CN1816348A - Improved heat shock protein-based vaccines and immunotherapies - Google Patents

Improved heat shock protein-based vaccines and immunotherapies Download PDF

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CN1816348A
CN1816348A CN 200480015764 CN200480015764A CN1816348A CN 1816348 A CN1816348 A CN 1816348A CN 200480015764 CN200480015764 CN 200480015764 CN 200480015764 A CN200480015764 A CN 200480015764A CN 1816348 A CN1816348 A CN 1816348A
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seq
leu
trp
phe
arg
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J·B·弗莱奇特纳
K·普赖恩斯－科汉
S·梅塔
P·斯卢萨勒奇茨
S·安德杰利克
B·H·巴伯
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Agenus Inc
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Antigenics LLC
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Abstract

Hybrid antigens comprising an antigenic domain and improved heat shock protein binding domains are described which are useful for the induction of an immune response to the antigenic domain and thus can be used to treat infectious diseases and cancers that express an antigen of the antigenic domain.

Description

The vaccine and the immunization therapy based on heat shock protein of improvement
The mutual reference of related application
The application requires the priority of u.s. patent application serial number of submitting on February 12nd, 2,004 10/776,521 and the PCT patent application PCT/US04/04340 that submitted on February 13rd, 2004, these two patent applications all by reference integral body be attached to herein.According to the 35th the 119th (e) money of United States code, the application also requires the provisional application sequence number 60/462 of submission on April 11st, 2003,469, the provisional application sequence number of submitting on April 18th, 2,003 60/463, the provisional application sequence number 60/503 of 746 and 2003 on JIUYUE submission in 16,, 417 priority, all these three provisional application all by reference integral body be attached to herein.
Introduction
The present invention relates to the method and composition of induce immune response in the patient, wherein said patient is given the heterozygosis antigen of at least a or various definitions of effective dose, and it is optional in conjunction with one or more heat shock proteins.These method and compositions can be used for treating infectious disease and cancer.
Background of invention
Heat shock protein is observed increment expression in the mammalian cell that is exposed to temperature rising suddenly at first, and the expression of most of cell protein significantly descends.Henceforth having determined these albumen can produce under all kinds of pressure (comprising glucose deprivation) effect.Term used herein " heat shock protein " had both comprised the albumen of clearly sorting out as described, also comprised other stress protein, comprised this albumen homology thing (promptly not having incentive condition) of constitutive expression.The example of heat shock protein comprises BiP (being also referred to as grp78), hsp70, hsc70, gp96 (grp94), hsp60, hsp40 and hsp90.
Heat shock protein can be in conjunction with nascent peptide that form or that be squeezed into endoplasmic reticulum on other albumen of non-natural attitude, particularly binding ribosomal body.Hendrick and Hartl，Ann.Rev.Biochem.62：349-384(1993)；Hartl，Nature 381：571-580(1996)。And heat shock protein has been shown that correct folding the and assembling in kytoplasm, endoplasmic reticulum and mitochondrion plays an important role to protein; Given this function, they are called as " molecular chaperones ".Frydman et al.，Nature 370：111-117(1994)；Hendrick and Hartl，Ann.Rev.Biochem.62：349-384(1993)；Hartl，Nature 381：571-580(1996)。
For example, protein B iP is a member that is called as in the class heat shock protein of hsp70 family, has found that it combines new synthetic folding μ heavy chain immunoglobulin prior to heavy chain and being assembled in of light chain in endoplasmic reticulum.Hendershot et al.，J.Cell Biol.104：761-767(1987)。Another heat shock protein gp96 is a member of hsp90 stress protein family, is positioned in the endoplasmic reticulum.Li and Srivastava，EMBO J.12：3143-3151(1993)；Mazzarella and Green，J.Biol.Cltem.262：8875-8883(1987)。Having proposed gp96 can help multi-subunit protein to assemble in endoplasmic reticulum.Wiech et al.，Nature358：169-170(1992)。
Having observed in laboratory animal can be with tumour-specific mode induce immune response by the heat shock protein of tumor preparation; That is to say, by the heat shock protein of specific tumors purification can be in laboratory animal induce immune response, this replying suppressed growth of tumor of the same race, but do not suppress other growth of tumor.Srivastava and Maki，Curr.Topics Microbiol.167：109-123(1991)。The gene of also not finding the coding heat shock protein has the tumor specific DNA polymorphism.Srivastava and Udono，Curr.Opin.Immunol.6：728-732(1994)。The high-resolution gel electrophoresis shows that gp96 may be inhomogenous on molecular level.Feldweg and Srivastava，Int.J.Cancer 63：310-314(1995)。The evidence prompting, the root of inhomogeneity may be the hundreds of little peptide group who adheres to heat shock protein.Ibid.The multiple peptide that has proposed to adhere to the synthetic heat shock protein of tumor can make these albumen excite immunne response in the patient with different HLA phenotypes, and by contrast, some HLA-is restricted a little on it is renderd a service for more traditional immunogen.Ibid.
Nieland et al., (Proc.Natl.Acad.Sci.U.S.A.93:6135-6139 (1996)) identify a kind of antigenic peptides that contains cytotoxic T lymphocyte (CTL) vesicular stomatitis virus (VSV) epi-position, the gp96 that it produces in conjunction with the VSV-infection cell.The method of Nieland has hindered identification also may be in conjunction with any other peptide or other chemical compound of gp96, and therefore can not further characterize also can be by the more high-molecular weight material of high performance liquid chroma-tography detection in conjunction with gp96.
Report, a kind of synthetic peptide that contains a plurality of malaria antigen NANP (Asp Ala Asp Pro) duplicate block, itself and immobilized mycobacteria hsp65 of glutaraldehyde or hsp70 chemical crosslinking can induce antibody to form (being humoral response) in mice under without any the situation of adding adjuvant; Use colibacillary heat shock protein also to observe similar effect.Del Guidice，Experientia 50：1061-1066(1994)；Barrios et al.，Clin.Exp.Immunol.98：224-228(1994)；Barrios et al.，Eur.J.Immunol.22：1365-1372(1992)。Antibody induction need synthesize peptide and heat shock protein is crosslinked, may also need the glutaraldehyde immobilization.Barrios et al.，Clin.Exp.Immunol.98：229-233。
PCT/US96/13363 has described the heterozygosis antigen that contains antigenic structure territory and heat shock protein binding structural domain, this antigen is inducing at antigenic immunne response with the composite form of heat shock protein, so this heterozygosis antigen can be used for treating cancer and infectious disease.PCT/US98/22335 has described the heat shock protein binding structural domain of other similar purposes, and the ability of heterozygosis antigen induce immune response when giving separately.Have now found that the peptide connector that exists between at least one the antigenic structure territory in the improvement heterozygosis antigen and at least one the heat shock protein binding structural domain can make biological activity increase.Find that also this increase increases the immunne response of inductive anti-heterozygosis antigen antigen part.The application relate to these improvement the peptide connector, contain improvement the peptide connector T1249 and having and do not having application under the heat shock protein situation.
Summary of the invention
The present invention relates to the method and composition of induce immune response in the patient, wherein the heterozygosis antigen of at least a definition is chosen wantonly and given the patient with composite form with heat shock protein.Heterozygosis antigen comprises at least one antigenic structure territory and at least one heat shock protein binding structural domain and at least one the peptide connector between them.Induce the immunne response of anti-disease (as infectious disease or tumor) related antigen to be used for the treatment of this disease.Antigenic antigenicity of heterozygosis or immunogenic structure territory can be complete albumen or peptide antigen, perhaps can only be a selected antigenic part, for example epitopes of Xuan Zeing.The heat shock protein binding structural domain is the peptide in conjunction with heat shock protein, be preferably 7-15 amino acid whose peptide in conjunction with heat shock protein, more preferably, most preferably be 7-15 amino acid whose hydrophobic peptide in conjunction with heat shock protein in conjunction with the hydrophobic peptide of heat shock protein.Connector has a kind of in the following sequence: Phe PheArg Lys (FFRK; SEQ ID NO:1000); Phe Arg Lys (FRK); Phe Arg LysAsn (FRKN, SEQ ID NO:1002); Arg Lys Asn (RKN); Phe Phe Arg LysAsn (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); LysAsn (KN); Arg Lys (RK); Or AA 1-AA 2-AA 3-leucine, wherein AA 1Be A, S, V, E, G, L or K, preferred V, more preferably S, most preferably A; AA 2Be K, V or E, preferred E, more preferably V, most preferably K; AA 3Be V, S, F, K, A, E or T, preferred F, more preferably S, most preferably V.In aforementioned connector, preferred Gln Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO:1001), Phe Phe Arg Lys (FFRK most preferably; SEQ ID NO:1000).
The invention provides the independent antigenic method of described heterozygosis that gives, and provide the method that gives heat shock protein/heterozygosis antigen composition, the latter comprises: (i) combination takes place and form under the condition of heterozygosis antigen/heat shock protein complex one or more heat shock proteins of external mixing and one or more heterozygosis antigens at heterozygosis antigen and heat shock protein; The heterozygosis antigen that (ii) needs patient's effective dose of this treatment in conjunction with heat shock protein.
Perhaps, can choose wantonly with the heat shock protein code nucleic acid, optional heterozygosis antigen in conjunction with heat shock protein is incorporated among the patient by giving patient's heterozygosis antigen encoding nucleic acid.
Therefore, aspect first, the present invention relates to a kind of heterozygosis antigen, it basic composition is: the antigenic structure territory of infectious agent or tumor antigen, non-covalent in conjunction with heat shock protein binding structural domain and the peptide connector of separating antigenic structure territory and binding structural domain, wherein the peptide connector is 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, wherein AA 1Be A, S, V, E, G, L or K, preferred V, more preferably S, most preferably A; AA 2Be K, V or E, preferred E, more preferably V, most preferably K; AA 3Be V, S, F, K, A, E or T, preferred F, more preferably S, most preferably V.In aforementioned connector, preferred Gln Leu Lys (QLK), Arg Lys (RK) and Ala LysVal Leu (AKVL; SEQ ID NO:1001), Phe Phe Arg Lys (FFRK most preferably; SEQ ID NO:1000).
Aspect second, the present invention relates to a kind of heterozygosis antigen, it basic composition is: a plurality of antigenic structures territory of one or more infectious agent or one or more tumor antigens, at least one non-covalent in conjunction with heat shock protein binding structural domain and at least one separate the peptide connector of antigenic structure territory and at least one binding structural domain, wherein at least one peptide connector is from Phe Phe Arg Lys (FFRK; SEQ ID NO:1000); Phe Arg Lys (FRK); PheArg Lys Asn (FRKN, SEQ ID NO:1002); Arg Lys Asn (RKN); Phe PheArg Lys Asn (FFRKN, SEQ ID NO:1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ IDNO:1001); Lys Asn (KN); Arg Lys (RK); Or AA 1-AA 2-AA 3-leucine, wherein AA 1Be A, S, V, E, G, L or K, preferred V, more preferably S, most preferably A; AA 2Be K, V or E, preferred E, more preferably V, most preferably K; AA 3Be V, S, F, K, A, E or T, preferred F, more preferably S, most preferably V.In aforementioned connector, preferred Gln Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQID NO:1001), Phe Phe Arg Lys (FFRK most preferably; SEQ ID NO:1000).In a specific embodiments, at least one the antigenic structure territory in the aforementioned heterozygosis antigen is the t helper cell epi-position.
