35 mm tissue culture plates with 1 ml serum-free Grace's medium
It was dropped on f9 insect cells (ATCC CRL 1711). The plate was tilted back and forth to mix the newly added solution. The plate is then incubated at 27 ° C for 5 hours. After 5 hours, the transfection solution was removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum was added. The plate was placed back in the incubator and the culture was continued at 27 ° C. for 4 days.

After 4 days, supernatants were collected and plaque assays performed as described by Summers and Smith, supra. By modification, "Blue Gal
(Life Technologies Inc., Gaithersburg)
The agarose gel containing g) was used to allow easy isolation of blue-stained plaques. (A detailed description of the “plaque assay” is also available at Life Techno
logs Inc. (Also found in the User's Guide for Insect Cell Culture and Baculovirology distributed at Gaithersburg, pp. 9-10).

Four days after serial dilution, virus was added to cells and blue-stained plaques were Ep
Picked up with the tip of a pendorf pipette. The agar containing the recombinant virus was then resuspended in an Eppendorf tube containing 200 ml Grace's medium. The agar is removed by brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect Sf9 cells seeded in a 35 mm dish. Four days later, the supernatants of these culture dishes are collected and then they are stored at 4 ° C.

Sf9 cells were grown in Grace's medium supplemented with 10% heat inactivated FBS. Cells were treated with recombinant baculovirus V-gp6 at a multiplicity of infection of 1 ("MOI").
7-VEGF-2 was infected. After 6 hours, the medium was removed and SF900 II medium (Life Technology) free of methionine and cysteine.
ies Inc. , Gaithersburg). 5 hours after 42 hours
Ci ³⁵ S-methionine and 5 mCi ³⁵ S-cysteine (Amersham)
) Was added. Cells were incubated for an additional 16 hours, then cells were harvested by centrifugation, and labeled protein was visualized by SDS-PAGE and autoradiography.

[0374] Proteins from the media and cytoplasm of Sf9 cells were analyzed by SDS-PAGE under non-reducing and reducing conditions. See FIGS. 8A and 8B, respectively. The medium was dialyzed against 50 mM MES, pH 5.8. After dialysis, a precipitate was obtained and resuspended in 100 mM Na citrate, pH 5.0. The resuspended precipitate was analyzed again by SDS-PAGE and stained with Coomassie brilliant blue. See FIG.

The culture supernatant was also diluted 1:10 in 50 mM MES, pH 5.8, and applied to a SP-650M column (1.0 × 6.6 cm, Toyopearl) at a flow rate of 1: 1.
It was applied at ml / min. The protein was prepared using 200, 300 and 500 mM NaC
Eluted with a step gradient of l. VEGF-2 was obtained using elution at 500 mM. The eluate was analyzed by SDS-PAGE in the presence or absence of the reducing agent b-mercaptoethanol and stained with Coomassie brilliant blue. See FIG.

Example 4 Expression of Recombinant VEGF-2 in COS Cells Expression of plasmid VEGF-2-HA was performed using the vector pcDNAI / Amp (I
nvitrogen), this vector pcDNAI / amp contains: (1) SV40 origin of replication, (2) ampicillin resistance gene, (3) E. coli. co
li replication origin, (4) CMV promoter, followed by polylinker region, SV
40 introns and polyadenylation sites. A DNA fragment encoding the entire VEGF-2 precursor and the HA tag fused in frame to its 3 'end was cloned into the polylinker region of this vector. Thus, expression of the recombinant protein is directed under the CMV promoter. The HA tag is (Wils
on et al., Cell 37: 767 (1984)), corresponding to an epitope derived from the influenza hemagglutinin protein previously described. Fusion of the HA tag to the target protein allows for easy detection of the recombinant protein using an antibody that recognizes the HA epitope.

The plasmid construction strategy is as described below.

The DNA sequence encoding VEGF-2 (ATCC No. 97149) was constructed by PCR using the following two primers: 5 ′ primer (CGC
GGA TCC ATG ACT GTA CTC TAC CCA) (SEQ ID NO: 14) contains a BamH1 site followed by a VEGF-
3 'sequence (CGC TCT AGA
TCA AGC GTA GTC TGG GAC GTC GTA TGG
GTA CTC GAG GCT CAT TTG TGG TCT 3 ')
(SEQ ID NO: 15) contains an XbaI site, an HA tag, an XhoI site, and VEGF.
-2 contains the complementary sequence to the last 15 nucleotides of the coding sequence (not including the stop codon). Thus, the PCR product was fused with a BamHI site, a coding sequence, followed by an XhoI restriction endonuclease site and an H-frame fused in frame.
It contains an A tag, a translation stop codon following the HA tag, and an XbaI site. PCR
The amplified DNA fragment and the vector pcDNAI / Amp were digested with BamHI and XbaI restriction enzymes and ligated. The ligation mixture is coli
SURE (Stratagene Cloning Systems, La
(Jolla, CA 92037), transformed cultures were plated on ampicillin medium plates and resistant colonies were selected. Plasmid DNA was isolated from transformants and tested by restriction analysis for the presence of the correct fragment. For recombinant VEGF-2 expression, COS cells were subjected to the DEAE-DEXTRAN method (J. Sambrook, E. Fritsch).
, T .; Maniatis, Molecular Cloning: A Labo
rattro Manual, Cold Spring Laboratory
Press (1989)). VE
Expression of GF-2-HA protein was determined by radiolabeling and immunoprecipitation (E. Har.
low and D.C. Lane, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory
y Press, (1988)). Cells were labeled with ³⁵ S-cysteine for 8 hours two days after transfection. The culture medium is then collected and the cells are washed with detergent (RIPA buffer (150 mM NaCl, 1% N
P-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM
Tris, pH 7.5) (Wilson et al., Cell 37: 767 (19
84)). Both cell lysate and culture medium were sedimented using an HA-specific monoclonal antibody. The precipitated protein was subjected to 15% SDS-PAG.
Analyzed on E-gel.

Example 5 Effect of Partially Purified VEGF-2 Protein on Proliferation of Vascular Endothelial Cells On day 1, human umbilical vein endothelial cells (HUVEC) were treated with 4% fetal bovine serum (FBS).
), 16 units / ml heparin and 50 units / ml endothelial cell growth complement (ECGS, Biotechnique, Inc.) in M199 medium at a density of 2-5 x 10 ⁴ cells / 35 mm dish. On day 2, the medium was replaced with M199 containing 10% FBS, 8 units / ml heparin. VEGF-2 protein of SEQ ID NO: 2, lacking the first 45 amino acid residues, (VE
GF) and basic FGF (bFGF) were added at the concentrations indicated. On days 4 and 6, the medium was replaced. On day 8, cell counts were determined using a Coulter Counter.
ter (see FIG. 12).

Example 6 Influence of Purified VEGF-2 Protein on Proliferation of Vascular Endothelial Cells On day 1, human umbilical vein endothelial cells (HUVEC) were treated with 4% fetal bovine serum (FBS).
), 16 units / ml heparin, 50 units / ml endothelial cell growth supplement (ECG
S, Biotechnique, Inc. ) In M199 medium containing ^Four Seeded at a density of cells / 35 mm dish. On day 2, the medium was replaced with M199 containing 10% FBS, 8 units / ml heparin. First 45 amino acids
Purified VEGF-2 protein of SEQ ID NO: 2 with the residues removed was added to the medium at this point
did. On days 4 and 6, the medium was replaced with fresh medium and supplements. 8
On the day, cell numbers were determined on a Coulter Counter (see FIG. 13).
thing).

Example 7 Expression via Gene Therapy Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue culture medium and divided into small pieces. Tissue chunks are placed on the wet surface of a tissue culture flask and approximately 10 pieces are placed in each flask. Turn the flask upside down,
Seal and leave at room temperature overnight. After 24 hours at room temperature, invert the flask,
The tissue mass is then left attached to the bottom of the flask and fresh media (eg,
Ham's F1 with 10% FBS, penicillin, and streptomycin
2 medium). It is then incubated for approximately one week at 37 ° C. At this point, fresh medium is added and replaced every few days thereafter. After an additional two weeks of culture, a monolayer of fibroblasts appears. The monolayer is trypsinized and scaled into a larger flask.

PMV-7 (Kirs) flanking the Moloney murine sarcoma virus long terminal repeat
chmeier, P .; T. DNA 7: 219-225 (1988)).
Digest with coRI and HindIII followed by treatment with calf intestinal phosphatase. The linear vector is fractionated on an agarose gel and purified using glass beads.

The cDNA encoding the polypeptide of the present invention is amplified using PCR primers corresponding to the 5 ′ terminal sequence and 3 ′ terminal sequence, respectively. The 5 'primer contains an EcoRI site and the 3' primer further contains a HindIII site. Equal amounts of Moloney murine sarcoma virus linear scaffold, and amplified Eco
The RI and HindIII fragments are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is used to transform bacterial HB101, which is then plated on kanamycin-containing agar for the purpose of verifying that the vector has the gene of interest properly inserted.

The amphotropic pA317 or GP + am12 packaging cells were
Du with 0% calf serum (CS), penicillin, and streptomycin
Grow to confluent density in tissue culture in lbecco modified Eagle's medium (DMEM). The MSV vector containing the gene is then added to the medium, and the packaging cells are transduced with the vector. At this point, the packaging cells produce infectious virions containing the gene (at this point, the packaging cells are called producer cells).

[0385] Fresh medium is added to the transduced producer cells, after which the medium is collected from a 10 cm plate of confluent producer cells. Spent media containing infectious virus particles is filtered through a millipore filter to remove detached producer cells, and the media is then used to infect fibroblasts. Media is removed from fibroblast subconfluent plates and immediately replaced with media from producer cells.
Remove this medium and replace with fresh medium. If the virus titer is high, virtually all fibroblasts are infected and selection is not required. If the titer is very low, it is necessary to use a retroviral vector with a selectable marker (eg, neo or his).

The engineered fibroblasts are then either alone or after being grown to confluence on cytodex 3 microcarrier beads,
Inject into host. At this point, the fibroblasts produce a protein product.

Example 8 Expression of VEGF-2 mRNA in Human Fetal and Adult Tissue Experimental Design Northern blot analysis was performed on VEGF-2 in human fetal and adult tissues.
Performed to test the level of mRNA expression. A cDNA probe containing the complete nucleotide sequence of the VEGF-2 protein was prepared according to the manufacturer's instructions.
rediprime ^o DNA labeling system (Amersham Life Sc ience) was labeled with ³² P using. After labeling, the probe was purified using a CHROMA SPIN-100 ^* column (Clontech Laboratories, Inc.) according to the manufacturer's protocol number PT1200-1. The purified labeled probe was then used to test various human tissues for VEGF-2 mRNA.

[0388] Various human tissues (fetal kidney, fetal lung, fetal liver, brain, kidney, lung, liver, spleen,
Multiple tissues northern (MT), including thoracic line, bone marrow, testicle, placenta, and skeletal muscle)
N) Blots were obtained from Clontech. MTN blots were tested with labeled probes using ExpressHyb ^* hybridization solution (Clontech) according to the manufacturer's protocol number PT1190-1. After hybridization and washing, the blot was exposed to film overnight at -70 ° C with an intensifying screen and developed according to standard procedures.

(Results) VEGF-2 mRNA expression was abundant in vascular smooth muscle and some highly vascularized tissues. VEGF-2 was expressed at significantly higher levels in tissues associated with hematopoietic and angiogenic activities, ie, fetal kidney, fetal lung, bone marrow, placenta, spleen and lung tissue. VEGF-2 expression levels are low in adult kidney, fetal liver, adult liver, testis; and almost undetectable in fetal and adult brain (see FIG. 14).

In primary cultures, VEGF-2 mRNA expression is abundant in vascular smooth muscle cells and dermal fibroblasts, but much lower in human umbilical vein endothelial cells (see FIG. 15). This mRNA distribution pattern is VEGF
Very similar to that of.

Example 9 Construction of Amino- and Carboxy-Terminal Deletion Mutants To identify and analyze biologically active VFGF-2 polypeptides,
A panel of EGF-2 deletion mutants was constructed using the expression vector pHE4a.

(1. Construction of VEGF-2 T103-L215 in pHE4) VEGF-2 T103- into E. coli protein expression vector pHE4
To enable amplification and subcloning of L215 (amino acids 103-215 of FIG. 1 or SEQ ID NO: 18) directed at the polymerase chain reaction, VEG
Two oligonucleotide primers complementary to the desired region of F-2 were synthesized using the following base sequence: 5 ′ primer (NdeI / START and 18 nucleotides of coding sequence): 5′-GCA GCA CAT ATG ACA GAA GAG ACT
ATA AAA-3 '(SEQ ID NO: 19) 3'-primer (Asp718, STOP, and 15 nucleotides of coding sequence): 5'-GCA GCA GGT ACC TCA CAG TTT AGA
CAT GCA-3 ′ (SEQ ID NO: 20) The 5 ′ primer (SEQ ID NO: 19) above incorporates an NdeI restriction site, and the 3 ′ primer (SEQ ID NO: 20) incorporates an Asp718 restriction site.
The 5 'primer (SEQ ID NO: 19) also contains an ATG sequence that is adjacent to and in frame with the VEGF-2 coding region;
The translation of the cloned fragment in E. coli allowed the 3 'primer (SEQ ID NO: 20) to flank the VEGF-2 coding region and
One stop codon in frame with the F-2 coding region (preferably E.c
oli, which is used in E. coli. Ensure correct translation termination in E. coli.

The polymerase chain reaction is performed using standard conditions well known to those skilled in the art, and as a template the nucleotide sequence for mature VEGF-2 (eg, amino acids 24 to SEQ ID NO: 18, as constructed in Example 3). 419). The resulting amplicon was restriction digested with NdeI and Asp718 and NdeI
/ Asp718 digested into pHE4a expression vector.

