Human receptor tyrosine kinase, KDR

An isolated nucleic acid molecule encoding a novel human receptor type tyrosine kinase gene, KDR, is disclosed. The isolation of this KDR cDNA sequence results in disclosure of purified forms of human KDR protein, recombinant vectors and recombinant hosts which express human KDR.

STATEMENT REGARDING FEDERALLY-SPONSORED R&D
 Not applicable.
 REFERENCE TO MICROFICHE APPENDIX
 Not applicable.
 FIELD OF THE INVENTION
 The present invention relates to an isolated nucleic acid molecule
 (polynucleotide) which encodes a human receptor tyrosine kinase, KDR,
 which is expressed on human endothelial cells. This receptor is activated
 by VEGF and mediates a mitogenic signal. The present invention also
 relates to recombinant vectors and recombinant hosts which contain a DNA
 fragment encoding human KDR, a DNA fragment encoding the intracellular
 portion of KDR, a DNA fragment encoding the extracellular portion of KDR
 with or without a membrane anchor sequence, substantially purified forms
 of associated human KDR, and human mutant forms of KDR.
 BACKGROUND OF THE INVENTION
 Vascular endothelial cells form a luminal non-thrombogenic monolayer
 throughout the vascular system. Mitogens promote embryonic vascular
 development, growth, repair and angiogenesis in these cells. Angiogenesis
 involves the proteolytic degradation of the basement membrane on which
 endothelial cells reside followed by the subsequent chemotactic migration
 and mitosis of these cells to support sustained growth of a new capillary
 shoot. One class of mitogens selective for vascular endothelial cells
 include vascular endothelial growth factor (referred to as VEGF or VEGF-A)
 and the homologues placenta growth factor (PlGF), VEGF-B and VEGF-C.
 Human VEGF exists as a glycosylated homodimer in one of four mature
 processed forms containing 206, 189 (see U.S. Pat. No. 5,240,848), 165
 (see U.S. Pat. No. 5,332,671), and 121 (U.S. Pat. No. 5,332,671) amino
 acids, the most prevalent being the 165 amino acid form. The 206 amino
 acid and 189 amino acid forms of human VEGF each contain a highly basic
 24-amino acid insert that promotes tight binding to heparin, and
 presumably, heparin proteoglycans on cellular surfaces and within
 extracellular matrices (Ferrara et al., 1991, J. Cell. Biochem.
 47:211-218).
 Human PlGF is also a glycosylated homodimer which shares 46% homology with
 VEGF at the protein level. Differential splicing of human PlGF mRNA leads
 to either a 170 or 149 amino acid residue precursor, which are
 proteolytically processed to mature forms of 152 or 131 amino acid
 residues in length, respectively (Maglione et al., 1993, Oncogene 8:
 925-931; Bayne and Thomas, 1992, EPO Publication No. 0 506 477 A1; Hauser
 and Weich, 1993, Growth Factors 9: 259-268).
 VEGF-B has been isolated and characterized (Grimmond et al., 1996, Genome
 Research 6: 124-131; Olofsson et al., 1996, Proc. Natl. Acad. Sci. USA 93:
 2576-2581). The full-length human cDNAs encode 188 and 207 amino acid
 residue precursors wherein the NH.sub.2 terminal portions are
 proteolytically processed to mature forms 167 and 186 amino acid residues
 in length. Human VEGF-B expression was found predominantly in heart and
 skeletal muscle as a disulfide-linked homodimer. However, human VEGF-B may
 also form a heterodimer with VEGF (id. @ 2580).
 VEGF-C has also been isolated and characterized (Joukov et al., 1996, EMBO
 J. 15: 290-298). A cDNA encoding VEGF-C was obtained from a human
 prostatic adenocarcinoma cell line. A 32 kDa precursor protein is
 proteolytically processed to generate the mature 23 kDa form, which binds
 the receptor tyrosine kinase, Flt-4.
 VEGF and its homologues impart activity by binding to vascular endothelial
 cell plasma membrane-spanning tyrosine kinase receptors which then
 activate an intracellular mitogenic signal. The KDR receptor family is the
 major tyrosine kinase receptor which transduces the mitogenic signal
 initiated by VEGF.
