CA2247827A1 - A novel disintegrin metalloprotease and methods of use - Google Patents

Abstract

This invention provides a method for identifying compounds capable of binding to the disintegrin protein, and determining the amount and affinity of a compound capable of binding to the disintegrin protein in a sample. This invention also provides a host cell comprising a recombinant expression vector to the disintegrin protein and a recombinant expression vector encoding to the disintegrin protein and the human disintegrin metalloprotease protein, fragment or mutant thereof, useful for these purposes. This invention also provides an in vivo or in vitro method for screening for osteoarthritis and other metalloprotease based diseases, capable of manufacture and use in a kit form.

Description

Wo 97131931 PCT/US97103217 A NOVEL D~SIN-IEGR~N METALLOPROTEASE AND hll~HODS OF USE

Field of the invention The invention relates to a novel protein, its L~ s and m~ tQ and to its use in d~tec~ g and testing drugs for ailments, inrl~rling osteod,lhl;l;s and otherschara~;te. -zed by up re~ tion of ~ olea~s.
Back~ ound A number of enzymes effect the breakdown of structural prolci~.s and are structurally related metallopro~eases. These include human skin ~brobla; l co!lag~n~Qe, human skin fibroblast gel~ P, human sputum collagenase and ~ ;n~tQ~ and human ~l.u.,lelysin. These are zinc-containing metalloprotea3e C.~l.PS, as are the angiotensin-converting enymes and the enkcph~;A~,s Collagenase, ~I.o.l.el~l and related c.~...es are i,.lpo.~l~ in lnc.~;-t;n~ the ~---p~c.llatoloD of a number of ~l:ce~cr~c~
in~ ing rhe -m.~t- d arthritis (~llllins~ D. E., et al., Biochim Biophys Acta (1983) 695:117-214); osteo~lhlh;s (IIc~de.~n, B., et al., Drugs ofthe Future (1990) 15:495-508); the ~ cl~c:c of tumor cells (ibid, Broadhurst, M. J., et al., European Patent ~,~'ic~ti~.)n 276,436 (puk!iched 1987), Reich, R., et al., 48 Cancer Res 3307-3312 (1988); and various ulcerated con~itionc Ulcerative con~);l;Qr~C can result in the cornea as the result of alkali burns or as a result of ;"r~l;on by Pseudo---onas aeruginosa, Acanthamoeba, Herpes simplex and vaccinia viruses.
Other con~ iQnC chara~.~d by u.ldesll~d metallopr~tease activity include p~.iodun~ disease, epid~,.-.lol~;s bullosa and scleritis. In view of the involvement of n~r~ roteases in a number of disease con~;t;onc~ c"~plc have been made to prepare inhibitors to these e~l..es. A number of such inhibitors are ~i~,losed in the literature.
The ~~ e~Lon s,eeks to provide novel inhibitors, preferably specific to this protease., that have e ~ nce~ activity in treating ~ c mediated or modlllsted by this protease.
Inhibitors of r~tslloprot~a3es are useful in ll~dtillg ~i~r~5 caused, at least in part, by breakdown of structural proteins. A variety of inhibitors have been pl~ pa~ed, but there is a cGl.~ need for metalloprot~ inhibitor screens to design drugs for -~l.l 8 such ~ ~r5~r5 Metalloproteases are a broad class of proteins which have widely varied filn~tionc Di~ e~-ns are zinc m~,tsllopn)t~rs abundant in snake venom. Alternate cloning strategies could be used. M~ ;Q -~1c~ s are a family of prole.ns with about 18 known subgroups. They act as cell adhesion disrupters and are also known to be active in reproduction (for t"~ "lc, in fertilization of the egg by the sperm, inc~Ajng fusion thereof, and in sperm maturation).
These proteases and many others are uncovered in mole,~ r biology and bioche~ As a result, Genbank, a reposito~ for gene seq~lPnces~ provides several se~uences of metalloproteases, in-~lvAin~ some said to encode L~.-e~ of r~icinte~ins.
For exarnple, GenBank a~c~~o;on # Z48444 dated February 25, 1994 d;crloses 2407 bases of a rat gene said to be a rat ~ evill metalloprotease gene; GenBank aCce~Q;on #Z48579 dated March 2, 1995 discloses 1824 bases of a partial sequence of a gene said to be a human AiC;~fevul rnet7ll0protease gene; ~n~Drlt~ ~c~sQion # Z21961 dated October25, 1994,d~r1os~c-2397basesofapartialsequen~eofagenesaidtobeabovine zinc metalloprotease gene.
It would be advDnt~g~o~c to i~ fe m~t~opl~edses in specific disease states, and to use these metallopr~teases as tools to detect and u~ tf,ly cure, control or design cures for such Ai~ c OBJECTS OF T~ INVENTION
It is an object of the present ,n~e.llion to provide a method for identifying co,..po~ -As capable of binding to the ~:r ~.1eu,;n protein.
It is also an object of the present inv~ ;on to provide a host cell COIll~ a recol.~hi~ e~.ress;on vector to the rl;c ~le~in protein and a lecolnl,;na h ~ ,ress;on vector el-r;o~l ~8 to the ~ u~ protein.
It is also an object of the present invention to provide a method for SCl~ Ung for metalloplo~esse ~~-ed;~t~ ~ e~es such as cancer, ~lLropvlhies (inchlriing anlylosing spondol,vtis, ~ liod arthritis, gouty &lluilis (gout), ;-.n~ toly arthritis, Lyrne disesse and o_s~al lL lis).
It is also an object of the present invention to provide an antibody to the protein useful in the screen, in the i~l~;o-~ ofthe protein or ss a t~5~,l~ moiety for the protein.
SUMMA~Y 0~ THE INVENl'ION
This invention provides a method for identifying compounds capable of binding tothe ~ te~rin protein, and determining the amount and affinity of a comro~n-l capable of binding to the 1; ~ protein in a sample.
This invention also provides a host cell cOll.~Jl;.u~g a recombinant cAples~;on vector to the ~ .n protein and a recc--.l.;~.1 cA~,le.~.on vector Pncoding to the tC~Jin protein and the human ~ tc!;-in metallop,ot~se protein, L~ cnl or mutant thereof, useful for these purposes.

