MXPA00001234A - Heparin-binding growth factor (hbgf) polypeptides - Google Patents

Abstract

Substantially pure heparin-binding growth factor polypeptides (HBGFs), nucleic acids encoding the HBGFs and antibodies which bind to the HBGFs of the invention are provided. The HBGF polypeptides are useful in methods for the induction of bone, cartilage and tissue formation, growth and development of the endometrium and in the acceleration of wound healing.

Description

POLYPEPTIDES OF THE GROWTH FACTOR LINKED TO HEPARINE (HBGF) 5 1. Field of the Invention This invention relates generally to the field of growth factors, and more specifically to growth factors that bind with heparin (HBGF). 2. Background of the Invention Growth factors are a class of polypeptides that stimulate target cells to proliferate, differentiate, and organize in developing tissues. The action of growth factors depends on their binding to specific receptors, which stimulates a signaling event inside the cell. Examples of growth factors include platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I, IGF-II), factor g ^ of transforming growth beta (TGF-β), factor increase Transformer alpha (TGF-a), epidermal growth factor (EGF), growth factors of acidic and basic fibroblasts (aFGF, bFGF), and connective tissue growth factor (CTGF), which are known to stimulate cells to proliferate. PDGF is a heat stable cationic protein that is found in the alpha granules of circulating platelets, and a mitogen and a chemotactic agent are known for connective tissue cells, such as fibroblasts, and smooth muscle cells. Due to the activities of this molecule, it is believed that PDGF is a major factor involved in • the normal healing of wounds, and that contributes pathologically to 5 conditions such as atherosclerosis and fibrotic conditions. PDGF is a dimeric molecule consisting of combinations of α and / or β chains. The chains form heterodimers and homodimers, and all combinations isolated to date are biologically active. Látk Studies on the role of different growth factors in tissue regeneration and repair have led to the discovery of proteins of the PDGF type. These proteins share both immunological and biological activities with PDGF, and can be blocked with antibodies specific for PDGF. Polypeptide growth factors and cytokines are emerging as an important class of uterine proteins that can form growth signaling pathways • between the maternal uterus and the developing embryo or fetus. The studies in a variety of species have suggested that EGF, the EGF-type growth factor that binds to heparin (HB-EGF), IGF-I, IGF-II, aFGF, bFGF, pleitrofine (PTN), factor leukemia inhibitor, colony-stimulating factor-1 (CSF-1), and TGF-oi, may be among the regulatory molecules of the uterine growth involved in these processes.

CTGF is a monomeric peptide rich in cysteine of Mr 38,000, which is a growth factor that has mitogenic and chemotactic activities for connective tissue cells. CTGF is secreted by cells, and is active after interaction with a specific cell surface receptor. CTGF is the product of a gene unrelated to the α- or β-chain genes of PDGF. It is a member of a family of growth regulators that includes mouse CTGF (also known as jfisp-12 or ßIG-M2) and human, Cyr61 (mouse), CeflO (chicken), and Nov (chicken). Based on the comparisons of the sequences, it has been suggested that the members of this family all have a molecular structure consisting of: (1) an insulin-like growth factor domain responsible for the link, (2) a factor domain von Willebrand responsible for complex formation, (3) a repeat type of thrombospondin I, possibly responsible for the binding of matrix molecules, and (4) a C-terminal module found in matrix proteins , which is postulated to be responsible for the receiver's link. The cDNA sequence for human CTGF (hCTGF) contains an open reading frame of 1047 nucleotides, with a start site at position 130, and a TGA termination site at position 1177, and encodes a peptide of 349 amino acids. There is only a sequence homology of 40 percent between the CTGF cDNA and the cDNA for any of the or chains. or ß of the PDGF. The open reading frame of hCTGF encodes a polypeptide containing 39 cysteine residues, indicating a protein with multiple intramolecular disulfide bonds. The amino terminus of the peptide contains a hydrophobic signal sequence that indicates a secreted protein, and there are two N-linked glycosylation sites in the asparagine 28 and 225 residues, in the amino acid sequence. There is a 45 percent overall sequence homology between the CTGF polypeptide and the polypeptide encoded by the transcription of the CEF-10 mRNA; the homology reaches 52 percent when a supposed alternative splice region is suppressed. CTGF is antigenically related to PDGF, although there is little, if any, sequence homology of the peptide. The anti-PDGF antibody has a high affinity with the non-reduced forms of PDGF or CTGF, and a 10-fold lower affinity with the reduced forms of these peptides, which lack biological activity. This suggests that there are regions of shared tertiary structure between the PDGF isomers and the CTGF molecule, resulting in common antigenic epitopes. The synthesis and secretion of CTGF are selectively induced by TGF-β, BMP-2, and possibly other members of the TGF-β protein superfamily. Although TGF-β can stimulate the growth of normal fibroblasts in soft agar, CTGF alone can not induce this property in fibroblasts. However, it has been shown that the synthesis and action of CTGF are essential for TGF-β to stimulate fibroblast growth independent of anchoring. CTGF is likely to function as a growth factor in wound healing. Pathologically, it has been postulated that CTGF is involved in conditions where there is an overgrowth of connective tissue cells, such as systemic sclerosis, cancer, fibrotic conditions, and atherosclerosis. The primary biological activity of the CTGF polypeptide is its mitogenicity, or its ability to stimulate target cells to proliferate. The final result of this mitogenic activity in vivo is the growth of the target tissue. CTGF also possesses chemotactic activity, which is the movement chemically induced cells as a result of interaction with particular molecules. SUMMARY OF THE INVENTION • dt The present invention is based on the discovery, purification, and characterization of growth factors that are bind with heparin (HBGFs) in uterine secretory fluids. These growth factor polypeptides bind with heparin, and exhibit many of the functional characteristics of full-length CTGF. In a first aspect, the present invention provides polypeptides that bind to heparin (HBGF polypeptides) that have been identified as having mitogenic activity and nucleic acids encoding these polypeptides. In still an additional aspect of this • invention, antibodies are provided that bind to HBGFs. In yet a further aspect of the present invention, nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to a nucleic acid sequence encoding HBGFs are also provided. In accordance with yet a further aspect of the invention, there is provided a method for using HBGFs, nucleic acid molecules encoding HBGFs, or anti-sense sequences for nucleic acid molecules encoding HBGFs, to affect wound healing, tissue formation, sclerotic or cellular proliferative disorders, atherosclerosis, or fibrotic disease. Brief Description of the Drawings The following drawings are illustrative of the embodiments of the invention, and are not intended to limit the scope of the invention as encompassed by the claims. Figure la is an illustration showing the results of heparin affinity chromatographic fractions of uterine luminal fluids that were tested to stimulate DNA synthesis.

