CA2132859A1 - Decorin fragments and methods of inhibiting cell regulatory factors - Google Patents

Abstract

The present invention provides a method of inhibiting an activity of a cell regulatory factor comprising contacting the cell regulatory factor with a purified polypeptide, wherein the polypeptide comprises a cell regulatory factor binding domain of a protein.
The protein is characterized by a leucine-rich repeat of about 24 amino acids. In a specific embodiment, the present invention relates to the ability of decorin, a 40,000 dalton protein that usually carries a glycosaminoglycan chain, and more specifically to active fragments of decorin or its functional equivalents to bind TGF.beta.. The invention also provides a cell regulatory factor designated MRF. Also provided are methods of identifying, detecting and purifying cell regulatory factors and proteins which bind and effect the activity of cell regulatory factors.

Description

2132~S!~ ~

Decorin Fraoments and Methods of Inhibitina Cell Re~ulatorv Factors ::
This invention was made with ~upport of government grants CA 30199, CA 42507 and CA 28896 from the National Cancer In~titute. Therefore, the United States government may have certain rights in the inventio~

FIE D OF T~E INVENTIOM

This invention relates to cell biology and more specifically to the control of cell proliferation by inhibiting cell r~gulatory factors.

BACK~OUND OF T~E INVENTION

Proteoglycans are protein~ that carry one or more glycosaminoglycan chains. The kno~n proteoglycans carry out a wide variety of functions and are found in a variety of cellular locations. ~any proteoglycans are compc~e~ts of extracellular matrix, where they participate in the acsembly of cells and effect the attachment of cells to the matrix.

Decorin, al~o known a~ PG-II or PG-40, is a ~mall 2G proteoglycan produced by fibrobla~ts~ Its core protein has a molecular weight of about 40,09U daltons. The core has been sequenced (Kru~ius and Ruoslahti, Proc. Natl. Acad.
Sci. USA 83:76B3 ~19863; Day et al. Biochem. J. 248:801 (1987), both of which are incorporated herein by reference) and it is known to carry a single glyco~amlnoglycan chain of a chondroitin sulfate/dermatan sulfate type (Pearson, et al., J. Biol. Chem. 258:15101 (1983), which is incorporated herein by reference). The only previously known function for decorin is binding to type I and type II collagen and its effect on the fibril formation by these collagens tVo~el, et al., Biochem. J. 223:587 ~1984); Schmidt et al., J. Cell Biol. 104:1683, ~1987)). Two proteoglycans, biglycan (Fisher et al., J. Biol~ Chem. 264:4571 (1989)) 2 1 3 2 S 3 ~
WOg3/20202 PCT/U~93/03171 and fibromodulin, (Oldberg et al., EMBO J. 8:2601, (1989) have core proteins the amino acid sequences of which are closely related to that of decorin and they, together with decorin, can be considered a protein f~mily. Each of their ~equences is characterized by the presence of a leucine-rich repeat of about 24 amino acids. Several other proteins contain similar repeats. Together all of these proteins form a superfamily of proteins (Ruoslahti, Ann.
Rev. Cell Biol. 4:229, (1988); McFarland et al., Science 245:494 (1989)).

Transforming growth factor ~s (TGF~) are a f~ily of multi-functional cell regulatory factors produced in various forms by many types of cells (for review see Sporn et al., J. Cell Biol. 105:1039, (1987)). Five different TGF~ are known, but the fu~ctions of only two, TGF~-1 and TGP~-2, have been characterized in any detail.
TGF~'s are the subject of U.S. Patent Nos. 4,863,899;
4,816,561; and 4,742,003 which are incorporated by reference~ TGF~-1 and TGF~-2 are publicly available through many commercial sources (e.g. R & D Systems, Inc., Minneapolis, MN). These two proteins have sLmilar functions and will be here collectively referred to as TGF~. TGFB
binds to cell surface receptors posse~sed by esg~ntially all types of cells, causing profound changes in them. In some cells, TGF~ promotes cell proliferation, in others it suppres~es proliferation. A marked effect of TGF~ is that it promotes the production of extracellular matrix proteins and their receptors by cells (for review see Keski-Oja et al., J~ Cell Biochem 33:~5 (19~7); Massague, Cell 4~:437 (1987); Roberts and Sporn in ~Peptides Growth Factors and Their Receptors~ (Springer-Verlag, Heidelberg (1989)).

While TGF~ has many essential cell regulatory functions, improper TGF~ activity can be detrimental to an organism. Since the growth of mesenchyme and proliferation of mesenchymal cells is stimulated by TGF~, some tumor -- 21328~
.. .. .

cells may use TGF~ as an autocrine growth factor.
Therefore, if the growth factor activity of TGFB could be prevented, tumor growth could be controlled. In other ca~es the inhibition of cell proliferation by TGFB may be detrLmental, in that it may prevent healing of injured tissues. The stimulation of extracellular matrix production by TGF~ is important in situations such as wound healing. However, in some cases the body takes this response too far and an excessive accumulation of extracellular matrix ensues. An example of excessive accumulation of extracellular matrix is glomerulonephritis, a di~ease with a detrimental involvement of TGF~.

Thus, a need exists to develop compounds that can modulate the effects of cell regulatory factors such as TGFB. The present invention satisfies this need and provides related advantages.

SUMMARY OF T~E INVENTI~N

The present invention provides active fragments of proteins having a cell regulatory factor binding domain.
The invention further provides a method of inhibiting an activity of a cell regulatory factor comprising contacting the cell regulatory factor with a purified polypeptide, wherein the polypeptide comprises a cell regulatory factor binding domain of a protein and wherein the protein is characterized by a leucine-rich repeat of about 24 amino acids. In a specific embodiment, the present invention relates to the ability of decorin, a 40,000 dalton protein that usually carries a glycosaminoglycan chain, and more specifically to active fragments of decorin or a functional equivalent of decorin to bind TGFB or other cell regulatory factors. The invention also provides a novel cell regulatory factor designated Morphology Restoring Factor, ~MRF). Also provided are methods of identifying, detecting and purifying cell regulatory factors and proteins that 213-~5~
W093/20202 . PCT/US93/03171 bind and affect the activity of cell regulatory factors.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows expression of decorin cDNA
containing a mutation of the serine acceptor ~ite to alanine. COS-1 cultures were transfected with cDNA coding for wild-type decorin (lane 1), decorin in which the serine-4 residue was replaced by an alanine (lane 2), or decorin in which the serine-4 residue was replaced by a threonine (lane 3)~ Immunoprecipitations were performed with an anti-decorin antibody and medium which was labeled with 35S-sulfate (A) or 3H-leucine (B). Lane 4 shows an immunoprecipitate from mock transfected COS-l cultures.
Arrow indicates top of gel. The numbers indicate Mr X 1 o-3 for molecular weight standards.

lS Figure 2 shows binding of [12sI~TGF~1 to decorin-Sepharose: (A) Fractionation of [l25I]-TGF~1 by decorin-Sepharose aff~nity chromatography~ r'2sI]TGF~l (5 x 105 cpm) was incu~ated in BSA-coated polypropylene tubes with 0.2 ml of packed decorin-Sepharose (~) or gelatin-Sepharose (o) in 2 ml of PBS p~ 7.4, containing 1 M ~aCl and 0.05% Tween 20.
After overnight incubation, the affinity matrices were transferred into BSA-coated di~posable columns (Bio Rad) and washed with the bindi~g ~uffer~ Elution was effected first with 3 M NaCl in the binding buffer and then with 8 M urea in the s~me buffer: (B) Analysis of eluents of decorin-Sepharose affinity chromatography by SDS-polyacrylamide gel under nonreducing conditions. Lane 1:
the original [l25I3-labeled TGF~1 sample; lanes 2-7: flow through and wash fractions; lanes 8-10: 3 M NaCl fractions; lanes 11-14: 8 M urea fractions. Arrows .
indicate the top and bottom of the 12% separating gel. -FigurP 3 shows the inhibition of binding of [~2sI~TGF~31 to decorin by proteoglycans and their core 213285~ :
W O 93/20202 P(~r/US93/03171 proteins: (A) Competition of [l25I]TGF~l binding to decorin-coated microtiter wells by recombinant decorin (~), decorin isolated from bovine ~kin (PGII) (~), biglycan isolated from bovine articular cartilage (PGI) (-), chicken S cartilage proteoglycan (o), and BSA (0). Each point represents the mean of duplicate determinants. (B) Competition of ['2sI]TGF~l binding with chondroitinase A~C-treated proteoglycans and BSA. The concentrations of competitors were expressed as intact proteoglycan. The ~ymbols are the same as in Figure 3A.

Figure 4 shows neutralization of the growth regulating activity of TGF~1 by decorin: (A) Shows inhibition of TGF~l-induced proliferation of CHO cells by decorin. t3H]Thymidine incorporation assay was performed in the presence of 5 ng/ml of TGF~-l and the indicated concentrations of purified decorin (~) or BSA (o). At the concentration used, TGF~-l induced a 50% increase of t3~]thymidine incorporation in the CHO cells. The data repre~ent percent neutralizatîon of this growth stimulation; i.e. [3H]thymidine incorporation in the absence of either TGF~l or decorin = 0~, incorporation in the presence of TGF~ but not decorin = 109%. Each point shows the mean ~ standard deviation of triplicate s2mples. (B~
Shows neutralization of TGF~1-induced growth inhibition in MvlLu cells by decorin. The assay was performed as in A
except that TGF~-1 was added at 0.5 ng/ml. This concentration of TGF~-1 induces 50~ reduction of [3H]thym1dine incorporation in the Mvl~u cells. The data represent neutralization of TGF~-induced growth inhibition;
i.e. [3H]thymidine incorporation in the presence of neither TGF~ or decorin = 100%; incorporation in the presence of TGF~ but not decorin = 0%.

Figure SA shows separation of growth inhibitory activity from decorin-expressing CHO cells by gel filtration. Serum-free conditioned medium of decorin 213~a~ ~-overexpre~or cëlls was fractionated by DEAE-Sepharose chromatography in a neutral Tris-HCl buffer and fractions containing growth inhibitory activity were pooled, made 4M
with guanidine-HCl and fractionated on a Sepharose CL-6 column equilibrated with the ~ame guanidine-HCl solution.
The fraction~ were analyzed for protein content, decorin content, and growth regulatory activities. Elution positions of marker proteins are indicated by arrows. BSA:
bovine ~erum albumin (Mr=66,000); CA: carbonic anhydrase (Mr=29,000); Cy:cytochrome c (Mr=12,400); Ap:aprotinin (Mr=6,500); TGF: ['25I]TGF~l (Mr=25,000).