Aspect the 3rd, the present invention relates to a kind of heterozygosis antigen, it comprises: the antigenic structure territory of infectious agent or tumor antigen, non-covalent in conjunction with the binding structural domain of heat shock protein and the peptide connector between them, and wherein at least one peptide connector is 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), GlnLeu 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 1Be A, S, V, E, G, L or K, preferred V, more preferably S, most preferably A; AA 2Be K, V or E, preferred E, more preferably V, most preferably K; AA 3Be V, S, F, K, A, E or T, preferred F, more preferably S, most preferably V.In aforementioned connector, preferred Gln Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO:1001), Phe Phe Arg Lys (FFRK most preferably; SEQ ID NO:1000).In a specific embodiments, aforementioned heterozygosis antigen has the peptide connector of separating antigenic structure territory and binding structural domain.
Aspect the 4th, the present invention relates to a kind of heterozygosis antigen, it comprises: a plurality of antigenic structures territory of one or more infectious agent or one or more tumor antigens, at least one is non-covalent in conjunction with the binding structural domain of heat shock protein and at least one the peptide connector between them, and wherein at least one peptide connector is from Phe Phe Arg Lys (FFRK; SEQ IDNO:1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ IDNO:1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ IDNO: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 1Be A, S, V, E, G, L or K, preferred V, more preferably S, most preferably A; AA 2Be K, V or E, preferred E, more preferably V, most preferably K; AA 3Be V, S, F, K, A, E or T, preferred F, more preferably S, most preferably V.In aforementioned connector, preferred Gln Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO:1001), Phe Phe ArgLys (FFRK most preferably; SEQ ID NO:1000).In a specific embodiments, at least one antigenic structure territory is the t helper cell epi-position.
Aspect the 5th, the present invention relates to a kind of compositions of inducing the immunne response of infectious agents or tumor antigen, it contains at least a heterozygosis antigen, this heterozygosis antigen comprises: the antigenic structure territory of infectious agent or tumor antigen, non-covalent in conjunction with the binding structural domain of heat shock protein and at least one the peptide connector between them, and wherein at least one peptide connector is from Phe Phe Arg Lys (FFRK; SEQ ID NO:1000); Phe Arg Lys (FRK); PheArg Lys Asn (FRKN, SEQ ID NO:1002); Arg Lys Asn (RKN); Phe PheArg Lys Asn (FFRKN, SEQ ID NO:1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), Ala Lys Val Leu (AKVL; SEQ IDNO:1001); Lys Asn (KN); Arg Lys (RK); Or AA 1-AA 2-AA 3-leucine, wherein AA 1Be A, S, V, E, G, L or K, preferred V, more preferably S, most preferably A; AA 2Be K, V or E, preferred E, more preferably V, most preferably K; AA 3Be V, S, F, K, A, E or T, preferred F, more preferably S, most preferably V.In aforementioned connector, preferred Gln Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQID NO:1001), Phe Phe Arg Lys (FFRK most preferably; SEQ ID NO:1000).In one embodiment, described compositions contains multiple heterozygosis antigen, and wherein a kind of heterozygosis antigen can comprise the t helper cell epi-position.
Aspect the 6th, the present invention relates to a kind of compositions of inducing the immunne response of infectious agents or tumor antigen, it contains at least a heterozygosis antigen, this heterozygosis antigen comprises: wherein at least one a plurality of antigenic structures territory from infectious agent or tumor antigen, at least one is non-covalent in conjunction with the binding structural domain of heat shock protein and at least one the peptide connector between them, wherein at least one peptide connector is from Phe Phe Arg Lys (FFRK; SEQ IDNO:1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ IDNO:1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ IDNO: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 1Be A, S, V, E, G, L or K, preferred V, more preferably S, most preferably A; AA 2Be K, V or E, preferred E, more preferably V, most preferably K; AA 3Be V, S, F, K, A, E or T, preferred F, more preferably S, most preferably V.In aforementioned connector, preferred Gln Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO:1001), Phe Phe ArgLys (FFRK most preferably; SEQ ID NO:1000).
Aspect the 7th, the present invention relates to a kind of compositions of inducing the immunne response of infectious agents or tumor antigen, it contains at least a heterozygosis antigen, this heterozygosis antigen basic composition is: the antigenic structure territory of infectious agent or tumor antigen, non-covalent in conjunction with heat shock protein binding structural domain and the peptide connector of separating antigenic structure territory and binding structural domain, wherein at least one peptide connector is from Phe Phe Arg Lys (FFRK; SEQ ID NO:1000); PheArg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO:1002); Arg LysAsn (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 1Be A, S, V, E, G, L or K, preferred V, more preferably S, most preferably A; AA 2Be K, V or E, preferred E, more preferably V, most preferably K; AA 3Be V, S, F, K, A, E or T, preferred F, more preferably S, most preferably V.In aforementioned connector, preferred Gln Leu Lys (QLK), Arg Lys (RK) and Ala LysVal Leu (AKVL; SEQ ID NO:1001), Phe Phe Arg Lys (FFRK most preferably; SEQ ID NO:1000).In one embodiment, foregoing comprises multiple heterozygosis antigen.On the other hand, at least a t helper cell epi-position that contains in the multiple heterozygosis antigen.
Aspect the 8th, the present invention relates to a kind of compositions of inducing the immunne response of infectious agents or tumor antigen, it contains at least a heterozygosis antigen, this heterozygosis antigen basic composition is: wherein at least one a plurality of antigenic structures territory from infectious agent or tumor antigen, at least one non-covalent in conjunction with heat shock protein binding structural domain and at least one peptide connector of separating antigenic structure territory and binding structural domain, wherein at least one peptide connector is from Phe PheArg Lys (FFRK; SEQ ID NO:1000); Phe Arg Lys (FRK); Phe Arg LysAsn (FRKN, SEQ ID NO:002); Arg Lys Asn (RKN); Phe Phe Arg LysAsn (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); LysAsn (KN); Arg Lys (RK); Or AA 1-AA 2-AA 3-leucine, wherein AA 1Be A, S, V, E, G, L or K, preferred V, more preferably S, most preferably A; AA 2Be K, V or E, preferred E, more preferably V, most preferably K; AA 3Be V, S, F, K, A, E or T, preferred F, more preferably S, most preferably V.In one embodiment, the t helper cell epi-position is contained at least one antigenic structure territory.
Aspect the 9th, the present invention relates to a kind of method of inducing the immunne response of infectious agents or tumor antigen, this method comprises and give patient's heat shock protein and the antigenic complex of heterozygosis that this heterozygosis antigen contains: the antigenic structure territory of at least one infectious agent or tumor antigen, at least one contains the binding structural domain of non-covalent peptide in conjunction with heat shock protein and the peptide connector between them; Wherein said heterozygosis antigen and the non-covalent combination of heat shock protein, wherein at least one peptide connector is from Phe Phe Arg Lys (FFRK; SEQ ID NO:1000); PheArg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO:1002); Arg LysAsn (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 1Be A, S, V, E, G, L or K, preferred V, more preferably S, most preferably A; AA 2Be K, V or E, preferred E, more preferably V, most preferably K; AA 3Be V, S, F, K, A, E or T, preferred F, more preferably S, most preferably V.In aforementioned connector, preferred Gln Leu Lys (QLK), Arg Lys (RK) and Ala LysVal Leu (AKVL; SEQ ID NO:1001), Phe Phe Arg Lys (FFRK most preferably; SEQ ID NO:1000).In one embodiment, complex comprises multiple heterozygosis antigen.In one embodiment, at least a heterozygosis antigen is the t helper cell epi-position.In another embodiment, heterozygosis antigen contains a plurality of antigenic structures territory, and wherein at least one antigenic structure territory can be the t helper cell epi-position.In another embodiment again, wherein complex contains multiple heterozygosis antigen, and wherein at least a heterozygosis antigen contains a plurality of antigenic structures territory.In the present invention's another embodiment in this respect, heat shock protein is hsp70.
Aspect the tenth, the present invention relates to a kind of method of inducing the immunne response of infectious agents or tumor antigen, this method comprises and gives patient's heat shock protein and the antigenic complex of heterozygosis, this heterozygosis antigen basic composition is: the antigenic structure territory of at least one infectious agent or tumor antigen, non-covalent in conjunction with heat shock protein binding structural domain and the peptide connector of separating antigenic structure territory and binding structural domain, wherein at least one peptide connector is 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), GlnLeu 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 1Be A, S, V, E, G, L or K, preferred V, more preferably S, most preferably A; AA 2Be K, V or E, preferred E, more preferably V, most preferably K; AA 3Be V, S, F, K, A, E or T, preferred F, more preferably S, most preferably V.In aforementioned connector, preferred Gln Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO:1001), Phe Phe Arg Lys (FFRK most preferably; SEQ ID NO:1000).In one embodiment, complex comprises multiple heterozygosis antigen.In another embodiment, at least a heterozygosis antigen is the t helper cell epi-position.In a further embodiment, heterozygosis antigen contains a plurality of antigenic structures territory.In another embodiment again, at least one antigenic structure territory is the t helper cell epi-position.Still again in another embodiment, complex contains multiple heterozygosis antigen, and wherein at least a heterozygosis antigen contains a plurality of antigenic structures territory.In the preferred embodiment in this regard, heat shock protein is hsp70.
Aspect the 11, the present invention relates to a kind of method of inducing the immunne response of infectious agents or tumor antigen, this method comprises and gives the patient at least a heterozygosis antigen, this heterozygosis antigen comprises: the antigenic structure territory of at least one infectious agent or tumor antigen, at least one contains the binding structural domain of non-covalent peptide in conjunction with heat shock protein and at least one the peptide connector between them, and wherein at least one peptide connector is from Phe Phe Arg Lys (FFRK; SEQID NO:1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ IDNO:1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ IDNO: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 1Be A, S, V, E, G, L or K, preferred V, more preferably S, most preferably A; AA 2Be K, V or E, preferred E, more preferably V, most preferably K; AA 3Be V, S, F, K, A, E or T, preferred F, more preferably S, most preferably V.In aforementioned connector, preferred Gln Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO:1001), Phe Phe ArgLys (FFRK most preferably; SEQ ID NO:1000).In one embodiment, complex comprises multiple heterozygosis antigen.In another embodiment, at least a heterozygosis antigen is the t helper cell epi-position.In another embodiment, heterozygosis antigen contains a plurality of antigenic structures territory.In a further embodiment, at least one antigenic structure territory is the t helper cell epi-position.In another embodiment again, complex contains multiple heterozygosis antigen, and wherein at least a heterozygosis antigen contains a plurality of antigenic structures territory.In the present invention's another embodiment in this respect, the peptide connector is separated antigenic structure territory and binding structural domain.
