EP1373299A2 - Facteur de croissance 2 de tissu conjonctif - Google Patents

Facteur de croissance 2 de tissu conjonctif

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Publication number
EP1373299A2
EP1373299A2 EP01954638A EP01954638A EP1373299A2 EP 1373299 A2 EP1373299 A2 EP 1373299A2 EP 01954638 A EP01954638 A EP 01954638A EP 01954638 A EP01954638 A EP 01954638A EP 1373299 A2 EP1373299 A2 EP 1373299A2
Authority
EP
European Patent Office
Prior art keywords
ctgf
polypeptide
antibodies
antibody
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01954638A
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German (de)
English (en)
Other versions
EP1373299A4 (fr
Inventor
Haodong Li
Mark D. Adams
Valerie Calenda
Virginie Fataccioli
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Transgene SA
Human Genome Sciences Inc
Original Assignee
Transgene SA
Human Genome Sciences Inc
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Filing date
Publication date
Application filed by Transgene SA, Human Genome Sciences Inc filed Critical Transgene SA
Publication of EP1373299A2 publication Critical patent/EP1373299A2/fr
Publication of EP1373299A4 publication Critical patent/EP1373299A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is connective tissue growth factor-2 sometimes hereinafter referred t ⁇ as "CTGF-2". The invention also relates to inhibiting the action of such polypeptides. In addition, the invention encompasses methods of using CTGF-2 polynucleotides and polypeptides to stimulate angiogenesis, i.e., formation of new blood vessels. The invention also encompasses methods of treatment of atherosclerosis, ischemia, restenosis.
  • the CTGF polypeptides are structurally and functionally related to a family of growth factors which include IGF (insulin-like growth factor), PDGF (platelet-derived growth factor), and FGF (f ⁇ broblast growth factor).
  • IGF insulin-like growth factor
  • PDGF platelet-derived growth factor
  • FGF f ⁇ broblast growth factor
  • This emerging family of secreted proteins are a group of cysteine-rich proteins.
  • This group of growth factors are important for normal growth, differentiation, morphogenesis of the cartilaginous skeleton of an embryo and cell growth.
  • the extended superfamily of growth factors include TGF (transforming growth factor), bone morphogenic factors, and activins, among others.
  • TGF-alpha can inhibit the differentiation of certain cells of mesodermal origin (Florini, J.R. et al., T Riol Chem ., 261:1659-16513 (1986) induced the differentiation of others (Seyedine, S.M. et al, PNAS TTSA 82:2267-2271 (1987) and potently inhibit proliferation of various types of epithelial cells, (Tucker, R.F., Sri ⁇ nne, 226:705-705 (1984)).
  • This last activity has led to the speculation that one important physiological role for TGF-" is to maintain the repressed growth state of many types of cells. Accordingly, cells that lose the ability to respond to TGF-" are more likely to exhibit uncontrolled growth and become tumorigenic.
  • polypeptide of the present invention is a member of this extended family of growth factors which has many effects on a variety of different tissues.
  • CTGF-2 polypeptide as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
  • the polypeptide of the present invention is of human origin.
  • nucleic acid molecules encoding such CTGF-2 polypeptide and polypeptide fragments, analogs, and derivatives of the present invention, including mRNAs, DNAs, cDNAs, genomic DNAs as well as analogs and biologically active and diagnostically or therapeutically useful fragments thereof.
  • a process for producing such polypeptide by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence encoding a polypeptide of the present invention, under conditions promoting expression of said protein and subsequent recovery of said protein.
  • a process for utilizing such polypeptide, or polynucleotide encoding such polypeptide for therapeutic purposes for example, enhancing the repair of connective and support tissue, promoting the attachment, fixation and stabilization of tissue implants and enhancing wound healing.
  • methods for stimulating angiogenesis i.e., formation of new blood vessels, using the polynucleotides and polypeptides of the invention are provided. Such methods include, but are not limited to, gene therapy of patients in need of new blood vessel formation.
  • polynucleotides and polypeptides of the invention are useful in the treatment of cardiovascular disease, including but not limited to atherosclerosis, restenosis, reperfusion injury
  • cardiovascular disease including but not limited to atherosclerosis, restenosis, reperfusion injury
  • cardiovascular disease including but not limited to atherosclerosis, restenosis, reperfusion injury
  • disorders include, but are not limited to, heart failure, angina, blood vessel (e.g. coronary artery) blockage and ischemia, inlcuding critical limb ischemia and refractory myocardial ischemia.
  • vectors are provided for use in administering the polynucleotides of the invention.
  • nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to a nucleic acid sequence of the present invention.
  • agonists which mimic the polypeptide of the present invention and binds to the receptors.
  • antagonists to such polypeptides which may be used to inhibit the action of such polypeptides, for example, in the treatment of CTGF dependent tumor growth.
  • diagnostic assays for detecting diseases or susceptibility to diseases related to mutations in the nucleic acid sequences encoding a polypeptide of the present invention.
  • Figures 1A-B Figures 1A-B depict the cDNA sequence and corresponding deduced amino acid sequence of CTGF-2. The standard one-letter abbreviation for amino acids is used.
  • FIG. 2 Migration of HDMEC across 5- ⁇ m pore size Transwell migration chamber incubated with supernatant of cells infected with Ad-Null, Ad-VEGF ⁇ 65 .or Ad-CTGF- 2.
  • Ad-CTGF-2 and Ad-NEGF 165 -infected cells supernatant significantly stimulated the migration of HDMEC compared with Ad- ⁇ ull.
  • FIG. 3 Calculated blood flow at rest (A) and after papaverine injection (B) in the ischemic limb.
  • Ad-VEGFi65 and Ad-CTGF-2-treated animals had a significant increase in flows when day 40 is compared with day 10 both at rest and after papaverine infusion.
  • rest flow as well as maximum flow were significantly improved in Ad-CTGF-2 animals compared with Ad- ⁇ ull group.
  • Ad-CTGF ⁇ 65 -treated rabbits there was no statistical difference between these two groups.
  • Figure 4 Serial assessement of internal iliac artery luminal diameter in the ischemic limb.
  • Figure 5 Representative angiograms of the ischemic limb recorded on day 10
  • Ad-Null Ad-Null
  • Ad-VEGF ⁇ 65 Ad-CTGF-2-treated animals showing collateral vessel formation through the 30 days follow-up period.
  • the morphology of the augmented collateral circulation was typically made of fine networks of so-called midzone collateral vessels.
  • serial angiograms disclosed progressive linear extension of the stem artery to the reentry (popliteal or saphenous arteries), predominently for Ad-CTGF-2-treated rabbits.
  • Figure 6 Quantitative angiographic analysis of collateral vessel development in the medial thigh of ischemic hindlimb evaluated by an angiographic score. The vascular density was significantly improved for each group at day 40 compared to day 10. Angiographic scores at day 40 were significantly higher in both treated groups compared to control animals and, in CTGF-2-treated animals it exceeded that of the VEGFi ⁇ s-treated group.
  • Figure 7 Capillary density evaluated on histological sections of adductor (A) and (B) semimembranous muscles harvested at the time of sacrifice (day 40).
  • the capillary-to-myocyte ratio was statistically increased in the Ad-CTGF-2 group compared with the Ad-Null and Ad-VEGFi ⁇ s-treated animals.
  • the ratio was increased for the Ad-VEGF ⁇ 65 compared to the Ad- ⁇ ull whereas the increase seen for Ad-CTGF-2 did not attain the level of significance.
  • Figure 8 shows the vector map of the transfer vector pTG13387.
  • This transfer vector contains the Ad5 1-458 region followed by the CMV enhancer/ promoter and a chimeric intron generated by combining the splice donor from the human ⁇ -globin intron 1 and the splice acceptor from the IgG intervening sequence obtained from pCI plasmid (Promega, Charbonnieres, France).
  • Figure 9 shows the vector map of the CTGF-2 transfer vector ptgl4488.
  • Figure 10 shows the vector map of the adenoviral vector ptgl4550.
  • Figure 11 depicts an alternative cDNA sequence and corresponding deduced amino acid sequence of CTGF-2.
  • the standard one-letter abbreviation for amino acids is used.
  • an isolated nucleic acid (polynucleotide) (SEQ ID NO:l) which encodes for the polypeptide having the deduced amino acid sequence of Figures 1A-B (SEQ ID NO: 2) or as encoded by the cDNA of the clone deposited as with the American Type Culture Collection (ATCC) on June 7, 1994 as Deposit No. 75804.
  • An alternative cDNA sequence (SEQ ID NO:6) and corresponding deduced amino acid sequence (SEQ ID NO:7) of CTGF-2 is demonstrated in Figures llA-C.
  • the Pasteur Institute Depository is located at the following address: Collection Nationale de Cultures de Microorganismes, INSTITUT PASTEUR, 25, rue du DOCTEUR ROUX, F-74724 Paris Cedex 15, France.
  • the polynucleotide of this invention was discovered in a cDNA library derived from human fetal lung. It is structurally related to the IGF and PDGF family. It contains an open reading frame encoding a protein of 382 amino acid residues of which approximately the first 23 or 24 amino acids residues are the putative leader sequence such that the putative mature protein comprises 358 amino acids (residues 24-382 or 25-382).
  • CTGF-2 contains an IGF Binding Domain that extends from about amino acid Ser-24 to about Ala-93; a von Willebrand Factor Type C Repeat Domain from about amino acid Arg-98 to about amino acid Asp- 164; a Sulfated Glycoconjugate Binding Motif from about amino acid Cys-229 to about amino acid Gly-273; and a C-Terminal Dimerization and Receptor Binding Domain from about amino acid Cys-286 to about amino acid His-361.
  • “about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
  • Cyr61 is a growth factor-inducible immediate early gene initially identified in serum-stimulated mouse fibroblasts. It encodes a member of an emerging family of cysteine-rich secreted proteins that includes a connective tissue growth factor (O'Brien and Lau, L.F., Cell Growth Differ., 3:645- 54 (1992)).
  • CTGF-2 is active in stimulating angiogenesis.
  • novel methods for stimulating angiogenesis i.e., formation of new blood vessels, using the polynucleotides and polypeptides of the invention are provided. Such methods include, but are not limited to, gene therapy of patients in need of new blood vessel formation.
  • the polynucleotides and polypeptides of the invention are useful in the treatment of cardiovascular disease, including but not limited to atherosclerosis, restenosis, reperfusion injury
  • cardiovascular disease including but not limited to atherosclerosis, restenosis, reperfusion injury
  • Such disorders include, but are not limited to, heart failure, angina, blood vessel (e.g. coronary artery) blockage and ischemia, including critical limb ischemia and refractory myocardial ischemia.
  • CTGF-2 polynucleotides and derivatives described herein below are useful, for example, in the production of CTGF-2 polypeptide and polypeptide derivatives (described below), as well as as agents in gene therapy to stimulate angiogenesis, as described in detail below.
