KR20050121721A - Improved heat shock protein-based vaccines and immunotherapies - Google Patents

Abstract

Hybrid antigens comprising at least one antigenic domain, at least one heat shock protein binding domain, and at least one improved peptide linker there between are described which are useful for the induction of an immune response to the antigenic domain when administered alone or in a complex with at least one heat shock protein. The hybrid antigens and complexes can be used to treat infectious diseases and cancers that express an antigen of the antigenic domain.

Description

IMPROVED HEAT SHOCK PROTEIN-BASED VACCINES AND IMMUNOTHERAPIES}

This application is a priority claims application for US Patent Application Nos. 10/776, 521, filed February 12, 2004, and PCT Patent Application No. PCT / US04 / 04340, filed February 13, 2004, where they are incorporated by reference. It became. This application also discloses Provisional Application No. 60 / 462,469, filed April 11, 2003; Application No. 60 / 463,746, filed April 18, 2003; And 35U to Application No. 60 / 503,417, filed September 16, 2003. A priority claim under S. C. 119 (e), all three of which are incorporated by reference.

The present invention relates to methods and compositions for inducing an immune response in a subject, wherein the subject is administered in combination with an effective amount of at least one defined hybrid antigen and optionally one or more heat shock proteins. These methods and compositions can be used to treat infectious diseases and tumors.

  Heat shock proteins were initially observed to have increased amounts of expression in mammalian cells exposed to sudden rises in temperature, while most cellular proteins are significantly reduced. Such proteins have been determined to be produced for several types of stress, such as glucose deficiency. The term "heat shock protein" will be used herein to include both such labeled proteins as well as other stress proteins, including homologues of such proteins that are expressed at all times (ie, even in stress-free conditions). Examples of heat shock proteins include BiP (or named grp78), hsp70, hsc70, gp96 (grp94), hsp60, hsp40 and hsp90.

Heat shock proteins have the ability to bind to other proteins in the non-natural state, especially early proteins coming out of ribosomes or pushing into the endoplasmic reticulum. Hendrick and Hartl, Ann. Rev. Biochem. 62: 349-384 (1993); Hartl, Nature 381: 571-580 (1996). In addition, heat shock proteins have been shown to play an important role in proper overlapping and binding of proteins in plasma, endoplasmic reticulum and mitochondria; In terms of this function they are named "molecular chefs" Frydman et al., Nature 370: 111-117 (1994); Henrick and Hartl, Ann. Rev. Biochem. 62: 349-384 (1993); Hartl, Nature 381: 571-580 (1996).

For example, the BiP protein, a member of the family of heat shock proteins in the hsp79 sequence, was found to bind to the newly synthesized nonoverlapping μ immunoglobin heavy chain before binding to the light chain in the endoplasmic reticulum. Hendershot et al., J. Cell Biol. 104: 761-767 (1987). Another thermal shock protein, gp96, is a member of the hsp90 family of stress proteins located in the endoplasmic reticulum. Li and Srivastava, EMBO J. 12: 3143-3151 (1993); Mazzarella and Green, J. Biol. Cltem. 262: 8875-8883 (1987). gp96 is known to support the binding of complex-unit proteins in endoplasmic reticulum. Wiech et al., Nature 358: 169-170 (1992).

It has been observed that heat shock proteins produced in tumors of experimental animals can induce tumor-specific forms of immune responses; In other words, the heat shock protein purified from a particular tumor can induce an immune response in experimental animals that can inhibit the growth of the same tumor rather than another tumor. Srivastava and Maki, Curr. Topics Microbiol. 167: 109-123 (1991). Genes encoding heat shock proteins were not found to exhibit tumor specific DNA polymorphism. Srivastava and Udono, Curr. Opin. Immunol. 6: 728-732 (1994). High resolution gel electrophoresis indicates that gp96 can be heterogeneous at the molecular level. Feldweg and Srivastava, Int. J. Cancer 63: 310-314 (1995). Evidence suggests that the source of heterogeneity can be a group of small peptides attached to a heat shock protein, which can be hundreds of numbers. Id. The wide variety of peptides attached to tumor synthesized heat shock proteins can enable such proteins to elicit immune responses in subjects with various HLA phenotypes, as compared to more traditional immunogens, which may be somewhat HLA-limited in their effects. Id.

Nieland et al. (Proe. Natl. Acad. Sci. USA 93: 6135-6139 (1996)) describe cytotoxic T lymphocyte (CTL) vesicular otitis virus (VSV) bound to gp96 produced by VSV-infected cells. Antigenic peptides containing epitopes were identified. Neiland's methods interfered with the identification of additional peptides or other compounds capable of binding to gp96 and thus were unable to further identify high molecular weight materials bound to gp96 and high pressure liquid chromatography. Was detected.

Synthetic peptides comprising multiple repeat NANPs (Asp Ala Asp Pro) malaria antigens that are chemically crosslinked to glutaaldehyde immobilized mycobacterial hsp65 or hsp70 can form antibodies (ie humoral response) in mice without any added adjuvant. Have been reported; Similar effects were observed using heat shock proteins from Escherichia coli bacteria. Del Guidice, Experientia 50: 1061-1066 (1994); Barrios et al., Clin. Exp. Imnzunol. 98: 224-228 (1994); Barrios et al., Eur. J. Immunol. 22: 1365-1372 (1992). Cross linking of synthetic peptides and possible glutaaldehyde immobilization to heat shock proteins was required for antibody induction. Barrios et al., Cliva. Exp. Imznunol. 98: 229-233.

PCT / US96 / 13363 describes hybrid antigens having antigenic and heat shock protein binding domains which induce immune responses to antigens in complex with heat shock proteins and are thus useful for the treatment of tumors or infectious diseases. PCT / US98 / 22335 describes additional heat shock protein binding domains for similar uses as well as the ability for hybrid antigens to be administered alone to induce an immune response. It has now been found that improvements in peptide linkers present between at least one antigen domain and at least one heat shock protein binding domain in hybrid antigens increase biological activity. This increase is also known to provide an increase in the induction of immune responses to the antigenic portion of the hybrid antigen. Improved Peptide Linkers With or Without Heat Shock Protein Hybrid peptides comprising them and their uses are provided herein.

1 shows the results of a tumor challenge study in which immunization was carried out using hybrid antigens or heat shock proteins and hybrid antigen complexes and challenged with tumors expressing the antigens 7 days later.

Summary of the Invention

The present invention relates to methods and compositions for inducing an immune response in a subject, wherein optionally at least one hybrid antigen in complex with a heat shock protein is administered to the subject. The hybrid antigen comprises at least one antigen domain and at least one heat shock protein binding domain with at least one peptide linker therebetween. Induction of an immune response to an antigen associated with a disease such as an infectious disease or a tumor is useful for the treatment of that disease. The antigenic or immunological domain of the hybrid antigen may be a whole protein or peptide antigen or only a portion of the selected antigen, for example a selected epitope of the antigen. The heat shock protein binding domain is a peptide that binds to a heat shock protein, preferably a peptide of 7-15 amino acids that binds to a heat shock protein, more preferably a hydrophobic peptide that binds to a heat shock protein and most preferably a heat shock protein. It is a hydrophobic peptide of 7-15 amino acids that binds to. The linker is Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); Or AA ₁ -AA ₂ -AA ₃ -leucine, where AA ₁ is A, S, V, E, G, L, or K, preferably V, more preferably S, most preferably A; AA ₂ is K, V, or E, preferably E, more preferably V and most preferably K; and AA ₃ is V, S, F, K, A, E, or T, preferably F More preferably S and most preferably V. In the above, Gln Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO: 1001) are preferred, and Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000) is most preferred.

The present invention provides a method of administering such a hybrid antigen alone as well as a heat shock protein / hybrid antigen composition, the latter of which (i) the binding of the hybrid antigen to the heat shock protein to form a hybrid antigen / heat shock protein complex In vitro combining one or more hybrid antigens with one or more heat shock proteins; (ii) administering an effective amount of said hybrid antigen bound to a heat shock protein to a subject in need of such treatment.

In addition, the hybrid antigen selectively bound to the heat shock protein may be introduced into the subject by administering to the subject a nucleic acid encoding the hybrid antigen, together with a nucleic acid encoding the heat shock protein.

Thus, in a first aspect, the present invention is directed to a hybrid antigen comprising essentially one antigen domain of an infectious or tumor antigen, one binding domain that binds non-covalently to a heat shock protein, and an antigen domain and a peptide linker that separates the binding domain. Wherein the peptide linker comprises Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gin Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); Or AA ₁ -AA ₂ -AA ₃ -leucine, where AA ₁ is A, S, V, E, G, L, or K, preferably V, more preferably S, most preferably A and AA ₂ Is K, V, or E preferably E, more preferably V, most preferably K; AA ₃ is one of V, S, F, K, A, E, or T, more preferably S, most preferably A, preferably F, more preferably S, most preferably V. . Among the above, Gin Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO: 1001) are preferred, and Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000) is most preferred.

In a second aspect, the invention provides a plurality of antigenic domains of one or more infectious agents or one or more tumor antigens, at least one binding domain that covalently binds to a heat shock protein, and at least one that separates the antigenic domain and the binding domain. A hybrid antigen is provided that essentially comprises a peptide linker of wherein the at least one peptide linker comprises Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gin Leu Lys (QLK), Gin Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); Or AA ₁ -AA ₂ -AA ₃ -leucine, where AA ₁ is A, S, V, E, G, L, or K, preferably V, more preferably S, most preferably A and AA ₂ Is K, V, or E preferably E, more preferably V, most preferably K; AA ₃ is one of V, S, F, K, A, E, or T, preferably F, more preferably S, most preferably V. Among the above, Gin Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO: 1001) are preferred, and Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000) is most preferred. In certain embodiments at least one of the antigenic domains in the hybrid antigen is a T helper epitope.

In a third aspect, the invention provides an infectious disease comprising an antigenic domain of an infectious or tumor antigen, a binding domain that covalently binds to a heat shock protein, and at least one hybrid antigen comprising at least one peptide linker therebetween. Or provides a composition for inducing an immune response against a tumor antigen, wherein at least one of said peptide linkers is Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); Or AA ₁ -AA ₂ -AA ₃ -leucine, where AA ₁ is A, S, V, E, G, L, or K, preferably V, more preferably S, most preferably A and AA ₂ Is K, V, or E preferably E, more preferably V, most preferably K; AA ₃ is one of V, S, F, K, A, E, or T, preferably F, more preferably S, most preferably V. Among the above, Gin Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO: 1001) are preferred, and Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000) is most preferred. In certain embodiments the composition may comprise a plurality of hybrid antigens, and one hybrid antigen may comprise a T helper epitope.

In a fourth aspect, the invention provides a hybrid comprising a plurality of antigenic domains of one or more infectious agents or one or more tumor antigens, at least one binding domain that covalently binds to a heat shock protein, and at least one peptide linker therebetween. Providing an antigen, wherein at least one said peptide linker is selected from the group consisting of Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); Or AA ₁ -AA ₂ -AA ₃ -leucine, where AA ₁ is A, S, V, E, G, L, or K, preferably V, more preferably S, most preferably A and AA ₂ Is K, V, or E preferably E, more preferably V, most preferably K; AA ₃ is one of V, S, F, K, A, E, or T, preferably F, more preferably S, most preferably V. Among the above, Gin Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO: 1001) are preferred, and Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000) is most preferred. In certain embodiments at least one antigenic domain is a T helper epitope. The hybrid antigen has a peptide linker that separates the antigenic domain and the binding domain.

In a fifth aspect, the present invention provides an infectious disease comprising at least one hybrid antigen comprising an antigenic domain of an infectious or tumor antigen, a binding domain that covalently binds to a heat shock protein, and at least one peptide linker therebetween. Or provides a composition for inducing an immune response against a tumor antigen, wherein at least one of said peptide linkers is Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); Or AA ₁ -AA ₂ -AA ₃ -leucine, where AA ₁ is A, S, V, E, G, L, or K, preferably V, more preferably S, most preferably A and AA ₂ Is K, V, or E preferably E, more preferably V, most preferably K; AA ₃ is one of V, S, F, K, A, E, or T, preferably F, more preferably S, most preferably V. Among the above, Gin Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO: 1001) are preferred, and Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000) is most preferred. In certain embodiments the composition may comprise a plurality of hybrid antigens, and one hybrid antigen may comprise a T helper epitope.

In a sixth aspect, the invention includes at least one plurality of antigenic domains derived from an infectious or tumor antigen, at least one binding domain that covalently binds to a heat shock protein, and at least one peptide linker therebetween. Providing a composition for inducing an immune response to an infectious disease or tumor antigen comprising at least one hybrid antigen, wherein at least one said peptide linker comprises Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); Or AA ₁ -AA ₂ -AA ₃ -leucine, where AA ₁ is A, S, V, E, G, L, or K, preferably V, more preferably S, most preferably A and AA ₂ Is K, V, or E preferably E, more preferably V, most preferably K; AA ₃ is one of V, S, F, K, A, E, or T, preferably F, more preferably S, most preferably V. Among the above, Gin Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO: 1001) are preferred, and Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000) is most preferred.

In a seventh aspect, the invention provides at least one comprising essentially an antigenic domain of an infectious or tumor antigen, a binding domain that covalently binds to a heat shock protein, and a peptide linker that separates the antigenic domain and the binding domain A composition for inducing an immune response to an infectious disease or tumor antigen comprising a hybrid antigen of: wherein at least one said peptide linker is selected from Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); Or AA ₁ -AA ₂ -AA ₃ -leucine, where AA ₁ is A, S, V, E, G, L, or K, preferably V, more preferably S, most preferably A and AA ₂ Is K, V, or E preferably E, more preferably V, most preferably K; AA ₃ is one of V, S, F, K, A, E, or T, preferably F, more preferably S, most preferably V. Among the above, Gin Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO: 1001) are preferred, and Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000) is most preferred. In one embodiment the composition comprises a plurality of hybrid antigens. In another aspect the at least one plurality of hybrid antigens comprises a T helper epitope.

In an eighth aspect, the present invention provides a method for separating at least one antigen domain derived from an infectious or tumor antigen, at least one binding domain that covalently binds to a heat shock protein, and separating the antigenic domain and the binding domain. Provided are compositions that induce an immune response against an infectious disease or tumor antigen comprising at least one hybrid antigen essentially comprising a peptide linker, wherein the at least one peptide linker comprises Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); Or AA ₁ -AA ₂ -AA ₃ -leucine, where AA ₁ is A, S, V, E, G, L, or K, preferably V, more preferably S, most preferably A and AA ₂ Is K, V, or E preferably E, more preferably V, most preferably K; AA ₃ is one of V, S, F, K, A, E, or T, preferably F, more preferably S, most preferably V. Among the above, Gin Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO: 1001) are preferred, and Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000) is most preferred. One at least one antigenic domain may comprise a T helper epitope.

In a ninth aspect, the invention provides a hybrid antigen comprising at least one antigenic domain of an infectious or tumor antigen, at least one binding domain comprising a peptide that covalently binds a heat shock protein, and a peptide linker therebetween. And inducing an immune response to an infectious disease or tumor antigen comprising administering to the subject a complex of heat shock proteins, wherein the at least one peptide linker comprises Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000). ); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); Or AA ₁ -AA ₂ -AA ₃ -leucine, where AA ₁ is A, S, V, E, G, L, or K, preferably V, more preferably S, most preferably A and AA ₂ Is K, V, or E preferably E, more preferably V, most preferably K; AA ₃ is one of V, S, F, K, A, E, or T, preferably F, more preferably S, most preferably V. Among the above, Gin Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO: 1001) are preferred, and Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000) is most preferred. In one embodiment the complex comprises a plurality of hybrid antigens. In one embodiment at least one hybrid antigen is a T helper epitope. In another embodiment the hybrid antigen may comprise a plurality of antigenic domains and at least one antigenic domain may be a T helper epitope. In another embodiment is a complex comprising a plurality of hybrid antigens of at least one hybrid antigen comprising a plurality of antigenic domains. In another embodiment of the invention the heat shock protein is hsp70.

In a tenth aspect, the present invention requires at least one antigenic domain of an infectious or tumor antigen, one binding domain that covalently binds to a heat shock protein, and a peptide linker that separates the antigenic and fraudulent binding domains thereof. A method of inducing an immune response to an infectious disease or tumor antigen comprising administering to a subject a complex of a hybrid antigen and a heat shock protein, wherein the at least one peptide linker is Phe Phe Arg Lys (FFRK). SEQ ID NO: 1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); Or AA ₁ -AA ₂ -AA ₃ -leucine, where AA ₁ is A, S, V, E, G, L, or K, preferably V, more preferably S, most preferably A and AA ₂ Is K, V, or E preferably E, more preferably V, most preferably K; AA ₃ is one of V, S, F, K, A, E, or T, preferably F, more preferably S, most preferably V. Among the above, Gin Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO: 1001) are preferred, and Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000) is most preferred. In one embodiment the complex comprises a plurality of hybrid antigens. In one embodiment at least one hybrid antigen is a T helper epitope. In another embodiment said hybrid antigen comprises a plurality of antigenic domains and at least one antigenic domain is a T helper epitope. In another embodiment the complex comprises a plurality of hybrid antigens of at least one hybrid antigen comprising a plurality of antigenic domains. In a preferred embodiment of the invention the heat shock protein is hsp70.

In an eleventh aspect, the invention provides at least one antigenic domain of an infectious or tumor antigen, at least one binding domain comprising a peptide that covalently binds to a heat shock protein, and at least one comprising a peptide linker therebetween. A method of inducing an immune response to an infectious disease or tumor antigen comprising administering a hybrid antigen of to a subject, wherein at least one of the peptide linkers comprises Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); Or AA ₁ -AA ₂ -AA ₃ -leucine, where AA ₁ is A, S, V, E, G, L, or K, preferably V, more preferably S, most preferably A and AA ₂ Is K, V, or E preferably E, more preferably V, most preferably K; AA ₃ is one of V, S, F, K, A, E, or T, preferably F, more preferably S, most preferably V. Among the above, Gin Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO: 1001) are preferred, and Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000) is most preferred. In one embodiment the complex comprises a plurality of hybrid antigens. In another embodiment at least one hybrid antigen is a T helper epitope. In another embodiment the hybrid antigen comprises a plurality of antigenic domains and at least one antigenic domain is a T helper epitope. In another embodiment the complex comprises a plurality of hybrid antigens of at least one hybrid antigen comprising a plurality of antigenic domains. In another embodiment of the invention the peptide linker separates the antigenic domain from the binding domain.

In a twelfth aspect, the present invention provides a method for separating at least one antigenic domain of an infectious or tumor antigen, one binding domain comprising a peptide that covalently binds to a heat shock protein, and separating the antigenic domain from the binding domain. A method of inducing an immune response to an infectious disease or tumor antigen comprising administering to a subject at least one hybrid antigen essentially comprising a peptide linker, wherein the at least one peptide linker comprises Phe Phe Arg Lys ( FFRK: SEQ ID NO: 1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); Or AA ₁ -AA ₂ -AA ₃ -leucine, where AA ₁ is A, S, V, E, G, L, or K, preferably V, more preferably S, most preferably A and AA ₂ Is K, V, or E preferably E, more preferably V, most preferably K; AA ₃ is one of V, S, F, K, A, E, or T, preferably F, more preferably S, most preferably V. Among the above, Gin Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO: 1001) are preferred, and Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000) is most preferred. In one embodiment the complex comprises a plurality of hybrid antigens. In another embodiment at least one hybrid antigen is a T helper epitope. In another embodiment the hybrid antigen comprises a plurality of antigenic domains and at least one antigenic domain is a T helper epitope. In another embodiment the complex comprises a plurality of hybrid antigens of at least one hybrid antigen comprising a plurality of antigenic domains.

In a thirteenth aspect, the present invention provides a peptide linker between at least one antigenic domain of an infectious disease or tumor antigen associated with an infectious disease or tumor, one binding domain comprising a peptide that covalently binds to a heat shock protein, and a peptide linker therebetween. A method of treating an infectious disease or a tumor antigen comprising administering to a subject a hybrid antigen and a heat shock protein complex comprising essentially, wherein said hybrid antigen and said heat shock protein are non-covalently bound, at least One such peptide linker is Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); Or AA ₁ -AA ₂ -AA ₃ -leucine, where AA ₁ is A, S, V, E, G, L, or K, preferably V, more preferably S, most preferably A and AA ₂ Is K, V, or E preferably E, more preferably V, most preferably K; AA ₃ is one of V, S, F, K, A, E, or T, preferably F, more preferably S, most preferably V. Among the above, Gin Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO: 1001) are preferred, and Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000) is most preferred. In one embodiment the complex comprises a plurality of hybrid antigens. In another embodiment at least one hybrid antigen is a T helper epitope. In another embodiment the hybrid antigen comprises a plurality of antigenic domains and at least one antigenic domain is a T helper epitope. In another embodiment the complex comprises a plurality of hybrid antigens of at least one hybrid antigen comprising a plurality of antigenic domains. In one embodiment of the invention the peptide linker separates the antigenic domain from the binding domain. In a preferred embodiment of the invention the heat shock protein is hsp70.

In a fourteenth aspect, the present invention provides at least one antigenic domain of an infectious disease or tumor antigen associated with an infectious disease or tumor, at least one binding domain that covalently binds to a heat shock protein, and the antigenic domain and the binding domain Provided is a method for treating an infectious disease or tumor antigen comprising administering to a subject a hybrid antigen and a heat shock protein complex essentially comprising a peptide linker that separates the peptide linker, wherein the at least one peptide linker is Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); Or AA ₁ -AA ₂ -AA ₃ -leucine, where AA ₁ is A, S, V, E, G, L, or K, preferably V, more preferably S, most preferably A and AA ₂ Is K, V, or E preferably E, more preferably V, most preferably K; AA ₃ is one of V, S, F, K, A, E, or T, preferably F, more preferably S, most preferably V. Among the above, Gin Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO: 1001) are preferred, and Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000) is most preferred. In one embodiment the complex comprises a plurality of hybrid antigens. In another embodiment at least one hybrid antigen is a T helper epitope. In another embodiment the hybrid antigen comprises a plurality of antigenic domains and at least one antigenic domain is a T helper epitope. In another embodiment the complex comprises a plurality of hybrid antigens of at least one hybrid antigen comprising a plurality of antigenic domains. In one embodiment of the invention the peptide linker separates the antigenic domain from the binding domain. In a preferred embodiment of the invention the heat shock protein is hsp70.