 Shibuya et al. (1990, Oncogene 5: 519-524) disclose a human receptor type
 tyrosine kinase gene fit, which comprises a 4.2 Kb open reading frame
 encoding a 1338 amino acid protein which comprises a glycosylated
 extracelluar domain, membrane spanning region and predicted tyrosine
 kinase domain.
 Pajusola et al. (1992, Cancer Res. 52: 5738-5743) disclose a human receptor
 type tyrosine kinase gene which, as noted above, binds human VEGF-C.
 Vascular endothelial growth factor (VEGF) binds the high affinity
 membrane-spanning tyrosine kinase receptors KDR and Flt-1. Cell culture
 and gene knockout experiments indicate that each receptor contributes to
 different aspects of angiogenesis. KDR mediates the mitogenic function of
 VEGF whereas Flt-1 appears to modulate non-mitogenic functions such as
 those associated with cellular adhesion. Inhibiting KDR thus significantly
 diminishes the level of mitogenic VEGF activity.
 Vascular growth in the retina leads to visual degeneration culminating in
 blindness. VEGF accounts for most of the angiogenic activity produced in
 or near the retina in diabetic retinopathy. Ocular VEGF mRNA and protein
 are elevated by conditions such as retinal vein occlusion in primates and
 decreased pO.sub.2 levels in mice that lead to neovascularization.
 Intraocular injections of either anti-VEGF mono-clonal antibodies or VEGF
 receptor immunofusions inhibit ocular neovascularization in rodent and
 primate models. Regardless of the cause of induction of VEGF in human
 diabetic retinopathy, inhibition of ocular VEGF is useful in treating the
 disease.
 Expression of VEGF is also significantly increased in hypoxic regions of
 animal and human tumors adjacent to areas of necrosis. Monoclonal and
 polyclonal anti-VEGF antibodies inhibit the growth of human tumors in nude
 mice. Although these same tumor cells continue to express VEGF in culture,
 the antibodies do not diminish their mitotic rate of most, if not all,
 tumor cells derived from cells other than vascular endothelial cells
 themselves. Thus tumor-derived VEGF does not function as an autocrine
 mitogenic factor for most tumors. Therefore, VEGF contributes to tumor
 growth in vivo by promoting angiogenesis through its paracrine vascular
 endothelial cell chemotactic and mitogenic activities. These monoclonal
 antibodies also inhibit the growth of typically less well vascularized
 human colon cancers in athymic mice and decrease the number of tumors
 arising from inoculated cells. Viral expression of a VEGF-binding
 construct of Flk-1, the mouse KDR receptor homologue, truncated to
 eliminate the cytoplasmic tyrosine kinase domains but retaining a membrane
 anchor, virtually abolishes the growth of a transplantable glioblastoma in
 mice presumably by the dominant negative mechanism of heterodimer
 formation with membrane-spanning endothelial cell VEGF receptors.
 Embryonic stem cells, which normally grow as solid tumors in nude mice, do
 not produce detectable tumors if both VEGF alleles are knocked out. Taken
 together, these data indicate the role of VEGF in the growth of solid
 tumors. KDR and Flt-1 are implicated in pathological neoangiogenesis, and
 inhibitors of these receptors are useful in the treatment of diseases in
 which neoangiogenesis is part of the overall pathology, e.g., diabetic
 retinal vascularization, various forms of cancer as well as forms of
 inflammation such as rheumatoid arthritis, psoriasis, contact dermatitis
 and hypersensitivity reaction.
 Terman et al. (1991, Oncogene 6: 1677-1683; 1992, Biochem. Biophys. Res.
 Commun. 187: 1579-1586) disclose a full-length cDNA encoding a form of
 KDR. However, the Terman et al. disclosures do not identify a novel,
 optimal nucleic acid fragment encoding the human form of the receptor type
 tyrosine kinase gene, KDR. It will be advantageous to identify and isolate
 a human cDNA sequence encoding an optimized form of human KDR. A nucleic
 acid molecule expressing the human KDR protein will be useful in screening
 for compounds acting as a modulator of the protein kinase domain of this
 protein. Such a compound or compounds will be useful in modulating the
 mitogenic signal of VEGF and VEGF-related proteins on vascular endothelial
 cells. The KDR nucleic acid sequence may be also useful for gene therapy
 encoding a portion of the KDR protein that would contain functional ligand
 binding and membrane anchoring moieties but not tyrosine kinase activity.
