Note: Descriptions are shown in the official language in which they were submitted.
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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
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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.
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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
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"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.
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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~.
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,
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.
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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
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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
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(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
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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
CA 02247827 1998-08-31
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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.
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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:
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13
C CAG ACC ACA GAC TTC TCC GGA ATC CGT AAC ATC AGT TTC ATG GTG 46
Gln Thr Thr Asp Phe Ser Gly Ile Arg Asn Ile Ser Phe Met Val
1 5 10 15
AAA CGC ATA AGA ATC AAT AGA ACT GCT GAT GAG AAG GAC CCT A Q AAT 94
Lys Arg Ile Arg Ile Asn Thr Thr Ala Asp Glu Lys Asp Pro Thr Asn
20 25 30
CCT TTC CGT TTC CCA AAT ATT AGT GTG GAG AAG TTT CTG GAA TTG AAT 142
Pro Phe Arg Phe Pro Asn Ile Ser Val Glu Lys Phe Leu Glu Leu Asn
35 40 45
TCT GAG Q G AAT CAT GAT GAC TAC TGT TTG GCC TAT GTC TTC AGA GAG 190
Ser Glu Gln Asn His Asp Asp Tyr Cys Leu Ala Tyr Val Phe Thr Asp
50 55 60
CGA GAT TTT GAT GAT GGC GTA CTT GGT CTG GCT TGG GTT GGA GCA CCT 238
Arg Asp Phe Asp Asp Gly Val Leu Gly Leu Ala Trp Val Gly Ala Pro
65 70 75
TCA GGA AGC TCT GGA GGA ATA TGT GAA AAA AGT AAA CTC TAT TCA GAT 286
Ser Gly Ser Ser Gly Gly Ile Cys Glu Lys Ser Lys Leu Tyr Ser Asp
80 85 90 95
GGT AAG AAG AAG TCC TTA AAC ACT GGA ATT ATT ACT GTT CAG AAC TAT 334
Gly Lys Lys Lys Ser Leu Asn Thr Gly Ile Ile Thr Val Gln Asn Tyr
100 105 110
