WO1995010610A9

WO1995010610A9 - BETAGLYCAN POLYPEPTIDES HAVING TGF-β BINDING ACTIVITY

Info

Publication number: WO1995010610A9
Application number: PCT/US1994/011648
Authority: WO
Filing date: 1994-10-14
Publication date: 1995-06-22

Abstract

This invention provides polypeptides that bind to TGF-β and that have an amino acid sequence of a portion of a mammalian betaglycan. These polypeptides enhance TGF-β binding to type II receptor and enchance the suppression of cell growth by TGF-β. This invention also provides isolated nucleic acid molecules encoding these polypeptides. This invention also provides methods of detecting and isolating TGF-β. This invention also provides pharmaceutical compositions having these peptides and therapeutic uses of them. This invention also provides decoy betaglycan polypeptides, anti-betaglycan-binding-site antibodies and anti-idiotypic antibodies against anti-betaglycan. It also provides uses of these compositions.

Description

BETAGLYCAN POLYPEPTIDES HAVING TGF-/3 BINDING ACTIVITY

BACKGROUND OF THE INVENTION

This invention relates to the field of polypeptides and, in particular, to betaglycan polypeptides that bind to TGF-β.

This invention was made partially with government support under CA 42507 and CA 30119 awarded by the National Cancer Institute. The government has certain rights in this invention.

Transforming growth factor-beta ("TGF-β") is a multifunctional cytokine that plays an important role in regulating repair and regeneration following tissue injury. Three isoforms of TGF-β (TGF-βl, 2 and 3) are expressed in mammals and to date show similar properties in vitro . Platelets contain high concentrations of TGF- β, and upon degranulation at a site of injury, release TGF-β into the surrounding tissue. TGF-β then initiates a sequence of events that promotes healing including (1) chemo-attraction of onocytes, neutrophils, and fibroblasts, (2) auto-induction of TGF-β production and stimulation of monocytes to secrete interleukin-1 (IL-1), tumor necrosis factor and other cytokines, (3) induction of angiogenesis and cell proliferation, (4) control of inflammation and cell toxicity by acting as a potent immunosuppressant and inhibitor of peroxide release, and (5) increased deposition of extracellular matrix. TGF-β also induces proliferation of macrophages exposed in combination with macrophage colony stimulating factor ("M-CSF") or granulocyte macrophage colony stimulating factor ("GM-CSF").

However, the excessive action of TGF-_/3 is associated with pathological scarring and has been implicated in glomerulonephritis, diabetic nephropathy, lung fibrosis, liver cirrhosis, intimal hyperplasia. cardiac cirrhosis after infarct, adult respiratory distress syndrome and other fibrotic pathologies.

Thus, the regulation of TGF-β activity, both up and down, has important therapeutic significance.

At least three proteins have been identified as part of the cell surface receptor system that transduces the signal from TGF-3 to the cell: type I receptor (RI), type II receptor (RII), and betaglycan (type III receptor) . Betaglycan is a transmembrane proteoglycan whose core protein has molecular weight of about 100 kD. The betaglycan core protein binds TGF-β with high affinity.

The betaglycan core protein has 853 amino acids and consists of four domains. The N-terminal one-third of the extracellular domain and the C—terminal cytoplasmic domain have similarities with endoglin. Endoglin is a TGF-3 binding protein expressed on the surface of endothelial cells. The middle part of the betaglycan extracellular domain has a short stretch similar to a portion of endoglin but otherwise bears no homology to any known protein. The 260 amino acids in the ectodomain closest to the membrane are related to a domain in a group of transmembrane proteins which include sperm receptors Zp2 and Zp3, the zymogen granule membrane protein, GP2, and uro odulin. This common domain occurs at a similar location relative to the transmembrane domain in these proteins.

Betaglycan is the major TGF-β-binding molecule on most cell types. However, many cell types which respond to TGF-β, e.g., hematopoietic cells, do not appear to have detectable amounts of betaglycan. This, together with evidence from TGF-β-resistant mutant cell lines, suggests that betaglycan is not directly involved in the TGF-β signal transduction pathway. Over- expression of betaglycan in Chinese hamster ovary cells enhances binding of TGF-β to type II receptor, suggesting that betaglycan acts by stockpiling TGF-β and presenting it to the signal transducing proteins in the receptor.

Thus, betaglycan has potential as a modulator of TGF-β bioactivity. However, the art does not disclose the portion of betaglycan that has TGF-3 binding activity and that enhances TGF-3 binding to the type II receptor. This invention satisfies this need and provides additional advantages by identifying a portion of betaglycan that binds TGF-/3, enhances TGF-3 binding to the type II receptor and enhances suppression of cell growth by TGF-3.

SUMMARY OF THE INVENTION

This invention provides polypeptides of at least 155 amino acids that bind to TGF-3 and that have a sequence consisting essentially of a sequence of a portion of a mammalian betaglycan within about one-third of the extracellular domain closest to the cell membrane. It also provides polypeptides having a sequence consisting essentially of a portion of a mammalian betaglycan wherein the portion is about one-fourth or about one-fifth of the extracellular domain of a mammalian betaglycan closest to the cell membrane. More particularly, it provides polypeptides wherein the sequence consists essentially of at least amino acids 543 to 769 of SEQ ID N0:2 to at most amino acids 501 to 853 of SEQ ID NO:2.

This invention also provides a soluble polypeptide having the formula A-B-C, wherein A is a sequence excluding amino acid sequences of more than 4 amino acids from amino acids 1 to 543 of SEQ ID N0:2; B is an amino acid sequence consisting essentially of at least amino acids 543 to 769 of SEQ ID NO:2 to at most amino acids 501 to 769 of SEQ ID NO:2; and C is an amino acid sequence.

This invention also provides a soluble polypeptide having the formula A-B-C, wherein A is an amino acid sequence excluding sequences of more than 4 amino acids from amino acids 1 to 543 of SEQ ID NO 2; B is at least 155 amino acids in a sequence consisting essentially of an amino acid sequence within amino acids 543 to 769 of SEQ ID NO:2; and C is an amino acid sequence.

This invention also provides isolated nucleic acid molecules encoding any of the polypeptides of this invention, expression vectors having an expression control sequence operatively linked to a nucleic acid molecule of this invention, and prokaryotic or eukaryotic cells transfected with an expression vector of this invention and capable of expressing a nucleic acid of this invention.

This invention also provides methods of detecting TGF-3 in a sample by contacting the sample with a polypeptide of this invention and determining the amount of TGF-3 bound to the polypeptide.

This invention also provides methods of isolating TGF-3 from a sample by contacting the sample with a polypeptide of this invention bound to a solid support to allow binding of TGF-/3 to the polypeptide and isolating the TGF-β from the polypeptide.

This invention also provides methods of enhancing the binding of TGF-β to a TGF-/3 receptor by contacting a cell bearing a TGF-3 receptor with TGF-3 and a polypeptide of this invention.

This invention also provides methods of enhancing suppression of cell growth by TGF-β comprising contacting a cell with TGF-β and a polypeptide of this invention.

This invention also provides pharmaceutical compositions comprising a polypeptide of this invention in a pharmaceutically acceptable carrier.

This invention also provides methods of treating a subject with a condition ameliorated by the enhanced binding of TGF-β to a TGF-3 receptor or by suppression of cell growth by TGF-β by administering a therapeutically effective amount of a pharmaceutical composition having a polypeptide of this invention.

This invention also provides decoy betaglycan polypeptides. It also provides methods of treating a subject with a condition ameliorated by the diminished binding of TGF-β to a TGF-β receptor or by the inhibition of the suppression of cell growth by TGF-β by administering to the subject a therapeutically effective amount of a pharmaceutical composition having a decoy betaglycan polypeptide. It also provides methods of suppressing TGF-β-induced deposition of extracellular matrix in a subject by administering to the subject a therapeutically effective amount of a pharmaceutical composition having a decoy betaglycan polypeptide.

This invention also provides anti-betaglycan- binding-site antibodies. It also provides methods of treating a subject with a condition ameliorated by the diminished binding of TGF-β to a TGF-β receptor or by the inhibition of the suppression of cell growth by TGF-β by administering to the subject a therapeutically effective amount of a pharmaceutical composition having an anti- betaglycan-binding-site antibody of this invention. It further provides methods of suppressing TGF-β-induced deposition of extracellular matrix in a subject by administering to the subject a therapeutically effective amount of a pharmaceutical composition having an anti- betaglycan-binding-site antibody of this invention.

This invention also provides anti-idiotypic antibodies that mimic the ability of a polypeptide of this invention to bind TGF-β but that lack the biological function of enhancing binding of TGF-β to the type II receptor or enhancing suppression of cell growth by TGF- β. It also provides methods of treating a subject with a condition ameliorated by the diminished binding of TGF-β to a TGF-β receptor or by inhibition of the suppression of cell growth by TGF-β by administering to the subject a therapeutically effective amount of a pharmaceutical composition having an anti-idiotypic antibody of this invention. It also provides methods of suppressing TGF- β-induced deposition of extracellular matrix in a subject by administering to the subject a therapeutically effective amount of a pharmaceutical composition having an anti-idiotypic antibody of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A and IB depict the coding region of rat betaglycan cDNA. The filled part is the transmembrane region. Restriction sites used to generate the cDNA fragments are shown. The brackets show the cDNA fragments that were expressed as proteins (bgl through bg4) . Figure IB depicts recombinant fragments of betaglycan analyzed by SDS-PAGE (4 % to 20 % gels) and Coomassie Blue staining. Figures 2A and 2B present results of a competition assay for the binding of ¹²⁵I-TGF-βl to Hep G2 cells by betaglycan fragments. Hep G2 cells on 24-well dishes were incubated with ¹²⁵I-TGF-βl (100 pM) and with indicated concentrations of betaglycan fragments that had been expressed as fusion protein in bacteria. The bound TGF-β was measured, and the results are expressed as a percent of total binding. Vertical bars show the S.D. of triplicate samples. T=total binding of ¹²⁵I-TGF-βl, NS = nonspecific binding measured as residual binding in the presence of 20 nM of unlabeled TGF-βl. Figure 2A presents the effect of fragments bgl, bg2, bg3, and bg4 and sub-fragments of bg3. Figure 2B presents the effect of bg3 and combinations of bg3 sub-fragments.

Figure 3 shows gel electrophoresis of bg3 betaglycan fragments on the binding of ¹⁵I-TGF-β to MV1 Lu cells in receptor affinity labeling. MV1 Lu cells on 6-well dishes were affinity labeled by using 100 pM of ¹²⁵I-TGF-βl at 37°C without or with various concentrations of the bg3 fragment. After cross-linking the cells were solubilized and analyzed in SDS-PAGE followed by autoradiography. The band at 71 kDa is RI, the bands at and below the 101 kDa marker represent RII and the wide band above the 208 kDa is betaglycan.

Figures 4A and 4B present results of binding studies of the bg3 betaglycan fragment to TGF-βl in a solid-phase binding assay. In Figure 4A, ¹²⁵I-labeled bg3 fragment was incubated in microtiter wells coated with increasing concentrations of TGF-βl in the buffer (•) and in the presence of unlabeled bg3 fragment (o) (lμM). In Figure 4B, ¹²⁵I-bg3 was incubated for the times indicated in microtiter wells coated with 1 μg/ml of TGF-βl. Binding is expressed as percent of the radioactivity added to the wells. Error bars show standard deviation of triplicate samples. Figure 5A and 5B show competition and Scatchard plots for the binding of ¹²⁵I-bg3 betaglycan fragment to immobilized TGF-βl. Figure 5A shows competition of ¹²⁵I- bg3 binding to TGF-βl by increasing concentrations of bg3 fragment. Figure 5B shows Scatchard plot of the binding data in Figure 5A.

Figure 6 shows results of a competition assay of the binding of ¹²⁵I-bg3 betaglycan fragment to immobilized TGF-βl by core proteins of decorin-type proteoglycans. ¹²⁵I-bg3 was incubated in microtiter wells coated with TGF-βl (lμg/ml) in the presence of various concentrations of unlabeled bg3 fragment (O) or fusion proteins of human decorin (■) , human biglycan (^A) , and human fibromodulin (•) core proteins, or maltose-binding protein (♦), the protein the proteoglycan core proteins were fused to. Binding is expressed as a percent of binding of the ¹²⁵I-bg3 in the absence of competitors.

