AU2003203658B2 - Conjugate heat shock protein-binding peptides - Google Patents

Description

-9

AUSTRALIA

Patents Act COMPLEM SPECIFICATION

(ORIGINAL)

Class Int. Glass Application Number: Lodged: Com plete Specification Lodged: Accepted: Pubfihed: Priority Related Art: Name of Applican: Sloan-Kettering Inttute For Cancer Research Actual InveritorCa): James E Rothman, Mark Meyhew, Mee H Hoe, Ousriell, Yoichi Moroi Alan N Houghiton. U~lrich Harti, Quiathek Address for Service and Correspondence: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Corrns Street Melbourne 30D0 AUSTRALIA I nverli ion ruie: CONJUGATE HEAT SHOCK PROTEIN-BINDING PEPTIDES Our Ref: 692167 POF Code: 14241112794 The following staftement Is a full descrption of this invention, including the best methrod of perfrmnrg Rt known to appflcant(s): 1la CONJUGATE HEAT SHOCK PROTEIN-BINDING PEPTIDES The present application is a divisional application fromn Australian patent application number 11 130/9 the entire disclosure of which Is incorporated herein by meferenas.

1. INTRODQUC1QN The present invention relates to conjugate peptides engineered to non-covalently bind to heat shock proteins; (II) to composions comprising such conjugate peptides, optionally bound to heat shock protein; and (III) to methods of using such Compositions to kiduce an immune response In a subject in need of such treatment It is based, at eIs in part. on the dscvery of peptie sequences which may be used to tthw anligenic peptides to heat shock proteins. The present invention almo provides for methods of identifying additional tethering peptides which may be omprised, together with antigenic sequences, In conjugate molecules.

2. BACKGROUND OF THE INVENTION Heat shock pmtins ccnsitute a highly conserved class of proteins selectively ecpressed in cells under stressful conditions, such as sudden inreases in temperature or glucose deprivation. Able to bind to a wide variety of other proteins in their non-ntive state, heat shoc* proteins participate in the genesis of these bound proteins, including their synthesis, folding, assembly, disassembly and translocation (Freeman and Morimoto, 1996. EMBO J. n: 2969-2979; Llndquist and Cralg, 1988, Annu. Rev. Genet.

22:031-877; Hendrick and Harti, 1993, Annu. Rev. Biochen. 2: 349-384). Because they guide other proteins through the biosynthetic parthway, heat shock pmteins are said to function as "molecular chaperones" (Frydman et aL., 1994, Nature M: 111-11T; Hendrick and Harli, Annu. Rev Bkxiochem. 349-384; Marti, 1996, Nature 381: 571- 580). Induction during stress is consistent with their chaperone function; for example, dnaK, the Eschechia coi hsp70 hornolog, is able to reactivate heat-inactivated RNA poymerase (Zlemienowcz et al.. 1993. J. Biol. Chem. 268: 25425-25341), The heat shock protein gp96 resides in the endoplasmic reticuium, targeted there by an amino-terminal signal sequece end retained by a carboxy-terminal KDEL arnino acdd motif (which promotes endopasmc retcukrn recapture; Srivastava et al.. 1987 Proc. Natl. Acad. Sci. U.S.A M: 3807-3811). Found in higher eukaryotes but not in Drsophila or yeast, gpSs appears to have evolved relatively WcMunteni4Q SdMla pinda recently, perhaps by a duplication of the gene enooding the cyxoolic heat shock protein hsp9O7 to which it is highly related (Li and Srivastava. 1993, EMBO 2:3143-315 1; identity between human hsp9O and murine gp96 is about 48 peent).

It has been proposed that gp96 may assist in the assembly of multi-subunir proteins in the endopiasmie reuculum (Wiech at al., 1992, Nature 33:1 69-170). hideed, gp96 has been obsenred Io assciate with unassembled immunoglobuIin chains, maor histocompatabiiity class 11 nokecuIes, end a mutant glycoprotein B from Herpes sirnpiex virus (MnIe k cii.. 1992, J. Bot. Chtin 262:21303-2136: Melnick at a..

1994, Naure f:3 73-3 75 Schaiff et i 1992, J. Exp. Med. 116:657-666; Raxakrishnan CL a, 1995, DNA and Cell Biol. 4:373384). Furth, expression of gp96 is induced by conditions which result in the accunulation of unfoldd proteins in the endoplasmic reticulun (Kazrsuii et alt, 193 8, Nare 332:462464). It has been rcporte4 tt gp%- appears t have ATPase activity (Li and Srivatava, 1993, 8MBO J, 12:3143-3151), but this observaion has been quesined (Weanch and Nicduitta, 1997,J. Biol. Chou, 22.5152-5156).

Unlike gp9O, hsp90 1.*i the signal peptide and KDEL sequence associated with iccalizatian in the edoplasmic rerclum. residing, instead, in the cytosal Although hsp90 has not been detected as a component of the transationai machinery (Frydmaun et al, 1994, Natue I:1 11- 116), it has been reported to be highly effective in converting a deatured protein in the absenc of nuc leodides such as ATP or AM, to a 'folding competent" state which can subsequently be refolded upon aldition of hup70, hdj-I and mrjecuid (Freman and Morimot, 1996, EMBO J. .Lt29 -2979; Schneider et at, 1996, Proc. Na. Ao& Sd. U.S.A. 2l: 14536- 1454). Hsp9O has been observed to smrve as a chapro to a number of biologically ihly relevant protens, including sternid aporc eptos, tubuhdm oncogenic tyrosine kinases, and ellulwar eimthenine kineses (Rose et a, 1957, Biocherisuy 26;6583-6587; Sanchez t al., 198, Mol. Eocingl. 2:756-760; Miymta and Yabaru, 1992,. Biol. ChinL 262:7042-7047; Doyle and Bishop, 1 93, Gnes Dv. :;633 63w; Smith and TAft, 1993, Mol, Endorinol. 7A4-11; Xu and Lindqui, 1993, Proc.

Nail Acad. Sci. U.S.A. 2:7074-707S; Stacata t al, 1993. J. Bid. Ch=m, 2Mr: 21711-21716 Cutfort and Rubin, I94, Cell 1Z:1027-1035; Pratt and WeLsh. 1994.

Semin. Cell Rio], j-:83-93; Wanm and Davis, 1 994, J. Bic]. Cirm. ZWt6695.

6701 Nathan and Ljndq uisx 1995, Mat. Cell. Biol. 11:3 917-3 925; Redmond et a., 1989, Ear. J1. Ccl, Biol, UQ:66-75). I-Ip 9 o has been observed to fimctiorn in concert with other proteins, some of which may act as tru chaperones, others serving only as acccssmics; for example, cellular assembly of the progesterone rceptor has bee reported to involve bsp9O and sevn other protins (Smit ct al., 199 M,Mol CCli, Dial. [U:6804-6812).

I-sp9O has been implicated Idl the rmhanisa of reverion of tranifrAdon by the anibiotics gelanmycin and IICiLin A (Whiteselle al-, 1994, Proc- Na4. Aced. Sci. U. S-A. 2X:8324-8328; for Structurs aee FIGURE 9A).

These antibiotics are members of a clas of compounds krwwn ws beunqnone ansamycius derived from actinomyccie and originatlyisoAed far their herbicidal activity (Omur et at,, 1979, 1, Antibiotics 2125 5-26 Exposure to herbfrnycin A and geidanamycin was obseved to revert the morphology of fibroblasts tranfonne via vafiuOUoncogenic tyrOsitt kiussa, lrMduding uc, &n3, Ick, bcr-b mid etbB2 (Uehar at al., 1988, ViroV 1(4.2%-299); as a result, thes compounds have been (mhcmr cmerrnusly. s=e infa refied to as tyrosine kinase inhibitors ad have beep tested as anti-cancer drugs (Yanedacet al., 19931 Cliii. invest 2:2791-2795 Houa at sa, 19M, Ih J. Cancer&6:685-638).

It was repoirted that herbimycin A treatment of Row sarcoma vzrm transformed cels reulted mn reduced kraus activity =nd inaed turnver of the tyrceine kuiiase pW're (Ueharu -t 1989, Canme Res. 4±ISO-785). However, benacquin aismmycins were subse*quwnriy found to have no direct uffect on tYFOSJDkifme activity (Witesell etal-, 1992, Can=crRe&fU;1721-1729); rather, their mechanism of action appear ma invoive iibition of hsp9O/tyrosne kinse heteroprotein complex formation and consequen incrased turnver of p 6 0" (Wbitesell et al., 1994, Proc. NMtI. Acad. Sci. U.SJLa.8:324-9328). Thes drugs have also been shown to interfere with the chaperont tbction of hsp9fl outside of the wrosine kiniase earnes; Smith et al. (1995, Mol. Cell. Biol. j1-6&04-68!2) rcport that geldanamycin rest progesteron receptor assmbly at an inanrmdiate step.

Inculation wnth heat shock protein prepared fr-om inmors of exPcfimezital imals has been show]' to induce immune responss in a nnor-spedfic nmnr; tha is to say.. heat shock protin bp9 puriled from a partcular tumor could induce a imnwmueponse wvhich wold inhibit the growt of cellis fro(wn t identical tumnor of origin, but not Other tmors, regardlns of Telmiedness (Srivastva and Maki, 1991, Cunr. Topics Microbiol. 162109-123). The narc of the tumor-specific nmunogcnic-ityr Wa not been confid. Genes enoding heat shock protems have not been found to exhibit tiLnor-spcitk DNA polymorphism (Srivastava and Udoflo, 1994, Ctwn. Opin. Immunol. f1728-732). High-resOludon gel electrophoresi has indicated thal tumor-derived gp96 mnay be hetergeneous at the mnolecular ]ee[; evidence suest OWt t awc of this hetuogeneity mnay be populatons of smal peptides adheret to the hea shock proteua, which may number in t hundred (Feidweg and Srivasz 1995, lInt. J, Cancer Ca.310-3 14). Indeed, an antigenic pepide of vesicula stoatilis vinn has been shown to associate with Vp9 in virus infected cells (NieLand at al., 1996. Proc. Not. Acid. Sci. U.S~A 9fl6135-61139). hi h=s ben suggested that this wuwulhtion of pcp-du is relaned to the localization of gp96 in the endoplasmic raficulurn, wher it may act as a peptde Wacept and accessory to pepti-de ludizig of mnajor histocompntabilliy omplex class I molecules (Ui and Sdrvagxava, 1993, EMBO J. 12:3143-315 1; Suto and Srivastava, 1995, S'cienc-e 26.21585-1588).

The use of heat shock proteins as djuvants to stimulate an imunem respons has been proposed (see, for example, Edgni 1995, Biciechnol, 1l: 1442-1444-; PCT Application IstrernaionAt Publiatio Number 1WO W429451) by the Whtdead Iwnstitt for Biomedical Reseach, Richard Young, inventr, and referencs iqra)- One of the best known adjuvanus Freunds complet adj uvant, contain a mixur of bea shock proteins denvcd from mycobucewia (the genus of t batuw which causcs tvberculsis); Frewids omp~let indjuvant has bea usd for yeas to boos the immune response to non-ncobarieal antigen. A number of referendes suggest, Iter aija, the use of isolated mycobacteral heut shock proteins for a simnilar purpose, including vaccination against tuberculosis itself (Lukrcs et al., 1993, J. Exp. Med. 123:343-3439; Lowri et aL., 1994, Vaccine 12:1537-1540; Silva and Lowrie, 1994, Immunology fl:244-248; Lowrie at alk, 1995, 3 C.ll, iochen.

SuppL. Q(XUb)20; Retzlaff et al, 1994. lafect immun, fZ:5689-5693, PCT App1icaiO1 Internatonu Publication Noc i WO 94111513 by the Medica Research Council, CoIm ton aL, inventor; FCT Appliction inr~nanal PUblication N-0. WO 93/17 71 by 3iocine Scavo Spa, Rappuoli et31.1 invot#9 Other reernces focu on t abiity -of heu shock proteins to naturally form asociation with angenic peptides, rather than the claessical adjuvant wtfivity 4cc, for example PCT A pplication No. PCT(U596 1323 3 by Sloan-Keteing hnsutc for Ctcer Rewar4 Rothnan et., inventor; Blachee aWx Srivflva, 19"5, Seminars in Cance Biology f-349-355; PCT Application Inteiational Publication No. WO 95/2492-3 by Mount Sinai School -of Medicine of the City University of New York, Srivastava 81 al.. inventrs). In one protocol used by Syivastava in a phase I Eropean clinical tria, cells prepared from a mrgily needted tumm wern used to prepare gp96, which was then renoculated into the sme potiont (Edgingiom 1995, Blot Tebnl. 111442-i1444). The Met that a new gp9E piuparation must be wade for each patient is a significant diuadvantq. PCT? Application Internationa] Publication.

No.. WO 95 P24923 (qwa) suggest tha peptides in hea shoc-k protin cmlpkexes may be isolated and then reincorporaed into heat shock protin complexes in vitro.

There is no evidence tat skis tie-conawning procedure would he mmsuccai beyond the treatmnt of the patient fom which the hea shock protin wa derived. Further, the preparaion of an effective quanity of heat shock proten require the harvest, fr-om the patient, of an anoimt of tissue which not every patlnt oud be able to provik. Morover, this approach bimit. the use of heat shock protein as pepude cam=io to those pqpdne wit Which a natural asoaton is ftiand in vtv, and the affinity of w6c peptides for heat shock protei may be inadequate to produe a desire imMUne respons using complexes generated M in 4f.

In autifpt to circumvent these luimuin, hea shock proins have bceitovatedy joined to mntigenic peptdes of choice For enpe, it has been repasti tha a aytbctic pa~de comprising multiple iteration ofVNANP f(Am Ala Am Pro) malarial antigen, chemicaly crogslinke to glumdehydc-fixed rnycohuceria] brat shock proteis hsp65 or hsplO was capble of inducing a hmiora] (antibody based) im mun qrsons in mile in the absece of fur*The adjuvant; -a simnilar Cffet Was Obserived using heal shock jpr<rein fon the bartrium ~E rcwihiO cobi (DeO Guidicc, 19"4, Experientia 2:106j -1 W66 Ban-jog et al., 1994, Clin. Exp.

lImmunol. 9-6:224-228; Barrios et at., 1I9M Eur. J- Tmmuriol. 2Z, 1365-13 72). Cross- Eitng of synthetic peptide to heat shock prouiin and possibly glumml~dehyde fixation were required for antibody i nduction (Barrios et al., 1994, Clin, Exp. Immunol.

f:2 29-233), and cellular immunity d=nnot appear tD be induced. In amother example, Young et al., in PCT Appiieaiof Intematona Publication Number

WO

9429, dces fusion proteins in which an antienic protein is joined to a hea sh~ock protein.

A potna disadvanragp of such coveleng linkage approaches i8 tha they tend to favor an anil dy-based, rather tha a celuljar, immune response In such come xt. the heat shbock protein may act as a carier to pmoat antibody respnses to covalently l inked proteins or peptides a wei? known adjuv&An function of Iimnogmnic proteigu. Fwihennmore, hea shock protin and antigen arm irreersibly linked; this may ite the solubility of elterprwi componentor may creat smructWn diswtoion which interferes with the association between atigen and critical major histocoinpatabilitY complex compones, The present invenat overcmas These limttions by usin conugat peptides comprisig the desired targe antgen and also aL tether which binds to heat shock protein without the need for covalent Attalat.L Rothmm et al., in PICT Application No- PCW9S6.R/13363 discloses such conjiute peptides including -a peptide camnring, 4s a *dw, a pciae scguer= meopizej by nIonm-guid el tx al, (1993, Cdl 1l:71 7-2 IS)a binding to the hwat shock proltein BiP (a mmber of the bsp7o protein famil). The preset invention. reiats to the idmntificajion of addition.

ttiem 'tich may be emxprisd, togethe with an aruige into conjugate peptides. In prefeired, nonlaidn embodimnents of the invention such tethes may be compried wn onugat pephides in order W noncovaientiy link ante with die beat Shock proteins bsp90 and/or p9. Funthermre, unlike prior at approahes which utilize hea shock proteins in their tradirgsj adjuvant roe, the presen invention coinpuses Ohe use of bea shock proteins found in the intended host specie, including eridcgezmw heat shock proteins Thmughout the description and the claims of this specification the word 'comprise" and variations of the word, such as tcomprising" anrd "comprises" is not Intended to exclude otler additivesu, components, intagers or steps.