Figure lb is an illustration showing the results of suuent heparin affinity chromatography on samples positive for DNA synthesis (from Figure la), where the major component peaks (marked as Pl and P2) represent the HBGF-0.8 polypeptides. Figure 2 is an illustration showing a gel filtration chromatography profile of the HBGF-0.8 polypeptides. Figures 3a and 3b are illustrations showing reverse phase high pressure liquid chromatography and the • SDS-PAGE of HBGF-0.8 polypeptides. Figure 4 is an illustration showing a Western blot analysis of unpurified uterine luminal fluxes. Figure 5 is an illustration showing the effect of the mitogenic activity of the HBGF-0.8 polypeptides. Figure 6 is an illustration showing the relationship between the HBGF-0.8 polypeptides and the CTGF primary translation product. "Detailed Description of Preferred Modes 20 Before the present methods, apparatuses, compositions, and formulations are described , it should be understood that this invention is not limited to the particular methods, apparatuses, compositions, and formulations described herein, because such methods, apparatus, compositions, and formulations, of course, may vary. that the terminology used herein is for the purpose of describing the particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. It should be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references, unless that the context clearly dictates otherwise. Therefore, for example, the reference to "an organism" includes one or more different organisms; the reference to "an amino acid" includes one or more of these amino acids, and the reference to "a method" includes reference to equivalent steps and methods known to those skilled in the art, and so on. Unless defined otherwise, all the The technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention pertains. Although J ^ k any methods and materials similar or equivalent to those described herein may be used in practice or In a test of the invention, preferred methods and materials are now described. The publications described above are provided exclusively by their disclosure prior to the date of filing of the present application. Nothing contained herein should be construed as an admission of that the invention is not entitled to a date prior to said disclosure by virtue of the prior invention. The present invention provides growth factors that bind to heparin (HBGF polypeptides, or HBGFs), which are mitogenic for fibroblasts and smooth muscle cells in vi tro. HBGFs are labile to heat and acid, and exist in two forms, HBGF-0.8-P1 and HBGF-0.8-P2, each of which has different heparin binding properties, and each of which has a Mr of approximately 10 kDa under reducing conditions by SDS-PAGE. HBGFs are structurally and functionally related to CTGF. Both HBGF-0.8-P1 and HBGF-0.8-P2 require the presence of 0.8 M NaCl for elution from an affinity column with heparin. Sequencing revealed that the N-terminal sequence of HBGF-0.8-P1 corresponded to the amino acid residues 247-262 of the predicted primary translation product of residue 349 of porcine connective tissue growth factor (ETGF), while the N-terminal sequence of HBGF-0.8-P2 corresponded to jf *? to amino acid residues 248-259 of CTGF. Accordingly, the HBGFs correspond to two highly truncated microheterogeneous N-terminal forms of the CTGF translation product, both of which are biologically active. HBGF-0.8-P2 is identical to HBGF-0.8-P1, except for the presence of an additional Glu residue at the N-terminus of HBGF-0.8-P1. The HBGFs of the invention are highly CTGF forms N-terminally truncated; however, there is no intron / exon limit that can directly result in the N terminus of the two proteins. HBGFs do not align with the proposed modular components of CTGF; the proteins of the invention do not * contain none of the sulfated glycoconjugate binding motifs of CTGF, termed a repeat of the thrombospondin I type, which is postulated to be responsible for the bonding of the matrix molecules. A C-terminal CTGF module found in matrix proteins, which is postulated to be involved in receptor binding, is entirely present in HBGF. The proposed binding motif for sulfated glycoconjugates between amino acid residues 206 and 214 of CTGF is absent from HBGFs, and yet HBGFs bind with heparin, and interactions with heparin are functionally significant. The N-terminus of HBGF-0.8-P1 and of HBGF-0.8-P2 may be involved in the heparin bond, because the two proteins of the invention differ only by an N-terminal Glu, and yet exhibit a differential link with heparin. The HBGFs of the invention are secreted from both cultured human and mouse fibroblasts. The production of HBGFs is not limited to a particular species or biological system. Preferably, the HBGFs of the invention are mitogenic and chemotactic for the mesenchymally derived cells (e.g., fibroblasts, chondrocytes, osteoclasts, osteoblasts, and astroglial); however, other cell types (eg, muscle cells, connective tissue cells, epithelial cells, and secretory cells) respond to HBGFs as well. HBGFs may have a significant role in the normal development, growth, and repair of human tissue. HBGFs are present in the uterine flows, and may have an additional role in the growth and remodeling of the endometrium, and during pregnancy, may affect the growth and development of extra-embryonic or placental membranes. Therapeutic agents derived from HBGFs may be useful for increasing the normal growth processes or • injured parties that involve connective tissues in certain clinical conditions (for example, wound healing). When these HBGFs are involved in pathological conditions, therapeutic developments can be used from these proteins to control or modulate uncontrolled tissue growth. The term "substantially pure", as used herein, refers to HBGFs that are substantially *** free from other proteins, lipids, carbohydrates, or other materials with which they are naturally associated. A substantially pure HBGFs polypeptide will produce a single larger band on a non-reducing polyacrylamide gel. The purity of the HBGFs can also be determined by analysis of the amino-terminal amino acid sequence. HBGFs, as defined herein, include functional fragments of the polypeptide, so long as the biological activity of HBGF is retained (eg, by inducing a biological response in the fibroblasts, as determined using conventional assays common in the art and as they are taught in the present). Smaller polypeptides containing HBGF biological activity are included in the invention. Additionally, the most effective HBGFs produced are included, for example, through site-directed mutagenesis of the HBGF polypeptide cDNA. "Recombinant" HBGFs refer to HBGF polypeptides produced by recombinant DNA techniques; that is, produced from cells transformed by an exogenous DNA construct encoding the desired HBGF polypeptide. "Synthetic" HBGFs are those prepared by chemical synthesis. A "DNA coding sequence of" or a "nucleotide sequence that encodes" a particular HBGF polypeptide, is A DNA sequence that is transcribed and translated into a HBGF polypeptide when placed under the control of appropriate regulatory sequences. á * k The invention provides nucleic acids encoding HBGFs polypeptides. These nucleic acids include sequences of DNA, cDNA, and RNA encoding HBGFs. It is understood that all nucleic acids encoding all or a portion of the HBGF polypeptides are also included herein, as long as they encode a polypeptide with biological activity of HBGF. These nucleic acids include nucleic acids so much that they occur naturally as intentionally manipulated. For example, HBGF polypeptides can be subjected to site-directed mutagenesis. The nucleic acids of the invention include sequences that degenerate as a result of the genetic code. There are only 20 natural amino acids, most of which are specified by more than one codon. Accordingly, provided that the amino acid sequence of an HBGF polypeptide does not change functionally, all degenerate nucleotide sequences in the invention are included. The fragment, derivative, or analog of the HBGF polypeptides can be: (i) one wherein one or more of the amino acid residues is substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), and this substituted amino acid residue may or may not be one encoded by the genetic code, or between Preferred variants are those that vary from a reference by conservative amino acid substitutions (these substitutions are those that substitute a given amino acid in • ^ *? a polypeptide for another amino acid of similar characteristics. Normally, conservative substitutions are replacements, for each other, between the aliphatic amino acids Ala, Val, Leu, e lie; exchange of the hydroxyl residues Ser and Thr, exchange of the acid residues Asp and Glu, substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg, and replacements among the aromatic residues Phe, Tyr); (ii) one in which one or more of the amino acid residues includes a substituent group; (iii) one in which a HBGF polypeptide is fused with another compound, such as a compound to increase the half-life of HBGF polypeptides (eg, polyethylene glycol); or (iv) one wherein additional amino acids are fused with HBGF polypeptides, such as a leader or secretory sequence, or a sequence that is used for the purification of HBGF polypeptides, or a protein sequence. These fragments, derivatives, and the like are considered within the scope of the experts in this field, from the teachings herein. The HBGFs of the present invention and • the nucleic acids that encode them, - are preferably provided in an isolated form, and preferably are purified to homogeneity. The DNA sequences that encode the polypeptides HBGF of the invention, can be obtained by various methods. For example, DNA can be isolated using well-known hybridization procedures. These include, but are not limited to: j4 ** k 1) hybridization of probes to genomic or cDNA libraries to detect shared nucleotide sequences (see, for example: Current Protocols in Molecular Biology, Ausubel F.M. and collaborators (EDITORS) Green Publishing Company Assoc. and John Wiley Interscience, New York, Current Edition), and 2) antibody screening of expression libraries to detect shared structural features. It is appreciated by an expert in the technique that nucleic acids (comprising at least 12 contiguous nucleotides) that encode HBGFs are particularly useful as probes. "Selective hybridization", as used herein, refers to hybridization under moderately astringent or highly astringent physiological conditions (see, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory (Current Edition) , which is incorporated herein by reference in its entirety), which distinguish the related HBGF from the unrelated, based on the degree of identity between the nucleotide sequences in proximity for hybridization to occur. Also, it is understood that a fragment of a sequence of 100 base pairs that is 95 base pairs in length, has 95 percent identity with the sequence of 100 base pairs from which it is derived. gets. As used herein, the first DNA (RNA) sequence is at least 70 percent, and preferably at least 80 percent identical to another DNA sequence ? * * (RNA) if there is at least 70 percent, and preferably at least 80 percent or 90 percent identity, respectively, between the bases of the first sequence and the bases of another sequence, when they are properly aligned with one another, for example, when they are aligned by BLASTN. "Identity", as the term is used herein, refers to a polynucleotide sequence comprising a percentage of the same bases as a reference polynucleotide. For example, a polynucleotide that is at least 90 percent identical to a reference polynucleotide, has bases of the polynucleotide that are identical in 90 percent of the bases that make up the reference polynucleotide (ie, when the sequences are properly aligned with each other using common conventional alignment and homology settings for those in the art (eg, NetBlast or GRAIL), and may have different bases in 10 percent of the bases comprising that polynucleotide sequence. Tracing procedures that rely on nucleic acid hybridization make it possible to isolate any genetic sequence from any organism, provided that the appropriate probe is available. For example, oligonucleotide probes, which correspond to a part of The sequence encoding the protein in question can be synthesized chemically. This requires that short stretches of oligopeptides of the amino acid sequence be known. The • **** DNA sequence that encodes the protein can be deduced from the genetic code; however, it must be taken into account the degeneracy of the code. It is possible to perform a mixed addition reaction when the sequence is degenerate. This includes a heterogeneous mixture of denatured double-stranded DNA. For this screening, the preferred hybridization is performed on any single-stranded DNA, or double-stranded DNA. Hybridization is particularly useful in the detection of derived cDNA clones from sources where an extremely low amount of mRNA sequences related to the polypeptide of interest is present. In other words, through the use of targeted hybridization conditions to avoid a non-specific binding, it is possible, for example, to allow the autoradiographic visualization of a specific cDNA clone by hybridizing the target DNA with that single probe of the mixture, which is its full complement (Wallace et al., Nucl eic Acid Research, 9: 879, 1981). It is also appreciated that these selective hybridization probes can be, and preferably are labeled with an analytically detectable reagent to facilitate identification of the probe. Useful reagents include, but are not limited to, radioactivity, fluorescent dyes, or enzymes capable of catalyzing the formation of a detectable product. Selective hybridization probes, therefore, are useful for isolating complementary copies of DNA from other sources, or for screening these sources and determining related sequences. A cDNA expression library, such as lambda gtll, can be screened indirectly for HBGFs having at least one epitope, using antibodies specific for HBGF polypeptides or antibodies to CTGF that cross-react with HBGF polypeptides, or antibodies to PDGF that react cross-wise with the HBGF polypeptides. These antibodies can be derived in a polyclonal or monoclonal fashion, and can be used to detect expression products that indicate the presence of the HBGF polypeptide cDNA. The DNA sequences encoding HBGF polypeptides can be expressed in vi tro by transfer of the DNA to a suitable host cell. The "host cells" are genetically engineered cells (transduced or transformed or transfected) with the vectors of this invention, which may be, for example, a cloning vector or an expression vector. The vector can be, for example, in the form of a plasmid, a viral particle, a phage, and so on. The cells • designed hosts can be cultured in a modified conventional nutrient medium as appropriate to activate promoters, select transformants, or amplify the genes of the present invention. The growing conditions, such as Temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to an ordinary expert. The term also includes tk any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell, or because there may be mutations that occur during replication. However, this progeny is included when the term "host cell" is used. The introduction of the construction into the host cell can be effected by transfection with calcium phosphate, transfection mediated by DEAE-5 dextran, electroincorporation, or any other method of the art (Davis, L. et al., Basi c Methods in Molecular Bi ol ogy, (Current Edition)). The nucleic acids of the present invention can be used for the production of HBGFs by recombinant techniques. Accordingly, for example, the polynucleotide can be included in any of a variety of expression vectors to express HBGF polypeptides. These vectors include chromosomal, non-chromosomal, and synthetic DNA sequences, for example, SV40 derivatives; bacterial plasmids; Phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA, such as vaccine, adenovirus, poultry varicella virus, and pseudo-rabies. However, any other vector can be used, provided that it can replicate and be viable in the host.