  • the polynucleotides 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 Figures 1 A-B or that of the deposited clone in ATCC accession no. 75804, 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 Figures 1A-B or the deposited cDNA.
  • the polynucleotide which encodes for the mature polypeptide of Figures 1 A-B 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 additional coding sequence such as a leader or secretory sequence or a proprotein sequence; 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/or 3' of the coding sequence for the mature polypeptide.
  • polynucleotide encoding a polypeptide encompasses a polynucleotide which 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 Figures 1A-B 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.
  • the present invention includes, polynucleotides encoding the same mature polypeptide as shown in Figures 1 A-B or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figures 1A-B or the polypeptide encoded by the cDNA of the deposited clone.
  • Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
  • the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figures 1A-B or of the coding sequence of the deposited clone.
  • an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition 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)).
  • 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).
  • 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 similarity 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 promoter regions, exons, 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.
  • the present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 90%, and more preferably at least 95% identity between the sequences.
  • the present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides.
  • polypeptides which hybridize to the hereinabove 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 Figures 1 A-B or the deposited cDNA.
  • the polynucleotide 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.
  • such polynucleotides may be employed as probes for the polynucleotide of Figures 1 A-B, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.
  • the present invention is directed to polynucleotides having at least a
  • the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, the complement of the CTGF-2 coding polynucleotide sequence disclosed herein or the cDNA clone contained in ATCC Deposit No. 75804.
  • stringent hybridization conditions is intended overnight incubation at 42EC in a solution comprising: 50% formamide, 5x SSC (750 mM NaCl, 75mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65°C.
  • a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide.
  • nt nucleotides
  • nt nucleotides
  • a polynucleotide which hybridizes only to a poly A sequence would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).
  • polynucleotides of the invention are less than
  • polynucleotides of the invention comprise at least 15, at least 30, at least 50, at least 100, or at least 250, at least 500, or at least 1000 contiguous nucleotides of CTGF-2 coding sequence, but consist of less than or equal to 1000 kb, 500 kb, 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, or 5 kb of genomic DNA that flanks the 5' or 3' coding nucleotide set forth in Figures 1A-B.
  • polynucleotides of the invention comprise at least 15, at least 30, at least 50, at least 100, or at least 250, at least 500, or at least 1000 contiguous nucleotides of CTGF-2 coding sequence, but do not comprise all or a portion of any CTGF-2 intron.
  • the nucleic acid comprising CTGF-2 coding sequence does not contain coding sequences of a genomic flanking gene (i.e., 5' or 3 1 to the CTGF-2 gene in the genome).
  • the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
  • CTGF-2 polypeptide may include, but are not limited to the coding sequence for the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional sequences, such as those encoding a leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing - including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA; additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
  • the polypeptide may be fused to a marker sequence, such as a peptide, which facilitates purification of the fused polypeptide.
  • the marker sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • the "HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al, Cell 57:767-778 (1984). As discussed below, other such fusion proteins include the CTGF-2 receptor fused to Fc at the N- or C-terminus.
  • the present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs, or derivatives of the CTGF-2 receptor. Variants may occur naturally, such as a natural allelic variant. By an "allelic variant” is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
  • variants include those produced by nucleotide substitutions, deletions or additions which may involve one or more nucleotides.
  • the variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions, or additions. Especially prefe ⁇ ed among these are silent substitutions, additions, and deletions, which do not alter the properties and activities of CTGF-2 or portions thereof. Also especially preferred in this regard are conservative substitutions.
  • nucleic acid molecules comprising or, alternatively, consisting of a polynucleotide having a nucleotide sequence at least 80%, 85%, or 90% identical, and more preferably at least 95%, 96%, 97%, 98%, or 99% identical to: (a) a nucleotide sequence encoding the polypeptide having the amino acid sequence in Figures 1A-B; (b) a nucleotide sequence encoding the polypeptide having the amino acid sequence in Figures 1A-B, but lacking the amino terminal methionine; (c) a nucleotide sequence encoding the polypeptide having the amino acid sequence in Figures 1 A- B, but lacking the N-terminal leader peptide (i.e.
  • nucleotide sequence encoding a CTGF-2 polypeptide is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five mismatches per each 100 nucleotides of the reference nucleotide sequence encoding the CTGF-2 polypeptide.
  • nucleotide sequence at least 95% identical to a reference nucleotide sequence
  • up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • These mismatches of the reference sequence may occur at the 5' or 3' te ⁇ ninal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the reference (query) sequence may be the entire CTGF-2 encoding nucleotide sequence shown in Figures 1A-B or any CTGF-2 polynucleotide fragment (e.g., a polynucleotide encoding the amino acid sequence of any of the CTGF-2 N- and/or C- terminal deletions described herein), variant, derivative or analog, as described herein.
  • 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence shown in Figures 1A-B or to the nucleotide sequence of the deposited cDNA clone can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482- 489 (1981), to find the best segment of homology between two sequences.
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
  • the identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
  • the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. A determination of whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of this embodiment.
  • a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for the purposes of this embodiment.
  • the present application is directed to nucleic acid molecules at least 80%,
  • nucleic acid sequence for example, shown in Figures 1 A-B, or to the nucleic acid sequence of the deposited cDNA, irrespective of whether they encode a polypeptide having CTGF-2 activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having CTGF-2 functional activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer.
  • PCR polymerase chain reaction
  • nucleic acid molecules of the present invention that do not encode a polypeptide having CTGF-2 activity include, inter alia: (1) isolating the CTGF-2 gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the CTGF-2 gene, as described in Verma et al, Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); and (3) Northern Blot analysis for detecting CTGF-2 mRNA expression in specific tissues.
  • FISH in situ hybridization
  • a polypeptide having CTGF-2 functional receptor activity is intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the CTGF-2 of the invention (either the full-length protein or, preferably, the mature protein), as measured in a particular biological assay.
  • CTGF-2 functional receptor activity can be measured using the cell proliferation or angiogenesis assays performed essentially as previously described in the Examples, below.
  • nucleic acid molecules having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, for example, the nucleic acid sequence of the deposited cDNA or the nucleic acid sequence shown in Figures 1A-B will encode a polypeptide "having CTGF-2 functional activity.”
  • degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay.
  • the present invention further relates to a polypeptide which has the deduced amino acid sequence of Figures 1 A-B or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptide.
  • fragment when referring to the polypeptide of Figures 1A-B or that encoded by the deposited cDNA, means a polypeptide which retains essentially the same biological function or activity as such polypeptide.
  • an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
  • the fragment, derivative or analog of the polypeptide of Figures 1A-B 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-conserved 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 residues 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 polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence.
  • 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.
  • isolated means that the material is removed from its original environment (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 system, is isolated.
  • polypeptides of the present invention include the full-length polypeptide, the mature polypeptide, as well as polypeptides which have at least 70% similarity (preferably at least 70% identity) to the polypeptide of Figures 1 A-B and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of Figures 1A-B and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of Figures 1A-B 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.
  • 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.
  • protein engineering may be employed. Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or muteins including single or multiple amino acid substitutions, deletions, additions or fusion proteins.
  • modified polypeptides can show, e.g., enhanced activity or increased stability.
  • they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.
  • one or more amino acids may be deleted from the N-terminus or C-terminus without substantial loss of biological function.
  • Ron et al., J. Biol. Chem., 268:2984-2988 (1993) reported modified KGF proteins that had heparin binding activity even if 3, 8, or 27 amino-terminal amino acid residues were missing.
  • deletions of N-terminal amino acids up to the Ile(I) residue at position 376 in Figures 1A-B may retain some biological activity.
  • the amino acids constituting the IGF-Binding Domain, von Willebrand Factor Type C Repeat Domain, Sulfated Glycoconjugated Binding Motif, and C-Terminal Dimerization and Receptor Binding Domain of the CTGF-2 protein are described above. However, even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities may still be retained.
  • the ability of the shortened protein to induce and/or bind to antibodies which recognize the complete or extracellular domain of the protein generally will be retained when less than the majority of the residues, of the complete or extracellular domain of the protein are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete protein retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.
  • the present invention further provides polypeptides having one or more residues from the amino terminus of the amino acid sequence of the CTGF-2 shown in Figures 1A-B, up to the isoleucine residue at position 376 (Ile-376 residue from the amino terminus), and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides having the amino acid sequence of residues n -381 of Figures 1 A-B, where n 1 is an integer in the range of the amino acid position of amino acid residues 2-376 of the amino acid sequence in Figures 1A-B.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues S-2 to D-381; S-3 to D- 381; R-4 to D-381; 1-5 to D-381; A-6 to D-381; R-7 to D-381; A-8 to D-381; L-9 to D-381; A-10 to D-381; L-ll to D-381; V-12 to D-381; V-13 to D-381; T-14 to D-381; L-15 to D- 381; L-16 to D-381; H-17 to D-381; L-18 to D-381; T-19 to D-381; R-20 to D-381; L-21 to D-381; A-22 to D-381; L-23 to D-381; S-24 to D-381; T-25 to D-381; C-26 to D-381; P-27 to D-381; A-28 to D-381; A-29 to D-381; C-30 to D-381;
  • the present application is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the CTGF-2 polypeptides described above.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence.
  • Polypeptides encoded by these nucleic acids and/or polynucleotide sequences are also encompassed by the invention, as are polypeptides comprising an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above, and polynucleotides that encode such polypeptides.
  • polypeptides comprising an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above, and polynucleotides that encode such polypeptides.
  • many examples of biologically functional C-terminal deletion muteins are known. For instance, Interferon gamma shows up to ten times higher activities by deleting 8-10 amino acid residues from the carboxy terminus of the protein (Dobeli et al., J Biotechnology 7:199-216 (1988).
  • the present invention further provides polypeptides having one or more residues from the carboxy terminus of the amino acid sequence of the CTGF-2 shown in Figures 1A-B, up to the histidine residue at position 361 (His-361) and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides having the amino acid sequence of residues 1-m of the amino acid sequence in Figures 1A-B, where m 1 is any integer in the range of the amino acid position of amino acid residues 6 to 380 in Figures 1A-B.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues M-l to R-380; M-l to F-379; M-l to K-378; M-l to H-377; M-l to 1-376; M-l to D-375; M-l to N-374; M-l to F-373; M-l to L-372; M-l to R-371; M-l to Y-370; M-l to F-369; M-l to P-368; M-l to F-367; M-l to A-366; M-l to A-365; M-l to
  • M-l to R-321 M-l to G-320; M-l to D-319; M-l to V-318; M-l to C-317 M-l to S-
  • M-l to Y-309 M-l to K-308; M-l to K-307; M-l to V-306; M-l to S-305 M-l to L-
  • M-l to D-254 M-l to N-253; M-l to T-252; M-l to V-251; M-l to R-250; M-l to T-249; M-l to S-248; M-l to 1-247; M-l to G-246; M-l to T-245; M-l to G-244; M-l to
  • M-l to W-236 M-l to S-235; M-l to T-234; M-l to T-233; M-l to Q-232; M-l to V-
  • M-l to 1-230 M-l to C-229; M-l to K-228; M-l to Q-227; M-l to G-226; M-l to Q-
  • M-l to L-224 M-l to P-223; M-l to N-222; M-l to Y-221; M-l to L-220; M-l to 1-219;
  • the present application is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the CTGF-2 polypeptides described above.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence.
