CA2000048A1 - Peptides and antibodies that inhibit integrin-ligand bindin g - Google Patents

Abstract

ABSTRACT
Polypeptides which are derived from the Arg-Gly-Asp (RGD) binding portion of an Integrin beta subunit are disclosed as are their use for modulation of Integrin ligand binding. Anti-peptide antibodies, hybridomas secreting these antibodies, as well as methods of making and using such antibodies, and recombinant DNA molecules that define the structural gene coding for the polypeptides are also contemplated as within the scope of the present invention.

Description

O c t . S ~ 3 13 ~ ~1 G G ~ 1 ~. ~ r i ~ ~ ~~ 1 c _ 5 d, ~ 3 ~:13 p . ~.
Z'30(~U~

PF:Pl'lV~:S ~Nr) ANTI130DIF:~5 T}I~T lNIII~TT
lN~ Gl~lN-Lï(;ANl) 13TNnTN(-.

Te~
Th~ present invention rela~e~ to ~ polypeptide derlved from the Ar~-GIy-~p (RGD) ~nd;T1~3 region Or t~c Int~qrin beta ~ubunit and to the use of that polypept~d~
to modulate Integrin-ligand binding. Also contemplated are antibodies that immunoreact with the RGD-binding 10 region of ~n Integrin beta 6ubunit and the use of tho~e antibodles ~o modulate or detect Integrin-ligand binding o~ detect ligand bindin~ sit~s within ~tegrin~.
Baa~ound Cell adhesion generally involves recognition of 15 spe~ific adhesive proteins by cell surface receptors. A
family of cell sur~ace receptors of particular interest to ~he pre~ent inventlo~ are the Inte~rins.
According to Hynes, Cell, 4~:549-554 (lg87), Integrin~ are a functionally and Rtruct~rally related 20 group of receptors that interact with a wide variety of ligands includlng ex~racellular matrix glycoproteins, comple~ent and other ~ells. Integrin~ participate in ~ell-matrix and cell-cell adhesion in many physiologically important proce~ses including 25 ~mbryological dev~lopment, hemo3ta~i~, thrombo~is, wound healing, immune and nonimmune defense mechanisms a~d oncogeni~ tran~formation. Two human genetic disea es, Glazmann's thrombasthenia and leukocyte adhesion deficiency, affect members of the Integrin 30 family.
Structurally, Inte~rin~ are heterodimerio complexes ~ompri~ed of noncovalently associated alphaand beta su~units. Within the Integrin family there are recognized ~ubfamilie~ rela~ed ~y the presencR of a ~imilar beta ~u~unit and memb~r~ with~n each group are ~I c t . ~ 3 ~3 [~ 5:~-1 5. ~ r i ~ -a e. I =--=~ 4 ~: ~ 1 3 53 ~ P .
Zt)OU0'~8 dictingui~h~d by unlque alpha ~ubunits~
For ln~tance, reeent ev:idence indicates that an Integrin found on ~he ~urface of platelet~ and known as GPIIb-IIIa is one of ~e~eral adhesion receptors that have unique alpha .~ubunit~ but s~are a similar ~eta subunit and the functional property of recognizing the tripeptide amino acid re~idu~ sequence Arg-Gly-A~p (using single letter symbol~, RG~). Pytela et al., Scienae, 231:1559-15~2 (1986) and Ruo~l~hti e~ al., Cell, 44:517-518 (1986). In addition to GPlIb-IIIa, thie group of relate~
receptors includes the vitronectin receptor (VnR) and fibronectln receptor ~FnR) isolated from osteo~arcoma cells. [Pytela et al., ~ , 40:191-198 (1~85), and Pytela et al., e ~L~e~ ~9~95i~ ~L~ ~ B2 5766-5770 (1985).
The ~imilar functional, structural, and antigeni~
proper~ieS of the~e proteins ~uggests GPIIb-~IIa and VnR
are member~ of an Integrin ~ubfamily for which the de~ignation "cytoadhesin" has been proposed. Plow et al., ~ , 83:6002-6006 (198~).
Within the cytoadhesin group, distinct alpha subunits comblne with a common or very gimi~ar beta subunit, resulting i~ func~onally distinguishable receptors.
~insberg et al., J. Bio~l ~he~-, 2~2:5437-5440 (1987).
Fo~ example, GPIIb-IIIa iB a heterodimer co~plex ~omprised of alphA and beta subunit~. Jennings et al., J. Biol. Che~., 257:10458-10466 (1~82). The alpha subunit, GPIIb, consi~t~ o~ a heavy chain and a light chain that are linked together by disulfide bonds. The beta subunit, GPIIIa is a single chain polypeptlde of about 100 kDa. Phillip~ et ~ 9iL_5 252:2121-2126 (1977~. Cell ~urf~ce moleoules im~unologically related to GPIIb-IIIa have been id~nti~ied on a varie~y of cell type3. See Thiagaraj an et al., J. Clin. Inve~., 75:896-901 (198S); Plow et al., ~n~c~Qs~l E~QS~-N~ 5~d. Sci~ ~S~, 83:~002-6006 (1986); and Fitzgerald et al., J. Biol. C~emL, 2~0:10893-10896 (1985).
GPIIb-IIIa contributes ~o platelet function through interactions with RGD-containing protein~, i.e., proteinS containing an Arg-Gly-~sp amino acld residue sequence, ~uch as ~ihrinogen [Bennett et al~ 99 ~5L_~LL, USA, ~0:2417-2421 ~1983)], fibronectin ~in6b~rg et al., ~5~ 5,~., 71:~1g-624 (1983)], and von WillQbrand ~actor [Ruggeri et nl-, Pr W- N~tLl Acadr~ç~ USA, 7~:603a-6041 (1~82)3, and therefore is a ~o~ponent o~ the common platelet adhesive protein receptor [Pytela et al., Science, 231:1559-1562 ~1986) a~d Plow et al.~ ~ Biol. Che~., 259:5388-5391 (1984)~.
At least 2 other group~ of heterodimeria adhesion receptors have been i~entlfied in which a common beta subunit combine~ with a number of di~tinct alp~a ~ub~nits. One ~roup i8 round on leukocytes and has been ref~rred to as the leukocyte adhesion (Leucam) family and include~ LFA-l, Mac-1, and P150,s5. Sanchez-Madrid et al., J. Exp._Med., 158:1785-1803 (1983) an~ Sprlhg~r at al., olb~ nd Svm~ , 118:102-1~ 8~. The oth~r group i~ ~ore widely distributed and has been re~erred to as thQ VLA ~amily. ~emler et al., ~ 5L__Çh~m~, 262:3300-3309 (1987). The beta subunit of the ~A family [~Qmler et al., ~ oL~_5h~m~, 262:3300-3309 (1987)] in the chic~en has been cloned, ~equenced and designated "Integrin" ~TamXun ~t al., Cell, 46:271-282 ~19B6)]. The sequence of chicken Integrin i5 similar to that o~ GPII~a ~Fit2gerald et al., ~. Biol. Chem., 262:3936-~939 (1987)]
and 'o the beta subunit of th~ leukocyte adhesion family [Xi~himoto et al., Cell, 4~:681-690 (1987)]. Mor~over, partial ~equence~ o~ several alp~ subunits also indicate similaritie3. Gins~erg et ~1., J. Biol. Chç~., 2~2:54~7-5440 (1987) Suzuki et al., ~9~ 99~ Y~

3 ~ G '; ~ 1 oe r i ~ ~ 4 .~.-- 1 3 ~ ~I F . :=1 ;~()O~Q48 ~:nfil~-~618 (198fi)J ~rld C~l~ro ~t ~1., Pt~oc. NALl. ~c~d.
, 83:~351-~356 (1986).
The ~ite3 on GPIIb-I~Ia, or the other cytoadhe~ins, that are ~rucial for their functions a~
adhe~ion receptors are presently unknown. Several observations suggest that a func~ionally ~ignificant site on GPIlb-IIIa ls near the epitope defined by the monoclonal antibody PMI-1~ Thi~ antibody binds to the he~vy chain o~ GPIIb ~Shadle et al., ~. Cell. ~iol , g9:2056-2060 (1984)~ ~nd defines a region o~ GPIIb tha~
i~ a~sociated wlth ~everal distinet functiona activitieS. For in~tance, PMI-l inhibits adhesion o~
washed platelets to collagen. Shadle et al., J. Cell.
Bi~ 9:2056-20~0 (lg~4).

The invention relates to polypeptides ~herein al~o referred to as sub~ect polypeptide(s)~ of about 11 to about 90 amlno acid r~sidue~ in leng~h which are characterized ~8 having an amlno acid residue sequence homologous to a port~oh of the RGD-bindin~ region of an Integrln beta subunit.
The invention also relates to a polyclonal antibody which immunoreact~ ~ith a subject polypeptide as well as monoclonal antibodies that immunoreact wi~h an epitope formed by the RGD-binding region of an ~ntegrin betR subunit, Al~o contemplated within the scope of the present inventlon ar~ the hybridomas having the capacity to p~oduce a subject m~noclonal anti~ody.
Me~hods are contemplated fo~ modulating the adhesion, in vivo, of cells expressing an Integrin beta subunit to which the subj~c~ polypeptides correspond. In thi~ ~ethod cell adhesion is modulated using eithe~ the polypeptides or anti-polypeptide antibodies of the 3s pre~ent invention.

O ~ t . 1 :3 ~ ~ 3 1 ~ o G ~=. = æ ~ I ~ r i ~ ~ ~. I c _ 5 4 ~ F
Z~ J~

Further oontemplated i~ a nucleotide segment compri~ing n~ more than about 12,000 nuoleotide ba~e pairs inclùding a sequence defin~ng a structural gen~
coding for a sub~eot polypeptide. Also con~emplate~
<~ recombin~nt DNA molecu3e compri~ing ~ veçtor Operatively linked to a DNA segmen~ that defines a structural ~ene coding ~or a ~ubj ect polypeptide .
~ri~ ~g3~rlEtion of t.he Dr~wlna~
In the drawing~ forming ~ portlon of this disclosure:
Fiqure 1 lllu~trates the nucleotide base sequence and deduced ~mino ~cid residue equence of a DNA segment coding ~or the RGD-binding region of GPIIIa. Amino acid re8idue and nucleotide bas~ po~itions are indicated in the le~t and right margins, respectively. Fitzge~ald et ~1., J. Bi~l. Çh~m., 262:3936-3939 (1987).
Figure 2 illustrates the results of the cros~lin~ing of an RGD peptide to dis~e~e sites in the beta ~ubunit (~PIIIa) of an Integrin adheslon receptor.
The 1~5I-labeled RGD peptide, Fn-7 (20 ~M), was bound to plat~let (6 x 1o8/ml) for 45 min at 22-C, and crosslinked with BS3 (O.2 mM). The fragmentation patterns after proteolysis were analyzed by SDS-PAGE ~Laemmli, ~h~Y~, 227:680 (197D)~ and autoradiography. Lane 1, a typically SDS-PAGE analy5is t7.5~ gel, nonreducing condition~), show1ng the predominance of 12sI-Fn-7 cro~linking to the beta ~bunit (GPI~Ia) and its more minor c~o6slinking to the alpha subunit (GP~Ib) on thrombin-stimulated platelets. Lanes 2-7, GPIIIa:Fn-7 bands were excised from gels, 6ubjected to proteoly~is and then analyzed by SDS-PAGE ( 15~ gels, reducing conditions). ~or lanes 2-5, GPIIIA:Fn-7 bands were excised and di~e~tionS with V-8 protease were performed within the gel slice~ as describ~d in Cleveland et al., J. Biol. Chem , 252:1102 (1977)-2 ~ 3 ~ = æ r i e ~ .~ I ~ _ ~; 4 .~ ~ F I 1~
2~ 4!3 Lane~ 2 and 4 are the i~ta~t GPII~a:Fn-7 compl~xes from thrombin stimulated and non~timula~ed c~
re~pe~tively.
Lanes 3 and 5 are the V8 protea~e (2 ~g/ml) digR3~8 from ~timula~d and non6timulated cellq, respect.ively.
For lane~ 6 and 7, GPIIIa:Fn-7 wa~ extracted f~om the gel ~lice~ ln 2% SDS, ~.2 M Tri~, pH 7.8, p~ecipitated with ~0% acetone, and then dige-~ted with ln chymotrypsin. Lane 6 i~ intact GPI~Ia:Fn-7. Lane 7 i~
a~tex digestion wi~h 20 ~ ChymotrypSih for 1 hr ~t 22~C.
Note that no bond3 within the pept~de are ~usceptible to V8 protease. If the tyro6yl-glycyl peptide b~nd within l25~-Fn-7 wer~ cl~aved by chymotryp~in, the radio~ctivity o~ the tyrogine re~idue would ~main cro6Glinked to GP~IIa via the ly~ine residue.
F~gure 3 illu~trate~ the RGD cro~slinking ~ite re~ide~ in the 34 kDa NH2-termihal portion of the beta subunit (GPIIIa). 125I-Fn-7 was cro661inked to platelet~, 20 a~d the intact cells were then digestsd w1~h chymotrypsin tO.5 mg/ml ~or 4 hours at 22-C). The cell~ were recovtred by centr~fugation and analyzed on SDS-PAGE (10%
gels under nonredu~ing condltion~).
~anes 1 and 2 ~how ~he position of the ~2sI-Fn-7 peptide oros~linked to platelets based upon autoradiography without and With chymotryp~ln digestion, re6peotively. Lanes 3-8 are immunoblots of these platelet~ after transfer onto PVDF membranes. Lanes 3, 5 and 7 are from undige~ted platelets, and Lanes 4, ~ and 8 ar~ from chymotrypsin-dige~ted platele~s. Th~
immunoblots, developed with horeeradish peroxldase con~ugate~, w~re prob~d with polyclonal antibody to GPIlb-IIIa (lane~ 3-4), a monoclonal anti~ody (22C4~ to GPIIIa ~Ginsberg et al., J. Biol. Chem. 262:5437 (1987)], 35 (lanes 5 and 6) and an anti-peptid~ antibody made to ~