GGG TCT QT GTA CCT CCC AAA GTC TCT QC ATT ACT TTT GCT QC GAA 382
Gly Ser His Val Pro Pro Lys Val Ser His Ile Thr Phe Ala His Glu
115 120 125
GTT GGA QT AAC TTT GGA TCC CCA QT GAT TCT GGA ACA GAG TGC A Q 430
Val Gly His Asn Phe Gly Ser Pro His Asp Ser Gly Thr Glu Cys Thr
130 135 140
CCA GGA GAA TCT AAG AAT TTG GGT QA AAA GAA AAT GGC AAT TAC ATC 478
Pro Gly Glu Ser Lys Asn Leu Gly Gln Lys Glu Asn Gly Asn Tyr Ile
145 150 155
ATG TAT G Q AGA G Q A Q TCT GGG GAC AAA CTT AAC AAC AAT AAA TTC 526
Met Tyr Ala Arg Ala Thr Ser Gly Asp Lys Leu Asn Asn Asn Lys Phe
160 165 170 175
TCA CTC TGT AGT ATT AGA AAT ATA AGC QA GTT CTT GAG AAG AAG AGA 574
Ser Leu Cys Ser Ile Arg Asn Ile Ser Gln Val Leu Glu Lys Lys Arg
180 185 190
AAC AAC TGT TTT GTT GAA TCT GGC CAA CCT ATT TGT GGA AAT GGA ATG 622
Asn Asn Cys Phe Val Glu Ser Gly Gln Pro Ile Cys Gly Asn Gly Met
195 200 205
GTA GAA QA GGT GAA GAA TGT GAT TGT GGC TAT AGT GAC QG TGT AAA 670
Val Glu Gln Gly Glu Glu Cys Asp Cys Gly Tyr Ser Asp Gln Cys Lys
210 215 220
GAT GAA TGC TGC TTC GAT G Q AAT CAA CCA GAG GGA AGA AAA TGC AAA 718
Asp Glu Cys Cys Phe Asp Ala Asn Gln Pro Glu Gly Arg Lys Cys Lys
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225 230 235
CTG AAA CCT GGG AAA CAG TGC AGT CCA AGT CAA GGT CCT TGT TGT ACA 766
Leu Lys Pro Gly Lys Gln Cys Ser Pro Ser Gln Gly Pro Cys Cys Thr
240 245 250 255
GCA CAG TGT GCA TTC AAG TCA AAG TCT GAG AAG TGT CGG GAT GAT TCA 814
Ala Gln Cys Ala Phe Lys Ser Lys Ser Glu Lys Cys Arg Asp Asp Ser
260 265 270
GAC TGT GCA AGG GAA GGA ATA TGT AAT GGC TTC ACA GCT CTC TGC CCA 8 62
Asp Cys Ala Arg Glu Gly Ile Cys Asn Gly Phe Thr Ala Leu Cys Pro
275 280 285
GQ TCT GAC CCT AAA CCA AAC TTC AQ GAC TGT AAT AGG CAT AQ CAA 910
Ala Ser Asp Pro Lys Pro Asn Phe Thr Asp Cys Asn Arg His Thr Gln
290 295 300
GTG TGC ATT AAT GGG CAA TGT GQ GGT TCT ATC TGT GAG AAA TAT GGC 958
Val Cys Ile Asn Gly Gln Cys Ala Gly Ser Ile Cys Glu Lys Tyr Gly
305 310 315
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
320 325 330 335
TTA TGC CAT GTA TGC TGT ATG AAG AAA ATG GAC CCA TCA ACT TGT GCC 1054
Leu Cys His Val Cys Cys Met Lys Lys Met Asp Pro Ser Thr Cys Ala
340 345 350
AGT AQ GGG TCT GTG CAG TGG AGT AGG CAC TTC AGT GGT CGA ACC ATC 1102
Ser Thr Gly Ser Val Gln Trp Ser Arg His Phe Ser Gly Arg Thr Ile
. 355 360 365
ACC CTG CAA CCT GGA TCC CCT TGC AAC GAT TTT AGA GGT TAC TGT GAT 1150
Thr Leu Gln Pro Gly Ser Pro Cys Asn Asp Phe Arg Gly Tyr Cys Asp
370 375 380