(2. Construction of VEGF-2 T103-R227 in pHE4) VEGF-2 T103- into E. coli protein expression vector pHE4
To enable amplification and subcloning of R227 (amino acids 103-227 of FIG. 1 or SEQ ID NO: 18) directed toward the polymerase chain reaction, VEG
Two oligonucleotide primers complementary to the desired region of F-2 were synthesized using the following base sequence: 5 ′ primer (NdeI / START and 18 nucleotides of coding sequence): 5′-GCA GCA CAT ATG ACA GAA GAG ACT
ATA AAA-3 '(SEQ ID NO: 19) 3'-primer (Asp718, STOP, and 15 nucleotides of coding sequence): 5'-GCA GCA GGT ACC TCA ACG TCT AAT
AAT GGA-3 ′ (SEQ ID NO: 21) In the case of the above primer, the NdeI or Asp718 restriction site was incorporated into the 5 ′ primer and the 3 ′ primer, respectively. This 5 'primer (
SEQ ID NO: 19) is also flanked by the VEGF-2 coding region and
A coding region and an in-frame ATG sequence; the translation of the cloned fragment in E. coli while the 3 'primer (SEQ ID NO: 2)
1) contains one stop codon (preferably a codon utilized in E. coli) that is adjacent to and in frame with the VEGF-2 coding region. Ensure correct translation termination in E. coli.

The polymerase chain reaction is performed using standard conditions well known to those skilled in the art, and as a template the nucleotide sequence for mature VEGF-2 (amino acids 24 to SEQ ID NO: 18 as constructed in Example 3). 419). The resulting amplicon was restriction digested with NdeI and Asp718 and NdeI
/ Asp718 digested into pHE4a protein expression vector.

3. Construction of VEGF-2 T103-L215 in pA2GP In this exemplary example, the plasmid shuttle vector pA2GP was replaced with a baculovirus leader and Summers et al., A Manual of M.
methods for Baculovirus Vectors and I
nsect Cell Culture Procedures, Texas
Agricultural Experimental Station Bu
Encode the VEGF-2 protein (amino acids 103-215 of FIG. 1 or SEQ ID NO: 18) with N-terminal and C-terminal deletions using standard methods as described by lletin number 1555 (1987). Cloned D
NA is used to insert into baculovirus to express the N-terminal and C-terminal deleted VEGF-2 protein. This expression vector is Auto
ograph californica nuclear polyhedrosis virus (AcMNPV)
And a secretory signal peptide (leader) for the baculovirus gp67 protein and convenient restriction sites such as BamHI, XbaI, and Asp718. Simian virus 40 ("SV4
0 ") is used for efficient polyadenylation. For easy selection of the recombinant virus, this plasmid is constructed with a weak Dro in the same orientation.
under the control of the E. sophila promoter. It contains the β-galactosidase gene from E. coli, followed by the polyadenylation signal of the polyhedrin gene.
The inserted gene is flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to produce a viable virus expressing the cloned polynucleotide.

[0397] Many other baculovirus vectors (eg, pAc373, pVL941
, And pAcIM1), as the skilled artisan will readily recognize such constructs as properly positioned signals for transcription, translation, secretion, etc., including signal peptides and in-frame AUGs as required. Can be used in place of the above vector as long as Such vectors are described, for example, in Luckow et al., V.
irology 170: 31-39 (1989).

In FIG. 1, the cDNA sequence encoding the VEGF-2 protein without the N-terminal 102 amino acids and without the C-terminal 204 amino acids was added to the 5 ′ and 3 ′ sequences of this gene. Amplification was performed using the corresponding PCR oligonucleotide primers.

The 5 'primer contains a BamHI restriction enzyme site (bold), followed by one spacer nucleotide to stay in frame with the signal peptide supplied by the vector, and 17 nucleotides of the coding sequence bases of the VEGF-2 protein. Array

[0400]

Embedded image Having. The 3 'primer was an XbaI restriction enzyme site (bold) followed by a stop codon and a sequence comprising 17 nucleotides complementary to the 3' coding sequence of VEGF-2.

[0401]

Embedded image Having.

[0402] The amplified sequence was converted to a commercially available kit ("Geneclean" BIO 101, I
nc. , La Jolla, CA) using a 1% agarose gel. The fragment was then converted to the endonucleases BamHI and Xba.
Digested with I and then purified again on a 1% agarose gel. This fragment was ligated at the BamHI and XbaI sites with the pA2 GP baculovirus transfer vector (Supplier). Through this connection, N-
VFGF-2 cDNA representing the terminal and C-terminally deleted VEGF-2 protein (amino acids 103-215 of FIG. 1 or SEQ ID NO: 18) was ligated to baculovirus G
It was cloned in frame with the signal sequence of the P gene and placed at the 3 'end of the vector signal sequence. This was designated as pA2GPVEGF-2. T1
It was named 03-L215.

4. Construction of VEGF-2 T103-R227 in pA2GP VEGF-2 protein in FIG. 1 without the N-terminal 102 amino acids and without the C-terminal 192 amino acids (ie, , SEQ ID NO: 18 amino acids 103-227) were amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' sequences of this gene.

[0404]

Embedded image The primers contained a BamHI restriction enzyme site (bold), followed by one spacer nucleotide to remain in frame with the signal peptide provided by the vector, and a sequence comprising 26 nucleotides of the coding sequence base of the VEGF-2 protein (sequence). No. 24). The 3 'primer is a sequence comprising an XbaI restriction enzyme site (bold) followed by a stop codon and 21 nucleotides complementary to the 3' coding sequence of VEGF-2.

[0405]

Embedded image Having.

The amplified sequence was prepared using a commercially available kit (“Geneclean” BIO 101, I
nc. , La Jolla, CA) using a 1% agarose gel. The fragment was then converted to the endonucleases BamHI and Xba.
Digested with I and then purified again on a 1% agarose gel. This fragment was ligated at the BamHI and XbaI sites with the pA2 GP baculovirus transfer vector (Supplier). Through this connection, N-
VFGF-2 cDNA representing the terminal and C-terminal truncated VEGF-2 protein (amino acids 103-227 of FIG. 1 or SEQ ID NO: 18) was cloned into Baculovirus G
It was cloned in frame with the signal sequence of the P gene and placed at the 3 'end of the vector signal sequence. This construct was constructed using pA2GPVEGF-2.
. It was named T103-R227.

5. Construction of VEGF-2 in pC1 The expression vectors pC1 and pC4 are compatible with the Rous sarcoma virus strong promoter (LTR) (Cullen et al., Molecular and Cellula).
r Biology, 438-447 (March 1985)) and fragments of the CMV enhancer (Boshart et al., Cell 41: 521-530 (1985)). Multiple cloning sites (e.g., those having restriction sites BamHI, XbaI, and Asp718) facilitate cloning of the gene of interest. This vector further contains the 3N intron of the rat preproinsulin gene, polyadenylation and termination signals.

The vector pC1 is used for the expression of VFGF-2 protein. Plasmid pC1 is plasmid pSV2-dhfr [ATCC accession number 37146].
Is a derivative of Both plasmids contain the mouse DHFR gene under the control of the SV40 early promoter. Chinese hamster ovary cells or other cells lacking dihydrofolate activity transfected with these plasmids were transformed into selective media (alpha minus ME) supplemented with the chemotherapeutic agent methotrexate.
M, Life Technologies). DHF in cells that are resistant to methotrexate (MTX)
R gene amplification has been well documented (eg, Alt, FW, Kel).
lems, R .; M. Bertino, J .; R. And Schimke, R .;
T. 1978; Biol. Chem. 253: 1357-1370, Ha
mlin, J.M. L. And Ma, C.I. 1990, Biochem. et Bio
phys. Acta, 1097: 107-143; Page, M .; L. And S
ydenham, M .; A. 1991, Biotechnology 9: 64-
68). Cells that grow at increased concentrations of MTX have a DHFR
By overgrowing the target enzyme DHFR as a result of gene amplification,
Develop resistance to drugs. If a second gene is linked to the DHFR gene, it is usually co-grown and overexpressed. Developing cell lines with more than 1,000 copies of the gene is a form of art. Subsequently, if methotrexate is removed, the cell line will contain the amplified gene integrated into the chromosome.

Plasmid pC1 is a strong promoter of the Rous sarcoma virus long terminal repeat (LTR) (Cullen et al., Molecula) for expression of the gene of interest.
r and Cellular Biology, March 1985: 438-4.
470) and the human cytomegalovirus (CMV) immediate-early gene enhancer (Boshart et al., Cell 41: 521-530 (1985)). Downstream of the promoter are the following single restriction sites that allow for gene integration: BamHI, PvuII, and NruI. After these cloning sites, the plasmid contains a translation stop codon in all three reading frames, followed by a 3N intron and a polyadenylation site of the rat preproinsulin gene. Other highly efficient promoters, such as the human b-actin promoter, the SV40 early or late promoter, or long terminal repeats from other retroviruses (HIV and HTLVI) can also be used for expression. For polyadenylation of mRNA, other signals from, for example, the human growth hormone gene or the globin gene can be used as well.

Stable cell lines with the gene of interest integrated into the chromosome are also gpt, G
418, or upon co-transfection with a selectable marker such as hygromycin. It is advantageous to first use one or more selectable markers (eg, G418 and methotrexate).

The plasmid pC1 was ligated with the restriction enzyme Bam by procedures known in the art.
Digest with HI and then dephosphorylate using calf intestinal phosphatase. The vector is then isolated from a 1% agarose gel.

The DNA sequence encoding VEGF-2 (ATCC Accession No. 97149) was
Constructed by PCR using two primers corresponding to the 5 'and 3' ends of the EGF-2 gene: 5 'primer (5'-GAT CGA TCC
ATC ATG CAC TCG CTG GGC TTC TTC TCT
GTG GCG TGT TCT CTG CTC G-3 ′ (SEQ ID NO: 26
)) Contains Klenow blunted BamHI site and 40 nucleotides of VEGF-2 coding sequence starting from the start codon; 3 ′ primer (5 ′
-GCA GGG TAC GGA TCC TAG ATT AGC TCA
TTT GTG GTC TTT-3 ′ (SEQ ID NO: 27)) contains 16 nucleotides of VEGF-2 coding sequence without a BamHI site and a stop codon.

The PCR-amplified DNA fragment is isolated from a 1% agarose gel as described above, then digested with the endonuclease BamHI, and then purified again on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated using T4 DNA ligase. Then, E. coli HB1
01 cells are transformed and bacteria containing the plasmid pC1 are identified. The sequence and orientation of the inserted gene is confirmed by DNA sequencing. This construct is called pC1VEGF-2.

(6. Construction of pC4SigVEGF-2 T103-L215) The plasmid pC4Sig is a plasmid pC4 (Accession No. 209646) containing the sequence of the human IgG Fc portion and the protein signal sequence.

Complementation of the desired region of VEGF-2 to allow amplification by polymerase chain reaction and subcloning into pC4Sig of VEGF-2 T103-L215 (amino acids 103-215 in FIG. 1 or SEQ ID NO: 18) Two unique oligonucleotide primers (with the following base sequences) were synthesized: 5 ′ primer (BamHI and 26 nt coding sequence): 5′-GCA GCA GGA TCC ACA GAA GAG ACT A
TA AAA TTT GCT GC-3 ′ (SEQ ID NO: 34): 3 ′ primer (XbaI, stop, and 15 nt coding sequence) 5′-CGT CGT TCT AGA TCA CAG TTT AGA C
AT GCA TCG GCA G-3 '(SEQ ID NO: 35).

The polymerase chain reaction was performed using standard conditions well known to those skilled in the art and, for example, the nucleotide sequence for mature VEGF-2 (amino acids 24-419) constructed in Example 3 as a template. The resulting amplicon was replaced with BamHI and Xb.
Restriction digested with aI and subcloned into pC4Sig vector digested with BamHI / XbaI.

7. Construction of pC4SigVEGF-2 T103-R227 To enable amplification by polymerase chain reaction of VEGF-2 T103-L215 (amino acids 103-227 in FIG. 1 or SEQ ID NO: 18) and subcloning into pC4Sig. 2 oligonucleotide primers (with the following base sequences) complementary to the desired region of VEGF-2 were synthesized: 5 ′ primer (BamHI and 26 nt coding sequence): 5′-GCA GCA GGA TCC ACA GAA GAG ACT A
TA AAA TTT GCT GC-3 ′ (SEQ ID NO: 34): 3 ′ primer (XbaI, stop, and 21 nt coding sequence) 5′-GCA GCA TCT AGA TCA ACG TCT AATA
AT GGA ATG AAC-3 '(SEQ ID NO: 25).

The polymerase chain reaction was performed using standard conditions well known to those skilled in the art and, for example, the nucleotide sequence for mature VEGF-2 (amino acids 24-419) constructed in Example 3 as a template. The resulting amplicon was replaced with BamHI and Xb.
Restriction digested with aI and subcloned into pC4Sig vector digested with BamHI / XbaI.

8. Construction of pC4VEGF-2 M1-M263 The expression vector pC4 is a strong promoter (LTR) of Rous sarcoma virus.
(Cullen et al., Molecular and Cellular Biol.
, 438-47 (March 1985) and fragments of the CMV enhancer (Boshart et al., Cell 41: 521-530 (1985)). Multiple cloning sites (eg, restriction enzyme cleavage sites BamHI, Xba
I and Asp718) facilitate cloning of the gene of interest. This vector further contains the 3N intron of the rat preproinsulin gene, a polyadenylation signal and a stop signal.

