Patent Publication Number: US-2004043419-A1

Title: Javelinization of protein antigens to heat shock proteins

Description:
1. INTRODUCTION  
       [0001] The present invention relates to antigenic complexes, wherein an antigenic complex comprises a peptide or protein containing a plurality of epitopes non-covalently joined to a heat shock protein via a molecular tether referred to as a “javelin”. Such complexes do not require that each epitope be defined, and may, in certain embodiments, elicit both antibody and cell-mediated immune reactions. The complexes of the invention may be used to induce therapeutic immune responses directed toward the treatment or prevention of infectious diseases and malignancies.  
       2. BACKGROUND OF THE INVENTION  
       [0002] Heat shock proteins (“hsps”) constitute a highly conserved class of proteins selectively induced in cells under stressful conditions, such as sudden increases in temperature or glucose deprivation. Able to bind to a wide variety of other proteins in their non-native state, heat shock proteins participate in the genesis of these bound proteins, including their synthesis, folding, assembly, disassembly and translocation (Freeman and Morimoto, 1996, EMBO J. 15:2969-2979; Lindquist and Craig, 1988, Annu. Rev. Genet. 22:631-677; Hendrick and Hartl, 1993, Annu. Rev. Biochem. 62:349-384). Because they guide other proteins through the biosynthetic pathway, heat shock proteins are said to function as “molecular chaperones” (Frydman et al., 1994, Nature 381: 111-117; Hendrick and Hartl, Annu. Rev. Biochem. 62:349-384; Hartl, 1996, Nature 381:571-580). Induction during stress is consistent with their chaperone function; for example, dnak, the  Escherichia coli  hsp70 homolog, is able to reactivate heat-inactivated RNA polymerase (Ziemienowicz et al., 1993, J. Biol. Chem. 268:25425-25341).  
       [0003] The heat shock protein gp96 resides in the endoplasmic reticulum, targeted there by an amino-terminal signal sequence and retained by a carboxy-terminal KDEL amino acid motif (Lys-Asp-Glu-Leu (SEQ ID NO: 1); referred to hereafter as the “KDEL” sequence, which promotes endoplasmic reticulum recapture; Srivastava et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84:3807-3811). Found in higher eukaryotes but not in Drosophila or yeast, gp96 appears to have evolved relatively recently, perhaps by a duplication of the gene encoding the cytosolic heat shock protein hsp90, to which it is highly related (Li and Srivastava, 1993, EMBO J. 12:3143-3151; identity between human hsp90 and murine gp96 is about 48 percent). It has been proposed that gp96 may assist in the assembly of multi-subunit proteins in the endoplasmic reticulum (Wiech et al., 1992, Nature 358:169-170). Indeed, gp96 has been observed to associate with unassembled immunoglobulin chains, major histocompatability class II molecules, and a mutant glycoprotein B from  Herpes simplex  virus (Melnick et al., 1992, J. Biol. Chem. 267:21303-21306; Melnick et al., 1994, Nature 370:373-375; Schaiff et al., 1992, J. Exp. Med. 176:657-666; Ramakrishnan et al., 1995, DNA and Cell Biol. 14:373-384). Further, expression of gp96 is induced by conditions which result in the accumulation of unfolded proteins in the endoplasmic reticulum (Kozutsumi et al., 1988, Nature 332:462-464). It has been reported that gp96 appears to have ATPase activity (Li and Srivastava, 1993, EMBO J. 12:3143-3151), but this observation has been questioned (Wearsch and Nicchitta, 1997, J. Biol. Chem. 272:5152-5156).  
       [0004] Unlike gp96, hsp90 lacks the signal peptide and KDEL sequence associated with localization in the endoplasmic reticulum, residing, instead, in the cytosol. Although hsp90 has not been detected as a component of the translational machinery (Frydmann et al., 1994, Nature 370:111-116), it has been reported to be highly effective in converting a denatured protein, in the absence of nucleotides such as ATP or ADP, to a “folding competent” state which can subsequently be refolded upon addition of hsp70, hdj-1 and nucleotide (Freeman and Morimoto, 1996, EMBO J. 15:2969-2979; Schneider et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:14536-14541). Hsp90 has been observed to serve as a chaperone to a number of biologically highly relevant proteins, including steroid aporeceptors, tubulin, oncogenic tyrosine kinases, and cellular serine-threonine kinases. (Rose et al., 1987, Biochemistry 26:6583-6587; Sanchez et al., 1988, Mol. Endocrinol. 2:756-760; Miyata and Yahara, 1992, J. Biol. Chem. 267:7042-7047; Doyle and Bishop, 1993, Genes Dev. 7:633-638; Smith and Toft, 1993, Mol. Endocrinol. 7:4-11; Xu and Lindquist, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:7074-7078; Stancato et al., 1993, J. Biol. Chem. 268: 21711-21716; Cuttforth and Rubin, 1994, Cell 77:1027-1035; Pratt and Welsh, 1994, Semin. Cell Biol. 5:83-93; Wartmann and Davis, 1994, J. Biol. Chem. 269:6695-6701; Nathan and Lindquist, 1995, Mol. Cell. Biol. 15:3917-3925; Redmond et al., 1989, Eur. J. Cell. Biol. 50:66-75). Hsp90 has been observed to function in concert with other proteins, some of which may act as true chaperones, others serving only as accessories; for example, cellular assembly of the progesterone receptor has been reported to involve hsp90 and seven other proteins (Smith et al., 1995, Mol. Cell. Biol. 15:6804-6812).  
       [0005] Inoculation with heat shock protein prepared from tumors of experimental animals has been shown to induce immune responses in a tumor-specific manner; that is to say, heat shock protein gp96 purified from a particular tumor could induce an immune response which would inhibit the growth of cells from the identical tumor of origin, but not other tumors, regardless of relatedness (Srivastava and Maki, 1991, Curr. Topics Microbiol. 167:109-123). The source of the tumor-specific immunogenicity has not been confirmed. Genes encoding heat shock proteins have not been found to exhibit tumor-specific DNA polymorphism (Srivastava and Udono, 1994, Curr. Opin. Immunol. 6:728-732). High-resolution gel electrophoresis has indicated that tumor-derived gp96 may be heterogeneous at the molecular level; evidence suggests that the source of this heterogeneity may be populations of small peptides adherent to the heat shock protein, which may number in the hundreds (Feldweg and Srivastava, 1995, Int. J. Cancer 63:310-314). Indeed, an antigenic peptide of vesicular stomatitis virus has been shown to associate with gp96 in virus infected cells (Nieland et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:6135-6139). It has been suggested that this accumulation of peptides is related to the localization of gp96 in the endoplasmic reticulum, where it may act as a peptide acceptor and accessory to peptide loading of major histocompatability complex class I molecules (Li and Srivastava, 1993, EMBO J. 12:3143-3151; Suto and Srivastava, 1995, Science 269:1585-1588).  
       [0006] Heat shock proteins have been used as adjuvants to stimulate an immune response (see, for example, Edgington, 1995, Bio/Technol. 13:1442-1444; PCT Application International Publication Number WO 94/29459 by the Whitehead Institute for Biomedical Research, Richard Young, inventor, and references infra). One of the best known adjuvants, Freund&#39;s complete adjuvant, contains a mixture of heat shock proteins derived from mycobacteria (the genus of the bacterium which causes tuberculosis); Freund&#39;s complete adjuvant has been used for years to boost the immune response to non-mycobacterial antigens. A number of references suggest, inter alia, the use of isolated mycobacterial heat shock proteins for a similar purpose, including vaccination against tuberculosis itself (Lukacs et al., 1993, J. Exp. Med. 178:343-348; Lowrie et al., 1994, Vaccine 12:1537-1540; Silva and Lowrie, 1994, Immunology 82:244-248; Lowrie et al., 1995, J. Cell. Biochem. Suppl. 0(19b):220; Retzlaff et al., 1994, Infect. Immun. 62:5689-5693; PCT Application International Publication No. WO 94/11513 by the Medical Research Council, Colston et al., inventors; PCT Application International Publication No. WO 93/1771 by Biocine Sclavo Spa, Rappuoli et al., inventors).  
       [0007] Other references focus on the ability of heat shock proteins to naturally form associations with antigenic peptides, rather than the classical adjuvant activity (see, for example PCT Application No. PCT/US96/13233 by Sloan-Kettering Institute for Cancer Research, Rothman et al., inventors; Blachere and Srivastava, 1995, Seminars in Cancer Biology 6:349-355; PCT Application International Publication No. WO 95/24923 by Mount Sinai School of Medicine of the City University of New York, Srivastava et al., inventors). In one protocol used by Srivastava in a phase I European clinical trial, cells prepared from a surgically resected tumor were used to prepare gp96, which was then reinoculated into the same patient (Edgington, 1995, Bio/Technol. 13:1442-1444). The fact that a new gp96 preparation must be made for each patient is a significant disadvantage. PCT Application International Publication No. WO 95/24923 (supra) suggests that peptides in heat shock protein complexes may be isolated and then re-incorporated into heat shock protein complexes in vitro. There is no evidence that this time-consuming procedure would be successful beyond the treatment of the patient from which the heat shock protein was derived. Further, the preparation of an effective quantity of heat shock protein requires the harvest, from the patient, of an amount of tissue which not every patient would be able to provide. Moreover, this approach limits the use of heat shock proteins as peptide carriers to those peptides with which a natural association is formed in vivo, and the affinity of such peptides for heat shock protein may be inadequate to produce a desired immune response using complexes generated in vitro.  
       [0008] Although the immunogenic potential of heat shock proteins and molecular chaperones has been clearly demonstrated (reviewed by Schild H. et al., Current Opinion in Immunology (1999) 11:109-113), whereby hsps are believed to deliver bound antigens to antigen presenting cells for subsequent display on MHC class I or class II molecules (thereby generating a T cell response), many antigens do not bind sufficiently well to heat shock proteins or molecular chaperones for them to be efficiently delivered.  
       [0009] In attempts to circumvent this limitation, heat shock proteins have been covalently joined to antigenic peptides of choice. For example, it has been reported that a synthetic peptide comprising multiple iterations of NANP (Asn Ala Asn Pro; SEQ ID NO: 2) malarial antigen, chemically crosslinked to glutaraldehyde-fixed mycobacterial heat shock proteins hsp65 or hsp70, was capable of inducing a humoral (antibody based) immune response in mice in the absence of further adjuvant; a similar effect was observed using heat shock protein from the bacterium  Escherichia coli  (Del Guidice, 1994, Experientia 50:1061-1066; Barrios et al., 1994, Clin. Exp. Immunol. 98:224-228; Barrios et al., 1992, Eur. J. Immunol. 22:1365-1372). Cross-linking of synthetic peptide to heat shock protein and possibly glutaraldehyde fixation were required for antibody induction (Barrios et al., 1994, Clin. Exp. Immunol. 98:229-233), and cellular immunity does not appear to be induced. In another example, Young et al., in PCT Application International Publication Number WO 94/29459, discloses fusion proteins in which an antigenic protein is joined to a heat shock protein.  
       [0010] A potential disadvantage of such covalent linkage approaches is that they tend to favor an antibody-based, rather than a cellular, immune response. In such context, the heat shock protein may act as a carrier to promote antibody responses to covalently linked proteins or peptides, a well known adjuvant function of immunogenic proteins. Furthermore, heat shock protein and antigen are irreversibly linked; this may alter the solubility of either protein component, or may create structural distortion which interferes with the association between antigen and critical major histocompatability complex components.  
       [0011] As an alternate approach to covalent linkage, antigenic proteins have been non-covalently bound to heat shock protein via a molecular tether which binds to heat shock protein under physiologic conditions. This tether is referred to as a “javelin” herein, and the process of complexing an antigenic protein or peptide with a heat shock protein is referred to as “javelinization”.  
       [0012] An example of the usefulness of “javelinization” is as follows. Heat shock protein 70 has been shown to be effective at delivering bound peptide antigens to antigen presenting cells for their display on MHC class I molecules. Immunization of mice with hsp70 bound antigens has resulted in the generation of strong cellular immune responses against the chosen antigen. However, in vitro experimentation has shown that many optimal MHC class I binding antigens, do not bind well to hsp70. In an attempt to optimize the binding of such antigenic peptides to hsp70, hybrid peptides engineered to contain an optimized hsp70 binding peptide (a “javelin” having a sequence Hy-x-Hy-x-Hy-x-Hy, where Hy corresponds to a hydrophobic amino acid and x corresponds to any amino acid, and more specifically His Trp Asp Phe Ala Trp Pro Trp; SEQ ID NO: 3), a linker (having the sequence GSG) and the antigenic peptide of choice, were synthesized. Such peptides bound well to hsp70 and when mice were immunized with these peptides bound to hsp70, the cellular immune responses generated were significantly stronger than those obtained from immunizations with un-javelinized versions of the antigenic peptides bound to hsp70 (International Patent Application No. PCT/US 96/13363, and Moroi Y. et al., (2000) Proc. Natl. Acad. Sci. 97:3485-90). From a product development point of view, however, the javelinization of short peptide antigens may be limited by the knowledge of which specific antigens to use. Further, it may be desirable, for therapeutic purposes, to produce an immune response toward more than one antigenic peptide, either in order to induce immunity of sufficient magnitude to eliminate a diseased cell or pathogen, or because different individuals may, by virtue of their major histocompatibility phenotype, be more or less responsive to particular antigens.  
       3. SUMMARY OF THE INVENTION  
       [0013] The present invention provides for an antigenic complex comprising a plurality of epitopes non-covalently bound to a heat shock protein via a javelin sequence. The plurality of epitopes are covalently bound to the javelin, optionally via a linker sequence. In certain embodiments, the epitopes are comprised in a larger protein, and may occur naturally in the context of said protein. As such, the characterization of particular epitopes within the protein is not required to practice the invention. In alternative embodiments, epitopes which may not occur together in nature are bound to a single javelin, optionally via a linker sequence, thereby providing a cocktail of antigens which, when non-covalently associated with heat shock protein in an antigenic complex, may be used to produce a therapeutic immune response in a subject.  
       [0014] Accordingly, the present invention overcomes a number of the limitations of the prior art. For example, specific knowledge of the T cell epitope (MHC class I binding peptide) is not required, and a specific product is not restricted to patients with a certain HLA haplotype as a larger protein will contain many T cell epitopes that can bind to the various HLA types. Furthermore, the same protein may contain MHC class II epitopes that can also be processed by antigen presenting cells to generate a helper T cell response as well as an antibody response. Yet another advantage of the present invention is that it may be used to identify antigens restricted to either MHC class I or class II, in that a javelin may deliver a protein to a cell, resulting in the binding of peptide to MHC components, and the bound peptide(s) may be eluted and sequenced. 
     