 Either all or a portion of the KDR protein is also useful to screen for
 VEGF antagonists. The KDR nucleic acid sequence can be transfected into
 cells for analysis of function in the absence of Flt-1. The KDR protein is
 also useful for x-ray structure analysis in the presence or absence of
 ligand and/or inhibitors. The present invention addresses and meets these
 needs by disclosing an isolated nucleic acid fragment which expresses a
 form of human KDR which is shown by computer modeling to be predictive of
 higher activity and functionality than the previously disclosed KDR.
 SUMMARY OF THE INVENTION
 The present invention relates to an isolated nucleic acid molecule
 (polynucleotide) which encodes a novel human receptor type tyrosine kinase
 gene, KDR. This specification discloses a novel, optimized DNA molecule
 which encodes, KDR, a receptor tyrosine kinase expressed on human
 endothelial cells.
 The present invention also relates to biologically active fragments or
 mutants of SEQ ID NO:1 which encodes mRNA expressing a novel human
 receptor type tyrosine kinase gene, KDR. Any such biologically active
 fragment and/or mutant will encode either a protein or protein fragment
 comprising at least an intracellular or extracelluar kinase domain similar
 to that of the human KDR protein as set forth in SEQ ID NO:2. Any such
 polynucleotide includes but is not necessarily limited to nucleotide
 substitutions, deletions, additions, amino-terminal truncations and
 carboxy-terminal truncations such that these mutations encode mRNA which
 express a protein or protein fragment of diagnostic, therapeutic or
 prophylactic use and would be useful for screening for agonists and/or
 antagonists for KDR function.
 The isolated nucleic acid molecule of the present invention may include a
 deoxyribonucleic acid molecule (DNA), such as genomic DNA and
 complementary DNA (cDNA), which may be single (coding or noncoding strand)
 or double stranded, as well as synthetic DNA, such as a synthesized,
 single stranded polynucleotide. The isolated nucleic acid molecule of the
 present invention may also include a ribonucleic acid molecule (RNA).
 The present invention also relates to recombinant vectors and recombinant
 hosts, both prokaryotic and eukaryotic, which contain the substantially
 purified nucleic acid molecules disclosed throughout this specification.
 The present invention also relates to subcellular membrane fractions of the
 recombinant host cells (both prokaryotic and eukaryotic as well as both
 stably and transiently transformed cells) comprising the nucleic acids of
 the present invention. These subcellular membrane fractions will comprise
 either wild-type or human mutant forms of KDR at levels substantially
 above wild-type levels and hence will be useful in various assays
 described throughout this specification.
 A preferred aspect of the present invention is disclosed in FIG. 1A and
 FIG. 1B and SEQ ID NO:1, a human cDNA encoding a novel receptor type
 tyrosine kinase gene, KDR.
 The present invention also relates to a substantially purified form of the
 receptor type tyrosine kinase gene, KDR which is disclosed in FIG. 2 and
 as set forth in SEQ ID NO:2.
 The present invention also relates to biologically active fragments and/or
 mutants of the KDR protein as initially set forth as SEQ ID NO:2,
 including but not necessarily limited to amino acid substitutions,
 deletions, additions, amino terminal truncations and carboxy-terminal
 truncations such that these mutations provide for proteins or protein
 fragments of diagnostic, therapeutic or prophylactic use and would be
 useful for screening for agonists and/or antagonists for KDR function.
 A preferred aspect of the present invention is disclosed in FIG. 2 and is
 set forth as SEQ ID NO:2, the amino acid sequence of the novel receptor
 type tyrosine kinase gene, KDR.
 The present invention also relates to polyclonal and monoclonal antibodies
 raised in response to either the human form of KDR disclosed herein, or a
 biologically active fragment thereof.