In this exemplary example, the cloned DNA encoding the C-terminally deleted VEGF-2 M1-M263 protein (amino acids 1-263 in FIG. 1 or SEQ ID NO: 18) was replaced with a C-terminally deleted VEGF-2 Insert into the plasmid vector pC4 to express the protein.

To enable amplification by the polymerase chain reaction of VEGF-2 M1-M263 and subcloning into the expression vector pC4, two oligonucleotide primers complementary to the desired region of VEGF-2 (the following bases) 5'-primer 5'-GAC TGG ATC CGC CAC CAT G
CA CTC GCT GGG CTT CTT CTC-3 ′ (SEQ ID NO: 28
3) Primer 5'-GAC TGG TAC CTT ATC ACAT
AA AAT CTT CCT GAG CC-3 '(SEQ ID NO: 29).

A BamHI restriction site was incorporated for the 5 ′ primer above, while an Asp718 restriction site was incorporated for the 3 ′ primer. The 5 'primer also contains 6 nt, 20 nt of the VEGF-2 coding sequence, as well as E. coli. The ATG sequence contains an ATG sequence that is adjacent to and in frame with the VEGF-2 coding region that allows translation of the cloned fragment in E. coli, while the 3 ′ primer
2 nt, 20 nt of the GF-2 coding sequence, and E. coli. flanking the VEGF-2 coding region, which ensures correct translation termination in E. coli, and contains one stop codon, in-frame, which is preferentially used in E. coli.

The polymerase chain reaction was performed using standard conditions well known to those skilled in the art and, for example, the nucleotide sequence for mature VEGF-2 (amino acids 24-419) constructed in Example 3 as a template. The resulting amplicon is converted to BamHI and As.
It was restriction digested with p718 and subcloned into a BamHI / Asp718 digested pC4 protein expression vector. This construct was cloned into pC4VEGF-2.
Named M1-M263.

9. Construction of pC4VEGF-2 M1-D311 In this illustrative example, a clone encoding the C-terminally deleted VEGF-2 M1-D311 protein (amino acids 1-311 in FIG. 1 or SEQ ID NO: 18). The modified DNA is inserted into the plasmid vector pC4 to express the C-terminally deleted VEGF-2 protein.

To enable amplification by polymerase chain reaction of VEGF-2 M1-D311 and subcloning into the expression vector pC4, two oligonucleotide primers complementary to the desired region of VEGF-2 (the following bases) 5'-primer 5'-GAC TGG ATC CGC CAC CAT G
CA CTC GCT GGG CTT CTT CTC-3 ′ (SEQ ID NO: 30
3) Primer 5'-GAC TGG TAC CTT ATC AGT C
TA GTT CTT TGT GGG G-3 ′ (SEQ ID NO: 31).

A BamHI restriction site was incorporated for the 5 'primer, while an Asp718 restriction site was incorporated for the 3' primer. The 5 'primer also contains 6 nt, 20 nt of the VEGF-2 coding sequence, as well as E. coli. flanking the VEGF-2 coding region that allows translation of the cloned fragment in E. coli and contains an ATG sequence that is in frame, while the 3 'primer
2 nt, 20 nt of the GF-2 coding sequence, and E. coli. flanking the VEGF-2 coding region, which ensures correct translation termination in E. coli and contains one stop codon in frame, which is preferentially used in E. coli.

The polymerase chain reaction was performed using standard conditions well known to those skilled in the art and, for example, the nucleotide sequence for mature VEGF-2 (amino acids 24-419) constructed in Example 3 as a template. The resulting amplicon is converted to BamHI and As.
It was restriction digested with p718 and subcloned into a BamHI / Asp718 digested pC4 protein expression vector.

10. Construction of pC4VEGF-2 M1-Q367 In this illustrative example, the cloned DNA encoding the C-terminally deleted VEGF-2 M1-D311 protein (amino acids 1-311 in SEQ ID NO: 18) was Plasmid vector p to express the C-terminally deleted VEGF-2 protein.
Insert into C4.

To enable amplification by the polymerase chain reaction of VEGF-2 M1-D311 and subcloning into the expression vector pC4, two oligonucleotide primers complementary to the desired region of VEGF-2 (bases below) 5'-primer 5'-GAC TGG ATC CGC CAC CAT G
CA CTC GCT GGG CTT CTT CTC-3 ′ (SEQ ID NO: 32
3) Primer 5'-GAC TGG TAC CTC ATT ACT G
TG GAC TTT CTG TAC ATT C-3 '(SEQ ID NO: 33).

A BamHI restriction site was incorporated for the 5 ′ primer above, while an Asp718 restriction site was incorporated for the 3 ′ primer. The 5 'primer also contains 6 nt, 20 nt of the VEGF-2 coding sequence, as well as E. coli. flanking the VEGF-2 coding region that allows translation of the cloned fragment in E. coli and contains an ATG sequence that is in frame, while the 3 'primer
2 nt, 20 nt of the GF-2 coding sequence, and E. coli. flanking the VEGF-2 coding region, which ensures correct translation termination in E. coli and contains one stop codon in frame, which is preferentially used in E. coli.

The polymerase chain reaction was performed using standard conditions well known to those skilled in the art and, for example, the nucleotide sequence for mature VEGF-2 (amino acids 24-419) constructed in Example 3 as a template. The resulting amplicon is converted to BamHI and As.
It was restriction digested with p718 and subcloned into a BamHI / Asp718 digested pC4 protein expression vector. This construct was cloned into pC4VEGF-2.
It is named M1-Q367.

Example 10 Transient Expression of VEGF-2 Protein in COS-7 Cells Experimental Design For example, expression of the VEGF-2-HA fusion protein from the construct made in Example 4 For example, Harlow and co-workers (Antibod
ies: A Laboratory Manual, 2nd edition; Cold Spr
ing Harbor Laboratory Press, Cold Spr
radiolabeling and immunoprecipitation using the method described in A. Harbor, New York (1988). To this end, two days after transfection, cells were plated in medium containing 35S cysteine for 8 days.
Labeled by incubation for hours. Cells and media are harvested and cells are washed, then detergent-containing RIPA buffer (150 mM NaCl, 1%, as described by Wilson and coworkers (supra)).
NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50m
M Tris (pH 7.5). Protein was precipitated from cell lysates and culture media using an HA-specific monoclonal antibody. The precipitated proteins were then analyzed by SDS-PAGE and autoradiography.

(Results) As shown in FIG. 16, cells transfected with pcDNA1 VEGF-2HA secreted 56 kd and 30 kd proteins. This 56 kd protein (but not the 30 kd protein) can also be detected in cell lysates, but not in controls. This means that 30kd protein is 56k
d suggests likely to result from cleavage of the protein. HA tag is VEG
Because it is at the C-terminus of F-2, this 30 kd protein must correspond to the C-terminal portion of the truncated portion, but the N-terminal portion of the truncated protein is not detected by immunoprecipitation. These data indicate that VEGF-2 protein expressed in mammalian cells is not secreted and processed.

Example 11 Stimulating Effect of VEGF-2 on Proliferation of Vascular Endothelial Cells Experimental Design VEGF-2 expression is abundant in highly vascularized tissues. Therefore, the role of VEGF-2 in regulating the proliferation of several types of endothelial cells was examined.

(Endothelial Cell Proliferation Assay) For evaluation of the mitotic activity of growth factors, colorimetric MTS (3- (4,5-dimethylthiazole-2-) was used together with the electronic coupling reagent PMS (phenazine methosulfate). Yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) 2H-tetrazolium) assay was performed (CellTiter).
96 AQ, Promega). Cells were seeded in 96-well plates (5,000 cells / well) in medium supplemented with 0.1 mL serum and allowed to attach overnight. After serum starvation in 0.5% FBS for 12 hours, conditions (bFGF, VEGF ₁₆₅ or VEGF-2 in 0.5% FBS, with or without heparin (8 U / ml) in 0.5% FBS) were added to the wells for 48 hours. Was added. 20 mg of MTS / PMS mixture (
1: 0.05) was added per well and allowed to incubate at 37 ° C. for 1 hour before measuring absorbance at 490 nm in an ELISA plate reader. Background absorbance from control wells (some media, no cells) was subtracted, and each condition was performed in parallel on 7 wells. Leak et al., I
n Vitro Cell. Dev. Biol. 30A: 512-518 (19
94).

(Results) VEGF-2 slightly stimulated the proliferation of human umbilical vein endothelial cells (HUVEC) and dermal microvascular endothelial cells (FIGS. 17 and 18). The stimulatory effect of VEGF-2 is more pronounced on endometrial and microvascular endothelial cell proliferation (
(FIG. 19). Endometrial endothelial cells (HEEC) showed the highest response to VEGF-2 (96% of the effect of VEGF on microvascular endothelial cells). VEGF-2
Response to microvascular endothelial cells (HMEC) was 73% compared to VEGF. The response of HUVEC and BAEC (bovine aortic endothelial cells) to VEGF-2 was significantly lower, at 10% and 7%, respectively. The activity of VEGF-2 protein varied between different purification runs, with the stimulatory effect of certain batches on HUVEC growth being significantly higher than the stimulatory effect of other batches.

Example 12 Inhibition of PDGF-Induced Vascular Smooth Muscle Cell Proliferation VEGF-2 expression is high in vascular smooth muscle cells. Smooth muscle is an important therapeutic target for vascular diseases such as restenosis. VEGF-2 on smooth muscle cells
To evaluate the potential effect of VEGF-2, we tested the effect of VEGF-2 on human aortic smooth muscle cell (HAoSMC) proliferation.

Experimental Design HAoSMC proliferation can be measured, for example, by incorporation of BrdUrd. Briefly, subconfluent resting cells growing on 4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP. The cells are then pulsed with 10% calf serum and 6 mg / ml BrdUrd. 2
Four hours later, immunocytology was performed using the BrdUrd Staining Kit (Zyme).
d Laboratories). Briefly, cells are incubated with a biotinylated mouse anti-BrdUrd antibody at 4 ° C. for 2 hours after exposure to a denaturing solution, and then with streptavidin-peroxidase and diaminobenzidine. After counterstaining with hematoxylin, cells are mounted for microscopic examination and BrdUrd positive cells are counted. B
The rdUrd index is calculated as the ratio of BrdUrd positive cells to the total cell number. In addition, simultaneous detection of BrdUrd staining (nuclei) and FITC uptake (cytoplasm) is performed on individual cells by joint use of bright-field and dark-field UV fluorescent illumination. Hayashida et al. Biol. Chem. 6; 271
(36): 21985-21992 (1996).

(Results) VEGF-2 has an inhibitory effect on PDGF-induced vascular smooth muscle cell proliferation, but VEGF-2 does not inhibit vascular smooth muscle cell proliferation induced by fetal bovine serum (FBS). No inhibitory effect (FIG. 20).

Example 13 Endothelial Cell Migration Stimulation Endothelial cell migration is an important step involved in angiogenesis.

Experimental Design Using this experiment, VEGF-2 was analyzed for lymphatic endothelial cell migration (lymphatic).
It explores the possibility of stimulating endothelial cell migration. At present, there are no published reports of such models. However, we have adopted a model of vascular endothelial cell migration for use with lymphatic endothelial cells essentially as follows: An endothelial cell migration assay was performed in a 48-well microchemotax.
is) using a chamber (Neuroprobe Inc., Cabin)
Jhon, MD; Falk, W.C. Goodwin, R .; H. J. , And Le
onard, E .; J. "A 48 well micro chemotaxi
s assembly for rapid and accurate me
assurement of leukocyte migration. "J.
Immunological Methods 1980; 33: 239-24.
7). A polyvinylpyrrolidone-free polycarbonate filter (Nucleopore Corp. Cambridge, MA) having a pore size of 8 μm is coated with 0.1% gelatin at room temperature for at least 6 hours and dried under sterile air. The test substance was M1 supplemented with 0.25% bovine serum albumin (BSA).
Dilute to the appropriate concentration in 99 and place 25 μl of the final dilution in the lower chamber of the modified Boyden apparatus. Subconfluent early passage (2-6)
HUVEC and BMEC cultures are washed and trypsinized for the minimum time required to achieve cell detachment. After placing the filter between the lower and upper chambers, suspended in 50 μl M199 containing 1% FBS 2
. 5 × 10 ⁵ cells are seeded in the upper compartment. The device is then incubated for 5 hours at 37 ° C. in a chamber humidified with 5% CO 2 to allow cell migration. After a period of incubation, the filter is removed and the top of the filter with non-migrated cells is scraped with a rubber policeman. The filter was fixed with methanol and Giemsa solution (D
if-Quick, Baxter, McGraw Park, IL). Migration is quantified by counting cells in three random high power fields (40 ×) in each well, and all groups are performed in quadruplicate.

Results In an assay to test HUVEC migration using a 48-well microchemotaxis chamber, VEGF-2 could stimulate HUVEC migration (FIG. 21).

Example 14 Stimulation of Nitric Oxide Production by Endothelial Cells Nitric oxide released by vascular endothelium is believed to be a mediator of vascular endothelial relaxation. VEGF-1 has been demonstrated to induce the production of nitric oxide by endothelial cells in response to VEGF-1. As a result, VEGF-2 activity
It can be assayed by determining the production of nitric oxide by endothelial cells in response to VEGF-2.

Experimental Design Nitric oxide was starved for 24 hours and then for 4 hours at various levels of VEG.
Measurements are made in 96-well plates of confluent microvascular endothelial cells after exposure to F-1 and VEGF-2. Nitric oxide in the medium
Determine the total amount of nitrite after reduction of nitric oxide-derived nitrate by nitrate reductase, determined by the use of s reagent. The effect of VEGF-2 on nitric oxide release was tested in HUVEC.