Figure 5B shows identification of the growth stimulatory material from gel filtration as TGF~l. The growth stimulatory activity from the late fractions from Sepharose 6B (bar in panel A) was ider~tified by inhibiting the act~vity with protein A-purified ~gG from an anti-TGF~
anti~erum. Data represent percent inhibition of growth ~timulatory activity in a [3H]thymidine incorporation assay.
Each point shows the mean +standard deviation of triplicate determinations. Anti-TGF~ , normal rabbit IgG (o).

Figure 6 is a schematic diagram of M9P-decorin fragment fusion proteins. LRR is a leucine rich repeat.
MBP is maltose binding protein.

Figure 7 shows the results of binding studies of l2sI-TGF~ to Lmmobilized recombinant decorin (DCl3) and MBP-decorin fragments PT-65, PT-71, PT-72 and PT-73.

Figure 8 shows the results of binding studies of I-TGF~ to immobilized decorin (DC-18v) and MBP-decorin fragments PT-7l, PT-72, PT-84, PT-85, PT-86 and PT-87.

Figuré 9 shows the results of binding studies of I-TGF~l to HepG2 cells in the presence of decorin fragments PT-65, PT-71, PT-72 and PT-78.

W093/20202 2 1 3 2 8 ~ 9 PCT/US93/03171 Figure 10 shows the results of binding studies of l2sI-TGF~ to ~-M(tk-) cells in the presence of decorin and decorin fragments PT-71, PT-72, PT-84 and PT-85.

Figure 11 shows the results of binding studies of ~25I-TGF~l to ~-M(tk-) cells in the pre~ence of decorin and recom~inant decorin fragments PT-71, PT-72, PT-86 and PT-87.

Figure 12 shows the results of binding studies of l25I-TGF~l to L-M(tk-) cells in the presence of synthetic decorin peptide fragments P25-Q36, ~3l-S3, and H3l L42 and a control peptide corresponding to the N-terminal 15 mer.

Figure 13 shows the re~ults of 125I-TGF-~ binding to immobilized decorin with or without the presence of synthetic decorin peptide ~ragments 16r" 16E, 16G and 16H
as well as a control peptide corresponding to the N-terminal 15-mRr.
~ .
DETAILED DESCRlPTION OF T~E INVXNTION

The invention provides a method of inhibit~ng an act.ivi~y of a cell regulatory factor comprising contacting the cell regulatory factor with a purified polypeptide, wherein ~he polypeptide comprises the cell regulatory factor bi~ding domain of a protei~. The protein can be characterized by a leucine-rich repeat of about 24 amino acid~. Since di~eases such as cancer result from uncontrolled cell proliferation, the invention can be used to treat such diseases.

~ y "cell regulatory factor~' is meant a molecule which can regulate an activity of a cell. The cell regulatory factors are generally proteins which bind cell surface receptors and include gr th factors. Examples of cell regulatory factors inclllde the flve TGFB's, platelet-~ 1 3 ~ 3 WO n/20202 PCT/US93/03171 derived growth factor (PDGF), epidermal growth factor,in~ulin like growth factor I and II, fibrobla~t growth factor, interleukin-2, nerve growth factor, hemopoietic cell growth factors (IL-3, GM-CSF, M-CSF, G-CSF, S erythropoietin) and the newly discovered Morphology Re~toring Factor, hereinafter ~MRF". Different regulatory factors can be bound by different proteins which can affect the regulatory factor~s activity. For example, TGF~-l is bound by decorin and biglycan, and MRF by decorin.

By Ucell regulatory factor binding domain~ is meant a fragment of a protein which binds to the cell regulatory factor. A protein fragment that retains the binding activity is included within the scope of the invention and is referred to herein as an active fragment.
Fragments that retain ~uch activity, such as active fragment~ of decorin or biglycan, can be recognized by their ability to competitively inhibit the binding of, for example, decorin to TGF~, or of other polypeptides to their cognate growth factorc.

Active fragments can be obtained by proteolytic digestion of the native polypeptide according to methods known in the art or as described, for example, in Example V~II. Alternatively, active fragments can be synthesized based on the ~nown amino acid sequence by methods known to 2~ those ~killed in the art or as described in Example VIII.
The fragments can also be produced recombinantly by methods known in the art or as described in Example V. Examples of active fragments are included in Tables 4-15.

Such fragments can then be used in a competitive assay to determine whether they retain binding activity.
For example, decorin can be attached to an affinity matrix, as by the method of Example II. ~abelled TGFB and an active fragment can then be contacted with the affinity matrix and the amount of TGFB bound thereto determined.

` ' W0~3/~0202 2 1 3 2 8 S ~ PCT/Usg3/~3171 As used herein, "decorin" refers to a proteoglycan having ~ubstantially the structural characteristics attributed to it in Krusius and Ruoslahti, supra . ~uman fibroblast decorin has substantially the S amino acid sequence pre~ented in Krusius and Ruoslahti, supra. "Decorin" refers both to the native composition and to modifications thereof which substantially retain the functional characteristics. Decorin core protein refers to decorin that no longer is ~ubstantially substituted with glycosaminoglycan and is included in the definition of decorin. Decorin can be rendered glycosaminoglycan-free by mutation or other means, such as by producing recombinant decorin in cells incapable of attaching glycosaminoglycan chains to a core protein.

lS Functional equivalents of decorin include modificatioils of decorin that retain its functional characteristics and molecules that are homologous to decorin, such as the decorin family members biglycan and fibromodulin, for example, that have the similar functional activity of decorin. Modifications can include, for example, the addition of one or more side chains that do not interfere with the functional activity of the decorin core protein.

Since the regulatory factor binding proteins each contain leucine-rich repeats of about 24 amino acids which can constitute 80~ of the protein, it is likely that the fragments which retain the binding activity occur in the leucine-rich repeats. However, it is possible the bi~ding activity resides elsewhere such as in the carboxy terminal amino acids or the junction of the repeats and the carboxy terminal amino acids.

The invention teaches a general method whereby one skilled in the art can identify proteins that can bind to cell regulatory factors or identify cell resulatory 21328S!~

' ' 10 factors that bind to a certain family of proteins. The invention also teaches a general method in which these novel proteins or known existing proteins can be assayed to determine if they affect an activity of a cell regulatory factor. Specifically, the invention teaches the discovery that decorin and biglycan bind TGF~-l and MRF and that such binding can inhibit the cell regulatory functions of TGF~-1. Further, both decorin and biglycan are about 80%
homologous and contain a leucine-rich repeat of about 24 amino acids in which the arrangement of the leucine residues is conserved. As defined, each repeat generally contains at least two leucine residues and can contain five or more. These prot~oglycans are thus considered members of the same protein family. See Ruoslahti, supra, Fisher et al., J. Biol. Chem., 264:4571-4576 (1989) and Patthy, J.
Mol. Biol., 198:567-577 (1987), all of which are incorporated by reference. Other known or later discovered proteins having this leucine-rich repeat, i.e., fibromodulin, would be expected to have a similar cell regulatory activity. The ability of such proteins to bind cell regulatory factors could easily be tested, for example by affinity chromatography or microtiter assay as set forth in Example II, using known cell regulatory factors, such as TGFB-1. Alternatively, any later discovered cell regulatory factor could be tested~ for example by affinity chromatography using one or more regulatory factor binding proteins. Once it is determined that such binding occurs, the effect of the binding on the activity of all regulatory factors can be determined by methods such as growth assays as set forth in Example III. Moreover, one skilled in the art could simply substitute a novel cell regulatory factor for TGF~ 1 or a novel leucine-rich repeat protein for decorin or biglycan in the Examples to determine their activities. Thus, the invention provides general methods to identify and test novel cell regulatory factors and proteins which affect the activity of these factors.

W O 93/20202 PC~r/US93/03171 The invention also provides a novel purified compound comprising a cell regulatory factor attached to a purified polypeptide wherein the polypeptide comprise~ the cell regulatory factor binding domain of a protein and the protein is characterized by a leucine-rich repeat of about 24 amino acids.

The invention further provides a novel purified protein, designated MRF, having a molecular weight of about 20 kd, which can be isolated from CH0 cells, copurifies with decorin under nondissociating condition~, separates from decorin under dissociating conditions, change~ the morphology of transformed 3T3 cells, and has an activity which is not inhibited with anti-TGF~-l antibody.
Additionally, MRF ~eparates from TGFB-l in ~PLC.

The invention still further provides a method of purifying a cell regulatory factor comprising contacting the regulatory factor with a protein which binds the cell regulatory factor and has a leucine-rich repeat of about 24 amino acids and to purify the regulatory factor which becomes bound to the protein. The method can be used, for example, to purify TGF~-1 by using decorin.

The invention additionally provides a method of treating a pathology caused by a TGFB-regulated activity comprising contacting the TGF~ with a puri~ied polypeptide, wherein the polypeptide comprises the TGF~ ~inding domain of a protein and wherein the protein is characterized by a leucine-rich repeat of about 24 amino acids, whereby the pathology-causing activity is prevented or reduced. While the method is generally applicable, specific examples of pathologies which can be treated include a cancer, a fibrotic disease, and glomerulonephritis. In cancer, for example, decorin can be used to bind TGF~-1, destroying TGF~-l's growth stimulating activity on the cancer cell.

2132~â3 -- ~
WOg3/20202 ~, PCT/US93/03171 Finally, a method of preventing the inhibition of a cell regulatory factor is provided. The method comprises contacting a protein which inhibits an activity of a cell regulator factor with a molecule which inhibits the activity of the protein. For example, decorin could be bound by a molecule, such as an antibody, which prevents decorin from binding TGF~-l, thus preventing decorin from inhibiting the TGF~-l activity. Thus, the TGF~-l wound healing activity could be promoted by binding TGF~
inhibitors.