GTT TTC ATG CGG TGC AGA TTA GTA GAT GCT GAT GGT CCT CTA GCT AGG 1198
Val Phe Met Arg Cys Arg Leu Val Asp Ala Asp Gly Pro Leu Ala Arg
385 390 395
CTT AAA AAA GCA ATT TTT AGT CCA GAG CTC TAT GAA AAC ATT GCT GAA 1246
Leu Lys Lys Ala Ile Phe Ser Pro Glu Leu Tyr Glu Asn Ile Ala Glu
400 405 410 415
TGG ATT GTG GCT CAT TGG TGG GQ GTA TTA CTT ATG GGA ATT GCT CTG 1294
Trp Ile Val Ala His Trp Trp Ala Val Leu Leu Met Gly Ile Ala Leu
420 425 430
ATC ATG CTA ATG GCT GGA TTT ATT AAG ATA TGC AGT GTT QT ACT CCA 1342
Ile Met Leu Met Ala Gly Phe Ile Lys Ile Cys Ser Val His Thr Pro
435 440 445
AGT AGT AAT CCA AAG TTG CCT CCT CCT AAA CCA CTT CCA GGC ACT TTA 1390
Ser Ser Asn Pro Lys Leu Pro Pro Pro Lys Pro Leu Pro Gly Thr Leu
450 455 460
AAG AGG AGG AGA CCT CCA CAG CCC ATT QG CAA CCC CAG CGT CAG CGG 1438
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Lys Arg Arg Arg Pro Pro Gln Pro Ile Gln Gln Pro Gln Arg Gln Arg
465 470 475
CCC CGA GAG AGT TAT CAA ATG GGA QC ATG AGA CGC TAACTGCAGC 1484
Pro Arg Glu Ser Tyr Gln Met Gly His Met Arg Arg
480 485 490
TTTTGCCTTG GTTCTTCCTA GTGCCTACAA TGGGAAAACT TCACTCCAAA GAGAAACCTA 1544
TTAAGTCATC ATCTCCAAAC TAAACCCTCA CAAGTAACAG TTGAAGAAAA AATGGCAAGA 1604
GATCATATCC TCAGACCAGG TGGAATTACT TAAATTTTAA AGCCTGAAAA TTCCAATTTG 1664
GGGGTGGGAG GTGGAAAAGG AACCCAATTT TCTTATGAAC AGATATTTTT AACTTAATGG 1724
CACAAAGTCT TAGAATATTA TTATGTGCCC C~l~lCCCT ~.Cl.C~.. GCTGCATTTT 1784
CTTCACTTGC AGGCAAACTT GGCTCTCAAT AAACTTTTCG GTCCAGACCA CAGACTTCTC 1844
CGGAATCCGT AACATCAGTT TCATGGTGAA ACGCATAAGA ATCAATACAA CTGCTGATGA 1904
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
1 5 10 15
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
100 105 110
Ser His Val Pro Pro Lys Val Ser His Ile Thr Phe Ala His Glu Val
115 120 125
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Gly His Asn Phe Gly Ser Pro His Asp Ser Gly Thr Glu Cys Thr Pro
130 135 140
Gly Glu Ser Lys Asn Leu Gly Gln Lys Glu Asn Gly Asn Tyr Ile Met
145 150 155 160
Tyr Ala Arg Ala Thr Ser Gly Asp Lys Leu Asn Asn Asn Lys Phe Ser
165 170 175
Leu Cys Ser Ile Arg Asn Ile Ser Gln Val Leu Glu Lys Lys Arg Asn
180 185 190
Asn Cys Phe Val Glu Ser Gly Gln Pro Ile Cys Gly Asn Gly Met Val
195 200 205
Glu Gln Gly Glu Glu Cys Asp Cys Gly Tyr Ser Asp Gln Cys Lys Asp
210 215 220
Glu Cys Cys Phe Asp Ala Asn Gln Pro Glu Gly Arg Lys Cys Lys Leu
225 230 235 240
Lys Pro Gly Lys Gln Cys Ser Pro Ser Gln Gly Pro Cys Cys Thr Ala
245 250 255
Gln Cys Ala Phe Lys Ser Lys Ser Glu Lys Cys Arg Asp Asp Ser Asp