  • Polypeptides encoded by these nucleic acids and/or polynucleotide sequences are also encompassed by the invention, as are polypeptides comprising an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above, and polynucleotides that encode such polypeptides.
  • polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini which may be described generally as having residues n -m of Figures 1A-B, where n and m are integers as defined above. Also included are a nucleotide sequence encoding a polypeptide consisting of a portion of the complete CTGF-2 amino acid sequence encoded by the deposited cDNA clone contained in ATCC Accession No.
  • polypeptides of the present invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or all fourteen of the immunogenic epitopes of the CTGF-2 protein shown in Figures 1A-B as residues: Glu-35 to Pro-41, Arg-47 to Gly-51, Gln-59 to Gly-75, Cys-91 to His-119, Cys-145 to Leu-150, Asp-164 to Asp-176, Gly-202 to Lys-208, Pro-223 to Lys-228, Cys-239 to Gly-244, Arg-250 to Glu-257, Ser-279 to Val-296, Lys-307 to Cys-314, Val-318 to Cys-323, and Cys-337 to Phe-343.
  • polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of Figures 1A-B, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in deposited clone (ATCC accession no.
  • polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention such as, for example, the sequence disclosed in Figures 1A-B
  • polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.
  • epitopes refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human, hi a preferred embodiment, the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide.
  • An "immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci.
  • antigenic epitope is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the . art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.
  • Fragments that function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Patent No. 4,631,211).
  • antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, and, most preferably, between about 15 to about 30 amino acids.
  • Prefe ⁇ ed polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length.
  • Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope.
  • Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).
  • immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985).
  • the polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as, for example, rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier.
  • a carrier protein such as an albumin
  • immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
  • Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985).
  • animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.
  • KLH keyhole limpet hemacyanin
  • peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N- hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde.
  • Animals such as, for example, rabbits, rats, and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 micrograms of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response.
  • booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody that can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.
  • the titer of anti- peptide antibodies in serum from an immunized animal may be increased by selection of anti- peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
  • polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to other polypeptide sequences.
  • the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CHI, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric polypeptides.
  • immunoglobulins IgA, IgE, IgG, IgM
  • CHI constant domain of immunoglobulins
  • IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995).
  • Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin ("HA") tag or flag tag) to aid in detection and purification of the expressed polypeptide.
  • an epitope tag e.g., the hemagglutinin ("HA") tag or flag tag
  • HA hemagglutinin
  • a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972- 897).
  • the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues.
  • the tag serves as a matrix-binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
  • DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol.
  • alteration of polynucleotides corresponding to Figures 1A-B and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling.
  • DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence
  • polynucleotides of the invention, or the encoded polypeptides may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide coding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • the present invention further relates to antibodies and T-cell antigen receptors
  • TCR which immunospecifically bind a polypeptide, preferably an epitope, of the present invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding).
  • Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab?) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.
  • the immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single- chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain.
  • Antigen-binding antibody fragments, including single-chain antibodies may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CHI, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CHI, CH2, and CH3 domains.
  • the antibodies of the invention may be from any animal origin including birds and mammals.
  • the antibodies are human, murine, donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.
  • "human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.
  • the antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al, J. Immunol. 147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
  • Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention that they recognize or specifically bind.
  • the epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-te ⁇ ninal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures.
  • Antibodies that specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
  • Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention.
  • Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. Further included in the present invention are antibodies that bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein). Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention.
  • Prefe ⁇ ed binding affinities include those with a dissociation constant or Kd less than 5X10 "2 M, 10 "2 M, 5X10 “3 M, 10 “3 M, 5X10 “4 M, 10 “4 M, 5X10 “5 M, 10 “5 M, 5X10 “6 M, 10 “ 6 M, 5X10 "7 M, 10 “7 M, 5X10 “8 M, 10 “8 M, 5X10 “9 M, 10 “9 M, 5X10 “10 M, 10 “10 M, 10 “10 M, 5X10 “ ⁇ M, 10 " ⁇ M, 5X10 "12 M, 10 "12 M, 5X10 "13 M, 10 "13 M, 5X10 “14 M, 10 "1 M, 5X10 "15 M, and 10 "15 M.
  • the invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein, i preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
  • Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention.
  • the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully.
  • the invention features both receptor-specific antibodies and ligand-specif ⁇ c antibodies.
  • the invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation.
  • Receptor activation i.e., signaling
  • receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra).
  • antibodies are provided that inhibit ligand or receptor activity by at least 90%, at least 80%, at least 70%, at least 60% ⁇ , or at least 50% of the activity in absence of the antibody.
  • the invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand.
  • neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor.
  • antibodies which activate the receptor may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation.
  • the antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein.
  • the above antibody agonists can be made using methods known in the art.
  • Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods.
  • the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).
  • the antibodies of the present invention may be used either alone or in combination with other compositions.
  • the antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions.
  • antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No.
  • the antibodies of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response.
  • the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
  • the antibodies of the present invention may be generated by any suitable method known in the art.
  • Polyclonal antibodies to an antigen-of- interest can be produced by various procedures well known in the art.
  • a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen.
  • adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties).
  • the term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • mice can be immunized with a polypeptide of the invention or a cell expressing such peptide.
  • an immune response e.g., antibodies specific for the antigen are detected in the mouse serum
  • the mouse spleen is harvested and splenocytes isolated.
  • the splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC.
  • Hybridomas are selected and cloned by limited dilution.
  • hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention.
  • Ascites fluid which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
  • the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
  • Antibody fragments that recognize specific epitopes may be generated by known techniques.
  • Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • F(ab')2 fragments contain the variable region, the light chain constant region and the CHI domain of the heavy chain.
  • the antibodies of the present invention can also be generated using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage including fd and Ml 3 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene HI or gene VIII protein.
  • Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below.
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
  • Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al, BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entireties.
  • Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non- human species and framework regions from a human immunoglobulin molecule.
  • CDRs complementarity determining regions
  • framework residues in the human framework regions will be substituted with the co ⁇ esponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Patent No.
  • Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Patent No. 5,565,332).
  • Human antibodies are particularly desirable for therapeutic treatment of human patients.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.
  • Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
  • the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
  • the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
  • the mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination, hi particular, homozygous deletion of the JH region prevents endogenous antibody production.
  • the modified embryomc stem cells are expanded and micromjected into blastocysts to produce chimeric mice.
  • the chimeric mice are then bred to produce homozygous offspring that express human antibodies.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention.
  • Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
  • Completely human antibodies which recognize a selected epitope can be generated using a technique refe ⁇ ed to as "guided selection.”
  • a selected non- human monoclonal antibody e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope.
  • antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)).
  • antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that "mimic" the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand.
  • anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand.
  • anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.
  • the invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof.
  • the invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of Figures 1A-B.
  • polynucleotides are used in accordance with the invention both to produce antibodies for isolation, and as components for use in antibody-based gene therapy regimens. Thus, these polynucleotides can be used in according to the procedures for using CTGF-2 encoding polynucleotides in gene therapy as described in detail below. [0115] The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art.
  • a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
  • chemically synthesized oligonucleotides e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)
  • a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody.
  • a suitable source e.g., an antibody cDNA library, or a
  • nucleotide sequence and co ⁇ esponding amino acid sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.
  • the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability.
  • CDRs complementarity determining regions
  • one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra.
  • the framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al, J. Mol. Biol.
  • the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention.
  • one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds.
  • Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
  • the antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.
  • Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, e.g., a heavy or light chain of an antibody of the invention requires construction of an expression vector containing a polynucleotide that encodes the antibody.
  • a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art.
  • methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein.
  • the invention provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter.
  • Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S.
  • Patent No. 5,122,464 and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
  • the expression vector is transfe ⁇ ed to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention.
  • the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, operably linked to a heterologous promoter.
  • vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
  • host-expression vector systems may be utilized to express the antibody molecules of the invention.
  • Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ.
  • These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mamm
  • bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
  • mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., 1986, Gene 45:101; Cockett et al., 1990, Bio/Technology 8:2).
  • a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed.
  • vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the antibody coding sequence maybe ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Jnouye & hiouye, 1985, Nucleic Acids Res.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • AcNPV is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • a number of viral-based expression systems may be utilized.
  • the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts, (e.g., see Logan & Shenk, 1984, Proc.
  • Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:51-544).
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the co ⁇ ect modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
  • cell lines which stably express the antibody molecule may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded intp cell lines.
  • This method may advantageously be used to engineer cell lines which express the antibody molecule.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine- guanine phosphoribosyltransferase (Szybalski & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt- cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci.
  • the expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)).
  • vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)).
  • a marker in the vector system expressing antibody is amplifiable
  • increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).
  • the host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes both heavy and light chain polypeptides. i such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197).
  • the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
  • an antibody molecule of the invention may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • centrifugation e.g., centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • the present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20 or 50 amino acids of the polypeptide) of the present invention to generate fusion proteins.
  • the fusion does not necessarily need to be direct, but may occur through linker sequences.
  • the antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20 or 50 amino acids of the polypeptide) of the present invention.
  • antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors.
  • Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S.
  • Polypeptides of the invention may also be fused to albumin (including but not limited to recombinant human serum albumin (see, e.g., U.S. Patent No. 5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S. Patent No. 5,766,883, issued June 16, 1998, herein incorporated by reference in their entirety)), resulting in chimeric polypeptides.
  • albumin including but not limited to recombinant human serum albumin (see, e.g., U.S. Patent No. 5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S. Patent No. 5,766,883, issued June 16, 1998, herein incorporated by reference in their entirety)), resulting in chimeric polypeptides.
  • polypeptides (including antibodies) of the present invention are fused with the mature form of human serum albumin (i.e., amino acids 1-585 of human serum albumin as shown in Figures 1 and 2 of EP Patent 0 322 094) which is herein incorporated by reference in its entirety
  • polypeptides and/or antibodies of the present invention are fused with polypeptide fragments comprising, or alternatively consisting of, amino acid residues 1-z of human serum albumin, where z is an integer from 369 to 419, as described in U.S. Patent 5,766,883 herein incorporated by reference in its entirety.