It is understood that modifications which do not substantially affect the activity of the various molecules of this invention including TGFB, MRF, decorin, biglycan and fibromodulin are also included within the definition of tho~e molecules. It is al80 understood that the core protein& of decorin, ~iglycan and fibromodulin are also included within the definition of those molecules.
."~.

The followin~ examples are intended to illustrate but not limit the invention. -~

W093/20202 2 1 3 2 8 ~ 9 PCT/US93/03171 EXAMPLE I
EXPRESSION AND PURIFICATION OF RECOMBINANT DECORIN
AND DECORIN CORE PROTEIN

Expression System The 1.8 kb full-length decorin cDNA described in Krusi-s and Ruoslahti, Proc. Natl. Acad. Sci. USA 83:7683 (198~ ) which is incorporated herein by reference, was used for the construction of decorin expression vectors. For the expression of decorin core protein, cDNA was mutagenized so the fourth codon, TCT, coding for ~;erine, was changed to ACT codi~g for threonine, or GCT coding for alanine. This was engineered by site-directed mutagenesis according to the method of Kunkel, Proc. Natl. Acad. Sci USA 82:488 (1985), which is incorporated herein by reference. The presence of the appropriate mutation was verified by DNA sequencing.

The m~mm~lian expression vectors pSV2-decorin and pSV2-decorin/CP-thr4 core protein were constructed by ligating the decorin cDNA or the mutagenized decorin cDNA
into 3.4 kb HindIII-Bam HI fragment of pSV2 (Mulligan and ~erg, Science 209:l423 (1980), which is incorporated herein by reference). -Dihydrofolate reductase (dhfr)-negative CHO cells (C~O-DG44~ were cotransfected with pSV2-decorin or pSV2-decorin/CP and pSV2dhfr by the calcium phosphatecoprecipitation method. The C~O DG44 cells transfected with pSV2-decorin are deposited with the American Type Culture Collection under Accession Number ATCC No. CRL
10~32. The transfected cells were cultured in nucleoside-minus alpha-modified minimal essential medium (~-MEM), (~IBCO, Long Island~ supplemented with 9% dialyzed fetal calf serum, 2 mM glutamine, l00 units/ml penicillin and l00 ~g/ml streptomycin. Colonies ar- ng from transfected 21328a9 cells were picked using cloning cylinders, expanded and checked for the expression of decorin by Lmmunoprecipitation from 35SO4-labeled culture ~upernatants.
Clones expressing a substantial amount of decorin were then subjected to gene amplification by stepwi~e increasing concentration of methotrexate (MTX) up to 0.64 ~M (Kaufman and Sharp, J. Mol. Biol. 159:601 (1982), which is incorporated herein by reference). All the amplified cell lines were cloned either by lLmiting dilution or by picking single MTX resistant colonies. Stock cultures of these established cell lines were kept in MTX-containing medium.
Before use in protein production, cells were subcultured in MTX-minus medium from stock cultures and passed at least once in this medium to elLminate the possible MTX ef~Eects.

Alternatively, the core protein was expressed in COS-l cells as dsscribed in Adams and Rose, Cell 41:1007, (1985), which is incorporated herein by reference.
Briefly, 6-well multiwell plates were seeded with 3-5xl05 cells per 9.6 cm2 growth area and allowed to attach and grow for 24 hour~. Cultures were transfected with plasmid DNA
when they were 50-70% confluent. Cell layers were washed briefly with Tris buffered saline ~TBS) containing 50 ~M
Tris, l50 mM NaCl pH 7.2, supplemented with 1 mM CaCl2 and 0.5 mM MgCl2 at 37C to prevent detachment. The wells were incubated for 30 minutes at 37C with 1 ml of the above solution containing ? ~g of closed circular plasmid DNA and 0.5 mg/ml DEAE-Dextran (Sigma) of average molecular mass of 500,000. As a control, cultures were transfected with the pSV2 expression plasmid lacking any decorin insert or mock transfec~ed with no DNA. Culture were then incubated for 3 hours at 37C with Dulbecco's Modified Eayle's medium (Irvine Scientific) containing 10% fetal calf serum and l00 yM chloroquine (Sigma), after removing the DNA/TBS/DEAE-~extran solution and rinsing the wells with TBS. The cell 3~ layers were then rinsed twice and cultured in the above medium, lacking any chloroquine, for approximately 36 WOg3/20202 213 2 8 5 9 PCT/US93/03171 hours. WI38 human embryonic lung fibroblasts were routinely cultured in the same medium.

COS-l cultures were radiolabeled 36-48 hours after transfection with the plasmid DNAs. All radiolabeled S metabolic precursors were purchased from New England Nuclear (Bosto~, MA). The isotopes used were 35S-sulfate (460 mCi/ml), L-13,4,5-3H(N)] -leucine (140 Ci/ml) and L-t~'C(U)] - amino acid mixture (product number 445E).
Cultures were labeled for 24 hours in Ham's F-12 medium (GIBCO Labs), supplemented with l0~ dialyzed fetal calf serum, 2 mM glutamine and 1 mM pyruvic acid, and containing 200 ~Ci/ml 35S-sulfate or 3H-leucine, or l0 yCi/ml of the ~C-amino acid mixture. The medium was collected, ~upplemented with 5 mM EDTA, 0.5 mM
phenylmethylsulfonylfluoride, 0.04 mg/ml aprotinin and l ~g/ml pepstatin to inhibit protease activity, fx~ed of cellular debris by centrifugation for 20 minutes at 2,000 x G and stored at -20C. Cell extracts were prepared by rin~ing the cell layers with TBS and then scraping with a rubber policeman into 1 ml/well of ice cold cell lysis buffer: 0.05 M Tris-HCl, 0.5 M NaCl, 0.1% BSA, 1% NP-40, 0O~% Triton X-l00, 0.l~ SDS, pH 8.3. The cell extracts were clarified by centrifugation for 1.5 hours at 13,000 x G at 4C.

Rabbit antiserum was prepared against a synthetic peptide based on the first 15 residues of the mature form of the human decorin core protein (Asp-Glu-Ala-Ser-Gly-Ile-Gly-Pro-Glu-Val-Pro-Asp-Asp-Arg-Asp). The synthetic peptide and the antiserum against it have been described elsewhere (Krusius and Ruoslahti, 1986 supra. ) Briefly, the peptide was synthesized with a solid phase peptide synthesizer (Applied Biosystems, Foster City, CA) by using the chemistry suggested by the manufacturer. The peptide was coupled to keyhole limpet hemocyanin by using N-succinimidyl 3-(2-pyridyldithio) propionate (Pharmacia Fine 21328~9 ~...:;

Chemlcals, Piscataway, NJ) according to the manufacturer's instructions. The resulting conjugates were emulsified in Freund's complete adjuvant and injected into rabbits.
Further injections of conjugate in Freund's incomplete adjuvant were given after one, two and three months. The dose of each injection was equivalent to 0.6 mg of peptide.
Blood was collected l0 day~ after the third and fourth injection. The antisera were tested against the glutaraldehyde-cross linked peptides and isolated decorin in ELISA (Engvall, Meth. Enzymol. 70:419-439 (1980)), in immunoprecipitation and Lmmunoblotting, and by staining cells in Lmmunofluorescence, as is well known in the art.

Immunoprecipitations were performed by adding 20 yl of antiserum to the conditioned medium or cell extract collected from duplicate wells and then mixing overnight at 4C. Immunocomplexes were isolated by incubations for 2 hours at 4C with 20 ~l of packed Protein A-agarose (Sigma). The beads were washed with the cell lysis buffer, with three tube changes, and then washed twice with phosphate-buffered saline prior to boiling in gel electrophoresis sample buffer containing 10%
mercaptoethanol. Immunoprecipitated proteins were separated by SDS-PAGE in 7.~-20% gradient gels or 7.5% non-gradient gels as is well known in the art. Fluorography was performed by usi~g Enlightning (New England Nuclear) with intensification screens. Typical exposure tLmes were for 7-l0 days at -70C. Autoradiographs were scanned with an LKB Ultroscan XL ~nhanced Laser Densitometer to compare the relative intensities and mobilities of the proteogIycan bands.

SDS-PAG~ analysis of cell extracts and culture medium from COS-l cells transfected with the decorin-pSV2 construct and metabolically radiolabeled with 35S-sulfate revealed a sulfated band that was not present in mock-transfected cells. Immunoprecipitation with the antiserum -- 213285~

rai~ed against a synthetic peptide derived from the decorin core protein showed that the new band was decorin.

Expression of the construct mutated such that the ~erine residue which is normally substituted with a glycosAminoglycan (serine-4) was replaced by a threonine residue by SDS-PAGE revealed only about 10% of the level of proteoglycan obtained with the wild-type construct. The rest of the immunoreactive material migrated at the position of free core protein.

The alanine-mutated-cDNA construct when expre~sed and analyzed in a similar manner yielded only core protein and no proteoglycan form of decorin. Figure 1 shows the expression of decorin (lanes 1) and its threonine-4 (lanes 3) and alanine-4 (lanes 2) mutated core proteins expressed in COS cell transfectants. 35SO8-labeled (A) and 3H-leucine labeled (B) culture supernatants were Lmmunoprecipitated with rabbit antipeptide antiserum prepared against the NH2-terminus of human decorin.

Purification of Decorin nd Decorin Core Protein from Spent Culture Media Cells transfected with pSV2-decorin vector and amplified as described above and in Yamaguchi and Ruo~lahti, Nature 36:244-246 (1988J, which is incorporated herein by reference, were grown to 90% confluence in eight culture flasks (175 cm2) in nucleoside minus a-MEM
supplemented with 9% dialyzed fetal calf serum, 2 mM
glutamine, 100 units/ml penicillin and 100 ~g/ml streptomycin. At 90% confluence culture media was changed to 25 ml per flask of nucleoside-free a-MEM supplemented with 6~ dialyzed fetal calf serum which had been passed through a DEAE Sepharose Fast Flow column (Pharmacia) equilibrated with 0.25 M NaCl in 0.05 M phosphate buffer, pH 7.4. Cells were cultured for 3 days, spent media was 2132~

collectet and, immediately made to 0.5 mM
phenylmethylsulfonyl fluoride, l yg/ml pepstatin, 0.04 mg/ml aprotinin and 5 mM EDTA.