This invention also provides an in vivo or in vitro method for scleelfmg for osteoarthrtis and other metalloprotease based di-Qe-q-ces such as cancer, capable of mqn--f ct~-re and use in a kit form.
DETAILED DESCRIPTION
As used herein, the terms "protein," "protease," and "metalloprotease" refer to a ~licintegrin. ~lerelably this is a human riicinte~in as desc.il,ed below.
The term "antibody" refers to an ~llibody to a rliQ;'te~in, or fragment thereo~
These many be molloclonql or polyclonal, . nd can be from any of several . ources. The invention also co~m .~pl~c5 L~ e.-l~ of these antibodies made by any method in the protein or peptide art.
The term "disease screen" refers to a screen for a disease or disease state. A
disease state is the physiole~r-s-l or cellular or b:cchenlical ..-9 .;~ ~,t~l;on of the disease.
îc~bly this screen is used on body tissues or fluids of an animal or cell culture, using ~and~d techniques, such as ELISA. It also Co~ 9~'eS "~"~pp- ~g~ of disease in a whole body, such as by labeled antibody as desc,il,ed above given ~ r~lly regardless of the detectio~ m.otho-l, preferable such detectio~ .od~ include fluoi~s~encç, X-ray (inchl~li~ CAT scan), N~ (Tnr~ li~ MRI), and the like.
The term "cGl"po~md screen" is related to the ...~.lho~ls and screens related tofinding compounds, determining their affinity for the protease, or decigr.i~ or se~e~ g co"l~)ollnds based on the screen. In another Pmho~ nt~ it co.~- .pl~es the use of the three dimensional structure for drug design, preferable "rational drug design", as undc.~lood by the art. It may be pl~.f~ cd that the prote~ is in "es~ ly pure form", which refers to a protein re--o~-~ly free of other impulilics, so as to make it useful for cA~e~im~ls or chara~ ion. Use of this sel~,ning method assists the skilled artisan in finding novel structures, whether made by the chemist or by nature, which bind to and preferably inhibit the protease. These "inhibitors" may be useful in re~Jl~tin~ or moch ting the activity of the protcase, and may be used to thus mod~ te the b-olo~
cascade that they fi-nctiQn in. This appl.a-h affords new pha, - eutiçslly useful compounds.
The term "disinte~in" refers to a diQ;nte~g~in, a fragment thereof, a mutant thereof or a homnl~-e which still retains its fi~nCtio~ This term conlen~ es aggrn-qr~qse, and other p~ot~s which are involved in or modul~e tissue r~mr)dellin~ This conte-..p!~es ~iQ~ .If~ins from di~cling, species, and those prepar. d by recol.lbinanl m~thodc, in vitro m~thod5, or ~landald peptide ~l~thes;s. Preferably the protein is a human dic; ~leg~in or mutant thereof. For the purposes of ~fini~ the mut~ntQ~ of the protein the p~cÇcl~,d W O 97/31931 PCT~US97/03217 "native" protein is described in t~nR~ accession # Z48579, inco.~lo.~led herein by r~;rer~. ce and le~.red to in the seqllPnce below. SEQ ID NO 1 de,s~ es a fragment of that DNA sequence and its transcript and SEQ ID NO 2 describes the coding region of the gene and its l-~ns~,-ipt. Homologue d;C n~ ;nS include whole proteins with at least 90% homology as unde.~lood by the art, Ot ~iAgJ~ r~S thereo~ For exarnple, a ratprotein which is 95% homologous to that of SEQ ID NO. I or SEQ ID NO. 2 based onthe peptide sequence derived from the DNA or cDNA se~Upnce~ and a bovine protein~5i.,~il&,1y derived) being 97-98% homologous, are both conQ;de~ed homologuPs Thus homologous cDNAs cloned from other o~ n~ s give rise to homologous proteins.
Likewise pr~te.ns may be co-- de~ed homolcel~es based on the amino acid sequenre alone. Practical lin.;~ ;ol-~ of amino acid seq~lencing would allow one to d.,tel.-une that a protein is homologous to another using for ~ rle Co~l~yaliSOn of the first 50 amino acids ofthe protein. Hence 90% l~nlol~gy in would allow for 5 di~,.ing an~ino acids in the chain of the first 50 amino acids of the hon~f ~OgO~lS protein.
The skilled artisan will al,p,~,~,;ate that the de&_ne~_ ~ of the genetic code provides for dilrt;ling DNA sequences to provide the same l.ans_~ipt, and thus the sarne peptide. In certain cases pr~_paring the DNA seq~eneç, which encodes for the same peptide, but differs from the native DNA include;
~- ease of seq~pn~;ng or synthesis;
--- ine,~sed e,.prejiion of the peptide; and .,f~,rence of certain heterologous hosts for certain codons over others.
These practical conQ;~Ierations are widely kno~,vn and provide elllbo-J; ~.. nls that may be adv~ ~t~eQus to the user of the invention. Thus it is clearly cont~Q"..p~ ed that the native DNA is not the only ~--, bod:- n~ envisioned in this invention.
In r ~itiQn it is ap~e,.lt to the skilled artisan that L~ ne .-l c of the protein may be used in screening, drug design and the like, and that the entire peptide may not be r~uir~ for the ~ oses of using the invention. Thus it is clearly conte..~ ted that the skilled artisan will unde-~L~nd that the ~icrlosvre ofthe peptide and its uses contemplates the useful peptide fr~ ntc The practical consid~,lalions of protein c.~l)re~.;on, purification yield, stability, solLhility, and the like, are cor~;dç~ed by the skilled artisan when choo~;l.g ~~fLelher to use a ~ 1 and the fi~"l~ to be used. As a result, using routine practices in the art, the artisan can, given this disclosure p. ~.c~ice the invention using rl ag...~ ~1 s of the protein as well.