O ~ ~ 5' ~:: 1 '=. C~ r C:~ G ~ 1 -. ~ r- i e ~ .~ 5 ~ ~.-- 1 3 ~: ~ F . I I
Z(~(JU~48 region (amino acid re~idues 636-~54) in the COOH-terminal a~pects o~ GPIIIa.(lanes 7 and ~).
~ igure 4 lllustrate~ localization of the RGD
cro6~1in~ing site wit~in the beta s~bunit (GPIIIa). The diagram illu~trate~ the variou~ proteolytic fragments of the GPIIIa:Fn-7 isolated, their determlned NH~-terminal amlno acid sequence~ and the.ir location within the known structure ~ GPIIIa. In ~top 2, the NH2-t.er~inal 3~ kDa localiza~ion i~ based upon the chy~otrypti~ cleavage of lo intact platele~ (see Figure 2). GPIIIa:Fn-7 was extracte~ from gels and concentrated as indicated in Figure 2. GPIIIa:Fn-7 wa~ solubilized ~n 1% SDS, PBS, pH
7.3, a~ then dige~ted with c~ymotryp~in at a 1:1 (w/w) ~ubstrate/enzyme ratio ~or 24 h at 22~. Sample~ were boil~d to inactivate the enzyme, and then appl.ied to a ~-18 ~PLC re~ersR pha~e column, equilibrated in 0.1~
tri~luoroacetic acid and 10 ~M dithiothreitol. The pep~ide3 were eluted with an acetonitrile gra~ient containing 0.08~ trifluoroacetic acid and 10 ~M
dit~iothreitol. RadioaCtiVe peaks were pooled, concentrated and separated on SDS-PAGE u~lng a 15~ gel under reduolng condltions. The gels were tran~ferr~d to PVDF membrane~ a~ described for sequ~nce analy~Q~
[Matsudaira, J. ~iol. ~hem., 2fi2:1~035 (1987)] and then autoradiographed. Radioactive bands, at 23 and 14 kDa from the ~ymotryptic digest ~Step 3) and 10 and 8 kDa from the YB protease digest tsteP 4) were exci~ed and ~ubjected to N~l2-termi~al ~equence analyci~ in ~n Applied Biosy~tem Model 47sA gas-phase seq~enator. The size~ of the ~ragmentS and the position of the NH2-terminal re~iduos of each gragment are drawn to scale relative ~o GPlIIa. The open triangle~ repre3ent potential glyoo~ylation ~ite~, X in amino acid ~equences indicate uhdetermined re.idues, and the heavy bar indicates the locatlon of RGD cros~linking ~egionO

1~1 C ~ c~ 2 ~ 0 ~3 ~ 5~ = ~ r i c~ 5 4 ~-- 1 :3 f~ P 1 ~

Figure 5 illustrate~ the results obtained by RGD-affinity chromatogr~phy of platelet~ incubated in the a~sehce ~lane~ 1 ~ 2) and pregence lane~ 3 ~ 4 of chymotrypsin~ After incubation, the platelets were ~olubilized in octylg~ucoside, and passed over an RGD-affinity column substahtially as de cribed in Pytela et al., Science, 231:155~ (1986) and Lam et al., J Biol.
Che~,, 262:~47 (1987). The pa~s-through (l~ne~ 1 & 3) and the bound matorial (lanes 2 & 4) were analyzed on SDS-P~GE (7% gel~, non~reducing conditionS~. Gels were t~ansferred to PYDF membranes and then probed with anti-GPIIIa monoclonal antibody 22~4.
Flgure 6 illustrates the deduced amino aci~
re~idu~ ~equences taken rrom references Fltz~erald et al., J. ~1Q1. Chem., 262:3926 (1987), ~ishimoto e~ al., Cell, 48:6B1 (1987), Argrave~ et al, ~ Linl~
105;1183 (1987), Tamkun et al., C211, 4~:Z71 (1986), and D~Simone et al., ~ QL__5he=~, 2~3:5333 (1988) and are ali~ned by the GAP and PRET~Y computer program. Devereux et al., University o~ Wi~con~in, Sequen~e AnalysiS
19~7. The conseh6us sequence ~equir~s exact ma~ahes of at least four of the five ~equence~
compared.

A. ~inl~l~n~
Amino Aci~ Re~idue: The ~mino acid re~idu~s de~cribed here~n are pre~erred to be in the "L" isomeric form. How~ver, residue~ in the "D" i~omeric for~ can be ~ubstituted for any L-amino acid residue, as long as the desired functional property i~ retained by the polypeptlde, NH2 refers to the free amino group pre~ent at ~he amino terminu~ of a polypeptide. ~OOH refers to the free carboxyl group pre~ent at the carboxy terminus of the polypeptide. In keep~ng with standard polypeptide nomenclature, J _2iÇ~ h~=~, 243:3557-59, (19~9), O c t . i ' ~ [~ 1 a ~ r i ~ ~ 5 4 ~ 3 8 ~ P . 1 :3 Z~?OU~4~
_ 9 _ abbreviation~ ~or amino acid ~e~idues are a5 shown in the ~ollowing ~able o~ ~orrespondence:

TABLE OF CO~RESPONDENC~
~XM~Q~ ANINQ AslD

Y Tyr tyrosine G Gly glycine F ~he phenylalanine M Met methionine A . Ala alanine S Ser ~erlne I Ile i~oleuclne L Leu leu~ine ~ Thr threonine V Val valine P Pro proline K Lys lysine H Hi~ hi6tidine Q Gln glutamine E Glu glutamic acid W Trp tryptophan R Arg arginine D A~p aspartic acid N Asn ~paragine C Cy~ cy6teine It ~hould be noted that all amino acid residue ~equences are ~eprecented herein by formulae who~e le~t to right orlentation i~ in the con~entional direction of amino-terminu~ to carboxy-terminus. Furthermore, it ~hould be noted th~t a da~h at the beginning or end of an amino acid re~idu~ ~equence indicate~ a peptide bond to a ~urther sequence of one or more amino acid re3idue~, or n ~3 ~-1 [~ G -. ~ , I c- ~ r . 1 .
~f)()V~

to an amino-terminal NH2 group or to a car~oxy-terminal COO~ group.
Polypeptide and E~id~: Polypeptide and peptide are t~rms used interchangeably herein to designate a linear ~erie~ of no more than about. 90 amino acid r~ldu~ ~onneated ona to th~ ot.h~r ~y ~t.1dh hon~lf;
hntw~c~n t.~ nlph~ mlno ~nd cnrboxy ~r~up3 ~ ~dj.~c~nt re~ldue~.
~ ide an_ ucleotide: A ~onomeric unit of DNA
or RNA con6~ting of a ~ugar moiety (pento~e~, a pho~phate, ~nd ~ n~trogenous hete~ocyclic base~ The base i~ linked to the ~ugar moiety via the glycosidic carbon (l' carbon of the pentose) and that combination of base and sugar i~ a nucleo~ide. When ~he nucleoside cont~in6 a phosphate group bonded to the 3~ or 5' position Or the pentose it ls ~ef~rred to as a nucleotide.
Base Pair (bp): A hydrogen-bonded partner~hlp of adenine (A) with thymine (T), or of cytosine (C) with guanin~ (G) in a double stranded DNA molecule.
~çceptor: Receptor and receptor protein are terms used herein to ind~ate a biologically active proteinaceous molecu~e that specirically binds to (or wl~h) other molecules, referrod to as ligands, to form a receptor-ligand protein ~omplex.
Ligand: ~.igand refers to a molecule that contains a ~tru~tural portion that i~ bound by specific interaction with a par~icular receptor protein. A
representative ligand and receptor are fibrinogen and the platelet glycoprotein GPIIb-IIIa.
B. Polv~e~ides A polypept~de of the present invention has at leaat about 11, and no more than a~oUt 90, amino acid re~idues. In addition, a sub j ect polypeptid~ is characterized as having an amin~ acid residue seguence homolo~ous to, i.e., derived ~rom the ~unc~ional region Clc t . l 9s~ Z: t~ 311~1~1 C~G':--:3~-1 s.~r i~-. ~ ~.4~ 3 F . l~i 2()0~V4~

o~, the RG~-bi"ding region OL ~PIIIa be~ween residues 110-170 as shown in Figure 1.
In pre~erre~ embodiment~, a ~ubject polypeptide ha~ at leaet a~out 60 am~no aci~ re~idues, and has an amin~ acid residue sequence that corresponds, and is pre~erably identical to, a ~ormula shown in Table 1.
Table 1 Formula esianation Amino Aci~ Residue Seauence pl DYPVDIYYLMDLSYS~XDDLWSIQNLGTKL-ATQM~KLTSNLRIGFGAFVDKl)vsPyMy:t:sppE

p2 DYPIDLYYLMDLSYSMXDDLENVKSLGTDL-MNEMRRITsDFRIGFGsFvEKTvMpyIsTTp LRALNEITESGRIGFGSFVDRTVLPFVNTHP

In another preferred em~odiment, a subject 20 polypep~ide ha6 a ~eguence that includes an amino aoid residue -~equence ~epresented by the formula:
--DDS~FSIQVRQ~EDYPV--, -EDYPVDIYYLM-, -I.MDLSYSMKDDL-, -DDLWSIQNLGTKL-, -TXLATQMRKLTS-, -1TSNLRIGFG~FVD-, -AFVDXPVSPYMYIS-, or -YISPPEALENPCYD-.
Exemplary of this embodiment i~ a polypeptide havlng an amino acid re~idue sequence that corre~p~nds, and is preferably identical to, a formula shown in Table 2.

O .= t . 1 - :~2 c ~: ~ :3 ~3 ~ 13 13 C:~ 13 ~ -. ~ r i ~- 3 ~. I ?--5 ~ OE-- t ~-: :3 13 P . 1 ~
2~0~

~le 2 sequence Formula Origin wit~in Am~no Acid Residue 5 De~ignation GP~ Se~UQnCe P4 0~5-112 DDS~FSIt~ 2VEDYPV

1 0 E) 7 1 2 6 - 1 3 ~ Dl)LW3 I ~N LGTKL

P9 145-158 LTSN1RI~FGAFVD

P11 166-17~ YISPP~A:C.ENPCYD
15 In pre~erred embodiments a subject polypeptide is further charaoter~zed by lts a~ility to compatitively inhi~it Integrin-mediated cell adhes~on such as the aggregatiOn of platelets, the adhe-~ion of fibrobla~t~ ~o a matrix, and the like. ~hat ls, a preferred qubject polypeptide is able to competitively inhib$t Integrln blnding to a nat1ve ligand, i.e., a ligand to which the Integrin binds in vivo.
In preferred embodiments a sub~ect polyp~ptide is al~o further characterized by lts ability, when used in an inoculum, to induce ~he production of a polyclonal antibody or mono~lonal antibody of the present invention.
Amino acid residue~ present in a ~ubject polypeptide, in addition to a s~quence corresponding ~o an above-described formula, can be any residue~ that do not materially affect the basic and novel characteristlcs o~ a polypepti~e as are discu~sed h~r~in. Such additional residues are usually added to One or both tar~ini of an enumerated peptide and can inelude repeats and partial repeats of an enumerated peptide sequence or contiguou~ ra-~idues of the Integrin beta subunit protein ~equenoe.
A ~ubject polypeptide has an amino acid r~sidue 0.= t . i ~a = 2 ~ C~G';~;q ~ l -.o r i o -. .S 1 c--5~1. C ~ F . 1 ~
zno~4&~