Aspect the 12, the present invention relates to a kind of method of inducing the immunne response of infectious agents or tumor antigen, this method comprises and gives the patient at least a heterozygosis antigen, this heterozygosis antigen basic composition is: the antigenic structure territory of at least one infectious agent or tumor antigen, non-covalent in conjunction with heat shock protein binding structural domain and the peptide connector of separating antigenic structure territory and binding structural domain, wherein at least one peptide connector is from Phe Phe Arg Lys (FFRK; SEQ ID NO:1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQID NO:1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQID 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 1Be A, S, V, E, G, L or K, preferred V, more preferably S, most preferably A; AA 2Be K, V or E, preferred E, more preferably V, most preferably K; AA 3Be V, S, F, K, A, E or T, preferred F, more preferably S, most preferably V.In aforementioned connector, preferred Gln Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO:1001), Phe Phe ArgLys (FFRK most preferably; SEQ ID NO:1000).In one embodiment, complex comprises multiple heterozygosis antigen.In another embodiment, at least a heterozygosis antigen is the t helper cell epi-position.In another embodiment, heterozygosis antigen contains a plurality of antigenic structures territory.In another embodiment again, at least one antigenic structure territory is the t helper cell epi-position.Still again in another embodiment, complex contains multiple heterozygosis antigen, and wherein at least a heterozygosis antigen contains a plurality of antigenic structures territory.
Aspect the 13, the present invention relates to a kind of treatment infectious disease or method for cancer, this method comprises and gives patient's heat shock protein and the antigenic complex of heterozygosis that this heterozygosis antigen comprises: the infectious agent that at least one is relevant with infectious disease or cancer or the antigenic structure territory of tumor antigen, the binding structural domain that contains non-covalent peptide in conjunction with heat shock protein and the peptide connector between them; The wherein non-covalent combination of heterozygosis antigen and heat shock protein, wherein at least one peptide connector is from Phe Phe Arg Lys (FFRK; SEQ ID NO:1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ ID NO:1002); ArgLys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ ID NO:1003); PheArg (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 1Be A, S, V, E, G, L or K, preferred V, more preferably S, most preferably A; AA 2Be K, V or E, preferred E, more preferably V, most preferably K; AA 3Be V, S, F, K, A, E or T, preferred F, more preferably S, most preferably V.In aforementioned connector, preferred Gln Leu Lys (QLK), Arg Lys (RK) and Ala LysVal Leu (AKVL; SEQ ID NO:1001), Phe Phe Arg Lys (FFRK most preferably; SEQ ID NO:1000).In one embodiment, complex comprises multiple heterozygosis antigen.In another embodiment, at least a heterozygosis antigen is the t helper cell epi-position.In another embodiment again, heterozygosis antigen contains a plurality of antigenic structures territory.In another embodiment again, at least one antigenic structure territory is the t helper cell epi-position.Still again in another embodiment, complex contains multiple heterozygosis antigen, and wherein at least a heterozygosis antigen contains a plurality of antigenic structures territory.In the present invention's embodiment in this respect, the peptide connector is separated antigenic structure territory and binding structural domain.In the present invention's preferred embodiment in this respect, heat shock protein is hsp70.
Aspect the 14, the present invention relates to a kind of treatment infectious agent disease or method for cancer, this method comprises and gives patient's heat shock protein and the antigenic complex of heterozygosis, this heterozygosis antigen basic composition is: the infectious agent that at least one is relevant with infectious disease or cancer or the antigenic structure territory of tumor antigen, at least one non-covalent in conjunction with heat shock protein binding structural domain and the peptide connector of separating antigenic structure territory and binding structural domain, wherein at least one peptide connector is 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, wherein AA 1Be A, S, V, E, G, L or K, preferred V, more preferably S, most preferably A; AA 2Be K, V or E, preferred E, more preferably V, most preferably K; AA 3Be V, S, F, K, A, E or T, preferred F, more preferably S, most preferably V.In aforementioned connector, preferred Gln Leu Lys (QLK), Arg Lys (RK) and Ala LysVal Leu (AKVL; SEQ ID NO:1001), Phe Phe Arg Lys (FFRK most preferably; SEQ ID NO:1000).In one embodiment, complex comprises multiple heterozygosis antigen.On the other hand, at least a heterozygosis antigen is the t helper cell epi-position.More on the other hand, heterozygosis antigen contains a plurality of antigenic structures territory.More on the other hand, at least one antigenic structure territory is the t helper cell epi-position.More on the one hand, complex contains multiple heterozygosis antigen, and wherein at least a heterozygosis antigen contains a plurality of antigenic structures territory.In a preferred embodiment, heat shock protein is hsp70.
Aspect the 15, the present invention relates to a kind of treatment infectious disease or method for cancer, this method comprises and gives the patient at least a heterozygosis antigen, this heterozygosis antigen comprises: the infectious agent that at least one is relevant with infectious disease or cancer or the antigenic structure territory of tumor antigen, the binding structural domain that contains non-covalent peptide in conjunction with heat shock protein and the peptide connector between them, and wherein at least one peptide connector is from Phe Phe Arg Lys (FFRK; SEQ IDNO:1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ IDNO:1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ IDNO: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 1Be A, S, V, E, G, L or K, preferred V, more preferably S, most preferably A; AA 2Be K, V or E, preferred E, more preferably V, most preferably K; AA 3Be V, S, F, K, A, E or T, preferred F, more preferably S, most preferably V.In aforementioned connector, preferred Gln Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO:1001), Phe Phe ArgLys (FFRK most preferably; SEQ ID NO:1000).In one embodiment, complex comprises multiple heterozygosis antigen.On the other hand, at least a heterozygosis antigen is the t helper cell epi-position.More on the other hand, heterozygosis antigen contains a plurality of antigenic structures territory.Still more on the other hand, at least one antigenic structure territory is the t helper cell epi-position.Still more on the other hand, complex contains multiple heterozygosis antigen, and wherein at least a heterozygosis antigen contains a plurality of antigenic structures territory.In the present invention's embodiment in this respect, the peptide connector is separated antigenic structure territory and binding structural domain.
Aspect the 16, the present invention relates to a kind of treatment infectious disease or method for cancer, this method comprises and gives the patient at least a heterozygosis antigen, this heterozygosis antigen basic composition is: the infectious agent that at least one is relevant with infectious disease or cancer or the antigenic structure territory of tumor antigen, non-covalent in conjunction with heat shock protein binding structural domain and the peptide connector of separating antigenic structure territory and binding structural domain, wherein at least one peptide connector is from Phe Phe ArgLys (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), GlnLeu 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 1Be A, S, V, E, G, L or K, preferred V, more preferably S, most preferably A; AA 2Be K, V or E, preferred E, more preferably V, most preferably K; AA 3Be V, S, F, K, A, E or T, preferred F, more preferably S, most preferably V.In aforementioned connector, preferred Gln Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO:1001), Phe Phe Arg Lys (FFRK most preferably; SEQ ID NO:1000).In one embodiment, complex comprises multiple heterozygosis antigen.In another embodiment, at least a heterozygosis antigen is the t helper cell epi-position.In another embodiment again, heterozygosis antigen contains a plurality of antigenic structures territory.Still again in another embodiment, at least one antigenic structure territory is the t helper cell epi-position.In another embodiment, complex contains multiple heterozygosis antigen, and wherein at least a heterozygosis antigen contains a plurality of antigenic structures territory.
Aspect the 17, the present invention relates to a kind of peptide, it is 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), GlnLeu 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 1Be A, S, V, E, G, L or K, preferred V, more preferably S, most preferably A; AA 2Be K, V or E, preferred E, more preferably V, most preferably K; AA 3Be V, S, F, K, A, E or T, preferred F, more preferably S, most preferably V.
Aspect the 18, the present invention relates to a kind of immunogenic polypeptide, it comprises: a plurality of antigenic structures territory, at least one heat shock protein binding structural domain and at least one the peptide connector between them, and wherein at least one peptide connector is 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), GlnLeu 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 1Be A, S, V, E, G, L or K, preferred V, more preferably S, most preferably A; AA 2Be K, V or E, preferred E, more preferably V, most preferably K; AA 3Be V, S, F, K, A, E or T, preferred F, more preferably S, most preferably V.In aforementioned connector, preferred Gln Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO:1001), Phe Phe Arg Lys (FFRK most preferably; SEQ ID NO:1000).
Aspect nineteen, the present invention relates to a kind of polynucleotide, its encode aforementioned first, second, third or fourth aspect in any heterozygosis antigen.
Aspect the 20, the present invention relates to a kind of method of inducing the immunne response of anti-infectious disease or cancer, this method comprises and gives the patient the antigenic polynucleotide of heterozygosis of encoding, this heterozygosis antigen comprises: the infectious agent relevant or the antigenic structure territory of tumor antigen, heat shock protein binding structural domain and the peptide connector between them with infectious disease or cancer, and wherein the peptide connector is from Phe Phe Arg Lys (FFRK; SEQ ID NO:1000); Phe ArgLys (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 1Be A, S, V, E, G, L or K, preferred V, more preferably S, most preferably A; AA 2Be K, V or E, preferred E, more preferably V, most preferably K; AA 3Be V, S, F, K, A, E or T, preferred F, more preferably S, most preferably V.In aforementioned connector, preferred Gln Leu Lys (QLK), Arg Lys (RK) and Ala LysVal Leu (AKVL; SEQ ID NO:1001), Phe Phe Arg Lys (FFRK most preferably; SEQ ID NO:1000).
Aspect the 21, the present invention relates to a kind of method of inducing the immunne response of anti-infectious disease or cancer, this method comprise give the patient encode the antigenic polynucleotide of above-mentioned heterozygosis and the coding heat shock protein polynucleotide.In a preferred embodiment, the heat shock protein of coding is hsp70.
Of the present invention any or all aforementioned aspect in, infectious disease antigen can derive from infectious agent, for example is antibacterial, virus, protozoacide, mycoplasma, fungus, yeast, parasite or Protein virus as limiting examples.As limiting examples, cancer relevant with cancer or tumor antigen can derive from sarcoma, lymphoma, leukemia or malignant tumor, melanoma, breast carcinoma, carcinoma of prostate, ovarian cancer, cervical cancer, colon cancer, pulmonary carcinoma, glioblastoma or astrocytoma.The antigen that the antigenic structure territory of infectious agent or cancer comprises derives from infectious disease or tumor antigen or relevant with it, and this makes the immunne response of inductive antigen of infectious agents respectively of institute or cancer antigen can be used for treating the infectious disease or the cancer of correspondence.
According to the 35th the 119th (e) money of United States code, the application requires the provisional application sequence number 60/462 of submission on April 11st, 2003,469, the provisional application sequence number of submitting on April 18th, 2,003 60/463, the provisional application sequence number 60/503 of 746 and 2003 on JIUYUE submission in 16,, 417 priority, all these three provisional application all by reference integral body be attached to herein.
Accompanying drawing describes in detail
Fig. 1 has shown the tumor challenge result of experiment, wherein uses the complex of heterozygosis antigen or heterozygosis antigen and heat shock protein to carry out immunity inoculation, uses after 7 days and expresses this antigenic tumor challenge.