  • Polypeptides and/or antibodies of the present invention may be fused to either the N- or C- terminal end of the heterologous protein (e.g., immunoglobulin Fc polypeptide or human serum albumin polypeptide).
  • heterologous protein e.g., immunoglobulin Fc polypeptide or human serum albumin polypeptide
  • human serum albumin-CTGF-2 fusion proteins may be used therapeutically in accordance with the invention to stimulate, for example, angiogenesis as indicated below with respect to adenoviral-vector delivered DNA encoding CTGF-2.
  • the present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions.
  • the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof.
  • the antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CHI domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof.
  • the polypeptides may also be fused or conjugated to the above antibody portions to form multimers.
  • Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions.
  • Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Patent Nos.
  • polypeptides of the present invention may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides of the present invention may be fused or conjugated to the above antibody portions to facilitate purification.
  • One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86 (1988).
  • polypeptides of the present invention fused or conjugated to an antibody having disulfide- linked dimeric structures may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone.
  • the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties.
  • EP A 232,262 Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired.
  • the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
  • human proteins such as hD -5
  • Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5.
  • the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitates their purification
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available.
  • a pQE vector QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311
  • hexa-histidine provides for convenient purification of the fusion protein.
  • peptide tags useful for purification include, but are not limited to, the HA tag, which co ⁇ esponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the flag tag.
  • the present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent.
  • the antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, biolummescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See, for example, U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase
  • suitable prosthetic group complexes include streptavidin biotin and avidin/biotin
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin
  • an example of a luminescent material includes luminol
  • examples of biolummescent materials include luciferase, luciferin, and aequorin
  • suitable radioactive material include I, I, In or Tc.
  • an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
  • Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
  • the conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a thrombotic agent or an anti- angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“E -l”), interleukin-2 (“IL-2”), interleukin-6 (“E -6”), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin
  • a protein such as tumor necrosis factor
  • Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.
  • An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.
  • the antibodies of the invention may be assayed for immunospecific binding by any method known in the art.
  • the immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
  • Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RDPA buffer (1% NP-40 or Triton X- 100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4°C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4°C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer.
  • RDPA buffer 1% NP-40 or Triton X- 100, 1% sodium deoxycholate, 0.1%
  • the ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis.
  • One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads).
  • immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Cu ⁇ ent Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
  • Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 1251) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen
  • ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen.
  • a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
  • a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
  • the antibody may be coated to the well, hi this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well.
  • ELISAs see, e.g., Ausubel et al, eds, 1994, Cu ⁇ ent Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.
  • the binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays.
  • a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., H or I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen.
  • labeled antigen e.g., H or I
  • the affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis.
  • Competition with a second antibody can also be determined using radioimmunoassays.
  • the antigen is incubated with antibody of interest is conjugated to a labeled compound (e.g., H or I) in the presence of increasing amounts of an unlabeled second antibody.
  • the present invention is further directed to antibody-based therapies which involve administering antibodies (e.g., CTGF-2 antagonists) of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the described disorders.
  • Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof as described herein).
  • the antibodies of the invention can be used to treat, inhibit or prevent diseases and disorders associated with abe ⁇ ant expression and/or activity of a polypeptide of the invention, including, but not limited to, inhibition of angiogenesis, as discussed in detail below.
  • the treatment and/or prevention of diseases and disorders associated with abe ⁇ ant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases and disorders.
  • Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
  • a summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below.
  • the antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
  • the antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is prefe ⁇ ed.
  • human antibodies, fragments derivatives, analogs, or nucleic acids are administered to a human patient for therapy or prophylaxis.
  • ⁇ include those with a dissociation constant or Kd less than 5 X 10 " M, 10 " M, 5 X 10 " M, 10 " 7 M, 5 X 10 "8 M, 10 “8 M, 5 X 10 "9 M, 10 "9 M, 5 X 10 "10 M, 10 “10 M, 5 X 10 "11 M, 10 " ⁇ M, 5 X 10 "12 M, 10 “12 M, 5 X 10 "13 M, 10 "13 M, 5 X 10 "14 M, 10 “14 M, 5 X 10 '15 M, and 10 "15 M.
  • CTGF-2 encoding DNA in the stimulation of angiogenesis by gene therapy, in accordance with the invention, is disclosed herein. (See, e.g., Example 12 below).
  • nucleic acids comprising sequences encoding CTGF-2 or functional derivatives thereof, are administered to stimulate angiogenesis.
  • Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid.
  • the nucleic acids produce their encoded protein that mediates a therapeutic effect.
  • the compound comprises nucleic acid sequences encoding CTGF-2 or polypeptide having CTGF-2 activity as described above, said nucleic acid sequences being part of expression vectors that express the CTGF-2 protein or derivative in a suitable host.
  • nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue- specific.
  • nucleic acid molecules are used in which the CTGF-2 coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody nucleic acids (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
  • the expressed polynucleotides include the polynucleotides of the invention described in detail, above.
  • Delivery of the nucleic acids int ⁇ a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid- carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
  • the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Patent No.
  • microparticle bombardment e.g., a gene gun; Biolistic, Dupont
  • coating lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432) (which can be used to target cell types specifically expressing the receptors), etc.
  • nucleic acid- ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
  • the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180 dated April 16, 1992 (Wu et al.); WO 92/22635 dated December 23, 1992 (Wilson et al.); WO92/20316 dated November 26, 1992 (Findeis et al.); WO93/14188 dated July 22, 1993 (Clarke et al.), WO 93/20221 dated October 14, 1993 (Young)).
  • the nucleic acid can be introduced mtracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932- 8935; Zijlstra et al., 1989, Nature 342:435-438).
  • viral vectors that contains nucleic acid sequences encoding a CTGF-2 polypeptide of the invention are used.
  • a retroviral vector can be used (see Miller et al., 1993, Meth. Enzymol. 217:581-599). These retroviral vectors have been to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA.
  • the nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient.
  • retroviral vectors More detail about retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302, which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
  • Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Cu ⁇ . Opin. in Genetics and Devel. 3:110-114.
  • Adenoviruses are other viral vectors that can be used in gene therapy as detailed in a prefe ⁇ ed embodiment in Example 12, below. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, 1993, Cu ⁇ ent Opinion in Genetics and Development 3:499-503 present a review of adenovirus-based gene therapy.
  • adenovirus vectors are used. [0165] hi a specific, prefe ⁇ ed embodiment, the CTGF-2 cDNA of ATCC deposit no.
  • Adeno-associated virus has also been proposed for use in gene therapy
  • Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection.
  • the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transfe ⁇ ed gene. Those cells are then delivered to a patient.
  • the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell.
  • introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc.
  • Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol.
  • the resulting recombinant cells can be delivered to a patient by various methods known in the art.
  • Recombinant blood cells e.g., hematopoietic stem or progenitor cells
  • the amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
  • Cells into which a nucleic acid can be introduced for purposes of gene therapy are prefereably endothelial cells, but encompass any desired, available cell type, and include but are not limited to epithelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
  • the cell used for gene therapy is autologous to the patient.
  • nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect.
  • stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication
  • the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.
  • 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 maybe, 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 transformants or amplifying the genes of the present invention.
  • the culture conditions such as temperature, pH and the like, 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.
  • 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 phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • 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 procedures.
  • the DNA sequence is inserted into an appropriate restriction endonuclease site(s) 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 is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
  • promoters there may be mentioned: LTR or SV40 promoter, the E coli. lac or t ⁇ , 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.
  • 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 E. 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.
  • bacterial cells such as F- coli, St ⁇ eptomyce ⁇ 3 Salmonella typhim ⁇ rinm
  • fungal cells such as yeast
  • insect cells such as Drosophila S2 and Spodoptera _f_
  • 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.
  • the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
  • 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, hi a prefe ⁇ ed aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • a vector such as a plasmid or viral vector
  • regulatory sequences including, for example, a promoter, operably linked to the sequence.
  • Bacterial pQE70, pQE60, pQE-9 (Qiagen), pBS, pDIO, phagescript, psiX174, pbluescript SK, pbsks, ⁇ NH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.
  • Promoter regions can be selected from any desired gene using CAT
  • bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL and trp.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • 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, DEAE-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.
  • the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
  • Mature proteins can be expressed in mammalian cells, yeast, bacteria, 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 DNA constructs of the present invention.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegaloviras early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of R coli and S r.previsi e TRPl gene, and a promoter derived from a highly- expressed gene to direct transcription of a downstream structural sequence.
  • 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.
  • PGK 3-phosphoglycerate kinase
  • the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.
  • 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 E.
  • 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).
  • plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).
  • commercial vectors include, for example, ⁇ KK223-3 (Pha ⁇ nacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA).
  • 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 well known to those skilled in the art.
  • Various mammalian cell culture systems can also be employed to express recombinant protein.
  • 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 example, the 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 ribosome 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 sites may be used to provide the required nontranscribed genetic elements.
  • the polypeptide can 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.
  • HPLC high performance liquid chromatography
  • 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.
  • This invention provides a method for identification of the receptor for the
  • CTGF-2 polypeptide The gene encoding the receptor can be identified by expression cloning. Briefly, polyadenylated RNA is prepared from a cell responsive to CTGF-2, 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 CTGF-2. Transfected cells which are grown on glass slides are exposed to labeled CTGF-2.
  • the CTGF-2 can be labeled by a variety of means including iodidation or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis.
  • labeled ligand can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE and exposed to x-ray film. The labeled complex containing the CTGF-2-receptor can be excised, resolved into peptide fragments, and subjected to protein microsequencing.
  • This invention also provides a method of screening compounds to identify those which bind to the CTGF-2 receptor and elicit a second messenger response (agonists) or do not elicit a second messenger response (antagonists).
  • a mammalian cell or membrane preparation expressing the CTGF-2 receptor would be incubated with a labeled compound.
  • the response of a known second messenger system following interaction of the compound and the CTGF-2 receptor is then measured.
  • Such second messenger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.
  • CTGF-2 stimulates angiogenesis.
  • CTGF-2 polynucleotides and polypeptides of the invention are useful in the treatment of disorders in which stimulation of new blood vessel development would ameliorate the disorder.
  • disorders include, but are not limited to, heart failure, angina, blood vessel (e.g. coronary artery) blockage and ischemia, inlcuding critical limb ischemia and refractory myocardial ischemia.
  • CTGF-2 encoding polynucleotides can be delivered to individuals to using the gene therapy embodiments described above.
  • CTGF-2 polynucleotides or polypeptides, or agonists CTGF-2 of the present invention may be employed in treatment for stimulating re-vascularization of ischemic tissues due to various disease conditions such as thrombosis, arteriosclerosis, and other cardiovascular conditions.
  • the polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed to stimulate angiogenesis and limb regeneration, as discussed above.
  • CTGF-2 polynucleotides or polypeptides, or agonists CTGF-2 can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues.
