CA2224093C - Human vascular endothelial growth factor 2 - Google Patents
Human vascular endothelial growth factor 2 Download PDFInfo
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- CA2224093C CA2224093C CA002224093A CA2224093A CA2224093C CA 2224093 C CA2224093 C CA 2224093C CA 002224093 A CA002224093 A CA 002224093A CA 2224093 A CA2224093 A CA 2224093A CA 2224093 C CA2224093 C CA 2224093C
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Abstract
Disclosed is a human VEGF2 polypeptide and DNA (RNA) encoding such VEGF2 polypeptides. Also provided is a procedure for producing such polypeptide by recombinant techniques and antibodies and antagonist against such polypeptide.
Also disclosed is a method of using such polypeptide for stimulating wound healing and for vascular tissue repair. Also provided are methods of using the antagonists to inhibit tumor growth, inflammation and to treat diabetic retinopathy, rheumatoid arthritis and psoriasis. Diagnostic methods for detecting mutations in the VEGF2 coding sequence and alterations in the concentration of VEGF2 protein in a sample derived from a host are also disclosed.
Also disclosed is a method of using such polypeptide for stimulating wound healing and for vascular tissue repair. Also provided are methods of using the antagonists to inhibit tumor growth, inflammation and to treat diabetic retinopathy, rheumatoid arthritis and psoriasis. Diagnostic methods for detecting mutations in the VEGF2 coding sequence and alterations in the concentration of VEGF2 protein in a sample derived from a host are also disclosed.
Description
Human Vascular gadothalial Gropth Factor 2 This invention relates to newly identified polynucleotides, polypeptides encoded. by such polynucleotidea, the use of such polynucleoti:des and polypeptides, as well as, the production of such polynucleotides and polypeptides. The polypeptide of the present invent=ion has been. ~ identified as a member ~ of the vascular endothelial growth factor family. More particularly, the poly~eptide of the present invention is vascular. :endot:helial growth . factor 2 , sometimes hex-einaf ter referred 'to ass "VBGF2 . " ~ The invention also relates to inhibiting the: action of. such polypeptide.
The formation of new blood vessels, or angiogenesis, is essential for embryonic development, subsequent growth, and tissue repair. Angiogenesis, however, is an essential part of certain pathological conditions such as neoplasia, for example, tvunox-s and gliomas, and abnozmal angiogenesis is associated with other diseases such as inflaaauation, WO 96/39515 ~ PCT/US96/09001 rheumatoid arthritis, psoriasis, and diabetic retinopathy (Folkman, J. and IClagsbrun, M., Science 235:442-447,(1987)).
Both acidic and basic fibroblast growth factor molecules are nutogens for endothelial cells and other cell types.
Angiotropin and angiogenin can induce angiogenesis, although their functions are unclear (Folkman, J., 1993, Cancer Medicine pp. 153-170, Lea and Febiger Press). A highly selective mitogen for vascular endothelial cells is vascular endothelial growth factor or V$GF (Ferrara, N., et al., $ndocr. Rev. 13:19-32, (1992)), also known as vascular permeability factor (VPF). Vascular endothelial growth factor is a secreted angiogenic mitogen whose target cell specificity appears to be restricted to vascular endothelial cells.
The murine VEGF gene has been characterized and its expression pattern in embryogenesis has been analyzed. A
persistent expression of V$GF was observed in epithelial cells adjacent to fenestrated endothelium, e.g., in choroid plexus and kidney glomeruli. The data was consistent with a role of V$GF as a multifunctional regulator of endothelial cell growth and differentiation (Breier, G. et a1.
Development, 114:521-532 (1992)).
VSGF is structurally related to the a and ~B chains of platelet-derived growth factor (PDGF), a ~togen for mesenchymal cells 'and placenta growth factor (PLGF), an endothelial cell triltogen. These three proteins belong to the same family and share a conserved motif. Sight cysteine residues contributing to disulfide-bond formation are strictly conserved in these proteins. Alternatively spliced mRNAs have been identified for both VEGF, PLGF and PDGF and these different splicing products differ in biological activity and in receptor-binding specificity. VEGF and PDGF
function as homo-dimers or hetero-dimers and bind to receptors which elicit intrinsic tyrosine kinase activity _ following receptor dimerization.
The formation of new blood vessels, or angiogenesis, is essential for embryonic development, subsequent growth, and tissue repair. Angiogenesis, however, is an essential part of certain pathological conditions such as neoplasia, for example, tvunox-s and gliomas, and abnozmal angiogenesis is associated with other diseases such as inflaaauation, WO 96/39515 ~ PCT/US96/09001 rheumatoid arthritis, psoriasis, and diabetic retinopathy (Folkman, J. and IClagsbrun, M., Science 235:442-447,(1987)).
Both acidic and basic fibroblast growth factor molecules are nutogens for endothelial cells and other cell types.
Angiotropin and angiogenin can induce angiogenesis, although their functions are unclear (Folkman, J., 1993, Cancer Medicine pp. 153-170, Lea and Febiger Press). A highly selective mitogen for vascular endothelial cells is vascular endothelial growth factor or V$GF (Ferrara, N., et al., $ndocr. Rev. 13:19-32, (1992)), also known as vascular permeability factor (VPF). Vascular endothelial growth factor is a secreted angiogenic mitogen whose target cell specificity appears to be restricted to vascular endothelial cells.
The murine VEGF gene has been characterized and its expression pattern in embryogenesis has been analyzed. A
persistent expression of V$GF was observed in epithelial cells adjacent to fenestrated endothelium, e.g., in choroid plexus and kidney glomeruli. The data was consistent with a role of V$GF as a multifunctional regulator of endothelial cell growth and differentiation (Breier, G. et a1.
Development, 114:521-532 (1992)).
VSGF is structurally related to the a and ~B chains of platelet-derived growth factor (PDGF), a ~togen for mesenchymal cells 'and placenta growth factor (PLGF), an endothelial cell triltogen. These three proteins belong to the same family and share a conserved motif. Sight cysteine residues contributing to disulfide-bond formation are strictly conserved in these proteins. Alternatively spliced mRNAs have been identified for both VEGF, PLGF and PDGF and these different splicing products differ in biological activity and in receptor-binding specificity. VEGF and PDGF
function as homo-dimers or hetero-dimers and bind to receptors which elicit intrinsic tyrosine kinase activity _ following receptor dimerization.
WO 96/39515 ~ PCT/US96/09001 VgGF has four different forms of 121, 165, 189 and 206 amino acids due to alternative splicing. V$GF121 and VSGF165 are soluble and are capable of promoting angiogenesis, whereas V$GF189 and VSGF206 are bound to heparin containing proteoglycans a.n the cell surface. The temporal and spatial expression of V$GF has been correlated with physiological proliferation of the blood vessels (Gajdusel~, C.M. , and Carbon, S.J., Cell Physiol., 139:570-579, (1989)); McNeil, P.L., Muthukrishnan, L., iaarder, 8., D'Amore, P.A., J. Cell.
Biol., 109:811-822, (1989)). Its high affinity binding sites are localized only on endothelial cells in tissue sections (Jakeman, L.B., et al., Clin. Invs~st. 89:244-253, (1989)).
The factor can be isolated from pituitary cells and several tumor cell lines, and has been :implicated in some human gliomas (Plate, K.H. Nature 359:845-848, (1992)).
Interestingly, expression of VBGF121 or V$GF165 confers on Chinese hamster ovary ce:ils the ability to form tumors in nude mice (Ferrara, N., et al., J. Clin. Invest. 91:160-170, (1993) ) . The inhibition of VSG1~ function by anti-VSGF
monoclonal antibodies was shown to inhibit tumor growth in immune-deficient mice I;Kim, K.J'., Nature 362:841-844, (1993)). Further, a dominant-negative mutant of the V$GF
receptor has been shown to inhibit growth of glioblastamas in mice . - . : _ -- ,_ Vascular per~a~eability factor, has also been found to be responsible for persistent microvasc:ular hyperpermeability to plasma proteins even after the cessation of injury, which is a characteristic feature of nornlal wound healing. This suggests that VPF is an :Lmportant factor in wound healing.
Brown, L.F. et al., J. Exp. Med., 1.76:1375-9 (1992).
The expression of VE(sF is high in vascularized tissues, (a. g., lung, heart, placenta and solid tumors) and correlates with angiogenesis both temporally and spatially. VBGF has also been shown to induce angiogenesis in vivo. Since angiogenesis is essential for the repair of normal tissues, especially vascular tissues, VBGF has been proposed for use in promoting vascular tissue repair (e.g., in atherosclerosis).
U.S. Patent No. 5,073,492, issued December 17, 1991 to Chen et al.. , discloses a method for synergistically enhancing endothelial cell growth in an appropriate environment which comprises adding to the environment, VEGF, effectors and serum-derived factor. Also, vascular endothelial cell growth factor C sub-unit DNA has been prepared by polymerase chain reaction techniques. The DNA encodes a protein that may exa.st as either a hetero-dimer or homo-dimer. The protein is a mammalian vascular endothelial cell mitogen and, as such, is useful for the promotion of vascular development and repair, as disclosed a.n $uropean Patent Application No.
92302750.2, published September 30, 1992.
The polypeptides of the present invention have been putatively identified as a novel vascular endothelial growth factor based on amino acid sequence homology to human VSGF.
In accordance with one aspect of the present invention, there are provided novel mature polypeptides, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The polypeptides of the present invention are of human origin.
In accordancewith another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the polypeptides of the present invention, including mRNAs, DNAs, cDNAs, genomic DNA as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
In accordance with still another aspect of the present invention, there are provided processes for producing such polypeptides by recombinant techniques comprising culturing recombinant prolzaryotic and/or eukaryotic host cells, containing a nucleic acid sequence encoding a polypeptide of _. ,! _.
Biol., 109:811-822, (1989)). Its high affinity binding sites are localized only on endothelial cells in tissue sections (Jakeman, L.B., et al., Clin. Invs~st. 89:244-253, (1989)).
The factor can be isolated from pituitary cells and several tumor cell lines, and has been :implicated in some human gliomas (Plate, K.H. Nature 359:845-848, (1992)).
Interestingly, expression of VBGF121 or V$GF165 confers on Chinese hamster ovary ce:ils the ability to form tumors in nude mice (Ferrara, N., et al., J. Clin. Invest. 91:160-170, (1993) ) . The inhibition of VSG1~ function by anti-VSGF
monoclonal antibodies was shown to inhibit tumor growth in immune-deficient mice I;Kim, K.J'., Nature 362:841-844, (1993)). Further, a dominant-negative mutant of the V$GF
receptor has been shown to inhibit growth of glioblastamas in mice . - . : _ -- ,_ Vascular per~a~eability factor, has also been found to be responsible for persistent microvasc:ular hyperpermeability to plasma proteins even after the cessation of injury, which is a characteristic feature of nornlal wound healing. This suggests that VPF is an :Lmportant factor in wound healing.
Brown, L.F. et al., J. Exp. Med., 1.76:1375-9 (1992).
The expression of VE(sF is high in vascularized tissues, (a. g., lung, heart, placenta and solid tumors) and correlates with angiogenesis both temporally and spatially. VBGF has also been shown to induce angiogenesis in vivo. Since angiogenesis is essential for the repair of normal tissues, especially vascular tissues, VBGF has been proposed for use in promoting vascular tissue repair (e.g., in atherosclerosis).
U.S. Patent No. 5,073,492, issued December 17, 1991 to Chen et al.. , discloses a method for synergistically enhancing endothelial cell growth in an appropriate environment which comprises adding to the environment, VEGF, effectors and serum-derived factor. Also, vascular endothelial cell growth factor C sub-unit DNA has been prepared by polymerase chain reaction techniques. The DNA encodes a protein that may exa.st as either a hetero-dimer or homo-dimer. The protein is a mammalian vascular endothelial cell mitogen and, as such, is useful for the promotion of vascular development and repair, as disclosed a.n $uropean Patent Application No.
92302750.2, published September 30, 1992.
The polypeptides of the present invention have been putatively identified as a novel vascular endothelial growth factor based on amino acid sequence homology to human VSGF.
In accordance with one aspect of the present invention, there are provided novel mature polypeptides, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The polypeptides of the present invention are of human origin.
In accordancewith another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the polypeptides of the present invention, including mRNAs, DNAs, cDNAs, genomic DNA as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
In accordance with still another aspect of the present invention, there are provided processes for producing such polypeptides by recombinant techniques comprising culturing recombinant prolzaryotic and/or eukaryotic host cells, containing a nucleic acid sequence encoding a polypeptide of _. ,! _.
5 ' PCT/(TS96/0900I
the present invention, under conditions promoting expression of said proteins and subsequent recovery of said proteins.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptide, or polynucleotide encoding such polypeptide for therapeutic purposes, for example, t:o stimulate angiogenesis, wound-healing, and to promote vascular tissue repair.
In accordance with yet another aspect of the present invention, there are provided antibodies against such polypeptides.
In accordance with yet another aspect of the present invention, there are provided antagonists to such polypeptides , which may be used to :inhibit the action of such polypeptides, for example:, to inhilbit the growth of tumors, to treat diabetic retinopathy, inflammation, rheumatoid arthritis and psoriasis.
In accordance with another aspect of the present invention, there are provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to nucleic acid sequences of the present invention.
In accordance with another aspect of the present invention, there are provided methods of diagnosing diseases or a susceptibility to diseases related to mutations in nucleic acid sequences of the present invention and proteins encoded by such nucleic acid sequences.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucl~eotides encoding such polypeptides, for in vitro purposes related to scientific research, synthesis of DNA and manufacture oi' DNA vectors .
These and other aspects of the: present invention should be apparent to those spilled in the art from the teachings herein.
The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.
Fig. 1 shows the cDNA sequence and the corresponding deduced amino acid sequence of the polypeptide of the present invention. The standard one letter abbreviations for amino acids are used. Sequencing was performed using 373 Automated DNA Sequencer (Applied Biosystems, Inc.). Sequencing accuracy is predicted to be greater than 97%.
Fig. 2 is an illustration of the amino acid sequence homology between the polypeptide of the present invention and other members .of the human PDGF/VSGF family. The boxed areas indicate the conserved sequences and the location of the eight conserved cysteine residues.
Fig. 3 shows a photograph of a gel after in vitro transcription, translation and electrophoresis of the polypeptide of the present invention. Lane 1: 1'C and rainbow M.W. marker; Lane 2: FGF control; Lane 3: VBGF2 produced by M13-reverse and forward primers; Lane 4: VEGF2 produced by M13 reverse and VEGF-F4 primers; Lane 5: VEGF2 produced by M13 reverse and VEGF-F5 primers.
Fig . 4 . VEGF2 polypeptide is expressed in a baculovirus system consisting of Sf9 cells. Protein from the medium and cytoplasm of cells were ana7.~r~ed by SDS-PAGB under reducing and non-reducing conditions.
Fig. 5. The medium from Sf9 cells infected with a nucleic acid sequence of the present invention was precipitated and the resuspended precipitate was analyzed by SDS-PAG$ and was stained with coomassie brilliant blue.
Fig. 6. VEGF2 was purified from the medium supernatant and analyzed by SDS-PAGE in the presence or absence of the reducing agent ~B-mercaptoethanol and stained by coomassie brilliant blue.
Fig. 7. Reverse phase IiPLC analysis of purified VEGF2 using a RP-300 column (0.21 x 3 cm, Applied Biosystems, Inc.). The column was equilibrated with 0.1% trifluoroacetic acid (Solvent A) and the proteins eluted with a~7.5 min gradient from 0 to 60% Solvent B, composed~of acetonitrile containing 0.07%"TFA. The protein elution was monitored by absorbance at 215 nm (Red line) and 280 nm (Blue line) . The percentage of Solvent B is shown by Green line.
Fig. 8 illustrates the effect of partially-purified VEGF2 protein on the growth of vascular endothelial cells in comparison to basic fibroblast growth factor.
Fig. 9 illustrates the effect of purified VBGF2 protein on the growth of vascular endothelial cells.
The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer)., as well as intervening sequences (introns) between individual coding segments (exons).
In accordance with one aspect of the present invention, there are provided isolated nucleic acid molecules (polynucleotides) which encode for the mature polypeptides .
having the deduced amino acid sequence of Figure 1 or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No 97149 ion May 12, 1995 at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, U.S.A., or for polypeptides which have fewer amino acid residues than those showing in Figure 1.
A polynucleotide encoding a polypeptide of the present invention may be obtained from early stage hcm~an embryo (week 8 to 9) osteoclastomas, adult heart or several breast cancer cell lines. The polynucleotide~ of this invention was discovered in a cDNA library derived from early stage human embryo week 9. It is structurally related to the tTBGF/PDGF
family.. VEGF2 contains an open reading frame encoding a protein of 419 amino acid residues of Which approximately the first 23 amino acid residues are the putative leader sequence such that the mature protein comprises 396 amino acids, and which protein exhibits the highest amino acid sequence _7_ homology to human vascular endothelial growth factor (30%
identity), followed by PDGFa (23%) and PDGF~ (22%).
It is particularly important that all eight cysteines are conserved within all four members of the family (see boxed areas of Figure 2). In addition, the signature for the PDGF/VEGF family, PXCVX~~XRCXGCCN, (S$Q ID N0:3) is conserved a.n VEGF2 (see Figure 2).
The VEGF2 polypeptide of the present invention is meant to include the full length polypeptide and polynucleotide sequence which encodes for any leader sequences and for active fragments of the full length polypeptide. Active fragments are meant to include any portions of the full length ama.no acid sequence which have less than the full 419 am3.no acids of the full length amino acid sequence as shown in S$Q ID No . 2 and Figure 2 , but still contain the eight cysteine residues shown conserved in Figure 2 and such fragments still contain VEGF2 activity.
There are at least two alternatively spliced VEGF2 mRNA
sequences present in normal tissues. The size of the two VEGF2 mRNA sequences which correspond to the full-length and truncated version respectively are shown in Figure 3, lane 5 shows two bands indicating the presence of the alternatively spliced~mRNA encoding the VEGF2 polypeptide of the present inventa.on. ' ' - _ -The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptide as the DNA of Figure 1 or the deposited cDNA.
_g_ The polynucleotide which encodes for the mature polypeptide of Figure 1 or for the 'mature polypeptide encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide (and optionally additional coding ' sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/a:r 3' of the coding sequence for the mature polypeptide.
Thus, the term "pol.ynucleotide encoding a polypeptide"
encompasses a polynucleotide wha.ch includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional. coding and/or non-coding sequence.
The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone. The variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
Thus, the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 or the same mature polypeptide encoded by the cDNA of the deposited clone as well as~wariants of such polpnucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 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 hereinabove indicated, the polynucleotide may have a coding sequence which .is a naturally occurring allelic variant of the coding sequence shown in Figure 1 or of the coding sequence of the deposited clone. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a, substitul=ion, deletion or addition WO 96/39515 ~ PCT/US96/09001 of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.
The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention. The marker sequence may be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).
The teen "gene's means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
Fragments of the full length gene of the present invention may be used as a hybridization probe for a cDNA
library to isolate the full length cDNA and to isolate other cDNAs which have a high sequence sima.larity to the gene or similar~biological activity. Probes of this type preferably have at least 30 bases and' may contain, for example, 50 or more bases. The probe may also be used to identify a cDNA
clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promotor regions, axons, and introns. An example of a screen comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the .
probe hybridizes to.
WO 96/39515 ~ PCT'/US96/0900I
The present invention further relates to polynucleotides which hyboidize to 'the hereinabove-described sequences if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences. The present invention particularly relates to polynucleotides which hybridine u~nde:r stringent conditions to the hereinabove-described polynucleotides. As herein used, the term "stringent conditions'° nneans hybridization will occur only if there is at least 95% and preferably at least 97% identity between the: sequences. The polynucleotides which hybridize to the hei:einabove described polynucleotides in a preferred embodiment: encode polypeptides which either retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of Figure 1 (S$Q ID NO:1) or the deposited cDNA(s).