The discussion of documents, acts, materials, devices, articles and the like Ies included in this speciflcation slely for the purpose of providing a oontexd for the present invention. It is not suggested or represented that any or all of these mallse formed padrt of the prior art base or were common general knowledge in the field relevant to the present invention as it dexisted In Australia before the priority date of each claim of this application.

IC)

3. SUMMARY OF THE INVENTION The present invention relates to onjugate peptldes comprising a portion which may be bound to a heat shoak protein under physiologic conditions, referred to hereafter as the "tther; and (lI) a portion whlich is anligenic (hereafter, the mantigenic peptide"). Both peptide and non-peptide tathers are provided for.

i addition to providing for aspecific tethers and conjgate peptides, the present invention also relates to methods of identifying fLrdr tethers. These methods utlie filamentus phage wxpression library panning, and are iryrovements over prior art phage panning protocols In that the methods d the invention siulate conditions found In the native wilular location for peptdehat shock protein binding: (ii) utilize compounds which fadiltat the binding of peptide to heat shock protein, such as ansamrycin antibitlvs andor isolate region of heat shock protein which are associated with peptide bindkng and use said isolated regions as the substrate in a phage panning protocol.

The present Invention provhdes a method of Identifying a peptide which binds to a heat shock proain, comprising: contacting a phage display library comprising a plurailty of bacteriophage which express. in a surface protein, a plurality of inserted peptildes with a hsp target in a physioogic binding buffer; isolating a phage which binds to the hsp target; and (iii) idenifyng the Inserted peptide expressed In the surface protein of the phage.

The present invention also provides a conjugate peptide comprising: a tater which comprises a peptide identified by a mrnethod including the steps ot contacting a phag.

display library rrmprising a plurality of bacterIophage which express, in a surface protein, a plurality ot Inserted pepilda, with a hsp target bound to EL benwpquinone ansamycin antibodc in a Mnding b~,le IsolaUng a phag which binds to the hap targe; and identying th Inrted pepid. expressed In tie surface protein of t ph"$s; and (ii) an anhleic paptide.

The invenioin furthe relates to the use of conjugate peptidee In Indu~cfg an Imrrwne response in a subject. The resulting Immune response mnay be dhecled towd, for example, a turnwr cell orea pathogen, and a6 such may be used In the pwntn or treatinent of an Infeclious or malignant disease. The coqugate peplides of te invention may be adrnilistered elber togeherwith or, alternaively, without, one or mare beat shock proteins. it has been discovered that a corjugate peptide. adnistered vifioul exogenous heat shoc protein 1 was capab~e of intduing an Immnrme rnsponise.

4. DESCRIPTION OF TI-E DRAWING0S FIGURE 1 respectively, show the distriuioui of amino acids at poeltkina 1-7 of hsptupeptides expressed by phage bound to gpG6 in the presence of herbirnycin A, where t bfidwg buffer used was 20 MiM HEPES pH 7.5, 100O mM KCI, 1 mM MgAcetate, and 0.1%, or TWEEN 2D depending on fte painning round. Amino avid sequences (SEQ ID NOS: 1-S7) and orresponding nucleic adid sequences (SEQ ID NOS: 38-74) of certaki binding pepIdes.

FIGURE 2A-H. respectively 1 s4ow the distribution Df Emirto acids at positions 1-7 of be pqptda expressed by phage bound to gp96 in the presence Of herbu~nys A. wher the biniding buffer ilia Wwl 20 ruM HEPES pH 100 mM KCI. I mM DTT, IcaM MgAcetc, and 0. or 0.5% TWEEN depending On the pianig roud. Amino acid sequen (SEQ ID NOS 75 -107) Mnd owrrsponding ucleic acdaequences (SEQ ID NOS: JOB3- 1 4 0 )asequenccocf =-Wn bWadin pqxides.

FIGURE 3A-B. Crotoxi-c ativity Of effector Tcel Is prepared fitim ik; ifmmized once w IchOVA POOaid (SJ~hIFEKL; SEQ ID NO: 141) plu TiterMax adjuvant, agis OVA-pdmffed EL-4 targe calls or unprimed EL -4 control calls In a careful compariwon of immune adjuvaina, TilciMax w25 shonn previously to be the optimal adjuvant fix induclion of cylotoxic T ME responses against OVA pept ind w other peptdes (DyaII et al,. 1995, lInternt. imnumol.

21205-1212).

FIGURE 4A-D.- Cynotxic activity of effecto T cells prepared from mice wammunized w~ith bsplO plus OVA-SilP conjugat peptida again OVA-primed ETA4 target cells or unprnned EL-4 ontrol *cell Each curve represents data 'obtaied from -a single mouse, Mice w eftber iMmunized onc (solid square and triangles> or twicc (ope. squre md rectagles).

FIGURE A-B. CYtotoxic activity of effector T ca~s prepared frm mic iniuunizd owce (solid square and tringle) or twice (open -sqtmm nsmd iectWWea) w'ith OVA-RiP Ccozj Lgat peptide (without added adjuvWA or apWOVA-pimed EL-4 target cells or urnpimne £14 contro cells FIGURE 6A-B. Cyzooxic activity of effector T cells prepared from mice inmmizd once (solid squares and triangles) or twice (ope squars a nd retanges) with Titerlax Plus OVA-BiP conjugou pepide agait OVA-primed EL> 4 tage celS ot unpuired EL-4 control cells FIGURE 7. Cywwcxic inctvizy of offcctor T cell prepared from mice immunized once .(Solid cir-ck14) or twice (open square and diamonds) with OVApeptde alone.- FIGUTRE SA-H. Tumor diameters iin mice inimize with (A) TherMal pius OVA-peptide; HsplO phas OVA-peptde; TiterN~x plus OVA- HiP; H&O7 plus OVA-HiP; control (no immunization; tumor cells only injected); OVA-pajade alone; or OVA-HiP alone prior to E0J7 tumor Cell cange. (14I) depicts the averug delay of onse of EG'7-OVA tumor rowth in mice immunized with ceber OVA peptide only, TiiwMax and OVA peptide, Hs~plO and OVA peptide or Hsp 70 or OVA-DiP.

FIGURE 9A-D. Strvgures of geldananycin

M

GDM") and berhimyiin A C'HAn. ReAction of.a primary amine wit geldanamycin at the carbon 17 position. Comparison of the reacdiviiesf herbimycinkA and geldanatnycn towards the same nucophile. Reaction of linker unth grldwaumvcin and herbimycin A, and diiem product obtained theefrom FIGURE I CA-F_ Conjugation of peptides, via their carboxyl tefini to geldanwnycin using at variety of linker molecules. Tinew pair of examples ae presente in which arm either schemtic C and E) -or which speifically utiliz the OVA peptide D and F).

FIGURE I ITA-F. Conjuption of pepddles, via their amino tcmini, to dwauny'Qn using a variet of inkzr molccis Three paur, of examples6 are presented in which are cidxthescmatc C and IS) or which specificay utilize the OVA peptide.

FIGURE 12. Affpohrnnt of Frnocprotected amino aid to TOT and chlcotrityl rfams.

FIGURE 13A-B. Synchais of protcte peptid on TOT resin to rroduce a fully protcte iritegediut whicha may be used for coupihg of gekianycin at the amnino terminus of.a peptide.

FIGURE 14A-B. Frotection of the last amino acid of pcpt synteis with Buc and removal of thec protected peptde from TOT rein to produc a pcptide with a icactive carboxyl ternmu for coupling to gcldawmycm.- FIGURE 15. Reaction of geldanamycin with the cmarbxyl terminus Of a peptde protcted at hts amino icrminw followed by deprotction usung nfifluoroacetic acid 2.5% methylene chloride (CHCl7) and uiiscprpysilew (MPSt and purificationm (using a po~yHYDROXYETHRYL Aspartamide cniumnL FIGURE i6A-B. Reion of geidanayci]n with the amjnok re nus of a peptde Proteled at it& carboxy trmfiinus followied by depretection and purfication..

FIGURE 1 7A-c. Conjugaw PePtidus co05mpi a gekianamycin anaog wnhb lower binding affintity far hea shock pimein. Prepsmtianof a geldanmmYain anlog with a known Iowar affinity for hsp0, Amnino terminal wCnjuse of a Jow affinity geldanamycin anaog. Carboxyl terninal conjugaze of a low affinity geidaanycin analog, FIGURE 18S. conjugat pepwiescomnpisinig antien ic peptide joincd to Jgelanar-nycjn viaa variet of cleavable linkers FIGURE Z91A-G. Melanma tworgrowi a mi chane vjxlwthe OVA~expesmng mclmnlma 041l Jime M04 after immuniAtion. with eidnr (A) TiwerMax pivs OVA pepfide-; tlsp7O and OVA pcptda Or Hap7O aW OVA- BiP pepide and E) show tumor grwt when eidwe OVA pepdde alone or HSP7Il and OVA-Bd' were adnini:sterrt 14 days aftr tumor chalenge. depkt the surv ivat ratios of mice icnmwiizej seven days beore Iwllage with mlanoma calls. depictS t survval. ratios of mice immauzed seven and fourteen dlays after Chalentge with melanoma cell&.

For purposes of ciiy of pres enrzian and not by way of limiftation 1 the deraled description of the invention is divided into the following subsections.

methods for identif u turban; 00i 0*qugfat peptidcs: and (iii) methods of using conjugate peptides.

1. MPTHODS FOR I DFrYENQ ThEjjg.j The present invent ion prov ides for methds for idendfying a tether which may be compflhul together with an anigenic peptide., in a onjugate peptide.

The conjugate pepride, via the tether, may then associate with a heat shock protin in viro 20dOr in vtvo.

Idedtification of suitable tthfem may be achieved through the technique of affinity paning, using an exjnssion library such as a filamentous phage expression library, to identify cloned pepxides which bind to a heat shock praicint Suitabe phage display libraries include, but are not limited to, the "Ph.D. Phage Display Peptide Library Kit" (Catalog #8100, New Englad BicLabs), the "Ph.D.-12 Phg Display 12-mci Peptide Library" (Catalog W8 I 10, New England RioLabs), the "TiSelct Phae Display Systm" (Novagmn Inc.) (se also, United Soles Patents 5423,409; 5,403,484; =nd 5,571,698) and libraries prepared as described in Blnd- Elguindi et at. (1993, Ce1 2j5-717-728, citing Cwirla et ak, 1990, Pr=. Mad. Acad.

Sri. U.S. A- 12:637M382) which reports the identification of peptides that bind to RP using phage pa4tm& For excampLe, and not by way of limitation, this technique may be practiced by expsing a phage expression library, each phage displaying a different peptide sequcne- to a solid substate coated with a heat shock prtein target (hmcn&oth die "lsp WrgeC). under oOditios which allow the binding of phage [a the hap target. Unbound phage is then washed away, and spccifmciy-lboimd phage is demd either osing a substnce which reeases pep ide frm the lsp target or by lowering the pH. The clud pool of phage may then be amplificd. and the prcss may than be repeated (prefeaby te or four time4 using the selected pbagt Then, iivididu clones may be isolated end seqenced to identify the peptidus which they contain. The idetifled peptides may the be synthesized in quantts which allow dinet testing of their ability to bind to hap targ.

As a specific, nonlnilting example, the "Ph.D. Phage Display Library" from New England Rioabs may be utilized to identify tthers, using the procol set forth l the coresponding instruction manaL The 'PKD. Phag. DispLay Library" is a combinaral librmy of rndom pepuide beptames fused to a minor cat protein (pl) of the filametous coliphage M1 3. The library consists of 2 x 1W elctrporated sequece'. amplified once, to yield an avrage of appvoxiaely 100 copin of each peptide sequence in 10 P1 of the phage libary- The displayed beptapeplides ar ewpressed diretly a the N-termins of pil, followed by a abort spacer (Gly y fly Sr SEQ ID NO: 142) and the native pill protein. Affinity paing using this library aye Cptrfonned aS Mows. A well (6 mm in diemner)-ofa 96 well polystyrene mtrotitel plhu may be coated with hap targt by adding 150 lit of a510W200 pg/mI.

solution of hvs target in 0. 1 M NaHCO3, pk 1.3-9.6, and swirling unetilte Well surface is completely wd. The plate rmay, dhe be incbaee overnight Bt 4-C on a rocr in a bwnidified ontinerr the wells may be covcred with cape or the plate may be placed in a sealable plastic box linecd with damp pepe tovitls). Plates conainitg wells prqurd in this man=e may be stored at 4-C in a bwnaidified cotainer VW~l neeed. Iznnrdaiely prior to ume, the coating solution is poured off, and meidua solution removed. The well may then be fiWe with "bloking buffem (0,1kM NaHCO3 (pH 5 mg/mi. bovine scrum ibumlin (BSA), 0.02% NaN,), ad incubaed at 4*C for at leas one hour The blocking solution may tme be discarded and the well washed rapdly abou six timnes with "iHSr" [50 mM Tdis-HCI (pMH ISO nmM NaCI. 0,1-0.5% (vfv) TWEEN-20 (the permetag of TWRJEN-20 may be icrased Drom 0. 1% to in successive ounds of panning~j. working quickly to avoid the well dryfngout 21x10" age may then be diluted in lO0 W1 of 'binding buffce (which =ay be TBST or wtticb may be vasied as discussed bi-a), and pipettd iuto he w~aled well, The plac may then be rocked genty, at room tcnpemturc or at 37-C, for I 0-6 minuses Then, the phage-contaiingt solution rumy be discarded. and the well washe about ten times with bindin buffer. Next; bound plugs may be elute by adding 100 Mpl 0.2 M glyciuc-HCI p1H 2.2 and incubeting for about ten minutes The resulting cluxe may then be pipened into a micracenirifige tube and neubied. wit 15 0i 1 i M Tria pH- 8.8-9. 1. The eluat may then be amplified by inocuatn a mid-loj phas cukure of 2R2;537 Erchqdchia coli (P lac!DEL TA (1aucZ)MI5praA +B-t-/ffi A~supDhtDEL TA fac-proB)DEL TA QttdMSC- (r~n4McrBC-) with the clined phig., and incubating at 37-C wit vigorous shaking for about 4.5 hafirs. If small nunibet of phuge elte from 2we hsp target. a second round of amplification using a fresh hocell culture iii mid-log phase, may be desirable. The culture may then be transferre to a centrifuge tube and spun for nms at 10,000 rpm (using, frT example, a Sorail: SS-34 rotor) at 4,C. The suprnazat may then be tsfred to a fresh centifuge tube Wn re-spun. The uipper prcent of the resultng supnuaan may the be transfrred te a fresh tube, and 1/6 vojune of PEGIN&CI (20% (wtv) polyethylene gyc4oOO8 2.5 M NaCI) may be added. The phage may the be arnwbd to precipitate at 4-C for at least 1 ho. and preferably oernight- The precipftawd solution may be ceifuged for 15 minutes at [0.000 rpm at 4C. after which the supemmuaant may he dcanted, the tube re-spun bnefiy. and residua] supernatani may be removed with a pipm. The resting pellet may 6c resusended in I ml TS (50 mM Tirs-HCi (pH 150 mM NaCI), Which may then be transferred to a ricroccntdfuge wue and spun for 5 minutes at 4C. The superaxani may be Lrmnsfrred to a fsh microcentifuge tube and reprecipitated by adding 116 volume PEG/NaCL, incubating on ice for 15-60 minutes, and cntrifuging in a raicrofug for 10 minutes at 4C, The supenwan: may be diecare4, the tube respun briefly, and residual superaant discarded as before. The pelet may be upenmded in 200 p TBS containing 0.02% NaN 3 and the resulting solution micrcentrifuged for abou one minute to embve any remaining insoluble mabeial.

The supernatant constittes amplified uase, which may be titered to determine the volume which contains 2 x 10" phi. The amplified elumt may then be used in a second round of biopanning. Preferably, three rounds of biopanning are used to idntif phae which specifically bind to hap target.