Detailed Description Of The Invention
For clearly describing but be not restriction the present invention, describe in detail and be divided into following chapters and sections:
(i) heterozygosis antigen;
(ii) heat shock protein; With
(iii) give method.
Heterozygosis antigen
Heterozygosis antigen of the present invention comprises: the peptide connector between at least one antigenicity (immunogenicity) domain, at least one heat shock protein binding structural domain and at least two these domains, wherein the peptide connector 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 Ash(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-AA 2-AA 3-leucine, wherein AA1Be A, S, V, E, G, L or K, preferred V, more preferably S, most preferably A; AA2Be K, V or E, preferred E, more preferably V, most preferably K; AA1Be V, S, F, K, A, E or T, preferred F, more preferably S, most preferably V.
In aforementioned connector, preferred Gln Leu Lys (QLK), Arg Lys (RK) and Ala Lys Val Leu (AKVL; SEQ ID NO:1001), Phe Phe Arg Lys (FFRK most preferably; SEQ ID NO:1000).
Therefore, heterozygosis antigen plays at least two effects, namely (i) its contain the epi-position that can induce required immune response; (ii) it can the physical bond heat shock protein. As described below, this combination can occur in vivo, can induce required immune response so that give separately heterozygosis antigen, and required therapeutic action is provided.
Term used herein " antigen " refers to can be by amino acid, sugar, nucleic acid or lipid separately or the compound that forms of any combination.
Term used herein " heterozygosis antigen " refers to this compound: it is in conjunction with one or more heat shock proteins, and representative needs the immunogene of immune response antagonism. For example, when immunogene was influenza virus, heterozygosis antigen can comprise the fragments of peptides of influenza virus stromatin. Term used herein " immunogene " is applicable to excite tumour cell, infection cell, pathogen or its component for its immune response, and heterozygosis antigen comprises that this immunogene can excite the part of required immune response and in conjunction with the part of one or more heat shock proteins. Specifically, select the antigenic structure territory of heterozygosis antigen to excite the immune response of anti-specified disease or pathogen, this antigenic structure territory comprises the peptide that derives from MHC molecule, mutant DNA gene outcome and DNA direct product (for example deriving from the DNA direct product of tumour cell).
Although the present invention can be used for the immunogene of any type, but concrete target immunogene is relevant with tumor disease, or derive from tumor disease, or expectation is relevant with tumor disease, described tumor disease includes but not limited to sarcoma, lymthoma, leukaemia or malignant tumour, specifically is melanoma, breast cancer, prostate cancer, oophoroma, cervix cancer, colon cancer, lung cancer, spongioblastoma or astrocytoma etc. As limiting examples, the melanoma antigen that select to be used for heterozygosis antigen of the present invention is found in PCT/US01/12449 (WO0178655), its by reference integral body be attached to herein. In addition, the mutant of cancer suppressor protein such as p53 or oncoprotein such as ras also can be provided for heterozygosis antigen of the present invention.
In a further embodiment, immunogene can be relevant with infectious diseases, can be as described bacterium, virus, protozoan, mycoplasma, fungi, yeast, parasitic animal and plant or prion. Without limitation, immunogene is such as can be HPV (vide infra), herpesviral such as herpes simplex virus or herpes zoster virus, retrovirus such as human immunodeficiency virus 1 or 2, hepatitis viruse, influenza virus, rhinovirus, respiratory syncytial virus (RSV), cytomegalovirus, adenovirus, mycoplasma pneumoniae (Mycoplasma pneumoniae), Salmonella (Salmonella) bacterium, staphylococcus (Staphylococcus) bacterium, streptococcus (Streptococcus) bacterium, enterococcus spp (Enterococcus) bacterium, fusobacterium (Clostridium) bacterium, Escherichia (Escherichia) bacterium, Klebsiella (Klebsiella) bacterium, vibrio (Vibrio) bacterium, mycobacterium (Mycobacterium) bacterium, amoeba, plasmodium, schizotrypanum cruzi (Trypanosoma cruzi) etc.
Can by directly former by sample, cell culture or the biological culture thing separation adaptive immune of tumour, infection cell, infection object, perhaps can pass through chemistry or recombinant technique synthetic immunogen. As limiting examples, suitable antigenic peptides, especially for heterozygosis antigen, be used for the antigenic peptides of antiviral, bacterium etc., can design by retrieving the MHC I class restricted peptides epi-position that contains the HLA binding sequence in its sequence, wherein said HLA binding sequence is such as but not limited to HLA-A2 peptide binding sequence:
Xaa (Leu/Met) XaaXaaXaa (Val/Ile/Leu/Thr) XaaXaa (Val/Leu) (SEQ ID NO:2), for example
Derive from 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 Asn Tyr 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 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 Lys 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 Gly Lys Lys Lys(SEQ ID NO：104)；
Glu Ile 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 Trp 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 Gly 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)；
Derive from antibacterial:
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)；
Derive from parasite:
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)；
Derive 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)；
Tyr Leu Glu Pro Gly 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 Tyr (SEQ ID NO:149); With
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 derive from 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)).
Aforementioned epi-position only is that the representativeness relevant with cancer with various infectious disease can be utilized selection, provides these epi-positions without any the restriction intention.
May also need to consider required type of immune response.For example, under some environment, humoral immunoresponse(HI) may be suitable.In other cases, in fact want to excite the immunne response of antitumor cell or infection cell, this moment, cellullar immunologic response was suitable especially.Therefore, but discriminated union select to activate relevant defined epitope with B cell, t helper cell or cytotoxic T cell, be integrated in the heterozygosis antigen.
May also need to utilize the heterozygosis antigen relevant with autoimmune disease or allergy.This heterozygosis antigen can give with one or more heat shock proteins, and administered dose is enough to the antigenic immunne response of anti-heterozygosis that tolerates or suppress to be pre-existing among the patient.Suppress the required heat shock protein amount expection of immunne response and will be significantly higher than the required amount of stimulation.
Although the antigenic big I of heterozygosis changes according to employed heat shock protein, but in non-limiting embodiments of the present invention, heterozygosis antigen can be the peptide with 10-500 amino acid residue, be preferably peptide, most preferably be peptide with 18-50 amino acid residue with 14-100 amino acid residue.Therefore, may need to produce immunogen fragment, perhaps by chemistry or the synthetic heterozygosis antigen of recombinant DNA method as the antigenic antigenic structure of heterozygosis territory.
According to aforementioned Consideration, can prepare heterozygosis antigen, test its ability then in conjunction with heat shock protein.In some cases, there is at least a other albumen that can be heat shock protein to have to be beneficial to combining of heterozygosis antigen and particular thermal shock protein.
For example, by coming labelling heterozygosis antigen with detectable label such as radioactivity, fluorescence, enzyme or pigment mark, under can making in conjunction with the condition that takes place, expection mixes heterozygosis antigen and heat shock protein, heat of dissociation shock protein then, remove any unconjugated heterozygosis antigen simultaneously, and measure and whether to have the heterozygosis antigen of any labelling to adhere to heat shock protein, thereby estimate combining of heterozygosis antigen and heat shock protein.As concrete limiting examples, estimate ability following carry out of heterozygosis antigen: contain 50mM Tris HCl (pH 7.5), 200mM NaCl and 1mM Na at final volume 50 μ l in 37 ℃ in conjunction with heat shock protein BiP 2Mixed 2 μ g BiP and maximum about 1150pmol radioactive label heterozygosis antigen in the buffer of EDTA 30 minutes.Then through 1ml Sephadex-G post with the centrifugal desalination of 100g 2 minutes, by removing unconjugated heterozygosis antigen in the bonded BiP-heterozygosis antigen.Penefsky，J.Biol.Chem.252：2891(1977)。For preventing and resin-bonded that pillar can be handled with 100 μ l bovine serum albumin in the identical buffer solution earlier, and as above centrifugal.Then by the quantitative bonded heterozygosis antigen of liquid scintillation counting.Consult Flynn et al., Science 245:385-390 (1989).
Because the ATP hydrolysis impels many known heat shock proteins to discharge peptide, so often can use ATP enzyme amount alive quantitatively in conjunction with the antigenic amount of the heterozygosis of heat shock protein.Flynn etal., Science 245:385-390 (1989) has proposed a example how to implement this experiment.
Select heat shock protein-binding structural domain, so that heterozygosis antigen is external or body in separately in conjunction with or unite with auxiliary heat shock protein such as hsp40 or hsp60 and to combine heat shock protein, as BiP, hsp70, gp96 or hsp90, or the member of aforementioned heat-shock protein family.
As Blond-Elguindi et al., Cell 75:717-728 (1993) is described, can be by the antigenic storehouse of known one or more heat shock proteins of good combination of elutriation, and authenticator is should the limiting examples of the peptide of standard:
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 Arp 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 Trp 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 Gln 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 Ile Asp Cys(SEQ ID NO：245)；
Ser Leu Val Ala Ala Arg Asn Leu(SEQ ID NO：246)；
Phe Thr Ile 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 Arg(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); With
Gly Ser Ser Met Val Val Gln Arg(SEQ ID NO：286).
Use this technology, Blond-Elguindi infers that heat shock protein BiP identification contains the polypeptide in the heptamer district with sequence Hy (Trp/X) HyXHyXHy (SEQ ID NO:29), wherein Hy represents hydrophobic amino acid residues, particularly tryptophan, leucine or phenylalanine (SEQ IDNO:30), and X is an arbitrary amino acid.High-affinity heat shock protein-the binding sequence that is integrated into this motif comprises:
His Trp Asp Phe Ala Trp Pro Trp (SEQ ID NO:1); With
Phe Trp Gly Leu Trp Pro Trp Glu(SEQ ID NO：4)。
Also identified other heat shock protein binding motif.For example, Auger et al., NatureMedicine 2:306-31O (1996) identify two kinds of pentapeptide binding motifs in conjunction with heat shock protein in the HLA-DR type relevant with rheumatoid arthritis:
Gln Lys Arg Ala Ala (SEQ ID NO:5) and
Arg Arg Arg Ala Ala(SEQ ID NO：6)。
Also identify the heat shock protein binding motif of forming by the long peptide of 7-15 residue, its enrichment hydrophobic amino acid.
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 peptide comprises:
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 Thr(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 Gly(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); With
Ser Ala Val Thr Asn Thr Gly(SEQ ID NO：338)；
As Gragerov et al., J.Molec.Biol.235:848-854 (1994) is described.
Other heat shock protein binding structural domain comprises Phe Tyr Gln Leu Ala Leu Thr (SEQ ID NO :), Phe Tyr Gln Leu Ala Leu Thr Trp (SEQ ID NO :), Arg LysLeu Phe Phe Asn Leu Arg (SEQ ID NO :), Arg Lys Leu Phe Phe Asn LeuArg Trp (SEQ ID NO :), Lys Phe Glu Arg Gln (SEQ ID NO :), Asn Ile ValArg Lys Lys Lys (SEQ ID NO :) and Arg Gly Tyr Val Tyr Gln Gly Leu (SEQ ID NO :).
In addition, other heat shock protein binding structural domain comprises WO 9922761 described heat shock protein binding structural domains.Xaa represents any aminoacid.