Four hundred milliliters of the spent media were first passed through gelatin-SepharoFie to remove fibronectin and materials which would bind to Sepharose.
The flow-through fraction was then mixed with DEAE-Sepharose pre-equilibrated in 50 mM Tris/HCl, pH 7.4, plus 0.2 M NaCl and batch absorbed overnight at 4 C with gentle mixing. The slurry was poured into a l.6 x 24 cm column, washed extensively with 50 mM Tris/~Cl, pH 7.4, containing 0.2 M NaCl and eluted with 0.2 M - 0.8 M linear gradient of NaCl in 50 mM Tris/HCl, pH 7.4. Decorin concentration was determined by competitive ELISA as described in Yamaguchi and Ruoslahti, supra. The fractions containing decorin were pooled and further fractionate~1 on a Sephadex gel filtration column equilibrated with 8 M urea in the Tris-HCl buffer. Fractions containing decorin were collected.

The core protein is purified from cloned cell lines transfected with the pSV2-decorin/CP vector or the vector containing the alanine-mutated cDNA and amplified as described above. The~e cells are grown to confluency as described above. At confluency the cell monolayer is washed four tLmes with serum-free medium and incubated in a MEM supplemented with 2 mM glutamlne for 2 hours. This spent medium is discarded. Cells are then incubated with a MEM supplemented with 2 mM glutamine for 24 hours and the spent media are collected and Lmmediately made to 0.5 mM
phenylmethylsulfonyl fluoride, l ~g/ml pepstatin, 0.04 mg/ml aprotinin and 5 mM EDTA as serum-free spent media.
The spent media are first passed through gelatin-Sepharose and the flow-through fraction is then batch-absorbed to CM-Sepharose Fast Flow (Pharmacia Fine Chemicals, Piscataway, NJ) preequilibrated in 50 mM Tris/HCl, pH 7.4 containing O.l M NaCl. After overnight incubation at 4C, the slurr~

W O 93/20202 P(~r/US93/03171 ~.

is poured into a column, washed extensively with the preequilibration buffer and eluted with O.lM - lM linear gradient of NaCl in 50 mM Tris/HCl, pH 7.4. The fractions containing decorin are pooled, dialyzed against 50 mM
NB~HCO3 and lyophilized. The lyophilized material is di~solved in 50 mM Tris, pH 7.4, containing 8M urea and applied to a Sephacryl S-200 column (1.5 X 110 cm).
Fractions containing decorin core proteins as revealed by SDS-polyacrylamide electrophoresis are collected and represent purified decorin core protein.

. E~ PLE II
BINDING OF TGF~ TO DECORIN

A. Affinity Chromatoqraphy of TGF~ on Decorin-Sepharose Decorin and gelatin were coupled to cyanogen bromide-activated Sepharose (Sigma) by using 1 mg of protein per ml of Sepharose matrix according to the manufacturer's instructions. Commercially obtained TGF~-1 (Calbiochem, La Jolla, CA) was ~2sI-labelled by the chlor~ine T method (Frolik et al., J. Biol. Chem.
2~9:10995-11000 (1984)~ which is incorporated herein by reference and the labeled TGF~ was separated from the unreacted iodine by gel filtration on Sephadex G-25, equilibrated with phosphate buffered saline (PBS~
containing 0.1% bovine serum albumin (BSA) (Figure 2).
[l25I}-TGF~1 (5 x 105 cpm) was incubated in BSA-coated polypropylene tubes with 0.2 ml of packed decorin-Sepharose (~) or gelatin-Sepharose (o) in 2 ml of PBS pH 7.4, containing 1 M NaCl and 0.05~ Tween 20. After overnigh;
incubation, the affinity matrices were transferred into BSA-coated disposable columns (Bio Rad) and washed with the binding buffer. Elution was effected first with 3 M NaCl in the binding ~uffer and then with 8 M urea in the same buffer. Fractions were collected, counted for radioactivity in a gamma counter and analyzed by SDS-PAGE

2132~9 - : ~ ;~
W O 93/20202 P ~ /US93/03171 under nonreducing condition using 12% gels.

Figu~e 2A show~ the radioactivity profile from the two columns and the SDS-PAGE analysis of the fractions is shown in Figure 2B. The TGF~-1 starting material contains a major band at 25 kd. This band represents the native TGF~-l dimer. In addition, there are numerous minor bands in the preparation. About 20-30% of the radioactivity binds to the decorin column and elutes with 8 M urea, whereas only about 2% of the radioactivity is present in the urea-eluted fraction in the control fractionation performed on gelatin-Sepharose (Figure 2A).
The decorin-Sepharose nonbound fraction contains all of the minor components and some of the 25 kd TGFB-1, whereas the bound, urea-eluted fraction contains only T~F~-1 (Figure 2B). These results show ~hat TGF~-1 binds specifically to decorin, since among the various components present in the original TGF~-l preparation, only TGF~-1 bound to the decorin-Sepharo~e affinity matrix and since there wa~ very little binding to the control gelatin-Sepharo~e affinity matrix. The TGF~-1 that did not bind to the decorin-Sepharose column may have been denatured by the iodination.
Evidence for this pos~i~ility was provided by affinity chromatography of unlabeled TGF~-1 as described ~elow.

In a second experiment, unlabeled TGFB-1 180 ng was fractionated on decorin-Sepharose as described above for 12sI-TGFB.

TGF~-1 (180 ng) was incubated with decorin-Sepharose or BSA-agarose (O.2 ml packed volume) in PBS (pH
7.4) containing 1% BSA. After overnight incubation at 4C, the resins were washed with 15 ml of the buffer and eluted first with 5 ml of 3 M NaCl in PBS then with 5 ml of PBS
containing 8 M urea. Aliquots of each pool were dialyzed against culture medium without serum and assayed for the inhibition of [~H]thymidine incorporation in MvlLu cells - 21328~3 (Example III). The amounts of TGF~-l in each pool were calculated from the standard curve of t3H]thymidine incorporation obtained from a parallel experLment with known concentration of TGFB-l. The results ~how that the TGF~-l bound e~sentially quantitatively to the decorin column, whereas there was little binding to the control c~lumn (Table 1). The partial recovery of the TGF~-l activity may be due to loss of TGF~-l in the dialy~es.

TABLE I

Decorin-Sepharose affinity chromatography of nonlabeled TGFB-l monitored by growth inhibition assay in MvlLu cells.

TGF~-l (ng) Elution Decorin-Seph~ro~e BS~-Sepharose Fl~w through & wash 2.7 ( 2-3%) 82.0 (93.9~) 3 M NaCl 2.2 ( 1.8%) 1~3 ( 1.5%) 8 M Urea 116.0 (95.9~) 4.0 ~ 4.6%~

B. Bindinq of TGF~-l to Decorin in a Microtiter Assav:
Inhibition bv Core Protein and Biqlycan The binding of TGFB-l to decorin was also examined in a microtiter binding a~say. To pexform the assayt the wells of a 96-well microtiter plate were coated overnight with 2yg/ml of recombinant decorin in 0.l M
sodium carbonate ~uffer, pH 9.5. The wells were washed with PBS containing 0.05% Tween (PBS/Tween) and samples containing 5 x 104 cpm of ['2sI3-TGFB-l and various concentrations of competitors in PBS/Tween were added to each well. The plates were then incubated at 37C for 4 3G hours (at 4C overnight in experiments with chondroitinase ABC-digested proteoglycans), washed with PBS/Tween and the bound radioactivity was solubilized with 1% SDS in 0.2 M
NaOH. Total binding without competitors was about 4% under 2132~

the conditions used. Nvnspecific binding, determined by adding l00-fold molar excess of unlabeled TGF~-l over the labeled TGFB-l to the incubation mixture, was about 13% of total binding. This assay was also u&ed to study the ability of other decorin preparations and related proteins to compete with the interaction.

Completion of the decorin binding was examined with the following proteins (Figure 3; symbols are indicated in the section of BRIEF DESCRIPTION OF T~E
10 FI~U~ES): ;

Decorin isolated from bovine skin and b;;glycan isolated from bovine articular cartilage (PGI and PGII, obtained from Dr. Lawrence Rosenberg, Monteflore ~edical Center, N o Y ~; and described in ~osenberg et al., J. Biol.
Chem. 2~0:6304-6313, (1985), incorporated by reference herein), chicken cartilage proteoglycan (provided by Dr.
Paul Goetinck, La Jolla Cancer Research Foundatio~, La Jolla, CA, and described in Goetinck, P.F., in T~E
GLYCOCONJUGATES, Vol. III, ~orwitz, M.I., Editor, pp. 197-2l7, Academic Press, NY) r For the preparation of coreproteins, proteoglyca~s were digested with chondroitinase AB~ (Seikagaku, Tokyo, Japan) by incubating 500 ~g of proteoglycan with Q.8 units of chondroiti~ase ABC in 250 yl of 0.l M Tris/Cl, pH 8~0, 30 mM sodium acetate, 2 mM PMSF, l0 mM N-ethylmalelmide, l0 mM EDTA, and 0.36 mM pepstatin for 1 hour at 37C. Recombinant decorin and decorin isolat~d from bovine skin (PGII) inhibited the binding of E 125I~-TGF~-l, as expected (Figure 3A). Biglycan isolated from bovine articular cartilage was as effective an inhibitor as decorin. Since chicken cartilage proteoglycan, which carries many chondroitin sulfate chains, did not show any inhibition, the effect of decorin and biglycan is unlikely to be due to glycosaminoglycans.
Bovine serum albumin did not shown any inhibition. This notion was further supported by competition experiments W O 93/20202 PC~r/US93/03171 with the mutated decorin core protein (not shown) and chondroitinase ABC-digested decorin and biglycan (Figure 3B). Each of th~se proteins was inhibitory, whereas cartilage proteoglycan core protein was not. The decorin and biglycan core proteins were somewhat more active than the intact proteoglycans. Bovine serum albumin treated with chondroitinase A~C did not ~hown any inhibition.
Additional binding experiments ~howed that [l2sI]-TGFB-1 bound to microtiter wells coated with biglycan or its chondroitinase-treated core protein. These results show that TGFB-l binds to the core protein of decorin and biglycan and implicates the leucine-rich repeats these proteins share as the potential binding sites.