s The protein or pro~ease itself can be used to determine the binding activity of small ms~le~ules to the protein. Drug scle.,.~il 8 using enymatic targets is used in the art and can be employed using all~o...aled, high throughput technologies.
The inh;bition of dicinte~in activity may be a predictor of efficacy in the l-c~n.~-~of oste-oa-llu;lis, and other d ~ ACe~: involving dege~c~alion of articular cartilage.
Gene therapy Without being bound by theory it is thought that the metalloprotease is up regulvsted during osteoarthritis in tissues. We have SII~ u~gly found that a human ~lisintegrin is up-re~ ed in human chond.o~,~rtes during o~leoa.lluilic conAitionc Inhibition of signal trncd~ctiQn ...ech~ .. is cn.c~cio -c in dis.u~)th~g the rqccade of events in osteo~lluilis and other A;cç~c~s involving ca.lilage degene.alion. The skilled artisan will ~cco~uLe that if up-re~llAtion is a cause of the onset of arthritis, then interfering with the activity ofthis gene may be useful in treating oslfJoa-lluilis.
This is done by any of several ~ hod~ inclu~ii~ gene (i.e., A~-1 ;~n.~) therapy.Inhibitors of the f~ f ~y ;n The plo~ease of the invention can be used to find inhibitors of the prot~.
Hence it is useful as a scree~ng tool or for rational drug design. Without being bound by theory, the protease may modulate cellular rc.n~ g and in fact may enhsnce extracellular matrix ren od~ling and thus e-~h~r~ee tissue breakdo vn.. Hence inhib;tion of tegrin provides a ~ peulic route for ll~ nl of diC~F,5 chara.,lcliLcd by these ~. ~c~ss~,s In sc.-,~fing, a drug compound can be used to dct.,...une both the quality and quantity of inhibition. As a result such scr~lullg provides i~lfu~ alion for selection of actives, preferably smaD c'~cule actives, which are useful in ~l~,aling these diseases.
In therapy, inh:bifion of ~ 1c~yin me~s~cp~ulease activity via binding of small sr weight, ~.l~h~,lic metalloplote~e inhibitors, such as those used to inhibit the matrLx m_taUoproteases would be used to inhibit ~"~ll r~ ls~ matrix remodPIing L~libodies to the protein Metalloprûl~s active at a puticululy undesired loc~ion (e.g., an orgul or certain types of cells) can be lalg~led by ~ u l, g,gti~ a metallop, .,l~sc inhibitor to a to an untibody or fragment thereo~ Conj--8,s-tion rnetho-lc ue known in the art.
The a.l~il,od~ of the invention CUI also be conjugated to solid s.~ )G.ls. TheseconJIlgates can be used as affinity ,~,ag~ s for the purifi~iQn of a desired metalloprotease, preferably a ~ e~,u~.

, In another aspect, the antibody of the invention is directly conjugs-ted to a label.
As the antibody binds to the metallop~oteas_, the label can be used to detect the p~sence of relatively high levels of metalloploteast in vivo or in vitro cell culture.
In addition, the metallopro~ease inhibiting conlroun~s can be cor~ ated to antibodies. Typical conjugation metho~c are known in the art. These antibodies are then useful both in therapy and in ~.~or.;lo,ing the dosage ofthe inhibitors.
Por example, l~gelillg ligand which s~e~;r~rally reacts with a marker for the int~nded target tissue can be used. Mell~o~2c for co~p1:~ the il~f nlion CG~pvO~ t to the tal~,~,lil.g ligand are well known and are similar to those desc,ibvd below for cou~ g to carrier. The conj~ ,qtçs are forrm.lqted and administered as de3v~il,ed above.
Preparation and Use of Antibodies:
~ ntibo~iRc may be made by several n~etho~1c for f ~ the protein may be injected into suitable (e.g." ~ 9n) s~ cts itlCI~]t~ g m-ice~ rabbits, and the like.
P~ef~,..vd protocols involve repeated injection of the imn ~nooen in the p~3ellce of adjuvants according to a s~hed~lle which boosts produ~ioll of ~ bod;ec in the serum.
The titers of the immlm~ serum can readily be ..-cas~lr~d using immlmo~s~s~qy procedures, now standard in the art.
The antisera oblained can be used directly or n~Q~ clor~ql antibodies may be ob~t~ined by harvesting the pc~iphc.~l blood Iymphocytes or the spleen of the immllni7ed animal and imrnortali~ing the antil,oJ~-prodtlc;~ cells, fo"..,.ed by ide.ltil~;.lg the suitable antibody producers using ~ dal~d immllnosccvsy techni~ues.
Polyclonal or ~nnnoclonsl preparations are useful in ".or..lG.ù,g therapy or prophylaxis reg~ ,n~ involving the compounAC of the invention. Suitable samples such as those derived from blood, serurn, unne, or saliva can be tested for the pre~nce of the protein at various times during the ~ ,ç ~1 protocol using ~,Landard imml~nnsccs,y techniques which employ the antibody p~ &alions ofthe inv~ ion.
These antibodies can also be coupled to labels such as sc;nti~..phic labels, e.g., t~l~f-~iv ~ 99 or I-131, using standard coupling . ~ o~ The labeled compounds are ad~ tf-c~ to subjects to dete.-,lille the locvstiQ~c of excess a-novnts of one or more metalloprotea_es in vivo. Hence a labelled &.lil,ody to the protein would operate a a scr~ning tool for such ~h~nce~l eA~l~ss;on, ;l.~) c~ g the disease.
The ability of the antibodies to bind n~t~slloprolease selectively is thus talcen advantage of to map the distribution of these enymes in situ. The techniques can also be employed in histological procedures and the labeled -s-r~tibo~lies can be used in co"~p.,t;~i~e lmml~ C~ S.