The lsp target used for affinity penning may be any heat shock protein or portion thereof, or any fuion protein compiing at learn a portion of a heat shock protein. The te N beat shock protein", as used herein, refers to s s prateos (including homologs therf expressed constitutively), including, but not limited to gp96, hp90, RiP, hsp70, pO, hp40. hsc70, and hap 10. lsp target may be prepared frm a natwUl mutate, expressed rorbinandy, or chemically synthesizd.

For example, recombinant expression of gp96 for use as a hsp target is described in Section 6, iir. cONAs which may be used to express other heat shock prteins include, but are oot limited to, gp96: humat Genebenk Acsio No.

X15187; Mai et al., Proc. Nall. Aad. Sci. U.S.A. j7:5651-5562; mouse: Gerebank Accio~ No. M16370; Srivanva 61 at.. Proc. Nad. Aemd. Sei. U.SA. M:3807- 3811; BiP: human: Genebauk Accession No. M19645, Ting et aL, 198, DNA :275- 286, mouse Genebank Accession No. U 16277, Haa et aL, 1988, Proc. Nail. Acad.

Sci. U.S.A. 11:225022$4; hsp70: hwnan. Genebank Accession No. M24743 Hunt et 4, "1985, Proc. Nali. Acad. Sci. U.S-A. fl: 6 4 5 5 -489; moin* Gncbok Acesion No. NO35D21, hun t alt, 190, Gemc 12:.199.204; and hsp4O: humaun: Ganebank Accesion No. 049547, Chrauka M93, BkOcIUCI. Bwoptys. Res. Caunmnu., M:235- 240, Such scqucncca may be expressed using any wropnaze expression vecWt known in the art. Suitable vectors include. but are not limited to, herpes simplex viral based vectors such as PHSV I (Ocere el at. 1990, Proc, Natl Acad. Seti US.A.

JJ;8950-895-4); rewrvimj3 vector such Is MPG (Jaffelc 1993, Cancr Ret.

51:22 21-2226), and in panicular Molo0ney ctovYa vcwrs such as.LN, LNSX, LNCX, and LXSN (Mfiler and Rosrnn 1989. Biatuclmiques 2:980 -989); vaccinia vim] vectOr such as MVA (Suuer and Moss, 1992, Proc. Nat Acid. Sci. U. S.A.

R2:10947-1085 adenvirus 'vector such as pJMl7 (All e-t BLh, 1994, (lecnt heapfy 1:367-384; Blerke, 1988, Biocbhniquca 6:616-6U4; Wand and Finer, 1996. Natur Medic 2V-74-7 16); adeno-anociated virus vector, such as AAV/ncO (Mura-Cacho vt at., 1992, 1 Immunoaher, 11:231.23 pCDNA.3 (IntVitroge); pEE? I I a pET3, pET I Id. pETXd pET22d, wnd pEfl2a (Navagen.); pland AH5 (which conbains -the origi and the adanovirw major afte promowe); pRC/CMV (lIlY iRmge); pCMIU7(Paabo et aI 946 ENB B 1: 1921-1927) fp~i~mSV (CeJCo et aL, 1984, Ccli U,1053-106M) sau pSRu: (DNAX, Palo Altc, CA).

The affility penning procdure may be varied in alterntive cinbodiment, of the presnt invention- For exampie. and as discussed More f[uly below, the binding bulft ft d to biwi *Se to hip targa and/or the hip turn itself may be modified chemically or by genefic enginerin technique.

hn a first ries of embodimenls, a low ionic strngt binding buffer, such as tha used in the Paming experiment of Bloud-ElguWn et at 1993 Pcell a,717-728, may be wued A specific, nonlimiting example of such a binding buffer is pHJ7.5, 2OIIM KCI, 1O mM(NI{4)SOM,2 mM MgC2, andc0-1s-0.5% TWEEN 20. It should be noted that when a paricumlar buffer such as HIEPES or detergent such as TWEEN 20 is refened to, other species of buffer and/or detcren may be subszMUnd by the skilled intisan.

In a second series of embodiments, a bining buffer haag a higher ionic sbrngth relative to the binding buffr of the foregoing pngraph may be used- Such highr ionic strength may more clowly dupicate binding conditions between hip target and peptde in vivo be "ph~ysioflgic 1 In that reprd, the ionic strength of the binding buffer, takio into onsideration the buffer system and mry salts present. may approximace the ionic strength of 100 -150 mM NCO. A nonlimnit example of a high ionic stregth, or "physiologic,'" buffer is 20 mM HEPES pH DomM KCL, 1 mM MgAcetate, and 0. 1-0.5% TWEEN In B thid. related sedecs of embodiments, a bhxl~ing buffer which creaws a molecular enviromnt similar to that occurring at the native suheeLlular locution of a lisp target may be used. For example, when the hap arge ymonaily resides in the endaplusmic reticulum,. the binding buffer may be desiged to approxiate the molecuar conditios presen in tOe cndoplasmi retictdum. Because the endioplasmic reticuium contains an abundance -of cailciu ions, a binding buffrr which coriscs calciwn ions (or one or snore othe species of divalent cation) may be'used. In particular nonlimidng embodimenMs fth concentraition of calcium ions may be 1-75 mM, preferably 1-50 ruM, and more preferably 1-2-5 mM. Specific mxmplcs of such bindling buffers inconde, but arm not limited to: i) 20 mM HEPES pH 7.5, W100mM KCL, 25 nmM Cad1 2 5mMW MgAcezate. and 0.1-0.5% TWEEN and (Ii) 20 mM 1-EPES pH1 7.5, 100 mM KC1, I raM CaAceate, 1 ruM MgAcerate and 0. 1-0.5% TWEMN In a fourth series of embodiments. the binding buffer may comprse a reducing agen or an oxidiing agent. Suitable reducing agat incke, but are =4D Umitad to, ditbiothreitol ("Dfl m reduced giutatione, and beta aweatoediowl; =table cndditg agent incudW -but are Mo limited to, oxidzd Sluwhione Specific nonlmitin examples of binsdias buffers which comprise a reducing agent include (i) mM IIEPES pH 7.5,100 mM KC1, 1 miM COl 1 2, I. mM DIT, 1 mM MgAcetate, and 0,1 -M1% TWEEN 20; and (ii) 20 mM HEPES pH 7.5, 100 mM KG, I mM DYE I mM MgAreuate, -and 0. 1-0.5% TWEEN 2D.

In a fift series of embodirmts, die binding buffer Maly comprise a rmleoride which may, alternaively, be hydrolyzable or numhydrolyzable. Such a binding buffer may be used to identify tethers whkh bind to a hsp target where the hV~ target binds or release peptides in associalion with nucleotide hydrolysis. For example. wher the hsp target release peptides i association with nucleotide hydrolysis. a non-hydroLyabke nucleotid may be comprised in the binding buffer.

suitable nucleotides include, but a r notLmited to, ATP, ADP. AMP, cAMP, AMP- PNF, GTh, CGDP, OMP, etc.. specific, n miting eamples of such binding buffers.

include 2 0 inM EPES pH 100 mM KCI, I mM CaCJ 2 I mM MgACetHe, I mM (a hydzclyzabk micleozide) and 0.l1-.S% TWEEN 20; and (ii) 20 mM HEPES pH 7-5, 100 mM KG, I mM CaCI 2 1 ruM NIgAcetnd 1 mM AMP-PNP (a non-hydrolyrail ucletide).

The present invention also prooides for methods of identifing wowes wherein the hap targe is a modified version of a mnatually occuuig beat shock Protein. such that the hap Waret provides a more effmciet me=r for identifying totbe relatve to the unmodified bea shock protein For example. the conformtion of a native heat shock pmoein my be alterd -to faciitat pepdde binding; such a confornmxia change my be effiected by binding the hea bdek protin to oCe or more addiina] molecules to produce a hap target. Such molecules may be other best shock prtfins or accesor molecul thereto. Alternatively, end particuly where peptides which hind to gp9E or hsp9ft at sought suitable molecles include rmmr of the beNuinone nmycin antibiotics, such as herbimycin A, geldmnmyew maciimyc-in I. rimcuumycin, and kuwuitimycin (Ommnet at., 1979,1 Antibiocics 32.-255-261 h or structmnfy related compowdt L-In specific, noniniting exapls..a I10-100u fold molar excess of a benzoquiaw antibiotic reative to heat shock protein may be either combind vith heat shock proten concurrnt vith adpioc ontoa a solid phas or. dltrnatively. may be presnt during binding of phag. For example, a So fold mola ex'es of berbinmycin A may be combined with P%6 or concurrnt with acdorpon onto a solid substm rior to affinity penning.

In reated embodiment, the smwutur of a hut shock proten MY be altered by tuncation or by kncrporador into a imsian protein to anct a hsp targe with enhance peptde binding properties. For cxample. becaus a benu shock protein which noma~ly act in concert w&ith othe molecules may contain ceruin domains associated w'ith binding those accessory molecules, and other domains whi ch actualy bind chaperoned peptdes. The isolation of the latter for use as hap target my provide 17 a=oc efficien mumn of identifying suitable tether. As a specific nonltiting exnnple, Wearseb and Nicehitm, 1996, Biochen U- 6760-i16769 have idenhfted a c-tenr naldan of S"~9 which appear to be, responsible for dinierizhton of that molecule; the remove] of this doma f1cm grp94 may produce a more efficient hap taget for identifying peptides that bind to grpQ4. Altemnatively. t C-terminal domain alone may be used as an hap tuargt for identifying gp4 bining pes bused on preliinairy evidence thm it has peptide binding capacity.

Phage-expressd peptides identified as Winding to a lisp taget using the above methods may then be equencd and the ontaied peptides synthsized or rrcombiuhiy expressed in order to detemin whetbar the expressed peptde itself binds to b# targe and may serve as an effeeive tether. Preerbly, thw -w binding buffer used in affinity panning is used to evaluat pqptde binding. A vaiey of techz'iue may be used 14 perfom such an evaluatica For example, radiolabdled g, iodine.- 125,* carr- 14. or tridium -labed) peptide may be exposed to hsp targe under suitable conditions and labelled pepjde/a target may the passed over a chroauapuc resin such as Superdex 75, Superdex 200, Sephurose S300 or Superose 6; if' binding bus occurrcd, the labelle peptide and hsp target should toinigraw. Strenth of bindng may be evauatd by determinin the conditions under which the association betwee the peptide and hap target is brokmn Peptide havng various binding affinities to lisp target may be used in divars clinical applations; it may be desirble to combine weakly mutgenc peptides wit strogly bound tethers.

Ahtenmtvly 1 ceatain pcptdes may become tolerogenic when Ikked to a tth AMd bound to an hap target and therefore it may be deiable to ouple these untigenic peptdes using weakly hound teter.

5.2. CONJUALfl&E1ES.

The preent mvemton relates to conjugat peptides comprising a portio whiclfiay be bond to a he-at shock proten under physiologic conditios, Minfed to hereafte as the 'tether"; and (Hi) a portion which is antigenic (hereafter, the "adgeic pepide"). The term "peptde" as used herein refmn to molecules which alight otherwise be conmsidered to be peptides or polypeptides within the aft The conjugat peptides of the invention may comprise portion which may or may not be peptides: such addiiona portions mazy improve stability, or Laget delivery, of the cionjugate pephde. For eample 1 in speific, nonliwitin embodiments of the invention, the tethr may comprise a bvnzoquinmw~csamyciui antibiotic sah a.s geldanamyc-in Or herbimyi A (asce FIGURE 9A); such Wehens may or may not furthr comprise an bip-binding piptid teter. The use of ten* cocnjuga denotes tint de conugate pePtides of the invention compiisc an .nitigenic peptide covalently linke to another compoun4 whi ch may or may not be anoter peptde, provide that the 000 igAt pcptde is not found in nature. mT.

1 peptides which natumlly bind to hea& shock protrin (and therfore contain an indigenous tether) and comprise a anuigenic regioni 53 not mconjzgafc peplides" according to the invention. However, much flaurally oocuning peptdas may be genetically engineered to Postion tlho indigemus tter in an altred posirtom relative to the autgeic regiac; in iw4ich came a conugat pepdde acoDng to the invention would be producei In particular monilimdtin speific embudhnur, the conjugae peptide my be an antiaic. peptide fBrom a natural swvclince4 toa beinoquinone mnsamycin antibiotic such as gelduaamycin or hfrbinycin A; such a composition may or may no compris additiona peplide sequence.

The term 1 'Phyaiologi condtions; as used herein, refes to conditons oaf 1cmpature pK ionic stmmgrfr and molecuar omposition as am found within living orgmnLiSnis For example, but not by way of limnitation, physiological conditions wuld include temaue of 4-55i{, and preferably 20-40nC; ampH of 3-12, and pmrnuly 5-A; aid ionic Mstnths approximatW die ionic strength of 50-300 mM and preferaby- 10 0 -200 nM MaCI. A specifi4, nonlimiting exampl of physiologki conditios includes pbosphate buffered saline (13 mM N.H 2 P0 1 13 7 riM NOCI, pH 7A) at 372C A conjugate pepde may bind WC at heat shock protein under such conditions; hawaer, a conjugat pepuide also mwsu the definition set foth above if lving beent bound to a heat shock proten under non-physiologc conditions, it remain bound under physiologic condifions, when, in preferred nalimiring embodimgns of die invention, said conjugate pepddehe m ok protein has a UAlff of at least I minute, preferably at least 10 mnuies and morm preferably 2-10 hours or longe.

The term 'antigenic", as used herein, refers to the capwality of tat portion of t conjugate pepuide. cithr alone or in conjunction with eithr the tether or a heat shock protein ar portion treof, to elicit a cellula or humoa! inmue response in an organism or culture cOrtSInint~g cells sensitized to respond to ffhe onespanding anigem n Mimune response is -defined herein as a cellular or humorul immune response which is at leas 2-fold greate. and preferaby att lent three-fold ireatcr than backgound levels, Tctucm which rmy be comprised in conugate peptides of the invemton may be identified usin the methods set forth in the preceding section. Such tethers may have aino acid ompositions which comprise a substantial proportion of hydrophobic amino acids such as phenylalanine and tryptophan, andor a substai mnber of mcrine threnine. or praline residnm In part5iular nonliming embodiment, tothers -of th~e inventin may compise amino acid sequene which have the geneal decription. hydrophobic basic hydrcphobic hydrophobic hydrophobic; Serurh, hydrophobic hydrophobic Scr'Thr; Scr/Thr Ser/Thi hydrophobic -hydrophobic Senflr Seir~ih and Ser/fTb SerThr hydrophobic hydrophobic hydropbhic. Alternatively, tathom may comprise hems shock binding pepides as dmrsaid in Blond-Elguai et Wl., 1993, Cell 71:71 7-7A% including the conens=us etne hydrophobic (TrpIX hydrophbic X hydrophobic X hydrophobic and the spcciflc peptides His Trp Asp The Ala Tip Pro Tip (SEQ MD NO 143) and Phe Trp Gly Leu TrP Piro Tip G~u (SEQ ID NO: 14-4>; Auge et al., 1996, Natue Mod& 2306-310, including Gin Lys Mrg Aim Ain (SEQ ID NO: 145) and AT& Arg Ag Ala Ala (SEQ ID) NO:146),, Flynn eal., 1989, Scienc 2!3 85-390; Gragerv et al, 1994, J. Mal. Bijol. 231:84-854: Terlwky et al., I99M,3. Biol. Chemt W,79202-9202, Lys Phe Gin Ass Gin (SEQ MI NO: 147); and Nielmid et 1996, Proc. Nodl. Acad. Sci. U.SA. 91:6135-6139, irluding the vsva peptide Arg G1y Tyr Val Tyr Gin fly Lau (SEQ ID NO, 143). In preferred embodinwn%, tztef cthe kavention may have a length of 4-50 amnino acid residues, and momn preferably 7-20 amino acid =idu=.

In specilk. nonlinuiting embodimets, he folloing amino acd scqumzces, discused more Mbly in dhe working examples which follow below, my be comprised, as tethers, in conjutc peptides according to dhe invention: Tyr Tr Leu Vat Thr Pro Asp fk

TIV

Asp Met Tyr Phe Ser Met Ser His HiL Gin Ser ie Lys Lau Ala V~al Glu Vat 3cr Gly Oly Am His4 Tyr Arg &-r Gin Pro Ser Ak Ser 3cr Leu.