In a fifteenth aspect, the present invention provides a peptide comprising at least one antigenic domain of an infectious disease or tumor antigen associated with an infectious disease or tumor, one binding domain comprising a peptide that covalently binds to a heat shock protein, and a peptide therebetween. A method of treating an infectious disease or tumor antigen comprising administering to a subject at least one hybrid antigen comprising a linker, wherein the at least one peptide linker comprises Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000) ; Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); Or AA ₁ -AA ₂ -AA ₃ -leucine, where AA ₁ is A, S, V, E, G, L, or K, preferably V, more preferably S, most preferably A and AA ₂ Is K, V, or E preferably E, more preferably V, most preferably K; AA ₃ is one of V, S, F, K, A, E, or T, preferably F, more preferably S, most preferably V. Among the above, Gin Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO: 1001) are preferred, and Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000) is most preferred. In one embodiment the complex comprises a plurality of hybrid antigens. In another embodiment at least one hybrid antigen is a T helper epitope. In another embodiment the hybrid antigen comprises a plurality of antigenic domains and at least one antigenic domain is a T helper epitope. In another embodiment the complex comprises a plurality of hybrid antigens of at least one hybrid antigen comprising a plurality of antigenic domains. In one embodiment of the invention the peptide linker separates the antigenic domain from the binding domain.

In a sixteenth aspect, the present invention relates to a subject comprising at least one antigenic domain of an infectious disease or tumor antigen associated with an infectious disease or tumor, one binding domain comprising a peptide that covalently binds to a heat shock protein, and the antigenic domain And administering to the subject at least one hybrid antigen essentially comprising a peptide linker separating said binding domain, wherein at least one said peptide linker is Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); Or AA ₁ -AA ₂ -AA ₃ -leucine, where AA ₁ is A, S, V, E, G, L, or K, preferably V, more preferably S, most preferably A and AA ₂ Is K, V, or E, preferably E, more preferably V, most preferably K; AA ₃ is one of V, S, F, K, A, E, or T, preferably F, more preferably S, most preferably V. Among the above, Gin Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO: 1001) are preferred, and Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000) is most preferred. In one embodiment the complex comprises a plurality of hybrid antigens. In another embodiment at least one hybrid antigen is a T helper epitope. In another embodiment the hybrid antigen comprises a plurality of antigenic domains and at least one antigenic domain is a T helper epitope. In another embodiment the complex comprises a plurality of hybrid antigens of at least one hybrid antigen comprising a plurality of antigenic domains.

In a seventeenth aspect, the invention provides Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); Or AA ₁ -AA ₂ -AA ₃ -leucine, where AA ₁ is A, S, V, E, G, L, or K, preferably V, more preferably S, most preferably A and AA ₂ Is K, V, or E preferably E, more preferably V, most preferably K; AA ₃ represents a peptide which is one of V, S, F, K, A, E, or T, preferably F, more preferably S, most preferably V.

In an eighteenth aspect, the present invention provides an immunogenic polypeptide comprising a plurality of antigenic domains, a binding domain to at least one heat shock protein, and at least one peptide linker therebetween, wherein at least one of said peptide linkers Is Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); Or AA ₁ -AA ₂ -AA ₃ -leucine, where AA ₁ is A, S, V, E, G, L, or K, preferably V, more preferably S, most preferably A and AA ₂ Is K, V, or E preferably E, more preferably V, most preferably K; AA ₃ is one of V, S, F, K, A, E, or T, preferably F, more preferably S, most preferably V. Among the above, Gin Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO: 1001) are preferred, and Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000) is most preferred.

In a ninth aspect the present invention provides a polynucleotide encoding any hybrid antigens in the first, second, third or fourth aspect.

In a twentieth aspect, the present invention relates to a polynucleotide encoding a hybrid antigen comprising one antigenic domain, an heat shock protein binding domain, and a peptide linker therebetween, of an infectious or tumor antigen associated with an infectious disease or tumor. A method of inducing an immune response to an infectious disease or tumor antigen comprising administering, wherein the hybrid antigen and the heat shock protein are noncovalently bound, and at least one of the peptide linkers is Phe Phe Arg Lys ( FFRK: SEQ ID NO: 1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); Or AA ₁ -AA ₂ -AA ₃ -leucine, where AA ₁ is A, S, V, E, G, L, or K, preferably V, more preferably S, most preferably A and AA ₂ Is K, V, or E preferably E, more preferably V, most preferably K; AA ₃ is one of V, S, F, K, A, E, or T, preferably F, more preferably S, most preferably V. Among the above, Gin Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO: 1001) are preferred, and Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000) is most preferred.

In a twenty-first aspect, the present invention provides a method of inducing an immune response to an infectious disease or tumor antigen comprising administering to a subject a polynucleotide encoding a hybrid antigen mentioned above and a polynucleotide encoding a heat shock protein. to provide. In a preferred embodiment the encoded heat shock protein is hsp70.

In any or all of the aforementioned aspects of the invention the infectious disease antigen may be derived from non-limiting examples from an infectious agent, such as bacteria, viruses, protozoa, mycoplasmas, fungi, yeasts, parasites or prions. Cancer or tumor antigens associated with the tumor may originate through non-limiting examples from sarcoma, lymphoma, leukemia or carcinoma, melanoma, breast cancer, prostate cancer, ovarian cancer, cervical cancer, colon cancer, lung cancer, glioblastoma or astrocytoma Can be. The antigenic domain of an infectious agent or tumor is associated with or derived from an infectious disease or tumor antigen so that induction of an immune response to each antigen of the infectious agent or tumor antigen is useful for treating the infectious disease or tumor.

This application claims the provisional application number 60 / 462,469, filed April 11, 2003; Application No. 60 / 463,746, filed April 18, 2003; And 35U to Application No. 60 / 503,417, filed September 16, 2003. A priority claim under S. C. 119 (e), all three of which are incorporated by reference.

(I) hybrid antigens, (ii) heat shock proteins which are subsections for the purpose of clarifying the invention but not for limiting the invention; And (iii) methods of administration are described in detail below.

hybrid  Antigen (Hybrid Antigen)

The hybrid antigen according to the invention consists of at least one antigenic (immunogenic) domain, at least one heat shock protein-binding domain, and a peptide linker between at least two of these domains, wherein The peptide linker in is one of

Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000),

Phe Arg Lys (FRK);

Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002);

Arg Lys Asn (RKN);

Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003);

Phe Arg (FR),

Gln Leu Lys (QLK),

Gln Leu Glu (QLE),

Ala Lys Val Leu (AKVL; SEQ ID NO: 1001),

Lys Asn (KN);

Arg Lys (RK); or

AA ₁ -AA ₂ -AA ₃ -leucine, wherein AA ₁ is A, S, V, E, G, L, or K, preferably V, more preferably S, and most preferably A; AA ₂ is K, V, or E, preferably E, more preferably V and most preferably K; AA ₃ is V, S, F, K, A, E, or T, preferably F, more preferably S and most preferably V.

Of the foregoing, Gln Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO: 1001) are preferred, and Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000) is most preferred. .

Thus, a hybrid antigen serves at least two functions, namely (i) it contains an epitope capable of eliciting a desired immune response, and (ii) it can physically bind to a heat shock protein. As described below, such binding can occur in vivo such that administration of the hybrid antigen alone will induce a desired immune response and produce a desired therapeutic effect.

As used herein, the term “antigen” refers to a compound which may consist of amino acids, carbohydrates, nucleic acids or lipids alone or in any combination.

As used herein, the term “hybrid antigen” refers to a compound that binds one or more heat shock proteins and represents an immunogen to which the immune response is preferably directed. For example, if the immunogen is an influenza virus, the hybrid antigen may comprise peptide fragments of the matrix protein of the influenza virus. As used herein, the term “immunogen” can be applied to tumor cells, infected cells, pathogens, or components thereof to which an immune response is directed. On the other hand, hybrid antigens include those that can bind to one or more heat shock proteins and some of the immunogens that can cause the desired response. In particular, the antigenic domain of the hybrid antigen is selected to elicit an immune response to certain diseases or pathogens, including direct DNA products such as peptides obtained from MHC molecules, mutant DNA gene products, and those obtained from tumor cells.

While the present invention can be applied to certain types of immunogens, immunogens of particular interest are tumor diseases, including but not limited to sarcoma, lymphoma, leukemia or carcinoma, in particular melanoma, breast carcinoma, prostate gland Carcinoma, ovarian carcinoma, cervix carcinoma, colorectal carcinoma, lung carcinoma, glioblastoma, astrocytoma, and the like, or the like derived from, or related to, and the like. Selection of melanoma antigens useful for the hybrid antigens of the present invention can be found in PCT / US01 / 12449 (W00178655) as a non-limiting example, which is incorporated herein by reference in its entirety. In addition, mutations in tumor suppressor gene products such as p53, or oncogene products such as ras can also provide hybrid antigens used in accordance with the present invention.

In other embodiments, the immunogen may be associated with an infectious disease, such as bacteria, viruses, protozoa, mycoplasma, fungi, yeast, parasites, or prions. For example, by way of example, the immunogen may be a human papilloma virus (see below), a herpes virus such as herpes simplex or herpes zoster, human immunodeficiency virus 1 or 2, and the like. Such as retrovirus, hepatitis virus, influenza virus, rhinovirus, respiratory syncytial virus, cytomegalovirus0, adenovirus, Mycoplasmapneumoniae, Salmonella , Staphylococcus , Streptococcus , Enterococcus , Clostridium , Escherichia , Klebsiella , Vibrio , Mycobacterium virus, amoeba, malaria parasite, Trypanosoma cruzi and the like.

Immunogens can be obtained through direct isolation from tumors, infected cells, samples from infected individuals, cell cultures, or organism cultures, or can be made through chemical or recombinant techniques. By way of non-limiting example, suitable antigenic peptides for viruses, bacteria and the like include, but are not limited to, HLA binding sequences, such as, but not limited to, the HLA-A2 peptide binding sequences below, for use in hybrid antigens. Can be designed through their sequence search for MHC class I restricted peptide epitopes:

Xaa (Leu / Met) XaaXaaXaa (Val / Ile / Leu / Thr) XaaXaa (Val / Leu) (SEQ ID NO: 2), for example

From the virus:

Ser Gly Pro Ser Asn Thr Pro Pro Glu Ile (SEQ ID NO .: 31);

Ser Gly Val Glu Asn Pro Gly Gly Tyr Cys Leu (SEQ ID NO: 32);

Lys Ala Val Tyr Asn Phe Ala Thr Cys Gly (SEQ ID NO: 33);

Arg Pro Gln Ala Ser Gly Val Tyr Met (SEQ ID NO .: 34);

Phe Gln Pro Gln Asn Gly Gln Phe Ile (SEQ ID NO .: 35);

Ile Glu Gly Gly Trp Thr Gly Met Ile (SEQ ID NO .: 36);

Thr Tyr Val Ser Val Ser Thr Ser Thr Leu (SEQ ID NO .: 37);

Phe Glu Ala Asn Gly Asn Leu Ile (SEQ ID NO .: 38);

Ile Tyr Ser Thr Val Ala Ser Ser Leu (SEQ ID NO .: 39);

Thr Tyr Gln Arg Thr Arg Ala Leu Val (SEQ ID NO .: 40);

Cys Thr Glu Leu Lys Leu Ser Asp Tyr (SEQ ID NO: 41);

Ser Asp Tyr Glu Gly Arg Leu Ile (SEQ ID NO: 42);

Glu Glu Gly Ala Ile Val Gly Glu Ile (SEQ ID NO: 43);

Val Ser Asp Gly Gly Pro Asn Leu Tyr (SEQ ID NO: 44);

Ala Ser Asn Glu Asn Met Glu Thr Met (SEQ ID NO: 45);

Ala Ser Asn Glu Asn Met Asp Ala Met (SEQ ID NO .: 46);

Lys Leu Gly Glu Phe Tyr Asn Gln Met Met (SEQ ID NO .: 47);

Leu Tyr Gln Asn Val Gly Thr Tyr Val (SEQ ID NO: 48);

Thr Tyr Val Ser Val Gly Thr Ser Thr Leu (SEQ ID NO: 49);

Phe Glu Ser Thr Gly Asn Leu Ile (SEQ ID NO: 50);

Val Tyr Gln Ile Leu Ala Ile Tyr Ala (SEQ ID NO: 51);

Ile Tyr Ala Thr Val Ala Gly Ser Leu (SEQ ID NO: 52);

Gly Ile Leu Gly Phe Val Phe Thr Leu (SEQ ID NO: 53);

Ile Leu Gly Phe Val Phe Thr Leu Thr Val (SEQ ID NO: 54);

Ile Leu Arg Gly Ser Val Ala His Lys (SEQ ID NO: 55);

Glu Asp Leu Arg Val Leu Ser Phe Ile (SEQ ID NO: 56);

Glu Leu Arg Ser Arg Tyr Trp Ala Ile (SEQ ID NO: 57);

Ser Arg Tyr Trp Ala Ile Arg Thr Arg (SEQ ID NO: 58);

Lys Thr Gly Gly Pro Ile Tyr Lys Arg (SEQ ID NO: 59);

Phe Ala Pro Gly Jtsn Ty T Pro Ala Leu (SEQ ID NO: 60);

Arg Arg Tyr Pro Asp Ala Val Tyr Leu (SEQ ID NO: 61);

Asp Pro Val Ile Asp Arg Leu Tyr Leu (SEQ ID NO: 62);

Ser Pro Gly Arg Ser Phe Ser Tyr Phe (SEQ ID NO: 63);

Tyr Pro Ala Leu Gly Leu His Glu Phe (SEQ ID NO: 64);

Thr Tyr Lys Asp Thr Val Gln Leu (SEQ ID NO: 65);

Phe Tyr Asp Gly Phe Ser Lys Val Pro Leu (SEQ ID NO: 66);

Phe Ile Ala Gly Asn Ser Ala Tyr Glu Tyr Val (SEQ ID NO: 67);

Tyr Pro His Phe Met Pro Thr Asn Leu (SEQ ID NO: 68);

Ala Pro Thr Ala Gly Ala Phe Phe Phe (SEQ ID NO: 69);

Ser Thr Leu Pro Glu Thr Thr Val Val Arg Arg (SEQ ID NO: 70);

Phe Leu Pro Ser Asp Phe Phe Pro Ser Val (SEQ ID NO: 71);

Trp Leu Ser Leu Leu Val Pro Phe Val (SEQ ID NO: 72);

Gly Leu Ser Pro Thr Val Trp Leu Ser Val (SEQ ID NO: 73);

Asp Leu Met Gly Tyr Ile Pro Leu Val (SEQ ID NO: 74);

Leu Met Gly Tyr Ile Pro Leu Val Gly Ala (SEQ ID NO: 75);

Ala Ser Arg Cys Trp Val Ala Met (SEQ ID NO: 76);

Lys Leu Val Ala Leu Gly Ile Asn Ala Val (SEQ ID NO: 77);

Phe Leu Arg Gly Arg Ala Tyr Gly Leu (SEQ ID NO: 78);

Arg Arg Ile Tyr Asp Leu Ile Glu Leu (SEQ ID NO: 79);

Ile Val Thr Asp Phe Ser Val Ile Lys (SEQ ID NO: 80);

Arg Arg Arg Trp Arg Arg Leu Thr Val (SEQ ID NO: 81);

Glu Glu Asn Leu Leu Asp Phe Val Arg Phe (SEQ ID NO: 82);

Cys Leu Gly Gly Leu Leu Thr Met Val (SEQ ID NO: 83);

Ser Ser Ile Glu Phe Ala Arg Leu (SEQ ID NO: 84);

Leu Tyr Arg Thr Phe Ala Gly Asn Pro Arg Ala (SEQ ID NO: 85);

Asp Tyr Ala Thr Leu Gly Val Gly Val (SEQ ID NO: 86);

Leu Leu Leu Gly Thr Leu Asn Ile Val (SEQ ID NO: 87);

Leu Leu Met Gly Thr Leu Gly Ile Val (SEQ ID NO: 88);

Thr Leu Gln Asp Ile Val Leu His Leu (SEQ ID NO: 89);

Gly Leu His Cys Tyr Glu Gln Leu Val (SEQ ID NO: 90);

Pro Leu Lys Gln His Phe Gln Ile Val (SEQ ID NO: 91);

Arg Leu Val Thr Leu Lys Asp Ile Val (SEQ ID NO: 92);

Arg Ala His Tyr Asn Ile Val Thr Phe (SEQ ID NO: 93);

Leu Leu Phe Gly Tyr Pro Val Tyr Val (SEQ ID NO: 94);

Ser Ala Ile Asn Asn Tyr Ala Gln Lyb Leu (SEQ ID NO: 95);

His Gln Ala Ile Ser Pro Arg Thr Leu (SEQ ID NO: 96);

Gln Met Val His Gln Ala Ile Ser Pro Arg Thr Leu (SEQ ID NO: 97);

Cys Lys Gly Val Asn Lys Glu Tyr Leu (SEQ ID NO: 98);

Gln Gly Ile Asn Asn Leu Asp Asn Leu (SEQ ID NO: 99);

Asn Asn Leu Asp Asn Leu Arg Asp Tyr (SEQ ID NO: 100);

Ser Glu Phe Leu Leu Glu Lys Arg Ile (SEQ ID NO: 101);

Ser Tyr Ile Gly Ser Ile Asn Asn Ile (SEQ ID NO: 102);

Ile Leu Gly Asn Lys Ile Val Arg Met Tyr (SEQ ID NO: 103);

Arg Leu Arg Pro Gly Cly Lys Lys Lys (SEQ ID NO: 104);

Glu Tle Lys Asp Thr Lys Glu Ala Leu (SEQ ID NO: 105);

Gly Glu Ile Tyr Lys Arg Trp Ile Ile (SEQ ID NO: 106);

Glu Ile Tyr Lys Arg Trp Ile Ile Leu (SEQ ID NO: 107);

Arg Tyr Leu Lys Asp Gln Gln Leu Leu (SEQ ID NO .: 108);

Arg Gly Pro Gly Arg Ala Phe Val Thr Ile (SEQ ID NO: 109);

Ile Val Gly Leu Asn Lys Ile Val Arg (SEQ ID NO: 110);

Thr Val Tyr Tyr Gly Val Pro Val Trp Lys (SEQ ID NO: 111);

Arg Leu Arg Asp Leu Leu Leu Ile Val Thr Arg (SEQ ID NO: 112);

Lys Arg Tip Ile Ile Leu Gly Leu Asn Lys (SEQ ID NO: 113);

Ser Phe Asn Cys Gly Gly Glu Phe Phe (SEQ ID NO: 114);

Gly Arg Ala Phe Val Thr Ile Gly Lys (SEQ ID NO .: 115);

Thr Pro Gly Pro Cly Val Arg Tyr Pro Leu (SEQ ID NO: 116);

Gln Val Pro Leu Arg Pro Met Thr Tyr Lys (SEQ ID NO: 117);

Thr Glu Met Glu Lys Glu Gly Lys Ile (SEQ ID NO: 118);

Ile Leu Lys Glu Pro Val His Gly Val (SEQ ID NO: 119);

Val Glu Ala Glu Ile Ala His Gln Ile (SEQ ID NO: 120);

Arg Gly Tyr Val Tyr Gln Gly Leu (SEQ ID NO: 121);

Tyr Ser Gly Tyr Ile Phe Arg Asp Leu (SEQ ID NO: 122);

Val Gly Pro Val Phe Pro Pro Gly Met (SEQ ID NO: 123);

Ile Ile Tyr Arg Phe Leu Leu Ile (SEQ ID NO .: 124)

From bacteria:

Lys Tyr Gly Val Ser Val Gln Asp Ile (SEQ ID NO: 125);

Ile Gln Val Gly Asn Thr Arg Thr Ile (SEQ ID NO: 126);

Thr Pro His Pro Ala Arg Ile Gly Leu (SEQ ID NO: 127);

From parasites:

Ser Tyr Ile Pro Ser Ala Glu Lys Ile (SEQ ID NO: 128);

Lys Pro Lys Asp Glu Leu Asp Tyr (SEQ ID NO: 129);

Lys Ser Lys Asp Glu Leu Asp Tyr (SEQ ID NO .: 130):

Lys Pro Asn Asp Lys Ser Leu Tyr (SEQ ID NO: 131);

Lys Tyr Leu Lys Lys Ile Lys Asn Ser Leu (SEQ ID NO: 132);

Tyr Glu Asn Asp Ile Glu Lys Lys Ile (SEQ ID NO: 133);

Asn Tyr Asp Asn Ala Gly Thr Asn Leu (SEQ ID NO: 134);

Asp Glu Leu Asp Tyr Glu Asn Asp Ile (SEQ ID NO .: 135);

Ser Tyr Val Pro Ser Ala Glu Gln Ile (SEQ ID NO: 136);

From cancer:

Phe Glu Gln Asn Thr Ala Gln Pro (SEQ ID NO: 137);

Phe Glu Gln Asn Thr Ala Gln Ala (SEQ ID NO: 138);

Glu Ala Asp Pro Thr Gly His Ser Tyr (SEQ ID NO: 139);

Glu Val Asp Pro Ile Gly His Leu Tyr (SEQ ID NO: 140);

Ala Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO: 141);

Thr Leu Glu Pro Cly Pro Val Thr Ala (SEQ ID NO: 142);

Ile Leu Asp Gly Thr Ala Thr Leu Arg Leu (SEQ ID NO: 143);

Met Leu Leu Ala Leu Leu Tyr Cys Leu (SEQ ID NO: 144);

Tyr Met Asn Gly Thr Met Ser Gln Val (SEQ ID NO: 145);

Leu Pro Tyr Leu Gly Trp Leu Val Phe (SEQ ID NO: 146);

Phe Gly Pro Tyr Lys Leu Asn Arg Leu (SEQ ID NO: 147);

Lys Ser Pro Trp Phe Thr Thr Leu (SEQ ID NO: 148);

Gly Pro Pro His Ser Asn Asn Phe Gly Tp (SEQ ID NO: 149); And

Ile Ser Thr Gln Asn His Arg Ala Leu (SEQ ID NO .: 150)

(Rammensee et al., Immunogenetics 41: 178-223 (1995)),

Xaa (Leu / Met) XaaXaaXaaXaaXaaXaaVal (SEQ ID NO: 3)

Tarpey et al., Immunology 81: 222-227 (1994),

Xaa (Val / Gln) XaaXaaXaaXaaXaaXaaLeu (SEQ ID NO: 28),

For example, from a virus:

Tyr Gly Ile Leu Gly Lys Val Phe Thr Leu (SEQ ID NO: 151);

Ser Leu Tyr Asn Thr Val Ala Thr Leu (SEQ ID NO .: 152);

(Barouch et al., J Exp . Med . 182: 1847-1856 (1995)).