The appropriate DNA sequence can be inserted into the vector by a variety of methods. In general, the DNA sequence is inserted into an endonuclease site of ^ * Proper restriction by known procedures in this field. These procedures and others are considered within the scope of the experts in the field. The DNA sequences encoding HBGFs can be expressed in vivo in prokaryotes or eukaryotes. Methods for expressing DNA sequences that have eukaryotic coding sequences in prokaryotes are well known in the art. Guests include organisms microbial, yeast, and mammalian.

Biologically functional viral and plasmid DNA vectors capable of expressing and replicating in a host are known in the art. These vectors are used to incorporate DNA sequences of the invention. In general, expression vectors containing promoter sequences that facilitate efficient transcription of the inserted eukaryotic gene sequence are used in relation to the host. The expression vector typically contains a replication origin, a promoter, and a terminator, as well as specific genes capable of providing phenotypic selection of the transformed cells.

In addition to the expression vectors known in the art, such as bacterial, yeast, and mammalian expression systems, baculovirus vectors can also be used. An advantage for the expression of foreign genes in this expression vector of invertebrate virus, is that it can express high levels of recombinant proteins, which are antigenically and functionally similar to their natural counterparts. The vectors of baculoviruses, and the appropriate insect host cells used in conjunction with the vectors, are known to those skilled in the art. The isolation and purification of the polypeptides expressed by the host cell of the invention, can be any conventional elements, such as, for example, chromatographic preparation separations, and immunological separations, such as those involving the use of monoclonal or polyclonal antibodies. .