[0445] Briefly, NO release from cultured HUVEC monolayers was measured using a NO-specific polarographic electrode (Iso-NO, World Pr.
etc. instruments Instruments Inc. ) (1049). Calibration of the NO component was performed according to the following equation: 2KNO ₂ + 2KI + 2H ₂ SO ₄ 62NO + I ₂ + 2H ₂ O + 2K ₂ SO ₄ . Standard KNO graded concentrations of a calibration curve ₂ (0,5,10,25,50,100, 250, and 500 nmol / L) was obtained by adding the calibration solution containing KI and H ₂ SO ₄ . The specificity of the Iso-NO electrode for NO is true (au
tentic) predetermined by measurement of NO from NO gas (1050). The culture medium was removed and the HUVECs were washed twice with Dulbecco's phosphate buffered saline. The cells are then added to 5 ml of the filtered Krebs-Henseleit solution in a 6-well plate, and the cell plate is placed on a slide warmer (Lab Line Instrument) to maintain the temperature at 37 ° C.
s Inc. ). The NO sensor probe was inserted vertically into the well before adding different conditions (holding the electrode tip 2 mm below the surface of the solution). S-Nitrosoacetylpenicillamine (SNAP) was used as a positive control. The amount of NO released was expressed as picomoles per 1 × 10 ⁶ endothelial cells. All values reported were the average of 4-6 measurements in each group (number of cell culture wells). Leak et al., Biochem. and B
iophys. Res. Comm. 217: 96-105 (1995).

(Results) VEGF-2 was able to stimulate nitric oxide release in HUVEC (FIG. 22) to a higher level than VEGF. This suggested that VEGF-2 could alter vascular permeability and vasodilation.

Example 15: Effect of VEGF-2 on cord formation in angiogenesis Another step in angiogenesis is cord formation that is prominent in endothelial cell differentiation. This bioassay measures the ability of microvascular endothelial cells to form capillary-like structures (hollow structures) when cultured in vitro.

(Experimental Design) CADMEC (microvascular endothelial cells) was prepared using Cell Applications
Inc. Purchased as proliferating (passage 2) cells from Cell Applic
cultures in CADATION growth medium and used at passage 5.
For in vitro angiogenesis assays, the wells of a 48-well cell culture plate are coated with Cell Applications' adherent factor medium (200 ml / well) for 30 minutes at 37 ° C. Seed the CADMEC-coated wells at 7,500 cells / well and culture overnight in growth medium. Then
The growth medium is either control buffer or HGS protein (0.1-100 ng).
/ Ml) and replace with 300 mg of Cell Applications' cord formation medium, and the cells are cultured for an additional 48 hours. The number and length of capillary-like cords are quantified by using a Boeckeler VIA-170 video image analyzer. All assays are performed in triplicate.

[0449] Commercially available (R & D) VEGF (50 ng / ml) is used as a positive control. β-estradiol (1 ng / ml) is used as a negative control. An appropriate buffer (no protein) is also used as a control.

(Results) VEGF2 was observed to inhibit IFN-like cord formation which also stimulates endothelial cell proliferation (FIG. 23). This inhibitory effect may be a secondary effect of endothelial cell proliferation that is incompatible with the cord formation process.

Example 16: Angiogenic Effect on Chick Chorioallantoic Membrane Chick chorioallantoic membrane (CAM) is a well-established system for testing angiogenesis. Angiogenesis in the CAM is easily visible and can be quantified. VEGF
-2 was able to stimulate angiogenesis in the CAM.

(Experimental Design) (Embrys) Fertilized eggs of White Leghorn chickens (Gallus gallus) and fertilized eggs of Japanese quail (Coturnix coturnix) were subjected to 37
. Incubated at 8 ° C and 80% humidity. Differentiated C of 16-day-old chicken
AM and 13 day old quail embryos were studied using the following method.

CAM Assay On day 4 of development, a window was made in the eggshell of a hen's egg. Embryos were checked for normal development and eggs were plugged with cellotape. They were further incubated until day 13. Thermanox cover slips (Nunc, Naperville)
e, IL) were cut into discs about 5 mm in diameter. Sterile and salt-free growth factors were dissolved in distilled water and about 3.3 mg / 5 ml was pipetted onto the disc.
After air drying, the inverted disc was applied to the CAM. Three days later, specimens were fixed with 3% glutaraldehyde and 2% formamide and rinsed with 0.12 M sodium cacodylate buffer. They were photographed with a stereomicroscope [Wild M8] and embedded for semi-ultra and ultra-slicing as described above. Control was performed with the carrier disk alone.

Results This data demonstrates that VEGF-2 can stimulate angiogenesis 9-fold in CAM assays compared to untreated controls. However, this stimulus
Only 45% of the level of VEGF stimulation (FIG. 24).

Example 17 Angiogenesis Assay Using Matrigel Implants in Mice Experimental Design To establish an in vivo model for angiogenesis, testing protein activity, mice and rats were tested. , 20 mg BSA (negative control) and 1
Methylcellulose disks containing either mg bFGF and 0.5 mg VEGF-1 (positive control) were implanted subcutaneously.

The positive control disc showed signs of angiogenesis, while the BSA disc appeared to contain little angiogenesis. On day 9, one mouse had bFG
A clear response to F was shown.

Results Both VEGF proteins appeared to enhance Matrigel cellularity by a factor of about 2 by gross evaluation.

An additional 30 mice received BSA, bFGF, and variable amounts of VEGF-1,
Discs containing VEGF-2-B8 and VEGF-2-C4 were implanted. Each mouse received two identical disks instead of one control and one experimental disk.

[0459] All collected disc samples were analyzed for von Willebrand factor for detecting the presence of endothelial cells in the disc, and for flk-1 and flt- to distinguish between vascular and lymphatic endothelial cells. 4 and immunostained. But,
Final histochemical analysis of neovascularization and lymphangiogenesis could not be determined.

Example 18: Rescue of ischemia in a rabbit lower limb model Experimental design To study the in vivo effect of VEGF-2 on ischemia, a rabbit hind limb ischemia model was previously described (Takeshita). , S. et al., Am.
Hol 147: 1649-1660 (1995)) by surgical resection of the femoral artery. Resection of the femoral artery results in retrograde growth of thrombus and occlusion of the external iliac artery. In conclusion, blood flow to the ischemic limb depends on collateral vessels derived from the internal iliac artery (Takeshita, S. et al., Am. J. Pathol 1).
47: 1649-1660 (1995)). The 10-day interval allowed the rabbit to recover post-operatively and develop endogenous collateral vessels. On the 10th day after the operation (day 0), basal angiography was performed, and then the internal iliac artery of the ischemic limb was described (Riesse).
n, R. Hum. Gene Ther. 4: 749-758 (1993);
Leclerc, G .; J. et al. Clin. Invest. 90: 936-944
(1992)), using a hydrogel-coated balloon catheter, transfected with 500 mg of the naked VEGF-2 expression plasmid by the arterial gene transfer technique. When VEGF-2 was used in the treatment, 500
A single bolus of mg VEGF-2 protein or control was delivered over 1 minute to the internal iliac artery of the ischemic limb using an infusion catheter. On day 30, various parameters were measured in these rabbits.

(Results) VEGF-2 protein (FIG. 25, upper panel) and naked expression plasmid (
Both (FIG. 25, middle panel) were able to recover the following parameters in the ischemic limb. The recovery of blood flow, angiographic score appears to be slightly greater with administration of 500 mg of plasmid when compared to that with 500 mg of protein (FIG. 25, lower panel). The extent of recovery is comparable to that of VEGF in a separate test (data not shown). Vasodilators failed to achieve the same effect.
This suggests that restoration of blood flow is not solely due to vasodilatory effects.

(A. BP ratio (FIG. 25a)) Contractile pressure of ischemic limb: ratio of blood pressure to contraction pressure of normal limb.

[0463] 2. Blood flow and blood flow reserve (FIG. 25b) Rest FL: blood flow during unexpanded state Maximum FL: blood flow during fully expanded state (also indirect measurement of vascular volume) Reflects the ratio of maximum FL: static FL.

[0464] 3. Angiographic score (FIG. 25c) This is measured by collateral angiography. The score is determined by the percentage of circles in the overlapping grid, where the crossing impermeable arteries are divided by the total number m of rabbit thighs.

[0465] 4. Capillary density (FIG. 25d) The number of collateral capillaries was determined by microscopic examination of a section taken from the hind limb with a light microscope.

As discussed, VEGF-2 is processed into co-purified N- and C-terminal fragments. N-terminal fragments contain an intact putative functional domain and may be responsive for biological activity.

Example 19: Effect of VEGF-2 on vasodilation As described above, VEGF-2 is a mediator of vascular endothelial cell expansion, NO
May stimulate release. Because the expansion of vascular endothelial cells is important in lowering blood pressure, the ability of VEGF-2 to affect blood pressure in spontaneously hypertensive rats (SHR) was tested. VEGF-2 caused a dose-dependent decrease in diastolic blood pressure (FIGS. 26a and b). When administered at a dose of 300 mg / kg, VEGF-2
As the dose of was increased, there was a steady decrease in diastolic blood pressure, which reached statistical significance. The changes observed at this dose were not as different as those seen with acetylcholine (0.5 mg / kg). A decrease in mean arterial pressure (MAP) was also observed (FIGS. 26c and d). VEGF-2 (300 mg / kg) and acetylcholine reduced MAP in these SHR animals relative to normal levels.

In addition, escalating doses (0, 10, 30, 100, 300, and 900 mg / k
g) B8, C5, and C4 prep VEGF-2 were administered to 13-14 week old spontaneously hypertensive mice (SHR). Data are expressed as mean +/- SEM. Statistical analysis was performed by paired T-test,
Statistical significance was then defined as p <0.05 (relative to response to buffer alone).

A study using VEGF-2 (C5 prep) showed that VEGF-2 (C5 prep) significantly reduced blood pressure, but the magnitude of the response was used at a dose of 900 mg / kg. Showed that it was not as large as observed with VEGF-2 (B8 prep).

Studies using VEGF-2 (C4 preparation) showed that this CHO-expressed protein preparation gave results similar to those seen with C5 (ie,
(Statistically significant, but much smaller than the size observed for the B8 preparation)
(See Figures 26A-D).

As a control and the C4 and C5 batches of VEGF-2 gave a small, but not statistically significant, change in blood pressure, the experiment was performed on a separate CH.
The experiment was performed using an O-expressed protein (M-CIF). 10-900mg / k
Administration of M-CIF at doses in the g range did not result in significant changes in diastolic blood pressure. Slightly statistically significant reduction in mean arterial pressure between 100 and 900 mg / kg
, But the dose response was not significant. These results indicate that VEG
The decrease in blood pressure observed with the C4 and C5 batches of F-2 suggests that it was specific (ie, VEGF-2 related).

Example 20 Rat Ischemic Skin Flap Model Experimental Design Evaluation parameters include skin blood flow, skin temperature, and Factor VIII, immunohistochemical or endothelial cell alkaline phosphatase response. VE during skin ischemia
GF-2 expression will be studied using in situ hybridization.

The study in this model is divided into three parts as follows: a) Ischemic skin b) Ischemic skin wound c) Normal wound Experimental protocols include: a) 3 x 4 cm (single) Pedicled full-thickness random flap) (muscle flap on lower back of animal) b) Resectable wound on ischemic skin (4-6 mm in diameter) (skin flap) c) Various dosage ranges below Treatment of excisional wounds with VEGF-2 (day 0, 1, 2, 3, 4 post wound): 1 mg to 100 mg d) for histological, immunohistochemical and in situ studies Wound tissue harvesting at 3, 5, 7, 10, 14, and 21 days after wounding.

Example 21: Peripheral artery disease model Angiogenesis therapy using VEGF-2 was developed as a novel therapeutic strategy to restore peri-ischemic blood flow in the case of peripheral artery disease Got.

Experimental Design The experimental protocol includes: a) One side of the femoral artery is ligated to create hind limb ischemic muscle, the other side of the hind limb serves as a control b) 20 mg ~ VEGF-2 protein at dosages ranging from 500 mg to 2 mg
3 times intravenously and / or intramuscularly per week for 3 weeks (possibly more)
Deliver c) Ischemic muscle tissue is harvested at one, two and three weeks after VEGF-2 ligation for analysis of VEGF-2 expression and histology. A biopsy is also performed on the other side of the normal muscle of the contralateral hind limb.

Example 22 Ischemic Cardiomyopathy Model VEGF-2 is evaluated as a potent mitogen that can stimulate the development of collateral vessels and reconstitute new vessels after coronary artery occlusion. Alterations of VEGF-2 expression will be investigated in situ.

Experimental Design The experimental protocol includes: a) The heart is exposed by opening the chest on the left side of the rat. Immediately occlude the left coronary artery with a thin (6-0) suture and close the thorax. B) VEGF-2 protein at a dosage in the range of
3 times intravenously and / or intramuscularly per week for 4 weeks (possibly more)
Deliver c) 30 days after surgery, hearts are removed and sectioned for morphological and in situ analysis.

Example 23: Rat Corneal Wound Healing Model This animal model demonstrates the effect of VEGF-2 on neovascularization.

Experimental Design The experimental protocol includes: a) making a 1-1.5 mm long incision from the center of the cornea to the stromal layer b) the incision margin facing the outer cornea of the eye Insert spatula below c) Create pocket (its base is 1-1.5 mm from eye margin) d) Place pellet containing 50-500 mg VEGF-2 in pocket e) VEGF- Two treatments can be applied topically to corneal wounds at dosages ranging from 20-500 mg (daily treatment for 5 days).

Example 24 Diabetic Mice and Glucocorticoid Deficient Wound Healing Model Experimental Design The experimental protocol includes the following: Diabetic db + / db + Mouse Model To determine that VEGF-2 promotes the healing process, we use wound healing in a genetically diabetic mouse model. The full thickness wound healing model in db + / db + mice is clinically clear and is well characterized as a reproducible model of wound healing defects. The healing of diabetic wounds is not a contraction, but rather the formation of granulating tissue and re-epithelialization.
(Gartner, MH, et al., J. Surg. Res.
52: 389 (1992); Greenhalgh, D.C. G. FIG. Et al., Am. J. Pa
thol. 136: 1235 (1990)).