Alternatively, the polynucleot:ide may have at least 20 bases, preferably 30 bases, and more preferably at least 50 bases which hybridize to~ a polynucleotide of the present invention and which has an identity thereto, as hereinabove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for the polynucleotide of S$Q ID NO:1, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR
primer . ~ ' ' - -Thus, the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% and more preferably at least a 95% identity to a polynucleotide which encodes the polypeptide of SEQ ID N0:2 as well as fragments thereof, which fragments have at least 30 bases and preferably at: least 50 bases and to polypeptides encoded by such polynucleotides.
The deposits) referred to herein will be maintained - under the terms of the Budapest Treaty on the International Recognition of the Deposit: of Micro--organisms for purposes of Patent Procedure. These deposits are provided merely as convenience to those of s:~cill in the art and are not an admission that a deposit is required under Section 38.1(2) of the Patent Act.
The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are controlling in the event of any conflict with any des~:ription of sequences herein. A license may be required to mz3ke, use or sell the deposited materials, and no such licentae is hereby granted.
The present invention further relates to a polypeptides which have the deduced amino acid sequence of Figure 1 or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptide.
The terms "fragment," "derivative" and "analog" when referring to t:he polypeptide of Figure 1 or that encoded by the deposited cDNA, means a polypeptide which'retains the conserved motif of V$GP proteins as shown in Figure 2 and essentially tt~e same biological function or activity.
The polypeptides of the present invention may be recombinant polypeptides, natural polypeptides or synthetic polypeptides, preferably recombinant.polypeptides.
The fragment, derivati'v~ or analog of the-polypeptide of Figure 1 or that encoded by the deposited cDNA may be (i)~
one in which one or.more of the amino acid residues are substituted with a conserved or non-consezved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid ref:idues includes a substituent. group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature -.12-polypeptide or (v) one in which comprises fewer amino acid residues shown in S$Q ID No. 2 and retains the conserved motif and yet still retains activity characteristic of the VEGF family of polypeptides. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
The tern "isolated" 'means than the material is removed from its original envirorunent (e. g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural systenn, is isolated. Such polynucleotides could be part oi= a vector and/or such polynucleotides or pol~rpeptides could be part of a composition, and still be isolated a.n that such vector or composition is not part of its natural environment.
The polypeptides of the present invention include the polypeptide of SSQ ID N0:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70%
similarity (preferably at" least 70% identity) to the polypeptide of S8Q ID N0:2 and more. preferably at least 90%
similarity (more preferably at least 95% identity) to the polypeptide of S$Q ID N0:2~ and still. more preferably at least 95% similarity (still more preferably at least 90% identity) to the polypeptide of SEQ ID N0:2 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
As known in the art "similarity" between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters , selecting transforn~a_nts or amplifying the V$GF2 genes of the present invention. The culture conditions, such as temperature, pH and the line, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and W~ 96/39515 PCT/US96/09001 phage DNA, viral DNA such. as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host.
The appropriate DNA sequence may be inserted into the vector by a variety of procedure~a. In general, the DNA
sequence is inserted into an appropriate restriction endonuclease sites) by procedures known in the art. Such procedures and others are: deemed to be within the scope of those skilled in the art.
The DNA sequence in the expression vector a.s operatively linked to an appropriate expression control sequences) (promoter) to direct mRI~TA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the F. c ~ lac or ~r , the phage lambda PL
promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses .
The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
The vector may also include appropriate sequences for amplifying expression.
In addition, the expression vectors preferably contain one or more selectable marker genes: to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in B. coli.
The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as B. coli, Streptomyces, Salmonella ~tvohimurimm; fungal cells, such as yeast; insect cells such as Droso.,phila S2 and Spodoutera Sf9;
animal cells such as CHO, CoS or Bowes melanoma;
adenoviruses; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. 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 by way of example. Bacterial: pQ$70, pQ860, pQ$-9 (Qiagen), pBS, pDlO, phagescript, psiX174, pBluescript SR, pBSKS, pNH8A, pNHl6a, pNHl8A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Phazmacia). $ukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Phazmacia). However, any other plasmid or vector may be used as long as they are replicable and viable a.n the host.
Promoter regions can be selected from any desired gene using CAT (chloramphenicol'~transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. .Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp.
Eukaryotic promoters include QMV immediate early, HSV
thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter a.s 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, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DBA,B-Dextran mediated transfection, or electroporation. (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).
The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
Mature proteins can be expressed in mammalian cells, yeast, bacte::ia, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the D,tZA constructs of the present invention.
Appropriate cloning and expression vectors for use with prokaryotic a~zd eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989).
Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an ~~nhancer sequence into the vector. Bnhancers~
are cis-acting elements of DNA; usually about from 10 to 300 by that act on a promoter to increase its transcription.
Bxam~ples including the SV40 enhancer on the late side of the replication o~:igin by 100 to 270, a cytomegalovirus early promoter~enhancer, the polyoma Pnhancer an the late side of the replication origin, and adenovirus enhancers.
Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation. of the host cell, e.g., the ampicillin resistance gene of B. coli and ~. cerevisiae TRP1 gene, and _x7_ a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and tezmination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include 8. coli, Bacillus subtilis, Salmonella twhimurium and var~.ous species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice .
As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC
37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GBM1 , (Promega Biotec, Madison, WI, USA). These pBR322 °backbone°°
b WO 96/39515 PCT/US96/0900i sections are combined with an appropriate promoter and the structural sequence to bye expressed.
Following transformation of a suitable host strain and growth of the host strain.to an appropriate cell density, the selected promoter is induced by appropriate means (e. g., temperature shift or chemical induction) and cells are cultured for an additional period.
Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
Microbial cells employed in Expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents , such methods are wel l know to those spilled in the art.
Various mammalian cell culture systems can also be employed to express rf~combinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for e.~cample, th.e C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ' ~ri.bosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences.. DNA sequences derived from the Sv40 splice, and polyadenylation sates may be used to provide the required nontranscri:bed genetic elements.
The polypeptides cyan be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated.
Polypeptides of the invention may also include an initial methionine amino acid residue.
As shown in Figures 8 and 9 , the VEGF2 polypeptide of SEQ ID No. 2, minus the initial 46 amino acids, is a potent mitogen for vascular endothelial cells and stimulates their growth and proliferation. The results of a Northern blot analysis performed for the VEGF2 nucleic acid sequence encoding this polypeptide wherein 20 ~.g of RNA from several human tissues were probed with 3aP-VEGF2, illustrates that this protein is actively expressed in the heart and lung which is further evidence of mitogenic activity.
Accordingly, VEGF2 ~ina~ ~ be employed ~o promote angiogenesis, for example, to stimulate the growth of transplanted tissue (where coronary bypass surgery a.s performed. VSGF2 may also be employed to promote wound healing, particularly to re-vascularize damaged tissues or stimulate collateral blood flow during ischemia and where new capillary angiogenesis is desired. VSGF2 may be employed to treat full-thickness wounds such as dermal ulcers, including pressure sores, venous ulcers, and diabetic ulcers. In addition, VSGF2 may be employed to treat full-thickness burns and injuries where a skin graft or flap is used to repair such burns and injuries. VEGF2 may also be employed for use in plastic surgery, for example, for the repair of lacerations from trauma and cuts in. association with surgery.
Along these same lines, VEGF2 may be employed to induce the growth of damaged bone, periodontium or ligament tissue.
VBGF2 may also be employed for regenerating supporting tissues of the teeth, including cementum and periodontal ligament, that have been damaged bay disease and trauma.
Since angiogenesis is important in keeping wounds clean and non-infected, vEGF2 may be employed in association with surgery and following tyre repair of cuts. It may also be employed for the treatments of abdominal wounds where there is a high risk of infection.
vEGF2 may be employed for the promotion of endothelialization in vascular graiEt surgery. In the case of vascular grafts using either t:cansplanted or synthetic material, VEGF2 can be applied to t:he surface of the graft or at the junction to promote the growth of vascular endothelial cells. vEGF2 may also be employed to repair damage of myocardial tissue as a result c~f myocardial infarction.
VEGF2 may also be employed to repair the cardiac vascular system after ischemia. 'VEGF2 may also be employed to treat damaged vascular tissue as a result of coronary artery disease and peripheral and CNS vascular disease.
VEGF2 may also be employed to coat artificial prostheses or natural organs which acre to be transplanted in the body to minimize rejection of the transplanted material and to stimulate vascularizatio:n of the transplanted materials.
VEGF2 may also be employed for vascular tissue repair, for example, that occurring during arteriosclerosis and required following balloon angioplasty where vascular tissues are damaged.
V>3GF2 nucleic acid faequences and V13GF2 polypeptides may also be employed for in ~rritro purposes related to scientific research, synthesis of I)NA and manufacture of DNA vectors, and for the production of diagnostics and therapeutics to treat human disease. For example, VEGF2 may be employed for in vitro culturing of vascular endothelial cells, where it is added to the conditional medium in a concentration from 10 pg/ml to 10 ng/ml.
Fragments of the full length VEGF2 gene may be used as a hybridization probe for a cDNA library to isolate other genes which have a high sequence similarity to the gene or similar biological activity. Probes of this type generally have at least 50 base pairs, although they may have a greater number of bases. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete vBGF2 gene including regulatory and pramotor regions, exons, and introns. An example of a screen comprises isolating the coding region of the VEGF2 gene by using the known DNA
sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
This invention provides methods for identification of vEGF2 receptors. The gene encoding the receptor can be identified by numerous methods known to those of skill a.n the art, for example, ligand panning and FAGS sorting (Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). Preferably,,expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to VEGF2, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to VSGF2. Transfected cells which are grown on glass slides are exposed to labeled VEGF2.
VEGF2 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.
WO 96139515 ~ PCT/US96/0900I
Positive pools are identified and sub-pools are prepared and retransfected using an iterative s:ub-pooling and rescreening process, eventually yielding a single clone that encodes the putative receptor.
As an alternative approach for receptor identification, labeled VEGF2 can be pho~toaffinity linked with cell membrane or extract preparations that express the receptor molecule.
' Cross-linked material a.s. resolved by PAG$ and exposed to X-ray film. The labeled. complex containing VBGF2 is then excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from ma.crosequencing would be used to design a set of degenerate oligonucleoti~de probes to screen a cDNA library to identify the gene encoding the pui_ative receptor.
This invention is also related to a method of screening compounds to identify lthose which are VEGF2 agonists or antagonists. An example of such a~ method takes advantage of the ability of VSGF2 to significantly stimulate the proliferation of human endothelial cells in the presence of the comitogen Con A. $ndothelial cells are obtained and cultured in 96-well flint-bottomed culture plates (Costar, Cambridge, MA) in a reaction mixture supplemented with Con-A
(Calbiochem, La Jolla, CA). ~ Con-A, polypeptides of the present invention and the compound to be screened are added.
After incubation at 37°C, cultures are pulsed with 1 ~.Ci of 3 [H] thymidine (5 Ci/mmol; 1 Ci = 37 BGq; NEN) for a sufficient time to incorporate the 3 [H] and ;harvested onto glass fiber filters (C:ambridge Teclvzology, Watertown, MA) . Mean ' [H] -thymidine inco~oration (cpm) of triplicate cultures is determined using a liquid scintillation counter (Beckman Instruments, Irvine, CA) . Significant ' [H] thymidine incorporation, as compared to a control assay where the compound is excluded, indicates stimulation of endothelial cell proliferation.
WO 96/39515 ~ PCT/US96/09001 To assay for antagonists, the assay described above is performed and the ability of the compound to inhibit ' [H] thymidine incorporation in the presence of VBGF2 indicates that the compound is an antagonist to VEGF2. Alternatively, VI3GF2 antagonists may be detected by combining VEGF2 and a potential antagonist with membrane-bound VEGF2 receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay. Vl3GF2 can be labeled, such as by radioactivity, such that the number of VEGF2 molecules bound to the receptor can determine the effectiveness of the potential antagonist.
Alternatively, the response of a known second messenger system following interaction of VEGF2 and receptor would be measured and compared in the presence or absence of the compound. 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 VEGF2 receptor a.s incubated with labeled VEGF2 in the presence of the compound.
The ability of the compound to enhance or block this interaction could then be measured.
Potential V13GF2 antagonists include an antibody, or a.n some cases, an oligonucleotide, which bind to the polypeptide and effectively eliminate VSGF2 function. Alternatively, a potential antagonist may be a closely related protein which binds to VEGF2 receptors, however, they are inactive forms of the polypeptide and thereby prevent the action of VSGF2.
$xamples of these antagonists include a negative dominant mutant of the VEGF2 polypeptide, for example, one chain of the hetero-da.meric form of vEGF2 may be dominant and may be mutated such that biological activity is not retained. An example of a negative dominant mutant includes truncated versions of a dimeric VI3GF2 which is capable of interacting with another dimer to form wild type VEGF2, however, the WO 96/39515 PCTlUS96/0900I
resulting homo-dimer a.s inact:ive and fails to exhibit characteristic VEGF activity.
Another potential. VSGF2 antagonist is an antisense construct prepared using antisense technology. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DZ~TA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl. Acids Res., 6:3073 (1979); Gooney et al, Science, 241:456 (1988);
and Dezvan et a1_, Science, 251: 1360 (1991)), thereby preventing transcription and the production of VJ3GF2. The antisense RNA oligonucl.eotide hybridizes to the mRNA in vivo and blocks translation of the mltNA molecule into the VSGF2 polypeptide (Antisense - Okano, J. Neurochem., 56:560 (1991);
Oligodeoxynucleotides as Antisense Inhibitors of Gene $xpression, CRC Press., Boca Raton, FL (1988)). The oligonucleotides descr:Lbed above can also be delivered to cells such that the antisense~RNA or DNA may be expressed in vivo to inhibit production of VSGF2.
Potential ~TEGF2 anttagonists also include small molecules which bind to and occupy the active site of the polypeptide thereby making the catalytic site inaccessible to substrate such that normal biological activity is prevented. $xamples of small molecules include but are not limited to small peptides or peptide-like molecules.
The antagonists may be employed to treat limit angiogenesis necessary for solid tumor metastasis.
The mRNA encoding for VBGF2 is found to be expressed at moderate levels in at leaast two breast tumor cell lines which is indicative of the role of VEGF2 polypeptides in the malignant phenotype. Gliomas are also a type of neoplasia which may be treated with the antagonists of the present invention.
The antagonists may also be used to treat chronic inflammation caused by increased vascular permeability. In addition to these disorders, the antagonists may also be employed to treat retinopathy associated with diabetes, rheumatoid arthritis and psoriasis.
The antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.
The VEGF2 polypeptides and agonists and antagonists may be employed 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 a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical composit~.ons of the invention. Associated with such containers) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the pharmaceutical compositions may be employed in conjunction with other therapeutic compounds.
The pharmaceutical compositions may be administered in a convenient manner such as by the topical, intravenous, intraperitoneal, intramuscular, intratumor, subcutaneous, intranasal or intrader<nal routes. The pharmaceutical compositions are administered in an amount which is effective for treating and/or prophylaxis of the specific indication.
In general, the pharmaceutical ccm~positions are administered in an amount of at least about :LO ~Cg/kg body weight and in most cases they will 7be admini~~tered in an amount not in excess of about 8 mg/Kg body weight per day. In most cases, the dosage is from about 10 ug/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.
The V13GF2 polypeptides, and agonists or antagonists which are polypeptides may also be employed in accordance with the present invention by expression of such polypeptide in~ vivo, which is often referred to as "gene therapy. "
Thus, for example, cells such as bone marrow cells may be engineered with a polynucleotide (DNA or RNA) encoding for the polypeptide ex vi:vo, the engineered cells are then provided to a patient to be treated with the polypeptide .
Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding the polypeptide of the present invention.
Similarly, cells may be engineered in vivo for expression of a polypeptide ~~in vivo, for example, by procedures known in the art: 'As :known in the art; a producer cell for producing a retroviral particle containing RNA
encoding a polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide .in vivo. These and other methods for administering a polypeptide 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, the expression, vehicle f_or engineering cells may be other than a retroviral particle, for example, an adenovirus, which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
WO 96/39515 ' PCT/US96109001 Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus , human immunodef iciency virus , adenovirus , Myeloproliferative Sarcoma Virus, and mammary tumor virus .
In one embodiment, the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.
The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (C3~tV) promoter described a.n Miller, et al. , Biotechnigues, Vol. 7, No. 9, 980-990 (1989), or any other promoter (e. g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and ~-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thyma.dine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art 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 which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or hetorologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described); the ~B-actin promoter; and human growth hormone promoters. The pramoter also may be the native promoter which controls the gene encoding the polypeptide.
The rEaroviral plasmid vector is employed to transduce packaging cell lines to fozm producer cell lines. $xamples of packaging cells which may be transfected include, but are not limited to, the P$501, PA317, ~-2, ~-AM, PA12, T19-14X, VT-19-17-H~;, ~C~tg, ~Cg,Ip, GP+$-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Theraov, Vol. 1, pgs. 5-14 x;1990), The vector may transduce the packaging cells through any means known in the art. Such means include, bu,t are not limited to, electroporation, the use of liposomes, and CaPO, precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequences) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or' in vivo. The transduced eukaryotic cells will express the nucleic . acid sequences) encoding the.
polypeptide. 8ukaryotic cells which may be transduced include, but are not limited~~to, .embryonic -stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocyt~s, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.
This invention is also related to the use of the V$GF2 gene as part of a diagnostic assay for detecting diseases or susceptibility to diseases related to the presence of mutations in VBGF2 nucleic acid sequences.
Individuals carrying mutations in the V8GF2 gene may be detected at the DNA level by a variety of techniques.
Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy and ' -29-WO 96/39515 ~ ' PCT/US96109001 autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR
(Saiki et al., Nature, 324:163-166 (1986)) prior to analysis.
RNA or cDNA may also be used for the same purpose . As an example, PCR primers complementary to the nucleic acid encoding VEGF2 can be used to identify and analyze VSGF2 mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype . Point mutations can be identified by hybridizing amplified DNA to radiolabeled VBGF2 RNA or alternatively, radiolabeled VEGF2 antisense DNA
sequences. 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 may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA
fragments of different sequences may be distinguished on denaturing forniamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see," e:g., Myers et al:, Science, 23:1242 (1985)).
Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).
Thus, the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e. g., Restriction Fragment Length Polymorphisms (RFLP)) and Southern blotting of genomic DNA.
WO 96/39515 ' ~ PCT/US96/0900I
In addition to more conventional gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.
The present invention also relates to a diagnostic assay for detecting altered levels of VBGF2 protein i.n various tissues since an over-expression of the proteins compared to normal control tissue samples ma.y detect the presence of a disease or susceptibility to a disease, for example, abnormal cellular differentiation. Assays used to detect levels of VSGF2 protein in a sample derivecl from a host are well-known to those of skill in the art and include radioimmunoassays, competitive-binding as~aays, Western Blot analysis, ELISA
assays and "sandwich" .assay. An $LISA assay (Coligan, et al., Current Protocol:a in Immunology, 1(2), Chapter 6, (1991) ) initially comprises preparing an antibody specific to the VBGFZ antigen, preferably a~ monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal antibody. To the reporter antibody is attached a detectable reagent such as radioactivity, fluorescence or, in this example, a horseradish pero:xidase enzyme. A sample is removed from a host and incubated on a solid support, e.g. a polystyrene dish, that hinds the~proteins in the sample. Any free protein binding s2tes on tree dish are then covered by incubating with a non-specific~protein, such as, bovine serum albumen. Next, the monoclonal antibody a.s incubated in the dish during which time the monoclonal antibodies attach to any VSGF2 proteins attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer. The reporter antibody linked to horseradish peroxidase is placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to VSGF2. Unattached reporter antibody is then washed out. Peroxidase substrates are then added to the dish and the amount of color developed in a given time period is a. measurement of the amount of VEGF2 protein present in a given volume of patient sample when compared against a standard curve.
A competition assay may be employed wherein antibodies specific to VBGF2 are attached to a solid support.
Polypeptides of the present invention are then labeled, for example, by radioactivity, and a sample derived from the host are passed over the solid support and the amount of label detected, for example by liquid scintillation chromatography, can be correlated to a quantity of VSGF2 in the sample.