    
    
     4. BRIEF DESCRIPTION OF THE FIGURES  
     [0015] FIGS.  1 A-D. Various embodiments of the invention. A. A protein comprising a plurality of epitopes (represented by a square, triangle, and circle) is covalently joined to a javelin molecule by a chemical linker (L). B. A plurality of epitopes originating from the same protein, comprised in isolated peptides, are covalently joined to a javelin by chemical linkers (L). C. A plurality of proteins (designated as an open shape and a cross-hatched shape, respectively) comprising a plurality of epitopes (represented by an open square, an open triangle, an open circle, a cross-hatched diamond, and a cross-hatched doughnut), are covalently joined to a javelin molecule by a chemical linker (L). D. A plurality of epitopes, as designated in (C), comprised in isolated peptides, are covalently joined to a javelin by chemical linkers. Where epitopes are comprised in isolated peptides, the peptides are designated by wavy lines.  
     [0016]FIG. 2. The protein sequence of ovalbumin (SEQ ID NO: 4). The bolded amino acids correspond to those of the domain 200-291. The major histocompatibility complex (“MHC”) class I epitope, SIINFEKL(SEQ ID NO: 5), is underlined and the MHC class II epitope, TEWTSSNVMEERKIKV(SEQ ID NO: 6), is double-underlined.  
     [0017]FIG. 3. The structure of ovalbumin. Ova200-291 is shown in blue and approximately circled, and SIINFEKL (SEQ ID NO: 5)is shown in red and approximately enclosed in a box.  
     [0018]FIG. 4. The nucleotide sequence of the ovalbumin cDNA(SEQ ID NO: 7). The ATG start codon is bolded as is the termination codon. Underlined are the 5′ and 3′ regions of the sequence that code for the OVA 200-291 domain.  
     [0019] FIGS.  5 A-D. Tumor growth curves for mice immunized with TiterMax and buffer (group A), TiterMax and SIINFEKL peptide (SEQ ID NO: 5; group B), Javelin-Ova200-291-Javelin alone (group C), and Javelin-Ova200-291-Javelin bound to mouse hsp70 (group D). 
    