[0481] Diabetic animals have many unique characteristics observed in type II diabetes. Homozygous (db + / db +) mice have their normal heterozygous (db +
/ + M) Obese compared to littermates. Mutant diabetic (db + / db +) mice have a single autosomal recessive mutation on chromosome 4 (db +) (Colem
et al., Proc. Natl. Acad. Sci. USA 77: 283-29
3 (1982)). Animals exhibit polyphagia, polydipsia and polyuria. Mutant diabetic mice (db + / db +) have elevated blood glucose, increased or normal insulin levels, and suppression of cell-mediated immunity (Mandel et al., J. Immun
ol. 120: 1375 (1978); Debray-Sachs, M; et al., C.
lin. Exp. Immunol. 51 (1): 1-7 (1983); Leit
er et al., Am. J. of Pathol. 114: 46-55 (1985)).
Peripheral neuropathy (peripheral neuropathy), myocardial complications,
And microvascular lesions, basement membrane thickening and glomerular filtration abnormalities are described in these animals (Norido, F. et al., Exp. Neurol. 83 (2): 2).
21-232 (1984); Robertson et al., Diabetes 29 (
1): 60-67 (1980): Giacomelli et al., Lab Inves.
t. 40 (4): 460-473 (1979); Coleman, D .; L. , D
iabetes 31 (supplement): 1-6 (1982)). These homozygous diabetic mice develop insulin-resistant hyperglycemia, similar to human type II diabetes (Mandel et al., J. Immunol. 120: 1375-1377 (1).
978)).

The characteristics observed in these animals suggest that the healing in this model may be similar to the healing observed in human diabetes (Greehalgh et al., Am. J. of Pathol. 136: 1235-1246 ( 1990))
.

Animals Animals with genetically diabetic female C57BL / KsJ (db + / db +) mice and their non-diabetic (db + / + m) heterozygous littermates were used in this study (Ja
ckson Laboratories). Animals were purchased at the age of 6 weeks and the study began at 8 weeks of age. Animals were individually housed and had unlimited access to food and water. All operations were performed using aseptic techniques. The experiment was Hum
an Genome Science, Inc. Institutional
Animal Care and Use Committee and t
he Guidelines for the Care and Useo
f Performed according to the rules and guidance of Laboratory Animals.

Surgical Wound The wound protocol is based on previously reported methods (Tsuboi, R. and Rif).
Kin, D.K. B. , J. et al. Exp. Med. 172: 245-251 (1990).
). Briefly, on the day of wounding, animals are anesthetized by intraperitoneal injection of Avertin (0.01 mg / mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized water. . The dorsal area of the animal is shaved and the skin is washed with a 70% ethanol solution and iodine. The surgical area is dried using sterile gauze before the wound. A full thickness wound of 8 mm is then made using a tissue punch from Key et al. Immediately after the wound, the surrounding skin is gently stretched to remove the enlargement of the wound. The wound is left open for the duration of the experiment. The treatment is applied topically for 5 consecutive days from the day of the wound. Prior to treatment, the wound is gently washed with sterile saline and sponge gauze.

The wounds are visually examined on the day of surgery and every two days thereafter and photographed at a fixed distance. Wound closure is determined by daily measurements on days 1-5 and 8. Wounds are measured horizontally and vertically using a graduated Jameson caliper. A wound is considered to have healed when the granulation tissue is no longer visible and the wound is covered with a continuous epithelium.

VEGF-2 was administered from 4 mg to 500 mg / wound / day of VEG in vehicle for 8 days.
Administer using different dose ranges of F-2. Vehicle control group: 50
Receive mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg / kg). The wound and surrounding skin are then harvested for histology and immunohistochemistry. Tissue samples are placed in 10% neutral buffered formalin in a tissue cassette between biopsy sponges for further processing.

Experimental Design Three groups of 10 animals (5 diabetic controls and 5 non-diabetic controls) were evaluated for 1) vehicle placebo control, 2) VEGF-2. did.

Measurement of Wound Area and Closure Wound closure is analyzed by measuring the area on the vertical and horizontal axes and obtaining the total square area of the wound. Contraction is then estimated by establishing the difference between the initial wound area (day 0) and the post-treatment wound area (day 8). The wound area on day 1 was 64 mm ² (corresponding to the size of the skin punch). The calculation was performed using the following formula: [open area on day 8]-[open area on day 1] / [open area on day 1].

Histology Samples are fixed in 10% buffered formalin and paraffin-embedded blocks are cut perpendicular to the wound surface (5 mm) and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin (H & E) staining is performed on bisected wound sections. Histological examination of the wound is used to assess whether the healing process and the morphological appearance of the repaired skin are altered by treatment with KGF-2. This assessment included examination of the presence of cell accumulation, inflammatory cells, capillaries, fibroblasts, reepithelialization and epithelial maturity (Greenhalgh, DG
. Et al., Am. J. Pathol. 136: 1235 (1990)). A mechanical observer uses a graduated lens micrometer.

Immunohistochemistry Re-epithelialization Tissue sections are immunohistochemically stained with a polyclonal rabbit anti-human keratin antibody using the ABC Elite detection system. Human skin is used as a positive tissue control, while non-immune IgG is used as a negative control. Keratinocyte proliferation is determined by assessing the degree of re-epithelialization of the wound using a calibrated lens micrometer.

(Cell Proliferation Marker) Proliferating cell nuclear antigen / cyclin (PCNA) in skin samples was assayed for ABC El
Demonstrated by use of anti-PCNA antibody (1:50) using the item detection system. Human colon cancer serves as a positive tissue control and human brain tissue is used as a negative tissue control. Each sample contained sections with shedding of primary antibody and replacement with non-immune mouse IgG. The ranking of these sections is based on the degree of growth on a scale of 0-8, from the lower side of the scale reflecting slight growth to the higher side reflecting strong growth.

Statistical analysis The experimental data is analyzed using a one-tailed t test. A p-value less than 0.05 is considered significant.

B. Steroid Impaired Rat Model Inhibition of wound healing by steroids has been well documented in various in vitro and in vivo systems (Wahl, SM Glucocorticoi).
ds and Wound hearing. Anti-Inflammato
ry Steroid Action: Basic and Clinical
Aspects. 280-302 (1989); Wahl, S .; M. J. et al.
Immunol. 115: 476-481 (1975); Werb, Z .; J
. Exp. Med. 147: 1684-1694 (1978)). Glucocorticoids inhibit angiogenesis, vascular permeability (Ebert, RH et al., An. In
tern. Med. 37: 701-705 (1952)), proliferation of fibroblasts,
And collagen synthesis (Beck, LS, et al., Growth Factors).
. 5: 295-304 (1991); Haynes, B .; F. J. et al. Clin
. Invest. 61: 703-797 (1978)) and the production of a transient decrease in circulating monocytes (Haynes, BF et al., J. Clin. Inves).
t. 61: 703-797 (1978); Wahl, S .; M. "Glucoco
rticoids and Wound hearing ”Antiinfla
mmary Steroid Action: Basic and Cli
medical Aspects, Academic Press, New Yor
k, 280-302 (1989)). Systemic administration of steroids to attenuated wound healing is a well-established phenomenon in rats (Beck, LS, et al., Growth Factors. 5: 295-3).
04 (1991); Haynes, B .; F. J. et al. Clin. Invest.
61: 703-797 (1978); Wahl, S .; M. "Glucocort
"Icoids and Wound Healing" Antiinflamm
attory Steroid Action: Basic and Clini
cal Aspects, Academic Press, New York,
280-302 (1989); Pierce, G .; F. Proc. Nat
l. Acad. Sci. USA 86: 2229-2233 (1989)).

To demonstrate that VEGF-2 could accelerate the healing process, systemic administration of methylprednisolone resulted in multiple localizations of VEGF-2 on full thickness resected wounds of impaired healing rat skin. Evaluate the effect of the application.

(Animals) Young adult male Sprague Dawley rats (Charles River Laboratories) weighing 250-300 g are used in this example. Animals were purchased at the age of 8 weeks and the study started at 9 weeks of age. The healing response of rats is impaired at the time of wounding by systemic administration of methylprednisolone (17 mg / kg / rat intramuscularly). Animals are housed individually and are given food and water ad libitum. All operations are performed using aseptic techniques. In this study, Human
Genome Sciences, Inc. Institutional A
natural Care and Use Committee and the
Guidelines for the Care and Use of
Perform according to Laboratory Animal's rules and guidance.

Surgical Wound The wound protocol is performed according to Section A above. On the day of the wound, the animals are anesthetized with an intramuscular injection of ketamine (50 mg / kg) and xylazine (5 mg / kg). The dorsal area of the animal is shaved and the skin is washed with a 70% ethanol solution and an iodine solution. The surgical area is dried using sterile gauze before the wound. A wound with a total thickness of 8 mm is then made using a Key tissue punch. The wound is left open for the duration of the experiment. Application of the test material is performed topically for 7 consecutive days from the day of wounding and once daily following administration of methylprednisolone. Prior to treatment, the wound is gently washed with sterile saline and sponge gauze.

The wound is visually examined at the day of the wound and at the end of the procedure and photographed at a fixed distance. Wound closure is determined by measuring daily for days 1-5 and 8 for the figures. Wound the graduated James caliper
Measure horizontally and vertically using er. A wound is considered to have healed when granulation tissue is no longer visible and the wound is covered with a continuous epithelium.

VEGF-2 was administered at 4 mg-500 mg / wound / day of VEG in vehicle for 8 days.
Administer using different dose ranges of F-2. Vehicle control group: 50
Receive mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg / kg). The wound and surrounding skin are then harvested for histology. Tissue samples are placed in 10% neutral buffered formalin in a tissue cassette between biopsy sponges for further processing.

Experimental Design Four groups of 10 animals (5 treated with methylprednisolone and 5 without glucocorticoids) received 1) untreated group, 2) vehicle placebo control, 3) Evaluated for VEGF-2 treated group.

Measurement of Wound Area and Closure Wound closure is analyzed by measuring the area of the vertical and horizontal axes and obtaining the total area of the wound. Closure is then estimated by establishing the difference between the starting wound area (day 0) and the post-treatment wound area (day 8). The wound area on day 1 was 64 mm ² (corresponding to the size of the skin punch). The calculation was performed using the following formula: [open area on day 8]-[open area on day 1] / [open area on day 1].

Histology Samples are fixed in 10% buffered formalin, and paraffin-embedded blocks are cut perpendicular to the wound surface (5 mm) and cut using an Olympus microtome. Routine hematoxylin-eosin (H & E) staining was performed on bisected wound sections. Histological examination of the wound allows assessment of whether the healing process and the morphological appearance of the repaired skin have been improved by treatment with VEGF-2. A calibrated lens micrometer was used by a mechanical observer to determine the distance of the wound gap.

Statistical Analysis The experimental data is analyzed using a one-tailed t-test. A p-value less than 0.05 is considered significant.

Example 25: Specific peptide fragments for VEGF-2 monoclonal antibody production Four specific peptides (SP-40, SP-41, SP-42 and SP-4)
3) was produced. These are used to produce monoclonal antibodies for analyzing VEGF-2 processing. This peptide is shown below: “SP-40”: MTVLYPEYWKMY (amino acid 70 of SEQ ID NO: 18)
~ 81) 2. 2. "SP-41": KSIDNEWWRKTQSMPREV (amino acids 120 to 136 of SEQ ID NO: 18 (note the C-> S mutation at position 131)) “SP-42”: MSKLDVYRQVHSIIRR (amino acids 212 to 227 of SEQ ID NO: 18) 4. "SP-43": MFSSDAGDDSTDGFHDI (amino acids 263-279 of SEQ ID NO: 18) (Example 26: animal model of lymphedema) The purpose of this experimental approach was to establish and re-establish lymphatic circulatory system in the rat hind limb Is to generate an appropriate and consistent model of lymphedema to test the therapeutic effects of VEGF-2 in E. coli. Efficacy is measured by volume expansion of affected hind limbs, quantification of lymphatic vessels, total blood plasma protein, and histopathology. Acute lymphedema is observed for 7-10 days. Perhaps more importantly, the chronic progression of edema lasts up to 3-4 weeks.

Experimental Procedures Prior to the start of surgery, blood samples were taken for protein concentration analysis. Approximately 350 g of male rats are administered Pentobarbital. Subsequently, the right foot was shaved from the knee to the hip. Gauze dipped in 70% ethanol is applied to the shaved area. Blood is collected for serum total protein test. Circumference and volume measurements were taken prior to infusion of the dye into the paw and after marking of two measurement levels (middle of the pt of the dorsal paw, 0.5 cm above the heel). Inject 0.05 ml of 1% Evan's Blue intradermally on the dorsal skin of both the right and left feet. Perimeter and volume measurements are then taken following injection of the dye into the paw.

Using the knee joint as a landmark, an incision in the groin of the mid-leg is made so that it can be located around the blood vessels in the thigh. The flap is dissected and separated using forceps and a hemostat. After finding the blood vessels in the thigh, find the lymph vessels that run along the sides and bottom of the blood vessels. The major lymphatic vessels in this area are then electrically coagulated or sutured.

Using a microscope, a smooth incision is made in the muscles behind the legs (near the semitendinosus and adductor muscles). The popliteal lymph nodes are then found. The two proximal and two distal lymph vessels of the popliteal node and the distal blood supply are then ligated by suturing. The popliteal lymph nodes and any accompanying fat tissue are then removed by cutting connective tissue.