Figure 7 shows the effect of bg3 betaglycan fragment on TGF-β activity in MV 1 Lu cells. MVl Lu assay was performed without (TGF-β-) or with (TGF-β+,0,1 ng/ml) added TGF-β and various concentrations of bg3 betaglycan fragment or an unrelated fusion protein prepared in the same way as bg3 as a control. The bars show ± standard error of mean (n=4).

Figure 8 shows the specificity of the effect of the bg3 betaglycan fragment on TGF-β induced growth suppression. MV 1 Lu assay was performed without or with TGF-β (0.1 ng/ml), bg3 (20 μg/ l) and neutralizing anti- TGF-β antibodies (20 μg/ml) as indicated in the figure. The bars show standard error of mean (n=4).

Figures 9A and 9B depict the nucleotide sequence [SEQ ID N0:1] and deduced amino acid sequence [SEQ ID NO:2] of a cDNA encoding rat betaglycan. Figure 10 depicts the nucleotide sequence [SEQ ID NO:3] and deduced amino acid sequence [SEQ ID NO:4] of a cDNA encoding human betaglycan.

DETAILED DESCRIPTION OF THE INVENTION

This invention identifies, for the first time, a binding site in betaglycan for TGF-β. Results of experiments reported herein demonstrate that a polypeptide containing about one-fourth of the extracellular domain of betaglycan closest to the cell membrane has the TGF-β binding site. In particular, a polypeptide containing amino acids 543 to 769 of rat betaglycan [SEQ ID NO:2] contains this binding site. This polypeptide enhances the binding of TGF-β to the TGF-β type II receptor, enhances suppression of cell growth by TGF-β and competes with decorin-type proteoglycans for TGF-β binding.

This invention provides polypeptides of at least 155 amino acids that bind to TGF-β and that have a sequence consisting essentially of a sequence from a portion of a mammalian betaglycan within about one-third of the extracellular domain closest to the cell membrane. As used herein, the term "sequence" in reference to a polypeptide means the amino acid sequence of the polypeptide. A sequence consists essentially of a sequence of a mammalian betaglycan if it corresponds, or corresponds except for minor modifications, to a portion of a sequence of a mammalian betaglycan.

As used herein, "minor modifications" refers to simple substitutions, additions or deletions that do not eliminate the TGF-β binding capacity of the polypeptide or its ability to enhance TGF-β bioactivity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutation in hosts having DNA encoding these polypeptides. Simple substitutions include the substitution of an amino acid for another having a side chain off the alpha carbon of the same class, i.e. non-polar (hydrophobic) , neutral, positively charged or negatively charged.

The polypeptides of this invention have a sequence from a mammalian betaglycan and, in particular, from human, rat or pig betaglycan. The nucleotide and amino acid sequences of rat betaglycan are given in Figures 9A-9B [SEQ ID N0S:1 and 2] and in Lopez-Casillas et al.. Cell , 67:785-95 (1991) (incorporated herein by reference) . The nucleotide and amino acid sequences of human betaglycan are given in Figure 10 [SEQ ID NOS:3 and 4] and in Moren et al., Biochem. Biophys . Res . Comm. , 189:356-362 (1992) (incorporated herein by reference).

Moren et al. also provides the amino acid sequence of pig betaglycan.

About one-third of the extracellular domain of betaglycan closest to the cell membrane corresponds to about amino acid 501 to 769 of rat betaclycan [SEQ ID N0:2]. This corresponds to about amino acid 497 to 765 [SEQ ID NO:4] of human betaglycan. Other corresponding amino acids are apparent from a comparison of these two sequences.

Polypeptides of this invention include those wherein the portion of a mammalian betaglycan is about one-fifth of the extracellular domain of a mammalian betaglycan closest to the cell membrane. A one-hundred- fifty-five amino acid polypeptide having the sequence of amino acids 615-769 of SEQ ID NO:2 is one such polypeptide.

Preferably, polypeptides of this invention include those wherein the portion of a mammalian betaglycan is about one-fourth of the extracellular domain of a mammalian betaglycan closest to the cell membrane. The two-hundred-twenty-seven amino acid polypeptide having the sequence of amino acids 543-769 of SEQ ID NO:2 is one such polypeptide.

Polypeptides of this invention include those having sequences consisting essentially of a portion of the sequence of rat betaglycan [SEQ ID N0:2], According to one embodiment of the invention, the polypeptide has a sequence consisting essentially of at least amino acids 615 to 769 of SEQ ID NO:2 to at most amino acids 501 to 769 of SEQ ID N0:2.

More preferably, the polypeptide has a sequence consisting essentially of at least amino acids 543 to 769 of SEQ ID NO:2 to at most amino acids 501 to 769 of SEQ ID NO:2. All of these polypeptides include amino acids in a sequence shown to bind to TGF-β. They exclude polypeptides having the sequence that Lopez-Casillas et al.. Cell , 73:1435-44 (1993) asserted bind to TGF-β.

This invention is also directed to soluble polypeptides having the formula: A-B-C, wherein A is an amino acid sequence that excludes sequences of more than 4 amino acids from amino acids 1 to 501 of SEQ ID NO 1; B is a sequence consisting essentially of at least amino acids 543 to 769 of SEQ ID NO:2 to at most amino acids 501 to 769 of SEQ ID NO:2; and C is an amino acid sequence. In one embodiment of the invention, neither A nor C have more than 100 amino acids. Soluble polypeptides are those lacking a transmembrane region, the hydrophobic region of the polypeptide that anchors it in the cell membrane.

This invention is also directed to soluble polypeptides having the formula: A-B-C, wherein A is an amino acid sequence that excludes sequences of more than 4 amino acids from amino acids 1 to 543 of SEQ ID NO 1; B is at least 155 amino acids in a sequence consisting essentially of an amino acid sequence within amino acids 543 to 769 of SEQ ID NO:2; and C is an amino acid sequence. In one embodiment of this invention, neither A nor C have more than 100 amino acids.

The polypeptides of this invention can be produced by synthesis on an automated peptide synthesizer, according to the manufacturer's instructions. For example, MODEL 430A, Applied Biosystems, Foster City, California, USA, is a synthesizer useful for this purpose. The polypeptides of this invention can also be produced by the expression of a nucleic acid molecule that encodes the polypeptide. Methods for expressing the nucleic acids of this invention are described below.

This invention is also directed to isolated nucleic acids that encode any of the polypeptides of this invention. Nucleic acid molecules of this invention can have nucleotide sequences for portions of rat or human betaglycan derived from Figures 9A-9B [SEQ ID NO:2] or Figure 10 [SEQ ID NO:4]. For example, a nucleic acid having a sequence of at least nucleotides 1961 to 2641 and at most 1835 to 2641 of SEQ ID NO:l encodes a polypeptide having a sequence of at least amino acids 543 to 769 of SEQ ID N0:2 to at most amino acids 501 to 769 of SEQ ID NO:2. Nucleic acid molecules of this invention include degenerate versions of sequences of mammalian genes.

This invention is further directed to expression vectors having an expression control sequence operatively linked to a nucleic acid of this invention. Expression vectors useful in this invention include plasmids, cosmids, phage and the like. An expression control sequence is operatively linked to a nucleic acid molecule when it directs the transcription and translation of that molecule in an appropriate host cell. Expression vectors and their use are well known to the art. This invention is further directed to prokaryotic and eukaryotic cells transfected with an expression vector of this invention and capable of expressing the nucleic acid of this invention.

The nucleic acids of this invention can be produced by organic synthesis on a commercial nucleic acid synthesizer or through PCR on a nucleic acid encoding a mammalian betaglycan. Nucleic acid sequences encoding mammalian betaglycans, can be identified by probing cDNA libraries with probes derived from rat betaglycan [SEQ ID N0:1] or human betaglycan [SEQ ID NO:3] and by analyzing cDNA expression libraries with antibodies against betaglycan. Alternatively, betaglycan from these mammals can be isolated and partially sequenced, and the sequence can be used to make sets of degenerate nucleic acid probes for probing gene libraries. Other methods for identifying and isolating genes are also known.

The construction of expression vectors and the expression of genes in transfected cells involves the use of molecular cloning techniques also well known in the art. Sambrook et al.. Molecular Cloning — A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1989) (incorporated herein by reference) provides many protocols in the art of molecular genetics.

The nucleic acids, expression vectors and cells of this invention are useful for producing the polypeptides of this invention. The nucleic acids of this invention also find use as probes for detecting a nucleic acid having a sequence encoding betaglycan.

The polypeptides of this invention find use in methods of detecting TGF-β in a sample. Since levels of TGF-β are altered upon injury or other pathologies, the level of TGF-β is a useful sign of these conditions. The methods involve contacting the sample with a peptide of this invention and determining the amount of TGF-β bound to the polypeptide. According to one embodiment of this method, the well of a microtiter plate is coated with a polypeptide of this invention. The sample is added to the well and incubated under conditions to allow binding of TGF-β to the polypeptide. Unbound sample is removed. Then, the amount of TGF-β bound is determined by, for example, contacting the microtiter well with an anti- TGF-β antibody bound to a reporter group. Reporter groups useful in this invention include chemiluminescent labels, fluorescent labels, radioactive labels, enzyme labels and the like. Variations on this method will be apparent to any person skilled in the art.

The polypeptides of this invention find use in methods of isolating TGF-β from a sample by contacting the mixture with a peptide of this invention bound to a solid support to allow binding and isolating the TGF-β from the polypeptide. According to one embodiment of this method, a polypeptide of this invention is bound to an insoluble matrix and made into an affinity column. The sample is passed over the column under conditions to allow the binding of TGF-β to the polypeptide. Unbound material is washed out of the column. TGF-β is recovered by washing the column with a solution and under conditions that allow TGF-β to become unbound from the polypeptide. Variations on this method will be apparent to any person skilled in the art. The polypeptides of this invention find use in methods of enhancing the binding of TGF-β to type II receptor. These methods involve, for example, contacting a cell bearing a type II receptor with TGF-β and a polypeptide of this invention. This invention also provides methods of enhancing suppression of cell growth, particularly epithelial cell growth, by TGF-β. These methods involve, for example, contacting a cell with TGF- β and a polypeptide of this invention. Embodiments of these methods are described in the Example.

The induction of these cellular events finds use in therapeutic methods in which the events are preventative or ameliorative. In any such therapeutic method, the polypeptides of this invention normally will be administered in a pharmaceutical composition. In one embodiment of the invention, the pharmaceutical compositions comprise the polypeptides of this invention in a pharmaceutically acceptable carrier. The pharmaceutical composition can further comprise TGF-β.

As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as a phosphate-buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. Suitable pharmaceutical carriers and their formulations are described in Martin, Remington 's Pharmaceutical Sciences, 15th Ed. (Mack Publishing Co., Easton 1975). Such compositions will, in general, contain a therapeutically effective amount of the polypeptide together with a suitable amount of carrier so as to prepare the proper dosage form for proper administration to the subject.

This invention provides methods of treating a subject with a condition ameliorated by the enhanced binding of TGF-β to type II receptor or by the enhanced suppression of cell growth by TGF-β by administering to the subject a therapeutically effective amount of a pharmaceutical composition having a polypeptide of this invention. The pharmaceutical composition can further include TGF-β.

As used herein, the term "therapeutically effective amount" is that amount necessary to alleviate the condition from which the subject suffers or prevent such a condition. As used herein, the term "subject" includes humans, other mammals or other vertebrates.

In the practice of the therapeutic methods of the present invention, an effective amount of a polypeptide of this invention, including derivatives or salts thereof, or a pharmaceutical composition containing the same, as described above, is administered via any of the usual and acceptable methods known in the art, either singly or in combination with other pharmaceutical agents.

In the practice of the therapeutic methods of the invention, the particular dosage of pharmaceutical composition to be administered to the subject will depend on a variety of considerations including the nature of the disease, the severity thereof, the schedule of administration, the age and physical characteristics of the subject, and so forth. Proper dosages may be established using clinical approaches familiar to the medicinal arts.

In the practice of the therapeutic methods of this invention, the pharmaceutical compositions can be administered via any of the usual and acceptable methods known in the art, for example orally, parenterally (e.g., intra-muscularly, intravenously, subcutaneously or locally to other tissues) or by inhalation, and in the form of solid or liquid dosage including tablets, suspensions, and aerosols.