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：9D)；
DALMWP 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)；
TPDITPK(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)；
ASAGYQI(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：183)；
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)；
ATLEPVR(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)；
FTNQQYH(SEQ ID NO：243)；
SHVPSMA(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)；
NTAPHMY(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); With
FHWWW(SEQ ID NO：317).
Aforementioned hot shock protein binding structural domain only is the example that can be used for implementing various peptides of the present invention in peptide and the non-peptide heat shock protein binding molecule.In other embodiments, the heat shock protein binding structural domain can be directly in conjunction with the different piece of aforementioned mammal heat shock protein, and heat shock protein-binding structural domain of the present invention is not limited to any specific part in conjunction with the heat shock protein molecule.In a limiting examples, peptide IFAGIKKKAERADLIAYLKQATAK (Greene et al., 1995, J.Biol.Chem.270:2967-2973; SEQ ID NO:331) or the heat shock protein-binding fragment of this peptide be used for any conjugate of the present invention, with combining of the molecule that promotes to select in advance and heat shock protein.Except aforementioned peptide, can also have heat shock protein by use and realize combination in conjunction with active organic molecule or chemical compound in conjunction with heat shock protein.For example, suitable molecule comprises benzoquinone Ansamycin antibiotic member, for example Antibiotic TAN 420F, geldanamycin, macmimycin I, mimosamycin (mimosamycin) and kuwaitimycin (kuwaitimycin) (Omura et al., 1979, J.Antibiotics 32:255-261; Other consults WO9922761, its by reference integral body be attached to herein), or structurally associated chemical compound and analog or derivant.These molecules can be conjugated to antigenic structure of the present invention territory through the peptide connector by the chemical method of having established, with produce can be external or body in conjunction with heat shock protein and excite the anti-wherein heterozygosis antigen of antigenic immunne response.
Pending trial and the patent application serial number of owning together 10/776 when submitting to as on February 12nd, 2004,521 (its integral body be attached to herein) by reference are described, find that C-end at heat shock protein binding structural domain (such as but not limited to discriminating as indicated above) adds trp residue (Trp or monamino acid password W) and strengthened and the combining of heat shock protein.No matter have been found that to strengthen and combining of heat shock protein strengthened the ability of the immunne response in the anti-heterozygosis antigen of heterozygosis antigen induction antigenic structure territory, be with the composite form of heat shock protein or be not always the case separately.Immunne response strengthens to be increased relevant with curative effect of disease.Other method case description of measuring affinity is in PCT/US96/13363 (WO9706821), its by reference integral body be attached to herein.
Formerly in the heat shock protein binding structural domain of Xuan Zeing, the present invention preferred heat shock protein binding structural domain a part of for heterozygosis antigen (it comprises antigenic structure territory and the peptide connector of the present invention between heat shock protein binding structural domain and antigenic structure territory) comprises following heat shock protein binding structural domain:
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 Gln Gly Leu(SEQ ID NO：).
Other limiting examples that is used for this heat shock protein binding structural domain of the terminal Trp residue of having of various aspects of the present invention comprises:
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 Ile 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); With
Asn Arg Leu Leu Leu Ala Trp(SEQ ID NO：348).
Other is used to implement heat shock protein binding structural domain of the present invention and comprises Phe Tyr GlnLeu Ala Leu Thr Trp (SEQ ID NO:501), Phe Tyr Gln Leu Ala Leu ThrTrp (SEQ ID NO:502), Arg Lys Leu Phe Phe Asn Leu Arg Trp (SEQ IDNO:503), Arg Lys Leu Phe Phe Asn Leu Arg Trp (SEQ ID NO:504), LysPhe Glu Arg Gln Trp (SEQ ID NO:505), Asn Ile Val Arg Lys Lys Lys Trp (SEQ ID NO:506) and Arg Gly Tyr Val Tyr Gln Gly Leu Trp (SEQ IDNO:507).
In addition, other heat shock protein binding structural domain comprises the heat shock protein binding structural domain that is described among the WO9922761, can add terminal Trp residue to achieve the object of the present invention.Xaa represents arbitrary 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 Asp Leu 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 Trp Gln 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 Ile 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 Leu 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 His Ala 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 Ila 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(AEQ 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 Trp(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 Ile Thr Trp(SEQ ID NO：1271)；
Phe Ala Pro Leu Ile Ala His Trp(SEQ ID NO：1272)；
Ser Trp Ile Gln Thr Phe Met Trp(SEQ ID NO：1273)；
Asn Thr Trp Pro His Met Tyr Trp(SEQ ID NO：1274)；
Glu Pro Leu 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 Ara 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 Xaa 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); With
His Gly Gln Ala Trp Gln Phe Trp(SEQ ID NO：1312).
In all aforementioned hot shock protein binding peptides, the heat shock protein binding structural domain in the heterozygosis antigen of the present invention is Asn Leu Leu Arg Leu Thr Gly Trp (SEQ IDNO:350) most preferably.But aforementioned hot shock protein binding structural domain only is the example that can be used for implementing various parts of the present invention in peptide and the non-peptide heat shock protein binding molecule.
Heterozygosis antigen of the present invention has been integrated at least one antigenicity (immunogenicity) domain and at least one heat shock protein binding structural domain, and they are separated by at least one peptide connector as herein described.Heterozygosis antigen of the present invention can use chemical method of peptide synthesis synthetic, perhaps can be synthetic by the express nucleic acid construction, and this construction contains the catenation sequence of coding for antigens domain and heat shock protein binding structural domain.A kind of suitable technology is utilized the DNA sections of independent two domains of coding of pre-separation pcr amplification reaction production, and each domain all has the connector fragment that is connected to an end, and then reuse PCR step merges the product of two amplifications.This technology is called as connector tailing (linker tailing).Suitable restriction site transformation can also be gone in the purpose zone, after this utilize restriction digestion to produce required heterozygosis antigen-coded sequence with being connected.
As described herein, by giving the patient, the antigenic nucleic acid of code book invention heterozygosis also is suitable for therapeutic use, and wherein the heterozygosis antigen of expression in vivo generation can induce immune response.
Heat shock protein
Term used herein " heat shock protein " is meant any albumen that increases of expressing in cell when cell bears pressure.In preferred non-limiting embodiments, heat shock protein derives from eukaryotic cell; In a more preferred embodiment, heat shock protein derives from mammalian cell.Without limitation, can be used for heat shock protein of the present invention for example comprise BiP (being also referred to as grp78), hsp70, hsc70, gp96 (grp94), hsp60, hsp40 and hsp90 with and the family member.Particularly preferred heat shock protein is BiP, gp96 and the hsp70 that hereinafter exemplifies.Hsp70 family member most preferably.Heat shock protein mutant natural or recombinant sources also can be used for the present invention.Without limitation, the present invention for example provide sudden change with impel its purposes of secreting the heat shock protein of cell (for example the sudden change or the disappearance impel endoplasmic reticulum to catch again element such as KDEL or its congener; This mutant is described in PCT application serial PCT/US96/13233 (WO 97/06685), and it is attached to herein by reference).
For directly giving patient's embodiment of the present invention with albumen/peptide complexes form with heat shock protein and heterozygosis antigen, can use standard technique to prepare heat shock protein by natural origin, for example as Flynn et al., Science 245:385-390 (1989) is described, perhaps uses recombinant technique to prepare heat shock protein as express the heat shock protein code carrier in suitable host cell (as antibacterial, yeast or mammalian cell).If worry heat shock protein pre-loaded the host living beings peptide, then can be, and then purification with heat shock protein and ATP incubation.Hereinafter proposed to prepare the limiting examples of method of heat shock protein of recombinating.
The nucleic acid of coding heat shock heat shock protein can effectively be connected to the element that expression must or need, and expresses the heat shock protein that needs with it then, with this production method as the heat shock protein that is used for protein vaccine, or is used for nucleic acid vaccine.Express the element that must or need and include but not limited to promoter/enhancer element, transcription initiation and terminator sequence, polyadenylation signal, translation initiation and terminator sequence, ribosome binding site, signal sequence etc.Without limitation, cloned and the various heat shock protein genes that check order for example include but not limited to gp96 (people: Genebank searching number X15187; Maki et al., Proc.Natl.Acad.Sci.U.S.A.87:5658-5562 (1990); Mice: Genebank searching number M16370; Srivastava et al., Proc.Natl.Acad Sci.U.S.A.84:3807-3811 (1987)), BiP (mice: Genebank searching number U16277; Haas et al., Proc.Natl.Acad.Sci.U.S.A.85:2250-2254 (1988); People: Genebank searching number M19645; Ting et al., DNA 7:275-286 (1988)), hsp70 (mice: Genebank searching number M35021; Hunt et al., Gene87:199-204 (1990); People: Genebank searching number M24743; Hunt et al, Proc.Natl.Acad.Sci.U.S.A.82:6455-6489 (1995)) and hsp40 (people: Genebank searching number D49547; Ohtsuka K., Biochem.Biophys.Res.Commun.197:235-240 (1993)).
Give method
Can use peptide type, protein type or nucleic acid vaccine, give the patient, make the amount of the complex that in the patient, produces effective inductive treatment immunne response in the patient the complex of heterozygosis antigen of the present invention or heterozygosis antigen and heat shock protein.
Described patient can be the mankind or non-human patients.
Term used herein " therapeutic immunization is replied " is meant according to standard technique and detects that antigenic body fluid of anti-heterozygosis and/or cellular immunization strengthen.Preferably but without limitation, the antigenic humoral immunity level of inductive anti-heterozygosis is higher 4 times than anti-this antigenic humoral immunity level before giving patient's present composition at least, preferably high at least 16 times.Can also be by the method detection by quantitative immunne response that is suitable for testing in external or the body, wherein patient's tumor or infectious disease progress is stagnated or is alleviated and is considered to represent that inducing therapeutic immunization replys.
The antigenic concrete administered dose of heat shock protein/heterozygosis depends on numerous factors, comprise immunogenicity, the patient of concrete vaccine combination immunocompetence, patient size and give approach.The suitable administered dose of measuring any given compositions is a conventional screening operation.
In addition, in the research that gives heterozygosis antigen (promptly not having heat shock protein) separately, identify significant immune efficacy.Although the applicant does not have obligation openly to implement theory of the present invention, be not limited to theory, the results suggest heterozygosis antigen of these researchs in conjunction with endogenous heat shock protein, therefore need not followed for effectiveness and give heat shock protein when the patient is given in injection.The present invention extends to the antigenic this application of heterozygosis of the present invention, and, extend to concomitant therapy or treatment, i.e. general or increase endogenous heat shock protein white level will giving the antigenic position of heterozygosis of the present invention.This concomitant therapy or treatment include but not limited to local application heat, or the medicament of part or systemic administration increase local organization heat shock protein expression.This medicament and method are known in the art.
The heterozygosis antigen that gives (promptly not giving with the composite form with heat shock protein) under the situation that does not give heat shock protein altogether comprises at least one antigenic structure territory and at least one heat shock protein binding structural domain, and comprises wherein a kind of peptide connector mentioned above.