260 265 270
Cys Ala Arg Glu Gly Ile Cys Asn Gly Phe Thr Ala Leu Cys Pro Ala
275 280 285
Ser Asp Pro Lys Pro Asn Phe Thr Asp Cys Asn Arg His Thr Gln Val
290 295 300
Cys Ile Asn Gly Gln Cys Ala Gly Ser Ile Cys Glu Lys Tyr Gly Leu
305 310 315 320
Glu Glu Cys Thr Cys Ala Ser Ser Asp Gly Lys Asp Asp Lys Glu Leu
325 330 335
Cys His Val Cys Cys Met Lys Lys Met Asp Pro Ser Thr Cys Ala Ser
340 345 350
Thr Gly Ser Val Gln Trp Ser Arg His Phe Ser Gly Arg Thr Ile Thr
355 360 365
Leu Gln Pro Gly Ser Pro Cys Asn Asp Phe Arg Gly Tyr Cys Asp Val
370 375 380
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
405 410 415
Ile Val Ala His Trp Trp Ala Val Leu Leu Met Gly Ile Ala Leu Ile
420 425 430
Met Leu Met Ala Gly Phe Ile Lys Ile Cys Ser Val His Thr Pro Ser
435 440 445
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Ser Asn Pro Lys Leu Pro Pro Pro Lys Pro Leu Pro Gly Thr Leu Lys
450 455 460
Arg Arg Arg Pro Pro Gln Pro Ile Gln Gln Pro Gln Arg Gln Arg Pro
465 470 475 480
Arg Glu Ser Tyr Gln Met Gly His Met Arg Arg
485 490
(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:
GGCGGCGGCA CGGAAG ATG GTG TTG CTG AGA GTG TTA ATT CTG CTC CTC 49
Met Val Leu Leu Arg Val Leu Ile Leu Leu Leu
495 500
TCC TGG GCG GCG GGG ATG GGA GGT QG TAT GGG AAT CCT TTA AAT AAA 97
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
520 525 530
QA AAA Q C QG CGT GCC AAA AGA G Q GTC TCA QT GAA GAC QA TTT 193
Gln Lys His Gln Arg Ala Lys Arg Ala Val Ser His Glu Asp Gln Phe
535 540 545 550
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
555 560 565
AAG AGG GAC ACT TCC CTT TTC AGT GAT GAA TTT AAA GTA GAA A Q T Q 289
Lys Arg Asp Thr Ser Leu Phe Ser Asp Glu Phe Lys Val Glu Thr Ser
570 575 580
AAT AAA GTA CTT GAT TAT GAT ACC TCT QT ATT TAC ACT GGA QT ATT 337
Asn LYR Val Leu Asp Tyr Asp Thr Ser His Ile Tyr Thr Gly His Ile
585 590 595
TAT GGT GAA GAA GGA AGT TTT AGC CAT GGG TCT GTT ATT GAT GGA AGA 385
Tyr Gly Glu Glu Gly Ser Phe Ser His Gly Ser Val Ile Asp Gly Arg
600 605 610
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18
TTT GAA GGA TTC ATC CAG ACT CGT GGT GGC ACA TTT TAT GTT GAG CCA 433
Phe Glu Gly Phe Ile Gln Thr Arg Gly Gly Thr Phe Tyr Val Glu Pro
615 620 625 630
G Q GAG AGA TAT ATT AAA GAC CGA ACT CTG CCA TTT CAC TCT GTC ATT 481
Ala Glu Arg Tyr Ile Lys Asp Arg Thr Leu Pro Phe His Ser Val Ile
635 640 645
TAT CAT GAA GAT GAT ATT AGT GAA AGG CTT AAA CTG AGG CTT AGA AAA 529