This invention also provides decoy betaglycan polypeptides. As used herein, a "decoy betaglycan polypeptide" is a polypeptide having a sequence corresponding to at least a portion of a mammalian betaglycan except for disabling modifications to the amino acid sequence. As used herein, "disabling modifications" refers to simple substitutions, additions or deletions that allow retention of the TGF-β binding capacity of the polypeptide but that eliminate its ability to enhance TGF-β bioactivity, such as the binding of TGF-β to a TGF-β receptor, such as the TGF-β type II receptor, or the suppression of cell growth by TGF-β.

Disabling modifications also allow the decoy to suppress TGF-β bioactivity.

Decoy betaglycan polypeptides contain disabling modifications to betaglycan within the region known to enhance TGF-β bioactivity, that is, within about one- fourth of the extracellular domain of a mammalian betaglycan closest to the cell membrane and, in particular, to amino acids 543 to 769 of SEQ ID NO:2. These modifications can be introduced deliberately, as through site-directed mutagenesis.

Disabling modifications include, for example, the deletion of one or more amino acids, substitution of an amino acid for another having a different class of side chain off the alpha carbon, the elimination of a cyεteine residue involved in disulfide bonding necessary for activity, the introduction of a proline or cysteine residue to alter the polypeptide's secondary structure and the like. Decoy betaglycan polypeptides can be identified by introducing likely disabling modifications into the amino acid sequence and testing the resulting polypeptides for activity in any of the assays known to the art or described herein.

Decoy betaglycan polypeptides find use in detecting TGF-β in a sample and in isolating TGF-β from a sample. They also find use in methods of treating a subject with a condition ameliorated by the diminished binding of TGF-β to TGF-β receptor, by the inhibition of the suppression of cell growth by TGF-β, or the suppression of any other bioactivity of TGF-β. In particular, decoy betaglycan polypeptides find use in suppressing TGF-β-induced deposition of extracellular matrix at a site of tissue injury. Thus, they are useful for preventing or ameliorating glomerulonephritis, diabetic nephropathy, lung fibrosis, liver cirrhosis, intimal hyperplasia, cardiac cirrhosis after infarct, adult respiratory distress syndrome and other fibrosis- related pathologies. These methods involve administering to the subject a therapeutically effective amount of a pharmaceutical composition having a decoy betaglycan polypeptide of this invention.

This invention also provides anti-betaglycan- binding-site antibodies that eliminate the ability of betaglycan to enhance TGF-β bioactivity. This includes the binding of TGF-β to a TGF-β receptor, such as the

TGF-β type II receptor, or the suppression of cell growth by TGF-β. Anti-betaglycan-binding-site antibodies find use in detecting betaglycan in a sample and in isolating betaglycan from a sample. They also find use in methods of treating a subject with a condition ameliorated by the diminished binding of TGF-β to TGF-β receptor, by the inhibition of the suppression of cell growth by TGF-β, or the suppression of any other bioactivity of TGF-β. In particular, anti-betaglycan binding site antibodies find use in suppressing TGF-β-induced deposition of extracellular matrix at a site of tissue injury. Thus, they are useful for preventing or ameliorating glomerulonephritis, diabetic nephropathy, lung fibrosis, liver cirrhosis, intimal hyperplasia, cardiac cirrhosis after infarct, adult respiratory distress syndrome and other fibrosis-related pathologies. These methods involve administering to the subject a therapeutically effective amount of a pharmaceutical composition having an anti-betaglycan-binding-site antibody of this invention.

Anti-betaglycan-binding-site antibodies can be made by inoculating an animal with a polypeptide of this invention. For example, an aminal can be inoculated with a polypeptide having the sequence of amino acids 543 to 769 of rat betaglycan [SEQ ID N0:2].

An idiotype represents the specificity of an antibody for its ligand at the binding site. An anti- idiotypic antibody is an antibody directed against the idiotype of another antibody. They are made by immunizing an animal with the other antibody. Anti- idiotypic antibodies can be made that have the internal image of the antigen against which the other antibody is directed and that mimic the binding characteristics of the antigen.

Accordingly, this invention is also directed to anti-idiotypic antibodies that mimic the ability of the polypeptides of this invention to bind TGF-β. Such anti- idiotypic antibodies lack the biological function of betaglycan to enhance binding of TGF-β to the type II receptor or to enhance the suppression of cell growth by TGF-β. They also suppress other bioactivities of TGF-β. Anti-idiotypic antibodies of this invention can be made by inoculating an animal with an anti-betaglycan-binding- site antibody of this invention. The anti-idiotypic antibodies of this invention find use in detecting TGF-β in a sample and in isolating TGF-β from a sample. They also find use in methods of treating a subject with a condition ameliorated by the diminished binding of TGF-β to a TGF-β receptor or by the inhibition of the suppression of cell growth by TGF-β. They also find use in methods of suppressing TGF-β- induced deposition of extracellular matrix at a site of tissue injury. Thus, they are useful for preventing or ameliorating conditions associated with this that are described above. The methods involve administering to a subject a therapeutically effective amount of a pharmaceutical composition having an anti-idiotypic antibody of this invention.

Methods of immunizing animals, isolating antibodies, and producing polyclonal or monoclonal antibodies are well known in the art and are described in, e.g., Harlow and Lane, Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1988). Kennedy et al.. Scientific American, 255:48-56 (July 1986) and Carlsson and Glad, Bio/Technology, 7:567-73 (June 1989) describe anti- idiotypic antibodies. All of the above-identified texts are incorporated herein by reference.

The following Example is intended to illustrate but not limit the invention.

EXAMPLE

The pBluescript SK(+) vector and XLl Blue host cells were purchased from STRATAGENE® (La Jolla, California). The expression vectors, pQE8, 10, 11 and M15 host cells and Ni-NTA-agarose came from QIAGEN® (Chatsworth, California) , the buffer for PCR reactions from IDAHO TECHNOLOGY® (Idaho Falls, Idaho) and the Taq polymerase from BOEHRINGER MANNHEIM®. GM 242 host cells (dam") were used in these experiments. Other dam" cell lines, for example, GM 48 or GM 163 cells (available from New England Biolabs, Beverly, Massachusetts) can also be used. The purification kits for PCR fragments and for the plasmid were from PROMEGA® (Madison, Connecticut). All other cloning reagents were from INTERNATIONAL BIOTECHNOLOGIES INC.® (La Jolla, California) and BIORAD® (Richmond, California) .

TGF-βl is commercially available from Genzyme

(Cambridge, Massachusetts). SDS-polyacrylamide gel electrophoresis (PAGE) pre-cast gels were from NOVEX® (San Diego, California) . Hep G2 and Mv 1 Lu cells were from American Type Culture Collection (ATCC HB 8065 and ATCC CCL 64, respectively). Fetal calf serum was purchased from TISSUE CULTURE BIOLOGICALS® (Tulare, California) and L-glutamine, antibiotics and antimycotic agents from IRVINE SCIENTIFIC® (Santa Ana, California). DMEM (Dulbecco's modified Eagle's medium) was from GIBCO® (Gaithersburg, Maryland) , carrier-free Na¹²⁵I and ³H- thy idine from NEW ENGLAND NUCLEAR® (Boston, Massachusetts), and IODO-GEN® from Pierce Chemical Co. (Rockford, Illinois). IMMULON 2 REMOVAWELL® strips came from Dynatech Laboratories Inc. (Chantilly, Virginia), and CENTRICON® micro-concentrator from Amicon (Danvers, Massachusetts). All chromatographic materials including pre-packed PD-10 columns were from PHARMACIA® (Uppsala, Sweden) . Neutralizing chicken anti-TGF-β antibody and normal chicken Ig were from R & D SYSTEMS® (Minneapolis, Minnesota) .

The following segments from the DNA sequence of rat betaglycan [SEQ ID NO:l] were first amplified by PCR with appropriate tags for cloning purposes: nucleotides 404-1439 (A) ; nucleotides 1126-2268 (B) ; nucleotides 1712-3042 (C) ; and nucleotides 1961-2641 (D) (Lopez- Casillas et al.. Cell , 67: 785-95 (1991)). PCR reactions were performed on cDNA derived from rat smooth muscle cells using a MODEL 1605® Air Thermo-Cycler (Idaho Technology, Idaho Falls, ID) . The fragments were ligated into the TA cloning site of the pBluescript SK vector (Marchuk et al., Nucl . Acids . Res . , 19:1154 (1990)) and the recombinant vector was transformed into XL1 Blue. The plasmid carrying PCR product B was re-transformed into GM 242 to recover a demethylated Bel I site. All transformations were carried out using the CELL-PORATOR® (BRL, Gaithersburg, MD) electroporation system, and positive colonies were selected by the addition of isopropyl-b-D-thiogalactopyranoside (IPTG) and X-gal. The sequences of the PCR-generated DΝAs completely agreed with the published sequence for betaglycan (Lόpez-

Casillas et al., 1991, supra ,- Viang et al.. Cell , 67:792- 805 (1991)).

From product A, fragment bgl was excised by Bam HI digestion. Fragment bg2 was obtained from product B by Bel I and Sal I digestion, and fragments bg3'Ν, bg3N and bg3C were prepared from product C by Bam HI, Bam HI and Xmn I, and Bam HI and Bgl II digestion, respectively. Product D was designated bg3. Fragments bgl, bg3, bg3'N and bg3N were ligated into the Bam HI site of the pQE8 expression vector. Fragments bg3C and bg4 were cloned into pQElO and bg2 into pQEll. The vectors were transformed into M15 host cells by electroporation. Positive clones were selected by testing for the expression of the protein product.

The expression and purification of the bacterially expressed proteins were carried out according to instructions on page 16 of Qiagen's manual. Briefly, the expression was induced by 2 mM of IPTG and continued for 3 hr, after which the bacteria were lysed in 6 M guanidine-HCl, Tris buffer, pH 8. The supernatant was loaded onto a column of Ni-NTA resin, and the column was eluted with 8 M urea in Tris buffer, pH 4.5, after several washing steps. The protein solution was neutralized with 1 M Tris buffer, pH 9, containing 8 M urea. The proteins were treated with dithiotreitol (50 °C for 30 min) and iodoacetamide (25 °C for 30 min) to reduce and alkylate cysteine residues. (Matsudaira, "A Practical Guide to Protein and Peptide Purification for Microsequencing," Academic Press, (1989)). The reaction mixture was loaded onto a PD-10 column equilibrated with 0.1 M ammonium bicarbonate containing 6 M urea. Elution of protein was monitored by protein assay (Bradford, Anal. Biochem., 72:248-54 (1976)), and the main fraction was concentrated with CENTRICON 10®.

Eighty-percent-confluent monolayers of cells maintained in DMEM containing 10% FCS were used for TGF-β binding assays and affinity labeling experiments. The conditions used were essentially as described earlier, except that the labeling reaction, which is usually performed at +4 °C, was also carried out at 37 °C

(Massague, Meth . Enz . , 146:174-195 (1987)). Competitors were dissolved in 6 M urea at 150-fold molar excess of solution. Final samples were prepared by diluting the sample 150-fold into assay buffer containing the ¹²⁵I-TGF- βl. The following buffers were used: 25 mM Hepes, pH 7.4, 125 mM NaCl, 5 mM MgS0₄, 5 mM KC1, 1 mM CaCl₂, 2 g/ml bovine serum albumin (BSA) ("binding buffer"); 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 % Triton X-100 ("solubilization buffer"); 10 mg/ml antipain, 10 mg/ml leupeptin, 2 mM benzamidine, 1 mM ethylenediamine- tetraacetic acid ("protease inhibitor cocktail," final concentrations) . A fresh stock of disuccinimidyl suberate (10 mg/ml in dimethyl sulfoxide) was prepared for each experiment, and a 1:200 dilution in binding buffer without BSA was used in cross-linking for 15 minutes on ice. The reaction was quenched by 10 mM Tris- HC1, pH 7.4, 150 mM NaCl and the samples were solubilized and analyzed.