In concrete non-limiting embodiments of the present invention, may need to comprise more than one heat shock protein and/or more than one heterozygosis antigen, so that make immunne response the best.This scheme may be characterized as the infection advantageous particularly that causes the sudden change fast development that immunne response escapes to treatment cancer or treatment.And heterozygosis antigen of the present invention can comprise more than one immunogenic structure territory or more than one epi-position.
Contain heterozygosis antigen/heat shock protein set forth above or separately the antigenic compositions of heterozygosis be called as " vaccine " at this paper.Term " vaccine " is used to represent that compositions of the present invention can be used for inducing preventative or therapeutic immunization is replied.Vaccine of the present invention can comprise the heterozygosis antigen with single antigenic structure territory or epi-position, or has the heterozygosis antigen of a plurality of antigenic structures territory or epi-position.And vaccine can comprise the antigenic mixture of the heterozygosis with single or multiple antigenic structures territory or epi-position, or aforesaid combination in any.As mentioned above, heterozygosis antigen or its mixture can be compound with one or more heat shock proteins before giving, and perhaps can give under the situation of heat shock protein not having.
But on the skin, in subcutaneous, the Intradermal, vein, intramuscular, parenteral, lung, vagina, rectum, intranasal or topical administration contain the antigenic vaccine combination of the present invention of one or more heterozygosis, wherein said heterozygosis antigen is chosen compound one or more heat shock proteins wantonly.Vaccine combination can be by injection, partickle bombardment, oral or aerosol transmission.
Incubation in solution in most of the cases is enough to antigen is loaded on the heat shock protein with heat shock protein and heterozygosis antigen.But, may need to add the material that can help antigen loaded in some cases.
Heating incubation heat shock protein and heterozygosis antigen generally can make antigen be loaded on the heat shock protein.But, may need to add other material in some cases and help to load.For example, hsp40 can promote that peptide is loaded on the hsp70.Minami et al.，J. Biol.Chem.271：19617-19624(1996)。Can use denaturant example hydrochloric acid guanidine or urea moiety or reversibly make the heat shock protein instability, so that the peptide binding pocket is easier near antigen.
Say that at length the vaccine of the present invention that contains heat shock protein preferably also comprises adenosine diphosphate (ADP), to promote heat shock protein and the combination before complex reaches its target of heat shock protein binding structural domain.Can be used in combination the chemical compound that other has similar ability separately or to ADP.
Vaccine combination of the present invention also can comprise various other materials, for example medicine acceptable carrier.Suitable carriers comprises the drug acceptable carrier of any standard, for example phosphate buffered saline(PBS), water, emulsion such as oil/aqueous emulsion or triglyceride emulsion, all kinds of wetting agent, tablet, coated tablet and capsule.Being used for the example that vein and intraperitoneal give the triglyceride the accepted emulsion of chemical compound is that trade name is the triglyceride emulsion of Intralipid .Usually this class carrier comprises excipient, for example the clay of starch, breast, sugar, some type, gelatin, stearic acid, Pulvis Talci, vegetable butter or vegetable oil, natural gum, ethylene glycol or other known excipient.This carrier can also comprise flavoring agent and coloring agent or other composition.
Vaccine combination of the present invention can also comprise suitable diluent, antiseptic, solubilizing agent, emulsifying agent, adjuvant and/or carrier.This compositions can be liquid or lyophilized form or other anhydrous formulation, can comprise and have various buffer compositions (Tris-HCl for example, acetate, phosphate), the diluent of pH and ionic strength, prevent to be adsorbed to additive such as the albumin or the gelatin on surface, (for example Tween 20 for denaturant, Tween 80, Pluronic F68, cholate), solubilizing agent (glycerol for example, Polyethylene Glycol), antioxidant (ascorbic acid for example, sodium metabisulfite), antiseptic (thimerosal for example, benzylalcohol, p-Hydroxybenzoate), implant or tension regulator (lactose for example, mannitol), polymer such as Polyethylene Glycol and albumen are covalently bound, with complexing of metal ion, or material joined specific polymerizable compound goods as poly-acetic acid, polyglycolic acid, among the hydrogel etc. or on, or material joined liposome, microemulsion, micelle, the single or multiple lift vesicle, on erythrocyte umbra or the spheroplast.This combination will influence rate of release and the interior clearance rate of body in physical state, dissolubility, stability, the body.The physics and the chemical characteristic of vaccine depended in the selection of compositions.For example, may need to contain the preparation of denaturant derived from the proteic product of film conjunction type.Controlled release or slow releasing composition are included in the preparation in the lipophilic storehouse (for example fatty acid, wax, oil).The present invention also comprises with polymer (for example poloxamer or Poloxamine) coated granules compositions, the anti-tissue specificity receptor of its coupling, part or antigenic antibody, or the part of coupling tissue specificity receptor.Other embodiment of the present composition has added and has been used for various protecting film, protease inhibitor or the penetration enhancers that give the particle form of approach (comprising intramuscular, parenteral, pulmonary, nose or mouth).
As the alternative method that directly gives heterozygosis antigen (optional compound heat shock protein), but can give the coding heterozygosis antigen of one or more expression-forms and the polynucleotide construction of optional coding heat shock protein.Use in the body of earlier external back or in the body method effable polynucleotide construction is imported in patient's cell.Suitable method comprises and is injected directly in tissue and the tumor, uses liposome transfection (Fraley et al., Nature 370:111-117 (1980)), receptor-mediated endocytosis (Zatloukal et al., Ann.NY Acad.Sci.660:136-153 (1992)), gene transfer (Eisenbraun et al., the DNA﹠amp of particle bombardment mediation; Cell Biol.12:792-797 (1993)) and use peptide to present transfection (Barry et al, the NatureMedicine 2:299-305 (1996) of phage.Also can import in the suitable cell polynucleotide vaccine is external, then it is imported among patient.
For making up effable polynucleotide, prepare coding heat shock protein and/or the antigenic zone of heterozygosis as mentioned above, be inserted in the mammalian expression vector, this carrier can effectively be connected to suitable promoter, for example SV40 promoter, cytomegalovirus (CMV) promoter or rous sarcoma virus (RSV) promoter.Then the construction that obtains is used as the genetic immunization vaccine.Also nucleic acid polymers can be cloned in the viral vector.Suitable carriers includes but not limited to retroviral vector, adenovirus vector, vaccinia virus vector, poxvirus vector and gland relevant viral vector.Be applicable to that concrete carrier of the present invention is pCDNA3 (In Vitrogen), plasmid AH5 (it contains SV40 origin of replication and adenovirus major late promoter), pRC/CMV (In Vitrogen), pCMU II (Paabo et al., EMBO is (1986) J.5:1921-1927), pZip-Neo SV (Cepko et al., Cell 37:1053-1062 (1984)) and pSR α (DNAX, Palo Alto, CA).
WO9706821 and WO9922761 disclose various preparation heat shock proteins and the antigenic method of heterozygosis, and these two documents integral body by reference are attached to herein.
In following embodiment and whole application, aminoacid can be represented with its following one-letter code:
The A alanine
The C cysteine
The D aspartic acid
E glutamic acid
The F phenylalanine
The G glycine
The H histidine
The I isoleucine
K lysine
The L leucine
The M methionine
The N agedoite
The P proline
The Q glutamine
The R arginine
The S serine
The T threonine
The V valine
The W tryptophan
Y tyrosine
The present invention may be better understood with reference to following non-limiting example, and the present invention is for providing this embodiment for example.Proposing following embodiment is in order more fully to set forth the preferred embodiments of the invention.But, the broad range that can not by any way it be construed as limiting the invention.
Embodiment 1
Prepare various heterozygosis antigens, each all comprises the I class H2-K of heat shock protein binding structural domain and cancer antigen epi-position or ovalbumin bEpi-position model SIINFEKL.Comprise the peptide connector between two domains.The heat shock protein binding structural domain that is used for these experiments is as follows: HWDFAWPW, NLLRLTGW, FYQLALTW and RKLFFNLRW.Connector is above-described connector.
Cancer and epi-position model such as following:
Come source protein The source tumor Aminoacid Popular name (aminoacid sequence)
Prostate specific membrane antigen Carcinoma of prostate 771-779 PSMA P2 (ALFDIESKV)
Gp100 Melanoma 209-217 IMD(210M) (IMDQVPFSV)
Tryrosinase Melanoma 368-376 YMD(370D) (YMDGTMSQV)
Human papillomavirus (HPV) strain 16 E7 Cervical cancer 86-93 HPV16 E7 86-93 (TLGIVCPI)
HPV strain 16 E7 Cervical cancer 11-20 HPV16 E711-20 (YMLDLQPETT)
Ovalbumin The tumor antigen model 257-264 Ova (SIINFEKL)
The solid-phase peptide of use standard is synthetic, uses the F-moc chemical method, the heterozygosis antigen that contains heat shock protein binding structural domain, cancer epi-position and the peptide connector between them of synthetic various directions.
Embodiment 2
Analyze by standard Scatchard, measure the labelling heterozygosis antigen (tritiate or fluorescence ALFDIESKVGSGHWDFAWPW) of reference and the binding affinity (Kd is respectively 22.64 μ M and 10.75 μ M) of hsp70, with respect to this binding affinity, by in conjunction with suppressing between experiment (Hill figure) mensuration recombined human or Mus heat shock protein 70 (hsp70) and various heat shock protein binding structural domain mentioned above and the antigenic peptides and and containing binding affinity between the heterozygosis antigen of above-mentioned antigenic peptides and heat shock protein binding structural domain.In conjunction with studying at 39%PBS; 20mMTHAM, pH 8; 37mM NaCl, 5mM MgCl 2With carry out among the 1mM ADP.
Embodiment 3
For in mice, carrying out immune Research, use H2-K (b) peptide RGYVYQGL (aminoacid 52-59) the preparation heterozygosis antigen that derives from Mus MHC H2-K (b) the epi-position SIINFEKL (aminoacid 257-264) of ovalbumin and derive from vesicular stomatitis virus (VSV) nucleoprotein.Following table provides the sequence independent and epi-position in heterozygosis antigen and to the affinity of hsp70.
The mice epi-position Independent epi-position The heterozygosis antigen that contains epi-position
Epitope sequences Affinity (μ M) to hsp70 The heterozygosis antigen sequence Affinity (μ M) to hsp70
Ovalbumin: aminoacid 257-264 SIINFEKL 235 NLLRLTGWGSGSI INFEKL 1.6
NLLRLTGWFFRKS IINFEKL 2.2
NLLRLTGWRKSII NFEKL 0.8
VSV nucleoprotein: aminoacid 52-59 RGYVYQG L 82 NLLRLTGWGSGR GYVYQGL 1.4
NLLRLTGWFFRK RGYVYQGL 1.0
NLLRLTGWRKRG YVYQGL 0.6
Embodiment 4
With the hsp70 of independent hsp70, composite S IINFEKL and the heterozygosis SIINFEKL peptide that is with or without HSP70 at the subcutaneous immunized mice of root of the tail portion.Adjust dosage, so that each inoculation all contains equal amounts of S IINFEKL, except the independent hsp70.After 7 days, collect spleen and enrichment CD8+T cell, it is carried out IFN-γ ELISPOT experiment in elder generation's external back body.Reply (" SIINFEKL ") with SIINFEKL is postimpulse and be recorded in following table, comprise dosage, per 4 * 10 5The speckle number (meansigma methods ± standard deviation) of cd8 t cell carries out the experiment more than 4 times or 4 times altogether, every group of at least 3 mices.Contrast comprises independent culture medium (" culture medium contrast "), no pulse T cell (" no pulse contrast "), uses non-immune peptide RGYVYQGL pulse T cell (" VSV contrast ") that derives from VSV and the positive control (" Con A positive control ") that contacts concanavalin A.