Care was taken to control for any mild bleeding caused by this procedure. After occlusion of the lymphatic vessels, the flap was removed from the liquid skin (Vetbond).
) (AJ Buck) to seal. Seal the edge of the detached skin to the underlying muscle tissue, but leave a gap of about 0.5 cm around the leg. The skin may also be fixed, if necessary, by ligating it to the underlying muscle.

Animals are housed individually (without bed) using nets to avoid infection. The recovering animals are identified via optimal edema peaks, which typically occur by 5-7 days.
I checked every day. A plateau edema peak was then observed. To assess the intensity of lymphedema, we measured the circumference and volume of the location on each of the two designated paws before surgery and daily for 7 days. The effects of plasma proteins having on lymphedema and the determination of whether protein analysis is a useful test perimeter will also be studied. The weight of both control and leg edema is assessed at two locations. The analysis is performed in a blind mode.

Perimeter measurement: Using cloth tape under short gas anesthesia to prevent leg movement
Measure the circumference of the leg. Measurements are taken by the two different people on the talus and dorsal foot, and then the two records are averaged. Recordings are taken from both control and edema legs.

Volumetric measurements: On the day of surgery, animals are anesthetized with Pentobarbital and tested before surgery. For daily measurements, the animals are anesthetized with a simple halothane (rapid fixation and immediate recovery), both legs are shaved and similarly marked with hydrophobic markers on the legs . First, put your feet in the water, then
The level of each mark is placed in the instrument and then measured by Buxco edema software (Chen / Victor). The data is recorded by one person, while the other feet on the marked area.

Blood plasma protein measurement: Blood is collected, allowed to fall, and serum separated before, and at the end of, the surgical procedure for total protein and Ca ²⁺ comparison.

[0514] Law Weight Comparison: After blood is drawn, the animals are prepared for tissue collection. Legs,
Cuts were made using the quiltine, then both the experimental and control legs were cut with a ligature and weighed. The second weighing was performed using the tibia-calcaneal joint (t
This was done when the ibio-cacanal joint was dissected and the paw was weighed.

Histological preparation: Dissecting the transverse muscles located in the area behind the knee (popliteal) and placing the metal skeleton, filling with frozen gel, immersing in chilled methyl butane and in a labeled sample bag Placed at -80EC until cut out. At the time of excision, the muscle was observed with a fluorescence microscope for lymph nodes. Other immuno / histological methods are currently being evaluated.

Example 27 Method of Treatment Using Gene Therapy for Production of VEGF-2 Polypeptide-In Vivo Another aspect of the invention is a method of treating disorders, diseases and conditions in vivo. The use of gene therapy methods. This method of gene therapy is directed to VEG operably linked to a promoter into an animal to increase VEGF-2 expression.
Naked nucleic acids (DNA, RNA, and antisense DNA or RN containing F-2)
Regarding the introduction of A). Such gene therapy and delivery techniques and methods are known in the art and include, for example, WO 90/11092, WO 98/11779; U.S. Pat. Nos. 5,693,622; 5,705,151; 5,580,859;
abata, H .; (1997) Cardiovasc. Res. 35 (3):
470-479, Chao, J et al. (1997) Pharmacol. Res. Three
5 (6): 517-522; Wolff, J .; A. (1997) Neuromu
scul. Disord. 7 (5): 314-318, Schwartz, B .;
(1996) Gene Ther. 3 (5): 405-411, Tsurum
i, Y. Et al., (1996) Circulation 94 (12): 3281-
See 3290 (hereby incorporated by reference).

Any method that delivers an injectable substance to the cells of an animal, such as injecting the VEGF-2 polynucleotide construct into the interstitial space of a tissue (heart, muscle, skin, lung, liver, intestine, etc.) Can be delivered. VEGF-2 polynucleotide constructs can also be delivered directly into arteries. VEGF-2 polynucleotide constructs can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA, acts to assist, facilitate, or enhance entry into cells, including viral sequences, viral particles, liposome formulations, lipofectin, or precipitants. Refers to sequences that do not contain any delivery vehicles. However, VEGF-2 polynucleotides can also be prepared in liposome formulations that can be prepared by methods well known to those skilled in the art (eg, Felgner PL.
(1995) Ann. NY Acad. Sci. 772: 126-139 and Abdallah B.A. (1995) Biol. Cell 85 (1): 1
-7 as taught in US Pat.

The VEGF-2 vector construct used in the gene therapy method is preferably a construct that is not integrated into the host genome but also does not contain sequences that allow it to replicate. Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transient nature of polynucleotide synthesis in the cell. Studies have shown that non-replicating DNA sequences can be introduced into cells and provide production of the desired polypeptide for periods of up to six months.

The VEGF-2 construct can be delivered to the interstitial space of a tissue in the animal,
Muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eyes, glands And connective tissue. The interstitial space of these tissues can be found in the intercellular fluid, between the reticulum fibers of organ tissues, between the elastic fibers in the walls of blood vessels or chambers, the mucopolysaccharide matrix between the collagen fibers of fibrous tissues, or in the connective tissue sheath muscle cells. Or the same matrix at the bone hiatus. It is likewise the space occupied by the circulating plasma and the lymph of the lymph channels. They can be conveniently delivered by injection into the tissue containing these cells. These are preferably delivered to persistent, differentiated, non-dividing cells and are expressed in the non-dividing cells, but are delivered and expressed in undifferentiated or fully differentiated cells (eg, (Such as blood stem cells or dermal fibroblasts). Preferably, they are delivered by direct injection into the artery.

For a naked polynucleotide injection, an effective dose of DNA or RNA will be about 0
. It ranges from 05 g / kg body weight to about 50 mg / kg body weight. Preferably, the dosage will be from about 0.005 mg / kg to about 20 mg / kg, and more preferably, from about 0.05 mg / kg to about 5 mg / kg. Of course, as the skilled artisan will appreciate, this dosage will vary according to the tissue site of the injection. Appropriate and effective doses of nucleic acid sequence can be readily determined by one skilled in the art, and will depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of the tissue or directly into the artery. However, other parenteral routes, such as, for example, inhalation of an aerosol formulation for delivery to the lung or bronchial tissue, mucosa of the throat or nose, may also be used. In addition, the naked VEGF-2 construct can be delivered to arteries during angioplasty by the catheter used in this procedure.

The in vivo dose response effect of the injected VEGF-2 polynucleotide construct in the arteries is determined as follows. Appropriate template DNA for the production of mRNA encoding VEGF-2 is prepared according to standard recombinant DNA methodology. The template DNA, which can be circular or linear, is either used as naked DNA or complexed with liposomes. The rabbit artery is then injected with various amounts of template DNA.

Rabbit hind limb ischemia is surgically induced as described in Example 18. Immediately after this, five different sites (adductor muscle (2 sites), medial large (
2) and semi-membrane-like muscle (1) through a small skin incision
The plasmid DNA encoding VEGF-2 is directly injected using a modified 3 ml syringe and a 2 gauge needle. The skin is then closed using 4.0 nylon.

The ability to rescue hindlimb ischemia was assessed by measuring the ischemic hindlimb from untreated rabbits, calf blood pressure measurement and flow intra-arterial Doppler guidewire.
wire) as determined by measuring the number of capillaries in light microscopy sections taken from the treated hind limb as compared to the measurements (Takeshita et al., J.
. Clin. Invest. 93: 662-670 (1994)). The results of the above experiments in rabbits can be used to infer appropriate dosages and other treatment parameters in humans and other animals using VEGF-2 polynucleotide naked DNA.

Example 28, Method of Treatment Using Gene Therapy-Ex Vivo One method of gene therapy involves transplanting fibroblasts, which can express VEGF-2 polypeptide, into a patient. is there. Generally, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue culture medium and separated into small pieces. A small piece of tissue is placed on the wet surface of a tissue culture flask and approximately 10 pieces are placed in each flask. The flask is inverted, stoppered, and left overnight at room temperature. After 24 hours at room temperature, the flask is inverted. And the tissue mass still remains fixed at the bottom of the flask, and fresh media (eg, 10% FBS
, Ham's F12 medium with penicillin and streptomycin). The flask is then incubated at 37 ° C. for about one week.

At this time, fresh medium is added and subsequently changed every few days. After another two weeks of culture, a monolayer of fibroblasts appears. This monolayer is trypsinized,
Then scale up to a larger flask.

PMV-7 (Kirschmeier, PT et al., DNA, 7: 219-25)
(1988) (adjacent to the Moloney murine sarcoma virus long terminal repeat).
It is digested with RI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on an agarose gel and purified using glass beads.

The cDNA encoding VEGF-2 was isolated from the 5 ′ and 3 ′ as shown in Example 1.
Amplification can be performed using PCR primers, each corresponding to a terminal sequence. Preferably, the 5 'primer contains an EcoRI site and the 3' primer is a HindI
II site. Equal amounts of Moloney murine sarcoma virus linear scaffold and amplified EcoRI and HindIII fragments are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is then used to transform bacterial HB101, and the bacterial HB101 is then transformed into VEGF with the vector properly inserted.
Plate on agar containing kanamycin for the purpose of confirming that it contains -2.

Amphotropic pA317 packaging cells or GP + am12 packaging cells to confluent density in tissue culture in Dulbecco's Modified Eagle Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. Proliferate. Next, MS containing the VEGF-2 gene
The V vector is added to the medium and the packaging cells are transduced with the vector. Here, the packaging cells produce infectious viral particles containing the VEGF-2 gene (where the packaging cells are referred to as producer cells).

[0530] Fresh media is added to the transduced producer cells, and the media is subsequently harvested from 10 cm plates of confluent producer cells.
Spent media containing infectious virus particles is filtered through a Millipore filter to remove detached producer cells. This medium is then used to infect fibroblasts. The medium is removed from the subconfluent plate of fibroblasts and quickly replaced with the medium from the producer cells. Remove this medium and replace with fresh medium. If the virus titer is high, then substantially all fibroblasts are infected and no selection is required. If this titer is very low, then it is necessary to use a retroviral vector with a selectable marker (eg, neo or his). Once the fibroblasts are efficiently infected, the fibroblasts are analyzed to determine if VEGF-2 protein is being produced.

The engineered fibroblasts are then transplanted onto the host, either as is or after growing to confluence on cytodex 3 microcarrier beads.

Example 29, Method of Treatment Using Gene Therapy, Homologous Recombination Another method of gene therapy according to the present invention is via homologous recombination, for example, as described below. Operably linking an endogenous VEGF-2 sequence to a promoter: US Pat. No. 5,642, issued June 24, 1997.
No. 1,670; International Publication No. WO 96/29 published September 26, 1996.
No. 411; International Publication No. WO 94/12650 published on Aug. 4, 1994.
No .; Koller et al., Proc. Natl. Acad. Sci. USA 86:
8932-8935 (1989); and Zijlstra et al., Nature.
342: 435-438 (1989). The method involves activating a gene that is present in the target cell but is not expressed in that cell, or that is expressed at a lower level than desired.

A polynucleotide construct is generated that includes a promoter and a target sequence, which are homologous to the 5 ′ non-coding sequence of endogenous VEGF-2 adjacent to the promoter. The target sequence is sufficiently close to the 5 'end of VEGF-2 so that the promoter is operably linked to the endogenous sequence upon homologous recombination. The promoter and target sequence can be amplified using PCR. Preferably, the amplified promoter contains different restriction sites at the 5 'and 3' ends. Preferably, the 3 'end of the first target sequence contains the same restriction enzyme site as the 5' end of the amplified promoter, and the 5 'end of the second target sequence is the 3' end of the amplified promoter. And the same restriction sites.

The amplified promoter and the amplified target sequence are digested with appropriate restriction enzymes and subsequently treated with calf intestinal phosphatase. The digested promoter and the digested target sequence are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The construct is size fractionated on an agarose gel and then purified by phenol extraction and ethanol precipitation.

In this example, the polynucleotide construct is administered as a naked polynucleotide via electroporation. However, the polynucleotide construct may also be administered with a transfection facilitating agent (eg, liposomes, viral sequences, viral particles, precipitants, etc.). Such methods of delivery are known in the art.

Once the cells have been transfected, homologous recombination occurs, which results in a promoter operably linked to the endogenous VEGF-2 sequence. This results in expression of VEGF-2 in the cell. Expression may be detected by immunological staining or any other method known in the art.

[0537] Fibroblasts are obtained from the subject by skin biopsy. The resulting tissue is placed in DMEM and 10% fetal calf serum. Exponentially growing or early stationary phase fibroblasts are trypsinized and rinsed from the plastic surface with nutrient media. An aliquot of the cell suspension is removed for counting and the remaining cells are subjected to centrifugation. The supernatant is aspirated and the pellet is washed with 5 ml of electroporation buffer (20 mM HEPES (pH 7.3), 137 mM NaCl, 5 mM
Resuspend in KCl, 0.7 mM Na ₂ HPO ₄ , 6 mM dextrose). The cells are centrifuged again, the supernatant is aspirated, and the cells are resuspended in electroporation buffer containing 1 mg / ml acetylated bovine serum albumin. The final cell suspension contains about 3 × 10 ⁶ cells / ml. Electroporation should be performed immediately after resuspension.

[0537] Plasmid DNA is prepared according to standard techniques. To construct a plasmid for targeting to the VEGF-2 locus, plasmid pUC18 (MBI
Fermentas, Amherst, NY) is digested with HindIII. C
The MV promoter is amplified by PCR that adds an XbaI site at the 5 'end and a BamHI site at the 3' end. Two VEGF-2 non-coding sequences are amplified via PCR: one VEGF-2 non-coding sequence (VEGF-2 fragment 1) is amplified by adding a 5'-end HindIII site and a 3'-end Xba site. The other VEGF-2 non-coding sequence (VEGF-2 fragment 2) is amplified by adding a BamHI site at the 5 'end and a HindIII site at the 3' end. C
MV promoter and VEGF-2 fragment were converted to appropriate enzymes (CMV promoter-XbaI and BamHI; VEGF-2 fragment 1-XbaI;
VEGF-2 fragment 2-BamHI) and ligate together. The resulting ligation product is digested with HindIII and ligated with the HindIII digested pUC18 plasmid.