The epitopes described above are merely examples of the options available in connection with various infectious diseases and cancers and are not intended to be limiting of the invention in any way.

It may also be desirable to consider the type of immune response desired. For example, under certain circumstances, a humoral immune response may be appropriate. In other cases, a cellular immune response is particularly preferred where it is really required to exert an immune response towards tumor cells or infected cells. Thus, specific epitopes associated with activation of B cells, T helper cells, or cytotoxic T cells can be identified and selected for inclusion in the hybrid antigen.

It is also desirable to use hybrid antigens associated with autoimmune diseases or allergies. Such hybrid antigens may be administered with one or more heat shock proteins in an amount sufficient to inhibit a pre-existing immune response to the hybrid antigen or to be tolerogenic in the individual. The amount of heat shock protein needed to suppress the immune response will be substantially greater than the amount required for stimulation.

The size of the hybrid antigen may vary depending on the heat shock protein used, but in a non-limiting embodiment of the invention, the hybrid antigen may be peptide size with 10 to 500 amino acid residues, preferably 14 to 100 amino acids. Peptide size with residues, most preferably peptide size with 18-50 amino acid residues. As such, it may be desirable to produce fragments of an immunogen to act as the antigenic domain of the hybrid antigen, or alternatively synthesize the hybrid antigen by chemical or recombinant DNA methods.

Based on the foregoing, hybrid antigens can be prepared and their ability to bind heat shock proteins can be tested. In some embodiments, binding of the hybrid antigen to a particular heat shock protein may be facilitated by the presence of at least one other protein that may be one heat shock protein.

For example, the binding of a heat shock protein to a hybrid antigen can be performed by labeling the hybrid antigen with a detectable label, such as radioactive, fluorescent, enzymatic or pigmented labels, and under conditions where binding is expected to occur. It can be assessed by binding the antigen, then separating the heat shock protein during removal of the unbound hybrid antigen, and measuring whether the labeled hybrid antigen is immobilized on the heat shock protein. In certain non-limiting embodiments, the ability of the hybrid antigen to bind to the heat shock protein BiP is characterized by the presence of 2 μg BiP and up to about 1150 pmole of radioactively labeled hybrid antigen in 50 mM Tris HCI (pH 7.5), 200. It can be assessed by binding for 30 minutes at 37 degrees Celsius so that the final volume is 50 μl in a buffer comprising mM NaCl, and 1 mM Na ₂ EDTA. Unbound hybrid antigen can then be removed from bound BiP-Hybrid antigen by centrifugation at 100 g and desalting for 2 minutes on a 1 ml Sephadex-G column. Penefsky, J. Biol . Chem. 252: 2891 (1977). To prevent binding to the resin, the column may first be treated with 100 μl of bovine serum albumin in the same buffer and centrifuged as above. Bound hybrid antigens can then be quantified by liquid scintillation counting. See Flynn et al., Science 245: 385-390 (1989).

Since ATP hydrolysis can induce release of peptides from many known heat shock proteins, the amount of ATPase activity can often be used to quantify the amount of hybrid antigen bound to the heat shock protein. Examples of how such an assay is performed are described in Flynn et al., Science 245: 385-390 (1989).

Heat shock protein-binding domains are selected so that the hybrid antigen is either biP, hsp70, gp96, or hsp90, or accessory columns such as hsp40, or hsp60, alone or in numerous of the aforementioned heat shock protein families in vitro or in vivo. Will combine together with the impact protein.

Examples of non-limiting peptides that meet this criterion include panning antigen libraries known to bind well to one or more heat shock proteins, as described in Blond-Elguindi et al., Cell 75: 717-728 (1993). Can be identified by:

Leu Phe Trp Pro Phe Glu Trp Ile (SEQ ID NO .: 153);

Asp Gly Val Gly Ser Phe Ile Gly (SEQ ID NO: 154);

Glu Ser Leu Trp Asn Pro Gln Cys (SEQ ID NO: 155);

Leu His Phe Asp Val Leu Trp Arg (SEQ ID NO: 156);

Cys His Leu Lys Met Val Pro Trp (SEQ ID NO: 157);

Asn Ser Val Leu Val Cys Glu Leu (SEQ ID NO: 158);

Asp Arg Gly His Ser Thr Tyr Ser (SEQ ID NO: 159);

Asp Val Trp Gly Trp Val Thr Trp (SEQ ID NO .: 160);

Ile Gln Phe Arg Val Glu Leu Phe (SEQ ID NO: 161);

Leu Trp Leu Glu Leu Ser Leu Ser (SEQ ID NO: 162);

Val Gly Ile Cys Ala Leu Phe Gly (SEQ ID NO: 163);

Pro Tyr Pro Ser Gly Leu Asp Ser (SEQ ID NO: 164);

Phe Trp Gly Val Leu Pro Tyr Pro (SEQ ID NO: 165);

Phe Thr His Gly Ile Ser Leu Tyr (SEQ ID NO: 166);

Asn His Ser Phe Gly Gly Ser Thr (SEQ ID NO: 167);

Val Asp Tyr Val Tyr Phe His His (SEQ ID NO: 168);

Phe Leu Asp Ile Ile Gly Tyr Gly (SEQ ID NO: 169);

Trp Asp Asp Leu Leu His Gly Arg (SEQ ID NO: 170);

Leu Arg Leu Leu Gly Thr Leu Asn (SEQ ID NO: 171);

Phe Glu Gln His Asn Gln Glu Pro (SEQ ID NO: 172);

Phe Val Gly Thr Val Thr Trp Ser (SEQ ID NO: 173);

Leu Trp Ala Leu Thr Tyr Arg Gly (SEQ ID NO: 174);

Ser Trp Gly Ser Asn Gly Gly Phe (SEQ ID NO: 175);

Asp Met Trp Arg Arg Ala Val Gln (SEQ ID NO: 176);

Cys Arg Val Ile Tyr His Ala Thr (SEQ ID NO: 177);

Met Val Val Ala Arg Cys Gly His (SEQ ID NO: 178);

His Met Trp Ile Asn Trp Val Gln (SEQ ID NO: 179);

Cys Ala Gly Arg Cys Phe Gly Tyr (SEQ ID NO: 180);

Cys Thr His Val Leu Ala Tyr Ser (SEQ ID NO: 181);

Ser Trp Met Pro Trp Leu Thr Met (SEQ ID NO: 182);

Leu Glu Trp Cys Ile Trp Arg Tyr (SEQ ID NO: 183);

Cys Leu Ala Cys Ile Ile His Ser (SEQ ID NO: 184);

Phe Trp Phe Pro Trp Asp Arg Ser (SEQ ID NO: 185);

Trp Arg Thr Gly Val Phe His Gly (SEQ ID NO: 186);

Met His Leu Arg Val Ala Asp Arg (SEQ ID NO: 187);

Ala Leu Asp Leu Tyr Leu Tyr Val (SEQ ID NO: 188);

Phe Phe Trp Phe Thr Leu Lys Glu (SEQ ID NO: 189);

Leu Ser Phe Ala Gly Trp Gly Val (SEQ ID NO: 190);

Met Met Met Leu Gly Arg Ala Pro (SEQ ID NO: 191);

Trp Ser Phe Tyr Thr Trp Leu Asn (SEQ ID NO: 192);

Phe Val Trp Met Arg Trp Ile Asp (SEQ ID NO: 193);

Met Gln Val Asn Thr Pro Asp Asn (SEQ ID NO: 194);

Phe Trp Gly Trp Leu Ile Pro Trp (SEQ ID NO: 195);

Trp Gly Trp Val Trp Trp Asp (SEQ ID NO: 196);

Trp Ile Phe Pro Trp Ile Gln Leu (SEQ ID NO: 197);

Trp Met Phe Asn Trp Pro Trp Tyr (SEQ ID NO: 198);

Met Asn Met Ile Val Leu Asp Lys (SEQ ID NO: 199);

Phe Trp Gly Trp Pro Gly Trp Ser (SEQ ID NO: 200);

Trp Leu Ile Arg Val Gly Thr Ala (SEQ ID NO: 201);

Gly Leu Leu Thr His Leu Ile Trp (SEQ ID NO: 202);

Leu Trp Trp Leu Asn Val His Gly (SEQ ID NO: 203);

Trp Trp Trp Ile Asn Asp Glu Ser (SEQ ID NO: 204);

Ala Asn Pro Ser Leu Ala Thr Tyr (SEQ ID NO: 205);

Trp Leu Gln Gly Trp Trp Gly Trp (SEQ ID NO: 206);

Met Met Pro Val Thr Ser Phe Arg (SEQ ID NO: 207);

Gly Trp Met Asp Trp Trp Tyr Tyr (SEQ ID NO: 208);

Leu Ala Ser Met Arg Asn Ser Met (SEQ ID NO: 209);

Asp Leu Met Arg Trp Leu Gly Leu (SEQ ID NO: 210);

Tyr Phe Tyr Ala Trp Trp Leu Asp (SEQ ID NO: 211);

Leu Gly His Leu Trp Thr Gln Val (SEQ ID NO: 212);

Leu Trp Trp Arg Asp Val Met Ala (SEQ ID NO: 213);

Phe Ile Trp Trp Ala Pro Leu Ala (SEQ ID NO: 214);

Gly Ser Val Gly Gly Gly Val Val (SEQ ID NO: 215);

Asp Ser His Asp Asp Trp Arg Met (SEQ ID NO: 216);

Phe Trp Arg Phe Asp Tyr Tyr Phe (SEQ ID NO: 217);

Trp Thr Trp Trp Glu Trp Leu Ala (SEQ ID NO: 218);

Trp Leu Tip Asp Trp Ile Val Val (SEQ ID NO: 219);

Gly Trp Thr Trp Phe Phe Asp Met (SEQ ID NO: 220);

Ala Trp Trp Gln His Phe Ile Val (SEQ ID NO: 221);

Leu Trp Trp Asp Ile Ile Thr Gly (SEQ ID NO: 222);

Phe Thr Tyr Gly Ser Arg Trp Leu (SEQ ID NO: 223);

Phe Ser Leu Trp Pro Leu Ala Trp (SEQ ID NO: 224);

Gly Ile Ile Leu Gly Tyr Asn Val (SEQ ID NO: 225);

Ser Trp Met Thr Trp Ile Glu His (SEQ ID NO: 226);

Gly Trp Trp Val Thr Trp Pro Trp (SEQ ID NO: 227);

Val Val Ser Pro Trp Trp Leu Gly (SEQ ID NO: 228);

Asn Val Leu Ser Arg Gly Phe Ser (SEQ ID NO: 229);

Ser Phe Glu Ser Leu Gly Gly Leu (SEQ ID NO .: 230);

Ile Thr Lys Gly Ser Ser Phe Pro (SEQ ID NO: 231);

Leu Asp Trp Ala Arg Lys Leu Arg (SEQ ID NO: 232);

Thr Ala Trp Asn Leu Leu Gly Tyr (SEQ ID NO: 233);

Phe Gly Gln Gly Ile Lys His Val (SEQ ID NO: 234);

Asp Val Val Trp Gin Arg Leu Leu (SEQ ID NO: 235);

Tyr Val Asp Arg Phe Ile Gly Trp (SEQ ID NO: 236);

Lys Met Ala Arg Pro Glu Gly Asn (SEQ ID NO: 237);

Leu Gly Arg Trp Gly His Glu Ser (SEQ ID NO: 238);

Ser Ile Trp Ser Leu Leu Val Leu (SEQ ID NO: 239);

Val Trp Leu Asp Leu Leu Leu Ser (SEQ ID NO: 240);

Tyr Leu Thr Asp Ser Leu Phe Gly (SEQ ID NO: 241);

Thr Trp Trp Pro Ser Ile Thr Trp (SEQ ID NO: 242);

Tyr Gly Leu Trp Trp Phe Pro Trp (SEQ ID NO: 243);

Phe Ser Pro Ala Asp Thr Arg Tyr (SEQ ID NO: 244);

Cys Asn Arg Leu Gln He Asp Cys (SEQ ID NO: 245);

Ser Leu Val Ala Ala Arg Asn Leu (SEQ ID NO: 246);

Phe Thr He His Asn Val Ala Val (SEQ ID NO: 247);

Met Gly Pro Leu Gly Pro Leu Leu (SEQ ID NO: 248);

Arg Gln Leu Ser Glu Leu Phe Val (SEQ ID NO: 249);

Arg Val Val Cys Gln Ala Leu Leu (SEQ ID NO: 250);

Trp Pro His Leu Trp Trp Leu Asp (SEQ ID NO: 251);

Trp Met Asp Trp Val Trp His Thr (SEQ ID NO: 252);

Trp Trp Gly Tyr Leu Ile Cys Gln (SEQ ID NO: 253);

Phe Arg Gly Leu Ser Glu Gly Pro (SEQ ID NO: 254);

Ser Trp Phe Asp Trp Leu Val Ala (SEQ ID NO: 255);

Val Val Met Trp Tyr Ser Val Asp (SEQ ID NO: 256);

Trp Gly Trp Ser Leu Ala Thr (SEQ ID NO: 257);

Leu Gly Trp Phe Asp Arg Phe Phe (SEQ ID NO: 258);

Ala Trp Trp Trp Pro Thr Tyr Val (SEQ ID NO: 259);

Gly Phe Leu Ser Ser Trp Phe Leu (SEQ ID NO: 260);

Gly Val Ile Asn Cys Ala Gly Thr (SEQ ID NO: 261);

Val Cys Ala Arg Ala Ala His Leu (SEQ ID NO: 262);

Gly Asn Ser Tyr Gly Asp Gly Gly (SEQ ID NO: 263);

Gly Phe Leu Ser Ser Trp Phe Leu (SEQ ID NO: 264);

Phe Asp Gln Pro Gly Arg Phe Leu (SEQ ID NO: 265);

Arg Ser His Ala Thr Gly Val Val (SEQ ID NO: 266);

Gly Tyr Trp Ala Met Met Ser Trp (SEQ ID NO: 267);

Cys His Ser Met Trp Asp Gly Leu (SEQ ID NO: 268);

Phe Ile Trp Arg Gly Trp Pro His (SEQ ID NO: 269);

Leu Ser Phe Leu Gly Gly Arg Leu (SEQ ID NO: 270);

Phe Ser Gly Val Arg Gln Pro Asn (SEQ ID NO: 271);

Trp Gly Trp Met Pro Phe Tyr Tyr (SEQ ID NO: 272);

Phe Thr Arg Pro Ala Val Val Asp (SEQ ID NO: 273);

Asp Leu Trp Thr Trp Leu Gly Leu (SEQ ID NO: 274);

Cys Asp Thr Ala Ala Val Ala Asp (SEQ ID NO: 275);

Trp Trp Val Lys His His Met Leu (SEQ ID NO: 276);

Ile Ala Phe Leu Arg Asp Asn Arg (SEQ ID NO: 277);

Leu Ala Arg Pro Asp His Tyr Ser (SEQ ID NO: 278);

Met Glu Ser Lys Arg Trp Thr Val (SEQ ID NO: 279);

Met Ile Leu Lys Gly Tyr Ser Aig (SEQ ID NO: 280);

Ala Pro Ser Asp Tyr Asp Glu Ser (SEQ ID NO: 281);

His Trp Leu Arg Ser Lys Arg Thr (SEQ ID NO: 282);

Gly Ala Arg Val Trp Asn Tyr Gln (SEQ ID NO: 283);

Leu Ser Asn Trp Asn Met Arg Leu (SEQ ID NO: 284);

Cys Gly Ala Ala Gln Gln Gly Met (SEQ ID NO: 285); And

Gly Ser Ser Met Val Val Gln Arg (SEQ ID NO: 286). Using this technique, Blond-Elguindi concluded that the heat shock protein BiP recognizes a polypeptide comprising a seven-hepatic region with the following sequence.

Hy (Trp / X) HyXHyXHy (SEQ ID NO .: 29)

Hy here means non-hydrophilic amino acid residues, in particular tryptophan, leucine or phenylalanine (SEQ ID NO: 30), and X can be any amino acid. High affinity heat-shock protein binding sequences comprising such motifs include His Trp Asp Phe Ala Trp Pro Trp (SEQ ID NO: 1); And Phe Trp Gly Leu Trp Pro Trp Glu (SEQ ID NO: 4).

Other heat shock protein binding motifs can also be identified. For example, Auger et al., Nature Medicine 2: 306-310 (1996) identified two pentapeptide binding motifs.

Type of HLA-DR associated with rheumatoid arthritis

Gln Lys Arg Ala Ala (SEQ ID NO .: 5) and

Arg Arg Arg Ala Ala (SEQ ID NO .: 6)

Combine with heat shock protein.

Heat shock binding motifs have also been identified as consisting of 7 to 15 residues of long peptides rich in non-hydrophilic amino acids.

Lys Arg Gln Ile Tyr Asp Leu Glu Met Asn Arg Leu Gly Lys (SEQ ID NO: 287);

Leu Ser Ser Leu Phe Arg Pro Lys Arg Arg Pro Ile Tyr Lys Ser (SEQ ID NO: 288);

Lys Leu Ile Gly Val Leu Ser Ser Leu Phe Arg Pro Lys (SEQ ID NO: 289);

Arg Arg Pro Ile Tyr Lys Ser Asp Val Gly Met Ala His Phe Arg (SEQ ID NO: 290);

Cys Lys Ile Gln Ser Thr Pro Val Lys Gln Ser (SEQ ID NO: 291);

Tyr His Cys Asp Gly Phe Gln Asn Glu (SEQ ID NO: 292);

Val Gly Ile Asp Leu Gly Thr Thr Tyr Ser Cys (SEQ ID NO: 293);

Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys (SEQ ID NO: 294)

(Flynn et al., Science 245: 385-390 (1989)),

In addition, other heat shock protein binding peptides include:

Gly Lys Trp Val Tyr Ile (SEQ ID NO: 295);

Ala Lys Arg Glu Thr Lys (SEQ ID NO: 296);

Lys Trp Val His Leu Phe (SEQ ID NO: 297);

Arg Leu Val Leu Val Leu (SEQ ID NO: 298);

Trp Lys Trp Gly Ile Tyr (SEQ ID NO: 299);

Ser Ser His Ala Ser Ala (SEQ ID NO: 300);

Trp Gly Pro Trp Ser Phe (SEQ ID NO: 301);

Ala Ile Pro Gly Lys Val (SEQ ID NO: 302);

Arg Val His Asp Pro Ala (SEQ ID NO: 303);

Arg Ser Val Ser Ser Phe (SEQ ID NO: 304);

Leu Gly Thr Arg Lys Gly (SEQ ID NO: 305);

Lys Asp Pro Leu Phe Asn (SEQ ID NO: 306);

Leu Ser Gln His Thr Asn (SEQ ID NO: 307);

Asn Arg Leu Leu Leu Thr (SEQ ID NO: 308);

Tyr Pro Leu Trp Val Ile (SEQ ID NO: 309);

Leu Leu Ile Ile Asp Arg (SEQ ID NO: 310);

Arg Val Ile Ser Leu Gln (SEQ ID NO: 311);

Glu Val Ser Arg Glu Asp (SEQ ID NO: 312);

Ser Ile Leu Arg Ser Thr (SEQ ID NO: 313);

Pro Gly Leu Val Trp Leu (SEQ ID NO: 314);

Val Lys Lys Leu Tyr Ile (SEQ ID NO .: 315);

Asn Asn Arg Leu Leu Asp (SEQ ID NO: 316);

Ser Lys Gly Arg Trp Gly (SEQ ID NO: 317);

Ile Arg Pro Ser Gly Ile (SEQ ID NO: 318);

Ala Ser Leu Cys Pro Thr (SEQ ID NO: 319);

Asp Val Pro Gly Leu Arg (SEQ ID NO: 320);

Arg His Arg Glu Val Gln (SEQ ID NO: 321);

Leu Ala Arg Lys Arg Ser (SEQ ID NO: 322);

Ser Val Leu Asp His Val (SEQ ID NO: 323);

Asn Leu Leu Arg Arg Ala (SEQ ID NO: 324);

Ser Gly Ile Ser Ala Trp (SEQ ID NO: 325);

Phe Tyr Phe Trp Val Arg (SEQ ID NO: 326);

Lys Leu Phe Leu Pro Leu (SEQ ID NO .: 327);

Thr Pro Thr Leu Ser Asp (SEQ ID NO: 328);

Thr His Ser Leu Ile Leu (SEQ ID NO .: 329);

Leu Leu Leu Leu Ser Arg (SEQ ID NO: 330);

Leu Leu Arg Val Arg Ser (SEQ ID NO: 331);

Glu Arg Arg ser Arg Gly (SEQ ID NO: 332);

Arg Met Leu Gln Leu Ala (SEQ ID NO: 333);

Age Gly Trp Ala Asn Ser (SEQ ID NO: 334);

Arg Pro Phe Tyr Ser Tyr (SEQ ID NO: 335);

Ser Ser Ser Trp Asn Ala (SEQ ID NO: 336);