  • the regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage.
  • Tissues that could be regenerated using the present invention include organs
  • CTGF-2 polynucleotides or polypeptides, or agonists CTGF-2, may increase regeneration of tissues difficult to heal. For example, increased tendon/ligament regeneration would quicken recovery time after damage.
  • CTGF-2 polynucleotides or polypeptides, or agonists CTGF-2, of the present invention could also be used prophylactically in an effort to avoid damage.
  • Specific diseases that could be treated include of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects.
  • a further example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.
  • nerve and brain tissue could also be regenerated by CTGF-2 polynucleotides or polypeptides, or agonists CTGF-2, to proliferate and differentiate nerve cells.
  • Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic disorders (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stoke).
  • diseases associated with peripheral nerve injuries, peripheral neuropathy (e.g., resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome) could all be treated using the CTGF-2 polynucleotides or polypeptides, or agonists CTGF-2.
  • CTGF-2 stimulates angiogenesis.
  • CTGF-2 may be useful in the treatment disorders in which inhibition of angiogenesis would amelioriate the disorder.
  • antagonists include, but are not limited to, antibodies, peptides, and other molecules that bind to CTGF-2, as well as ribozymes, antisense molecules, and triple helix forming oligonucleotides that interfere with the transcription and translation of the CTGF-2 genes, and are described in further detail, below.
  • such antagonists may be used in accordance with the invention to treat cancers, such as solid tumors, including prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primary tumors and metastases; melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non- small cell lung cancer; colorectal cancer; advanced malignancies; blood born tumors (such as leukemias); benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; artheroscleric plaques; ocular angiogenic diseases, for example diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft
  • antagonists i.e., peptides, antibodies, antisense, ribozyme, triple helix forming molecules and other CTGF-2 inhibitory molecules
  • CTGF-2 activity are useful in inhibiting the angiogenesis of proliferative cells or tissues, either alone, as a protein fusion, or in combination with other polypeptides directly or indirectly, as described elsewhere herein.
  • anti-angiogenic effect may be achieved indirectly, for example, through the inhibition of hematopoietic, tumor-specific cells, such as tumor-associated macrophages (See Joseph IB, et al.
  • angiogenesis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as that characterizing solid tumor growth, these regulatory controls fail. Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases.
  • a number of serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis. See, e.g., reviews by Moses et al, Biotech. P:630-634 (1991); Folkman et al, N. Engl. J. Med., 555:1757-1763 (1995); Auerbach et al, J. Microvasc. Res. 2P.401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz, Am. J.
  • the present invention provides for treatment of diseases or disorders associated with neovascularization by administration of CTGF-2 antagonists (e.g., antibodies or peptides) of the present invention.
  • CTGF-2 antagonists e.g., antibodies or peptides
  • Malignant and metastatic conditions which can be treated with the antagonists of the invention include, but are not limited to, malignancies, solid tumors, and cancers described herein and otherwise known in the art (for a review of such disorders, see Fishman et al, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)).
  • the present invention provides a method of treating an angiogenesis-related disease and/or disorder, comprising administering to an individual in need thereof a therapeutically effective amount of a CTGF-2 antagonist of the invention.
  • CTGF-2 antagonists may be utilized in a variety of additional methods in order to therapeutically treat a cancer or tumor.
  • Cancers which may be treated with CTGF-2 antagonists include, but are not limited to solid tumors, including prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primary tumors and metastases; melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non- small cell lung cancer; colorectal cancer; advanced malignancies; and blood born tumors such as leukemias.
  • polynucleotides, polypeptides, antagonists and/or agonists may be delivered topically, in order to treat cancers such as skin cancer, head and neck tumors, breast tumors, and Kaposi's s
  • CTGF-2 antagonists may be utilized to treat superficial forms of bladder cancer by, for example, intravesical administration.
  • CTGF-2 antagonists may be delivered directly into the tumor, or near the tumor site, via injection or a catheter.
  • the appropriate mode of administration will vary according to the cancer to be treated. Other modes of delivery are discussed herein.
  • methods for treating hypertrophic scars and keloids comprising the step of administering a CTGF-2 antagonist of the invention to a hypertrophic scar or keloid.
  • antagonists are directly injected into a hypertrophic scar or keloid, in order to prevent the progression of these lesions.
  • This therapy is of particular value in the prophylactic treatment of conditions which are known to result in the development of hypertrophic scars and keloids (e.g., burns), and is preferably initiated after the proliferative phase has had time to progress (approximately 14 days after the initial injury), but before hypertrophic scar or keloid development.
  • the present invention also provides methods for treating neo vascular diseases of the eye, including for example, corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroplasia and macular degeneration.
  • neo vascular diseases of the eye including for example, corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroplasia and macular degeneration.
  • ocular disorders associated with neovascularization which can be treated with the polynucleotides and polypeptides of the present invention (including agonists and/or antagonists) include, but are not limited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of prematurity macular degeneration, corneal graft neovascularization, as well as other eye inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, e.g., reviews by Waltman et al, Am. J. Ophthal 55:704-710 (1978) and Gartner et al, Surv. Ophthal 22:291-312 (1978).
  • neovascular diseases of the eye such as corneal neovascularization (including corneal graft neovascularization)
  • corneal neovascularization including corneal graft neovascularization
  • a CTGF-2 antagonist to the cornea, such that the formation of blood vessels is inhibited.
  • the cornea is a tissue which normally lacks blood vessels.
  • capillaries may extend into the cornea from the pericorneal vascular plexus of the limbus.
  • the cornea becomes vascularized, it also becomes clouded, resulting in a decline in the patient's visual acuity. Visual loss may become complete if the cornea completely opacitates.
  • corneal neovascularization e.g., corneal infections (e.g., trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (e.g., graft rejection and Stevens-Johnson's syndrome), alkali burns, trauma, inflammation (of any cause), toxic and nutritional deficiency states, and as a complication of wearing contact lenses.
  • corneal infections e.g., trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis
  • immunological processes e.g., graft rejection and Stevens-Johnson's syndrome
  • alkali burns trauma, inflammation (of any cause)
  • toxic and nutritional deficiency states e.g., as a complication of wearing contact lenses.
  • prefe ⁇ ed embodiments of the invention may be prepared for topical administration in saline (combined with any of the preservatives and antimicrobial agents commonly used in ocular preparations), and administered in eyedrop form.
  • the solution or suspension may be prepared in its pure form and administered several times daily.
  • anti-angiogenic compositions prepared as described above, may also be administered directly to the cornea.
  • the anti-angiogenic composition is prepared with a muco-adhesive polymer which binds to cornea.
  • the anti-angiogenic factors or anti-angiogenic compositions may be utilized as an adjunct to conventional steroid therapy.
  • Topical therapy may also be useful prophylactically in corneal lesions which are known to have a high probability of inducing an angiogenic response (such as chemical burns), h these instances the treatment, likely in combination with steroids, may be instituted immediately to help prevent subsequent complications.
  • the compounds described above may be injected directly into the corneal stroma by an ophthalmologist under microscopic guidance.
  • the prefe ⁇ ed site of injection may vary with the morphology of the individual lesion, but the goal of the administration would be to place the composition at the advancing front of the vasculature (i.e., interspersed between the blood vessels and the normal cornea). In most cases this would involve perilimbic corneal injection to "protect" the cornea from the advancing blood vessels.
  • This method may also be utilized shortly after a corneal insult in order to prophylactically prevent corneal neovascularization.
  • neovascular glaucoma comprising the step of administering to a patient a therapeutically effective amount of a CTGF-2 antagonists to the eye, such that the formation of blood vessels is inhibited.
  • the compound may be administered topically to the eye in order to treat early forms of neovascular glaucoma.
  • the compound may be implanted by injection into the region of the anterior chamber angle.
  • the compound may also be placed in any location such that the compound is continuously released into the aqueous humor.
  • methods are provided for treating proliferative diabetic retinopathy, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eyes, such that the formation of blood vessels is inhibited.
  • proliferative diabetic retinopathy may be treated by injection into the aqueous humor or the vitreous, in order to increase the local concentration of the CTGF-2 antagonist in the retina.
  • this treatment should be initiated prior to the acquisition of severe disease requiring photocoagulation.
  • methods for treating retrolental fibroplasia comprising the step of administering to a patient a therapeutically effective amount of a CTGF-2 antagonist to the eye, such that the formation of blood vessels is inhibited.
  • the compound may be administered topically, via intravitreous injection and/or via intraocular implants.
  • disorders which can be treated with CTGF-2 antagonists include, but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osier- Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.
  • disorders and/or states, which can be treated with be treated with the the CTGF-2 antagonists include, but are not limited to, solid tumors, blood born tumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound healing, endometriosis, vascluogenesis, granulations, hypertrophic scars (keloids), nonunion fractures, scleroderma, trachoma
  • an amount of the compound sufficient to block embryo implantation is administered before or after intercourse and fertilization have occu ⁇ ed, thus providing an effective method of birth control, possibly a "morning after" method.
  • CTGF-2 antagonists may also be used in controlling menstruation or administered as either a peritoneal lavage fluid or for peritoneal implantation in the treatment of endometriosis.
  • CTGF-2 antagonists of the present invention may be incorporated into surgical sutures in order to prevent stitch granulomas.
  • CTGF-2 antagonists may be utilized in a wide variety of surgical procedures.
  • compositions in the form of, for example, a spray or film
  • a compositions may be utilized to coat or spray an area prior to removal of a tumor, in order to isolate normal su ⁇ ounding tissues from malignant tissue, and/or to prevent the spread of disease to su ⁇ ounding tissues.
  • compositions e.g., in the form of a spray
  • surgical meshes which have been coated with anti- angiogenic compositions of the present invention may be utilized in any procedure wherein a surgical mesh might be utilized.
  • a surgical mesh laden with an anti-angiogenic composition may be utilized during abdominal cancer resection surgery (e.g., subsequent to colon resection) in order to provide support to the structure, and to release an amount of the anti-angiogenic factor.
  • methods for treating tumor excision sites, comprising administering a CTGF-2 antagonists to the resection margins of a tumor subsequent to excision, such that the local recu ⁇ ence of cancer and the formation of new blood vessels at the site is inhibited.
  • the anti-angiogenic compound is administered directly to the tumor excision site (e.g., applied by swabbing, brushing or otherwise coating the resection margins of the tumor with the anti-angiogenic compound).
  • the anti-angiogenic compounds may be incorporated into known surgical pastes prior to administration.
  • the anti-angiogenic compounds are applied after hepatic resections for malignancy, and after neurosurgical operations.
  • CTGF-2 antagonists may be administered to the resection margin of a wide variety of tumors, including for example, breast, colon, brain and hepatic tumors.
  • anti-angiogenic compounds may be administered to the site of a neurological tumor subsequent to excision, such that the formation of new blood vessels at the site are inhibited.