 The present invention also relates to isolated nucleic acid molecules which
 are fusion constructions expressing fusion proteins useful in assays to
 identify compounds which modulate wild-type human KDR activity. A
 preferred aspect of this portion of the invention includes, but is not
 limited to, glutathione S-transferase (GST)-KDR fusion constructs. These
 fusion constructs include, but are not limited to, either the
 intracellular tyrosine kinase domain of human KDR as an in-frame fusion at
 the carboxy terminus of the GST gene or the extracellular ligand binding
 domain fused to an immunoglobulin gene by methods known to one of ordinary
 skill in the art. Soluble recombinant GST-kinase domain fusion proteins
 may be expressed in various expression systems, including Spodoptera
 frugiperda (Sf21) insect cells (Invitrogen) using a baculovirus expression
 vector (pAcG2T, Pharmingen).
 The present invention also relates to isolated nucleic acid molecules which
 encode human KDR protein fragments comprising a portion of the
 intracellular KDR domain. The protein fragments are useful in assays to
 identify compounds which modulate wild-type human KDR activity. A
 preferred aspect of this portion of the invention includes, but is not
 limited to, a nucleic acid construction which encodes the intracellular
 portion of human KDR, from about amino acid 780-795 to about amino acid
 1175-1386.
 Therefore, the present invention relates to isolated nucleic acid molecules
 which encode human KDR protein fragments comprising a portion of the
 extracellular KDR domain. These isolated nucleic acid proteins may or may
 not include nucleotide sequences which also encode the transmembrane
 domain of human KDR. These KDR extracellular and/or KDR
 extracellular-transmembrane domain protein fragments will be useful in
 screening for compounds which inhibit VEGF binding as well as utilizing
 these isolated nucleic acids as gene therapy vehicles to inhibit
 VEGF-mediated mitogenic activity. Expression of either a soluble version
 of KDR (extracellular) or membrane bound form
 (extracellular-transmembrane) will inhibit in vivo VEGF/KDR mediated
 angiogenesis.
 Therefore, the present invention relates to methods of expressing the
 receptor type tyrosine kinase gene, KDR, and biological equivalents
 disclosed herein, assays employing these receptor type tyrosine kinase
 genes, cells expressing these receptor type tyrosine kinase genes, and
 compounds identified through the use of these receptor type tyrosine
 kinase genes and expressed human KDR protein, including one or more
 modulators of the human KDR-dependent kinase either through direct contact
 with the kinase domain of human KDR or a compound which prevents binding
 of VEGF to human KDR, or appropriate dimerization of the KDR receptor
 antagonizing transduction of the normal intracellular signals associated
 with VEGF-induced angiogenesis.
 The present invention also relates to gene therapy applications, especially
 for nucleic acid fragments which encode soluble extracelluar protein
 fragments of human KDR. It is disclosed herein that such methods will be
 useful especially in the treatment of various tumors as well as diabetic
 retinopathy.
 It is an object of the present invention to provide an isolated nucleic
 acid molecule which encodes a novel form of human KDR, or human KDR
 fragments and KDR mutants which are derivatives of SEQ ID NO:2 and
 preferably retain Val at position 848, and especially preferable is
 retention of Val at position 848, Glu at position 498, Ala at position
 772, Arg at position 787, Lys at position 835 and Ser at position 1347.
 Any such polynucleotide includes but is not necessarily limited to
 nucleotide substitutions, deletions, additions, amino-terminal truncations
 and carboxy-terminal truncations such that these mutations encode mRNA
 which express a protein or protein fragment of diagnostic, therapeutic or
 prophylactic use and would be useful for screening for agonists and/or
 antagonists for KDR function.
 It is a further object of the present invention to provide the human KDR
 proteins or protein fragments encoded by the nucleic acid molecules
 referred to in the preceding paragraph.
 It is also an object of the present invention to provide biologically
 active fragments or mutants of human KDR which comprise an intracellular
 kinase domain similar to that of the human KDR protein as set forth in SEQ
 ID NO:2, preferably retaining Val at position 848, and especially
 preferable is retention of Val at position 848, Glu at position 498, Ala
 at position 772, Arg at position 787, Lys at position 835 and Ser at
 position 1347.
 It is a further object of the present invention to provide recombinant
 vectors and recombinant host cells which comprise a nucleic acid sequence
 encoding human KDR or a biological equivalent thereof.