In same experiment, use the target cell of SIINFEKL pulse to carry out above-mentioned 51The Cr-release experiment.When the effector lymphocyte/when the target cell ratio was 200: 1, the deadly percentage result of acquisition was shown in the rightest hurdle of following table.
(200-10)
Immunogen The speckle number of per 400,000 cells CTL experiment: the dead % during 200: 1 E/T
SIINFE KL The culture medium contrast The no pulse contrast The VSV contrast Con A positive control
4.4μg Hsp70 0.00± 0.00 1.50± 2.12 0.67± 0.58 0.33± 0.58 834±28.3 0％
4.4μg 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％
4.4μg Hsp70+2.0 μg NLLRLTG- WGSGSIINFEKL 80.0± 17.0 0.00± 0.00 1.50± 0.71 1.50± 0.71 1170± 56.5 38％
4.4μg Hsp70+2.4 μg NLLRLTGW- FFRKSIINFEKL 222± 17.7 0.00± 0.00 0.67± 0.58 1.33± 1.53 1010± 56.5 52％
Embodiment 5
Carry out one and be similar to above-mentioned experiment, it also comprises does not have the heterozygosis of hsp70 antigen.
(200-11)
Immunogen Per 4 * 10 5The speckle number of individual cd8 t cell
SIINFEKL The culture medium contrast The no pulse contrast The VSV contrast Con A positive control
4.4μg Hsp70 0.33±0.58 1.00±1.73 1.67±1.15 4.00±1.00 965±62.6
4.4μg Hsp70+0.9 μg SIINFEKL 1.67±0.58 1.00±1.00 2.00±0.00 2.67±2.08 591±48.1
4.4μg Hsp70+2.0 μg NLLRLTGW- GSGSIINFEKL 12.0±5.2 2.67±0.58 1.67±1.15 2.00±2.65 748±58.6
4.4μg Hsp70+2.4 μg NLLRLTGWF- FRKSIINFEKL 770±80.6 3.33±1.53 3.67±1.53 4.33±1.53 742±72.6
2.4 μ g NLLRLTG-WFFRKSIINFEKL (no hsp70) 151±20.7 1.00±1.00 1.67±0.58 0.00±0.00 459±149
Embodiment 6
Carry out one again and be similar to above-mentioned experiment.
(200-12)
Immunogen Per 4 * 10 5The speckle number of individual cd8 t cell CTL experiment: the dead % during 200: 1 E/T
SIINFE KL The culture medium contrast The no pulse contrast The VSV contrast Con A positive control
4.4μg Hsp70 0.67± 0.58 0.00± 0.00 0.50± 0.71 1.00± 1.41 552± 24.0 8.45±41.3
4.4μg Hsp70+0.9μg SIINFEKL 3.33± 2.52 0.00± 0.00 0.33± 0.58 0.33± 0.58 450± 69.0 43.0±21.2
4.4μg Hsp70+2.00μg NLLRLTGWGS- GSIINFEKL 134± 4.16 1.33± 1.53 0.67± 1.15 1.00± 1.00 865± 93.0 31.9±5.41
4.4μg Hsp70+2.4μg NLLRLTGWFFR- KSIINFEKL 680± 23.0 0.00± 0.00 0.00± 0.00 1.67± 058 801± 56.6 84.6±1.70
2.4μg NLLRLTGW- FFRKSIINFEKL 211± 17.0 0.00± 0.00 0.50± 0.71 1.00± 0.00 688± 41.7 9.91±5.57
Embodiment 7
As experiment in the previous body, with the subcutaneous immunity inoculation B6 mice of various dose, to estimate hsp70 and the antigenic complex of heterozygosis that uses other small peptide connector preparation, wherein said small peptide connector comprises (using the single-letter amino acid code) FFRK, RK, AKVL, QLK and FR.Carry out IFN-γ ELISPOT experiment in elder generation's external back body as mentioned above.The result who comprises control value is as follows.
(200-13)
Immunogen The speckle number of per 300,000 cells
SIINFEKL The culture medium contrast The no pulse contrast The VSV contrast
4.4μg Hsp70+2.4μg NLLRLTGWFFRKSIINFEKL 114±21 1.0±1.2 1.0±0 0.67±0.41
4.4μg Hsp70+2.4μg NLLRLTGWRKSIINFEKL 70±8.5 1.3±1.1 0.67±0.82 2.7±1.1
0.9μg Hsp70+0.48μg NLLRLTGWFFRKSIINFEKL 98±0.41 0.67±0.82 1.3±1.1 4.3±2.3
0.9μg Hsp70+0.48μg NLLRLTGWRKSIINFEKL 29±2.2 0±0 1±0 0±0
2.4μg NLLRLTGWFFRK- SIINFEKL 11±1.8 0.67±0.82 0±0 0.67±0.82
200-21
Immunogen The speckle number of per 400,000 cells
SIINFEKL The culture medium contrast The no pulse contrast The VSV contrast
4.4μg Hsp70+2.4μg NLLRLTGWFFRKSIINF EKL 124±8.8 0.33±0.41 0.67±0.82 2.67±2.68
4.4μg Hsp70+2.4μg NLLRLTGWAKVLSIINF EKL 95±12 1.3±0.82 1.0±1.2 0.67±0.41
200-23
Immunogen The speckle number of per 400,000 cells
SIINFEKL The culture medium contrast The no pulse contrast The VSV contrast
4.4μg Hsp70+2.4μg NLLRLTGWFFRKSIINFEKL 318±17 0.67±0.51 0.67±0.58 0.67±0.58
4.4μg Hsp70+2.4μg NLLRLTGWQLKSIINFEKL 174±18 0.0±0.0 0.0±0.0 3.7±2.5
4.4μg Hsp70+2.4μg NLLRLTGWFRSIINFEKL 53±2.9 0.0±0.0 0.67±0.58 1.0±1.0
2.4μg NLLRLTGWFRSIINFEKL 31±5.7 1.0±1.7 0.0±0.0 0.67±0.58
Embodiment 8
Using the preparation that does not add hsp70 to be similar in the above-mentioned body in the B6 mice studies.The result is as follows.
(200-17)
Immunogen The speckle number of per 400,000 cells
SIINFEKL The culture medium contrast The no pulse contrast The VSV contrast Con A positive control
10μg SIINFEKL 2.33±0.41 0.33±0.41 1.33±0.82 1.7±0.41 928±72
0.5μg NLLRLTGWFFR KSIINFEKL 22±7.2 1.33±0.41 1.67±1.1 1.0±0.71 906±17
2.5μg NLLRLTGWFFR KSIINFEKL 28±2.7 1.0±1.7 0.33±0.41 2.0±1.2 930±23
25μg NLLRLTGWFFR KSIINFEKL 46±4.3 2.0±0.41 1.33±1.1 3.0±0.71 1007±17
Embodiment 9
The preparation that use is with or without hsp70 is similar in the above-mentioned body in the B6 mice to be studied.In addition, carry out the research that heterozygosis antigen and free heat shock protein binding structural domain peptide (NLLRLTGW) give altogether.The result is as follows.
(VSV-72-02)
Immunogen The speckle number of per 4000,000 cells CTL experiment: the dead % during 200: 1 E/T
SIINFEK L The culture medium contrast The no pulse contrast The VSV contrast Con A positive control
4.4μg Hsp70+2.0μg NLLRLTGWFFRKSIINFEK L 48±11 0.0±1.0 0.0±1.0 4.0±2.0 588±151 32％
2.0μg NLLRLTGWRKSIINFEKL 24±1 1.0±1.0 1.0±1.0 5.0±3.0 842±73 24％
2.0 L+50 times of excessive N LLRLTGW of μ g NLLRLTGWFFRKSIINFEK 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％
Embodiment 10
VSV epi-position RGYVYQGL is with comparing in many previous experiments, uses it as epi-position when other heterozygosis antigen of the present invention of preparation, and to be similar to above-mentioned experimental evaluation inducing immunne response.
(VSV-72-02)
Immunogen The speckle number of per 400,000 cells
SIINFEKL The culture medium contrast The no pulse contrast VSV (RGYVYQGL)
4.4μg HSP+2μg NLLRLTGWFFRKSIINF EKL 48±11 1.0±1.0 0.0±1.0 4.0±2.0
4μg HSP+2μg NLLRLTGWFFRKRGYV YQGL 1.0±1.0 1.0±1.0 4.0±2.0 20±1.0
4μg HSP+6μg NLLRLTGWFFRKRGYV YQGL 6.0±3.0 2.0±2.0 12±3.0 104±13
Embodiment 11
For estimate aforementioned heterozygosis antigen and and the complex of hsp70 to the effectiveness of treatment disease, use will be modified into the model of the subcutaneous implantation B6 of 20,000 E7 tumor cells (the being called E.G7) mice of expressing ovalbumin.Each treatment group is used 10 mices.This model description is in for example Moroi et al., 2000, Proc.Nat.Acad.sci.USA 97:3485-3490.The time dependent Fig. 1 that the results are shown in of mice number that suffers from tumor.After 31 days, tumor all do not occur with 10 mices of hsp70:NLLRLTGWFFRKSIINFEKL inoculation, tumor does not appear in the mice that inoculates with the SIINFEKL of Titermax adjuvant emulsion yet.10 mices with independent NLLRLTGWFFRKRSIINFEKL (not having hsp70) inoculation have 3 tumor to occur.10 mices with the hsp70:SIINFEKL inoculation have 5 tumor to occur, and 10 mices that inoculate separately with Titermax and buffer have 9 tumor to occur.
Embodiment 12
Further use above-mentioned exo-antigen to offer experiment, so that estimate preparation of the present invention.No matter be with dosage form or can endogenous acquisition form providing, more specifically say so its antigen when entering the angtigen presentation path at heterozygosis antigen, and heterozygosis antigen of the present invention all needs hsp70, is to confirm this point, experimentize with following preparation, and shown the result.
(200-MF-41)
Preparation The Pg/ml IL-2 that the B3Z cell produces
0.5ng SIINFEKL 2690±369
5ng NLLRLTGWFFRKSIINFEKL 46±11
5ng NLLRLTGWFFRKSIINFEKL+1.4μg hsp70 3920±344
1.4μg Hsp70 0.0±0.0
Embodiment 13
Firat et al., 1999, " H-2 class I knockout; HLA-A2.1-transgenic mice:a versatile animal model for preclinical evaluation of antitumorimmunotherapeutic strategies; " Eur J Immunol.29:3112-21 has described the HHD II mouse model of carrier HLA-A2 complex, uses this model to estimate people HLA-A2 epi-position in the heterozygosis antigen of the present invention in following experiment." IMD " peptide epitopes IMDQVPFSV with heterozygosis antigen of the present invention appraiser's melanoma antigen gp100 in HHD II model of low and high dose.Use is similar to method described above and carries out the ELISPOT experiment, and test peptides is the IMD peptide, contrasts the peptide YMDGTMSQV (" YMD ") for deriving from the melanoma antigen tryrosinase.The results are shown in following table.