  • CTGF-2 antagonists of the present invention may also be administered along with other anti-angiogenic factors.
  • anti-angiogenic factors include: Anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel, Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of MetalIoproteinase-2, Plasminogen Activator Inhibitor- 1, Plasminogen Activator Inhibitor-2, and various forms of the lighter "d group" transition metals.
  • Lighter "d group" transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.
  • Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate.
  • Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.
  • tungsten and molybdenum complexes also include oxo complexes.
  • Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes.
  • Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid.
  • Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide.
  • Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes.
  • Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates.
  • Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (NI) oxide, and molybdic acid.
  • Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate.
  • Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.
  • Representative examples include platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex (SP- PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix metabolism, including, for example, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)- oxazolone; Methotrexate; Mitoxantrone; Heparin; h terferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J.
  • the present invention is also directed to antagonists molecules of the polypeptides of the present invention, and their use in reducing or eliminating the function of
  • an antagonist is an antibody or in some cases, an oligonucleotide, which binds to the CTGF-2 polypeptide.
  • antagonists include closely related proteins that have lost biological function and thereby prevent the action of
  • CTGF-2 since receptor sites are occupied.
  • Antisense technology may be employed to decrease the level of in vivo circulation of CTGF-2.
  • 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.
  • 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 DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl.
  • RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into CTGF-2
  • oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of CTGF-2.
  • Another example of an antagonist is a small molecule which binds to the
  • CTGF-2 receptors such that normal biological activity is prevented.
  • small molecules include but are not limited to small peptides or peptide-like molecules.
  • the antagonists may be employed to prevent scar formation due to excess proliferation of connective tissues and to prevent CTGF-2 dependent tumor growth.
  • the antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinabove described.
  • compositions comprise a therapeutically effective amount of the polypeptide, agonist or antagonist, and a pharmaceutically acceptable carrier or excipient.
  • 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 compositions of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Associated with such container(s) 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, hi addition, the polypeptides, agonists and antagonists of the present invention may be employed in conjunction with other therapeutic compounds.
  • compositions may be administered in a convenient manner such as by the topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes.
  • CTGF-2 is administered in an amount which is effective for treating and/or prophylaxis of the specific indication.
  • CTGF-2 will be administered in an amount of at least about 10 Fg/kg body weight and in most cases CTGF-2 will be administered in an amount not in excess of about 8 mg/Kg body weight per day. hi most cases, the dosage is from about 10 Fg/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.
  • polypeptides may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often refe ⁇ ed to as "gene therapy.”
  • cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
  • a polynucleotide DNA or RNA
  • cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.
  • cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art.
  • a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo.
  • the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
  • 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 immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
  • 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 cytomegaloviras (CMV) promoter described in Miller, et al., Biotechniques, 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 HI, and a-actin promoters).
  • Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvoviras promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
  • Suitable promoters which may be employed include, but are not limited to, adeno viral 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 a-actin promoter; and human growth hormone promoters.
  • adeno viral promoters such as the adenoviral major late promoter
  • hetorologous promoters such as the cytomegalovirus (CM
  • the promoter also may be the native promoter which controls the gene encoding the polypeptide.
  • the retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ⁇ -2, ⁇ -AM, PA12, T19-14X, VT-19-17-H2, ⁇ CRE, ⁇ CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs. 5-14 (1990), which is incorporated herein by reference in its entirety.
  • the vector may transduce the packaging cells through any means known in the art.
  • 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 sequence(s) 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 sequence(s) encoding the polypeptide.
  • Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.
  • This invention is also related to the use of the gene of the present invention as a diagnostic. Detection of a mutated form of the gene will allow a diagnosis of a disease or a susceptibility to a disease which results from underexpression of CTGF-2.
  • Individuals carrying mutations in the gene of the present invention may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a patient's cells, including but not limited to blood, urine, saliva, tissue biopsy and 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.
  • PCR primers complementary to the nucleic acid encoding CTGF-2 can be used to identify and analyze 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 RNA or alternatively, radiolabeled antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures. [0249] Sequence differences between the reference gene and genes having mutations may be revealed by the direct DNA sequencing method, hi addition, cloned DNA segments may be employed as probes to detect specific DNA segments.
  • a sequencing primer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-tags.
  • 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 formamide 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, 230:1242 (1985)).
  • Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (e.g., Cotton et al, PNAS, USA, 85:4397-4401 (1985)).
  • 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.
  • restriction enzymes e.g., Restriction Fragment Length Polymorphisms (RFLP)
  • mutations can also be detected by in situ analysis.
  • the present invention also relates to a diagnostic assay for detecting altered levels of the polypeptide of the present invention in various tissues since an over-expression of the proteins compared to normal control tissue samples can detect the presence of disorders of the host.
  • Assays used to detect levels of the polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art and include radioimmunoassays, competitive-binding assays, Western Blot analysis and preferably an ELISA assay.
  • An ELISA assay initially comprises preparing an antibody specific to the CTGF-2 antigen, preferably a monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal antibody.
  • a detectable reagent such as radioactivity, fluorescence or in this example a horseradish peroxidase enzyme.
  • a sample is now removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin.
  • the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attached to the polypeptide of the present invention attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer.
  • the reporter antibody linked to horseradish peroxidase is now placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to the polypeptide of the present invention. 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 the polypeptide of the present invention present in a given volume of patient sample when compared against a standard curve.
  • a competition assay may be employed wherein antibodies specific to the polypeptide of the present invention are attached to a solid support and labeled CTGF-2 and a sample derived from the host are passed over the solid support and the amount of label detected attached to the solid support can be co ⁇ elated to a quantity of the polypeptide of the present invention in the sample.
  • 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.
  • Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location.
  • the mapping of DNAs to chromosomes according to the present invention is an important first step in co ⁇ elating those sequences with genes associated with disease.
  • sequences can be mapped to chromosomes by preparing PCR primers
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
  • sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner.
  • Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA 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.
  • FISH Fluorescence in situ hybridization
  • 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 polypeptides co ⁇ esponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). [0266] Techniques described for the production of single chain antibodies (U.S.
  • Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention.
  • transgenic mice may be used to express humanized antibodies to immunogenic polypeptide products of this invention.
  • 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 available plasmids in accord with published procedures, addition, equivalent plasmids 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 enzyme that acts only at certain sequences in the DNA.
  • the various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan.
  • plasmid or DNA fragment typically 1 ⁇ g of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
  • isolating DNA fragments for plasmid construction typically 5 to 50 ⁇ ig of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37EC are 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. [0271] Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D.
  • 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.
  • Ligase 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 to T4 DNA ligase ("ligase”) per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated.
  • ligase T4 DNA ligase
  • CGCGGGATCCTGCGCGACACAATGAGCT (SEQ ID NO: 3) and contains a BamHI restriction enzyme site (in bold) followed by 18 nucleotides resembling an efficient signal for the initiation of translation in eukaryotic cells (J. Mol. Biol. 1987, 196, 947-950, Kozak, M.).
  • the initiation codon for translation "ATG” is underlined.
  • the 3' primer has the sequence
  • CGCGGGTACCAGGTAGCATTTAGTCCCTAA (SEQ ID NO:8) and contains the cleavage site for the restriction endonuclease Asp781 and 20 nucleotides complementary to the 3' non-translated sequence of the CTGF-2 gene.
  • the amplified sequences were isolated from a 1% agarose gel using a commercially available kit ("Geneclean", BIO 101 Inc., La Jolla, Ca.). The fragment was then digested with the endonucleases BamHI and Asp781 and then purified by isolation on a 1% agarose gel. This fragment is designated F2.
  • the vector pRGl (modification of pVL941 vector, discussed below) is used for the expression of the CTGF-2 protein using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.E. 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555).
  • This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the recognition sites for the restriction endonucleases BamHI and Asp781.
  • the polyadenylation site of the simian virus (SV)40 is used for efficient polyadenylation.
  • the beta- galactosidase gene from E.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 cofransfected wild-type viral DNA.
  • Many other baculovirus vectors could be used in place of pRGl such as pAc373, pVL941 and pAc l (Luckow, V.A. and Summers, M.D., Virology, 170:31-39).
  • the plasmid was digested with the restriction enzymes BamHI and Asp781 and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The DNA was then isolated from a 1% agarose gel. This vector DNA is designated V2. [0280] Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNA ligase. E.coli HB101 cells were then transformed and bacteria identified that contained the plasmid ( ⁇ BacCTGF-2) with the CTGF-2 gene using the enzymes BamHI and Asp781. The sequence of the cloned fragment was confirmed by DNA sequencing.
  • the cells were infected with the recombinant baculovirus V-CTGF-2 at a multiplicity of infection (MOI) of 2.
  • MOI multiplicity of infection
  • the medium was removed and replaced with SF900 E medium minus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hours later 5 ⁇ Ci of 35 S-methionine and 5 ⁇ Ci 35 S 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.
  • CTGF-2 HA The expression of plasmid, CTGF-2 HA is derived from a vector pcDNAI/Amp (Invifrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site.
  • a DNA fragment encoding the entire CTGF-2 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 co ⁇ espond to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, H.
  • CTGF-2 ATCC accession no. 75804
  • 5' primer 5' AAAGGATCCACAATGAGCTCCCGAATC 3' contains a Bam HI site followed by 18 nucleotides of CTGF-2 coding sequence starting from the -3 position relative to the initiation codon; the 3' sequence 5'
  • CGCTCTAGATTAAGCGTAGTCTGGGACGTCGTATGGGTATTGGAACAGCCTGTAG AAG 5' contains complementary sequences to an Xba I site, translation stop codon, HA tag and the last 19 nucleotides of the CTGF-2 coding sequence (not including the stop codon). Therefore, the PCR product contains a Bam HI site, CTGF-2 coding sequence followed by an HA tag fused in frame, a translation termination stop codon next to the HA tag, and an Xba I site.
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp were digested with Bam HI and Xba I restriction enzymes and ligated.
  • the ligation mixture was transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North To ⁇ ey Pines Road, 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 co ⁇ ect fragment.
  • CTGF-2 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 CTGF-2 HA protein was detected by radiolabelling and immunoprecipitation method. (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
  • Fibroblasts are obtained from a subject by skin biopsy. The resulting 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, approximately 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 in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.
  • fresh media e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin
  • 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 Hindi ⁇ and subsequently treated with calf intestinal phosphatase.
  • the linear vector is fractionated on agarose gel and purified, using glass beads.
  • the 5' primer containing an EcoRI site and the 3' primer further includes a Hindi ⁇ site.
  • Equal quantities of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and Hindi ⁇ fragment are added together, in the presence of T4 DNA ligase.
  • the resulting mixture is maintained under conditions appropriate for ligation of the two fragments.