 It is an object of the present invention to provide a substantially
 purified form of the receptor type tyrosine kinase gene, KDR, as set forth
 in SEQ ID NO:2.
 It is an object of the present invention to provide for biologically active
 fragments and/or mutants of the KDR protein, such as set forth in SEQ ID
 NO:2, including but not necessarily limited to amino acid substitutions,
 deletions, additions, amino terminal truncations and carboxy-terminal
 truncations such that these mutations provide for proteins or protein
 fragments of diagnostic, therapeutic or prophylactic use.
 It is also an object of the present invention to provide for KDR-based
 in-frame fusion constructions, methods of expressing the receptor type
 tyrosine kinase gene, KDR, and biological equivalents disclosed herein,
 related assays, recombinant cells expressing these receptor type tyrosine
 kinase genes, and agonistic and/or antagonistic compounds identified
 through the use of these receptor type tyrosine kinase genes and expressed
 human KDR protein.
 As used herein, "VEGF" or "VEFG-A" prefers to vascular endothelial growth
 factor.
 As used herein, "KDR" or "FLK-1" refers to kinase insert domain-containing
 receptor.
 As used herein, "FLT-1" refers to fms-like tyrosine kinase receptor.
 As used herein, the term "mammalian host" refers to any mammal, including a
 human being.

DETAILED DESCRIPTION OF THE INVENTION
 The present invention relates to isolated nucleic acid and protein forms
 which represent human KDR. This specification discloses a DNA molecule
 encoding human KDR, a receptor tyrosine kinase expressed on human
 endothelial cells. The receptor is activated by vascular endothelial
 growth factor (VEGF) and mediates a mitogenic signal. This activation and
 subsequent mitogenesis leads to an angiogenic response in vivo. The
 nucleic acid molecule disclosed in the specification as SEQ ID NO:1
 encodes a human KDR protein (SEQ ID NO:2) which results in six amino acid
 differences from the published sequence (Terman et al., 1992, Biochem.
 Biophys. Res. Commun. 187: 1579-1586, Terman et al., International PCT
 application number WO 92/14748, International application number
 PCT/US92/01300). These changes are position 498 (Ala to Glu), 772 (Thr to
 Ala), 787 (Gly to Arg), 835 (Asn to Lys), 848 (Glu to Val), and 1347 (Thr
 to Ser). These six amino acid changes affect the activity of the receptor.
 Val 848 is conserved throughout most of the tyrosine kinase family and
 appears to be important for the binding of ATP and presumably ATP
 competitive inhibitors to the KDR receptor kinase as inferred by computer
 modeling. A change to Glu at this position results in a non-functional
 kinase as a consequence of impaired ATP binding. The other changes may
 also cause activity differences.
 The present invention also relates to either biologically active fragments
 or mutants of SEQ ID NO:1 which encodes mRNA expressing a novel human
 receptor type tyrosine kinase gene, KDR. Any such biologically active
 fragment and/or mutant will encode a protein or protein fragment
 comprising at least an intracellular kinase domain similar to that of the
 human KDR protein as set forth in SEQ ID NO:2 and preferably retain Val at
 position 848. It is also envisioned that other intracellular-based KDR
 domains will result in a soluble protein fragment which mimics wild-type
 intracellular domain structure and function. Any such protein fragment may
 be a fusion protein, such as the exemplified GST-KDR fusion, or may be
 solely comprised of the KDR intracelluar domain, with increasing deletions
 in from the COOH-terminal region. It is especially preferable that the
 following amino acids be retained, if this domain encompasses the
 respective protein or protein fragment: Val at position 848, Glu at
 position 498, Ala at position 772, Arg at position 787, Lys at position
 835 and Ser at position 1347. Therefore, any such polynucleotide includes
 but is not necessarily limited to nucleotide substitutions, deletions,
 additions, amino-terminal truncations and carboxy-terminal truncations
 such that these mutations encode mRNA which express a protein or protein
 fragment of diagnostic, therapeutic or prophylactic use and is useful for
 the identification of modulators of KDR receptor activity.