AL

M~a met Pro Ala Ala Pro Scr

TILF

Hia 3cr Thu Gin Pro Thr 731 *PMo Thr Thr His Ph.

Pbe pro The Tyr Tb:

AL

Pro Arg Pro Pro His Ala Pro Pro Set His Trp Pro Lou Pm Asp His Thr Ala er Pro 3cr Pro Am His Gin His Gl Tyr His Val Trp (My Lys

MCI

P~w Phe lrp His Pro 3cr p v

T

L

T'

r Fl

VI

Se

GI

se Gin Pro 11w pro Leu Arg Ala Thr Phe Tyr Val Gin A Ser Pro Lou Pin Pro Asp Lau V9 Leu r Gin ;erArg jis 11C 7o* Let [is Leu lot Thi ip Al eu Lou p Gin hr Gly ro Val al 'lu if rp r Pr in Ap i* Thr Lcu (SEQ ID NO: ]49); LyS (SEQ ID NO: 150); TYr (SE ID Ser (SEQ ID NO: 152); 5cr (SEQ ID NO: 153); Thr (SEQ ID NO: 154); Thor (SEQ ID NO: 155); Asp (SEQIDNO: 156); His (SEQ ID NO; 157); Leu (SEQ ID NO: 158); Pro (SEQ ID NO: 159); Arg (SEQ ID Noi6O); Ser (SEQ ID NO: 161);.

(My (SEQ ID NO: 162); lie (SEQ ID NO: 163); le (SEQ ID NO: 164); Sc (SEQ ID NO: 165); Ser (SEQ ID NO: 166): Ile (SEQIDNO: 167); Ile (SEQ ID NO: 168); 5cr (SEQ ID NO: 169): Leu (SEQ ID NO: 170); Ser (SEQ ID NO: 171); Pro (SEQ IDNO: 172): Asp (SEQ IDO: 173); Ser (SEQ ID NO: 174); le (SEQ ID NO: 175); VaJ (SEQ ID NO.~ 176); Tyr Gly Am Pro Lcu Gin Pro (SEQDNO 177); Phe His Trp lie ThI LIM Ple His Trp Tin Ala Gir Ph. His Trp Phe His Trp Glu Pro Phi Tiff Trp Tip Phe His Trp Crn Pro Sir Ser Pro Alb The His Tip His Pm Ser.

Am Ser Ala Gin Lu

T

mrp Ser Tip Pro ASp TIM Th Trp is Trp Asp Ser The Thr Sr PrO Ala Tyr Asn Ag Pro Lou Trp Pro Ser Ala Thi Leu Ser Met Thr Gin II. Gly Ala Pro Asp Arg Met Aa Lys Ala Tk Pro Pro Le Pro Ile cTrp Lys Pro Asp Trp Trp Phe Len Trp His S4T Trp Asn Pro Sew The Leu Gin Mg Len Tyr His Thr Olu Val Ala Leu Pro Tbn Lau His Trp Gin Tyr Pro Trp Leu Scr l Tp Gin Av. Trp Arg Met Asn Tyr Tip Gin Leu Aig Pro Val Trp Gin Gin Ala Afg Pro lie Tyr Phe Asp Pro Le Met Len lbr Pro Ser LZu Val. Sor Asp pmo Thr Leu Pm Val Ltu Arg Pro 5cr Tyr Val Lu Lem Pro 01w Arg Ile Ala Pro Pro (SEQ ID NO: 178); Lou (SEQ ID NO: 179); the (SEQ ID NO: 180); Gly (SEQ ID NO: 181); Pro SEQ ID NO: 182); Trp (SEQ I NO: 193); Gin (SEQ ID NO: 184); Arg (SEQ ID NO: 185); Pro (SEQ ID NO: 186); Tip (SEQ ID NO: 187); Tyr (SEQ ID NO: 188); Pro (SEQ ID NO: 189); Ser (SEQ ID NO: 190); Vai (SEQ ID NO: 191); Pm (SEQ ID NO: 192); Lu (SEQ ID NO: 193); His (SEQ MDNO: 194); Trp (SEQ ID NO: 195); Val (SEQ IT NO: 196); Lea (SEQ ID NO: 197); Asp (SEQ ID NO: 9B); Mtt (SEQ ID NO: 199); Phe (SEQ ID NO: 200); rg (SEQ ID NO: 201); Pro (SEQ ID NO: 202): Irp (SEQ ID NO 203); Pr (SEQ ID NO: 204); Pro (SEQIDNO: 205); His (SEQ ID NO: 206); Asn (SEQ 1D NO: 207); His (SEQ ID NO: 208); Asp Lcu Asn Ala Tyr Thr His (SEQ ED NO; 209); Val Thy Lea ro Asn Phe His (SEQ ID NO, 210); Asn Sir Ag Lcu Pro Thr Leu (SEQ ID NO: 211); Tyr Pro His PrO Arg Ser (SEQ ID NO: 212); Gly Thr Ala His the Met Tyr fSEQID NO. 213); Tyr Ser Leu Isv Pro Thr rg (SEQ ID NO: 214); LZU Po Ag Arg Thr Lau Leu(SEQTED NO: 215); Thr 8r Tbr Len Lau Trp Lys (SEQ ID No: 216); 1kw Set Asp Met Lys Pr His (SEQ ID NO: 217); Thr Sr Scr Tyr Lem Ala Leu (SEQ ID NO: 218); Asn Lu Tyr GIY Pro Is Asp (SEQ ID NO: 219); Leu Glu Thy Tyr Thr Ala Sr (SEQ ID NO: 220); Ma Tyr Lys Soy La, Thr Gin (SEQ ID NO: 22j1); Scr Thr Sa Val T yr Ser Ser (SEQ ED NO: 222); Cl:u Gly Pr La,0 Arg Ser PrO (SEQ ID NO: 223); Thr Thr Tyr His Ala bmi Guy (SEQ W NO: 224); Vil 5cr lie Gly His Pro Sir (SEQ ID NO: 225); Thr His Scr His Arg Pro Sn (SEQIDNO: 226); II Thr AM Pro Lea Th Thr (SEQ ID NO: 227); Ser lie Gin Ala His His Sir (SEQ D NO: 228); Lcu Asa Trp Pr Arg Val Leu (SEQ ED NO: 229); Tyr Tyr Tyr Ala Pro Pro Pu (SEQ ID NO: 23o); Icr Lou Tip Thr Ag Leu Pm (SEQED NO: 231); Am Val Tyr His 5cr Sr Leu (SEQ IDNO: 232); Am Sir Pro His Pro Pro flu (SEQ ID NO: 233); Val PrO Al. Lys Pro Arg His (SEQ ID NO: 234); H's Au Leu Is Pm Am Mg (SEQ ID NO: 235); Tyr Thr 11%1 His Aig Trp Leu (SBQ D NO: 236); AU VaJ Thy Ala Ala 1r Val (SEQID NO 237); hr Lea Met His Asp Arg Val (SEQID NO: 23s); Thr Pro Lou Lys V.1 Pro Tyr (SEQ ID NO: 239); Phe Thr Am Gln Sor His Vai Pro His Tbr Tr. Val Th Gb Thr LeU Thr Iht Gly Val Pro Lys Leu Pro Cys Arg Phe lyr Thr Arg Ser SeT Aia Sec Leu lie Asp Ala Leu Ser Ser UKN Phe An Thr Thr Val OIn AspTq Sar Phe UKN Pro Ala Pro Pro Ile Ala Thr Lys Pro Pro Tyr His Thr r Ty lie Ser Ser Phc lir Th Pro 1le Sor Leu LeuPro An Lu eu Asp Gin Asn Ser His Phe itk Pro Gin Pro pro eCu Thr His Asp Ala Gin Met Ser Set His Phe Met Met Arg Lys Lcu Ala Gin Ala Pro Asp Len Lys Glu Len GLa Tyr 5cr Met Tyr GLy Tyr Pro Phe Ser Thr met Val Leu (ly Asn Plic Glu (lly Fro Tyr Ser Tip Pro Len Tyr T11w Arg Val Ala Pro Pro Gin Set His (SEQ ID NO: 240); Ala (SEQ ID NO: 241); Ala (SEQ ED N: 242); Tin (SEQ ID NO: 243); Ser (SEQ ID NO: 244); Asp (SEQ ID NO: 245); Arg (SEQ ID NO: 246); Arg (SEQ ID NO-,247); Ala (SEQ ID NO: 248); Arg (SEQ ID NO: 249); Arg (SEQ ID NO: 250); UCN (SEQ ID NO: 251); Ile (SEQ ID NO- 252); Tim (SEQ ID NO: 253); Phe (SEQ ID NO: 254); Leu (SEQ ID NO: 255); Len (SEQID NO: 256); Ile UKN Sc (SEQIDNO-257); Val ThT Met (SEQ ID NO: 251); Thr Po Ly(SEQfIDNO 259); Ph. Gin Ile (SEQ ID NO: 260); His Asn Met(SBQIDNO: 261); Ala Thr His (SEQ ID NO:262); Thr Ph Lu (SEQ IDNO: 263) Asn Pha Aim (SEQ ID NO: 264); Pro Arg Met (SEQ ID NO: 265); Phc Gly AL& (SEQ ID NO: 266); Thr Tr Len (SEQ ID NO. 267); Pro Arg Aig (SEQ ID NO: 268)- Gin Lou Leu (SEQ IDNO: 269); His His Gly (SEQ ID NO: 270); Ala Pro Leu Asp Arg Ile Phe Ser Aso lu His Tyr His 8cr Asn Tyr His Leu LZu

LYS

Ala Tyr Scr Gly 01y Lys Tin His Va] Lem Ala Arg

LYS

Ala ThI Ala Pro lip U Thr Trp Pro Leu Cily Pro Lem Asn Lou [i Lu Pro Ser Pro Gin Val Pro Tr Pro Ala LCU Pro Ciii Tbr Tyr Val Pro Leu Gin Ser His Ala Lau Ala Ala lbr Pr Pro Lou Tyr Aa Pro Ala Phe Pro Thr Set Phe Met Agn Met im A-in Ala lie Trp Leu Gin Pm~ Pro His So Ser His Arg Asp Pro Ala His Tyr Pro Ala Lou Ala Thr Set Gly Val Phw Arg M.p Tin Pro rhr Ile Thr Ifis Tib, Len Th Pro Arg Git Thr Leu The So, Tir LCu L4u Lo= 5Cr vel Phe Pro Ser Lys Arg Thr Lou

GLL

Lou Pro

PTO

Ala Phe Met Tbr Phe Lou Ser Leu Val Ala Lcu

PM

Scr Ala Pro His Thr Gly Lys ely Val ely set Lou Pro Lou Pro LoU Pro Tr (SEQ ID NO: 271); His (SEQ ID NO: 272); Met (SEQ ID NO: 273); Tyr (SEQ ID NO: 274); LOu (SEQ ID NO: 275); APn (SEQ ID NO: 276); Ala (SEQ ID NO: 277); GMy (SEQ ID NO: 278); Am (SEQ ID NO: 279); His (SEQ ID NO: 280); Arg (SEQ ID NO: 281); Thi (SEQ ID NO: 292); Trp (SEQ ID NO: 283); Ala (SEQ ID NO: 284); Sa (SEQ ID NO: 285); Pm (SEQ ID NO: 286); Ala (SEQ ID NO: 287); Ly (SEQ ID NO: 285); Thr (SEQ ID NO: 289); Ser (SEQ II NO: 290); Len (SEQ IDNo. 291); Sc, (SEQ ID NO: 292); Am (SEQ ID NO: 293); Oin (SEQ ID NO: 294); Pro (SEQ ID NO: 295): Ala (SEQ IDNO: 296); le (SEQ ID NO: 297); Pro (SEQ ID NO: 298); Thr (SEQ ID NO: 299); Leu (SEQ I) NO: 300); Val (SEQIDNO: 301); Gi~n Thr Lys Val Met Tin Tbz (SEQ ID NO: 3 02); Asn His Ala Val Phe Ala Set (SEQ IDI NO: 303); Lec Hlis Ala Ala UFKN

T

ln Ser (SEQ ID NO: 304); Thr Trp Gin Pro Tyr Phe His (SEQ ID NO: 305); Ala ?ro [Aeu Ala Lea His Ala (SEQ ID NO- 306), Thir Ala His Asp Lou Thu VaI (SEQ ID NO. 307); Asa Met Thr Ann Met Leu Thr (SEQ ID -NO: 308); Gly Sir Gy [Au Ser Gin Asp (SE ID NO: 309); Thr Pro Ik Lys Tin lie Tyr (SEQ ID NO: 310); Ser Msi Leni Tyr Arg Ser 5cr (SEQ IDNO. 3 11): anid His (ily GhP Me. Tip Gin Ph. (SEQ ID NO: 312).

(UKN undcathat the sc qies of amino acid at that residue is not known).

IW a series ofnonlimidang embodimans, conjugwe pepticles of the invevtinn my comprise a benzoqinouw warnycin antibiotic molecuje ad an antdgenic pepide. Such conjugate pepkies may be pwduccd by covalendy liking a benicquinn anmmycin antibiotic to an antigenic peptde. Suitabe beazoquinon ensnmycin antibiotics include, but are not limited to, herbirnycin A, geldusamycii.

niimosnycin, macrnimycin I and kuwaitimycin, as well as .nalogs and dedivatives chereoI in nonlimtn embodfiments, it may be desirable to utilize a bnzaquinww .nsauiycin antibioti havng greate or lem& inffinity for heat shock pr"e relatve to berbimycin A or geldannycin: a spccilk nonlimiting example of such a conmoud is 8-decabamoyl Oedanaycin, which has a lower affinity for heat ock protein, and which may be produced hy recting geldanamycin with potassium tenbutyloxide in dimethylforrunmidc (wee FIGURE 17).

A chemieui structue 'which6 if present connects beaoquinon ansmyein antibiotic and anfigenic pptide is refere to herein as a "i*et.1b linker may or, alteratively, may not be a pepride, or may compris both peptide as well as non-peptide components. The linker may be designed to provide an optimized assocmaton beeemn the conjugate pepuide and a heat shock protein. Fetixc of a linker which may be relevant in this regard incLde not only its length 4 but also its poLadit. bYdrophobicity (for example, as puovided by aliphatic or aroatic side chains),, hewterrom composiiog the Premmnc of ether andor amife, (primazy, secondazy, r tertiary) the presence of sulfur dci vadves (d4g. sulfide,, Sulfoxi and Sul fcnes) andtor plaospboom derivatives egphshns Phosphtes phosphinates.

and phosphates) end the like. in spccific, noulimiting exaples of the invenlion, a cleavabte linker, for examPle, a I inkrwhich is acid ueaithv w serscumive. light senitive, =seitive TO reduction Or Oxidatidnj or to cicavAge by. acellular enzyme maRy be used (see FIGURE 18), A Peptde comprising an anuigenie pepid maly beg covaicady bound to the beflFAXuinm isanycin andi bladec by either its amino or cwboxyi teminus or via reative side ch"M The binding affinity of the reutig oqj gate pepida for bhaw shoc Protein mawy be evahauod in order tD select the opeimna linkage sit. FIGURE I OAF depict asugetr peptdes covuicudy bound to a. bcnzoquinoe anwniycin antibiotic (ge Idanmyoin is shown in the figure) via t pepddts earboxyl tenninus.

Alternativey, the beuzoquinone annarycin antibiotic ga be covuiendy budto the amnino terminus of fhe peptide, as shomm in FIGURE

IHA-F,

In a specfic,, nonlimting cnbcdimnem of die iventon, conjugate Peptides omprisin knzoquine~ ansamycin anibiowis May be prepared accordng to the following schem. In view of the X-M struanu of the site of itnrcton beee geianamycin and hsp9o, it May be dcuimbke to lin geldaamycin or herbimycin A to anigenic peptde aC carbon 17 of these antibiotics. Primary 'mines afloa to rect readiy with geldarwnycin at ibis positio to produce 17-clmax y- 17 alk am i edanawycin, as shown in FIGURE 9B. Althoug the reactivity of berbiraycin A is quit. sim~lar to that of geldanaanycin, the reaction of ally] amine wit geidanamycin give rise to a sinle compound, l 7 -allyluznc 17demethoxyg.1damnyci, whema aliylazine nems with herbimycin A at a higher tempertr and for a longe reacion timew 0produce two derivaties naely 17ullIminO herbimycn and 19 -aflylanina bherbiniyicn, in a ratio of apprximaately 3Jto 2, respectively (FIGURE 9C2). l?-allylan'Jno herbimnycin is m~ore actieg tha 19allylarnino herbirnycin, which is consistent with the X-ray diffiaction pawn= of geldawninp9o (Siebbins m? al., 1 t997, Cell 12. 239-250).

Because himycin A is lea ractive than geidanamycin towards amnine nuclwophilus, it is desluable to form a linker between hcrbimycan A and antisenic pcptde as follows, Herbimycin A may be rend with a mnopricte aikajiediai in chloroform, at 40-4W for 8 -24 hours in the dark to produ a mnixtur of the 17 and 19 -monoprotted alkndiumino herbimycin. These two compouinds may tn be sepuated by clraography, and the desired 17-derivative oliecnd+ deprotctd and then submitted to the sanw ditions used to pepare antigeic peptide linked to geidmmycir (se= FIGURE 9).

For the preparatio of a conjugat pe-ptide compising a beroquinone ansamycin antibiotic, a synthetic schan moy he utilzed such tt both the amino end and the carboxyl end of the antigenic peptde my be furrricnuized using the sam proted peptide precursor; in other words, the same protcted peptid may be used in the preparation of either amino.1 inked or carboxyl-linked conjugate "epides. For example, the peptide may be prepaed on a solid support, such as a rein, to improve efficiey. In choosing a resin, it should be onsidered that Mt the end of the synthesis Ln order to prpare carbox y1-linked conjugate peptides the caroxylic acid group should be seectivrly hydrolyzeti so that the rwhide is rlmd from the resin without dcprowcinj any amino acid in thie peptide (Boiagen ot al, 1994, J. Chem. SOC., Chew. Corn. 2559; Cog t at 1990. Tetrahed. Let. 31:205; Pkov=et c 1993, Tetra1cd. Let 34:2199; Cazrpino and EJ-Fahum. 1995,1. Org& Chem. 6D:3561; Sieber and Riniker, 199 1, Tztrahed. Let. 22:739; DoHin et a, 1994. L Charm Soc. Chem.

Coma=o. 853; Lapetsais at 91., JL Chem. Soc. Ciinn Commun. 671; Badmu et W1., 199 1, I. L. Feptide Protein Res. Uf'51 3; Noughimn et 1936, lax.J. Peptide Protein PRm. 7J:653; Riniker et i993, Tethed. 42-93M0), This also enntses tha the soqueneC doe not contain any contamunation or impurimie that often result fr-om the reaction of periphera functiouiien theO pepide chain. As specific, noniimnitiu .xamplem Novaeiocem TOT or ClTnt resins may bc wsed (me FIGURE 12): thew art polynmic resins With tinyl or aWorotrityl end protelng proups respetively.

Where a TOT resin is use. the first amino acid is acmhed to the resin n= acid sensitive whiy] eate. In tact, this functionalit is very sensitive even to mild acids, thereby eancing the selectivity in the eventua deprcnecton of the peptide. An analogousq Produr my be applied wsing CITrt rusin. IE should ftter be noted that the- prOteting griips OD the Pppide chain are desiaby compatible wit the couplig and deprotectiori condition dit am applied thrughout the synthesis of thke pepdde [A norlinmi ig emnbodiments of the inventicu a thaxenyniethoxy carbonate stug imay be used, wherein al deprotecfions and couplinp are perfomed under basic conditions comnpablb with the resin. FIGURE 1 JA-B depict the synthesis of a protected peptide on TOT main using Fnm protrug gmnps (wTyBop nreferW t o t zjlytrsprodnapo~cw hexafluomophophar old "DiPEA" refer to diaappyledylamine). The resWiing peptide is procte at both amino and carboxyl tetmni, and therefore may be used as a common intermediate ifor conjuLgation to bazoquno asunyf vin either cenninus. FIGURE 1 6-A-B depict a scheme in whtdch a fully prowtecd peptide, as produced according to FIGURE 1 3A-B, is derTecte at the amino teninus and d=e reacte with a primasy amine linker and gekdanUnycin.

However 1 wher adgenic peptide is to be conjugatd to benzoquinone annibiotic via its carbaxyl terminus it has bee fond to be preferable to add the last amino, acid of the peptide as a N-Boy protected amino acid instead ofta N-Fnioc protced amin acid (FIGURE 14A). The resu~in peptide Wu both carboxyl and amin termini protected (FIGURE l4BX, and thus may serve as a cunce anterunzdirn for ouugiou wo antibiotic via eithe teminus. In FIGURE 1 4B, t peptde ws released from the resin, and its carboxyl terminti ezpoued, by rrcaune~ with I% TEA, GC1A, and then pyridine/metbnowl (1 9, volunrvyohne A schem wherby the rwl~iing varboxyl-ftumnus depmoteced (amlx tcminu protected) pepde is conjugated to geldanamycin is shown in FIGURE 15S. The N-Boc.-basd mthod has bee fotund to greedy enhanc the yields at the fin epotection stzp, probably becnus geldanmycin my be sensitive to excess piperidine required to remove the Fmaoc. As shown in the Last step of FIGURE 1 5, once acnic peptide has bee= conjugated to linker and gelanantycin via. die pcptice's carboxyl terrnin us, the remaining Hoc protecting group on thie nMino cerrninw oflthe poptide may bc removed wiiouit the use of piperidine It my also he useful to note tha geldanamycin may be sensitive to eymestve expommr to strong acids such as crifinoroceti acid CTFA"). For intance, s tirring peptide Fiig 8ecidamycin achetd at its carboxyl wemiows for four hours at room ucmpcrtue in 50% TPA, 10% rriisopropyluitne in CH 2

CT

2 yielded only tin smoisms of the deprotected conugae because of extensive product decmposition. It view of thiis probker, it may desirable to use thm following procedue iS t finsi deprtetion step (see FIGURE 15). Firm, a conjugat peptid having a &rnpratctd aminio terminus may be meated with 95%lzifttwroecdc acid triisopropyisilanc, 2.5% (11t12 for less than 1 houw. The above reagent shouild be inideily added on ice and the reactions should be illowod to gradually warm to room termnrnn After addition of watr, tine crue mixture may then be evaporutd to dryness under high vacuum. The resulting purple solid mnay than be washed with chioruform and dissolved in waer to ptoduce a purple soluxtion which may be pH adjusted to abouat 5 with Iiiediylammaniwn hicarbwiate, filtered and subratud to

IIPLC.

The resuling oonjugsc ppide may be puriied using any method known in the art (see Nihin et id, 1992. Tethedron Lefts. U7007; Kurodae Ar, 1992, Int- 1. Peptide Prot. Rn. IQ294-, Alps., 1990.1J. Chromatography 422l77).

Cure should be takun not to us conditions which would suvbOantiLly imar the Wwogical function of cither the hap-binding pordc'n or antigmnic portion of the molecule. A specific nonlimhing example of a method fr~ the purification of coupua peptid is as folowa. The foregoing filterd solution, at pH S, may be, injectd ino a pronditioned 1{PLC colum such as a rolyHYDROXYETHYL AcpflndeTb from PolyLC. Columbwa MD. The- conjugate peptid may then be cluted using a two-component elution systm: ehaemt A- 6.M% trieftylunnoium actae in 92% ancctoid and 12% hexfluoroisopopmnowl; chati R- 10% 1lOmMotiethylanmiuin acetate, 10% acetonitrile in water. Reactian product may be injected into the column in 100% thuaw A, eluemt A may be kept isocatic at 3.2 mihnin for tan minutes, and tin the proportion of chuat B may he inceased over 40) minutes to 3 At this stage the product eluted with aretention time of about 60 niinuts Antipmeni peptida aocording to the invntion may be capable of inducing aif inmue res'pons t) anY anigen Of h tt. Antiges of interest inclu&e, but are =o limited tu antigens associated wit neopasis suLch as weams. lMphoma, leukemia. melwomt- cA.-=O=x of the breast, carcinoma of ame prostate, avanann cardinma, carinom of the cervix, werine Carinoma, oak,, carcina, carinoa of the lung, gi obULvasma and astrcytt, antigen ussocmied with &dectve tumor sizpresso genes suc ms p5.3; anutigcn. assocatedvorith oncogeuc such as r&e, mr., er*9, foss 4bl, and mYc: antigen Wsoiwted with infecticia diseasc caused by a bacterium, vinis, prcoYm mycopaSnUL fungUs, ycat' paasite or prior; and ancigen associated wit an alierg or autoimmune diseas. Bxamples of Sourme Of antgens assciated wMt infetious disase include, but are not limited to, a human Papilloin AirM (see beLow), a herpes viis such as herpe simplex or herpe zosr, a reuovirus such as humwi imm undcfu envy virus I or 2, s hepatitis virus, an influenza vin~s, a rhirus, a rspifory Syncylial Arum a cywmegalovirus, an alaawvnas, A& Yprmoie a batru of tie genus Salmmgjla SIWhWMOccW. &reWccuA~ £nerocaccw ClosMuriin Esriflda, Kiebsiefla.

Vibrio. Or Atycbtwtcnrn, and a protozoan such as an amoeba a malazial paraite, and Twpanoom crud.

Specific 1 nonlimiLng exaMPleS of humn pailoma viru anrienic popides which may be omprised in a conjugatvpeptideoftlue invention ame as follows: Lcu Lou Leu Gly Tin LU Am. le Val (SEQ ID NO: 313); Lcu LOW Ma Lily Thr Leu tly He Val (SEQ ID) NO: 314); Tbr Lcu Gin Asp Ile Val Len His Leu (SBEQLD NO.

315); (Ily Leu Hi. Gym Tyr Glu Gin Loeu Val (SEQ ID NO: 316); and Pro Lax Lys Gint His Phe G~n le Vat (SEQ ID NO: :317).

Conjugate pepties of the invention masy be prepared chemicnily or usingrecoMbinan tchniques, TpjQin tethier and emigenic peptide, cacth pcptie may be prcpand separatly and lat eovaiaidly joined or, preferbly, the two may be synthesized sequwanial ly (although anotr pptde sequence may reside betwee tether and antigenic peptides P cornprised i a single molecule. In preferrd, nonlimiting embcdhncmu. the coi~uguf peptides may contin 15-40 amin-o acids, and more preferably 1$-25 amino adds, and my further convpnse lipid or carbohydrate moieties.

The present invention provides for thewupeiti compobstoc cown ing conjugate peptides which may or my not also ompise heat shock protein for compositions which result in the production of conjugate peptides in a subject, and for methods of using such compositionis.

In pertiailr emnbodimnt~s, ompoitons of the invetin comprise.a tbcnpcuticaly ecti=vc anotm ofa conjugat peptde in a sutable pharnmutical carrier. Such compositions may further comprise other biologically wetive substancs, including but not limited to cylokines and adjuvant compoun&s In further cntodimuta, ompositions of the invention omprisesa snrkei acid encodingsa cmnjugat peptid comiprisd in a suitable exprmsion vectr, such that when the composition is administered to a subjct the conjugate peptide is ex-w In relafted embodiments, composhions of the invention compxrise.a cell containing a irleic mid uncoding a coojugate pcpdidc. such that vwn the call lis intoduced ino a subjec the conjugate peptide is cxprtssed and releaed. in the sutject Suitable clis include eukaryoti as well as prokaryotic cells According to addtiona embodiments, oompooitions of the invention comprise a conjugate peptide and a beat shock protein. Such compositions may further compflse one or more additiona heat sho4. protein or prvocin which serves as an accesaory in the chupcrne process, and/or may omrprise a lymphokioc. In preferred monhiniting emabodiments of the inventon, in such compositions the coqjugSt peptide is boun to the heat shock protein. Such bitning may be achieved, in general uwkdr condhjtops wher the suit concentrations may be betwen 20-350 mM, preerably between 50-250 mM, and mor preferably betwee 10-200 mMI (of; for exsmpleNaCi or KCJ); (iij) emnpcruur may be between 4-SOM, preferably bcrwcn i10-4W, and more preferably between 20-17-C; and (Wi) pH4 may be betwen 4-10, and preferably betwee &S (all ruges inclusive of endpoints), In a specific, nndimn example of the invnui4On M uflagte pepde may be bound to hea shock Protein by mbin a molar ratio of 1: 1 to 100: 1 or cOqugte ppddc-.heaj shocd prinK on icein abuffer w~eb is 2OIM HBFES pH 7r.0, 150rnM KCI, 10 mM (NIJ~d 2 2 mM MgCI, and 2 mM MgADP, pH 7.0, mud then incubatin the imur far 30 mninutes at 37C. A woring exampkof mmh binding is Ms forth in Section 7, below.

In other nonlimideng specific exampies, the prsent invaition. provides for comapo-sitions omprising a conjugate peptide, a heat shock protein, aid a beazoqinon anamycin antibioti- such as habimycrn A or geidaamycin. The molar raio of antibi.,tjc to hea shocij protein in such compasition may be I prufraby 1-3 0-fold and more prefaubiy I 0-20-fold.

Adingly, one or mmr of 4we ftweoing ompositions may be adinistoed wo a subjec in ordr to mrat or prevent a neopatc disase, an infhcdous duease or imnunoiogic diseas or disordet In pofticujar, such compositions mary be used to inuce a therapeutic immune rsponse in a subjec suffering ftom :I neosic dims.e, an iai'ctious disease, or an immunologi disease or disorder.

WhMi thme composifimn we used to induce or augmen( a hurnral. or celluLar immune reapcmw in -a subject the increase in immunity (measuzuj, for exmAinpl by manibody UWwxqkic aetjvk 9tkne reee,ofby increase in cell1 or T cc po polatins assoiate with the desired respons) may be at leas 2-fold, prfabl~y at kain 3.foid, and mom preferably at leas 4-ohL The Compositions of the inmenton may be ajhniisred by any suitabe moute, including but not limited to subekmzowiy, intaemaly, inkumurulAdy, iflhYUvenously, orally, imranasafly, or topically, Neoplastc diseases wich may be mmsed according to thec invention inclde, but are not imita urcoms, lymphoma,~ leukemia, melanoma, carcinoma of the breast, cam-inonia of the prostat, ovarian carcinm, carcinoma of the cerix.

uterine carcinoma, colon carinoma, carcinoma of the lung, glioblastoma, and Infectiou disease which may be treaic accordin to the invention include, but arc not limited to, diseases caused by a bacterium, virus, protozoan.

mycopiasmia, fungus, yeast parsite or prion, such as a human plapima. virns a herpe virus such as herpes simplex or herpes zoner, a retrvirus such as human inunmodeficiency virus I or 2, a hepattis virus, an influenz virus, a rhinvirus, a respiratory syncyial. virus, a cywomegalovnrus, an adenovirus. Atycopimsma pnewnnniae, a bacten of the gams &a/mon.IW Staphyiococcw, &nmpwvcoca.

Emverococcw, Clauiridhem, Escherkcha, Klebsiella, Vido, or Atobacerum. or a protozoan such w a amoeba, a malairial parasite, or Trypaojwcm err.

Disease of the imune system '&tich may be tented according to the invention iuchide, but arm nol limited to, inherited or mauird imune deficiencies wine the capacity of the subjec to mount an irmne response is impaied- ]Examples of acquired immune deficiencies incude AIDS and ARC andihe impuinnenrof immunity associated with various cancers. Alternatively, the method of die invention mnay be used toD lin wauoirrune diseases, such as rheumatoid arthitis, systemic lupus erytliematosis, diabetes muellitus, thyroiditis. and multiple sclerosis, In such embodimnts, the "o ugate peptde and its intemction with beat shock protein, mnior the immunization protcl, may be designe such tha imimunization results in a decreased inmum rsponse; for example, the inimufe resporse may be deresd if repeated or prolonged oxosw of the subject to onz ugale peptide occin 6. EXaML Un DFTE1CAUWQE flT 6. 1. MAJB&AL& AiD 1MJ Preparation of a p 9 6 exprusio vector. The mouse cDNA encoding roamiw gpO6 wherein the endoplasnie retculum signa peptide has bee removed) was iixxxporatcd into thec pET I1I a expresion vector (Novagn) as follows. 0yp96 CDNA inen was prepared by polymeruse chair reaction (PCR) of a pRcJCMvV clone containing the eDNA using the following oligcnumuckd primers: AGdATATACAT'ATOO(ATGrATGAAOTCGACG;TGG (SEQ ID NO: 318) and TCIACTAATCATCTTTiAAT (SEQ ID No: 31 9)- The resulting 9p96 inser was then cut with NdeI and &nnHl and repwzified. and ligated into pET I I ia which also had be=n cut with N&/e and BamHIl and repuried, to form fthe expression vactor pET I lgp%- Expreusiom oflg^f pET!1gp9V% ws tmsnsfrmed into BL21 &ckerichia coli cells, and pLated arn LB plates contaning ampicilin (SCOgm). One oflthe resulting colonies was used to inocuLate a 2D0m1 ovemnir cuhue of 2x TY medium conrang mpxilrin (150 jag/nil). The falbwin day, the reulting cultur was spun down mnd the havested bacteria were reuwended in 1 ral of fresh niediwn., Tmone I fter cuture. were than each inocuiated with 0.5 ml of the barvasted cclhs and allowed to growsa 37&C until the optical density, measurd at 600 rim, was 0.

Then, JPTG was added to a coneeirudon of I mM a&d the od Is wcre cultured for anothe 3 ho=r before bein bar-sted by PenaiiUpn The resung cell pelle was resuspeded in 20 ml of 50 mM HEPES pH 7.5, 50 mM KU, 5 mK4 MgAceate, sucrose and 1 =M PMSF Cell extrac, mwe prepared by presure shearing in a French Press The lysates we!re then spun at I100,000 x g fbe 1.5 hours and the supernsiant, wich consiUted crude gp96 extract, ww collaecl.

Furftion of gpSE. The ibllowing Aqep were all performed at tO.

A 12.3 cm x 3 .2 cm coiwunof DE52 'msin (Whtima) was equilibratcd in a solution of 50 mM MOPS pH 7.4, 10 mM NeC!, 5 mM MgAcae (herefte,

T

Hfluffcr A N).

The crud. gp96 ennma was diluted 2-fcM and immediaely loaded ont the colum at a flow rate of 2 nil/unin Elation fr-om the clum was achieved usin a gradiem -of a solution of 50 mM MOPS pH 7.4, 1IM NCI, J rnM MgAcetmw (hereate, -Buffer B") from 0%to 100% Bufe B ovar 1000 nil. The elution profile was eamined by subjecting &acn colleced trin the column io SDS-PAGE analysis. Fracfions contaning gp 9 6 were pooled and diluted 2-fold. with cold wate, and watt immodianely rn onto the nex column (see beow).

A 10 arn x I cm coiwnn of hydroxyapataja (BioRad) was washed 'with 100 mIO 0. 2P4 0m C H74an hneulbudwt 0m

KAHPO

4 Sf0 nM KOI pH 7A4, The pooled diuted fmecdoms from die DE52 colum w=r loaded onto this column a; a flow rate of I mi/ruin. The gp96 protein na ciuted in a gradient of 10-500 mM KHPO 4 over 800 m]l. Fmdcioms containng gp9 were pOoled and loaded onto the phenyLsepharose colun descaibcd bel ow.

A 9 cm x 3 cm column of phmnLsepharo (Phannacia) was equilibrated with 500 mM NaCI, 50 TaM MOPS pH 7.4, The pooled fractons Colitlizung V%9 from the bydroxyapatie wer loaded ont this columnm 1t mi/mm and the gp96 was ehtud in a gradient of 5000 mM NaCi over SW ml. The gp96 conuainng fractions collected fr-om the olmn were identified by SDS-PAGE, pooled 1 and mcnne.

The gp96 was then loaded onto Hi Tapd 26/60 Supe-rdx-200 colun (Pharznacia) equiLibiutcd with 100 niM NaCI, 5 M MgAoeuaf, 50 mM MOPS pH 3 ml fractioc were collected, mid the fmctious containing the most pue gp96 (as identified by SDS PAGE using a 12 pentan reducing gel) and pooled. To the, pooledfr-actions glycerel was added to 1W% an then die 6whtons were concettd wo 2 1 mg/mI on a Cencricon-SO oncenan (Amicc'u. froe using liuid nitrgen and stord at -SOC.

Afiwty pauimg The Pih Phage Display Library Kit (New Eagland Beverly, MA), was used for infinity panning. For eah panziing experiment, a well of a 96-well polystyen imicrolite plate (each wel lying a 6 MM dintz was filled with 150 pl. of.a solutom of 200W=l of gp9G in 0.1 M NaHCOD pH 8.3, If berbimycin A was wo be inclded in the expenmeuI ipl. of TO nip/mi hrbiznycin A omICO) in DM50 was added to each well, couespcmding to aL 50-fold mor excess relaive to gp96. The plowe wa then held at 4mC overnigh in the dark (htrbinwcin is light sensiive). The rax day, the 2p96 solution was removed hum the weU and 200 pi of blocking buffer (0.1 M Nal-CO, (pH 5 mg/mI bovine =en ulbomin (BSA), 0.02% was added, and the plat containin the well was incubated at 4-C for a fiier hour The well ws then washed sAx times with TES (50 mM Irisl4id (pH 150 mM MaCI) furthe ovntani eithe 0. 03% or 0.5% TWEEN depending on whither the firs, second, or &H nd, respectively, of panning was being performed. 2 x 1011 phage wecre then diluted into OD p1 of the appropriae binding buffer (we below), containin the appropriat amont of TWEEN 20 for dha pIudCUI*TYuJW of panning and the phage were icubate in die 'wD at 3 7C for hour. NOn-bound phage were then emoved from the wait, and the weU was washed ten times with the Pwtlcular binding buffer used for Opg bidng containn the appropriate amomt of TW 20 for tba round of par*g Bound pbage were then elated by a 1 min, inicubfionin 100OA of 0.2M gJyeepuz 2-2hil*elnu wasilde neutralized by addinii 15p11.5 Mv Ti pH B.S. These phage were then amplified in two cycLes of amplilkauan titred and used in the next rounld of panin. Three rounds of punning were performed. After the lIs round of panriin& between ten arnd fiftY pig. clones from eath exernnt were sequence4 and the corresponding peptide seywnee were deduced.

Affinty panning was pfawne using a diveniy of binding bufer, which -differed in elctroyte concentiin caicim ion concenzrxdon, md/o0r the presece or emwe of hesbimycin dithiohrehol ("DMfl Or nucleodde, As discussedheLow, whuen the cmposition of binding buffern va ried, tbe comiouition of bound phage-aprused pepfides was timid to chang.

Using the binding buffer udlizd in Blond-Elguindfi et gLI., 1993, Cell l7 17-72S (20 mM HPES pH 7-5, 20 .iN 10 mM (NI-L,O. 2 nM MgCL and 0. 0.3% or 0.5% TWEEN-20, dependin on the panning round), piag expressing the pepdes set tint x Table I w=r found to bind to gp96. The pifeeiag of specific amin acid oc~auring in themupepdec s mcompredwt the eWce percetage (basa on the occurrence of =ah amino acid in the exession I ibmy ina whole, provided by. the mnufacnrer) in Table H, From the. reults, and wot considering the reLadve posiios of eah amine acid in the bound pepdes it appeurs tha binding to pcpddes containng asputc aeid threonn, praline. zyrsine and phenylsain (and, Lo a lesser extent mine) -a favore. Conversey, pepdew conlinin 91y6in, SIUtMii, aspuagine, Icucine 1 isoleucbne ad. Wo leksser extent, alanine and nitine vkv selectd against.

IABW I.

Tyr 1Kr Leu Val Gin Pro Leu (SEQ ID NO: 149) Thr Pr Asp lie Thr Pro Ly& (SEQ ID NO: 150) Thi Tyr Pro Asp Lcu Arg Tyr (SEQ DNO: 151) Asp Arg Thr His Ala Thr 5cr (SEQ ID NO: 152) Met Sir Thr Thi Phi Tyr Scr (SEQ ID NO: 153) Tyr GLn His Ala Val Gin Tbr (SEQ ID NO: 154) Phi Pro PbW Ser Ala 5c Thi (SEQ ID NO: 155) Sec Set Phe Pro Pr Ecu Asp (SEQ If) NO: 156) Met Ala Pro Sur Pro Pro His (SEQ [D NO: 157) Ser Sir Pht Pro Asp Leu Leu (SEQ ID NO; 158) TABLE li %AaebMI MWMWc His 4-29 4.3 Arg 2.85 3-9 LYs IA 1.7 GhL 421 6.4 In 0 4.1 Asp 7.14 2.1 GLU 0 12 ecu 8.57 11.5 Ala 5,7 7.2 Val 215 4.3 Ile 1.43 5.4 (fy 0 3.7 14.28 11.4 Tin 14.28 9.3 pro 15.7 12 Tyr 7.14 2.S Ph. 7.14 2.9 Tp 0 1 0 -0.8 Met 2.85 3.3 Tables IA and ILA repetively, show tha phg ctssi g peptides of a ditrcnt compositon bound to gp96 when de same idind buffer was usd, but hebiyiin A was prent (where harbiznyc A was dded to gp 9 6 during binding o the polysymne well). The composition of boud pepde appmd to be enriched in histid, alanih, and isdeucine (and to a ler extant s=int, urqgnine and yrosin) residues Ser LCa Ala Ala Met Pro Ala Ala Pro Scr Tyr Thr Ala Thr pro Arg Pro Pro His Ala TARLE TA.

AsnArg His 3cr His His Glu Pro Tyr His His Met Val Tip Trp Leu Gly Ty-r Leu Gin Arg le Leou Lou Thr Ala Lou Ginl Pro (SEQ ID NO. 159) Arg (SEQI1D NO: 160) Sa (SEQ ID NO: 161) Ciy (SEQ IIDNO 162) Lie (SEQ ID NO, 1631 11e (SEQ ID NO: 164) Ser (SEQ ID NO: 165) Ser (SEQ ID NO: i66} Ile (SEQ ID NO: 167) Ile (SEQ ID NO: 168) His Arg Lys Gin Asn Asp Glu Lou ma Val

II.

Gly Ser Thr PMo Tyr Trp C"3 Met 11.4 5.7 1.4 5.7 1.4 0 1.4 10.0 11A 1.4 8.5 7 12.85 4.3 10.D 4.28 1.4 2.85 Li 2,85 exomcted 4.3 39 1.7 6.4 41 2.1 1.2 11.8 7.2 4.3 5A4 3.7 11.4 9.3 12 2,9 2,9 11, 0 4 3 When te bding buffer was modified to contain the ecrolytc KC in physioogic conenfruion (20 mM HEPES pH 7.5, 100 mM KC1, 1 mM Mg~cctac and 0. 1I 03% or 0.5% YWEEN20* depemnn d on the panizg round), and berbimycin A was present the omposition of pge-expresaed peptides bound was found to be amiched in tonine, phenylanie and histidie. and relaveiy depleted for glutamine, inolucine, and aarrsidue. TabLe Hi conaiz the scqences of 46 bound peptides; the deee of eruicmcn for cetain avutno acids in these 46 peptides is set forth ii Table IV, FIGURE lH depicts the nucleic acid sequences encoding 37 of theme pepuides. FIGURE I A- depits the distbution of amino acids a positins 1-7, rcspectively, of the expressed Pptic in all those phage sequnced, and shows dbw the occrrmnc of seine appeared to be favored at posion 1, proline ww favored at position 3, &d iueniac was favowd at position TABLU-I F Val Thr Pro Lys i Gii His Pro Met Pro 'Val 8cr Ser PhW Vai Ser Thr His Ple Thr Gly Gin Tip TIp Ser Gly Pro Pro His Gin Asn Tin Lou Pro Sei His Gin ro Ser Arg Tyr Gty An Pro Leu His It The Val Tyr Thr GLu Thr Pro Tyr Lcu Tbr Thr Pro Ph Gly Val Pro Lgu Thi Lys Lu Pro Thr Val Cys Ag Pbs His (Ny Tyr Th Arg Asp Pit Sr Ser Ala Ala Gly Se Leu ie- Gin Tyr Asp Ala Leo Met Trp Ser Ser UKN SR Lau PWh Am Ti2 Sef Thr lbr Val Gin His Va1 Asp Tyr Ser Phe Pm Val Giy Ser Met Olu Ph. UKN Pro Met Ile Ala Pro Pro Arg Val le Ala Thr Lys Tin Lye Pro Pro Leu Phe Tyr His Thr Ala His Tyr Ie Gin Ala Ser Ser Phe Ala Thr Thr Thy Pro Pro Asn ie Set Leu Asp Pro Scr i= Pro Lau Phw Gly Set (SEQ ID NO: 169) Val Lcu (SQID NO:170) Thr 8cr (SEQ ID O: 171) Trp Pro (SEQ ID NO: 172) Pro Asp (SEQIDNO: 173) Asp Ser (SEQJID NO; 174) Thr tic (SEQ 1D NO: 175) Trp Va] (SEQ ID NO- 176) GIr Pm (SEQ ID NO: 177) Gly Ala (SEQ ID NO: 242) Pro Thr SEQ ID NO 243) Scr S (SEQ ID NO: 244) Met Asp (SEQ TD NO: 245) Lcu Aig (SEQ ID NO: 246) Asa Azg (SEQ ID NO: 247) Glu Ala (SEQ It NO: 248) Pro Arg (SEQ ID NO: 249) Ser Arg (SEQ ED NO: 250) Pro U104(SEQ ID NO 251) Tyr le (SEQ ID NO: 252) Arg Thr (SEQ ID NO: 253) Ala Lbs (SEQ IDNO: 254) Pro leu (SEQ ID NO: 255) Ser Leu (SEQ ID NO: 2S6) UKN Ser (SEQ ID NO: 257) Thr Met (SWQ ID NO: 258) Pro Lys (SEQ ID NO: 259) Gn Ile (SEQ ID NO: 260) Asn Met (SEQ ID NO,261) The His (SEQ ID NO: 262) Fir Lou (SEQ ID No: 263) Pit AlR (SEQ ID NO: 264) Arg Met (SEQ ID NO: 265) Gly Ala (SEQ ID NO; 266) AS Lcu Lou Lys Thr Thr LCu (SEQ rID NO: 267) Asp Gin Asn Lu Pro Arg Arg (SEQ ID NO: 268) Sr His Pb. Glu Gin Len Lcu (SEQ 1 NO: 269) Thr PMp GIn Leu His His GlSy (SEQID NO: 270) Ala Pm Lou Asp Arg le Thr (SEQ W NO:2 71) Pht Ala Pt Lau lie. Ala His (SEQ ID NO: 272) Set Trp le TER Thr Phi Met (SEQ U) NO: 273) A"n Tbr Tip ro -s MO Tyr (SEQJID NO. 274) Giti Pro Leu Pro Thy Thy Lou (SEQ ID NO: 275) His Gly Pr [tis Leu Phe AMn (SEQID NO: 276) Tyr Le Amn Ser Thi Lou Ala (SEQ IDNO: 277) His Leu His Set Pro Ser 01y (SEQ DD NO: 27g)

IABLEJ~.

a& jawl %irwh 5.7 43 Arg 3,49 3.9 Lys 1.9 1.7 Gin 3.8 6.4 Asn 3.17 4.1 Asp 2.86 2.1 ii 1.9 12 L&u 10.5 11.8 Ala 5.39 7.2 Val 3.8 4.3 Lie 5.4 Gy 3. 3.7 Sir 10,47 11.4 Thr 12.06 9.3 Pro 12.38 12 Tyr. 3.49 2.9 Pb. 5.39 2.9 Trp 2.22 1 Cys 0 0-8 Met 3.17 3.3 The binding buffer used to geneate the data Of Tabls U ad V was further modified o inclde 25 mM CaCl, (in order to simulem the high cakitn concnauimon found in the endopasnic reficu rn), to pmducc a binding butfer having 20mM HEPES pH 7 5, 100 mM KCI, 25 mM CaC6 &d 5 mM MaAzeatc and 0.1% 02% or 0.5% TWEEN-20 1 depending on te panting n'md. The rmuli of slthity penning using this binding bfr and gp96, in the pcmsence of herbimycin A depiCtd in Tables V and VI. The dat indicates that binding of phge exprssing pepdCs conmining phenyluinin hisiine, and tryptphan residues was favored.