Binding of ¹²⁵I-labeled proteins to immobilized TGF-βl was determined using a solid-phase binding assay (Mooradian et al., J. Cell . Biochem. , 41:189-200

((1989)). Briefly, 96-well IMMULON 2® plates were coated with 1 mg/ml TGF-bl (100 ml, 0.1 M carbonate buffer, pH 9.5) at 4 °C for 16 hr. The wells were blocked by incubating with the blocking buffer containing 2 % BSA, 0.005 % Tween 20, 0.02 % NaN₃ for 2 hr at 37 °C. An equal volume of varying concentrations of competitors, diluted in 6 M urea, was mixed in the assay buffer (same as the blocking buffer) containing the labeled proteins. Since the binding was found to be sensitive to urea, the final urea concentration was always kept under 100 mM. Duplicate 100 ml samples were incubated in the wells for 5 hr at 37 °C. After four washings with the same buffer, the bound radioactivity was measured in a Beckman Gamma- 4000 counter.

Data from the binding assays were analyzed using a LIGAND computer program (Munson and Rodbard, Anal . Biochem. , 107:220-239 (1980)) on an Apple Macintosh II computer.

The mink lung epithelial cell growth inhibition assay was done as described (Danielpour et al., J. Cell . Physiol . , 138:79-86 (1989)). Briefly, cells were grown in Dulbecco's medium containing 10 % fetal calf serum, 10 mM L-glutamine, 100 IU/ml penicillin and 100 mg/ml streptomycin. For the experiments, the cells were plated on 96-well plates (20,000 cells/well) in the same medium. After one day the medium was replaced by Dulbecco's medium containing 1 % fetal calf serum with glutamine and antibiotics. The experiments were started the next day by adding the controls and effectors to the cells in the above medium. The fusion proteins were dialyzed against assay medium. After 24 hours of incubation the cells were pulsed with ³H-thymidine for 3 hours. The cells were fixed with methanol and extracted 3 times with 10 % trichloracetic acid, solubilized with 1 % SDS-0.3 N NaOH and the radioactivity was counted.

SDS-PAGE was performed according to Laemmli (Nature , 227:680-685 (1970)) using vertical precast gels. For autoradiography after electrophoresis the gels were fixed in 10 % isopropanol, 10 % acetic acid for 15 minutes and dried. Kodak X-OMAT/AR® film with an enhancing screen was used for autoradiography.

Each of the pairs of PCR primers from the published rat betaglycan sequence generated a DΝA fragment of the expected size. Shorter DΝA fragments were prepared from the primary PCR products as shown in Figure 1A and ligated into a vector that expresses the cloned protein as a fusion protein with a cassette of six histidines. The expression system yielded 0.5 mg to 1.0 mg each of the betaglycan protein fragments from a 250 ml culture. The bgl fragment contained a second band that may represent a fragment of the fusion protein; the other fusion protein products were essentially homogeneous. (Figure IB.)

Betaglycan fragments bgl, bg2 and bg3 were tested for their ability to inhibit the binding of ¹²⁵l- TGF-bl to Hep G2 cells; in these cells most of the specific binding of TGF-β is to betaglycan. Hep G2 cells on 24-well dishes were incubated with ¹²⁵I-TGF-βl (100 pM) and with indicated concentrations of betaglycan fragments that had been expressed as fusion protein in bacteria. The bound TGF-β was measured. Ten percent of the total labeled protein bound to the cells in the absence of any competitor, and more than 90 % of this binding was displaced by the addition of 40 nM of unlabeled TGF-βl.

Among the betaglycan fragments, only bg3 inhibited the binding of ¹²⁵I-TGF-β to Hep G2 and the inhibition was concentration dependent. (Figure 2A. )

The inhibitory activity of the bg3 fragment was lost upon further fragmentation. (Figure 2A. ) Combinations of the sub-fragments also showed little or no effect. (Figure 2B. ) There was some apparent enhancement of TGF-β binding by the bgl and bg2 fragments.

In affinity cross-linking of ¹²⁵I-TGF-β to the cell surface receptors of MV 1 Lu cells, the bg3 fragment enhanced the binding of TGF-β to the bands representing the type II receptor and to betaglycan itself at low concentrations (up to 50 nM) of the fragment. (Figure 3.) Higher concentrations competed for the binding of TGF-β to these binding sites. The cross-linking of TGF-β to RI was unaffected by bg3 concentrations up to 500 nM, but higher concentrations appeared to be inhibitory.

The availability of the TGF-β-binding betaglycan fragment were used to study various parameters of its binding to TGF-β. A solid-phase binding assay in which ¹²⁵I-labeled betaglycan fragment binds to immobilized TGF-β showed that saturation of binding was approached at the coating concentration of 10 mg/ml of TGF-βl. (Figure 4A. ) The binding was inhibited by 1 mM of unlabeled bg3 fragment. The binding of the bg3 fragment to TGF-β reached a maximum (about 25 % of total added) after 5 hours of incubation when 1 mg/ml of TGF-βl was used for the coating. (Figure 4B.)

Homologous competition for the binding of ¹²⁵I- bg3 to immobilized TGF-βl by various concentrations of unlabeled bg3 was performed. (Figure 5A. ) Scatchard analysis of these results showed an excellent theoretical fit with a two-site binding model giving two dissociation constants, K_d = 3.9 nM and K_d = 145 nM. (Figure 5B. )

The core proteins of extracellular matrix proteoglycans decorin, biglycan and fibromodulin bind TGF-β. Solid-phase binding assay, used to compare the TGF-β binding characteristics of these proteoglycans and betaglycan, showed that fusion proteins representing the core proteins of decorin, biglycan and fibromodulin each inhibited the binding of the bg3 betaglycan fragment to TGF-β, whereas the fusion partner, maltose-binding protein, showed no significant effect. (Figure 6.) The 50 % inhibitory concentrations for the extracellular matrix proteoglycans were similar or slightly higher than for the betaglycan fragment. In a reverse experiment, the binding of labeled biglycan and fibromodulin core proteins to immobilized TGF-βl was inhibited by unlabeled betaglycan fragment.

The bg3 betaglycan fragment, when administered together with a sub-maximally effective concentration of TGF-βl, enhanced the activity of TGF-βl in a concentration-dependent fashion. (Figure 7.) The fragment alone had no effect on DNA synthesis of MV 1 Lu cells. An unrelated fusion protein used as a negative control had no effect. The TGF-β-promoting effect of bg3 could be blocked by adding neutralizing anti-TGF-β antibodies into the assay. (Figure 8.) Non-immune IgG, which was used as a control, did not have this effect. >

Little activity was found in the subfragments of the 30 kilodalton bg3 fragment in the assay that measures the ability of the fragments to inhibit the binding of TGF-β to cell surface receptors. This suggests that the TGF-β binding site in betaglycan may be an extended structure or that the binding site had lost its proper conformation in the subfragments, resulting in a decrease of affinity.

Assays described above showed that betaglycan and the decorin-type small extracellular matrix proteoglycans interfered with one another's binding to TGF-β. This result suggests that the decorin-type proteoglycans bind to the same or overlapping site in TGF-β as betaglycan. That this is the case is also indicated by the finding that the decorin-type proteoglycans compete with the binding of TGF-β to betaglycan in receptor affinity cross-linking experiments.

Even though endoglin and betaglycan share sequence similarities and bind to TGF-β in a similar manner, the region in betaglycan where we have localized the TGF-β binding site shows no apparent similarity with any part of endoglin. However, the decorin-type proteoglycans also show no sequence similarity with betaglycan or endoglin. Moreover, the binding specificity of endoglin is different from that of betaglycan in that endoglin does not bind TGF-β2, whereas betaglycan does. It may be that the sequence similarities in betaglycan and endoglin relate to shared functions of these molecules other than the TGF-β binding.

One secondary function of betaglycan and possibly also endoglin, is the modulation of TGF-β binding to the signal transduction receptors. We found that the active betaglycan fragment could increase the binding of TGF-β to the type II receptor in cell surface affinity labeling and that the fragment enhanced the bioactivity of TGF-β when added to cell cultures as a soluble protein. The enhancement of the type II receptor binding is in agreement with the results of Lopez- Casillas et al. (1991), supra , who found that expression of recombinant betaglycan in a cell line that originally had little of it increased the TGF-β binding activity of the type II receptor.

It is noteworthy that our affinity labeling experiments showed inhibition of TGF-β binding to the type II receptor at high concentrations of the betaglycan fragment, even though the bioassay showed increased activity at those same concentrations. The explanation for these results may be that the two assays are done under different conditions; we were able to eliminate some of the differences in the test conditions but not others. We found that when the affinity labeling was performed at +4°, which is customary, the betaglycan fragment inhibited the affinity labeling of the type II receptor at all concentrations of the fragment (result not shown) . However, when the temperature was raised to the 37° bioassay temperature, low and moderate concentrations of the fragment, now in agreement with the bioassay results, enhanced type II receptor labeling. The remaining major difference in the two assays, the time of incubation (10 minutes versus 24 hours), may explain the discrepancy of the results at the high fragment concentrations.

Although the invention has been described with reference to the presently-preferred embodiments, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the claims that follow. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: La Jolla Cancer Research Foundation

(ii) TITLE OF INVENTION: BETAGLYCAN POLYPEPTIDES HAVING TGF-BETA BINDING ACTIVITY

(iii) NUMBER OF SEQUENCES: 4

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Campbell and Flores

(B) STREET: 4370 La Jolla Village Drive, Suite 700

(C) CITY: San Diego

(D) STATE: California

(E) COUNTRY: USA

(F) ZIP: 92122

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: 14-OCT-1994

(C) CLASSIFICATION:

(Viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Campbell, cathryn A.

(B) REGISTRATION NUMBER: 31,815

(C) REFERENCE/DOCKET NUMBER: FP-LA 1185

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (619) 535-9001

(B) TELEFAX: (619) 535-8949

(2) INFORMATION FOR SEQ ID Nθ:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3237 base pairs

(B) TYPE: nucleic acid

(C) STRANDΞDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 241..2799

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

CAGGAGGTGA AAGTCCCCGG CGGTCCGGAT GGCGCAGTTG CACTGCGCTG CTGAGCTCGC 60

GGCCGCCTGC GCACACTGGG GGGACTCGCT TCGGCTAGTA ACTCCTCCAC CTCGCGGCGG 120

ACGACCGGTC CTGGACACGC TGCCTGCGAG GCAAGTTGAA CAGTGCAGAG AAGGATCTTA 180

AAGCTACACC CGACTTGCCA CGATTGCCTT CAATCTGAAG AACCAAAGGC TGTTGGAGAG 240 ATG GCA GTG ACA TCC CAC CAC ATG ATC CCG GTG ATG GTT GTC CTG ATG 288 Met Ala Val Thr Ser His His Met He Pro Val Met Val Val Leu Met 1 5 10 15

AGC GCC TGC CTG GCC ACC GCC GGT CCA GAG CCC AGC ACC CGG TGT GAA 336 Ser Ala Cys Leu Ala Thr Ala Gly Pro Glu Pro Ser Thr Arg Cys Glu 20 25 30

CTG TCA CCA ATC AAC GCC TCT CAC CCA GTC CAG GCC TTG ATG GAG AGC 384 Leu Ser Pro He Asn Ala ser His Pro Val Gin Ala Leu Met Glu Ser 35 40 45

TTC ACC GTT CTG TCT GGC TGT GCC AGC AGA GGC ACC ACC GGG CTG CCA 432 Phe Thr Val Leu Ser Gly Cys Ala Ser Arg Gly Thr Thr Gly Leu Pro 50 55 60

AGG GAG GTC CAT GTC CTA AAC CTC CGA AGT ACA GAT CAG GGA CCA GGC 480 Arg Glu Val His Val Leu Asn Leu Arg Ser Thr Asp Gin Gly Pro Gly 65 70 75 80

CAG CGG CAG AGA GAG GTT ACC CTG CAC CTG AAC CCC ATT GCC TCG GTG 528 Gin Arg Gin Arg Glu Val Thr Leu His Leu Asn Pro He Ala Ser Val 85 90 95

CAC ACT CAC CAC AAA CCT ATC GTG TTC CTG CTC AAC TCC CCC CAG CCC 576 His Thr His His Lys Pro lie Val Phe Leu Leu Asn Ser Pro Gin Pro 100 105 110