 The isolated nucleic acid molecule of the present invention may include a
 deoxyribonucleic acid molecule (DNA), such as genomic DNA and
 complementary DNA (cDNA), which may be single (coding or noncoding strand)
 or double stranded, as well as synthetic DNA, such as a synthesized,
 single stranded polynucleotide. The isolated nucleic acid molecule of the
 present invention may also include a ribonucleic acid molecule (RNA).
 It is known that DNA sequences coding for a peptide may be altered so as to
 code for a peptide having properties that are different than those of the
 naturally occurring peptide. Methods of altering the DNA sequences include
 but are not limited to site directed mutagenesis. Examples of altered
 properties include but are not limited to changes in the affinity of an
 enzyme for a substrate or a receptor for a ligand.
 As used herein, "purified" and "isolated" are utilized interchangeably to
 stand for the proposition that the nucleic acid, protein, or respective
 fragment thereof in question has been substantially removed from its in
 vivo environment so that it may be manipulated by the skilled artisan,
 such as but not limited to nucleotide sequencing, restriction digestion,
 site-directed mutagenesis, and subcloning into expression vectors for a
 nucleic acid fragment as well as obtaining the protein or protein fragment
 in pure quantities so as to afford the opportunity to generate polyclonal
 antibodies, monoclonal antibodies, amino acid sequencing, and peptide
 digestion. Therefore, the nucleic acids claimed herein may be present in
 whole cells or in cell lysates or in a partially purified or substantially
 purified form. A nucleic acid is considered substantially purified when it
 is purified away from environmental contaminants. Thus, a nucleic acid
 sequence isolated from cells is considered to be substantially purified
 when purified from cellular components by standard methods while a
 chemically synthesized nucleic acid sequence is considered to be
 substantially purified when purified from its chemical precursors.
 The present invention also relates to recombinant vectors and recombinant
 hosts, both prokaryotic and eukaryotic, which contain the substantially
 purified nucleic acid molecules disclosed throughout this specification.
 The present invention also relates to subcellular membrane fractions of the
 recombinant host cells (both prokaryotic and eukaryotic as well as both
 stably and transiently transformed cells) comprising the nucleic acids of