~equenae that correYpond3 to a portion of the RGD-binding region of an Integr~n beta subunit s~quence. Thus, a polypeptide o~ the pre~ent invention need not be identical to the aminv acid residue sequence of the R~D-binding portion of an Integrin bet~ subunit, so long as it is able to exhibit at lea~t one of the above preferr~d characteri5tics of a 6ubject polypeptide. There~ore, a sub~ect polypeptide can be sub~ect to various change~, ~uch as insertion~, delations and ~ub~tltution~, either conservatlve or non con3er~ative, where such changes provide for certaln advantages in their use.
Conservative substitutions are those where one amino acid re9idue is replaced by another, biologically similar residue. Examples ~f con~ervative ~ubstitution~
include the ~ubstitution vf one hydrophobic residue ~uch a3 i~oleucine, valine, leucine or met~ionine for another, or the ~ubstitution of one polar residue for another ~ch as between arg1nlne and lysine, between glutamic and a~partic acids or between glu~amine and aRparagihe and the lik~. The term ~'conservative subs~itUtionl' also includes the use o~ a 6ubstitute~ amino acid in place o~
an unsub~tituted parent amino acid provided that such a polypep~e al~o di~plays the requi~lte binding or lnoculum activity.
When a polypeptide of the pre~ent invention ha6 a 6equenGe that is not identical to th~ ~equence of an Integrin beta ~ubunit becau~e one or more conservative or non-con3ervative sub6~itution~ have been made, u~ually no more than about 20~ and more usually no more than 10% of the amino acid regidue6 are sub~tituted. An exception is where addi~ional residue~ hav~ been ~dded at either terminus for the purpose o$ providing a "linker" by which the polypep~ides o~ thi invention can be conveniently affixed to a label or ~olid ma~rix, or antigenlc carrier.
Label~, 60l1d matxices and carriers ~hat can be used wi~h ~ ~ t . ~ 2 13 ~ ~3 1~ 13 [` ~ 5 ~ ~ ~ 13 P ~ 1 ~
Z00~4!3 th~ polypep~i~es of thi~ lnvention are de~cribed hereina~ter.
Amlno acid residue linker3 are usually at lea~t one residue and can be 40 or more re~idue~, more o~ten 1 to 10 re~idue~. Typical amlno acid re~idue~ u3ed for llnking are tyrOGine~ cysteine, ly~ine, glutamic and a~partic ~ci~, or the like. ~ repre8ent:at~ve linker i~ a Cys-Gly-Gly (CG~-) tripep~ide attached to the amino terminus of a subject polypeptide by the carboxy terminal glycine re~idue of the linker, a~ is ~hown in ~xample 2.
In addition, a polypeptid~ sequence of this invention can dif~er from th~ natural ~equenc~ by the sequence be~ng modi~ied by terminal-NH2 acylation, e.g., acetylation, o~
thioglycolic acid amidation, terminal-car~oxlyamidation, e.~., ammonia, methylamine, etc.
When coupled to a carrier via a l~nker t~ form what is known ln the art a3 a carrier-hapten conjuga~e, a subject polypeptide is capable Or inducing antibodies that immunDreact with the Integrin b~ta Rubunit to which the amino acid re~idue ~equenae o~ the polypeptide corresponds. In v~ew of the well established principle of immunologic cros~-reactivity, the present invention therefore contemplates antigenically related var~nts of a polypeptide havlng an amino acld residue seguence correspond~ng to a polypeptide having a formula shown in Table 1 or 2. An "antigenically related variant" is a polypeptide ~hat immunoreact~ wi~h an antibody induced by a polypeptide according to formula ~hown in ~able 1 Qr 2.
A subject polypeptide ~an be synthe~ized by any t~chniques that ar~ known to tho~e ~kille~ in the polypeptide art. An ex~ellent summary of the many technlque6 available may be found ~n J.M. Stew~rd and J.D. Young, "Solid Phase Peptide Synthesis", W.H. Freeman Co., San Francisco, 1969, and J. MeienhOfer, "Hormonal Proteins and Pep~de~", Vol. 2, p. 4~, Academ1c Pres~

O c 1, 1 :3=~ n ~3l3~3~3 [:~GSS:~-1 ~r i~-~ .~ 1c~--54S-- 13:3~ P . 1c.
zn(~u~

(New York), 19~3 for ~olld phase peptide synthesis, and E. Schroder and K. Ku~ke, "The Peptides", Vol. 1, Academ~c Press (New York), 1965 for classical solution synthe~
C. Inocula In anoth~r ~mbodiment, a polypeptide of thi~
invention, prefera~ly a peptide corre~ponding to a formula shown in Table 1 or 2 or an antigenically related variant th~reof ls used ln a pharmaceutically acc~pta~le aqueou diluent compositlon to form an inoculum that, when ~dminis~e~ed in an effective ~mount, is c~pa~le of inducing antibodies that immunoreact with an Integrin beta ~ubunit to which the amino acid residue ~eguence of the polypeptide corre~ponds.
The word "inoaulum" in its various grammatical form~ is used herein to describe a compo3ition containlng a polypepti~e of ~hi~ inve~tion as an aotive ingredient u~ed for the preparation of antibodie~ aga~n~t an In~egrin b~ta subunit.
When a polypeptide i~ used to ~nducs antibodiss lt i~ to be under~tood that the polypeptide can be u~ed alone, or linked to a carrler as a conjugate, or a~ a p~lypeptide polymer, but for ease of expre~ion the various embodime~ts of ths polypeptide6 of this invention are collectively ~e~erred to herein by the term "polypeptide~, and its variou~ grammatical form~.
AS alr~ady noted, one or more additional amino acid regidue~ can be added to the amino~ or carboxy-~ermini o~ the polypeptide to as~ist in bin~ing the polypeptlde to a carrier. Cy~teine resi~ues added at the amino- or carboxy-termini of ths polypeptide h~ve been found to be particularly u~eful for forming conjuga~es via disulfide bonds. ~owever, other method~ well known in the art ~or preparing conjuga~es can also be used.
Exemplary additional linking prccedures inalude the use ~'c ~ 2: 2 1 ~3 ~3 13 l:1 D 13 5 .=.~ r 1 c i= r i ~ ~. o 1 c~---5 1~.-- 1 3 s3 r1 F . ~ ~1 Z(~O~
- 16 ~

of Mich~el addition reaction products, di-aldehydes such as glutaraldehy~e, Klipstein et al., _. In~ec~. Dis ., g~ 318-326 (1983) and the li~e, or ~he u e of carbodimlde t~ch~ology a~ in the u~e of a water-soluble carbodiimide to form amide links to the carrier. For a review of protein con~ugation or coupling through activated functional group~, see Aurameas, et al., Scand J. Immunol., Vol. 8, Suppl. 7:7-23 (1g78).
U~eful carriers are well known in the art, and are lo generally protein~ them~elves. Exemplary of ~uch carrierQ are keyhole limpet hemocyanin ~KLH), ede~tin, thyroglobulin, albu~ins ~uch as bovine serum albumin (BSA~ or human serum alhumin (HSA), red blood cells ~uch a sheep erythrocyte-~ (SRBC), tetanus toxoid, cholera toxoid as well as polyamino acid~ such as poly (D-lysl-ne:
D-glutamic acid), and the like.
The choice of carrier i6 more dependent upon the ultimate use o~ th~ inoculum and ig based upon criteria not particularly involved in the present invention. For example, a carrier that doe~ not genexate an untoward reaction in ~he particular animal to be lnoculated ~hould be selected.
The present inoculum contain~ an ef~ective, immunogenic amount of a polypeptide of this invention, typically as a coniugate lin~ed to a carrier. The e~ective amount of polypeptide or protein per unit do~
depends, among other things, on the ~pecies oi animal inoculated, the body weight of the animal and the chosen inoculatiQn re~imen as 1~ well ~nown in the ar~. Inocula ~ypically contain polypeptide or pro~ein concentrations of ~bout 10 microgram~ to about S00 milligram~ per inoculatio~ (do~e), pre~erably about 50 micrograms to abou~ 50 milligram~ per dosR.
Th~ term "unit dose" as it pertains to the inocula of the present invention refer~ to phy-~ically discrete ~I c t . ~ 3 13 ~3 u D C. S S~ l -. ~ r i ~ 5 ~ '-- ~ 3 :~: ~ F' ~
2~
~ ~7 -units ~uitable as unitary do~age~ for anim~ls, each unit containing a pr~determined quantlty of active material calculated ~o produce the desired immunogenic eE~ect in a~oclatlon with the required diluent; i.e., carrier, or vehicle. Tlle ~peci~ications for th~ novel unit dose of an lnoculum of thi~ inventlon are ~ctated by and are directly deperldent on (a) the unique character1~tics ~f the activs material and the par~icular immunologic ~ffect to be a~hieved, and (b) the liml~ations inherent in the art of compoundlng such active material for immunolo~ic u~e in animals, as di~closed in detail ~lerein, the~e boing ~eature~ of the present invention.
Inocula ar~ typically prepared from the dried solid polypeptlde~conjugate by di~persing the polypeptide-conjugate in a physiologically tole~able (acceptable) diluent or vehicle such as water, 6alins or pho~ph~te-buI~ered ali~e to form an aqueou~ oomposition.
Such d~luentg are well ~nown in the art and are di~cu6~ed, for example, in ~
~Ç~DSe~, 16th Ed., Mack Publl~hing Company, Easton, PA
(1980) at page~ 1465-1467.
Inocula can also include an adjuvant a~ part of the ~iluent. Ad~uvan~s ~uch a~ complete Freund's adjuvant (CFA), incomplet~ Freund's ad~uvant (IFA) and alum ars materials well known in the art, an~ are available commercially from several sourcss.
D.
an~ibod' The t~rm "antibody'l in its variou~
grammatical forms i~ used herein to refer to immunoglobulin molecules and i~munologically a~tive portionQ o~ immunoglobulin molesule~, i.e., molecule~
that contain an antibody oombinin~ ~ite or paratope.
Exemplary antibody moleGule~ are intact immuno~lobulin ~5 molecule~, substantially in~act immunoglobulin molecule6 O c t . 1 ~ 3 ~ C~ ~i s ~sxt~l ~ e r i e ~- c. 1 ~ 0 P . ~
Z~)U'~8 and thosa portions of an immunoglobulin molecule that contaLn the paratope, including tho~e portion~ known in the art a9 Fab, Fab', F(ab')2 and F(v).
An "antibody combining site" is that structural portion ~f an anti~ody molecule compri~ed of a heavy and 11ght chain variable ~nd hypQrvariable regions ~h~t ~pscifically binds (immunoreacts with) antigen. The ter~
"immunoreac~ in its variou~ form~ means binding between an antigenic determinant-con~aining molecule and a mole~ule containing an antibody com~ining ~i~e such as a whole antibody molecule or a port$on th~reof.
"Antigenic determinantll refers to the actual ~tructural portlon of the antigen that immunologically bDund by an antibody combining ~ite. ~h~ t~rms is als~
u~ed inter~hangeably with "epitope".
1. Poly~l~l Anti~odies A polyolonal antibody o~ the precent invention immunoreaats with a gubject polypeptide, pre~erably a polypeptide corresponding in amino acid residue sequence to a for~ula ~hown in Table 1 or 2. A
~ubject polyclonal antibody is ~urther characterized as not ~ubstantially immunoreacting with any Integrin alpha ~ubunit or a polypeptide having an amino acid re~idue sequence identi~al to the 50 carboxy-terminal residue~ of the Integrln to which the amino acid re~idue sequence of the s~bject polypeptide corresponds.
A pre~err~d ~ly~lonal ~ntibody i~ ch~raotori~ed a~ h~ving the ability to immunoreact with an Integrin beta subunit and inhibit tho capacity of the Integrin to 6pecifically bind to its ligand by an interaction wlth an RGD-containing protein.
ThU~ a preferred polyclonal an~body that immunoreact wi~h a subjeot polypeptide wh~e C~quence is derived from the RG~-binding re~ion of ~PIIIa has t~
capacity to inhibit fibrlnogen-GPIIb-IIIa ligand-~eceptor O c t . I ~ 3 1~ 13 13 1~ c-c.c =æ r 1 ~ o r i ~. .'. 1 = ~ 1 3:3 1~l F . :~3 4~3 complex-me~iat~d events, ~uch a~ plat~l~t ~gqre~ation and ~hrombus ~ormation.
A polyclonal antlbody of the present invention i5 typically produce~ by imm~nizing a mammal w~th an inoculum of the present invention, pre~erably an inoculum ~ontaining a peptide corre~ponding to a formula ~hown in Table 1 or 2, and thereby induce in ~he mammal antibody molecule~ having the appropriate polypeptide i~munospec~ficlty. The antibody mole¢ules ~re then collectRd ~rom the mammal and i~olated to th~ extent desired by well known technique~ ~uch a~, for example, by immunoaffinity chromato~aphy ~sing the immunizing polypeptide in the ~olid phase. The polyclonal antibody so produced can b~ used in, in~r ~lia, the diagnostic methods and ~yRtems of thQ present invention to di~criminate betw~en activated and nonactivated platelets or nucleated cells and in therapeutic method~ for the purpoqe of modula~ing c~ll adhesion, such as inhibiting platelQt adhesion.
2. Monoçlonal. Antibodies A monoclonal antlbody of the present invention is characterized as immunoreacting with an epitope ~or~ed by the RGD-binding region of an Integrin beta subunit th~t i~ homologous to re~idues 110-170 of GPIIIa. Preferably, a subject monoclonal antibody i~
further characterized aa immunoreacting with a ~ubj~ct polypeptide, preferably a polyp~ptide corresponding to a formul~ ~hown in Table l or ~.
A pre~erre~ monoclonal antibody is also cha~a~terized a~ haviny the ability to inhibit the specific binding between GPIII~ and it~ ligand, fibr1nogen, ~uch as is de~cribed before for polyclonal antibodies, Thus, in one embodimen~, a monoclonal antibody is contemplated compri~ing antibody molecules that = t . i S '~ ' ~ 3 ~ q C:~ C~ 1 ~. e r i ~ ~ .~ 1,--5 '1.~ 1 3 æ ~3 F .
Z()O~Q4~

immunoreact with a) GPIIIa, and ~) a polypeptide corresponding to the formula: DYPVDIYYLMDLSYSMKDDLWSIQ-NLGTKLATQMRKLTSNLRIG~GAFVDKPUSPYMYISPPE.
A related embodim~n~ contemplates a monoclonal antibody comprl6iny monoclonal antibody molecules that i~munoreact with a) the ~eta subunlt of the LeuCam Integrin and b) a polypeptide corresponding to the formula: GYPIDLYYLMDLSYSMLDDLRNVXKLGGDLLRALNEITESGRIGY-GSFVDKTVLPFVNTHP.
~0 Another related embodiment contemplates a monoclonal antibody comprising antibody molecules that immunor~act with a) the beta su~unit of the VLA ~ntegrin, and b) a polypeptide correQponding to the ~ormula:
DYPIVLYYLMDLsysMKDDLENVKSLGT~LMNEMRRITsDFRIGFGSFVEKTVMP-YIST~P.
The phrase "monoclonal antibody" in it~ various grammatical forms re~ers to a population of antibody molecules ~hat contain only one ~pecle~ o~ antibody combining si~e ~apable Or immunoreacting with a particular antigen. A monoclonal antibody composition thu~ typically di~play~ a single binding affinity for any antigen wi~h which it immunoreactc. A monoclonal antibody composition may therefore contain an anti~ody molecule having a plurality of antibody combining sites, each immuno~p~cific for a different antigen, ~.g., a bispec~flc monoclonal antibody.
A monoclonal antibody is typically co~po~ed of antibodies produced by clone~ of a ~ingle cell called a hybridoma that secrete3 (produce5) bu~ one kind of antibody molecule. The hyl~ridom~ c~ll is formed by fusing an antibody-producing cell and a myeloma or other ~elf-perpetuating cell line. Such antibodie~ were first describ~d by Kohler and Mll~tein, Nature 256:495-4~7 (1975), which de~cription ic. incorporated by reference.