Tyr His Glu Asp Asp Ile Ser Glu Arg Leu Lys Leu Arg Leu Arg Lys
650 655 660
CTT ATG TCA CTT GAG TTG TGG ACC TCC TGT TGT TTA CCC TGT GCT CTT 577
Leu Met Ser Leu Glu Leu Trp Thr Ser Cys Cys Leu Pro Cys Ala Leu
665 670 675
CTG CTT CAC TCA TGG AAG AAA GCT GTA AAT TCT CAC TGC CTT TAC TTC 625
Leu Leu His Ser Trp Lys Lys Ala Val Asn Ser His Cys Leu Tyr Phe
680 685 690
AAG GAT TTC TGG GGC TTT TCT GAA ATC TAC TAT CCC CAT AAA TAC GGT 673
Lys Asp Phe Trp Gly Phe Ser Glu Ile Tyr Tyr Pro His Lys Tyr Gly
695 700 705 710
CCT CAG GGC GGC TGT GCA GAT CAT TCA GTA TTT GAA AGA ATG AGG AAA 721
Pro Gln Gly Gly Cys Ala Asp His Ser Val Phe Glu Arg Met Arg Lys
715 720 725
TAC CAG ATG ACT GGT GTA GAG GAA GTA ACA CAG ATA CCT CAA GAA GAA 769
Tyr Gln Met Thr Gly Val Glu Glu Val Thr Gln Ile Pro Gln Glu Glu
730 735 740
CAT GCT GCT AAT GGT CCA GAA CTT CTG AGG AAA AGA CGT ACA ACT TCA 817
His Ala Ala Asn Gly Pro Glu Leu Leu Arg Lys Arg Arg Thr Thr Ser
745 750 755
GCT GAA AAA AAT ACT TGT CAG CTT TAT ATT CAG ACT GAT CAT TTG TTC 865
Ala Glu Lys Asn Thr Cys Gln Leu Tyr Ile Gln Thr Asp His Leu Phe
760 765 770
TTT AAA TAT TAC GGA ACA CGA GAA GCT GTG ATT GCC QG ATA TCC AGT 913
Phe Lys Tyr Tyr Gly Thr Arg Glu Ala Val Ile Ala Gln Ile Ser Ser
775 780 785 790
CAT GTT AAA GCG ATT GAT ACA ATT TAC CAG ACC ACA GAC TTC TCC GGA 961
His Val Lys Ala Ile Asp Thr Ile Tyr Gln Thr Thr Asp Phe Ser Gly
795 800 805
ATC CGT AAC ATC AGT TTC ATG GTG AAA CGC ATA AGA ATC AAT ACA ACT 1009
Ile Arg Asn Ile Ser Phe Met Val Lys Arg Ile Arg Ile Asn Thr Thr
810 815 820
GCT GAT GAG AAG GAC CCT ACA AAT CCT TTC CGT TTC CCA AAT ATT AGT 1057
Ala Asp Glu Lys Asp Pro Thr Asn Pro Phe Arg Phe Pro Asn Ile Ser
825 830 835
GTG GAG AAG TTT CTG GAA TTG AAT TCT GAG QG AAT QT GAT GAC TAC 1105
Val Glu Lys Phe Leu Glu Leu Asn Ser Glu Gln Asn His Asp Asp Tyr
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840 845 850
TGT TTG GCC TAT GTC TTC ACA GAC CGA GAT TTT GAT GAT GGC GTA CTT 1153
Cys Leu Ala Tyr Val Phe Thr Asp Arg Asp Phe Asp Asp Gly Val Leu
855 860 865 870
GGT CTG GCT TGG GTT GGA GCA CCT TCA GGA AGC TCT GGA GGA ATA TGT 1201
Gly Leu Ala Trp Val Gly Ala Pro Ser Gly Ser Ser Gly Gly Ile Cys
875 880 885
GAA AAA AGT AAA CTC TAT TCA GAT GGT AAG AAG AAG TCC TTA AAC ACT 1249
Glu Lys Ser Lys Leu Tyr Ser Asp Gly Lys Lys Ly~ Ser Leu Asn Thr
890 895 900
GGA ATT ATT ACT GTT CAG AAC TAT GGG TCT CAT GTA CCT CCC AAA GTC 1297
Gly Ile Ile Thr Val Gln Asn Tyr Gly Ser His Val Pro Pro Lys Val
90S 910 915