EXAMPL~ III
ANALYSIS OF THE EFFECT OF DECORIN ON CELL PROLIFERATION
STIMULATED OR INHIBITED BY TGFB-l The ability of decorin to modulate the activity of TGF~-l was examined in [3~]thymidine in~orporation as~ays. In one assay, an unamplified C~O cell line transfected only with pSV2dhfr (control cell line A in reference 1, called CHO ~ells here3 was used. The cells were maintained in nucleoside-free alpha-modified minLmal essential medium (a-MEM, GIBCO, Long Island, -NY) supplemented with 9% dialyzed fetal calf serum (dFCS) and [3H]thymidine incorporation was assayed as described (Cheifetz et al., Cell 48~409-415 ~1987)). TGF~-l was added to the CH0 rell cultures at S ng/ml. At this concentration, it induced a 50% increase of [3H]thymidine incorporation in these cells. Decorin or BSA was added to the medium at different concentrations. The results are shown in Figure 4A. The data represent percent neutralization of the TGF~ induced growth stimulation, i.e., [3H]thymidine incorporation, in the absence of either TGF~-1 or decorin = 0%, incorpor~tion in the presence of TGFB-1 but not decorin = 100~. Each point shows the mean 213285~
W093/20202 PCT/US93/03171''"' "

+ standard deviation of triplicate samples. Decorin (~
~SA (o).

Decorin neutralized the growth stimulatory activity of TGFB-l with a half maximal activity at about 5 ~g/ml. Moreover, additional decorin suppressed the [3H]-thymidine incorporation below the level observed without , any added TGF~-l, demonstrating that decorin al80 inhibited TGF~ made by the CHO cells themselves. Both the decorin-expressor and control C~O cells produced an apparently active TGF~ concentration of about 0.25 ng/ml concentration into their conditioned media as determined by the inhibition of growth of the mink lung epithelial cells.
(The assay could be performed without interference from the decorin in the culture media because, as shown below, the effect of TGF~ on the mink cells was not ~ub~tantially inhibited at the dec~rin concentrations pre~ent in the decorin-producer media.) -,~, ~xperLments in MvLu mink lung epithelial cell~
(American Type Culture Collection CCL64) also revealed an effect by decorin on the activity of TGFn-l. Figure 4B
shows that in these cells~ the growth of which is measured by thymidine incorporation, had been suppre~se~ by TGF~
Assay was performed as in Figure 4A, except that TGF~ was -, added at 0.5 ng/ml. This concentration of TGEB induces 50%
reduction of [3H]-thymidine incorporation in the MvlLu cells. The data represent neutralization of T~F~-induced growth inhibition; i.e., [3H]-thymidine incorporation in the ,presence of neither TG~ or decorin = lO0%; incorporation in the presence of TGF~ but not decorin = 0%. ' EXAMPLE IV
NEW DECORIN-BINDING FACTOR THAT CONTROLS CELL SPREADING
AND SAT~RATION DENSITY

Analysis of the decorin contained in the W O 93/20202 P(~r/US93/03171 overexpressor culture media not only uncovered the activities of decorin de6cribed above, but also revealed the presence of other decorin-a~ociated growth regulatory activitie~. The overexpres~or media were found to contain a TGF~-like growth inhibitory activity. This was shown by gel filtration of the DEAE-i~olated decorin under dissociating conditions. Serum-free conditioned medium of decorin overexpressor CHO-DG44 cells tran~fected with decorin cDNA was fractionated by DEAE-Sepharose chromatography in a neutral Tris-HCl buffer and fractions containing growth inhibitory activity dialyzed against 50 mM NH,HCO3, lyophilized and dissolved in 4 M with guanidine-FCl in a ~odium acetate buffer, pH 5.9. The dissolved material was fractionated on a 1.5 x 70 cm Sepharose CL-6B
column equilibrated with the same guanidine-~Cl solution.
The fractions were analyzed by SDS-PAGE, decorin ELISA and cell growth a~says, all described above. Three protein peaks were obtained. One contained high molecular weight proteins such as fibronectin (m.w. 500,000) and no detectable growth regulatory activities, the second was decorin with the activities described under Example III and the third was a low molecular wei~ht (10,000-30,000-dalton~
fraction that had a growth inhibitory activity in the mink cell as~ay and stLmulated the growth of the CHO cells.
Figure 5 summ~rizes these results. Shown are the ability of the gel filtration fractions to affect [3H]-thymidine incorporation by the C~O cells and the concentration of decorin as determined by enzyme Lmmunoassay. Shown also (arrows) are the elution positions of molecular size markers: BSA, bovine serum albumin (Mr=66,000); CA, carbonic anhydrase (Mr=29,000); Cy, cytochrome c (Mr=12,400); AP, aprotinin (Mr=6,500); TGF, ['2sI]TGF~-l ~Mkz25,000).
.~,, The nature of the growth regulatory activity detected in the low molecular weight fraction was examined with an anti-TGF~-l antiserum. The antiserum was prepared 213~3a9 ~ : ~

against a synthetic peptide from residues 78-lO9 of the human mature TGF~-l. Antisera raised by others against a cyclic form of the same peptide, the terminal cy~teine residues of which were disulfide-linked, have previously been shown to inhibit the binding of TGF~-l to its receptors (Flanders et al., Biochemistry 27:739-746 (1988), incorporated by reference herein). The peptide was synthesized in an Applied Biosystems solid phase peptide ~ynthesizer and purified by ~PLC. A rabbit was immunized subcutaneously with 2 mg per injection of the peptide which was mixed with 0.5 mg of methylated BSA (5igma, St. Louis, MO) and emulsified in Freund's complete adjuvant. The injections were generally given four weeks apart and the rabbit was bled approximately one week after the second and every successive injection. The antisera u~ed in this work has a titer (50% binding) of l:6,000 in radioimmunoassay, bound to TGFB-l in immunoblots.

This antiserum was capable of inhibiting the activity of purified TGF~-l on the CHO cells. Moreover, hS
5hown in Figure 5, the antiserum also inhibited the growth stimulatory activity of the low molecular weight fraction as determined by the t3~]-thymidine incorporation assay on the CHO cells. Increasing concentrations of an IgG
fraction prepared from the anti-TGFB-l antiserum suppressed the stLmulatory effect of the low molecular weight fraction in a concentration-dependent manner (0)- IgG from a normal rabbit serum had no effect in the assay (o).

The above result identified the stLmulatory factor in the low molecular weight fraction as TGF~
However, TGF~-l is not the only active compound in that fraction. Despite the" restoration of thymidine incorporation by the anti-TGF~-l antibody shown in Figure 5, the cells treated with the low molecular weight fraction were morphologically different from the cells treated with the control IgG or cells treated with antibody alone. This -` 2132859 -WOg3/20202 PCT/U~93/03171 effect was particularly clear when the antibody-treated, low molecular weight fraction was added to cultures of H-ras transformed NIH 3T3 ~elle (Der et al., Proc. Natl.
Acad. Sci. USA 79:3637-3640 (1982)). Cells treated with the low molecular weight fraction and antibody appeared more spread and contact inhibited than the control cells.
This result shows that the CHO cell-derived recombinant decorin is associated with a cell regulatory factor, MRF, distinct from the well characterized TGF~'s.

Additional evidence that the new factor is distinct from TGF~-l came from ~PLC experiments. Further ~eparations of the low molecular weight from the Sepharose CL-6B column was done on a Vydac C4 reverse phase column (1 x 25 cm, 5 ~m particle size, the Separations Group, ~e~peria, CA) in 0.1% trifluoroacetic acid. Bound proteins were eluted with a gradient of acetonitrite (22-40%) and the factions were assayed for growth-inhibitory activity in the mink lung epithelial cells and MRF activity in ~-ras 3T3 cells. The result showed that the TGF~-1 activity eluted at the beginning of the gradient, whereas the MRF
activity eluted toward the end of the gradient.

EXAMPLE V
CONSTRUCTION AND EXPRESSION OF MBP-DECORIN
FRAGMENT FUSION PROq~EINS

MBP-Decorin fragment fusion proteins of varying lengths were engineered such that the Maltose Binding Protein (MBP) was attached to the amino terminus of the gene encoding mature decorin as shown in Figure 6. The techniques incorporated for such construction are described in F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons (1987) and Maniatis et al., ~olecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (lg82), which are incorporated herein by reference.

2132~a9 The decorin-encoding DNA fragments were generated by polymerase chain reaction (PCR), Scharf et al., Science 233:1076-1078 (1986), which is incorporated herein by reference. The prLmers, synthetic oligonucleotides S obtained from Geno~ys (Houston, Texas), incorporate an Eco RI restriction site at the 5' end and an Xba I restriction sit~ at the 3~ end of the PCR product. In some instances, the prLmers also included a base change to code for a different amlno acid. The primers used to generate specific inserts are identified in Table 2, while the prLmer sequences are identified in Table 3. The template DNA was a large scale CsCl prep of pPG-40 described in Krusius and Ruoslahti, Proc. N~tl. Acad. Sci. ~SA 83:7683-7687 ( 1986 ), incorporated herein by reference. The DNA
amplification reaction was done in a thermal cycler according to manufacturer's recommendations (Perkin-Elmer Cetus; Norwalk, Conneticut) using the Vent~ DNA Polymerase (New England Biolabs; Beverly, Massachussets). The decorin-encoding DNA fraqments cycled 35-40 times at 94, 40, and 72C.
-.
The PCR products were analyzed by agarose gel electrophoresis, Ausubel et al., supra , and Maniatis et al., supra, to identify and determine the decorin-encoding DNA fragments (see Table 2 under "Insert Size"). The-PCR
products less than 200 ba~e pairs (bp) in size were purified by electrophoresis onto DEAE-cellulose paper, Ausubel et al., supra,; the PCR products greater than 200 bp were purified by using Prep-A-Gene~ DNA Purification Kit (Bio-Rad; Richmond, California~ according to manufacturer's ~0 instructions.