A "sandwich" assay is similar to an SLISA assay. In a "sandwich" assay VBGF2 is passed over a solid support and binds to antibody attached to a solid support. A second antibody is then bound to the VSGF2. A third antibody which is labeled and specific to the second antibody is then passed over the solid support and binds to the second antibody and an amount can then be quantified.
The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome . Few chromosome marking reagents based on actual sequence data (repeat polymorphism's) are presently available for marking chromosomal location. The~'mapping of DNAs to -chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA.
Computer analysis of the cDNA is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing inda.vidual human chromosomes. Only those , hybrids containing the human gene corresponding to the primer will yield an amplified fragment .
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome .
Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous nnanner. Other mapping strategies that can similarly be used tto map to its chromosome include in situ hybridization, pr~sscreening~ with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDiz4 libraries .
Fluorescence in situ hybridization (FISH) of a cDNA
clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used With cDNA as short as 50 or 60 bases.
For a review of this techniques see Verma et al., Human Chromosomes: a Manual of Basic Techniques. Pergamon Press, New York (1988) .
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for Example, :Ln V. McKusick, Mendelian Inheritance in Man (availa3~le~'on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
Next, it is necessary to deaermine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but- not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb ) .
The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies.
The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
Antibodies generated against the polypeptide corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptide into an animal or by administering the polypeptide to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptide itself. In this manner, even a.sequence encoding only a fragment of the polypeptide can be used to generate antibodies binding the whole native polypeptide.
Such antibodies can then be' used to isolate the~polypeptide from tissue expressing that polypeptide. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. $xamples include the hybridoma technique (Rohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Rozbor et al., 1983, Immunology Today 4:72), and the $BV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., PP. 77-96).
Techniques described for the production of single chain antibodies (U. S. Patent 4,946,77x) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. Also, transgenic mice may be used to express humanized antibodies to immunogenic polypeptide products of this invention.
The present invent=ion will be further described with reference to the following examples; however, it is to be understood that the pre~aent invention is not limited to such examples. All parts or amounts, ~.uiless otherwise specified, are by weight.
In order to facilntate understanding of the following examples, certain frequently occurring methods and/or tezms 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, publicly available on an unrestricted basis, or can be constructed from availalble plasmi,ds in accord with published procedures. In addition, equ~!valent plasmi.ds to those described are known in the art and will be apparent to the ordinarily skilled artisan.
"Digestion" of DNA. refers to catalytic cleavage of the DNA with a restriction enzymes 'that acts only-at certain sequences in the DNA. The various restriction enzymes used herein are commercia7_ly available and their reaction conditions, cofactors ~3nd other requirements were used as would be known to they ordinarily skilled artisan. For analytical purposes, t=ypically 1 ~g of plasmid or DNA
fragment is used with aabout 2 units of enzyme in about 20 ~,1 of buffer solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 ~.g of DNA are digested with 20 to 250 r.~nits of enzyme in a larger . volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the WO 96/39515 PC'T/US96/09001 manufacturer. Incubation times of about 1 hour at 37~C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D.
et al., Nucleic Acids Res., 8:4057 (1980).
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 ~Cg of approximately equimolar amounts of the DNA'fragments to be ligated.
Unless otherwise stated,' transformation was ZSerformed as described by the 'method of Graham, F. and Van der 8b, A. , Virology, 52:456-457 (1973).
$~cample 1 Expression uattern of yEGF2 in human tissues and breast oancer cell lines Northern blot analysis was carried out to examine the levels of expression of the V8GF2 gene in human tissues and human breast cancer cell lines. Total cellular RNA samples were isolated with RNAzol1''' B system (Biotecx Laboratories , Inc . ) . About 10 ~.g of total RNA isolated from each breast tissue and cell line specified was separated on ~.% agarose gel and blotted onto a nylon filter, (Molecular Cloning, Sambrook Fritsch, and Maniatis, Cold Spring Harbor Press, 1989). The labeling reaction was done according to the TM
Stratagene Cloning Systems, Inc., Prime-It kit with 50 ng DNA
fragment. The labeled DNA was purified with a Select-G-50 column from 5 Prime -- 3 Prime, Inc, Boulder, CO, USA. The filter was then hybridized with radioactively labeled full length VEGF2 ~~ene at 1,000,000 cpm/ml in 0.5 M NaP04and 7 %
SDS overnight: at 65°C. After washing twice at room temperature acid twice at 60°C with 0.5 X SSC, 0.1 % SDS, the filters were then exposed at -70°C overnight with an intensifying :screen. A message of 1.6 Rd was observed in 2 breast cancer cell lines.
$xample 2 Cloning and expression of VEGFZ usincx the baculovirus expression s~rstem The DNA ;sequence encoding the VBGF2 protein without 46 amino acids at the N-terminus, see ATCC I 9'~1'~9 was amplified using PC'R. oligonucleotide primers corresponding to the S' and .
3' sequences of the gene:
The 5' primer has the sequence TGT AAT ACG~ACT CAC TAT
AGG GAT CCC GC'.C ATG GAG GCC ACG GCT TAT GC (SBQ ID N0:4) and contains a BarnHl restriction enzyme site (in bold) and 174 nucleotide nucleotide sequence complementary to the 5' sequence of VE;GF2 (nt. 150-166) .
The 3' primer has the sequence GATC TCT AG,A TTA GCT CAT
TTG T'GG TCT (SBQ ID N0:5) and contains the cleavage site for the restz'iction enzyme XbaI and 18 nucleotides complementary.
to the 3' sequence of VSGF2, including the stop codon and 15 nt sequence before stop codon. .
The amplified sequences were isolated from a 1% agarose gel using a cocamercially available kit ( "Geneclea~ M!' BIO 101, Inc., La Jolla, CA). The fragment was then digested with the endonuclease HamH1 and XbaI and then purified again on a 1%
agarose ge.l. This fragment was liaated to pAcGP67A
baculovirus transfer vector (PHarmingenjMat the BamHl and XbaI sites. Through this ligation, V$GF2 cDNA was cloned in frame with the signal sequence of baculovirus gp67 gene and was located at the 3' end of the signal sequence in the vector. T'h:is is designated pAcGP67A-V$GF2.
To clone VBGF2 with the signal sequence o~ gp67 gene to the pRG1 vector for expression,, VSGF2 with the signal sequence and some upstream sequence were excised from the pAcGP67A-VBG~F2 plasmid at the Xho restriction endonuclease site located upstream of the VBGF2 cDNA and at the Xbal restriction endonuclease site by XhoI and Xbal restriction enzyme. This fragment was separated from the rest of vector on a 1% agarose gel and was purified using "Geneclean"Mkit.
It was desigrnated F2.
The PRG~1 vector (modification of pVL941 vector) is used for the expression of the V8GF2 protein using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.B. 19S7,.y manual of methods for baculovirus.vectars and insect cell culture procedures, Texas Agricultural ..
$xperimental Station Bulletin No. 1555). This expression vector contains the strong polyhedrin promoter of the Autographa t:alifornica nucTeaf polyhedrosis virus (AcMNpV) followed by the recognition sites for the restriction .
endonucleases BamHl, Smal, XbaI, HglII and Asp718. A site for restriction endonuclease Xho1 is located upstream of HamHl site. The sequence between Xhol and BamHI is the same as that in PAcGp67A (static on tape) vector. The polyadenylat:ion site of the simian virus (SV) 40 is used for efficient polyadenylation. - For an easy selection of recombinant virus the beta-galactosidase gene from 8.coli is .
inserted in 'the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sides by viral sequences for the cell-mediated homologous recombination of cotransfected wild-type viral DNA. Many other baculovirus vectors could be used in place of pRGl such as pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D., Virology, 1~~0:31-39). ' The plasmid was digested with the restriction enzymes XboI and XbaI and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The DNA was then isolated from a 1% agarose gel using the commercially TM
available k:i.t ("Geneclean" BIO 101 Inc., La Jolla, Ca.).
This vector DNA,ie designated V2.
Fragmea,t F2 and the dephosphorylated plasmid V2 were ligated with T4 DNA ligase. $.col3 HB101 cells were then transformed and bacteria identified that contained the plasmid (pHac gp67-VSGF2) with the VBGF2 gene using the enzymes HamHl and XbaI. The sequence of the cloned ,fragment was confirmed by DNA sequencing.
~Cg of the plasmid pane gp67-VgGF2 was cotransfected with 1.0 erg of a commercially available linearized baculovirus ("HaculoGold° baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Felgner et al.
Proc. Natl. :~lcad. Sci. USA, 84:7413-7417 (1987)).
l~Cg of BaculoGold" virus DNA -and 5 ~Cg of the plasmid pBac gp67-vBt~F2 were mixed~in a sterile well of a microtiter plate containing 50 ~1 of serum free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Afterwards 10 ~,1 Lipofectin plus 90 ~tl Grace' s medium were added, mixed and incubated fc~r 15 minutes at room temperature. Then the transf action mixture was added dropwise to the Sf9 insect cells ('ATCC C~tL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate was rocked back and forth to mix the newly added solution. The plate was then incybated for 5 hours at 27°C. After~5 hours the transfection solution was removed from the plate and 1 ml of Grace's inseca medium supplemented with 10% fetal calf serum was added. The plate was put back into an incubator and cultivation continued at 27°C for four days, After four days the supernatant was collected and a plaque assay performed 'similar as described by Summers and Smi.th~(supra). As a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) was used which allows an easy isolation of blue stained p7.aques. (A
detailed desccription of a "plaque assay" can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10) .
Four days after the serial dilution, the virus was added to the cellt~, blue stained plaques were picked With the tip of an Sppend.orf pipette: The agar containing the recombinant TM
viruses was then resuspended in an Bppendorf tube containing 200 ~1 of G:cace's medium. The agar was removed by a brief centrifugation and the supernatant containing the recombinant baculovi.rus was used to infect Sf9 cells seeded in 35 mm dishes . Fo~.ir days later the supernatants of these culture dishes were harvested and then stored at 4°C.
Sf 9 cells were grown in Grace ' s medium supplemented with 10% heat-inactivated FBS. The cells were infected with the ..
recombinant baculovirus V-gp6T-V8GF2 at a multiplicity of infection (MOI) of 1. Six hours later the medium-was removed and rep2aced with SF900 II medium minus methionine and cysteine (L9.fe Technologies Inc. , Gaithersburg) . 42 hours later 5 ~eCi of "S-methionine and 5 ~CCi 'SS cysteine (Amersham) were added. The cells were further incubated for 16 hours before they were harvested by centrifugation and the labelled proteins visualized by SDS-PAGE and autoradiography.
Protein. from the medium and cytoplasm of the Sf9 cells was analyzed by SDS-PAGE under reducing and nori-reducing conditions. See Figure 4. The medium was dialyzed against 50 mM MSS, pH 5.8. Precpitates were obtained after dialysis and resuspended in 100 mM NaCitrate, pH 5Ø The resuspended precipitate was analyzed again by SDS-PAGE and was stained with Coomassie Brilliant Blue. See Figure 5.
The medium supernatant was also diluted 1:10 in 50 mM
MES, pH 5.8 and applied to an SP-650M column (1.0 x 6.6 cm, Toyopearl) at a flow rate of 1 ml/min. Protein was eluted with step gradients at 200, 300 and 500 mM NaCl. The VEGF2 was obtained using the elution at 500 mM. The eluate was analyzed by SDS-PAGE in the presence or absence of reducing agent, f3-mercaptoethanol and stained by Coommassie Brilliant Hlue. See Figure 6.
Example 3 E ression of Recombinant VEGF2 in COS cells The expression of plasmid, VBGF2-HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV4U origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, an SV40 intron and polyadenylation site. A DNA
fragment encoding the entire VBGF2 precursor and a HA tag fused in frame to its 3' end was cloned into the polylinker region of the vector, therefore,. the recombinant protein expression is directed under the CMV promoter. The HA tag corresponds to an epitope derived fr~n the influenza hemagglutinin protein as previously described (I. Wilson, H.
Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37:767, (1984)). The infusion of HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
The plasmid construction strategy is described as i follows The DNA sequence encoding VBGF2, ATCC # 97149 was constructed by PCR using two primers : the 5' primer (CGC GGA
TCC ATG ACT GTA CTC TAC CCA) (SEQ ID N0:6) contains a BamH1 site followed by 18 nucleotides of VEGF2 coding sequence starting from the initiation codon; the 3' sequence (CGC TCT
WO 96/39515 ' PCT/US96/09001 AGA TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA CTC GAG GCT CAT
TTG TGG TCT 3') (SBQ ID N0:7) contains complementary sequences to an XbaI site, HA tag, XhoI site, and the last 15 nucleotides of the VSGF2 coding sequence (not including the stop codon). Therefore, the PCR product contains a BamHI
site, coding sequence followed by an XhoI restriction endonuclease site and HA tag fused in frame, a translation termination stop codon next to the HA tag, and an XbaI site. ' The PCR amplified DNA fragment and the vector, pcDNAI/Amp, were digested with BamH1 and XbaI restriction enzyme and ligated. The ligation mixture was transformed into 8. coli strain SURE (Stratagene Cloning Systems, La Jolla, CA 92037) the transformed culture was plated on ampicillin media plates and resistant colonies were selected. Plasmid DNA was isolated from transformants and examined by restriction analysis for the presence of the correct fragment. For expression of the recombinant VEGF2, COS cells were transfected with the expression vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A
Laboratory Manual, Cold Spring Laboratory Press, (1989)).
The expression of the VEGF2-HA protein was detected by radiolabelling and immunoprecipitation method (B. Harlow, D.
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1-988)) ."Cells were labelled-for 8 hours with 3sS-cysteine two days post transfection. Culture media was then collected and cells were lysed with detergent (RIPA
buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5) (Wilson, I . et al. , Id. 37:767 (1984) ) .
Both cell lysate and culture media were precipitated with an HA specific monoclonal antibody. Proteins precipitated were analyzed on 15% SDS-PAG$ gels.
$~le 4 The effect of partially-purified VSGF2 protein on the Qrowth Qf vascular endothelial cells On day 1, human umbilical vein endothelial cells (HUVBC) were seeded at 2-5x10' cells/35 mm dish density in M199 medium containing 9:% fetal bovine serum (FHS), 16 units/ml heparin, and 50 unit;s/ml endothelial cell growth supplements (BCGS, Biotechnique, Inc.). On day 2, the medium was replaced with M199 containing 10% FBS, 8 units/ml heparin. VEGF2 protein of SBQ ID NO. 2 minus the initial 45 amino acid residues, (~TSGF) a.nd basic FGF (bFGF) were added, at the concentration shown. On days 4 & 6, the medium was replaced. On day 8, cell number was determined with a Coulter Counter (See Figure 8) .
Example 5 The effect of aurified V8GF2 protein on the growth of vascular endothelial cells On day :L, human umbilical vein endothelial cells (BUVBC) were seeded at 2-5 x 10' cells/35 min dish density in M199 medium containing 4% fetal bovine serum (FBS), 16 units/ml heparin, 50 units/ml endothelial cell growth supplements (BOGS, Hiotechnique, Inc.). On day 2, the medium was replaced with M199 containing 10% FBS, 8 units/ml heparin. ., Purified V8G:B2 protein of SBQ ID No. 2 minus initial 45 amino acid residues was added to the medium at this point. On days 4 & 6, the medium' was replaced with fresh -medium and r supplements. On day 8, cell number was determined with a Coulter Counter (See Figure 9).
Example 6 Bxaressi_on v:~a.Gene Thera Fi~roblasts are obtained from a subject by skin biopsy.
The resultin~~ tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are , placed on a wet surface of a tissue culture flask, approximatel;t ten pieces are placed in each flask. The flask is turned upside. down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin, is added.
This is then incubated at 37°C for approximately one week.
At this time, fresh media is added and subsequently changed every several days. After an additional two weeks a.n culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.
pMV-7 (Kirschmeier, P.T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.
The cDNA encoding a polypeptide of the present invention is amplified using PCR primers which correspond to the 5' and 3' end sequences respectively. The 5' primer containing an EcoRI site and the 3' primer further includes a HindIII site.
$qual quantities of the Moloney murine sarcoma virus linear backbone and the amplified BcoRI and HindIII fragment are added together, in the presence of T4 DNA lipase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments . The ligatiorr mixture is used to transform bacteria HB101, which are then plated onto agar-containing kanamycin for the purpose of confizmi.ng that the vector had the gene of interest properly inserted.
The amphotropic pA317 or GP+aml2 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMBM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells are transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells).
Fresh media is added to the tra.nsduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent: producer cells. The spent media, containing the infectious viral particles, is filtered through a milliporeTM
filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the mE~dia from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection. is required. Zf the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such. as neo or his.
The engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence TM
on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product.
Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, within the scope of the appended .claims, the invention ma.y be practiced otherwise than as particularly..
described.
SEQUENCE LISTING
(1) GENERAL INFORMATTON:
(i) APPLICANT: Human Genome Sciences, Inc.
(ii) TITLE OF INVENTION: Human Vascular Endothelial Growth Factor 2 (iii) NUMBER OF SEQUENCES: 10 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: MBM & C0.