Antibodies are advvntageol~sly c~rled to other compounds or materials using known methods. For example, materials having a carboxyl functionality, the carboxyl ~ residue can be reduced to an aldehyde and co~Fl~d to carrier through reaction with side chain amino groups, optionally followed by reducflon of imino linkage formed. The carboxyl residue can also be reacted with side chain amino groups using cQ~ e.~p.
agents such as dicyclohexyl carbodiimide or other carbodiimide dehydrating agents.
Linker compo~n~le can also be used to effect the coupling; both ho...ob-filnctional and h.,t~ .ob;r~ ;Q~ linkers are available from Pierce Chemical Company, Rockford, Iil.
These antibodies, when conjugated to a suitable chro..,alography material are useful in isolating the protein. Separation .~ o~ls using affinity cluol..alography are well known in the art, and are within the purview of the skilled artisan.
Disease marker Without being bound by theory, e.~.re~ ;cn of genes, and preferably this gene may have a restricted tissue distribution and its e.~ s;,;on is up regl~l?ted by potential osteoalllllil;s mediators. Fnh~l~Ge~ e.~yl~;on of this gene (and hence its protein) for e~_np'~ in articular chor.dlo.,ytes provides a marker to monitor the deve1~!pment ~ hl~1ine the earliest, as~ tG~Ilalic stages, and the pro~,s;.;on of osteoarthritis. Hence an antibody raised to the protein would operate a a s~;l-,c.-.n8 tool for such enhqnced e,~,ress;on, inA;~ cqti~ the disease.
In ad~1iti~n, when used in a disease screen, anlil,od;cs can be conju~ted to clu~nlophole or ~uorophole containing materials, or can be conjugated to er~...es which produce chro.-.opho- es or fluorophGres in certain con~litioll~ These conjngD-ti~
materials and m~tho~s are well known in the art. When used in this manner detection of the protein by immlll~nqr~-~ is ;.l~ htr lward to the skilled artisan. Body fluids, for example can be scr~ned in this manner for calibration, and d~,t~l;ol of distribution of metallopr~ Q~-~, or i..~ ased levels ofthese prot~D~Q
When used in this way the inv~nlion is a useful diagnostic and/or clinical marker for met~'lopr~te~e mediated f~ eP5~ such as osteoarthritis or other articular cartilage de~,ene~ali./e ~ ~D~S When disease is detecte~l it may be treated before the onset of syrnptom or debilit~tion Fu.lhe.ll.ore, such antibodies can be used to target di-~~ e~ tissue, for dete~,l;on or 11.~ It as desclibed above.
EXA~LES
The following non-limiting examples illustrate a pref,.led embodim~nt of the present invention, and briefly des~,libe the uses of the present invention. These W O 97/31931 PCTrUS97/03217 e~mples are provided for the guidance of the skilled artisan, and do not limit the invention in any way. Armed with this disclosure and these examples the skilled artisan is capable of making and using the c1simed invention.
Standard starting materials are used for these eYsmpla~ Many of these .l~alel;als are known and co,-u".,~-,;ally available. For eY-s-nlple E. coli CJ236 and JM101 are known strains, pUB110 is a known plasmid and Kunkel method mlltsgPnPci~ is also we11 known in the art.
Variants may be made by ~,Al"esa;on systems and by various metho~c in various hosts, these mPthods are within the scope of the practice of the skilled artisan in mole~ biology, b och~ y or other arts related to biotechnolaOy.
EA~n~le1 RNA was i~olsted from l~nstim~ ted and interleukin-1 stimlllsted cultures of normal human articular chor.dlo~les. The RNA was reverse l-ansc,il,ed into cDNA.The cDNA was subjected to a mo-lified d;~cr~nlial display procedure using a series of random primers.
PCR samples generated from both stimulated and ur ctimlll-s~ted chondrocytes were ele~ phoreses in u~jac~nt lanes on polyacrylamide gels. The di~.~nli~lly eAI,.,ssed band was excised from the gel, cloned, and sequenr~ The dilF~,.e.ltial eA~,.e,,i,;on ofthe gene was ccsnL."ed by RNase prole.,lion and nuclear run on e.~eri",e~ls.
Example 2 A novel partial human cDNA coding the protein is cloned from pli~ llu,~,s of interleukin-l stim~llsted human articular (femoral head) cl~ondru~es, using known mP,th~l~, The same se~l~enee is found, and the gene completed by S~ClCe h.l,~ of human cDNA
lib,~nes to obtain full length clones.
Exunple 3 The cloned DNA of example 2 was placed in pUB 110 using known mPtho~lC
This plasmid is used to l,~ sfo,l,. E. coli and plovides a t., ~ e for site-d;r~,~,led ml~ e~ ;s to create new n.vt~rts Kunkel method mllt~,~ c pe~,l,.ed altering GLN
1 ALA.
Example 4 [125Il d ~ ~ewi~ &l~il,od~ is pr~pared using IODOBEADS (Pierce, Rockford, IL;
immobilized chlo-,u-ane-T on ncsnpûrous pol~ e beads). LyophiliL~ antibody (2 ~g) is taken up in 50 Ill of 10 mM acetic acid and added to 450 ~l of phosphate-buffered saline (PBS) (Sigma, St. Louis, MO) on ice. To the tube is added 500 ~Curie Of ~25I

(A l.c.sh~n, Arlington ~Pight~ IL) (2200Ci/mmol) in 5 Ill, and one IODOBEAD. Thereaction is in~llb~ted on ice for l0 min with occ?~ionql shq~i~ The reaction is then te....; .~ed by remov. l ofthe re~~,tio~ from the IODOBEAD. To remove ul~e?~ed 125I, the mixture is applied to a PD-10 gel filtration ~D!--tnn Example 5 A fluorogenic peptide (~-hem Guelph Mills, King of Prussia, Pa) is mixed with the ~ egJ in and change in the fluorccence is evaluated at 2 min, as a control.. Then the ~uorogenic peptide is mixed with the ~;r:~ tr~in in the pl.,3ence ofthe compol~nd in ev~ qtior in a se~,a ale run, with evaluation at 2 mi~n~tes Data are evaluated using standald mPthodolc3y to provide relative binding ofthe evaluated CGI~.pO~nd F- r--ple 6 0.5ml of synovial fluid from the le~ knee of a patient is ~hithdla~l and tested for elevated levels dic:-~t~ n by ELISA. The results in~lic?te higher than normal .1;~ in level. The patient is ple..~..il~ed a prophylactic dose of a d;s;~ee,;n inhibitor, and is a~ ;s~ered an injection of same in the le~ knee before leaving the r~ ;ri~-'s office.
Example 7 Inhibition of eAlll,ce~ qr matrix ren~ode~ g is explored via inhibition of d:r~ e~ in metalloprotease activity. Using a small ~le~ qr weight, synthetic metalloprotease inhibitor, such as those used to inhibit the matrix metalloprot~ses, tissue ihllegrtly and pro~coglycsn is l..onilGled.
A sample of mouse derived articular cartilage is grown in a 1 miclo..lolar solution of a small ~ q~ weight d;~-.te~in inhibitor. The c.~pcl;n.e.~l is controlled andC~ U Cd to an identical culture grown with no inhibitor.
The assay of the culture after 7 days shows that the ;~-h-bj~ed culture has lesstissue breakdown and less ploteoglycan present in the serum of the culture. The result is cQr. ~ 1 with the inhibited agglcc~ ~e activity. Inhibition of a~giccanase would inhibit tissue breakdown and reduce the release of proteoglycan.
Example 8 Inhibition of proteolytic proceo~o;~ ~f --'I';~B in the release from the Illeml)l~e bound form of the ~io-integrin m~tq-lloprol~ domain inhibits "second .~.esse~
g ofthe .nen~ ne bound ~licinte~in m~'ewle. Such second ~PSs~pr~grr o~ignqlil~
would result in cellular phenotypic cl-~nges, ç~ es in gene e A~,res;,;on, rh~ges in mitotic activity, and the like.
Cells known to contain dio-inte~in are treated with a serine protease. Proteins released from the cell are meas.lred by s~ndard m~tho~s Speçific~lly the W O 97/31931 PCT~US97/03217 metalloprotease activity is Inon,lol c~ via literature m~thn~e. The amount of metalloprotease released is correlated to the amount of serine protease used to treat the cells Il,clcseses, versus control, in src tyrosine kinase activity a re measured by Western blot analysis of intracelh~l~qr proteins using mon~cl~nql anlil,odies specific for phosphotyrosine following cleavage and release of the dir; ~tegrin metalloprotea e. Controls are cells that have not been treated with serine protease.
src tyrosine kinase activity in the cell (or is it ceL culture) is measured by liler~lu,e me~q,thoAc Release ofthe meta!l~protease domain ofthe ~ lc~,n is also monitored via literature m~thoAc.. There is a direct co..~lalion b~ .~n release of the metalloprotease domain and illc~cases in intra.~ src tyrosine kinase activity This result is cor.~
with stimlllqtion of A ~ f~;n-...e l;~ted cell signaUing by c-timl~lqtion of the src tyrosine kinase cq~P~le.
E~amDle g Tnhi~ hiQn of ,nltlcdlular n~h~ n motec~ s~ or extracellular matrix co"~poncnls results in the inhibition of phenotypic cl~ B~ c, in~ ding ch~ge5 in cell shape, acsoçiq~ed with such interactions, as desc.;bed in Example 8.
Integrin binding is ...eas,lred with a peptide con~ g the sequen~e RGD, using the protocol of Example 8. Integrin binding is ...c~c~ ,d via com~,_t;l;~., assay, using cellular chq~g s in shape visible via ll,icl~scopy The peptide inhibits the ceUular ch~ s . s in Exarnple 8.
This result is CQ~C Ic ~I with c~.."~ ;on with or b'cc~ing of the interaction ofAicintegrin. The RGD peptide inhibits ceLular cl~ Bes in chondrwytes The osteoarthritis phe.~ot~e, characle.-Le~ by inc.~d matrix ~nlhe~.:s and acccle~lcd matrix metalloprotease activity does not occur. Other readily assayable cellular ch~r~eFs can be used to l..on-ter this result, incll~.1i~ gene .,,~p.es~;on, changes in mitotic activity, and the like.
Example 10 A small ~ r weight metallopr~,L~se inhibitor is used to treat a tissue culture accordu.~ to the method of Exarnple 7 The release of TNF{~ from the cellne is measured by literature metho-l$ The inhibitor of Example 7 also de~ s the ?~no~mt of TNF~ secreted from the cell membrane.
This is cQnCict~nt with the theory that inhibition of ~ 1e~;n metallop.o~ease activity will result in the inh:~ition of a .1i~ c~;n ~csoc~ted ;..n~ ...o-I;on ~ ç and W O 97/31931 PCTrUS97/03217 secretase activity. It is contemplated that monitoring the release of cytokines or IL-l from the cell l"~ I ane, and the like will produce the same result.
re~ences described herein are hereby incol},ola~ed by It;rerence.
While particular embod; ..~ s of the subject invention have been des_lil.ed, it will be obvious to those skilled in the art that various c~ ges and mo~lifi~tionc of the subject invention can be made without departing from the spirit and scope of the invention. It is intended to cover, in the appended claims, all such m~ fi~q~iorl~ that are vithin the scope of this invention.