The invention provides antibodies that are specifically reactive with HBGF polypeptides or fragments thereof. Although this polypeptide can cross-react with antibodies to PDGF or CTGF, not all antibodies to HBGFs will also react with PDGF, and not all antibodies to CTGF will react with HBGFs. Antibodies are provided which consist essentially of clustered monoclonal antibodies with different epitopic specificities, as well as preparations of different monoclonal antibodies. Monoclonal antibodies are made from fragments containing antigen from the • protein by methods well known in the art (Kohier et al., Nature 256: 495, 1975; Current Protocol s in Molecul ar Biology, Ausubel et al., 1989). Polyclonal antibodies are also included for the HBGFs of the invention, using common methods for experts in this field (see Harlow and Lane, 1988, Antibodies, A Labora tory Manual, Cold Spring Harbor Laboratory, New York, Current Edition). Monoclonal antibodies specific for HBGFs can be selected, for example, by screening for culture supernatants from Hybridoma that reacts with HBGF polypeptides, but does not react with PDGF. The antibodies generated against HBGFs corresponding to the present invention can be obtained by direct injection of the polypeptides in an animal, or by administration of the polypeptides to an animal, of preference a non-human. The antibody thus obtained will then bind to the polypeptide itself. In this way, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies that bind to the original polypeptides. Then these antibodies can be used to isolate the polypeptides from cells expressing that polypeptide. For the preparation of monoclonal antibodies, any technique that provides antibodies produced by continuous cell line cultures can be used. The ltf) examples include the hybridoma technique (Kohier et al., Nature 256: 495, 1975), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunol ogy Today 4:72), and the EBV hybridoma technique to produce human monoclonal antibodies (Colé et al. 1985, in Monoclonal Antibodi es and Cancer Therapy, Alan R. Liss, Inc., pages 77-96). The techniques described for the production of single chain antibodies (U.S. Patent No. 4,946,778) can be adapted to produce single chain antibodies for immunogenic peptide products of this invention. Additionally included within the scope of the invention are the production and use for diagnostic and therapeutic applications of both "human" and "humanized" antibodies directed to HBGF polypeptides, or fragments thereof. Humanized antibodies are antibodies, or antibody fragments, which have the same binding specificity as the parent antibody (ie, usually of mouse origin), but which have increased human characteristics. The • Humanized antibodies can be obtained by chain blending, or using phage display technology. For example, a polypeptide comprising a heavy or light chain variable domain of a non-human antibody specific for an HBGF, is combined with a repertoire of human complementary chain (light or heavy) variable domains. Hybrid pairs are selected that are specific for the antigen of '• interest. Then the human chains can be combined from the selected pairs, with a repertoire of human complementary variable domains (heavy or light), and then the antibody polypeptide dimers can be selected. humanized for the binding specificity for an antigen. These techniques are described in U.S. Patent No. 5,565,332, or can be obtained commercially (Scotgene, 4 Scotland, or Oxford Molecular, Palo Alto, CA, USA). In addition, the techniques described for the production of "human" 0 antibodies (i.e., de novo antibodies with human constant region sequences) in transgenic mice (U.S. Patent No. 5,545,806 and U.S. Patent No. 5,569,825) they can also be adapted to produce antibodies or fragments of "human" HBGF antibodies, or they can also be commercially contracted (GenPharm International, Inc., Mountain View, CA, USA). The antibodies generated against the polypeptides of the present invention can be used in the screening of similar HBGF polypeptides from other organisms and samples. These tracking techniques are known in the art. The invention provides a method for accelerating the healing of wounds in a subject, for example, a human, by applying to the wound a therapeutically effective amount of a composition containing purified HBGF polypeptides, PDGF, PDGF-related molecule, or combinations " thereof. The HBGF polypeptides of this invention are valuable as a therapy in cases where there is a healed wound from skin wounds, or there is a need to increase the normal healing mechanisms. The polypeptides HBGF, or the functional fragments thereof, are more stable and less susceptible to protease degradation than PDGF and other growth factors that are known to be involved in wound healing. In addition, the HBGF polypeptides may have a specific biological activity higher than the CTGF. The HBGF polypeptides are derived from fibroblast cells, which are present in an injured site. Accordingly, agents that stimulate the production of HBGF polypeptides can be added to a composition that is used to accelerate wound healing. Preferably, the agent is a member of the family of growth factors, such as insulin-like growth factor (IGF-I), platelet-derived growth factor (PGF), epidermal growth factor (EGF). , transforming growth factor beta (TGF-5 ß), and basic fibroblast growth factor (bFGF). More preferably, the agent is transforming growth factor beta (TGF-β), or another member of the TGF-β superfamily. Additionally, the biological effect of HBGF can be modulated by the addition of heparin at a concentration at the lflfc scale of about 1 microgram / milliliter to 100 microgram / milliliter. The HBGF compositions of the invention help to heal the wound, in part, by promoting connective tissue growth. The HBGF compositions are prepared by combining, in any pharmaceutically acceptable carrier substance, for example inert or liquid gels, the purified HBGF polypeptides of the invention. Other modulator compositions, such as heparin, or growth factors may be included, J "^ such as TGF-β, in HBGF compositions The term" cell proliferative disorder "refers to to a condition characterized by an abnormal number of cells. The condition may include both hypertrophic cell growth (the continuous multiplication of cells resulting in an overgrowth of a cell population within a tissue) and hypertrophic growth (a lack or deficiency of cells). inside a tissue), or an influx or excessive migration of cells to an area of a body. The cell populations are not necessarily transformed, tumorigenic or malignant cells, but they can include normal cells as well. For example, HBGFs may be involved in a pathological condition by inducing a proliferative lesion in the intimal layer of an arterial wall, resulting in atherosclerosis. Instead of trying to reduce the risk factors for the condition, for example, lowering blood pressure or reducing high cholesterol levels, the inhibitors or antagonists of the HBGF polypeptide of the invention would be useful to interfere with the activity in I live from the HBGFs associated with atherosclerosis. Antagonists of the HBGF polypeptide are also useful in the treatment of other disorders associated with an overgrowth of the connective tissues, such as different fibrotic conditions, include scleroderma, arthritis, and liver cirrhosis. These diseases, disorders, or modulated discomforts J ^ *** by HBGF, include tissue repair subsequent to injuries or traumatic conditions, including arthritis, osteoporosis, and other skeletal disorders, and burns. Because these problems are due to a poor growth response of fibroblasts, stem cells, chondrocytes, osteoblasts, or fibroblasts at the site of injury, the addition of an active biological agent that stimulates or induce the growth of these cells. The term "induce" or "induction", as used herein, refers to the activation, stimulation, improvement, initiation, and / or maintenance of the cellular mechanisms or processes necessary for the formation of any of the tissue, process of repair, or development, as described herein. The present invention further provides a method for modulating the function of the female reproductive tract. Growth factors have been shown to play a role in cyclic mitosis and in the differentiation of endometrial cell components, in the recruitment of macrophages in the • Decidualization of the endometrium, in endometrial-trophoblast interactions, in the early maintenance of pregnancy, and in functional endometrial regeneration. The term "modular", as used herein, denotes a modification of an existing condition or biological state. The modulation of a condition as defined herein, encompasses both an increase and a decrease in the determinants that affect • J * k the existing condition. For example, the administration of HBGFs could be used to increase uterine functions in a condition where the promotion of growth is desired. For example, the uterus can be treated with HBGFs to promote the growth and development of placental membranes or endometrial growth. In addition, treatment with HBGFs can be used to promote and maintain a pregnancy, facilitating endometrial-trophoblast interaction. Alternatively, antagonists are administered for HBGFs, in order to modulate the conditions of excessive endometrial growth, where the level of HBGF is excessive compared to a condition • normal biological. The invention also provides a method for the treatment of conditions characterized by a cell proliferative disorder, by treating the condition using a therapeutically effective amount of an HBGF reactive agent. The term "treat" denotes to lessen the detrimental effect of the condition on the subject receiving the reactive agent. When the condition is due to cell overgrowth, an HBGF antagonist is therapeutically effective to reduce the amount of growth factor that can bind to a specific HBGF receptor on a cell. This antagonist can be an antibody specific for HBGF, or functional fragments thereof (for example, Fab, F (ab) 2). The treatment requires contacting or supplying to the site of the condition, the HBGF polypeptide antagonist. When the cell proliferative disorder is due to a reduced amount of cell growth, a reactive agent of HBGF that is stimulant is contacted with, or delivered to, the site of the condition. For example, this reactive agent can be TGF-β (or another member of the TGF-β superfamily). Other biological agents will be known to those skilled in the art. When a cell proliferative disorder is associated with the expression of HBGFs, a therapeutic approach is possible that directly interferes with the transcription of HBGF into the mRNA, or the translation of the HBGF mRNA into protein. For example, • Anti-sense nucleic acid or ribosomes that bind to or dissociate HBGF mRNA are also included within the invention. The anti-sense RNA or DNA molecules specifically bind to an RNA message of the target gene, disrupting the expression of that gene protein product. The antisense binds to the mRNA, forming a double-stranded molecule that can not be translated by the cell. The preferred ones are • anti-sense oligonucleotides of approximately 15 to 25 nucleotides, because they are easily synthesized, and have an inhibitory effect, just like anti-sense RNA molecules. In addition, chemically reactive groups, such as iron-linked ethylenediaminetetraacetic acid (EDTA-Fc) can be attached to an anti-sense oligonucleotide, causing dissociation of the RNA at the site of hybridization. These and other uses of anti-sense methods to inhibit the in vivo translation of genes are well known in the art (eg, De 0 Mesmaeker et al., 1995, Backbone modifications in oligonucleotides and peptide nucleic acid systems, Curr. Opinion Struct. Biol. 5: 343-355; Gewirtz, AM et al., 1996. Facilitating delivery of antisense oligodeoxynucleotides: Helping antisense deliver on its promise; Proc. Na ti. Acad. Sci. USA 5 93: 3161-3161; Stein, CA A discussion of G-tetrads 1996.

Exploting the potential of antisense: beyond phosphorothioate oligodeoxynucleotides. Chem. And Biol. 3: 319-323). Another therapeutic approach included within the invention involves the direct administration of reagents or compositions that include the HBGFs of the invention, by any conventional administration technique (eg, but not restricted to, local injection, inhalation, or systemic administration), a subject with a sclerotic or proliferative cell disorder, atherosclerosis. The administration of HBGFs as described above, accelerates the healing of wounds, can induce the formation of tissue repair or regeneration, or the growth and development of the endometrium. The reagent, formulation, or composition, can also be targeted to specific cells or receptors by any The method described herein, or by any method known in the art of delivery, targeting, and expression of genes encoding HBGF. The actual dosage of the reagent, tfk formulation, or composition that modulates a fibrotic disorder, a sclerotic disorder, a cell proliferative disorder, or atherosclerosis, or wound healing, depends on many factors, including the size and health of an organism. However, one of ordinary skill in the art can use the following teachings which describe the methods and techniques for determining clinical dosages (Spilker B., Guide to Clini cal 5 Studies and Developing Protocols, Raven Press Books, Ltd., New York, 1984, pages 7-13, 54-60, Spilker-B., Guide to Clini cal Triai, Raven Press, Ltd., New York, 1991, pages 93-101; Craig C, and R. Stitzel, editors, Modern Pharmacology, 2nd Edition, Little, Brown and Co., Boston, 1986, pages 127-33; T. Speight, editor, Avery's Drug Trial: Principle and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, Williams and Wilkins, Baltimore, 1987, pages 50-56; R. Tallarida, R. Raffa and P. McGonigle, Principi is in General Pharmacology, Springer-Verlag, New York, 1988, pages 18-20), or to determine the appropriate dosage that should be used; but in general, the scale of about 0.5 micrograms / milliliter to 500 micrograms / milliliter inclusive of final concentration per day is administered to an adult in any pharmaceutically acceptable vehicle. The present invention also provides a method for detecting the presence of abnormal levels of HBGFs in a subject, to be used diagnostically, for the purpose of determi-a? The presence of conditions or pathologies associated with abnormal levels of HBGFs. These conditions include, but not are restricted to cell proliferative disorders, different fibrotic conditions, including scleroderma, arthritis, liver cirrhosis, and uterine fibroids. For example, a sample suspected of containing HBGFs from a subject is obtained, the level of HBGFs polypeptide is determined, and compared to the level of HBGF polypeptide in a normal tissue sample.