3. ~=

Oc ~ ! C~C~ ~ r i ~ l C' ~ 13 :3 ~3 ~ . :~
2(~0~48 The pre6ent invention contemplate~ a method of ~orming a monoclonal an~ibody that (a) immunoreact~
with (a) ~ ~ub~ect polypeptide, and (b) the Integrin be~a ~ubunit to which t.he amino acid residue sequence corresponds. The method comprige~ the steps of:
(a) Immunizing an animal with an Inteqrin heta subuni~ or a sub~ ect polypeptidQ .
Thi~ is typically ac~omplished by administering an immunologically e~fective amount i . e ., an amount ~ufficient to prod~ce an immune response, of the immunogen to an immunologlcally competent mammal.
Preferably, the mammal i~ a rodent such a~ a rabbit, rat or mouse. The mammal i6 then maintained for a time period sufficient for the mammal to produce cells secre~ing anti~dy molecules that immunoreact with the ~m~unogen.
(b) A suspen610n of antibody-producing cells removed from the immunized mammal 1~ then prepared.
This is typically accompli~hed by removing the spleen o~
the mammal and mechanically separatlng the individual ~pleen ~ells i~ a physiologically tolerable medium using methods well ~nown in the art.
(c) The su~pended antibody producing cells are treated with a tran~orming agent capable of producing a t~an~formed ("immortalized") cell line.
Tran6$0rming agents and their use ~o produce immortalized cell lines are well known in the art and include DNA
viruses ~uch ~8 Ep~tein Bar Viru~ (EBV), simian Virus 40 (SV40), Polyoma Virus and the like, RNA viruses such as Moloney Murine Leukemia Virus (Mo-Mu~V~, Rous Sarcoma Virus and the like, myeloma cells such as P3X~3-Ag8.653, Sp2~0~Agl4 and the like.
In preferred embodimen~s, treatmen~ with the tran~formlng agent re~ult~ in the production o~ a hybridoma by fusi~g the ~u~pended spleen cells with mou~e O c t . 1 .~: 5' 2: 2, ~ ~3 ~ ~ . S ~ r--1 ~ ~ r i q~ 5 ~ ~.-- 1 3 :3 ~ F . -:
~OU~)~)98 myeloma cell6 from a s~ltable c~ll line by the use of a ~uitable rusion promoter. The preferred ratio ls about 5 spleen cell~ per myeloma cell. A total volume of about 10~ ~pl~nocytes.
The cell line u~ed should preferablY be o~ the so-called "drug re~16tant" type, ~o that unfu~ed myeloma cell~ will not survive in a selective medium, while hybrids will Survi~e. The most common clas~ is ~-azaguanine re~i~tant cell lineg, which lack the enzyme hypoxanthine guanins phophorlbo~yl transferase and hence will not be ~upported by HAT (hypoxanthine, aminopterin, and thymldine) medium. It i~ al o generally preferred that ~he myeloma cell line u~ed be of the so-called "non-secreting" type, in that it doe~ not lt~elf produce any antibody, although secreting types may be used. In certain cases, however, ~ecreting myeloma lines may be preferred. While the preferred fusion promoter i~
polyethylene glycol having an average molecule weight from about 1000 to about 4~0 (commercially avalla~le as PEG 1000, etc.), o~her fusion promoters known in the art may~a employed.
(d) The transformed cells are then cloned, preferably to monoclonality. The cloning is preferably performed in a tis~ue culture medium that will not support non-tran~formed cell~. When the tran~formed Cell8 are hybridomas, this is typically performed by diluting and culturing in separate containers, the mixture of unfu~ed ~pleen cells, unfused myeloma cells, and fused cells (hybridomas) in a 6elective medium which will not suppor~ the unfu~ed myeloma cells for a ti~e sufficient to allow death of the unfused cells (about one week). The dilution may be a type of limiting one, in which the volume of diluent is ~tatistically calculated to i601ate a certain num~er of cells (e.g., 1~4) in each separate container (e.g., each well of a microtiter O c ~ . s - :3 ~ 2 ~ r~ [~ G S =. .~ c ~ 1 --. ~ r i ~= ~ ~ 1 5--5 ~ ~-- 1 3 s~: ~ F . ~
2(~ 48 plate). ~he medium i~ one (e.g., IIAT medi~m) whlch will not ~upport the drug-re~tant (e.g., 8-a~aguanine resi~tant) unfus~d myeloma cell line.
(e) The tissue culture medium of th~ cloned ~ran~forma~ts is evaluated for the presence of secreted antibody molQ~ules that imm~noreact with the im~noge~
and its corresponding subjec~ polypeptlde or Integrin beta ~ubunit.
(f) Once a de~ired transformant has been identi~ied in step ~e~, it is gelected and ~rown in a suitable tissu~ culture med~um for a suitable length of time, followed by recovery of the de3ired an~ibody fro~
the culture supernatant. The sultable medium and suitable length of culturing tim~ are known or are readily determined.
To produce a much qrea~er concentration o~
slightly less pure monoclonal antibody, the desired hybridoma ~ay ~e in~ected into mice, preferably -~yngenic or ~emisyngenic mice. The hybridoma will cause ~ormation of antlbody-produclng tumors a~ter a uitable incubation time, which will result in a high concen~ra~ion of the d~ir~d antibody (about 5-2~ mg/ml) ~n the bloodstream and peritoneal exudate (ascites) o~ the host mouse Media useful for ~he preparatlon o~ these compositions are both well known in the art and commercially available and include ~ynthetic culture media, inbred mice and the like. An Qxemplary synthetic medium is Dulbecco's ~inimal e~sent~al medium ~DMEM;
Dulbecco et al., Y1EQ1~ 8:396 tl959)] supplemented With 4.5 gmJ1 gluco e, 20 mm glutamine, and 20~ fetal calf serum. An exemplary inbred mouse strain is the Balb/c.
A monoclonal antibody of the pre~ent in~ention can also be further purified by well known immunoaffinity chromatography method~ by u~ing in the ~olid pha e a sub~ect polypeptide with which the antibody immunoreacts.

- C' '7 2 ': ~ [:~ C~ r1 ~ r i ~ ~ .~ 5 4 .~-- 1 3 ~ p ~
f~nO~Q~8 A monoclonal antibody produoed by the above method can be u6ed, for example, in ~iagno6tic and therapeutic mo~alities wherein formation of an Integrin beta subunit immunoreactiO~ product ls de~ired. Exemplary reaction products include a GPIIIa-containing immunore~ction product~
E. ybrldo~a~ and Methods of Prep~x~l~n Hybridoma~ of the present invention are those which are characterized as having the capacity to produce o a sub~ect mono~lonal antibody.
A preferred hybridoma of the present lnventlon is characterized as producing antibody molecules that also lmmunoreact wlth a cytoadhe~ion, preferably GPIIIa~
Method~ for producing hybri~omas producing ~eo~eting) anti~ody molecules haviny a desired immunospecificity, i.e~, having the ability to immunoreact with a particular protein, an identi~iable epitope on a particular protein and/or a polypeptide, are well known i~ the art. Par~lcularly applicable is t~e hybridoma technolo~y described by Niman et al., Proc.
Nat~, A~L Sci. USA, ~:4~49-4g53 (1983), and by Galfre et al., ~h _En3ym~1~, 73:3-46 (1981), which descriptions are incorporated herein by reference.
F . ~ ~
A subject polypeptide can be used to modulate the adhesi~n in vivo of cells expressing the Integrin beta subunit to which the polypeptide correspond~.
For in~tance, a ~ubject polypeptide corresponding to formula pl can be used in a pharmaceutiCallY
a~ceptable ¢ompo~ition that, when administered to a human subject in an effective amount, i~ capable of competitively inhibiting the ~ggregation of platelets.
That inhibition iB believed to regult in a decreased rate of thrombus formation. Thu~, v~vo administration of a subiect polypeptide ~an be used to modulate any n.= t . 1 ~:o - ~ 13,~ [:.~.=.s~ r1 ~=~ r i~ 5 .5 1C-5.~ 3.æ~-1 p . .'.
Z'~()UQ~3 ~}-y~i~lcg1cal re~ponse lnitiated by adhesion such a5 coagulation and ~ome inflammatory re~ponses.
In ~nother embodiment, the aggregation of platelets can be inhibited by intravenOu~ administration of an effe.ctive a~ount of a pharmaceutically acceptable compo~ition comprising a cubject polyclon~l antibody that immunoreacts with a polypeptide corresponding to a portion of th~ RGD-binding region of GPIIIa, ~uch as a polypeptide according to formula pl, o~ formula ~hown in o Table 2.
A preferred method of modulatlng platelet adhesion contemplate~ admini~tering a platelet aggregatlon-inhibiting amount of a ~ubje~t monoclonal antibody that immunoreactS with the RGD-binding region (residues llo-170) of GPIIIa. More prefera~ly, the monoclonal antibody used in a platelet-inhibiting therapeutic method ls further characterized a3 immunoreacting with a polypeptlde corresponding to formula p~., or a formulas shown in Table 2.
In~ofar as polyclonal or monoclonal antibodies can be used therapeutically to modulate cell adhe~ion-medlated events, the present inven~ion also contemplates the u~e of a gubject polypeptide as an antidote to neutralize the modulating effect of therapeutically administered antibodie~.
In thi~ emobodiment, an anti-thrombotic antibody-containing therapeutic reagent is first admini~tered to a patient to modulate cell adhesion, platelet aggregation or thrombus formation. Thereafter, upon the onsQt of a 3~ bleeding complication, or when it becomes de~irable to neutralize ~he anti-~hrombotie e~ects of the admini~tered antibody, an amount o~ a sub~ect polypeptide i9 admini tered that is effective to immunoreact with the administered antibodY and thereby neutralize the modula~ing effect of the antibody.

O c ~ 3 ~ 2: ~ c ~ 1 [:1 l3 -; _; r~ 1 =, c=. r i ~ - ~ 5 ~ .S ~ 3 ~3 p . ~
~00~ 48 The choice of polypepti~e to b~ administered a6 an antidote depends upon the antibody to be neutralized, and requires that the admini~tered polypeptid~ have the capacit.y to $mmunoreact wlth the administered antibody.
The polypeptide- or antibody molecule-containing compositions administcred take ~he form of ~olutions or suspensions, however, polypeptide~ can also take the form of tablets, pills, capsules, gustained release formulation~ or powder~. In any cas~, the polypeptide-containing compo6itlons typically contain about 0.1 uM to about 1.0 M of polypeptide as active lngredient, preferably about 1.0 uM to about 10 m~lllmOlar ~mM), whereas the antibody ~olecule-containing compositions typically contain about 10 ug/ml to about 20 mg/ml of antibody ag a~tive ingredient, preferably about 1 mg/ml to abou~ 10 mg~ml.
The preparation of a therapeutic composition that contain~ polypeptides or antibody molecules as active in~redient6 i~ ~ell under~tood in the art. Typically, such compositions are prepared a~ injectable~, either as liquid solutions or suspensions, however, ~olid forms suitable for solution in, or ~u~pen~ion in, liquid prio~
to injection can al~o be prepared. The preparation can also be emulsified. The active therapeutiC ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingr~dient as are well known. Suitable excipients are, for example, water, saline, dextro~e, glycerol, ethanol, or the 1 ike and combination~ ther~of. In addition, if de-Qired, the composition can contain minor amounts o~ auxiliary substances such as wetting or emulsifying agents, p~
buffering agents which enhance the effectivene~ of t~e active ingredient.
A polypeptide or anti~ody can be formul~ted into the therapeutic composition as neutralized c\ ~ t . 1 - ~: c~ r- ~ e r i e = .~. I s--5 4 .~ 3 ~1 P . s~::