    
     5. DETAILED DESCRIPTION OF THE INVENTION  
     [0020] The present invention provides for an antigenic complex comprising a plurality of epitopes non-covalently bound to a heat shock protein via a javelin sequence. The plurality of epitopes are covalently bound to the javelin, optionally via a linker sequence.  
     [0021] In a first set of embodiments (FIG. 1A and FIG. 1B), the present invention provides for an antigenic complex comprising a plurality of epitopes, non-covalently joined to a heat shock protein by a tethering molecule (“javelin”) having affinity for the heat shock protein, wherein the epitopes are covalently joined to the tethering peptide and wherein the epitopes are derived from a single antigenic protein. The epitopes may be comprised in the protein (FIG. 1A) or may be comprised in isolated peptides (FIG. 1B), and joined to the javelin via a chemical linker, which may be a covalent bond or may comprise one or more atoms (e.g. a peptide bond or a peptide linker).  
     [0022] In a second set of embodiments, the present invention provides for an antigenic complex comprising a plurality of epitopes, non-covalently joined to a heat shock protein by a tethering molecule (“javelin”) having affinity for the heat shock protein, wherein the epitopes are covalently joined to the tethering peptide and wherein the epitopes are derived from more than one antigenic protein. The epitopes may be comprised in the proteins (FIG. 1C) or may be comprised in isolated peptides (FIG. 1D), and joined to the javelin via a chemical linker, which may be a covalent bond or may comprise one or more atoms (e.g. a peptide bond or a peptide linker).  
     [0023] In a third set of embodiments, the present invention provides for an antigenic complex comprising a plurality of epitopes, non-covalently joined to a heat shock protein by a tethering molecule (“javelin”) having affinity for the heat shock protein under physiologic conditions, wherein the epitopes are covalently joined to the tethering peptide and wherein one epitope is a Class I epitope and the other epitope is a Class II epitope. For example, in FIG. 1A and FIG. 1B, the open square could represent a Class I epitope and the open circle could be a Class II epitope, and in FIG. 1C and FIG. 1D, the open square could represent a Class I epitope and the cross-hatched doughnut could represent a Class II epitope (it should be understood, however, that the MHC restrictions of the representative epitopes are only specified by way of explanation for this third set of embodiments and do not necessarily apply to all embodiments).  
     [0024] In a fourth set of embodiments, the present invention provides for one or more epitope, as comprised in an antigenic peptide, non-covalently joined to a heat shock protein by a plurality of tethering molecules (“javelins”), wherein the epitope or epitopes are covalently joined to the tethering molecule. The javelins may have the same or different chemcial structures (e.g. may have different peptide sequences).  
     [0025] As used herein, the term “heat shock protein” or “hsp” refers to any protein that has the capability to bind peptides or proteins and whose intracellular concentration increases (i.e., is “inducible”) when the cell is stressed, including non-inducible homologs of such proteins. Examples of heat shock proteins include but are not limited to gp96(grp94), hsp90, BiP, hsp70, hsp60, hsp40, hsc70, calnexin, calreticulin and hs10. Heat shock protein for use according to the invention may be prepared from a natural source, expressed recombinantly, or chemically synthesized.  
     [0026] The term “javelin” as used herein refers to a peptide or non-peptide sequence which non-covalently binds to heat shock protein under physiologic conditions. For example, but not by way of limitation, physiological conditions would include temperatures of 4-55° C., and preferably 20-40° C.; a pH of 3-12, and preferably 5-8; and ionic strengths approximating the ionic strength of 50-300 mM NaCl, and preferably 100-200 mM NaCl. A specific, nonlimiting example of physiologic conditions includes phosphate buffered saline (13 M NaH 2 PO 4 , 137 mM NaCl, pH 7.4) at 37° C. Such javelins may have amino acid compositions which comprise a substantial proportion of hydrophobic amino acids such as phenylalanine and tryptophan, and/or a substantial number of serine, threonine, or proline residues. In particular, nonlimiting embodiments, javelins of the invention may comprise amino acid sequences which have the general description hydrophobic-x-hydrophobic-x-hydrophobic-x-hydrophobic, where “hydrophobic” denotes a hydrophobic amino acid and x denotes any amino acid; more particularly, such javelins may have the sequence hydrophobic-basic-hydrophobic-hydrophobic-hydrophobic; Ser/Thr-hydrophobic-hydrophobic-Ser/Thr; Ser/Thr-Ser/Thr-hydrophobic-hydrophobic-Ser/Thr-Ser/Thr; and Ser/Thr-Ser/Thr-hydrophobic-hydrophobic-hydrophobic. Alternatively, javelins may comprise heat shock binding peptides as described in Blond-Elguindi et al., 1993, Cell 75:717-728, including the consensus sequence hydrophobic-(Trp/X)-hydrophobic-X-hydrophobic-X-hydrophobic and the specific peptides His Trp Asp Phe Ala Trp Pro Trp (SEQ ID NO: 3) and Phe Trp Gly Leu Trp Pro Trp Glu (SEQ ID NO: 8); Auger et al., 1996, Nature Med. 2:306-310, including Gln Lys Arg Ala Ala (SEQ ID NO: 9) and Arg Arg Arg Ala Ala (SEQ ID NO: 10); Flynn et al., 1989, Science 245:385-390; Gragerov et al., 1994, J. Mol. Biol. 235:848-854; Terlecky et al., 1992, J. Biol. Chem. 26:9202-9202, Lys Phe Glu Arg Gln (SEQ ID NO: 11); and Nieland et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:6135-6139, including the VSV8 peptide, Arg Gly Tyr Val Tyr Gln Gly Leu (SEQ ID NO: 12). In preferred embodiments, javelins of the invention may have a length of 4-50 amino acid residues, and more preferably 7-20 amino acid residues.  
     [0027] An epitope is defined as a molecule or region of a molecule against which an immune response is raised (cellular and/or antibody). In the case of peptidic epitopes, the epitope could constitute an MHC Class I binding peptide or an MHC Class II binding peptide ranging in size from 6 to 20 amino acids. Epitopes of the invention may be derived from virus proteins, bacterial proteins, protozoan proteins, fungal proteins, parasite derived proteins, intracellular pathogen derived proteins and proteins from diseased cells such as those derived from malignant tissue. According to the invention, an epitope need not be identified or characterized; its functional presence alone is required. Therefore, for example and not by way of limitation, a protein which is known to comprise a plurality of epitopes by virtue of the immune responses it induces may be javelinized according to the invention regardless of whether the peptide sequences or other characteristics of those epitopes are known.  
     [0028] A plurality of said epitopes may be comprised in a larger protein, which is in turn covalently linked to a javelin, optionally via a linker sequence. Epitopes, which may be comprised in larger molecules (such as larger peptides), may be covalently linked to javelin either in series (i.e., as part of a linear peptide molecule) or some epitopes may be linked to the javelin in parallel (i.e., via an amino acid side chain). Said plurality of epitopes may occur naturally in the same protein, or may occur in different proteins. For example, epitopes of proteins derived from a plurality of genetic variants of a virus may be linked to a javelin and incorporated into a heat shock protein complex of the invention.  
     [0029] In particular non-limiting embodiments of the invention, a protein antigen comprising a plurality of epitopes may have greater than 20 amino acids and may have one or more natural or heteroclitic MHC class I and/or MHC class II binding peptides, and may incorporate any additional immunogenic sequences including, but not limited to antibody recognition sites. Such a protein could constitute a naturally occurring protein or it could constitute a synthetic protein generated to contain one or more copies of MHC class I and/or MHC class II binding peptides.  
     [0030] The javelinization of epitopes can be carried out in a number of ways. For example, but not by way of limitation, a javelin can be chemically or photochemically crosslinked to one or more epitopes, or may be produced by genetic engineering techniques.  
     [0031] The molecule comprising the one or more epitopes is referred to herein as the “antigen”, such that the antigen is linked to a javelin.  
     [0032] Between a javelin and the antigen a linker molecule may be used. If this linker is peptidic, it could correspond to but is not limited to a 1-10 amino acid sequence. Such a linker could have the sequence but is not limited to GSG, GGSGG (SEQ ID NO: 13), GGPGG (SEQ ID NO: 14), SGPGS (SEQ ID NO: 15).  
     [0033] Antigens can be attached (by any of the methods described above) to one or more javelins. The javelin(s) can be placed at any point on a antigenic surface. In the case of peptidic antigens and a peptidic javelin, the javelin can be at the amino-terminus of the protein, the carboxyl terminus of the protein, or one or more javelins can be introduced at any point within the amino acid sequence of the protein antigen or any combination of the above.  
     [0034] Complexes between javelinized antigens and hsps and/or molecular chaperones can be generated by many methods. Javelinized antigens can be mixed with the hsps and/or molecular chaperones at molar ratios varying from but not limited to 0.01:1 to 100:1, although more preferably in molar ratios of 0.1:1 to 10:1. These mixtures are made in an aqueous solution that is buffered in the range between pH 4.5and pH 9 and more preferably in the range pH 5.5 to pH 8. The buffering compounds could include but are not limited to Tris base, phosphate based buffers, bicarbonate based buffers, succinate based buffers. The concentrations of these buffering compounds range from but is not limited to 1 mM to 500 mM, and more preferably range from 10 mM to 200 mM. Salts may also be added to the solution. These salts include but are not restricted to sodium chloride, potassium chloride, ammonium chloride, ammonium sulfate, magnesium chloride, magnesium acetate, potassium acetate, sodium acetate. The concentrations of these salts may fall in the range, but are not limited to, 1 mM to 500 mM, more preferably 20 mM to 200 mM. The formation of complexes between Javelinized antigens and hsps and/or molecular chaperones may also involve the addition of one or more salt to the complex formation solution. Other compound, not always referred to as salts may also be included in the complex formation solution. Such compounds may include but are not limited to adenosine 5′ diphosphate (ADP) and analogues thereof, adenosine 5′ triphosphate (ATP) and analogues thereof and DMSO. Such compounds may be added at concentrations ranging from but not limited to 0.001 mM to 500 mM, more preferably 0.1 mM to 100 mM.  
     [0035] An example of a complex formation solution is as follows, but is not in any way limiting:  
     [0036] hsp70 0.25 mg/ml  
     [0037] Javelinized antigen peptide at either 0.25 mg/ml in a buffer comprising:  
     [0038] 25 mM Tris (Tris) pH 8.0  
     [0039] 50 mM NaCl  
     [0040] 5 mM MgCl 2    
     [0041] 6.7 mM Acetate  
     [0042] 1 mM ADP  
     [0043] 0.26 mM KCl  
     [0044] 0.518 mM Na 2 HPO 4    
     [0045] 0.173 mM KH 2 PO 4    
     [0046] Final DMSO concentration 1%.  
     [0047] The complex formation reaction should then be incubated at a temperature ranging from, but not limited to 4° C. to 65° C., more preferably from 20° C. to 55° C. This incubation will be carried out for a time period ranging from, but not limited to 1 minute to 4 hours, more preferably from 20 minutes to 1 hour.  
     [0048] Immunizations of javelinized antigens bound to an hsp can be carried out in numerous ways. Immunization can be carried out using a single javelinized antigen bound to a heat shock protein or a plurality of javelinized antigens may be bound to a heat shock protein. Alternatively, a single javelinized antigen can be bound to numerous heat shock proteins or a plurality of javelinized antigens can be bound to a plurality of heat shock proteins. Additionally, one or more antigen can be variously javelinized and bound to a single or a plurality of heat shock proteins. Immunization can be carried out by methods including, but not limited to intradermal injection, subcutaneous injection, intraperitoneal injection and intramuscular injection. Immunization may also involve the treatment of patient derived antigen presenting cells with javelinized antigens bound to heat shock protein or molecular chaperone in vitro, followed by readministration of the antigen presenting cells into the patient.  
     [0049] Administration of javelinized antigen(s) bound to heat shock protein(s) may induce either a killer T cell response or a helper T cell response or an antibody response. More preferably, such an administration will induce both a helper T cell and killer T cell response or both a helper T cell and antibody response or both a killer T cell response and an antibody response. Even more preferably, such an administration will induce a killer T cell response, a helper T cell response and an antibody response.  
     [0050] Examples of javelinized antigens include but are not restricted to:  
     [0051] MHC Class I peptide antigen derived from ovalbumin containing one or two javelins, such as:  
                              (SEQ ID NO:16)                               SIINFEKL   GSG   HWDFAWPW ;                                     (SEQ ID NO:17)                               HWDFAWPW   GSG   SIINFEKL ; and                                     (SEQ ID NO:18)                               HWDFAWPW   GSG   SIINFEKL GSGHWDFAWPW              
 