Leu Gly His Leu Glu Glu (SEQ ID NO .: 337);

Ser Ala Val Thr Asn Thr (SEQ ID NO: 338);

Leu Arg Arg Ala Ser Leu (SEQ ID NO .: 339);

Leu Arg Arg Trp Ser Leu (SEQ ID NO: 340);

Lys Trp Val His Leu Phe (SEQ ID NO: 341);

Asn Arg Leu Leu Leu Thr (SEQ ID NO .: 342);

Ala Arg Leu Leu Leu Thr (SEQ ID NO .: 343);

Asn Ala Leu Leu Leu Th. (SEQ ID NO: 344);

Asn Arg Leu Ala Leu Thr (SEQ ID NO: 345);

Asn Leu Leu Arg Leu Thr (SEQ ID NO: 346);

Asn Arg Leu Trp Leu Thr (SEQ ID NO: 347);

Asn Arg Leu Leu Leu Ala (SEQ ID NO: 348);

Met Gln Glu Arg Ile Thr Leu Lys Asp Tyr Ala Met (SEQ ID NO: 349);

Leu Arg Arg Trp Ser Leu Gly (SEQ ID NO .: 353);

Lys Trp Val His Leu Phe Gly (SEQ ID NO: 354);

Asn Arg Leu Leu Leu Thr Gly (SEQ ID NO: 355);

Ala Arg Leu Leu Leu Thr Gly (SEQ ID NO: 356);

Asn Ala Leu Leu Leu Thr Gly (SEQ ID NO: 357);

Asn Arg Leu Ala Leu Thr Gly (SEQ ID NO: 358);

Asn Leu Leu Arg Leu Thr Gly (SEQ ID NO: 359);

Asn Arg Leu Trp Leu Thr Gly (SEQ ID NO: 360);

Asn Arg Leu Leu Leu Ala Gly (SEQ ID NO .: 361);

Gly Lys Trp Val Tyr Ile Gly (SEQ ID NO: 295);

Ala Lys Arg Glu Thr Lys Gly (SEQ ID NO: 296);

Lys Trp Val His Leu Phe Gly (SEQ ID NO: 297);

Arg Leu Val Leu Val Leu Gly (SEQ ID NO: 298);

Trp Lys Trp Gly Ile Tyr (SEQ ID NO: 299);

Ser Ser His Ala Ser Ala (SEQ ID NO: 300);

Trp Gly Pro Trp Ser Phe (SEQ ID NO: 301);

Ala Ile Pro Gly Lys Val (SEQ ID NO: 302);

Arg Val His Asp Pro Ala Gly (SEQ ID NO: 303);

Arg Ser Val Ser Ser Phe Gly (SEQ ID NO: 304);

Leu Gly Thr Arg Lys Gly Cly (SEQ ID NO: 305);

Lys Asp Pro Leu Phe Asn Gly (SEQ ID NO: 306);

Leu Ser Gln His Thr Asn Gly (SEQ ID NO: 307);

Asn Arg Leu Leu Leu Thr Gly (SEQ ID NO: 308);

Tyr Pro Leu Trp Val Ile Gly (SEQ ID NO: 309);

Leu Leu Ile Ile Asp Arg Gly (SEQ ID NO .: 310);

Arg Val Ile Ser Leu Gln Gly (SEQ ID NO: 311);

Glu Val Ser Arg Glu Asp Gly (SEQ ID NO: 312);

Ser Ile Leu Arg Ser Thr Gly (SEQ ID NO: 313);

Pro Gly Leu Val Trp Leu Gly (SEQ ID NO: 314);

Val Lys Lys Leu Tyr Ile Gly (SEQ ID NO: 315);

Asn Asn Arg Leu Leu Asp Gly (SEQ ID NO: 316);

Ser Lys Gly Arg Trp Gly Gly (SEQ ID NO: 317);

Ile Arg Pro Ser Gly Ile Gly (SEQ ID NO .: 318);

Ala Ser Leu Cys Pro Thr Gly (SEQ ID NO: 319);

Asp Val Pro Gly Leu Arg Gly (SEQ ID NO: 320);

Arg His Arg Glu Val Gln Gly (SEQ ID NO: 321);

Leu Ala Arg Lys Arg Ser Gly (SEQ ID NO .: 322):

Ser Val Leu Asp His Val Gly (SEQ ID NO: 323);

Asn Leu Leu Arg Arg Ala Gly (SEQ ID NO: 324);

Ser Gly Ile Ser Ala Trp Gly (SEQ ID NO: 325);

Phe Tyr Phe Trp Val Arg Gly (SEQ ID NO: 326);

Lys Leu Phe Leu Pro Leu Gly (SEQ ID NO .: 327)

Thr Pro Thr Leu Ser Asp Gly (SEQ ID NO: 328);

Thr His Ser Leu Ile Leu Gly (SEQ ID NO .: 329);

Leu Leu Leu Leu Ser Arg Gly (SEQ ID NO: 330);

Leu Leu Arg Val Arg Ser Gly (SEQ ID NO: 331);

Glu Arg Arg ser Arg Gly Gly (SEQ ID NO: 332);

Arg Met Leu Gln Leu Ala Gly (SEQ ID NO: 333);

Age Gly Trp Ala Asn Ser Gly (SEQ ID NO: 334);

Arg Pro Phe Tyr Ser Tyr Gly (SEQ ID NO: 335);

Ser Ser Ser Trp Asn Ala Gly (SEQ ID NO: 336);

Leu Gly His Leu Glu Glu Gly (SEQ ID NO .: 337); And

Ser Ala Val Thr Asn Thr Gly (SEQ ID NO: 338); Gragerov et at., J. Molec. Biol. 235: 848-854 (1994).

Other heat shock protein binding domains are Phe Tyr Gln Leu Ala Leu Thr (SEQ ID NO:), Phe Tyr Gln Leu Ala Leu Thr Trp (SEQ ID NO:), Arg Lys Leu Phe Phe Asn Leu Arg (SEQ ID NO:), Arg Lys Leu Phe Phe Asn Leu Arg Trp (SEQ ID NO:), Lys Phe Glu Arg Gln (SEQ ID NO:), Asn Ile Val Arg Lys Lys Lys (SEQ ID NO:), and Arg Gly Tyr Val Tyr Gln Gly Leu (SEQ ID NO: ).

Moreover, other heat shock protein binding domains include those described in W09922761. Xaa can be any amino acid.

HTTVYGAG (SEQ ID NO: 82);

TETPYPTG (SEQ ID NO: 83);

LTTPFSSG (SEQ ID NO: 84);

GVPLTMDG (SEQ ID NO: 85);

KLPTVLRG (SEQ ID NO: 86);

CRFHGNRG (SEQ ID NO: 87);

YTRDFEAG (SEQ ID NO: 88);

SSAAGPRG (SEQ ID NO: 89);

SLIQYSRG (SEQ ID NO: 90);

DALMWPP Xaa G (SEQ ID NO: 91);

SS Xaa SLYIG (SEQ ID NO: 92);

FNTSTRTG (SEQ ID NO: 93)

TVQHVAFG (SEQ ID NO: 94);

DYSFPPLG (SEQ ID NO: 95);

VGSMESLG (SEQ ID NO: 96);

F Xaa PMI Xaa SG (SEQ ID NO: 97);

APPRVTMG (SEQ ID NO: 98);

IATKTPKG (SEQ ID NO: 99);

KPPLFQIG (SEQ ID NO: 100);

YHTAHNMG (SEQ ID NO: 101);

SYIQATHG (SEQ ID NO: 102);

SSFATFLG (SEQ ID NO: 103);

TTPPNFAG (SEQ ID NO: 104);

ISLDPRMG (SEQ ID NO: 105);

SLPLFGAG (SEQ ID NO: 106);

NLLKTTLG (SEQ ID NO: 107):

DQNLPRRG (SEQ ID NO: 108);

SHFEQLLG (SEQ ID NO: 109);

TPQLHHGG (SEQ ID NO: 110);

APLDRITG (SEQ ID NO: 111);

FAPLIAHG (SEQ ID NO: 112);

SWIQTFMG (SEQ ID NO: 113);

NTWPHMYG (SEQ ID NO .: 114);

EPLPTTLG (SEQ ID NO: 115);

HGPHLFNG (SEQ ID NO: 116);

YLNSTLAG (SEQ ID NO: 117);

HLHSPSGG (SEQ ID NO: 118);

TLPHRLNG (SEQ ID NO: 119);

SSPREVHG (SEQ ID NO: 120);

NQVDTARG (SEQ ID NO: 121);

YPTPLLTG (SEQ ID NO: 122);

HPAAFPWG (SEQ ID NO: 123);

LLPHSSAG (SEQ ID NO: 124);

LETYTASG (SEQ ID NO: 125);

KYVPLPPG (SEQ ID NO: 126);

APLALHAG (SEQ ID NO: 127);

YESLLTKG (SEQ ID NO: 128);

SHAASGTG (SEQ ID NO: 129);

GLATVKSG (SEQ ID NO: 130);

GATSFGLG (SEQ ID NO: 131);

KPPGPVSG (SEQ ID NO: 132);

TLYVSGNG (SEQ ID NO: 133);

HAPFKSQG (SEQ ID NO: 134);

VAFTRLPG (SEQ ID NO: 135);

LPTRTPAG (SEQ ID NO: 136);

ASFDLLIG (SEQ ID NO: 137);

RMNTEPPG (SEQ ID NO: 138);

KMTPLTTG (SEQ ID NO: 139);

ANATPLLG (SEQ ID NO: 140);

TIWPPPVG (SEQ ID NO: 141);

QTKVMTTG (SEQ ID NO: 142);

NHAVFASG (SEQ ID NO: 143);

LHAA Xaa TSG (SEQ ID NO: 144);

TWQPYFHG (SEQ ID NO: 145);

APLALHAG (SEQ ID NO: 146);

TAHDLTVG (SEQ ID NO: 147);

NMTNMLTG (SEQ ID NO: 148);

GSGLSQDG (SEQ ID NO: 149);

TPIKTIYG (SEQ ID NO: 150);

SHLYRSSG (SEQ ID NO: 151);

YTLVQPL (SEQ ID NO: 152);

TPDITPTK (SEQ ID NO: 153);

TYPDLRY (SEQ ID NO: 154);

DRTHATS (SEQ ID NO: 155);

MSTTFYS (SEQ ID NO: 156);

YQHAVQT (SEQ ID NO: 157);

FPFSAST (SEQ ID NO: 158);

SSFPPLD (SEQ ID NO: 159);

MAPSPPH (SEQ ID NO: 160);

SSFPDLL (SEQ ID NO: 161);

HSYNRLP (SEQ ID NO: 162);

HLTHSQR (SEQ ID NO: 163);

QAAQSRS (SEQ ID NO: 164);

FATHHIG (SEQ ID NO: 165);

SMPEPLI (SEQ ID NO: 166);

IPRYHLI (SEQ ID NO: 167);

SAPHMTS (SEQ ID NO: 168);

KAPVWAS (SEQ ID NO: 169);

LPHWLLI (SEQ ID NO: 170);

ASAGYQl (SEQ ID NO: 171);

VTPKTGS (SEQ ID NO: 172);

EHPMPVL (SEQ ID NO: 173);

VSSFVTS (SEQ ID NO: 174);

STHFTWP (SEQ ID NO: 175);

GQWWSPD (SEQ ID NO: 176);

GPPHQDS (SEQ ID NO: 177);

NTLPSTI (SEQ ID NO: 178);

HQPSRWV (SEQ ID NO: 179);

YGNPLQP (SEQ ID NO: 180);

FHWWWQP (SEQ ID NO: 181);

ITLKYPL (SEQ ID NO: 182);

FHWPWLF (SEQ ID NO: 153);

TAQDSTG (SEQ ID NO: 184);

FHWWWQP (SEQ ID NO: 185);

FHWWDWW (SEQ ID NO: 186);

EPFFRMQ (SEQ ID NO: 187);

TWWLNYR (SEQ ID NO: 188);

FHWWWQP (SEQ ID NO: 189);

QPSHLRW (SEQ ID NO: 190);

SPASPVY (SEQ ID NO: 191);

FHWWWQP (SEQ ID NO: 192);

HPSNQAS (SEQ ID NO: 193);

NSAPRPV (SEQ ID NO: 194);

QLWSIYP (SEQ ID NO: 195);

SWPFFDL (SEQ ID NO: 196);

DTTLPLH (SEQ ID NO: 197);

WHWQMLW (SEQ ID NO: 198);

DSFRTPV (SEQ ID NO: 199);

TSPLSLL (SEQ ID NO: 200);

AYNYVSD (SEQ ID NO: 201);

RPLHDPM (SEQ ID NO: 202);

WPSTTLF (SEQ ID NO: 203);

ATLEPYR (SEQ ID NO: 204);

SMTVLRP (SEQ ID NO: 205);

QIGAPSW (SEQ ID NO: 206);

APDLYVP (SEQ ID NO: 207);

RMPPLLP (SEQ ID NO: 208);

AKATPEH (SEQ ID NO: 209);

TPPLRIN (SEQ ID NO: 210);

LPIHAPH (SEQ ID NO: 211);

DLNAYTH (SEQ ID NO: 212);

VTLPNFH (SEQ ID NO: 213);

NSRLPTL (SEQ ID NO: 214);

YPHPSRS (SEQ ID NO: 215);

GTAHFMY (SEQ ID NO: 216);

YSLLPTR (SEQ ID NO: 217);

LPRRTLL (SEQ ID NO: 218);

TSTLLWK (SEQ ID NO: 219);

TSDMKPH (SEQ ID NO: 220);

TSSYLAL (SEQ ID NO: 221);

NLYGPHD (SEQ ID NO: 222);

LETYTAS (SEQ ID NO: 223);

AYKSLTQ (SEQ ID NO: 224);

STSVYSS (SEQ ID NO: 225);

EGPLRSP (SEQ ID NO: 226);

TTYHALG (SEQ ID NO: 227);

VSIGHPS (SEQ ID NO: 228);

THSHRPS (SEQ ID NO: 229);

ITNPLTT (SEQ ID NO: 230);

SIQAHHS (SEQ ID NO: 231);

LNWPRVL (SEQ ID NO: 232);

YYYAPPP (SEQ ID NO: 233);

SLWTRLP (SEQ ID NO: 234);

NVYHSSL (SEQ ID NO: 235);

NSPHPPT (SEQ ID NO: 236);

VPAKPRH (SEQ ID NO: 237);

HNLHPNR (SEQ ID NO: 238);

YTTHRWL (SEQ ID NO: 239);

AVTAAIV (SEQ ID NO: 240);

TLMHDRV (SEQ ID NO: 241);

TPLKVPY (SEQ ID NO: 242);

FINQQYH (SEQ ID NO: 243);

SIHPSMA (SEQ ID NO: 244);

HTTVYGA (SEQ ID NO: 245);

TETPYPT (SEQ ID NO: 246);

LTTPFSS (SEQ ID NO: 247);

GVPLTMD (SEQ ID NO: 248);

KLPTVLR (SEQ ID NO: 249);

CRFHGNR (SEQ ID NO: 250);

YTRDFEA (SEQ ID NO: 251);

SSAAGPR (SEQ ID NO: 252);

SLIQYSR (SEQ ID NO: 253);

DALMWP Xaa (SEQ ID NO: 254);

SS Xaa SLYI (SEQ ID NO: 255);

FNTSTRT (SEQ ID NO: 256);

TVQHVAF (SEQ ID NO: 257);

DYSFPPL (SEQ ID NO: 258);

VGSMESL (SEQ ID NO: 259);

F Xaa PMI Xaa S (SEQ ID NO: 260);

APPRVTM (SEQ ID NO: 261);

IATKTPK (SEQ ID NO: 262);

KPPLFQI (SEQ ID NO: 263);

YHTAHNM (SEQ ID NO: 264);

SYIQATH (SEQ ID NO: 265);

SSFATFL (SEQ ID NO: 266);

TTPPNFA (SEQ ID NO: 267);

ISLDPRM (SEQ ID NO: 268);

SLPLFGA (SEQ ID NO: 269);

NLLKTTL (SEQ ID NO: 270);

DQNLPRR (SEQ ID NO: 271);

SHFEQLL (SEQ ID NO: 272);

TPQLHHG (SEQ ID NO: 273);

APLDRIT (SEQ ID NO: 274);

FAPLIAH (SEQ ID NO: 275);

SWIQTFM (SEQ ID NO: 276);

NTWPHMY (SEQ ID NO: 277);

EPLPTTL (SEQ ID NO: 278);

HGPHLFN (SEQ ID NO: 279);

YLNSTLA (SEQ ID NO: 280);

HLHSPSG (SEQ ID NO: 281);

TLPHRLN (SEQ ID NO: 282);

SSPREVH (SEQ ID NO: 283);

NQVDTAR (SEQ ID NO: 284);

YPTPLLT (SEQ ID NO: 285);

HPAAFPW (SEQ ID NO: 286);

LLPHSSA (SEQ ID NO: 287);

LETYTAS (SEQ ID NO: 288);

KYVPLPP (SEQ ID NO: 289);

APLALHA (SEQ ID NO: 290);

YESLLTK (SEQ ID NO: 291);

SHAASGT (SEQ ID NO: 292);

GLATVKS (SEQ ID NO: 293);

GATSFGL (SEQ ID NO: 294);

KPPGPVS (SEQ ID NO: 295);

TLYVSGN (SEQ ID NO: 296);

HAPFKSQ (SEQ ID NO: 297);

VAFTRLP (SEQ ID NO: 298);

LPTRTPA (SEQ ID NO: 299);

ASFDLLI (SEQ ID NO: 300);

RMNTEPP (SEQ ID NO: 301);

KMTPLTT (SEQ ID NO: 302);

ANATPLL (SEQ ID NO: 303);

TIWPPPV (SEQ ID NO: 304);

QTKVMTT (SEQ ID NO: 305);

NHAVFAS (SEQ ID NO: 306);

LHAA Xaa TS (SEQ ID NO: 307);

TWQPYFH (SEQ ID NO: 308);

APLALHA (SEQ ID NO: 309);

TAHDLTV (SEQ ID NO: 310);

NMTNMLT (SEQ ID NO: 311);

GSGLSQD (SEQ ID NO: 312);

TPIKTIY (SEQ ID NO: 313);

SHLYRSS (SEQ ID NO: 314);

HGQAWQF (SEQ ID NO: 315); And

FHWWW (SEQ ID NO: 317).

The aforementioned heat shock protein binding domains are merely examples of various peptides among peptides and non-peptide heat shock protein binding molecules that can be used in the use of the present invention. In other embodiments, the heat shock protein binding domain can be directed to bind to other portions of a mammalian heat shock protein as described above, and the heat shock protein-binding domain of the invention binds to any particular portion of the heat shock protein molecule. It is not limited to doing. In a non-limiting example, the peptide IFAGIKKKAERADLIAYLKQATAK (Greene et al., 1995, J. Biol. Chem. 270: 2967-2973; SEQ ID NO: 331) or a heat shock protein-binding fragment of this peptide is previously associated with a heat shock protein. -Can be used anywhere in the conjugates of the present invention to facilitate binding of selected molecules. In addition to the aforementioned peptides that bind to heat shock proteins, binding can be accomplished through the use of organic molecules or compounds with heat shock protein binding activity. For example, suitable molecules are benzoquinone ansamycins such as herbimycin A, geldanamycin, macmimycin I, mimosamycin, and kuwaitimycin. ansamycin) antibiotic members (Omura et al., 1979, J. Antibiotics 32: 255-261; see also W09922761, which is incorporated herein by reference in its entirety), or structurally related compounds, and analogs thereof Or derivatives. These molecules utilize chemical means established in the antigenic domains of the invention by peptide linkers to produce hybrid antigens that can bind heat shock proteins in vivo or ex vivo and can exert an immune response against the antigens presented herein. Can be bonded through.

As filed on February 12, 2004, and currently filed together, and described in commonly-owned application number 10 / 776,521, the document is incorporated herein in its entirety by reference. Inclusion, including a tryptophan residue (Trp, or single amino acid code W) at the C-terminus of a heat shock protein binding domain, such as but not limited to those identified above, may indicate binding to a heat shock protein. Promote. Increased binding to heat shock proteins has been shown to increase the ability of hybrid antigens to induce an immune response to the antigenic domains of hybrid antigens, whether administered in combination with heat shock proteins or alone. Increased immune response is associated with increased efficiency of disease treatment. Another example of a method of determining affinity is described in PCT / US96 / 13363 (W09706821), which is incorporated herein in its entirety by reference.