The decorin-encoding DNA fraqments (insert) were ligated between the Eco RI and Xba I restriction sites of the polylinker in the vector pMAL-p (Protein Fusion and Purification Sytem; New England Biolabs). The ligation had a total of 500 ng of DNA and the molar ratio of 2 1~285~
W093/20202 PCT/US93/0317t insert:vector was 3:1. The ligations were then transformed into Escherichia coli (E. coli) DH5a cells (Gibco BRL;
Gaither~burg, Maryland), genotype: F- ~80dlacZ~M15, ~(lacZYA-argF)U169, deoR, recAl, endAl, hsdR17(r~~, nk~), supB44 A-~ thi-l, gyrA96, relAl, or E. coli Sure~ cells (Stratagene, La Jolla, California), genotype: el4-(mcrA), ~(mcrCB-hsdSMR-mrr~171, sbcC, recB, recJ, umuC::Tn5(kanr), uvrC, supE44, lac, syrA96, relA1, thi-l, endAl lF'prQAB, lacIqZ~M15, TnlO, (tetr)]. The ~80dlacZ~N15 marker of the E.
coli DH5a ~train provides a-complementation of the ~
galactosida~e gene from pMAL-p. Colonies containing pMAL-p with the decorin-encoding DNA fragments were colorless on plates containinq 5-Bromo-4-chloro-3-indolyl-~-D-galactoside (X-gal) due to the interruption of the ~-galato~idase gene. Host cells containing pMAL-p only produces blue colonies.
.
Minipreps of colorless colonies were then made as described ~n Ausubel et al., supra, and Maniatis et al., supr~. Plasmids encoding MBP-decorin fragment fusion proteins PT-73, -74, -75, -77, and -78, were then digested with restriction endonucleases Eco RI and Xba I
(both from Promega; Madison, Wisconsin) and the presence of specific in~erts confirmed by agaro~e gel elec~rophore~is.
The other plasmids encoding fusion protein~, PT-72, -76; -84, -85, -86, and -~7, had inserts confirmed by sequencing using 5equena~e~ Version 2.0 DNA Sequencing Kit (U. S.
Biochemical; Cleveland, Ohio~ according to manufacturer's instructions.

Test expression of MBP-Decorin fragment fusion proteins were performed in the host bacterial strain (see Table 2). An overnight culture of E. coli DH5~ or E. coli Sure~ cells containing the MBP-decorin fragment fusion protein plasmids were made by taking a stab of a frozen stock and inoculating L-Broth, Ausubel et al., supra, containing 100 ~g/ml ampicillin at 37C with rapid shaking.

2l32~

The following morning, l ml was used to inoculate lO ml of prewarmed medium (L-Broth containing ampicillin). After 1 hour at 37C, lO0 ~l of O.l M IPTG were added per culture and the induced cultures were allowed to incubate for an additional 2-3 hours. The cells were ly~ed by resuspending in PAGE Sample Buffer (~ovex Experimental Technology;
Encinitas, California) with 0.8~ ~-mercaptoethanol and ~hearing lO times with a l cc tuberculin ~yringe. The sample was run on an 8-16% gradient SDS-PAGE gel ~Novex Experimental Technology) and a Western Blot (Novex ExperLmental Technology) was performed a~cording to manufacturer's recommendations. The blot was developed, Ausubel et al., supra , with Rabbit anti-PG40 serum (Telios Pharmaceuticals, Inc.; La Jolla, California) to test for PT-65, -73, -74, -75, -76, -77, and -78, and Rab~it anti-MBP serum (made in-house) to test for P~-72, -84~ -85, -86, and -87. The results are indicated in Table 2 under "MW."

Large scale CsCl preps, Ausubel, et al., supra, and Maniatus, et al., supra, of the plasmids encoding MBP-Decorin fragment fusion proteins PT-84, -85, -86, and -87 were made and used to transform E. coli. DH5a. Expression of the fusion proteins was confirmed by doing a test expression as described above.

Production batches of the fusion proteins were prepared as follows. An overnight culture of E. coli DHS~
cells contsining the MBP-Decorin fragment fusion protein plasmids was made by taking a stab of the frozen stock and inoculating L-Broth containing lO0 ~g/ml ampicillin at 37C
with rapid shaking. From this culture, 5 ml were used to inoculate a larger 50 ml overnight culture. The following morning, 50 ml of the larger culture were added to 500 ml of pre-warmed media. Typically 1-4 liters were prepared for each batch. After l hour at 37C, 5 ml of O.l M IPTG
were added per flask and the induced cultures were allowed to incubate for an additional 2-3 hours. The cells were --i 2132859 harvested by centrifugation at 5,000 rpm for lO minutes at 10C using either a GSA or GS-3 rotor in an RCSB centrifuge (DuPont Instruments; Wilmington, Delaware). The pellets were resuspended in O.l volume of lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, O.l M PMSF, and 0.25 mg/mllysozyme) and incubated for 10-15 minutes on ice. The suspension was freeze/thawed three times by repeated cycling through a dry ice/ethanol bath and a room temperature shaking water bath. The suspen6ion was sheared by homogenization using a dounce homogenizer. ~he lysate was pre-cleared by centrifugation at 12,000 rpm for 30 minute~ in a SA-600 rotor (DuPont). The cleared ~upernatant was decanted and saved. A final clarification step was done by centrifuging for 30 minutes at 4C in an RC-80 ultracentrifuge using an AH-629 rotor (DuPont). The final cleared lysates were stored either at 4C or -20C
until ready to be purified.

Affinity purifications of the MBP-Decorin fusion proteins were done using an amylose resin (New England Biolabs). 9riefly, six to seven ml of resin were packed into a 2.5 x lO cm glass column in MBP column buffer (lO mM
Tris-HCl, pH 8.4, 1 mM EDTA, 0.5 M NaCl). The resin was pre-equilibrated with at least 3 column volumes of MBP
column buffer containing 0.25% Tween 20. Cleared lysate as prepared above was diluted l part lysate to 1 part 2X MBP
column buffer containing 0.5% Tween 20 and added to the column at a flow rate of ~0-lO0 ml/ hr. Typically, 100-150 ml of diluted lysate were passed over each column. Non-specific material was removed by washing with at least 3 column volumes each of MBP column ~uffer containing 0.25%
Tween 20 and MBP column buffer. The purified MBP-Decorin fragment fusion protein was eluted with 5 x 4 ml aliquots of MBP column buffer containing lO mM maltose. Peak fractions containing the fusion protein were pooled, assayed to determine protein quantity (Bio-Rad Protein Kit;
Richmond, California or Pierce BCA Protein Kit; Rockford, 213~ ~S~
W093/20202 PCT/US93/03171`

Illinois), run on an 8-16% SDS-PAGE gel (Novex Experimental Technology), and stained with Coomasie Blue (Novex ExperLmental Technology) to check for purity. The results of the fusion protein are in Table I under "MW". The purified fusion protein was stored at 4C or -20C in aliquots until ready to be tested for activity.

The pMAL-p vector also was engineered such that a termination codon was incorporated between the Eco RI and Xba I sites. During this process, the original Eco RI site in the vector was destroyed and replaced at a position downstream from a second Factor Xa cleavage site. The second Factor Xa site was incorporated to facilitate subsequent cleavage of the decorin fusion protein from the MBP carrier. The construction involved annealing complLmentary oligos (OT-98 and OT-99; sequences in Table 3) a~d ligating into pMAL-p at the Eco RI and Xba I sites, Ausubel, et al., supra, and Maniatis, et al., supra. The ligation (PT-71) was transformed into E. coli DH5a cells, mini-preps were made from colorless colonies and the clones were sequenced for insert. Expression of the protein followed the same procedure as the MBP-Decorin fragment fusion proteins above. The results are indicated in Table 2 under "MW, W O 93/20202 2 1 3 2 8 ~ ~ PC~r/US93/03171 '''~' : -;

TAE~LE 2 _ CLoNE 5' 3' INSERr HOST # A~ MW
PRIMER PRIMER SIZE EALl~]Rl5L OF (K~) (bp) STR~ ~ ..
. . _ _ ..
Pl-65 Oq-83 OT'85 990 DH5a 330 76 Mature Wh~le Decorin (29ner) (38mer) . _ _ PT'i2 Oq-83 Oq-102137 D~5a 46 45 N~ =inal: (29mer) (3~mer) C m~tated to Y
Fq~73 Oq-83 CT'103 285 D~5a 95 50 N~ cm. to LRR 2 (29mer) (36ner) PT'74 ~q-83 Cq-104 420 DH5a 140 55 N~ m. to IRR 4 (2gmer) (32mer) .
Pq-75 Cq-83 05-105 561 DH5~ 187 60N~ cm. to LRR 6 (2gmer) (32mer) _ _ . .
Pq-76 CT'83 05-106 705 ~Sa 235 65 :~
~ m. to LRR 8 (29ner) (3~nPr) . _ .
Pl-77 Oq~83 CT~107 ~43 DS5a 281 70 N~ 3n. to IRR 10 (2gmer) (3.~mer~
. ~ . . _ PI-78 oq-83 C~'108 918 DB~a 306 73 N~Erm. to % C're~m. (29mer) (32mer) _ _ _ Pq'84 CI-83 CT'118 137 Sure~ 46 45 -:
N~ ~cm: (29mer) (77~er) C nutated to S
1-4 _ . . ~
~I'85 Oq-83 Cq'll9 137 Sure~ 46 45 N~lerm: (29mer) (65~er) . :~
2~ C~_ ~utated to S
_ 3 _ PT'~6 Cq-120 0l-85 165 Sure~ 54 46 C'rerminal ~29mer~ (38mer) . . _ __ -87 Oq-121 Oq-85 165 Sure~ 54 46 C'Term: (29me~) (3~mer~
C1 Dnr;ated to S .
. _ . _ _ . , . .
PT'71 ~ . . _ DH5a _ 40 .
ME~P
-= - . - - - .
C1 = the first cysteine Cl_4 = the f-rst thrcugh the fcurth cvsteine C2 3 = the second and third cvsteine SUBSTITUTE SHEET

2 1 ~ ~ ~j 3 ~

WO 93/20202 PC~`/US93/03171 `

Gl~.G~A.TQG. . . 3 ' C~103 5'.. (3G.q~T.AG~.q~.A~3G.A~T.A~C.m.~CT.AP~.m.~T~G.. 3' ~104 s~.. GG.T~r.A~.q~.m.Tc~.cAc.l~T.~r~G~.Qc... 3~

~106 5 ~ . . .GG.I~l~.A~.l~.A~T.~IC.A~.Al;C.A~.GP~.GCT. . . 3 ' C~107 5 ' . . .GG.~CT.AG~.I~.TCC.A~.I~.AG~.GP~.A~.(~IT.G. . . 3 ' OT-120 5'.. ~G.G~.llC.~.A~.G~C.qlC.TGC.C~.a~T.o.3' C~98 5 ' . . .A~.m.P~C.GP.G.GOE .A~.~.G~A.lTC.q~A.T., . 3 ' aI~99 5 ' . . .Cr.A~.l~.G~A.llC.A~.CCT.AOC.~C.G~.A. . . 3 ' ~` 2132859 ` -i ~ 1 W093/20202 PCT/USg3/03171 .
Tables 4-15 below provide the nucleotide and corresponding amino acid sequences of the decorin fragment fusion proteins prepared as described above. Each table also identifies the Eco RI and Xba I ligation sites.