CTG GTG TGG CAT CTG AAG ACG GAG AGA CTG GCC GCT GGT GTC CCC AGA 624 Leu Val Trp His Leu Lys Thr Glu Arg Leu Ala Ala Gly Val Pro Arg 115 120 125

CTC TTC CTG GTT TCG GAG GGT TCT GTG GTC CAG TTT CCA TCA GGA AAC 672 Leu Phe Leu Val Ser Glu Gly ser Val Val Gin Phe Pro Ser Gly Asn 130 135 140

TTC TCC TTG ACA GCA GAA ACA GAG GAA AGG AAT TTC CCT CAA GAA AAT 720 Phe Ser Leu Thr Ala Glu Thr Glu Glu Arg Asn Phe Pro Gin Glu Asn 145 150 155 160

GAA CAT CTC GTG CGC TGG GCC CAA AAG GAA TAT GGA GCA GTG ACT TCC 768 Glu His Leu Val Arg Trp Ala Gin Lys Glu Tyr Gly Ala Val Thr ser 165 170 175

TTC ACT GAA CTC AAG ATA GCA AGA AAC ATC TAT ATT AAA GTG GGA GAA 816 Phe Thr Glu Leu Lys He Ala Arg Asn He Tyr He Lys Val Gly Glu 180 185 190

GAT CAA GTG TTT CCT CCT ACG TGT AAC ATA GGG AAG AAT TTC CTC TCA 864 Asp Gin Val Phe Pro Pro Thr Cys Asn He Gly Lys Asn Phe Leu Ser 195 200 205

CTC AAT TAC CTT GCC GAG TAC CTT CAA CCC AAA GCC GCC GAA GGT TGT 912 Leu Asn Tyr Leu Ala Glu Tyr Leu Gin Pro Lys Ala Ala Glu Gly Cys 210 215 220

GTC CTG CCC AGT CAG CCC CAT GAA AAG GAA GTA CAC ATC ATC GAG TTA 960 Val Leu Pro Ser Gin Pro His Glu Lys Glu Val His He He Glu Leu 225 230 235 240

ATT ACC CCC AGC TCG AAC CCT TAC AGC GCT TTC CAG GTG GAT ATA ATA 1008 He Thr Pro Ser ser Asn Pro Tyr ser Ala Phe Gin Val Asp He He 245 250 255

GTT GAC ATA CGA CCT GCT CAA GAG GAT CCC GAG GTG GTC AAA AAC CTT 1056 Val Asp He Arg Pro Ala Gin Glu Asp Pro Glu Val Val Lys Asn Leu 260 265 270 GTC CTG ATC TTG AAG TGC AAA AAG TCT GTC AAC TGG GTG ATC AAG TCT 1104 Val Leu He Leu Lys Cys Lys Lys Ser Val Asn Trp Val lie Lys Ser 275 280 285

TTT GAC GTC AAG GGA AAC TTG AAA GTC ATT GCT CCC AAC AGT ATC GGC 1152 Phe Asp Val Lys Gly Asn Leu Lys val He Ala Pro Asn Ser He Gly 290 295 300

TTT GGA AAA GAG AGT GAA CGA TCC ATG ACA ATG ACC AAA TTG GTA AGA 1200 Phe Gly Lys Glu Ser Glu Arg Ser Met Thr Met Thr Lys Leu Val Arg 305 310 315 320

GAT GAC ATC CCT TCC ACC CAA GAG AAT CTG ATG AAG TGG GCA CTG GAC 1248 Asp Asp He Pro Ser Thr Gin Glu Asn Leu Met Lys Trp Ala Leu Asp 325 330 335

AAT GGC TAC AGG CCA GTG ACG TCA TAC ACA ATG GCT CCC GTG GCT AAT 1296 Asn Gly Tyr Arg Pro Val Thr Ser Tyr Thr Met Ala Pro Val Ala Asn 340 345 350

AGA TTT CAT CTT CGG CTT GAG AAC AAC GAG GAG ATG AGA GAT GAG GAA 1344 Arg Phe His Leu Arg Leu Glu Asn Asn Glu Glu Met Arg Asp Glu Glu 355 360 365

GTC CAC ACC ATT CCT CCT GAG CTT CGT ATC CTG CTG GAC CCT GAC CAC 1392 Val His Thr lie Pro Pro Glu Leu Arg He Leu Leu Asp Pro Asp His 370 375 380

CCG CCC GCC CTG GAC AAC CCA CTC TTC CCA GGA GAG GGA AGC CCA AAT 1440 Pro Pro Ala Leu Asp Asn Pro Leu Phe Pro Gly Glu Gly Ser Pro Asn 385 390 395 400

GGT GGT CTC CCC TTT CCA TTC CCG GAT ATC CCC AGG AGA GGC TGG AAG 1488 Gly Gly Leu Pro Phe Pro Phe Pro Asp He Pro Arg Arg Gly Trp Lys 405 410 415

GAG GGC GAA GAT AGG ATC CCC CGG CCA AAG CAG CCC ATC GTT CCC AGT 1536 Glu Gly Glu Asp Arg He Pro Arg Pro Lys Gin Pro He Val Pro Ser 420 425 430

GTT CAA CTG CTT CCT GAC CAC CGA GAA CCA GAA GAA GTG CAA GGG GGC 1584 Val Gin Leu Leu Pro Asp His Arg Glu Pro Glu Glu Val Gin Gly Gly 435 440 445

GTG GAC ATC GCC CTG TCA GTC AAA TGT GAC CAT GAA AAG ATG GTC GTG 1632 Val Asp He Ala Leu Ser Val Lys cys Asp His Glu Lys Met Val Val 450 455 460

GCT GTA GAC AAA GAC TCT TTC CAG ACC AAT GGC TAC TCA GGG ATG GAG 1680 Ala Val Asp Lys Asp Ser Phe Gin Thr Asn Gly Tyr Ser Gly Met Glu 465 470 475 480

CTC ACC CTG TTG GAT CCT TCG TGT AAA GCC AAA ATG AAT GGT ACT CAC 1728 Leu Thr Leu Leu Asp Pro Ser cys Lys Ala Lys Met Asn Gly Thr His 485 490 495

TTT GTT CTC GAG TCT CCC CTG AAT GGC TGT GGT ACT CGA CAT CGG AGG 1776 Phe Val Leu Glu Ser Pro Leu Asn Gly Cys Gly Thr Arg His Arg Arg 500 505 510

TCG ACC CCG GAT GGT GTG GTT TAC TAT AAC TCT ATT GTG GTG CAG GCT 1824 Ser Thr Pro Asp Gly Val Val Tyr Tyr Asn Ser He Val Val Gin Ala 515 520 525

CCG TCC CCT GGG GAT AGC AGT GGC TGG CCT GAT GGC TAT GAA GAC TTG 1872 Pro Ser Pro Gly Asp Ser Ser Gly Trp Pro Asp Gly Tyr Glu Asp Leu 530 535 540 GAG TCA GGC GAT AAT GGA TTT CCT GGA GAC GGG GAT GAA GGA GAA ACT 1920 Glu Ser Gly Asp Asn Gly Phe Pro Gly Asp Gly Asp Glu Gly Glu Thr 545 550 555 560

GCC CCC CTG AGC CGA GCT GGA GTG GTG GTG TTT AAC TGC AGC TTG CGG 1968 Ala Pro Leu Ser Arg Ala Gly Val Val Val Phe Asn Cys Ser Leu Arg 565 570 575

CAG CTG AGG AAT CCC AGT GGC TTC CAG GGC CAG CTC GAT GGA AAT GCT 2016 Gin Leu Arg Asn Pro Ser Gly Phe Gin Gly Gin Leu Asp Gly Asn Ala 580 585 590

ACC TTC AAC ATG GAG CTG TAT AAC ACA GAC CTC TTT CTG GTG CCC TCC 2064 Thr Phe Asn Met Glu Leu Tyr Asn Thr Asp Leu Phe Leu Val Pro Ser 595 600 605

CCA GGG GTC TTC TCT GTG GCA GAG AAC GAG CAT GTT TAT GTT GAG GTG 2112 Pro Gly Val Phe Ser Val Ala Glu Asn Glu His Val Tyr Val Glu Val 610 615 620

TCT GTC ACC AAG GCT GAC CAA GAT CTG GGA TTC GCC ATC CAA ACC TGC 2160 Ser Val Thr Lys Ala Asp Gin Asp Leu Gly Phe Ala He Gin Thr Cys 625 630 635 640

TTT CTC TCT CCA TAC TCC AAC CCA GAC AGA ATG TCT GAT TAC ACC ATC 2208 Phe Leu Ser Pro Tyr Ser Asn Pro Asp Arg Met Ser Asp Tyr Thr He 645 650 655

ATC GAG AAC ATC TGT CCG AAA GAC GAC TCT GTG AAG TTC TAC AGC TCC 2256 He Glu Asn He Cys Pro Lys Asp Asp Ser Val Lys Phe Tyr Ser Ser 660 665 670

AAG AGA GTG CAC TTT CCC ATC CCG CAT GCT GAG GTG GAC AAG AAG CGC 2304 Lys Arg Val His Phe Pro He Pro His Ala Glu Val Asp Lys Lys Arg 675 680 685

TTC AGC TTC CTG TTC AAG TCT GTG TTC AAC ACC TCC CTG CTC TTC CTG 2352 Phe Ser Phe Leu Phe Lys Ser Val Phe Asn Thr Ser Leu Leu Phe Leu 690 695 700

CAC TGC GAG TTG ACT CTG TGC TCC AGG AAG AAG GGC TCC CTG AAG CTG 2400 His Cys Glu Leu Thr Leu Cys Ser Arg Lys Lys Gly Ser Leu Lys Leu 705 710 715 720

CCG AGG TGT GTC ACT CCT GAC GAC GCC TGC ACT TCT CTC GAT GCC ACC 2448 Pro Arg Cys Val Thr Pro Asp Asp Ala Cys Thr Ser Leu Asp Ala Thr 725 730 735

ATG ATC TGG ACC ATG ATG CAG AAT AAG AAG ACA TTC ACC AAG CCC CTG 2496 Met He Trp Thr Met Met Gin Asn Lys Lys Thr Phe Thr Lys Pro Leu 740 745 750

GCT GTG GTC CTC CAG GTA GAC TAT AAA GAA AAT GTT CCC AGC ACT AAG 2544 Ala Val Val Leu Gin Val Asp Tyr Lys Glu Asn Val Pro Ser Thr Lys 755 760 765

GAT TCC AGT CCA ATT CCT CCT CCT CCT CCA CAG ATT TTC CAT GGC CTG 2592 Asp Ser Ser Pro He Pro Pro Pro Pro Pro Gin He Phe His Gly Leu 770 775 780

GAC ACG CTC ACC GTG ATG GGC ATT GCA TTT GCA GCA TTT GTG ATC GGA 2640 Asp Thr Leu Thr Val Met Gly lie Ala Phe Ala Ala Phe Val He Gly 785 790 795 800

GCG CTC CTG ACG GGG GCC TTG TGG TAC ATC TAC TCC CAC ACA GGG GAG 2688 Ala Leu Leu Thr Gly Ala Leu Trp Tyr He Tyr Ser His Thr Gly Glu 805 810 815 ACA GCA CGA AGG CAG CAA GTC CCT ACC TCG CCG CCA GCC TCG GAG AAC 2736 Thr Ala Arg Arg Gin Gin Val Pro Thr Ser Pro Pro Ala Ser Glu Asn 820 825 830

AGC AGC GCG GCC CAC AGC ATC GGC AGC ACT CAG AGT ACC CCC TGC TCT 2784 Ser Ser Ala Ala His Ser He Gly Ser Thr Gin Ser Thr Pro cys Ser 835 840 845

AGC AGC AGC ACA GCC TAGGTGGACA GACAGACGCC CGCCCACCGC AGCCAGGGCA 2839 Ser ser ser Thr Ala 850

GGGCCCGATG CCAGTGCTGC GTGTCCACAG TCAGAAGTCT TGATCTGGGC TCCCTGTAAA 2899

GAAAGAGTGA ATTTCAGTAT ACAGACAGCC AGTTCTACCC ACCCCTTACC ACGGCCCACA 2959

TAAATGTGAC CCTGGGCATC TGTCACACGA AAGCTAAGCT GGTGGCCTTC CCCACCAGCC 3019

CCTCGCAGGA TGGGGGTTTC AATGTGAAAC ATCTGCCAGT TTTGTTTTGT TTTTTTAATG 3079

CTGCTTTGTC CAGGTGTCCA AACATCCATC ATTTGGGGTG GTCTGTTTTA CAGAGTAAAG 3139

GAGGCGGTGA AGGGACGTCA GCTAGTGTGT AGAGCCAAGG GGAGACAGCT AGGATTCTCG 3199

CCTAGCTGAA CCAAGGTGTA AAATAGAAGA CACGCTCC 3237

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 853 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