Among the aforementioned choices of heat shock protein binding domains, those which are preferred in the present invention as part of a hybrid antigen comprising the antigenic domain and peptide linker of the invention include the following heat shock protein binding domains:

Gly Lys Trp Val Tyr Ile Gly Trp (SEQ ID NO:);

Ala Lys Arg Glu Thr Lys Gly Trp (SEQ ID NO:);

Lys Trp Val His Leu Phe Gly Trp (SEQ ID NO:);

Arg Leu Val Leu Val Leu Gly Trp (SEQ ID NO:);

Trp Lys Trp Gly Ile Tyr Gly Trp (SEQ ID NO:);

Ser Ser His Ala Ser Ala Gly Trp (SEQ ID NO:);

Trp Gly Pro Trp Ser Phe Gly Trp (SEQ ID NO:);

Ala Ile Pro Gly Lys Val Gly Trp (SEQ ID NO:);

Arg Val His Asp Pro Ala Gly Trp (SEQ ID NO:);

Arg Ser Val Ser Ser Phe Gly Trp (SEQ ID NO:);

Leu Gly Thr Arg Lys Gly Gly Trp (SEQ ID NO:);

Lys Asp Pro Leu Phe Asn Gly Trp (SEQ ID NO:);

Leu Ser Gln His Thr Asn Gly Trp (SEQ ID NO:);

Asn Arg Leu Leu Leu Thr Gly Trp (SEQ ID NO:);

Tyr Pro Leu Trp Val Ile Gly Trp (SEQ ID NO:);

Leu Leu Ile Ile Asp Arg Gly Trp (SEQ ID NO:);

Arg Val Ile Ser Leu Gln Gly Trp (SEQ ID NO:);

Glu Val Ser Arg Glu Asp Gly Trp (SEQ ID NO:);

Ser Ile Leu Arg Ser Thr Gly Trp (SEQ ID NO:);

Pro Gly Leu Val Trp Leu Gly Trp (SEQ ID NO:);

Val Lys Lys Leu Tyr Ile Gly Trp (SEQ ID NO:);

Asn Asn Arg Leu Leu Asp Gly Trp (SEQ ID NO:);

Ser Lys Gly Arg Trp Gly Gly Trp (SEQ ID NO:);

Ile Arg Pro Ser Gly Ile Gly Trp (SEQ ID NO:);

Ala Ser Leu Cys Pro Thr Gly Trp (SEQ ID NO:);

Asp Val Pro Gly Leu Arg Gly Trp (SEQ ID NO:);

Arg His Arg Glu Val Gln Gly Trp (SEQ ID NO:);

Leu Ala Arg Lys Arg Ser Gly Trp (SEQ ID NO:);

Ser Val Leu Asp His Val Gly Trp (SEQ ID NO:);

Asn Leu Leu Arg Arg Ala Gly Trp (SEQ ID NO:);

Ser Gly Ile Ser Ala Trp Gly Trp (SEQ ID NO:);

Phe Tyr Phe Trp Val Arg Gly Trp (SEQ ID NO:);

Lys Leu Phe Leu Pro Leu Gly Trp (SEQ ID NO:);

Thr Pro Thr Leu Ser Asp Gly Trp (SEQ ID NO:);

Thr His Ser Leu Ile Leu Gly Trp (SEQ ID NO:);

Leu Leu Leu Leu Ser Arg Gly Trp (SEQ ID NO:);

Leu Leu Arg Val Arg Ser Gly Trp (SEQ ID NO:);

Glu Arg Arg ser Arg Gly Gly Trp (SEQ ID NO:);

Arg Met Leu Gln Leu Ala Gly Trp (SEQ ID NO:);

Age Gly Trp Ala Asn Ser Gly Trp (SEQ ID NO:);

Arg Pro Phe Tyr Ser Tyr Gly Trp (SEQ ID NO:);

Ser Ser Ser Trp Asn Ala Gly Trp (SEQ ID NO:);

Leu Gly His Leu Glu Glu Gly Trp (SEQ ID NO:);

Ser Ala Val Thr Asn Thr Gly Trp (SEQ ID NO:);

Phe Tyr Gln Leu Ala Leu Thr (SEQ ID NO:);

Phe Tyr Gln Leu Ala Leu Thr Trp (SEQ ID NO.),

Arg Lys Leu Phe Phe Asn Leu Arg (SEQ ID NO:),

Arg Lys Leu Phe Phe Asn Leu Arg Trp (SEQ ID NO:),

Lys Phe Glu Arg Gln (SEQ ID NO.),

Asn Ile Val Arg Lys Lys Lys (SEQ ID NO.), And

Arg Gly Tyr Val Tyr Gin Gly Leu (SEQ ID NO:).

Other non-limiting examples of heat shock protein binding domains having Trp residues at the ends useful in various aspects of the present invention include:

Asn Leu Leu Arg Leu Thr Gly Trp (SEQ ID NO: 350);

Phe Tyr Gln Leu Ala Leu Tyr Trp (SEQ ID NO .: 351);

Arg Lys Leu Phe Phe Asn Leu Arg Trp (SEQ ID NO: 352);

Gly Lys Trp Val Tyr Ile Gly Trp (SEQ ID NO: 295);

Ala Lys Arg Glu Thr Lys Gly Trp (SEQ ID NO: 296);

Lys Trp Val His Leu Phe Gly Trp (SEQ ID NO: 297);

Arg Leu Val Leu Val Leu Gly Trp (SEQ ID NO: 298);

Trp Lys Trp Gly Ile Tyr Gly Trp (SEQ ID NO: 299);

Ser Ser His Ala Ser Ala Gly Trp (SEQ ID NO: 300);

Trp Gly Pro Trp Ser Phe Gly Trp (SEQ ID NO: 301);

Ala Ile Pro Gly Lys Val Gly Trp (SEQ ID NO: 302);

Arg Val His Asp Pro Ala Gly Trp (SEQ ID NO: 303);

Arg Ser Val Ser Ser Phe Gly Trp (SEQ ID NO: 304);

Leu Gly Thr Arg Lys Gly Gly Trp (SEQ ID NO: 305);

Lys Asp Pro Leu Phe Asn Gly Trp (SEQ ID NO: 306);

Leu Ser Gln His Thr Asn Gly Trp (SEQ ID NO: 307);

Asn Arg Leu Leu Leu Thr Gly Trp (SEQ ID NO: 308);

Tyr Pro Leu Trp Val Ile Gly Trp (SEQ ID NO: 309);

Leu Leu Ile Ile Asp Arg Gly Trp (SEQ ID NO .: 310);

Arg Val Ile Ser Leu Gln Gly Trp (SEQ ID NO: 311);

Glu Val Ser Arg Glu Asp Gly Trp (SEQ ID NO .: 312);

Ser Ile Leu Arg Ser Thr Gly Trp (SEQ ID NO: 313);

Pro Gly Leu Val Trp Leu Gly Trp (SEQ ID NO: 314);

Val Lys Lys Leu Tyr He Gly Trp (SEQ ID NO: 315);

Asn Asn Arg Leu Leu Asp Gly Trp (SEQ ID NO: 316);

Ser Lys Gly Arg Trp Gly Gly Trp (SEQ ID NO: 317);

Ile Arg Pro Ser Gly Ile Gly Trp (SEQ ID NO: 318);

Ala Ser Leu Cys Pro Thr Gly Trp (SEQ ID NO: 319);

Asp Val Pro Gly Leu Arg Gly Trp (SEQ ID NO: 320);

Arg His Arg Glu Val Gln Gly Trp (SEQ ID NO: 321);

Leu Ala Arg Lys Arg Ser Gly Trp (SEQ ID NO .: 322);

Ser Val Leu Asp His Val Gly Trp (SEQ ID NO: 323);

Asn Leu Leu Arg Arg Ala Gly Trp (SEQ ID NO: 324);

Ser Gly Ile Ser Ala Trp Gly Trp (SEQ ID NO: 325);

Phe Tyr Phe Trp Val Arg Gly Trp (SEQ ID NO: 326);

Lys Leu Phe Leu Pro Leu Gly Trp (SEQ ID NO: 327);

Thr Pro Thr Leu Ser Asp Gly Trp (SEQ ID NO: 328);

Thr His Ser Leu Ile Leu Gly Trp (SEQ ID NO: 329);

Leu Leu Leu Leu Ser Arg Gly Trp (SEQ ID NO: 330);

Leu Leu Arg Val Arg Ser Gly Trp (SEQ ID NO: 331);

Glu Arg Arg ser Arg Gly Gly Trp (SEQ ID NO: 332);

Arg Met Leu Gln Leu Ala Gly Trp (SEQ ID NO: 333);

Age Gly Trp Ala Asn Ser Gly Trp (SEQ ID NO: 334);

Arg Pro Phe Tyr Ser Tyr Gly Trp (SEQ ID NO: 335);

Ser Ser Ser Trp Asn Ala Gly Trp (SEQ ID NO: 336);

Leu Gly His Leu Glu Glu Gly Trp (SEQ ID NO .: 337);

Ser Ala Val Thr Asn Thr Gly Trp (SEQ ID NO: 338);

Leu Arg Arg Ala Ser Leu Trp (SEQ ID NO .: 339);

Leu Arg Arg Trp Ser Leu Trp (SEQ ID NO: 340);

Lys Trp Val His Leu Phe Trp (SEQ ID NO: 341);

Asn Arg Leu Leu Leu Thr Trp (SEQ ID NO: 342);

Ala Arg Leu Leu Leu Thr Trp (SEQ ID NO: 343);

Asn Ala Leu Leu Leu Thr Trp (SEQ ID NO: 344);

Asn Arg Leu Ala Leu Thr Trp (SEQ ID NO: 345);

Asn Leu Leu Arg Leu Thr Trp (SEQ ID NO: 346);

Asn Arg Leu Trp Leu Thr Trp (SEQ ID NO: 347); And

Asn Arg Leu Leu Leu Ala Trp (SEQ ID NO: 348).

Other heat shock protein binding regions useful in the practice of the present invention include Phe Tyr Gln Leu Ala Leu Thr Trp (SEQ ID NO: 501), Phe TyrGln Leu Ala Leu Thr Trp (SEQ ID NO: 502), Arg Lys Leu Phe Phe Asn Leu Arg Trp (SEQ ID NO: 503), Arg Lys Leu Phe Phe Asn Leu Arg Trp (SEQ ID NO: 504), Lys Phe Glu Arg Gln Trp (SEQ ID NO: 505), Asn Ile Val Arg Lys Lys Lys Lys Trp (SEQ ID N0: 506), and Arg Gly Tyr Val Tyr Gln Gly Leu Trp (SEQ ID NO: 507).

In addition, other heat shock protein binding regions include those disclosed in PCT Publication No. W09922761, and may have terminal Trp residues added to achieve the object of the present invention. Xaa represents any amino acid.

Tyr Thr Leu Val Gln Pro Leu Trp (SEQ ID NO: 1149);

Thr Pro Asp Ile Thr Pro Lys Trp (SEQ ID NO: 1150);

Thr Tyr Pro Asp Leu Arg Tyr Trp (SEQ ID NO: 1151);

Asp Arg Thr His Ala Thr Ser Trp (SEQ ID NO: 1152);

Met Ser Thr Thr Phe Tyr Ser Trp (SEQ ID NO: 1153);

Tyr Gln His Ala Val Gln Thr Trp (SEQ ID NO: 1154);

Phe Pro Phe Ser Ala Ser Thr Trp (SEQ ID NO: 1155);

Ser Ser Phe Pro Pro Leu Asp Trp (SEQ ID NO: 1156);

Met Ala Pro Ser Pro Pro His Trp (SEQ ID NO: 1157);

Ser Ser Phe Pro Aspleu Leu Trp (SEQ ID NO: 1158);

His Ser Tyr Asn Arg Leu Pro Trp (SEQ ID NO: 1159);

His Leu Thr His Ser Gln Arg Trp (SEQ ID NO: 1160);

Gln Ala Ala Gln Ser Arg Ser Trp (SEQ ID NO: 1161);

Phe Ala Thr His His Ile Gly Trp (SEQ ID NO: 1162);

Ser Met Pro Glu Pro Leu Ile Trp (SEQ ID NO: 1163);

Ile Pro Arg Tyr His Leu Ile Trp (SEQ ID NO: 1164);

Ser Ala Pro His Met Thr Ser Trp (SEQ ID NO: 1165);

Lys Ala Pro Val Trp Ala Ser Trp (SEQ ID NO: 1166);

Leu Pro His Trp Leu Leu Ile Trp (SEQ ID NO: 1167);

Ala Ser Ala Gly Tyr Gln Ile Trp (SEQ ID NO: 1168);

Val Thr Pro Lys Thr Gly Ser Trp (SEQ ID NO: 1169);

Glu His Pro Met Pro Val Leu Trp (SEQ ID NO: 1170);

Val Ser Ser Phe Val Thr Ser Trp (SEQ ID NO: 1171);

Ser Thr His Phe Thr Trp Pro Trp (SEQ ID NO: 1172);

Gly Gln Trp Trp Ser Pro Asp Trp (SEQ ID NO: 1173);

Gly Pro Pro His Gln Asp Ser Trp (SEQ ID NO: 1174);

Asn Thr Leu Pro Ser Thr Ile Trp (SEQ ID NO: 1175);

His Gln Pro Ser Arg Trp Val Trp (SEQ ID NO: 1176);

Tyr Gly Asn Pro Leu Gln Pro Trp (SEQ ID NO: 1177);

Phe His Trp Trp TrpGln Pro Trp (SEQ ID NO: 1178);

Ile Thr Leu Lys Tyr Pro Leu Trp (SEQ ID NO: 1179);

Phe His Trp Pro Trp Leu Phe Trp (SEQ ID NO: 1180);

Thr Ala Gln Asp Ser Thr Gly Trp (SEQ ID NO: 1181);

Phe His Trp Trp Trp Gln Pro Trp (SEQ ID NO: 1182);

Phe His Trp Trp Asp Trp Trp Trp (SEQ ID NO: 1183);

Glu Pro Phe Phe Arg Met Gln Trp (SEQ ID NO: 1184);

Thr Trp Trp Leu Asn Tyr Arg Trp (SEQ ID NO: 1185);

Phe His Trp Trp Trp Gln Pro Trp (SEQ ID NO: 1186);

Gln Pro Ser His Leu Arg Trp Trp (SEQ ID NO: 1187);

Ser Pro Ala Ser Pro Val Tyr Trp (SEQ ID NO: 1188);

Phe His Trp Trp Trp Gln Pro Trp (SEQ ID NO: 1189);

His Pro Ser Asn Gln Ala Ser Trp (SEQ ID NO: 1190);

Asn Ser Ala Pro Arg Pro Val Trp (SEQ ID NO: 1191);

Gln Leu Trp Ser Ile Tyr Pro Trp (SEQ ID NO: 1192);

Ser Trp Pro Phe Phe Asp Leu Trp (SEQ ID NO: 1193);

Asp Thr Thr Leu Pro Leu His Trp (SEQ ID NO: 1194);

Trp His Trp Gln Met Leu Trp Trp (SEQ ID NO: 1195);

Asp Ser Phe Arg Thr Pro Val Trp (SEQ ID NO: 1196);

Thr Ser Pro Leu Ser Leu Leu Trp (SEQ ID NO .: 1197);

Ala Tyr Asn Tyr Val Ser Asp Trp (SEQ ID NO: 1198);

Arg Pro Leu His Asp Pro Met Trp (SEQ ID NO: 1199);

Trp Pro Ser Thr Thr Leu Phe Trp (SEQ ID NO .: 1200);

Ala Thr Leu Glu Pro Val Arg Trp (SEQ ID NO: 1201);

Ser Met Thr Val Leu Arg Pro Trp (SEQ ID NO: 1202);

Gln Ile Gly Ala Pro Ser Trp Trp (SEQ ID NO: 1203);

Ala Pro Asp Leu Tyr Val Pro Trp (SEQ ID NO: 1204);

Arg Met Pro Pro Leu Leu Pro Trp (SEQ ID NO .: 1205);

Ala Lys Ala Thr Pro Glu His Trp (SEQ ID NO: 1206);

Thr Pro Pro Leu Arg Ile Asn Trp (SEQ ID NO .: 1207);

Leu Pro He His Ala Pro His Trp (SEQ ID NO .: 1208);

Asp Leu Asn Ala Tyr Thr His Trp (SEQ ID NO: 1209);

Val Thr Leu Pro Asn Phe His Trp (SEQ ID NO .: 1210)

Asn Ser Arg Leu Pro Thr Leu Trp (SEQ ID NO .: 1211)

Tyr Pro His Pro Ser Arg Ser Trp (SEQ ID NO .: 1212)

Gly Thr Ala His Phe Met Tyr Trp (SEQ ID NO .: 1213)

Tyr Ser Leu Leu Pro Thr Arg Trp (SEQ ID NO .: 1214)

Leu Pro Arg Arg Thr Leu Leu Trp (SEQ ID NO .: 1215)

Thr Ser Thr Leu Leu Trp Lys Trp (SEQ ID NO .: 1216)

Thr Ser Asp Met Lys Pro His Trp (SEQ ID NO .: 1217)

Thr Ser Ser Tyr Leu Ala Lue Trp (SEQ ID NO .: 1218)

Asn Leu Tyr Gly Pro His Asp Trp (SEQ ID NO .: 1219)

Leu Glu Thr Tyr Thr Ala Ser Trp (SEQ ID NO .: 1220)

Ala Tyr Lys Ser Leu Thr Gln Trp (SEQ ID NO .: 1221)

Ser Thr Ser Val Tyr Ser Ser Trp (SEQ ID NO .: 1222)

Glu Gly Pro Leu Arg Ser Pro Trp (SEQ ID NO .: 1223)

Thr Thr Tyr Hisala Leu Gly Trp (SEQ ID NO .: 1224)

Val Ser Ile Gly His Pro Ser Trp (SEQ ID NO .: 1225)

Thr His Ser His Arg Pro Ser Trp (SEQ ID NO .: 1226)

Ile Thr Asn Pro Leu Thr Thr Trp (SEQ ID NO .: 1227)

Ser Ile Gln Ala His His Ser Trp (SEQ ID NO .: 1228)

Leu Asn Trp Pro Arg Val Leu Trp (SEQ ID NO .: 1229)

Tyr Tyr Tyr Ala Pro Pro Pro Trp (SEQ ID NO .: 1230)

Ser Leu Trp Thr Arg Leu Pro Trp (SEQ ID NO .: 1231)

Asn Val Tyr His Ser Ser Leu Trp (SEQ ID NO .: 1232)

Asn Ser Pro His Pro Pro Thr Trp (SEQ ID NO .: 1233)

Val Pro Ala Lys Pro Arg His Trp (SEQ ID NO: 1234)

His Asn Leu His Pro Asn Arg Trp (SEQ ID NO .: 1235)

Tyr Thr Thr His Arg Trp Leu Trp (SEQ ID NO .: 1236)

Ala Val Thr Ala Ala Ile Val Trp (SEQ ID NO .: 1237)

Thr Leu Met His Asp Arg Val Trp (SEQ ID NO .: 1238)

Thr Pro Leu Lys Val Pro Tyr Trp (SEQ ID NO .: 1239)

Phe Thr Asn Gln Gln Tyr His Trp (SEQ ID NO .: 1240)

Ser His Val Pro Ser Met Ala Trp (SEQ ID NO .: 1241)

His Thr Thr Val Tyr Gly Ala Trp (SEQ ID NO: 1242);

Thr Glu Thr Pro Tyr Pro Thr Trp (SEQ ID NO .: 1243);

Leu Thr Thr Pro Phe Ser Ser Trp (SEQ ID NO .: 1244);

Gly Val Pro Leu Thr Met Asp Trp (SEQ ID NO: 1245);

Lys Leu Pro Thr Val Leu Arg Trp (SEQ ID NO .: 1246);

Cys Arg Phe His Gly Asn Arg Trp (SEQ ID NO: 1247);

Tyr Thr Arg Asp Phe Glu Ala Trp (SEQ ID NO .: 1248);

Ser Ser Ala Ala Gly Pro Arg Trp (SEQ ID NO: 1249);

Ser Leu Ile Gln Tyr Ser Arg Trp (SEQ ID NO: 1250);

Asp Ala Leu Met Trp Pro XAA XAA (SEQ ID NO: 1251);

Ser Ser XAA Ser Leu Tyr Ile Trp (SEQ ID NO: 1252);

Phe Asn Thr Ser Thr Arg Thr Trp (SEQ ID NO: 1253);

Thr Val Gln His Val Ala Phe Trp (SEQ ID NO: 1254);

Asp Tyr Ser Phe Pro Pro Leu Trp (SEQ ID NO .: 1255);

Val Gly Ser Met Glu Ser Leu Trp (SEQ ID NO: 1256);

Phe XAA Pro Met Ile XAA Ser Trp (SEQ ID NO: 1257);

Ala Pro Pro Arg Val Thr Met Trp (SEQ ID NO: 1258);

Ile Ala Thr Lys Thr Pro Lys Trp (SEQ ID NO: 1259);

Lys Pro Pro Leu Phe Gln Ile Trp (SEQ ID NO: 1260);

Tyr His Thr Ala His Asn Met Trp (SEQ ID NO: 1261);

Ser Tyr Ile Gln Ala Thr His Trp (SEQ ID NO: 1262);

Ser Ser Phe Ala Thr Phe Leu Trp (SEQ ID NO: 1263);

Thr Thr Pro Pro Asn Phe Ala Trp (SEQ ID NO .: 1264);

Ile Ser Leu Asp Pro Arg Met Trp (SEQ ID NO: 1265);

Ser Leu Pro Leu Phe Gly Ala Trp (SEQ ID NO: 1266);

Asn Leu Leu Lys Thr Thr Leu Trp (SEQ ID NO .: 1267);

Asp Gln Asn Leu Pro Arg Arg Trp (SEQ ID NO: 1268);

Ser His Phe Glu Gln Leu Leu Trp (SEQ ID NO .: 1269);

Thr Pro Gln Leu His His Gly Trp (SEQ ID NO: 1270);

Ala Pro Leu Asp Arg He Thr Trp (SEQ ID NO: 1271);

Phe Ala Pro Leu Ile Ala His Trp (SEQ ID NO: 1272);

Ser Trp Ill Gln Thr Phe Met Trp (SEQ ID NO: 1273);

Asn Thr Trp Pro His Met Tyr Trp (SEQ ID NO .: 1274)

Glu Pro Lue Pro Thr Thr Leu Trp (SEQ ID NO .: 1275)

His Gly Pro His Leu Phe Asn Trp (SEQ ID NO .: 1276)

Tyr Leu Asn Ser Thr Leu Ala Trp (SEQ ID NO .: 1277)

His Leu His Ser Pro Ser Gly Trp (SEQ ID NO .: 1278)

Thr Leu Pro His Arg Leu Asn Trp (SEQ ID NO .: 1279)

Ser Ser Pro Arg Glu Val His Trp (SEQ ID NO .: 1280)

Asn Gln Val Asp Thr Ala Arg Trp (SEQ ID NO .: 1281)

Tyr Pro Thr Pro Leu Leu Thr Trp (SEQ ID NO .: 1282)

His Pro Ala Ala Phe Pro Trp Trp (SEQ ID NO .: 1283)

Leu Leu Pro His Ser Ser Ala Trp (SEQ ID NO .: 1284)

Leu Glu Thr Tyr Thr Ala Ser Trp (SEQ ID NO .: 1285)

Lys Tyr Val Pro Leu Pro Pro Trp (SEQ ID NO .: 1286)

Ala Pro Leu Ala Leu His Ala Trp (SEQ ID NO .: 1287)

Tyr Glu Ser Leu Leu Thr Lys Trp (SEQ ID NO .: 1288)

Ser His Ala Ala Ser Gly Thr Trp (SEQ ID NO .: 1289)

Gly Leu Ala Thr Val Lys Ser Trp (SEQ ID NO .: 1290)

Gly Ala Thr Ser Phe Gly Leu Trp (SEQ ID NO .: 1291)

Lys Pro Pro Gly Pro Val Ser Trp (SEQ ID NO .: 1292)

Thr Leu Tyr Val Ser Gly Asn Trp (SEQ ID NO .: 1293)

His Ala Pro Phe Lys Ser Gln Trp (SEQ ID NO .: 1294)

Val Ala Phe Thr Arg Leu Pro Trp (SEQ ID NO .: 1295)

Leu Pro Thr Arg Thr Pro Ala Trp (SEQ ID NO .: 1296)

Ala Ser Phe Asp Leu Leu Ile Trp (SEQ ID NO .: 1297)

Arg Met Asn Thr Glu Pro Pro Trp (SEQ ID NO .: 1298)

Lys Met Thr Pro Leu Thr Thr Trp (SEQ ID NO .: 1299)

Ala Asn Ala Thr Pro Leu Leu Trp (SEQ ID NO .: 1300)

Thr Ile Trp Pro Pro Pro Val Trp (SEQ ID NO: 1301)

Gln Thr Lys Val Met Thr Thr Trp (SEQ ID NO: 1302)

Asn His Ala Val Phe Ala Ser Trp (SEQ ID NO .: 1303)

Leu His Ala Ala Zaa Thr Ser Trp (SEQ ID NO .: 1304)

Thr Trp Gln Pro Tyr Phe His Trp (SEQ ID NO .: 1305)

Ala Pro Leu Ala Leu His Ala Trp (SEQ ID NO .: 1306)

Thr Ala His Asp Leu Thr Val Trp (SEQ ID NO .: 1307)

Asn Met Thr Asn Met Leu Thr Trp (SEQ ID NO .: 1308)

Gly Ser Gly Leu Ser Gln Asp Trp (SEQ ID NO .: 1309)

Thr Pro Ile Lys Thr Ile Tyr Trp (SEQ ID NO .: 1310)

Ser His Leu Tyr Arg Ser Ser Trp (SEQ ID NO .: 1311) and

His Gly Gln Ala Trp Gln Phe Trp (SEQ ID NO .: 1312).