20~ 4~3 pharmaceutically acceptable salt forms. Pharmaceutlcally accep~able salts include th~ acid addition salt~ (formed with the ~ree amino groups o~ the polypeptlde or antibody molecule) that ~re forme.d with inorganic acid~ 6uch as, for example, hydrochloric or pho8phoric acids, or ~uch organic acid~ a~ aaetia, tar~aric, mandelic, ~nd the llke. Salts ~ormed with the free carboxyl groups can also be derived from inorganic baseg such as, f~r example, ~odium, pota6sium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropyl~mine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
The therapeutic polypeptide- or antibody containing ~ompositions are conventionally admini~tered 5 intravenously, AS by in~ection of a unit dose, for example. The term "unit doss" when u~ed in reference to a therapeutic composition of the presen~ invention refers ~o physically di~crete units ~uitable as unltary dosages for human~, each unit contalnlng a predetermined 20 quantity of active material calculated to produce the desired ~herapeutic effect in association with the required diluent, i.e., carrier, or vehicle.
The composltion~ are administered in a manner compatible with th~ dosage formulation, and in a therape~tically ef~eotive amount. The quantity to be adm$nl~tered depend~ on the subject to be treated, capaCity of the ~ubject to uti~ize the active ingredient, and degree of inhibitlon of receptor-ligand bindlng de61red. Preci~e amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual.
However, suitable dosage ranges are of the order of one to several milligrams of active ingredient per individual per day and depend on the route of adminl~tr~tion.
~5 ~uitable regimes for initial adminis~ration and booster I-J c t . I - ,~ C ~ 13 13 [.1~'= C;y~ ~ I -. e r i ~ -~ ~ 1 g--54 6 ~ 8 1-1 F . c Z~)0~04~3 shots are also variable, but are typifled by an initial administration followed by repea~ed doses at one or more hour intervals by a ~ubsequent in~eetion or other admin~stration. Alternatively, continuou~ intravenous infusion sufficient to maintain therapeutically effective concentrations tn the blood are contemplated. For a subject polypeptide, therapeutically effective blood concetltratiOn~ ~re in the range of about 0.1 mM to about 10 mM, pre~erably about 1.0 mM. ~erapeuticallY
o effective blood concentrations of antibody molecules of the p~esent invention are in the range of about 0.1 uM to about 10 uM, pre~erably 1.0 uM.
G. ~i~qnostic Sv~tems A diagno~tic sy~tem in kit form of the present invention includes, in an amount ~u~ficient for at least one a~say, a polypeptide, polyçlonal ~ntibody or monoclonal antibody of the present invention, as a separately packaged reagent. Instructions for use of the packaged reagent are also typlcally included.
"Instructions for use" typically include a tanglble expression describing th~ reagent concentration or at least one assay method parameter such as the relative amount~ of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, b~ff~r condi~ion~ and the like.
In one embodiment, a diagnostio sy~tem for assaying for activated platelets in a platelet-containing vascular fluid ~ample, such ao blood or plas~a, comprises a pa~kage containing a cubject polyclonal antibody that immunoreact~ with a polypeptide ~orre~ponding to formula pl or a ~ormula s~own in Table 2. In another embodimsnt, a diagnostic system for assaying ~or activated platelets in a platelet-containing vascular fluid ~ample comprise5 a packa~e containing a subiect monoclonal antibody that immunoreacts with an epitope formed by the RGD-binding O c ~ c ;~ 3 rl ~3 1-~ [~ G -~ . e r i ~ 3. ~. 1 ~---5 ~ .'. ~ F . ~ ~

Z00~8 -- 2g --region (re~idues 11~ 0) of ~;PIIIa, and preferably also immunoreact~ with a polypeptlde corresponding to formula pl or a fo~m~la shown in Table 2. Further preferred are kits wherein the antibody molecules of the polyclonal or monoclonal antibady are linked to a label.
Thu~, in preferred embodiments, a diagnostic 3ystQm of the present invention further includes a label or indicating means capable of signaling the formation of a complex cont~lnlng the antibody molecules of a o polclonal or monoclonal antibody of the present invention.
The word "complex" as used herein referq to the product o~ a speclfic bindinq reaction such as an antibody-~ntigen or receptor-ligand reaction. Exemplarly complexes are immunoreaction produc~.
As used hereln, the terms ~labal" and "indicating means" in their various grammatical for~s ref~r to sinyle atoms and moleaules th~t are either directly or indirectly involved in the production of a detectahle signal to indicate the presence of a complex. "In vivo"
la~els or indicating means are those useful within the body of a human sub~ect and include ~l1In, ~Tc, 67Ga, t~Re, and l32I. Any label or indicating means can be linked to or incorporated in an expressed protein, polypeptide, or antibody molecule that is part of an antibody or monoclonal antlbody composition ~f the present invention, or u~ed ~eparately, and those atoms or molecule~ can be used alone o~ in conjunction with additional reagents.
Such labels are themselves well-known in clinical diagnostic chemistry and Cons~itute a part of this invention only insofar a~ they are u~ilized with otherwise novel protein ~e~hods and/or ~ystems^
The linking of labels, i.e., labeling of, polypeptides and proteins is well known in the a~t. For in~tance, antibody molecules produced by a hybridoma can zno~Q4~
be labeled by meta~ollc lncorporation of radioisotope-containing amino acids provided a6 a ~omponent in the culture medium. See, for example, Galfre et al., M~h.
E~zy~ol~ 73:3-46 tl~l). The techniques o~ protein con~ugation or coupling through activated functional gro~ps are particularly applicable. See, ~or example, Aurameas, et al., _and. J. I~munol., Vol. 8 suppl. 7 : 7-23 (1978), Rodwell et al., Biotech., 3:889-~94 ~1984), and U.S. Pat. No. 4,4~3,7~5.
lo The diagnostic systems can al-~o include, preferably as a separate pack~ge, a specific binding agent. A ~specific binding agent" is a m~lecular entity capable of ~electively binding a reagent ~pecie~ of the pre~ent invention ~ut is not itoelf a protein expression produc~, polypeptide, or antibody molecule of the present invention. ~xemplaxy spe~iflc binding agents are antibody mole~ules, complement proteins or fragments thereo~, protein A and ~he like. Preferably, the specific binding agent can bind the antibody molecule or polypeptide Or this invention when it is present as part of a complex.
In preferred embodiments the specific binding a~ent i~ labeled~ However, when the diagnostic system includes a specific binding a~ent that is not labeled, the ag~nt i~ ~ypically used as an amplifying means or reagent. In these embodiments, the labeled specific bind~i nq a~ent i6 capa~le of speci~ically binding the amp,ifyin~ meAns when the amplifying means is ~ound to a reagent 6pe¢ies-containing complex.
The diagnostic kits of the pre6ent invention can be used in an "ELlSA" format to detect the pre~ence or yuantity of fibrinogen-~ound platelets in a body fluid sample such ag serum, plasma or urine. "ELISA" refers to an enzyme-linked immunosorbent assay that employ~ an antibody or anti~en bound to a solid pha6e and an enzyme-2~U~C~

antigen or enzyme-antibody conjugate to detect and quantify the amount of a~ antigsn or antlbody present in a ~ample. A description of the ELISA technique is found in Chapter 22 of the 4th Edition of B~siç and Cl~ni¢al Immunoloqy by D.P. ~1tefi et al., publi~hed by Lange Medical Publication~ of ~o~ Altos, CA in 1~82 and in U.S.
Patents No. 3,654,0~0 No. 3,850,75~ and No. 4,016,043, which are all incorporated hsrein by reference.
Thu~, in preferred embodiment~, the expre~6ed protein, polypeptide, or antibody molecule of the present inventlon can be affixed to a ~olid matrix to form a solid support that i~ ~eparately packaged in the subje~t diagno~tic sy~tem~.
The reagent ls typically afflxed to the solid matrix by adsorption from an aqueous medium alt~ough other mode~ of affixation, well known to those skilled in the art can be used.
Use~ul solid matric~s are well known in ~he art.
Such materlals inalude the cross-linked dextran available under the trademark SEP~ADEX from Pharmacia Fine Chemical~ (Piscataway, NJ); agarose; beada of polystyrene beads about l micron to about 5 mlllimeter~ in diameter available from Abbott Laboratorie~ o~ North Chicago, IL;
polyvinyl chloride, polystyrene, cross~linked ~5 polyacrylami~e, nitrocellulose- or nylon-based webs such a6 sheet~, ~trips or pa~dles: or tubes, plates or the wells of ~ microtiter plate such as those made from polystyrene or polyvinylchloride.

The reagent specie~, labeled specific binding agent or amplifyin~ reagent of any diagnostic system described herein can be provlded ln ~olution, as a liguid dicpersion or as a ~ubstantially dry power, e.g., in lyophilized form. Where the indl¢ating mean~ is an enzyme, the enzyme' fi ~ub~t~ate can al~o be provided in a separate package of a sy~tem. A solid support such as ;~o~o~

the be~ore-described microtite:r plate and one or more buffers can also be included a~ separately packayed elements in this diagnostic a6say 6y~tem.
The packages discussed herein in relation to diagnostic systems are those cu~tomarily util.ized in diagnostic ~y~tems. 5uch pac~ages includ~ glass and plastic ~e.g., polyethylene, polypropylene and polyoarbonate) bottle~, vials, pla~tic and plastic-foil laminated env~lopes and the like.
H. As6a~ Methods The pre6ent invention contemplatec any method that result in detecting an Integrin beta subunit, and particularly GPIIIa, by producing a complex containlng an antibody molecule contained in a polyclonal antibody or monoclonal antibody of the pr~sent invention. Those ~killed in the art w~ll under6tand that there are numerous well known clinical diagnostic chemistry procedures that can be utilized to form those complexes.
Thu~, while exemplary assay method~ are described herein, the invention i6 not ~o limlted.
For example, a heparin-pre~erved (non-clotte~) blood sample and 125I-labeled antibo~y molecules are admixed. Th~ immunoreaction admixture th~s formed is maintained under immunological aCsay conditions for a time period sufficient for any activated platelets ~o immunoreact with the labeled an~i~odie~ and form a labeled immunoreaction product. The labeled immunoreaction products are then separated from the non-reacted labeled-antibodies, typically by centri~ugation sufficient to pellet all platelet6 pre6ent in the sample.
The amount of labeled immunoreaction product formed is then a6sayed.
Immunological assay conditions are tho~e that maintain the immunological activity of the antibody 35 molecule~ contalned in a polyclonal or monoclonal ~lq~

antihody of this lnvention and the Integrin m~lecules sought to b~ as~ay~d. Tho~e cc,nditions in~lude a temperatur~ range of abo~t 4 degre~ c t~ about 45 degrees C, preferably abou~ 37 degree6 c, a pH value range of abou~ 5 to about 9, pre~erably about 7 and an ionic strength varying from that of dlstilled wat~r to that of about one molar sodium chloride, preferably about that of phy.iological galine. Methods for optimizing such conditions are well known in the art.
In living o~ganisms, ~he amino acid residue sequence of a protein or polypeptide is direatly related via the ~enetlc co~e to the deoxyribonuclei~ aaid (DNA) sequence of the 6tructural gene that code-~ for the protein. ~hus, a 6tructural gene can be defined in terms of the amino acid residue ~equence, i.e., protein or polypeptide, for which it codes.
An important and well known ~eature of the genetlc code ls lt~ redundancy. That is, for mo6~ of the amino acids used to make pro~elns, more than one coding nucleotide triplet (codon) can code for or designate a partic~lar amino acid residue. Therefore, a number of different nucleotide 6equences can code for a particular amino acid residue ~equence. Such nucleotide sequences are considered functionally equi~alent slnce they can result in the production of the same amino acid residue s.equence in all orgDniSms. Occasionally, a methylated variant o~ a purine or pyrimidine may be incorporated into a given nucleotide ~equence. However, ~uch methylations do not af~ect the coding relatlonship in any way.
A DNA -qegment of the present invention includes a structural gene that encodes a gubject polypeptide containing an Integrin beta ~ubunit amino aci~ re~idue seqUenCe homolo~ou~ to the GPIIIb ~equence located oc ~ . 1 æ~ =~ 5 ~ ~G~;5~ ~r ie-. ~ 5~.-- 1~a~1 p .
~()V~048 between re~idue~ 110-170 a~ ~hown in Figure 1.
A prefsrred DNA segment of the pre~ent invention includes a ~NA ~equence that codes for an amino acid re~idue ~equence corresponding to, and preferably identical to, a ~equence repre3ented by pol~peptide a fo~mula a~ ~hown ln ~able 1 or 2. Pre~erably, the DNA
sequence is present a~ an un~nterrupted linear series of codon~ where each codon code~ for an amino aald re~idue found in the abo~e de~crlbe~ amino acid re~idue sequences, i.e., a DNA ssquQnc~ contalning no introns.
Thus, a DNA segment consi~ting essentially of the nucleotide gequence ~hown in Figure 1 ~rom about base 423 to about ba~e 611 constitutefi one embodiment of th~
pre~ent invention.
A DNA ~egment o~ the present invention can ea~ily be ~ynthe~ized ~y Ghemical techniques, for example, the pho~photrieqter method of Matteucci et al., J. Am. Chem.
~Q_, 103:3185 (lg81). O~ cour~e, by chemically synthesizing the codin~ ~equence, any desired 20 modifications can be made simply by sub~tituting the appropriate bafie~ f or those encoding the native amino acid residue ~equencq. However, DNA molecules including ~equence~ exactly homologous to tho~e shown in Figure 1 are preferred.
The DNA molecule~ of the present invention typically have cohefii~e texmini, i.e., "overhanging"
single-~tranded portion3 that ex~end beyond the double-stranded portion of ~he molecule. ~he pre~ence of cohe~ive ta~mini on the DNA molecules of the prefient 30 invention i~ preferred.
Al60 contemplated by the present invention are ribonucleic acid (RNA) eguivalent~ of the ~bove de3cribed DNA ~egment~.
J. Re~Qmbin~ U~Molecules A reaombinant DNA molecule of the pre~ent Oc t . 1 f~ 5 ~3 ~3 l3 l~ Dl~S =:P~ ~ r i ~ ~ ~. I ?--~;4.~ 3~ 3 P . 12 ZnVU(~LB

