CA2084992A1 - Tissue-derived tumor growth inhibitors, methods for preparation and uses thereof - Google Patents

Abstract

The present invention provides methods for producing and purifying proteins comprising TGF-.beta.3 precursor, pro region of the TGF-.beta.3 precursor and mature TGF-.beta.3. Moreover, this invention also provides methods for producing and purifying novel homodimeric proteins with enhanced production and heightened therapeutic utility which comprises mutations, signal peptide deletions, and protease cleavage site substitutions.

Description

~09t~0031X PCT/US91/04541 20~4992 9l:~l3L~DCa S~O~ oaQ~L~9~ Qpa~ N~T~OD8 ~OR
P~PAR~IO~ A~D ~8~ ER~Q~

Thi~ application is a continuation-in-part of U.S. Serial No.
5353,410, filed May 17, 1989, whiGh is a continuation-in-part of U.S. Serial No. 183,224, filed April 20, 1988, which is a continuation in part of U.S. Serial No. 111,022, filed October 20, 1987, which i8 a continuation-in-part of U.s. Serial No.
922,121, filed October 20, 1986, now abandoned, which was a continuation-in-p~rt of U.S. Serial No. 847,931, filed April 7, 1986, now abandoned, which wa8 a continuation-in-part of 5 U.S. Serial No. 725,003, filed April 19, 1985, now abandoned, the content~ of each are hereby incorporated by reference into the prQsQnt application.

Backg~gund of the Invention Throughout thi~ application, variou~ publication~ are 10 referenced by Arabic numerals within parentheses. Full cit~tion~ for the~e publications may be found at the end of the specification im~ediately preceding the claim~. The disclosure~ of th~se publications in their entireties are hereby incorporated by reference into this application in 15 oxder to more ~ully dQscribe the state of the art as known to thos~ skill~d therein as o~ the date o~ the invention de~cribQd and clai~d herein.

Transfor~ing growth factor B tTGF-A) i~ part of a family of multi~unctional proteins which appear to modulate, alone or in 20 co~bin~tion with other ~olecule~, cell proliferation and di~f~rentiation. Reportedly, TGF-B, which comprises both a mature, a pro and a preGursor form, includes several isofor~s (i.e. TG~-Bl, -B2. -B3, -B4, and -B5) coded for by different .

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W092/0031~ PCT/US9l/04~
208~9~ 2 gene~. ~he TGF-B2 and TGF-B3 cDNA have been found from sever~l mammalian ~ources (14). TGF-B4 cDNA has only been isolated rrOm chicken Cell8 ~17). Mature TGF-B1, -B2, -B~
cDNAs share an amino acid sequence identity of abo~t 75-80%
5 whereas the T~F-Bl, -B2, -B3 precur~or exhibit only 25-35%
identity. Surprisingly, despite the high degree of homology among the TGF-B'~, it appears that these proteins have distinct differences in potenciQs (14).

Mature TGF-B ha~ been isolated from various species. Murine, 10 bovine, human, and porcine TGF-B have been isolated and show very little difference in amino acid co~position (5, 8, 11, 14 24).

The cDNA sequence of mature TGF-B,---its expression in both normal and tran~formed cells, ~nd methods for producing 15 biologically active mature TGF-B in eucaryotic cells have been described (2, 37, 8, 11, 38).

R. Derynck et al. (38) hava dQscribed a ~ethod comprising (a~
cons~ructing a vector whlch includes nucleic acld encoding TGF-B3, (b) transforming a h~terologou~ host eucaryotic cell 20 wlth th~ vector, (c) culturing the trans~orDed cell, and (d) r~covering TGF-B3 from thQ culture mediu~

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The present invention provides mQthods for producing and purifying proteins comprising TGF-B3 precurQor, pro region of the TGF-B3 precursor and mature TGF-B3. Moreover, this 5 invention also provide~ ~ethods for producing and purifying novel homodimeric proteins with enhanced production and heightened therapeutic utility which compri~es mutations, signal peptide dQletions, a~d protease cleavage site substitutions.

W092~003l8 PCT/US91/0 ~

2 0 ~ ~ 9 9 2 B~ o~lDtio~ o~ t~ lour~

rlgur- 1 shows the nucl~otide ~equence encoding TGF-B3 and lt~
deduced amino acid sequence. Putative glycosylation ~ites and polyadenylation signal are underlined. The start of the 5 mature TGF-B3 is marked by an asterisk at alanine at nucleotide positions 1163-1165.

~lgur- 2 shows a Southern blot analy~is of human tumor DNAs hybridized with a PvuII-PYuII TGF-~1 cD~A probe.

~ igur- 3 shows a Northern blot analysis of A673, A549, and 10 A498 cell lines using an EcoRI-BglII 1.7kb cDNA fragment of the gene encoding TGF-B3 a~ a probe.

~lgur- 4 shows a Northern blot analy~is of A673, A549, and A498 cQll lin~s using a PvuII-T~qI probe from genomic qequences o~ TGE-B3. -15 ~lgur~ 5 show~ a Northern blot analysi of A673, A549, and A498 cell line u~ing a PstI-BalI TGF-~1 probe.

rigur- 6 show~ a Northern blot analycis of A673, A549, and A498 cell lines using TGF-~1 cDNA containing the complete coding sequence o~ TGF-~1 precur~or as a probe.

20 risur- 7 shows ~ Northern blot analysi~ of mRNA fro~ umbilical cord ~nd A673 cell lines using an EcoRI-BglII cDNA fragment of the gene encoding TGF-B3 as a probe.

rlsu~- 8 qhows the production of ~X~E::TGE-B3 fusion protein o~ three lysates by SDS polyacryla~ide gel electrophore~is.
25 (A) corre~ponds to TGF-B3.

~igur- 9 shows whole cell bacterial lysates containing .. . : ...

~n 92/00318 PCT/US9l/04541 f 2!D~49~)2 trpE::TGF-~l fusion proteins (lanes 1 and 4), trpE::(A) fusion proteins (lAnes 2 and 5), and the TGF-~1 protein (purcha~ed from R&D Systems~ (lanes 3 a~d 6) were ~eparated on a 12.5%
SDS-polyacrylamide gel. The proteins were electrophoretically 5 transferred to a nitrocellulose filter (1 ~m pore size) and incubated w~th 100 ~g of af~inity purified anti-peptide antibody elther in the ab~ence (lanes 1, 2 and 3) or presence of a 300 fold molar exces~ of the antigenic peptide (lanes 4, 5, and 6). The antibodi¢s were detected u~ing alkaline 10 phosphatase conjugated to goat anti-rabbit antibody (Promega) according to the manufacturers instruction.

rlgur- lo shows a schematic diagram of mRNA encoding TGF-B3 with the coding sequence boxed. The relative extension of the cDNA insertQ obtained from placenta (1.7 kb), umbilical cord 15 (1.9 kb) and A673 (1.7 kb) libr~ris~ i8 indicated. The dashed part o~ th~ box represents th~ C-ter~nal r2gion ~howing high homology to TGF-~s. The 5' EcoRI-Bg II restriction fragment of the placenta cDNA is indicated by a bar.

rlgur- 11 A/B shows a comparison of the nucleotide ~equence 20 and predicted amino acid saquenc~ of the g~ne encoding TGF-~3 with TGF-~l and TGP-~2. Identic~l a~ino acids are boxed. The matur~ TGF-B3 amino acid s~quenc~s start at position 315. (~) corr~Rpond~ to TGF-B3.

~ gur- 12 i~ a schematic repr~sentation of the construction of 25 th~ pC~V-TGF-~3 expression plas~id from pORFX and p81ue-TGF-~3 pla~ids.

rlgur- 13 ~how~ the level o~ TGF-~3 ~RNA expres~ion, d~ter~ined by Northern hybridization using a TGF-~3 specific probe, o~ parental CHO cells (l~ne 1), CHO cells transfected 30 with TGF-~3 cDNA (CHO 6.35) (l~nQ 2) and CHO 6.35 amplified with 20nM ~TX (CHO 6.35/~OnM (l~ne 3).

WO92/00318 PCT/US91/04 ~
2 ~ 9 9 ~ 6 -~
rlgur- 14 (A) shows the dose re~ponse of mink cell growth inhibition using purified TGF-~l. Cell growth was quantitat~d by the metabolism of MTT 3-[4,5-Dimethylthiazol-~-yl]-2,5-5 diphenyltetraazolium bromide; Thiazolyl blue) (148).
~B) shows the dose response of mlnk cell growth inhibition using acid activation ~erum free supernatants CH0 6.35/20nM
transfectant and CH0 6.35 transfectant. Cell growth was qu~ntitated by the metabolism o~ MTT.

10 ~lguro 15 shows ~he relative location of the various TGF-~3 pQptides used as antigen3.

rigur- 16 shows the immunoprecipitation o~ native recombinant TGF-~3 protein by ~3V antibody.

~lgur- 17 15 ~A) shows the immunoblot of TGF-~3 from conditioned media of CH0 6.35/20nM transfectant using ~3III and ~3~ antlbodie3 for detection from gels under reducing conditions.
(B) shows the immunoblot o~ TGF-~3 from conditioned media of CHO 6.35/20nH tr~n~fsctant using ~3I~I ~nd ~3V antibodies for 20 detection frou gels under non-reducing conditions.

rigur- 18 ~hows a ~estern blot or cell extract (18A) and cond~tioned m dia (18B) o~ the CX0 6.35/20nH transfectant u~ing ~3V ~ntibody ~or detection.

rlsuse 19 A,B,C,D shows the staining to paraffin ~ections of 25 hum~n umbilical cord by ~3V antibody and control antibody. A
and C show fibroblast and epithelial staining and smooth mu w lQ ~iber staln~ng, respectively, by ~3V antibody. 8 and D ~how no st~ining by control rabbit polyclonal antibody.

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~,~092/00318 PCT/US91/04541 7 2 ~9 9 2 riguS- 20 is ~ ~ilver stained gel of purified TGF-B3 and TGF-rigur- 21 A, ~, C show~ spocific antibody neutralization of TGF-~3 inhibition of mink cell growth by B3V.

5 7~gur- 22 shows the location of the variou~ protease sites genetically engineered into the TGF-B3 precur~or.

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WO92/00~18 PCT/US91/045~
2~'19~2 8 ~ j t~ cript~o~ o~_tho Invention In accordance with the invention, mature TGF-B3 is defined as a recombinant homodimeric protein which comprises two polypeptides each of which consists essentially of 112 amino 5 acids a~d has a seguence substantially identical to the amino acid sequence shown in Figur- 1 begin~ing with an alanine encoded by nucleotides 1163-1165 and ending with a serine encoded by nucleotides 1496-1498.

Moreover, as used herein TGF-B3 precur~or i8 a recombinant 10 homodimeric protein which comprises two polypeptides, each polypeptide encoded by a sequence substantially identical to the amino acid sequence shown in Figuro 1 beginning with a methionine encoded by nucleotides 263-265 and ending with a serine encoded by nucleotides 1496-1498.

15 Further, as used herein the pro region of the TGF-B3 precursor is a recombinant protein which compriqes the TGF-B3 precursor without the mature TGF-B. In particular, the pro region of the TGF-B3 precursor is a protein encoded by a sequence substantially identical to the amino acid sequence shown in 20 F~gur- 1 beginning with an methionine encoded by nucleotides 263-265 and ending with a arginine encoded by nucleotides 1160-1162.

Also, as used herein, reference to TGF-B means either mature TGF-B (e.g. TGF-B1, -B2, -B3), TGF-B precursor (e.g. TGF-Bl 25 precursor, TGF-B2 precur~or, TGF-B3 precursor), or the pro region o~ the TGF-B (e.g. TGF-~1, -B2, -B3) precursor.

The present invention provides a method of recovering purified, non-denatured mature TGF-B3 from a mixture of mammalian cell-derived polypeptides. The method comprises 30 contac~ing the mixture with an antibody which specifically - . , .

,-~ 92/00318 PCT/US91/04541 9 2 ~ 2 binds to mature TGF-B3 but exhibits sub~tantially no cros~
reactivity with mature TGF-Bl and mature TGF-B2.

In one example, the mixtur~ of mammalian cell-derived polypeptidea is a mixture of non-human mammalian polypeptides 5 from non-human cells in which TGF-B3 ha~ been expres3ed.

Additionally, in another example of the subject invention, the antibody may be directed to an epitope defined by the amino acid sequencQ YLRSADT~THSI'VLGLYNTLNPEASASY. E3y way of example, the previou~ly descrlbed antibody may be immobilize~ on a 10 ~olid support under condition~ such that TGF-B3 i~ isolated and purified.

Additionally, this invention provides a method for producing sub~tantially purified TGF-B3 precursor having an anchorage m~mbrane sequence which comprises: (a) preparing DNA encoding 15 a TGF-B3 precursor having the m~mbrane anchorage sequence; (b) inserting the DNA into an expre~ion vector linked to a suitable promot~r compatible with a host cell; (c) transforming the host cell with the vector in order to induce ~xpression o~ the DNA of ~t~p (b) such that a TGF-B3 precur~or 20 is ~xpressed and subsequent translocation of the expres~ed TGF-B3 precur~or having th~ ~embrane anchorage sequence; (d) culturing th~ host cell in medlum; (e~ ~parating the ho~t c~ll rrO~ the medium; (f) di~rupting thQ c~ll such that a lyJat- cont~lnlng th~ TGF-B3 pr~c~r~or having the membrane 25 anchorage sequence i~ produc~d; and (g) purifying the TGF-B3 procursor having a membran~ anchorage sequence from the lyRate under conditions ~uch that the sub~tantially purified TGF-B3 precursor is produced.

The present invention also provides a method for producing 30 substantially purified TGF-~3 precursor having an anchorage membrane sequence. The method compri~es: (a) preparing DNA

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W092/0031X PCT/US91/04~ ~
20~992 lO
encoding the ~GF-B3 precursor having a membrane anchorage sequQnce; (b) inserting the DNA into an expre~sion vector linked to a suitable promoter compatible with a host cell; (c) tran~rorming the host cell with the vector in order to induce 5 expression of the DNA of step (b) such that a TGF-B3 precursor is expressed and sub~equent occlusion o~ the expressed ~GF-B3 precur~or having a membrane anchorage sequence in occlusion bodies; (d) culturing the host cell in culture medium; (e) separating the occlusion bodies from the host cells and the 10 culture medium; (~) difirupting the occlusion bodies to produce a solution containinq the TGF-B3 prQcursor having a membrane anchorage sequenee; and (g) purifying the resulting TGF-B3 preeursor having a membrane anehorage sequence from the lysate under condition~ such that the substantially purl~ied T~F-B3 15 precursor i8 produced. The above-deseribed method further compri~es: (a) treating the purified TGF-~3 precursor 80 recovered with an ~etivating agent to sQp~rat~ a mature TGF-B3 from the precur~or, and (b) raeovering the separated mature TGF-B3 of step (a).

20 In accordanee with the pr~ent invention, the anchorage linkage seguence ~ay be a phosphatidyl inositol linkage.
Alternatively, the anchoragQ linkage sequenee i5 a hydrophobic tran~embrana peptide 3equenee.

Al~o, the present invention provide~ a m2thod ~or producing a 25 sub~tantially purifiad mutant TGF-B3 precur~or. The method eompri~e~ (a) pr~p~ring a DNA compri~ing a first DNA sequene~
eneoding an a~ino acid ~equenc~ substAntially identical to the amlno aeid ~eguenee shown in rigur- 1 baginning with a ~thionine eneod~d by nuel~otides 263-265 and ~nding wtth 30 gluta~ine eneoded by nueleotides 1148-1150, a seeond DNA
seguenee whieh i8 linked to nueleotide 1150 eneoding a protease cleavage sequenee, and a-third DNA seguence, linked to the ~econd DNA ~equence, encoding an a~no acid sequence .
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W092/0031~ PCT/US91/04541 11 2~49~2 ~ubstantially ldentical to ~the ~mino acid ~equence shown in ~igur- 1 beginning with a alanine encoded by nucleotides 1163-1165 and ending with a serlne encoded by nucleotides 1496-1498; (b) inserting the DNA of step (a) into an expre~ion 5 vector linked to a suitable promoter compatible with a host cell; (c) transforming the ho~t cell of step (b) i~
tran~or~ed with the vec or in order to induce expre~sion of the DNA of step (b) such that a mutant TGF-B3 precursor i8 expre~sed; (d) culturing the host cell in medium under 10 conditions such that thQ mutant TGF-B3 precursor 80 expres~ed i5 secreted into the msdium; (e) ~eparating the ho~t call from the cultuxe medium containing the mutant TGF-B3 pr~ursor 80 secreted; and (~) puri~ying the mutant TGF-B3 precursor such that a substantially purified ~utant TGF-B3 precur~or i8 15 produced. The above-described method furthar co~pri~e~: (a) treating the purified mutant TGF-B3 pr2cursor 80 recovered with an activating agent to separate a mature TGF-B3 ~rom thQ
precursor; and (b) recovering the separated mature TGF-B3 of tep (a)-20 Moreover, this invention also provides a ~ethod for producinqa sub~tantially purifiod ~utant TGF-B3 precursor. The ~ethod compri~es: ta) preparing a DNA co~prising a first DNA sequence encod~ng an a~ino acid sequence ~ubstant~ally identical to the amino acid sequence shown in rlgur- 1 beginning with a 25 methionine ancoded by nucleotides 263-265 and ending with glutaaine encoded by nucleotides 1148-1150, a ~econd DNA
~equ~nce which i8 linked to nucleotide 1150 encoding a protea~e cl~a~ag~ s~quence, and a third DNA ~equ~nce, linXed to ths second DNA sequence, encoding an a~ino acid sequence 30 ~ub~tantially id~ntical to the ~nino ~cid ~quence shown in ~lgu~- 1 b~ginning with an alanine encoded by nucleotides 1163-1165 and ending ~ith a serine encoded by nucleotides 1496-1498; in~ertinq the DNA o~ step (a) into an expression vector linkod to a ~uitable pro~oter compatible with a hoat - , :
, , 2 0 ~ 003t8 12 PCT/US91/04 r cell; (c) transforming the host cell with th~ vector in order to induce expres6ion of the DNA of qtep (b) such that a ~utant TGF-B3 is expressed; (d) sQparating the host c~ll containing the expres~Rd, mutant TGF-B3 precursor from the cult~re S medium; (e) di~rupting the host cell such that a ly~ate containing th~ ~utant TGF-B3 precur~or i8 produced and (f) purifying the mutant TGF-~3 precursor ~uch that a substantially purified mutant TGF-B3 precursor i~ produced.
Additionally, th~ above-de~cribad method further comprises (a) 10 tr~ating the purified mutant TGF-B3 pre~ur or 80 recovered with ~n activating agent to ~eparate a mature TGF-B3 from the prQcursor and (b) recovering the separated mature TGF-B3 of step (a)-~ , Additionally, this invention provides a method ~or producing15 a substantially purified mutant T5F-B3 precursor. The method comprises: (a) preparing a DNA comprising a ~irst DNA sequence encoding an amino acid sequence sub~tantially identical to the amino acid ~equence shown in r~g~r- 1 beginning with a m~thionine encoded by nucl~otides 263-265 and ending with 20 glutamine encoded by nucleotides ll4a-l~5o~ a 3econd DNA
sequence comprising ATG which i~ linkQd to nucleotide 1150, a third DNA sequence, link~d to the s6cond DNA sequence, encodlng an 2mino acid o~qu~nc~ ffubstantially identical to the am~no acid s~quencs ~hown in Tlgus- 1 beginning with a alanine 25 encodcd by nucl~otides 1163-1165 and ~nding with a asparagine encod~d by nucleotide~ 1469-1471, a fourth DNA sequence, link~d to tho third DNA sequence, comprising X, and a ~irth DNA ~quence, link~d to the ~ourth DNA ~equence, encoding an amino acid ~equence sub~tantially identical to the amino acid 30 ~quence shown in rlgur- 1 beginning with a valine encoded by nucleotide~ 1475-1477 and end~ng with a 3erine encoded by nucleotide~ 1496-1498; (b) in~erting ths DNA of ~tep (a) into an expreasion vector linked to a suitable promoter compatible with a ho~t cell; (c) transforming the ho~t cell w~th the - ~
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W092/003lX PCT/US91/04S41 " 13 208l~99~
voctor in order to induce ~xpre~ion o~ the DNA o~ st~p (b) 8UC~ that a mutant TGF-B3 precursor 18 expre~sed; (d) culturing the host cell in mediu~ under condition~ such that the expressed mutant TGF-B3 precursor i~ secreted into the 5 medtum; (e) separating the cell from the culture mediu~
containing the mutant TGF-B3 precursor so secreted; and (f) purifying the mutant TGF-B3 precursor such that a ~ubstantially purlfied ~utant TGF-83 precursor is produced.
The method ~urther comprlsee: (a) treating the purified 10 ~utant TGF-B3 precursor 30 recovered with cyanogen bro~ide to separata a ~ature TGF-B3 from the precursor; and (b) recovering the ~eparated mature TGF-B3 of ~tep Sa).

Further, the invention al80 provides a method for producing a substantially purified mutant TGF-B3 precursor which 15 co~pri~e~: (a~ preparing a DNA conprising a ~irst DNA sequence encoding an aMino acid ~equence substantially identical to the amino acid sequence shown in rigux- 1 beginning wlth a methionine encoded by nucleotides 263-265 and ending with glutamine encoded by nucleotides 1148-1150, a second DNA
20 sequence co~pri~ing ATG which i~ linked to nucleotide 1150, a third DNA ~equence, linked to the sQcond DNA sequence, encoding an amino acid ~equence 8ub8tantially identical to the auino acid segu~nce shown in ~lyur- 1 baginning with a alanine encoded by nucl~otides 1163-1165 and endlng with a asparagine 25 encoded by nucleotides 1469-1471, a ~ourth DNA sequence, linked to the third DNA sequence, co~prising X, and a f if th DNA soquenco, llnked to th~ fourth DNA sequ~ncQ, encoding an a~ino ~cid equenco sub~t~ntially idQntic~l to the amino acid soqu~nco shown in ~lgur- 1 beginning with a valine encoded by 30 nucleotid~ 1475-1~77 ~nd onding with a ~rine encoded by nucl~otide~ 1496-1498; (b) in~ertlng the DNA o~ step ~a) into an expr~ssion vector linked to a suitable pronoter co~patible with a host cell; (c) tr~nsfor~ing the host cell with the vector in order to induce expression of the DNA of step (b) ~ 3 ' W092/0031# PCT/US9l/04 2 0 8 ~ ~ 2 14 - ~uch th~ a mutant TGF-B3 precursor is expressed; (d) sQparating the host cell containing the expres~ed, ~utant TGF-B3 precursor from the culture medium; (ej disrupting the host cell ~uch that a lysate containing a mutant TGF-B3 precursor 5 i~ produced; and (f) purifying the mutant TGF-B3 precur~or such that a ~ub~tantially purifled mutant TGF-B3 precursor i8 produced. The method further compris~s: (a) trsating the purified mutant TGF B3 precur80r ~o recovered with cyanogen bro~ide to separate a ~ature TGF-B3 from the precursor; and 10 (b) recovering the separated mature TGF-B3 of step (a).

Further, in accordance with the practice oS the above-de~cribed methods, X in step (a) may b2 ~elected from a group of tri-nuclQotidQs consi~ting oS TTT, TTC, TTA, TTG, TCT, TCC, TCA, TCG, TAT, TAC, TGT, TGC, T&G, CTT, CTC, CTA, CTG, CCT, 15 CCC, CCA, CCG, CAT, CAC, CM , C~G, CGT, CGC, CGA, CGG, ATT, ATC, ATA, ACT, ACC, ACA, ACG, AAT, AAC, AAA, AAG, AGT, AGC, AGA, AGG, GTT, GTC, GTA, GTG, GCT, GCC, GCA, GCG, GAT, GAC, GAA, GAG, GGT, GGC, G~A, and GGG.

In on~ embodi~ent of the sub~ect invention, puriSication i~
20 effected by a~inity chromatography. one example of affinity chromatography is antibody column chromatography. Another ~xample of affinity chro~tography i8 lectin column chromatography. L~ctin column chromatography allows isolation o~ the glyco~yl~ted precursor form of TGF-~3.

25 Also, in one example of the invention, in step (a) oS the abovo-d~cribed ~ethod, the prot~ase cleavage sequence may be a coll~gen~e rec~gnition sequ~nce. Altern~tively, ~he prote~se cle~vage ~equence may be a Factor Xa recognition sequence. The prote~se cleavage sequence allows for the 30 production o~ a ~e~brane anchorad TGF-~3 preGursor.

This invention also provide~ a method ~or producing mutant . - ~
- - , 92/003t8 PC1/US91/04541 1S 2~4992 TGF-B3. The method compri~e~ (a) preparing a DNA comprising a ~equence substantially identical to the amino acid ~equence shown in ~lgur~ 1 beginning with leucine encoded by nucleotides 332-334 and ending with serine encoded by 5 nucleotides 1496-1498; (b) inserting the DNA of step (a) into an expression vector op~rably linked to a suitable pro~oter compatible with a host cell; (c) trans~orming the host cell with the vector in order to induce ~xpr~ssion of the DNA of step (b) ~uch that a ~utant T&F-B3 i8 expressed; ~d) culturing 10 the host cell in mediu~; (e) separating the ho~t cell~
containing tha mutant TGF-B3 80 Qxpressed ~rom the mediu~; (f) disrupting th~ cells to produce a lysate containing the ~utant TGF-B3; and (g) purifying the mutant TGF-B3. The above-described method further coDpri~e~ (a) treating the purifisd 15 mutant TGF-B3 BO recovered wlth an activating agent to s~parate a mature TGF-B3 from the mutant TGF-B3; and (b) recovering the separated matur~ TGF-B3 of ~tep (a).

T~i4 invention additionally provides a proc~s which compri~es: (a) contacting a TGF-B3 precursor with a 20 precipitating agent thereby concentrating the TGF-B3 precur~or in a precipitate; (b) extracting the p~ t of step (a) with an acidified organic solution under such condition~ that mature TGF-B3 i8 ~eparated from the pell~t; and (c) recoverinq the ~ture TGF-~3 ~o s~p~ratod in ~tep (b).

25 In ~ccordance with the ~ub~ect invention, the acidified organic ~olution in ~tep (b) ~ay be an acidiried acetonitrile.
Additionally, th8 organic solution may comprise 50%
acetonltril~ and 1.0~ acetic acid. A1BO in accordance with the ~ub~ect inv~ntion, the precipitating agent in step (a) may 30 be a~monium sulfate.

Thi~ invention also providec a method for producing and identifying a mutant, mature TGF-B3 exhibiting reduced binding .

WO~2/0031X PCT/US91/04 ~
20~'1992 16 .-~r~inlty to sQrum blndlng proteln~. Th~ method comprises (a)preparlng a DNA encodlng the TGF-B3; (b) performing mutagQnesis (for example random mutagQnesis) on the DNA of step (a) thereby obtaining a ~utant DNA; (c) in~erting the 5 mutant DNA lnto an expression vector linked to a ~uitable promoter compatible with a host cell; (d) transforming the host cell with the vector in order to induce expression o~ the mutant DNA of step tc) under conditions such that a mutant TGF-B3 is expr~sed; (e) culturing the host cell in medium 10 under conditions such that the expressed ~utant TGF-B3 i~
secreted into the medium;. (f) ~eparating the ho~t cell from the culture medium containing the mutant TGF-B3 so expresfied;
(g) purifying the mutant TGF-B3; (h) ~ctivating the mutant TGF-B3 80 expre~ed under conditions such that a mutant, 15 mature TGF-~3 i8 separated ~rom the ~utant TGF-B3; and (i) assaying the culture medium ~or the ~utant mature TGF-B3 thereby identifying a mutant mature TGF-B3 exhibiting reduced binding affinity to seru~ binding proteins.

Additionally, thi8 invention ~urther provides a method for 20 producing and identifying a mutant, mature TGF-B3 exhibiting reduced binding a~finity to serum binding protein~. The method comprises: (a) preparing a DNA encoding the TGF-B3;
(b) performing mutagenesis (for example random mutagenesis) on the DNA o~ stRp (a) thereby obtaining a mutant DNA; (c) 25 inserting the ~utant DNA lnto an expression vec~or linked to a suitable promotQr comp~tible with a ho~t cell; (d) tr~n~or~ing ~he ho~t cell wlth th~ v~ctor in order to induce expr-~sion o~ the ~utant DNA of ~tep (c) under conditions such th~t ~ ~ut~nt TGF-B3 i8 expres~ed; (e) culturing the host cell 30 in medium under condition~ such that the expres~ed mutant TGF-B3 is produced in the host cell; (f) ~eparating the host cell eont~ining the mutant TGF-B3 80 expres~ed from the culture ~ediu~; (g)di~rupting the cells to produce a lysate containing the mutant TGF-B3; (h) purifying the mutant TGP-B3;

-: -, . ~ . , , , ,.: . ,, ~.'Q92/00318 PCT/US91/04541 `;` 17 20~992 (i) actlvating the uutant TGF-B3 8C expr~ed under cond.'.ions such that a mature TGF-B3 i~ ~eparated from the mutant TG~- 3;
and (~) ~ssaying the culture medium for thQ mutant mature T~
B3 exhibitinq reduced bindin~ af f inity to seru~ binding 5 proteins theroby identifying a ~utant, matur~ TGF-B3 exhibiting reduced binding a~finity to seru~ binding protQins.

In one example of the abov~-de~cribed method, the serum binding protein i~ ~2-~acroglobulin. Alternatively, in another example, the seru~ binding protein i8 type III TGF-B
10 raceptor, e.g. betaglyc~n. Further alternatively, the serum binding protein may be a pro region of the TGF-B precursor.

Thi~ invention further provides a method for producing a substantially purified pro region o~ the TGF-B3 precur~or.
Th~ me~hod compri~es: (a) prop~ring DNA encoding the pro 15 region of the TGF-B3 precur~or; (b) inserting the DNA into an axpros~ion vector llnked to a ouitable promoter compatible wlth ~ host cell; (c) transrorming th~ ho~t c~ll with the vector in ord~r to induce ~xpre~ion o~ the D~A o~ 8tep (b) und~r condltion~ such that a pro region of the TGF-B3 20 preCUrBOr i8 QxprQssed; (d) culturing tho ho~t cQll in medium;
(e) 3Qpar~ting host cells ~ro~ the mediu~; (f) disrupting the host cell~ to produce a ly~ate containing the pro region of ~he TGF-B3 precur~or; and (g) puri~ying the pro region o~ the TGF-B3 pre~ur~or fro~ tha lysata ~uch that t~e substantially 25 puri~iad pro region of the TGF-B3 precursor i5 produced.

Additionally, thi~ invent~on provides a ~ethod ~or producing a ~ub~tantially purified pro region of the TGF-B3 precursor which co~pri~ea: (a) preparing DNA encoding the pro region of the TGF-B3 precur~or; (b) inserting the DNA into an expre~ion 30 v~ctor llnked to a ~uitable pro~oter co~patlble wlth a ho~t cell; (c) tran~orming the ho~t cell w~th the vector ln order to induce expre6sion of the DNA of step (~) under conditions -W092/003lX 18 PCT/US91/04 2 0 ~ 9 such that a pro region of the TGF-B3 precursor i~ expres~ed;
(d) culturing th~ ho~t cQll in medium under conditions ~uch that the expre~sed pro ragion of the TGF-B3 precursor is secreted into the mQdium; (e) separating host cell~ from the 5 ~ediu~ containing the pro r~gion of th~ TGF-B3 precur~or ~o secreted; and (f) purifying the pro region of the TGF-B3 precursor such that the substantially purified pro region of the TGF-B3 precursor iB produced.

10 Further, in each o~ the above-describad method~, the host cell may be either a eucaryotic cell or a procaryotic cell.

This invention is illustrated in the Experimental Details section which follows. This section i8 set forth to aid in an understanding of the invention but i8 not intended to, and 15 should not be construed to, limit in any way the invention a8 ~et forth in the claims which follow.

-' ' ' ~ .
,: ' ~ ', -92/00318 PCT/US9l/04541 2~9~2 Abb~ç~i~tion~ and Technical-Terms:

AL (acute leuk~mia) ANLL (adult non-lymphocytic leukemia) 5 APRT (adenosylphosphoribosyl transf~rase) BFU-E (burst ~orming unit-erythroid) BSA (bovine s~rum albumin) CL (chronic leukem~a) CLL (chronic lymphocytic leukemia) 10 CML (chronic my~logenous lsukemia) CNBr (cyanogen bromide) CFU (colony forming unit) CFU-E (colony forming unit-erythroid) CFU-GEMM (colony forminq unit-granulocyte, erythroid, 15 macrophage, monocyte) CFU-GM (colony for~ing unit-granulocyte/macrophage) CFU-meg (colony forming unit-~gakaryocy~e) CHO (chinsse hamster ovary) CHV (cytomegaloviru~) 20 CSF (colony ~ti~ulating factor) DHFR (dihydrofolate reductase) D~EH (Dulbecco's ~odiri~d ~gle's ~dium) ~F (di~othyl ~or~mide) D~SO (dim~thyl ~ul~oxid~) 25 DNA (d-oxyribonucl~ic acid~
EPO (orythropoietin) FCS (~etal cal~ ~rum) G-CSF (granulocyte-colony sti~ulating factor~
G~-CSF ~granulocyte/macrophage-colony stimulating factor) 30 kb (kilob~se pair ) kDa (kilo Daltons) HPLC (high pre~ure liquid chromatography) .

wos2/0031X PCTtUS91/0 ~

o~ll 9~2 IL-3 (interleukin-3) 20 IL-4 (lnt~rleukin-4) MEM (modified Eagle's ~edium) mRNA (mes~enger ribonucleic acid) 5 RNA (ribonucleic acid) TGF-B (tran~forming growth factor-beta) TIF (tumor inhibitory factor) WBC (white blood cell) ~A~PLB ~: c~o~INa T~ CO~INC T~F-B3 10 To identify sequences with homology to TGF-~l a Pvu II-Pvu II
prob~, containing most o~ the ~ature form of the TGF-~l cDNA
~equence, was 32p labelled and u~ed to Rcreen a Southern blot (34) o~ total human DNA~ digested with Eco RI, Hind III or Sst I using 3tandard ~ethods. In each digest, two bands were 15 pre~ent at a low ~tringency wash (2.5 x SSC, 65C) (~igur- 2).
When the w~h stringency WA 8 increa~ed (O.Ol x SSC, 65C) only one hybridizing band re~ained in each digest (rlgur- 2). The strongly hybridizing b~nd is TGF-~l, and the waakly hybridizing ~and i8 a related gene which al~o encode~ TGF ~3.
20 The nucleotide seguence enco~ing TGF-B3 and its amino acid sequence are shown in (r~gu~ l).

To isolate the gene encoding TGF-B3 with ho~ology to TGF~
th~ TGF-~l clonQ was u~d to scr2en a hu~an phage library constructed ~ro~ the DNA of a chronic myelocytic leukemia cell 25 line (~562). Two geno~ic loci, which correRpond to TGF-01 and tho r~l~tod g~ne encoding TGF-~3 (rlgure l), w~re cloned.
Conctruction ~ the R562 library, ~creening and isolation of reco~binant clones W~8 c~rried out e~sentially according to th~ proc~dures o~ Grosveld, et al. t15).

30 The phage DNA clone containing the sequence encoding TGF ~3, was cut with Sau 3A and the restriction fragments cloned into -, - ' - ~ . , .
, -. -.
.
,:. , - , .
, s2/oo3lN PCT/US91/04~1 ``~` 21 2 ~ 9~ 2 Ml3. T~e recombinant plaques were screened with the SmaI-PvuII probe of TGF-~l. Six hybridizing genomic clones were saquenced by the method o~ Sanger, et al. (33) and a region of approximately 130 bp wa~ found to b~ homologou~ to TGF-~l S cDNA. When the amino ac~d ~qu~nce o~ TGF~l and TGF ~3 cloned in the~e experiments were compared they wsre found to be 82%
homologous.

To obtain a rep~at free probe Or TGF-~3 various restriction fragments from BamHI-BamHI ~ubclone of this gene were 10 hybridized to TGF-~l cDNA, as well as to total human DNA. A
~UI-TaqI fragment of the gene, i.e. TGF ~3, was found to cro~s hybridize with the TGF-~l cDNA but did not hybridlze to repetitive sequence element~ in human DNA.

The Ba~HI-TaqI unique probe o~ the aequence encoding TGF-B3 15 was used to screen the lambda-gtll human placenta cDNA library (Clonetech). Two ~trongly hybridizing clones, a~ well as four w~akly hybridizing clone6, w~re isolated. By DN~ ~equence analy~is the weakly hy~ridizing clones were shown to correspond to TGF-~l. One ~trongly hybridizing clone wa~
20 isolated and a l.7kb ~coRI in~rt wa~ ~u~cloned into pUC 8.

Re6triction frag~ont~ for th~ clone wQr~ subcloned into ~13 and s~quenced by the method o~ S~ng~r, et al. The deduced aoino acid seguance of this gene exhibits extensive ho~ology wlth a r~ily o~ genes (24) including TGF-~l, TGF-~2, 25 glioblasto~ T-cell suppressor factor (G-T~F), inhibln/activin, Mullerian Inhibiting Substance (MIS) and docapontaplagic tran~crip~ co~plex o~ Dro~ophila with the ~ix C-terminal cysteine residues being conserved throughout.

A 17 kb g~no~ic DNA frag~ent containing the sequence of the 30 g~ne, i.e. TGF-~3, has be~n cloned. Hybrldizing 5' and 3' portions of the l.7 kb cDNA clone which encode3 TGF-B3 with ' ~ , .

. . ~ . . ~ ,, ::. :: .. .. . ..

W092/003t8 PCT/US91/04 ~

2 ~ the genomtc locua of TGF-B3 revealed that the 1.7 kb ~DNA
sequence is completely contained in the genomic clone. Taking into account that the full length message of TGF-B3 i~ 3.5 kb, additional 5' and 3' flanking sequences need to be i~olated to 5 obtain the complete gene. This is done by screening genomic phage and cosmid librarie~ with probas unique to the gene encoding TGF-B3.
.
In TGF-~l thQ sequ~nce R-R repre~ent~ thQ proteolytic ~leavage site which generates the mature protein. In TGF-B3 the 10 ~equence R-K-K-R represents the coxrecponding cleavage site.

In tho rQgion N-t~rm$nal to the cleavag~ ~it~, TGF-~1 and TGF-B3 ~xhibit only 35% homology. However, both protein~ contain the qequencQ R-G-D in the N-t~r~inal region which i8 :
recognized by thQ fibronectin receptor.

15 In order to determine which cell types expre~s TGF-B3, Northern hybridization wa~ carri~d out using a 5' terminal Eco RI-Bgl II probe (~igur- 3). Th~ r~sult of Northern ~; hybridization revealed a ~RNA of approxi~ately 3.5 kb in A673 (a rhabdo~yosarco~a), A498 ( kidn~y c~rcino~a) and a faintly 20 hybridizing sign~l in A549 (a lung ~denoc~rcino~
. , .
; A genomlc prob~ from tho 3' region of TGF ~3 (corre~ponding to sequ~nc~s downstream of the presumed ~ite of proteolytic : cl-a~ag~) was th-n used to ~cr~n tho ~ame Northorn blot.
Thr~ strong hybridization ~ignals w~re ob~erved in both A673 25 and A498, corro~ponding to TGF-~1 (2.5 kb), TGF-~3 (3.5 kb) and anothcr r~lat~d g~ne (4.2 kb) (rlg~r- 4). Tho~e results are consi~tent with the notion that thi~ probe cro~s react3 with ~quences ho~ologou~ to TGF-B3.

Northern blot analy~is o~ A673, A549 and A498 cell line~ using 30 a PstI-BalI TGF-~1 probe was then performed. The PstI-BalI
.~
.
:
. . .
, , . :, : . ... ... , .. . . : ,, :.
. , . . ~ , . . .

~ 092/00318 PCT/US91/04541 23 20~992 TGF-Bl probe strongly hybridized to a 2.5 kb mRNA band in all thr-~ cell line~. S~veral weakly hybridlzing bands ar~ al~o observod at 4.2 kb and 3.5 kb (rlgur- 5). This probe i~
highly speci~ic for TGF~ ince it contains sequences 5 corre~ponding to tho8e re~idue~ N-ter~in~l to the prot~olytlc cleav~ge site, a region whera TGF~ xhibits little homology to other members o~ this gene family.

A Northern blot of A673, A549 and A498 cell lines were then screened using TGF-~l cDNA containing the complete coding 10 sequence of the TGF-~l precursor. This probe croa~ hybridized with homologous sequences to TGF-~l. Sp~cifically, there was strong hybridization to a 2.5 kb mRNA band corrasponding to TGF-~l (rlgur- 6).

Northern blot analysis of ~RNA ~rom human u~bilical cord ~nd 15 the A673 c~ll line was ~l~o ~cr~en~d u~ing an EcoRI - BglII
cDNA fragment o~ TGF-B3 ~ ~ probe (~lgus- 7). rlgur- 7 al~o illu~tr~tes the re~ult of a Northern blot using an actin probe a~ a control to nor~alizQ mRNA lovels in e~ch l~ne. When norm~lized to actin ~RNA l~vel~, the umbilical cord expre~es 20 the highe~t l~vel of ~RNA of the genQ encoding TGF-B3 in compari~on to other mRNA sourcQs ~o ~r examined.

Southern blot analysis was performed on a variety o~ different tu~or DNAs dige~ted with EcoRI and hybridized with ~ S~aI-AvaI
cDNA ~ragnent o~ TGF-~3 as a proba. Hybridization wa~
25 effe~tod at both low (2.5 X SSC, 65C) and high (0.3 X SSC, 65C) ~tringency washes. Southern blot an~ly~is indicated the po~siblo pr~a~nc~ o~ oth~r loci rel~t~d to TGF-B3. The probe hybridized wlth two banda (3 kb and 12 kb) which were observed only i~ washed with low ~tr~ngency.

, ., .

WO92/00318 PCT~US91/04 2 0 ~ 2 24 ~oductiQnLof ~n~Q~ with ~ ~

Chimeric bacterial proteins, l.e. fusion proteins, have been constructed which con~ain the C-terminal l50 amino acid~ o~
TGF-B3 fused to a small reglon o~ the trpE gena. The fusion 5 protein w~8 recognized by an antibody produced again~t a peptide derived from a~ino acid numbers 9 to 28 of the ~ature form of TGF-B3. Tha antibody recognized the trpE::TGF-B3 fu lon protein and the peptide specifically competes with TGF-B3 for the binding o~ the antibody.

10 DNA ~equences that code for TGF-B3 were cloned into a pKS
vector. This vector is à pATH II derivative that contains the inducible ~p promoter and a multiple cloning site. The ra ulting constructs produca a chimeric protein consisting of the ~irst 22 a~ino acid~ o~ the trp~ gane, the C-terminal 150 ; 15 amino acids of TGF-B3.

Trans~or~ant~ contalnlng these clones wer~ screened primarily by re~triction endonuclea~e analysi~ and ulti~ately for production of the chi~ric protein by SDS polyacryla~ide gel electrophore8i~. The protein products of 3 clone~, pll6, 20 p~34, and pl35, are shown in ~gur- 8. These cell~ were grown in de~ined m~din unt~l they raached early log pha~e and then incubated for 3 hour~ either in the pre~ence or ab~ence o~ the trpE inducer indoleacrylic ~cid (IAA). The cells were then collected, ly~ed and their protQins electrophore~ed on a 12.5%
25 SDS polyacryla~ide gel. rlgur- 8 i8 a photograph of one such gel that had b0en st~ined with Coomas~ie blue. As can be seen, lysates pll6 and pl35 produce a prot~in o~ about l9,000 Dalton ~olecular waight who~e relative abundance increases in the pre~ence o~ IAA. In contrast, pl34 doe~ not produce this 30 protein species. Both pll6 and pl3S contain pla3mids that, by restriction analy~is, h~ve the sequences of TGF-~3 cloned in ' : `

.
: .
~ , ' ;i ~92/003t8 PCT/US91/04341 2 ~ 99 2 the orientation that ~hould produce a 19,500 Dalton molecular welght ~usion protein. The pl34 pla~id was found to have the sequence~ of TGF-B3 in the opposito orientation.

The trpE::TGF-B3 fusion protein was used to test the 5 specificity of an antibody that used a peptide homologou~ to part o~ TGF-B3 a~ an antlgQn. A polypeptide w~ synthesizod corresponding to residuss 9 through 28 of mature TGF-B3 excQpt that residuQ 9 in the ~equen~e, arginine, was replaced by sorine. The peptide w~8 purified by rever~e pha~e HPLC and 10 coupled to keyhole limpet hemocyanin for u~e a~ an i~munogen in rabbits.

Thirty-three day~ ~ollowing the ~irst in~ection (500 ~g), the antisera w~re ~creened by ~tandard E$ISA u~ing l00ng of peptide per wall. One rabbit demonstratQd a signal o~ l.0 OD
15 unit~ at a 1:25 dilution o~ the Anti~era. Ten d~y~ a~ter this rabbit was first bl~d, a boo~t of 250~g o~ coupled antig~n w~5 given. The following bleed 20 day~ after the ~irst bleed showad a 20-~old increaso in antibody responsQ to the peptide antig~n. Forty days a~ter the initial bleed ~3rd bleed) a 20 signal o~ l.0 OD unit w~8 ~chiev0d at a l:B000 dilution of the antibody, a 16-~old increAsQ in antibody titer over the ~econd bleod. Thi8 antibody ~how~d little cross-re~ctivity with a ho~ologous p~ptide derlved ~ro~ ~GF-~l sequQnce8. Th~ TGF-~l ~` deriv~d peptid~ consi~ted o~ a~ino acid nu~bers 4 to l9 o~ the 25 ~tur~ ~GF-~l protein. Of the ll co~Don a~ino acids, i.9.
r~idu~ 9-l9, 7 ar~ cons~r~ed botwoen TG~-B3 and TGF~

To d~t~rmine i~ the peptide recognizing antibody could recognize TGF-~3, the antibody was u~ed ln Western blot analy~i~ again~t a trpE:mature TGF-B3 and trpE:~ature TGF-~l 30 ~usion protein~. As asen in ~lgus- 9, the anti-peptide antibody reacted 3trongly with the fu~ion protein o~ TGF-B3 while it reacted only weakly with a trpE::TGF-~l fusion .. . . . .
. - , ~ .
. .
' --wos2/ov3lX PCT/US9l/04 ~ ~, 2 ~ 2 protein. Both fu~lon protQin~ were recognized by a commerc~ally available antl-TGF-~l antibody (R and D 8y~tem8) 9 ) .

A~ can ~e seen in Flgur- 9, the anti-peptide antibody 5 recognizing TGF-B3 also has a high level of bacXground reactivity to bacterial proteinR. To reduce this cross r~activity, we purified the antibody on a CNBr-Sepharo~e column containing the original peptide used as an antigen.
The antibody retained it~ high titer to th~ peptide o~ TGF-B3 10 and low cross reactivity to the homologous peptide derived from TGF-~l. The purified antibody reacts very ~trongly with the fu~ion protein of TGF-B3.

EUc~ryQ~ic E~ession of TÇF-81 Fused Wi~h TÇE-~3 Human recombin~nt TGP-~l has been exprQssed in monkey COS
15 c~lls. Sequenc~ ~nroding the complete pr~cursor of the TGF-~1 cDNA were cloned down stre~ ~rom a SV40 promoter using the pSVL eukaryot1c expres~ion vector (obtain~d from Pharmacia~.
Thi8 construct was trans~ected into COS cell~ u8ing a ~tandard calcium phosphate precipitation method (13). After 20 trans~ection, approximately 4 x 106 cells were grown in serum free media ~or two day~. Th~ condltioned ~edia was then coll~cted, ~cidified and t~sted for biological activity.
Conditioned media from TGF-~l transfected cells was found to inhiblt tha growth of a monolayer ~ink lung test cell line 25 (CC~ 6~) by 59% a~ compared to conditionQd media ~rom COS
c~118 trans~ected with the pSVL v~ctor alone which inhibited growth o~ CCL 64 cells by only 32%.

A chimeric pro region o~ the TGF-~l pr~cur~or: :mature TGP-B3 ru~ion construct wa~ ~ade by ~ubstituting 5' ~e~uence~ o~ the

3 O T~F-~1 precur~or w~ th sequ~nces e~coding TGF-B3. Glven the homology betw~en the two proteins and the con~erved position .,....... , . - .
-: - . -.

:, - ~
.

~092/003tX PCT/US91/04541 ' ~' 27 2 ~ 93 2 o~ their cysteine residue~, when such a construct i~
trans~Qcted into COS cell~ the novel fu~ion protein is proce~sed lnto the biologically active mature TGF-B3.
Additlonal con~tructs, which consi~t o~ the trpE::TGF-B3 5 fusion cloned under the regulator ~equences of either the SV40 promoter of the long terminal repeat of the mou~e mam~ary tumor virus (MMTV) have be~n made and te~ted for biological activity in tran~ient tran~ection experiment~.

~l1GL5_Zs ~ur~r -~u~4--~tt~r~i~4~io~ of a g-3- aoo~i~g o ~r-B3 Screening a lambda gtll human placenta cDNA library (Clontech 1.2 x 106 independent clone~) with a repeat free probe of the genQ encoding TGF-B3, rQ~Ult~d in the isolation of a 1.7 kb cDNA clone. On Northern analy~is, the ~RNA for TGF-B3 was 15 found to be approximately 3.5 kb, indicating that a full length cDNA had not be~n obtained.

To obtain additional 5' sQquQnc~ information,- a lambda gtll hu~an umbilical cord cDNA library (Clontech, 1.5 x 106 independent clone~) wa~ ~creen~d with a 5' EcoRI-BglI~
20 ro~trict$on ~rag~ent derivRd fro~ the plac~nta cDNA clone.
Thi~ re~ult~d in thQ i~olation o~ a 1.9 kb cDNA. Sequence analysis revealed this clone contained an addltional 180 nucl~otide~ o~ 5' sequence infor~ation~ The i~olation of this cDNA rrom an u~bilical cord libr~ry again confirms that thi~
25 gono i- actively tr~nscribed in thi~ ti~sue.

To obtain furthQr cDNA ~qu~nce in~or~ation for the gene ~ncoding T~F-B3, ~RNA was ieolated fro~ A673 cQll~ and a cDNA
llbrary pr~p~red. St~rting with 5 ~g poly (A)+ RNA, a random primed cDNA library of approximately 2 x 1o6 clones was 30 con~tructed ln lnmbda gtlO, using the Acer~ha~ cDNA ~ynthe~iq ~ystem plus according to the manu~acturer's procedure~.

.
' : .
: . , .

W092/003lX PCT/US91tO45 2~
2 ~9 2 Approximately 0.7 x 106 unamplified cDNA clones were screened with a 25-mer oligonucleotide probe (5' ATATAGCGCTGTTTGGCAATGTGCT 3') corresponding to a sequ~nce near the 5' end o~ the l.9 kb cDNA clone and a single positive 5 clone containing a 1.7 kb in~ert wa8 identifiQd.

; Analysis of the three ovQrlapping cDNAs (r~gur~ 10) revealed a sQquence of 2529 ba~es, with ~he largest open reading frame being 1236 base~. No sequencQ difference were found in the overlapping cDNAs indicating they ware derived from 10 transcripts of the same gene. Overlapping sequences comprise a complete 3' untranslated region of 1031 bp with a polyadenylation signal 25 bp upstream from the poly(A) tract.
The 5' untranalated region co~pri~Qs 262 bp but lacks approximately 1 kb, as ~udg~d fro~ tho size o~ the ~RNA
15 esti~ated by Northern analysis. The predicted amino acid sequenca of the gene encoding TGF-B3 shows extensive homology to TGF-~l and ~2 (~igur- 11) (8, 9, 22~.

TGF-~l and TGF-~2 ~re produced in precursor for~s of 390 and 414 a~ino acid residues re~p2ctively (8, 9). The cDNA
20 soquence obt~ined ~or the gon~, i.e. TG~-~3, encoding TGF-B3 (~lgur- 1) contain~ an op~n ro~ding ~ra~e coding for 412 a~ino acid~, with the first ATG preceded by a stop codon, 162 nucleotides up~tre~m. A~ found w~th TGF-~l (10), the initiating codon ~or the protein having tu~or inhibitory 25 activity does not for~ part of a Rozak consensus (20).

Intere~tingly/ 8iX nucleotides downstra~ there i8 a second AT~, with an A at po~ition -3, which aligns with the initiating codon in TGF-~2 (9~. Ho~od$mers o~ the C-tarminal 112 residue~ o~ TGF-~1 nd ~2 represent ~h2 biologically 30 active ~0r~8 of these proteins. Preceding the slte of cleavage to their mature for~s, TG,F-~l and -~2 have stretches o~ 4 and 5 ba~ic residues, r~specti~ly. In TGF-B3 there are , . .
.
- .

.

w092/003l8 PCTtUS91/04~41 ; ~
29 20~'~992 5 bas~c residues preceding the cleavage site marked by the asterisk (rlgur- 10). The mature forms of TGF-~1 and -~2 sharQ aO/112 identical re~idue~. The corre~ponding 112 C-terminal amino acids in TGF-B3 exhibit 86/112 and 89/112 5 identical residues compared to TGF-~1 and -~2, r~pectively (~gur- 11). ~any o~ the remaining differences represent conservative ~ubstitutions. All three proteins show a strict conservation of the cysteine residues in this region. The N-terminal domain of TGF-B3 precursor exhibits approximately 35%
10 homology to TGF-~1 and 45% ho~ology to TGF-~2. By compari~on, the corresponding sequences of the TGF-~1 and -~2 precursors have 33~ sequence homology (~lgur- 11) (8, 9). Four potential glycosylation sites arQ contained in the N-termi~al part of TGF-B3 prQcursor, one of which is contained in all three 15 proteins. All three protein~ also po~se~s hydrophobic N-termini which may represent prosecretory ~ignal peptide sequQnces (31). Interestingly, both TGF-~1 ~nd TGF-~3 (but not TGF-~2) contain th~ ~ibronectin binding ~equonce R-G-D
(32). By analogy to TGF-~1 and -~2, -33 i8 nynth~siz~d a TGF-20 83 precursor which und~rgoos proteolytic cle~vage to producethe mature polypeptid~. B~ed on the functional and ~tructural ho~oloqy to TGF-~1 ~nd -~2, -~3 likely has therap4utic activity in cancer therapy, wound healing and imQuno~uppre~sion.

IO~ O~ SG7-~3 TGF~ Ex~re~siQ~ Construct A ~SOO ~p Alul-Hg~1 restriction rragment o~ TGF-~3 cDNA (site~
are indicated in Ylgur- l) which encodes the complete TGF-~3 prot~in wa~ cloned into the Bluescript pla~id (Strategene, La 30 Jolla, CA) to yleld th~ plasmld pBlue-TGF-~3. The fl intergenic rogion o~ thi8 vector allows the production of single ~tranded DNA via infection of its host bacteria with fl ''~ ~ ' ~, '' -, ~
.

WO 92/00318 PCI`/US91/045~
20g~992 helper phage. The initiation codon of TGF-~3 does not fo;~
part of a Xoz~k con~en~u~ ~equence (CCACC~ATG]~) (23), which has been shown to influence th~ efficiQncy o~ translation. In order to promote high yiQlds of the recombinar.t TGF-~3 5 protein, the flanking ~equenc~ of the initiation codon was mutagenized to a more e~ficient translation ~equence by changing CACACtATG]A into CCACC[ATG~A using the method of Nakamaye and Eckstein (26). Mutagene~is was confirmed by sequence analysis. Expression yields are ~urther opti~ized by 10 deletion of TGF-B3 5' and 3' untranslated [non-coding]
sequence~. Sub~equently, the mutagenized pBlue-TGF-~3 w~s cut with KpnI and SpeI, two polylinker re8triction 8ite8 ~lanking the cDNA insert. This`frag~ent was cloned lnto the eukaroytic expression vector pORFEX ~3~ cut with KpnI ~nd XbaI. In thi8 15 construct (pCMV:TÇF-~3) the TGF-~3 cDNA sequence iB
transcriptionally regulated by ~he cytonegalovlrus immediate e~rly promoter (rlgus- 12).
:`
D~A Tr~nsfection an~ Gene AJG~ ation Stable transfor~ants expr~s~ing TGF-~3 were obtained by 20 cotran~fecting tAe pCMV-TGF-~3 con~truct (Yigur- 12) with the dihydrofolate reducta~e (DHFR) gene (the pDCHIP pla~mid containing ha~ter DHPR minigenQ drivan by its own pro~oter) in~o Chine~e Ha~ster ovary (CHO) cells, which lack the DHFR
gene (35).

25 A ~t~ndard C~P04.DNA precipitation method (13) ~3 used for DN~ trhnsrection. pCMY:TGF-~3 (5.7 kb) and pDCHIP (2.5 kb~
were copr~cipitated with CaP04 in a ratio o~ 10 ~g to 50ng re~pectively ~nd the precipitate added to 0.5 X 106 CHO(DHFR-) cell~. Selection Or tr~n~ormant~ with a DHFR+ phenotypa w~8 30 per~orDed in alpha M~ (Glbco, Grand I31and, NY) ~upplemented with 10% dialyzed fetal calf ~erum. The colonies that appeared after culturing for 10-14 days in ~election medium '' ' . :

wos2/0o3lff P~/US91/04541 were l~olated by standard methods and expanded.

For gene amplification, tha primary transfectants were sub~ected to ~tepwise selection with incxea~ing concentrations of methotrexate (MTX; Sig~a Chemical Co., St. Louis, M0). The 5 fir~t round of ~lection was carried out at 20nM ~TX. TGF-~3 Qxpression level~ were measured by RNA cytodot hybridization normalizing the expres~ion of TGF-~3 ~RNA to that of actin.
Two of the three clon~s with initial high Qxpression (clone6 CHO 6.35 and CH0 9.1) showed increas~d TGF-~3 ~RNA expression 10 at 20nH M~X concentration (rlgur- 13). Total RNA (75 ~g) from CHO calls (lane~ 1), CH0 6.35 (lane 2), and CH0 6.35/20nM
(lan~ 3), werQ fraCtionatQd on a 1.2% agarosQ-formaldehyde gel, blott~d on~o nitrocellulose and probQd with a TGF-~3 spec$fic probe (EcoRI-SmaI cDNA re~tr$ction fragment of a 15 partial TGF-~3 cDNA clone isolated from umbilical cord; se~
~gur- 10). CHO 6.35/20nM (primary tran~fectant CH0 clone 6.35 at 20nM MTX), which had the highest level of expre~sion, was chosen for initial protein purification from conditioned m~dia and for furth~r gene amplification.

20 The best clone from further HTX ~election (10 ~H MTX) w~
~xp~nd-d and a bank Or ~rozen ~tock~ Q~tnbll~h~d. Thi~ clone, 6.36H, was u~d in all subsequent production of TGF-~3 and wa~
~aintained in T225 ~la~ka (225 ca2) in alphA MæH supplemented with lOS dialyzed fetal bo~lne seru~. TGP-~3 production 25 involved ~eeding Nunc cell factorie~ (600~ cm2 of ~ur~ace area p*r ~actory), with cell~ from three confluent T225 flasks of 6.35R in alpha MEM supplemented with 10% dialyzed FBS. The cQll~ wsre allowed to grow to 80% confluence in the cell factoriRs. Media wa~ then replaced wi~h HB CH0, a serum-free 30 media from HANA (Hana Biologics). After 72 hour~, ~edia wa~
remo~ed and replaced with rresh HB CH0 for a total of 5 coll~ctions of conditioned HB CH0 media. The rir~t collection of conditioned HB CH0 ~edia contained low levels o~ TGF-~3 .
~ ' :. ' .

`~ . '' :. ' ' ' . . ' `, . :. ,, ' , : ~
,, '' 2 ~ 9 2 32 with the ~aximum amounts produced in the 4th through 6th collections. Nunc cell factories provide sufficient ~urface area ~or the large scale growth of monolayer cell lines such as CHO, yi~lding a total of 7.5 lit~rs of condltioned media 5 por factor (3 collections, 2.5 liters per collection) with acceptable ease of u~e in a sterile environment. Using more advanced exprQssion vector ~yst~ms, it ~hould be pos3ible by one skilled in the art to significantly increa~e production : yield~.

lO Alternatively, cell lines could be adapted to suspenslon growth and produced in either a stirred tank fermentation ~y~t~o or in an air li~t ferm~ntator. The UBe 0~ porous gla~s cylinder support~ as a moan~ of adapting nonolayer cells (i.e.
CHO c~ll81 to stirred or air~ uspen~ion culture has al~o 15 been evaluat~d and shown to give acceptable yields o~ TG~-~3.

ExD~çs~ion of a mUtant TGF-~3 Precur~or The mutant TGF-~3 prQcursor is exprQssed a~ a ~ingle hbmodimeric polypeptide in a host cell by nutation of the R-K-K-R cleavage site botw~en th~ TGF-B3 pro region and mature 20 TGF-~3 to a protea~e cleavagQ ~ite, e.g. factor Xa cleavage ~equence (Il~-Glu-Gly-~rg) or a collagenas0 cleavage ~equence (Pro-X-Gly-Pro) (~lgur- 22) using standard site directed mutagene~is procedures, followed by insertion of ~he ~utant TGF-~3 nucl~ic acid into an ~xpression v~ctor and ~ransfection 25 o~ th- ~utant TGF-~3/voctor DNA into a host eell (~.g.
ÇQ~ ny ~a~malian eell~, e.g. CHO or HaLa cells, non-mammalian vortebr~te eell~, e.g. chiek c~lls, and invertebrate eella, a.g. insaet eell~ together with DN~ encoding a ~eleet~ble marker (e.g. neo, dhfr).

30 Additionally, a mutant TGF-B3 preeursor ean be produced by preparing DNA comprising a first DNA seguence encoding an W092/00318 PCT/US9ltO4541 ~, 33 2~9~2 amlno acid sequence ~ub~tantially id~ntical to the amino acid sequence ~hown in rlgur- 1 beginning with a ~ethionine encoded by nucleotid~s 263-265 and endinq with gluta~ine encoded by nucl~otides 1148-1150, a second DNA sQquence comprising ATG
5 wh~ch i~ linked to nucleotide 1150, a third DNA sequence, linked to the second DNA ~equence, encoding an amino acid ~aquenca substantially identical to the amino acid sequence shown in ~lgur- 1 ~sginning with a alanine encoded by nuclQotides 1163-1165 and ending with an asparagine encoded by 10 nucl~otides 1469-1471, a fcurth DNA seguence, linked to the third DNA sequence, comprising any tri-nuclQot$de sequence fro~ the list consisting of TTT, TTC, TTA, TTG, TCT, TCC, TCA, TCG, TAT, TAC, TGT, TGC, TGG, CTT, cTC, CTA, CTG, CCT, ccC~
CCA, CCG, CAT, CAC, CAA, CAG, CGT, CGC, CGA, CGG, ATT, ATC, 15 ATA, ACT, ACC, ACA, ACG, AAT, AAC, ~AA, AAG, AGT, AGC, AGA, AGG, GTT, GTC, GTA, GTG, GcT, GCC, GCA, GCG, GAT, GAC, GAA, GAG, GGT, GGC, GGA, and GGG. Th~ DNA ~160 Gomprise~ a ~irth DNA ~equence, linked to the fourth DNA ~aqu~nce, encoding an ~ulno acid sequence sub~tantially id~ntical to the amino acid 20 ~equence shown in ~igur- 1 b~ginning with ~ valine encGded by nucleotides 1475-1477 and ~nding with a ser~ne encoded by nucleotid~ 1496-1498. Such DNA ~y be in~erted in a suit~ble expro~sion vector and trane~ected into a host cell along with a ~lectibl~ marker (eith~r link~d or unlinked to the 25 expre~ion plas~id) by one ordinarily ~kill~d in the art.

Tran~0Cted C~118 may bs cult~red in an appropriate medium sQlecting ~or cells which expres~ a eelectible marker and such cell~ are ~urther charactorized ~or expr~sslon of mutant TGF-B3. Csll~ derived in thi~ w~y may bs us~d to produce mutant 30 TGF-~3 for subsequent purification. Mature TGF-B3 may be rel~ed rrom the precursor by proteolytic cleavage when a protea~e cl~avag~ site ~epar~tæs the mature and the pro r~gion Or th~ TGF-B3 precuraor. Si~ilarly, cyanogen bro~ide tr~atoent may be used to release ~utant, mature TGF-B3 when ,, ~ - . . . , ~ ,, ' .... : .

- , : . ~ .
~ ' ' .
: - .

W9~9~0031X PCT/~IS91/045 ~ethion~ne s~parateR~ the mutant, mature TGF-B3 lacking methlonine at nucl¢otideR 1472-1474 and the pro region of the TGF-B3 precur~or.

Biological Assay ~or ~onditio~ Media 5 Conditioned media waR treated with acetic acid to a final concentration of 0.1 H and serial dilutions tested for biological activity. CCL 64, a c~ll line deriv~d ~ro~ M$nk lung (AmericAn Type Culture Collection, RocXville, MD), wa~
found to be extre~ely sensltive to the naturally occurring 10 TGF-~3 isolated from u~bilical cord. This cell line w~s initially cho~en, there~ore, to test conditioned media for biological activity of the recombinant TGF-~3 protein according to the method of Iwata, et al. (19). Growth inhibition of CCL 64 mink lung cells produced by TGF-~l 15 (purified) or TGF-~3 ~from conditioned media~ i8 hown in 71gur- 14 A/8.

rigur~ 14A ~hows a dose response of growth inhibition using : purified TGF-~1 (C~lbiochQ~); a 50% inhibition was obtained with O.lng TGF-~1. An incr~sQ in mink cell growth inhibitory 20 activity was found co~paring conditioned media form the tran~ f ectant ~lected ~t 20nM MTX versus media ~rom the parental tran~r~ctant. rlgur- 14B ~how~ the biological ac~ivity o~ ~cid ~ctiY~ted ~rum free sup~rnatantR o~ CH0 6.35/20 n~ tran~fectant (clo~ed circles) and CH0 6.35 25 trans20ctan~ (op~n circles); 50% inhibit~ion wa~ obtained equival~nt to 30 and 5 ng/ml TGF-~1 activity, re~pectively.
Condit~on~d m~dium from parental C~0 (DHFR-) posse~sed much lower growth inhibition than eithar trans~ectant. These result~ cl~arly show that thQ ~GF-~3 cDNA i~ transcribed and 30 th~t TGF-~3 mRNA i8 tran~lated and produces biologically active protein.

,. , ~. . -, W092/00318 ~ ~ 41 ~` 35 In the prsse-ce Or EGF, acidified conditioned media from ~H0 6.35, containing TGF-~3 was able to promote soft agar growth Or NRK cells. Growth of NRK cell5 in soft agar ha~ bssn ~hown to be indUciblQ by stimulatin~ the production o~ extracellular S matrix proteins, an important parameter in wound healing.

Immunodetection Peptides corre~ponding to various partial amino acid aequences o~ the TGF-~3 prot~ln w~re synth~iz~d on an Applied Biosysts~s psptide synthe~iz~r (Mod~l 430A) using tBoc 10 chemi~try (rigur~ 15). Peptid~ w~re couplQd to keyhole limp~t hamocyanin with glutaraldehyde and used ~or im~unization of rabbits. Enzym~-linked immunosorbent assays wsrs u~ed initi~lly to ch~racteriz~ th~ antibody titsr~ (Table 1~. For thi~, and the following immunological exp~rim~nts~
15 ~tandard tQchniqu~s were ~ployed (17). High titer antibody ~rom ~mun~zed rabbits in~ected with ~3V or ~3III peptid~s were purifi~d using an ~inity ~atrix compo~ed of the re~pective peptide ~3 antigen conjugat~d to Affi-prep 10 (Bio R~d, Richmond, CA).

P~ptid~ S~quenc~21isa Titer I EEMHGERE~GC~QENTESEY1:6,000 IIL GDIL~NIH~VMEIRRXGVDNEDD1:10,000 IIs G~ILENIHEVn~IK 1:19,000 III DTNYCFRNLEENC 1:26,000 IV CVRPLYIDFRQDLGWRWVHEPKGYYANFC 1:19,000 V YLRSADTTHSTVIGLYNTLNP~ASASY 1:26,000 VI CVPQDLRPLTILYYVGRTPRV~QLSNMVVKSC 1:4,000 ~9 !~00318 36 PCT/US91/0454 The affinity purified ~3III antibody exhibit~ greater than 300 fold specificity for the ~3III peptide compared to the cognate peptide sequences from either the TGF-~1 or -B2. Furthermore, no significant cross reactivity of thi3 antibody has been 5 observHd against either the TGF-~1 or -~2 proteins. However, this antibody shows only a very limited ability to immunoprecipitats the native recombinant ~GF-~3 protein from conditioned media. The affinity purified ~3V antibody exhibits at least a 400-fold selectively for the ~3V peptide 10 compared to the corresponding peptide sequence form TGF~
This antibody can also efficiently i~munoprecipitate the n~tive TGF-~3 protein (~igur- 16).

rlgur- 1~ A/B show an immunoblot of TGF-~3 in condition~d media produced by the CH0 6.35/20nM trAnsfectant u~ing ~3III
15 and ~3V antibodie~ for detection. For peptide blocking experiment~, the antibody was preincubated with 80-fold molar Qxcess of peptide prior to incubation with the blot. For detection, alkaline phosphata~e (Zym6d, San Franci~co, CA) con~ugated to go~t anti-rabblt IgG W~E used a~ a second 20 antibody. ~lgur- 17A shows æ W~tern blot of a gel where the ~ample was sub~ected to r0duction prior to electrophoresi~
while ~lguro 17B ~hows the Western blot o~ the ~ample under non-reducing conditions. In ~ch figure, lanes 1-3 and 4-6 corresponds to conditioned ~edia immunoblotted with ~3V and 25 ~3III ~ntibody, re~pectively, lan~s 2 and 5 i~munoblots carri~d out in the pre~ence of ~xces~ cognate peptide, while lane~ 3 and 6 represent i~munoblots in the pregence of an exc-~s unrelated p~ptido sqqu~nce.

W~tern blotting of conditioned media from CH0 6.35/20nM cells 30 under reducing conditions, u~ing af~inity purified ~3III and ~3V ant~body, detected a 50 kDa and a 12 kDa band. These bands correapond to the TGF-B3 precursor and ~ature TGF-~3, by analogy to the proce~sing of TGF-~l and TGF-~2 described by - .
,, :~ .
-: . ' ~092/00318 PCT/US91/04541 37 203~992 Gentry et al. (ll) and Madisen et al. (22) (~lgur- 17A/B).

Und~r non-reducing conditions, lOo kDa and 24 kDa bands were observed, which we beliQve to correspond to homodi~eric forms of the TGF-B3 precursor and mature TGF-~3. The apparent 5 precursor appears as a broad b~nd, characteristic of so~e glycosylated proteins. Following cle~vage of tha signal peptide sequence of th~ precursor for~ of TGF-~3, one would expect a protein with ~W of 43 kDa (under reduced condition~).

Ba~ed on the primary sequencQ of TGF-~3, there are four N-lO linkad glycosylation sit~s, further indicating th~t th~detected precursor protain i8 glycosylated. ~lgur- 18 A/B
show Western blot of cell extract (rlgur- 18A) and conditioned media (~lgur~ 18B) of the CH0 6.35/20nM tran~fectant using ~3V
anti~ody ~or dotection. For prQp~ration o~ cell extract~, 15 cells were ~irst washed with phosph~t~ bu~fered saline then lysed directly wi~h SDS/~-mercapthoethanol prior to gel electrophore~i~. For p~ptida blocking (lanes 2 and 4), the antibody wa~ incubated wi~h a lO0-fold molar exces~ of ~peci~ic peptide prior to incukation with the blot (125I
20 protein-A was us~d ~or detection). In cQll ~xtract3 of CH0 6.35/20nM und~r r~duclng condltions, only the 50 kDa band corre~ponding to a potentlal pracursor ~orm is d~tect~d (Ylgur- 18 A/~). T~ ~pec~icity of the antibody wa~
d~onstratod by preab~orbing the antibodies with peptide 25 i~uunQg~n prior to W~tern blotting (~lgUr-8 17 A/B and 18 A/B). A~ exp~ctod, based on ~RNA and biological activity data, thQ ant~body d id not d~tect any TGF-~3 protein in condltioned ~edia o~ ~h~ pairent~l CH0 (DHFR-) c~lls.

Both antibody were al~o te~ted ~or immunoprecipitation of 30 native recombinant TGF-~3 protain (~lgur- 16). CH0 6.35/20nM
were 9rown to confluency ~nd labeled with ~35S] ~ethionine for 24 hours in methionine-free DMEM in the presence of 5%

, - . . . ..
- - : . - ~ - : . , .. ' .

, ,: . . , ~V092/0031~ PCT/US91/0454 2 ~ ~ - dialyzed plU8 5% nQn-dialyzed ~etal calf ~erum. The medlu wa~ collQcted and lm~unopr~clpitated wlth 10 ~g/ml ~inity puri~ied ant~body and 20 ~g/ml (1:2 dllution) protQin A
agaros~, for 2 hour~ at 4C. Separation of the 5 immunopr~cipitated proteins on a 12.5% SDS polyacrylamide gel revealed two protein~ migrating identically to the matur~ TGF-B3 (12 XDa) and precursor TGF-~3 (50 kDa) (~gur- 16).
However, one extra immunoprecipitated protein was found at 43 kDa.

10 Th~ 43 kDa protein may corr~spond to either the non-glyco~ylated precur~or or a prot~olytic breakdown product.
The ~3V antibody, in co~pari~on to th~ ~3III antibcdy, pro~ed to b~ muc~ more efficient in i unoprecipitating the TGF-~3 protein. The ~pecificity of the immunoprecipitation was 15 determined by preincubating the antibody with a 80-fold molar ~xcess of either the cognat~ p~ptide or an unrelated peptide sequence. The speci~ic peptide showed comple~e competition of all three band~ wherea~ the unrelated peptide did not. As ~xpected, bnsed on the amino acld composition and distribution 20 of m~thionine in ~he TGF-~3 protein, the 50 kDa contain~
significantly more 35S lab~l.

The ~3V a~inity purifi~d antibody w~8 also u~ed in par~fin ~oction~ o~ huoan u~bilical cord (r~gur-s 19 A/B/C/D).
~ibrobl~st~ 2nd ~pith~llal c~lls stained (~lgur- 19A) as did 25 th~ ~ooth ~uscle fib~r~ o~ thQ cord va~culature (rlgus- lOC) whsr~a~ n~ither the connective tis~ue nor the extracellular ~tr~x ~tain~d wlth this antibody. A control rabbit polyclon~l antibody (Ig ag~inst P210~h~ OSI catalog ~PC02) ~howed no ~taining (~lgur-~ 19 ~ and D). The ~trong staining 30 in umbilical cord tissue agr~eB with earlier data showing extracts from umbi~ical cord po~sessed high levels of ~RNA.

Prçp~tion of TGF-~3 ~onoclon~l Antibody '' :
. ~
:
- . ~
, :' , "~092/00318 PCT/US91tO4541 39 2~ 2 A TrpE-TGF-~3 fusion wa~ produced in E. çoli which had the following characteristics, i.e. amino acids l to l9 are coded by the TrpE and poly linker segment and amino acid~ 20 to 170 corre~pond to amino acida 273 to 412 o~ the TGF-~3 precursor S (containing the full m~ture TGF-~3 ~equence). Th~ fusion protein remain~d in the insoluble fr~ction after ~onication in PBS. Sub~equently, the protein was purified by ~eparation on a SDS-polyacrylamide gel and isolated by electroelution. This m~t~ri~l wa~ u~ed for inmuniz~tion of mice by the following lO protocol:

. Balb/C ~male mice were i~munized intraperi~oneally with lOO~g o~ Trp~-TGF-~3 in RIBI adjuvant on days O, 7 and 14;
b. on day 24 test bleed~ indic~ted high titer~ against TrpE-TGF-~3 and puri~ied TGF-~3 protein;
c. The mice were then boosted with lOO~g of the ~ame antigen on day~ 28, 29 and 30;
d. Spleen fusions were performed the following day;
and c. Sub~equent method~ of hybridoma selection, culture and subcloning wero per~ormed following standard procRdur~3 (17).

Five ~t~blq hybrido~as wcre produc~d and their characteri~tics aro ~hown in T~bls 2. All o~ the clones produced antibodies 25 o~ th- IgG ~ cl~. Th~ monoclonal antibodies immunoblotted with puriflsd TGF-p3. All five monoclonal antibodie~ ~howed no reactivity wlth TGF-~l by ELISA, but cro~sreacted with TGF-~2.

Analysls o~ the epitopes r~cognized by the monoclonal 30 antibodie~ u~ing TGF-~3 ~ynthetic peptide~ showed that all antibodies reacted with amino ~cid~ re~idue~ 380 to 412.

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, : :

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.. . . . . . .
.
.. :, . -Wos2/no3tx PCT/US91/045 2~ 1992 UJJL$lL__4t A ~S~OD ro~ R~R~r~5~Q~ O~ T~F-d3 rR3 COND~T~ DIA

Conditioned medium was prepared from CHO 6.35/20nM cells grown to confluence in the presence of 20nM methotrexate. The cslls 5 were washed with phosphate buffered saline and incubated with qerum free medium for 2 hours to eliminate carryover of serum proteins. Conditioned media was derived ~rom cells incubated with fresh serum-free medium ~or 48 hours. TGE-B3 was purified from conditioned media using the following protocol.

10 i. Conditioned media wa~ filtered through a 1 ~m glass fiber ~ilter (Micron Sep~ration~ NC CG 20000-A01) and stored in plastic containQrs at 4C a~ter addition of 0.1~M PMSF, 4~M EDTA, l~M ~GTA, 0.02%
sodium azide and 10~M Tris HCl pH 7.5.

15 ii. Media ~g concentrated approximately 100 fold using a high capacity, low protein binding Millipore ~Pellicon~ membrane cartridge (Millipore PLGC
RegQneratQd CQ11U108e MW cutof~ 10,000).

iii. The protein concentration wa~ adjusted to 10 mg/ml and a~oniu~ sul~ate (90%, pH 7) i3 added to 45~, pH ad~usted to 7.6, the material incubated for 4 hour~ at 0C (or ovornight). The precipitate pelleted by centrifugation (10,000 x g for 30 ~in) and the pellet le~t to drain ~or 10 ~inute~.

25 iv. The pellet w~ extracted with acetonitrile, 50%
(v/v)/acetic ~cid (L~ t 0C. 25 ~1 of extraction bur~er wa0 used per gram o~ starting protein in (iii) above. The 3u~pension was treated to centrifugation at 10,000 x g for 30 minutes.

,. . - .

-.
.

~W~092/00318 PCT/US91/W54l k;'i, 41 2084992 v. The extraction ~upern~t~nt was further concentrated uslng a Minitan concentrator (Millipor~) us~ng identical m~mbr~ne5 as dQ~Gribed in (ii). lM
acetic acid wa~ added during concentration to prevent protein precipitation and change th~ bu~Qr to lM ac~tic acld.

vi. The concentrated ~aterial i8 chromatographed by P-60 gel filtratton using a IM acetic aoid moblle pha~e, and peak ~ractions concentrated using the Minitan concentrator.

vii. The concQntrate w~s made 1% in Triton X-100, the pH
ad~usted to 7.5 with ~olid Tri~ Base (Sigma) and clari~ied by centri~ugation ~t 10,000 x g for 30 minut~. Thi8 ~teri~l was then chro~atogr~phed --tat 4C) on ~ ~3V anti-peptide antibody affinity colu~n ~12.5 cm x 0.8 c~), the colu~n wa~h~d xtQnEiv~ly with O.lM Tri~ HCl pH 7.5, lGmM EGTA, l~M PMSF, 1% Triton X-100, lM NaCl until no protein could be datected in the eluate. TGF-~3 was then ~
elutQd in 50~M glycina, pH 2 into ~iliconized ~-pla~tic Yi~l~. Thi~ material was pooled, acetonitrile wa~ added to a ~inal concentration of ; 2S% and stored ~rozen at -20C.

viii. C18 revar~e phase wa~ imple~ented at thi~ stage prior to in vivo testing. The TGF-~3 eluted fro~
tho antibody af~inity oolu~n was applied to a Water~ C18 re~er~e pha~e EPLC column and developed u~ing a 0-60~ acetonltrile 0.1% TFA gradient, a ~low ratQ 0.5 ~l/~inute and monitor~d using a 210nm ~low through det~ctor. Matsrial w~ al~quoted and stored in the elution 301vsnt at -20C. ~
~ :

W092/0031X PCTtUS91~04541 208'1992 42 ~
Quantlt~tion of chromatographic yield~ was acco~pli~hed by We~t~rn blot using ~3V antl-peptide antibody or by antigen capture as~ay. The CCL-64 cell bioassay was utilized to test conditioned media and purified chromatographic fraction~ for 5 biological activity of the recombinant TGF-~3 protein. The growth inhibition a3~ay i~ ba~ed on meaeure~ent of DNA
synthesis (18) using the decrea~e in 5'[12sI]-iodo-2'-deoxyuridine incorporation of treated compared to nontreated cells as a measure for growth inhibition. Conditioned media 10 wa~ acid activated and ts~ted at sevQral dilutions.

Fractions were analyzed by ~ilver 3tain and Western blot and peaX fraction~ pooled. A silv~r ~tained gel identified a single band of 12 kDa and 24 kDa under reducing and non-reducing conditions, re~pectively (rigur~ 20). The detaction 15 of ~ ~ingle silver staining band indicates that the preparation i~ gr~ater than 95% ho~ogeneous.

We further describ~ a noYel purific~tion protocol which allow~
the isolation o~ tho glyco~ylatod pr~cursor form o~ TGF-~3 or the pro region of th~ TGF-B3 prscursor u~ing a l~ctin affinity 20 column. An ~x~mple of thi~ i~ to u~e comm~rcially available immobilized lectin ~uch as wh~at gsr~ agglutinin ~ound to agaro~e (e.g. Sig~). The lectin column i8 washed and equilibratQd with 5 column ~oluoQs of binding buffer (0.15 M
NaCl S0 ~ HQp-a pH 7.6, 0.1% Triton X-100). Cell lysate~ or 25 c~ll cond~tionod ~edia containing the glyco~ylated precursor forc Or $GF-~3 i~ ~uspended in binding buffer and 810wly lo~d-d onto the lectin affinity coluon. It may be necessary to pa~s thc buf~r cont~ining TGF-~3 up to 3 tim85 to maximize binding of the protein to the coluun. The colu~n i~ wa~hed 30 wlth 5 column volu~e~ of the binding buffer to remove unbound ~terial. The colu~n i8 eluted with binding buffer containing 0.3 ~ N-ac~tylgluco~a~ine ~o olute the bound glyco~yl~ted TGF-~3 prec~-~r~or protein ~ro~ the colu~n. Further, TGF-~3 i~

.

~092tO031R ~Y~42 1 ~aparatad ~ro~ th~ other glycoproteins and treated to r~lea~e the bioloqlc~lly aCtiVQ homodimer ~rom th~ precur~or complax.
Othar exa~plas o~ lQctins which could be used include but not ba limited to ricin, abrin, ~nd Con A.

5 The precursor form of TGP-B3 or the pro region of the TGF-B3 precursor may further purified using chromatographLc ~tep~
known to those ~killed in the art. For example, by sequential gel filtration chromatography, ion exchange chro~atoqraphy, antibody-column chromatography, and high pres~ure liquid 10 chromatography, or co~binations thereor.

Mutants o~ the TGF-B3 precursor or mut~hts of mature TGF-B3 can ba puri~ied u~ing the ~athod~ described for t~e TGF-B3 precurQor and mature TGF-B3 a~ dsscribQd herein.

J~ L~ L~TxAh~8~ Tar-B3 aC~I~IT~
.
15 Hu~an platelet TGF-B1 (Collaborative Re~earch, MA), porcine TGF-B2 (R&D, ~innesota) or puri~ied recombinant human TGF-~3, at concentration~ ~rom 3.125 to 0.049 ng/~l, wa incubated with 5 ~g/~l of af~inity purified polyclonal rabbit antibodie6 (~3V ant~ body and snti-TGF~ D, Minnesota) for 3 hours at 20 37C. Control TGF-~3, TGF-B2 or TGF-B1 was incubated w~thout antibodi~. Gro~h inhibition o~ ~ink cQlls by antibody treat~d and control untrsated TG~-~3, TGF-B2 or TGF-B1 was dQt~rnin~d a~ de~eribed abovQ. r~gur-s 21A, 2lB and 2lC show th~t th- p3V antibody tclos~d ~quare~) n~utraliz~s the growth 25 inhibitory ~etivity of TGF-~3, but not TGF-B2 or TGF-B1 on ~nk eells r~l~t~v~ to the growth inhibitory activity of id~ntieal eoneentratlons o~ TGF~ in th~ ab~enc- o~ antibody (op~n eirclo~. Anti-T~F-B (R~D, ~innssot~) neutraliz~s TGF-B3, TGF-B2 and TGF-Bl (~gur~s 21A, 21~ and 21C)(elo~ed 30 eirel~s). Neither antibody ~ad ~ny ~ignificant eff~et on the growth o~ CCL-64 c~lls in th~ ab~nca o~ TGF-~3. Antibodie~

. ~ .,~ . . . .
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W092~00318 4 PCT/USgl/04 0 8 4 9 9 2 against the TGF-B3 peptide ~3V apparently specifically n-utral~zes tho growth inhibitory activlty o~ TGF-~3.

~SAXPL~ 6s BIO~O~IÇA~ ÇIAaYG33aaL&~I0~ or ~aF-B3 T~ V~R0 Growth was d~termined using a modification of the monolayer 5 assay for TGF-B3 described by Iwata, R.K., et al. (19). Non-leukemic cells were subcultured on 96-w~ll tissue culture plates in 100~1 of media ~t a YQeding dQnsity of 2x10-3 cells per well. Cells.were maintained and assayed in Dulbecco's modified Eagle's medium containing 10% fetal bovine and 2% L-10 gluta~in~. Tho~e cells were trsat~d with 25 ng/ml (-lnH) of TGF-~3, puls~d 24 hour~ with l~Ci/ml 5-~125I]-iodo-2'deoxyuridine wh~n cells in the untreated control wells were 90~ con~luent and harve~ted.

LQuke~ic cell~ (R562, RG-1, ~G-l~, HuT 78 and U937 ? were 15 ~eeded in 50~1 of medi~. R562 wa~ ~eeded at ~ density of lx103 cell~ per well in RPMI supple~ented with 10% fe~al bovine seru~. ~G-1 and KG-la were ~eeded at a density of 3.5x103 cells per well in Iscovs's media supplemented with 10%
fetal bovine ~eru~. But 78 and U937 were se~ded ~t a density 20 of 3.5x103 cella por cell ~n RPHI ~upple~ent~d with 10% fetal bovine serum. Cell growth wa~ determined by microscopic ox~in~tion. Exa~ples are shown in Table 2, showing inhibition of some hu~n tumor lin~ by TGF-~3.

~aYP~B 7s D~V~OP~B~$ OF A~ E~ CAP$~ AB~AY rOR ~r-B3 25 Plat~ aro co~t~d with 50 ~1 o~ ar~inity-puri~i~d rabbit polyclonal ~ntibody (5~g~1 in 0.1M N~HCO3, p~ 9.1) ~ade to TGF-~3 p~ptid~ ~3V. Pl~t~ w~re incub~ted overnight at 4C.
Unbound ~ntibvdy i8 romov~d by a~piration. Plates are blocked with 100 ~1 PBS containing 1% BSA (PBS-BSA) for 1 hr at room 30 temperature. The plate~ are then wa~hed twice with phosphate-, .

:: .
,: . . ', . . ' ~092/00318 PCT/US91/~4~1 ,~ 45 2 ~ ~4 9~ 2 bu~rod saline (PBS) containing 0.05% Tween 20 (PBST).

Sample~ in a ~inal volu~e o~ 50 ~1 o~ PBS-BSA are added to the appropriatQ well8 and incubated ~or l hr at roo~ te~perature.
Unbound protain is removed and the plate i8 wached four times 5 with PBST. All wells receive 50 ~l o~ mou~e monoclonal antibody again~t TGF-~3 (5~g/ml in PB8). After incubation for 1 hr at room temperature, unbound antibody i8 re~oved and the plate is washed four times w~th PBST. All wellR receive 50 ~l of an appropriate dilution of alkaline phosphatase conjugated 10 to goat anti-mouse antibody. A~tor incubation for 1 hr at room temperature, the plate Wa8 wa~hed four times with PBST.

Substrate for alkaline phosphatase (5-bromo-4-chloro-3-indolyl pho~phate) in 100 ~ dded to all o~ the well~ and incub~ted for 15 min at room temperature. Ab~orbance ~n efich 15 well mea~ured at 490 no. U~ing this assay, we detected between 3-5ng/ml reco~binant TGF-~3.

~$a~P~ 8s ~S~R~IO~ OF C~LL~LAR A~D ~R~A~E-A~C~OR~D T~ 3 Expre~ion o~ biologically actiYo and inactive TGF-~3 ~ay be achi~ved in eukaryotic cell~ such that the final protoin 20 product i8 retained by th~ cell and not released into the cell culture ~edi~. Thi~ has advantage~ oYer relea~e o~ TGF-~3 into ~sdia in concentrating TGF-~3 to thfi cell me~brane during recovory. Protoin ~orting of TGF-~3 within a c~ll to the ~ndopla~ic roticuluo (ER~, the pl~s~a ~bran~ or 25 extracollular ~atrix i~ achieved through incorporation of ~p-cl~ic targ-ting ~ignals into th~ ~GF-~3 precur~or or mature TGF-B3 ~uch th~t targeting ~ignal ~quonces can be removed - :-: - - : :

- ' ' ' ' , .
-: . : ~ .- : , . . . .
.. ' :" ~ ' '' ' .

20~'~992 46 ~ABLE 2 Effects of TGF-~3 (lnM) on the Growth Or Hu~an Cell Line~ in culturs CEL~ ~INE ~ IY~II3I

Hu~an Tumor A549 (lung ad~nocarcino~a) 46 A375 (melanoma) 47 A2058 (molanoma) ~8 WiD~ (colon adenoc~rcinoma) 24 MCF 7 (brsa3t carcino~) 57 Human L~uk~mic Coll~
' "
~562 (CML) 55 RG-l (A~h) 50 : XG-la (AML) 50 HuT 78 (T cell lympho~a) 50 `: U937 (histiocytic lymphoma) 50 ~orm~l Hu~an Hu~ (~oreskin fibroblasts) 6 -s, . , , . . :

~; - '" ~ ' ' ~ ', ~W,092/0031X PCT/US91/04541 47 2~ 92 during recovery and purification. For exa~plQ, TGF-~3 cDNA
eould be modi~ied so as to produce a TGF-~3 precursor protein which is membrane anchored via a hydrophobic trans~e~brane sequence. Brie~ly, such TGF-~3 proteins are ~xpre~d a~
5 follo~s.

Th~ molecular biology techniqu~s u~e standard method3 (23).
High level expreqion vectors Por production of TGF-~3 are eonstructed wlth the following propertie~. Transcription of TGF-~3 derives fro~ a strong promoter coupled with the 10 enhaneer~/replieation origin~ for SV40 virus and polyo~a viru8 to further enhane~ tran~eription and allow replieation in COS
eells or polyo~a trans~or~Qd e~ll lines for short ter~
analy~is of the con8truct8. Expre~sion vectors additionally eontain ~e~uene~s ~tabilizing tho RNA and increasing ~t~ half-15 life (e.g. untr~nslated sequene~ ~rom the rabbit ~-globin 5'and bovine growth hormone 3' regions), and contain splicing signals to additionally ~tabilize the RNA transcript (i~munoglobulin intron sequeneQs or SV40 s~all t intron ~qu~nees). Th~ veetor~ eontain a Kozak eonsen~us s~quence 20 ~urrounding th~ initizting mathionin~ to promote ~f~iei~nt tr~n~lation o~ th~ ~RNA. TGF-~2 and TGF-~3 laek a eonsen~u~
~ignal p~ptido cl~avage ~equencQ a~ dQfined by Blobel (a ba~ic a~ino acld, pr-~rably Ly~ or Arg, ~ollowed by an ~ino acid with a ~11 ~ide ch~in, co~oonly Gly or Ala). Th~ TGF-~3 25 propropr~cur~or i8 proca~ed to the precur~or for~ by cleavage ar a 22-24 ~ino acid hydropho~ic 3ignal pept$de co~ml~ant to tran~locAtion acro~s th~ andopla~ic rsticulu~ prQsumably at a cryptic cl~avage sit~. Thi~ ~ite can be mutated to a con~nsus Arg-Ala cleavago ~quence using standard site 30 Rp~cific ~utagenesis tachnlqu~ to promot~ optimal s~cretion/tran~location or deleted to promote cytopla~mic localization.

- - , - . . .

WO92/0031X PCTtUS91/045*~
2 0 8 !~ 9 9 2 4B ~
At the carbaxyl-terminus of the precur~or protein, DNA
encodlng a hydrophobic tran~membrane a~ino ac1d seguence (e.g.
that encoded by the epidsrmal growth ~actor receptor cDNA or c-erbB2 cDNA) ~ollowed by a highly charged 'stop tran~f2r' 5 amino ~cid ~equence i8 ins2rted such ~hat a TGF-~3 precursor protein modifled in this way binds to the plasma membrane. An amino acid recognition site for a speci~ic protease (e.g.
factor X or collagena~e) i~ included between the C-terminal transmembrane sequence and the N-terminal TGF-~3 precur~or to 10 allow efficient cleavage Or the TGF-~3 from the membrane by ~pecific protea~es (e.g. factor Xa or collagenase) (~igur-22). An example o~ thi~,procsdure is as follows:

1. A mutation oligonucl~otidQ [5' CTCTGTCGCACGT~GATCCTCAGCTA
3'] is u~ed to engineer a 8amHI restriction endonuclea3e claavage ~its 3' of th~ TGF-~3 t~r~ination codon.

2. A second mutation oligonucleotide i~ used by one skilled in the art to remove the TGF-~3 termination codon.

3. Synthetic oligonucleotides are con~tructed to introduce the protea~ cleavage site (for example Factor Xa:
IleGlu~lyArg or collagQnase: Pro-X-Gly-Pro) ~ollowed by the ~equence encoding the c-erbB2 transmembrane sequence a n d B t Op t r a n ~ ~ e r 8 e q u e n c e 8 ~ThrSerIleValSerAlaVa,lV~lGlYIleLeuLeuValValValLeuGly ValValPheGlyIle~euIleLysArgArgGlnGlnLysIleArgLysTyrThr ~t) such that sequences ~re inserted in the ~ame tran~lated r~ading fra~e a~ th~ TGF-03 precursor. The nucl~ic acid ~olecule pr~duced i~ in~erted in an oxpres~ion v~ctor and introducsd into a host cell line (~g., CHO and HaLa cQll~).

30 Alternativaly~ linkage of the tran~membrane and cytopla~ic domain~ of vesicular stomatius viru~ glycoprotein to the C-`~ 49 20~992 terminus o~ the TGF-~3 precur~or with an intervening protea~e cleavage ~equ~nce (e.g. Fhctor Xa or collagenase) allows ~or the production of membran~ anchored TGF-~3 prQcur50r, as described for rat growth hor~one ~l6).

5 A nu~ber o~ protein~ are anchored to the cQll ~embrans via covalont pho~phatidyl innsitol linkage (a.g. Qa-2, decay accelerating factor (DAF), Thy-l) (21). Expre~sion of mQ~brane anchored TGF-~3 ~ay al80 be achieved by inclu~ion of pho~phatidyl ino itol linkage sequence~. It i~ preferable, 10 though not required, that the pho~photidyl inositol linked TGF-~3 precursor protein msy be freed from the me~brane by treat~ent with phospholipa~Q C.

For exa~ple, inclu~ion oP the C-terminal 37 amino acid~ o~ ~AF
(PNRGSGTTSGTTRLLSGHTCFTLTGLLGTLVlMGLLT) (4) linked by a Factor - 15 Xa protease cleav~g~ sequence (I-D-G-R) or collagenase cleavage ~equence (P-X-G-P) to the TGF-~3 precursor equence would link TG~-~3 to the call in which it ~ 8 produc~d by a glycophospholipid membrane anchor attached to the DAF
~equence.

20 The C-terminal sequQnces o~ variant ~ur~c~ glycoprotein (VSG) o~ Trypano~o~a brucei hav~ been shown to be linked to the plasma ~embran~ by a pho~pholipase C ~en~itiv~ anchor (21).
Linkngs o~ thi~ ~quenc~ to C-t~r~inus of th~ TGF-~3 precur~or : via a protease cleavage ~equence a~ previou~ly described 25 enabl~ the ~ynthe~i 8 of ~embrane bound TGF-~3 precur~or protein.

Alternatively, TGF-~3 precur~or protein ~ay ~e targe~ed to the cndopl~s~ic reticulum by del~tion of TGF-B3 signal peptide s~quonce~ ~uch th~t thc rssulting protein begins with leucine 30 at nucleotid~ poKitions 332-334 and by inclu0ion of an endoplasmic reticulum targeting sequence, for example, the ,. -.
- . ., .

~ ' ' .
:.

WO92~00318 PCT/US91/045~
2 ~ 2 50 rotavirus S~II glycoprot~in VP7 signal ~equence (36) into the TGF-~3 precursor sequence. Alternatively, th~ VP7 signnl sequence ~y be linked to the C-terminu~ of the mature TGF-~3 such that a protea~e cleavage s~quence (e.g. Factor Xa or 5 collagenase) separates th~ ~ature TGF-~3 from the VP7 ~ignal ~equence.

R~F 9s O M I~8ATION Ql_CZlF~8~0~ A~D BXOLOBICAL AC~IVI~Ys ~R~ÇT~R~ r~c~o~ ~8L~IO~ 8 TGF-~ ex~rt th~lr biological eff~ct~ through rec~ptor 10 bindlng. Three putative receptor~ have been identi~ed by chemical cros~-linkin~, and ar~ de~ignated type I, type II, and type III receptors with molecular weights 65 kD, 85 kD and -280 kD, respectively (5, 6). Tho biological effect~ of TGF-2, and -3 ~ppear to b~ m~diated by type I and/or typ~ II
15 receptors. Cells whic:h lack the type III receptor still re~pond to rGF-,~s; thUR, type III receptor binding does not appear e~sential for at lea~t many o~ th~ biological activities Or the TGF-Bs.

Type III receptor i8 pxe~ent at high concentration in the 20 body, relative to type I and II receptors. Type III receptor~
exl~t~ in two form~ ~brAne proteoglycan which binds TGF~
vla th~ glycooylated core protein and b~taglycan, a ~ecret-d, 801uble foml a~60ciated ~ith the extracellular ~atrix (l). Accordingly, it may be possible to mutate TGF-~3 25 to prevent binding specifically to the type III receptors, thereby incre~ing the hal~ lifa in vivo of retained activity.

TGF-~ 2re al~o known to bind ~eru~ protein~, ~uch a~ ~2 macroglobulin t29, 18) . Thu8, . binding of T~F-~3 to ~2 30 ~acroglobulin or to other serum proteirls may al80 seguester, :

51 2 ~ 9 2 inActivate or prouot~ clearance o~ TGF-~3~ TGF-B~ are also known to blnd th~ TGF-B pro r~gion ~nd are inactiY~ted in that complex. TGF-~ have a short hal~-life in ~ivo (estim~ted at 2.2 minute~) (7).

S ~eaenerate Oll~onuçleotide Directed Nu~genesl~

In order to inVestigatQ binding of TGFo~3 to proteins involved in clearance or inactivation the region~ of TGF-~3 that interact with the~e protein~ (such a~ ~2 uacroglobulin and the secretad form of the b~taglycan receptor of TGF-~) are 10 sub~ected to mutagen~sis u~ing degenerata oligonucleotide~ (5, 25) and, sub~equently, recombinant mutant TGT-~3 are tQsted for their affinity for such protein~.

ThQ deg~nQrate oligonuclaotido~ ~panning the regioni~ involved with binding or clo~rance protoin~ are do~igned ~uch that the 15 3' ends contain an 8 nucleotide palindromic ~equence ~nco~p~ssing ~ restrict~on endonuclea~e cleAvage ~ite.
~ Pre~erably, the 5' end o~ tho oligonucleotidQ coneists of ;, s~quence~ encompas~ing ano~her restriction endonucle~se ~ite.
The central reqion (20-100 bp) contains the mutagenized 20 ~equence of the TGF-~3 interaction it~. In order to introduce point ~utation~ in the dosired regions, the ~ynthesis of th~ oligonucleotid~ are programmed such that pooition~ wh~ro mutation aro not do~ired (~.g., the re~tr~ct~on Bite~) are syntho~iz~d u~ing ~olution~ of the 25 individual pho~phoramidlt~s, while thQ rsgion~ wher~ ~utations ~r- d~ir-d are synth~siz~d u~ing a de~ined mixturo o~
phosphora~idit-~. U~ing thi~ procedur~, a 10% mutation rate can b~ achi~ved by a mixture o~ 90~ wild type nucleotide ~nd 3.3~ of each of the thre~ ~incorrect~ nucl~otide~. Following 30 ~yn~ho8i~ the oligonuclsotid~ are purified eith~r with S~p Pak C18 cartridg~ ~or ~hort oligonucl~otide~ (<50 nt~ or by denaturing pr~parative polyacryl~ide electrophorasis for ' :.
, W092/0~3t8 52 PCT/US91/0454~

2 0 ~ ~ 9 9 ~onger oligonuclQotid~s.

The conver~ion to double-strandad DNA i8 achiev~d by ~el~
Annealing o~ th~ ol~gonucleotide~ u~ing the 3' (and 5') ~nd palindromic sequences and treatment with DNA polymerase Klenow 5 fragment in the presence of the ~our dNTPs. After digestion with the re~triction enzymes, r~cognizing th~ 5' and 3' outside sites, the double ~tranded oligonucleotide mixture is purified by non-denaturing polyacrylamide gel~. The oligonucl~otide mixture i~ then b~ u~d to repIace the 10 corre~ponding fragment in the TGF-~3 expre~3ion plaqmid. A
doubl~ ~trand sQquencing method i8 used to directly determine the genotype of the mutants.

If the mutation frequency i8 too low, the ~utant clones are id~nti~ied by dlff~rential colony ~ilter hybridization using 15 the kinasQd S' 32p labeled oligonucleotide as a prob~. The TGF-03 mutant ~xpre~sion constructs are transfected into COS
cell~. Sub~equently, the TGF-~3 protein i8 p~rti~lly purl~led u~ing a TGF-~3 i~unoaf~inity colu~n and t~t~d for ability to intsract with TGF-~3 binding/inactiv~ting prot~in~.

20 Sa~urA~ion M~qenesis In ord~r to cre~t~ ~ largQ nu~ber of randomly di~tributed nucleotide ~ubstitution~ in th~ cDN~ ~r~g~ent encoding the complote m~ture TGF-~3, a ~aturation ~utagen~ mathod is appliod. The rQ~ulting TGF-~3 mutants are tested for 25 biological ~ctivity, raceptor binding capaclty and affinity for TGF-~ binding protein~. Finally, the in vivo half-life of a ~elected group of ~utants i~ test~d.

First the ~lu~cript (Str~tagene) pla~id containing the TGF-~3 cDNA i~ ~ub~cted to ~tQ direct0d ~utagenesi~ in order to 30 creAte a F~pI site at R-R-R-R proce~ing site o~ TGF-~3 .
, ~092/00318 PCT/VS91/045q1 20~992 (praceding the mature prot~in) and a Ba~HI ~it~ 3' o~ TGF-~3 stop codon. Ths ~ollowing mutation qligonucleotldRs wlll be used:

5' GTAATTGGTGTC~AATGCGCACTTCITCCTCTG 3' and FBpI ` ! ~

5' CTC~G'rCt;CACGTGGATCCTCAGCTA 3 ' BamHI

10 Th~e site~ ~acilitate removal ~rom and rein~ertion of the mutant ~ragment into the TG~-~3 cDNA plasmid. The FspI - ~
B~m~I TGF-~3 cDNA target fragment i3 subsloned in ~13 into ~ :
both orientation~ relative to a Ml3 origin o~ replication.
.~
Three di~erent re~ctions are per~ormed on e~ch o~ the strands 15 of the mutagQniz~d t~rget ~equence ~40 ~g o~ 1 mg/ml sin~le ~tranded DNA p~r tube).
:, l. lO ~l o~ Sodlu~ acQtate, pH 4.3 and 50 ~l 2 M
80dlu~ nitrit~ 1~ added and incubated for 60 min at room tomperatura.
. , 2. 60 ~l concontratod (18 M3 ~or~ic acld i~ add~d ~nd incub~ted ~or lO ~in room ta~perature.

- 3. 60 ~l o~ concentrat~d (12 ~) hydrazin~ i~ added and incubated for lO ~in at room te~peratur~.

After incubation, DNA'~ are ethanol precipitated, wa~hed with 25 70% ethanol and re~u~pended in T~ buffer. The 2nd ~trand are pr~parod by pri~r ~xtension u~ing AMV r~v~rs~ tran~ript~e (d~purination inhibits DNA synthesi~ by E.coli DNA pol I but :

:.. . . . .. . .. .

~ . ' ' .. . . .

, . ' ',. ~.- . , wos2/oo3l8 PCT/US91/0454L
2 ~ 9 2 54 not by revQr~e transcriptaoe). Mutant~ can be identified directly by DNA sequencing o~ random clones.

indinq of TGF-~3 to 2 ma~FQ~lQk~lin The binding o~ a2-macroglobulin to TGF-~3, modified TGF-~3 or 5 TGF-~3 bound to anti-TGF-~3 antibody i8 mea~ured u~ing a modification of the m~thod d~acribod by O'Conner-McCourt, et al. (29). Brie~ly, 125I TGF-~3 i3 incubated with ~2-macroglobulin in PBS with ~ither unlabel~d T~F-~3, 20di~ied TGF-~3 or TGF-~3 bound to anti-TGF-~3 antibody for 5 hr on 10 ice. Non-~pkctfic ~inding will be dotermined using a 400-fold molar Qxcess o~ unlabeled growth factor. Cro~slinking of ths -macrcglobulin to the 125I TGF-~3 i8 accomplished with the addition o~ a ~ volu~ o~ 5 mM bis(~ulfo~uccinimidyl)suberate (BS3; Pierce) in PBS and the reaction i8 ~topped a~ter 2 min 15 at 4C by the addition o~ 1/20 volu~e o~ 2.5 M glycine. An equal volume of SDS-PAGE sample bu~r (2X) i~ added to the ~ample will be heated in a boiling water bath for 3 min.
,~ .
Electrophore~is i~ p~rfor~d on the sample3 and destained gels iE dried and Qxpo~-d at -70-C to X-ray ~ u~ing intens~ying 20 ~creens. Alternatively, co.~ rcially available anti-~2-macroglobulin (Sig~ ) ~ 8 u~d to i~munopr~cipitate the ~2-~acroglobulin 125I TGF-~ couplex ~ith or without cros~llnking and quantitat-d dirQctly by a ga~Ja count~r.

~ Lina ~8~ay o~ TGF-~3 to ty7e III receDtors 25 Type I~I rocQptor~ Qxist ~n both a ~e~brane bound (prot~oglycan) and ~olubl~ (betaglycan) for~. The binding of : betaglyc~n to TG~-~, ~odl~iQd TGF-~ or TGF-~ bound to antl-TGF-~ antibody i~ mea~ured u~ing the ~ethod de~cribed by Andr~, et al (i). Non-specif~c binding i~ deter~ined using 30 a 400-~old molar exces~ o~ unlabeled growth factor. Briefly, ; , ................. . ........ ~ . :~ .

- ' , ' .

~092/00318 PCTtUS9ltO4541 ~..
2 ~ 2 soluble betaglycan is made fro~ ssrum-free conditioned ~edia form 3T3-L1 adipocyt2s. ~ou~e 3T3-L1 adipocyt~s i~ grown to confluence or near confluency. The cell monolayer~ ar~ wa~hed twice and incubated for 3 days in s~rum-free Waymouth 1 8 5 medium. A concentratad stock of frQsh PMSF ~ added to a final concentration of 0.2 mM immedi~tely after collection.
The collected conditioned media i8 c~ntri~uged at 2,000 x g for 15 min at 4C followed by ultracentrifugation at 2000,000 x g ~or 40 min at 4C. NaCl i~ added to the conditioned media 10 to a ~lnal concentration o~ 0.25 m NaCl and brought to pH 6.0 by ~he addition of 1 M BIS-Tris, pH 6.0 and pre-Qquilibrated with binding buffer (50 m~ NaCl, 10 ~M MgC12, 5 mM XCI, 25 mM
Hepe3, pH 7.5 containing 1 mg/~l o~ BSA). 125I TGF-~ is mixed with incubated with unlabeled TGF-~, ~odified TGF-~ or TGF-~
15 bound to anti TGF-~ antibody and soluble betaglycan (appropriately diluted) in binding buffer. Thi~ mixture is added to the pre-equilibrated DEAE-Sepharo~e Fast Flow and incubated for 3.5 hour~ at 4C with continued mixing. The be~ds are then washed 5 times with cold binding bu~er 20 (wlthout albu~in). The ~mount o~ radioactivity remaining on the beads will be quantitated. For analysis of 3pecific binding to the typ~ III prot~oglycan rRceptors, 125I TGF-~3 iB
cros~-linked to intact c~ (6), the receptors separated by standard SDS-~AGE, and 125I-binding quantita~ed by 25 dQnsito~etry.

The cha~ical hal~-life of TGF-~3 i8 determined in the serum of mice following bolu~ injections (O.1-lO~g/mouse) via i.v., i.p., and s.c. route~, using internally labelled TGP-~3 30 (lakelled ~etaboli~ally with 35S cysteine) or 125I TGF-~3.
Tl~u~ di~tribution o~ labell~d material ~ ~easured in variou~ org~ns with particular 6mphasis on liver, spleen and bon~ marrow ~ites. If the biological half~ e of TGF-~3 ~n ,.. .
'' ' ' ~ "

;~''' ~ ' ~ , , W092/003l8 56 pCT/US9l/04 2 ~ 9 9 ~ is found to be unacc~ptably short regional admlnistration by diroct intrasplenic in~ection (through the body wall) i8 employed or u~lng the surgical technique reportQd by Goey et al (12) involving in~ection into the femoral art~ry. Thi~
5 latter approach ha~ been r~ported to be effective in localizing TGF-~l to the marrow with rssultlng inhibitlon o~
early Bte~ c~ll and progenitor cell proli~eration.

k~U~ Os E~r-~-lo~ o~ Pro R-g~on ~ th~ Ta~-B3 P~ou~o~

The TGF-~3 pro region proteln a~sociat~s with the mature TGF-10 ~3 and modifiQs the hal~ life and biological activity of mature TGF-B3. Nucleic acid ~ncoding thQ TGF-~3 precur~or beginning with ~athionine at nucleotide positions 263-265 and ending with arginine at position 1160-1162 is engineered by mutagQne~is o~ the nucleic acid in rigu~- 1 to introduce a 15 translation ter~ination codon (~GA, TAG, TAA) at position 1163-1165. m e resulting nucleic acid i8 in~erted in an expre~ion vector and trans~ected into a ~uitable host cell with an addltional ~elect~ble ~arker, a~ previou~ly describQd.
TGF-B3 pro r~gion protein i~ recovered from the culture 20 madium. Thi~ prot~in ~tably bind~ ~atur~ TG~-B3 and thereby sQquester~ and modi~ies th8 hal~ life and biological activity o~ the m~turs TGF-B3.

~inding As ~y_of TGF-~3 to the ~GF-B3 ~ro reqion ~rotein The bindin~ Or TGF-~3 pro region to TGF-~3 or mutant TGF-~3 i8 25 ~ea~ured hy the ~ollowlng. 125I TGF-03 is incubated with puri~isd IGF-~3 pro region in P~S with either unlabeled TGF-~3 or ~utant TGF-~3 ~or 5 hr on ice. Non-specific binding will ba d~termined u~ing a 400-fold molar excess of unlabeled growth factor. Cro~linking o~ the ~GF-B3 pro region to l25I
30 TGF-~3 i8 ~cco~pli~hed with thQ addition o~ A ~ volu~o o~ 5 mM
bi~(sul~o3uccinimidyl)suber~te (BS3; Pi~rce) in PBS and the - . , -;. . ~. ~

.

W~092/00318 PCT/US91/04541 2 a ~ 2 reaction i8 stopped after 2 min at 4C by the addition o~ l/20 volume o~ 2.5 M glycine. An equal volu~e of SDS-PAGE ~ample bu~er (2X) i~ added to th~ sample will be heated in a boiling water bath ~or 3 min.

5 Electrophoresi~ i8 performed on the 8~mple~ and de~tained gels i~ dried and expo~ed at -70C to X-ray ~ilm using intensifying screens. Alt~rnatively, antI-TGF-B3 i8 u~ed to imnunoprecipitate the TGF-B3 pro region co~plexed with 125I
TGF-~ complex with or without crosslinXing and quantitated 10 dir~ctly by a g~mma counter.

. -, .

~vos2/00318 PCT/US91/0454~ ;
2 0 ~ 2 58 `' ~ C~8 1. Andres, J.L., et al. (1989) J. C~ll Biol. LQ~:3137-3145.

2. A~soian, et al. (1983) J. ~iol. Chem, ~5~: 7155.

3. Barker, C.R., ~orman, C.P., and Smith, J.L (1975) Immunolooy 29:765-777.

4. Cara~ et al. (1989) J. ~çll Biol. 108:1387-1396.

5. Chei~etz, S., et al. (1988) J~iol. Che~, 263:16984-16991.

6. Chei~eti, S. et al. (1987) Ç~ 409-415.

10 7. Coffey, R.J. ~t al. (1987) J. Clin. Iny~ 80:750-757.

8. R. Derynck et al. (1985) Nature, 316:701.

9. de Martin, et al. (1987) E~aQ_I 6:3673-3677. -10. Derynck et al. EM~O J. 6:3673-3677.

11. G~ntry, L.~. ~t al. (1987) Mol. Cell. BiQl. 7:3418-3427.

15 12. Gosy, ~. at al.~l989) J. ~ nol. 143:877-880.

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20~99~
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38. U.S. Patent No. 4,886,747, i~ued December 12, 1989 to R.
Derynck and D. Goeddell.

: . . . - - - . .: - - -- . - . :, -, - -.: . .

.

Claims (37)

What is claimed is:

1. A method for recovering purified, non-denatured mature TGF-.beta.3 from a mixture of mammalian cell-derived polypeptides which comprises contacting the mixture with an antibody which specifically binds to mature TGF-.beta.3 but exhibits substantially no cross reactivity with mature TGF-.beta.1 and mature TGF-.beta.2.

2. The method of claim 1, wherein the mixture of mammalian cell-derived polypeptides includes a mixture of non-human mammalian polypeptides from non-human cells in which TGF-.beta.3 has been expressed.

3. The method of claim 1, wherein the antibody is directed to an epitope defined by the amino acid sequence .

4. The method of claim 3, wherein the antibody is immobilized on a solid support.

5. A method for producing substantially purified TGF-.beta.3 precursor having an anchorage membrane sequence which comprises:
(a) preparing DNA encoding a TGF-.beta.3 precursor having the membrane anchorage sequence;
(b) inserting the DNA into an expression vector linked to a suitable promoter compatible with a host cell;
(c) transforming the host cell with the vector in order to induce expression of the DNA of step (b) such that a TGF-.beta.3 precursor is expressed and subsequent translocation of the expressed TGF-.beta.3 precursor having the membrane anchorage sequence;
(d) culturing the host cell in medium;

separating the host cell from the medium;
(f) disrupting the host cell such that a lysate containing the TGF-.beta.3 precursor having the membrane anchorage sequence is produced; and (g) purifying the TGF-.beta.3 precursor having a membrane anchorage sequence from the lysate under conditions such that the substantially purified TGF-.beta.3 precursor is produced.

6. A method for producing substantially purified TGF-.beta.3 precursor having an anchorage membrane sequence which comprises:
(a) preparing DNA encoding the TGF-.beta.3 precursor having a membrane anchorage sequence;
(b) inserting the DNA into an expression vector linked to a suitable promoter compatible with a host cell;
(c) transforming the host cell with the vector in order to induce expression of the DNA of step (b) such that a TGF-.beta.3 precursor is expressed and subsequent occlusion of the expressed TGF-.beta.3 precursor having a membrane anchorage sequence in occlusion bodies;
(d) culturing the host cell in culture medium;
(e) separating the occlusion bodies from the host cells and the culture medium;
(f) disrupting the occlusion bodies to produce a solution containing the TGF-.beta.3 precursor having a membrane anchorage sequence; and (g) purifying the TGF-.beta.3 precursor having a membrane anchorage sequence from the lysate such that the substantially purified TGF-.beta.3 precursor is produced.

7. The method of any of claims 5 or 6, wherein the anchorage linkage sequence includes a phosphatidyl inositol linkage.

8. The method of any of claims 5 or 6, wherein the anchorage linkage sequence includes a hydrophobic transmembrane peptide sequence.

9. A method for producing a substantially purified mutant TGF-.beta.3 precursor which comprises:
(a) preparing a DNA comprising a first DNA sequence encoding an amino acid sequence substantially identical to the amino acid sequence shown in Figure 1 beginning with a methionine encoded by nucleotides 263-265 and ending with a glutamine encoded by nucleotides 1148-1150, a second DNA
sequence which is linked to nucleotide 1150 encoding a protease cleavage sequence, and a third DNA sequence, linked to the second DNA sequence, encoding an amino acid sequence substantially identical to the amino acid sequence shown in Figure 1 beginning with an alanine encoded by nucleotides 1163-1165 and ending with a serine encoded by nucleotides 1496-1498;
(b) inserting the DNA of step (a) into an expression vector linked to a suitable promoter compatible with a host cell;
(c) transforming the host cell with the vector in order to induce expression of the DNA of step (b) such that a mutant TGF-.beta.3 precursor is expressed;
(d) culturing the host cell in a medium under conditions such that the expressed mutant TGF-.beta.3 precursor is secreted into the medium;
(e) separating the cell from the medium containing the mutant TGF-.beta.3 precursor so secreted; and (f) purifying the mutant TGF-.beta.3 precursor thereby producing the substantially purified mutant TGF-.beta.3 precursor.

10. A method for producing a substantially purified mutant TGF-B3 precursor which comprises:
(a) preparing a DNA comprising a first DNA sequence encoding an amino acid sequence substantially identical to the amino acid sequence shown in Figure 1 beginning with a methionine encoded by nucleotides 263-265 and ending with a glutamine encoded by nucleotides 1148-1150, a second DNA
sequence which is linked to nucleotide 1150 encoding a protease cleavage sequence, and a third DNA sequence, linked to the second DNA sequence, encoding an amino acid sequence substantially identical to the amino acid sequence shown in Figure 1 beginning with an alanine encoded by nucleotides 1163-1165 and ending with a serine encoded by nucleotides 1496-1498;
(b) inserting the DNA of step (a) into an expression vector linked to a suitable promoter compatible with a host cell;
(c) transforming the host cell with the vector in order to induce expression of the DNA of step (b) such that a mutant TGF-B3 precursor is expressed;
(d) separating the host cell containing the expressed, mutant TGF-B3 precursor from the culture medium;
(e) disrupting the host cell such that a lysate containing the mutant TGF-B3 precursor is produced;
and (f) purifying the mutant TGF-B3 precursor thereby producing the substantially purified mutant TGF-B3 precursor.

11. A method for producing a substantially purified mutant TGF-B3 precursor which comprises:
(a) preparing a DNA comprising a first DNA sequence encoding an amino acid sequence substantially identical to the amino acid sequence shown in Figure 1 beginning with a methionine encoded by nucleotides 263-265 and ending with a glutamine encoded by nucleotides 1148-1150, a second DNA
sequence comprising ATG which is linked to nucleotide 1150, a third DNA sequence, linked to the second DNA sequence, encoding an amino acid sequence substantially identical to the amino acid sequence shown in Figure 1 beginning with an alanine encoded by nucleotides 1163-1165 and ending with an asparagine encoded by nucleotides 1469-1471, a fourth DNA sequence, linked to the third DNA sequence, comprising X, and a fifth DNA
sequence, linked to the fourth DNA sequence, encoding an amino acid sequence substantially identical to the amino acid sequence shown in Figure 1 beginning with a valine encoded by nucleotides 1475-1477 and ending with a serine encoded by nucleotides 1496-1498;
(b) inserting the DNA of step (a) into an expression vector linked to a suitable promoter compatible with a host cell;
(c) transforming the host cell with the vector in order to induce expression of the DNA of step (b) under conditions such that a mutant TGF-B3 precursor is expressed;
(d) culturing the host cell in medium under conditions such that the expressed mutant TGF-B3 precursor is secreted into the medium;
(e) separating the cell from the culture medium containing the mutant TGF-B3 precursor so secreted;
and (f) purifying the mutant TGF-B3 presursor such that a substantially purified mutant TGF-B3 precursor is produced.

12. A method for producing a substantially purified mutant TGF-B3 precursor which comprises:
(a) preparing a DNA comprising a first DNA sequence encoding an amino acid sequence substantially identical to the amino acid sequence shown in Figure 1 beginning with a methionine encoded by nucleotides 263-265 and ending with a glutamine encoded by nucleotides 1148-1150, a second DNA
sequence comprising ATG which is linked to nucleotide 1150, a third DNA sequence, linked to the second DNA sequence, encoding an amino acid sequence substantially identical to the amino acid sequence shown in Figure 1 beginning with an alanine encoded by nucleotides 1163-1165 and ending with an asparagine encoded by nucleotides 1469-1471, a fourth DNA sequence, linked to the third DNA sequence, comprising X, and a fifth DNA
sequence, linked to the fourth DNA sequence, encoding an amino acid sequence substantially identical to the amino acid sequence shown in Figure 1 beginning with a valine encoded by nucleotides 1475-1477 and ending with a serine encoded by nucleotides 1496-1498;
(b) inserting the DNA of step (a) into an expression vector linked to a suitable promoter compatible with a host cell;
(c) transforming the host cell with the vector in order to induce expression of the DNA of step (b) under conditions such that a mutant TGF-B3 precursor is expressed;
(d) separating the host cell containing the expressed, mutant TGF-B3 precursor from the culture medium;
(e) disrupting the host cell such that a lysate containing the mutant TGF-B3 precursor is produced;
and (f) purifying the mutant TGF-B3 precursor such that a substantially purified mutant TGF-B3 precursor is produced.

13. The method of any of claims 11 or 12, wherein in step (a) X is selected from a group of tri-nucleotides consisting of .

14. The method of any of claims 5, 6, 9, 10, 11, or 12, wherein purification includes affinity chromatography.

15. The method of any of claims 5, 6, 9, 10, 11, or 12, wherein affinity chromatography comprises lectin column chromatography.

16. The method of claim 14, wherein affinity chromatography comprises antibody column chromatography.

17. The method of any of claims 9 or 10, wherein in step (a) the protease cleavage sequence includes a collagenase recognition sequence.

18. The method of any of claims 9 or 10, wherein in step (a) the protease cleavage sequence includes a Factor Xa recognition sequence.

19. The method of any of claims 5 or 6 which further comprises:

(a) treating the purified TGF-B3 precursor so recovered with an activating agent to separate a mature TGF-B3 from the presursor; and (b) recovering the separated mature TGF-B3 of step (a).

20. The method of any of claims 9 or 10 which further comprises:
(a) treating the purified mutant TGF-B3 precursor so recovered with an activating agent to separate a mature TGF-B3 from the precursor; and (b) recovering the separated mature TGF-B3 of step (a).

21. The method of any of claims 11 or 12 which further comprises:
(a) treating the purified mutant TGF-B3 precursor so recovered with cyanogen bromide to separate a mature TGF-B3 from the precursor; and (b) recovering the separated mature TGF-B3 of step (a).

22. A method for producing mutant TGF-B3 which comprises:
(a) preparing a DNA encoding an amino acid sequence substantially identical to the amino acid sequence shown in Figure 1 beginning with a leucine encoded by necleotides 332-334 and ending with a serine encoded by nucleotides 1496-1498;
(b) inserting the DNA of step (a) into an expression vector operably linked to a suitable promoter compatible with a host cell;
(c) transforming the host cell with the vector in order to induce expression of the DNA of step (b) under conditions such that a mutant TGF-B3 is expressed;
(d) culturing the host cell in medium;
(e) separating the host cell containing the mutant TGF-B3 so expressed from the medium;
(f) disrupting the cell to produce a lysate containing the mutant TGF-B3; and (g) purifying the mutant TGF-B3.

23. The method of claim 22 which further comprises:
(a) treating the purified mutant TGF-B3 so recovered with an activating agent to separate a mature TGF-B3 from the mutant TGF-B3; and (b) recovering the separated mature TGF-B3 of step (a).

24. A process which comprises:
(a) contacting a TGF-B3 precursor with a precipitating agent thereby concentrating the TGF-B3 precursor in a precipitate;
(b) extracting the pellet of step (a) with an acidified organic solution under such conditions that mature TGF-B3 is separated from the pellet; and (c) recovering the mature TGF-B3 so separated in step (b).

25. The process of claim 24, wherein the acidified organic solution in step (b) includes acidified acetonitrile.

26. The process of claim 25, wherein the acidified acetonitrile comprises 50% acetonitrile and 1.0M acetic acid.

27. The process of claim 24, wherein the precipitating agent in step (a) includes ammonium sulfate.

28. A method for producing and identifying a mutant, mature TGF-B3 exhibiting reduced binding affinity to serum binding proteins which comprises:
(a) preparing a DNA encoding the TGF-B3;

(b) performing mutagenesis on the DNA of step (a) thereby obtaining a mutant DNA;
(c) inserting the mutant DNA into an expression vector linked to a suitable promoter compatible with a host cell;
(d) transforming the host cell with the vector in order to induce expression of the mutant DNA of step (c) under conditions such that a mutant TGF-B3 is expressed;
(e) culturing the host cell in medium under conditions such that the expressed mutant TGF-B3 is secreted into the medium;
(f) separating the hose cell from the culture medium containing the mutant TGF-B3 so expressed;
(g) purifying the mutant TGF-B3;
(h) activating the mutant TGF-B3 so expressed under conditions such that a mutant mature TGF-B3 is separated from the mutant TGF-B3; and (i) assaying the culture medium for the mutant, mature TGF-B3 thereby identifying a mutant, mature TGF-B3 exhibiting reduced binding affinity to serum binding proteins.

29. A method for producing and identifying a mutant, mature TGF-B3 exhibiting reduced binding affinity to serum binding proteins which comprises:
(a) preparing a DNA encoding the TGF-B3;
(b) performing mutagenesis on the DAN of step (a) thereby obtaining a mutant DNA;
(c) inserting the mutant DNA into an expression vector linked to a suitable promoter compatible with a host cell;
(d) transforming the host cell with the vector in order to induce expression of the mutant DNA of step (c) under conditions such that a mutant TGF-B3 is expressed;
(e) culturing the host cell in medium under conditions such that the expressed mutant TGF-B3 is produced in the host cell;
(f) separating the host cell containing the mutant TGF-B3 so expressed from the culture medium;
(g) disrupting the cells to produce a lysate containing the mutant TGF-B3;
(h) purifying the mutant TGF-B3;
(i) activating the mutant TGF-B3 so expressed under conditions such that a mature TGF-B3 is separated from the mutant TGF-B3; and (j) assaying the culture medium for the mutant, mature TGF-B3 exhibiting reduced binding affinity to serum binding proteins thereby identifying a mutant, mature TGF-B3 exhibiting reduced binding affinity to serum binding proteins.

30. The method of any of claims 28 or 29, wherein the serum binding protein is .alpha.2-macroglobulin.

31. The method of any of claims 28 or 29, wherein the serum binding protein is type III TGF-B receptor.

32. The method of claim 31, wherein the type III TGF-B
receptor is betaglycan.

33. The method of any of claims 28 or 29, wherein the serum binding protein is a pro region of the TGF-B precursor.

34. A method for producing a substantially purified pro region of the TGF-B3 precursor which comprises:
(a) preparing DNA encoding the pro region of the TGF-B3 precursor;
(b) inserting the DNA into an expression vector linked to a suitable promoter compatible with a host cell;
(c) transforming the host cell with the vector in order to induce expression of the DNA of step (b) under conditions such that a pro region of the TGF-B3 precursor is expressed;
(d) culturing the host cell in medium;
(e) separating host cells from the medium;
(f) disrupting the host cells to produce a lysate containing the pro region of the TGF-B3 precursor so expressed; and (g) purifying the pro region of the TGF-B3 precursor from the lysate such that the substantially purified pro region of the TGF-B3 precursor is produced.

35. A method for producing a substantially purified pro region of the TGF-B3 precursor which comprises:
(a) preparing DNA encoding the pro region of the TGF-B3 precursor;
(b) inserting the DNA into an expression vector linked to a suitable promoter compatible with a host cell;
(c) transforming the host cell with the vector in order to induce expression of the DNA of step (b) under conditions such that a pro region of the TGF-B3 precursor is expressed;
(d) culturing the host cell in medium under conditions such that the expressed pro region of the TGF-B3 precursor is secreted into the medium;
(e) separating host cells from the medium containing the pro region of the TGF-B3 precursor so secreted;
and (f) purifying the pro region of the TGF-B3 precursor such that the substantially purified pro region of the TGF-B3 precursor is produced.

36. The method of any of claims 5, 6, 9, 10, 11, 12, 22, 28, 29, 34, or 35, wherein the host cell is a eucaryotic cell.

37. The method of any of claims 5, 6, 9, 10, 11, 12, 22, 28, 29, 34, or 35 wherein the host cell is a procaryotic cell.