The level of HBGFs can be determined by immunoassays using anti-HBGF polypeptide antibodies, for example. Other variations of these assays include radioimmunoassay (RIA), ELISA, and immunofluorescence. Alternatively, nucleic acid probes can be used to detect and quantify the HBGF polypeptide mRNA for the same purpose. The following examples are presented to provide ordinary experts in the field with a disclosure and complete description of how to make and use the HBGFs of the present invention, and are not intended, nor should they be construed, to limit the scope of the invention. that the inventors consider as their invention. Efforts have been made to ensure accuracy with respect to the numbers used (for example, quantities, time, temperature, etc.), but errors and experimental deviations must be taken into account. Unless indicated otherwise, the parts are parts by weight, the molecular weight is weight average molecular weight, the temperature is in degrees centigrade, and the pressure is at or near atmospheric. EXAMPLE 1 CHARACTERIZATION AND PURIFICATION OF HBGF POLYPEPTIDES Uteruses were randomly collected from trace pigs that were approximately 8 months or less in age. Each uterine tube was filled with cold phosphate buffered serum (PBS) (4 ° C) to collect the uterine luminal components.

The growth factor was purified in groups of 4 liters of the uterine luminal fluxes obtained from up to 120 animals. The uterine luminal fluxes (ULF) were clarified by centrifugation at 13,500 X g for 30 minutes at 4 ° C, and the supernatant was passed through glass wool. Samples of four liters of uterine luminal flux supernatant rinsed at 4 ° C were applied to a BioRex 70 cation exchange column (5 x 6 centimeters; BioRad), which had previously been equilibrated in phosphate buffered serum, 0.2 M NaCl. After the application of the sample, the column was washed with 500 milliliters of phosphate-buffered serum, 0.2M NaCl, and the bound proteins were eluted using a gradient of 500 milliliters of 0.2-2 M NaCl in phosphate-buffered serum. The flow rate was 3.5 milliliters / minute all the time, and fractions of 10 milliliters were collected during the treatment of the column with the NaCl gradient. Fractions demonstrating microgenic activity were selected for Balb / c 3T3 fibroblasts for additional use. All subsequent chromatographic steps were performed at room temperature. The ion exchange chromatograph of the uterine luminal flux showed the presence of cationic growth factor activity for Balb / c 3T3 cells eluted from BioRex 70 columns by NaCl 0.3-0.6 M. Affinity chromatography with heparin revealed the presence of an additional unidentified HBGF polypeptide that required 0.8 M NaCl to be eluted from an EconoPac heparin column. In terms of the amount of bioactivity recovered from the column, the fraction that required 0.8 M NaCl for elution appeared to be a major cationic heparin binding growth factor for 3T3 cells. The position of the elution of the HBGF polypeptides from the heparin affinity columns was clearly distinct from PDGF, HB-EGF, PTN, aFGF, bFGF, and amphiregulin. The mitogenic activity of HBGF was destroyed by exposure to heat (100 ° C for 2 minutes, or 56 ° C for 30 minutes) or acid (pH 2.0 for 2 minutes). Gel filtration chromatography was used to demonstrate that the HBGFs had an apparent relative molecular mass of approximately 10,000 Daltons. For these studies, 0.5 milliliters of a fraction containing the 0.8 M NaCl eluate were applied from the EconoPac heparin affinity FPLC of the uterine luminal flux of 30 animals at 0.5 milliliters-minute / minute to a FPLC column TSK G2000 SW (30 centimeters x 8 millimeters, particle size of 10 microns, range of fractionation M, 500-100,000; TosoHaas) equipped with a guard column SW (4 centimeters x 8 millimeters, 10 microns, TosoHaas). The proteins were eluted with phosphate-buffered serum containing 0.3 M NaCl. Fractions of 200 microliters were collected and tested to determine their capacity for stimulate the synthesis of DNA in 3T3 cells. Column calibration was performed using EGF (molecular weight of 6,000), lactalbumin (molecular weight of 14,200), trypsin inhibitor (molecular weight of 20,100), and ovalbumin (molecular weight of 45,000). The fractions were tested for their ability to stimulate DNA synthesis in 3T3 cells at 40 microliters / milliliter, as described above. Fractions containing HBGF activity (fractions 16-19 collected after cation exchange chromatography and heparin affinity chromatography) were pooled, diluted, and subjected to a second FPLC cycle of heparin affinity. using a 5PW column of TSK heparin. To perform the second affinity purification step with heparin, the biologically active HBGF fractions containing the 0.8 M NaCl eluate From the heparin purification step EconoPac were pooled, diluted three times with 20 mM Tris-HCl (pH 7.4), and rinsed by passing them through a 0.2 micron filter. The • 4b sample was applied at 2 milliliters / minute to a 5PW column of TSK heparin (0.8 x 7.5 centimeters; TosoHaas, Philadelphia, PA), 0 which was washed and eluted as described above, except that the CHAPS of the samples was omitted. regulators, and fractions of 0.5 milliliters were collected. Fractions containing proteins that were eluted by 0.8 M NaCl, and that demonstrated mitogenic activity of 3T3 cells, were divided into two groups 5 consisting of fractions 31-34 (peak 1), and fractions 35 and 36 (peak 2) . Again the HBGF polypeptide was eluted by 0.8 M NaCl (fractions 31-36), but resolved as two peaks of mitogenic activity that had different heparin binding properties. The peaks of activity were designated HBGF-0.8-P1 for fractions 31-34, and HBGF-0.8-P2 for fractions 35 and 36. HBGF-0.8-P1 and -P2 were adjusted to 10 percent acetonitrile, and 0.1 percent trifluoroacetic acid, and individually subjected to C8 reverse phase high pressure liquid chromatography. The reverse phase high pressure liquid chromatography was performed in a Hitachi high pressure liquid chromatography system (Hitachi Instruments Inc., Danbury CT) using a C8 column (0.46 x 25 centimeters, 5 micron particle size; Instruments Co., Woburn, MA), which was equilibrated with water containing 10 percent (volume / volume) of acetonitrile, and 0.1 percent (volume / volume) of trifluoroacetic acid. The grouped fractions • dk containing peaks 1 and 2 from the TSK heparin purification step, were individually adjusted, such that 0 contained 10 percent acetonitrile and 0.1 percent trifluoroacetic acid, and cleared by passing them through of a 0.2 micron filter. The conditions for the elution of the bound proteins were 10 percent acetonitrile from 0 to 10 minutes, after the injection of the sample, and from 10 to 90 percent from 10 minutes to 146 minutes. The flow rate was 1 milliliter / minute at all times, and the chromatogram (A214) was filed as described (Bray and Brigstock, (1994) Amer. Lab. 26, 38). The eluate was collected as fractions of 0.5 milliliters in siliconized tubes containing 50 microliters of 125 mM NaOH, to immediately neutralize the trifluoroacetic acid. The 80 microliter aliquots of the selected fractions were evaporated to dryness in a SpeedVac concentrator (Savant Instruments, Farmingdale, NY), and reconstituted in 25 microliters of 10 mM Tris-HCl (pH 7.4). 10 microliters of this concentrate were tested for its stimulation of DNA synthesis of 3T3 cells, and 10 microliters were used for the analytical SDS-PAGE. For the second purification step with C8 high pressure liquid chromatography, two active fractions of the first chromatography step were grouped of high-pressure liquids (total volume of 1 milliliter), were diluted five times with water, 0.1% trifluoroacetic acid, and were subjected to the same elution conditions < ^ * k chromatographic which were described herein. The elution positions of HBGF-0.8-P1 and -P2 were determined by bioassay of the aliquots of the fractions containing the eluate from the column after they had evaporated and reconstituted in phosphate-regulated serum, demonstrating that there was sufficient activity in the purified HBGF samples to allow their detection and further characterization, despite of the prolonged exposure (approximately 30 to 40 minutes) at pH = 2 during the high pressure liquid chromatography step. Following high-pressure liquid chromatography, the silver-stained SDS-PAGE analysis of fractions containing HBGF-0.8-P1 or -P2 was performed under reducing conditions using 18 percent polyacrylamide minigeles, as described ( Kim, GY et al., 81995) Bi ol. Reprod. 52, 561-571). Subsequently, the silver staining of the proteins was carried out as described (Wray, W., et al. (1981) Anal Biochem. 118, 197-203). SDS-PAGE was performed • on: (i) purified growth factors with high pressure liquid chromatography, (ii) 8 microliters of unfractionated uterine luminal flux, or (iii) 100 microliters of uterine luminal flux after passing through beds of 20 microliters of heparin-sepharose in the presence of 10 mM Tris-HCl, 0.5 M NaCl (pH 7.4), and the subsequent extraction of the heparin granules with SDS-PAGE sampling regulator. The gels were then prepared as described (Kim G.Y. et al., (1995).