(B) STREET: P.O. BOX 809, STATION B
(C) CITY: OTTAWA
(D) PROVINCE: ONTARIO
(E) COUNTRY: CANADA
(F) POSTAL CCDE: K1P 5P9 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: FlopMpy disk (B) COMPUTER: IBM PC compatible TM
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIriMVer. 2.0 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,224,093 (B) FILING DATE: 6-JUNE-1996 (C) CLASSIF.IC'.ATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/465,968 (B) FILING DF,TE : 6-JUNE-1995 (C).CLASSIFIC:ATION:
(viii) ATTORNEY/AGE1~IT INFORMATION:
(A) NAME: S~NAIN, Margaret (B) REGISTRATION NUMBER: 10926 (C) REFEREN~~E/DOCKET NUMBER: 306-127 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 613/567-0762 (B) TELEFAX: 613/563-7671 (2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1674 (B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
GTCCTTCCAC CATGCACTC'G CTGGGCTTCT TCTCTGTGGC GTGTTCTCTG CTCGCCGCTG 60 CGCTGCTCCC GGGT'CC'TCGC GAGGCGCCCG CCGCCGCCGC CGCCTTCGAG TCCGGACTCG 120 ACCTCTCGGA CGCGGAGCCC GACGCGGGCG AGGCCxICGGC TTATGCAAGC AAAGATCTGG 180 ATTGGAAAAT GTACAAGTC~'T CAGCTAAGGFa AAGGAGGCTG GCAACATAAC AGAGAACAGG 300 CCAACCTCAA CTC31,AGGACA GAAGAGACTA TAAAATTTGC TG~CA~T TATAATACAG 360 AGATC7:TGAA AAGTATTGAT AATGAG'T'GGA GAAAGACTCA ATGCATGCCA C'GGGACiGTGT 420 GTATAGATGT GGGG~AAGGAG TTTGGAGTCG ~CGACAAACAC CTTCTTTAAA CCTCCATGTG 480 TGTCCGTCTA ~CAGF.TGTGGG GGTTGCTGCA ATAGTGAGGG GCTGCAGTGC ATGAACACCA 540 TTTACAGACA AGTT'CATTCC ATTATTAGAC GTTCCCTGCC AGC31ACACTA CCACAGTGTC 720 AGGCAGCGAA CAAGiACCTGC CCCACCAATT ACATGTGGAA TAATCACATC TGCAGATGCC 780 TGGCTCAGGA RGATZ'ITATG TTTTCCTCGG ATGCTGGAGA TGACTCAACA GATGGATTCC 840 ATGACATCTG TGGA,CCAAAC AAGGAGCTGG ATGAAGAGAC CTGTCAGTGT GTCTGCAGAG 900 CGGGGCZTCG GCCT'GCCAGC TGTGGACCCC AC~1~AAGAACT AGACAGAiIAC TCA~GCCAGT 960 GTGTCTGTAA AAAC:AAACTC TTCCCCAGC:C AATGTGGGGC CAACCGAGAA TTTGATGAAA 1020 WO 96/39515 PCTlUS96/09001 ACACATGCCA GTGT'GTATGT AAAAGAACCT GCCCCAGAAA TCAACCCCTA AATCCTGGAA1080 AATGTGCCTG TGAA,TGTACA GAAAGTCCAC AGAAATGCTT GTTAAAAGGA AAGAAGTTCC1140 ACC3~CCAAAC ATGC31GCTGT TACpGACGGC CATGTACGAA CCGCCAGAAG 1200 GCT!'GTGAGC
CAGCATTTTC ATR,TAGTGAA GAAGTGTGTC GTTGTGTCCC TTCATATTGG CAAAGACCAC1260 AAAGTCTGTC TTTC.'CTGAAC CATGTGGATA ACTTTACAGA AATGGAC,'TGG 1440 AGCTCATCTG
TTGTGATTTC TIT3~AAAGAA TGACTATATA ATTTATTTCC ACTAAAAATA TTGTTTCTGC1560 ATTCATTTTT ATAGCAACAA CAATTGGTAA AACTCACTGT GATC3~ATATT TTTATATCAT1620 GCAAAATATG TTT3iAAATAA AATGAAA)~TT GTATTTATAA AAAAAAAAAA 1674 AAAA
( 2 ) INFORMA'.CION POR SBQ ID NO : 2 i ) S$QU8P7CE C~A,R.ACTBRISTICS
(A) LENGTH: 419 AMINO ACIDS
(B) TYPE: AMINO ACID
( C) STRAPTDEDNESS
(D) TOPOLOGY: LINBAR
(ii) MOLBCOLB TYPE: PROTEIN
(xi) SEQUENCE DESCRIPTION: SBQ ID N0:2:
Met His Ser Leu Gly Phe Phe 5er Val Aln Cys Ser Leu Leu Ala Ala Ala Leu Leu~Pro Gly Pro Arg Glu 3rla Pro Ala Ala Ala Ala Ala-Phe a Glu Ser Gly Leu Asp Iaeu Ser Asp Ala Glu Pro Asp Ala Gly Glu Ala Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu Gln Leu Arg Ser Val Ser Ser Val Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met 20 ~: 25 30 Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln Ala Asa Leu Aaa Ser Arg Thr Glu Glu Thr Ile Lys Phe Aln Ala Ala His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asa Glu Tzp Arg Lys WO 96/39515 ' PCT/US96/09001 Thr Gln Cps Met Pro Arg Glu 'Val Cars Ile Asp Val Gly Lys Glu Phe Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro C'ys Val Ser Val Tyr Arg t'~ss Gly Gly t'~rs t'~ys Asn ,Ser Glu Gly Leu Gln tars Met Asn Thr Ser Thr Ser Tyr Leu Ser Lys 'Thr Leu Phe Glu Ile Thr Val Pro Leu Ser Gln Gly Pro Lys Pro Val 'Thr Ile Ser Phe Ala Asn His Thr Ser fuss Arg Cys Met Ser Lys Leu :Asp Val Tyr Arg Gln Val His Ser Ile 165 ;170 I75 Ile Arg Arg Ser Leu Pro Ala 'Thr Leu Pro Gln Cys Gln Ala Ala Asn Lys Thr t'=ys Pro Thr Asn Tyr IKet Trp Asn Asn His Ile Cys Arg Cys Leu Ala Gln Glu Asp Phe Met IPhe Ser Ser Asp Ala Gly Asp Asp Ser Thr Asp Gly Phe His Asp Ile I~rs Gly Pro Asn Lys Glu Leu Asp Glu Glu Thr Cars Gln Cps Val Cps :Arg Ala Gly Leu Arg Pro Ala Ser Cars 245 :250 255 Gly Pro His Lys Glu Leu Asp ~Arg Asn Ser Cys Gln Cars Val Cars Lys Asn Lys Leu Phe Pro Ser Gln ~ys Gly Ala Asn Arg Glu Phe Asp Glu Asn Thr Cys Gln Cys Val tars 7Lys Arg Thr Cys Pro Arg Asn Gln Pro w- 295 , _ 300 305 Leu Asn Pro Gly Lys Cps Ala ~.'~rs Glu Cys Thr Glu Ser Pro Gln Lys Cps Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cps Ser t'~rs Tyr Arg Arg Pro tars Thr Asn Arg t;ln Lys Ala Cuss Glu Pra Gly Phe Ser Tyr Ser Glu Glu Val Cys Arg 1':ys Val Pro Ser Tyr Trp Gln Arg Pro Gln Met Ser ( 2 ) INFORMATION FOR SES2 ID NO : 3 ( i ) S$QL18NC$ CHAR~z~CTERISTICS
(A) LENGTH: 7.4 AMINO ACIDS
(B ) TYPE : AM7:N0 ACID
WO 96/39515 ~ PCT/US96/09001 (C) STRANDEDNESS:
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PEPTIDE
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Pro Xaa Cys Val Xaa Xaa Xaa Arg Cys Xaa Gly Cys Cys Asn (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE QiARACTERISTICS
(A) LENGTH: 50 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
( a.i ) MOLECUL$ TYPE : oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTEF~ISTICS
(A) LENGTH: 28 BASE PAIRS
(B) TYPE: NUCLEIC ACID
( C ) STRi~INDEDNESS : S INGLE
(D) TOPOLOGY : LINEAR
(ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
GATCTCTAGA TTAGCTCATT TGTGGTCT 2g (2) INFORMATION FOR SEQ ID N0:6:
( 1 ) SEQUENCE CHAR.~1,CTSRISTIC,'S
(A) LENGTH: 27 BASE PAIRS
(B) TYPE: NUC~BIC ACID
(C) STRANDEDNESS : SING1LE
(D) TOPOLOGY: LINEAR
(ii) MOL$CUL$ TYPE: oligonuCleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHAR~,CTERISTIC,S
(A) LENGTH: 60 BASE PAIRS
(B) TYPE : NUC2,EIC ACID
( C ) STRANDEDNES S : S ING:LE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: oligonu~leotide (xi) SEQUENCE DESCT:IPTION: SEQ ID N0:7:
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARF~C'.TERISTICS
(A) LENGTH : 1.96 AMINO .ACIDS
(B) TYPE: AM~:NO ACID
( C) STRANDEDNE:SS
(D) TOPOLOGY: LINRAR
(ii) MOLECOLE TYPE: PROTEIN
(xi) SEQU~TCE D$SCF:IPTION: SEQ ID N0:8:
Met Arg Thr Leu Ala Cys Leu Leu Leu Leu Gly C'.ys Gly Tyr Leu Ala His Val Leu Ala Glu Glu Ala Glu Ile Pro Arg Glu Val Ile Glu Arg Leu Ala Arg Ser Gln Ile His Ser Ile Arg Asp Leu Gln Arg Leu Leu Glu Ile Asp Ser Val Gly Ser Glu Asp Ser Leu Asp 50 55' 60 Thr Ser Leu Arg Ala His Gly Val His Ala Thr Lys His Val Pro Glu Lys Arg Pro Leu Pro Ile Arg Arg Lys Arg Ser Ile Glu Glu Ala Val Pro Ala Val Cys Lys Thr Arg Thr Val Ile Tyr Glu Ile Pro Arg Ser Gln Val Asp Pro Thr Ser Ala Asn Phe Leu Ile Trp Pro Pro Cys Val Glu val Lys Arg Cys Thr Gly Cys Cys Asn Thr Ser Ser Val Lys Cys Gln Pro Ser Arg Val His His Arg Ser Val Lys Val Ala Lys Val Glu Tyr Val Arg Lys Lys Pro Lys Leu Lys Glu Val Gln Val Arg Leu Glu Glu His Leu Glu Cys Ala Cys Ala Thr Thr Ser Leu Asn Pro Asp Tyr Arg Glu Glu Asp Thr Asp Val (2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 241 AMINO ACIDS
(B) TYPE: AMINO ACID
(C) STRANDEDNESS:
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN
(xi) SEQUENC$ DESCRIPTION: SEQ ID N0:9:
Met Asn Arg Cys Trp Ala Leu Phe Leu Ser Leu Cys Cys Tyr Leu Arg Leu Val Ser Ala Glu Gly Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu Ser Asp His Ser Ile Arg Ser Phe Asp Asp Leu Gln Arg Leu Leu His Gly Asp Pro Gly Glu Glu Asp Gly Ala Glu Leu Asp Leu Asn Met Thr Arg Ser His Ser Gly Gly Glu Leu Glu Ser Leu Ala Arg Gly Arg Arg Ser Leu Gly Ser Leu Thr Ile Ala Glu Pro Ala Met Ile Ala Glu Cys Lys Thr Arg Thr Glu Val Phe Glu Ile Ser Arg Arg Leu Ile Asp Arg Thr Asn Ala Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gln Arg Cys Ser Gly Cys Cys Asn Asn Arg Asn Val Gln Cys Arg Pro Thr Gln Val Gln Leu Arg Pro Val Gln Val Arg Lys Ile Glu Ile Val Arg Lys Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu Ala Cys Lys Cys Glu Thr Val Ala Ala Ala Arg fro Val Thr Arg Ser fro Gly Gly Ser Gln Glu GIn Arg Ala Lys Thr Pro Gln Thr Arg Val Thr Ile Arg Thr Val Arg Val Arg Arg Pro Pro Lys Gly Lys His Arg Lys Phe Lys His Thr His Asp Lys Thr Ala Leu Lys Glu Thr Leu Gly Ala ( 2 ) INFORMATION FOR SE(~ ID NO :10 (i) SBQUBNCB CHARAC'1'BRISTICS
(A) LENGTH: 232 AMINO ACIDS
(B) TYPE: AMINO ACID
(C) STRANDEI)NESS:
( D ) TOPOLOG7.' : LINEAR
(ii) MOLECULE TYPE: PROTEIN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr G1n Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu 65 70 75 g0 Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Va1 Pro Thr Glu Glu Se:r Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Lys Lys Ser Val Arg Gly Lys Gl:y Lys Gly Gln Lys Arg Lys Arg Lys Lys Ser Arg Tyr Lys Ser Trp Ser Val Tyr Val Gly Ala Arg Cys Cys Leu Met Pro Trp Ser Leu Pro Gl:y Pro His Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro G1n Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys As:p Lys Pro Arg Arg
the present invention, under conditions promoting expression of said proteins and subsequent recovery of said proteins.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptide, or polynucleotide encoding such polypeptide for therapeutic purposes, for example, t:o stimulate angiogenesis, wound-healing, and to promote vascular tissue repair.
In accordance with yet another aspect of the present invention, there are provided antibodies against such polypeptides.
In accordance with yet another aspect of the present invention, there are provided antagonists to such polypeptides , which may be used to :inhibit the action of such polypeptides, for example:, to inhilbit the growth of tumors, to treat diabetic retinopathy, inflammation, rheumatoid arthritis and psoriasis.
In accordance with another aspect of the present invention, there are provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to nucleic acid sequences of the present invention.
In accordance with another aspect of the present invention, there are provided methods of diagnosing diseases or a susceptibility to diseases related to mutations in nucleic acid sequences of the present invention and proteins encoded by such nucleic acid sequences.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucl~eotides encoding such polypeptides, for in vitro purposes related to scientific research, synthesis of DNA and manufacture oi' DNA vectors .
These and other aspects of the: present invention should be apparent to those spilled in the art from the teachings herein.
The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.
Fig. 1 shows the cDNA sequence and the corresponding deduced amino acid sequence of the polypeptide of the present invention. The standard one letter abbreviations for amino acids are used. Sequencing was performed using 373 Automated DNA Sequencer (Applied Biosystems, Inc.). Sequencing accuracy is predicted to be greater than 97%.
Fig. 2 is an illustration of the amino acid sequence homology between the polypeptide of the present invention and other members .of the human PDGF/VSGF family. The boxed areas indicate the conserved sequences and the location of the eight conserved cysteine residues.
Fig. 3 shows a photograph of a gel after in vitro transcription, translation and electrophoresis of the polypeptide of the present invention. Lane 1: 1'C and rainbow M.W. marker; Lane 2: FGF control; Lane 3: VBGF2 produced by M13-reverse and forward primers; Lane 4: VEGF2 produced by M13 reverse and VEGF-F4 primers; Lane 5: VEGF2 produced by M13 reverse and VEGF-F5 primers.
Fig . 4 . VEGF2 polypeptide is expressed in a baculovirus system consisting of Sf9 cells. Protein from the medium and cytoplasm of cells were ana7.~r~ed by SDS-PAGB under reducing and non-reducing conditions.
Fig. 5. The medium from Sf9 cells infected with a nucleic acid sequence of the present invention was precipitated and the resuspended precipitate was analyzed by SDS-PAG$ and was stained with coomassie brilliant blue.
Fig. 6. VEGF2 was purified from the medium supernatant and analyzed by SDS-PAGE in the presence or absence of the reducing agent ~B-mercaptoethanol and stained by coomassie brilliant blue.
Fig. 7. Reverse phase IiPLC analysis of purified VEGF2 using a RP-300 column (0.21 x 3 cm, Applied Biosystems, Inc.). The column was equilibrated with 0.1% trifluoroacetic acid (Solvent A) and the proteins eluted with a~7.5 min gradient from 0 to 60% Solvent B, composed~of acetonitrile containing 0.07%"TFA. The protein elution was monitored by absorbance at 215 nm (Red line) and 280 nm (Blue line) . The percentage of Solvent B is shown by Green line.
Fig. 8 illustrates the effect of partially-purified VEGF2 protein on the growth of vascular endothelial cells in comparison to basic fibroblast growth factor.
Fig. 9 illustrates the effect of purified VBGF2 protein on the growth of vascular endothelial cells.
The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer)., as well as intervening sequences (introns) between individual coding segments (exons).
In accordance with one aspect of the present invention, there are provided isolated nucleic acid molecules (polynucleotides) which encode for the mature polypeptides .
having the deduced amino acid sequence of Figure 1 or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No 97149 ion May 12, 1995 at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, U.S.A., or for polypeptides which have fewer amino acid residues than those showing in Figure 1.
A polynucleotide encoding a polypeptide of the present invention may be obtained from early stage hcm~an embryo (week 8 to 9) osteoclastomas, adult heart or several breast cancer cell lines. The polynucleotide~ of this invention was discovered in a cDNA library derived from early stage human embryo week 9. It is structurally related to the tTBGF/PDGF
family.. VEGF2 contains an open reading frame encoding a protein of 419 amino acid residues of Which approximately the first 23 amino acid residues are the putative leader sequence such that the mature protein comprises 396 amino acids, and which protein exhibits the highest amino acid sequence _7_ homology to human vascular endothelial growth factor (30%
identity), followed by PDGFa (23%) and PDGF~ (22%).
It is particularly important that all eight cysteines are conserved within all four members of the family (see boxed areas of Figure 2). In addition, the signature for the PDGF/VEGF family, PXCVX~~XRCXGCCN, (S$Q ID N0:3) is conserved a.n VEGF2 (see Figure 2).
The VEGF2 polypeptide of the present invention is meant to include the full length polypeptide and polynucleotide sequence which encodes for any leader sequences and for active fragments of the full length polypeptide. Active fragments are meant to include any portions of the full length ama.no acid sequence which have less than the full 419 am3.no acids of the full length amino acid sequence as shown in S$Q ID No . 2 and Figure 2 , but still contain the eight cysteine residues shown conserved in Figure 2 and such fragments still contain VEGF2 activity.
There are at least two alternatively spliced VEGF2 mRNA
sequences present in normal tissues. The size of the two VEGF2 mRNA sequences which correspond to the full-length and truncated version respectively are shown in Figure 3, lane 5 shows two bands indicating the presence of the alternatively spliced~mRNA encoding the VEGF2 polypeptide of the present inventa.on. ' ' - _ -The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptide as the DNA of Figure 1 or the deposited cDNA.
_g_ The polynucleotide which encodes for the mature polypeptide of Figure 1 or for the 'mature polypeptide encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide (and optionally additional coding ' sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/a:r 3' of the coding sequence for the mature polypeptide.
Thus, the term "pol.ynucleotide encoding a polypeptide"
encompasses a polynucleotide wha.ch includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional. coding and/or non-coding sequence.
The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone. The variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
Thus, the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 or the same mature polypeptide encoded by the cDNA of the deposited clone as well as~wariants of such polpnucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 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 hereinabove indicated, the polynucleotide may have a coding sequence which .is a naturally occurring allelic variant of the coding sequence shown in Figure 1 or of the coding sequence of the deposited clone. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a, substitul=ion, deletion or addition WO 96/39515 ~ PCT/US96/09001 of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.
The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention. The marker sequence may be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).
The teen "gene's means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
Fragments of the full length gene of the present invention may be used as a hybridization probe for a cDNA
library to isolate the full length cDNA and to isolate other cDNAs which have a high sequence sima.larity to the gene or similar~biological activity. Probes of this type preferably have at least 30 bases and' may contain, for example, 50 or more bases. The probe may also be used to identify a cDNA
clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promotor regions, axons, and introns. An example of a screen comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the .
probe hybridizes to.
WO 96/39515 ~ PCT'/US96/0900I
The present invention further relates to polynucleotides which hyboidize to 'the hereinabove-described sequences if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences. The present invention particularly relates to polynucleotides which hybridine u~nde:r stringent conditions to the hereinabove-described polynucleotides. As herein used, the term "stringent conditions'° nneans hybridization will occur only if there is at least 95% and preferably at least 97% identity between the: sequences. The polynucleotides which hybridize to the hei:einabove described polynucleotides in a preferred embodiment: encode polypeptides which either retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of Figure 1 (S$Q ID NO:1) or the deposited cDNA(s).
Alternatively, the polynucleot:ide may have at least 20 bases, preferably 30 bases, and more preferably at least 50 bases which hybridize to~ a polynucleotide of the present invention and which has an identity thereto, as hereinabove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for the polynucleotide of S$Q ID NO:1, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR
primer . ~ ' ' - -Thus, the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% and more preferably at least a 95% identity to a polynucleotide which encodes the polypeptide of SEQ ID N0:2 as well as fragments thereof, which fragments have at least 30 bases and preferably at: least 50 bases and to polypeptides encoded by such polynucleotides.
The deposits) referred to herein will be maintained - under the terms of the Budapest Treaty on the International Recognition of the Deposit: of Micro--organisms for purposes of Patent Procedure. These deposits are provided merely as convenience to those of s:~cill in the art and are not an admission that a deposit is required under Section 38.1(2) of the Patent Act.
The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are controlling in the event of any conflict with any des~:ription of sequences herein. A license may be required to mz3ke, use or sell the deposited materials, and no such licentae is hereby granted.
The present invention further relates to a polypeptides which have the deduced amino acid sequence of Figure 1 or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptide.
The terms "fragment," "derivative" and "analog" when referring to t:he polypeptide of Figure 1 or that encoded by the deposited cDNA, means a polypeptide which'retains the conserved motif of V$GP proteins as shown in Figure 2 and essentially tt~e same biological function or activity.
The polypeptides of the present invention may be recombinant polypeptides, natural polypeptides or synthetic polypeptides, preferably recombinant.polypeptides.
The fragment, derivati'v~ or analog of the-polypeptide of Figure 1 or that encoded by the deposited cDNA may be (i)~
one in which one or.more of the amino acid residues are substituted with a conserved or non-consezved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid ref:idues includes a substituent. group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature -.12-polypeptide or (v) one in which comprises fewer amino acid residues shown in S$Q ID No. 2 and retains the conserved motif and yet still retains activity characteristic of the VEGF family of polypeptides. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
The tern "isolated" 'means than the material is removed from its original envirorunent (e. g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural systenn, is isolated. Such polynucleotides could be part oi= a vector and/or such polynucleotides or pol~rpeptides could be part of a composition, and still be isolated a.n that such vector or composition is not part of its natural environment.