TCT CAC ATT ACT TTT GCT CAC GAA GTT GGA CAT AAC TTT GGA TCC CCA 1345
Ser His Ile Thr Phe Ala His Glu Val Gly His Asn Phe Gly Ser Pro
920 925 930
CAT GAT TCT GGA ACA GAG TGC ACA CCA GGA GAA TCT AAG AAT TTG GGT 1393
His Asp Ser Gly Thr Glu Cys Thr Pro Gly Glu Ser Lys Asn Leu Gly
935 940 945 950
CAA AAA GAA AAT GGC AAT TAC ATC ATG TAT GCA AGA GCA ACA TCT GGG 1441
Gln Lys Glu Asn Gly Asn Tyr Ile Met Tyr Ala Arg Ala Thr Ser Gly
955 960 965
GAC AAA CTT AAC AAC AAT AAA TTC TCA CTC TGT AGT ATT AGA AAT ATA 14 B 9
Asp Lys Leu Asn Asn Asn Lys Phe Ser Leu Cys Ser Ile Arg Asn Ile
970 975 980
AGC CAA GTT CTT GAG AAG AAG AGA AAC AAC TGT TTT GTT GAA TCT GGC 1537
Ser Gln Val Leu Glu Lys Lys Arg Asn Asn Cys Phe Val Glu Ser Gly
985 990 995
CAA CCT ATT TGT GGA AAT GGA ATG GTA GAA CAA GGT GAA GAA TGT GAT 1585
Gln Pro Ile Cys Gly Asn Gly Met Val Glu Gln Gly Glu Glu Cys Asp
1000 1005 1010
TGT GGC TAT AGT GAC CAG TGT AAA GAT GAA TGC TGC TTC GAT GCA AAT 1633
Cys Gly Tyr Ser Asp Gln Cys Lys Asp Glu Cys Cys Phe Asp Ala Asn
1015 1020 1025 1030
CAA CCA GAG GGA AGA AAA TGC AAA CTG AAA CCT GGG AAA CAG TGC AGT 1681
Gln Pro Glu Gly Arg Lys Cys Lys Leu Lys Pro Gly Lys Gln Cys Ser
1035 1040 1045
CCA AGT CAA GGT CCT TGT TGT ACA GCA CAG TGT GCA TTC AAG TCA AAG 1729
Pro Ser Gln Gly Pro Cys Cys Thr Ala Gln Cys Ala Phe Lys Ser Lys
lOS0 lOSS 1060
TCT GAG AAG TGT CGG GAT GAT TCA GAC TGT GCA AGG GAA GGA ATA TGT 1777
Ser Glu Lys Cys Arg Asp Asp Ser Asp Cys Ala Arg Glu Gly Ile Cys
1065 1070 1075
AAT GGC TTC ACA GCT CTC TGC CCA GCA TCT GAC CCT AAA CCA AAC TTC 1825
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Asn Gly Phe Thr Ala Leu Cys Pro Ala Ser Asp Pro Lys Pro Asn Phe
1080 1085 1090
ACA GAC TGT AAT AGG CAT ACA CAA GTG TGC ATT AAT GGG CAA TGT GCA 1873
Thr Asp Cys Asn Arg His Thr Gln Val Cys Ile Asn Gly Gln Cys Ala
1095 1100 1105 1110
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
1115 1120 1125
TCT GAT GGC AAA GAT GAT AAA GAA TTA TGC CAT GTA TGC TGT ATG AAG 1969
Ser Asp Gly Lys Asp Asp Lys Glu Leu Cys His Val Cys Cys Met Lys
1130 1135 1140
AAA ATG GAC CCA TCA ACT TGT GCC AGT ACA GGG TCT GTG CAG TGG AGT 2017
Lys Met Asp Pro Ser Thr Cys Ala Ser Thr Gly Ser Val Gln Trp Ser
1145 1150 1155
AGG CAC TTC AGT GGT CGA ACC ATC ACC CTG CAA CCT GGA TCC CCT TGC 20
Arg His Phe Ser Gly Arg Thr Ile Thr Leu Gln Pro Gly Ser Pro Cys
1160 1165 1170
AAC GAT TTT AGA GGT TAC TGT GAT GTT TTC ATG CGG TGC AGA TTA GTA 211
Asn Asp Phe Arg Gly Tyr Cys Asp Val Phe Met Arg Cys Arg Leu Val