Of all the heat shock protein binding peptides, the heat shock protein binding region Asn Leu Leu Arg Leu Thr Gly Trp (SEQ ID NO: 350) is most preferred in the hybrid antigen of the present invention. However, the above-described heat shock protein binding regions merely exemplify various residues that can be used in the practice of the present invention among peptide or non-peptide heat shock protein binding molecules.

The hybrid antigens of the present invention comprise one or more antigenic (immunogenic) regions and one or more heat shock protein binding regions, which are separated by one or more peptide linkers as disclosed herein. Hybrid antigens of the invention can be synthesized using chemical peptide synthesis, or by expressing a nucleic acid construct comprising a bound sequence encoding an antigenic region and a heat shock protein binding region. One suitable technique uses initial separate PCT amplification reactions to generate separate DNA fragments encoding the two regions, each fragment having a linker fragment attached to one end, followed by two further PCR steps. Fuse the amplified product. This technique is referred to as linker tailing. Suitable restriction sites can also be engineered at the target site, after which restriction cleavage and ligation are used to generate the desired hybrid antigen-encoding sequence.

As noted above, nucleic acids encoding hybrid antigens of the present invention are also suitable for treatment by administration to a subject and produce a hybrid antigen having the ability to induce an immune response by expression in vivo.

Thermal shock  protein

The term “thermal shock protein” as used herein refers to any protein whose expression is increased in a cell when the cell is under stress. In a preferred non-limiting embodiment, the heat shock protein is originally derived from eukaryotic cells; In a more preferred embodiment, the heat shock protein is originally derived from mammalian cells. For example, heat shock proteins that may be used in accordance with the present invention include, but are not limited to, BiP (also referred to as grp78), hsp70, hsc70, gp96 (grp94), hsp60, hsp40 and hsp90, and members of these classes. Do not. Particularly preferred heat shock proteins are BiP, gp96, and hsp70 as exemplified below. Most preferred is a member of the hsp70 family. Variants of naturally occurring or recombinantly derived heat shock proteins can also be used in accordance with the present invention. For example, the present invention relates to mutated heat shock proteins (eg, heat shock proteins with mutations or deletions of elements that promote vesicle recapture, such as KDEL or analogues thereof, for promoting their secretion in cells; Such variants provide, but are not limited to, the use of PCT Application No. PCT / US96 / 13233 (WO 97/06685, which is incorporated herein by reference).

In an embodiment of the invention in which heat shock proteins and hybrid antigens are administered directly to a subject in the form of a protein / peptide complex, the heat shock proteins can be prepared using standard techniques, for example, in Flynn et al., Science 245: 385-390 (1989), or may be prepared from natural sources, or using recombinant techniques such as expressing a heat shock-encoding vector in a suitable host cell, such as a bacterial, yeast or mammalian cell. . It is of interest to pre-load a heat shock protein with a peptide from the host organism, which heat shock protein can be repurified after incubation with ATP. Non-limiting examples of methods for making recombinant heat shock proteins are disclosed below.

Nucleic acids encoding heat shock proteins can be operatively associated with the elements necessary or suitable for expression, and then the desired heat shock protein can be used as a means to prepare heat shock proteins for use in protein vaccines or alternatively nucleic acid vaccines. Can be used to express. Required or preferred elements for expression include, but are not limited to, promoter / enhancer elements, transcription initiation and termination sequences, polyathenylation myths, translation initiation and termination sequences, ribosomal binding sites, signal sequences, and the like. For example, genes of various heat shock proteins have been cloned, but are not limited to these sequences, including, but not limited to, gp96 (human: Genebank Accession No. X15187; Maki et al., Proc. Natl. Acad. Sci. USA 87): 5658-5562 (1990); Mice: Genebank Accession No. M16370; Srivastava et al., Proc. Natl. Acad Sci. USA 84: 3807-3811 (1987)), BiP (Mouse: Genebank Accession No. U16277; Haas et al., Proc. Natl. Acad. Sci. USA 85: 2250-2254 (1988); humans: Genebank Accession No. M19645; Ting et al., DNA 7: 275-286 ( 1988))), hsp70 (mouse: Genebank Accession No. M35021; Hunt et al., Gene 87: 199-204 (1990); humans: Genebank Accession No. M24743; Hunt et al, Proc. Natl Acad. Sci. USA 82: 6455-6489 (1995)], and hsp40 (human: Genebank Accession No. D49547; Ohtsuka K., Biochem. Biophys. Res. Commun. 197: 235-240 (1993) ]), But it is not limited to this.

Dosing method

Hybrid antigens or complexes of hybrid antigens and heat shock proteins of the present invention are administered to a subject using one of a peptide-based, protein-based or nucleic acid vaccine to produce in the gas an amount of complex effective to induce a therapeutic immune response in the subject. Can be.

The subject can be a human or non-human subject.

The term "therapeutic immune response" as used herein refers to an increase in humoral and / or cellular immunity, as measured by standard techniques associated with hybrid antigens. Preferably, the degree of induction of humoral immunity associated with the hybrid antigen is, but is not limited to, at least four times, preferably at least 16 times, the degree of humoral immunity to the antigen prior to administration of the composition of the invention to a subject. . In addition, the immune response can be measured quantitatively by means suitable for in vitro or in vivo analysis, wherein interruption or alleviation of the progression of tumor disease or infectious disease in the subject is considered to indicate the induction of a therapeutic immune response.

The specific dosage of the heat shock protein / hybrid antigen may vary depending on many factors including the immunity of the particular vaccine composition, the immunity of the individual, the size of the individual and the route of administration. Determining an amount suitable for administering any given composition is a matter of general selection.

In addition, significant immunological efficacy was confirmed in studies in which the hybrid antigen was administered alone, for example without a heat shock protein. The Applicant is not obliged to disclose the principle by which the invention works and, although not related to it, the results of these studies show that the hybrid antigen binds to an endogenous heat shock protein when injected into a subject and thus exhibits efficacy. It is suggested that there is no need to administer heat shock proteins at the same time. The present invention encompasses such uses of the hybrid antigens of the present invention and also includes ancillary treatment of increasing the endogenous heat shock protein systemically or at the intended site of administration of the hybrid antigens of the present invention. Such incidental treatments include, but are not limited to, fever that increases the expression of heat shock proteins in local tissues, or local application of local or systemic therapeutic agents. Such therapeutic agents are known in the art.

A hybrid antigen administered without co-administration of a heat shock protein comprising at least one antigenic region and at least one heat shock protein binding region (eg, not administered in complex with the heat shock protein) may be modified by one of the peptide linkers mentioned herein. Include.

In certain non-limiting embodiments of the invention, it may be desirable to include one or more heat shock proteins and / or one or more hybrid antigens to optimize the immune response. Such techniques can be particularly effective in the treatment of cancer or infections characterized by rapid progression of mutations that avoid immune responses. In addition, the hybrid house of the invention may comprise one or more immune regions or one or more epitopes.

As mentioned above, a composition comprising a hybrid antigen / heat shock protein or comprising a hybrid antigen alone is referred to herein as a “vaccine”. The term "vaccine" is used to indicate that the compositions of the present invention can be used to induce a prophylactic or therapeutic immune response. Vaccines of the invention may comprise hybrid antigens having a single antigenic region or epitope, or hybrid antigens having a plurality of antigenic regions or epitopes. In addition, the vaccine may comprise a mixture of hybrid antigens having single or multiple antigenic regions or epitopes, and combinations thereof. As mentioned above, the hybrid antigen or mixtures thereof may be complexed with one or more heat shock proteins prior to administration, or may be administered without heat shock proteins.

Vaccine compositions comprising one or more hybrid antigens optionally complexed with one or more heat shock proteins according to the present invention may be transdermal, subcutaneous, intradermal, intravenous, intramuscular, parenteral, pulmonary, intravaginal, rectal, intranasal, or Topical administration. The vaccine composition may be delivered by injection, particle bombardment, oral or aerosol.

Incubation of the heat shock protein in a solution with a hybrid antigen is sufficient in most cases to load the antigen on the heat shock protein. In some cases, however, it may be desirable to add a medicament that may aid in loading the antigen.

When incubated with a heat shock protein with the hybrid antigen, the antigen will generally be loaded onto the heat shock protein. In some cases, however, it may be desirable to add additional agents to aid in the loading. For example, hsp40 can encourage the loading of peptides on hsp70 (Minami et al., J. Biol. Chem. 271: 19617-19624 (1996)). A denaturant such as guanidinium-HCl or urea can be used to partially and reversibly destabilize the heat shock protein that makes the peptide binding bag more accessible to the antigen.

In particular, the vaccine of the present invention comprising a heat shock protein also preferably comprises adenosine diphosphate (ADP) to encourage binding between the heat shock protein and the complex of said heat shock protein binding region before reaching its destination. . Other compounds with similar abilities can be used alone or in combination with ADP.

The vaccine composition according to the present invention may further comprise various additional substances such as pharmaceutically acceptable carriers. Suitable carriers include phosphate buffered saline solutions, any pharmaceutically acceptable standard carrier such as water, emulsions such as oil / water emulsions or triglyceride emulsions, various types of wetting agents, tablets, coated tablets and capsules. An example of an acceptable triglyceride emulsion useful for intravenous and intraperitoneal administration of the compound is a triglyceride emulsion commercially available as Intralipid®. Typically, such carriers include excipients such as starch, milk, sugar, gums, glycols, or other known excipients. Such carriers may also include perfumes, color additives or other ingredients.

Vaccine compositions of the invention may also comprise suitable diluents, preservatives, solubles, emulsions, adjuvants and / or carriers. Such compositions may be in liquid form, or lyophilized formulations, or otherwise dry formulations, and may contain various buffer contents (eg, tri-HCl, acetate, phosphate), pH and ionic strength diluents, surface absorption. Additives such as albupine or gelatin to block, detergents (e.g. Tween 20, Tween 80, Pluronic F68, bile salts), solubilizers (e.g. glycerol, polyethylene glycerol), antioxidants (e.g. Metals of polymers such as ascorbic acid, sodium metabisulfite), preservatives (eg thimersal, benzyl alcohol, parabens), extenders or toxic modifiers (eg lactose, mannitol), polyethylene glycol Covalent attachment points to proteins that form complexes with ions, or on particulate formulations of polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, and the like. It may comprise a mouth part or liposomes, mayikeuroyi emulsion, micelle, a single film or a multi-layered film package, red blood cell ghost (ghost erythrocyte) or Spanish incorporated ropeul sites on the last. Such compositions will affect the physical state, solubility, stability, rate of in vivo release and rate of in vivo removal. The choice of composition will depend on the physicochemical characterization of the vaccine. For example, products derived from proteins in membrane-bound form may require formulations containing detergents. Controlled or sustained release compositions include the formulation in a lipophilic reservoir (eg, fatty acid, wax, oil). In addition, particle compositions coated with polymers (eg, poloxamers or poloxamines) and compositions bound to tissue-specific receptors, antibodies to ligands or antigens, or compositions bound to tissue-specific receptors It is also contemplated by the present invention. Other embodiments of the compositions of the present invention include particulate protective coatings, protease inhibitors or permeation thickeners for various routes of administration, including intramuscular, parenteral, pulmonary, nasal and oral administration.

As an alternative to the method of directly administering the hybrid antigen, which optionally forms a complex with the heat shock protein, optionally one or more polynucleotide constructs encoding the hybrid antigen in an expressible form can be administered with the heat shock protein. An expressible polynucleotide construct is introduced into a cell of an individual using ex vivo or in vivo methods. Suitable methods include direct injection into tissues and tumors, transduction with liposomes (Fraley et al., Nature 370: 111-117 (1980)), receptor-mediated endocytosis (Zatloukal et al. , Ann. NY Acad. Sci. 660: 136-153 (1992)), incident injection-mediated gene transfer (Eisenbraun et al., DNA & Cell Biol. 12: 792-797 (1993)) and Transduction using peptide-presenting bacteriophages (Barry et al., Nature Medicine 2: 299-305 (1996)). The polypeptide vaccine can also be introduced into appropriate cells in vitro and then introduced into a subject.

In order to construct an expressible polynucleotide, a region encoding the heat shock protein and / or hybrid antigen was prepared as described above, and the SV40 promoter, cytomegalovirus (CMV) promoter or Laos sarcoma virus (RSV) promoter It is inserted into a mammalian expression vector operably linked to the same suitable promoter. The resulting construct can be used as a vaccine for genetic immunization. The nucleic acid polymer (s) may be cloned into a viral vector. Suitable vectors include, but are not limited to, retrovirus vectors, adenovirus vectors, vaccinia vectors, smallpox virus vectors, and adenovirus-binding viruses. Specific vectors suitable for use in the present invention include pCDNA3 (In Vitrogen), plasmid AH5 (which includes SV40 origin and adenovirus major terminal promoters), pRC / CMV (In Vitrogen), pCMU II (Paabo et al., EMBO J. 5: 1921-1927 (1986)], pZip-N vs. SV (Cepko et al., Cell 37: 1053-1062 (1984)) and pSRα (DNAX, Palo Alto, CA), but it is not limited to this.

Various methods for preparing heat shock proteins and hybrid antigens are disclosed in WO9706821 and WO9922761, which are incorporated herein by reference in their entirety.

In the following examples and throughout the specification, amino acids may be represented as follows using single letters:

A: Alanine C: Cytosine

D: Aspartic Acid E: Glutamic Acid

F: Phenylalanine G: Glycine

H: histidine I: isoleucine

K: Lysine L: Leucine

M: Methionine N: Aspartic Acid

P: Proline Q: Glutamine

R: Arginine S: Serine

T: threonine V: valine

W: Tryptophan Y: Tyrosine

The invention may be better understood with reference to the following non-limiting examples which serve as examples of the invention. The following examples are presented to illustrate the examples of the invention in more detail. However, it is to be understood that the embodiments of the invention do not depart from the broad scope of the invention.

Example  One

Various hybrid antigens were prepared, each of which comprises a heat shock protein binding region and a model I type H2-Kb epitope SIINFEKL from cancer antigen epitopes or egg white albumin. A peptide linker is included between the two regions. The heat shock protein binding regions used in these experiments are as follows: HWDFAWPW, NLLRLTGW, FYQLALTW and RKLFFNLRW. Linker is one of those described above.

Cancer and model epitopes are one of those listed in the table below.

table XX

Protein sources Tumor source amino acid Name (amino acid sequence) Prostate-specific membrane antigen Prostate cancer 771-779 PSMA P2 (ALFDIESKV) Gp100 Melanoma 209-217 IMD (210M) (IMDQVPFSV) Tyrosinase Melanoma 368-376 YMD (370D) (YMDGTMSQV) Human Papillomavirus (HPV) Variant 16 E7 Cervical cancer 86-93 HPV16 E7 86-93 (TLGIVCPI) HPV variant 16 E7 Cervical cancer 11-20 HPV16 E7 11-20 (YMLDLQPETT) Egg white albumin Model tumor antigen 257-264 Ova (SIINFEKL)

Using standard solid-phase peptide synthesis using F-moc chemistry methodology, hybrid antigens including heat shock protein binding regions, cancer epitopes and linkers between them were synthesized in various directions.

Example  2

Binding affinity between recombinant human or mouse heat shock protein 70 (hsp70) and various heat shock protein binding regions and antigenic peptides described above, as well as the binding affinity between hybrid antigens comprising antigenic peptides and the heat shock protein binding regions described above Or in a binding inhibition assay (Hill plot) for binding affinity for hsp70 measured by Scatchard assay (Kd: 22.64 μM and 10.75 μM, respectively) of standard labeled hybrid antigen (tritiated or fluorescent ALFDIESKVGSGHWDFAWPW). Was measured. Binding studies consisted of 39% PBS; 20 mM THAM, pH 8; 37 mM NaCl, 5 mM MgCl ₂ ; And 1 mM ADP.

Example  3

In the immunological studies of mice, the mouse MHC H2-K (b) epitope, SIINFEKL (amino acids 257-264), and the vesicular stomatitis virus (VSV) from nucleoproteins from egg protein, K (b) peptide RGYVYQGL (amino acids 52-59) was used for the preparation of hybrid antigens. The table below lists the affinity for hsp70 and the sequences in epitope alone and hybrid houses.

table XX

Mouse Epitope Epitope alone Hybrid Antigens Containing Epitopes Epitope sequences Affinity for hsp70 (μM) Hybrid antigen sequence Affinity for hsp70 (μM) Egg white protein: amino acid SIINFEKL 235 NLLRLTGWGSGSIINFEKL 1.6 NLLRLTGWFFRKSIINFEKL 2.2 NLLRLTGWRKSIINFEKL 0.8 VSV nucleoprotein: amino acid 52-59 RGYVYQGL 82 NLLRLTGWGSGRGYVYQGL 1.4 NLLRLTGWFFRKRGYVYQGL 1.0 NLLRLTGWRKRGYVYQGL 0.6

Mice were immunized subcutaneously in the lower tail of mice using hsp70 alone, hsp70 complexed with SIINFEKL, and hybrid SIINFEKL peptides with or without HSP70. Dosage was adjusted to include the same amount of SIINFEKL for each immunity except hsp70. After a few days, the spleens were harvested and concentrated on CD8 + T cells, which were used for in vitro IFN-γ ELISPOT analysis. Post-pulse response with SIINFEKL (“SIINFEKL”) is reported in the table below, which shows the dose per spot of 4 × 10 ⁵ CD8 T cells in four or more experiments with doses and three or more mice per group. Numbers (mean ± standard error) are shown. In control, medium alone (“medium control”), non-pulse T cells (“non-pulse control”), T cells pulsed with non-immunized peptide RGYVYQGL derived from VSV (“VSV control”) and cone as positive control. Exposure to cannavaline A (“Con A positive control”) is shown. In the same experiment, a ⁵¹ Cr-release assay as described above was performed using SIINFEKL-pulse target cells. At a ratio of 200: 1 effector: target cells, the results of% death obtained are shown in the rightmost column of the table below.

(200-10)

Immunogen Number of spots per 400,000 cells CTL analysis:% mortality at 200: 1 E / T SIINFEKL Medium control Non-pulse control VSV control Con A positive control 4.4 μg Hsp70 0.00 ± 0.00 1.50 ± 2.12 0.67 ± 0.58 0.33 ± 0.58 834 ± 28.3 0% 4.4 μg Hsp70 + 0.9 μg SIINFEKL 33.7 ± 7.09 0.00 ± 0.00 0.33 ± 0.58 0.00 ± 0.00 1000 ± 33.7 19% 4.4 μg Hsp70 + 2.0 μg NLLRLTGWGSGSIINFEKL 80.0 ± 17.0 0.00 ± 0.00 1.50 ± 0.71 1.50 ± 0.71 1170 ± 56.5 38% 4.4 μg Hsp70 + 2.4 μg NLLRLTGWFFRKSIINFEKL 222 ± 17.7 0.00 ± 0.00 0.67 ± 0.58 1.33 ± 1.53 1010 ± 56.5 52%

Example  5

Experiments were carried out as described above, where the hybrid antigen without hsp70 was also included.