The polypeptides of the present invention include the polypeptide of SSQ ID N0:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70%
similarity (preferably at" least 70% identity) to the polypeptide of S8Q ID N0:2 and more. preferably at least 90%
similarity (more preferably at least 95% identity) to the polypeptide of S$Q ID N0:2~ and still. more preferably at least 95% similarity (still more preferably at least 90% identity) to the polypeptide of SEQ ID N0:2 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
As known in the art "similarity" between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters , selecting transforn~a_nts or amplifying the V$GF2 genes of the present invention. The culture conditions, such as temperature, pH and the line, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and W~ 96/39515 PCT/US96/09001 phage DNA, viral DNA such. as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host.
The appropriate DNA sequence may be inserted into the vector by a variety of procedure~a. In general, the DNA
sequence is inserted into an appropriate restriction endonuclease sites) by procedures known in the art. Such procedures and others are: deemed to be within the scope of those skilled in the art.
The DNA sequence in the expression vector a.s operatively linked to an appropriate expression control sequences) (promoter) to direct mRI~TA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the F. c ~ lac or ~r , the phage lambda PL
promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses .
The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
The vector may also include appropriate sequences for amplifying expression.
In addition, the expression vectors preferably contain one or more selectable marker genes: to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in B. coli.
The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as B. coli, Streptomyces, Salmonella ~tvohimurimm; fungal cells, such as yeast; insect cells such as Droso.,phila S2 and Spodoutera Sf9;
animal cells such as CHO, CoS or Bowes melanoma;
adenoviruses; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. 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 by way of example. Bacterial: pQ$70, pQ860, pQ$-9 (Qiagen), pBS, pDlO, phagescript, psiX174, pBluescript SR, pBSKS, pNH8A, pNHl6a, pNHl8A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Phazmacia). $ukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Phazmacia). However, any other plasmid or vector may be used as long as they are replicable and viable a.n the host.
Promoter regions can be selected from any desired gene using CAT (chloramphenicol'~transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. .Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp.
Eukaryotic promoters include QMV immediate early, HSV
thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter a.s 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, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DBA,B-Dextran mediated transfection, or electroporation. (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).
The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
Mature proteins can be expressed in mammalian cells, yeast, bacte::ia, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the D,tZA constructs of the present invention.
Appropriate cloning and expression vectors for use with prokaryotic a~zd eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989).
Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an ~~nhancer sequence into the vector. Bnhancers~
are cis-acting elements of DNA; usually about from 10 to 300 by that act on a promoter to increase its transcription.
Bxam~ples including the SV40 enhancer on the late side of the replication o~:igin by 100 to 270, a cytomegalovirus early promoter~enhancer, the polyoma Pnhancer an the late side of the replication origin, and adenovirus enhancers.
Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation. of the host cell, e.g., the ampicillin resistance gene of B. coli and ~. cerevisiae TRP1 gene, and _x7_ a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and tezmination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include 8. coli, Bacillus subtilis, Salmonella twhimurium and var~.ous species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice .
As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC
37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GBM1 , (Promega Biotec, Madison, WI, USA). These pBR322 °backbone°°
b WO 96/39515 PCT/US96/0900i sections are combined with an appropriate promoter and the structural sequence to bye expressed.
Following transformation of a suitable host strain and growth of the host strain.to an appropriate cell density, the selected promoter is induced by appropriate means (e. g., temperature shift or chemical induction) and cells are cultured for an additional period.
Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
Microbial cells employed in Expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents , such methods are wel l know to those spilled in the art.
Various mammalian cell culture systems can also be employed to express rf~combinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for e.~cample, th.e C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ' ~ri.bosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences.. DNA sequences derived from the Sv40 splice, and polyadenylation sates may be used to provide the required nontranscri:bed genetic elements.
The polypeptides cyan be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated.
Polypeptides of the invention may also include an initial methionine amino acid residue.
As shown in Figures 8 and 9 , the VEGF2 polypeptide of SEQ ID No. 2, minus the initial 46 amino acids, is a potent mitogen for vascular endothelial cells and stimulates their growth and proliferation. The results of a Northern blot analysis performed for the VEGF2 nucleic acid sequence encoding this polypeptide wherein 20 ~.g of RNA from several human tissues were probed with 3aP-VEGF2, illustrates that this protein is actively expressed in the heart and lung which is further evidence of mitogenic activity.
Accordingly, VEGF2 ~ina~ ~ be employed ~o promote angiogenesis, for example, to stimulate the growth of transplanted tissue (where coronary bypass surgery a.s performed. VSGF2 may also be employed to promote wound healing, particularly to re-vascularize damaged tissues or stimulate collateral blood flow during ischemia and where new capillary angiogenesis is desired. VSGF2 may be employed to treat full-thickness wounds such as dermal ulcers, including pressure sores, venous ulcers, and diabetic ulcers. In addition, VSGF2 may be employed to treat full-thickness burns and injuries where a skin graft or flap is used to repair such burns and injuries. VEGF2 may also be employed for use in plastic surgery, for example, for the repair of lacerations from trauma and cuts in. association with surgery.
Along these same lines, VEGF2 may be employed to induce the growth of damaged bone, periodontium or ligament tissue.
VBGF2 may also be employed for regenerating supporting tissues of the teeth, including cementum and periodontal ligament, that have been damaged bay disease and trauma.
Since angiogenesis is important in keeping wounds clean and non-infected, vEGF2 may be employed in association with surgery and following tyre repair of cuts. It may also be employed for the treatments of abdominal wounds where there is a high risk of infection.
vEGF2 may be employed for the promotion of endothelialization in vascular graiEt surgery. In the case of vascular grafts using either t:cansplanted or synthetic material, VEGF2 can be applied to t:he surface of the graft or at the junction to promote the growth of vascular endothelial cells. vEGF2 may also be employed to repair damage of myocardial tissue as a result c~f myocardial infarction.
VEGF2 may also be employed to repair the cardiac vascular system after ischemia. 'VEGF2 may also be employed to treat damaged vascular tissue as a result of coronary artery disease and peripheral and CNS vascular disease.
VEGF2 may also be employed to coat artificial prostheses or natural organs which acre to be transplanted in the body to minimize rejection of the transplanted material and to stimulate vascularizatio:n of the transplanted materials.
VEGF2 may also be employed for vascular tissue repair, for example, that occurring during arteriosclerosis and required following balloon angioplasty where vascular tissues are damaged.
V>3GF2 nucleic acid faequences and V13GF2 polypeptides may also be employed for in ~rritro purposes related to scientific research, synthesis of I)NA and manufacture of DNA vectors, and for the production of diagnostics and therapeutics to treat human disease. For example, VEGF2 may be employed for in vitro culturing of vascular endothelial cells, where it is added to the conditional medium in a concentration from 10 pg/ml to 10 ng/ml.
Fragments of the full length VEGF2 gene may be used as a hybridization probe for a cDNA library to isolate other genes which have a high sequence similarity to the gene or similar biological activity. Probes of this type generally have at least 50 base pairs, although they may have a greater number of bases. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete vBGF2 gene including regulatory and pramotor regions, exons, and introns. An example of a screen comprises isolating the coding region of the VEGF2 gene by using the known DNA
sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
This invention provides methods for identification of vEGF2 receptors. The gene encoding the receptor can be identified by numerous methods known to those of skill a.n the art, for example, ligand panning and FAGS sorting (Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). Preferably,,expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to VEGF2, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to VSGF2. Transfected cells which are grown on glass slides are exposed to labeled VEGF2.
VEGF2 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.
WO 96139515 ~ PCT/US96/0900I
Positive pools are identified and sub-pools are prepared and retransfected using an iterative s:ub-pooling and rescreening process, eventually yielding a single clone that encodes the putative receptor.
As an alternative approach for receptor identification, labeled VEGF2 can be pho~toaffinity linked with cell membrane or extract preparations that express the receptor molecule.
' Cross-linked material a.s. resolved by PAG$ and exposed to X-ray film. The labeled. complex containing VBGF2 is then excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from ma.crosequencing would be used to design a set of degenerate oligonucleoti~de probes to screen a cDNA library to identify the gene encoding the pui_ative receptor.
This invention is also related to a method of screening compounds to identify lthose which are VEGF2 agonists or antagonists. An example of such a~ method takes advantage of the ability of VSGF2 to significantly stimulate the proliferation of human endothelial cells in the presence of the comitogen Con A. $ndothelial cells are obtained and cultured in 96-well flint-bottomed culture plates (Costar, Cambridge, MA) in a reaction mixture supplemented with Con-A
(Calbiochem, La Jolla, CA). ~ Con-A, polypeptides of the present invention and the compound to be screened are added.
After incubation at 37°C, cultures are pulsed with 1 ~.Ci of 3 [H] thymidine (5 Ci/mmol; 1 Ci = 37 BGq; NEN) for a sufficient time to incorporate the 3 [H] and ;harvested onto glass fiber filters (C:ambridge Teclvzology, Watertown, MA) . Mean ' [H] -thymidine inco~oration (cpm) of triplicate cultures is determined using a liquid scintillation counter (Beckman Instruments, Irvine, CA) . Significant ' [H] thymidine incorporation, as compared to a control assay where the compound is excluded, indicates stimulation of endothelial cell proliferation.
WO 96/39515 ~ PCT/US96/09001 To assay for antagonists, the assay described above is performed and the ability of the compound to inhibit ' [H] thymidine incorporation in the presence of VBGF2 indicates that the compound is an antagonist to VEGF2. Alternatively, VI3GF2 antagonists may be detected by combining VEGF2 and a potential antagonist with membrane-bound VEGF2 receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay. Vl3GF2 can be labeled, such as by radioactivity, such that the number of VEGF2 molecules bound to the receptor can determine the effectiveness of the potential antagonist.
Alternatively, the response of a known second messenger system following interaction of VEGF2 and receptor would be measured and compared in the presence or absence of the compound. 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 VEGF2 receptor a.s incubated with labeled VEGF2 in the presence of the compound.
The ability of the compound to enhance or block this interaction could then be measured.
Potential V13GF2 antagonists include an antibody, or a.n some cases, an oligonucleotide, which bind to the polypeptide and effectively eliminate VSGF2 function. Alternatively, a potential antagonist may be a closely related protein which binds to VEGF2 receptors, however, they are inactive forms of the polypeptide and thereby prevent the action of VSGF2.
$xamples of these antagonists include a negative dominant mutant of the VEGF2 polypeptide, for example, one chain of the hetero-da.meric form of vEGF2 may be dominant and may be mutated such that biological activity is not retained. An example of a negative dominant mutant includes truncated versions of a dimeric VI3GF2 which is capable of interacting with another dimer to form wild type VEGF2, however, the WO 96/39515 PCTlUS96/0900I
resulting homo-dimer a.s inact:ive and fails to exhibit characteristic VEGF activity.
Another potential. VSGF2 antagonist is an antisense construct prepared using antisense technology. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DZ~TA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl. Acids Res., 6:3073 (1979); Gooney et al, Science, 241:456 (1988);
and Dezvan et a1_, Science, 251: 1360 (1991)), thereby preventing transcription and the production of VJ3GF2. The antisense RNA oligonucl.eotide hybridizes to the mRNA in vivo and blocks translation of the mltNA molecule into the VSGF2 polypeptide (Antisense - Okano, J. Neurochem., 56:560 (1991);
Oligodeoxynucleotides as Antisense Inhibitors of Gene $xpression, CRC Press., Boca Raton, FL (1988)). The oligonucleotides descr:Lbed above can also be delivered to cells such that the antisense~RNA or DNA may be expressed in vivo to inhibit production of VSGF2.
Potential ~TEGF2 anttagonists also include small molecules which bind to and occupy the active site of the polypeptide thereby making the catalytic site inaccessible to substrate such that normal biological activity is prevented. $xamples of small molecules include but are not limited to small peptides or peptide-like molecules.
The antagonists may be employed to treat limit angiogenesis necessary for solid tumor metastasis.
The mRNA encoding for VBGF2 is found to be expressed at moderate levels in at leaast two breast tumor cell lines which is indicative of the role of VEGF2 polypeptides in the malignant phenotype. Gliomas are also a type of neoplasia which may be treated with the antagonists of the present invention.
The antagonists may also be used to treat chronic inflammation caused by increased vascular permeability. In addition to these disorders, the antagonists may also be employed to treat retinopathy associated with diabetes, rheumatoid arthritis and psoriasis.
The antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.
The VEGF2 polypeptides and agonists and antagonists may be employed 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 a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical composit~.ons of the invention. Associated with such containers) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the pharmaceutical compositions may be employed in conjunction with other therapeutic compounds.
The pharmaceutical compositions may be administered in a convenient manner such as by the topical, intravenous, intraperitoneal, intramuscular, intratumor, subcutaneous, intranasal or intrader<nal routes. The pharmaceutical compositions are administered in an amount which is effective for treating and/or prophylaxis of the specific indication.
In general, the pharmaceutical ccm~positions are administered in an amount of at least about :LO ~Cg/kg body weight and in most cases they will 7be admini~~tered in an amount not in excess of about 8 mg/Kg body weight per day. In most cases, the dosage is from about 10 ug/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.
The V13GF2 polypeptides, and agonists or antagonists which are polypeptides may also be employed in accordance with the present invention by expression of such polypeptide in~ vivo, which is often referred to as "gene therapy. "
Thus, for example, cells such as bone marrow cells may be engineered with a polynucleotide (DNA or RNA) encoding for the polypeptide ex vi:vo, the engineered cells are then provided to a patient to be treated with the polypeptide .
Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding the polypeptide of the present invention.
Similarly, cells may be engineered in vivo for expression of a polypeptide ~~in vivo, for example, by procedures known in the art: 'As :known in the art; a producer cell for producing a retroviral particle containing RNA
encoding a polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide .in vivo. These and other methods for administering a polypeptide 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, the expression, vehicle f_or engineering cells may be other than a retroviral particle, for example, an adenovirus, which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
WO 96/39515 ' PCT/US96109001 Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus , human immunodef iciency virus , adenovirus , Myeloproliferative Sarcoma Virus, and mammary tumor virus .
In one embodiment, the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.
The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (C3~tV) promoter described a.n Miller, et al. , Biotechnigues, Vol. 7, No. 9, 980-990 (1989), or any other promoter (e. g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and ~-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thyma.dine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art 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 which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or hetorologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described); the ~B-actin promoter; and human growth hormone promoters. The pramoter also may be the native promoter which controls the gene encoding the polypeptide.
The rEaroviral plasmid vector is employed to transduce packaging cell lines to fozm producer cell lines. $xamples of packaging cells which may be transfected include, but are not limited to, the P$501, PA317, ~-2, ~-AM, PA12, T19-14X, VT-19-17-H~;, ~C~tg, ~Cg,Ip, GP+$-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Theraov, Vol. 1, pgs. 5-14 x;1990), The vector may transduce the packaging cells through any means known in the art. Such means include, bu,t are not limited to, electroporation, the use of liposomes, and CaPO, precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequences) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or' in vivo. The transduced eukaryotic cells will express the nucleic . acid sequences) encoding the.
polypeptide. 8ukaryotic cells which may be transduced include, but are not limited~~to, .embryonic -stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocyt~s, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.
This invention is also related to the use of the V$GF2 gene as part of a diagnostic assay for detecting diseases or susceptibility to diseases related to the presence of mutations in VBGF2 nucleic acid sequences.
Individuals carrying mutations in the V8GF2 gene may be detected at the DNA level by a variety of techniques.
Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy and ' -29-WO 96/39515 ~ ' PCT/US96109001 autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR
(Saiki et al., Nature, 324:163-166 (1986)) prior to analysis.
RNA or cDNA may also be used for the same purpose . As an example, PCR primers complementary to the nucleic acid encoding VEGF2 can be used to identify and analyze VSGF2 mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype . Point mutations can be identified by hybridizing amplified DNA to radiolabeled VBGF2 RNA or alternatively, radiolabeled VEGF2 antisense DNA
sequences. 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 may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA
fragments of different sequences may be distinguished on denaturing forniamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see," e:g., Myers et al:, Science, 23:1242 (1985)).
Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).
Thus, the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e. g., Restriction Fragment Length Polymorphisms (RFLP)) and Southern blotting of genomic DNA.
WO 96/39515 ' ~ PCT/US96/0900I
In addition to more conventional gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.
The present invention also relates to a diagnostic assay for detecting altered levels of VBGF2 protein i.n various tissues since an over-expression of the proteins compared to normal control tissue samples ma.y detect the presence of a disease or susceptibility to a disease, for example, abnormal cellular differentiation. Assays used to detect levels of VSGF2 protein in a sample derivecl from a host are well-known to those of skill in the art and include radioimmunoassays, competitive-binding as~aays, Western Blot analysis, ELISA
assays and "sandwich" .assay. An $LISA assay (Coligan, et al., Current Protocol:a in Immunology, 1(2), Chapter 6, (1991) ) initially comprises preparing an antibody specific to the VBGFZ antigen, preferably a~ monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal antibody. To the reporter antibody is attached a detectable reagent such as radioactivity, fluorescence or, in this example, a horseradish pero:xidase enzyme. A sample is removed from a host and incubated on a solid support, e.g. a polystyrene dish, that hinds the~proteins in the sample. Any free protein binding s2tes on tree dish are then covered by incubating with a non-specific~protein, such as, bovine serum albumen. Next, the monoclonal antibody a.s incubated in the dish during which time the monoclonal antibodies attach to any VSGF2 proteins attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer. The reporter antibody linked to horseradish peroxidase is placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to VSGF2. Unattached reporter antibody is then washed out. Peroxidase substrates are then added to the dish and the amount of color developed in a given time period is a. measurement of the amount of VEGF2 protein present in a given volume of patient sample when compared against a standard curve.
A competition assay may be employed wherein antibodies specific to VBGF2 are attached to a solid support.
Polypeptides of the present invention are then labeled, for example, by radioactivity, and a sample derived from the host are passed over the solid support and the amount of label detected, for example by liquid scintillation chromatography, can be correlated to a quantity of VSGF2 in the sample.
A "sandwich" assay is similar to an SLISA assay. In a "sandwich" assay VBGF2 is passed over a solid support and binds to antibody attached to a solid support. A second antibody is then bound to the VSGF2. A third antibody which is labeled and specific to the second antibody is then passed over the solid support and binds to the second antibody and an amount can then be quantified.
The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome . Few chromosome marking reagents based on actual sequence data (repeat polymorphism's) are presently available for marking chromosomal location. The~'mapping of DNAs to -chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA.
Computer analysis of the cDNA is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing inda.vidual human chromosomes. Only those , hybrids containing the human gene corresponding to the primer will yield an amplified fragment .
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome .
Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous nnanner. Other mapping strategies that can similarly be used tto map to its chromosome include in situ hybridization, pr~sscreening~ with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDiz4 libraries .
Fluorescence in situ hybridization (FISH) of a cDNA
clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used With cDNA as short as 50 or 60 bases.
For a review of this techniques see Verma et al., Human Chromosomes: a Manual of Basic Techniques. Pergamon Press, New York (1988) .
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for Example, :Ln V. McKusick, Mendelian Inheritance in Man (availa3~le~'on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
Next, it is necessary to deaermine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but- not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb ) .
The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies.
The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
Antibodies generated against the polypeptide corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptide into an animal or by administering the polypeptide to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptide itself. In this manner, even a.sequence encoding only a fragment of the polypeptide can be used to generate antibodies binding the whole native polypeptide.
Such antibodies can then be' used to isolate the~polypeptide from tissue expressing that polypeptide. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. $xamples include the hybridoma technique (Rohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Rozbor et al., 1983, Immunology Today 4:72), and the $BV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., PP. 77-96).
Techniques described for the production of single chain antibodies (U. S. Patent 4,946,77x) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. Also, transgenic mice may be used to express humanized antibodies to immunogenic polypeptide products of this invention.
The present invent=ion will be further described with reference to the following examples; however, it is to be understood that the pre~aent invention is not limited to such examples. All parts or amounts, ~.uiless otherwise specified, are by weight.
In order to facilntate understanding of the following examples, certain frequently occurring methods and/or tezms 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, publicly available on an unrestricted basis, or can be constructed from availalble plasmi,ds in accord with published procedures. In addition, equ~!valent plasmi.ds to those described are known in the art and will be apparent to the ordinarily skilled artisan.