in~ention can be produced by operatively linking a vector to a DNA ~egme~t of the present invention, preferably a DNA segment coding for a subject polypeptide corre~ponding to a formula ~hown in Table 1 or 2.
A u~ed herein, the t~rm "vector" refers to a DNA
molecule capa~le of autonomou6 repllcation in a cell and to wh;.ch ~nother ~NA ~egment can be operatively linked ~o a~ to bring abou~ replication of the attached segment.
vectors capable o~ directing the expre6~ion of gene~
lo encoding proteins ~vln~ ~PIIIa-related amino acid residue sequenc~3 are referred to herein a~ "expression vectors". Thus, a recom~inant DNA molecule (rDNA) is a hybrid DNA mol~cule comprising at lea~t two nucleotide sesuences not normally found together in nature.
The choice o~ ~ector to which a DNA ~egment of the pre~ent invention ls operatively linked depends directly, as is w~ll known in the ar~, on the functional properties desired, e.~., protein expression, and the host cell to be tran~formed, these heing limita~ion~ inherent in the art of constructing recombinant DNA molecules. However, a vector contemplated by the present ~nventlon is at least capable of directing the replica~lon, and preferably also expression, of the gene encoding a polypeptide having an Integrin beta subunit-rela~ed amino acid residue sequence included in DNA segments to which it l~ opera ively linked.
In pre~erred embodiments, a vector contemplated by the present inven~ion include~ a procaryotic replicon, i.~., a DNA sequence having the abil~ty to direct autonomou5 replication and maintenance of the recombinant DNA molecule extrachromosomally in a procaryotic host cell, Yuch a~ a bacterial host cell, transfo~med therewith, Such r~pliconR are well known in the art. In addi~ion, those embo~lments that incl~de a proc~ryo~ic replicon al~o include a gene whose expression confer~

3 ~ [ ~ r~ ~ 5 :~ e 1 - i e ~ .s I o _ 5 c~ I 3 S n p . I :~.
2'~)V048 dru~ resistance to a ~acterial hos~ transformed therewith. Typical bacterial drug resistance genes are those that con~er resi~tance to ampiclllin or tetracyal~ne.
Tho~e vectors that include a procaryotic replicon can al~o include a procaryotic promoter capable of d~ectin~ the expresçion (transcription and translation) of the ~ene encoding a GPIIb-related amino acid re idue sequence in a bacterial host cell, such as E. coli, 10 tranY~orme~ therewith. A promoter ls an expres3ion control element ~ormed by a DNA Qequ~nce that permits binding Or RNA polymerase and transcrlpt~on to occur.
Promoter sequences aompatlble with bacterial hosts are typically pro~ided in pla~mid vectorfi containing 15 convenient restriction site6 ~or in~ertion o~ a DNA
segment of the present invention. Typical of auch vector pla~mids a~e pUC8, pUC9, pBR322 and ps~329 availabl~ from Bio-Rad LaboratorieS, (Richmond, CA) and pPL and pXK223 a~ailable from Pharma~ia, Pi~cataway, NJ.
Expression vectors compatible with eucaryotic cells, pre~erably thofie compati~le with vertebrate cells, can also be us~d to ~orm the recombinant DNA molecules of the present inve~tion. ~ucaryotic cell expre~sion vectors are well known in the art and are available from Qsveral commexo~al ~ources. ~ypically, such vectors are provided containing co~Venient restriction sites ~or in~ertion of the de~ired DNA segment. Typical o~ such ~ectors are pSVL and pXSV-10 (Pharmacia), pBPV-l/pML2d (International Biotechnologies, Inc. ), and pTDTl (AT~C, #31255), In preferred embodiments, the eucaryotic cell eXpresfiion vector~ used to construct the recombinant DNA
molecules of the pre~ent in~ention ~nclude a selection marker that is ef~ective in a eucaryotic cell, preferably a drug resi tance selectlon marker. A preferred drug n -= t . I ~ ~? ~ 3 ~ ~ [~ ; c a r 1 -. ~ r i ~æ 5 ~ .S - I ~ : 11 P . 1 4 ~f~oU048 resistance marker is the gene ~hose expression results in neomycin re~istanc~, i.e., the neomycin phosphotransferase (neo) gene. Southern et al., J. Mol.
A~pl. ~ 327-341 (1982).
The use of retroviral expression vectors to form the rDNAs of the present invention is also contemplated.
As used herein, the term ~'retroviral expresslon vector"
refer~ to a DNA molecule that include~ a promoter sequence derived from the long terminal repeat (LT~) region of a retroviru~ genome.
~n preferred embodiments, the expres6ion vector is typically a retroviral expression vector that i~
preferably replication-incompeten~ in eucaryotic cells.
The construction and use of retrovl~al vectors has ~een described by Sorge et al., MQl. Cell. Biol., 4:1730-37 (1984).
A variety of methods have been developed to operatively link ONA to ve~tors via complement~ry cohesive termlni. For instance, complementarY
homopolymer tracts can be added to the DNA ~egment to be inserted and to the vector DNA. The vector and DNA
~eqment are then joined by hydrogen bonding between the complementary homopolymeric tail~ to form recombinant DNA
molecules.
Synthetic linker~ ~on~alning one or more restriction site provide an alternative method o~
~oinlng the DNA ~egmen~ to vectors. A D~A seqment having cohesive termini is treated with bacteriophage T4 DNA
poly~era~e or E~ coli DNA polymerase I, enzymes tha~.
remove protruding, 3', single-strandad termini with their 3'-5' exonucleolytic acti~ities and fill in rece~sed 3' ends with their polymerizing activities. The combination of ~hese activities therefore generates blunt-ended ~N~
segments. The blunt-ended segments are then incubated with a large molar excess of linker mol~cule~ in the ~ 5` 2: 3 ~ 3 ~ 1 D G ~ 1 1 ~ ~ r i e ~ ~ 1 ,~--5 4 ~-- 1 3 8 0 p . 1 5 ~(3~)0~4~3 pre~ence of an enzyme that i5 able to catalyze the ligation of blunt-ende~ ~NA molecule~, such ag bacteriophage T4 DNA liga~e. Thu~, the products of the reac~ion are DNA segment~ carrying polymeric linker sequ~na~ t the1r endF.. ThaF.e ~N~ ~egmenkR ~re the cleaved with the appropriate re~triction enzyme and ligated ~o an expres~ion veator that ha~ been cleaved with ~n enzyme that produce~ termini compatible with those of the DNA segment.
Synthetic linker~ containing a variety o~
re~trictiOn endonuclea6e ~ite~ ~re commerciall~ available from a number o~ ~ources i~cluding Internati~nal Biotechnologies, Inc., NQW Haven, CN.
Also contemplated by the pre~ent invention are RNA
equival~nts of the above de~cribed recombinant DNA
mole~ule~.
X. Trans~ormed Cell and Cultures The preY~nt invention al~o rela~es to a host cell tranaformed with a recombinant DNA molecule of the pre~ent inven~ion. The ho~t cell can be elther procaryotic or eucaryotic. Bacterial cell~ are preferred procaryotic hQRt cell~ and typically are a ~train of E.
coli such a~, for example the E. coli strain DH5 a~ able from Be~hesda Research L~boratorie~, Inc., Bethecda, MD. Preferred eucaryo~ic ho~t cells include yea6t ~nd ~ammalian CR~lS~ prefera~ly ver~ebxa~e cell6 sUch as those from a mou~e, rat, monkey or human fibroblastic cell line. Preferred eucaryotic host ~ells in~lude ~hinese hamster ~vary (C~O) cellS a~aila~le ~om the A~CC as CCL61 and NIH 6WiS3 ~ou~e e~b~yo cell~
NIH/3T3 availabl~ from the A~CC ~S CRI, 165~.
~ra~s~ormation of approp~ia~e host cells with a r~combinant DNA molecule o~ the present inv~ntion i~
aCcomplished by well known m~thod~; that typically depend on the type of vector used. With regard to O C t . I R S ' ~ s :3 ~ r~ l~i S 5P~ r i c~ ~ ~L 1 S _ ~r, 4 ,~. _ 1 ~ F . I ~.
Z(~0004~3 tran~formation of procaryotic ho5t cells, ~ee, for example, C~hen et al., Proc. N~ltl. ~cad. Sci. USA, 69:2110 (1972): and Maniati~ ~t al., ~L~L ~L15~ ~9_~
Laboratory Ma~mal, Cold Spring Harbor ~aboratory, Cold,i Spring Harbor, NY (1982). With regard to tranc~formatibn of vertebrate cells with r~troviral vectorS cont~inin~
rDNAs, see, ~or example, Sorge et al., Mol. Cell. Biol., 4:1730-37 ~1984); Graham et al., Virol! ~ 52:456 (1973);
and Wlgler et al., Proc. Natl. Acad. Sci. USA, 76:1373-76 lo (1979~.
S~ccessfully transf~rmed cell~, i.e., cells that cont-ain a recombinant DNA molecule of the pre~ent invention, can be identified by well known techniques.
For example, cell~ resulting from the introductlon of an rDNA of the present lnvention can be cloned to produce monoclonal colonie~. Cells from tho~e colonies can he harvested, lysed and their DNA content examined for the pre~ence of the rDNA u~lng a method such as that described by southern, J. Mol. Biol., g~:503 (195) or Ber~nt et al., ~LglÇCh_, 3~208 (19~5).
In addition to directly acsaying for the presence of rDNA, successful transformation can ~e confirmed by well known immunologiaal methods when the rDNA i~ capable of directinq the expres~ion o~ a subject polypeptide.
For example, cells ~ucce-cs~ully transformed with a 6ub~ect rDNA containing an expre~6ion vector produce a polypeptide displaying a characteristic antigenicity.
Samples of a ~ulture containing cells suspected of being transformed are harvested and ass~yed for a ~ubject polypeptide u~ing antibodie6 specific for that polypeptlde antigen, ~U~h a~ tho~e produced by hybridoma of the present invention.
Thus, in addition to the transformed ho~t cells themselves, ~he present inventi~n al~o contemplate~ a culture o~ those cells, preferably a monoclonal (clonally 0 ,= t . 1 ~ 3 ? ~ ~ cl .~3 L~ l . 5 ~-~ 1 ~. ~ r 1 e--. ~. 1 c ~ 1 3 8 ~1 F . 1 .--;Z000(~

~omog~neous) culture, or a culture derived from a monoclonal culture, in a nutri,ent madium. Preferably, the culture also contain~ a protein di~playing Integrin beta subunit ~ntigeni~ity.
Nutrient media u~ef~l for culturing tra~sformed host cells arQ well known in the art ~nd can be o~tained from ~:everal commercial sources. In embodiments wherein the host cell is mammalian, a ~serum-free" medium i~
preferably u~ed.
L. M~thQds ~or Produçing ~ $ubieot PolY~e~3tide Another a~pect of the present invent~on pert.ain~ t~ a method for ~roducing a subject polypeptidc useful f or raicing anti~odie~ which can be used in the diagno~tic ~y~t~ms and methods o~ the preseht invention.
The present method entails initiating a culture compri~ing a nutrient medium containing host cell~
transformed with a recombinant DNA molecule of the pre~ent invention that is capable of express~ng a gene encoding a 6ub~ect polypeptide, preferably a polypeptide corresponding to a formula shown in Tahle 1 or 2. The culture is maintained for a time period sufficient for the transformed cells to exprgss t~le subject polypeptide.
The expressed polypeptide i~ then recovered from the culture.
Method~ for recovering an exprQssed polypeptide from a culture are w~ll known in the art and inclu~e fra~tionation of the polypeptide-con~aining portion of the culture using well known bioohemical techniques. For instance, the method~ of gel filtration, gel ch~om~to~raphy, ultrafiltration, electrophoresiS, ion exchange, affinity chromatography and the like, such as are known for protein fractionations, can be used to i~olate the expressed protein~ fouhd in the culture. In addition, immunochemical methods, such a~ immunoa~finity, z~ c~