• Bi ol. Reprod. 52, 561-571). The subsequent analysis revealed the presence of a single 10 kDa protein that was co-purified with mitogenic Balb / c 3T3 activity. Mitogenic activity levels correlated directly with those of the 10 kDa protein, which was completely pure, as shown by silver staining. The results of 18 purifications individuals from high pressure liquid chromatography confirmed a direct causative relationship between the 10 kDa proteins and the mitogenic activity of HBGF-0.8-P1 and -P2. Analysis of the individual purification steps showed that 0.5 to 1.1 micrograms of HBGF-0.8-P1 or -P2 were purified from 342 milligrams of crude uterine luminal flux protein, and that they were recovered from 10 to 22 units of activity for HBGF-0.8-P1 or -P2 after the first step of high pressure liquid chromatography, comparing 66.666 units in 1 liter of starting material (Table 1). It should be noted that the apparent low recovery of HBGF-0.8 peptide activity was attributable to: (i) a greater contribution by IGF, EGF, PDGF, bFGF, HB-EGF, and PTN to the overall 3T3 mitogenic cell activity of the raw and partially purified samples (2, 8, 9, 12, 25-27), and (ii) acid lability of the mitogenic activity of the peptide HBGF-0.8 during the separation steps by high pressure liquid chromatography. Although alternative strategies to recover purified growth factors of a higher specific activity were tested, it was not possible to avoid the use of high-performance liquid chromatography. reverse phase pressure or trifluoroacetic acid for the ion pairing without compromising the purity of the final product. Although in terms of its biological activity, the recovery of HBGF-0.8-P1 and -P2 was somewhat compromised, the structural characterization of the proteins was easily achieved, because they retained sufficient activity to be unequivocally attributed to a single homogeneous band of 10 kDa in SDS-polyacrylamide gels, and sufficient amounts of each protein were isolated from several liters of uterine luminal flux (Table 1).

TABLE 1 Concentration of the HBGF-0.8 preparation required to give a maximum synthesis of 50 percent DNA. bAn activity unit is the amount of HBGF-0.8 required to give the ED50. Comparing with the crude uterine luminal flux. d Bioactivity decreased due to acid exposure.

EXAMPLE 2 SEQUENCING OF THE HBGF POLYPEPTIDE The fractions containing the growth factors purified by high pressure liquid chromatography were pooled, dried, and subjected to preparation SDS-PAGE. Proteins in the gel were transferred for 90 minutes at 50 mA to a polyvinylidene difluoride membrane using 10 mM CAPS regulator (pH 11). The location of the proteins of interest was determined by staining the spots with Coomassie R250 jlO at 0.1 percent in 50 percent methanol for 2 minutes, followed by destaining with 50 percent methanol, 10 percent acetic acid. Half of each 10 kDa protein band was separated and subjected to N-terminal amino acid sequencing in a gas phase sequencer model 470A (Applied BioSystems, Foster City, CA). Phenylthiohydantoin derivatives were identified by C18 reverse phase high pressure liquid chromatography. A sequence of 16 residues was obtained for HBGF-0.8-P1 with a residue not determined at position 10, and a sequence of 12 residues was obtained for HBGF-20 0.8-P2 with a residue not determined at position 9 (Table 2 ). These data showed that HBGF-0.8-P1 and -P2 were N-terminally identical, except for the presence of an additional Glu residue in the N-terminus of HBGF-0.8-P1. A search of GenBankMR revealed that these sequences were perfectly aligned with the predicted internal sequences of hCTGF and mouse fisp-12 (also called ßIG-M2), the murine homologue of CTGF (Bradham, DM et al., (1991) J. Cell Bi ol. 114, 1285-1294; Ryseck, RP et al., (1991) Cell Growth Differ.2, 225-233; Brunner, A. et al., (1991) DNA Cell Biol. 10, 293-300). The unassigned residue in cycle 10 of HBGF-0.8-Pl, and in cycle 9 of HBGF-0.8-P2, corresponded to Cys256 of hCTGF and Cys255 of fi sp-12 (Table 2).

• • TABLE 2 HBGF-0.8-Pla (SEQ ID NO: 1) Glu-Glu-Asn-Ile-Lys-Lys-Gly-Lys-Lys-Xaa-Ile-Arg-Thr-Pro-Lys-Ile HBGF-0.8-P2b (SEQ ID NO: 2) Glu-Asn-Ile-Lys-Lys-Gly-Lys-Lys-Xaa-Ile-Arg-Thr CTGF- (247-262) Humanoc Glu-Glu-Asn-Ile-Lys-Lys-Gly- Lys-Lys-Cys-Ile-Arg-Thr-Pro-Lys-Ile flsp-12- (246-261) d Glu-Glu-Asn-Ile-Lys-Lys-Gly-Lys-Lys-Cys-Ile-Arg -Thr-Pro-Lys-Ile CTGF- (247-262) Porcine "Glu-Glu-Asn-Ile-Lys-Lys-Gly-Lys-Lys-Cys-Ile-Arg-Thr-Pro-Lys-Ile ^ Repetitive performance = 88 percent; Initial performance - 7 picomoles. bRend? m? ento repetitive = 90 percent; Initial yield - 3 picomoles. c See Bradha et al., J. Cell Biol. 114: 1285-1294, 1991. dVer Ryseck et al., CeJl Growth Differ. 2: 225-233, 1991. ß From the cDNA analysis in this study.