The plasmid DNA was placed in a sterile cuvette with a 0.4 cm electrode gap (Bi
o-rad). The final DNA concentration is generally at least 120 μg /
ml. Then 0.5 ml of the cell suspension (containing about 1.5 × 10 ⁶ cells) is added to the cuvette and the cell suspension and DNA solution are mixed gently. Electroporation is performed using a Gene-Pulser device (Bio-rad). The capacitance and voltage are set to 960 μF and 250-300 V, respectively. As the voltage increases, cell survival decreases, but the percentage of surviving cells that stably integrates the introduced DNA into the genome of the cell increases dramatically. Given these parameters, a pulse time of about 14-20 mSec should be observed.

The electroporated cells are maintained at room temperature for about 5 minutes, then the contents of the cuvette are gently removed using a sterile whole pipette. The cells are added directly to 10 ml of pre-warmed nutrient medium (DMEM with 15% calf serum) in a 10 cm dish and incubated at 37 ° C. (EC).
The next day, the medium is aspirated and replaced with 10 ml of fresh medium and an additional 1
Incubate for 6-24 hours.

The engineered fibroblasts are then injected into the host, either as such or after they have been grown to confluence on cytodex 3 microcarrier beads. Here, fibroblasts produce a protein product.

Example 30, VEGF-2 transgenic animals VEGF-2 polypeptides can also be expressed in transgenic animals.
Animals of any species (including mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, including, for example, baboons, monkeys, and chimpanzees, (Not limited to these)
Can be used to create transgenic animals. In certain embodiments, the techniques described herein or otherwise known in the art are used to express a polypeptide of the invention in humans as part of a gene therapy protocol.

[0539] Any technique known in the art can be used to introduce a transgene (ie, a polypeptide of the invention) into an animal to create a founder line of the transgenic animal. Such techniques include, but are not limited to: pronuclear microinjection (Paterson et al., Appl. Microbio
l. Biotechnol. 40: 691-698 (1994); Carver.
Et al., Biotechnology (NY) 11: 1263-1270 (1993)
); Wright et al., Biotechnology (NY) 9: 830-834.
(1991); and Hoppe et al., US Pat. No. 4,873,191 (198).
9))); reproductive line (Van der Putten et al., Proc. Natl. A
cad. Sci. USA 82: 6148-6152 (1985)), retrovirus-mediated gene transfer into blastocysts or embryos; gene targeting in embryonic stem cells (Thompson et al., Cell 56: 313-321 (1989)); Electroporation of embryos (Lo, 1983, Mol Cell. B
iol. 3: 1803-1814 (1983)); Introduction of a polynucleotide of the present invention using a gene gun (eg, Ulmer et al., Science 259: 1).
745 (1993)); introducing the nucleic acid construct into embryonic pleuripotent stem cells and transferring the stem cells into blastocysts; and sperm-mediated gene transfer (Lavitrano et al., Cell 57: 717-723 (
1989); For an overview of such techniques, see Gordon, "Tra.
nsgenic Animals "Intl. Rev .. Cytol. 115: 1
71-229 (1989), which is hereby incorporated by reference in its entirety.

[0544] Any art-known technique can be used to generate transgenic clones containing the polynucleotides of the present invention (eg, cultured embryonic, fetal or adult cells that have been induced to quiescent state). Nuclear transfer of nuclei of origin into enucleated oocytes) (Campell et al., Nature 380: 64-66 (1996); Wi).
Imut et al., Nature 385: 810-813 (1997)).

The present invention relates to transgenic animals that carry the transgene in all these cells, as well as transgenic animals that carry the transgene in some but not all of these cells. (That is,
Mosaic animals or chimeras). The transgene can be integrated as a single transgene or as multiple copies (eg, in a concatamer (eg, head-to-head or head-to-tail tandem)). This transgene is also described, for example, in Lasko et al. (Lasko et al., Proc. Natl. Acad. Sc.
i. USA 89: 6232-6236 (1992)), and can be selectively introduced into and activated in particular cell types. The regulatory sequences required for such cell type-specific activation will depend on the particular cell type of interest, and will be apparent to those skilled in the art. If it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred.

[0546] Briefly, if such a technique were to be employed, a vector containing some nucleotide sequences homologous to the endogenous gene would be transferred to the endogenous gene through homologous recombination with chromosomal sequences. For the purpose of incorporating into the function of the nucleotide sequence of the gene,
And designed for the purpose of destroying this function. The transgene can be selectively introduced into a particular cell type, and thus, for example, the gene can be expressed by Gu et al. (Gu et al., Sc.
inactivate the endogenous gene only in that cell type, according to the teachings of Science 265: 103-106 (1994). The regulatory sequences required for such cell type-specific inactivation depend on the particular cell type of interest and will be apparent to those skilled in the art.

[0547] Once the transgenic animals are created, the expression of the recombinant gene can be assayed using standard techniques. To confirm that transgene integration has occurred, initial screening can be accomplished to analyze animal tissues by Southern blot analysis or PCR techniques. The expression level of the transgene mRNA in the tissue of the transgenic animal also includes, but is not limited to, Northern blot analysis, in situ hybridization analysis, and reverse transcriptase PCR (rt-PCR) of tissue samples obtained from the animal. Can be evaluated using techniques that are not used. A sample of the transgenic gene-expressing tissue can also be evaluated immunocytochemically or immunohistochemically, using antibodies specific for the transgene product.

Once founders have been produced, they can be propagated, inbred, outbred,
Alternatively, they may be crossed to produce colonies of a particular animal. Examples of such reproductive strategies include, but are not limited to: outcrossing founders with more than one integration site to establish a segregation line; the effect of further expression of each transgene Therefore, inbreeding of segregants to produce complex transgenics expressing higher levels of the transgene; predetermined integration both to enhance expression and eliminate the need for animal screening by DNA analysis Mating of heterozygous transgenic animals to produce animals homozygous for the site; mating of isolated homozygous lines to produce a composite heterozygous or homozygous line; and Mating to place the transgene on a different background appropriate to the experimental model.

The conditions and / or disorders associated with abnormal VEGF-2 expression were studied and VEG
Animal model systems useful in, but not limited to, screening for compounds that are effective in increasing the biological function of the F-2 polypeptide and in alleviating such conditions or disorders. The transgenic animals of the present invention have been used.

Example 31, VEGF-2 Knockout Animal Endogenous VEGF-2 gene expression was also determined using targeted homologous recombination in VEG.
It can be reduced by inactivating or "knocking out" the F-2 gene and / or its promoter (eg, Smithies et al., Nature).
317: 230-234 (1985); Thomas and Capecchi.
Thompson et al., Cell 51: 503-512 (1987).
15: 313-321 (1989); each of which is incorporated herein by reference in its entirety). For example, DNA homologous to an endogenous polynucleotide sequence (either the coding region or the regulatory region of this gene)
A variant, non-functional polynucleotide (or completely unrelated DNA sequence) of the invention, flanked by or without a selectable marker and / or a negative selectable marker, It can be used to transfect cells expressing the polypeptide. In another embodiment, knockouts are generated in cells containing, but not expressing, the gene of interest using techniques known in the art. Insertion of this DNA construct via targeted homologous recombination results in inactivation of the targeted gene. Such an approach is particularly suitable in the research and agricultural fields where modifications to embryonic stem cells can be used to generate progeny of animals with inactive targeting genes (eg, Thomas and C.)
apechi 1987 and Thompson 1989, supra). However, this approach is not suitable for use in humans when the recombinant DNA construct is administered directly in vivo using an appropriate viral vector apparent to those of skill in the art, or is targeted to the required site. It can be customarily adapted.

In a further embodiment of the invention, cells engineered to express a polypeptide of the invention or cells (eg, knockouts) that are engineered to not express a polypeptide of the invention are obtained in vivo. To be administered to patients. Such cells may be obtained from a patient (ie, an animal including a human) or an MHC compatible donor, and these cells may include fibroblasts, bone marrow cells, blood cells (eg, lymphocytes), adipocytes, muscle cells , Endothelial cells, etc., but are not limited thereto. The cells can be transformed, for example, by transduction (using a viral vector, and preferably a vector that integrates the transgene into the genome of the cell) or transfection procedure (plasmid, cosmid, YAC, naked DNA, electroporation, (Including but not limited to the use of liposomes, etc.) to introduce a coding sequence for a polypeptide of the invention into cells, or alternatively, a coding and / or endogenous regulatory sequence associated with a polypeptide of the invention. Are engineered in vitro using recombinant DNA technology to destroy DNA. The coding sequence of a polypeptide of the present invention may be placed under the control of a strong constitutive promoter or a strong inducible promoter or promoter / enhancer to achieve expression and preferably secretion of the VEGF-2 polypeptide. Engineered cells that express and preferably secrete a polypeptide of the present invention can be introduced into a patient systemically (eg, in the circulation or intraperitoneally).

Alternatively, the cells can be incorporated into a matrix and implanted into the body, for example, genetically engineered fibroblasts can be implanted as part of a skin graft; engineered endothelium Cells may be transplanted as part of a lymphatic or vascular graft (eg, Anderson et al., US Pat. No. 5,399,349;
And Mulligan and Wilson, US Patent No. 5,460,959.
See issue. Each of these is hereby incorporated by reference in its entirety).

If the cells to be administered are non-autologous compatible cells or non-MHC compatible cells, these cells can be administered using well-known techniques, which will Prevents the development of a host immune response. For example, the cells can be introduced in an encapsulated form, which allows for the exchange of components with the initial extracellular environment, but does not allow the introduced cells to be recognized by the host immune system.

The conditions and / or disorders associated with abnormal VEGF-2 expression can be studied to
Animal model systems useful in, but not limited to, increasing the biological function of the GF-2 polypeptide and screening for compounds effective in ameliorating such conditions and / or disorders. The knockout animal of the present invention is used.

Many modifications and variations of the present invention are possible in light of the above teachings,
Within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

The complete disclosure of all publications (including patents, patent applications, journal articles, laboratory manuals, books or other documents) cited herein is hereby incorporated by reference. You.

[0557]

[Table 2]

[0558]

[Table 3]

[Sequence list]

[Brief description of the drawings]

1A-1E show the full length nucleotide (SEQ ID NO: 1) and deduced amino acid (SEQ ID NO: 2) sequences of VEGF-2. This polypeptide contains approximately 419 amino acid residues, approximately 23 represent the leader sequence. Use the standard one-letter abbreviations for amino acids. Sequencing was performed using a model 373 automated DNA sequencer (Applied Biosystems, Inc.). Sequencing accuracy is expected to be greater than 97%.

2A-2D show the nucleotide (SEQ ID NO: 3) sequence and the deduced amino acid (SEQ ID NO: 4) sequence of a shortened biologically active form of VEGF-2. This polypeptide has approximately 3 amino acids, with the first 24 amino acids representing the leader sequence.
It contains 50 amino acid residues.

FIGS. 3A and 3B show PDGFa (SEQ ID NO: 5) and PDGFb (SEQ ID NO: 6).
7 is an illustration of the amino acid sequence homology between VEGF (SEQ ID NO: 7) and VEGF-2 (SEQ ID NO: 4). The box region indicates the conserved sequence and the position of the eight conserved cysteine residues.

FIG. 4 shows, in table form, PDGFa, PDGFb, VEGF and VEG.
The percentage of homology with F-2 is shown.

FIG. 5 shows the presence of VEGF-2 mRNA in a human breast tumor cell line.

FIG. 6 shows the results of Northern blot analysis of VEGF-2 in adult human tissues.

FIG. 7 shows a photograph of an SDS-PAGE gel after in vitro transcription, translation, and electrophoresis of a polypeptide of the invention. Lane 1: ¹⁴ C and rainbow molecular weight markers; Lane 2: FGF control; Lane 3: VEGF-2 produced by M13 reverse and M13 forward primers; Lane 4
: VEGF-2 produced by M13 reverse primer and VEGF-F4 primer; Lane 5: VEGF-2 produced by M13 reverse primer and VEGF-F5 primer.

FIG. 8A shows a photograph of an SDS-PAGE gel. VEGF-2 polypeptide was expressed in a baculovirus system consisting of Sf9 cells. Proteins from cell media and cytoplasm were analyzed by SDS-PAGE under non-reducing conditions.

FIG. 8B shows a photograph of an SDS-PAGE gel. VEGF-2 polypeptide was expressed in a baculovirus system consisting of Sf9 cells. Proteins from cell media and cytoplasm were analyzed by SDS-PAGE under reducing conditions.

FIG. 9 shows a photograph of an SDS-PAGE gel. S infected with the nucleic acid sequence of the present invention
The medium from f9 cells was precipitated. The resuspended precipitate was analyzed by SDS-PAGE and stained with Coomassie brilliant blue.

FIG. 10 shows a photograph of an SDS-PAGE gel. VEGF-2 was purified from the culture supernatant and analyzed by SDS-PAGE in the presence and absence of the reducing agent, b-mercaptoethanol, and stained with Coomassie brilliant blue.

FIG. 11 shows an RP-300 column (0.21 × 3 cm, Applied Bio).
systems, Inc. 3) shows a reverse phase HPLC analysis of purified VEGF-2 using). The column was equilibrated with 0.1% trifluoroacetic acid (solvent A) and the protein was eluted with a gradient of 0% to 60% solvent B (consisting of acetonitrile with 0.07% TFA) for 7.5 minutes. Eluted. Protein elution was measured at 215 nm ("red").
Line) and 280 nm ("blue" line) absorbance. The percentage of solvent B is indicated by the "green" line.