immunoab~orption and the like can be performed using well known meth~ds.
E~ples The follo~ing exampl~s ar~ intended to illustrate, but not limit, the pre~ent invention.
1. Identification o~ an Adhesion Protein Bindina Site on an Inte~rin Chemical cros~linking has been u~ed extensively to ~tudy the interac~ions of ~GD-containing ligand~ with GPI~b-~IIa. Bennett et al., J. Biol. Chem., Z57:804~ 8~). Most recently, cro6slinking approaches have be~n used to examine the interaction of small RGD
peptides of ~ix to fourteen amino acids with GPIIb-IIIa as a mean~ of characterizing the topography of the RGD
recognition ~ite. Santero et al., Ç~ll, 4~:~67 (1987) and D'Souza et al., J. Biol. Chem., 263:3943 (198~).
The6e studies have shown that platelet activation with agonist, an event necessary for binding of adhesive proteins such as fibrino~en an~ fibronectin to GPIIb-IIIa, markedly and selec~ively enhances the ~ro~slinkingof the RGD-peptide~ to GPIIIa, t~e ~eta cubunit of the Integin GPIIb-IIIa.
The pre~ent study defines a dlBCretR site within GPIIIa to which a ~mall RGD-peptide can be chemically ~ro~slinked. That site is b~liev~d to define the func~ional site for ligand binding to Integrin, and is referred to herein as the RGD-binding region. The amlno aGid re~idue sequ~nce of this region is con~erved in other members of the Integrin family (Figure 6~
indicating it plays a critical role in the function of thl fi family of adhe~ion receptor~.
A. ~G~= ~e~ 3n~91~
Th~ RGD-peptl~e u~e~ ln ~hl~ at~ y, designated Fn-7, has the amino acid residue sequencs KYGR~GDS. This peptide was design~d to contain a ly~ine Z~OV~4~

residu~ (K) to ~acilitate crosslinking and a tyrosine re~idue (~) to provide a site ]Eor radioiodlnatlon~ Fn~7 was prepared by solid-phase ~ynthesis on an Applied Biosystems model 43~ peptide sy~thesizer using peptidylglycine a-amidating monooxygenase resins and t-Boc amino acids purcha~ed from Applied Bio3y~tems. The peptide was analyzed for homogeneity by high performance llquid chroma~ography u3ing a C18~ Bondapak column with a linear gradient o~ 0-~0~ acetonitrile in 0.1%
trifluoroa~etic acld and was found to be >~5%
homogeneous. The amino acid composition of ~he peptide was determined after about a 24 hour period, the hydrol~sates ~eing in a6 N HCl, and the results were con~istent witll theoretical yields. Peptides were di6601v~d in phosphate buffered galine (PBS) prior to use and the pH was ad~sted to 7.2.
Fn-7 was radioiodinated by a modified lactoperoxidase-glucoce oxidase method see Lam et al., J.
Biol. Chem., 2~2:947-950 (1987). ~riefly glucose (20 ~g in 40 ~1 of 0.2 ~ æodium phoæphate, pH 7.4), carrier-free Na125I and the other reagentc by gel filtration on a Bio-Gel P-2 c~lumn. The conditions for radioiod~nation were selected to minimize ligand heterogeneity, and >80~ of the iodinatad peptide was in the monoiodotyrosinated form u6ing this protocol. The concentra~ion o~ the labeled peptide was determined b~ ab~orbance at ~80 nm, using extinction coef~icients derived rrom the amino acid compositions. The æpecific activity of the peptide was 5-8 mci/mg.
B. Platelet Preparation and Chemi~al Ç~o~slinkinq Of Peptide Fn-7 to Discrete Sites os~ GPIII~
Platelets were i~olated from fresh human blood collected into acid~citrate/dextrose by '~OOUQ~

~ifferential cen~rifugation fol.lowed by gel filtration on Sepharose 2s in dival~nt ion-fre~ Tyrode'R buffer, pH
7.3, containing o.1~ ~ovine serum albumln. See, Narguerie et al., ~B~Ql, Chem., 225:154-161 (1980).
Platelet binding o~ Fn-7 ~ollowed the protocols previously de~erlbed for mea~uring platelet lnteractlon~
with adhesive proteins and with thi~ and other peptides.
See, Ginsbery et al., ~, ~iol~ Chem,, 2~0:3~31-3936 (1985); Lam et al., Fed. Proc, Fed, Am. Soc. Exp. Biol., 44:1126 (19~; and Mar~uerie et al., ~u~ra. Briefly, platelets ~ere suspended at 4 X l08/ml in divalent i~n-free Tyrode's albumin buffer. Unless otherwi~e ~pecified, Ca2+ was added to a final coneentration of 1 mM. The platelet stimulus used was 0,5 unit/ml alpha-thrombin, Th~ radivlabeled peptide wa~ then added to 6 X
108 cell~/ml ~timulated or nonstimulated platelets at a concentration of 20 ~M, and binding proceeded for 45 min at 22~C. The ~elected cros~-linking agent was then added. The cross-linking a~ent u~ed ln this ~tudy, bis~sulfocuccinimidyl) suberate ~BS3), purchased from Pierce Chemical Co. was dissolved in ~BS immediately prior to use and admixed with the platelets to a final concentration o~ 0.2 mM. The cross-linking reactions were terminated after 10 min at 22C by addition of 10 mM
Tri~, pH 7Ø
The cell-bound ligand was recovered by centrifugation through 20% sucro6e, and thé cell~ were extracted in PBS containing 1% Nonidet P40 and 10 mM N-ethylmaleimide (Sigma). Extrated proteinS were precipitated with 10% trichloroacetic acid, and the pellet obta~ ned after centriguation wa~ wa6hed three times with cold 85% ethanol. The cross-linked samples were analyz~d by Rl~ctrophor~sis (SDS-PAGE) on polyacrylamide verti~al lab ~els in the bu~fer system of Laemmli, Na~u~, 227 6~0-635 (lS70), For disulfide bond 2nOO(~48 reduction, the sample~ were treated with 5~ 2-mercaptoethanol. Gels were dried and autoradiograms were de~eloped with Kodak X-Omat AR films. MolecUlar weights were estimated on the ba~i~ of eleGtrophoretic mobility rel~tive to ~a~dards ob~ained either from Sigma Chemical or from Bethesda Research L~boratories.
c. Immun~lotti~a Procedures Cro~s-linked sample~ wers immunoprecipltated u~ing a monoclonal antibody designated 22C4, which recognizes GPIIIa, Gin~berg et al., J. Biol.
Chem., 26~:5437 (1987). Washed acid-precipitates, obtained from the cro~s-linked sample~ as described above, were dissolved in 250 ~1 of immunoprecipitation buffer (IPB) which contained 0.15 M Na~l, 0.01 M EDTA, 10 mM benzamidine-HCl, ~oybean trypsin inhibitor (10 ~g/
ml), 0.2 m~ phenylmethane~ul~onyl fluoride, 1~ (v~v~
Triton X-100, 0.05% Tween 20, 0.02~ Na~3, and ~rasylol (5 units/ml) in 0.02 M Tris-~C1, Ph ~.4. The IPB ha~ been found to dissociate the complex of GPIIb-IIIa. The ~ample~ we~e precleared by adding 15 ~1 of heat-inactivated normal rabbit ~erum followed by protein A
reagent (Pansorbin, Behring Diagno~Cs). The cleared ly~ates were then ~upplemented with 1~ bovine serum albumin and 150 ~1 o~ IPB containing 10 ~1 of the above monoclonal antibody. Sample6 were incubated overnight at 4-C, and Pansorbin was then added. A~ter 1 h at 22-C, samples were centrifuged, and the recovered immunopreCipitate~ were washed th~ee time~ by centrifugation in IPB. The precipitates were solubiliz~d by heat for 3 min at 100-C in Laemmli sample buffer and then ~ubjected to SDS-PAGE as described above. For immunoblotting, protein ~amples were resolved on SDS-PAGE
as indicated above. After electrophore6is, the resolved protein6 were tran~ferred onto polyvinylidene difluoride 3S membrane~ (PVDF). Th~ transfers were probed with the zno~o4~

anti-GPXIIa monoolonal antibody, 22C4, an anti -GPIIIa polyclonal antibody or with a rabbi~ antiserum raised to a peptide having a ~equence corresponding to residues 636-654 of GPIIIa. The bound anti~odles were detected using anti-mou~e IgG conjugated to ho~seradiSh peroxidase (Blo-Rad) and 4-chloro-1-napthol as substrate.
D. ChYmQ~rvP~i~ Fraqmentation ÇP~
hymotryptic cleavage o~ platelets lo deg~ades the 100 kDa GPIIIa ~ubunit to release approximately one-third of the molecule from the cell and to ~orm a core comprl~ed of a ~ kDa degradation product which can then be ~urther degraded ~o 55-66 kDa fragments that remain cell-associated. Kornecki et al., J. Biol.
~ , 258:8349 tl983), McGowan ~t al., ~. B~Ql. Chem., 258:11243 (1983) and McGregor et al., Eur. J. Biochem., 148:37 (1985).
Initial studle~ were preformed to determine if Fn-7 crosslinked to GPIIIa with BS3 was retained or released from the cell by chymotryp~in. lZ5I-Fn-7 wa~ crosslinked to thrombin-~timulated platelets as d~scri~ed he~ein before in Exampl~ iB. Crosslinked cell~ were then admlxed with chymotryp6in (0.5 mg/ml final concentration) and maintained for 4 hour~ at 22C. The radioactivity relea~d from the cells was quantitated.
In each of four chymotryptic digests, 50-70~ of the cell~a~soc~ated 12sI-Fn-7 was released from the platelets by chymotrypsin treatment. When the super~atan~ from the dige~ted platelet~ was treated with 10% trichloroacetic acid, only 6-8% of the radioactivity was prec~pitated, indicatin~ that Fn-7 was r~leased in asso~iation with ~mall peptides. When the superna~ant or its acid precipitate wa~ analyzed by SDS-PAGE on 15%
acrylamid~ ggls, ~he rad~oactivity migrate~ more rapidly than an 8 kDa molecular weight standard.

Zn~Q~3 The ~ell-as30ciated radloactivity after chymotrypsin treatment was recovered by centri-fu~ation and analyzed by sDS-PAGE as described hereinbe~ore. As ~hown in Figure 2 (lane 2), the only radioactive band detected ~rom the dige~ted platelets was re3idual, intake ~PI~Ia. Chymotryp~in dimini~hed the Fn-7 radloactivity associated wlth GPIIIa but did not generate di~crete radioactive degradation product~, particularly at the ~6 kDa position.
o Immunoblotting tFigure ~) of the same digest with eithe~ a polyclonal antiserum to GPIIb-IIIa (lane 4) or a monoclonal antibody to GPIIIa (lane 6) demonstrated that the enzyme had generated major derivatives at 66 and 55 kDa. As neither of these band~ were radioactive, the Fn-7 crosslinking site does not re~ide ln the 66 kDa domain of GPIIIa and is released from the cell bY chymotryptic cleavage.
As the puta~ive membrans spanning domain of GPIIIa lie~ close to its COOH-terminus, the 66 kDa fragments immunoblotta~ with an antipeptide antibody raiQed to an amino acid sequence of GPIIIa (re idues ~3~-654), proximal to the putat~ve membrane spanning region (be~inning at residue 693). ~herefore, the Fn-7 crosslinXing site is not present in the 6~ kDa COOH-terminal region of the GPIIIa and reside6 in the 34 kDa N~2-terminal regio~ of the subunit a~ diagramically shown in Figure 4 (steps 1 and 2).
As previou~ly ~hown in Figure 2, chymotryptic clea~age of isolated GPIIIa yields two Fn-? l~beled fragments. To determine the origin o~ these fragments from within GPIIIa, their N~z-terminal amino acid residue sequen~eS were identified. To this end, preparative ~rosslinking of l2sI-Fn-7 to thrombin-3timulated platelets was undertaken; and, ultimately, the GPIIIa:Fn-7 complex ~rom 6 units of blood was i~olated by SDS-PAGE followed ~nO0~4~
C)c t . 1 : ~ 2: 4 ~ c"3 c _:a r -1 5 ~ r i ~ = .~ 5 4 ~-- I 3 ~ F . 1 5' by elution. The isolated product was digested with chymotryps1n and subjected to HPLC rever~e phase chromatography on a ClB column. The radioactivity was poole~ lnto two ~raction3 enriche~ in either the 23 or the 14 kDa fragment~, each of which wa~ subjected to SDS-PA~ on a 15% gel under reduclng ~ondition~, and transferred to polyvinylidene difluroide (PVDF) membranes.
Autoradiograms o~ the transfers containing the 125I-Fn-7 labeled GPIIIa fragment6 ar~ shown in Figure 4 (Step ~), and both the 23 and 14 kDa fragments are apparent. The bands were cut from the transfers and subjected to N~z-terminal sequence analy~i~ u~ing ~tandard automated amino ~cid residue 6eguencing technology, A single predominant sequence was obtained for the 23 kDa fragm~nt tFigure 4). The regidues identlfied at the fir3t four po~itions accounted for 72 to 87~ of the total yield at the~e positions. At all of the determined 20 po8ition6 for the nine cycles performed, the amino acids were identical to the NHz-terminal sequence of GPIIIa.
This places tha Fn-7 crosslinking site within the 23 kDa NHz-terminal region of GPIIIa.
In the 14 kDa fragment, a ma~or sequence predominated for 22 cycles and coincide~ to residues 91-112 of GPIIIa. (A second sequence was detected at a lower yield ~ut wa~ identical to an internal sequence with ch~motrypsin). The residue at position 90 of GPIIIa is leucine, a preferred chymot~ypsin cleavage site. At 14 kDa, the ~maller GPIIIa fragm~nt i~ predicted to extend to residues 200-220 of the glycoprotein, and a 23 kDa fragm~nt extending from the NH2-terminUs ~hould also termlnate within the ~ame region of GPITIa. Thu~" thes~
po~itionings o~ the Fn-7 cro~slinking site are consis~ent with one anothQr and with the localizatio~ to the NHz-~no~