2132`~S~
WO 93/20202 36 PCI`/US93/03171 o o o o o o o o U + ~ _I ~ + h ~ + O .¢ + E~

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I ~ I E~
I E~ I E~
I E~ I E~
I C~ ~ I E~
E~ I ~ + E~ + a;
~7 1 ~ I ~ E~ I ~ W .¢ I E~
I ~ ~ I C~ .C
t~ I C~ I E~ C` ~ I C~ O I
I t~ I ~ 0 o I ~
I C~ I ~~ .' ~7 1 0 ~ I ~¢
I E - 1~ I t~ ~ Cl I
~ ~ ~ I C~ ~ ~
U I C I C~ ' V I t~ ~ I V '' t~ + ~ I ~ t) + ~ +
t) I ~ D~ U I ~
t~ I C~ t~ t7 I t~ ~ I U
t~ I U t) I t7 ~ m a~
t) t7 Ua ~
I t I U ~ i ~ U I ~ ~ i U I C~ -~ I
~ I ~ t7 1 U I E~
U + Cr) t~ + ~ O + C7 U I ~ ,~
U I t~ U I ~--~ ~ I V E~
C~
I t) t~ t) I V f~
U I t7 --I U I C~ ~ I ~ a: I E~ vY -U I ~ t) I t~
U I t!~ U I ~
U + t!i 1~ + ~ i + E~ ~ + t) U I ~ C E I ~¢
t) I U 11 U I ~ U I t7 ~; I E~ ~
t7 1 0 t~ I t7 C~- ¢ I E~
~C I E~ U I t7 ~1: I E~ C a; I E~ -C~ U ~D U t~ U t~ ~ ~ ~ :
C U I ~ u I ~a. E~ I ¢ ~ I E~
E~ I ~i E~ I U
-i E~ E~ I ¢ E~ U
... ~ U I ~ U I V --I E~ I a; .e i E~
0 ~ + E- E~ + ~C U + U _i U +
~ tq ~ I u ri .~¢ 3 E~ E~ I .¢ t~ I C~
Z t) I ~ t7 I U ~ E~ ; ~ I U
C7 1 U V I U ~¢ I E~
U I ~ ~ ~ t7 I t) E~ I ~ ~ ::
U ~ ~i E ,Y E~ ~ E
E~ a I 1~ .¢ I E~ E~ i I E~
P~ c~ I t) ~a v I ~ ~ I V
.) I t~ ~ t) I C7 I U
t7 I UV 1 5) ta ~ I E~
t~ + (~ + ~ V + 1~ ~ + E~ Yi E - I ~¢ t~ I U P ¢ I ~ ~ I U
U I C~ a: I E~ U I V
t) I t~ ~ ~ I h E~ I ~ ~ I C7 h I ~ Xh I ~ ~71 h ~ 7 ~ ~
C71 t) ~ t7 U I ~7 C~' I E~ V I U~
,¢ I h V I U ~ I h t71 U Cl E~ I ¢ t71 t) ¢ I h c: -¢ + h U + V,id + h t7 ~ U
t) I t7 h I ~: ~C I h .Y ¢ I h t) I t7 ~ ~71 V t71 tJ ~: I ~ Y
t) I C~ ~71 t) ~ U I t~
C~ I U V I U t) I ~ --I V I ~`7 V t) ~ h ~¢ U V E~ .¢ m ¢ I E~ h I S_Ist I E~ t710 h I ~¢ h I ~¢ ~ I h ~i E~
t71 U C71 t~ ~ h I ~ --V ~ + ~ ~; + h ~: ~ + E~
t71 t~ V I t7 h I ~i P I
h I ~alh I ~ h I
t) I C7 ~ I ~ h I ~
h I ~5 ~ V I U U I t7 tJ I t7 _I
h ~ h ~ h U I t7 C71 t~ t71 V ~ V
~71 U ~ I h o t71 U tJ I t7 tr t71 t) ¢ I h ~: I h V I V c710 t~
t7 + V a)E~ + ,~: h + ~i t7 + O a h I ~ V 1 Vi~~ I h t71 I h t~ I U t~ I V ~ E~
t7 1 V ~ E~ l C L I E~
V I t7 ~ I V -~ I h t7 I U
h ~ ~¢ h I I W t7 1 ~ V I t7 ~i I E~
I E O I V~ ¢ t7 1 ~
~ I E~ h I ~: ~ I h ~I h I ~¢
1~1 U ~ --~ I V ~U h I ~ ~ I p t7 1 u~

WO g3/20202 37 2 1 3 2 8 5 9 PCI-/US93/03171 o o o o o o o o o o U + ~ E~ + ~ d + E~ C ¢ + ~ E~ +
Id I E ~ m I t~ E~ I ~1 t~ I t~ 'a E~ I d ~ I O X m ~:
E~ C~; U P a;
C E~ I E~ I ~1 ~ I ~¢ ~ ~ l ~
d I E~ E~ I~ tJ
d I E ~1: 1 ~ I U 0 d I E~
CT + ~ ~ ~ + E~ ¢ .¢ f E

~ + ~ E~ + ~: m ~: + ~ ~5 + ~ ~ +
t~ I U E~ d I E~ C E~ I ~ U I C~
m ~ I ~ U I
a~ I
h I d d I E~ m ~ I U
t~ + t~ E~ + d E~ + ~ ~ +
I E~ C a~ t7 I t) ~ I t) t7 tJ t7 tr ~ ~ t> t7 .C
t7 I t7 E~ I a ~ I --~ t7 I t~ I h ~ t) I t7 t7 1 ~ V I t~
7 t~ I t7D. ~ I E~ t~ I ~ t7 ~ tJ t~
t7 ~ C t~ I t~
t7 + U ~ h + ~ + ~ ¢ + E~ ~ t7 + t7 t7 E~ I V I t7 ~ :
I ~ I t7 t7 1 U ~ E~ I
t7 1 ~ t~ I t~ ~ I t~ --~ V I t7 ¢ I h d I E~ m h I t7 1 U t~ I t~
t~ _1 ~ E~ O t7 ~7 1 ~ ~ U I ~ ~ I ~ I E~ X t7 I t) i t I t~ ~ I ~ o '.', t~ I t7 ~ C ~ t~ I t7 ~¢ I hrl E~ I d h I ~ C E~ I d U + t7 d + h ~ ~C + E~ ~) + t7 d d I E~ C ~ U I t7 ::
I V ~ 7 I t~
; I h t~ ¢ V I t7 t7 I t7 'a U I t7 t~ I t7 ~
d h --I t7 ~ E~ t7 ~ t7 a t7 ~ U
I ~ d~ I ~t7 I t~ t7 I U d I h ~ ~
~ I ~7 I t~t~' E~ I ~ a I ~
d I E~ d I E~ E~ I ~ ~ U I t7 t,7 1 ~7 t7 I rJ 0 d I ~ U I t7 E~ I ~ rL ::
d + E~ E ~ + d ~ + E~ + P ¢ + E : -U I t7t~' t.~ I t7 t7 1 ~ U I V.C t~
I ~7 t7 I t~ 0 E~ I ~C E l I .¢ t7 I t~ tll : ::
d I ~ E~ I d U I t7 C E - I ~ u I t7 ~ I t~
t7 U ~ 1t7 ~ u t~ U t7 U:~ -U I t7 ~ ro I t7 --~ t7 1 U E~ 7 I V a~ E~ I .¢
U I t~ ~U I t~ t7 1 U t~
(~ I t~ t~ I ~7 t,q` I U al ~ I ~ E~ I ~C `
5-7 + t~ t~ + ~ ~ + E l .¢ + E~ h U ~ V

~5 t~ I t7 t7 I t7 r~ 7 ~ rn ~1; I E~ C ~ U I r t~ I t7 E~
a t~ . t~ t7 . t~ ~ ,¢ E~ E~ . a V I t7 E~ I U I t7 .C
t~ I t~ U I t7 t~ I ~ I t7 V I t~ ~
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I ~; t~ I U ¢ I h ~ U
t7 I tJ > ~ I E~ U I t7 ~ I h ~ ~ I E~
I ~ h ~ t7 1 U U I ~ ~ I h c:
t7 I U ~ I h m ~ I E1 E I ~
~C h.Y ¢ h U ~ ~: h C ~ h U ~ ~ U I ~ S
t7 I V t7 1 U d I h ~,1 U I ~ h I
tJ I t7 ~ C I E~ ~ I u ra h I
t7 1 U ~: I h ~ ~; I h ~ I U U I t7 h + ~: ¢ + ~ ~ + h ~: E~ + ¢ U + ~
t~ I U ~ I U u I t7 U I ~ --I ¢ 7 h t~ I tJ ~ ~ I E~ E~
I ~ t~ I t~ ~ I ~ C U I t~ U I t7 :
I ~ .Y d I h U I t7 h I a: rn h I d tJ t~ tJ t~ ~ V u u ~ ~ u tJ I t7 tJ I ~ t7 1 U t~ t7 1 U h I .1!
¢ I E~ ~ h I E~ t7 1 u D~ h I ~
~ I t7 h I~ ¢ I h h I ~ I U P
h I ~ I V I t,) ~ ¢ I h ~ I U

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W0 93/20202 41 PCI`/US93/03171 o o o o o o o ~, + ~ ~ + E~ ~7 + ~ + E~
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V I U (a ~ h V I ~ U I V ~ t~
a ~ ~ U I ~ V I
+ ~) ~ ~ + ~ E~ + ~: ~ + CJ

U I V V I U ~ ¢ I ~ V I t~
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SUBSTITUTE SHEET

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EXAMPLE VI
BlNDING STUDIES OF 125I-TGF-~
TO IMMOBILIZED DECORIN AND FRAGMENTS

Immulon wells were coated with 0.5 yg/ml S recombinant decorin at 50 yl/well. The wells were placed in a 37C incubator overnight and thereafter washed 3 times with 200 yl P~S t0.15 M NaCl) per well to remove unbound decorin. TGF-~ labeled with l25I (400 pM, New England Nuclear, Bolton-~unter Labeled) was pre-incubated with or without competitors in 200 ~l PBS/0.05% Tween-20 for l hour and 45 minutes at room temperature. Competitors included recombinant human decorin preparations (DC-l3 and DC-18v), decorin fragments, and MBP as a negative control. DC-13 and DC-18v are different preparations of recombinant human decorin; PT-71 or MBP (malto~e-binding protein) is a negative control; PT-65 i~ MBP-whole decorin; PT-72 is MBP-decorin N-terminus; PT-73 is PT-72 + 2 LRR; PT-84 and PT-85 are cysteine to ~erine mutant of PT-72; PT-86 is decorin C
terminu~; PT-87 is cy~teine to ~erine mutant of PT-86.