 the present invention. These subcellular membrane fractions will comprise
 wild-type or human mutant forms of KDR at levels substantially above
 wild-type levels and hence will be useful in various assays described
 throughout this specification.
 A preferred aspect of the present invention is disclosed in FIG. 1A and
 FIG. 1B and SEQ ID NO:1, a human cDNA encoding a novel receptor type
 tyrosine kinase gene, KDR, disclosed as follows:
 ATGGAGAGCAAGGTGCTGCTGGCCGTCGCCCTGTGGCTCTGCGTGGAGACCCGGGCCGCCTCTGTGGGT
 (SEQ ID NO: 1)
 TTGCCTAGTGTTTCTCTTGATCTGCCCAGGCTCAGCATACAAAAAGACATACTTACAATTAAGGCTAAT
 ACAACTCTTCAAATTACTTGCAGGGGACAGAGGGACTTGGACTGGCTTTGGCCCAATAATCAGAGTGGC
 AGTGAGCAAAGGGTGGAGGTGACTGAGTGCAGCGATGGCCTCTTCTGTAAGACACTCACAATTCCAAAA
 GTGATCGGAAATGACACTGGAGCCTACAAGTGCTTCTACCGGGAAACTGACTTGGCCTCGGTCATTTAT
 GTCTATGTTCAAGATTACAGATCTCCATTTATTGCTTCTGTTAGTGACCAACATGGAGTCGTGTACATT
 ACTGAGAACAAAAACAAAACTGTGGTGATTCCATGTCTCGGGTCCATTTCAAATCTCAACGTGTCACTT
 TGTGCAAGATACCCAGAAAAGAGATTTGTTCCTGATGGTAACAGAATTTCCTGGGACAGCAAGAAGGGC
 TTTACTATTCCCAGCTACATGATCAGCTATGCTGGCATGGTCTTCTGTGAAGCAAAAATTAATGATGAA
 AGTTACCAGTCTATTATGTACATAGTTGTCGTTGTAGGGTATAGGATTTATGATGTGGTTCTGAGTCCG
 TCTCATGGAATTGAACTATCTGTTGGAGAAAAGCTTGTCTTAAATTGTACAGCAAGAACTGAACTAAAT
 GTGGGGATTGACTTCAACTGGGAATACCCTTCTTCGAAGCATCAGCATAAGAAACTTGTAAACCGAGAC
 CTAAAAACCCAGTCTGGGAGTGAGATGAAGAAATTTTTGAGCACCTTAACTATAGATGGTGTAACCCGG
 AGTGACCAAGGATTGTACACCTGTGCAGCATCCAGTGGGCTGATGACCAAGAAGAACAGCACATTTGTG
 AGGGTCCATGAAAAACCTTTTGTTGCTTTTGGAAGTGGCATGGAATCTCTGGTGGAAGCCACGGTGGGG
 GAGCGTGTCAGAATCCCTGCGAAGTACCTTGGTTACCCACCCCCAGAAATAAAATGGTATAAAAATGGA
 ATACCCCTTGAGTCCAATCACACAATTAAAGCGCGGCATGTACTGACGATTATGGAAGTGAGTGAAAGA
 GACACAGGAAATTACACTGTCATCCTTACCAATCCCATTTCAAAGGAGAAGCAGAGCCATGTGGTCTCT
 CTGGTTGTGTATGTCCCACCCCAGATTGGTGAGAAATCTCTAATCTCTCCTGTGGATTCCTACCAGTAC
 GGCACCACTCAAACGCTGACATGTACGGTCTATGCCATTCCTCCCCCGCATCACATCCACTGGTATTGG
 CAGTTGGAGGAAGAGTGCGCCAACGAGCCCAGCCAAGCTGTCTCAGTGACAAACCCATACCCTTGTGAA
 GAATGGAGAAGTGTGGAGGACTTCCAGGGAGGAAATAAAATTGAAGTTAATAAAAATCAATTTGCTCTA
 ATTGAAGGAAAAAACAAAACTGTAAGTACCCTTGTTATCCAAGCGGCAAATGTGTCAGCTTTGTACAAA
 TGTGAAGCGGTCAACAAAGTCGGGAGAGGAGAGAGGGTGATCTCCTTCCACGTGACCAGGGGTCCTGAA
 ATTACTTTGCAACCTGACATGCAGCCCACTGAGCAGGAGAGCGTGTCTTTGTGGTGCACTGCAGACAGA
 CAAGACAGGAAGACCAAGAAAAGACATTGCGTGGTCAGGCAGCTCACAGTCCTAGAGCGTGTGGCACCC
 ACGATCACAGGAAACCTGGAGAATCAGACGACAAGTATTGGGGAAAGCATCGAAGTCTCATGCACGGCA
 TCTGGGAATCCCCCTCCACAGATCATGTGGTTTAAAGATAATGAGACCCTTGTAGAAGACTCAGGCATT
 GTATTGAAGGATGGGAACCGGAACCTCACTATCCGCAGAGTGAGGAAGGAGGACGAAGGCCTCTACACC
 TGCCAGGCATGCAGTGTTCTTGGCTGTGCAAAAGTGGAGGCATTTTTCATAATAGAAGGTGCCCAGGAA