     [0052] MHC Class II peptide antigen derived from ovalbumin containing one or two javelins, such as:  
                              (SEQ ID NQ:19)                                       HWDFAWPW   GSG   TEWTSSNVMEERKIKV ;                                         (SEQ ID NO:20)                                       TEWTSSNVMEERKIKV   GSG   HWDFAWPW ; and                                         (SEQ ID NO:21)                                       HWDFAWPW   GSG   TEWTSSNVMEERKIKV   GSG   HWDFAWPW ;              
 
     [0053] ID NO: 21);  
     [0054] MHC Class I peptide antigen derived from herpes simplex virus containing one or two javelins, such as:  
                                            HWDFAWPW   GSG   SSIEFARL;     (SEQ ID NO:22)                             SSIEFARL   GSG   HWDFAWPW ; and   (SEQ ID NO:23)                         HWDFAWPW   GSG   SSIEFARL   GSG   HWDFAWPW ;   (SEQ ID NO:24)          
 
     [0055] according to the present invention, a second epitope-containing peptide or a plurality of epitope-containing peptides, originating from the same herpes simplex protein or a different protein, may be linked to the javelin molecule(s) in peptides SEQ ID NOS: 22-24.  
     [0056] MHC class I mutant peptide antigen derived from gp100 containing one or two javelins, such as:  
                                HWDFAWPW   GSG   IMDQVPFSV ;   (SEQ ID NO:25)                     IMDQVPFSV   GSG   HWDFAWPW ; and   (SEQ ID NO:26);                 HWDFAWPW   GSG   IMDQVPFSV   GSG   HWDFAWPW ;   (SEQ ID NO:27)          
 
     [0057] according to the present invention, a second epitope-containing peptide or a plurality of epitope-containing peptides, originating from the same gp100 protein or a different protein, may be linked to the javelin molecule(s) in peptides SEQ ID NOS: 25-27.  
     [0058] and  
     [0059] Ovalbumin derived protein domain containing both an MHC class I and MHC class II epitope and one javelin with or without the linker GSG, such as:  
                              (SEQ ID NO:28)                               HWDFAWPW VTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMS           MLVLLPDEVSGLEQLE SIINFEKL   TEWTSSNVMEERKIKV YLPRMKMEE       KY;                                 (SEQ ID NO:29)                               HWDFAWPW   GSG VTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASG           TMSMLVLLPDEVSGLEQLE SIINFEKL   TEWTSSNVMEERKIKV YLPRMK       MEEKY;                                 (SEQ ID NO:30)                             VTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDE           VSGLEQLE SIINFEKL   TEWTSSNVMEERKIKV YLPRMKMEEKY HWDFAW           PW ;                                 (SEQ ID NO:31)                             VTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDE           VSGLEQLE SIINFEKL   TEWTSSNVMEERKIKV YLPRMKMEEKY GSG   HWD           FAWPW ;                                 (SEQ ID NO:32)                               HWDFAWPWV TEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMS           MLVLLPDEVSGLEQLE SIINFEKL   TEWTSSNVMEERKIKV YLPRMKMEE       KY HWDFAWPW ; and                                 (SEQ ID NO:33)                               HWDFAWPW   GSG VTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASG           TMSMLVLLPDEVSGLEQLE SIINFEKL   TEWTSSNVMEERKIK VYLPRMK       MEEKY HWDFAWPW ;          
 
     [0060] where MHC class I epitopes are singly underlined, MHC class II epitopes are doubly underlined, the Javelin sequence is bolded and the linker is italicized.  
     6. EXAMPLE  
     [0061] Induction of a Protective Immune Response using a Javelinized Portion of Ovalbumin Containing both Class I and Class II Epitopes  
     [0062] Good CTL responses have been generated when mice were immunized with a javelinized version of SIINFEKL (SEQ ID NO: 5; a peptide located from residues 257-264 in the ovalbumin protein) bound to hsp70 (see PCT/US 96/13363, and Moroi Y. et. al., (2000) Proc Natl. Acad. Sci. 97:3485-90). Thus, in the experiments described herein, the ovalbumin system was used to test the javelinization of large proteins. Ovalbumin is a ˜42.9 kDa protein with 386 amino acids that is secreted as a disulfide bonded molecule. Thus it is not very convenient to express this protein in  E. coli . Furthermore, there have been no literature reports of soluble domains or fragments of ovalbumin that have been expressed. Therefore, in these experiments, a small 92 amino acid domain of ovalbumin (amino acids 200-291) was used, that contains the SIINFEKL (SEQ ID NO: 5) epitope from our Javelinization studies as well as an MHC class II peptide TEWTSSNVMEERKIKV (SEQ ID NO: 6) corresponding to residues 265-280 (Maecker H. T. et al., (1998) J. Immunol. 161:6532-6) The protein sequence of ovalbumin is shown in FIG. 2.  
     [0063] The bolded amino acids correspond to those of the domain 200-291. The MHC class I epitope, SIINFEKL (SEQ ID NO: 5), is underlined and the MHC class II epitope, TEWTSSNVMEERKIKV (SEQ ID NO: 6), is double underlined. This domain was chosen based on the fact that it contains both an MHC class I and II epitope, but also because structurally, the domain was considered to be relatively compact. The relatively compact nature of this domain may make it easier to work with (i.e. the domain would be relatively stable and be less susceptible to proteolysis or rapid aggregation even at low concentration). The structure of this domain in the context of the whole ovalbumin protein is shown in FIG. 3.  
     [0064] Manufacture of the Javelinized Ovalbumin Domains:  
     [0065] The nucleotide sequence of the ovalbumin mRNA is shown in FIG. 4. The ATG start codon is bolded as is the termination codon. Underlined are the 5′ and 3′ regions of the sequence that code for the OVA 200-291 domain. For your orientation, the first 9 bases of the 5′ sequence GTGACTGAG (SEQ ID NO: 34) codes for V-T-E and the last 9 bases of the 3′ sequence GAAAAATAC (SEQ ID NO: 35) codes for E-K-Y.  
     [0066] The following oligonucleotides were synthesized:  
                          (SEQ ID NO:36)                         5′ AACCCC ATG TGACTGAGCAAGAAAGC 3′;                                 (SEQ ID NO:37)                         5′ GCAAGGATCC TTA GTATTTTTCCTCC 3′;                                 (SEQ ID NO:38)                         5′ GGAATTCCATATG CACTGGGACTTCGCGTGGCCGTGG GTGACTGAGC           AAGAAAGCAA 3′; and                             (SEQ ID NO:39)                         5′ GGAGGATCCTTA CCACGGCCACGCGAAGTCCCAGTG GTATTTTTCCT           CCATCTTCATGCGA 3′.          
 