1175 1180 1185 1190
GAT GCT GAT GGT CCT CTA GCT AGG CTT AAA AAA GCA ATT TTT AGT CCA 2161
Asp Ala Asp Gly Pro Leu Ala Arg Leu Lys Lys Ala Ile Phe Ser Pro
1195 1200 1205
GAG CTC TAT GAA AAC ATT GCT GAA TGG ATT GTG GCT CAT TGG TGG GCA 2209
Glu Leu Tyr Glu Asn Ile Ala Glu Trp Ile Val Ala His Trp Trp Ala
1210 1215 1220
GTA TTA CTT ATG GGA ATT GCT CTG ATC ATG CTA ATG GCT GGA TTT ATT 2257
Val Leu Leu Met Gly Ile Ala Leu Ile Met Leu Met Ala Gly Phe Ile
1225 1230 1235
AAG ATA TGC AGT GTT CAT ACT CCA AGT AGT AAT C Q AAG TTG CCT CCT 2305
Lys Ile Cys Ser Val His Thr Pro Ser Ser Asn Pro Lys Leu Pro Pro
1240 1245 1250
CCT AAA CCA CTT CCA GGC ACT TTA AAG AGG AGG AGA CCT CCA CAG CCC 2353
Pro Lys Pro Leu Pro Gly Thr Leu Lys Arg Arg Arg Pro Pro Gln Pro
1255 1260 1265 1270
ATT CAG CAA CCC CAG CGT CAG CGG CCC CGA GAG AGT TAT CAA ATG GGA 2401
Ile Gln Gln Pro Gln Arg Gln Arg Pro Arg Glu Ser Tyr Gln Met Gly
1275 1280 1285
CAC ATG AGA CGC T AACTGCAGCT .,,GC~GG ~ C~AG TGCCTACAAT 2454
His Met Arg Arg
1290
GGGAAAACTT CACTCCAAAG AGAAACCTAT TAAGTCATCA TCTCCAAACT AAACCCTCAC 2514
AAGTAACAGT TGAAGAAAAA ATGGCAAGAG ATCATATCCT CAGACCAGGT GGAATTACTT 2574
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AAATTTTAAA GCCTGAAAAT TCCAATTTGG GGGTGGGAGG TGGAAAAGGA ACCCAATTTT 2634
CTTATGAACA GATATTTTTA ACTTAATGGC ACAAAGTCTT AGAATATTAT TATGTGCCCC 2694
GTGTTCCCTG TTCTTCGTTG CTGCATTTTC TTCACTTGCA GGCAAACTTG GCTCTCAATA 2754
AACTTTTCG 2763
(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
1 5 10 15
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
100 105 110
Ser Phe Ser His Gly Ser Val Ile Asp Gly Arg Phe Glu Gly Phe Ile
115 120 125
Gln Thr Arg Gly Gly Thr Phe Tyr Val Glu Pro Ala Glu Arg Tyr Ile
130 135 140
Lys Asp Arg Thr Leu Pro Phe His Ser Val Ile Tyr His Glu Asp Asp
145 150 155 160
Ile Ser Glu Arg Leu Lys Leu Arg Leu Arg Lys Leu Met Ser Leu Glu
165 170 175
Leu Trp Thr Ser Cys Cys Leu Pro Cys Ala Leu Leu Leu His Ser Trp
180 185 190
Lys Lys Ala Val Asn Ser His Cys Leu Tyr Phe Lys Asp Phe Trp Gly
195 200 205
CA 02247827 1998-08-31
W O 97/31931 PCT~US97/03217
Phe Ser Glu Ile Tyr Tyr Pro His Lys Tyr Gly Pro Gln Gly Gly Cys
210 215 220
Ala Asp His Ser Val Phe Glu Arg Met Arg Lys Tyr Gln Met Thr Gly
225 230 235 240
Val Glu Glu Val Thr Gln Ile Pro Gln Glu Glu His Ala Ala Asn Gly
245 250 255
Pro Glu Leu Leu Arg Lys Arg Arg Thr Thr Ser Ala Glu Lys Asn Thr
260 265 270
- 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