(200-11)

Immunogen Number of spots per 400,000 CD8 T cells SIINFEKL Medium control Non-pulse control VSV control Con A positive control 4.4 μg Hsp70 0.33 ± 0.58 1.00 ± 1.73 1.67 ± 1.15 4.00 ± 1.00 965 ± 62.6 4.4 μg Hsp70 + 0.9 μg SIINFEKL 1.67 ± 0.58 1.00 ± 1.00 2.00 ± 0.00 2.67 ± 2.08 591 ± 48.1 4.4 μg Hsp70 + 2.0 μg NLLRLTGWGSGSIINFEKL 12.0 ± 5.2 2.67 ± 0.58 1.67 ± 1.15 2.00 ± 2.65 748 ± 58.6 4.4 μg Hsp70 + 2.4 μg NLLRLTGWFFRKSIINFEKL 770 ± 80.6 3.33 ± 1.53 3.67 ± 1.53 4.33 ± 1.53 742 ± 72.6 2.4 μg NLLRLTGWFFRKSIINFEKL (no hsp70) 151 ± 20.7 1.00 ± 1.00 1.67 ± 0.58 0.00 ± 0.00 459 ± 149

Example  6

Additional tests were conducted similar to the experiments described above.

(200-12)

Immunogen Number of spots per 300,000 CD8 T cells CTL analysis:% mortality at 200: 1 E / T SIINFEKL Medium control Non-pulse control VSV control Con A positive control 4.4 μg Hsp70 0.67 ± 0.58 0.00 ± 0.00 0.50 ± 0.71 1.00 ± 1.41 552 ± 24.0 8.45 ± 41.3 4.4 μg Hsp70 + 0.9 μg SIINFEKL 3.33 ± 2.52 0.00 ± 0.00 0.33 ± 0.58 0.33 ± 0.58 450 ± 69.0 43.0 ± 21.2 4.4 μg Hsp70 + 2.00 μg NLLRLTGWGSGSIINFEKL 134 ± 4.16 1.33 ± 1.53 0.67 ± 1.15 1.00 ± 1.00 865 ± 93.0 31.9 ± 5.41 4.4 μg Hsp70 + 2.4 μg NLLRLTGWFFRKSIINFEKL 680 ± 23.0 0.00 ± 0.00 0.00 ± 0.00 1.67 ± 0.58 801 ± 56.6 84.6 ± 1.70 2.4 µg NLLRLTGWFFRKSIINFEKL 211 ± 17.0 0.00 ± 0.00 0.50 ± 0.71 1.00 ± .0.00 688 ± 41.7 9.91 ± 5.57

practice Yes  7

As in previous in vivo experiments, h670 was hybridized with hybrid antigens prepared using other short peptide linkers, including FFRK, RK, AKVL, QLK, and FR (using single letter amino acid codes) by immunizing B6 mice subcutaneously. Complexes with were evaluated at different doses. In vitro IFN-γ ELISPOT analysis was performed as described above. The results including control values are as follows.

(200-13)

Immunogen Number of spots per 300,000 cells SIINFEKL Medium control Non-pulse control VSV control 4.4 μg Hsp70 + 2.4 μg NLLRLTGWFFRKSIINFEKL 114 ± 21 1.0 ± 1.2 1.0 ± 0 0.67 ± 0.41 4.4 μg Hsp70 + 2.4 μg NLLRLTGWRKSIINFEKL 70 ± 8.5 1.3 ± 1.1 0.67 ± 0.82 2.7 ± 1.1 0.9 μg Hsp70 + 0.48 μg NLLRLTGWFFRKSIINFEKL 98 ± 0.41 0.67 ± 0.82 1.3 ± 1.1 4.3 ± 2.3 0.9 μg Hsp70 + 0.48 μg NLLRLTGWRKSIINFEKL 29 ± 2.2 0 ± 0 1 ± 0 0 ± 0 2.4 µg NLLRLTGWFFRKSIINFEKL 11 ± 1.8 0.67 ± 0.82 0 ± 0 0.67 ± 0.82

200-21

Immunogen Number of spots per 400,000 cells SIINFEKL Medium control Non-pulse control VSV control 4.4 μg Hsp70 + 2.4 μg NLLRLTGWFFRKSIINFEKL 124 ± 8.8 0.33 ± 0.41 0.67 ± 0.82 2.67 ± 2.68 4.4 μg Hsp70 + 2.4 μg NLLRLTGWAKVLSIINFEKL 95 ± 12 1.33 ± 0.82 1.0 ± 1.2 0.67 ± 0.41

200-23

Immunogen Number of spots per 400,000 cells SIINFEKL Medium control Non-pulse control VSV control 4.4 μg Hsp70 + 2.4 μg NLLRLTGWFFRKSIINFEKL 318 ± 17 0.67 ± 0.51 0.67 ± 0.58 0.67 ± 0.58 4.4 μg Hsp70 + 2.4 μg NLLRLTGWQLKSIINFEKL 174 ± 18 0.0 ± 0.0 0.0 ± 0.0 3.7 ± 2.5 4.4 μg Hsp70 + 2.4 μg NLLRLTGWFRSIINFEKL 53 ± 2.9 0.0 ± 0.0 0.67 ± 0.58 1.0 ± 1.0 2.4 µg NLLRLTGWFRSIINFEKL 31 ± 5.7 1.0 ± 1.7 0.0 ± 0.0 0.67 ± 0.58

Example  8

In vivo studies in B6 mice similar to those described above were performed using the formulation without hsp70. The result is as follows.

(200-17)

Immunogen Number of spots per 400,000 cells SIINFEKL Medium control Non-pulse control VSV control Con A positive control 10 μg SIINFEKL 2.33 ± 0.41 0.33 ± 0.41 1.33 ± 0.82 1.7 ± 0.41 928 ± 71 0.5 μg NLLRLTGWFFRKSIINFEKL 22 ± 7.2 1.33 ± 0.41 1.67 ± 1.1 1.0 ± 0.71 906 ± 17 2.5 μg NLLRLTGWFFRKSIINFEKL 28 ± 2.7 1.0 ± 1.7 0.33 ± 0.41 2.0 ± 1.2 930 ± 23 25 μg NLLRLTGWFFRKSIINFEKL 46 ± 4.3 2.0 ± 0.41 1.33 ± 1.1 3.0 ± 0.71 1007 ± 17

Example  9

In vivo studies in B6 mice similar to the methods described above were performed using preparations with or without hsp70. In addition, one study was conducted in which the hybrid antigen was co-administered with the free heat shock protein-binding region peptide (NLLRLTGW). The result is as follows.

(VSV-72-02)

Immunogen Number of spots per 400,000 cells CTL analysis:% mortality at 200: 1 E / T SIINFEKL Medium control Non-pulse control VSV control Con A positive control 4.4 μg Hsp70 + 2.00 μg NLLRLTGWFFRKSIINFEKL 48 ± 11 0.0 ± 1.0 0.0 ± 1.0 4.0 ± 2.0 588 ± 151 32% 2.0 µg NLLRLTGWRKSIINFEKL 24 ± 1 1.0 ± 1.0 1.0 ± 1.0 5.0 ± 3.0 842 ± 73 24% 2.4 μg NLLRLTGWFFRKSIINFEKL + 50 times excess NLLRLTGW 2.0 ± 1.0 1.0 ± 1.0 0.0 ± 1.0 1.0 ± 1.0 422 ± 54 18% SIINFEKL 1.0 ± 1.0 0.0 ± 0.0 0.0 ± 1.0 1.0 ± 1.0 478 ± 67 6%

Example  10

The VSV epitope RGYVYQGL, used as a control in many of these experiments, was used as an epitope in the preparation of additional hybrid antigens of the present invention and evaluated for induction of immune responses in a similar experiment as described above.

(VSV-72-02)

Immunogen Number of spots per 400,000 cells SIINFEKL Medium control Non-pulse control VSV control 4 μg HSP + 2 μg NLLRLTGWFFRKSIINFEKL 48 ± 11 1.0 ± 1.0 0.0 ± 1.0 4.0 ± 2.0 4 μg HSP + 2 μg NLLRLTGWFFRKRGYVYQGL 1.0 ± 1.0 1.0 ± 1.0 4.0 ± 2.0 20 ± 1.0 4 μg HSP + 6 μg NLLRLTGWFFRKRGYVYQGL 6.0 ± 3.0 2.0 ± 2.0 12 ± 3.0 104 ± 13

Example  11

In order to measure the efficacy of the hybrid antigen, and the complex with hsp70 for the treatment of diseases, a model in which 200,000 E7 tumor cells (named E.G7) modified to express egg protein are subcutaneously injected in B6 mice is used. It was. Ten mice were used for each treatment group. Such models are described, for example, in Moroi et al., 2000, Proc. Nat. Acad. Sci. USA 97: 3485-3490. Results for the number of mice with tumors over time are shown in FIG. 1. After 31 days, none of the 10 mice immunized with hsp70: NLLRLTGWFFRKSIINFEKL progressed into tumors, nor did they progress into tumors in mice immunized with SIINFEKL emulsified in Titermax adjuvant. Three of 10 mice vaccinated with NLLRLTGWFFRKSIINFEKL alone (no hsp70) progressed to tumors. Five of 10 mice vaccinated with hsp70: SIINFEKL advanced to tumors, and nine of 10 mice immunized with Titermax and buffer alone progressed to tumors.

Example  12

The in vitro antigen presentation assay described above was further used to evaluate the formulations of the present invention. To demonstrate the requirement of the hybrid antigen of the present invention for hsp70 whether the hybrid antigen and more specifically the antigen of hsp70 is supplied in the form of an agent for entry into the antigen presentation pathway or is endogenously available. The analysis was performed with the following formulations, and the results are shown below.

(200-MF-41)

Formulation Pg / ml IL-2 produced by B3Z cells 0.5ng SIINFEKL 2690 ± 369 0.5ng NLLRLTGWFFRKSIINFEKL 46 ± 11 1.4μg Hsp70 + 0.5ng NLLRLTGWFFRKSIINFEKL 3920 ± 344 1.4 μg Hsp70 0.0 ± 0.0

Example  13

Firat et al., 1999, "H-2 class I knockout, HLA-A2.1-transgenic mice: a versatile animal model for preclinical evaluation of antitumor immunotherapeutic strategies," Eur Immunol. 29: 3112-21, the HHD II mouse model with the human HLA-A2 complex disclosed in the following experiment was used to evaluate human HLA-A2 epitope in the hybrid antigen of the present invention. The "IMD" peptide epitope IMDQVPFSV from the human melanoma antigen gp100 was measured in small and large doses of the hybrid antigens of the present invention in the HHD II model. A similar method to that described above was used for ELISPOT analysis, in which the test peptide was an IMD peptide and the control peptide YMDGTMSQV (“YMD”) from melanoma antigen tyrosinase was used. The results are shown in the table below.

(HHD II 200-72-02)

Immunogen Number of spots per 400,000 cells IMD Medium control Non-pulse control YMD control 4 μg Hsp70 + 5 μg NLLRLTGWFFRKIMDQVPFSV 139 ± 11 0.67 ± 0.58 1.0 ± 1.0 3.7 ± 0.58 4 μg Hsp70 + 10 μg NLLRLTGWFFRKIMDQVPFSV 217 ± 3.2 0.67 ± 0.58 4.0 ± 6.0 2.7 ± 1.5 2 μg NLLRLTGWFFRKIMDQVPFSV 27 ± 5.1 0.0 ± 0.0 0.0 ± 0.0 2.0 ± 2.0

Similar experiments in HHD II mice carried out using YMD as epitope in the hybrid antigen were performed in complex with hsp70 and the results are as follows.

(200-72-01)

Immunogen Number of spots per 400,000 cells YMD Medium control Non-pulse control IMD control 4 μg Hsp70 + 5 μg NLLRLTGWFFRKYMDGTMSQV 33 ± 7.8 1.0 ± 0.0 1.0 ± 0.0 10. ± 1.4 4 μg Hsp70 + 10 μg NLLRLTGWFFRKYMDGTMSQV 323 ± 44 0.0 ± 0.0 1.5 ± 0.71 1.5 ± 0.71

Example  14

FAPGNYPAL, an epitope of Sendai virus (SdV), was evaluated as a hybrid antigen of the present invention in B6 mice similar to the above. The result is as follows.

(200-18)

Immunogen Number of spots per 400,000 cells SdV Medium control SIINFEKL control 4 μg hsp70 and 2 μgNLLRLTGWFFRKSIINFEKL 1.3 ± 1.2 1.0 ± 1.0 197 ± 27 2μgNLLRLTGWFFRKRGYVYQGL 0.33 ± 0.58 0.0 ± 0.0 87 ± 20 4 μg hsp70 and 2 μgNLLRLTGWFFRKFAPGNYPAL 38 ± 17 0.33 ± 0.58 1.0 ± 1.0 13 μg hsp70 and 7 μgNLLRLTGWFFRKFAPGNYPAL 169 ± 32 4.3 ± 1.5 7.0 ± 3.5

Example  15

Co-administration of two hybrid antigens of the present invention with hsp70 in B6 mice was performed in-vivo experiments. Hybrid antigens containing SIINFEKL of obbualbumin and RGYVYQGL of VSV were mixed and immunized with hsp70. The result is as follows.

(OVA-VSV-72-01)

Immunogen Number of spots per 400,000 cells VSV Medium control Non-pulse control OVA 2µg hsp702µNLLRLTGWFFRKSIINFEKL 2µg NLLRLTGWFFRKRGYVYQGL 77 ± 19 2.0 ± 1.0 2.0 ± 1.0 366 ± 19 2μg hsp706μNLLRLTGWFFRKSIINFEKL 6μg NLLRLTGWFFRKRGYVYQGL 185 ± 9 1.0 ± 1.0 4.0 ± 2.0 349 ± 10

Example  16

In aspects of the invention as described above, an agent comprising a plurality of hybrid antigens containing multiple antigen epitopes may be combined with one or more heat shock proteins for immunization in humans to induce an effective immune response for treating or preventing a disease. It may be formulated. For example, to treat human melanoma, a formulation containing eight different melanoma epitopes can be prepared as a hybrid antigen, for example formulated with hsp70. In this particular formulation, the N-terminal heat shock protein region NLLRLTGW is used for all epitopes and is linked to the epitope at the C-terminus using the peptide linker FFRK. Other binding regions and linkers are included here. Such specific agents are useful for treating patients with HLA-A2 haplotypes. The formulation comprises the following hybrid antigens with hsp70:

Antigen Resource and Amino Acid Sequences Hybrid antigen sequence gp 100: amino acids 209-217 (modified 210M) NLLRLTGWFFRKIMDQVPFSV Tyrosinase: amino acids 368-376 (modified 370D) NLLRLTGWFFRKYMDGTMSQV Melan-A: amino acids 26-35 (modified 27L) NLLRLTGWFFRKELAGIGILTV NY-ESO-1: amino acids 157-165 (modified 165 V) NLLRLTGWFFRKSLLMWITQV TRP-2: amino acids 180-188 NLLRLTGWFFRKSVYDFFVWL MAGE-10: amino acids 254-262 GLYDGMEHLGSGNLLRLTGW gp100: amino acid 280-288 (288V) YLEPGPVTVGSGNLLRLTGW SSX-2: Amino Acids 41-49 KASEKIFYVGSGNLLRLTGW

In one embodiment, approximately the same amounts of the eight hybrid antigens described above can be mixed with hsp70 and administered in saline. In another embodiment, the formulation comprises the first five hybrid antigens listed. The aforementioned formulations containing heat shock proteins in saline contain ADP to stabilize the complexes as well as other components such as the excipients, diluents and carriers mentioned above. In another embodiment, a mixture of the eight hybrid antigens described above or the first five listed are formulated in saline for administration without a heat shock protein.

Example  17

The first sponsored protocol was evaluated in this experiment. The five protocols using NLLRLTGWFFRKSIINFEKL hybrid antigen or without co-administered hsp70 are as follows: 1) 0 day administration, 7 day analysis; 2) 0 and 7 day administration, 21 day analysis; 3) 0 day dosing, 21 day analysis; 4) 0 and 14 day administration, 28 day analysis; And 5) day 0 administration and 28 day analysis. The results at spots per 400,000 cells are as follows.

(200-28-72-Ola, -Olb, -Olc)

Immunized Protocol Date 0 0, 7 0 0, 14 0 Protocol date analyzed 7 21 21 28 28 SIINFEKL 3.0 ± 2.0 2.0 ± 1.0 0.0 ± 1.0 1.0 ± 1.0 1.0 ± 1.0 2 μg hsp70, 4 μg SIINFEKL 3.0 ± 1.0 6.0 ± 1.0 22 ± 8.0 3.0 ± 2.0 20 ± 5.0 2μgNLLRLTGWFFRKSIINFBKL 72 ± 5.0 24 ± 6.0 42 ± 7.0 25 ± 9.0 82 ± 11 2 μg hsp70 and 4 μgNLLRLTGWFFRKSIINFBKL 99 ± 12 98 ± 11 141 ± 14 398 ± 18 27 ± 2.0 2 μg hsp70 5.0 ± 6.0 3.0 ± 2.0 3.0 ± 0.0 1.0 ± 1.0 4.0 ± 3.0

Example  18

Still other experiments were performed with a mixture of hybrid antigens to account for the induction of an immune response to the antigenic components as described above. In this experiment, a hybrid antigen containing SIINFEKL and VSV peptide RGYVYQGL was used.

(VSV / OVA-72-02)

Immunogen Number of spots per 300,000 cells SIINFEKL Medium control Non-pulse control VSV (RGYVYQGL) 3.7μg hsp702μNLLRLTGWFFRKSIINFEKL 238 ± 27 0.0 ± 0.0 1.0 ± 1.0 5.0 ± 2.0 11.2 μg hsp706 μg NLLRLTGWFFRKSIINFEKL 330 ± 45 1.0 ± 1.0 0.0 ± 0.0 4.0 ± 1.0 3.7μg hsp702μNLLRLTGWFFRKRGYVYQGL 1.0 ± 1.0 1.0 ± 1.0 0.0 ± 0.0 61 ± 11 11.2 μg hsp706 μg NLLRLTGWFFRKRGYVYQGL 2.0 ± 2.0 2.0 ± 1.0 2.0 ± 0.0 147 ± 20 3.7μg hsp702μNLLRLTGWFFRKSIINFEKL 2μNLNLLTLTGWFFRKRGYVYQGL 179 ± 4.0 2.0 ± 2.0 1.0 ± 1.0 165 ± 11 11.2 μg hsp706 μg NLLRLTGWFFRKSIINFEKL 6 μg NLLRLTGWFFRKRGYVYQGL 310 ± 13 1.0 ± 1.0 1.0 ± 1.0 242 ± 52

Example  19

The binding affinity for hybrid antigens containing the antigenic region (ofbualbumin) SIINFEKL or of the heat shock protein (linking the RGYVYQGL) of region NLLRLTGW and the various linkers set up in Example 32 as described in Example 17 are as described in Example 17. It was done together. Antigen regions alone had Kd for hsp70 binding of 235 μM and 82 μM, respectively. The results are as follows.

hybrid  antigen hsp70 To join Kd NLLRLTGWGSGSIINFEKL 1.6 μM NLLRLTGWFFRKSIIMFBKL 2.2 μM NLLRLTGWRKSIINFEKL 0.8 μM NLLRLTGWAKVLSIINFEKL 2.0 μM NLLRLTGWQLKSIINFEKL 0.4 μM NLLRLTGWFRSIINFEKL 1.5 μM NLLRLTGWGSGRGYVYQGL 1.4 μM NLLRLTGWFFRKRGYVYQGL 1.0 μM NLLRLTGWRKRGYVYQGL 0.6 μM

Example  20

Further studies were conducted to assess the immunogenicity of hybrid antigens when administered alone to B6 mice without co-administration of hsp70. The evaluation method using IFN-γ ELISPOT is as described above.

(Controls 200-24 and 200-30)

Immunogen Number of spots per 300,000 cells SIINFEKL SWDFITV badge Non-pulse splenocytes 25μgNLLRLTGWFFRKSIENFEKL 109 ± 14 NT 0 ± 0 3.0 ± 2.0 24.9µgNLLRLTGWFFRKSSWDFITV NT 26 ± 5 0.67 ± 0.58 0.33 ± 0.58 201 μg NLLRLTGWFRSIINFEKL 12 ± 2 NT 0.67 ± 0.58 0.67 ± 0.58 NT: not tested

Example  21

Hybrid antigens were prepared to comprise two antigens, and the hybrid antigens were separated by the linkers described above as having the following general structure.

(Thermal shock protein binding region)-(linker)-(antigen 1)-(linker)-(antigen 2)

In this embodiment, the heat shock protein binding region is in the N-terminal portion of the hybrid antigen, whereas the case is not essential, and hybrid antigens having a heat shock protein connection region between the C-terminal or two antigenic regions are provided in the present invention. Included by. In addition, although the linker peptide is used between the antigenic region and between the heat shock protein binding region and the close antigenic region in the examples below, this is not essential, and other linker peptides may be used. In addition, the presence of the linker in one or both positions is optional. In addition, three or more antigenic peptides may be used. Briefly, hybrid antigens having two or more such antigenic regions are called tandem hybrid antigens. Such single-strand hybrid antigen compositions, complexes of one or more single-strand hybrid antigens and heat shock proteins, and one or more single-strand hybrid antigens or at least one heat shock protein and at least one single-line hybrid antigen are administered to induce an immune response or prevent disease Methods for treating or treating are encompassed herein in their entirety. The following experiments compare immunogenicity and dose response studies of mixtures of two hybrid antigens and single-line hybrid antigens containing the same antigen. In one embodiment, a peptide comprising two linkers and epitopes without a heat shock protein binding region was included.

(Control-200-72-01)

Immunogen Number of spots per 300,000 cells SIINFEKL Medium control Non-pulse control RGYVYQGL 19.2µgNLLRLTGWFFRKSIINFEKLFFRKRGYVYGL 390 ± 56 1.7 ± 1.1 3.0 ± 1.9 146 ± 13 19.2µgNLLRLTGWFFRKRGYVYQGLFFRKSIINFEKL 180 ± 11 1.3 ± 1.1 2.7 ± 1.1 321 ± 5.8

(S200-72-02)

Immunogen Number of spots per 300,000 cells SIINFEKL Medium control RGYVYQGL 7.3 μgFFRKSIINFEKLFFRKRGYVYQGL 8.3 ± 1.1 1.7 ± 0.4 31 ± 5.5 9.6µgNLLRLTGWFFRKSIINFEKLFFRKRGYVYQGL 713 ± 13 9.0 ± 1.2 207 ± 8.2 9.6µgNLLRLTGWFFRKRGYVYQGLFFRKSIINFEKL 69 ± 12 0.7 ± 0.4 460 ± 14

(S200-72-12)

Immunogen Spots per 300,000 Cells SIINFEKL Medium control RGYVYQGL 20μgNLLRLTGWFFRKSIINFEKLFFRKRGYVYQGL 410 ± 49 0.3 ± 0.4 250 ± 11 10μgNLLRLTGWFFRKSIINFEKLFFRKRGYVYQGL 360 ± 13 0.3 ± 0.4 100 ± 10 5ugNLLRLTGWFFRKSIINFEKLFFRKRGYVYQGL 130 ± 3.3 0 ± 0 35 ± 6.6 20μgNLLRLTGWFFRKRGYVYQGLFFRKSIINFEKL 150 ± 6 0 ± 0 380 ± 12 10μgNLLRLTGWFFRKRGYVYQGLFFRKSIINFEKL 30 ± 3 0 ± 0 83 ± 5

In this and other examples, the epitopes proximate to the heat shock protein binding region showed the strongest immune response, so the positioning of epitopes selected for the vaccine formulations of the present invention was administered as complexes having heat shock proteins or administered under conditions without heat shock proteins. Whatever was administered may be placed to maximize the overall immunity of the agent.

Example  22

In the following experiments, mixtures of single-line hybrid antigens were measured for immunogenicity. In addition to the H2-K ^b class I peptides from obbualbumin (SIINFEKL) and VSV (RGYVYQGL), the H2-K ^b β-casein peptide IAYFYPEL and the Sendai virus peptide FAPGNYPAL were also used. In another embodiment, two single line hybrid antigens with the same antigenic peptide in an alternate configuration were mixed. A strong immune response to four epitopes was induced. All formulations included 1 mM ADP herein. One embodiment has been described as follows.

(200-72-04)

Immunogen Spots per 300,000 Cells SIINFEKL RGYVYQGL IAYFYPEL FAPGNYPAL 9.6µgNLLRLTGWFFRKSIINFEKLFFRKRGYVYQGL 537 ± 16 150 ± 10 4.7 ± 0.8 5.7 ± 2.5 9.7 μg NLLRLTGWFFRKIAYFYPELFFRKFAPGNYPAL 1.7 ± 1.1 1.7 ± 0.8 128 ± 9.2 136 ± 6.6 9.6µgNLLRLTGWFFRKSIINFEKLFFRKRGYVYQGL + 9.7µgNLLRLTGWFFRKIAYFYPELFFRKFAPGNYPAL 363 ± 31 256 ± 5.3 127 ± 7.9 155 ± 28

5 S200-72-13

Immunogen Spots per 300,000 Cells SIINFEKL RGYVYQGL IAYFYPEL FAPGNYPAL 9.6µgNLLRLTGWFFRKIAYFYPELFFRKFAPGNYPAL + 9.6µgNLLRLTGWFFRKSIINFEKLFFRKRGYVYQGL 388 ± 6.8 72 ± 5.0 402 ± 17 379 ± 30 9.6µgNLLRLTGWFFRKRGYVYQGLFFRKSIINFEKL + 9.6µgNLLRLTOWFFRKIAYFYPELFFRKFAPGNYPAL 76 ± 1.9 159 ± 8.3 115 ± 20 172 ± 5.9

S200-72-13

Immunogen Spots per 300,000 Cells SIINFEKL RGYVYQGL IAYFYPEL FAPGNYPAL 9.6µgNLLRLTGWFFRKSIINFEKLFFRKRGYVYQGL 450 ± 10 273 ± 12 3.0 ± 1.4 0.33 ± 0.41 9.6µgNLLRLTGWFFRKRGYVYQGLFFRKSIINFEKL 82 ± 4 445 ± 30 1.3 ± 0.41 0 ± 0 9.6µgNLLRLTGWFFRKSIINFEKLFFRKRGYVYQGL + 9.6µgNLLRLTGWFFRKRGYVYQGLFFRKSIINFEKL 202 ± 7.6 188 ± 24 1.0 ± 0.7 0.67 ± 0.41

S200-72-13, no ADP

Immunogen Spots per 300,000 Cells SIINFEKL RGYVYQGL IAYFYPEL FAPGNYPAL 9.6µgNLLRLTGWFFRKSIINFEKLFFRKRGYVYQGL 228 ± 2.5 126 ± 2.9 1.7 ± 0.4 0 ± 0 9.6µgNLLRLTGWFFRKRGYVYQGLFFRKSIINFEKL 83 ± 9 189 ± 19 13 ± 15 0.33 ± 0.41 9.6µgNLLRLTGWFFRKSIINFEKLFFRKRGYVYQGL + 9.6µgNLLRLTGWFFRKRGYVYQGLFFRKSIINFEKL 115 ± 7.8 86 ± 11 0.33 ± 0.41 0 ± 0

Example  23

In the following experiments, when a mixture of two-line hybrid antigens and a single hybrid antigen are administered to B6 mice, up to five antigenic peptides are delivered and induce immunogenicity without co-administered HSP70. The single-line hybrid antigens included VSV and obbualbumin peptides on one side and β-casein and Sendai virus peptides on the other. Single hybrid antigen contained NS2-114 influenza peptide (RTFSFQLI).

S200-72-15

Immunogen Spots per 300,000 Cells SIINFEKL RGYVYQGL IAYFYPEL FAPGNYPAL RTFSFQLI 9.6µgNLLRLTGWFFRKRGYVYQGL FFRKSIINFEKL +19 µgNLLRLTGWFFRKIAYFYPELF FRKFAPGNYPAL 67 ± 6.1 205 ± 20 229 ± 28 266 ± 33 0 ± 0 9.6µgNLLRLTGWFFRKRGYVYQGL FFRKSIINFEKL +19 µgNLLRLTGWFFRKIAYFYPELF FRKFAPGNYPAL +12.2 µgNLLRLTGWFFRKRTFSFQLI 156 ± 3.3 299 ± 18 175 ± 12 125 ± 3.3 33 ± 4.7

Example  24

The immunogenicity of the aforementioned single hybrid antigen administered without a heat shock protein was measured in parallel with the helper T cell epitope in the presence of the hybrid antigen. In most experiments, the H2-K ^b class II epitope of aminobubumin, amino acids 323-339, TEWTSSNVMEERKIKV was used (e.g., the hybrid antigen had the sequence NLLRLTGWFFRKTEWTSSNVMEERKIKV). Inclusion of a class II peptide-containing hybrid antigen increased the response to class I epitopes at an average of about seven foldings.

(250-72-08)

Immunogens Containing Class I Hybrid Peptides Spots per 300,000 Cells Responds to Class I Epitopes When Class I Hybrid Antigens Are Administered Responds to class I epitopes when class I and class II hybrid antigen mixtures are administered badge Non-cell 24.2μgNLLRLTGWFFRKDAPIYTNV 2 ± 19 13 ± 3.9 0.7 ± 0.4 0 ± 0 24.9µgNLLRLTGWFFRKSSWDFITV 18 ± 0.7 98 ± 5.8 0.7 ± 0.8 0.7 ± 0.4 25.4 µg NLLRLTGWFFRKRTFSFQLI 5.3 ± 1.5 43 ± 7.6 0.3 ± 0.4 0 ± 0 25.5 µg NLLRLTGWFFRKIAYFYPEL 11 ± 3 73 ± 9.8 0 ± 0 0 ± 0

Example  25

In the presence of heat shock proteins, the effect on the immunogenicity of hybrid antigens co-administered with various hybrid antigens containing H2-Kb class II peptides was measured. Class I peptides are SSWDFITV or DAPIYTNV; Class II peptides included the above-mentioned Obualbumin peptide, class II peptide NNFTVSFWLRVPKVSASHL (eg, the hybrid antigen has the sequence NLLRLTGWFFRKNNFTVSFWLRVPKVSASHL) or HBV (amino acids 128-140) peptide, TPPAYRPPNAPIL from the tetanus albumin peptide, tetanus toxin.

250-72-13

Immunogen Spots per 300,000 Cells badge SSWDFITV 24.9µgNLLRLTGWFFRKSSWDFITV 3.0 ± 0.7 78 ± 3.9 24.9 μg NLLRLTGWFFRKSSWDFITV +27.4 μg NLLRLTGWFFRKTPPAYRPPNAPIL 8.0 ± 3.1 84 ± 7.1 24.9 μg NLLRLTGWFFRKSSWDFITV +33.6 μg NNFTVSFWLRVPKVSASHLGSGNLLRLTGW 3.7 ± 1.1 315 ± 15 24.9 μg NLLRLTGWFFRKSSWDFITV 36.4 μg HWDFAWPWNGSGNNFTVSFWLRVPKVSASHL 2.7 ± 2.0 135 ± 5.7 24.9 µg NLLkLTGWFFRKSSVVDFITV +34.7 µg NLLRLTGWFFRKTEWTSSNVMEERKIKV 1.7 ± 0.4 229 ± 12

Thus, helper T cell epitopes can be administered in a hybrid antigen as epitope only and as a mixture with other hybrid antigens containing Class I epitopes, or helper T cell epitopes can be included in a single-line hybrid antigen as one of the epitopes. have. These are not possible with many of the variants typical of the hybrid antigen composition of the present invention.

Example  26

In a manner similar to the experiment described above, the immunogenicity of single-line hybrid antigens was measured with and without co-administration of hybrid antigens containing Obualbumin class II peptides.

S250-72-12

Immunogen Spots per 300,000 Cells IAYFYPEL FAPGNYPAL badge 19㎍NLLRLTGWFFRKIAYFYPELFFRKFAPGNYPAL 9.3 ± 4.7 17 ± 9 0.7 ± 0.6 19µgNLLRLTGWFFRKIAYFYPELFFRKFAPGNYPAL +20.8 µgNLLRLTGWFFRKTEWTSSNVMEERKIKV 44 ± 5.1 58 ± 5.2 0.7 ± 0.6

250-72-15

Immunogen Spots per 300,000 cells IAYFYPEL 25.5 µg NLLRLTGWFFRKIAYFYPEL 3.7 ± 3.1 25.5 µg NLLRLTGWFFRKIAYFYPEL +34.7 µg NLLRLTGWFFRKTEWTSSNVMEERKIKV 133 ± 11 25.5 μg NLLRLTGWFFRKIAYFYPEL + 25 MgNLLRLTGWFFRKSIINFEKL 88 ± 9.9

Example  27

Similar experiments were also performed with hybrid antigens comprising adjuvant T cell epitopes co-administered with at least one single-line hybrid antigen in the presence of co-administration of heat shock proteins.

250-72-12

Immunogen Spots per 300,000 Cells badge SIINFEKL RGYVYQGL IAYFYPEL FAPGNYPAL 24㎍NLLRLTGWFFRKIAYFYPBLF FRKFAPGNYPAL 0.7 ± 0.6 NT NT 9.3 ± 4.7 17 ± 8.7 24ugNLLRLTGWFFRKIAYFYPELF FRKFAPGNYPAL + 21ugNLLRLTGWFFRKTEWTSSNV MEERKIKV 0.7 ± 0.6 NT NT 44 ± 5.1 67 ± 5.5 15μgNLLRLTGWFFRKFAPGNYPAL 0 ± 0 NT NT 0.3 ± 0.6 4.3 ± 3.2 15 µg NLLRLTGWFFRKFAPGNYPA L +21 µg NLLRLTGWFFRKTEWTSSNV MEERKIKV 0 ± 0 NT NT 2.3 ± 2.1 58 ± 5.2

Example 28

Immunization studies with hybrid antigens comprising human class I (HLA-A2) epitopes were performed in the HHDII mice described above. Animals were immunized with complexes made with 5 μg hsp70 and 33 μg NLLRLTGWFFRKYMDGTMSQV. The ELISPOT results in cells per 300,000 were: medium, 1.33 ± 0.58; Splenocytes 1 ± 0; Splenocytes + YMDGTMSQV 123 ± 13: and Splenocytes + IMDQVPFSV 4 ± 1.

Example  29

In another experiment with HHDII mice, immunogenic HLA-A2 epitopes from Trp-2 were used (SVYDFFVWL). Since these epitopes are also H2-Kb epitopes and HHDII mice are B6 mice (H2-Kb) background, the immune response induced against Trp-2 peptides indicates a breakdown of resistance to self-epitopes in the mouse model. The results of this experiment demonstrated that resistance to such self-epitopes was destroyed, and the present invention also relates to a method of destroying resistance by administering the hybrid antigen and the complex of the present invention.

HHDⅡ-200-72-03

Immunogen Spots per 300,000 Cells badge SVYDFFVWL IMDQVPFSV YMDGTMSQV 4.33 μg NLLRLTGWFFRKSVYDFFVWL +25 μgsp70 0.5 ± 0.71 166 ± 25 2.0 ± 1.4 3.5 ± 0.71 8.66 µg NLLRLTGWFFRKSVYDFFVWL +25 µgsp70 3.5 ± 0.71 114 ± 11 7.7 ± 2.1 11 ± 3.1 4.1μgNLLRLTGWFFRKYMDGTMSQV + 25μgsp70 3.0 1.0 2.0 ± 1.4 74 ± 2.8 4.1μgNLLRLTGWFFRKIMDQVPQV + 25μgsp70 1.0 ± 1.4 2.0 ± 2.0 984 ± 26 2.3 ± 1.5

Example  30

HHDII mice were used to determine the immunogenicity of three hybrid antigens comprising hsp70 and the fourth set of Example 27 specific HIV viral component epitopes.

HHDⅡ-200-72-07

Immunogen Spots per 300,000 Cells badge ILKEPVHGV VIYQYMDDL SLYNTVATL 36μgNLLRLTGWFFRKILKEPVHGV + 25μg hsp70 1.0 ± 1.0 34 ± 12 0 ± 0 NT 36μgNLLRLTGWFFRKVIYQYMDDL + 25μg hsp70 0 ± 0 0.67 ± 0.58 24 ± 6.1 NT 36μgNLLRLTGWFFRKSLYNTVATL + 25μg hsp70 0.67 ± 0.58 NT NT 140 ± 6.7 NT = not tested

Example  31

A mixture of hybrid antigens containing H2-Kb epitope mixed with hsp70 was measured for immunogenicity in B6 mice as described above.

OBS- / 2-01

Immunogen Spots per 300,000 Cells badge Non-cell SIINFEKL FAPGNYPAL IAYFYPEL 2 μg NLLRLTGWFFRKSIINFFKL + 13.7 μg hsp70 One 2 ± 1 148 ± 11 7 ± 2 3 ± 2 10 μg NLLRLTGWFFRKIAYFYPEL + 13.7 μg hsp70 0 2 ± 2 3 ± 1 8 ± 2 47 ± 13 10μgNLLRLTGWFFRKFAPGNYPAL + 13.7μg hsp70 3 3 ± 3 3 ± 2 83 ± 6 6 ± 1 2 μg NLLRLTGWFFRKSIINFEKL 10 μg NLLRLTGWFFRKIAYFYPEL + 27.4 μg hsp70 2 4 ± 2 94 ± 4 9 ± 3 29 ± 4 2 μg NLLRLTGWFFRKSIINFEKL + 10 μg NLLRLTGWFFRKFAPGNYPAL + 27.4 μg hsp70 3 3 ± 0 169 ± 7 157 ± 27 4 ± 2 10 µg NLLRLTGWFFRKIAYFYPEL + 10 µg NLLRLTGWFFRKFAPGNYPAL ++ 27.4 µg hsp70 3 3 ± 3 4 ± 3 46 ± 8 39 ± 2 2 μg NLLRLTGWFFRKSIINFEKL + 10 μg NLLRLTGWFFRKIAYFYPEL + 10 μg NLLRLTGWFFRKFAPGNYPAL + 41 μg hsp70 One 5 ± 2 149 ± 19 61 ± 5 60 ± 7

Example  32

The immunogenicity of single-line hybrid antigens mixed with hsp70 was studied in B6 mice.

S200-72-01

Immunogen Spots per 300,000 Cells Medium control x Non-pulse control SIINFEKLRGYVYQGL 5.6 μg hsp70 + 3 μg NLLRLTGWFFRKSIINFEKL 0.33 ± 0.41 0.67 ± 0.41 43 ± 9.2 1 ± 0 11.2 μg hsp70 + 5.9 μg NLLRLTGWFFRKRGYVYQGL 0.33 ± 0.41 0.33 ± 0.41 1 ± 0.71 102 ± 16 11.2 μg hsp70 + 3 μg NLLRLTGWFFRKSIINFEKL + 5.9 μg NLLRLTGWFFRKRGYVYQGL 0.67 ± 0.82 1.7 ± 0.41 182 ± 11 113 ± 10 5.6 μg hsp70 + 4.8 μg NLLRLTGWFFRKSIINFEKLFFRKRGYVYQGL 0 ± 0 4 ± 1.4 456 ± 19 113 ± 1.1 11.2 μg hsp70 + NLLRLTGWFFRKSIINFEKLFFRKRGYVYQGL 0 ± 0 10 ± 3.3 505 ± 57 90 ± 11 22.4 μg hsp70 + 9.6 μg NLLRLTGWFFRKSIINFEKLFFRKRGYVYQGL 0.67 ± 0.82 1.7 ± 0.41 289 ± 26 130 ± 12 5.6 μg hsp70 + 4.8 μg NLLRLTGWFFRKRGYVYQGLFFRKSIINFEKL 0.33 ± 0.41 2.3 ± 0.41 72 ± 9.5 98 ± 9.2 11.2 μg hsp70 + 9.6 μg NLLRLTGWFFRKRGYVYQGLFFRKSIINFEKL 2 ± 0 2.3 ± 1.5 370 ± 16 617 ± 23 22.4 μg hsp70 + 19.3 μg NLLRLTGWFFRKRGYVYQGLFFRKSIINFEKL 0.67 ± 0.41 4.0 ± 2.1 336 ± 7.8 728 ± 12

Many modifications and variations of the present invention can be made without departing from the spirit and scope of the invention, which is apparent to those of ordinary skill in the art. The specific embodiments described herein are provided by way of example only, and the invention is limited only in the full scope of the equivalents to which such claims are entitled, along with the appended claims.

Various publications are cited herein and the contents of which are incorporated herein by reference in their entirety.

The present invention relates to methods and compositions for inducing an immune response in a subject, wherein the subject is administered in combination with an effective amount of at least one defined hybrid antigen and optionally one or more heat shock proteins. These methods and compositions can be used to treat infectious diseases and tumors.

Claims (21)

At least one antigenic domain of an infectious or tumor antigen, at least one binding domain that covalently binds to a heat shock protein, and between them Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); And AA 1 is A, S, V, E, G, L, or K, AA 2 is K, V, or E; And AA3 is at least one peptide linker selected from the group consisting of AAl-AA2-AA3-leucine, which is V, S, F, K, A, E, or T. A composition inducing an immune response against an infectious agent or tumor antigen comprising at least one hybrid antigen of claim 1. A composition for inducing an immune response against an infectious agent or tumor antigen comprising a complex of at least one hybrid antigen and at least one heat shock protein of claim 1. The composition of claim 3, wherein the heat shock protein is hsp70. A method of inducing an immune response against an infectious agent or tumor antigen comprising administering at least one hybrid antigen of claim 1 to a subject. Non-covalently coupled hybrid antigen and heat shock protein (a) the hybrid antigen of claim 1; And (b) inducing an immune response against an infectious agent or tumor antigen comprising administering to the subject a complex of heat shock proteins. The method of claim 6, wherein the heat shock protein is hsp70. A method of treating an infectious disease or tumor wherein at least one antigen domain comprises administering to the subject at least one hybrid antigen of claim 1 derived from an infectious disease or tumor. Non-covalently coupled hybrid antigen and heat shock protein (a) at least one hybrid antigen of claim 1, wherein at least one antigen domain is from an infectious disease or tumor; And (b) A method of treating an infectious disease or tumor comprising administering a complex of heat shock proteins to a subject. 10. The method of claim 9, wherein the heat shock protein is hsp70. At least one antigenic domain of an infectious or tumor antigen, at least one binding domain that covalently binds to a heat shock protein, and between them Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gin Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); And AA1 is A, S, V, E, G, L, or K, AA2 is K, V, or E; and AA3 is AAl-AA2-AA3, which is V, S, F, K, A, E, or T. A hybrid antigen essentially comprising at least one peptide linker selected from the group consisting of leucine. A composition inducing an immune response against an infectious agent or tumor antigen comprising at least one hybrid antigen of claim 11. A composition inducing an immune response against an infectious agent or tumor antigen comprising a complex of at least one hybrid antigen of claim 11 and at least one heat shock protein. The composition of claim 13, wherein the heat shock protein is hsp70. A method of inducing an immune response against an infectious agent or tumor antigen comprising administering at least one hybrid antigen of claim 11 to a subject. Non-covalently coupled hybrid antigen and heat shock protein (a) the hybrid antigen of claim 11; And (b) inducing an immune response against an infectious agent or tumor antigen comprising administering to the subject a complex of heat shock proteins. The method of claim 16, wherein the heat shock protein is hsp70. A method of treating an infectious disease or tumor wherein the at least one antigen domain comprises administering to the subject at least one hybrid antigen of claim 11 derived from an infectious disease or tumor. Non-covalently coupled hybrid antigen and heat shock protein (a) at least one hybrid antigen of claim 1, wherein at least one antigen domain is from an infectious disease or tumor; And (b) A method of treating an infectious disease or tumor comprising administering a complex of heat shock proteins to a subject. The method of claim 19, wherein the heat shock protein is hsp70. Phe Phe Arg Lys (FFRK; SEQ ID NO: 1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO: 1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO: 1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO: 1001); Lys Asn (KN); Arg Lys (RK); or AA1 is A, S, V, E, G, L, or K, AA2 is K, V, or E; And AA3 is a peptide that is AA1-AA2-AA3-leucine, which is V, S, F, K, A, E, or T.