W O97/31931 PCTrUS97/03217 SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT: TINDAL, MICHAEL H.
HAQQI, TARIQ M.
(ii) TITLE OF INVENTION: VSE OF A NOVEL DISINTEGRIN
METALLOPROTEASE, ITS MUTANTS, FRAGMENTS AND THE LIRE
(iii) NUMBER OF SEQUENCES: 4 ~iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: THE PROCTER ~ GAMBLE COMPANY
(B) STREET: 8700 MASON-MONTGOMERY ROAD
(C) CITY: MASON
(D) STATE: OHIO
(E) COUNTRY: USA
(F) ZIP: 450go-9462 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
~D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLI QTION NUMBER: US 60/012,679 (B) FILING DATE: 01-MAR-1996 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: HAKE, RICHARD A.
(B) REGISTRATION NUMBER: 37,343 iC) REFERENCE/DOCKET NUMBER: 5980 (ix) TELECOMMUNI QTION INFORMATION:
(A) TELEPHONE: 513/622-0087 (B) TELEFAX: 513/622-0270 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1961 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME~KEY: CDS
(B) LOCATION: 2..1474 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

W O 97/31931 PCTnUS97/03217 Gln Thr Thr Asp Phe Ser Gly Ile Arg Asn Ile Ser Phe Met Val Lys Arg Ile Arg Ile Asn Thr Thr Ala Asp Glu Lys Asp Pro Thr Asn Pro Phe Arg Phe Pro Asn Ile Ser Val Glu Lys Phe Leu Glu Leu Asn Ser Glu Gln Asn His Asp Asp Tyr Cys Leu Ala Tyr Val Phe Thr Asp Arg Asp Phe Asp Asp Gly Val Leu Gly Leu Ala Trp Val Gly Ala Pro Ser Gly Ser Ser Gly Gly Ile Cys Glu Lys Ser Lys Leu Tyr Ser Asp Gly Lys Lys Lys Ser Leu Asn Thr Gly Ile Ile Thr Val Gln Asn Tyr Gly Ser His Val Pro Pro Lys Val Ser His Ile Thr Phe Ala His Glu Val Gly His Asn Phe Gly Ser Pro His Asp Ser Gly Thr Glu Cys Thr Pro Gly Glu Ser Lys Asn Leu Gly Gln Lys Glu Asn Gly Asn Tyr Ile Met Tyr Ala Arg Ala Thr Ser Gly Asp Lys Leu Asn Asn Asn Lys Phe Ser Leu Cys Ser Ile Arg Asn Ile Ser Gln Val Leu Glu Lys Lys Arg Asn Asn Cys Phe Val Glu Ser Gly Gln Pro Ile Cys Gly Asn Gly Met Val Glu Gln Gly Glu Glu Cys Asp Cys Gly Tyr Ser Asp Gln Cys Lys Asp Glu Cys Cys Phe Asp Ala Asn Gln Pro Glu Gly Arg Lys Cys Lys CA 02247827 l998-08-3l Leu Lys Pro Gly Lys Gln Cys Ser Pro Ser Gln Gly Pro Cys Cys Thr Ala Gln Cys Ala Phe Lys Ser Lys Ser Glu Lys Cys Arg Asp Asp Ser Asp Cys Ala Arg Glu Gly Ile Cys Asn Gly Phe Thr Ala Leu Cys Pro Ala Ser Asp Pro Lys Pro Asn Phe Thr Asp Cys Asn Arg His Thr Gln Val Cys Ile Asn Gly Gln Cys Ala Gly Ser Ile Cys Glu Lys Tyr Gly TTA GAG GAG TGT ACG TGT GCC AGT TCT GAT GGC AAA GAT GAT AAA GAA 10 0 6Leu Glu Glu Cys Thr Cys Ala Ser Ser Asp Gly Lys Asp Asp Lys Glu Leu Cys His Val Cys Cys Met Lys Lys Met Asp Pro Ser Thr Cys Ala Ser Thr Gly Ser Val Gln Trp Ser Arg His Phe Ser Gly Arg Thr Ile . 355 360 365 Thr Leu Gln Pro Gly Ser Pro Cys Asn Asp Phe Arg Gly Tyr Cys Asp Val Phe Met Arg Cys Arg Leu Val Asp Ala Asp Gly Pro Leu Ala Arg Leu Lys Lys Ala Ile Phe Ser Pro Glu Leu Tyr Glu Asn Ile Ala Glu Trp Ile Val Ala His Trp Trp Ala Val Leu Leu Met Gly Ile Ala Leu Ile Met Leu Met Ala Gly Phe Ile Lys Ile Cys Ser Val His Thr Pro Ser Ser Asn Pro Lys Leu Pro Pro Pro Lys Pro Leu Pro Gly Thr Leu CA 02247827 l998-08-3l W O 97/31931 PCTrUS97/03217 Lys Arg Arg Arg Pro Pro Gln Pro Ile Gln Gln Pro Gln Arg Gln Arg Pro Arg Glu Ser Tyr Gln Met Gly His Met Arg Arg CACAAAGTCT TAGAATATTA TTATGTGCCC C~l~lCCCT ~.Cl.C~.. GCTGCATTTT 1784 GAAGGACCCT ACAAATCCTT .CC~-~.lCCC AAATATTAGT GTGGAGAAGT TAAACAA 1961 ~2) INFORMATION FOR SEQ ID NO:2:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 491 amino acids (B) TYPE: amino acid ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein ~xi~ SEQUENCE DESCRIPTION: SEQ ID NO:2:
Gln Thr Thr Asp Phe Ser Gly Ile Arg Asn Ile Ser Phe Met Val Lys Arg Ile Arg Ile Asn Thr Thr Ala Asp Glu Lys Asp Pro Thr Asn Pro Phe Arg Phe Pro Asn Ile Ser Val Glu Lys Phe Leu Glu Leu Asn Ser Glu Gln Asn His Asp Asp Tyr Cys Leu Ala Tyr Val Phe Thr Asp Arg Asp Phe Asp Asp Gly Val Leu Gly Leu Ala Trp Val Gly Ala Pro Ser Gly Ser Ser Gly Gly Ile Cys Glu Lys Ser Lys Leu Tyr Ser Asp Gly Lys Lys Lys Ser Leu Asn Thr Gly Ile Ile Thr Val Gln Asn Tyr Gly Ser His Val Pro Pro Lys Val Ser His Ile Thr Phe Ala His Glu Val W O 97/31931 PCT~US97/0321 Gly His Asn Phe Gly Ser Pro His Asp Ser Gly Thr Glu Cys Thr Pro Gly Glu Ser Lys Asn Leu Gly Gln Lys Glu Asn Gly Asn Tyr Ile Met Tyr Ala Arg Ala Thr Ser Gly Asp Lys Leu Asn Asn Asn Lys Phe Ser Leu Cys Ser Ile Arg Asn Ile Ser Gln Val Leu Glu Lys Lys Arg Asn Asn Cys Phe Val Glu Ser Gly Gln Pro Ile Cys Gly Asn Gly Met Val Glu Gln Gly Glu Glu Cys Asp Cys Gly Tyr Ser Asp Gln Cys Lys Asp Glu Cys Cys Phe Asp Ala Asn Gln Pro Glu Gly Arg Lys Cys Lys Leu Lys Pro Gly Lys Gln Cys Ser Pro Ser Gln Gly Pro Cys Cys Thr Ala Gln Cys Ala Phe Lys Ser Lys Ser Glu Lys Cys Arg Asp Asp Ser Asp Cys Ala Arg Glu Gly Ile Cys Asn Gly Phe Thr Ala Leu Cys Pro Ala Ser Asp Pro Lys Pro Asn Phe Thr Asp Cys Asn Arg His Thr Gln Val Cys Ile Asn Gly Gln Cys Ala Gly Ser Ile Cys Glu Lys Tyr Gly Leu Glu Glu Cys Thr Cys Ala Ser Ser Asp Gly Lys Asp Asp Lys Glu Leu Cys His Val Cys Cys Met Lys Lys Met Asp Pro Ser Thr Cys Ala Ser Thr Gly Ser Val Gln Trp Ser Arg His Phe Ser Gly Arg Thr Ile Thr Leu Gln Pro Gly Ser Pro Cys Asn Asp Phe Arg Gly Tyr Cys Asp Val Phe Met Arg Cys Arg Leu Val Asp Ala Asp Gly Pro Leu Ala Arg Leu 3~5 390 395 400 Lys Lys Ala Ile Phe Ser Pro Glu Leu Tyr Glu Asn Ile Ala Glu Trp Ile Val Ala His Trp Trp Ala Val Leu Leu Met Gly Ile Ala Leu Ile Met Leu Met Ala Gly Phe Ile Lys Ile Cys Ser Val His Thr Pro Ser W O 97131931 PCTrUS97103217 Ser Asn Pro Lys Leu Pro Pro Pro Lys Pro Leu Pro Gly Thr Leu Lys Arg Arg Arg Pro Pro Gln Pro Ile Gln Gln Pro Gln Arg Gln Arg Pro Arg Glu Ser Tyr Gln Met Gly His Met Arg Arg (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2763 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
~A) NAME/KEY: CDS
(B) LOCATION: 17..2414 (xi) S~uu~ DESCRIeTION: SEQ ID NO:3:

Met Val Leu Leu Arg Val Leu Ile Leu Leu Leu Ser Trp Ala Ala Gly Met Gly Gly Gln Tyr Gly Asn Pro Leu Asn Lys 505 510 SlS
TAT ATC AGA CAT TAT GAA GGA TTA TCT TAC AAT GTG GAT TCA TTA CAC l45 Tyr Ile Arg His Tyr Glu Gly Leu Ser Tyr Asn Val Asp Ser Leu His Gln Lys His Gln Arg Ala Lys Arg Ala Val Ser His Glu Asp Gln Phe TTA CGT CTA GAT TTC CAT GCC QT GGA AGA QT TTC AAC CTA CGA ATG 24l Leu Arg Leu Asp Phe His Ala His Gly Arg His Phe Asn Leu Arg Met Lys Arg Asp Thr Ser Leu Phe Ser Asp Glu Phe Lys Val Glu Thr Ser Asn LYR Val Leu Asp Tyr Asp Thr Ser His Ile Tyr Thr Gly His Ile Tyr Gly Glu Glu Gly Ser Phe Ser His Gly Ser Val Ile Asp Gly Arg Phe Glu Gly Phe Ile Gln Thr Arg Gly Gly Thr Phe Tyr Val Glu Pro Ala Glu Arg Tyr Ile Lys Asp Arg Thr Leu Pro Phe His Ser Val Ile Tyr His Glu Asp Asp Ile Ser Glu Arg Leu Lys Leu Arg Leu Arg Lys Leu Met Ser Leu Glu Leu Trp Thr Ser Cys Cys Leu Pro Cys Ala Leu Leu Leu His Ser Trp Lys Lys Ala Val Asn Ser His Cys Leu Tyr Phe Lys Asp Phe Trp Gly Phe Ser Glu Ile Tyr Tyr Pro His Lys Tyr Gly Pro Gln Gly Gly Cys Ala Asp His Ser Val Phe Glu Arg Met Arg Lys Tyr Gln Met Thr Gly Val Glu Glu Val Thr Gln Ile Pro Gln Glu Glu His Ala Ala Asn Gly Pro Glu Leu Leu Arg Lys Arg Arg Thr Thr Ser Ala Glu Lys Asn Thr Cys Gln Leu Tyr Ile Gln Thr Asp His Leu Phe Phe Lys Tyr Tyr Gly Thr Arg Glu Ala Val Ile Ala Gln Ile Ser Ser His Val Lys Ala Ile Asp Thr Ile Tyr Gln Thr Thr Asp Phe Ser Gly Ile Arg Asn Ile Ser Phe Met Val Lys Arg Ile Arg Ile Asn Thr Thr Ala Asp Glu Lys Asp Pro Thr Asn Pro Phe Arg Phe Pro Asn Ile Ser Val Glu Lys Phe Leu Glu Leu Asn Ser Glu Gln Asn His Asp Asp Tyr Cys Leu Ala Tyr Val Phe Thr Asp Arg Asp Phe Asp Asp Gly Val Leu Gly Leu Ala Trp Val Gly Ala Pro Ser Gly Ser Ser Gly Gly Ile Cys Glu Lys Ser Lys Leu Tyr Ser Asp Gly Lys Lys Ly~ Ser Leu Asn Thr Gly Ile Ile Thr Val Gln Asn Tyr Gly Ser His Val Pro Pro Lys Val Ser His Ile Thr Phe Ala His Glu Val Gly His Asn Phe Gly Ser Pro His Asp Ser Gly Thr Glu Cys Thr Pro Gly Glu Ser Lys Asn Leu Gly Gln Lys Glu Asn Gly Asn Tyr Ile Met Tyr Ala Arg Ala Thr Ser Gly Asp Lys Leu Asn Asn Asn Lys Phe Ser Leu Cys Ser Ile Arg Asn Ile Ser Gln Val Leu Glu Lys Lys Arg Asn Asn Cys Phe Val Glu Ser Gly Gln Pro Ile Cys Gly Asn Gly Met Val Glu Gln Gly Glu Glu Cys Asp Cys Gly Tyr Ser Asp Gln Cys Lys Asp Glu Cys Cys Phe Asp Ala Asn Gln Pro Glu Gly Arg Lys Cys Lys Leu Lys Pro Gly Lys Gln Cys Ser Pro Ser Gln Gly Pro Cys Cys Thr Ala Gln Cys Ala Phe Lys Ser Lys lOS0 lOSS 1060 Ser Glu Lys Cys Arg Asp Asp Ser Asp Cys Ala Arg Glu Gly Ile Cys W O 97t31931 PCT~US97103217 Asn Gly Phe Thr Ala Leu Cys Pro Ala Ser Asp Pro Lys Pro Asn Phe Thr Asp Cys Asn Arg His Thr Gln Val Cys Ile Asn Gly Gln Cys Ala GGT TCT ATC TGT GAG AAA TAT GGC TTA GAG GAG TGT ACG TGT GCC AGT l9Z1 Gly Ser Ile Cys Glu Lys Tyr Gly Leu Glu Glu Cys Thr Cys Ala Ser Ser Asp Gly Lys Asp Asp Lys Glu Leu Cys His Val Cys Cys Met Lys Lys Met Asp Pro Ser Thr Cys Ala Ser Thr Gly Ser Val Gln Trp Ser Arg His Phe Ser Gly Arg Thr Ile Thr Leu Gln Pro Gly Ser Pro Cys Asn Asp Phe Arg Gly Tyr Cys Asp Val Phe Met Arg Cys Arg Leu Val Asp Ala Asp Gly Pro Leu Ala Arg Leu Lys Lys Ala Ile Phe Ser Pro Glu Leu Tyr Glu Asn Ile Ala Glu Trp Ile Val Ala His Trp Trp Ala Val Leu Leu Met Gly Ile Ala Leu Ile Met Leu Met Ala Gly Phe Ile Lys Ile Cys Ser Val His Thr Pro Ser Ser Asn Pro Lys Leu Pro Pro Pro Lys Pro Leu Pro Gly Thr Leu Lys Arg Arg Arg Pro Pro Gln Pro Ile Gln Gln Pro Gln Arg Gln Arg Pro Arg Glu Ser Tyr Gln Met Gly CAC ATG AGA CGC T AACTGCAGCT .,,GC~GG ~ C~AG TGCCTACAAT 