  • the ligation mixture is used to transform bacteria HB101, which are then plated onto agar-containing kanamycin for the purpose of confi ⁇ ning 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 (DMEM) with 10% calf serum (CS), penicillin and streptomycin.
  • DMEM Dulbecco's Modified Eagles Medium
  • CS calf serum
  • 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 refe ⁇ ed to as producer cells).
  • Fresh media is added to the transduced 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 millipore 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 media 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. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as n o or his.
  • the engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads.
  • the fibroblasts now produce the protein product.
  • a step in angiogenesis is cord formation, marked by differentiation of endothelial cells.
  • This bioassay measures the ability of microvascular endothelial cells to form capillary-like structures (hollow structures) when cultured in vitro.
  • CADMEC microvascular endothelial cells
  • CADMEC Growth Medium and used at passage 5.
  • the wells of a 48-well cell culture plate are coated with Cell Applications' Attachment Factor
  • CTGF-2 polynucleotides e.g., gene therapy
  • agonists e.g., agonists
  • antagonists of CTGF-2 e.g., anti-proliferatives
  • Example 5 Angiogenic Effect on Chic Chorioallantoic Membrane
  • Chick chorioallantoic membrane is a well-established system to examine angiogenesis. Blood vessel formation on CAM is easily visible and quantifiable. The ability of CTGF-2 to stimulate angiogenesis in CAM can be examined.
  • Fertilized eggs of the White Leghorn chick (Gallus gallus) and the Japanese quail (Coturnix coturnix) are incubated at 37.8°C and 80% humidity. Differentiated CAM of 16-day-old chick and 13-day-old quail embryos is studied with the following methods. [0301] On Day 4 of development, a window is made into the egg shell of chick eggs.
  • the embryos are checked for normal development and the eggs sealed with cellotape. They are further incubated until Day 13.
  • Thermanox coverslips (Nunc, Naperville, IL) are cut into disks of about 5 mm in diameter. Sterile and salt-free growth factors, and the protein to be tested, are dissolved in distilled water and about 3.3 mg/ 5 ml are pipetted on the disks. After air-drying, the inverted disks are applied on CAM. After 3 days, the specimens are fixed in 3% glutaraldehyde and 2% formaldehyde and rinsed in 0.12 M sodium cacodylate buffer. They are photographed with a stereo microscope [Wild M8] and embedded for semi- and ultrathin sectioning as described above. Controls are performed with carrier disks alone. [0302] The studies described in this example test the activity in CTGF-2 protein.
  • CTGF-2 polynucleotides e.g., gene therapy
  • agonists e.g., agonists
  • antagonists of CTGF-2 e.g., anti-proliferatives
  • Example 6 Angiogenesis Assay Using a Matrigel Tmplant in Mouse [0303] hi order to establish an in vivo model for angiogenesis to test CTGF-2 protein activities, mice and rats are implanted subcutaneously with methylcellulose disks containing either 20 mg of BSA (negative control), 1 mg of CTGF-2 or 0.5 mg of VEGF-1 (positive control). The negative control disks should contain little vascularization, while the positive control disks should show signs of vessel formation.
  • CTGF-2 polynucleotides e.g., gene therapy
  • agonists e.g., agonists
  • antagonists of CTGF-2 e.g., anti-proliferatives
  • a rabbit hindlimb ischemia model is created by surgical removal of one femoral arteries as described previously (Takeshita, S. et al., Am J. Pathol 147:1649-1660 (1995)).
  • the excision of the femoral artery results in retrograde propagation of thrombus and occlusion of the external iliac artery. Consequently, blood flow to the ischemic limb is dependent upon collateral vessels originating from the internal iliac artery (Takeshita, S. et al., Am J. Pathol 147:1649-1660 (1995)).
  • CTGF-2 When CTGF-2 is used in the treatment, a single bolus of 500 mg CTGF-2 protein or control is delivered into the internal iliac artery of the ischemic limb over a period of 1 min. through an infusion catheter.
  • various parameters are measured in these rabbits: (a) BP ratio - The blood pressure ratio of systolic pressure of the ischemic limb to that of normal limb; (b) Blood Flow and Flow Reserve - Resting FL: the blood flow during undilated condition and Max FL: the blood flow during fully dilated condition (also an indirect measure of the blood vessel amount) and Flow Reserve is reflected by the ratio of max FL: resting FL; (c) Angiographic Score - This is measured by the angiogram of collateral vessels.
  • a score is determined by the percentage of circles in an overlaying grid that with crossing opacified arteries divided by the total number m the rabbit thigh; (d) Capillary density - The number of collateral capillaries determined in light microscopic sections taken from hindlimbs.
  • CTGF-2 polynucleotides e.g., gene therapy
  • agonists e.g., agonists
  • antagonists of CTGF-2 e.g., anti-proliferatives
  • the evaluation parameters include skin blood flow, skin temperature, and factor Vi ⁇ immunohistochemistry or endothelial alkaline phosphatase reaction.
  • CTGF-2 expression, during the skin ischemia, is studied using in situ hybridization.
  • the experimental protocol includes:
  • CTGF-2 polynucleotides e.g., gene therapy
  • agonists e.g., agonists
  • antagonists of CTGF-2 e.g., anti-proliferatives
  • Angiogenic therapy using CTGF-2 is a novel therapeutic strategy to obtain restoration of blood flow around the ischemia in case of peripheral arterial diseases.
  • the experimental protocol includes:
  • CTGF-2 protein in a dosage range of 20 mg - 500 mg, is delivered intravenously and/or intramuscularly 3 times (perhaps more) per week for 2-3 weeks.
  • the ischemic muscle tissue is collected after ligation of the femoral artery at 1,
  • CTGF-2 polynucleotides e.g., gene therapy
  • agonists e.g., agonists
  • antagonists of CTGF-2 e.g., anti-proliferatives
  • CTGF-2 is evaluated as a potent mitogen capable of stimulating the development of collateral vessels, and restructuring new vessels after coronary artery occlusion. Alteration of CTGF-2 expression is investigated in situ.
  • the experimental protocol includes:
  • the heart is exposed through a left-side thoracotomy in the rat. Immediately, the left coronary artery is occluded with a thin suture (6-0) and the thorax is closed.
  • CTGF-2 protein in a dosage range of 20 mg - 500 mg, is delivered intravenously and/or intramuscularly 3 times (perhaps more) per week for 2-4 weeks.
  • CTGF-2 polynucleotides e.g., gene therapy
  • agonists e.g., agonists
  • antagonists of CTGF-2 e.g., anti-proliferatives
  • This animal model shows the effect of CTGF-2 on neovascularization.
  • the experimental protocol includes:
  • CTGF-2 treatment can also be applied topically to the corneal wounds in a dosage range of 20mg - 500mg (daily treatment for five days).
  • CTGF-2 polynucleotides e.g., gene therapy
  • agonists e.g., agonists
  • antagonists of CTGF-2 e.g., anti-proliferatives
  • Connective tissue growth factor 2 is a secreted, cystein-rich heparin- binding protein that is associated with extracellular matrix and cell surface.
  • CTGF-2 Connective tissue growth factor 2
  • VEGFi ⁇ s vascular endothelial growth factor
  • CTGF-2 gene transfer demonstrates a stronger stimulus for limb revascularization compared to VEGFi ⁇ s , thereby promoting a greater improvement in tissue perfiision in the ischemic limb.
  • vascular endothelial growth factor (NEGF) " vascular endothelial growth factor
  • FGF fibroblast growth factor
  • Connective tissue growth factor 2 (also named cyr ⁇ l) is a member of the CC ⁇ family which comprises six distinct proteins (Fispl2/CTGF, Cyr ⁇ l, ⁇ ov, Elm-1, Cop-l/Wisp2, and Wisp3) involved in cell growth and differentiation .
  • CTGF-2 is encoded by a growth factor-inducible immediate-early gene that is transcriptionally activated by serum and OWth factors (bFGF, PDGF and TGF ⁇ ) 12"14 .
  • CTGF-2 is a 42-kDa secreted, cysteine-rich heparin- binding protein that is associated with extracellular matrix and cell surface 11 ' 5 ' 6 .
  • Recombinant CTGF-2 protein mediates adhesion of vascular endothelial cells, fibroblasts and lung epithelial cells " , stimulates migration of fibroblasts and vascular endothelial cells and synergizes
  • the purpose of the present study was to examine the angiogenic effects of adenoviral-mediated gene transfer of human CTGF-2 in the rabbit ischemic hindlimb
  • CTGF-2 appeared more prone to stimulate limb revascularization in comparison with VEGF 165 , indicating that CTGF-2 represents a promising candidate for therapeutic gene-based angiogenesis for promote, for example, perfiision in the ischemic limb in severe peripheral ischemic diseases.
  • Adenoviral vectors [0342] The recombinant El/E3-deleted adenoviral vectors containing the human
  • CTGF-2 cDNA (cDNA clone ID HLFBE49; Ad-CTGF-2) or the human cDNA sequence encoding the VEGF 165 protein (RT-PCR from HUVEC; Ad-VEGF ⁇ 65 ) under the control of the
  • Cytomegalovirus promoter were obtained by homologous recombination . Virus propagation, purification and tifration of infectious units (i.u.) by indirect immunofluorescence of the viral
  • DNA binding were carried out as described previously .
  • the HLFBE49 cDNA clone contained a BamHI restriction enzyme site at the 5' and 3' sequences.
  • the Bam/Bam fragment was isolated from a 1% agarose gel using a commercially avalaible kit ("Qiaquick", Qiagen, Courtaboeuf, France) and cloned in the Bam HI site of a pBluescript phagemid vector (Stratagene, La Jolla, CA). Ligation mixture was transformed into DH5 ⁇ strain. Plasmid DNA was isolated from transformants and examined by restriction analysis for the presence of the co ⁇ ect fragment.
  • the candidate clone was sequenced, and then, digested with Eco Rl/Xba I restriction enzymes for the ligation with the transfer vector (named pTG13387).
  • This transfer vector contains the Ad5 1-458 region followed by the CMV enhancer/ promoter and a a chimeric intron generated combining the splice donor from the human ⁇ -globin intron 1 and the splice acceptor from the IgG intervening sequence obtained from pCI plasmid (Promega, Charbonnieres, France).
  • the recognition sites for the restriction endonucleases Xbal and EcoRI was inserted upstream of the bovine growth hormone polyadenylation site followed by the Ad5 3511-5788 and 3512-5788 regions.
  • This vector contains the ampicillin resistance gene.
  • AdTG14550 was obtained by homologous recombination in 293 cells , between the CTGF-2 transfer vector (named pTG14488) and the genomic Clal DNA fragment isolated from the H5dl324 virus.
  • Virus propagation, purification and tifration of infectious units (iu) by indirect immunofluorescence of the viral DNA binding protein were carried out as described previously 24 .