( i) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Ala Val Thr Ser His His Met He Pro Val Met Val Val Leu Met 1 5 10 15

Ser Ala cys Leu Ala Thr Ala Gly Pro Glu Pro Ser Thr Arg Cys Glu 20 25 30

Leu Ser Pro He Asn Ala Ser His Pro Val Gin Ala Leu Met Glu Ser 35 40 45

Phe Thr Val Leu Ser Gly Cys Ala Ser Arg Gly Thr Thr Gly Leu Pro 50 55 60

Arg Glu Val His Val Leu Asn Leu Arg ser Thr Asp Gin Gly Pro Gly 65 70 75 80

Gin Arg Gin Arg Glu Val Thr Leu His Leu Asn Pro He Ala Ser Val 85 90 95

His Thr His His Lys Pro He Val Phe Leu Leu Asn Ser Pro Gin Pro 100 105 110

Leu Val Trp His Leu Lys Thr Glu Arg Leu Ala Ala Gly Val Pro Arg 115 120 125

Leu Phe Leu Val Ser Glu Gly Ser Val val Gin Phe Pro ser Gly Asn 130 135 140

Phe ser Leu Thr Ala Glu Thr Glu Glu Arg Asn Phe Pro Gin Glu Asn 145 150 155 160 Glu His Leu Val Arg Trp Ala Gin Lys Glu Tyr Gly Ala Val Thr Ser 165 170 175

Phe Thr Glu Leu Lys lie Ala Arg Asn He Tyr He Lys Val Gly Glu 180 185 190

Asp Gin Val Phe Pro Pro Thr Cys Asn He Gly Lys Asn Phe Leu Ser 195 200 205

Leu Asn Tyr Leu Ala Glu Tyr Leu Gin Pro Lys Ala Ala Glu Gly Cys 210 215 220

Val Leu Pro Ser Gin Pro His Glu Lys Glu Val His He He Glu Leu 225 230 235 240

He Thr Pro Ser Ser Asn Pro Tyr Ser Ala Phe Gin Val Asp He He 245 250 255

Val Asp He Arg Pro Ala Gin Glu Asp Pro Glu Val Val Lys Asn Leu 260 265 270

Val Leu He Leu Lys Cys Lys Lys Ser Val Asn Trp val He Lys Ser 275 280 285

Phe Asp Val Lys Gly Asn Leu Lys Val He Ala Pro Asn Ser He Gly 290 295 300

Phe Gly Lys Glu Ser Glu Arg Ser Met Thr Met Thr Lys Leu Val Arg 305 310 315 320

Asp Asp He Pro Ser Thr Gin Glu Asn Leu Met Lys Trp Ala Leu Asp 325 330 335

Asn Gly Tyr Arg Pro Val Thr Ser Tyr Thr Met Ala Pro Val Ala Asn 340 345 350

Arg Phe His Leu Arg Leu Glu Asn Asn Glu Glu Met Arg Asp Glu Glu 355 360 365

Val His Thr He Pro Pro Glu Leu Arg He Leu Leu Asp Pro Asp His 370 375 380

Pro Pro Ala Leu Asp Asn Pro Leu Phe Pro Gly Glu Gly Ser Pro Asn 385 390 395 400

Gly Gly Leu Pro Phe Pro Phe Pro Asp He Pro Arg Arg Gly Trp Lys 405 410 415

Glu Gly Glu Asp Arg lie Pro Arg Pro Lys Gin Pro He Val Pro Ser 420 425 430

Val Gin Leu Leu Pro Asp His Arg Glu Pro Glu Glu Val Gin Gly Gly 435 440 445

Val Asp He Ala Leu ser Val Lys Cys Asp His Glu Lys Met Val Val 450 455 460

Ala Val Asp Lys Asp ser Phe Gin Thr Asn Gly Tyr Ser Gly Met Glu 465 470 475 480

Leu Thr Leu Leu Asp Pro Ser Cys Lys Ala Lys Met Asn Gly Thr His 485 490 495

Phe Val Leu Glu ser Pro Leu Asn Gly Cys Gly Thr Arg His Arg Arg 500 505 510 Ser Thr Pro Asp Gly Val Val Tyr Tyr Asn Ser He Val Val Gin Ala 515 520 525

Pro Ser Pro Gly Asp Ser ser Gly Trp Pro Asp Gly Tyr Glu Asp Leu 530 535 540

Glu Ser Gly Asp Asn Gly Phe Pro Gly Asp Gly Asp Glu Gly Glu Thr 545 550 555 560

Ala Pro Leu Ser Arg Ala Gly Val Val Val Phe Asn Cys Ser Leu Arg 565 570 575

Gin Leu Arg Asn Pro Ser Gly Phe Gin Gly Gin Leu Asp Gly Asn Ala 580 585 590

Thr Phe Asn Met Glu Leu Tyr Asn Thr Asp Leu Phe Leu Val Pro Ser 595 600 605

Pro Gly Val Phe Ser Val Ala Glu Asn Glu His Val Tyr Val Glu Val 610 615 620

Ser Val Thr Lys Ala Asp Gin Asp Leu Gly Phe Ala He Gin Thr Cys 625 630 635 640

Phe Leu ser Pro Tyr Ser Asn Pro Asp Arg Met Ser Asp Tyr Thr He 645 650 655

He Glu Asn He Cys Pro Lys Asp Asp Ser Val Lys Phe Tyr Ser Ser 660 665 670

Lys Arg Val His Phe Pro He Pro His Ala Glu Val Asp Lys Lys Arg 675 680 685

Phe Ser Phe Leu Phe Lys Ser Val Phe Asn Thr Ser Leu Leu Phe Leu 690 695 700

His Cys Glu Leu Thr Leu Cys Ser Arg Lys Lys Gly Ser Leu Lys Leu 705 710 715 720

Pro Arg Cys Val Thr Pro Asp Asp Ala Cys Thr Ser Leu Asp Ala Thr 725 730 735

Met He Trp Thr Met Met Gin Asn Lys Lys Thr Phe Thr Lys Pro Leu 740 745 750

Ala Val Val Leu Gin Val Asp Tyr Lys Glu Asn Val Pro ser Thr Lys 755 760 765

Asp Ser Ser Pro He Pro Pro Pro Pro Pro Gin He Phe His Gly Leu 770 775 780

Asp Thr Leu Thr Val Met Gly He Ala Phe Ala Ala Phe Val He Gly 785 790 795 800

Ala Leu Leu Thr Gly Ala Leu Trp Tyr He Tyr Ser His Thr Gly Glu 805 810 815

Thr Ala Arg Arg Gin Gin Val Pro Thr Ser Pro Pro Ala Ser Glu Asn 820 825 830

Ser Ser Ala Ala His Ser He Gly Ser Thr Gin Ser Thr Pro Cys Ser 835 840 845

Ser Ser Ser Thr Ala 850

(2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4213 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 622..3168

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GCGGACGCGA TCGCAACCTC CGCTTCCCGG GTTCAAGGGA TTCTCCTGCC TCAGCCTCCT 60

GAGTAGCTGG GACTACAGGC GCACCACTAC ACCCGGCCAA TTTTTGTATT TATTTATTTT 120

ATTTTATTTT ATTTTTTAGT AGAGACGGGG TTTACCATGT TGGCCAGAAC AGTCTCGATC 180

TCCTAACCTC GTGATCCAAC GCCTCGGCTC CCAAAGTGTT GAGTTACAGG CGTGAGCCAC 240

CGCGCCCGTC CAGAATTATC TTTAAAATTT ATTTCTTTAA GATTTGTAGC TACTAAGAAA 300

GAAAGGAGCT TTTTTTCCTT GGGCCTTCAA ACTGAAAGAA CCGCATGAGC CTGACGGCGC 360

ATGGTCTTAA CATCAGGCTG TGCAGGAAGA AGCTATCTGC AGATGGATGC CAGCACACAC 420

AAGGAAGCAG AGCTCTGGCA ACATTGAGTC AAAGCAAGGA CACAACATCA GAGGGACGGC 480

AGAGAATCCT TGTGTGTAGT CTTTGGTGGC AGTTTGAAAA TTGCAAGGAG GGACTTTAAG 540

ACTACTTCTG ATTTGCAAAG ATGGTCTGTG CTCCGAGCAG GCTAAAGTGA CTGGACGAGA 600

CGCACTGTTG GAGAAATAAA A ATG ACT TCC CAT TAT GTG ATT GCC ATC TTT 651

Met Thr Ser His Tyr Val lie Ala lie Phe 1 5 10

GCC CTG ATG AGC TTC TGT TTA GCC ACT GCA GGT CCA GAG CCT GGT GCA 699 Ala Leu Met ser Phe Cys Leu Ala Thr Ala Gly Pro Glu Pro Gly Ala 15 20 25

CTG TGT GAA CTG TCA CCT GTC AGT GCC TCC CAT CCT GTC CAG GCC TTG 747 Leu Cys Glu Leu Ser Pro Val Ser Ala Ser His Pro Val Gin Ala Leu 30 35 40

ATG GAG AGC TTC ACT GTT TTG TCA GGC TGT GCC AGC AGA GGC ACA ACT 795 Met Glu Ser Phe Thr Val Leu Ser Gly Cys Ala Ser Arg Gly Thr Thr 45 50 55

GGG CTG CCA CAG GAG GTG CAT GTC CTG AAT CTC GCA CTG CGC CAG GGG 843 Gly Leu Pro Gin Glu Val His Val Leu Asn Leu Ala Leu Arg Gin Gly 60 65 70

CCT GGC CAG CTA CAG AGA GAG GTC ACA CTT CAC CTG AAT CCC ATC TCC 891 Pro Gly Gin Leu Gin Arg Glu Val Thr Leu His Leu Asn Pro lie Ser 75 80 85 90

TCA GTC CAC ATC CAC CAC AAG TCT GTT GTG TTC CTG CTC AAC TCC CCA 939 Ser Val His lie His His Lys Ser Val Val Phe Leu Leu Asn Ser Pro 95 100 105

CAC CCC CTG GTG TGG CAT CTG AAG ACA GAG AGA CTT GCC ACT GGG GTC 987 His Pro Leu Val Trp His Leu Lys Thr Glu Arg Leu Ala Thr Gly Val 110 115 120 TCC AGA CTG TTT TTG GTG TCT GAG GGT TCT GTG GTC CAG TTT TCA TCA 1035 Ser Arg Leu Phe Leu Val Ser Glu Gly Ser Val Val Gin Phe Ser Ser 125 130 135

GCA AAC TTC TCC TTG ACA GCA GAA ACA GAA GAA AGG AAC TTC CCC CAT 1083 Ala Asn Phe Ser Leu Thr Ala Glu Thr Glu Glu Arg Asn Phe Pro His 140 145 150

GGA AAT GAA CAT CTG TTA AAT TGG GCC CGA AAA GAG TAT GGA GCA GTT 1131 Gly Asn Glu His Leu Leu Asn Trp Ala Arg Lys Glu Tyr Gly Ala Val 155 160 165 170

ACT TCA TTC ACC GAA CTC AAG ATA GCA AGA AAC ATT TAT ATT AAA GTG 1179 Thr Ser Phe Thr Glu Leu Lys He Ala Arg Asn He Tyr He Lys Val 175 180 185

GGG GAA GAT CAA GTG TTC CCT CCA AAG TGC AAC ATA GGG AAG AAT TTT 1227 Gly Glu Asp Gin Val Phe Pro Pro Lys cys Asn He Gly Lys Asn Phe 190 195 200

CTC TCA CTC AAT TAC CTT GCT GAG TAC CTT CAA CCC AAA GCA GCA GAA 1275 Leu Ser Leu Asn Tyr Leu Ala Glu Tyr Leu Gin Pro Lys Ala Ala Glu 205 210 215