The sequence Phe.HEq-Trp-Trp-Trp (SEQ ID NO: 320) appcumd to e vwd.

TABLEXV

Phe His Trp Tip Trp Gin Pro (SEQ ID NO: 178) Ile Th Lcu Lys Tyr Pro Leu (SEQ IDPNO: 179) Ph. His Tip, Pro Trp Len Ph* (SEQ ID NO: ISO) Thr ALa Gin Asp Sr Thr 3y (SEQ ID NO; I81) Ph. His Tip Trp Tip in Pro (SEQ ID NO: 182) Ph. His Tip Trp Asp Trp Trp (SEQIL NO 133) iu Pro P. Ph. Arg Met Gin (SEQ ID NO: 184) Tbr Trp Trp Leu Ain Tyr Arg (SEQ ID NO: 185) Phe His Trp Tip Tip Gn Prn(SEQ ID NO. 186) GLn Pro Ser His Leu Avg Tp (SEQ ID NO. 187) TABLE VT.

His 8.6 4.3 Arg 43 3.9 Lys I A 1.7 Gin 8,6 6.4 Amn 1.4 4.1 Asp 2.85 2.1 0hz 1lA 1.2 Leu 7.1 1 L8 Ala 1.4 7.2 Val 0 4.3 Ile 1.4 54 Oly 1.4 3.7 Ser 2.85 11.4 TIV 5.7 9.3 Pro l0.0 12 Tyr 2.85 2.9 Phi 11.4 2.9 Trp 25. 1 Cys 0 0.8 Met 1.4 3.3.

When the sam bldg buffer was used, but crbimiycin A was not pmcnt, the composition of phage-exprsed bound pqptides w ated (Tables VA and VA). In particlar, the amount of smina and pinle reiues increased subnndaly 1 wile nihe amo=n of nryptphn thaug SUBMYa decresed, remned high ireistive to its excte occIrrn The WUoun of phnylalnn decresed rngnificandy but was mill presen at a frequency presterthan expected.

Po Ala -9&r PrO Vail Tyr (SEQ ID No: 13) The His TrP TrP Trp Gin Pmv(SEQ IDNO: 189) Hi. NrO Ser Am Gin Ala Sor(SEQ ID NO:l19)) Amw Scr Ala PrO Aug Pro Val (SE-Q ID NO. 191) (36 L4U TMp 5cr 11C TYr Pro (SEE) M NO: 192) r TlrpPr ft he Plm Asp Lou (SEQ ID NO: 193) Asp Thr Thrw a11 pro Leu HMs(SEQWIDNO: [94) Trp H" TsP Glt M"t Lou TYp (SEQ ID NO: 195) Amp The Arg Thr pro Val (SEQIt)NO):196) Thr Scr Pro Lfta Ser Let, Len (SEQ [D NO: 197) TALEfI Arg 2.8 3.9 LYN 0 1.7 Gin 5.7 6.4 Asn 2.8 4.

ATp 4.3 2. 1 GAg 0 1.2 Leu 11.A 11.8 Ala 4,3 7.2 Val 4.3 4.3 lie 1.4 SA4 Oly 0 3.7 [4-3 11.4 Thr 5,7 9.3 Pro 15.7 12 Tyr 1.8 2.9 2.9 TZp 11.4 1 Cys 0 0.8 met 1.4 2.3 When an othewise compa ble bhding buffer having a Lower caliuml ion ccofcefttO was used, the prevalenc of trypmaphan and pheyluinine residues ae-crased substwniaLfy, whees the pecrtage of praline residues remained elevated [i paricrdar. the use of a binding buffer having 20 mM HEFES pH 7.5. 100 mM KCI, 1 iM CaAcetSZ mM MAVc, and I 1 0, or 0.5%,Of T EEN depending on the paring round, and gp 9 6 in the presence of bbimycin, yielded the eults set foti in Tables VU and VIII.

TABLE Iil.

Ala Tyr As Tyr Val Sw Asp (SEQ IDNO: 198) Ar Pro Lou H Asp Pro Mat (SEQ ID NO: 199) Trp Pro &cr Thr Thr Leu Phe (SEQ ID NO: 200) ALa Thr Lcu (Mu Pr VSI Arg (SEQ ID No:- 201) SLr Met Th Val Leu Mrg Pro (SEQ ID NO: 202) Gin lie Gly Ala Pro Ser Trp (SEQ ID NO: 203) Ala Pro Asp Len Tyr Vat Po (SEQID NO 204) Aq Met Pro Pr Leu Lou Pro (SEQ ID NO: 205) Ala vy Ala Thr Pr Glu His (SEQ ID NO: 206) TARLP Y111, AJL %?&MUML mce His 3,17 4.3 Arg 6.35 3.9 LYS 1.59 17 Gin 1.58 6.4 An 1.58 4.1 Asp 4.76 2.1 (Mu 3.7 1.2 LCu 11A 11.8 Ala. 9.5' 7.2 Val 6.35 4-3 1k 1.58 5.4 (My 115$ 3.7 Ser 6.35 11-4 Thi 7.94 9.3 Pro 19.0 12 Tyr 4376 2.9 Phe 1.58 2.9 Trp 3.17 1 Cys 0 0.8 Met 4.76 3.3 Affiiy paiming expemanu w=r also caniied out usin a bindin buffer having, in addidon wo pysiologc electrolyu levels. and a jow calcium Concentatin, flT (in order to create a rcducLng enviramecs). The rcsults -of such cpctfl fltS.ff as ndigbuff, 2mMKEPES pH 7.$o100m KC1j mM CaC1 2 1 mnMDTT, and 1 m MMgAceme, witbO.I1%,O.3% or 0.5% TWEEN 2O, dependingaon the pennig round, and w%6 with herbimycin A. as hap target, we showni in Tables IX and x Phige-exprese pepddec bidn to gp96 under Chess conditions wem erniched for histidine, .rginhne, leuine and pmoliuc icaidue, and were somewhnt ericheti for aspwagin and tyrosie reSidues ThAILE I Thr Pro Pro Lu AMg le Asn (SEQ ID NO: 207) Len Pro Ile isi Aim Pro His (SRQ ID NO: 208) Asp Lcu Am Ala Tyr Thr Hi6 (SEQ ID NO: 209) VI Thr Lau Pro Ami Plu His (SEQ ID NO: 210) Ami Ser Mrg Lau Pro Thr Leu (SEQ ID NO:211) Tyr Pro His Pro Sr Ail Ser (SEQ IDNO: 212) GlY l Ala M3s Phe Mat Tyr (SEQ IDNO.-213) Tyr 8cr Leu Lau Pro Thr Aiz (SEQ IDNO. 214) Lau Pro Arg Arg Thr Lze Leu (SEQ ID NO:2i15) TAB3LELX

WE

Azg

LYS

GIB

Asa Asp Olu Lou

MA

Val le Ser Thr Pro Tyr Phe Trp H1MnI 9. 5

M.

0 a 6.3 1.58 0 17.4 4.76 i.58 1.17 138 6.3 I1.l 15.87 63 3.17 0 4.3 3.9 1.7 6A4 4.' 2.1 1-2 11.8 7.2 4.3 3,7 11.4 9-3 12 2S tAA Al51W h C=Qnwd Cys 0 0.8 met 1.58 3.3 When calcium was eliminated From the binding buffer, surh that uliwity pnning was carried out using, as hap Large', gp96 an herbimycin A. an. as binding buffer, 20 mM HEPES pH 7.5, 100 mM KC]. I mM DTT, 1 mM Mog~ctate.

and 0-1 or 0.5% TWEEN 20 depending on the paning round, and 42 phage.

expressed peptides wcrC sequced. resuls as set fot in Tables XI and XII wNe obwained. The binding of pbagexpresscd peptides containing htonine. senne, tyrosine, and, to a lesser extent, lyuine, glutuinic aid and leucine, appeared to be favoed. When the distribution of ami acids at each of the xven positions of the expressed hqnapcptide of all phage inserts sequencd were analyzed (se FIGURE 2A-G, positions 1-7, respetively), the occurrence of hreni e at positios I and 3, leucine at position 5 and senine at position 7 were favored. FIGURE 2H shows nuceic acid seque=ces encoding 33 of Ihese peptidcs.

TABLE )a Tr Ser Thr Len Leu TIrp Lys (SEQ ID NO: 216) Thr Ser Asp Met Lys Pm His (SEQ ID NO:.217) Thr Sr Sir Tyr Leu Ala Ln (SEQ ID NO: 218) An Lou Tyr Gly Pr His Asp (SEQ ID NO: 219) Leu Gli Tr Tyr Thr Ala Sar (SEQID NO: 220) Ala Tyr Lys &r Le The Gin (SEQ ID NO: 221) Ser Thr 5cr V.1 Tyr Ser Sr (SEQID NO.;222) Glu Gly Pr Lii Arg 5cr Pm (SEQ ID NO: 223) lir Thr Tyr His Ala Leu Gly (SEQIDNO:-224) Thr Leu Pr His Arg Leu An (SEQ TD NO: 279) Ser Sir Pr Arg Gii Val His (SEQ IIDNO: 280) Asn Gin vw Asp The Ala Mg (SEQ ID NO: 281) Tyr Pro The Pro Leu Leu Thr (SEQ ID NO 282) His pm Ala Ala Phe pro Trp(SEQ ID NO- 283) Lau Leu Pr His Scr 5cr Ala (SEQ IDNO: 24) Lou' Glu Thr Tyr Thr Ala Sir (SEQ I)N: 285) Lys Tyr Val Pro Leu Pro Pro (SEQ W1140, 286) Ala Pro Ecu Ala Len His Ala (SEQ ID NO:,287) Tyr GLu Sir Leu Leu Tha Lys (SEQ ID NO: 288) Sr His Ala Ala Sir Gly Thi (SEQ ID NO: 299) fly Leu Aim Thr Val Lys Sir (SEQ ID NO: 290) Gly Ala Ttr Scr Phe cly LIu (SEQ ID NO: 291) Lys Pro Pro Thr Lu Thy His Ala Pr Val Ala Pe Lau Pro Thr Ala Ser b Arg Me Asn Lys M Tr Ala Asa Ala Tin lie Trp Gin Tbr Ly ASn His Mla Leu His Ala Tlb Trp oin Ala Pr Leu Thr Ala His Am Met Thy Gly &M Gly Thr Pm ie Ser His Lau oly vx' The Thr Axg Asp Thr Pto Thf pro Val Val Ata Al As Lys Tyr Pro Val Scr (SEQ ID NO: 292) Ser Oly Am (SEQ ID NO: 293) Lys Scr Gin (SEQ ID NO: 294) Arg Lou Pm (SEQ ID NO:295) Thr Pro Ala (SEQ [DNO: 296) Leu Leu Hie (SEQ ID NO: 297) Glu Pro Pro (SEQ W1 NO; 29S) Len Thr Thi (SEQ ID NO: 299) Pro Lm Leu (SEQ ID NO: 300) Pm Pm Val (SEQ IDPNO: 301) Met Tr Thr(SEQLID NO: 302) Ph. Aka Sw (SEQ ID NO: 303) LKN Thr Ser (SEQIDNO: 304) Tyr PMc HisSEQInDNO:,305) Lcu His AIa (SEQ ID NO:306) Leu Thr Val (SEQ ID NO: 307) Met Lcu Thr(SEQ IDNO:308) Scr GIn Asp (SEQ ID NO: 309) Thr Ile Tyr (SEQID NO: 3oJ Arg 5cr Ser {SEQIDNO:Jl3)

TABLEXIIL

His Arg Lys Gin Am Asp

GIU

Ala Val 'Ie Gly 8cr Thr Pm Tyr PMg Trp Cys Met 5.44 2.72 3:74 2-0 3.06 2.D4 2.0 12.92 4.08 1.36 3.74 10.88 1195 10.88 4.76 2.38 1.36 0 210 rxDQ~hd 4.3 3,9 1.7 6.4 4.1 2.1 1.2 11.8 7.2 4.3 3.7 11 A 9.3 12 2.9 2.9 1 0.8 3.3 Affinity panning was also prformcd using gp 9 6 in the prease of Irbimycin A. as hip target and, as binding buffer, the following solutio contaiing AW: 20 rM HEPES pH 7.5, 100 rmM KC1. I mM CaCI 2 1 rtM MgActaw~, 1 mM ATP. and 0. 1% 0.3% or 0.5% TWEEN 20, depending an dhe round of panming. The results ar presented in Tabes XII and XI. Phe-cqpsscd pepdds bound by p96lherhimyin A under these conditons were enrichd in histidie, tyosine and swrim (imd to a lssr extnt roline and typtophin) residues.

TABLE XUL Vat Ser Ile lly His Pro 5r (SEQ ID NO: 225) Ti uHis cr His Arg Pro Setr (SEQ ID NO: 226) Ile Thr Amn Pro Leu Thr. Tr (SEQ ID NO: 227) Set le Gin Ala His His Ser (SEQIDNO, 228) Len Am Trp Pro Arg Vpi Lcu (SEQ I NO: 229) Tyr Tyr Tyr Ala Pro Pro Pro (SEQ ID NO: 230) Scr Leu Trp Thr Arg Lc Pro (SEQIDNO: 231) m Val Tyr His Set S= Lou (SEQ ID NO: 232) TABLE V.

His 10.7 4.3 Arg 5.35 3.9 (P D I Gin 1.78 6.4 Asn 5.3 441 Asp (P 1 Glu 0 1.2 LAU 10.7 11.8 Ala 3.57 7.2 'Val 5.3 43 le 5.35 5.4 kly 1.78 3.7 Ser 16.1 11A Thr &9 9.3 Pro 14.2 12 Tyr 71 29 Phe 0 2.9 Trp 3.57 1 (4S 0 0.1 Met 0 3.3 Whe, instead of ATP, the binding buffer conained AMP-PNP rmM HEPES pH 7.5, 100 mM KCI, 1 mi CaCI 2 1 ruM MgActa z 1 mM AMP- PNP, and 0.3% or 0.5% TWEEP 20 depending on te panning roundt as shw in Tabies XV mnd XVI, binding ofphmgt peptides coirnuing hisridine and vihac. Position 4 appcas tD favor basic residues.

Mn Swt Pro His No Pro Tbr (SEQ ID N0233) Val Pn Ala Lys Po Arg His (SEQIDNO:234) His Amn Lou His Pro Am Arg (SEQ [D NO: 235) Tyr lb Thr His Ar Trp Leu (SEQWID NO, 236) Ala Val Thz ALa Ala He Vel (SEQIDNO:237) Thr Inu Met His Asp Mj Val (SEQ ID NO: 238) Thr Pm Lau Lys Va Pro Tyr (SEQIDNO: 239) Phe 1kr An Gin Gin Tyr His (SEQIDN:-240) Sc His V Pro Ser Met Ala (SEQIDNO: 241) His Gly Cin Ala Trp Gin Phe (SEQ IDNO. 312)

IABLEXVIL

His 12.8 4.3 Arg 5.7 3.9 Lys 2.85 1.7 Gin 5.7. 6.4 Am 5.7 4.1 Asp 1A 2.1 Oli 0 1.2 Leu 5.7 L8 Ala S.5 7.2 Val 8.5 4.3 le 1.4 5A4 Oly I4 31 Se 4.23 11.4 Thr 10 9.3 PM 12.8 12 Tyr 4.28 2.9 Phe 2,85 2.9 Trp 2,85 1 Cp 0 0.8 Met 2.85 3.3 7. EXAMPLE. CONJUGATE PEPTIDE ADMINJST-ERED Preparatim of hWOO. Puified muse cytornolc hsplO was prepared troin Escherichia coil DH5aL Cells crusforrmd with pMS23 6 (Hunt and Calderwood.