To verify that partial sequences of HBGF-0.8-P1 and -P2 were actually present in the porcine CTGF molecule (pCTGF), a full-length pCTGF cDNA was isolated by hybridization screening of a pig endometrial cDNA library, using an hCTGF probe labeled with 32P. For these studies, the total pig endometrial RNA was obtained as described (Kim, GY et al., (Biol Reprod 52, 561-571 (1995)). A poly (A) Tract mRNA isolation system was used. Promega, Madison, Wl) to isolate RNA poly (A +), 5 micrograms of which were subjected to a cDNA synthesis of the first strand, using Moloney murine leukemia virus reverse transcriptase, and the oligo (dT) linker-primer containing Xhol. The synthesis of the second chain was primed by treating the mRNA-cDNA complex with RNAse. The cDNA of The double chain was made blunt using a Klenow fragment, and ligated with EcoRl adapters, which were subsequently phosphorylated with T4 polynucleotide kinase. 100 nanograms of cDNA digested with XhoI, purified on a Sephacryl S-400 column, were ligated into 1 microgram of arms of the Uni-ZAP XR vector in the XhoI-EcoRl multiple cloning site, and the product was packaged using the Gigapack II packaging extract (Stratagene, La Jolla, CA). The primary library was amplified in XL1-Blue MRF 'cells at a titre of 1.4x10 0 plaque-forming units / milliliter. A verified 32 P-labeled CTGF probe corresponding to the 3 'end of the predicted primary hCTGF translation product was obtained by reverse transcriptase chain reaction of the human skin fibroblast RNA using the forward primers and Inverse, 5 'GCCGTCTAGAGCGGCCGCATGGAAGAGAACATTAAGAAGGG3' (SEQ ID NO: 3) and 3 '-CCTCTGTACCGTACTTAAGCGCCGGCGACC-5' (SEQ ID NO: 4) respectively. The probe was used to screen 106 plates, two of which showed reproducible hybridization, and were isolated using a Rapid Excision Kit (Stratagene). They obtained two pig CTGF clones of pBluescript SK of approximately 5.0 kilo-base pairs, designated pBSK-pCTGF1 and pBSK-p-pCTGF2, and were used for the initial sequencing reactions. The pBSK-pCTGF1 was then completely sequenced by a combination of sequencing of Manual and automated dideoxy terminator (Sanger, F. et al, Proc.Nat.Acid Sci. E.U.A. 74, 5463-5467 (1977)). Sequence data were obtained from both strands of the DNA. The sequences of HBGF-0.8-P1 and -P2 are listed in Table 2. The cloned pig CTGF cDNA was determined to be 1.51 kilo-base pairs, with an open reading frame of 1.047 base pairs. The primary translation product of pCTGF is predicted to comprise 349 amino acids, and contains the peptide sequence HBGF-0.8 between residues 247 and 262 (Table 2). At the amino acid level, pCTGF is approximately 92 percent identical to f i sp-12 and hCTGF. After dissociation of its 26 residue budget signal peptide, pCTGF is predicted to comprise 323 amino acids, and contains 38 Cys residues that are fully conserved 5 in hCTGF and fisp-12. EXAMPLE 3 PRODUCTION OF HBGF ANTIBODY Because HBGFs represent microheterogeneous forms of truncated CTGF, the ratio of ilO HBGF to CTGF was investigated. The presence of the 10 kDa protein in the starting material was confirmed by Western blot of unfractionated uterine luminal flux samples, using a CTGF antibody that reacted with purified HBGF polypeptides by high pressure liquid chromatography. 15 To produce the antibody, a peptide was produced CTGF- (247-260) four-branched multiple antigen comprising the sequence EENIKKGKKCIRTP (residues 247-260) (SEQ ID NO: 5) on a Synergy 432A peptide synthesizer (Applied BioSystems), and purified by liquid chromatography of high pressure reverse phase using a C18 column (0.46 x 36 centimeters, Rainin Instruments) that was developed with an acetonitrile gradient of 5 to 95 percent for 90 minutes in water, 0.1 percent trifluoroacetic acid. The fractions containing the purified polypeptides were pooled, evaporated to dryness, and reconstituted in sterile water. Two New Zealand white rabbits (rabbits A and B), which had been bled to collect preimmune serum, were injected subcutaneously with 1 milligram of polypeptide in complete Freund's adjuvant, followed 3 weeks later by an intramuscular injection of 250 microliters of polypeptide in Freund's incomplete adjuvant. The animals were bled 7 days later for the collection of the antiserum. The reactivity of the antiserum was validated by Western blot and immunoprecipitation. In these experiments the pre-immune serum and rabbit antiserum A were used. EXAMPLE 4 GENERATION OF 10 kDa HBGF POLYPEPTIDES Western blot was performed as described previously (Harlow and Lane, 1988, Antibodi is, A Labora tory Manual, Cold Spring Harbor Laboratory, New York, Current Edition). Briefly stated, SDS-PAGE was performed under reducing conditions, using 18 percent polyacrylamide minigeles as described (Kim, G. Y. et al, Biol. Reprod. 52, 561-571 (1995)). The silver staining of the proteins was carried out as described (Wray, W. et al., Anal. Biochem. 118, 197-203 (1981)). The Western blot was performed on: (i) purified growth factors by high pressure liquid chromatography, (ii) 8 microliters of unfractionated uterine luminal flux, or (iii) 100 microliters of uterine luminal flux after passing through beds of 20 microliters of heparin-sepharose in the presence of 10 mM Tris-HCL, 0.5 M NaCl (pH 7.4), and the subsequent extraction of the heparin granules with SDS-PAGE sampling regulator. The genes were stained and blocked as described (Kim, GY et al, Biol. Reprod 52, 561-571 (1995)), and incubated with a 1: 1000 dilution of rabbit preimmune serum, or a dilution of 1: 1000 of rabbit anti-pCTGF- (247-260) peptide antiserum (rabbit A). The immunoreactive bands were visualized using goat anti-rabbit IgG conjugated with alkaline phosphatase, followed by chromogenic substrates of nitro blue tetrazolium / 5-bromo-4-chloro-3-indolyl phosphate. In addition to the 10 kDa protein, two additional mass forms of CTGF (16 and 20 kDa) were also present in the uterine luminal flux, but no convincing evidence was obtained for the 38 kDa CTGF. The Western blot also verified that HBGF purified by high pressure liquid chromatography comprised a single immunoreactive protein of 10 kDa. Comparing the intensity of HBGF staining from defined volumes of undiluted uterine fluid (ie, from 0.7 to 2.3 microliters) with the intensity of staining of mitogenic amounts of purified HBGF, indicated that mitogenic concentrations of HBGFs exist in the uterine fluid in vivo. Taken together, the data showing that the uterine luminal flux did not contain detectable levels of CTGF of 38 kDa, but did contain HBGFs in possibly mitogenic amounts, demonstrate that HBGF occur naturally in vivo, and are not the result of a breakdown of the 38 kDa CTGF during its purification. EXAMPLE 5 5 HEPARIN LINKED PROPERTIES OF HBGF POLYPEPTIDES The presence of an additional Glu acid residue at the N-terminus of HBGF-0.8-P1 polypeptide correlated with the lower affinity with heparin of this molecule, compared to HBGF-0.8 -P2, suggesting that the N-terminus of the peptide HBGF-0.8 jttilO may be part of a heparin binding domain. To test the heparin binding properties of the N-terminal region, as well as other portions of the CTGF molecule, we investigated the ability of 18 polypeptides that span all 103 C-terminal residues of hCTGF to bind to [3H] heparin. 18 synthetic polypeptides were synthesized extending through all 103 C-terminal residues of CTGF, and were received as a PepSetMR dissociated from Chiron Mimotopes (Clayton, Victoria, Australia). All polypeptides were synthesized with N-acetylated terms and amidated C-terms, except CTGF- (247-255) and -CTGF- (247-260), which were synthesized with free N-terminal amines, and CTGF- (326-349) and CTGF- (339-349), which were synthesized with C acid terms (Table 3). All polypeptides contained one or no Cys residue; Cys292 in CTGF (285-292), and Cys325 in CTGF- (318-328) were replaced with Ser to prevent intrachain chain disulfide bridging with Cys287 or Cys323 within the respective polypeptides. The binding properties with heparin were determined using an adaptation of the method of Baird et al. (Baird, A. et al., Proc. Na ti. Acad. Sci. E.U.A. 85, 2324-2328 (1988)). Briefly stated, 37.5 nanomoles of each polypeptide were duplicated in nitrocellulose using a dot-spot apparatus. The blot was blocked for 30 minutes with 10 mM Tris-HCl, 0.15 M NaCl, 0.1 percent bovine serum albumin (pH 7.4), and then incubated for 3 hours at room temperature in this solution containing 10 μCi / ml of [3H] heparin (NEN Life Science Products). The stain was washed four times with 10 mM Tris-HCl, 0.15 M NaCl, and individual spots were mixed with scintillation fluid for the [3 H] count. 15 Table 3 summarizes the results obtained with the synthetic polypeptides. The highest level of heparin binding was obtained for the polypeptides containing the residues 247-260, 274-286, and 305-328. It should be noted that none of these polypeptides had agonist or antagonist activity HBGF polypeptide in a DNA synthesis assay of 3T3 cells.

TABLE 3 a N-terminal free album, b C acid term.