2454 His Met Arg Arg W O 97/31931 PCT~US97/03217 (2) INFORMATION FOR SEQ ID NO:4:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 799 amino acids ~B) TYPE: amino acid ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Val Leu Leu Arg Val Leu Ile Leu Leu Leu Ser Trp Ala Ala Gly Met Gly Gly Gln Tyr Gly Asn Pro Leu Asn Lys Tyr Ile Arg His Tyr Glu Gly Leu Ser Tyr Asn Val Asp Ser Leu His Gln Lys His Gln Arg Ala Lys Arg Ala Val Ser His Glu Asp Gln Phe Leu Arg Leu Asp Phe His Ala His Gly Arg His Phe Asn Leu Arg Met Lys Arg Asp Thr Ser Leu Phe Ser Asp Glu Phe Lys Val Glu Thr Ser Asn Lys Val Leu Asp Tyr Asp Thr Ser His Ile Tyr Thr Gly His Ile Tyr Gly Glu Glu Gly Ser Phe Ser His Gly Ser Val Ile Asp Gly Arg Phe Glu Gly Phe Ile Gln Thr Arg Gly Gly Thr Phe Tyr Val Glu Pro Ala Glu Arg Tyr Ile Lys Asp Arg Thr Leu Pro Phe His Ser Val Ile Tyr His Glu Asp Asp Ile Ser Glu Arg Leu Lys Leu Arg Leu Arg Lys Leu Met Ser Leu Glu Leu Trp Thr Ser Cys Cys Leu Pro Cys Ala Leu Leu Leu His Ser Trp Lys Lys Ala Val Asn Ser His Cys Leu Tyr Phe Lys Asp Phe Trp Gly W O 97/31931 PCT~US97/03217 Phe Ser Glu Ile Tyr Tyr Pro His Lys Tyr Gly Pro Gln Gly Gly Cys Ala Asp His Ser Val Phe Glu Arg Met Arg Lys Tyr Gln Met Thr Gly Val Glu Glu Val Thr Gln Ile Pro Gln Glu Glu His Ala Ala Asn Gly Pro Glu Leu Leu Arg Lys Arg Arg Thr Thr Ser Ala Glu Lys Asn Thr - Cys Gln Leu Tyr Ile Gln Thr Asp His Leu Phe Phe Lys Tyr Tyr Gly 275 280 285 Thr Arg Glu Ala Val Ile Ala Gln Ile Ser Ser His Val Lys Ala Ile Asp Thr Ile Tyr Gln Thr Thr Asp Phe Ser Gly Ile Arg Asn Ile Ser Phe Met Val Lys Arg Ile Arg Ile Asn Thr Thr Ala Asp Glu Lys Asp Pro Thr Asn Pro Phe Arg Phe Pro Asn Ile Ser Val Glu Lys Phe Leu Glu Leu Asn Ser Glu Gln Asn His Asp Asp Tyr Cy5 Leu Ala Tyr Val Phe Thr Asp Arg Asp Phe Asp Asp Gly Val Leu Gly Leu Ala Trp Val Gly Ala Pro Ser Gly Ser Ser Gly Gly Ile Cys Glu Lys Ser Lys Leu Tyr Ser A~p Gly Lys Lys Lys Ser Leu Asn Thr Gly Ile Ile Thr Val Gln Asn Tyr Gly Ser His Val Pro Pro Lys Val Ser His Ile Thr Phe Ala His Glu Val Gly His Asn Phe Gly Ser Pro His Asp Ser Gly Thr Glu Cys Thr Pro Gly Glu Ser Lys Asn Leu Gly Gln Lys Glu Asn Gly Asn Tyr Ile Met Tyr Ala Arg Ala Thr Ser Gly Asp Lys Leu Asn Asn Asn Lys Phe Ser Leu Cys Ser Ile Arg Asn Ile Ser Gln Val Leu Glu Lys Lys Arg Asn Asn Cys Phe Val Glu Ser Gly Gln Pro Ile Cys Gly Asn Gly Met Val Glu Gln Gly Glu Glu Cys Asp Cys Gly Tyr Ser Asp W O 97131931 PCTrUS97/03217 Gln Cys Lys Asp Glu Cys Cys Phe Asp Ala Asn Gln Pro Glu Gly Arg Lys Cys Lys Leu Lys Pro Gly Lys Gln Cys Ser Pro Ser Gln Gly Pro Cys Cys Thr Ala Gln Cys Ala Phe Lys Ser Lys Ser Glu Lys Cys Arg Asp Asp Ser Asp Cys Ala Arg Glu Gly Ile Cys Asn Gly Phe Thr Ala Leu Cys Pro Ala Ser Asp Pro Lys Pro Asn Phe Thr Asp Cys Asn Arg His Thr Gln Val Cys Ile Asn Gly Gln Cys Ala Gly Ser Ile Cys Glu Lys Tyr Gly Leu Glu Glu Cys Thr Cys Ala Ser Ser Asp Gly Lys Asp Asp Lys Glu Leu Cys His Val Cys Cys Met Lys Lys Met Asp Pro Ser Thr Cys Ala Ser Thr Gly Ser Val Gln Trp Ser Arg His Phe Ser Gly Arg Thr Ile Thr Leu Gln Pro Gly Ser Pro Cys Asn Asp Phe Arg Gly Tyr Cys Asp Val Phe Met Arg Cys Arg Leu Val Asp Ala Asp Gly Pro Leu Ala Arg Leu Lys Lys Ala Ile Phe Ser Pro Glu Leu Tyr Glu Asn Ile Ala Glu Trp Ile Val Ala His Trp Trp Ala Val Leu Leu Met Gly Ile Ala Leu Ile Met Leu Met Ala Gly Phe Ile Lys Ile Cys Ser Val His Thr Pro Ser Ser Asn Pro Lys Leu Pro Pro Pro Lys Pro Leu Pro Gly Thr Leu Lys Arg Arg Arg Pro Pro Gln Pro Ile Gln Gln Pro Gln Arg Gln Arg Pro Arg Glu Ser Tyr Gln Met Gly His Met Arg Arg