290 295 300
Asp Thr Ile Tyr Gln Thr Thr Asp Phe Ser Gly Ile Arg Asn Ile Ser
305 310 315 320
Phe Met Val Lys Arg Ile Arg Ile Asn Thr Thr Ala Asp Glu Lys Asp
325 330 335
Pro Thr Asn Pro Phe Arg Phe Pro Asn Ile Ser Val Glu Lys Phe Leu
340 345 350
Glu Leu Asn Ser Glu Gln Asn His Asp Asp Tyr Cy5 Leu Ala Tyr Val
355 360 365
Phe Thr Asp Arg Asp Phe Asp Asp Gly Val Leu Gly Leu Ala Trp Val
370 375 380
Gly Ala Pro Ser Gly Ser Ser Gly Gly Ile Cys Glu Lys Ser Lys Leu
385 390 395 400
Tyr Ser A~p Gly Lys Lys Lys Ser Leu Asn Thr Gly Ile Ile Thr Val
405 410 415
Gln Asn Tyr Gly Ser His Val Pro Pro Lys Val Ser His Ile Thr Phe
420 425 430
Ala His Glu Val Gly His Asn Phe Gly Ser Pro His Asp Ser Gly Thr
435 440 445
Glu Cys Thr Pro Gly Glu Ser Lys Asn Leu Gly Gln Lys Glu Asn Gly
450 455 460
Asn Tyr Ile Met Tyr Ala Arg Ala Thr Ser Gly Asp Lys Leu Asn Asn
465 470 475 4B0
Asn Lys Phe Ser Leu Cys Ser Ile Arg Asn Ile Ser Gln Val Leu Glu
485 490 495
Lys Lys Arg Asn Asn Cys Phe Val Glu Ser Gly Gln Pro Ile Cys Gly
500 505 510
Asn Gly Met Val Glu Gln Gly Glu Glu Cys Asp Cys Gly Tyr Ser Asp
515 520 525
CA 02247827 1998-08-31
W O 97131931 PCTrUS97/03217
Gln Cys Lys Asp Glu Cys Cys Phe Asp Ala Asn Gln Pro Glu Gly Arg
530 535 540
Lys Cys Lys Leu Lys Pro Gly Lys Gln Cys Ser Pro Ser Gln Gly Pro
545 550 555 560
Cys Cys Thr Ala Gln Cys Ala Phe Lys Ser Lys Ser Glu Lys Cys Arg
565 570 575
Asp Asp Ser Asp Cys Ala Arg Glu Gly Ile Cys Asn Gly Phe Thr Ala
580 585 590
Leu Cys Pro Ala Ser Asp Pro Lys Pro Asn Phe Thr Asp Cys Asn Arg
595 600 605
His Thr Gln Val Cys Ile Asn Gly Gln Cys Ala Gly Ser Ile Cys Glu
610 615 620
Lys Tyr Gly Leu Glu Glu Cys Thr Cys Ala Ser Ser Asp Gly Lys Asp
625 630 635 640
Asp Lys Glu Leu Cys His Val Cys Cys Met Lys Lys Met Asp Pro Ser
645 650 655
Thr Cys Ala Ser Thr Gly Ser Val Gln Trp Ser Arg His Phe Ser Gly
660 665 670
Arg Thr Ile Thr Leu Gln Pro Gly Ser Pro Cys Asn Asp Phe Arg Gly
675 680 685
Tyr Cys Asp Val Phe Met Arg Cys Arg Leu Val Asp Ala Asp Gly Pro
690 695 700
Leu Ala Arg Leu Lys Lys Ala Ile Phe Ser Pro Glu Leu Tyr Glu Asn
705 710 715 720
Ile Ala Glu Trp Ile Val Ala His Trp Trp Ala Val Leu Leu Met Gly
725 730 735
Ile Ala Leu Ile Met Leu Met Ala Gly Phe Ile Lys Ile Cys Ser Val
740 745 750
His Thr Pro Ser Ser Asn Pro Lys Leu Pro Pro Pro Lys Pro Leu Pro
755 760 765
Gly Thr Leu Lys Arg Arg Arg Pro Pro Gln Pro Ile Gln Gln Pro Gln
770 775 780
Arg Gln Arg Pro Arg Glu Ser Tyr Gln Met Gly His Met Arg Arg
785 790 795
