CA2176208A1 - Surface membrane proteins and their effect on immune response - Google Patents

Abstract

p74 is a protein found in T-cells and other cells, which when bound with specific agents results in inhibition of cytolytic activity and differentiation of CTLs. p74 can be isolated from T-cells and other cells using palindromic HLA-B2702.84-75-84 peptide by affinity binding of a cell 1ysate.

Description

~VO 95/13288 2 1 7 6 2 0 8 PCT/US9.1/1298~

SURFACE MEMBRANE PROTEINS AND THEIR EFFECT O~;
IMMUNE RESPONSE
INTRODUCTION

Technical Field The field of this invention is, ---~ l; r~ of immune response.
Back~round The immune system is the subject of ever increasing scientific scrutiny. Despitethe enormous interest in the immune system and the contin~o~lcly expanding number of investigators, both academic and industtial, a complete ulld~,laL~ ding of the system continues to remain elusive. One of the major blt~Lll~uu6lls was i-IPntifi~tinn of the interaction between the T-cell receptor and a major l,;~ ;l ,;lity complex antigen.
15 The identification that both class I and class II major l~ ihility complex antigens have a cleft which binds a small peptide provided a significant key to ",.1~.,~l ,.I;"g~T-cellspecificityandT-cellresttiction.
The llnfil r~tAn~in~ that the MHC served macrophages and B-cells in their role as antigen presenting cells, where the peptide served to define which T-cells could bind 20 to the MHC-peptide complex still did not explain many other aspects of the T-cell response. It appeared that the binding of the T-cell receptor to the antigen presenting cell was not enough by itself to result in T-cell activation. The process of anergy or Lul~ iul- could not be explained by an activation process solely involving the T-cell receptor and the MHC antigen. In addition, there was the distinction between helper 25 cells (CD4+) and aulJ~)Icajul/~yLulu~iC cells (CD8+). Some m~hAnicrn was necessary to associate the helper cells with class Il MHC and the au~ ;aaul/~,yLuLù~-iC cells with class I MHC. It was ~ . ."lly found that CD4 and CD8 I~uLh,;~ Lul in the MHC
TCR complex by binding a loop of the MHC, enhancing the stability of the complex.

2 1 7 6 2 0 8 PCT/IJS9~ 98~ 0 ln addi~ion, other interactions were uncovered, apparently not directly associated with the T-cell receptor/MHC complex, where CD28 and B7 were found to bind. Thus, there appear to be a number of different interaCLions invo~ved with the association of the antigen presenting cell and the T-cell, which could result in tolerization or acLivaLion of the T-cel~.
Because of the crucial role that the T-cell plays at the center of a major component of the immune system, it remains of great importance to be able to understand how T-cells are selected, activated or tolerized. By lln~Prctl~n~iing the role thal various participanls play in T-cell activation, there will be ~ " ~ to regulate the immune system, either enhancing the immune response, where one is dealing with vaccines, pathogens, neoplasia, or the like, or ~liminiching the immune response, whereoneisdealingwithA~ yororganla~ alltaLiOII.
A number of il",ll",l.~S"l.~lCS~allL~ have been shown to have specific cellular proteins as targets. Cyclosponn A binds to cyclophilin, FK506 to FK binding protein, and d.,~ a~ udlin to both Hsc70 and Hsp70. Hsp 70 has been shown to be a member of a multigene fan~ily, having at least one constitutively expressed cognate, Hsc70. The effect of DSG seems to be specific for antigen presenting cells; interfering with antigen presentation and/or processing. The action of imml~nf cllrpressants is mediated by their cellular binding proteins, and there is, therefore, interest in identifying targets and other agents binding to the targets which will provide modulation of Lhe immune response.
RelGvant T.iter~rllre Wan er al. (1986) J. Immunol 137:3671-4 describes the Bw4/Bw6 epitopes.
Clayberger, el a~. (1987) Nature 330:763-765 dc.~ sL, - that HLA-A2 peptides canreguiate cytolysis by human allogeneic T IYll.L I~.~Y~t;S. WO93/17699 describes the activity of peptides from the HLA-A and -B L~l and a2 helices in mf)~ tin~ CTL
activity. HLA-B2702.6U-84 peptide is described therein.
The molecular CllalA~ lr~ I;on of the heai shock protein Hsp 70 is described in ~mmllno~enetics (1990) 32 242-251, and its constitutively expressed homologue inMol. Cell. Bio. (1988) 8:2925-2932.
,1, r~ .;,,.li.,l~ of the i"""",l..s,~ cs~ive effect of d~ y~ ,u~lin (DSG) may be found in Hoeger, er al. (1994) J. Immungl, 153:3908. The binding of DSG to Hsp70 and Hsc70 is described in Nadler et al. (1992) Science 258:484 and Nadeau et al. (1994) BiochPmictrY 33:2561.

~vo 95113288 2 1 7 6 2 0 8 PCTIUS9~11298~

-3-SUMMARY OF THE INVENTION
Methods are provided for preparing and isolating T-cell proteins which participate in T-cell activation upon antigen presenting cell - T-cell interaction. The proteins are .,I.~ i~d by being associated with T cell activation and binding to the 5 HLA-B2702 ctl helix peptide, and are immllnt~ ir~lly cross-reactive with the heat shock protein cognate, Hsc70. The proteins, fragments thereof and nucleic acids encoding the proteins and ~ onll(~ o~ are provided, as well as antibodies which bind thereto. By employing agents which bind to the proteins or inhibit binding of other agents to the proteins, T~cell activation may be modulated.
DESCRIPTION OF SPECIFIC EMBODIMENTS
Methods and ~,UIIl~o~i~iulls are provided for isolating proteins expressed by T
cells as surface membrane proteins associated with T-cell activation in m~mm~ lnT-cells, which proteins bind to the HLA-B2702 otl helix peptide, nucleic acid encoding 15 such proteins or fragments thereof, and agents for mr)~ in~ the signal associated with the proteins in the modulation of activation of T-cells The protein of interest is referred to as p74. It is a surface membrane protein which is expressed in T cells and a limited number of other cells. It can be readily isolated by extracting a Iysate of T cells with the peptide of the t~l-helix of ~LA-20 B2702, namely the RENLRIALRY sequence, peptide extensions thereof, or dimersthereof, particularly palindromic dimers, having the amino acid sequence YRLAIRLNERRENLRIALRY. The affinity of p74 for the HLA-B2702 palindrome is at least about 104 M. The extraction may occur with the peptide on a surface, such as particles, microtiter well walls, etc. or in solution, where the peptide may be labeled 25 with a label which allows for separation of complexes with the peptide. For example, one may label the amino terminus of the peptide with a ligand, or biotin and precipitate or sequester complexes with antibody or avidin, respectively. The protein may be freed from the peptide and ~ ,J as appropriate.
p74 is a protein found in a limited number of cells, particularly activated CTLs, 30 the T cell tumor PEER, and the EBV ~ rull-l.,d cell line JY, in effect, Iymphoid T
and B cells. The p74 protein can be obtained by Iysis with an amphooeric detergent, such as CHAPS. The Iysate may then be combined with a peptide or protein having an , Bw4 epitope, for example, HLA-B 2702.84-75-84 palindromic peptide.
By preparing an affinity column, having the peptide covalently bound to appropnate 35 particles, the Iysate may be passed through the column, followed by elution with Iysis buffer or other convenient buffer. Effective elution can be achieved using relatively Wo 95113288 2 1 7 6 2 0 8 PCT/USg~/1298S O