GGG TGT GTG ATG TCC AGC CAG CCC CAG AAT GAG GAA GTA CAC ATC ATC 1323 Gly Cys Val Met Ser Ser Gin Pro Gin Asn Glu Glu Val His He He 220 225 230

GAG CTA ATC ACC CCC AAC TCT AAC CCC TAC AGT GCT TTC CAG GTG GAT 1371 Glu Leu He Thr Pro Asn Ser Asn Pro Tyr Ser Ala Phe Gin Val Asp 235 240 245 250

ATA ACA ATT GAT ATA AGA CCT TCT CAA GAG GAT CTT GAA GTG GTC AAA 1419 He Thr He Asp He Arg Pro Ser Gin Glu Asp Leu Glu Val Val Lys 255 260 265

AAT CTC ATC CTG ATC TTG AAG TGC AAA AAG TCT GTC AAC TGG GTG ATC 1467 Asn Leu He Leu He Leu Lys Cys Lys Lys Ser Val Asn Trp Val He 270 275 280

AAA TCT TTT GAT GTT AAG GGA AGC CTG AAA ATT ATT GCT CCT AAC AGT 1515 Lys Ser Phe Asp Val Lys Gly Ser Leu Lys He He Ala Pro Asn Ser 285 290 295

ATT GGC TTT GGA AAA GAG AGT GAA AGA TCT ATG ACA ATG ACC AAA TCA 1563 He Gly Phe Gly Lys Glu Ser Glu Arg ser Met Thr Met Thr Lys Ser 300 305 310

ATA AGA GAT GAC ATT CCT TCA ACC CAA GGG AAT CTG GTG AAG TGG GCT 1611 He Arg Asp Asp He Pro Ser Thr Gin Gly Asn Leu Val Lys Trp Ala 315 320 325 330

TTG GAC AAT GGC TAT AGT CCA ATA ACT TCA TAC ACA ATG GCT CCT GTG 1659 Leu Asp Asn Gly Tyr Ser Pro He Thr ser Tyr Thr Met Ala Pro Val 335 340 345

GCA ATA GTA TTT CAT CTT CGG CTT GAA AAT AAT GAG GAG ATG GGA GAT 1707 Ala He Val Phe His Leu Arg Leu Glu Asn Asn Glu Glu Met Gly Asp 350 355 360

GAG GAA GTC CAC ACT ATT CCT CCT GAG CTA CGG ATC CTG CTG GAC CCT 1755 Glu Glu Val His Thr He Pro Pro Glu Leu Arg He Leu Leu Asp Pro 365 370 375

GGT GCC CTG CCT GCC CTG CAG AAC CCG CCC ATC CGG GGA GGG GAA GGC 1803 Gly Ala Leu Pro Ala Leu Gin Asn Pro Pro He Arg Gly Gly Glu Gly 380 385 390 CAA AAT GGA GGC CTT CCG TTT CCT TTC CCA GAT ATT TCC AGG AGA GTC 1851 Gin Asn Gly Gly Leu Pro Phe Pro Phe Pro Asp He Ser Arg Arg Val 395 400 405 410

TGG AAT GAA GAG GGA GAA GAT GGG CTC CCT CGG CCA AAG GAC CCT GTC 1899 Trp Asn Glu Glu Gly Glu Asp Gly Leu Pro Arg Pro Lys Asp Pro Val 415 420 425

ATT CCC AGC ATA CAA CTG TTT CCT GGT CTC AGA GAG CCA GAA GAG GTG 1947 He Pro Ser He Gin Leu Phe Pro Gly Leu Arg Glu Pro Glu Glu Val 430 435 440

CAA GGG AGC GTG GAT ATT GCC CTG TCT GTC AAA TGT GAC AAT GAG AAG 1995 Gin Gly Ser Val Asp He Ala Leu Ser Val Lys Cys Asp Asn Glu Lys 445 450 455

ATG ATC GTG GCT GTA GAA AAA GAT TCT TTT CAG GCC AGT GGC TAC TCG 2043 Met He Val Ala val Glu Lys Asp Ser Phe Gin Ala Ser Gly Tyr Ser 460 465 470

GGG ATG GAC GTC ACC CTG TTG GAT CCT ACC TGC AAG GCC AAG ATG AAT 2091 Gly Met Asp Val Thr Leu Leu Asp Pro Thr cys Lys Ala Lys Met Asn 475 480 485 490

GGC ACA CAC TTT GTT TTG GAG TCT CCT CTG AAT GGC TGC GGT ACT CGG 2139 Gly Thr His Phe val Leu Glu Ser Pro Leu Asn Gly Cys Gly Thr Arg 495 500 505

CCC CGG TGG TCA GCC CTT GAT GGT GTG GTC TAC TAT AAC TCC ATT GTG 2187 Pro Arg Trp ser Ala Leu Asp Gly Val Val Tyr Tyr Asn ser He Val 510 515 520

ATA CAG GTT CCA GCC CTT GGG GAC AGT AGT GGT TGG CCA GAT GGT TAT 2235 He Gin Val Pro Ala Leu Gly Asp Ser Ser Gly Trp Pro Asp Gly Tyr 525 530 535

GAA GAT CTG GAG TCA GGT GAT AAT GGA TTT CCG GGA GAT ATG GAT GAA 2283 Glu Asp Leu Glu Ser Gly Asp Asn Gly Phe Pro Gly Asp Met Asp Glu 540 545 550

GGA GAT GCT TCC CTG TTC ACC CGA CCT GAA ATC GTG GTG TTT AAT TGC 2331 Gly Asp Ala ser Leu Phe Thr Arg Pro Glu He Val Val Phe Asn Cys 555 560 565 570

AGC CTT CAG CAG GTG AGG AAC CCC AGC AGC TTC CAG GAA CAG CCC CAC 2379 Ser Leu Gin Gin Val Arg Asn Pro ser ser Phe Gin Glu Gin Pro His 575 580 585

GGA AAC ATC ACC TTC AAC ATG GAG CTA TAC AAC ACT GAC CTC TTT TTG 2427 Gly Asn He Thr Phe Asn Met Glu Leu Tyr Asn Thr Asp Leu Phe Leu 590 595 600

GTG CCC TCC CAG GGC GTC TTC TCT GTG CCA GAG AAT GGA CAC GTT TAT 2475 Val Pro Ser Gin Gly Val Phe Ser Val Pro Glu Asn Gly His Val Tyr 605 610 615

GTT GAG GTA TCT GTT ACT AAG GCT GAA CAA GAA CTG GGA TTT GCC ATC 2523 Val Glu Val ser Val Thr Lys Ala Glu Gin Glu Leu Gly Phe Ala He 620 625 630

CAA ACG TGC TTT ATC TCT CCA TAT TCG AAC CCT GAT AGG ATG TCT CAT 2571 Gin Thr Cys Phe He Ser Pro Tyr Ser Asn Pro Asp Arg Met ser His 635 640 645 650

TAC ACC ATT ATT GAG AAT ATT TGT CCT AAA GAT GAA TCT GTG AAA TTC 2619 Tyr Thr He He Glu Asn He Cys Pro Lys Asp Glu Ser Val Lys Phe 655 660 665 TAC AGT CCC AAG AGA GTG CAC TTC CCT ATC CCG CAA GCT GAC ATG GAT 2667 Tyr Ser Pro Lys Arg Val His Phe Pro He Pro Gin Ala Asp Met Asp 670 675 680

AAG AAG CGA TTC AGC TTT GTC TTC AAG CCT GTC TTC AAC ACC TCA CTG 2715 Lys Lys Arg Phe ser Phe Val Phe Lys Pro Val Phe Asn Thr ser Leu 685 690 695

CTC TTT CTA CAG TGT GAG CTG ACG CTG TGT ACG AAG ATG GAG AAG CAC 2763 Leu Phe Leu Gin cys Glu Leu Thr Leu Cys Thr Lys Met Glu Lys His 700 705 710

CCC CAG AAG TTG CCT AAG TGT GTG CCT CCT GAC GAA GCC TGC ACC TCG 2811 Pro Gin Lys Leu Pro Lys Cys Val Pro Pro Asp Glu Ala Cys Thr Ser 715 720 725 730

CTG GAC GCC TCG ATA ATC TGG GCC ATG ATG CAG AAT AAG AAG ACG TTC 2859 Leu Asp Ala Ser He He Trp Ala Met Met Gin Asn Lys Lys Thr Phe 735 740 745

ACC AAG CCC CTT GCT GTG ATC CAC CAT GAA GCA GAA TCT AAA GAA AAA 2907 Thr Lys Pro Leu Ala Val He His His Glu Ala Glu Ser Lys Glu Lys 750 755 760

GGT CCA AGC ATG AAG GAA CCA AAT CCA ATT TCT CCA CCA ATT TTC CAT 2955 Gly Pro Ser Met Lys Glu Pro Asn Pro He Ser Pro Pro He Phe His 765 770 775

GGT CTG GAC ACC CTA ACC GTG ATG GGC ATT GCG TTT GCA GCC TTT GTG 3003 Gly Leu Asp Thr Leu Thr Val Met Gly He Ala Phe Ala Ala Phe Val 780 785 790

ATC GGA GCA CTC CTG ACG GGG GCC TTG TGG TAC ATC TAT TCT CAC ACA 3051 He Gly Ala Leu Leu Thr Gly Ala Leu Trp Tyr He Tyr Ser His Thr 795 800 805 810

GGG GAG ACA GCA GGA AGG CAG CAA GTC CCC ACC TCC CCG CCA GCC TCG 3099 Gly Glu Thr Ala Gly Arg Gin Gin Val Pro Thr Ser Pro Pro Ala Ser 815 820 825

GAA AAC AGC AGT GCT GCC CAC AGC ATC GGC AGC ACG CAG AGC ACG CCT 3147 Glu Asn Ser Ser Ala Ala His ser He Gly Ser Thr Gin Ser Thr Pro 830 835 840

TGC TCC AGC AGC AGC ACG GCC TAGCCCAACC CAGGCCCAAC CCGGCCCAAC 3198

Cys Ser Ser Ser ser Thr Ala 845

CCAGCCCAGC CCAGCTCAGC TCAGCTACTC CAAGGGCAGG ACCAATGGCT GAGCCTCGTG 3258

TCCAGACTCA GAGGGCTGGA TTTTGGTTCC CTTGTAAAGA CAGAGTGAAT TTCAGTATAA 3318

AGATCACCCG TTGTATTCAC CCCACACCCA GGGCTAGTAT AAACATGACC CTGGGCTTCT 3378

GTACCACACT AGAATTCATG TGAGAAAGCT AAAATGGTGG TCTTCTCCAC CAGCCCCTCA 3438

CAGGCTTGGG GGTTTTCTAT GTGAAACACA TGCCAGTTTT TAAAATGCTG CTTTGTCCAG 3498

GTGAGAACAT CCATAATTTG GGGCCCTGAG TTTTACCCAG ACTCAAGGAG TTGGTAAAGG 3558

GTTAATAGCC AGATAGTAGA ACCAGTGAGG AGATGCGGCC AAAGATTCTT TATATCTGAA 3618

CCAAGATGTA AAACAAGAAA TGCTTTGAGG CTTTCTAAGC GATCCTCCTG TCTAATTTGC 3678

ACCTTTGTCT GGATGCACTC TTCTGACCTT GCTGCCACAA CCTGTGGGGT CTGATGTGTC 3738

CCAAGATGGG TGCTGCCCTC AGGGACTGCA CCCTGACAAG TGTTAAGGCA ACATTCCTTG 3798 CTTGTGCCCT GGGCCAAAAC CAATGCTGAT GACCTTATCA GCTTCCTGTT TCTTCCCATA 3858

CTGCATACAC CACTGCAAAA TGTCTTAATG CAAATTTTGT ATTTCTTACA GGCCTACAGA 3918

AATTGAAAAT GACCAAAATC AGGAACCACA GATTTGTGCC CATTCCTAAT ATTTTGTTCT 3978

GCAAATTAAT GTATAATTTG AGGTGAAATT CAGTTATAAA GTCAAGGACG AATTTGCACA 4038

GTGATATATT TCTATGTGTA TGCAAGTACA AGTATATAAT ATGTCACCTG GCACATTCAT 4098

TTTCTCAGTT GAAGAAGAGA AAATTTGAAA ATGTCCTTAT GCTTTTAGAG TTGCAACTTA 4158

AGTATATTTG GTAGGGTGAG TGTTTCCACT CAAAATATGT CAACTTAAAA AAAAA 4213

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 849 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:4:

Met Thr Ser His Tyr Val He Ala He Phe Ala Leu Met Ser Phe cys 1 5 10 15

Leu Ala Thr Ala Gly Pro Glu Pro Gly Ala Leu Cys Glu Leu Ser Pro 20 25 30

Val Ser Ala Ser His Pro Val Gin Ala Leu Met Glu Ser Phe Thr Val 35 40 45

Leu Ser Gly Cys Ala Ser Arg Gly Thr Thr Gly Leu Pro Gin Glu Val 50 55 60

His Val Leu Asn Leu Ala Leu Arg Gin Gly Pro Gly Gin Leu Gin Arg 65 70 75 80

Glu Val Thr Leu His Leu Asn Pro He Ser Ser Val His He His His 85 90 95

Lys Ser Val Val Phe Leu Leu Asn Ser Pro His Pro Leu Val Trp His 100 105 110

Leu Lys Thr Glu Arg Leu Ala Thr Gly Val Ser Arg Leu Phe Leu Val 115 120 125

Ser Glu Gly ser Val Val Gin Phe Ser Ser Ala Asn Phe Ser Leu Thr 130 135 140

Ala Glu Thr Glu Glu Arg Asn Phe Pro His Gly Asn Glu His Leu Leu 145 150 155 160

Asn Trp Ala Arg Lys Glu Tyr Gly Ala Val Thr Ser Phe Thr Glu Leu 165 170 175

Lys He Ala Arg Asn He Tyr He Lys Val Gly Glu Asp Gin Val Phe 180 185 190

Pro Pro Lys cys Asn He Gly Lys Asn Phe Leu Ser Leu Asn Tyr Leu 195 200 205

Ala Glu Tyr Leu Gin Pro Lys Ala Ala Glu Gly cys Val Met ser Ser 210 215 220 Gin Pro Gin Asn Glu Glu Val His He He Glu Leu He Thr Pro Asn 225 230 235 240

Ser Asn Pro Tyr Ser Ala Phe Gin Val Asp He Thr He Asp lie Arg 245 250 255

Pro Ser Gin Glu Asp Leu Glu Val Val Lys Asn Leu He Leu He Leu 260 265 270

Lys Cys Lys Lys Ser Val Asn Trp Val He Lys Ser Phe Asp Val Lys 275 280 285

Gly Ser Leu Lys He lie Ala Pro Asn Ser He Gly Phe Gly Lys Glu 290 295 300

Ser Glu Arg Ser Met Thr Met Thr Lys Ser He Arg Asp Asp He Pro 305 310 315 320

Ser Thr Gin Gly Asn Leu Val Lys Trp Ala Leu Asp Asn Gly Tyr Ser 325 330 335

Pro He Thr Ser Tyr Thr Met Ala Pro Val Ala He Val Phe His Leu 340 345 350

Arg Leu Glu Asn Asn Glu Glu Met Gly Asp Glu Glu Val His Thr He 355 360 365

Pro Pro Glu Leu Arg He Leu Leu Asp Pro Gly Ala Leu Pro Ala Leu 370 375 380

Gin Asn Pro Pro He Arg Gly Gly Glu Gly Gin Asn Gly Gly Leu Pro 385 390 395 400

Phe Pro Phe Pro Asp lie Ser Arg Arg Val Trp Asn Glu Glu Gly Glu 405 410 415

Asp Gly Leu Pro Arg Pro Lys Asp Pro Val He Pro Ser He Gin Leu 420 425 430

Phe Pro Gly Leu Arg Glu Pro Glu Glu Val Gin Gly Ser Val Asp He 435 440 445

Ala Leu Ser Val Lys Cys Asp Asn Glu Lys Met He Val Ala Val Glu 450 455 460

Lys Asp ser Phe Gin Ala ser Gly Tyr Ser Gly Met Asp Val Thr Leu 465 470 475 480

Leu Asp Pro Thr Cys Lys Ala Lys Met Asn Gly Thr His Phe Val Leu 485 490 495

Glu Ser Pro Leu Asn Gly Cys Gly Thr Arg Pro Arg Trp Ser Ala Leu 500 505 510

Asp Gly Val Val Tyr Tyr Asn Ser He Val He Gin Val Pro Ala Leu 515 520 525

Gly Asp Ser Ser Gly Trp Pro Asp Gly Tyr Glu Asp Leu Glu ser Gly 530 535 540

Asp Asn Gly Phe Pro Gly Asp Met Asp Glu Gly Asp Ala Ser Leu Phe 545 550 555 560

Thr Arg Pro Glu He Val Val Phe Asn Cys Ser Leu Gin Gin Val Arg 565 570 575 Asn Pro ser Ser Phe Gin Glu Gin Pro His Gly Asn lie Thr Phe Asn 580 585 590

Met Glu Leu Tyr Asn Thr Asp Leu Phe Leu Val Pro ser Gin Gly Val 595 600 605

Phe Ser Val Pro Glu Asn Gly His Val Tyr Val Glu Val ser Val Thr 610 615 620

Lys Ala Glu Gin Glu Leu Gly Phe Ala He Gin Thr cys Phe He Ser 625 630 635 640

Pro Tyr Ser Asn Pro Asp Arg Met Ser His Tyr Thr lie He Glu Asn 645 650 655

He Cys Pro Lys Asp Glu ser Val Lys Phe Tyr Ser Pro Lys Arg Val 660 665 670

His Phe Pro He Pro Gin Ala Asp Met Asp Lys Lys Arg Phe Ser Phe 675 680 685

Val Phe Lys Pro Val Phe Asn Thr Ser Leu Leu Phe Leu Gin Cys Glu 690 695 700

Leu Thr Leu Cys Thr Lys Met Glu Lys His Pro Gin Lys Leu Pro Lys 705 710 715 720

Cys Val Pro Pro Asp Glu Ala Cys Thr Ser Leu Asp Ala Ser He He 725 730 735

Trp Ala Met Met Gin Asn Lys Lys Thr Phe Thr Lys Pro Leu Ala Val 740 745 750

He His His Glu Ala Glu Ser Lys Glu Lys Gly Pro ser Met Lys Glu 755 760 765

Pro Asn Pro He Ser Pro Pro He Phe His Gly Leu Asp Thr Leu Thr 770 775 780

Val Met Gly He Ala Phe Ala Ala Phe Val He Gly Ala Leu Leu Thr 785 790 795 800

Gly Ala Leu Trp Tyr He Tyr Ser His Thr Gly Glu Thr Ala Gly Arg 805 810 815

Gin Gin Val Pro Thr Ser Pro Pro Ala Ser Glu Asn Ser Ser Ala Ala 820 825 830

His ser He Gly Ser Thr Gin Ser Thr Pro cys Ser Ser Ser Ser Thr 835 840 845

Ala

Claims

We claim:

1. A polypeptide of at least 155 amino acids that binds to TGF-/3 and that has a sequence consisting essentially of a sequence from a portion of a mammalian betaglycan within about one-third of the extracellular domain closest to the cell membrane.

2. The polypeptide of claim 1 wherein the portion of a mammalian betaglycan is about one-fifth of the extracellular domain of a mammalian betaglycan closest to the cell membrane.

3. The polypeptide of claim 1 wherein the portion of a mammalian betaglycan is about one-fourth of the extracellular domain of a mammalian betaglycan closest to the cell membrane.

4. The polypeptide of claim 1 wherein the mammalian betaglycan is from a human, rat or pig.

5. The polypeptide of claim 1 wherein the sequence consists essentially of at least amino acids 615 to 769 of SEQ ID NO:2 to at most amino acids 501 to 769 of SEQ ID N0:2.

6. The polypeptide of claim 1 wherein the sequence consists essentially of at least amino acids 543 to 769 of SEQ ID NO:2 to at most amino acids 501 to 769 of SEQ ID NO:2.

7. The polypeptide of claim 1 wherein the sequence consists essentially of amino acids 543 to 769 of SEQ ID NO:2.

8. A soluble polypeptide having the formula A-B-C, wherein A is a sequence excluding amino acid sequences of more than 4 amino acids from amino acids 1 to 543 of SEQ ID NO: 2; B is an amino acid sequence consisting essentially of at least amino acids 543 to 769 of SEQ ID NO:2 to at most amino acids 501 to 769 of SEQ ID NO:2; and C is an amino acid sequence.

9. The polypeptide of claim 8 wherein A and C have no more than 100 amino acids.

10. A soluble polypeptide having the formula

A-B-C, wherein A is an amino acid sequence excluding sequences of more than 4 amino acids from amino acids 1 to 543 of SEQ ID NO 2; B is at least 155 amino acids in a sequence consisting essentially of an amino acid sequence within amino acids 543 to 769 of SEQ ID NO:2; and C is an amino acid sequence.

11. The polypeptide of claim 10 wherein A and C have no more than 100 amino acids.

12. An isolated nucleic acid molecule encoding any of the polypeptides of claims 1 to 11.

13. An expression vector comprising an expression control sequence operatively linked to a nucleic acid of claim 12.

14. A prokaryotic or eukaryotic cell transfected with an expression vector of claim 13 and capable of expressing a nucleic acid of claim 12.

15. A method of detecting TGF-β in a sample comprising contacting the sample with a polypeptide of claim 1 and determining the amount of TGF-β bound to the polypeptide.

16. A method of isolating TGF-β from a sample comprising contacting the sample with a polypeptide of claim 1 bound to a solid support to allow binding of TGF- β to the polypeptide and isolating the TGF-β from the polypeptide.

17. A method of enhancing the binding of TGF-β to type II receptor comprising contacting a cell bearing a type II receptor with TGF-β and a polypeptide of claim 1.

18. A method of enhancing suppression of cell growth by TGF-β comprising contacting a cell with TGF-/3 and a polypeptide of claim 1.

19. A pharmaceutical composition comprising a polypeptide according to claim 1, 8 or 10 in a pharmaceutically acceptable carrier.

20. The pharmaceutical composition of claim 19 further comprising TGF-β.

21. A method of treating a subject with a condition ameliorated by the enhanced binding of TGF-β to type II receptor or by the enhanced suppression of cell growth by TGF-β comprising administering a therapeutically effective amount of a pharmaceutical composition having a polypeptide of claim 1.

22. The method of claim 21 wherein the pharmaceutical composition further comprises TGF-β.

23. A decoy betaglycan polypeptide.

24. The decoy betaglycan polypeptide of claim

23 having a disabling modification within about one- fourth of the extracellular domain of a mammalian betaglycan closest to the cell membrane.

25. The decoy betaglycan polypeptide of claim

24 having a disabling modification within the sequence of amino acids 543 to 769 of SEQ ID NO:2.

26. A method of treating a subject with a condition ameliorated by the diminished binding of TGF-β to a TGF-β receptor or by the inhibition of the suppression of cell growth by TGF-β comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition having a decoy betaglycan polypeptide.

27. A method of suppressing TGF-β-induced deposition of extracellular matrix in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition having a decoy betaglycan polypeptide.

28. An anti-betaglycan-binding-site antibody.

29. A method of treating a subject with a condition ameliorated by the diminished binding of TGF-β to a TGF-β receptor or by the inhibition of the suppression of cell growth by TGF-β comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition having an anti- betaglycan-binding-site antibody of claim 28.

30. A method of suppressing TGF-β-induced deposition of extracellular matrix in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition having an anti-betaglycan-binding-site antibody of claim 28.

31. An anti-idiotypic antibody that mimics the ability of a polypeptide of claim 1 to bind TGF-β but that lacks the biological function of enhancing binding of TGF-β to a TGF-β receptor or suppressing cell growth.

32. A method of treating a subject with a condition ameliorated by the diminished binding of TGF-β to a TGF-β receptor or by the inhibition of the suppression of cell growth by TGF-β comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition having an anti- idiotypic antibody of claim 31.

33. A method of suppressing TGF-β-induced deposition of extracellular matrix in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition having an anti-idiotypic antibody of claim 31.