"Digestion" of DNA. refers to catalytic cleavage of the DNA with a restriction enzymes 'that acts only-at certain sequences in the DNA. The various restriction enzymes used herein are commercia7_ly available and their reaction conditions, cofactors ~3nd other requirements were used as would be known to they ordinarily skilled artisan. For analytical purposes, t=ypically 1 ~g of plasmid or DNA
fragment is used with aabout 2 units of enzyme in about 20 ~,1 of buffer solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 ~.g of DNA are digested with 20 to 250 r.~nits of enzyme in a larger . volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the WO 96/39515 PC'T/US96/09001 manufacturer. Incubation times of about 1 hour at 37~C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D.
et al., Nucleic Acids Res., 8:4057 (1980).
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 ~Cg of approximately equimolar amounts of the DNA'fragments to be ligated.
Unless otherwise stated,' transformation was ZSerformed as described by the 'method of Graham, F. and Van der 8b, A. , Virology, 52:456-457 (1973).
$~cample 1 Expression uattern of yEGF2 in human tissues and breast oancer cell lines Northern blot analysis was carried out to examine the levels of expression of the V8GF2 gene in human tissues and human breast cancer cell lines. Total cellular RNA samples were isolated with RNAzol1''' B system (Biotecx Laboratories , Inc . ) . About 10 ~.g of total RNA isolated from each breast tissue and cell line specified was separated on ~.% agarose gel and blotted onto a nylon filter, (Molecular Cloning, Sambrook Fritsch, and Maniatis, Cold Spring Harbor Press, 1989). The labeling reaction was done according to the TM
Stratagene Cloning Systems, Inc., Prime-It kit with 50 ng DNA
fragment. The labeled DNA was purified with a Select-G-50 column from 5 Prime -- 3 Prime, Inc, Boulder, CO, USA. The filter was then hybridized with radioactively labeled full length VEGF2 ~~ene at 1,000,000 cpm/ml in 0.5 M NaP04and 7 %
SDS overnight: at 65°C. After washing twice at room temperature acid twice at 60°C with 0.5 X SSC, 0.1 % SDS, the filters were then exposed at -70°C overnight with an intensifying :screen. A message of 1.6 Rd was observed in 2 breast cancer cell lines.
$xample 2 Cloning and expression of VEGFZ usincx the baculovirus expression s~rstem The DNA ;sequence encoding the VBGF2 protein without 46 amino acids at the N-terminus, see ATCC I 9'~1'~9 was amplified using PC'R. oligonucleotide primers corresponding to the S' and .
3' sequences of the gene:
The 5' primer has the sequence TGT AAT ACG~ACT CAC TAT
AGG GAT CCC GC'.C ATG GAG GCC ACG GCT TAT GC (SBQ ID N0:4) and contains a BarnHl restriction enzyme site (in bold) and 174 nucleotide nucleotide sequence complementary to the 5' sequence of VE;GF2 (nt. 150-166) .
The 3' primer has the sequence GATC TCT AG,A TTA GCT CAT
TTG T'GG TCT (SBQ ID N0:5) and contains the cleavage site for the restz'iction enzyme XbaI and 18 nucleotides complementary.
to the 3' sequence of VSGF2, including the stop codon and 15 nt sequence before stop codon. .
The amplified sequences were isolated from a 1% agarose gel using a cocamercially available kit ( "Geneclea~ M!' BIO 101, Inc., La Jolla, CA). The fragment was then digested with the endonuclease HamH1 and XbaI and then purified again on a 1%
agarose ge.l. This fragment was liaated to pAcGP67A
baculovirus transfer vector (PHarmingenjMat the BamHl and XbaI sites. Through this ligation, V$GF2 cDNA was cloned in frame with the signal sequence of baculovirus gp67 gene and was located at the 3' end of the signal sequence in the vector. T'h:is is designated pAcGP67A-V$GF2.
To clone VBGF2 with the signal sequence o~ gp67 gene to the pRG1 vector for expression,, VSGF2 with the signal sequence and some upstream sequence were excised from the pAcGP67A-VBG~F2 plasmid at the Xho restriction endonuclease site located upstream of the VBGF2 cDNA and at the Xbal restriction endonuclease site by XhoI and Xbal restriction enzyme. This fragment was separated from the rest of vector on a 1% agarose gel and was purified using "Geneclean"Mkit.
It was desigrnated F2.
The PRG~1 vector (modification of pVL941 vector) is used for the expression of the V8GF2 protein using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.B. 19S7,.y manual of methods for baculovirus.vectars and insect cell culture procedures, Texas Agricultural ..
$xperimental Station Bulletin No. 1555). This expression vector contains the strong polyhedrin promoter of the Autographa t:alifornica nucTeaf polyhedrosis virus (AcMNpV) followed by the recognition sites for the restriction .
endonucleases BamHl, Smal, XbaI, HglII and Asp718. A site for restriction endonuclease Xho1 is located upstream of HamHl site. The sequence between Xhol and BamHI is the same as that in PAcGp67A (static on tape) vector. The polyadenylat:ion site of the simian virus (SV) 40 is used for efficient polyadenylation. - For an easy selection of recombinant virus the beta-galactosidase gene from 8.coli is .
inserted in 'the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sides by viral sequences for the cell-mediated homologous recombination of cotransfected wild-type viral DNA. Many other baculovirus vectors could be used in place of pRGl such as pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D., Virology, 1~~0:31-39). ' The plasmid was digested with the restriction enzymes XboI and XbaI and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The DNA was then isolated from a 1% agarose gel using the commercially TM
available k:i.t ("Geneclean" BIO 101 Inc., La Jolla, Ca.).
This vector DNA,ie designated V2.
Fragmea,t F2 and the dephosphorylated plasmid V2 were ligated with T4 DNA ligase. $.col3 HB101 cells were then transformed and bacteria identified that contained the plasmid (pHac gp67-VSGF2) with the VBGF2 gene using the enzymes HamHl and XbaI. The sequence of the cloned ,fragment was confirmed by DNA sequencing.
~Cg of the plasmid pane gp67-VgGF2 was cotransfected with 1.0 erg of a commercially available linearized baculovirus ("HaculoGold° baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Felgner et al.
Proc. Natl. :~lcad. Sci. USA, 84:7413-7417 (1987)).
l~Cg of BaculoGold" virus DNA -and 5 ~Cg of the plasmid pBac gp67-vBt~F2 were mixed~in a sterile well of a microtiter plate containing 50 ~1 of serum free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Afterwards 10 ~,1 Lipofectin plus 90 ~tl Grace' s medium were added, mixed and incubated fc~r 15 minutes at room temperature. Then the transf action mixture was added dropwise to the Sf9 insect cells ('ATCC C~tL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate was rocked back and forth to mix the newly added solution. The plate was then incybated for 5 hours at 27°C. After~5 hours the transfection solution was removed from the plate and 1 ml of Grace's inseca medium supplemented with 10% fetal calf serum was added. The plate was put back into an incubator and cultivation continued at 27°C for four days, After four days the supernatant was collected and a plaque assay performed 'similar as described by Summers and Smi.th~(supra). As a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) was used which allows an easy isolation of blue stained p7.aques. (A
detailed desccription of a "plaque assay" can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10) .
Four days after the serial dilution, the virus was added to the cellt~, blue stained plaques were picked With the tip of an Sppend.orf pipette: The agar containing the recombinant TM
viruses was then resuspended in an Bppendorf tube containing 200 ~1 of G:cace's medium. The agar was removed by a brief centrifugation and the supernatant containing the recombinant baculovi.rus was used to infect Sf9 cells seeded in 35 mm dishes . Fo~.ir days later the supernatants of these culture dishes were harvested and then stored at 4°C.
Sf 9 cells were grown in Grace ' s medium supplemented with 10% heat-inactivated FBS. The cells were infected with the ..
recombinant baculovirus V-gp6T-V8GF2 at a multiplicity of infection (MOI) of 1. Six hours later the medium-was removed and rep2aced with SF900 II medium minus methionine and cysteine (L9.fe Technologies Inc. , Gaithersburg) . 42 hours later 5 ~eCi of "S-methionine and 5 ~CCi 'SS cysteine (Amersham) were added. The cells were further incubated for 16 hours before they were harvested by centrifugation and the labelled proteins visualized by SDS-PAGE and autoradiography.
Protein. from the medium and cytoplasm of the Sf9 cells was analyzed by SDS-PAGE under reducing and nori-reducing conditions. See Figure 4. The medium was dialyzed against 50 mM MSS, pH 5.8. Precpitates were obtained after dialysis and resuspended in 100 mM NaCitrate, pH 5Ø The resuspended precipitate was analyzed again by SDS-PAGE and was stained with Coomassie Brilliant Blue. See Figure 5.
The medium supernatant was also diluted 1:10 in 50 mM
MES, pH 5.8 and applied to an SP-650M column (1.0 x 6.6 cm, Toyopearl) at a flow rate of 1 ml/min. Protein was eluted with step gradients at 200, 300 and 500 mM NaCl. The VEGF2 was obtained using the elution at 500 mM. The eluate was analyzed by SDS-PAGE in the presence or absence of reducing agent, f3-mercaptoethanol and stained by Coommassie Brilliant Hlue. See Figure 6.
Example 3 E ression of Recombinant VEGF2 in COS cells The expression of plasmid, VBGF2-HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV4U origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, an SV40 intron and polyadenylation site. A DNA
fragment encoding the entire VBGF2 precursor and a HA tag fused in frame to its 3' end was cloned into the polylinker region of the vector, therefore,. the recombinant protein expression is directed under the CMV promoter. The HA tag corresponds to an epitope derived fr~n the influenza hemagglutinin protein as previously described (I. Wilson, H.
Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37:767, (1984)). The infusion of HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
The plasmid construction strategy is described as i follows The DNA sequence encoding VBGF2, ATCC # 97149 was constructed by PCR using two primers : the 5' primer (CGC GGA
TCC ATG ACT GTA CTC TAC CCA) (SEQ ID N0:6) contains a BamH1 site followed by 18 nucleotides of VEGF2 coding sequence starting from the initiation codon; the 3' sequence (CGC TCT
WO 96/39515 ' PCT/US96/09001 AGA TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA CTC GAG GCT CAT
TTG TGG TCT 3') (SBQ ID N0:7) contains complementary sequences to an XbaI site, HA tag, XhoI site, and the last 15 nucleotides of the VSGF2 coding sequence (not including the stop codon). Therefore, the PCR product contains a BamHI
site, coding sequence followed by an XhoI restriction endonuclease site and HA tag fused in frame, a translation termination stop codon next to the HA tag, and an XbaI site. ' The PCR amplified DNA fragment and the vector, pcDNAI/Amp, were digested with BamH1 and XbaI restriction enzyme and ligated. The ligation mixture was transformed into 8. coli strain SURE (Stratagene Cloning Systems, La Jolla, CA 92037) the transformed culture was plated on ampicillin media plates and resistant colonies were selected. Plasmid DNA was isolated from transformants and examined by restriction analysis for the presence of the correct fragment. For expression of the recombinant VEGF2, COS cells were transfected with the expression vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A
Laboratory Manual, Cold Spring Laboratory Press, (1989)).
The expression of the VEGF2-HA protein was detected by radiolabelling and immunoprecipitation method (B. Harlow, D.
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1-988)) ."Cells were labelled-for 8 hours with 3sS-cysteine two days post transfection. Culture media was then collected and cells were lysed with detergent (RIPA
buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5) (Wilson, I . et al. , Id. 37:767 (1984) ) .
Both cell lysate and culture media were precipitated with an HA specific monoclonal antibody. Proteins precipitated were analyzed on 15% SDS-PAG$ gels.
$~le 4 The effect of partially-purified VSGF2 protein on the Qrowth Qf vascular endothelial cells On day 1, human umbilical vein endothelial cells (HUVBC) were seeded at 2-5x10' cells/35 mm dish density in M199 medium containing 9:% fetal bovine serum (FHS), 16 units/ml heparin, and 50 unit;s/ml endothelial cell growth supplements (BCGS, Biotechnique, Inc.). On day 2, the medium was replaced with M199 containing 10% FBS, 8 units/ml heparin. VEGF2 protein of SBQ ID NO. 2 minus the initial 45 amino acid residues, (~TSGF) a.nd basic FGF (bFGF) were added, at the concentration shown. On days 4 & 6, the medium was replaced. On day 8, cell number was determined with a Coulter Counter (See Figure 8) .
Example 5 The effect of aurified V8GF2 protein on the growth of vascular endothelial cells On day :L, human umbilical vein endothelial cells (BUVBC) were seeded at 2-5 x 10' cells/35 min dish density in M199 medium containing 4% fetal bovine serum (FBS), 16 units/ml heparin, 50 units/ml endothelial cell growth supplements (BOGS, Hiotechnique, Inc.). On day 2, the medium was replaced with M199 containing 10% FBS, 8 units/ml heparin. ., Purified V8G:B2 protein of SBQ ID No. 2 minus initial 45 amino acid residues was added to the medium at this point. On days 4 & 6, the medium' was replaced with fresh -medium and r supplements. On day 8, cell number was determined with a Coulter Counter (See Figure 9).
Example 6 Bxaressi_on v:~a.Gene Thera Fi~roblasts are obtained from a subject by skin biopsy.
The resultin~~ tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are , placed on a wet surface of a tissue culture flask, approximatel;t ten pieces are placed in each flask. The flask is turned upside. down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin, is added.
This is then incubated at 37°C for approximately one week.
At this time, fresh media is added and subsequently changed every several days. After an additional two weeks a.n culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.
pMV-7 (Kirschmeier, P.T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.
The cDNA encoding a polypeptide of the present invention is amplified using PCR primers which correspond to the 5' and 3' end sequences respectively. The 5' primer containing an EcoRI site and the 3' primer further includes a HindIII site.
$qual quantities of the Moloney murine sarcoma virus linear backbone and the amplified BcoRI and HindIII fragment are added together, in the presence of T4 DNA lipase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments . The ligatiorr mixture is used to transform bacteria HB101, which are then plated onto agar-containing kanamycin for the purpose of confizmi.ng that the vector had the gene of interest properly inserted.
The amphotropic pA317 or GP+aml2 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMBM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells are transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells).
Fresh media is added to the tra.nsduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent: producer cells. The spent media, containing the infectious viral particles, is filtered through a milliporeTM
filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the mE~dia from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection. is required. Zf the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such. as neo or his.
The engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence TM
on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product.
Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, within the scope of the appended .claims, the invention ma.y be practiced otherwise than as particularly..
described.