Claims (22)

WHAT IS CLAIMED IS:

1. A DNA fragment encoding a human disintegrin expressed differentially during arthritis development, capable as being used as a screen disintegrin antagonism, drug design and screening.

2. A human disintegrin according to Claim 1 of a molecular weight, and solubility useful as a drug screening agent.

3. A human disintegrin according to Claim 1 in essentially pure form.

4. A screening method for compounds capable of binding to a human disintegrin, comprising the disintegrin of Claim 1.

5. A screening kit for compounds capable of binding to a human disintegrin, comprising the disintegrin of Claim 1.

6. An antibody, or fragment thereof, to human disintegrin of Claim 1.

7. A screening method for a metalloprotease mediated disease comprising the administration of an antibody according to Claim 6 and observing its effect.

8. A screening method for osteoarthritis comprising the administration of an antibody according to Claim 7 and observing its effect.

9. A screening method for osteoarthritis according to Claim 7, where blood, synovial fluid or other body fluids are screened.

10. A screening kit for osteoarthritis comprising an antibody, or fragment thereof, to human disintegrin of Claim 6.

11. A screening method, according to Claim 4, useful in determining relative potency in treating osteoarthritis.

12. DNA encoding the disintegrin of Claim 1 (Seq ID NO 2).

13. An expression vector or plasmid comprising the DNA of Claim 12.

14. A cell comprising the DNA of Claim 12.

15. A cell comprising the expression vector or plasmid of Claim 13.

16. The cell of Claim 14 where the DNA is foreign to that cell.

17. An inhibitor of the human disintegrin of Claim 2.

18. A method of treating a disease state associated with disintegrin activity.

19. The disintegrin of Claim 2, wherein the disintegrin is aggrocanase.

20. A method of treating a disease state according to Claim 18 wherein the disease is an arthropothy.

21. A method according to Claim 20, wherein the disease is osteoarthritis.

22. The disintegrin of Claim 2, wherein the disintegrin modulates tissue remodeling or breakdown.