     [0067] These oligonucleotides have the following features:  
     [0068] 1. Forward oligo coding for an NcoI site (CCATGG; SEQ ID NO: 40), the start codon ( ATG ) reading straight into the ovalbumin sequence (i.e. no bases coding for Javelin added);  
     [0069] 2. Reverse oligo coding for a Bam HI site (GGATCC; SEQ ID NO: 41), a stop codon ( TTA ) immediately following ovalbumin sequence (remember this is a reverse complementary oligonucleotide);  
     [0070] 3. Forward oligo coding for an NdeI site (CATATG; SEQ ID NO: 42), a start codon (ATG), 24 bases that code for the sequence of the Javelin ( CACTGGGACTTCGCGTGGCCGTGG : (SEQ ID NO: 43) followed by ovalbumin sequence; and  
     [0071] 4. Reverse oligo coding for a Bam HI site (GGATCC;SEQ ID NO: 44), a stop codon (TTA) which follows the sequence coding for the Javelin ( CCACGGCCACGCGSAGTCCCAGTG (SEQ ID NO: 45). This immediately following ovalbumin sequence (remember this is a reverse complementary oligonucleotide).  
     [0072] Thus, these oligo&#39;s were used to generate, using the polymerase chain reaction, sequences corresponding to:  
     [0073] Ova 200-291 (no Javelinization)  
     [0074] Javelin-Ova 200-291 (One Javelin coding sequence at the 5′ end)  
     [0075] Ova200-291-Javelin (One Javelin coding sequence at the 3′ end)  
     [0076] Javelin-Ova200-291-Javelin (A Javelin coding sequence at both the 5′ and the 3′ end)  
     [0077] These sequences were subsequently cloned into pET vectors. The sequences corresponding to Ova 200-291 and Ova200-291-Javelin were cut with the restriction enzymes NcoI and Bam HI according to the enzyme manufacturers (New England Biolabs) instructions and cloned into pET28 (which had been similarly cut). The sequences corresponding to Jav-Ova and Jav-OVA-Jav were cut with NdeI and Bam HI according to the manufacturers instructions and cloned into pET27 (which had been cut with the same enzymes). The ligated vectors were transformed into the  E.coli  cell line HMS174. The resulting vectors were sequenced to confirm that no mutations had been introduced as a result of handling. All sequences were correct.  
     [0078] Expression and Purification of Ova200-291, Javelin-Ova200-291, Ova200-291-Javelin and Javelin-Ova200-291-Javelin:  
     [0079] This procedure is identical for the expression and purification of all of these proteins. A colony of HMS174 containing the vector of interest is picked and grown in LB medium supplemented with 30 g/ml of kanamycin. After overnight culture, the cells are spun out and resuspended in 5 ml of fresh LB medium. 1 ml of the resuspended cells is then used to innoculate a liter of LB medium which has been supplemented with 30 g/ml kanamycin. The cells are grown at 37° C. until the optical density at 600 nm is between 0.4-0.8 but preferably 0.6. At this point the culture is supplemented with IPTG at a final concentration of 1 mM. The culture is grown for an additional 3 hours and the cells spun down. Cell pellet can be stored frozen at this point.  
     [0080] The proteins are all found in inclusion bodies. Thus an inclusion body preparation is carried out. The cell lysis reagent Bugbuster™ sold by Novagen is used according to the manufacturers recommendation. Briefly, 5 ml of the Bugbuster™ reagent per gram of cell paste (pellet) is used to resuspend the pellet. 25 units of Benzonase (Novagen) is added for every ml of Bugbuster™ added. The cells are incubated at room temperature with gentle rotation for 10-20 minutes. The suspension is spun at 16000×g for 20 minutes at 4° C. and the pellet kept. The pellet is then resuspended in the same volume of Bugbuster™ as the original cell pellet was. Lysozyme is added to 200 g/ml, the sample vortexed and incubated for a further 5 minutes. 6 volumes of 1:10 fold diluted Bugbuster™ are then added and the suspension vortexed once again. The pellet is once again harvested by centrifugation (as above). The pellet is then resuspended in 20-30 ml of 1:10 fold diluted Bugbuster™ per liter of culture. The suspension is vortexed, spun and the pellet wash repeated 2 more times. The final pellet, containing purified inclusion bodies is dissolved in 50 mM Mops, pH6.5, 8M Urea, 0.1 mM DTT, 0.1 mM EDTA. The pellet goes into solution slowly over the course of about 1 hour (rotating at room temperature). The volume of urea is added such that the final concentration of protein is between 20-50 mg/ml. For the doubly Javelinized Ova200-291, the protein was not very soluble at pH 6.5 so KOH was added until the protein went back into solution. The pH after adjustment was approximately 10.  
     [0081] Immunization Studies:  
     [0082] An immunization study was initiated using the doubly Javelinized Ova200-291. The following solutions were set up:  
     [0083] 100 μl TiterMax™ +100 μl buffer (50 mM NaCl, 20 mM Tris pH 8.0, 5 mM MgAc).  
     [0084] 100 μl TiterMax™ +100 μl buffer containing 100 μg SIINFEKL (SEQ ID NO: 5) peptide.  
     [0085] 1000 μl buffer containing 100 μg Javelin-Ova200-291-Javelin, 1 mM ADP.  
     [0086] 1000 μl buffer containing 300 μg mouse hsp70, 100 μg Javelin-Ova200-291-Javelin, 1 mM ADP.  
     [0087] In samples C and D the buffer containing the other compounds listed was added very rapidly (by pipeting) onto the peptide (which is 8M urea). The TiterMax™ samples were vortexed for 30 minutes prior to immunization and the aqueous samples were incubated for a minimum of 30 minutes before immunization (The exact time may be more due to the time between formulating the solutions and actually administering the immunization).  
     [0088] 10 mice were each immunized with one of the above mixtures. Thus 40 mice were immunized in total. For the TiterMax™ samples mice were immunized with 10 μl intradermally while for the aqueous preparations, each mouse was immunized with 50 μl intradermally. Thus mice in the following groups received the following moles of active ingredients:  
     [0089] Group A: no peptide, no hsp7O  
     [0090] Group B: 5000 pmoles SIINFEKL (SEQ ID NO:S), no hsp70  
     [0091] Group C: 365 pmoles Javelin-OVA200-291-Javelin, no hsp70  
     [0092] Group D: 365 pmoles Javelin-OVA200-291-Javelin, 214 pmoles mouse hsp70  
     [0093] After 7 days, the mice were each challenged with 1×10 6  EG7 cells intradermally. EG7 is a tumor cell line (derived from EL4) that has been stably transfected with the ovalbumin gene. Thus, if our immunizations result in the development of good immune responses to ovalbumin, the mice should be able to clear the tumor. Tumor growth measurements are then carried out at regular 2 day intervals. The responses observed for this experiment can be seen in FIG. 5 (note: two mice from the Hsp70: Javelin-Ova200-291-Javelin group (group D) died during anesthesia, hence only 8 mice are represented in the graph). These data clearly indicate that while all the mice immunized with TiterMax™ +buffer (group A) succumbed to the tumors, the mice immunized with Javelin-OVA200-291-Javelin bound to hsp70 (group D) had either no tumor ({fraction (6/8)}) or had prolonged times to onset of disease ({fraction (2/8)}). Mice immunized with TiterMax™ +SIINFEKL (SEQ ID NO: 5) peptide (group B) also, as expected, resisted disease well, while mice immunized only with Javelin-Ova200-291-Javelin (group C) had slightly increased survival times, but the response was not as significant in the group with hsp70. This clearly indicates the potential of this therapeutic approach.  
     7. EXAMPLE  
     [0094] Expression of Peptides or Proteins in Mammalian Cells  
     [0095] The foregoing example section utlized a bacterial system to express a protein for use according to the invention. However, typically expression of mammalian proteins in bacterial expression systems can be problematic in view of the lack of various modification systems (e.g. for glycosylation) that are lacking in bacterial cells. It therefore may be preferable to use a mammalian system to express a peptide or protein for use according to the invention. The following is a specific, non-limiting example of how a mammalian expression system may be used to produce a javelinized ovalbumin protein.  
     [0096] To create an expressible vector encoding the Javelinized and non-Javelinized Ova domains above in a mammalian cell, a Hind III may be created at the 5′ end of the Nco I restriction sites of the Nco I-BamH I fragments encoding Ova 200-291 and Ova200-291-Jav by PCR cloning from the bacterial expression system described in the section above. Replacement with a Hind III site will facilitate cloning into most mammalian expression vector systems such as pCDNA3.1 (In-Vitrogen) using standard techniques in Molecular Biology.(Molecular Cloning, Sambrook). Similarly, a Hind III site may be added to 5′end of the Nde I restriction sites of the Nde I-Bam-HI fragments encoding Jav-Ova and Jav-Ova-Jav.  
     [0097] 5′ primers which may be used are:  
     [0098] 5′ CCCAAGCTTGGGCCATGGTGACTGAGCAAGAAAGC 3′  (SEQ ID NO: 46; addition of Hind III restriction site at the 5′ end of the Nco I site of primer 1 (supra)  
     [0099] 5′ CCCAAGCTTGGGCATATGCACTGGGACTTCGCGTGGCCGTGGGTGACTG AGCAAGAAAGCAA 3′ (SEQ ID NO; 47; addition of Hind III restriction site at the 5′ end of the Nde I site of primer 3 (supra)  
     [0100] Plasmids encoding Ova200-291, Javelin-Ova200-291, Ova200-291-Javelin and Javelin-Ova200-291-Javelin may each be transfected into a mammalian cell line such as CHO cells. Cells may be transfected with 2 μg mammalian expression vector encoding Ova 200-291, Ova200-291-Jav, Jav-Ova and Jav-Ova-Jav using methods well known in the art such as Lipofectamine (Gibco BRL) according to the manufacturer&#39;s directions. Briefly, the expression vector and 6 μL of Lipofectamine may be diluted separately in 100 μL serum-free medium (OPTI-MEM I Reduced Serum Medium, Gibco BRL). The two solutions may then be mixed and incubated at room temperature for 45 minutes to allow formation of DNA-liposome complexes. 800 μL OPTI-MEM may be added to the complexes, mixed, and overlaid onto rinsed cells. After a 6-hour incubation at 37° C., 1 mL growth medium containing 20% FCS may be added. Fresh medium may be added to the cells 24 hours post-transfection.  
     [0101] Stable clones may be selected by adding 800 μg/mL Geneticin (Gibco BRL) to the cells 72 hour later. The selection medium may be changed every 3 days. Colonies of stably transfected cells may be expected to be seen after 10-14 days. Expression of the desired javelinized Ova proteins may be assayed for by radiolabeling. Newly synthesized Ova may be detectable by immunoprecipitation and gel electrophoresis. Alternately, expression of the Ova producing clones may be confirmed by fluorescent-labeling of the permeabilized fixed cells and analyzed by fluorescent activated cell sorter (FACS analysis). Since the Ova protein is a secretable protein, Brefeldin A (BFA) may be added to the cells to prevent transport and hence secretion of the Ova protein prior to fluorescent labeling.  
     [0102] Analogous procedures may be used to express other peptides or proteins for use according to the invention.  
     8. EXAMPLE  
     [0103] Determination of Immunogenicity of HSP/Javelin—Protein Complexes  
     [0104] The following is a protocol which may be used to determine the immunogenicity of hsp/javelinized protein complexes (HSP/Jav-Protein).  
     [0105] Purified javelinized protein may be complexed with recombinant hsp in vitro at molar ratios of 10:1 to 500:1, and preferably at molar ratio of 10:1 to 100:1. The mixture may be incubated in a salt containing buffer in the range of pH4.5 and pH9 and more preferably in the range of pH6 to pH8. Examples of buffering systems include Tris-based buffer, phosphate-based buffer, citrate based buffer, succinate based buffer, bicarbonate based buffer and Hepes based buffer. The concentrations may range from 1 to 500 mM and preferably range from 10 to 100 mM. The mixture may be incubated at 25° C. for 20-120 minutes. The resulting HSP/Jav-Protein complexes may be assayed for immunogenicity using cytotoxic T cell assay.  
     [0106] Mice may be immunized intra-dermally once with 10-50 μL of HSP/Jav-Protein complex. Ten days after immunization, the spleens may be removed and the lymphocytes may be cultured with restimulation in vitro by the addition of whole protein or taansfected cells expressing the javelinized protein. If a pathogen protein is javelinized, the stimulation cells can be inactivated pathogen infected cells. Inactivation can be achieved by irradiation at 3000 rads or by treatment with mitomycin C.  
     [0107] Cytotoxicity of spleen cells from vaccinated mice may be assayed with either protein-pulsed cells or target cells expressing the protein. CTL may be generated by culturing in vivo immunized spleen cells for 5-6 days at a concentration of 1-10×10 6  cells/mL in RPMI medium containing 10% FCS, penicillin-streptomycin and 2 mM glutamine, together with 1-5×10 4  gamma irradiated stimulator cells/mL. Target cells may be prepared by culturing cells for 1 h in the presence of 200 mCi  51 Cr (sodium chromate)/mL (NEN) in Tris-phosphate buffer, pH 7.4 at 37° C. After washing, 10 4 51 Cr-labeled target cells may be mixed with effector lymphocytes to yield several different Effector/Target (E/T) ratio and incubated for 4 h. The cells may be pelleted by centrifugation at 200×g for 5 minutes and the amount of  51 Cr released into the supernatant determined using a gamma counter. Percent specific lysis may be calculated as 100%×[(cpm released by CTL−cpm spontaneous release)/(cpm maximal release−cpm spontaneous release)]. Maximal release may be determined by addition of 1% Triton X-100. Spontaneous release by target cells in the absence of effector cells is typically less than 25% of themaximal release.  
     [0108] Various publications are cited herein, the contents of which are hereby incorporated by reference in their entireties.  
    
     
       
         1 
         
           
             47  
           
           
             1  
             4  
             PRT  
             Homo sapiens  
           
            1 

Lys Asp Glu Leu 
 1 

 
           
             2  
             4  
             PRT  
             Plasmodium falciparum  
           
            2 

Asn Ala Asn Pro 
 1 

 
           
             3  
             8  
             PRT  
             Aritifical Sequence  
             
               insert in M13 coliphage  
             
           
            3 

His Trp Asp Phe Ala Trp Pro Trp 
 1               5 

 
           
             4  
             386  
             PRT  
             Gallus gallus  
           
            4 

Met Gly Ser Ile Gly Ala Ala Ser Met Glu Phe Cys Phe Asp Val Phe 
 1               5                  10                  15 

Lys Glu Leu Lys Val His His Ala Asn Glu Asn Ile Phe Tyr Cys Pro 
            20                  25                  30 

Ile Ala Ile Met Ser Ala Leu Ala Met Val Tyr Leu Gly Ala Lys Asp 
        35                  40                  45 

Ser Thr Arg Thr Gln Ile Asn Lys Val Val Arg Phe Asp Lys Leu Pro 
    50                  55                  60 

Gly Phe Gly Asp Ser Ile Glu Ala Gln Cys Gly Thr Ser Val Asn Val 
65                  70                  75                  80 

His Ser Ser Leu Arg Asp Ile Leu Asn Gln Ile Thr Lys Pro Asn Asp 
                85                  90                  95 

Val Tyr Ser Phe Ser Leu Ala Ser Arg Leu Tyr Ala Glu Glu Arg Tyr 
            100                 105                 110 

Pro Ile Leu Pro Glu Tyr Leu Gln Cys Val Lys Glu Leu Tyr Arg Gly 
        115                 120                 125 

Gly Leu Glu Pro Ile Asn Phe Gln Thr Ala Ala Asp Gln Ala Arg Glu 
    130                 135                 140 

Leu Ile Asn Ser Trp Val Glu Ser Gln Thr Asn Gly Ile Ile Arg Asn 
145                 150                 155                 160 

Val Leu Gln Pro Ser Ser Val Asp Ser Gln Thr Ala Met Val Leu Val 
                165                 170                 175 

Asn Ala Ile Val Phe Lys Gly Leu Trp Glu Lys Thr Phe Lys Asp Glu 
            180                 185                 190 

Asp Thr Gln Ala Met Pro Phe Arg Val Thr Glu Gln Glu Ser Lys Pro 
        195                 200                 205 

Val Gln Met Met Tyr Gln Ile Gly Leu Phe Arg Val Ala Ser Met Ala 
    210                 215                 220 

Ser Glu Lys Met Lys Ile Leu Glu Leu Pro Phe Ala Ser Gly Thr Met 
225                 230                 235                 240 

Ser Met Leu Val Leu Leu Pro Asp Glu Val Ser Gly Leu Glu Gln Leu 
                245                 250                 255 

Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Trp Thr Ser Ser Asn 
            260                 265                 270 

Val Met Glu Glu Arg Lys Ile Lys Val Tyr Leu Pro Arg Met Lys Met 
        275                 280                 285 

Glu Glu Lys Tyr Asn Leu Thr Ser Val Leu Met Ala Met Gly Ile Thr 
    290                 295                 300 

Asp Val Phe Ser Ser Ser Ala Asn Leu Ser Gly Ile Ser Ser Ala Glu 
305                 310                 315                 320 

Ser Leu Lys Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn 
                325                 330                 335 

Glu Ala Gly Arg Glu Val Val Gly Ser Ala Glu Ala Gly Val Asp Ala 
            340                 345                 350 

Ala Ser Val Ser Glu Glu Phe Arg Ala Asp His Pro Phe Leu Phe Cys 
        355                 360                 365 

Ile Lys His Ile Ala Thr Asn Ala Val Leu Phe Phe Gly Arg Cys Val 
    370                 375                 380 

Ser Pro 
385 

 
           
             5  
             8  
             PRT  
             Gallus gallus  
           
            5 

Ser Ile Ile Asn Phe Glu Lys Leu 
 1               5 

 
           
             6  
             16  
             PRT  
             Gallus gallus  
           
            6 

Thr Glu Trp Thr Ser Ser Asn Val Met Glu Glu Arg Lys Ile Lys Val 
 1               5                  10                  15 

 
           