SEQUENCE LISTING
(1) GENERAL INFORMATTON:
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(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
GTCCTTCCAC CATGCACTC'G CTGGGCTTCT TCTCTGTGGC GTGTTCTCTG CTCGCCGCTG 60 CGCTGCTCCC GGGT'CC'TCGC GAGGCGCCCG CCGCCGCCGC CGCCTTCGAG TCCGGACTCG 120 ACCTCTCGGA CGCGGAGCCC GACGCGGGCG AGGCCxICGGC TTATGCAAGC AAAGATCTGG 180 ATTGGAAAAT GTACAAGTC~'T CAGCTAAGGFa AAGGAGGCTG GCAACATAAC AGAGAACAGG 300 CCAACCTCAA CTC31,AGGACA GAAGAGACTA TAAAATTTGC TG~CA~T TATAATACAG 360 AGATC7:TGAA AAGTATTGAT AATGAG'T'GGA GAAAGACTCA ATGCATGCCA C'GGGACiGTGT 420 GTATAGATGT GGGG~AAGGAG TTTGGAGTCG ~CGACAAACAC CTTCTTTAAA CCTCCATGTG 480 TGTCCGTCTA ~CAGF.TGTGGG GGTTGCTGCA ATAGTGAGGG GCTGCAGTGC ATGAACACCA 540 TTTACAGACA AGTT'CATTCC ATTATTAGAC GTTCCCTGCC AGC31ACACTA CCACAGTGTC 720 AGGCAGCGAA CAAGiACCTGC CCCACCAATT ACATGTGGAA TAATCACATC TGCAGATGCC 780 TGGCTCAGGA RGATZ'ITATG TTTTCCTCGG ATGCTGGAGA TGACTCAACA GATGGATTCC 840 ATGACATCTG TGGA,CCAAAC AAGGAGCTGG ATGAAGAGAC CTGTCAGTGT GTCTGCAGAG 900 CGGGGCZTCG GCCT'GCCAGC TGTGGACCCC AC~1~AAGAACT AGACAGAiIAC TCA~GCCAGT 960 GTGTCTGTAA AAAC:AAACTC TTCCCCAGC:C AATGTGGGGC CAACCGAGAA TTTGATGAAA 1020 WO 96/39515 PCTlUS96/09001 ACACATGCCA GTGT'GTATGT AAAAGAACCT GCCCCAGAAA TCAACCCCTA AATCCTGGAA1080 AATGTGCCTG TGAA,TGTACA GAAAGTCCAC AGAAATGCTT GTTAAAAGGA AAGAAGTTCC1140 ACC3~CCAAAC ATGC31GCTGT TACpGACGGC CATGTACGAA CCGCCAGAAG 1200 GCT!'GTGAGC
CAGCATTTTC ATR,TAGTGAA GAAGTGTGTC GTTGTGTCCC TTCATATTGG CAAAGACCAC1260 AAAGTCTGTC TTTC.'CTGAAC CATGTGGATA ACTTTACAGA AATGGAC,'TGG 1440 AGCTCATCTG
TTGTGATTTC TIT3~AAAGAA TGACTATATA ATTTATTTCC ACTAAAAATA TTGTTTCTGC1560 ATTCATTTTT ATAGCAACAA CAATTGGTAA AACTCACTGT GATC3~ATATT TTTATATCAT1620 GCAAAATATG TTT3iAAATAA AATGAAA)~TT GTATTTATAA AAAAAAAAAA 1674 AAAA
( 2 ) INFORMA'.CION POR SBQ ID NO : 2 i ) S$QU8P7CE C~A,R.ACTBRISTICS
(A) LENGTH: 419 AMINO ACIDS
(B) TYPE: AMINO ACID
( C) STRAPTDEDNESS
(D) TOPOLOGY: LINBAR
(ii) MOLBCOLB TYPE: PROTEIN
(xi) SEQUENCE DESCRIPTION: SBQ ID N0:2:
Met His Ser Leu Gly Phe Phe 5er Val Aln Cys Ser Leu Leu Ala Ala Ala Leu Leu~Pro Gly Pro Arg Glu 3rla Pro Ala Ala Ala Ala Ala-Phe a Glu Ser Gly Leu Asp Iaeu Ser Asp Ala Glu Pro Asp Ala Gly Glu Ala Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu Gln Leu Arg Ser Val Ser Ser Val Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met 20 ~: 25 30 Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln Ala Asa Leu Aaa Ser Arg Thr Glu Glu Thr Ile Lys Phe Aln Ala Ala His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asa Glu Tzp Arg Lys WO 96/39515 ' PCT/US96/09001 Thr Gln Cps Met Pro Arg Glu 'Val Cars Ile Asp Val Gly Lys Glu Phe Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro C'ys Val Ser Val Tyr Arg t'~ss Gly Gly t'~rs t'~ys Asn ,Ser Glu Gly Leu Gln tars Met Asn Thr Ser Thr Ser Tyr Leu Ser Lys 'Thr Leu Phe Glu Ile Thr Val Pro Leu Ser Gln Gly Pro Lys Pro Val 'Thr Ile Ser Phe Ala Asn His Thr Ser fuss Arg Cys Met Ser Lys Leu :Asp Val Tyr Arg Gln Val His Ser Ile 165 ;170 I75 Ile Arg Arg Ser Leu Pro Ala 'Thr Leu Pro Gln Cys Gln Ala Ala Asn Lys Thr t'=ys Pro Thr Asn Tyr IKet Trp Asn Asn His Ile Cys Arg Cys Leu Ala Gln Glu Asp Phe Met IPhe Ser Ser Asp Ala Gly Asp Asp Ser Thr Asp Gly Phe His Asp Ile I~rs Gly Pro Asn Lys Glu Leu Asp Glu Glu Thr Cars Gln Cps Val Cps :Arg Ala Gly Leu Arg Pro Ala Ser Cars 245 :250 255 Gly Pro His Lys Glu Leu Asp ~Arg Asn Ser Cys Gln Cars Val Cars Lys Asn Lys Leu Phe Pro Ser Gln ~ys Gly Ala Asn Arg Glu Phe Asp Glu Asn Thr Cys Gln Cys Val tars 7Lys Arg Thr Cys Pro Arg Asn Gln Pro w- 295 , _ 300 305 Leu Asn Pro Gly Lys Cps Ala ~.'~rs Glu Cys Thr Glu Ser Pro Gln Lys Cps Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cps Ser t'~rs Tyr Arg Arg Pro tars Thr Asn Arg t;ln Lys Ala Cuss Glu Pra Gly Phe Ser Tyr Ser Glu Glu Val Cys Arg 1':ys Val Pro Ser Tyr Trp Gln Arg Pro Gln Met Ser ( 2 ) INFORMATION FOR SES2 ID NO : 3 ( i ) S$QL18NC$ CHAR~z~CTERISTICS
(A) LENGTH: 7.4 AMINO ACIDS
(B ) TYPE : AM7:N0 ACID
WO 96/39515 ~ PCT/US96/09001 (C) STRANDEDNESS:
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PEPTIDE
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Pro Xaa Cys Val Xaa Xaa Xaa Arg Cys Xaa Gly Cys Cys Asn (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE QiARACTERISTICS
(A) LENGTH: 50 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
( a.i ) MOLECUL$ TYPE : oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTEF~ISTICS
(A) LENGTH: 28 BASE PAIRS
(B) TYPE: NUCLEIC ACID
( C ) STRi~INDEDNESS : S INGLE
(D) TOPOLOGY : LINEAR
(ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
GATCTCTAGA TTAGCTCATT TGTGGTCT 2g (2) INFORMATION FOR SEQ ID N0:6:
( 1 ) SEQUENCE CHAR.~1,CTSRISTIC,'S
(A) LENGTH: 27 BASE PAIRS
(B) TYPE: NUC~BIC ACID
(C) STRANDEDNESS : SING1LE
(D) TOPOLOGY: LINEAR
(ii) MOL$CUL$ TYPE: oligonuCleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHAR~,CTERISTIC,S
(A) LENGTH: 60 BASE PAIRS
(B) TYPE : NUC2,EIC ACID
( C ) STRANDEDNES S : S ING:LE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: oligonu~leotide (xi) SEQUENCE DESCT:IPTION: SEQ ID N0:7:
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARF~C'.TERISTICS
(A) LENGTH : 1.96 AMINO .ACIDS
(B) TYPE: AM~:NO ACID
( C) STRANDEDNE:SS
(D) TOPOLOGY: LINRAR
(ii) MOLECOLE TYPE: PROTEIN
(xi) SEQU~TCE D$SCF:IPTION: SEQ ID N0:8:
Met Arg Thr Leu Ala Cys Leu Leu Leu Leu Gly C'.ys Gly Tyr Leu Ala His Val Leu Ala Glu Glu Ala Glu Ile Pro Arg Glu Val Ile Glu Arg Leu Ala Arg Ser Gln Ile His Ser Ile Arg Asp Leu Gln Arg Leu Leu Glu Ile Asp Ser Val Gly Ser Glu Asp Ser Leu Asp 50 55' 60 Thr Ser Leu Arg Ala His Gly Val His Ala Thr Lys His Val Pro Glu Lys Arg Pro Leu Pro Ile Arg Arg Lys Arg Ser Ile Glu Glu Ala Val Pro Ala Val Cys Lys Thr Arg Thr Val Ile Tyr Glu Ile Pro Arg Ser Gln Val Asp Pro Thr Ser Ala Asn Phe Leu Ile Trp Pro Pro Cys Val Glu val Lys Arg Cys Thr Gly Cys Cys Asn Thr Ser Ser Val Lys Cys Gln Pro Ser Arg Val His His Arg Ser Val Lys Val Ala Lys Val Glu Tyr Val Arg Lys Lys Pro Lys Leu Lys Glu Val Gln Val Arg Leu Glu Glu His Leu Glu Cys Ala Cys Ala Thr Thr Ser Leu Asn Pro Asp Tyr Arg Glu Glu Asp Thr Asp Val (2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 241 AMINO ACIDS
(B) TYPE: AMINO ACID
(C) STRANDEDNESS:
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN
(xi) SEQUENC$ DESCRIPTION: SEQ ID N0:9:
Met Asn Arg Cys Trp Ala Leu Phe Leu Ser Leu Cys Cys Tyr Leu Arg Leu Val Ser Ala Glu Gly Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu Ser Asp His Ser Ile Arg Ser Phe Asp Asp Leu Gln Arg Leu Leu His Gly Asp Pro Gly Glu Glu Asp Gly Ala Glu Leu Asp Leu Asn Met Thr Arg Ser His Ser Gly Gly Glu Leu Glu Ser Leu Ala Arg Gly Arg Arg Ser Leu Gly Ser Leu Thr Ile Ala Glu Pro Ala Met Ile Ala Glu Cys Lys Thr Arg Thr Glu Val Phe Glu Ile Ser Arg Arg Leu Ile Asp Arg Thr Asn Ala Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gln Arg Cys Ser Gly Cys Cys Asn Asn Arg Asn Val Gln Cys Arg Pro Thr Gln Val Gln Leu Arg Pro Val Gln Val Arg Lys Ile Glu Ile Val Arg Lys Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu Ala Cys Lys Cys Glu Thr Val Ala Ala Ala Arg fro Val Thr Arg Ser fro Gly Gly Ser Gln Glu GIn Arg Ala Lys Thr Pro Gln Thr Arg Val Thr Ile Arg Thr Val Arg Val Arg Arg Pro Pro Lys Gly Lys His Arg Lys Phe Lys His Thr His Asp Lys Thr Ala Leu Lys Glu Thr Leu Gly Ala ( 2 ) INFORMATION FOR SE(~ ID NO :10 (i) SBQUBNCB CHARAC'1'BRISTICS
(A) LENGTH: 232 AMINO ACIDS
(B) TYPE: AMINO ACID
(C) STRANDEI)NESS:
( D ) TOPOLOG7.' : LINEAR
(ii) MOLECULE TYPE: PROTEIN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr G1n Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu 65 70 75 g0 Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Va1 Pro Thr Glu Glu Se:r Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Lys Lys Ser Val Arg Gly Lys Gl:y Lys Gly Gln Lys Arg Lys Arg Lys Lys Ser Arg Tyr Lys Ser Trp Ser Val Tyr Val Gly Ala Arg Cys Cys Leu Met Pro Trp Ser Leu Pro Gl:y Pro His Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro G1n Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys As:p Lys Pro Arg Arg
Claims (107)
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. An isolated polynucleotide, or one which is substantially identical to said polynucleotide, encoding a proprotein portion of a protein comprising SEQ ID NO:2.
2. An isolated polynucleotide, or one which is substantially identical to said polynucleotide, which encodes a polypeptide comprising amino acids +1 to +373 of SEQ ID NO:2.
3. The isolated polynucleotide of claim 2, or one which is substantially identical to said polynucleotide, wherein the polypeptide comprises amino acids -23 to +373 of SEQ ID
NO:2.
NO:2.
4. The isolated polynucleotide of claim 2, or one which is substantially identical to said polynucleotide, wherein the polypeptide comprises amino acids -46 to +373 of SEQ ID
NO:2.
NO:2.
5. An isolated polynucleotide, or one which is substantially identical to said polynucleotide, encoding a VEGF-2 polypeptide, which hybridizes to a polynucleotide consisting of the complement of SEQ ID NO:1 under the following conditions: hybridization followed by a final wash using 0.5 × SSC, 0.1% sodium dodecyl sulfate (SDS) at 60°C.
6. An isolated polynucleotide, or one which is substantially identical to said polynucleotide, encoding a VEGF-2 polypeptide, which hybridizes to the cDNA contained in ATCC
Deposit No. 97149 under the following conditions: hybridization followed by a final wash using 0.5 × SSC, 0.1% sodium dodecyl sulfate (SDS) at 60°C.
Deposit No. 97149 under the following conditions: hybridization followed by a final wash using 0.5 × SSC, 0.1% sodium dodecyl sulfate (SDS) at 60°C.
7. An isolated polynucleotide, or one which is substantially identical to said polynucleotide, comprising a polynucleotide that encodes a polypeptide fragment of SEQ ID
NO:2, wherein said polypeptide fragment has angiogenic activity and wherein said polypeptide fragment contains at least one amino acid from amino acid residues -46 to 23 of SEQ ID
NO:2, inclusive.
NO:2, wherein said polypeptide fragment has angiogenic activity and wherein said polypeptide fragment contains at least one amino acid from amino acid residues -46 to 23 of SEQ ID
NO:2, inclusive.
8. An isolated polynucleotide, or one that is substantially identical to said polynucleotide, comprising a polynucleotide that encodes a polypeptide fragment encoded by the cDNA
contained in ATCC Deposit No. 97149, wherein said fragment has angiogenic activity and wherein said polypeptide fragment contains at least one amino acid from the first 69 amino acids in the protein encoded by the cDNA contained in ATCC Deposit No.
97149, inclusive.
contained in ATCC Deposit No. 97149, wherein said fragment has angiogenic activity and wherein said polypeptide fragment contains at least one amino acid from the first 69 amino acids in the protein encoded by the cDNA contained in ATCC Deposit No.
97149, inclusive.
9. An isolated polynucleotide, or one which is substantially identical to said polynucleotide, comprising a polynucleotide that encodes a polypeptide fragment of SEQ ID
NO:2, wherein said polypeptide fragment has endothelial cell proliferative activity and wherein said polypeptide fragment contains at least one amino acid from amino acid residues -46 to 23 of SEQ ID NO:2, inclusive.
NO:2, wherein said polypeptide fragment has endothelial cell proliferative activity and wherein said polypeptide fragment contains at least one amino acid from amino acid residues -46 to 23 of SEQ ID NO:2, inclusive.
10. An isolated polynucleotide, or one which is substantially identical to said polynucleotide, comprising a polynucleotide that encodes a polypeptide fragment encoded by the cDNA
contained in ATCC Deposit No. 97149, wherein said fragment has endothelial cell proliferative activity and wherein said polypeptide fragment contains at least one amino acid from the first 69 amino acids in the protein encoded by the cDNA
contained in ATCC Deposit No. 97149, inclusive.
contained in ATCC Deposit No. 97149, wherein said fragment has endothelial cell proliferative activity and wherein said polypeptide fragment contains at least one amino acid from the first 69 amino acids in the protein encoded by the cDNA
contained in ATCC Deposit No. 97149, inclusive.
11. An isolated polynucleotide, or one which is substantially identical to said polynucleotide, encoding a protein encoded by the cDNA contained in ATCC Deposit No. 97149.
12. An isolated polynucleotide, or one which is substantially identical to said polynucleotide, encoding a proprotein portion of a protein encoded by the cDNA contained in ATCC
Deposit No. 97149.
Deposit No. 97149.
13. An isolated polynucleotide, or one which is substantially identical to said polynucleotide, encoding a polypeptide fragment of at least 50 contiguous amino acids of SEQ
ID NO:2 having VEGF-2 activity, wherein said polypeptide fragment contains at least one amino acid from amino acid residues -46 to 23 of SEQ ID NO:2, inclusive.
ID NO:2 having VEGF-2 activity, wherein said polypeptide fragment contains at least one amino acid from amino acid residues -46 to 23 of SEQ ID NO:2, inclusive.
14. An isolated polynucleotide, or one which is substantially identical to said polynucleotide, contained in ATCC Deposit No. 97149, encoding a polypeptide fragment of at least 50 contiguous amino acids having VEGF-2 activity, wherein said polypeptide fragment contains at least one amino acid from the first 69 amino acids in the protein encoded by the cDNA contained in ATCC Deposit No. 97149, inclusive.
15. An isolated polynucleotide, or one which is substantially identical to said polynucleotide, encoding a polypeptide fragment of at least 30 contiguous amino acids of SEQ
ID NO:2 having VEGF-2 activity, wherein said polypeptide fragment contains at least one amino acid from amino acid residues -46 to 23 of SEQ ID NO:2, inclusive.
ID NO:2 having VEGF-2 activity, wherein said polypeptide fragment contains at least one amino acid from amino acid residues -46 to 23 of SEQ ID NO:2, inclusive.
16. An isolated polynucleotide, or one which is substantially identical to said polynucleotide, contained in ATCC Deposit No. 97149, encoding a polypeptide fragment of at least 30 contiguous amino acids having VEGF-2 activity, wherein said polypeptide fragment contains at least one amino acid from the first 69 amino acids in the protein encoded by the cDNA contained in ATCC Deposit No. 97149, inclusive.
17. An isolated polynucleotide which is at least 70 % identical to a nucleic acid having a nucleotide sequence as set forth in SEQ ID NO:1, said polynucleotide encoding a polypeptide having at least one characteristic selected from the group of:
i) exhibits VEGF2 activity;
ii) specifically binds an antibody which specifically binds VEGF2;
iii) prevents VEGF2 activity; and iv) binds a receptor, agonist or antagonist of VEGF2, wherein said polynucleotide is not identical to the cDNA contained in ATCC
Deposit No.
75698.
i) exhibits VEGF2 activity;
ii) specifically binds an antibody which specifically binds VEGF2;
iii) prevents VEGF2 activity; and iv) binds a receptor, agonist or antagonist of VEGF2, wherein said polynucleotide is not identical to the cDNA contained in ATCC
Deposit No.
75698.
18. An isolated polynucleotide according to claim 17, wherein said polynucleotide is at least 90 % identical to said nucleic acid.
19. An isolated polynucleotide according to claim 17, wherein said polynucleotide is at least 95 % identical to said nucleic acid.
20. An isolated polynucleotide according to claim 17, wherein said polynucleotide is said nucleic acid.
21. The polynucleotide, or one which is substantially identical to said polynucleotide, of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, fused to a heterologous polynucleotide.
22. The polynucleotide, or one which is substantially identical to said polynucleotide, of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, fused to a polynucleotide which encodes a heterologous polypeptide.
23. A vector comprising the polynucleotide, or one which is substantially identical to said polynucleotide, of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
24. A vector comprising the polynucleotide, or one which is substantially identical to said polynucleotide, of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, operatively associated with a regulatory sequence that controls gene expression.
25. The polynucleotide, or one which is substantially identical to said polynucleotide, of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, fused, in frame, to a polynucleotide sequence encoding a polypeptide which facilitates purification of the resultant fusion polypeptide.
26. A recombinant host cell transformed, transfected or transduced with the polynucleotide, or one which is substantially identical to said polynucleotide, of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
27. A recombinant host cell comprising the polynucleotide, or one which is substantially identical to said polynucleotide, of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, operatively associated with a heterologous regulatory sequence that controls gene expression.
28. A method for producing a VEGF-2 polypeptide, comprising:
(a) culturing the host cell of claim 26 or 27, under conditions suitable to produce a polypeptide; and (b) recovering the polypeptide from the cell culture.
(a) culturing the host cell of claim 26 or 27, under conditions suitable to produce a polypeptide; and (b) recovering the polypeptide from the cell culture.
29. The isolated polynucleotide, or one which is substantially identical to said polynucleotide, of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, wherein the polynucleotide is DNA.
30. The isolated polynucleotide, or one which is substantially identical to said polynucleotide, of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, wherein the polynucleotide is RNA.
31. A method for producing a VEGF-2 polypeptide, comprising:
(a) culturing a genetically engineered host cell comprising an isolated mammalian RNA or cDNA which hybridizes to the complement of SEQ ID NO:1 under the following conditions; hybridization followed by a final wash using 0.5 ×
SSC, 0.1% sodium dodecyl sulfate (SDS) at 60°C, under conditions suitable to produce the polypeptide; and (b) recovering the polypeptide from the cell culture.
(a) culturing a genetically engineered host cell comprising an isolated mammalian RNA or cDNA which hybridizes to the complement of SEQ ID NO:1 under the following conditions; hybridization followed by a final wash using 0.5 ×
SSC, 0.1% sodium dodecyl sulfate (SDS) at 60°C, under conditions suitable to produce the polypeptide; and (b) recovering the polypeptide from the cell culture.
32. An isolated polypeptide, or one that is substantially identical to said polypeptide, comprising a proprotein portion of a protein comprising the amino acid sequence of SEQ
ID NO:2.
ID NO:2.
33. An isolated polypeptide, or one which is substantially identical to said polypeptide, comprising a protein encoded by the cDNA contained in ATCC Deposit No. 97149.
34. An isolated polypeptide, or one which is substantially identical to said polypeptide, comprising a proprotein portion of a protein encoded by the cDNA contained in ATCC
Deposit No. 97149.
Deposit No. 97149.
35. An isolated polypeptide, or one which is substantially identical to said polypeptide, comprising amino acids +1 to +373 of SEQ ID NO:2.
36. The isolated polypeptide, or one which is substantially identical to said polypeptide, comprising amino acids -23 to +373 of SEQ ID NO:2.
37. The isolated polypeptide, or one which is substantially identical to said polypeptide, comprising amino acids -46 to +373 of SEQ ID NO:2.
38. An isolated polypeptide, or one which is substantially identical to said polypeptide, comprising a polypeptide fragment of SEQ ID NO:2, wherein said fragment has angiogenic activity and wherein said fragment contains at least one amino acid from amino acid residues -46 to 23 of SEQ ID NO:2, inclusive.
39. An isolated polypeptide, or one which is substantially identical to said polypeptide, comprising a polypeptide fragment encoded by the cDNA contained in ATCC
Deposit No. 97149, wherein said fragment has angiogenic activity and wherein said fragment contains at least one amino acid from the first 69 amino acids in the protein encoded by the cDNA contained in ATCC Deposit No. 97149, inclusive.
Deposit No. 97149, wherein said fragment has angiogenic activity and wherein said fragment contains at least one amino acid from the first 69 amino acids in the protein encoded by the cDNA contained in ATCC Deposit No. 97149, inclusive.
40. An isolated polypeptide, or one which is substantially identical to said polypeptide, comprising a polypeptide fragment of SEQ ID NO:2, wherein said fragment has endothelial cell proliferative activity and wherein said fragment contains at least one amino acid from amino acid residues -46 to 23 of SEQ ID NO:2, inclusive.
41. An isolated polypeptide, or one which is substantially identical to said polypeptide, comprising a polypeptide fragment encoded by the cDNA contained in ATCC
Deposit No. 97149, wherein said fragment has endothelial cell proliferative activity and wherein said fragment contains at least one amino acid from the first 69 amino acids in the protein encoded by the cDNA contained in ATCC Deposit No. 97149, inclusive.
Deposit No. 97149, wherein said fragment has endothelial cell proliferative activity and wherein said fragment contains at least one amino acid from the first 69 amino acids in the protein encoded by the cDNA contained in ATCC Deposit No. 97149, inclusive.
42. An isolated VEGF-2 polypeptide, or one which is substantially identical to said polypeptide, comprising a polypeptide fragment of at least 50 contiguous amino acids of SEQ ID NO:2, wherein said fragment contains at least one amino acid from amino acid residues -46 to 23 of SEQ ID NO:2, inclusive.
43. An isolated VEGF-2 polypeptide, or one which is substantially identical to said polypeptide, comprising a polypeptide fragment of at least 50 contiguous amino acids encoded by the cDNA contained in ATCC Deposit No. 97149, wherein said fragment contains at least one amino acid from the first 69 amino acids in the protein encoded by the cDNA contained in ATCC Deposit No. 97149, inclusive.
44. An isolated VEGF-2 polypeptide, or one which is substantially identical to said polypeptide, comprising a polypeptide fragment of at least 30 contiguous amino acids of SEQ ID NO:2, wherein said fragment contains at least one amino acid from amino acid residues -46 to 23 of SEQ ID NO:2, inclusive.