terminal 34 kDa reglon released from intact platelets by chymotrypsin, E. V8 ~rotea e ~ L_ntation o~ GPIIIa.
A similar approach wa~ taken to characterize the V8 protease ~ra~ment~ of GPIIIa contalning the Fn-7 cro~linking ~ite. After the steps o~ enzymatic digestion of GPIIIa:Fn-7 complex~ HPLC chromatography, S~S-pAGE and transfer, the 8 and 10 kDa doublet was observed on tha autoradiogram of the tran~fer (Figure 4, ~tep 4). These bands were exci ed from the transfer and ~equenced separately. ~he 10 kDa fragment yielded a ~ingle NH2-terminal sequence that extended for 16 re~idue$. This gequence corre~ponded precisely to that predicted for reqidue~ 109-124 o~ ~PIIIa. The amino acid residue at position 108 of GPIIIa i~ glutamic acid, a preferred cleavage ~ite for V8 protease. Interestingly, altho~gh a strong gignal was obtained for the methionihe a~ position 124, no ~ignal was obtained at the next residue, predicted ~o be a lysine. Thus, the lysine at the 125 position is a candidate for direct cro~slinking to the lysine in Fn-7.
The 10 kDa frayment extending from residue 109 is predicted to te~mlnate in th~ vicinity of residue 200.
The cequen~e signal of the 8 kDa fragment was n~t a~
~trong as that of th~ 10 kDa derivative, but the first four positions were clear and were identical to those of the 10 kDa fragm~nt. With the same NH2-terminu6 at residue 109 but being 2 kDa smaller, the B kDa fragment should terminate in th~ 170-180 region; two ylutamic acid~ occur within thls 1~ amino ac$d fi~retch. It is notewor~hy that th~ N~lz-terminal sequence of the 8 and 10 kDa fragments was also detected within the sequence determined for the 14 kDa chym4tryptic fragment, providing independent confirmation of the localization of the Fn-7 crossllnking sit~. ThereforQ, the ~n-7 0 ~ t . 1 S ~ = : 4 a, 13 ~t ~3 ~3 C ~ ~3 C~ c~ 1 ~ ~ r i e s ~ 5 4 '-- 1 3 ~: ~3 P . ~2 13 ZO~}OU~

- 4g -crosslinking ~ite is local$zed ~y the~e ragmentation ~tudies to a region defined by residues 109 and the first ~luta~io ~cid re~idues at po~ition 171 in the 170-1~0 reglon o~ GPIIIa.
F. ~ GD-~in~in~

The above ob~ervations indicate that the Fn-7 crosslinking ~ite re~ide~ in the NH2-terminal region of GPIIIa. ~ffinity chromatography experiments were lo und~rtaken to determine if the RGD binding site al~o resided in thi~ region. Previous studies have documented that GP~Ib-IIIa can be selectively bound to RGD affinity column3 from detergent extract~ of platelets and ~pecifically eluted by free R~D peptide. Pytela et al., 15 Sai~e, 231:155~ (1986).
Platelet~ were either untreated or dige6ted with chymotrypsin. The chymotrypsin conditions were chosen ~uch that approximatoly 50% of the ~PIIIa was deg~aded to the 66 kDa derivatlve.
~he two platelet preparations were then fiolubilized with octylglucoside, and the extracta were passed over an R~D affinity column. After washinq to obtain a passed-through fraction, the column was elute~
with free RGD peptide. T~e pa~s-through and the eluate 25 we~e subj~cted to SDS-PAGE on 7~ gel~ under non-reducing condition~, transferred, and immunoblotted with anti-GPIIIa monoclOnal ahtibody 22C4.
A~ shown in Figure 5, lntact GPIIIa was observed in the pase-through from the undige~ted platelet and 30 mixture of intact GPIIIa and the fi6 kDa GPIIIa fragment were present in the pa~-th~ough from the chymotrypsin-treated platelet~. In the RG~ eluates from both columns, only intake GPI~Ia was detected. ~hus, the 66 kDa fragment was not retained on the RGD affinity aolumn.
35 This result is compatible with the interpretati~n that n c t . 1 ~ 5 ~ 3 C.~ ~i C; '~ e ~ i ~? S ~ 5 ~ 3 æ ~ P . ~ 1 2(~0~8 - 5~ -the 6ite which RGD peptides bind, and to which they cros31ink, coinclde and reside in the NH2-termihal aspectS of GPIIIa. Thi~ c~nclu~ion is also consistent wi~h a report of the inhibition of platelet aggregation by a monoclonal antibody to the NH2-terminal region of . C~lv~t.~ nt ~1., nlQL~m- J., 250~ 7 (19~n).
lntion~hl~.Ql~ tl-n R~
Cros~link~ it~ in ~.~Il.Ia in~
GPIlIn i~ a pro~ein 752 amino acid re~idues in leng~h. Fitzg~rald et al., J. BioI Chem.
2~;3926 (1987). Based upon the amino acid residu~
~equences of the chymotryptic and Y8 protease frag~ents, the RGD cro~slinking site has ~een confined to a region, lS of nbout 63 amino acids (the first glutamic acid residue in the GPIIIa 170-180 region), extending COOH-terminal from residue 109.
Insof~r as the site was identified by chemical crosslinking to proteolytic fragments, it is believed that the precise boundaries of the RGD-binding 6ite can vary by as much as about 8 to 15 amino acid residues.
~he~efore the site, for convenience, will be re~erred to a~ an RGD-binding region encompaRsing residue~ from about position 110 to about po~ition 170.
In view of the broad utilization of the RGD
recognition spec~ficity throu~hout the Integrin family, a comparison of the prlmary struc~ure o f this region among the three be~a ~ubunits o~ the human Integrins is of considerable interest.
An alignment of the amino acid residue ~equences of this re~ion of the beta subunits of the LeuCam, the VL~ and the Cytoadheein ~ubf~ilies is shown in Figure 6.
In addition, the deduced sequences of the beta subunits of avian and Xenopus Integrins are included. The amino acid sequence wlth in this region of the Integrins is Oc t . 1 ~ 31~:3~3 D~ 5~ ~r ie~ ~ 1~--5~-- 13~:~3 P . 2~
Z()0~48 remarkably conserved, not only in the human protein~ but also in th~ avian and amphi~ian proteins. In the con~ensus sequence, requiring an amino acid identity at ~ach position in at least four of the five proteins, 47 of the fi3 re~idues t75%) are ~pe~ified. GPIIIa fits this con~en~u~ 6equence at 38 o~ the 63 r~ldue~- ~he conserved nature of this reyion of the Integrins greatly exceed~ the ove~all identlty among Integrins. Such conservation i~ clearly compatible with a contribution of o thi~ reg~on to the function of the~e IntegrinS as adhesion receptors.
As noted, members of each of the three familié~ of human Integrins ~Pytela et al., S~ience, 231:1559 (1~86), Ruoslahti et al., ~ , 44:517 (1986), Pytela et al., J.
~elL~iQl~l 102;4~2 (1986) and Wright et al., Proo. Natl.
Acad. Sci., U.S.A. 84 (1987)] and the avian Integrin tHorwitz et al., J. Cell ~ol., 103:2421 (1986~] can bind ligand~ via an RGD reco~nition -~peCi~icitY. The localization of the RGD cros~linking site in GPIIIa to thi~ region is the first direct implication of the re~ion in Integrin function. Within th~ 63 amino aci~ stretGh of GPIIIa, there are four lysine residues that could be directly involved in the crosslinking reaction: and, based on the amino acid sequence analy~l~ of the 10 kDa ~5 V8 protease ~r~gment, the lysine at position 125 i6 a prime can~idate for thi~ function.
The members of the LeuCam family apparently exhibit the lowe~t a~finity o~ the three human Integrin families for RGD peptides and can blnd ligands that lack RGD ~equence~ AM, a li~and for LFA-l, lacks an RGD
sequence ~orwitz et al., J. Cell Biol~, 103:2421 (1~8~)].
The LeuCam beta chain i~ the only one of the five determined beta chain ~e~uences that does not have a ly~ine at the position corre~ponding to residue 125 of GPIIIa; a non-conservative l~ucine substitution ocour~

Oc 1~ 3C 2 ~ 3 ~ iSS~... r~1 ~ ~ i c~e, ~ 1 <~--54~'~-- 1 3~ 3 ;~)00C~48 for the lysine. It iG al60 noted in comparing the ~equence of GPIIIa to the other Integrins that a non-conserved region, GPIIIa residue~ 129-149, is flanked by two very highly con~erved regions. This non-conserved region is ~ clear candidate for imparting functions to GPIIb~ a, such a~ its high a~finity ~or multiple RGD
ligands, that di~tingulsh it from th~ other Integrins.
Furthermore, it ~houl~ be noted that the RGV
crosslinking ~ite on GPIIIa certainly need not constitute the complet~ recogni~ion ~lte(s) involved in the binding of adhesive protein~ such as ~ibrinogen to ~PIIb-IIIa or other Integrins. Indeed, it ha~ ~een sugge~ted that fibrinogen may ~ind to additional regions of GPIIb-IIIa tKornecki et al., J. Biol. Chem., 258:8349 (1983), Parise et al., slood~ Su~l., 70:357a ~1987)] via non-RGD
~equences, namely the extreme COOH-terminal aspects of the gamma chain of fi~rino~en Kloczewiak et al., Blochemiætrv, 23:1767 (1984). Although g~mma chain peptide6 can inhibl~ the binding and cro~31inking of RGD
peptides to GPIIIa, Santoro et al.,LÇÇll, 4~:867 ~1987)]
have shown that these peptides become crosslinked to GPIIb. The presen~ studies have confir~ed these results.
2.
A ~eries of eight overlapping polypeptide~
from the identified R~D-bind$ng region were synthesized u~ing the cla~ical solid-phase technique de~ribed ~y Merri~ield, Adv. Enzymol., 32:221-96, ~19~9) aC adapted for use with a model 430 automated peptide sy~thesizer (Applied Biosystems, Foster City, CA~. The polypeptides, shown in Table 2, were synthesized with an additional Cys-Gly-Gly (CGG) tripeptide (not shown in Table 2) as a linker attached be~ween the amino terminus of ea¢h polypeptide and the carboxy terminal glycine of the tripept1de, ~o allow for thiol coupling of the polypeptide to a carrier protein. Polypeptide re~ins C~ c ~ 5 2: 4 ,~ 3 ~ Ci c; ~ 1 s . ~ r i ~ 3 ~ e~ 4 ~,-- 1 3 ~. 1 P . ~ ~
~ OV04~

were cleaved by hydroyen ~louride, extracted and analy~ed for purity ~y high-performance liqui~ chromatography (~PLC) using a rever~e~pha~e C18 column ~anufactured by Water~ A~30ciates, Milford, MA.
3. Preuaration of Polyclonal An~i era The synthetic polypept1des prepared in Example 2 were coupled to keyhole limpet hemocyanin (KLH) through t~e thiol residue present on the cysteine reQidue linker to form polypeptide-KLH c~njugate~. Balb/c mice w~re immunized with loO microyrams (ug) of con~ugate~
first intraperitone~lly (IP) in complete Freund's adjuvant, and boo~ter~ wsre then given subcutaneou~ly and/or intraperito~eally in incomplete Freund's adjuvant.
After three or more booster6, antisera is collectad from the responding mice. The collected antisera contain~ polyclonal antibody molecUle~ that immunoreact with the immunizing polypeptide3.
The foregoing speci~ication, including the 3pecific embodiment6 and examples, i~ intended to be illustrative of the present inven~ion and is not to be taken a~ limiting. Numerous other variatiOns and modificationg can be effec~ed without departing from the true ~pirit and scope of the pregent invention.

Claims (12)

1. A polypeptide of no more than 90 amino acid residues in length having a sequence that includes an amino acid residue sequence represented by the formula:
, , , , , , , or .

2. A polypeptide of about 60 to about 90 amino acids residues in length having a sequence homologous to the GPIIIa amino acid residue sequence represented by the formula;
.

3. A polypeptide corresponding in amino acid residue sequence to the formula:
a) , b) , or c) .

4. A nucleotide segment comprising no more than about 12,000 nucleotide base pairs including a sequence defining a structural gene coding for a polypeptide according to claim 2.

5. A polyclonal antibody comprising antibody molecules that immunoreact with a polypeptide according to claim l, but do not substantially immunoreact with an Integrin alpha subunit or with a polypeptide whose amino acid residue sequence consists essentially of a sequence that corresponds to the sequence shown in Figure 1 from residue 191 to residue 240.

6. A monoclonal antibody comprising antibody molecules that immunoreact with a) GPIIIa, and b) a polypeptide according to claim 1.

7. A monoclonal antibody comprising antibody molecules that immunoreact with a) a polypeptide according to claim 2 and b) the beta subunit of an Integrin to which the amino acid residue sequence of said polypeptide corresponds.

8. A method of modulating cell adhesion in a patient comprising administering to said patient a therapeutically effective amount of a polypeptide according to claim 1.

9. A method of modulating cell adhesion in a patient comprising administering to said patient a therapeutically effective amount of a polypeptide according to claim 3.

10. A method of modulating cell adhesion in a patient comprising administering to said patient a therapeutically effective amount of a polyclonal antibody according to claim 5.

11. A method of modulating cell adhesion in a patient comprising administering to said patient a therapeutically effective amount of a monoclonal antibody according to claim 6.

12. A method of modulating cell adhesion in a patient comprising administering to said patient a therapeutically effective amount of a monoclonal antibody according to claim 7.