             7  
             1873  
             DNA  
             Gallus gallus  
           
            7 

gacatacagc tagaaagctg tattgccttt agcactcaag ctcaaaagac aactcagagt     60 

tcaccatggg ctccatcggc gcagcaagca tggaattttg ttttgatgta ttcaaggagc    120 

tcaaagtcca ccatgccaat gagaacatct tctactgccc cattgccatc atgtcagctc    180 

tagccatggt atacctgggt gcaaaagaca gcaccaggac acagataaat aaggttgttc    240 

gctttgataa acttccagga ttcggagaca gtattgaagc tcagtgtggc acatctgtaa    300 

acgttcactc ttcacttaga gacatcctca accaaatcac caaaccaaat gatgtttatt    360 

cgttcagcct tgccagtaga ctttatgctg aagagagata cccaatcctg ccagaatact    420 

tgcagtgtgt gaaggaactg tatagaggag gcttggaacc tatcaacttt caaacagctg    480 

cagatcaagc cagagagctc atcaattcct gggtagaaag tcagacaaat ggaattatca    540 

gaaatgtcct tcagccaagc tccgtggatt ctcaaactgc aatggttctg gttaatgcca    600 

ttgtcttcaa aggactgtgg gagaaaacat ttaaggatga agacacacaa gcaatgcctt    660 

tcagagtgac tgagcaagaa agcaaacctg tgcagatgat gtaccagatt ggtttattta    720 

gagtggcatc aatggcttct gagaaaatga agatcctgga gcttccattt gccagtggga    780 

caatgagcat gttggtgctg ttgcctgatg aagtctcagg ccttgagcag cttgagagta    840 

taatcaactt tgaaaaactg actgaatgga ccagttctaa tgttatggaa gagaggaaga    900 

tcaaagtgta cttacctcgc atgaagatgg aggaaaaata caacctcaca tctgtcttaa    960 

tggctatggg cattactgac gtgtttagct cttcagccaa tctgtctggc atctcctcag   1020 

cagagagcct gaagatatct caagctgtcc atgcagcaca tgcagaaatc aatgaagcag   1080 

gcagagaggt ggtagggtca gcagaggctg gagtggatgc tgcaagcgtc tctgaagaat   1140 

ttagggctga ccatccattc ctcttctgta tcaagcacat cgcaaccaac gccgttctct   1200 

tctttggcag atgtgtttcc ccttaaaaag aagaaagctg aaaaactctg tcccttccaa   1260 

caagacccag agcactgtag tatcaggggt aaaatgaaaa gtatgttctc tgctgcatcc   1320 

agacttcata aaagctggag cttaatctag aaaaaaaatc agaaagaaat tacactgtga   1380 

gaacaggtgc aattcacttt tcctttacac agagtaatac tggtaactca tggatgaagg   1440 

cttaagggaa tgaaattgga ctcacagtac tgagtcatca cactgaaaaa tgcaacctga   1500 

tacatcagca gaaggtttat gggggaaaaa tgcagccttc caattaagcc agatatctgt   1560 

atgaccaagc tgctccagaa ttagtcactc aaaatctctc agattaaatt atcaactgtc   1620 

accaaccatt cctatgctga caaggcaatt gcttgttctc tgtgttcctg atactacaag   1680 

gctcttcctg acttcctaaa gatgcattat aaaaatctta taattcacat ttctccctaa   1740 

actttgactc aatcatggta tgttggcaaa tatggtatat tactattcaa attgttttcc   1800 

ttgtacccat atgtaatggg tcttgtgaat gtgctctttt gttcctttaa tcataataaa   1860 

aacatgttta agc                                                      1873 

 
           
             8  
             8  
             PRT  
             Aritifical Sequence  
             
               insert in M13 coliphage  
             
           
            8 

Phe Trp Gly Leu Trp Pro Trp Glu 
 1               5 

 
           
             9  
             5  
             PRT  
             Aritifical Sequence  
             
               insert in M13 coliphage  
             
           
            9 

Gln Lys Arg Ala Ala 
 1               5 

 
           
             10  
             5  
             PRT  
             Aritifical Sequence  
             
               insert in M13 coliphage  
             
           
            10 

Arg Arg Arg Ala Ala 
 1               5 

 
           
             11  
             5  
             PRT  
             vesicular stomatitis virus  
           
            11 

Lys Phe Glu Arg Gln 
 1               5 

 
           
             12  
             8  
             PRT  
             vesicular stomatitis virus  
           
            12 

Arg Gly Tyr Val Tyr Gln Gly Leu 
 1               5 

 
           
             13  
             5  
             PRT  
             Aritifical Sequence  
             
               synthetic peptide linker  
             
           
            13 

Gly Gly Ser Gly Gly 
 1               5 

 
           
             14  
             5  
             PRT  
             Aritifical Sequence  
             
               synthetic peptide linker  
             
           
            14 

Gly Gly Pro Gly Gly 
 1               5 

 
           
             15  
             5  
             PRT  
             Aritifical Sequence  
             
               synthetic peptide linker  
             
           
            15 

Ser Gly Pro Gly Ser 
 1               5 

 
           
             16  
             19  
             PRT  
             Aritifical Sequence  
             
               javelinized peptide from Gallus gallus 
      ovalbumin  
             
           
            16 

Ser Ile Ile Asn Phe Glu Lys Leu Gly Ser Gly His Trp Asp Phe Ala 
 1               5                  10                  15 

Trp Pro Trp 

 
           
             17  
             19  
             PRT  
             Aritifical Sequence  
             
               javelinized peptide of Gallus gallus ovalbumin  
             
           
            17 

His Trp Asp Phe Ala Trp Pro Trp Gly Ser Gly Ser Ile Ile Asn Phe 
 1               5                  10                  15 

Glu Lys Leu 

 
           
             18  
             30  
             PRT  
             Aritifical Sequence  
             
               javelinized peptide of Gallus gallus ovalbumin  
             
           
            18 

His Trp Asp Phe Ala Trp Pro Trp Gly Ser Gly Ser Ile Ile Asn Phe 
 1               5                  10                  15 

Glu Lys Leu Gly Ser Gly His Trp Asp Phe Ala Trp Pro Trp 
            20                  25                  30 

 
           
             19  
             27  
             PRT  
             Aritifical Sequence  
             
               javelinized peptide of Gallus gallus ovalbumin  
             
           
            19 

His Trp Asp Phe Ala Trp Pro Trp Gly Ser Gly Thr Glu Trp Thr Ser 
 1               5                  10                  15 

Ser Asn Val Met Glu Glu Arg Lys Ile Lys Val 
            20                  25 

 
           
             20  
             27  
             PRT  
             Aritifical Sequence  
             
               javelinized peptide of Gallus gallus ovalbumin  
             
           
            20 

Thr Glu Trp Thr Ser Ser Asn Val Met Glu Glu Arg Lys Ile Lys Val 
 1               5                  10                  15 

Gly Ser Gly His Trp Asp Phe Ala Trp Pro Trp 
            20                  25 

 
           
             21  
             38  
             PRT  
             Aritifical Sequence  
             
               javelinized peptide of Gallus gallus ovalbumin  
             
           
            21 

His Trp Asp Phe Ala Trp Pro Trp Gly Ser Gly Thr Glu Trp Thr Ser 
 1               5                  10                  15 

Ser Asn Val Met Glu Glu Arg Lys Ile Lys Val Gly Ser Gly His Trp 
            20                  25                  30 

Asp Phe Ala Trp Pro Trp 
        35 

 
           
             22  
             19  
             PRT  
             Aritifical Sequence  
             
               javelinized peptide of Herpes Simplex virus  
             
           
            22 

His Trp Asp Phe Ala Trp Pro Trp Gly Ser Gly Ser Ser Ile Glu Phe 
 1               5                  10                  15 

Ala Arg Leu 

 
           
             23  
             19  
             PRT  
             Aritifical Sequence  
             
               javelinized peptide of Herpes Simplex virus  
             
           
            23 

Ser Ser Ile Glu Phe Ala Arg Leu Gly Ser Gly His Trp Asp Phe Ala 
 1               5                  10                  15 

Trp Pro Trp 

 
           
             24  
             30  
             PRT  
             Aritifical Sequence  
             
               javelinized peptide of Herpes Simplex virus  
             
           
            24 

His Trp Asp Phe Ala Trp Pro Trp Gly Ser Gly Ser Ser Ile Glu Phe 
 1               5                  10                  15 

Ala Arg Leu Gly Ser Gly His Trp Asp Phe Ala Trp Pro Trp 
            20                  25                  30 

 
           
             25  
             20  
             PRT  
             Aritifical Sequence  
             
               javelinized peptide of gp100 human melanoma 
      antigen  
             
           
            25 

His Trp Asp Phe Ala Trp Pro Trp Gly Ser Gly Ile Met Asp Gln Val 
 1               5                  10                  15 

Pro Phe Ser Val 
            20 

 
           
             26  
             20  
             PRT  
             Aritifical Sequence  
             
               javelinized peptide of human gp100 melanoma 
      antigen  
             
           
            26 

Ile Met Asp Gln Val Pro Phe Ser Val Gly Ser Gly His Trp Asp Phe 
 1               5                  10                  15 

Ala Trp Pro Trp 
            20 

 
           
             27  
             31  
             PRT  
             Aritifical Sequence  
             
               javelinized peptide of gp100 melanoma antigen  
             
           
            27 

His Trp Asp Phe Ala Trp Pro Trp Gly Ser Gly Ile Met Asp Gln Val 
 1               5                  10                  15 

Pro Phe Ser Val Gly Ser Gly His Trp Asp Phe Ala Trp Pro Trp 
            20                  25                  30 

 
           