1990, Goe17-l99-204) enodinj mous cytoslic hsp70. The cells w=tr grown to an optica density of 0,6 at 600 urn at 27'C, m4d expresion was induced by the addition of IPTO to a- il commctrsgjon of I MM.i Cells were havested. by ccntrfugahin at 2hour past-induction, and the cell pellets were respcndcd to a voluin, of 20 nil with BuffierX (20 mM HEPES pH 7.0,25 mM KCI, 1 mM DTTJ 10 mM (NH J ,S 4 I mM PMSFj- The cells were lysed by passag (thre times) fthough a French press.

The lysam was cleared by low speed cennifligaxion followed by cenriion at 100,000 x g for 30 minuses. The resting cleared Lysa= was applied to a Phamac XK26 oiwim pecked wit 100 ml DEAE Scphactl (Phurnacia) and equiibr&We with Buffer X at a flow rate of 0.6 cm/ruin. The column was washed to stble baseline with Bufe X and elute with Buffer X containng 175 ruM KC1. The chase was appl ied to a 25 al ATP-agaroc column (Sit=a Chemical Co., A2767), washed to baseine with Buffer X, and dAuted with Buffer X conraindog I mM MgATP preadjusted to pH EDTA was added to thie clet to -a final. caoonwdou of 2 mM. The diate, which ecortsane4 essntisly pure hsp70, was precipitated. by addition of (N1430 4 to percetit axnuratton The precipitate was resuspadml in Buffer X cornainig I mM MgC17 and dialyzed "gis the sain buffer with multpe chages For storage, the was froze in aul aliquota at -RC.C Peptdez. The folowin peptides we preprd- OVA pepsid (Ser He Ile Asm Phe ilutLys Leu- SEQ ID)1NO: and (1i) OVA peptide, joined, via a tuipcptidc linker (gly ser gly> to the BiP-binng tether peptide His Tip Asp Pit All. Tip Pro Trp (Blond.-Elguindi et al., 1993 Cell 71.-717-721; SEQ ID NO:) 1 X to form the co~ugue peptide OVA-RiP (Ser Tic Ile Am. Phe Glu Lys Lwu Gly Scr Gly HisTpAs PkcAla.Trp Pm Tny SEQID NO) Preparaton of hsp7S sad/r pupilde for urse in immnunization.

Approximately 15 jig 6 sp 7 0 and 12 Ag OVA-fil were mixed. on ice, to a final so volume of 10 P.L in Buffer Y (to produce a final concenndofc 21.5 pM hSP70, nM OVA-BiP, 20 mM HEPES pH 7.0, 150 aiM KC 1 10 mM (N}14,S0 4 2mnM MgCI 2 and 2 MiM MgADP, pH The mixtur was incubated for 30 minutes at 37-C and was used fur hi viva immunizutons- Shiila incubation. were carred out with 5 pi TiterMax adjuvant (Vaxcei, Norcrsa, GA) and 12 P& OVA-HiP 00i S P1 TiterMax and 5Spg OVA peptide iw Pi) L2 I.tg OVA-BiP alone.

Prepanalon of ceb for -bromluam niew awsay. Female C57BLI6 rc 8-10 weeks old (two per assay). were immunizzd intniraemay o=c (xlI) or twice (x2) at a one-week inerv.] with 10 ya of eithe 6i) hsp7O/OVA-BiF; (ii) Tiudr&YJOVA-BiP; (iii) TiterMaxOVA; or (iv) OVA-BiP. One week after the 1wt immunizaton the rmce were sacdficed 1 their sptrns removed, and used to prepare monnuceareffcto cels. 8- 10 x 107 of these effeckxr clls wore the cultured with 4 x IlCV gumma-irradiaWe 3000 zad) sinmiator cells end feeder COil (which wec obta ieum the spleen of nave mice and senstized 1 vi ro, wit 10 4gmI OVA pepddce for 30 minutes at roam tempernre prior to gam&a indadon) in RPM 1640 medium contaiing um percen fetvl calf serum 100 Uhmi penicillin (GIRCO, CaL No. 15140-12fl), 100 4gm] strqptwmycin. and 2 mM L-glutamine. After culturing in Woro for five days the cyoloxic activity of the result effector cells w~s assayed as set forth below. CTL. lines were maintaind by stimula with kira d stixulators, gygenkc splenic feedr calU plus T cell growth fithoo.

Chromuiam relis *msy. The cytorxiefty of spleen cells from irwiD~UzM mce, cuhttnd as set forth in the preceding pargraph, was assycd in a 4 hour "Cr release assa using, as =igut cells. eidhe OVA-peptide pulse EL4 cells or (i1) ufivc EL4 clls, which were chronus labaled. Effactr cells were prepared as set forth above, Target cells were prepared as follows. EtA =Uls were washed with PBS hree times. To prepare naive "eLs 5 x 10' EtA4 cells vwe incubated with 100 pit 21Cr (sodits chromame, DuPoin. Bostn4 MA) in 1 ml of 10% PCS/RPMJ madium for 1 houunt 3X'. To prepae pudsed calls, 5 x 10' EL4 cells mme incubated Mi& 1 R/m1 of OVA-pqptdec and IOU 110 "Cr in 1 mlk of 10% FC&dRPMI mnedium for 1 hour at 3n<. The target cetls were then washed tbm times with RPM! and resuspandalto a final cell vomt of 1 x 10' cells/mi in l0% FCSYM I 1W of the

I

5 ICr-abcled EL-4 cells wer mixce wit effeor lymphocytes Lo yied scvaul cfietor to target cell (EM ratios, mud then mpubaae for 4 hour, Supenitants werr harvested and radioactivitiy relcasal by cyzoboxic acMW was measuwl in a gamm counter.

The percent specific lyui 9 waW calculad as 100 x Ucpm re leas by CTL cpm spontaneously reeuedY(cpm maxima] release qnff spontanously released)].

Maximal receae was determined by adding I NP-40 to lyse all. cells& Spontaneous rlease of all target in the absence of effector cells (nured in a cu hure of target cells (in the absence c ffceo celis) maiantaqi ned in parallel fo~r the duTion of' tie assy) was less 2W% of the maimal nice IFIGURE 3A-B depicts the cytotoxic activity of'effecwr cell prepared from mice inunimized onc with ThweMax plus OVA peptade (which does we mprise a lelber) agaim OVA-primed EL-4 targt ce~ls (FIGUJRE 3A) or rmprmd EL-4 control cells (FIGURE 3B). The two curvs represnt data obtained with two &ffezcni maicc. These resUs indicate dint TiterMux adjvant together with OVA poptid was able to iuchn a OVA-Specific cytAonc immune response.

FIGUJRE 4A-B shows the resus of immunization of mice with hsplO plus OVA-1B iP ccpte peptide. Each curve represets data obtained fom a sinle mouse. Mice wee either immunized owce (solid sqiwm and triagles) or twice (open squre and rectangls). Percent killing of O'VA-prime EL-4 target colls (FIGURE 4A) or wiprimed control cells (FIGURE 4B) was measured As shown in FIGURE 4A, a skngl immunization with hsp7WOVA.B3iP was able to induce a OVA-specii eyooi innu respons which appeared to he peathanim that indwWe by The~Ax/YOVA (FIGURE 3A) and as least as good as tha induced by TiterMax/OVA.&P (FIGURE 6A). Mice receiving two irnmunizadiorts appeared to manifest a somewhat smalkr response. A simila respons was obtained when mice were immunized once or twic with TiterMax/OVA-BiP (FIGURE 6A)ffiterstngy. mnice immunized with OVA-HIP alone were also Found to exhibit a significant anti-OVA irmm respons,- as shown in FIGURE 5A. Effecro cells produced frucm mice imunized once ur twice with the conijugate pepde. alone 52 wer Wtetd agains OVA-pcimad EL-4 targcc. cells 1 significam cel lysi. occuirod (relative tO lysis of naive EL-4 cells. as shown in FIGURE 5B), Thus, the conjugate pcpuide OVA- i P was capable of elicitng a cytocoxic unmime mnsu in the absence of added adjavam- jFIGURE 7 shows the reslts whten mice wer= immunized cxe or twice vith OVA-pepide alone.

EXAMPLE: IMMUNIZATION WITH

CONJUGATE

CS7BL/6 mice, g- I) weeks old, wart immunized irdradermaily wit one of t following (eigh mnice in eah group): (8)5 p1 TirerMax and 5 Vg OVA pepdde; (b 15 gig hsp7O and 5 jgg OVA peptde; 5 p1 TiterMax and 12 gil (OVA- BiP); 15 !a6 hsp7O and 22 pg OVA-RliP; controi (ibu ainimal -only ini this grrup); (05 pg OIVA pcptide or 12 At OVA-RiP. The mice then were injected with 4 X 10' E07 Ceilt Tumor nze Was evaluated over time by nmsung rwo diatetem, the gretest dimea, and the diameter perpendicuin to the gretst dianor, and then c-alculatin the aveag diametr. The remats arm hwna in FGURE &A-0 (correspondng to gpsM as act forthi above).

The data indicate that when admiiaeed with TiterMax adjuvam, OVA-BiP (FIGURE SC) was superior to OVA pepide (FIGURE HA) in reducin tMowr diameoter and in prevaiting detectble tmor formtio altogedwe. Further, tuMor SLe in mice imatumd with hsp70 and OVA.BIP (F!GjUREi SD) was I=s tha inL mice iinmunizrd with hsp70 mnd OVA-peptide (FGURE SB). I Mice receiving pepede .1cmg (without erMax or hs70), while io anmals w=r mnor-free when OVA-peptid. was the soiek unumogcn(FIGURE 8F), 2i8 animals immunnized wilh OVA-NIP wer tumor-free and the averag tumor diameters were MATae (FIGURE 8G). It therefore appors tha the conjugate peptide associated with hSP7O wa mare effecajve than the antigaic peptde alme at Preventing or reducing tumor faimaton in vivro (FIGURE ft1 FIGU'RES 19"- show the reukts -of analogous experimen in which mice %wr challnged with at second tumor cell line, ngamely the ovajbwnin-expresint melam cell 1=n M04. Mice were immunized wit either 5 p4 rwiMax ptus jig OVA pe-ptide, 15 pjg Hsp7O plus 0.5 jig OVA peptide. or 15 pg Hsp70 plus 1.2 pg OVA-BIP seven days before chalene with 1 x 100 M404 cells. FIOGURES]I9A- C show. tmor growth OWe time, measured as the average tumor diameter for grups of mice A-C, respectively. FIURES 1 9D-E show dw results of experinmts i which mice wer first challenged wish t x 1 0' M04 cells to estblish a palpable tumor before immunizaton (fourtesa days afte tbsllertg) wit either 5 iag OVA peptide alouor 15SpgIHsp7O plus Ilp)g OVA-BiP. FIGURE S 19F and 190 show, respectively, the su-vival ratios of mice immwdized seven days before challenge v~th melanoma cells and the srvival ratios of mice immunized seven and fourtee days after melanma tumoer cell ciwliee As shown above wi4th the EO7-OYA tumor model, Hsp70 plus OVA- BiP immunizatio confenrd supetior protetion against M04 tumor growth relatve to inwwnization wikh either TiterMax plus OVA peptd.e or 1 1 p 70 plus OVA peptide, Two of eigbt mice inmunized with HspJO plus OVA-BiP wae of mnmzr whereas none of sixtence immunized with either TiterMax phi OVA pepuide or Hsp7W plus OVA peptids were tumor be 77= namo trwnd was abvered w imuization ocwrcd after tumor challenge; that is to my, tumor growth wn sqlowe~st in the HspYG plus OVA-BiP? immunized group.

Various publications arc cited hcremn the contens of whkh arc hereby incoprae by rcfcrc in their enties

Claims (24)

1. A method of inducing an immune response in a subject in need thereof, Scomprising administering, to the subject, a composition comprising a conjugate peptide, wherein the conjugate peptide comprises 00 5 a first portion which binds to a heat shock protein under physiologic V) conditions, and (ii) a second portion comprising an antigenic peptide, wherein said antigenic peptide is associated with a neoplasia or a pathogen, wherein a heat shock protein is not concurrently administered with the conjugate peptide, and whereby an immune response to said second portion is induced in said subject, said immune response being to an antigen of said pathogen or said neoplasia.

2. A method of inducing an immune response in a subject in need thereof, comprising administering, to the subject, a composition comprising a nucleic acid expression vector encoding a conjugate peptide, wherein the conjugate peptide comprises a first portion which binds to a heat shock protein under physiologic conditions, and (ii) a second portion comprising an antigenic peptide, wherein said antigenic peptide is associated with a neoplasia or a pathogen wherein an immune response to said second portion is induced in said subject, said immune response being to an antigen of said pathogen or said neoplasia..

3. The method of claim 1 wherein the conjugate peptide is purified.

4. The method of claim 1 or 3, wherein the composition further comprises one or more adjuvants.

The method of any one of claims 1, 3 and 4, wherein the first portion is covalently bound to the second portion.

6. The method of any one of claims 1 to 5, wherein the first portion is a peptide of 7 to 20 amino acids. W:ANge650000 699999\6921 57\692157 Clairrs Jun OBdoc 0

7. The method of any of one of claims 1 to 6, wherein said conjugate peptide is in the Srange of 15 to 40 amino acids. I

8. The method of any one of claims 1 to 7, wherein the first portion is HWDFAWPW (SEQ ID NO: 143). 00 5

9. The method of any one of claims 1 to 7, wherein the first portion is ID c HyXHyXHyXHy where Hy represents a hydrophobic amino acid and X is any CI amino acid. 0

10. The method of any one of claims 1, 3 and 4, wherein the first portion comprises a benzoquinone ansamycin antibiotic.

11. The method of claim 10, wherein the benzoquinone ansamycin antibiotic is geldanamycin or herbimycin A.

12. The method of any one of claims 1 to 11, wherein the conjugate peptide further comprises a peptide linker, said linker separating the first and second portion of the conjugate peptide.

13. The method of claim 12, wherein said peptide linker is cleavable, and wherein the linker is sensitive to cleavage by a cellular enzyme.

14. The method of claim 12, wherein said linker is cleavable, and wherein the linker is acid sensitive, base sensitive, light sensitive, sensitive to reduction or sensitive to oxidation.

15. The method of any one of claims 1 to 14, wherein said antigenic peptide is associated with a neoplasia and said administering induces an immune response to an antigen associated with said neoplasia.

16. The method of any one of claims 1 to 14, wherein said antigenic peptide is associated with a pathogen and said administering induces an immune response to an antigen associated with said pathogen.

17. The method of claim 15, wherein the neoplasia is selected from the group consisting of sarcoma, lymphoma, leukemia, melanoma, carcinoma of the breast, WANiQeffi50000 699999\692151'a92157 Clairn Jun 06.doc 0 carcinoma of the prostate, ovarian carcinoma, carcinoma of the cervix, uterine Scarcinoma, colon carcinoma, carcinoma of the lung, glioblastoma, and astrocytoma.

18. The method of claim 16, wherein the pathogen is selected from the group oO 5 consisting of a bacterium, a virus, a protozoan, a mycoplasma, a fungus, a yeast, a V) parasite, and a prion. INO rC

19. The method of claim 18, wherein the pathogen is a bacterium.

The method of claim 19, wherein the bacterium is selected from the group consisting of Salmonella, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Escherichia, Klebsiella, Vibrio, Mycobacterium, and Mycoplasma pneumoniae.

21. The method of claim 18, wherein the pathogen is a virus.

22. The method of claim 21, wherein the virus is selected from the group consisting of a human papilloma virus, herpes virus, retrovirus, hepatitis virus, influenza virus, rhinovirus, respiratory syncytial virus, cytomegalovirus, adenovirus, herpes simplex virus, herpes zoster virus, human immunodeficiency virus 1, and human immunodeficiency virus 2.

23. The method of claim 18, wherein the pathogen is a protozoan.

24. The method of claim 23, wherein the protozoan is selected from the group consisting of an amoeba, a malarial parasite, or Trypanosoma cruzi. The method of any one of claims 1 to 24, wherein the administering is repeated at least once. DATED: 13 June 2006 Phillips Ormonde Fitzpatrick Attorneys for: Sloan-Kettering Institute For Cancer Research N:NigeO650000 699999\692157\692157 Claims Jun O6.doc