  • HDMEC Human dermal microvascular endothelial cells
  • PromoCell (Heidelberg, Germany). Cells were grown and maintained in Endothelial Cell Growth Medium (ECGM) supplemented with 5% Fetal Calf Serum (FCS), lOng ml Epidermal Growth Factor and 0.4% Endothelial Cell Growth Supplement. Cells were infected with recombinant adenovirus at a multiplicity of infection of 50 for 12 hours and cell supernatants were then collected. For the migration assay, 1x10 cells were added to the upper side of 5 ⁇ m pore size Transwell migration chambers (Costar) in ECGM containing 5% FCS. Adenoviral- infected HDMEC supernatants were added in the lower compartment and cell migration was allowed to proceed at 37°C for 3.5 hours. Cells that migrated to the underside were stained with 0.5% crystal violet in 70% methanol followed by a brief rinse in PBS and counted on nine randomly selected microscopic fields (magnification x 20) for each condition.
  • ECGM Endothelial
  • Angiography and Doppler-derived blood flow at rest and after papaverine infusion were performed before adenoviral injection (day 10) and 30 days after injection (day 40) as described previously .
  • a 4-French infusion catheter introduced through the common carotid artery was positioned to the aortoiliac bifurcation.
  • nitroglycerin 0.25mg
  • a bolus of contrast media was injected and serial images of the aortoiliac bifurcation were recorded to obtain angiograms.
  • a 0.014-in Doppler guide wire (Cardiometrics, Mountain view, CA) was positioned in the proximal segment of either the internal iliac artery supplying the ischemic limb or the common iliac artery supplying the non-ischemic limb.
  • Average peak velocities (APV) were recorded at rest and after bolus injection of 2mg of papaverine (maximum APV).
  • Angiographic luminal diameter of the internal iliac artery in the ischemic limb and of the common iliac artery in the normal limb were determined with an automated edge-detection system. The luminal diameter was measured at the site of the Doppler wire position.
  • a morphometric angiographic analysis of collateral vessel development in the ischemic limb was performed using the angiograms recorded at day 10 and day 40.
  • a grid overlay composed of 5-mm squares was placed over the angiogram at the level of the medial thigh area, positioned always at the same place considering anatomic references.
  • the number of contrast-opacified arteries crossing over rows in two sides were counted in a single blind fashion. This angiographic score reflects vascular density in the medial thigh.
  • tissue samples were harvested from the adductor and semimembranous muscles of the ischemic limb. Muscles were frozen in iced-isopentane and stored at -80°C. Transverse tissue sections (7 ⁇ m) were stained for alkaline phosphatase with an
  • Ad-CTGF-2 and Ad-VEGF ⁇ 65 -infected cell supernatants enhanced significantly the migration of HDMEC in comparison with Ad-Null (P ⁇ 0.05), validating adenoviral-mediated gene expression and angiogenic activities of the gene product.
  • Ad-VEGF ⁇ 65 or by Ad-CTGF-2 was similar.
  • Ad-VEGF 165 13.1+1.4 versus 16.4+2.5 ml/min, NS
  • Ad-CTGF-2 18.6+2.8 versus 19.6+4.5 ml/min, NS
  • Ad-Null 10.8+1.7 versus 17.9+2.3 ml/min, P ⁇ 0.01
  • maximum blood flow was also restored in Ad-CTGF-2 group (34.2 ⁇ 5.5 versus 50.8+10.7ml/min, NS) whereas it was not in Ad-VEGF 16 5 or in Ad-Null groups (P ⁇ 0.05).
  • Angiographic luminal diameters of the common iliac artery showed no significant modification in diameter at day 10 and day 40 between the three groups (data not shown).
  • FIG.6 Representative angiograms of rabbit ischemic hindlimb showed that collateral vessel development during the 30 days of the follow-up period was more marked in the Ad- VEGF 165 and the Ad-CTGF-2-treated groups, over control groups (Fig.5).
  • the quantitative analysis of collateral blood vessel development in rabbit hindlimb is summarized in Fig.6.
  • Angiographic scores did not differ significantly among groups before treatment at day 10. An increase in vascular density was measured in each group from day 10 to day 40 (Ad-Null: +26.3%; P ⁇ 0.01; Ad-VEGF ⁇ 65 : +76.4%; P ⁇ 0.0001 and Ad-CTGF-2: +100.7%; P ⁇ 0.001).
  • VEGF ⁇ 65 The choice of VEGF ⁇ 65 as a reference has been suggested by the extensive literature demonstrating its potent angiogenic effect. Nevertheless, adenoviral-mediated gene transfer of VEGF165 has never been used in the rabbit ischemic hindlimb model.
  • CTGF-2 appears to be more efficient than VEGF ⁇ 65 to enhance the collateral growth as suggested by the greater angiographic score. This could explain that in the CTGF-2 group, the maximal blood flow was restored in the ischemic limb compared with the non ischemic limb, but not in VEGFies-treated animals.
  • CTGF-2 may stimulate the revascularization by acting as an angiogenic inducer upon endothelial cells as VEGF does, and additionally by acting as a chemotactic, mitogenic and
  • CTGF-2 enzymes known to play a role in angiogenesis.
  • Involvement of CTGF-2 in angiogenesis can also be attributed to its interaction with different integrins: ⁇ v ⁇ 3-dependent pathway for adhesion and migration of endothelial cells ' , d ⁇ i and ⁇ v ⁇ 3 for adhesion and migration of fibroblasts, respectively 30.
  • CTGF-2 is not mitogenic by itself, it may synergize with growth factors like bFGF to enhance mitogenesis of endothelial cells or fibroblasts ' ' .
  • CTGF2 angiogenic properties appears closely related to bFGF, with respect to induction of MMP-1 and MMP-3, to its interaction with integrin ⁇ v ⁇ 3 ,
  • CTGF-2 gene has been presented as the main angiogenic factor implicated in arteriogenesis .
  • the interplay between the actions of bFGF and CTGF-2 is likely to be complex and remains to be more extensively investigated.
  • VEGF-C/VEGF-2 promotes angiogenesis in the setting of tissue ischemia.
  • Quyyumi AA Basic fibroblast growth factor in patients with intermittent claudication: results of a phase I trial. J Am Coll Cardiol 2000;36:1239-44.
  • Cyr ⁇ l and Fispl2 are both ECM-associated signaling molecules: activities, metabolism, and localization during development. Exp Cell Res. 1997;233:63-77.
  • CTGF-2 polynucleotides and polypeptides of the present invention were disclosed in U.S. provisional application numbers 60/217,402, filed July 11, 2000, and 60/291,642, filed May 18, 2001, the specifications and sequence listings of which are herein incorporated by reference in their entireties.
  • the applicant hereby requests that the application has been laid open to public inspection (by the Norwegian Patent Office), or has been finally decided upon by the Norwegian Patent Office without having been laid open inspection, the furnishing of a sample shall only be effected to an expert in the art.
  • the request to this effect shall be filed by the applicant with the Norwegian Patent Office not later than at the time when the application is made available to the public under Sections 22 and 33(3) of the Norwegian Patents Act. If such a request has been filed by the applicant, any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on the list of recognized experts drawn up by the Norwegian Patent Office or any person approved by the applicant in the individual case.
  • the applicant hereby requests that, until the application has been laid open to public inspection (by the Danish Patent Office), or has been finally decided upon by the Danish Patent office without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert in the art.
  • the request to this effect shall be filed by the applicant with the Danish Patent Office not later that at the time. when the application is made available to the public under Sections 22 and 33(3) of the Danish Patents Act. If such a request has been filed by the applicant, any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on a list of recognized experts drawn up by the Danish Patent Office or any person by the applicant in , the individual case.
  • the applicant hereby requests that, until the application has been laid open to public inspection (by the Swedish Patent Office), or has been finally decided upon by the Swedish Patent Office without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert in the art.
  • the request to this effect shall be filed by the applicant with the International Bureau before the expiration of 16 months from the priority date (preferably on the Form PCT/RO/134 reproduced in annex Z of Volume I of the PCT Applicant's Guide). If such a request has been filed by the applicant any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on a list of recognized experts drawn up by the Swedish Patent Office or any person approved by a applicant in the individual case.
  • the applicant hereby requests that the application has been laid open to public inspection (by the Norwegian Patent Office), or has been finally decided upon by the Norwegian Patent Office without having been laid open inspection, the furnishing of a sample shall only be effected to an expert in the art.
  • the request to this effect shall be filed by the applicant with the Norwegian Patent Office not later than at the time when the application is made available to the public under Sections 22 and 33(3) of the Norwegian Patents Act. If such a request has been filed by the applicant, any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on the list of recognized experts drawn up by the Norwegian Patent Office or any person approved by the applicant in the individual case.
  • the applicant hereby requests that, until the application has been laid open to public inspection (by the Danish Patent Office), or has been finally decided upon by the Danish Patent office without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert in the art.
  • the request to this effect shall be filed by the applicant with the Danish Patent Office not later that at the time when the application is made available to the public under Sections 22 and 33(3) of the Danish Patents Act. If such a request has been filed by the applicant, any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on a list of recognized experts drawn up by the Danish Patent Office or any person by the applicant in the individual case.
  • the applicant hereby requests that, until the application has been laid open to public inspection (by the Swedish Patent Office), or has been finally decided upon by the Swedish Patent Office without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert in the art.
  • the request to this effect shall be filed by the applicant with the International Bureau before the expiration of 16 months from the priority date (preferably on the Form PCT/RO/134 reproduced in annex Z of Volume I of the PCT Applicant's Guide). If such a request has been filed by the applicant any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on a list of recognized experts drawn up by the Swedish Patent Office or any person approved by a applicant in the individual case.
  • the applicant hereby requests that until the date of a grant of a Netherlands patent or until the date on which the application is refused or withdrawn or lapsed, the microorganism shall be made available as provided in the 31F(1) of the Patent Rules only by the issue of a sample to an expert.
  • the request to this effect must be furnished by the applicant with the Netherlands Industrial Property Office before the date on which the application is made available to the public under Section 22C or Section 25 of the Patents Act of the Kingdom of the Netherlands, whichever of the two dates occurs earlier.

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Abstract

La présente invention concerne un polypeptide CTGH-2 humain et l'ADN (ARN) codant ce polypeptide, une méthode de production d'un tel polypeptide au moyen de techniques de recombinaison, ainsi que des anticorps et des antagonistes/des inhibiteurs de ce polypeptide. Cette invention a également trait à des méthodes d'utilisation du polypeptide à des fins thérapeutiques pour stimuler l'angiogenèse, améliorer la réparation de tissus conjonctifs et de soutien, promouvoir l'attache, la fixation et la stabilisation des implants, et améliorer la guérison des blessures. Ladite invention concerne aussi des dosages diagnostiques permettant d'identifier des mutations dans une séquence d'acide nucléique codant un polypeptide susmentionné et de détecter des niveaux modifiés dudit polypeptide.
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