             28  
             100  
             PRT  
             Aritifical Sequence  
             
               javelinized peptide of Gallus gallus ovalbumin  
             
           
            28 

His Trp Asp Phe Ala Trp Pro Trp Val Thr Glu Gln Glu Ser Lys Pro 
 1               5                  10                  15 

Val Gln Met Met Tyr Gln Ile Gly Leu Phe Arg Val Ala Ser Met Ala 
            20                  25                  30 

Ser Glu Lys Met Lys Ile Leu Glu Leu Pro Phe Ala Ser Gly Thr Met 
        35                  40                  45 

Ser Met Leu Val Leu Leu Pro Asp Glu Val Ser Gly Leu Glu Gln Leu 
    50                  55                  60 

Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Trp Thr Ser Ser Asn 
65                  70                  75                  80 

Val Met Glu Glu Arg Lys Ile Lys Val Tyr Leu Pro Arg Met Lys Met 
                85                  90                  95 

Glu Glu Lys Tyr 
            100 

 
           
             29  
             103  
             PRT  
             Aritifical Sequence  
             
               javelinized peptide of Gallus gallus ovalbumin  
             
           
            29 

His Trp Asp Phe Ala Trp Pro Trp Gly Ser Gly Val Thr Glu Gln Glu 
 1               5                  10                  15 

Ser Lys Pro Val Gln Met Met Tyr Gln Ile Gly Leu Phe Arg Val Ala 
            20                  25                  30 

Ser Met Ala Ser Glu Lys Met Lys Ile Leu Glu Leu Pro Phe Ala Ser 
        35                  40                  45 

Gly Thr Met Ser Met Leu Val Leu Leu Pro Asp Glu Val Ser Gly Leu 
    50                  55                  60 

Glu Gln Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Trp Thr 
65                  70                  75                  80 

Ser Ser Asn Val Met Glu Glu Arg Lys Ile Lys Val Tyr Leu Pro Arg 
                85                  90                  95 

Met Lys Met Glu Glu Lys Tyr 
            100 

 
           
             30  
             100  
             PRT  
             Aritifical Sequence  
             
               javelinized peptide of Gallus gallus ovalbumin  
             
           
            30 

Val Thr Glu Gln Glu Ser Lys Pro Val Gln Met Met Tyr Gln Ile Gly 
 1               5                  10                  15 

Leu Phe Arg Val Ala Ser Met Ala Ser Glu Lys Met Lys Ile Leu Glu 
            20                  25                  30 

Leu Pro Phe Ala Ser Gly Thr Met Ser Met Leu Val Leu Leu Pro Asp 
        35                  40                  45 

Glu Val Ser Gly Leu Glu Gln Leu Glu Ser Ile Ile Asn Phe Glu Lys 
    50                  55                  60 

Leu Thr Glu Trp Thr Ser Ser Asn Val Met Glu Glu Arg Lys Ile Lys 
65                  70                  75                  80 

Val Tyr Leu Pro Arg Met Lys Met Glu Glu Lys Tyr His Trp Asp Phe 
                85                  90                  95 

Ala Trp Pro Trp 
            100 

 
           
             31  
             103  
             PRT  
             Aritifical Sequence  
             
               javelinized peptide of Gallus gallus ovalbumin  
             
           
            31 

Val Thr Glu Gln Glu Ser Lys Pro Val Gln Met Met Tyr Gln Ile Gly 
 1               5                  10                  15 

Leu Phe Arg Val Ala Ser Met Ala Ser Glu Lys Met Lys Ile Leu Glu 
            20                  25                  30 

Leu Pro Phe Ala Ser Gly Thr Met Ser Met Leu Val Leu Leu Pro Asp 
        35                  40                  45 

Glu Val Ser Gly Leu Glu Gln Leu Glu Ser Ile Ile Asn Phe Glu Lys 
    50                  55                  60 

Leu Thr Glu Trp Thr Ser Ser Asn Val Met Glu Glu Arg Lys Ile Lys 
65                  70                  75                  80 

Val Tyr Leu Pro Arg Met Lys Met Glu Glu Lys Tyr Gly Ser Gly His 
                85                  90                  95 

Trp Asp Phe Ala Trp Pro Trp 
            100 

 
           
             32  
             108  
             PRT  
             Aritifical Sequence  
             
               javelinized peptide of Gallus gallus ovalbumin  
             
           
            32 

His Trp Asp Phe Ala Trp Pro Trp Val Thr Glu Gln Glu Ser Lys Pro 
 1               5                  10                  15 

Val Gln Met Met Tyr Gln Ile Gly Leu Phe Arg Val Ala Ser Met Ala 
            20                  25                  30 

Ser Glu Lys Met Lys Ile Leu Glu Leu Pro Phe Ala Ser Gly Thr Met 
        35                  40                  45 

Ser Met Leu Val Leu Leu Pro Asp Glu Val Ser Gly Leu Glu Gln Leu 
    50                  55                  60 

Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Trp Thr Ser Ser Asn 
65                  70                  75                  80 

Val Met Glu Glu Arg Lys Ile Lys Val Tyr Leu Pro Arg Met Lys Met 
                85                  90                  95 

Glu Glu Lys Tyr His Trp Asp Phe Ala Trp Pro Trp 
            100                 105 

 
           
             33  
             111  
             PRT  
             Aritifical Sequence  
             
               javelinized peptide of Gallus gallus ovalbumin  
             
           
            33 

His Trp Asp Phe Ala Trp Pro Trp Gly Ser Gly Val Thr Glu Gln Glu 
 1               5                  10                  15 

Ser Lys Pro Val Gln Met Met Tyr Gln Ile Gly Leu Phe Arg Val Ala 
            20                  25                  30 

Ser Met Ala Ser Glu Lys Met Lys Ile Leu Glu Leu Pro Phe Ala Ser 
        35                  40                  45 

Gly Thr Met Ser Met Leu Val Leu Leu Pro Asp Glu Val Ser Gly Leu 
    50                  55                  60 

Glu Gln Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Trp Thr 
65                  70                  75                  80 

Ser Ser Asn Val Met Glu Glu Arg Lys Ile Lys Val Tyr Leu Pro Arg 
                85                  90                  95 

Met Lys Met Glu Glu Lys Tyr His Trp Asp Phe Ala Trp Pro Trp 
            100                 105                 110 

 
           
             34  
             9  
             DNA  
             Artificial Sequence  
             
               primer designed based on Gallus gallus 
      ovalbumin cDNA  
             
           
            34 

gtgactgag                                                              9 

 
           
             35  
             9  
             DNA  
             Artificial Sequence  
             
               primer based on Gallus gallus ovalbumin cDNA  
             
           
            35 

gaaaaatac                                                              9 

 
           
             36  
             27  
             DNA  
             Artificial Sequence  
             
               primer based on Gallus gallus ovalbumin cDNA  
             
           
            36 

aaccccatgg tgactgagca agaaagc                                         27 

 
           
             37  
             26  
             DNA  
             Artificial Sequence  
             
               primer based on Gallus gallus ovalbumin cDNA  
             
           
            37 

gcaaggatcc ttagtatttt tcctcc                                          26 

 
           
             38  
             57  
             DNA  
             Artificial Sequence  
             
               primer based on Gallus gallus ovalbumin cDNA  
             
           
            38 

ggaattccat atgcactggg acttcgcgtg gccgtgggtg actgagcaag aaagcaa        57 

 
           
             39  
             61  
             DNA  
             Artificial Sequence  
             
               primer based on Gallus gallus ovalbumin cDNA  
             
           
            39 

ggaggatcct taccacggcc acgcgaagtc ccagtggtat ttttcctcca tcttcatgcg     60 

a                                                                     61 

 
           
             40  
             6  
             DNA  
             Artificial Sequence  
             
               primer based on Gallus gallus ovalbumin cDNA  
             
           
            40 

ccatgg                                                                 6 

 
           
             41  
             6  
             DNA  
             Artificial Sequence  
             
               primer based on Gallus gallus ovalbumin cDNA  
             
           
            41 

ggatcc                                                                 6 

 
           
             42  
             6  
             DNA  
             Artificial Sequence  
             
               primer based on Gallus gallus ovalbumin cDNA  
             
           
            42 

catatg                                                                 6 

 
           
             43  
             24  
             DNA  
             Artificial Sequence  
             
               artificial sequence in M13 coliphage  
             
           
            43 

cactgggact tcgcgtggcc gtgg                                            24 

 
           
             44  
             6  
             DNA  
             Artificial Sequence  
             
               primer based on Gallus gallus ovalbumin cDNA  
             
           
            44 

ggatcc                                                                 6 

 
           
             45  
             24  
             DNA  
             Artificial Sequence  
             
               artificial sequence in M13 coliphage  
             
           
            45 

ccacggccac gcgaagtccc agtg                                            24 

 
           
             46  
             35  
             DNA  
             Artificial Sequence  
             
               primer based on Gallus gallus ovalbumin cDNA  
             
           
            46 

cccaagcttg ggccatggtg actgagcaag aaagc                                35 

 
           
             47  
             62  
             DNA  
             Artificial Sequence  
             
               primer based on Gallus gallus ovalbumin cDNA  
             
           
            47 

cccaagcttg ggcatatgca ctgggacttc gcgtggccgt gggtgactga gcaagaaagc     60 

aa                                                                    62