FIG. 12 is a bar graph illustrating the effect of partially purified VEGF-2 protein on vascular endothelial cell proliferation as compared to basic fibroblast growth factor.

FIG. 13 is a bar graph illustrating the effect of purified VEGF2 protein on vascular endothelial cell proliferation.

FIG. 14 shows VEGF-2 mRNA in human fetal tissues and adult human tissues.
1 shows the expression of.

FIG. 15 shows VEGF-2 mRNA expression in primary human cultured cells.

FIG. 16A shows transient expression of VEGF-2 protein in COS-7 cells.

FIG. 16B shows transient expression of VEGF-2 protein in COS-7 cells.

FIG. 17 shows VEGF-2 stimulated proliferation of human umbilical vein endothelial cells (HUVEC).

FIG. 18 shows VEGF-2 stimulated proliferation of skin microvascular endothelial cells.

FIG. 19 shows the stimulatory effect of VEGF-2 on proliferation of microvessels, umbilical cord, endometrium, and bovine aortic endothelial cells.

FIG. 20A shows inhibition of PDGF-induced proliferation of vascular (human aortic) smooth muscle cells.

FIG. 20B shows inhibition of PDGF-induced proliferation of vascular (human aortic) smooth muscle cells.

FIG. 21 shows VEGF-2 induced HUVEC and bovine microvascular endothelial cells (BM
EC) shows stimulation of migration.

FIG. 22 shows stimulation of HUVEC nitric oxide release by VEGF-2 and VEGF-1.

FIG. 23 shows the inhibition of cord formation of microvascular endothelial cells (CADEC) by VEGF-2.

FIG. 24 shows VEGF, VEGF-2, and bFG in CAM assays.
F shows stimulation of angiogenesis by F.

FIG. 25A shows VEGF-2 protein (FIG. 25, a, d, e, j, m) and naked expression plasmid (FIG. 25, b, f, g, k, n) in ischemic limbs. Indicate restoration of certain parameters: BP ratio (FIGS. 25a-c).

FIG. 25B shows VEGF-2 protein (FIGS. 25, a, d, e, j, m) and naked expression plasmids (FIGS. 25, b, f, g, k, n) in ischemic limbs. Indicate restoration of certain parameters: BP ratio (FIGS. 25a-c).

Figure 25C shows VEGF-2 protein (Figure 25, a, d, e, j, m) and naked expression plasmid (Figure 25, b, f, g, k, n) in ischemic limbs. Indicate restoration of certain parameters: BP ratio (FIGS. 25a-c).

FIG. 25D shows VEGF-2 protein (FIG. 25, a, d, e, j, m) and a naked expression plasmid (FIG. 25, b, f, g, k, n) in ischemic limbs. Indicate the restoration of certain parameters: blood flow and flow storage (FIGS. 25d-i).

FIG. 25E shows VEGF-2 protein (FIGS. 25, a, d, e, j, m) and naked expression plasmids (FIGS. 25, b, f, g, k, n) in ischemic limbs. Indicate the restoration of certain parameters: blood flow and flow storage (FIGS. 25d-i).

Figure 25F shows VEGF-2 protein (Figure 25, a, d, e, j, m) and naked expression plasmid (Figure 25, b, f, g, k, n) in ischemic limbs. Indicate the restoration of certain parameters: blood flow and flow storage (FIGS. 25d-i).

FIG. 25G shows VEGF-2 protein (FIGS. 25, a, d, e, j, m) and naked expression plasmids (FIGS. 25, b, f, g, k, n) in ischemic limbs. Indicate the restoration of certain parameters: blood flow and flow storage (FIGS. 25d-i).

Figure 25H shows VEGF-2 protein (Figure 25, a, d, e, j, m) and naked expression plasmid (Figure 25, b, f, g, k, n) in ischemic limbs. Indicate the restoration of certain parameters: blood flow and flow storage (FIGS. 25d-i).

FIG. 25I shows VEGF-2 protein (FIG. 25, a, d, e, j, m) and naked expression plasmid (FIG. 25, b, f, g, k, n) in ischemic limbs. Indicate the restoration of certain parameters: blood flow and flow storage (FIGS. 25d-i).

Figure 25J shows VEGF-2 protein (Figure 25, a, d, e, j, m) and naked expression plasmids (Figure 25, b, f, g, k, n) in ischemic limbs. Indicate the restoration of certain parameters: angiographic score (FIGS. 25j-l).

FIG. 25K shows VEGF-2 protein (FIGS. 25, a, d, e, j, m) and naked expression plasmids (FIGS. 25, b, f, g, k, n) in ischemic limbs. Indicate restoration of certain parameters: angiographic score (FIGS. 25j-l).

FIG. 25L shows VEGF-2 protein (FIG. 25, a, d, e, j, m) and naked expression plasmid (FIG. 25, b, f, g, k, n) in ischemic limbs. Indicate the restoration of certain parameters: angiographic score (FIGS. 25j-l).

FIG. 25M shows in the ischemic limb with VEGF-2 protein (FIG. 25, a, d, e, j, m) and a naked expression plasmid (FIG. 25, b, f, g, k, n). Indicate the restoration of certain parameters: density of capillaries (Fig. 25mo).

Figure 25N shows VEGF-2 protein (Figure 25, a, d, e, j, m) and naked expression plasmid (Figure 25, b, f, g, k, n) in ischemic limbs. Indicate the restoration of certain parameters: density of capillaries (Fig. 25mo).

FIG. 25O shows VEGF-2 protein (FIG. 25, a, d, e, j, m) and naked expression plasmid (FIG. 25, b, f, g, k, n) in ischemic limb. Indicate the restoration of certain parameters: density of capillaries (Fig. 25mo).

FIG. 26A shows the ability of VEGF-2 to affect diastolic blood pressure in spontaneously hypertensive rats (SHR). FIG. 26a shows the dose-dependent reduction achieved with VEGF-2 in diastolic blood pressure.

FIG. 26B shows the ability of VEGF-2 to affect diastolic blood pressure in spontaneously hypertensive rats (SHR). FIG. 26b shows the dose-dependent reduction achieved with VEGF-2 in diastolic blood pressure.

FIG. 26C shows the ability of VEGF-2 to affect diastolic blood pressure in spontaneously hypertensive rats (SHR). FIG. 26c shows the decrease in mean arterial pressure (MAP) observed with VEGF-2.

FIG. 26D shows the ability of VEGF-2 to affect diastolic blood pressure in spontaneously hypertensive rats (SHR). FIG. 26d shows the decrease in mean arterial pressure (MAP) observed with VEGF-2.

FIG. 26E shows the ability of VEGF-2 to affect diastolic blood pressure in spontaneously hypertensive rats (SHR). Panel E shows mean arterial pressure (MAP) of SHR rats.
4 shows the effect of increasing doses of VEGF-2 on GF.

FIG. 26F shows the ability of VEGF-2 to affect diastolic blood pressure in spontaneously hypertensive rats (SHR). Panel F shows VE versus diastolic pressure in SHR rats.
4 shows the effect of GF-2.

FIG. 26G shows the ability of VEGF-2 to affect diastolic blood pressure in spontaneously hypertensive rats (SHR). Panel G shows V versus diastolic blood pressure in SHR rats.
4 shows the effect of EGF-2.

FIG. 27 shows inhibition of VEGF-2N = and VEGF-2-induced proliferation.

FIG. 28 shows a schematic depiction of the pHE4a expression vector (SEQ ID NO: 16).
Kanamycin resistance marker gene, linker regions for multiple cloning sites, o
The positions of the riC sequence and the lacIq coding sequence are shown.

FIG. 29 shows the nucleotide sequence of the regulatory element of the pHE4a promoter (SEQ ID NO: 17). Two lac operator sequences, the Shine-Dalgarno sequence (S / D), and the terminal HindIII and NdeI restriction sites (italics) are shown.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 17/06 A61P 19/02 19/02 C07K 14/50 C07K 14/50 16/22 16/22 C12Q 1 / 68 A C12N 5/10 1/70 15/09 ZNA A61K 37/24 C12Q 1/68 C12N 5/00 B 1/70 15/00 ZNAA (81) Designated countries EP (AT, BE, CH, CY, DE , DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML) , MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD) , RU, T , TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW , MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW ( 71) Applicant 9410 Key West Avenue, Rockville, Maryland 20850, United States of America (72) Inventor Kao, Liang Hong Kong South Horizons, Block 13A, Flat 32D (72) Invention , Jin-shan United States 94086, Sunnyvale, Lakeside Drive Number 3034 1247 F term (reference) 4B024 AA01 BA21 CA04 DA02 DA06 EA02 EA04 GA11 HA01 4B063 QA01 QQ79 QR77 QR79 QS33 QS34 QX01 4B065 AA90A AAAAAAAAAAAAAAAAAAAAAAAAA93 CA44 4C084 AA02 AA13 BA23 BA35 DB57 ZA331 ZA332 ZA361 ZA362 ZA451 ZA452 ZB111 ZB112 ZB151 ZB152 ZB261 ZB262 ZB351 ZB352 4H045 AA10 BA10 CA40 DA01 EA24 FA74

Claims (26)

[Claims] 1. An isolated polynucleotide comprising: (a) an N-terminal deletion fragment described by general formula m-396 of SEQ ID NO: 2; (b) SEQ ID NO: 2 (C) N-terminal and C-terminal deleted fragments described by general formulas mn of SEQ ID NO: 2; and (d) sequence An isolated polynucleotide comprising a nucleic acid sequence that is at least 95% identical to a polynucleotide encoding a polypeptide selected from the group consisting of: a C-terminal deletion fragment represented by the general formula + 9-n of No. 2 . 2. The polypeptide according to claim 1, wherein the polypeptide is amino acid residue S-205 of SEQ ID NO: 2.
2. The isolated polynucleotide of claim 1, comprising -S-396 and / or amino acid residues F-9-R203 of SEQ ID NO: 2. 3. A composition comprising the isolated polynucleotide of claim 1. 4. The isolated polynucleotide of claim 1, wherein said polynucleotide encodes a biologically active fragment of VEGF-2. 5. The isolated polynucleotide of claim 1, wherein said polynucleotide encodes a polypeptide that binds an antibody for VEGF-2. 6. The polynucleotide of claim 1, further comprising a polynucleotide encoding a heterologous polypeptide. 7. A vector comprising the polynucleotide according to claim 1. 8. The vector of claim 7, wherein said polynucleotide is operably linked to a heterologous regulatory sequence. 9. A host cell comprising the vector according to claim 7 or the polynucleotide according to claim 1. 10. A method for producing a VEGF-2 polypeptide, comprising the steps of: (a) under conditions suitable for producing the polypeptide, the host of claim 9. Culturing cells; and (b) recovering the polypeptide from the cell culture. 11. A polypeptide produced by the method of claim 10. 12. An isolated polypeptide, comprising: (a) an N-terminal deletion fragment described by general formula m-396 of SEQ ID NO: 2; (C) N-terminal and C-terminal deletion fragments described by general formulas mn of SEQ ID NO: 2; and (d) SEQ ID NO: An isolated polypeptide, comprising a polypeptide that is at least 95% identical to an amino acid sequence selected from the group consisting of: a C-terminal deletion fragment described by 2 general formulas + 9-n. 13. The polypeptide of claim 2, wherein the polypeptide is amino acid residue S-20 of SEQ ID NO: 2.
5-S-396, and / or amino acid residues F-9-R-203 of SEQ ID NO: 2
13. The isolated polypeptide of claim 12, comprising: 14. A composition comprising the isolated polypeptide of claim 12. 15. A first polypeptide fragment comprising amino acid residues S-205 to S-396 of SEQ ID NO: 2, and amino acid residues F-9 to R of SEQ ID NO: 2
A second polypeptide fragment comprising -203. 16. The isolated polypeptide of claim 12, wherein said polypeptide is a biologically active fragment. 17. The isolated polypeptide of claim 12, wherein said polypeptide is antigenic. 18. The isolated polypeptide of claim 12, further comprising a heterologous polypeptide. 19. An antibody against the polypeptide according to claim 12. 20. A compound that activates the polypeptide of claim 12. 21. A compound that inhibits the polypeptide of claim 12. 22. A method for preventing, treating, or ameliorating a medical condition, wherein said method comprises treating a mammalian subject with a therapeutically effective amount. A method comprising administering the polypeptide of claim 13, the composition of claim 3, 14-15, or the polynucleotide of claim 1-2. 23. A method for diagnosing a pathological condition or susceptibility to a pathological condition in a subject associated with the expression or activity of a secreted protein, the method comprising the following steps: (a) Determining the presence or absence of a mutation in the described polynucleotide; (b) diagnosing a pathological condition or susceptibility to the pathological condition based on the presence or absence of the mutation. Method. 24. A method for diagnosing a pathological condition or susceptibility to a pathological condition in a subject associated with the expression or activity of a secreted protein, the method comprising the following steps: (a) a biological sample Determining the presence or amount of expression of the polypeptide of claim 12; (b) diagnosing a pathological condition or susceptibility to the pathological condition based on the presence or amount of expression of the polypeptide. A method comprising: 25. A method for identifying a binding partner to a polypeptide according to claim 10, comprising: (a) contacting the polypeptide according to claim 12 with a binding partner. Process;
And (b) determining whether the binding partner affects the activity of the polypeptide. 26. A method for identifying activity in a biological assay, comprising:
The method comprises the following steps: (a) expressing the polypeptide of claim 12 in cells; (b) isolating the supernatant; (c) detecting activity in a biological assay. And d) identifying a protein in said supernatant having said activity.