Fifty ~l/well of the pre-incubated 125I-$GF-~
mixture or control were added and incubated o~ernight at 0C. Following the incubation, 50 ~l of the free TGF-~
æupernatants were transferred to labeled tubes. The plate was washed 3 tLmes with 0~05% Tween-20 in PBS (200 25 ~1/w811). The wells were then transferred into tubes for counting in a gamma counter. The results of the binding studies with Lmmobilized decorin are summarized in Figures 7 and 8.

Recombinant human decorin (DC-13), MBP-whole 30 decorin (PT-65), MBP-decorin N-terminus (PT-72) and MBP-decorin N-terminus ~ 2 leucine rich repeats (PT-73) inhibited l25I-TGF-~ binding to immobilized decorin as shown in Figure 7. MBP alone (PT-7l~-had no effect on 12sI-TGF-~binding to immobilized decorin. These results demonstrate 2 132~s'~

that the N-termlnus of decorin is capable of binding TGF-~in ~olution and preventing it from binding to immobilized decorin. Thus, two portions of the molecule appear to contain part of the binding site in decorin for TGF-~.

As shown in Figure 8, recombinant human decorin (DC-18V) + M~P-decorin N-terminus (PT-72) inhibited 12sI-TGF-binding to immobilized decorin. In addition, cysteine to ~erine mutants of PT-72, (C-24, C-28, C-30, C-37 to serine, PT-84; C-28 and C-30 to serine, PT-85) did not inhibit l2sI-TGF-~ binding to decorin. MBP-decorin C-terminus (PT-86) and a cy~teine to serine mutant (PT-87) of PT-86 also inhibited l25I-TGF-~ binding_to decorin. These results demonstrate that the C-terminus of decorin is also capable of binding TGF-~ and that the first cysteine residue in the C-terminus i8 not required for TGF-~ bin~ing.

EXAMPLE VII
BINDING OF l25I-TGF-~ TO ~EPG2 CELLS

About 2.5 x lO' HepG2 cells or L-M(tk-) cells were incub~ted with 250 pM[l25I]TGF-~ in the presence of recombinant human decorin (DC-12), PT 71 ~MBP), decorin fragments (PT-72, -73, -84, -85, -86 and -87) or anti-TGF-~ antibodies for 2 hours at room temperature. Cells were washed with washing buffer ~l28 mM NaCl, 5 mM XCl, 5 mM Mg2SO~, 1.2 mM CaCl2, 50 mM HEPES, p~ 7.5) four times before determination of bound CPM. The results are ~ummarized in Figures 9, lO and ll.

Recombinant human decorin, MBP-whole decorin (PT-65), MBP-decorin N-terminus (PT-72) and MBP-decorin N-terminus + 2 leucine rich repeats ~PT-78) inhibited l'5I-TGF-~ binding to ~epG2 cells. MBP alone (PT-71) had no effect on 12sI-TGF-~ binding to HepG2. These results shown in Figure 9 demonstrate that the N-terminus of decorin is capable of preventing TGF-~ from binding to its receptor on HepG2 cells.

As shown in Figure 10, recombinant hu~an decorin a~d MBP-decorin N-terminus (P~-72) inhibited l25I-TGF-~binding to L-N(tk-) cells. An anti-TGF-Bl antibody also inhibited TGF-~ binding to these cell~. Cysteine *o ~erine mutants of PT-72 (C 24, C-28, C-30, C-37 to serine, PT-8~-C-28 and C-30 to serine, PT-85) did not inhibit ~2sI-TGF~
binding to L-M(tk-) cells.

Recombinant h-lm~n decorin, MBP-decorin N-terminus lo (PT-72) and anti-TGF-~ antibodies inhibited '2sI-TGF-~binding to L-M(tk-) cells. MBP-decorin C-terminus (PT-86) and a cysteine to serine mutant (PT-87) of PT-86 also inhibited ~25I-TGF-~ binding to L-M(tk-) cells. As shown in Figure 11, these results demonstrate that the C-terminus of decorin also i~ capable of i~hibiting TGF-~ binding to its receptor, and that the first cysteine residue of the C-terminu~ is not required for inhibition.
':"
EXAMPI~ VIII
SYNTE~ETIC PEPTIDES

The following peptides were synthesized and tested for their ability to inhibit binding of TGFB 1 to L- -M(tk-) cells:

Name Sequence ' ~3~ - S37 ~LR W QS
P2s - Q36 PFRSQSHLRVVQ
H31 - ~42 HLR W QSSDLGL
- nP2s ~ Q36 PFRCQC~LRWQ

Peptide H3, - S37 corresponds to the same decorin sequence between His-31 and Cys-37 except the Cysteine at position 37 is replaced with a serine. Peptide P2s ~ Q36 corresponds W093~ ~322~ ~ PCT/US93/03171 to the sequence reported in Krusius and Ruoslahti, supra, from position Pro-25 through Gln-36, except the native Cysteine residues at positions 28 and 30 are each replaced with a serine. Peptide H3, - L,2 alco correæponds to decorin between ~is-3l and Leu-42 except the cysteine at position 37 is replaced with a serine. Peptide nP2s-Q36 corresponds exactly with decorin from position Pro-25 through Gln-36.

The peptides were synthesized using the applied Biosystems, Inc. Model 43OA or 43lA automatic peptide synthesizer and the chemistry provided by the manufacturer.

The activity of the peptides was evaluated using the L-M~tk-) TGF~l binding inhibition assay described in Example VlI, except various concentrations of peptide were incubated with the cells and TGF~l instead of decorin and rec~mbinant decorin fragments. The negative control wa~ a synthetic peptide corresponding to the first 15 ~mino acids of decorin, which has the sequence DEASGIGPEVPDDRD.

Figure 12 provides the binding data for peptides P2S - Q36 ~ ~31 - S37 ~ and H31 - L~2 and the control peptide.
All three test peptides inhibited binding of TGF~l to L-M(tk-) cells. Peptide nP2s - Q36~ in which the native Cys residues remain, also demonstrated inhibitory activity, al~eit to a lesser extent. Table 16 lists the test peptides in the order of decreasing inhibitory acti~ity, i.e., peptide ~3l-S3, was found to show the highest inhibitory activity.

Further binding studies were conducted with soluble N-terminal decorin peptide fragments synthesized and tested for their ability to inhibit TGF-~l binding to immobilized decorin as described above. The N-terminal peptide fragments are listed in Table 17.

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W093/20202 PCT/US~3/03171 ss -.

Peptide Sequence 16D VPDDRDFEPSL&

~he results of the binding studies are shown in -Fig~re 13, which shows that the peptide 16G inhibited TGF-~
binding to i~mo~ilized decorin.

lo 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 l;m;ted only by the following claim~.

Claims (22)

We claim:

1. An active fragment of a protein having a cell regulatory factor binding domain.

2. The active fragment of claim 1, wherein the cell regulatory factor is TGF.beta..

3. The active fragment of claim 2, wherein TGF.beta.
is TGF.beta.l.

4. The active fragment of claim 2, wherein TGF.beta.
is TGF.beta.2.

5. The active fragment of claim 1, wherein the cell regulatory factor is MRF.

6. The active fragment of claim 1, wherein said protein is decorin.

7. The active fragment of claim 1, wherein said protein is a functional equivalent of decorin.

8. The active fragment of claim 7, wherein said functional equivalent is biglycan,

9. The active fragment of claim 1, wherein said fragment is a recombinant DNA peptide.

10. The active fragment of claim 9, wherein said fragment is PT-72, PT-73, PT-78, PT-86, PT-87 or PT-65.

11. The active fragment of claim 9, wherein said fragment is PT-78.

12. The active fragment of claim 1, wherein said fragment is a synthetic peptide.

13. The active fragment of claim 12, wherein said synthetic peptide is H31-S37, P25-Q36, H31-L42, P25-Q36 or 16G.

14. A purified compound comprising a cell regulatory factor attached to an active fragment of a protein having a cell regulatory factor binding domain.

15. The purified compound of claim 14, wherein said cell regulatory factor is TGF-.beta..

16. A method of inhibiting an activity of a cell regulatory factor comprising contacting the cell regulatory factor with an active fragment of a protein having a cell regulatory factor binding domain.

17. The method of claim 16, wherein said protein is decorin.

18. A method of detecting a cell regulatory factor in a sample, comprising:
(a) contacting the sample with an active fragment of a protein having a cell regulatory factor binding domain; and (b) detecting the binding of said cell regulatory factor to said active fragment, wherein binding indicates the presence of said cell regulatory factor.

19. The method of claim 18, wherein said protein is decorin.

20. A method of treating a pathology associated with the activity of a cell regulatory factor, comprising administering to an individual an effective amount of an active fragment of a protein having a binding domain corresponding to said cell regulatory factor to prevent or treat said pathology.

21. The method of claim 20, wherein said protein is decorin.

22. The method of claim 20, wherein said cell regulatory factor is TGF-.beta..