45. An isolated VEGF-2 polypeptide, or one which is substantially identical to said polypeptide, comprising a polypeptide fragment of at least 30 contiguous amino acids encoded by the cDNA contained in ATCC Deposit No. 97149, wherein said fragment contains at least one ami acid from the first 69 ami acids in the protein encoded by the cDNA contained in ATCC Deposit . 97149, inclusive.
46. An isolated polypeptide which is at least 70 % identical to a polypeptide as set forth in SEQ ID NO:2, said polypeptide having at least one characteristic selected from the group of:
i) exhibits VEGF2 activity;
ii) specifically binds an antibody which specifically binds VEGF2;
iii) prevents VEGF2 activity; and iv) binds a receptor, agonist or antagonist of VEGF2, wherein said polypeptide is not the protein encoded by the cDNA contained in ATCC
Deposit No. 75698.
i) exhibits VEGF2 activity;
ii) specifically binds an antibody which specifically binds VEGF2;
iii) prevents VEGF2 activity; and iv) binds a receptor, agonist or antagonist of VEGF2, wherein said polypeptide is not the protein encoded by the cDNA contained in ATCC
Deposit No. 75698.
47. The isolated polypeptide according to claim 46, wherein said polypeptide is at least 90 %
identical to a polypeptide as set forth in SEQ ID NO:2.
identical to a polypeptide as set forth in SEQ ID NO:2.
48. The isolated polypeptide according to claim 46, wherein said polypeptide is at least 95 %
identical to a polypeptide as set forth in SEQ ID NO:2.
identical to a polypeptide as set forth in SEQ ID NO:2.
49. The isolated polypeptide according to claim 46, wherein said polypeptide is a polypeptide as set forth in SEQ ID NO:2.
50. The polypeptide, or one which is substantially identical to said polypeptide, produced by the method of claim 28 or 31.
51. A method for producing a VEGF-2 polypeptide, comprising:
(a) performing cell-free translation of mRNA encoding the polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50; and (b) recovering the polypeptide, or one which is substantially identical to said polypeptide, from the translation mixture.
(a) performing cell-free translation of mRNA encoding the polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50; and (b) recovering the polypeptide, or one which is substantially identical to said polypeptide, from the translation mixture.
52. The polypeptide, or one which is substantially identical to said polypeptide, produced by the method of claim 51.
53. A fusion protein comprising the polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52, fused to a heterologous polypeptide.
54. The polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52, within a homodimer or a heterodimer.
55. The polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52, which is glycosylated.
56. An isolated polynucleotide, of 20 to 50 bases in length, that hybridizes specifically to a unique region of a nucleic acid encoding the polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52, wherein said polynucleotide is used as a hybridization probe for the diagnosis of a disease.
57. An isolated polynucleotide, of 20 to 50 bases in length, that hybridizes specifically to a unique region of a nucleic acid encoding the polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52, wherein said polynucleotide is used as a hybridization probe for the diagnosis of susceptibility to a disease.
58. The polynucleotide of claim 56 or 57, wherein the polynucleotide is single stranded DNA.
59. The polynucleotide of claims 56 or 57, wherein the polynucleotide is RNA.
60. The use of the polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52, in a conditional medium for culturing vascular endothelial cells in vitro.
61. The use according to claim 60, wherein said polypeptide, or one which is substantially identical to said polypeptide, is added to conditional medium in a concentration from 10 pg/ml to 10 ng/ml.
62. The use of the polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52, in a conditional medium for culturing lymphatic endothelial cells in vitro.
63. The use of the polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52, to stimulate the endothelial cell proliferation in a patient in need of such therapy.
64. The use of the polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52, to stimulate angiogenesis in a patient in need of such therapy.
65. The use of the polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52, to coat artificial prostheses or natural organs which are to be transplanted into the body of a patient.
66. The use according to claim 65, wherein said polypeptide stimulates vascularization.
67. The use of the polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52, to stimulate the proliferation of lymphatic endothelial cells in a patient in need of such therapy.
68. The use of the polynucleotide, or one which is substantially identical to said polynucleotide, of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 to express a polypeptide, in vivo, which when expressed will stimulate endothelial cell proliferation in a patient in need of such therapy.
69. The use of the polynucleotide, or one which is substantially identical to said polynucleotide, of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 to express a polypeptide, in vivo, which when expressed will stimulate angiogenesis in a patient in need of such therapy.
70. A retroviral particle comprising the polynucleotide, or one which is substantially identical to said polynucleotide, of claim 30.
71. A method for preparing a producer cell wherein the retroviral particle of claim 70 is used to transduce a packaging cell.
72. The method of claim 71, wherein said packaging cell is chosen from the group of cell lines comprising: PE501, PA317, .PSI.- 2, .PSI.- AM, PA12, T19-14X, VT-19-17-H2, .PSI.CRE, .PSI.CRIP, GP+E-86, GP+envAml2, and DAN.
73. A producer cell prepared by the method of claim 71 or 72, for the production of the retroviral particle of claim 70.
74. An adenovirus comprising the polynucleotide, or one which is substantially identical to said polynucleotide, of claim 29.
75. The use of the retroviral particle of claim 70 to transduce cells ex vivo, wherein said cells become capable of in vivo expression of a polypeptide with VEGF-2 activity.
76. The use of the retroviral particle of claim 70 to express in vivo a polypeptide, or one which is substantially identical to said polypeptide, which, when expressed will stimulate endothelial cell proliferation in a patient in need of such therapy.
77. The use of the retroviral particle of claim 70 to express in vivo a polypeptide, or one which is substantially identical to said polypeptide, which, when expressed will stimulate angiogenesis in a patient in need of such therapy.
78. The use of the producer cell of claim 73 to express in vivo a polypeptide, or one which is substantially identical to said polypeptide, which, when expressed will stimulate endothelial cell proliferation in a patient in need of such therapy.
79. The use of the producer cell of claim 73 to express in vivo a polypeptide, or one which is substantially identical to said polypeptide, which, when expressed will stimulate angiogenesis in a patient in need of such therapy.
80. The use of the adenovirus of claim 74 to express in vivo a polypeptide, or one which is substantially identical to said polypeptide, which, when expressed will stimulate endothelial cell proliferation in a patient in need of such therapy.
81. The use of the adenovirus of claim 74 to express in vivo a polypeptide, or one which is substantially identical to said polypeptide, which, when expressed will stimulate angiogenesis in a patient in need of such therapy.
82. The use according to any one of claims 63, 68, 76, 78 or 80, wherein said endothelial cell proliferation leads to angiogenesis.
83. The use according to any one of claims 63, 64, 68, 69, 76, 77, 78, 79, 80, 81 or 82, wherein said patient has a wound.
84. The use according to any one of claims 63, 64, 68, 69, 76, 77, 78, 79, 80, 81 or 82, wherein said patient has tissue damage.
85. The use according to any one of claims 63, 64, 68, 69, 76, 77, 78, 79, 80, 81 or 82, wherein said patient has bone damage.
86. The use according to any one of claims 63, 64, 68, 69, 76, 77, 78, 79, 80, 81 or 82, wherein said patient has ischemia.
87. The use according to any one of claims 63, 64, 68, 69, 76, 77, 78, 79, 80, 81 or 82, wherein said patient has had a myocardial infarction.
88. The use according to any one of claims 63, 64, 68, 69, 76, 77, 78, 79, 80, 81 or 82, wherein said patient has had coronary bypass surgery.
89. The use according to any one of claims 63, 64, 68, 69, 76, 77, 78, 79, 80, 81 or 82, wherein said patient requires revascularization of damaged tissue.
90. The use according to any one of claims 63, 64, 68, 69, 76, 77, 78, 79, 80, 81 or 82, wherein said patient requires growth of one or more chosen from the group comprising:
bone; peridontium; and ligament tissue.
bone; peridontium; and ligament tissue.
91. The use according to any one of claims 63, 64, 65, 66, 67, 68, 69, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90, wherein said patient is human.
92. A method for identifying receptors for the polypeptide, or one which is substantially identical to said polypeptide, of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52, comprising the sequential steps:
(a) preparing a cDNA library from mRNA isolated from VEGF-2 receptive cells;
(b) dividing the library into pools for transfecting into cells not receptive to VEGF-2;
(c) identifying receptor-producing, transfected cells using a polypeptide, or one which is substantially identical to said polypeptide, of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52;
(d) repeatedly preparing sub-pools of cDNA from identified transfected cells and retransfecting; and (e) recovering a single clone that encodes the putative receptor.
(a) preparing a cDNA library from mRNA isolated from VEGF-2 receptive cells;
(b) dividing the library into pools for transfecting into cells not receptive to VEGF-2;
(c) identifying receptor-producing, transfected cells using a polypeptide, or one which is substantially identical to said polypeptide, of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52;
(d) repeatedly preparing sub-pools of cDNA from identified transfected cells and retransfecting; and (e) recovering a single clone that encodes the putative receptor.
93. A method for identifying receptors for the polypeptide, or one which is substantially identical to said polypeptide, of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52, comprising the sequential steps:
(a) preparing cell membranes or extracts expressing the VEGF-2 receptor molecule;
(b) labeling said polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52;
(c) photoaffinity linking the labeled polypeptide to said membranes or extracts to form complexes;
(d) isolating the resultant polypeptide complexes and preparing peptide fragments;
(e) sequencing peptide fragments;
(f) preparing degenerate oligonucleotide probes from deduced sequences; and (g) using said probes in identifying the gene encoding the putative receptor.
(a) preparing cell membranes or extracts expressing the VEGF-2 receptor molecule;
(b) labeling said polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52;
(c) photoaffinity linking the labeled polypeptide to said membranes or extracts to form complexes;
(d) isolating the resultant polypeptide complexes and preparing peptide fragments;
(e) sequencing peptide fragments;
(f) preparing degenerate oligonucleotide probes from deduced sequences; and (g) using said probes in identifying the gene encoding the putative receptor.
94. A method for identifying a compound which demonstrates antagonist activity for the polypeptide, or one which is substantially identical to said polypeptide, of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52, comprising the sequential steps:
(a) labeling said polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52;
(b) using a competitive inhibition assay wherein membrane bound receptors are mixed with the labeled polypeptide and said compound to be tested;
(c) measuring label bound to said receptors; and (d) comparing measured bound label to the amount of bound label measured in a control assay, wherein said compound is excluded, as a means of indicating reduction of endothelial cell proliferation.
(a) labeling said polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52;
(b) using a competitive inhibition assay wherein membrane bound receptors are mixed with the labeled polypeptide and said compound to be tested;
(c) measuring label bound to said receptors; and (d) comparing measured bound label to the amount of bound label measured in a control assay, wherein said compound is excluded, as a means of indicating reduction of endothelial cell proliferation.
95. A method for identifying a compound with agonist or antagonist activity for the polypeptide, or one which is substantially identical to said polypeptide, of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52, comprising the sequential steps:
(a) culturing endothelial cells in a medium supplemented with said polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52, and said compound to be screened;
(b) pulsing with labeled nucleotide;
(c) harvesting cells;
(d) measuring incorporated label; and (e) relating measured label to the amount of label incorporated in a control assay, wherein said compound is excluded, as a means of indicating stimulation or reduction of endothelial cell proliferation.
(a) culturing endothelial cells in a medium supplemented with said polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52, and said compound to be screened;
(b) pulsing with labeled nucleotide;
(c) harvesting cells;
(d) measuring incorporated label; and (e) relating measured label to the amount of label incorporated in a control assay, wherein said compound is excluded, as a means of indicating stimulation or reduction of endothelial cell proliferation.
96. A method for identifying a compound with agonist or antagonist activity for the polypeptide. or one which is substantially identical to said polypeptide, of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52, comprising the sequential steps:
(a) mixing said polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52, with cells expressing the VEGF-2 receptor, in the presence of said compound being tested;
(b) monitoring the response of a known second messenger system; and (c) comparing said response to the response of the second messenger system in a control assay, wherein said compound is excluded, as a means of indicating stimulation or reduction of endothelial cell proliferation.
(a) mixing said polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52, with cells expressing the VEGF-2 receptor, in the presence of said compound being tested;
(b) monitoring the response of a known second messenger system; and (c) comparing said response to the response of the second messenger system in a control assay, wherein said compound is excluded, as a means of indicating stimulation or reduction of endothelial cell proliferation.
97. A method for identifying a compound with agonist or antagonist activity for the polypeptide, or one which is substantially identical to said polypeptide, of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52, comprising the sequential steps:
(a) preparing cell membranes or extracts expressing the VEGF-2 receptor;
(b) labeling said polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52;
(c) adding labeled polypeptide to said membranes or extracts in the presence and absence of said compound being tested;
(d) monitoring interactions between said receptor and labeled polypeptide; and (e) comparing the interactions with interactions observed in a control assay, wherein said compound is excluded, as a means of indicating stimulation or reduction of endothelial cell proliferation.
(a) preparing cell membranes or extracts expressing the VEGF-2 receptor;
(b) labeling said polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52;
(c) adding labeled polypeptide to said membranes or extracts in the presence and absence of said compound being tested;
(d) monitoring interactions between said receptor and labeled polypeptide; and (e) comparing the interactions with interactions observed in a control assay, wherein said compound is excluded, as a means of indicating stimulation or reduction of endothelial cell proliferation.
98. A composition comprising a pharmaceutically acceptable carrier and one or more polypeptides selected from the group: the polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52.
99. A composition comprising a pharmaceutically acceptable carrier and one or more nucleic acids selected from the group: the polynucleotide, or one which is substantially identical to said polynucleotide, of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
100. A composition according to any one of claims 98 or 99, wherein said carrier is chosen from the group comprising: saline, buffered saline, dextrose, water, glycerol, ethanol and any combination thereof.
101. A composition according to any one of claims 98 or 99, designed for administration by a convenient method chosen from the group comprising: topical, intravenous, intraperitoneal, intramuscular, intratumor, subcutaneous, intranasal and intradermal routes.
102. A pharmaceutical kit comprising:
(a) one or more containers containing a composition according to any one of claims 98 or 99; and optionally (b) a notice, in the form prescribed by a governmental agency regulating the manufacture, sale or use of pharmaceutical or biological products, which reflects approval by the agency of manufacture, use or sale for human administration.
(a) one or more containers containing a composition according to any one of claims 98 or 99; and optionally (b) a notice, in the form prescribed by a governmental agency regulating the manufacture, sale or use of pharmaceutical or biological products, which reflects approval by the agency of manufacture, use or sale for human administration.
103. A diagnostic kit, for use with an antibody with specificity for the polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52, including more than one element chosen from the list comprising:
(a) a receptacle for sample collection;
(b) a solid support for immobilization of any polypeptides present within said sample;
(c) a contained amount of a blocking solution comprising a protein, a buffer and a preservative, wherein said protein will bind in any unoccupied sites of the solid support;
(d) a contained amount of a wash solution comprising a buffer and a preservative;
(e) a contained amount of a signal developing reagent whereby said reagent will react with said antibody to generate a signal as a positive result;
(f) a contained amount of a standard solution of a polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52 in a buffer; and (g) instructions for use.
(a) a receptacle for sample collection;
(b) a solid support for immobilization of any polypeptides present within said sample;
(c) a contained amount of a blocking solution comprising a protein, a buffer and a preservative, wherein said protein will bind in any unoccupied sites of the solid support;
(d) a contained amount of a wash solution comprising a buffer and a preservative;
(e) a contained amount of a signal developing reagent whereby said reagent will react with said antibody to generate a signal as a positive result;
(f) a contained amount of a standard solution of a polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 52 in a buffer; and (g) instructions for use.
104. An isolated polypeptide, or one which is substantially identical to said polypeptide, comprising a polypeptide fragment of at least 50 contiguous amino acids of SEQ
ID
NO:2, wherein said polypeptide is used as an immunogen for the production of anti-VEGF-2 antibodies and wherein said polypeptide fragment contains at least one amino acid from amino acid residues -46 to 23 of SEQ ID NO:2, inclusive.
ID
NO:2, wherein said polypeptide is used as an immunogen for the production of anti-VEGF-2 antibodies and wherein said polypeptide fragment contains at least one amino acid from amino acid residues -46 to 23 of SEQ ID NO:2, inclusive.
105. An isolated polypeptide, or one which is substantially identical to said polypeptide, comprising a polypeptide fragment of at least 50 contiguous amino acids encoded by the cDNA contained in ATCC Deposit No. 97149, wherein said polypeptide is used as an immunogen for the production of anti-VEGF-2 antibodies and wherein said polypeptide fragment contains at least one amino acid from the first 69 amino acids in the protein encoded by the cDNA contained in ATCC Deposit No. 97149, inclusive.
106. An isolated polypeptide, or one which is substantially identical to said polypeptide, comprising a polypeptide fragment of at least 30 contiguous amino acids of SEQ
ID
NO:2, wherein said polypeptide is used as an immunogen for the production of anti-VEGF-2 antibodies and wherein said polypeptide fragment contains at least one amino acid from amino acid residues -46 to 23 of SEQ ID NO:2, inclusive.
ID
NO:2, wherein said polypeptide is used as an immunogen for the production of anti-VEGF-2 antibodies and wherein said polypeptide fragment contains at least one amino acid from amino acid residues -46 to 23 of SEQ ID NO:2, inclusive.
107. An isolated polypeptide, or one which is substantially identical to said polypeptide, comprising a polypeptide fragment of at least 30 contiguous amino acids encoded by the cDNA contained in ATCC Deposit No. 97149, wherein said polypeptide is used as an immunogen for the production of anti-VEGF-2 antibodies and wherein said fragment contains at least one amino acid from the first 69 amino acids in the protein encoded by the cDNA contained in ATCC Deposit No. 97149, inclusive.
Priority Applications (1)
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CA002413012A CA2413012A1 (en) | 1995-06-06 | 1996-06-06 | Human vascular endothelial growth factor 2 |
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CA002413012A Division CA2413012A1 (en) | 1995-06-06 | 1996-06-06 | Human vascular endothelial growth factor 2 |
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CA2224093C true CA2224093C (en) | 2003-03-25 |
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CA (1) | CA2224093C (en) |
DE (1) | DE69636752T2 (en) |
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1995
- 1995-06-06 US US08/465,968 patent/US6608182B1/en not_active Expired - Fee Related
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1996
- 1996-06-06 JP JP9501427A patent/JPH11513883A/en not_active Withdrawn
- 1996-06-06 DE DE69636752T patent/DE69636752T2/en not_active Revoked
- 1996-06-06 KR KR1019970709003A patent/KR100511692B1/en not_active IP Right Cessation
- 1996-06-06 CA CA002224093A patent/CA2224093C/en not_active Expired - Fee Related
- 1996-06-06 AT AT96918130T patent/ATE347595T1/en not_active IP Right Cessation
- 1996-06-06 AU AU60467/96A patent/AU714484B2/en not_active Withdrawn - After Issue
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1999
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2002
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2007
- 2007-04-13 JP JP2007106593A patent/JP2007244388A/en active Pending
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US20020120123A1 (en) | 2002-08-29 |
AU6046796A (en) | 1996-12-24 |
CN1186518A (en) | 1998-07-01 |
WO1996039515A1 (en) | 1996-12-12 |
AU714484B2 (en) | 2000-01-06 |
DE69636752D1 (en) | 2007-01-18 |
CA2224093A1 (en) | 1996-12-12 |
EP0837934A4 (en) | 2000-11-02 |
US20100143360A1 (en) | 2010-06-10 |
MX9709243A (en) | 1998-03-31 |
KR19990022522A (en) | 1999-03-25 |
US7115392B2 (en) | 2006-10-03 |
US6608182B1 (en) | 2003-08-19 |
JPH11513883A (en) | 1999-11-30 |
ATE347595T1 (en) | 2006-12-15 |
JP2007244388A (en) | 2007-09-27 |
EP0837934A1 (en) | 1998-04-29 |
EP0837934B1 (en) | 2006-12-06 |
CN1552855A (en) | 2004-12-08 |
DE69636752T2 (en) | 2007-10-11 |
JP2000069982A (en) | 2000-03-07 |
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