Previous studies have shown that heparin modulates receptor binding and the biological activity of several HBGF polypeptides, including bFGF, HB-EGF, and amphiregulin • (Besner, G.E. and collaborators, Growth Factors 1, 289-296 (1992); Higashiyama, S. et al., J. Cell Bi ol. 122, 933-940 (1993); Rapraeger, A.C. and collaborators, Sci ence 252, 1705-1708 (1991); Olwin, B.B. and collaborators, J. Cell Bi ol. 118, 631-639 (1992); Cook, P. and collaborators, J. Cell Physio. 163, 418-429 (nineteen ninety five); Yayon, A. et al., Cell 64, 841-848 (1991); klO Aviezer, D. and collaborators Proc. Nati Acad. Sci. E.U.A. 91, 12173-12177 (1994)). Because the peptide HBGF-0.8 exhibited a strong affinity for heparin, we examined the effect of this glycosaminoglycan on the mitogenic activity of the peptide HBGF-0.8. The activity of a high stimulant dose of the peptide HBGF-0.8 was potentiated in a significant way with 1 to 3 micrograms / milliliter of heparin, but was inhibited with 30 to 100 micrograms / milliliter of heparin. The same dosages of heparin had no effect on basal DNA synthesis or stimulated by calf serum in 3T3 cells. EXAMPLE 6 HBGF MYTHOGENIC ASSAY To evaluate the relative mitogenic capacity of the HBGFs with IGF-I, EGF, bFGF, and PDGF-AB, DNA synthesis assays were performed on 3T3 cells (Table 4). The fractions biologically active samples containing the 0.3-0.6 M NaCl eluate from the Bio-Rex column were pooled, diluted three times with 20 mM Tris-HCl (pH 7.4) containing 0.1 percent CHAPS, passed through of a 0.45 micron membrane filter were placed in a siliconized polypropylene container and applied with a peristaltic pump to an EconoPac heparin column (0.7 x 3.6 centimeters; BioRad) at 2 milliliters / minute. The heparin column was then washed with 50 milliliters of 20 mM Tris-HCl buffer, 0.2 M NaCl, 0.1 percent CHAPS, and developed at 1 milliliter / minute with a gradient of 40 milliliters of 0.1-2.0 M NaCl in Tris. -HCl 20 mM, 0.1 percent CHAPS (pH 7.4), using a rapid protein liquid chromatography (FPLC) system (Pharmacia Biotech Inc.). The fractions (1 milliliter) were collected in siliconized tubes during the elution of the NaCl gradient, and were tested for the mitogenic activity of 3T3 cells. Fractions from the column were tested for their ability to stimulate DNA synthesis, as measured by incorporation of [3H] thymidine into the DNA of confluent passive Balb / c 3T3 cells cultured in 200 microliters of Dulbecco's modified Eagle's medium, serum of 10 percent bovine calf in 96-well culture plates, as described (Kim, GY et al., Bi ol. Reprod. 52, 561-571 (nineteen ninety five)) . Dose response curves were established for the growth factors purified from the uterine luminal flux by assaying each dose in triplicate, and the data were calculated as the mean + SD The statistical significance of the effects from 1 to 100 micrograms / milliliter of porcine heparin (Sigma) on the activity of the growth factor, was determined by Student's t test. Incorporation of [3 H] thymidine by the HBGF peptide was comparable to that of purified calf serum or PDGF or bFGF, rather than that of weaker mitogens such as IGF or EGF. In addition, it was found that the mitogenic and biological activity in 3T3 of the HBGFs was synergistically enhanced by 10 milligrams / milliliter of IGF-I, 10 nanograms / milliliter of PDGF, 3 nanograms / milliliter of EGF, or 0.3 nanograms / milliliter of bFGF. The specificity of the target cells was studied using Balb / c 3T3 cells, endothelial cells, bobbin coils (BCECs), and vascular smooth muscle cells. The 3T3 cells were used as described above. The BCECs were obtained from Dr. J. Folkman (Children's Hospital, Boston, MA), and kept in gelatinized culture flasks in Dulbecco's modified Eagle's medium containing 3 nanograms / milliliter of bFGF, and inactivated bovine calf serum. by heat at 10 percent. Smooth muscle cells were isolated from a 2 to 3 centimeter stretch of pig thoracic aorta, using established procedures (Weich, HA et al., Growth Factors 2, 313-320 (1990)), and were maintained in Eagle medium modified by Dulbecco at 10 percent, fetal bovine serum at percent. BCEC DNA and smooth muscle cells assays were performed in 48- or 96-well plates essentially as described (Besner, G.E., Higashiyama, S. and Kagsbrun, M. Cell Regul. 1, 811-819 (1990)). BCEC DNA synthesis assays were also performed in the presence of 100 micrograms / milliliter of porcine heparin. It was found that HBGF is mitogenic for smooth muscle cells, and produced • A10 a level of stimulus that exceeded that of a maximum amount of EGF, but was lower than that of bFGF. HBGFs lacked mitogenic activity for endothelial cells when tested alone or in the presence of 100 micrograms of heparin (See Table 4). fifteen TABLE 4 Although the invention has been described with reference to the currently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. In accordance with the foregoing, the invention is limited only by the following claims.

Claims (35)

1. CLAIMS 1. A substantially pure polypeptide, characterized by: a) having an amino acid sequence corresponding to carboxy terminal amino acids of a connective tissue growth factor protein (CTGF); b) bind to heparin and be eluted from heparin with salt around 0.8M; and c) have a molecular weight of about 10 kDa per kilogram reducing SDS-PAGE analysis.
2. 2. The polypeptide of claim 1, wherein the polypeptide has an amino acid sequence that begins at amino acid residue 247 from the N-terminus of CTGF.
3. 3. The polypeptide of claim 1, wherein the polypeptide has an amino acid sequence that begins at amino acid residue 248 from the N-terminus of CTGF. .
4. An isolated polynucleotide sequence encoding a polypeptide having an amino acid sequence as in any one of claims 1, 2 or 3.
5. 5. A recombinant expression vector containing the polynucleotide of claim 4.
6. 6. A host cell that contains the expression vector of claim 5.
7. 7. The host cell of claim 6, which is a prokaryotic cell.
8. 8. The host cell of claim 6, which is a eukaryotic cell.
9. 9. An antibody that binds to the polypeptide of claim 1, and which is ligated with its immunoreactive fragments.
10. 10. The antibody of claim 9, wherein the antibody is polyclonal.
11. 11. The antibody of claim 7, wherein the antibody is monoclonal.
12. A method of treating atherosclerosis or a fibrotic, sclerotic or cellular proliferative disorder, comprising contacting a cell with a therapeutically effective amount of an antagonist of a polypeptide characterized by: a) having an amino acid sequence derived from carboxy amino acids; terminals of a connective tissue growth factor protein (CTGF); b) bind to heparin and be eluted from heparin with salt around 0.8M; and c) having a molecular weight of about 10 kDa by reducing SDS-PAGE analysis.
13. A method for accelerating the healing of wounds in a subject in need of such treatment, comprising contacting a cell with a therapeutically effective amount of a composition containing a polypeptide characterized by: a) having an amino acid sequence derived from the carboxy terminal amino acids of a connective tissue growth factor protein (CTGF); b) bind to heparin and be eluted from heparin with salt around 0.8M; and c) having a molecular weight of about 10 kDa by reducing SDS-PAGE analysis.
14. The method of claim 12 or 13, wherein the cell is selected from the group consisting of an epithelial cell, a muscle cell, a connective tissue cell, and an endothelial cell.
15. The method of claim 14, wherein the connective tissue cell is selected from the group consisting of an astroglia cell, a fibroblast cell, an osteoclast cell, an osteoblast cell, and a chondrocyte cell.
16. 16. The method of claim 14, wherein the muscle cell is a smooth muscle cell.
17. 17. The method of claim 14, wherein the muscle cell is a cardiac muscle cell.
18. 18. The method of claim 14, wherein the endothelial cell is a capillary endothelial cell.
19. 19. The method of claim 14, wherein the epithelial cell is a secretory epithelial cell.
20. The method of claim 12 or 13, further comprising contacting a cell with growth factor selected from the group consisting of: insulin-like growth factor (IGF-I), platelet-derived growth factor (PDGF) , epidermal growth factor (EGF), beta-transforming growth factor (TGF-β), and basic fibroblast growth factor (bFGF).
21. 21. The method of claim 20, further comprising contacting a cell with heparin.
22. 22. The method of claim 21, wherein the heparin is in a concentration in the range of about 1 to 100 μg / ml.
23. 23. A method for identifying a compound that affects the activity of a polypeptide of claim 1, comprising: a) incubating the compound with a polypeptide of claim 1, or with biologically active fragments thereof, or with a recombinant cell that expresses a polypeptide of claim 1, under conditions sufficient to allow the components to interact; and b) determining the effect of the compound on the activity or expression of a polypeptide of claim 1.
24. 24. The method of claim 23, wherein the effect is inhibition of the activity or expression of a polypeptide of claim 1.
25. The method of claim 23, wherein the effect is stimulating the activity or expression of a polypeptide of claim 1.
26. 26. A method for diagnosing a HBGF condition in a subject suspected of having a condition characterized by associated with HBGF, which comprises obtaining from the subject a sample suspected of containing HBGF; determine the level of HBGF in the sample; and comparing the level of HBGF in the sample with the level of HBGF in a normal standard sample.
27. 27. The method of claim 26, wherein the condition is selected from the group consisting of an arteriosclerotic, fibrotic, sclerotic or cell proliferative disorder.
28. 28. A method for treating a condition associated with HBGF, which comprises administering to a subject having the 15 condition a therapeutically effective amount of a reactive agent to HBGF in a pharmaceutically acceptable carrier.
29. 29. The method of claim 28, wherein the condition is a cell proliferative disorder characterized by an excess of cell growth.
30. 30. The method of claim 29, wherein the excess cell growth is due to an excess of connective tissue cells.
31. 31. The method of claim 28, wherein the HBGF reactive agent is an HBGF antagonist.
32. 32. The method of claim 31, wherein the antagonist is an antibody that specifically binds to HBGF polypeptides.
33. 33. The method of claim 28, wherein the condition is a cell proliferative disorder characterized by a cell growth deficiency.
34. 34. A pharmaceutical composition comprising a therapeutically effective amount of HBGF in a pharmaceutically acceptable carrier.
35. 35. A method of modulating the growth of endometrial or placental fcpLO membranes.