-4-high pH, conveniently in the range of about 11-1~. See, for example, (Ey (1978) Imm~mochemistr,v 15:429-436). Alternatively, a different detergent may be used as described by Mescher er al. (1983) Meth. Enz~mol. 92:86-109. Other separation techniques employing the peptides bound to a support may also be employed, such as S panning, magnetic beads, or the like. Usually, after each contacting of the peptide witn the p74 sample, the supernatant will be removed and the solid support washed to remove non-specifically bound proteins and other co~ Washes may be any convenient buffer solution, varying in salt content. The washing should not adversely affect the complex between the peptide and p74. In this way successive enrichments may be employed to provide for a protein .,v.lll,o~;lio.~ having at least about 50 weight % of total protein as p74 protein, preferably at least about 75 weight %, more preferably at least about 90 weight %, up to 100 % pure.
As an alternative purification scheme, the imml-noln~ir~l cross-reactivity of p74 with the heat shock protein cognate, Hsc70, may be exploited. Antibodies directed against Hsc70 react with p74. Various affinity pl-rifi~ Ati~n methods are known in the art. Antibodies may be bound to beads or another insoluble support, and combinedwith the cellular Iysate. The bound protein is washed free of the unbound Iysate. The bound protein is eluted from the antibody by a number of methods, e.g. heat, salt gradients, mild detergents, elc.
Instead of affinity Clll~lllAll~A~ Y~ gel clc~ u~l-v-csis of the Iysate may be performed, where the Bw4 epitope containing peptide, suitably labeled, may be used to identify the presence of p74. The Bw4 peptide may be labeled with fluorescer, rA~ icotope, or the like, non-specifically bound peptide washed away, and the appropriate band developed.
Instead of binding on a solid or in a gel, binding can be ~rcr,mrlich~ in solution, using a suitably labeled soluble form of the peptide, e.g. N-terminal bivLillyld~ivll, e~c. By incubating an appropriate cell with the peptide or dimeric form thereof, pa~ Luly palindromic form, at a mildly elevated t~ diUlC, usually about25 - 40-C. for sufficient time for complexes to form, one obtains specific binding of the peptide to cell surface proteins. The complexed cells may then be Iysed as described above. After Iysis, the complexes may be isolated by means of the label, e.~g. biotin, isolated by means of avidin bound to a solid surface. Alternatively, the label-1 ,., y binding member complex may be preformed and used to extract specific protein binding to the peptide as described above. The samples may then be washed extensively, boiled in SDS and separated using SDS-PAGE gel ele~,~lu~llvlc~;s.

~vo gs/13288 2 1 7 6 2 0 8 PCTiUSg~/12985

-5-Pnor ~o the affin;ty purificat;on or gel electrophores;s, one may enrich for th~desired p74 protein by using the peptide sequence of the B7.84-75-84 palindrome,YGRLNRLSERRESLRNLRGY, to remove any proteins which bind to that sequence, as distinct from the B270~.84-75-84 palindrome sequence to which the p74 binds. In this manner, p74 may be greatly enriched and obtained cllhst~nti~lly free of other proteins.
The subject proteins may be obtained from any m:~mm~ n species, such as rodent, bovine, canine, primate, particularly human, or the like.
Using the proteins individually in a sample of at least about 80 weight % punty,based on protein, the sequence of the proteins may be readily determined in accordance with conventional ways. The protein purified as described above may be sequencedusing ~,u~ -Liollal sequence equipment. The purified protein may be cleaved, using mild proteolytic flr ~ u ;~ ," . cyanogen bromide, or other mode of cleavage, to provide fragments under about 20 amino acids. The fragments may then be fr:~rtif~n~t~d e.8.
reverse phase-HPLC or preparative SDS-PAGE and electroelution, and the fragmentssequenced. The sequences of ~he fragments may then be used to deduce t~le ~equence of the respective protein.
By employing conventional technifll-f-s, degenerate probes may be produced from the known amino acid sequence of the proteins. Using DNA probes of at leastabout 15 nllrlr~r>tiflrs. usually of at least about 20 nllr1f-r tiflr s, one may screen a cDNA
library of cells known to express the proteins, particularly T-cells. Those cDNAS
which bind to the probes may then be isolated and sequenced. Where the sequence encodes the ~lu~lidt~ peptide sequence, if the isolated cDNA is not a complete cDNA, the isolated cDNA may be used as a probe to isolate a complete cDNA which encodes the entire protein. If desired, the cDNA gene may then be used to isolate the genomic gene in accordance with conventional te~hni~l~lec (See, for example, Molecular Cloning: A Laboratory Manual, 2nd ed., J. Sambrook, E. F. Fritsch, T.
Maniatis, CSHL, Cold Spring Harbor, NY, 1989).
The gene or fragments thereof, usually of at least about 25 nucleotides, preferably of at least about 30 nucleondes and not more than about 60 ~llrlf oti~lrC, may be used for probing cells to determine whether a subject protein ;s expressed, when in the cell cycle the protein is expressed, and at what level of dirr~ ialion it may be expressed, and quantitate the amount of message RNA during activation or deactivation of T-cells.
The gene may be introduced into an expression vector for expression in a wide variety of hosts, both prokaryotic and eukaryotic. A large number of promoters are Wo 95/13288 2 1 7 6 2 0 8 PCT/US9~11298~ ~

-6-commercial~y available today which are included upstream (in the direction of Ll.~ JLiull) of a polylinker for insertion of a gene to be under the transcnptional and rranslational control of a transcriptional and translational initiation regulatory region functional in an expression host. Besides the L ~ and ~ l initiation 5 regulatory region, the expression vector may include dUWI~LI~ I from the polylinker, a trRncrrir~ nRl and translational t~rrninRtinn regulatory region, functional in the expression host. Thus, the gene may be inserted into the vector after lil~ iull of the vector with a restriction enzyme which cuts at a convenient site in the polylinker. In addition to the L~ 1 and trRn~tRtirmRl regulatory regions, the expression vector1û may also include a gene which allows for selection of hosts comprising the expression vector. For the most part, these genes will provide for antibiotic resistance, but may also serve to ~--",1 1 "...1l an 21U~U~lU~ host, or other mode of selection. The vector may also include an origin for episomal Ill~ or the vector may allow for integration into the expression host. The vector may be introduced into the expression host by any convenient means, including el~ lu~ul~Liull, fusion, calcium ~ iUi~l.t~
DNA, transfection, etc., the particular mode employed not being cntical tû this invention and depending upon the choice of expression host.
In addition to using the expression construct for production of the subject proteins, the expression construct can also be used in the rll~ri~lRtil.n of T-cell activation. These constructs may be used, for example, in c(-".l~ studies.
For example, cells lacking one or both of the subject proteins, as well as other surface member proteins associated with T-cell activation, may be modified so as to express one or both of the subject proteins. The effect of p74 binding on various pathways in the cell may then be studied. These studies may also examine the effect of mutations on the activity of p74. In addition, one may choose to use the cytoplasmic portion of p74 and bind it to a different extracellular portion, so as to provide a fused protein, which will transduce the signal when a ligand or other binding molecule for the -YtrRrt lllllAr portion binds to the t ~ctrRr~ lRr portion. For example, one may use various receptors with known ligands, such as the EGF receptor, IL2 receptor, insulin receptor, CD4, or the like, where binding of the protein, particularly ~ lu~ hil~g on the surface, will result in signal trRnC~llrriOn Thus, one may use a natural ligand or antibodies to the receptor, so as to provide for the signal Llrll~ l A soluble form of p74 may be uul~LIu~ by deletion of the ~ lle region. The soluble protein is used as a competitor for the native protein, thereby modulating the cellular immune response.
The expression constructs may also be used to produce fusion proteins, where p74 may be fused to a marker, such as 13-gal, CAT, ~acZ, and the like. The fusion ~WossJL3288 2 1 7 6208 PCT/USg~/12985

-7 -proteins ma~ serve as markers for translational e~pression, for production of antibodies, in assays, or the like. The fusion proteins may serve to aid in the of a source of p14, where p74 0r truncated portion thereof may be joined to a sequence which allows for ,UUI ;r.~ ,.. in the Qiagen process or other process.
p74 binds to HLA-B 2702.75-84, particularly as the palindromic dimer. and not to HLA-B7 75-84 or its palindromic dimer. It is associated with the process of T-cell activation. Binding to p74 can result in inhibition of cytolysis by CTL as well as CTL
dirr~,.c-~Li~-Lion. By employing agents which bind to the p74 of CTL, the immuneresponse can be significantly inhibited. Alternatively, by inhibiting binding agents, either naturally occurring or synthetic to p74, CTL activity may be mAin~inf~rl, other aspects necessary for CTL activity being present. For inhibition of CTL activity.
peptides having the Bw4 epitope may be employed in an amount sufrlcient to inhibit differentiation or cytolysis of target ce~ls. Alternatively, antibodies may be employed which bind to p74, where the antibody may provide for activation or inhibition.
Depending upon the antibody, it may inhibit binding of an agent to p74 allowing for acti tion of the CTL, it may provide for activation of the CTL in conjunction wit}l other;,,~ A~ llC of surface member proteins or other signals being transduced into the cell, or it may i",l, .l)....l. ,~lly inactivate the CTL through the binding of p74. Whole antibodies need not be used, binding fragments thereof, such as Fab, F(ab')2, Fv, or 20 the like, or other binding entities, either naturally occumng or synthetic.
p74 when bound to the B2702 peptide results in a large calcium influx in .."",."~r""". ~1 T cells. The magnitude and kinetics are essentially the same as that induced by anti-CD3 ant~odies, supporting the conclusion that the rise in intracellular calcium is involved in the dCVCI~ L of anergy. This response appears to be specific to the B2702.75-84 peptide.
Soluble p74 may be prepared by truncating the respective gene and removing the ~ e sequence and the intracellular sequence. The truncation can be achieved in A~ e with conventional methods, using restriction enzymes, primer repair, exonucleases, or synthesis of the extracellular portion of the protein. The soluble p74 may be used for a variety of purposes. The soluble p74 may be used in diagnostic assays to determine the presence and amount of p74 in a sample, may be used for screening ~,ull~poulld~ which bind to p74 in accordance with conventional binding assays, for prophylactic or therapeutic purposes (i.e. to inhibit binding of agents to the native protein, as well as in research to elucidate the, 'f ~ 1 l ' ' ;`" ' of T cell activation.

8 2 1 7 6 2 0 8 PCT/US9.1/1298S O

Soluble p74 may be used for identifying APCs which bind to p74 as well as agents which interfere with such binding. Using labeled p74, cells binding ~o p74 can be readily detected by magnetic separation, nuulc~cll~c~ etc. Soluble p74 may also be used to modulate CTL response in vivo or in vitro in a mixture of T cells and antigen S presenting cells, such as Illa.,luplla~ and B cells.
Antibodies to the subject proteins may be prepared in aucul-lall-,c with conventional methods. p74, native or soluble form, may be used as an ;IIIIIIUI10~
being injected intravascularly,; " l, AI,r,; 1~ ,. ,. Ally,; " ~ Iy, ~1 l1). I l l A l~r ~ y~ or the like normally in conjunction with an adjuvant, into an a~J~uulvlidlc mammal, e.g.
10 mouse, rat, guinea pig, or the like. Usually, one or more booster injections will be employed to enhance the specificity of the antisera. The immunized host may then be sacrificed, the spleen isolated and ~ o~yLcs immortali~ed by any convenient means, e.g. fusion with a hybridoma. The hybndomas may then be grown using limiting dilution and screened for binding characteristics with p74, as appropnate. Hybridomas 15 which show binding affinity for p74, may then be expanded and the ~ulJ~,IIlàLallL used for isolation of mnn-lrlonAI an~ibodies, or the hybridomas used for the preparation of ascites fluid as a source of mr~norlnlnsl antibodies. The llyblidulllds may then be further subcloned and screened to identify antibodies which have a desired binding affinity. These antibodies may be then be further screened with T-cells having p74 20 under conditions where the physiological effect of the antibodies can be determined.
For procedures for preparing m~-nr,rl--nsl antibodies, see Antibodies: A Laboratory Manual, Eds., Ed Harlow and David Lane, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988.
For use as ~JIu,ull~la~,LiC or therapeutic reagents in a host, the antibodies may be 25 modified by replacing constant regions and framework regions of the antibody with sequences utilized by the host to be treated. Techniques for isolating the variable rcgions of the heavy and light chains and fusing them to variable regions native to the host, as well as :,ub~liLuLiull of framework regions native to the host, have been described in numerous publications. See, for example, EPA 173,494 and 30 WO92/16562.
The subject therapeutic agents may be prepared as formulations in p~ 1y acceptable media, for example saline, PBS, in glucose, generally at a pharmacologically effective dose, the conr~n~rsAtinn~ of which will be determined empirically in accordance with .UII V'~,llLi(Jlldl procedures for the particular purpose. The 35 filrrn-llAAtirm~ may include ~srt~ riri~Al agents, stabilizers, buffers or the like. The amount ddlllillisL~Icd to the host will vary depending on what is being a.llllilli,u,lcd, the ~NO 9sll3288 2 1 7 6 2 0 8 PCTIUS9-1112985 purpose of the adn1lnistration, such as prophylaxis or therapy, whether inhibition or activation is desired, the state of the host, the manner of a.l..~ Li~,--, and the like. In order to enhance the half life of the subject peptide agents, the peptides may be r~i, introduced into the lumen of liposomes, prepared as a colloid, modified 5 by binding to stable peptides, such as the constant region of IgG, or polyalkyleneoxy groups, or other conventional techniques.
The role of p74 in the mixed Iylll~llo~ , reaction, B cell activation, and other cellular or compound i., l r, ,~ may be investigated. For example, in culture, the p74 or biologically active fragment thereof may be added to the culture medium in from about I to 500, Lg per I x 105 cells, where inhibition of T cell activation is studied. In addition, p74 may be used for screening novel compounds for agonist or antagonist activity to CTL activation. Thus, one can combine the protein bound to a peptide or other agent which binds to the protein, where the peptide or other agent is labeled, so as to be detectable. By carrying out a ~mr~titi-)n between the complex involving p74 and 15 the labeled agent with the compound being analyzed, and fh~tl~rminin~ the rate of release of the labeled agen~ from p7~, one can measure the affinity of the candidate compollnd for p74. Alternative~y, one may use CTLs where the peptide or other agent competes for binding to the protein with the candidate compound and measure the effect of the candidate compound on CTL ~lirr~ ion or cytolytic activity as compared to a 20 standard, where the candidate compound is not present.
The following examples are offered by way of illustration and not by way of limitation.
EXPERIMENTAL
Effect of a Varietv of HLA Per)tide,s of Amino Acids 60-84 and HLA- B
2702/0~.145-169 on Lvsis The following peptides were :,y--ll-.,;,i~.
HLA-B2702.60-84 WDRETQICKAKAQTDRE~LR~LRY
HLA-B2705.60-84 WDRETQICKAKAQTDREDLRTLLRY
HLA-Bw46.60-84 WDRETQKYKRQAQTDRVSLRNLRGY
HLA-Bw62.60-84 WDRETQISKTNTQTYRESLRNLRGY
HLA-A2. 1.60-84 WDGETRKVKAHSQTHRVDLGTLRGY
HLA-B2702/05.145-169 RKWEAARVAEQLRAYLEGECVEWLR
HLAB38.6084 WDRNTQICKTNTQTYRENLRL~LRY

WO 95113288 2 1 7 6 2 0 8 PCT/US9~/1298~ 0 The effect of the above sequences on Iysis of long-term CTL speciflc for HLA-A2, -B2705, -Bw46, -Bw62, and -Cw4 was rlr-lr-lTninr-rl, including CTL specific for HLA-B27 and the HLA-Cw4. None of the peptides inhibited or enhanced Iysis with the exception of the B2702.60 84 peptide. This peptide blocked Iysis by all CTL, 5 regardless of their HLA specificity. This effect was due to interaction with the CTL
and not the target cell as shown by pre-treatment ~
These peptides were tested for effects on the dirrclclllia~iul~ of CTL from CTL
precursors in limiting dilution assay. The procedure was modified from Skinner and Marbrook (J. Exp. Med. 143:1562; 1976) as follows: PBL from normal HLA-typed 10 donors were punfied over Ficoll-Hypaque and co-cultured in round bottom microtiter wells with irradiaoed (10,000 R) EBV ~al~rullll~d B-lymphoblasts expressing the HLA allele of interest. Responder PBL were added at 3000, 600û, lOOûO and 30000 cells per well while stimulators were added at 6000 cells per well. 20-4 replicates were setupforeach..,"...,~ ;r.)lofrespondercellsinRPMI-1640medium~u~ l.t~"1 15 with lû% fetal bovine serum plus L-glutamine. Plates were incubated for six days in â
5% C02/95% air humidifled incubator at which time the contents of each well were mixed by pipetting five times with a multi-channel pipette. Fifty microliter aliquots were transferred to the V-bottom microtiter wells to which 1000 5ICr labeled targets of known HLA type were then added. Lysis was determined in a four-hour cyLulu~icily20 assay. Wells were designated positive if specific Iysis was >10%. CTL precursor frequency was determined by linear regression analysis using a computer program.The B2702.60-84, Bw46.60-84 and Bw62.60-84 peptides all blocked the d;rrclcll~;ali~ll of CTL, whereas the other peptides had no effect.
25 Effect of Peptides C~JIIC~IJOIId~ to HLA Re~ions on CTL Precursor Frequencv as Determined by Limitin~ Dilution Analysis Peptide l/CTL Precursor Frequency B2705.60-84 1 64,245 B2702.60-84 349.99 B38.60-84 334,937 A2. 160-84 164,245 Bio46.60-84: : 995,400 Bio62.60-84 995,400 B27.145-169 164,245 PBL from a normal donor (HLA-A3; B-7, 38: Cw4; DR4,6) were cultured wi~l ty (HLA-A2; B7; DR4,6) or HOM2 (HLA-A3; B27) in the presence of 10-100 ~Lg/ml peptide. After 6 days, Iysis was tested on 5ICr-labeled CIR cells expressing either HLA-A2.5 or HLA-B2705. Results are shown for HLA-A2 specific Iysis but similar 5 results were obtained for HLA-B27 specific Iysis.
'rhe effect was not allele specific since the lirrL~ id~iOI. of CTL specific for a number of different HLA molecules was inhibited. None of the peptides affected Class Il restricted responses, including mixed Iy.~ L~y~e responses and mitogen induced ~ILlir~l~Liull.
PBL from normal donors were cultured at sx105 cells/round bottom microtiter well in RPMI-1640 supplemented with 10% fetal bovine serum and L-glutamine.
Cultures were ~ with either 5x lO3 irridiated (10,000 R) pBV l, ... ,~r~ ", .~ ~B Iymphoblasts or 10 ~g/ml phyt~hpm~e~ tinin P (PHA-P). Cells were incubated at 37-C for 3 days for PHA-P and 5 days for alloantigen at which point 3H-thymidine15 was added (2 lali/well). After 16 hours wells were harvested and 3H-thymidine incorporation deter~nined by srilltill~tir)n counter.
EffectofTruncatedSe~uencesonL-rsisandDirr~li..,li~l,."~
Since the B2702.60-84 and B2705.60-84 peptides differed by only 3 amino acids, additional peptides were prepared to investigate the effect of these differences.
Three addihonal peptides were ~y~ l.,s;~d~
HLA-B2702.75-84 RENLRIALRY
HLA-B2705.75-84 REDLRTLLRY
HLA-B2702/05.60-69 WDRETQICKA
The peptide l,L~ ;~LII~dillg to residue 60-69 of HLA-B2702/05 had no effect on the assays described above. The peptide LollL~vlldillg to residue 75-84 of HLA-B2702 blocked all Class I specific CTL responses, whereas the peptide ~, ." ~ e to the same region of HLA-B27o5 did not~
To determine which residue(s) mediated the inhibitory effects, 3 more peptides were synthesized in which single amino acid changes were introduced at residues 77, 80 and 81 to convert the B2702 sequence into the B2705 sequence at that position. The B2702.75-84(D) and B2702.75-84(L) peptides still blocked Iysis by existing CTL and ;rr~ of pre-CTL while the B2702.75-84(T) peptide had no inhibitory activity.
Thus, the isoleucine at position 80 is required for inhibition.
HLA-B2702.75-84(D) REDLRIALRY
HLA-B2702.75-84(T) RENLRIALRY

Wo 95/13288 2 1 7 6 2 0 ~ PCT/US9~/12985 o HLA-B~70~.75-84(L~ RENLRLLEY
It was also found by the following assay that B2702.60-84, B38.60-84 and B2702.75-84 when pre-bound to plastic caused cells to bind. None of the other peptides were found to have this effect. However, when the B2702.60-84 peptide was 5 conjugated to bovine serum albumin or to beads via the cysteine at residue 67, the blocking effect and the ability to bind cells to plastics were lost.
The plastic binding procedure was as follows: peptide (100 ~Lg/ml) was dissolved in PBS and 50 111 was added to round bottom microtiter wells or 5-10 1ll to petri dishes. After 60 minutes at ~7 C or overnight at 4', the solution was removed and the plates washed twice in RPMI-1640 s~rrl~-m~ nt~ with 10% fetal bovine serum.
Cells were added and incubated at 4- for 30 minutes. Binding to petri dishes wasdetermined by inspecting the dishes under a Illil,lU~l_U~lC following gentle agitation.
Binding to microtiter wells was determined after centrifugation at 500 rpm for 3minutes. Cells which did not bind formed a small pellet at the bottom of the well 15 whereas cells that did bind did not form a pellet.
Binding occurred equally well at 4, 25, or 37' and was not dependent on exogenously added divalent cations since binding was observed in medium containing EDTA. However, if cells were preincubated with 1% NaN3 or fixed with ~,a,Arulllldldcll~de, no binding was observed, indicating that viable cells and most likely 20 generation of ATP were required.
Isolation and CI.aIA~ ~r~ Of p74 The amino terminal amino groups of the B2702.60-84, B2702.84-75-84, B2702.84-79/79-84, B2702.84-75T17S-84T, B7.60-84, and B7.84-75nS-84 peptides 25 were conjugated to biotin-(CH)12-for use with strepavidin-agarose (SAA) to isolate the peptide receptor from 35S-methionine and cysteine labeled cells.
HLA-B2702.60-84 WDRETQICKAKAQTDRENLRIALRY
B2702 84-75-84 Palilld.u~ ; YRLAIRLNERRENLRIALRY
B2702 84-79-84 Palindrome YRLAIRRIALRY
30 B2702 84-75TnS-84T Palindrome YRLAIRLNETRENLRIALRT
B7.60-84 WDRETQICKAKAQTDRESLRNLRGY
B7.84-75nS-84 Palindrome YGRLNRLSERRESLRNLRGY
Two different protocols were used. In the first, the biotinylated peptide was complexed to the SAA and allowed to bind to labeled cells at 4 C for 30 minutes. The 35 cells were washed free of excess complex and Iysed by addition of CHAPS containing Iysis buffer. This method ~lcrtlc~ lly ~.c,,i~ ..t., material from the cell surface. In ~VO 95/13288 2 1 7 6 2 0 8 PCT/US9.1/12985~
Ihe second protocol cell ~ysates were prepared in CHAPS ~ysis buffer and the ~ysate was incubatcd with biotiny~ated peptide/SAA comp~exes for 30 minutes at 4-C fol~owed by extensive washing. This method preferential~y precipitates inn ArP~ r material For both methods proteins wcre separated by SDS-PAGE and visua~iæd by nu J~ . The B2702.84-75nS-84 peptide bnngs down 2 bands of 70 and 74 kD
whi~e the c~osely re~ated B7.84-75J7S-84 peptide does not. The 70 and 74 kD bands were a~so broughl down with the B2702.60-84 and B2702.84-79/7g-84 peptides but not with the B7.60-84 peptide. These two bands cou'd be ~ iL. t~ d from the surface or Iysate of CTL ~ines and periphcral blood Iymphocytes but on~y from the Iysate of most other cel~s tested. Substitution of either iso~eucine with threonine ~ed to a signif1cant decrease in the intensity of both bands and the doub~e threonine substituted peptide B2702.84-75TnS-84T did not precipitate either band.
The 70 and 74 kD bands were subsequently shown to be reactive with antibodies specific for members of the heat shock protein family. Biotin-conjugated B2702.84-75/75-84 or B7.84-75/75-84 peptides were used to precipitate proteins from CTL Following separation by SDS-PAGE proteins were electrophoreticall~
transferred to a ny~on membrane and the Westem b~ot was probed with a m-7m-~Ir-nAI
antibody specific for Hsp70. It is seen that the antibody reacts specifica~y with the p74 protein.
Isoe~ectric focusing on the p74 protein as we~ as on the Hsc70 after JiLdLiOl~ with either peptide or anti-Hsc70 mAb shows an identica~ molecu~ar weight and isoelectric points. The B2702 peptide a~so binds to the heat inducible fomm commonly referred to as Hsp70 although the binding to the Hsc70 is much more VUIIC~:l. Both forms are expressed on the cell surface and are upregulated following treatment of cells at 43- for one hour. Expression of the Hsc70 protein corre~ates with peptide induccd ca~cium increase and the induction of unresponsiveness.
Binding of the B2702 peptide to ~ r -" "~ rl T cel~s was found to result in a large calcium infLux having a magnitude and kinetics l - 1~ O that induced by anti-CD3 antibodies in support of the peptide binding to a surface membrane protein and being involvcd in the dcvcl~--- .-L of anergy. Other Class I HLA ccl helix derived peptides did not induce this response under ~ conditions.
The correlation between the peptide induced increase in calcium and iL lLiull of the 70 and 74 kD bands from the surface for a number of different cel~
types is shown in Tab~e 1. The largest increase in intrace~ular calcium is observed in thehumanCTL PBL Jurkat andratsp~eence~s. WiththeexceptionofJurkat these Wo 95/13288 2 1 7 6 2 0 8 PCT/US9~/12985 0 cells also express the highest levels of the 70 and 74 kD bands. Six ~Lg of p70 and p74 have been purified.
Table 1 Effect of B2702.84-75-84 peptide on intr~rPlllllrr calcium and correlation with ~ J;Ldliull of 70 ar d 74 kD bands fronr the cell surface.
CELL CALClUM[nM] 70 kD BAND 74 kD BAND
C~L - CD8+ T cells 500 +++ +++
PBL- Human 250 ++ +++
Jurkat - al3 T cell tumor 400 - +
HUT-78 - a~ T cell tumor 50 - +/-Peer - ~3 T cell tumor None HSB - al3 T cell tumor None NK clones - Human 200 ND ND
JY - B Iy~ Jhul,ldst None - +/-MS - Burkitt's 100 - +
Daudi - ~2m-Burkitt's 75 - +
K562 - Pre-erythrocyte None HEL - Pre-erythrocyte None Rat Iy.. l~Jl,~yLc~ -ACI spleen 200 - +++
An increase in ;"l. ,.r~ Ca2+ induced by peptide was observed in only some of the cell lines tested. The greatest changes in in~r~rrll~ r Ca2+ were ,~ l inPBL, CTL lines, and the human T cell tumor Jurkat. A smaller but significant increase in intrAr~ r Ca2+ was induced by the inverted repeat B2702.84-75~75-84 peptide in two Burkitt's Iymphoma cell lines, Daudi and MS. The B2702.84-75/75-84 peptide did not cause a Ca2+ flux in the human T cell lines Peer, HUT-78, HSB; human Epstein Barr virus tr~ncforrnrr1 B cell lines, including JY and 721.221; the natural killer cell line YT2C2; or several pre-erythrocytic cell lines, including K562 and HEL.Neither the B2702.75-84 peptide nor the inverted repeat B7.84-75/75-84 peptide initiated Ca2+ Illubili~Liull in any of the cells tested.
The threonine substituted peptides, which failed to cause IIIIIC:~)Ull~ l in T
cells, were also unable to promote Ca2+ mobilization. The maximum intr~r~ r Ca2+ level induced by the B2702.84-75/75-84T and B2702.84-75T/75-84 peptides was only 10-30% of that achieved with the lln~llh~ti~l~l~l B2702.84-75n5-84 peptide.
The double substituted B2702.84-75T/75-84T peptide did not initiate any Ca2+ flux.
These results suggest that the B2702 peptides cause an increase in intr~r~ lAr Ca2+
_ _ _ ,, , ~!0 9sl~3288 2 1 7 6 2 0 8 PCT/US9 ~1298~i that results in T cell unresponsiveness. The B270~.84-75T/75-84T pept;de, which did inhibit any T cell responses, did not cause a rise in inrr~rP~ r Ca2+.
Mobili~ation of intracellular Ca2+ is mediated by at least two major mf ch~nicmc IP3 is one messenger molecule that links surface receptor activation to S the release of Ca2+ from internal stores by binding to a receptor on the Pnrir~rl~cmir reticulum. The level of IP3 in cells is elevated following the activation of either G
protein linked receptors or by tyrosine kinase linked receptors. The other major"~ ,- is Ca2+ induced Ca2+ release (CICR). It has recently been rl~mr~nctr~tPd that a molecule called cyclic adenosine .l,l.l,b,l.l, . ribose (cADPR), a metabolite of 10 .l;~ padenine~ lrlF~ll;flF-(NAD+)~isthemessenger~ Jb~l~;L/lcforregulating this channel through the ryanodine receptor. Using the T cell line Jurkat, we observed that stim~ tirm with anti-TCR mAb produced a substantial increase in IP3 whereastreatment with the B2702.84-75n5-84 peptide had no effect on levels of IP3. The peptide induced increase in intracellular Ca2+ is likely to be mediated through the 15 ryanodine receptor.
Another early event in T cell activation is a change in the patterns of phosphorylation of many proteins. Therefore, it was determined whether the peptides that inhibited T cell function affected protein ~ llulyldP~oll or the level of;..., ~, . 11"1~.
calcium. None of the peptides examined thus far has had any effect on the pattern of 20 tyrosine phosphorylation observed by Western blot analysis in whole Iysates from T
cells.
A C~ dli~J-- of the effects of the B2702 84-75-84 palindrome with other known ;II,Il~ dlll~ f' a novel pattern of activity. The results are ~.~....A.;,..lil~T~ble2 Wo 95113288 2 1 7 6 2 0 8 PCT/US9~/1298~ 0 Tab~- 2 CSA FK~06 Rapamycin d~u~ya~ udlin B2702 Block T cell +++ +++ +++ +++ +
proliferation Block Cytotoxiciy - - - - +++
Block Cytokine +++ +++ - ND ND
Block IL-2 +++ ND - ND ND
T"" . ~
Induce Ca++ flux - - - - +++
Block calcineurin +++ +++ - ND ND
Cellular Receptor Cyclophilin FKBP FKBP Hsc70 p74 The data d~ oll~LIaL~s that B2702 haâ a novel pattern of activity, as compared to the known immunusu~ ;s~ànts~ cyclosporin A, FK~06, rapamycin and 5 deoxyspergualin.
It is evident from the above results, that p74 plays an important role in the mt~ tirm of CTL activity. By controlling protein interactions resulting in signal transduction or the absence of signal ~ lc ~ 111, CTL ~irr~ .c~id~iul~ and cytolytic activity can be modulated.
All ~ c and patent A~ Al;llllC cited in this cre~ rir)n are herein i~lcullJuldl~;d by reference as if each individual publication or patent application were specifically and individually indicated to be i~,u~ ,d by reference.
Although the fo}egoing invention has been described in some detail by way of illustration and example for purposes of clarity of ulld~,laLdlldillg, it will be readily 15 apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and " " K 1; r~ may be made thereto without departing from the spirit or scope of the appended claims.

~VO 95/13288 2 1 7 6 2 0 8 PCT/US9 ~/12985 SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) A?PLICANT The Board of Trustees for the Leland Stanford Junior University (ii) TITLE OF ~NVENTION SURFACE MEMBR~NE PROTEINS AND THEIR
EFFECT ON IMM[~NE RESPONSE
(iii) NtlMBER OF SEQUENCES 20 (iv) ~U~ UN~4N ~ ADDRESS
A) T~nnVRCCRR FLEHR, HOHBACH, TEST, ALBRITTON ~/ ~ERBERT
B) STREET 4 r ' C~dT LU Center, Suite 3400 C) CITY San Francisco D) STATE l"Alil'nrrliA
, E ) COUNTRY USA
F) ZIP 94111-4187 (v) COM?UTER READABLE FORM
(A MEDIUM TYPE Floppy disk (B COMPUTER ~BM PC compatible (C OPERATING SYSTEM PC-DOS/MS-DOS
(D SOFTt\TARE PatentTn Release #1 0, Version #1 25 (vi) CURRENT A?PLICATION DATA
(A) A?PLICATION N0MBER PCT/USs4/
(B) FILING DATE 10-NûV-1994 ( C) CLASSIFICATION
(vii) PRIOR A?PLICATION DATA
(A) A?PLICATION N[~MBER 08/150,493 (B) FILING DATE 10-NOV-1993 (viii) ATTORNEY/AGENT INFORMATION
(A) NAME Rowland, Bertram I
(B) REGISTRATION NtlMBER 20, 015 (C) ~~ /DOCl~ET N~MBER FP-58976/BIR
(ix) TEL~;~VI_ JNl~TION INFORMATION
(A) TELEPHONE (415) 781-1989 (B) TELEFAX (415) 398-3249 (C) TELEX 910 277299 (2) INFORMATION FOR SEQ ID NO 1 ( i ) SEQUENCE ~TD ~ A ~
A LENGTH 2 0 amino acids B TYPE amino acid C sTR7lT~RnNEcc single ~ D TOPOLOGY linear (ii) MOLECULE TYPE peptide (xi) SEQUENCE ~ U~ SEQ ID NO 1 Tyr Arg Leu Ala Ile Arg Leu Asn Glu Arg Arg Glu Asn Leu Arg Ile Ala Leu Arg Tyr (2) INFûRMATION FOR SEQ ID NO 2 (i) SEQUENOE r~rTv~ ;1lU j:
(A) LENGTH 20 amino acids -WO 95113288 - 2 1 7 6 2 0 8 PCTII~S9~/1298~ 0 (B) TYPE: amino acid (C) STR~ nNRcq single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Tyr Gly Arg Leu ~sn Arg Leu Ser Glu Arg Arg Glu Ser Leu Arg Asn eu A~g Gly Tyr (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE rp~ DrTR~r.qTIcs A) LENGTB: 25 amino aclds B) TYPE: amino acid C) ST~ r~n~Rqs single D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE L'~;S~ llUlY: SEQ ID NO:3:
Trp Asp Arg Glu Thr Gln Ile Cys Lys Ala Lys Ala Gln Thr Asp Arg lD l5 Glu Asn Leu Arg Ile Ala Leu Arg Tyr (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE r~DrTR~TqTICS:
A LENGTB: 20 amino acids B' TYPE: amino a~id C sT~D~nRnN~.q~: single ,D TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Tyr Arg Leu Ala Ile Arg Leu Asn Glu Arg Arg Glu A~n Leu Arg Ile ~ 10 15 la Leu Arg Tyr (2) INFORMATION FOR SEQ ID NO:5:
(i) sEgJENcE rp~ rTRl7rqTIcs A LENG~: 12 amino acids B TYPE: amino acid C sT~Rn~Ecq: single 1 D TOPOLQGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Tyr ~rg Leu Ala Ile Arg Arg Ile Ala Leu Arg Tyr ~VO 95/13288 2 1 7 6 2 0 8 PCT/US94/1298~

(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE r'BZ~PrTT;'RTCTICS:
(A) LENGTH: 20 amino acid6 (B) TYPE: amino acid (C) 5T~NDRn~Rqq 6ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Tyr Arg Leu Ala Ile Arg Leu A6n Glu Thr Arg Glu A6n Leu Arg Ile la Leu Arg Thr (2) INFORMATION FOR SEQ ID NO:7:
( i ) SEQUENCE r~TP~ p ~ LW
A) LENGTH: 25 amino acids B) TYPE: amino acid C) ST~ ~CC: 6ingle 'D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Trp A6p Arg Glu Thr Gln Ile Cy6 Lys Ala Ly6 Ala Gln Thr A6p Arg Glu Ser Leu Arg A6n Leu Arg Gly Tyr (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQ-~ENCE rup~ T7~TcTIcs AI LENGTH: 20 amino acids B TYPE: amino acid C ST~ nN~qq ~ingle D I TOPOLOGY: lir~ear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE l~ ~LlUN: SEQ ID NO: 8:
Tyr Gly Arg Leu A6n Arg Leu Ser Glu Arg Arg Glu Ser Leu Arg Asn eu Arg Gly Tyr (2) INFORMATION FOR SEQ ID NO:g:
(i) SEQUENCE ~P~Pr~TF~TCTICS:
(A I LENGTH: 10 amino acid6 (B TYPB: amino acid (C sT~ n~c~: single (Dl TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE J~;S~l:Clrll~?N: SEQ ID NO:g:

WO 95113288 2 1 7 6 2 0 8 PCT/US9~1112985 0 Ary Glu Asp Leu Arg Ile Ala Leu Arg Tyr ~2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE r~T~TPrT~TCTIC5:
~A LENGT~ 10 amino acids B TYPE: amino acid C ST~Nn~n~ECC: single D TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQIJENCE ~ b~ lUN: SEQ ID NO:10:
Arg Glu Asn Leu ~ Thr Ala Leu Arg Tyr rg 10 (2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENOE rT~ rT~TqTICS:
A) LENGTH: 10 amino acids B) TYPE: amino acid C) ST~NnRnl~T~cc: ~ingle D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Arg Glu Asn Leu Arg Ile Leu Leu Glu Tyr (2) INFORMATION FOR SEQ ID NO:12:
~i) SEQ-JENCE rF~ rT~Z~TqTICS:
A LENGTH: 10 amino acids B TYPE: amino i~cid C sT~ nNRqc: ~ingle ID. TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Arg Glu Asn Leu Arg Ile Ala Leu Arg Tyr (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE r~ rT~TcTIcs (~ LENGTE: 10 amino acids (B TYPE: amino acid (C ST~ ~TlnNF~c single (D, TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
rg Glu Asp Leu Arg Thr Leu Leu Arg Tyr (2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE r~T~rT~7TCTICS:
(A) LENGTH: 10 amino acids ~WO 95/13288 2 1 7 6 2 0 8 PCT/US9~/12985 (B) TYPE: amino acid (C) sT~zlNn~nN~c.~: single (D) TOPOLOGY: linear ~-:
(ii) MOLBCULE TYPE: peptide (xi) SEQUENCE ~ ~lrLlUN: SEQ ID NO:14:
Trp Asp Arg Glu Thr Gln Ile Cys Lys Ala (2) INFORMATION POR SEQ ID NO:15:
(i) SEQUENCE r~7\DprT~TcTIcs:
(A LENGTH: 25 Lmino acids (B, TYPE: amino acid (C sTRLNn~nN~ cc ~ingle (D TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Trp Asp Arg Glu Thr Gln Ile Cys Lys Ala Lys Ala Gln Thr Asp Ar Glu Acp Leu Arg T}Lr Leu Leu~Arg Tyr 15 (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE rT~p~prTR~TcTIcs:
Al LENGTH: 25 amino acids B TYPE: amino acid C sTRpl~Tnli:nN~!cs: ~ingle I D TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi ) SEQUENCE DESCRIPTION: SEQ ID NQ :16:
Trp Asp Arg Glu Thr Gln Lys Tyr Ly~ Arg Gln Ala Gln Thr Asp Arg Val Ser Leu Arg Asn Leu Arg Gly Tyr (2) INFORMATION' FOR SEQ ID NO:17:
(i) SEQUENCE rrPl~prT~TcTIcs~
A) LENGTH: 25 amino acids B) TYPE: amino acid C) sT~Nn~nN~:.C: single ID) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE l,l~ llUN: SEQ ID NO:17:
Trp Asp Arg Glu Thr Gln Ile Ser Ly~ Thr Asn Thr Gln Thr Tyr Arg lu Ser Leu Arg Asn Leu Arg Gly Tyr (2) LNr1 --TrN FOR SEQ ID NO:18:
(i) SEQ~ENCE rFrP~P~ 5:

WO95113288 21 7 6 2 0 8 PCTIUS9~/1298: 0 (A) LENGT~: 25 amino acids (B) TYPE: amino acid (C) sTl7~Mn~nN~cq: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Trp A~p Gly Glu Thr Arg Lys Val Lys Ala His Ser Gln Thr His Arg 5 l0 15 Val A6p Leu Gly Thr Leu Arg Gly Tyr (2) ~NFORMATION FOR SEQ ID NO:l9:
( i ) SEQUENCE r~
A) I.ENGTH: 25 amino acids B) TYPE: amino acid C) ~ bb: single D) TOPOLOGY- liDear (ii) MOLECI~LE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9:
Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Leu Arg Ala Tyr Leu Glu Gly Glu Cys Val Glu Trp Leu Ar (2) INFO~MATION FOR SEQ m NO:20:
(i) SEQ-~ENCE r~T3~'T~.~rCTICS:
A LENGTH: 25 amino acids B TYPE: amino acid C 1 ~ ~ single ~ D' TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQ~ENCE L~ ~: SEQ ID NO: 20:
Trp Asp ~rg Asn Thr Gln Ile Cys Lys Thr Asn Thr Gln Thr Tyr Arg Glu Asn Leu Arg Ile Ala Leu Arg Tyr

Claims (10)

WHAT IS CLAIMED IS:

1. A composition comprising at least 50 weight % of a lymphoid surface membrane protein designated as p74 and characterized by:
having an affinity to HLA-B2702.84-75-84 peptide of at least about 10-4 M, having a molecular weight of about 74 kD as determined by SDS-PAGE gel electrophoresis, being expressed on B and T cells; and when present as a surface membrane protein of a nontransformed T cell upon binding to HLA-B2702.60-84 induces calcium influx and inhibits at least one of CTL
differentiation or cytolysis by said T cell, or fragment thereof comprising at least the portion of said protein binding to said HLA-B2702,84-75-84, the transmembrane region, the intracellular region or the extracellular region.

2. A composition according to Claim 1 comprising a fragment of said protein according to Claim 1, comprising said extracellular region and lacking atransmembrane region.

3 . A composition according to Claim 1, comprising at least 90 weight % of said protein.

4. A composition according to Claim 1, wherein said protein is obtained by affinity purification with a polypeptide comprising a Bw4 epitope.

5. A method of screening compounds for their effect on cytolytic activity of T cells, said method comprising:
combining a candidate compound with the extracellular portion of p74; and determining the amount of binding of said compound to p74 .

6. A method according to Claim 5, wherein said extracellular portion of p74 is present on the surface membrane of a T cell.

7. A method according to Claim 4, wherein said binding is related to the cytolytic activity of said T cell.

8. A method for enriching a composition for p74 from a lysate from cells expressing p74, said method comprising:
combining said lysate with the palindromic peptide HLA-B2702.84-75-84 bound to a solid support;
separating said lysate from said solid support;
washing said solid support to remove non-specifically bound proteins; and isolating protein bound to said support to provide a composition enriched for p74.

9. A method for inhibiting the modulation of CTL activity of in a cellular composition comprising T cells and antigen presenting cells (APC), said method comprising:
adding to a cellular mixture of CTLs and APCs the extracellular portion of p74 free of cellular surface membrane in an amount sufficient to compete with p74 for binding of ligand to p74, whereby the activity of said CTLs is modulated.

10. A method according to Claim 9, wherein an immunosuppressant is added to said cellular mixture up to an amount to immunosupress said CTLs.