 AAGACGAACTTGGAAATCATTATTCTAGTAGGCACGGCGGTGATTGCCATGTTCTTCTGGCTACTTCTT
 GTCATCATCCTACGGACCGTTAAGCGGGCCAATGGAGGGGAACTGAAGACAGGCTACTTGTCCATCGTC
 ATGGATCCAGATGAACTCCCATTGGATGAACATTGTGAACGACTGCCTTATGATGCCAGCAAATGGGAA
 TTCCCCAGAGACCGGCTGAAGCTAGGTAAGCCTCTTGGCCGTGGTGCCTTTGGCCAAGTGATTGAAGCA
 GATGCCTTTGGAATTGACAAGACAGCAACTTGCAGGACAGTAGCAGTCAAAATGTTGAAAGAAGGAGCA
 ACACACAGTGAGCATCGAGCTCTCATGTCTGAACTCAAGATCCTCATTCATATTGGTCACCATCTCAAT
 GTGGTCAACCTTCTAGGTGCCTGTACCAAGCCAGGAGGGCCACTCATGGTGATTGTGGAATTCTGCAAA
 TTTGGAAACCTGTCCACTTACCTGAGGAGCAAGAGAAATGAATTTGTCCCCTACAAGACCAAAGGGGCA
 CGATTCCGTCAAGGGAAAGACTACGTTGGAGCAATCCCTGTGGATCTGAAACGGCGCTTGGACAGCATC
 ACCAGTAGCCAGAGCTCAGCCAGCTCTGGATTTGTGGAGGAGAAGTCCCTCAGTGATGTAGAAGAAGAG
 GAAGCTCCTGAAGATCTGTATAAGGACTTCCTGACCTTGGAGCATCTCATCTGTTACAGCTTCCAAGTG
 GCTAAGGGCATGGAGTTCTTGGCATCGCGAAAGTGTATCCACAGGGACCTGGGGGCACGAAATATCCTC
 TTATCGGAGAAGAACGTGGTTAAAATCTGTGACTTTGGCTTGGCCCGGGATATTTATAAAGATCCAGAT
 TATGTCAGAAAAGGAGATGCTCGCCTCCCTTTGAAATGGATGGCCCCAGAAACAATTTTTGACAGAGTG
 TACACAATCCAGAGTGACGTCTGGTCTTTTGGTGTTTTGCTGTGGGAAATATTTTCCTTAGGTGCTTCT
 CCATATCCTGGGGTAAAGATTGATGAAGAATTTTGTAGGCGATTGAAAGAAGGAACTAGAATGAGGGCC
 CCTGATTATACTACACCAGAAATGTACCAGACCATGCTGGACTGCTGGCACGGGGAGCCCAGTCAGAGA
 CCCACGTTTTCAGAGTTGGTGGAACATTTGGGAAATCTCTTGCAAGCTAATGCTCAGCAGGATGGCAAA
 GACTACATTGTTCTTCCGATATCAGAGACTTTGAGCATGGAAGAGGATTCTGGACTCTCTCTGCCTACC
 TCACCTGTTTCCTGTATGGAGGAGGAGGAAGTATGTGACCCCAAATTCCATTATGACAACACAGCAGGA
 ATCAGTCAGTATCTGCAGAACAGTAAGCGAAAGAGCCGGCCTGTGAGTGTAAAAACATTTGAAGATATC
 CCGTTAGAAGAACCAGAAGTAAAAGTAATCCCAGATGACAACCAGACGGACAGTGGTATGGTTCTTGCC
 TCAGAAGAGCTGAAAACTTTGGAAGACAGAACCAAATTATCTCCATCTTTTGGTGGAATGGTGCCCAGC
 AAAAGCAGGGAGTCTGTGGCATCTGAAGGCTCAAACCAGACAAGCGGCTACCAGTCCGGATATCACTCC
 GATGACACAGACACCACCGTGTACTCCAGTGAGGAAGCAGAACTTTTAAAGCTGATAGAGATTGGAGTG
 CAAACCGGTAGCACAGCCCAGATTCTCCAGCCTGACTCGGGGACCACACTGAGCTCTCCTCCTGTTTAA.
 The present invention also relates to a substantially purified form of the
 receptor type tyrosine kinase gene which comprises the KDR amino acid
 sequence disclosed in FIG. 2 and as set forth in SEQ ID NO:2, which
 includes Glu at position 498, Ala at position 772, Arg at position 787,
 Lys at position 835, Val at position 848 and Ser at position 1347,
 disclosed as follows:
 MESKVLLAVALWLCVETRAASVGLPSVSLDLPRLSIQKDILTIKANTTLQITCRGQRDLDWLWPNNQSG
 (SEQ ID NO:2)
 SEQRVEVTECSDGLFCKTLTIPKVIGNDTGAYKCFYRETDLASVIYVYVQDYRSPFIASVSDQHGVVYI
 TENKNKTVVIPCLGSISNLNVSLCARYPEKRFVPDGNRISWDSKKGFTIPSYMISYAGMVFCEAKINDE
 SYQSIMYIVVVVGYRIYDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRD
 LKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCA