CA2237900A1 - Type ix collagen and chimeras - Google Patents

Abstract

The present invention relates to novel collagens, polynucleotide sequences encoding these novel proteins, and to the use of these novel proteins and polynucleotides in the diagnosis and treatment of disease. The present invention further relates to specific collagens and derivatives, specifically fusion proteins of type IX collagen with type II and/or type XI collagens, and their use as therapeutic agents.

Description

TYPE IX COLLAGEN AND CH~IERAS

5 I. ~IELD OF THE INVENTION
The present h~ ioll relates to novel human collagen p~ s~
polym-rleQti~e sequrnr~s which encode these novel collagens ~ st and to the use of these novel ~r~tems in the ~ no~i~ and 1~ of disease. More specifir~lly, the present invention relates to polynucleotides enro~1in~ human 10 cY3(IX) collagen and d~livalives hereof, fusion proLeil~s of type IX collagen with type II and/or type XI cQll~gen ~ubu~ and dc~i~alivt:s, and use of these ~r~
and polymlrle~ s as ~ gnnstir and Ille.alJ~ulic agents.

II. BACKGROUND
Collagen fibrils, proteoglycan ag~.~gatcs and glyc~ vte.lls are critical col~ k. of the cartilage eYtr;-~ell~ r ma~c that, collectively, resist cc,.~..,s:,ion and the tensile and shear forces that are g~ during artirnl~tion ~L;.1~~.~ and Oldberg, FASEB J. 3:2042-20~1 (1989); Mayne and ]3-~,vV~
Ca~lage Degradation: Basic and Clinical Aspects (Woessner, J.P. and Howell, 20 D.S., eds.) Marcel Dekker, Inc., New York, pp. 81-108 (1993). Mut~ti~n~ in cartilage matrix genes that affect the b}o~y~ f ~ ass~ bly or ;..~ ons ~cL~
t_ese various matrix coln~ol..,.lLs may co~ to deg.~ .on of t_e cartilage matrix and the loss of normal cartilage ru.l;~ioll. Mllt~tions in human collagens have been shown to cause a series of chO~l~dy~lasiâs l~lging in seie.ily from 25 lethal ~cllo~ uge.~is type II to Stickler arthroorhth~ llly and early onset f~mili~l o~,Ga,Ll liLis (reviewed by S~ . et al., Eur. J. Pediatr. 153:56-65 (1994); Vikkula et al., Ann. Medicine 26:107-114 (1994); P~ck~ and Kivi~kko, Annu. ~ev. Biochem. 64:403434 (1995)).
Analyses of type IX collagen d~ e this molecule is located on the 3û surface of type II coll~gen-co-~ fibrils in hyaline cartilage and other tissues, inrTnrlin~ the vitreous humor (~,vi~ ~,d by I~CVVL~>n and Mayne, E~tr~el~ r MatrzJc Assemb~ and Structure (Yu~ co, P.D., Birk, D.E., Mr.c~ . R.P., eds) ";~-- Press, Inc., San Diego, pp. 129-170 (1994)). Type IX collagen is a L~rl)L,~er co~osed of three polypeptide ~Jl~ l(IX), a2aX) and cY3(IX), W O 97/17988 PCT~US96/18149 that are products of distinct genes and that contain ~lt ~ non-triple-helical or nrmfQll~g~noll~ domains (NC14) and triple-helical or collag~.luus f1~m~in~
(COL1-3). I~he three polypeptide ~lbul~iL~i are assembled into a mature collagenmolec~ with the sllu.;~c cx(IX)~2aX)a3(IX) (van der Rest and Mayne, Structure 5 and Function of Collagen Iypes (Mayne, R. and Bulgesoll, R., eds.) ~r~rle ~;r Press, Orlando, FL, pp. 195-221 (1987). In addition to type II and type IX
collagen, hyaline cartilage from a variety of sources also co~l~ s ~
~IlllUUlll~i of at least three other collagen mol~ es, types VI, X and XI. Thomas et al., Ann. Rheuma~. Diseases 53:488496 (1994); ~ayne and I3~ on, Cartilage 0 Degradation: Basic and Clinical Aspects (Woes~ " J.F. and Howell, D.S., eds) Marcel Dekker, Inc., New York, pp. 81-108 (1993). Type XI collagen, li~e type IX collagen, is a ~ty~a)L~ L comrose~l of three different polypeptide ~
cYl(XI), cr2(XI), and cY3(XI). Collagen types XII and X~V were also isolated from bovine articular cartilage. Watt et al., J. Biol. Chem. 267:20093-20099 (1992).
Native type IX collagen mol,o ~ s ill~,a~;l with type II collagen molecules in a highly ~l~e~il;r lna"l-er so that the ~om~in~ NC1, COLl, NC2, COL2 and NC3 lie along the surface of the collagen fibril. The i~lle.a~;liol~s b~,Lween type IX
and type II collagen are stabilized by multiple covalent crosslinks derived fromsreci~1r Iysine .e~ 1ues. See van der Rest and Mayne, J. Biol. Chem.
263:1615-1618 (1988); S ~ nokom~ki et al., Ann. N.Y. Acad. Sci. 580:1-7 (1990);
Wu et al., J. Biol. Chem. 267:23007-23014 (1992). The perio~lir loc~ tion of type IX collagen along type II collagen fibrils can ~e readily vi~l~li7~-1 by rotary s_adowing bec~ e the collag~nous domain COL3 and the large globular ~o~n~in NC4 project from the surface of the fibril. Vaughan et al., J. Cell Biol.
106:991-997 (1988); Shima'- m~ki et al., Ann. N.Y. Acad. Sci. 580:1-7 (1990).
II1 cn~ I the type XI CQll~gPn ~t.,.~ ~ is th~lght to reside in the central portion of the fibril. ~Pnlllpr et al., J. Cell Biol. I08: 191-97 (1989).
Cloning and sc~ of the human type II collagen gene and the three human ~rpe XI collagen genes has been reported. The cn...plFtc human t~pe II
30 collagen gene seqll~on~e was reported by Baldwin et al., Biochem. J. 262:521-28 (1989) and by Su et al., Nucleic Acids Res. 17:9473 ~1989). Of the three ~rpe XIcoll~gen ~ul~u~, the o~3~) cbslin is believed to be the product of the type II
collagen gene. I~ d et al., J. Biol. Chem. 263:17159-66 (1988) cli~eloses W O 97/17988 PCT~US96/18149 cDNA ~e~ .re ~u~ lA,dly enro~lin~ the proc~1(XI) collagen. ~ e coding for the a2(XI) gene was r~ol~d by Kiml-r~ et al., J. Biol. Chem. 264:13910-16 (1989).
The genes e~coding the three chains of type IX collagen are eYcell~nt 5 c~ntli~l~tPs for cholldrody~lasias and ~leg~ e diso~ that affect the joints and/or vi~ u~ humor becau~e type IX co~ en is a .~ ir,~,.." bllu~ al molecule m both of these tissues. Th~.',r~ , cloning of the genes enro~ the three type IX collagen s.lb~ has been the object of ill~iVt; l~se~;h. ~lr~ ki et al., Eur. J. Biochem. 192:703-8 (199O),~ tu~d the co...~ cDNA seq~l~n~e of 10 both ~ iv~ from the human ~1aX) gene. The ll~jo~;~y of the human cr2(IX) collagen cDNA was l~ol~d by Perala et al., FEBSLett.
319:177-80 (1993) and completed by Warman et al., Genomics 23:158-62 (1994).
The complete human seq lenre for the cY3(lX) subunit was unavailable until ~e.lLly . As descr he~l in the colh~ull~.lLly ~iled applir~tinn (U.S. Applir~tion to be 15 S~C'~;~".F/~!), Drs. R.W. ~ ,n and R. Mayne have itllo-ntifi~cl and e1~S ".c~ ~ ;,~1 the full ler~h s~ coll.,~ol~.l~ to human a3(IX). The i~o...~;on co.~ F~ in the 13r.,~ I~>n and Mayne provisional applir~tion is ~col~olaLed herein by l~L~ell~.
F.~ tili7in~ mice suggest that type IX collagen plays an lL~lL role in ...Z;..~ the LL~,~,1;LY of hyaline cartilage. Animals that either 20 express a mini~n~ C~lyillg a ~ tif n in the ~l(IX) chain (Nakata et al., Proc.
Natl. Acad. Sci. U.S.A. 90:2870-2874 (1993)) or that carry dislu~Led a1~X) genes(Fèssler et al., Proc. Natl. Acad. Sci. U.S.A. 91:5070-5074 (1994)) d~lo~
~Pg~~'F~ joint disease 1hat resembles human os~ G~L~ . The i~ olL~lce of type IX collagen in human disease was verified by the i(lentifi~tion of a ml-ts ti~n 25 in COL9A2 (Mt~ ki et al., submitted for ~-lblirS ~ , (1995)) that results in the ski~ of exon 3 and that causes Multiple ~i~hyb_al Dysplasia (EDM2).

m. Sul~lMAl~y OF ~l ~; INVENTION
The present i~v~lllion relates to novel collagen del;~a~iv~ protei~ and the 30 polynucleotide s~ ,s which encode them. Also desclibed herein are mPth~c ~ for ~is~nosic Of ~ r~c~sresllltin~ from abnotmS~litiPs in collagen synthesis or W O 97/17988 PCT~US96/18149 One aspect of this iu~nlioll is the di~co.~ that fusion ~lot~ s of human type IX coll~n may be produced in which a human type IX coll~n subunit is covalently linlced to human type II coll~çn and/or a human type XI collagen suhnnit In one embo~lim~nt of this iu~ u, the fusion ~,o~i~ls are 5 lecuulbhJal.lly p~ ced as chi~ as by linking polymlr3eoti~le coding se~ rc; for a human type IX collagen subunit in frame to polynucleotide coding se.~ re for human type II coll~en and/or a human type XI collagen. This cl~ ic coding se~ re is h~s~d into an e,.~ ion vector and used to lla~Çolln a~lo~ia~
host cells. The host cells are then in-~ured to express the ch;~..P- ;~ coding sey~ re 10 and ~cl~ produce the chilllc ic colla~n fusion ~rus_ I~S. These fusion ~r~te~s are useful in the ~ of collagen related ~ a~es and co~
Irhe present invention also relates, in part, to nucleotide seq~enres and ,ssion vectors enrodir~ the c~ P- ;r collagens of the invention.
Also disclosed herein are mPthf~c of L,~ ..f -~ of ~ ,eA'~ or c~ Jus a~coci~t~d with ~bn~)rmqliti~s in collagen proAllction or ~uto;.. ~.;ly to collagen.
Such abnG..~.AIitiPs can result in, for eY~mple, .l.- .~ t~ id ~Ll~iliD, o~t~,oalLlJIilis, reactive A~LIlliLiD, aUIO;~ mP h~qri~ di~e~se, cartilage ;~ 9~ A~;OIl due to b~-lt~;~l or viral ;..r~-1;ol-~ (e.g. Lyme's disease), ~ P~e, bursitis, corneal ~ es~ and ankylosing s~(J~lylilis (fusion of the spine). The novel 20 ~lOkil~S of the invention are used in these methods of ~l~,aLill~, col~a~en related A;,~F~S.

IV. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Salt r~A~ ul;on of recombinant human type IX collagen 2~ he~.~u~iul~. of al~X), a2~CX~, and a3~GY) in llOllC~uClug 8% sodium dodecyl sulfate pol~aclyl~ide gel clectlo~h(,l-,~is (SDS-PAGE).
Figure 2. Salt frartion~tion of recombinant human type IX collagen h~ iulc~ of alaX:), a2(~X), and a3aX) in re~ cing 10% SDS-PAGE.

30 V. DETAIL~:D DESCRIPTION
The present invention relates to the polynucleotide and nucleic acid s~ es encoding the recombinant fusion proteins of type IX co11~gerl dclivalivcs with type II collagen protein and/or type XI collagen protein and such fusion W O 97/17988 PCT~US96/18149 eills. Also within the scope of the hl~nlic,ll are mf th~ls of using these collagen fusion lJ~Ut~ ..IS to treat crJll~gen related disur~., and co~ n~

A. De~mitions The term "collagen subunit" refers to the amino acid se~lue~ e of one subunit of a collagen protein enro~led by a single gene, as well as de.ivalives, inrlllrlin~ ~f~lfti~n d~ iv~s, Cc~llsf,~ iv~ sll~ etc.
A "fusion protein" is a protein in which peptide se.ll~f ~~rf,s from dirf~ L
ploleills are covalently linked together.
Tne term "Ç~ lf ~ or "cllim~rlc" refers to fusion protein produced by operably linking polylulclf ulide coding seqlif nre for two or more collagen protein subunits in frame and recom~i.,alllly e,~ essLg the linked coding se~llenf e as a single peptide chain.
"Active human type IX collagen" refers to the native trimeric protein complex, and may be recoml)illalllly ~lu~ C~
The phrase "~I.~l cnnrliti~-n~" as used herein re~ers to those hyl lid~mg co.~ that (1) employ low ionic ~ and high t~ c for washing, for ~Y ~ ,lc, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50~C; (2) employ during h~ n a ~ agent such as fc....~ e, for e,~ le, 50%
(vol/vol) f~.. ~."i-l~ with 0.1% bovine serum albumin/0.1% Ficoll/0.1%
polyv--lyl~yllolidone/50 rnM sodium ~hn~ .; buffer at pH 6.5 with 750 mM
NaCl, 75 mM sodium citrate at 42~C; or (3) employ 50% ~....~ ide, 5 x SSC
(0.75 M NaCl, 0.075 M Sodium pyroph~ .h~, 5 x Denl~ dL~S solution, so.~;r~
salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% A~P~ n sulfate at 42~C, with 25 washes at 42~C in 0.2 x SSC and 0.1% SDS.
In accor~ce with the invention, any nucleotide se~ e which Pnrorlps the amino acid se~lv~--re of cl~im~od fusion protein can be used to g~
_~bi~ m~ s which direct the CA~l~S~ioll of the fusion protein.
The term "~ ~" as used herein in ~f,.e~ce to coll~n~ ~ennte~ that the 30 j,~ rA mrlPclllps are present in the snbst~nt~ bs- ~-re of other biological ll~aclolllolecules, e.g., polymlrkPoti~l~Ps~ plolei~, and the }ike. The term ~.,lir.
as used herein preferably means at least 95% by weight, more preferably at least99.8% by weight, of the ;~ r~P,~l biological l~acroll.olecules present (but water, buffers, and other small mole ~IPS~ e.speciqlly moleclll~ having a molerlllsr weight of less than 1000 daltons can be present). The te~n "icolstPd" as used herein refers to a protein mnlpclll~ s~p~ rd not only from other pl'uLc~l15 that are present in the natural source of the protein, but also from other ~luteills, and preferably S refers to a protein found in the prcsel~c of (if anything) only a solvent, buffer, ion, or other col~oucll~ non~ally present in a solution of the same. The tenns "iCQ~9te-l~ and "pnrifi~" do not C~ o~ ccl~loLells present in their natural source.

B. Expression Of The Col~agen Eusion E~tP;nc Of The ~vention 1. ~o~ir~ Sequences In acc(sl-l~lce with the invention, polynnrl~oti~lc seq~l~nres which encode type IX, type TI, and type XI collagen ~lGt~,ins, or ru~ io~al e~uivalents thereof, may be used to g~lleldLe lcculllbil~t DNA mnleclll~s that direct the 15 cA~l~,ssion of fusion ~llUL~ ; of type IX collagen subunit with type II collagen and/or type XI collagen ~ubulli~, or a Çu~ io~al equivalent thereof, in a~r~ ialc host cells. Such collagen polyllucle~,lide se.~ os, as well as other polynucleotides which scl~ ely hyl~lidi~ to at least a part of such collagen polynucleotides or their col~ , may also be used in nucleic acid 20 h~ .. assays, Southern and Northern blot analyses, etc.
Due to the i~ .,nt ~leg~ r of the genetic code, other DNA sc~ .res which encode s~bs~ ly the same or a filnrtion~lly equivalent amino acid se~ rP, may be used in the ~l~.elice of the invention for the cloning and ion of these collagen IJr )t~s. Such DNA se~ -r~s include those which 25 are capable of hybridizing to the ~ u~iaLe hurnan collagen se~ ..re under Sl~ g~ ~t ctm-1ition~.
Altered DNA se~ rf,~ which may be used in acconla~lce with the invention include dcl.,Lons, ~ iti~m~ or sllb~ )..c of dirr~e,~t mlr~otil1e residues resllhin~ in a sequenre that enrodes the same or a ~l~;Liol~lly equivalent 30 gene product. The gene product itself may contain deletions, additions or sn~l;l..l;o.~ of amino acid residues within a collagen se4~ r~, which result in a silent change thus producing a filnrtion~lly equivalent collagen. Such amino acid l;ol~s may be made on the basis of simi~rity in polarity, charge, solubility, lly~hobicity, hydlo~ ilicity-, and/or the ~ pl~ nature of the l~si~lues involved. For example, ~ Li~ y Cl~h~5.,d amino acids include a~,p~Lic acid and ...ir acid; po;,i~ ly charged amino acids include lysine and al~ e; amino acids with u~hal~;ed polar head groups having si7nilar hy~op~ilicity values 5 include the followmg: leucine, isolen< inP, valine, glycine, alanine; ~gp~r~ginP, ~l--~i----;--~; serine, L~ ; phenyl~lAnin~ ylo~ine.
The DNA seq~n~es of the invention may be ~ ,.i"~ ed in order to alter the collagen coding seq~lert~e for a variety of ends in-,lu~iing but not l~ited to AltP7~ti-~ng which modify proce~ and tA~ 3n of the gene plu-luct. For 10 eA~Ic, ~ .llA~ , se.;l~,~uly signals may be sllh~ti~l~t~l for the native human sec.~t~l~r signal and/or ~llul~liGl~S may be illL,~luced using t~r~ es which arewell known in the art, e.g., site~il~d mntA~ siC~ to insert new l~sL.;eLioll sites, to alter glycosylation ~ , phosl~holyla~ioll, etc. Additionally, when eA~r~ssi~ in non-human cells, the polyl,ucleolides ~n~o~7i~ the collagens of thelS invention may be mor.7if7P~ in the silent ~osilion of any triplet amino acid codon so as to better CO~J~I11 to the codon ~l~f~ ,e,~ of the particular host Or~
In ~u~JLh~l embo~li...- -.~ of the invention, a collagen se~lu- ~.~e may be ligated to a heterologous s~ e to encode a fusion protein. For example, a fusion protein may be P.~in~,ed to contain a cleavage site located beL~.~n an c~3(IX) 20 collagen se~ r~ and the heterologous protein se(lu- ~ c, so that the ~3(IX) collagen may be cleaved away from the heterologous moiety.
In a particularly ~ f~l~d embo-limPnt chi...- . ir fusion l,lUt~,ilJs are constructed by lig~tit~ s~lu - ~rG ~ .o~ a type IX collagen s~b~lnit or d~ ali~
thereof, to se~P-~r~ en~oriing type II collagen and/or tyE~e XI coli~n s~bnnit 2S One of skill in the art will recognize that several te~lmiqnes are available which allow one to ligate all or any part of the coding sequenre of a type IX collagensubunit to all or any part of the coding se.lu l~,s for type II and type XI coll~g~
For r~Y~mp1e, one may ligate coding se.~v~ s to~th~-r at &~1~ ly chosen io~ endonn~le~ce sites. However, in order to ensure that the coding 30 se-l~ es of the chosen collagens are ligated in the correct trAnclstir)n~l frame, it may be ~ e~ to f, ~ ion sites by site ~riirlc .~ An even more al~,anL~geous m~th~ to join two or more polynnclPQtirle se~ e~-res makes use of the polylll~lase chain reaction and &~prOpl;à~ly ~çs;~-r~ as ' CA 02237900 1998-05-13 W O 97/17988 PCT~US96/18149 des~lil)f d in Section 3.17.1 of Ausubel et al., Current Protocols in Molecular Biology, Greene p-lhlichirl~ Associates and Wiley Interscience, N.Y., (1990).
Using this mfth~, one of skill in the art may join t~wo or more polym~rlPoti~le se~ res in any e onfi~lrAtif~n S In an ~It~rnqt~ embo~...... r-~l of ~e invention, the coding se~ re of ~he collagens of the invention could be :-y.~l.f~ ;d in whole or in part, using ch-mf~th~ful~ well known in the art. See, for example, C~ulL.,.~ et al., Nuc. AcidsRes. Symp. Ser. 7:215-233 ~1980); Crea and Horn, Nuc. Acids Res. 9(10):2331 (1980); lU~ ci and C~.~ , Tetra*edron Letters 21:719 (1980); and Chow 10 and Kempe, Nuc. Acids ~es. 9(12):2807-2817 (1981). ~Ih.~ ,ly, the protein itself could be pl~nluced using ch....if~ fllc to :jy..~l.f ~ the desired coll~n amino acid seql~enr~ at least in part. For eA~lc, peptides can be ~..~ ;,fA by solid phase oec-hni~ es, cleaved from the resin, and ~u~irled by ~ a,~ e high ~.r~ r~ uid chn.ll~dtoglalJl~. (e.g., see Creigllloll, Proteins Structures And MolecularPrinciples, W.F~ and Co., N.Y., pp. 50-60 ~1983). The cc,l~l~osiLiol, of the ~ ,lic peptides may be co--l;~ by amino acid analysis or se -~ril~g (e.g., the Edman A~.,A,1;r.~pl ~ dule; see Cr~ , Proteins, Structures and Mol~ ar Principles, W.H. ~ n~ and Co., N.Y., pp. 34-49 (1983).
In order to eA~l.,i~S the collagens of the hl~enliull, the nucleotide se~c-~re .-nro~lin~ the cQll~g~n, or a r~ ;o~l equivalent, is inserted into an a~r~liale eA~l~ssi ll vector, i.e., a vector which coll~ ; the .~ocesC-~ rl,~ lC for the lioll and tr~n~l~tinn of ~e ~elt,d coding seq~e 2. E~pression Systems M~th~l~ which are well known to those skilled in the art can be used to co~llu~;~ eAL,l~i,siol- vectors co~ .;n~ a collagen coding se~ .re for tlhe collagens of the invention and ~r~ial~ IlA~ ti~n~l/t~n~l~ti-~nAI control signals. These mrth~c include in vitro l~,CO llbi.~ DNA t~hni~ es, ~ylllh~,~ic tcrl~ es and in vivo l.,colllbi~alioll/genetic lccolllbil.~ See, for example, the te( Imi~ es ~es~ ~ iberl in M~ni~ti~ et al., Molecular Cloning A Laboratory Manu~l, Cold Spring Harbor Labolat~ly~ N.Y. (1989) and Ausubel et al., Current W O 97/17988 PCTrUS96/18149 Protocols in Molecular Biology, Greene Publishing ~.~soc;-~t~ s and Wiley T..t~ ci~..re., N.Y. (1989).
A variety of host-t;A~l~,,,sion vector ~.1l~ may be utilized to express a collagen coding seq~c- ~re. These include but are not limited to microol2,~ c 5 such as ba~ ;A ~ sro~ cd with l~,col~i~ll b~cteriophage DNA, plasmid DNA
or cosmid DNA eA~r~ssion vectors c~,..l t;";,~ a collagen coding seq~lenre; yeast LLa~,fol~ed with l~,c(sllll,h a 1~ yeast eA~ ion vectors co~t~inin~ a collagen coding se.~ - .re; insect cell ~,y~L~nls i~,.,Led with l~ombi ~ virus eA~ie~"ionvectors (e.g., baculovirus) cont~inin~ se~ ~re enrotlin~ the collagens of the 10 invention; plant cell ~y~.lls infected with recombinant virus eA~les~ioll vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, I~V) or L.~lllled with l~:colll~ plasmid eA~lessioll vectors (e.g., Ti plasmid) cont~inin~ a collagen coding se.l~e~-re; or animal cell ~y~nls. ~ ition~lly, thecollagens of the invention may be ~ essed in ~.",~sg~.~;r non-human ~nim~l.c 15 wL~ the desired collagen product may be recovered from the miLlc of the ~ ~ n~ ..ir animal. The eA~ ion c~ of these ~y~llls vary in their ~llell~
q-ncl Speeifiriti~s ~ on the host/vector system ~1tili7~A~ any of a number of suitable l~n~ J" and L,n.~ ;on el ...l .,1~, inr~ iin~ CO..~ ;V~ and inducible ~JlllOh~;7, may be used in the eA~ ,ion vector. For example, when 20 cloning in baclf- ;~1 sy~t~,lls, inrll)cihl~ ~l.J~Ot~,., such as pL of b~c=le~ hage l, plac, p~p, ptac ~php-lac hybrid promoter) and the like may be used; when cloningin insect cell sy~ ls, promoters such as the baculovirus polyl~e~.~n promoter may be used; when rlonin~ in plant cell :iy~s, plOlllUt~.~ derived from the gen~ of plant cells (e.g., heat shock ~lu~ ~..; the ~ O~ for the small subunit of 25 RUBISCO; the promoter for the chloro~ll a/b l)i~ g protein) or from plant viruses (e.g., the 35S RNA ~l.J~O~, of CaMV; the coat protein ~ lllOt'_. of TMV) may be used; when cloning in ,,,~.",,,~liqn cell ~y~,lls, ~lO~ Jt~ derived from the ~ u,l~c; of .. A-.. ~liqn cells (e.g., metallofhio~in ~ llloLer) or from - ,"-",",~liqn viruses (e.g., the ade.luvil-ls late ~r~lllo~., the vaccinia virus 7.5 K
30 ~lU~Ot~,~) may be used; when gpl~f ~~ , cell lines that contain multiple copies of a collagen DNA, SV40-, BPV- and EBV-based vectors may be used with an ap~ a~ s~rcl~le marker.

W O 97/17988 PCT~US96/18149 In ba~ y~t~s a mlmh~r of c~ ,i,sion vectors may be advantageously s~l~eLf,d ~e~ upon the use i~r~lriPd for the collagen e~c~ ,d. For ç~ ple, when large ~ s of the coll~n~ of the invention are to be produced for the ge.l~lalion of ~ntibo~ s~ vectors which direct the e"~-eision of high levels of 5 fusion protein ~r~lu;~ that are readily purified may be desirable. Such vectors include but are not limited to the E. coli ~ ,ssion vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the collagen coding se~ Pn~e may be ligated into the vector in frame with the lac Z coding region so that a hybrid AS-lac Z
protein is produced; pIN vectors ~nouye ~c Inouye, Nucleic Acids Res. 13:3101-10 3109 (198S); Van Heeke & Schnst~r, J. Biol. Chem. 264:5503-5509 (1989)); and the like. pGEX vectors may also be used to express foreign poly~ ides as fusion pl~lcills with ~l-)t~thioll~ S-LI~Ç.,.ase (GST). In general, such fusion pru~i~s are soluble and can easily be purified from Iysed cells by adsorption to -ag~,.ose beads followed by elution in the p~s~,.~e of free glllt~thione.
15 The pGEX vectors are ~lPsign-~l to include thrombin or factor Xa ~lol~,as~, cleavage sites so ~at the cloncd polypeptide of interest can be l~leased from the GST
moiety.
A pl~,f~ cd ~ n,~.~ioll system is a ye~t e~lci.siull system. In yeast, a e~ of vectors cr~ ;~.;n~ cn~ ;vt; or inducible plUlllUt~:!i may be used. For 20 a review see, Current Protocols in Molecular Biology, Vol. 2, Ed. Ausubel et al., Greene Publish. Assoc. & Wiley T..t~ Cif '~''P, Ch. 13 (1988); Grant et al.l Expression and Secretion Vectors for Yeast, in Methods fn Er~ymology, Ed. Wu &
Gru~ , Acad. Press, N.Y. 153:516-544 (1987); Glover, DNA Cloning, Vol. ll, IRL Press, Wash., D.C., Ch. 3 (1986); and Bitter, Heterologous Gene Expression 25 in Yeast, Methods in Enymology, Eds. Berger & K;m ~1, Acad. Press, N.Y.
152:673-684 (1987); and The Molecular Biology of the Ye~st Sacc~ "yc~s, Eds.
t~thP~rn et al., Cold Spring Harbor Press, Vols. I and II (1982).
A particularly pl~,f~ .,d system useful for cloning and e~y~ ;ull of the co11~en ~uL~ of the h~ lLiull uses host cells from the yeast Pichiu. Species of30 non-Saccharomyces yeast such as Pichia pastoris appear to have special advantages in producing high yields of l~COl~ protein in scaled up ~locedu~,s.
1ition~11y, a Pichia e~ ssioll kit is available from Invitrogen Col~o.aLion (SanDiego, CA).

W O 97/17988 11 PCT~US96n8149 There are a ..~ . of ...~ o~ivc; genes in methylullu~hic yeasts such as Pzchia pastoris, the eA~ ioll of each being controlled by ~ u)l .~,~o~iv~ regulatory regions (also lefc.,~,d to as ~olln)~). Any of such 3sive promoters are ~uilhble for use in the ~l~cli_e of the present S invention. FY~mrle~ of specific regulatory regions include the ~ llOI. ~ for the hy alcohol oxidase gene from Pichia pastons AOXl, the promoter for the sec~l,da-y alcohol oxidase gene from P. pastoris AX02, the ~ ot~,l for the dihyLo~y-~e~ol-P :~yll~se gene from P. pastons (DAS), the ~l~lllGt~l for the P40gene from P. pastoris, the pl~,llu~ for the c~t~ e gene from P. pastons, and the10 l;ke.
Typical e,.~l.,;,sion in Pichia pastoris is obtained by the promoter from the tightly regulated AOX1 gene. See Ellis et al., Mol. Cell. Biol. 5:1111 (1985) and U.S. Patent No. 4,855,231. This promoter can be in~luced to produce high levels of lecoll.~ protein after r'l'litinll of ,.,~ ml to the culture. By ~..hs~ nt 15 msnip~ tiQn~ of the same cells, ~ essioll of genes for the collagens of the invention desclil)ed herein is achieved under co~ "-.~ where the r~,CoL~ t protein is ~le~ ely llydr~ylated by prolyl 4-hydroxylase and, l~ fol~ can fold into a stable helix that is required for the normal biological function of the ~,oteins in Ç~lLuillg fibrils.
Another particularly pl~E~ d yeast c~ ,S;,iOll system makes use of the methylotrophic yeast Hansenula po~y"~o,~lla. Growth on ..~ l results in the in~ tiQn of key e~yL les of the ~--~ metabolism, namely MOX (.... I~ nl oxidase), DAS (dihyd~ cya~Lol~e synthase) and FMHD (folLLL~Lc dchydlogenase).
These e~yul,s can COLL~I1LULe UP to 30~0% of the total cell protein. I'he genes 25 e~u~o~ g MOX, DAS, and FMDH pro~lurti~n are controlled by very strong promoters which are in.l...~ed by growth on ...~ u)l and l~ ssed by growth on glucose. Any or all three of these promoters may be used to obtain high level e~ ,ssion of heterologous genes in H. po~morpha. The gene ~ nf~o~ a collagen - of the invention is cloned into an e~r_~ion vector under the control of an 30 i-klucil~k H. pol~".o,~l~ promoter. If secretion of the product is desired, apolymll~leol;~les en~o~iin~ a signal sequence for S~l~,liOl~ in yeast, such as the S.
cerevisiae prepro-mating factor ~1, is fused in frame with the coding seqllen~e for the collagen of the hL~_llLioll. The cA~Lcssion vector pl~,f~,làbly conL~iLls an W O 97/17988 PCT~US96/18149 ~u~Lo~ hic marker gene, such as URA3 or LEU2, which may be used to complement the ~ ry of an aux~llo~llic host.
The tA~ ion vector is then used to ~l~rv",l H. po~morpha host cells using tec~ eC known to those of skill in the art. An illt~ ~g and useful S feature of H. poly"~,~ha ~,~Ço~ dLion is the spo~ u~c integration of up to 100 copies of the eA~ iL,n vector into the y~ ~e~ In most cases, the integrated DNA folms m7lltimPrfi eAhibiLi~ a head-to- tail ~l~ e~l~P~I The ;l~t~ IPCI
foreign DNA has been shown to be ll,ilolically stable in several l~CC~
strains, even under non-selective col~di~iol~s. This phP..~ of high copy 10 ;..I~;.,ll;on fur~er adds to the high productivity potential of the system.
In cases where plant eA~l~s;,ioll vectors are used, the eA~ ion of S~ ~.r~s enro~in~ the collagens of the h,~ tioll may be driven by any of a llulllbcl of promoters. For ~;A~Ie, viral promoters such as the 35S RNA and 19S
RNA ~,~..~ut~-~ of CaMV (Brisson et al., Nature 310:511-514 (1984), or the coat 15 protein promoter of TMV (T:~k,..~A~ . et al., EMBO J. 6:307-311 (1987)) may be used; ~ ly, plant promoters such as the small subunit of RUBISCO
(Coruzzi et al., EMBO J. 3:1671-1680 (1984); Broglie et al., Science 224:838-843 (1984); or heat shoclc prs~.not~~~, e.g., s~l,eal~ hspl7.5-E or hspl7.3-B (Gurley et al., Mol. Cell. Biol. 6:559-565 (1986) may be used. These 20 C(J~l1U~;~ can be introduced into plant cells using Ti pl~cmi-lc, ~i pl~mi~s~ plant VilUS vectors, direct DNA L~r~ ion, lllicro;~-jec~ n, cl~L,(;~uldtioll, etc. Forof such ~ e~ see, for ~ , We:~açl- & Wc~ cl-, Methodsfor Plan~ Molecular Biology, Academic Press, NY, .Sect~ vm, pp. 421463 (1988);
and ~ ;- -..ol- & Corey, Plant Molecular Biology, 2d Ed., Blackie, T onfion, Ch.2~ 7-9 (1988).
~ ;vc e~l~i,sioll system which could be used to express the coll~enc of the invention is an insect system. In one such system, Autographa califc"nica nuclear polyhidrusis vims (AcNPV) is used as a vector to express foreign genes. The vims grows in Spodoptera frugiperda cells. Coding se~ e~ e 30 for the cnllag~on.~ of the invention may be cloned into non- e~e-~ regions (for example the polyhe llvll gene) of ~e virus and placed under control of an AcNPV
promoter (for e ,.a~ )lc, the polyhedron l,lo~ t. .). .C~ ce~j~rul i~ of a collagen coding se~ e will result in h~d~;Liv~lioll of the polyhed,vn gene and pro~ ction of non-occ~lnrl~rt l.,combi~ll virus (i.e., virus lacking the pl'ùleiuaceous coat coded for by the polyL~dlun gene). These recombinant viruses are then used fo infect Spodoptera frugiperda cells in which fhe inserted gene is e~,esscd.
(e.g., see Smith et al., J. Virol. 46:584 (1983); Smith, U.S. Patent No.
5 4,215,û51).
In ".~.".~.~liqn host cells, a llulllb~ of viral based cA~lc~sion ~yb~ls may be ntili7~A In cases where an ade-luvilus is used as an C~ ,S~iiUIl vector, coding seq~ re for fhe collagens of fhe in~ lioll may be ligated to an adenovirus Ll~clil~ion/~r~qnClqfion control comrhPY, e.g., the late promoter and Lli~allilc10 leader se.~ ~.r~. This cl.;...- ~ ie gene may then be inserted in the adenovirus g~-~n.~r by in vitro or in vivo lecolllbinalioll. Insertion in a non~ e~ l region of fhe viral g~-~....t~ (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of C~)l-,~Sillg collagen in infçcteft hosts. (e.g., See Logan &
Shenk, Proc. Natl. Acad. Sci. fUSA) 81:3655-3659 (1984)). ~ ivcly, the 15 vacc~ia 7.5 K promoter may be used. (See, e.g., l~Aqrl~t tt et al., Proc. Natl.
Acad. Sci. (USA) 79:7415- 7419 (1982); Mq-r~P-tt et al., J. Virol. 49:857-864 (1984); Panicali et al., Proc. Natl. Acad. Sci. 79:4927-4931 (1982).
~ perifir j";~;a~;o~, signals may also be r~luil~,d for crrirle-~l trqnclqtion of ~ ;c.~d collagen coding se.~e-~-r~s. These signals include the ATG i~ ;on 20 codon and qftjqcçnt seq~lenrPc. In cases where the entire collagen gene, inr~ iin~
its own inhiqtion codon and atTjqrPnt seqnPnres, is illscl~d into the appruL,lialc e~.ci,sion vector, no -q-d~titi~nql tr~q~n~t~q-tir)nal control signals may be nPefl~P(l However, in cases where only a portion of a collagen coding se~ll,c~.re is inserted, exoge.~ous tr~qnelqtional control signals, inrlllrting the ATG i~ . codon, must 25 be provided. ~,.llI,.,.~o~, the initiqtion codon must be in phase with the reading frame of the collagen coding s~ e to ensure ~ n of the entire insert.
These exogenous tran~lqtit nql control signals and ;..;~ n codons can be of a variety of origins, both natural and ~yl~ll~clic. The erl;~ .ry of e~ s~ion may be - e-~ Gr1 by the inrlllcion of a~ol)lidL~ r~ o~ t~ CtP~
30 ~ c~ ;l.Lion l~-- ",;~ , etc. (see Bittner et al., Methods in En~mol. 153:516-544 (1987)).
One ~ .cd ei~n_~ion system for the ~.,co..-bil~L production of the collq-gen~ of the invention is in ~ S~ ;r non-hDan qnimqt~, wl~r~l ~e desired ' CA 02237900 1998-05-13 W O 97/17988 PCT~US96/18149 collagen may be lGco~ ,d from the milk of the ~ ge~ animal. Such a system is constructed by operably linldng the DNA se~lur~e ~ o~lig the coll~ns of the invention to a lJL.~ ot~ and other lc~uil~d or optional regulatory SP~ f s capable of err~C~ e~ ,ssioll in ~.. z~.. ~.y glands. I,ikewise, l~luil~d or 5 optional post-tr~nQl~tior~1 e~y.J.~s may be produced ~im~ u~ly in the target cells, employing suitable c~ sion systems, as disclosed in, in~er alia, U.S.
Applir~tion~ Serial No. 08/037,728, op~.~.ble in the targeted miL~c protein producing l~A-~ gland cells.
For e~ c,ssion in miLk, the plUl~Ut~,. of choice would p.~ ~ly be from 10 one of the ~b~ln~l~nt millc- specific ~r~)lcills, such as alpha Sl-casein, or b-lactoglobulin. For example, S' and 3' regulatory seq~ es of alpha S1-casein have been s~r~eQQfillly used for the e~lei~ion of the human laclofc.-il. cDNA, and similarly, the b-lactoglobin ~rvlllot~ has effect.otl the el~-cSSiOn of human ~ y~sin gene fr~nrnt~ in sheep millc pro~ cells. Wright et al., 15 Biotechnology 9:830-833 (1991). In t~ g~..ir goats, the whey acid plUIllOt~. has been used for the cA~ression of human tissue pl~mim-gen acli~alol, resnlfin~ in the sec.elio~ of human tissue pl~minngen a~ atcl in the miLlc of the l~lallSg~.~iCS.
Ebert et al., Biotechnology 9:835-838 (1991). Using such tA~l~.,Siol- ~y~ s, anim~l~ are ob~i~d which secrete the collagens of llhe Lu~nLioll into m~Lk. Using 20 ~iocedu,es well-known by those of the cldil~y skill in the art, the gene encoding the desired collagen chain can simply be ligated to suitable control se.lu~,.lces which ru,~ Oll in the .. ~.. ~.~ cells of the chosen animal .speci~?S EApression ~ ellls for the genes e-,ro~l;..p the ~~uh~,d post-l~d.~ Qnal ~yllles are co~L
analogously.
Preferably, the collagens of the i~ liun are eAy~ ed as sec~ d ~ s.
When the P..~;l-.?~ xl cells used for eA~.es~iu,. of the ~rott,~ are non- human host cells, it is often ~lva.,~geous to replace the human s~l~,toly signal peptide of Ihe collagen prote?,in with an ~ vt; secl tUl,y SigIldl peptide which is more errir~ lly r~coy,,.i~ed by the host cell's sccl~tloly ~eliug ...~. k;.._l~. Ihe 30 a~lùplidle sec~ signal SG~ G iS particularly i ll~ol~l in obtai~ g optimal fungal eA~ sio~ of i~ n genes. For eY~m~l~, in methylc,L-upl~ic yeasts, a DNA s~ u~--.r~ enrot~ the in-reading frame S. cerevisiae a- mating factor pre-pro se~ may be inserted at tne amino t~ l end of tne coding s~l~e~.re. The _ W O 97/17988 PCT~US96/18149 aMF pre-pro se~l..< nre is a leader se~ c~ co"~ r~l in the aMF p~;ul~or m~ cnle, and inrh-ries the lys-arg e.lcodi~ ~y~ re which is .-~ce~ ~. y for proteolytic proc~sC-in~ and secretion (see, e.g., Brake et al., Proc. Nat'l. Acad.
Sci. USA, 81:4642 (1984~).
!; In ~Mition~ a host cell strain may be chosen which mo~ t~c the e~lc~SiOn of the inserted sequences, or m~ifi~s and ~lOCe5i~ieS the gene product in the s~e~irlc fashion desired. Such mo~ifir~tions (e.g., glycosylation) and ~rvce~ p (e.g., cleavage) of protein products may be h~l~o~ for the rul~;liOll of the protein. Dirr~ host cells have f~ cl~.islic and ~lJec-;l;r ~ rl~n;~...c for 10 the post- t~nc1~tion~ CÇ~ and mo~1ifir~tion of ~lote~. A~r~ Lc cells lines or host ~ ,~s can be chosen to ensure the correct m~jfir~tion and procescin~ of the foreign protein e~l~i~sed. To this end, eu~yolic host cells which possess the cellular ~ rk;~ for proper prvc~;n~ of the plil~al~
tl ~ lycos~latiorl, and phos~ho,ylation of the gene product rnay be used.
Such .. ~.. Ali~n host cells include but are not li_ited to CHO, VE~O, BHK, HeLa, COS, MDCK, 293, WI38, etc. Additionally, host cells may be ~ ,d to express various c ~yl~les to ensure ~e proper plOC~ g of the collagen mo1~PC~ Ps. For ~ , the gene for prolyl~-hy~llv~ylase may be co~A~l~,ssed with the collagen gene in the host cell.
For long-terrn, high-yield production of lc;cullll~hla~L plOL~,~, stable expression is ~l~f~ d. For e~lplc, cell lines which stably express the collagensof the invention may be e.~ .cd. Rather than using e~.~ion vectors which contain viral origins of replir~ti- n, host cells can be Ll~rulllled with collagen el~ro l;n~ DNA controlled by app~ ialG e~lession control elPmPnt~ (e.g., 25 promoter, enhA~ , se.~ s, I.i.n~. . ;plion ~- -,llj,~A'Ql~, polyadeylaLiull sites, etc.), and a sc~ lc marker. Following the introd~ ction of foreign DNA, c ng;..P~ d cells may be allowed to grow for 1-2 days in an c.l.ichcd media, andthen are ~wi~ched to a scle~LivG media. The se1e,ct~hle marker in the l~,COll~
plasmid confers 1~ nre to the sekPction and allows cells to stably hlt.,glalG the 30 plasmid into their chromosomes and grow to form foci which in tum can be cloned and ~xl~ e~l into cell lines. This method may advantageously be used to engil~loeL
cell lines which express a desired collagen.

W O 97/11988 16 PCT~US96/18149 A mlmh~r of scle~;lio~ ~y~t~,~s may be used, il)rl~ but not limited to t~e herpes simplex virus thymidine kinase (Wigler et al., CeU 11:223 (1977)), hypo~ .r-gua~ hn~h~ o~ylLId~re~dse (Szybalska & Szybalski, Proc.
Natl. Acad. Sci. USA 48:2026 (1962)), and ~ inP phosl~ho~ o~l~ ~r~l~e 5 (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt-cells, lcsp~ ly. Also, ~ntim~-~holite ~ re can be used as the basis of sel~ilion for dhfr, which confers l~ s~ re to lllc~ xale (Wigler et al., Natl.
Acad. Sci. USA 77:3567 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA
78:1527 (1981)); gpt, which confers ~ n~e to mycophenolic acid (Mllllig~n &
10 Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers ~ e to the aminogly~;~side G418 (Colberre- Garapin et al., J. Mol. Biol. 150:1 (1981)); and hygro, which confers lc~ e to lly~ y~;in (Santerre et al., Gene 30: 147 (1984)). Recently, ~ hion~l selPct~hle genes have been described, namelytrpB, which allows cells to utilize indole in place of Llypk,~ ; hisD, which 15 allows cells to utilize histinol in place of hictidinP (Harhnan & l!~ lli~n, Proc.
Natl. Acad. Sci. USA 85:8047 (1988)); and ODC (o~ decarboxylase) which confers ~-~ ;..-re to the ~ P decarboxylase i,lh~ or, 2-(difluc,l~Jnl~ D~o...;~l.;.,P, DFMO (McConlogue L., In: Current Communications in Molecular Biology, Cold Spring Harbor Labo, 20 (1987).

C. Identific~tion of Transfectants or Transformants that Express the pn ~ ;nc of the Invention and Purification of the Expressed l?~.)t~s The host cells w~ich contain the coding se(~ re and which express the biologically active gene product rnay be ~ ;r~d by at least four general approaches; (a) DNA-DNA or DNA-RNA h~l,li.li~alion; (b) the yl~ ce or ~hse~-~e of ''lllcu~ll' gene fuDctions; (c3 ~e~:e~ the level of Ll~sc~i~Lion as ,.le&,u~,d by the eA~es~ion of collagen rnRNA L~ in the host cell; and (d) ~letecticn of the gene product as Illeasuled by ;~ y or by its biological ~ctivity.
In the ~lrst approach, the ~,~,s~lce of the collagen coding s~q~n~e inserted in the eAy~ vector can be ~et.ectP-d by DNA-DNA or DNA-RNA

hyb~ ;oll using probes colll~li..ing ..~eleo~ se~vFn~es that are homologous to the collagen coding se~llenre~ ~~DpF~Iiv~ly, or portions or de.iv~ ,s thereof.
In the second ~y~loach, the l~ollll)inall~ e"~ Dsion vector/host system can be ;t1F~ ;r;Pd and SC1~CI~A based upon the p~e..ellce or ~bsen~e of certain "malL~l"
5 gene rul~lionD (e.g., thymidine kinase activity"~ to alllil,iolics, le~ re to lllCIllU~ ale, L,~.îullllalion ~h~loly~e, occlllsion body formation in baculovirus, etc.). For example, if the collagen coding s~F~ is inD~,.~d within a marker gene se~ e of the vector, lCCOlll~ cells co~ collagen coding se~l~,f.-~e can be if~ lir~d by the ~bsen~-e of the marker gene r~
10 ~ ,ly, a marker gene can be placed in tandem with the collagen seqnenre under the control of the same or dirr~.e.l~ ~rol~o~, ~sed to control the eA~l~,i,sion of the collagen coding sequence. EAyl~Dsion of the marker in 1~DL~UIIDe to in~llction or selection i".l;r~l.s ~y~eSSiO~ of the collagen coding sequPnre.
In the third ay~luach~ n~ ;plional activity of the collagen coding region lS can be ~c~ ,s,e-~ by hy'~. ;.1;,;.~ ioll assays. For PY~mple, RNA can be isolated and analyzed by Northern blot using a probe homologous to the collagen coding sequenr~ or particular portions thereof. ~ "li~,ly, total nucleic acids of the host cell may be eY~ P~1 and assayed for h~.;.1;,~ion to such probes.
In the fourth ayyruacll, the ~yl~s7sion of a collagen protein product can be S7~ nlogically, for example by Western blots, ;.. ~.oa~c~y~, such as radio;----------n-p~.,iy;~Lion, cllLylllc-linked ;.. --.~n.q~s~ys and the like.
The e,~ylc~sed collagen of the i-l~e,llion, which is preferably secl~lcd into the culture ...P.I;~ , is yulirlcd to homoge~iLy, e.g., by cl~ulllà~u~,layhy. In one ~,.llbo~ the recombinant collagen protein is pnrifir~l by size eYrlllcion 2S cl~lllalc~layhy. However, other y~ I ;oll Ir~ C known in the art can also be used, inrln~1in~ ion ~Y~ A.~, ch,ulllatogla~hy, and reverse-phase clnulllalugraphy.
. Uses of The Cr~ 'Iq~n~ of the Inven~ion snd Engineered Cell Lines 1. A~ G~ "d~.~lion and ~e~
Various plucedulcs known in the art may be used for the pro~cti- n of antibodies to cyiluyeS of the 1~ Colll~ allLly produced collagens.

Such antibodies include but are not liIILited to polyclonal, mon~clon~l,cl~ ic~
single chain, Fab r.~ and fr~m~ntc produced by a Fab ~ ",s~ion library.
For the pro~h~ctir)n of antibodies, various host ~nim~l~ may be ;.-.. ;,- ~1 by injection with a collagen protein in~ in~ but not limited to rabbits, mice, rats, 5 etc. Var.ious aljuv~ may be used to i~ ease the immllnological ~ ol~se, ;-¢ on the host speci~S, in~l~lrling but not limited to Freund's (colll~lctc andincomplete), minPr~l gels such as ~II.. i.. ~ hydroxide, surface active s~ ec such as lysole~ilh;.., pluronic polyols, poly~iolls, peptides, oil ~m~ ionC, keyhole limpet ~ ~-ocy~ , diniLIo~ ol, and ~ot~ y useful human adjuv~ such as 10 BCG (bacilli Calm~otte-Guerin) and Corynebactenum parvum.
I~onoclon~l antibodies to a co11agen may be pl~ued by using any l~h~ e which provides for the ~ of antibody molecules by contin~ollc cell lines in culture. These include but are not limited to the hybridoma f~c~lni~ue originally ~ ;l~cl by K ~hl~r and ~ilc~in, (Nature, 256:495-497 (1975)), the 15 human B-cell hybridoma t~- hniql~e (Kosbor et al., Immunology Today, 4:72 (1983)); Cote et al., Proc. Natl. Acad. Sci., 80:2û26-2030 (1983) and the EBV-hybri~ m~ r~ (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985). In ~d~lhion, t~-hniql~es developed for thepl~lu~;lion of "chi~elic antibodies" (l\lorn~on et al., Proc. Natl. Acad. Sci.
20 81:6851-6855 (1984); Nc.lbc,ge~ etal., ~alure, 312:604-608 (1984); Takeda et al., Nature 314:452454 (1985)) by splicing the genes from a mouse antibody m~lecllle of ~ u~lia~ antigen ~ecirlcily together with genes from a human antibody molecule of a~rl,~l.a~ biological activity can be used. ~lt-~ ;v-ely, t~hniqne~ ;hed for the pro~lllrtion of single chain antibodies (U.S. Patent No.
25 4,946,778) can be adapted to ~l~luce a collagen- sl~e;l ;r single chain antibodies.
Antibody r.,.~ which contain dcleliolls of specifir binding sites may be ~f - ~ Cl by Imown ~I h~ P-s~ For eY~nlrle, such fr~mf nf~ include but are not limited to: the F(ab')2 fr~ nt~ which can be ~I nluccd by pepsin ~ estion of thean$ibody m~ cllle and the Fab 1i~ nt~ which can be g~l-- ",l~-ll by i~ ci.~ the 30 ~ ltfi t~ bridges of the F(ab')2 fr~ ont~ I;V~1Y, Fab e~yl sion lil)-~ies may be co~L~ue~cd (~use et al., Science 246: 1275-1281 (1989)) to allow rapid and easy i~l~ontifir~tion of mon~lclon~l Fab rla~ with the desired ~l eeili~ y for the coll~çrl of interest.

W O 97/17988 PCT~US96/18149 2. Th~ lic Uses Of The C'~'la~n Proteins Of The Invention Another aspect of the invention is to provide m~thor1.c of ll~,àlhlg ;.. ..P system~ p~ A. ~;.C~CeS using the collagen protel. s of the 5 invention. The terms ~ A~ I or "ll~alhlg" as used herein with r.,f~ e to a disease refer both to plo~hylaxis and to the AmP1ior~Ation of ~.y~ tO---C already present in an illdividual. It will be apy~ciated by the person of ordinary skill in the art that a 1l~ Al...F--l need not be co~ll,~ly effective in ~ ,nli~ the onset of a disease or in ~ -c;.~ the sy~ ""~c a~soe;~'~l with the disease. Any reduction 10 of the s~ y of ~Y~ , delay in the onset of ..~ s, or delay in the progression of i..,~.,.ily of sy~ llls is desirable to a patient. re...o~s at risk of developing a given ;.. ~ system-.~-fA;AIr~l disease may be treated ~r~llyl~ irAlly based on any of a variety of factors s~ es~ the possible onset of an ;..-...~ system- I~PA;~t~A A;C~ACe, e.g., family history, genetic I~A-I~
15 early ~1ll~l~., and the like.
T....... ----~ system--,~ iA~ 1;CeAC~7S that may be treated by the subject m~th~lc include, but are not limited to, e.g., ~ l -- ~ -- - - ~loid a ~l~iLis, osteo~~ is, n,a_Live ar~itis, auL~ h~,rin~ Aice~ce, cartilage ;.. ll~.. ;.l;n~- due to b~et -;~l or viral infFctionc ~e.g. Lyme's disease), y_~t~c;~ disease, bursitis,20 corneal ~ e~r s, and ankylosing s~oll lylilis ~fusion of the spine). The subject mPthr~s of the h~ ll co---l" ;.~e the step of ~ g an err~;live ~mollnt of a com~o~.ilioll of the invention, e.g., collagens, collagen dc~i~/aliv~s. P~,f~ d colll~osiLions for use in tl.,ali~g specific ;....~ .-f system~ (1 rliee~ce~ are fusion ~r~t~ s of type IX coll~gen, ~ felably cl~illl.,.~. of a type IX collagen25 subunit with type II collagen and/or type XI collagen, and de~ivalivt;s and ~b~
thereof, as well as as cl~s~ 1 in the p.~c~ g sectir)nc. In a ~1.,~ll~1 ~,.llbo~ --.l of the subject methods, the coll,~o~.;';nne ~ d to the subject c~...l.~ ;~e variably glycosylated coll~n~. The compo~hion~ d in the subject ~-.- ~ ; are ~ ~ it~led so that the active Colll~)Ul~ lS, i.e., collagens 30 andlor collagen del;vaLi~_s, contact the ly~phoid tissue of the gut, e.g., Peyer's patches or other similar sites, so that i~ F tol~ e is ;..~ cerl Such n may be err~t~ ~1 by among many possible m~th~le, through the use of form~ ti~n~ COlll~ i~3 the sul)je~led compositions that are ~ 1 for oral W O 97/17988 PCT~US96tl8149 a~ nl;r,.~ i.e., the active components are not de~ o~,d or i~ ;Liv~t~d in the mouth, ~/o"~ tl, or other portions of the dige,~ive system prior to eo~ the ~pr~lid~ gut Iy~lloid tissue. The l~ n~l methorl~ of the invention may also c~ c; the steps of ~-l.";.,;.~t. .;,~g ~cl-litinn~l ph~-.--~.~c;--l;. ~1 cc,~uu"ds for the 5 L,e~ .t of i.~ system-mPAi~te(~ , such as anti-infl~ ol~ agents and the like.
The dosage at which the subject compositions are ~ d may vary within a wide range and will depend on various factors such as for example the s~ y of the infl5.. ~.~l;.. , the age of the patient, etc., and may have to be 10 ill~lividually a~jv~l~A. As a y~5sil)le range for the amount of collagen(s) and/or collagen(s) de~ àtives which may be ~ I~.. ;n~ d per day may be in ~e range of from about 0.001 mg to about 200 mg. ~,fe.~ly, the ~mo~mt of collagen and/or coll~en d~ ,s ~l...i"i~t...~d iS lûW, thereby favoring ~e i"~ of ;~
tole.~ce by Ou~?l~O~ion rather than clonal anergy. The ph~ r~
co~ oS;l;on~ contAinin~ the collagen(s) and/or collagen(s) de~iva~ ,s may suitably be forrmllst~ so that they provide doses within these ranges either as single dosage units or as multiple dosage units.
The optirnal dosage of tOlC.al~Ce in~ pi Cul~OX;l;O~ for use in the mPthnA~ of the invention will vary in accold~ce with a IlU~- of factors. The 20 terms "dosage" and "dose" as used herein, unless inllirAted olLe.-lvise, may refer not only to a single A~ lion of a co...~o~ but may be used to refer to the total Am~unt of a given ph~rm~reUtir~icuLl~ooilion ~ e~ over a s~lçct~A.
period of time and involving multiple individual A.l~ ;onO Factors arre.;lillg the optimal dosage irlclude the choice of collagen molecule or lllGI~ s (and/or 25 collagen de~i~aLi~S) A.l.~i"i~ d to ~he patient, the ~c;~r mllcoc~ ~Ll~L~
molecllles s~clF~it~A the age of the patient, the ~ iLy of the disease, other A~S that may be present in the patient, inert cc,~ oll~,.L~ in the folmnlAtion, adju~,all~, and the like. There may be co..!~;A~ ~~hle variation in the range ofdosages that are err~clivc in ll.,al~g a given ;~",,,~"r disorder. Dirr.,,e~ osA~s 30 of the same ph~ ;r~l coll~osiLion may produce the desired tolc,allcc effect by dirr,,~ c!~A~;x~ lthmlgh the operation of the h~ llioll is not lle~el~e~
upon a particular theory of operation~ the person of c~di~y skill in tlle art will better understand the invention and provide additional e~ lx by &~ cC ;~

W O 97/17988 21 PCT~US96/18149 that there are bcl;~d to be two l~lh~y Illfc~ by which oral tol~ .lrc is ;l Oral tGl l~ce may be l"Prl;.t~A by active cellular Ou~l,sOion in which re~ tory T cells that bU~l.,Ss the a~;livaLion and proli~lalios~ of lymphocytes s~irlc for tolerized ~nti~ll Another mPrh_ ;A I of oral tOl~a~cc in~ ctinn is 5 clonal anergy in which T lymphocytes having a suitable le~ul are rendered LL~o~U~oi~ T~nPr~lly active oU~lCosivll tolc"~ e is ravorcd by "low" doses of a tol~ ;,; .g antigen and clonal anergy is favored by c~ p ~ ;vely "high" doses of the same tol~ antigen A review of the principles and techni~ s for oral t~,le~ .~ -e inAllrti~n can be found in Weiner et al., Annual Review of Irnmunology, 10 pp 809-835, Annual Reviews (1994) The subject compositions may be forml~l~t~ as l,h_ ",~ ;ri~l co~ oOiLions so as to be ~ pt~d for cerhin types of ~rl ~ t"io to II ~cos~l -O--- ri ~S~ e g, oral, topical, and inh~ tion The ~l~f,ll~d form of form~ tion for oral ~A. ~;. ;AI alion in a form where the collagen and/or collagen de.;~,a~i~. s in the 15 culll~osilion come into contact with i ~ l lylll~hoid tissue, e g, Peyer's ~ s C~GOi~iG~ of the hl~,~,.lLioll may be ~ At~- ~d topically, orally, intran~ ly, by injeetiorl or by inh~l~ti~n in the form of a ~h_....~ ;r~l co~ osi~ions co ~ g a collagen(s) and/or co11~gen(s) d~i~a~ ,s in the forsn of the f~ri~ini~1 cwllyound or optionally in the form of a ph_....~ A11y acce~Lable20 salt thereof, in ~Csociqtion wit_ a ph_i...Arelltir~lly accepLable carrier which may be a solid, semi-solid or liquid diluent or an in~e~ le cArsnle, and such ~lCp-~Cl!~ .1;AC a fur~er aspect of the invention. The collagen(s~ and/or collagen(s)~ /aLi~,s and mllcoc~ ldi~g collagen conj~g~qt~C may also be used with carrier m~trri~l. As examples of pl-~ A~ ;r~l pl~aLions may be mentioned tablets, 25 drops such as nasal drops, ~ ;u~c for topical application such as o;l~
jellies, crea_s and O..A~ nc, ae,osûls for inh~1~tion, nasal spray, li~osolll~s,etc. Usually the coll~en(s) and/or collagen(s) d~,.iva~ives will c~ .. ;se belweell 0.05 and 99%, or ~ ,.,n 0.1 and 99% by weight of the ~lep~.~lL~-, for e~ lc be~.l 0.5 and 20% for p~ ;onC ;~ for injectit-n and ~h.~,~n 0.1 and 30 50% for ~ p~ A int~n-lecl for oral a~
To produce ph~l ..l~rel-tir~l pl~ ns in this form of dosage units for oral applir~tion co..~ a co~ ou.ld of the invention the active hlgl.,di~ maybe mixed with a solid, pulverulent carrier, for example lactose, saccl~o.,e, ~ CA 02237900 1998-05-13 sorbitol, ~ ul, a starch such as potato starch, corn starch, amylopectin, o~v~le~ or citrus pulp ~.w~ , a cç~ tose dc~i~valivc or gel~tin~ and also may include lul)lic~ls such as mA~F~ or c~ inm ste~r~t~o or a Carbowax-- or other polyethylene glycol waxes and are co~ ,sed to form tablets or cores for S dragees. If ~l.t.~, es are ~ uil~d, the cores may be coated, for example, withcol-~c~ ~ sugar solutions which may contain gum arabic, talc and/or ~ ";,.", dioxide, or al~ ly with a film fo,ming agent dissolved in e~ily volatile organic solvents or ~ lu~S of organic solvents. D~.,.,lurrs can be added to these CQ-I;.~, for c~lc, to ~ l;"~ 1 dirr~,lell~ cc...~ of active .,~W~ e.
10 For the pf~s~ n of soft gel~tinP c~rs~lles cone;.~ o of gel~tin~ and, for "lJle, glycerol as a pl~ctiri7Pr, or similar closed caps~ e, the active ~ulJ~,~ce may be ~AmiYP~ with a C~lJGWaX- or a suitable oil as e.g. sesame oil, olive oil,or arachis oil. Hard gelatine capsules may contain gramll~t~e of the active sll~stanre with solid, pulverulent c~riers such as lactose, saccl~ose, sorbitol,15 ~~ ,hes (for ex~ r) potato starch, corn starch or amylopectin), cellntose d~ a~ s or 8~ r- and may also include m~..~ S~aldt~ or stearic acid as lul~lican~,.
The CG~ o~ iol~s of the il~ ioll may also be fo~nlat~o~l so as to provide a ~"~ lf~1 release. By using several layers of the active drug, ~ 1 by slowly 20 dissolving co-~ s--~ PA release tablets may be oblail ed. Another way of p~,pa~h~g ~ Gd release tablets is to divide the dose of ~e active drug into ~mllPs with co"~ of dirre~ ir} ~f q~.~ c and COlll~ S~i the granules into tablets together with the carrier s.lbs~ e. The collagen(s) and/or collagen(s) de~ , and ~....~n:~;. bil d~ coll~n conjugates may also be illco.~oL~t~d in 2~ slowly dissolving tablets made, for ;~ .,re, of fat and wax ~.,b~ Ps or evenly di~LLit,ut~d in a tablet of an insoluble ~b~ e; such as a physiologically inert plastic ~
~ n order to obtain dosage units of oMl ~ ~alions -- tablets, c~rs~lhPs, etc.
-- which are rleci~nPd so as to ~ .ll release of and possible ~eco...l.o~ilion of the 30 active sllb~ r~ in the gastric juice, the tablets, ~lr~es etc. may be enteric-coated, that is provided with a layer of gastric juice-res~ l enteric film or coating having such pl'~ LieS that it is not dissolved at the acidic pH in the gastric juice.
Thus, the active .~ e will not be released until the ~ " l ion l~,a~ es the W O 97/17988 PCT~US96/18149 ;~t ~ FS. As .,~le of s~ch known enteric CG~ g,~ may be ,l,~ llio,~ed c~ ose acetate phth~l~t~ r~oAy~r~ methylcellulose phth~l~t~s such as those sold under the trade names HP SS and HP S0, and FA~it~L and Fl~ it~S.
Li~uid ~ ;oll~ for oral applir~tinn may be in the folm of elixirs, syrupsor~ ;nl.~, fore~ solutionsco..~ fromabout0.1~ to20%
by weight of active s~h~ re, sugar and a "~ Lu,. or ethanol, water glycerol, propylene glycol and optionally aroma, !,~rc-l~ F and/or ca-l,o~y~ llylc~ lnse as a ~ ;n~ agent.

10 VI. F'.XAl~PLES
The ill~nlion will be further ~-.~ tood by l~,fe.e~e to the following e~camples, which are i"~ 1 to be purely elrPmpl~ry of the hl~ ,lion.

A. EXAMPL13 1: Expression Of Recombinant a3(I2~ C~ g Sub~it Jn Pichia rt ~ ~
PCR p~ ;, for the amp1if1r~tion of the a3(IX) col1a~en cl:)NA
coding se~ .r~ from the plasmid pS45 and the cDNA library clone RB4lO are a.~i. The ~ are ~1P~i~nt~tl such that they i~ ~lucc an Eco RI site at the S' and the 3' termini of the cY3(IX~ collagen coding se~ re, and a unique 20 If ~7~ 1 ion site is used to join the two halves of the coding ~quPn~e found in these two clones.
A Primer 1 and a Primer 2 are used to amplify the mature amino~ ...;..At coding se~lu~ e for cY3(IX) collagen from pl~"llid pS45 using ~,l~dal~l PCR
con~1itinnc as dese,;l~d in Ausubel et al., Current Protocols in Molecular Biology, 25 Greene P~ h¢ A;,sociat~,s and Wiley T-.t"~ , N.Y. ~19gO). A Primer 3 and Primer 4 are used to amplify the l....~i..;,~ cDNA coding ~ e~ e, inr1n~ing the stop codon, from the cDNA clone RB410 as described above. The resl11tin~
PCR product is di~s~1 with the chosen unique ~ oll en~on~r~ ce and with EcoR I.
C~n~ idlly available eA~ sicu vector pPIC9 an~ ogell, San Diego, CA), which directs S~Cl~ A~ Si~3n in Pic*ia pastons is ~ cl with ;OI~ rl~c~ EcoR I, followed by calf ;..I~h.~1 I?hn~l~h l_c~
(Pl._....~- j~), and then heat t1e~ lion at 70~C for S .~h.~ s. The Ai~st~l PCR

~l~lu~;ls and the pPIC9 vector are gel purfflled as desclil~d in Example 3 and athree-way lig~tit n iS ~-rOllllcd. After L.~ru~ - into co--~ Escherichica colit coll~ y ligated ~ c are jllt ~~ ;rd by le~LIi~Li~ll analysis and co.~li....~1 by se~ rin~ using the co.. ~l.;ially available Pichia se~ t';l~, ~L
S (IllviLlu~,en, San Diego, CA).
The o~3(~X) Pichia e~ ssion vector is l;"r~.;,. (~ and used to ~
~h~,~lasts of a his4 Pichia pastoris strain which also e"~l~.,ses prolyl 4-hy~o~ylase. Tla~Çu~ ..l; are i~ r~ on hi~ti~1int~ deftri~nt media and are co..l;....~ by a~ayi~ for the loss of tbe AOX1 gene by slow growth on ..~h~.~nl 10 media. ]~ iOll of the ~3(IX) gene is ~ ce~ by ~;lUWillg cells on ~ 1 as the sole carbon source. o~3~X) collagen subunit protein is s~el~d into the grow~",~ and ~vbs~ lly purified using ~ l c~ ~ ;r"g~tion~ filtt~ti~ , and cl~ulllillo~rhir t~ ni~ s.

B. ~XAl~rPLE 2: Expression Of Trimeric Human Type IX Cc~a~n In PichuI r,~
In a similar nlal~ the Pzchia pastons strain which produces ~3(IX) cûllagen subunit is en~ f ~d to coc~ sthe cYl(IX) and c~2(IX) collagen ~..1...,.;1~ in the same cell.

C. ~XA~PLE 3: Expression Of Trimeric Human Type IX Collagen In ~~pQ'J'~ptrra F~ la Sf9 Insect Cells Three ~cco~l~bi~an~ viruses were ~,~ne-~cd by co~ r~ clic~n of l~col~i~l al(IX~, cr2(IX), and ~3(IX) Cû~ Cl~ and a m~ifiçd Autographa 2~; cali~JCornica nuclear polyl,edlu~is vws DNA ~to Spodoptera frugiperda Sf9 insect cells using the R~rlllo~old L,~ f.,.,lioll kit (Pl.A....;ne~r~ The sPcrl~nr~s used tO
co~L,u~ the three a(IX) chains are ~l;~loseA in van der Rest and Mayne, Structu~
and FunetiQn of Collagen Iypes (Mayne, R. and ~ Sull, R. eds.3 ~e~ ;r Press, Orlando, FL, pp. 185-221 (1987). Ihe reSlllt~nt viral pûûls were collected, 30 ~.~.pl;l;~d and pla~ue- ~u,i~ as ~1esc~ A by C~ l~ald, S. and Heitz, J., Baculovirus E:xpression System: Procedures & Methods Manual, Pl.a- ~ , SaII
Diego, CA (1993).

W O 97/17988 25 PCT~US96/18149 Spodoptera frugiperda Sfg insect cells were cultured in TNH-FH ~
suppl~ l with 10% fetal bovine serum (BioClear) as monolayers at 27~C.
Appro~ ely, 5 x 106 insect cells were infect~ with recombinant human cY1(IX), a2(IX), and ~x3~IX) co~LI.u;~ and with the ~ virus for human prolyl 5 4-llydl~ylase (~ c,~ in ~ r~l;Oll). Viruses for the type IX collagen cY-c_ains were used in 2 to 3 fold excess over the prolyl 4-hy~u~ylase virus.
ASCOllJale of 80 ,ug/ml was added to the culture ".~.1i.-.., daily. The culture media was removed 72 hours after illr~lion, and the cell layer was washed once with a soll~tion of 0.15 NaCl and 0.02 M ph-~s~ at pH of 7.4. T~e cells were 10 hal~ ~l by sc~ , them in 1.4 ml of ice cold solution of 0.5 acetic acid, 0.75 M NaCl, 10mM EDTA, and 1mM PMSF at pH of 2.5. The cells were then homo~ ;,"A, and c~,n~,iruged at 15000 x g for 20 l~ yt~s. .CIJ1~ was ~.c.,~i~d with NaCl at the final co~cc.lLlaLion of 1.2 M by mixing the sample for 12 hours at 4~C. The ~l~ci~i~lc was cen~Liruged at 15000 x g for 20 ...i.~les 15 at 4~C. The reY~1tin~ pellet was dissolved in 500 ~1 of cold 50mM acetic acid for three hours at 4~C. A sample of 15 ~l was analyzed by either non-l~3.r;.~ or ~J~c;..~ SDS-PAGE followed by ~ ;n~ with Co(~ Rrilli~nt Blue. Irhe ... ~t~ 1 was also rii~ d with pepsin for 4 hours at 22~C, and the thf~rrn~l stability of the pepsin-l~;x~ .,omb~ type IX collagen was l~,ea~u,e~ by 20 rapid l~;~r ~1 ;ol~ with a l~~ of tryps~n and c}~lllu~ as ~f ~ e(~ lr~r et al, Anal. Biochem 110:360-368 (1981). The resnltin~ m~teri~l was analyzed on --r;"~ SDS-PA~E followed by Western Blotting with an antibody to a ~iple-helical coll~gon The results i~ ic~ e~ ion of human type IX collagen as a 25 h~r~ of about 300 kDA (Figure 1) co~;xli~g of equal ~ JIIIIIx of al(IX), cx2aX), and ~3(I~ chains as i~ tl ~1 in Figure 2. The t~ermal stability of the l~olll~i~L human type IX collagen was analyzed after a brief ~lut,asc f~i~stion.The mPnn~l stability of the l~colllbina~L human type IX cQll~Pn was over 40~C.

D. ~XAMPLE 4: Clo~ing And Expression Of A Chimeric Type II/~12~ Collagen Molecule l'he a!3~1X) Pichia eA~l~;.sion vector ~e~c~ ;l~l above is modified so as to direct CA~ iOll of a ~ type II/type IX/type Xl molPclllP.

W O 97/17988 PCTrUS96/18149 Specifi~lly, the vector is cleaved either 5' or 3' to the cx3(IX) collagen coding s~ re and coding s~ for type II collagen is i~,.t~l in-fila~e.
.~ltlition~lly, ~e vector may be again cleaved, either 5', 3', or 1~ the type IIand type IX coding se~ e-~e, and se.~ n~e encoding type Xl collagen is also 5 i~;.t~,d in the correct reading fiame so as to express a C]~i~llC.iC type II/IXtXI
coll~gen molecule. Tla~Ço....~ of co...pet~ E. coli are sc.~ ed for pl~cmirle with the desired o~ by l~slliclion Ai~ ;nn and Co~r~ by se4~ -ihlg as ~e 5C~ C~ above.
Various m~~ lh~l-c of the i~ n, in ~lrlition to those shown and 10 described herein, will becollle~ a~ ll to those skilled in the art from the Ç(,l.,~oh~g cle s~ .lif n. Such mo~ are int~n-lP~l to fall within the scope ofthe appended claims. It is also to be ~ ood that all base pair sizes given for nucleotides are approximate and are used for ~ul~oses of de.,~ ion.
~ r~.ences cited herein are hereby hlcol~ul~t~d by l.,Ç~r,_.lce in their 15 ~ y.

Claims (7)

WHAT IS CLAIMED IS:

1. A fusion protein comprising a human type IX collagen linked to heterologous peptide sequence.

2. The fusion protein of Claim 1 wherein the heterologous peptide sequence comprises a type II collagen.

3. The fusion protein of Claim 1 wherein the heterologous peptide sequence comprises a type XI collagen.

4. The fusion protein of Claim 1 wherein the heterologous peptide sequence comprises type II and type XI collagen.

5. A method for producing recombinant human fusion protein comprising:
(a) culturing a host cell transformed with the recombinant DNA
expression vector which expresses said fusion protein; and (b) recovering the fusion protein from the cell culture.

6. A protein comprising human recombinant type IX collagen.

7. A method for producing recombinant human type IX collagen comprising:
(a) culturing a host cell transformed with the recombinant DNA
expression vector which expresses said type IX collagen; and (b) recovering the type IX collagen from the cell culture.