(HDD II 200-72-02)
Immunogen The speckle number of per 400,000 cells
IMD The culture medium contrast The no pulse contrast The YMD contrast
4 μ g hsp70 and 5 μ g NLLRLTGWFFRK IMDQVPFSV 139±11 0.67±0.58 1.0±1.0 3.7±0.58
4 μ g hsp70 and 10 μ g NLLRLTGWFFRK IMDQVPFSV 217±3.2 0.67±0.58 4.0±6.0 2.7±1.5
2μg NLLRLTGWFFRK IMDQVPFSV 27±5.1 0.0±0.0 0.0±0.0 2.0±2.0
Use YMD as the epi-position in the heterozygosis antigen, to carry out similar experiment with the complex of hsp70 in the HDDII mice, the result is as follows.
(200-72-01)
Immunogen The speckle number of per 400,000 cells
YMD The culture medium contrast The no pulse contrast The IMD contrast
4 μ g hsp70 and 5 μ g NLLRLTGWFFRKY MDGTMSQV 33±7.8 1.0±0.0 1.0±0.0 1.0±1.4
4 μ g hsp70 and 10 μ g NLLRLTGWFFRKY MDGTMSQV 323±44 0.0±0.0 1.5±0.71 1.5±0.71
Embodiment 14
With above similar, in the B6 mice, estimate Sendai virus (SdV) epi-position FAPGNYPAL with heterozygosis antigen of the present invention.The result is as follows.
(200-18)
Immunogen The speckle number of per 400,000 cells
SdV The culture medium contrast The SIINFEKL contrast
4 μ g hsp70 and 2 μ g NLLRLTGWFFRKSII NFEKL 1.3±1.2 1.0±1.0 197±27
2μg NLLRLTGWFFRKRG YVYQGL 0.33±0.58 0.0±0.0 87±20
4 μ g hsp70 and 2 μ g NLLRLTGWFFRKFA PGNYPAL 38±17 0.33±0.58 1.0±1.0
13 μ g hsp70 and 7 μ g NLLRLTGWFFRKFA PGNYPAL 169±32 4.3±1.5 7.0±3.5
Embodiment 15
Carry out experiment in the body, give the B6 mice altogether two kinds of heterozygosis antigens of the present invention and hsp70.Mix the heterozygosis antigen of the RGYVYQGL of the SIINFEKL contain ovalbumin and VSV, inoculate with hsp70.The result is as follows.
(OVA-VSV-72-01)
Immunogen The speckle number of per 400,000 cells
VSV The culture medium contrast The no pulse contrast OVA
2μg hsp70 2μg NLLRLTGW- FFRKSIINFEKL 2μg NLLRLTGW- FFRKRGYVYQGL 77±19 2.0±1.0 2.0±1.0 366±19
2μg hsp70 6μg NLLRLTGW- FFRKSIINFEKL 6μg NLLRLTGW- FFRKRGYVYQGL 185±9 1.0±1.0 4.0±2.0 349±10
Embodiment 16
As mentioned above, in one aspect of the invention, containing the multiple antigenic preparation of heterozygosis with different epitopes can prepare with one or more heat shock proteins, is used to inoculate the people, to excite effective treatment or prophylactic immunne response.For example, be the treatment human melanoma, the formulation preparation that will contain 8 kinds of different melanoma epi-positions is a heterozygosis antigen, with for example hsp70 preparation.In this concrete preparation, the N-of all epi-positions end all uses heat shock protein binding structural domain NLLRLTGW, uses peptide connector FFRK to be connected to the C-end of epi-position.This paper comprises other binding structural domain and connector.This concrete preparation can be used for treating HLA-A2 haplotype patient.Preparation contains following heterozygosis antigen and hsp70:
Antigenic source and aminoacid sequence The heterozygosis antigen sequence
Gp 100: aminoacid 209-217 (modifying 210M) NLLRLTGWFFRKIMDQVPFSV
Tryrosinase: aminoacid 368-376 (modifying 370D) NLLRLTGWFFRKYMDGTMSQV
Melanin-A: aminoacid 26-35 (modifying 27L) NLLRLTGWFFRKELAGIGILTV
NY-ESO-1: amino acid/11 57-165 (modifying 165V) NLLRLTGWFFRKSLLMWITQV
TRP-2: amino acid/11 80-188 NLLRLTGWFFRKSVYDFFVWL
MAGE-10: aminoacid 254-262 GLYDGMEHLGSGNLLRLTGW
Gp 100: aminoacid 280-288 (288V) YLEPGPVTVGSGNLLRLTGW
SSX-2: aminoacid 41-49 KASEKIFYVGSGNLLRLTGW
In one embodiment, can the aforementioned 8 kinds of heterozygosis antigens and the hsp70 of about equivalent is compound, in saline, give.In another embodiment, preparation comprises listed preceding 5 kinds of heterozygosis antigens.Optional ADP and other component that comprises stable compound of saline solution that contains the previous formulations of heat shock protein, excipient for example mentioned above, diluent and carrier.In another embodiment, aforementioned 8 kinds of heterozygosis antigens or listed preceding 5 kinds of antigenic mixture of heterozygosis are formulated in the saline, give under the situation of heat shock protein not having.
Embodiment 17
Scheme is just exempted from this experimental evaluation.Use NLLRLTGWFFRKSIINFEKL heterozygosis antigen, perhaps do not give hsp70 altogether, 5 kinds of following schemes are: 1) gave in the 0th day, analyzed in the 7th day; 2) gave in the 0th day and the 7th day, analyzed in the 21st day; 3) gave in the 0th day, analyzed in the 21st day; 4) gave in the 0th day and the 14th day, analyzed in the 28th day; With 5) the 0th day give, analyzed in the 28th day.It is as follows that the speckle of per 400,000 cells is counted the result.
(200-28-72-01a、-01b、-01c)
Scheme inoculation day 0 0、7 0 0、14 0
Program analysis day 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
2μg hsp70，4μg SIINFEKL 3.0±1.0 6.0±1.0 22±8.0 3.0±2.0 20±5.0
2μg NLLRLTGWFFRKS IINFEKL 72±5.0 24±6.0 42±7.0 25±9.0 82±11
2 μ g hsp70 and 4 μ g NLLRLTGWFFRKS IINFEKL 99±12 98±11 141±14 398±18 27±2.0
2μg hsp70 5.0±6.0 3.0±2.0 3.0±0.0 1.0±1.0 4.0±3.0
Embodiment 18
As above experimentize again with the heterozygosis antigen mixture, to confirm to inspire immunne response at fraction antigen.In this experiment, use the heterozygosis antigen that contains SIINFEKL and VSV peptide RGYVYQGL.
(VSV/OVA-72-02)
Immunogen The speckle number of per 300,000 cells
SIINFEKL The culture medium contrast The no pulse contrast VSV (RGYVYQGL)
3.7μg hsp70 2μg NLLRLTGW- FFRKSIINFEKL 238±27 0.0±0.0 1.0±1.0 5.0±2.0
11.2μg hsp70 6μg NLLRLTGW- FFRKSIINFEKL 330±45 1.0±1.0 0.0±0.0 4.0±1.0
3.7μg hsp70 2μg NLLRLTGW- FFRKRGYVYQGL 1.0±1.0 1.0±1.0 0.0±0.0 61±11
11.2μg hsp70 6μg NLLRLTGW- FFRKRGYVYQGL 2.0±2.0 2.0±1.0 2.0±0.0 147±20
3.7μg hsp70 2μg NLLRLTGW- FFRKSIINFEKL 2μg NLLRLTGW- FFRKRGYVYQGL 179±4.0 2.0±2.0 1.0±1.0 165±11
11.2μg hsp70 6μg NLLRLTGW- FFRKSIINFEKL 6μg NLLRLTGW- FFRKRGYVYQGL 310±13 1.0±1.0 1.0±1.0 242±52
Embodiment 19
As described in embodiment 17, the antigenic binding affinity of heterozygosis that contains the various connectors that heat shock protein binding structural domain NLLRLTGW, antigenic structure territory SIINFEKL (deriving from ovalbumin) or RGYVYQGL (deriving from VSV albumen) and embodiment 32 propose is detected.Independent antigenic structure territory is respectively 235 μ M and 82 μ M to the bonded Kd of hsp70.The results are shown in following.
Heterozygosis antigen Kd in conjunction with HSP70
NLLRLTGWGSGSIINFEKL 1.6μM
NLLRLTGWFFRKSIIMFEKL 2.2μM
NLLRLTGWRKSIINFEKL 0.8μM
NLLRLTGWAKVLSIINFEKL 2.0μM
NLLRLTGWQLKSIINFEKL 0.4μM
NLLRLTGWFRSIINFEKL 1.5μM
NLLRLTGWGSGRGYVYQGL 1.4μM
NLLRLTGWFFRKRGYVYQGL 1.0μM
NLLRLTGWRKRGYVYQGL 0.6μM
Embodiment 20
Further study and estimate that heterozygosis antigen gives the B6 mice separately and immunogenicity when not giving altogether with hsp70.IFN-γ ELISPOT is used in evaluation methodology as mentioned above.
(contrast 200-24 and 200-30)
Immunogen The speckle number of per 300,000 cells
SIINFEKL SWDFITV Culture medium The no pulse splenocyte
25μg NLLRLTGWFFRKSIINFEKL 109±14 NT 0±0 3.0±2.0
24.9μg NLLRLTGWFFRKSSWDFITV NT 26±5 0.67±0.58 0.33±0.58
2.1μg NLLRLTGWFRSIINFEKL 12±2 NT 0.67±0.58 0.67±0.58
NT does not test
Embodiment 21
Preparation contains two kinds of antigenic heterozygosis antigens being separated by above-mentioned connector, so that heterozygosis antigen has following universal architecture:
(heat shock protein binding structural domain)-(connector)-(antigen 1)-(connector)-(antigen 2).
Although the heat shock protein binding structural domain is in the antigenic N end parts of heterozygosis in this embodiment, this situation not necessarily, the present invention comprises that also the heat shock protein binding structural domain is at C-end or the heterozygosis antigen between two antigenic structure territories.And although use identical connector peptide between the heat shock protein binding structural domain of between the antigenic structure territory and antigenic structure territory and vicinity in following embodiment, this situation not necessarily can be used different connector peptides.And, choose wantonly in one or two position and have connector.Further again, can use three or more antigenic peptides.For the sake of simplicity, this heterozygosis antigen with two or more antigenic structure territories is called series connection heterozygosis antigen.The complex of this series connection heterozygosis antigen composition, one or more series connection heterozygosis antigens and heat shock protein, and, be completely contained in herein by giving the method that one or more series connection heterozygosis antigens or at least a heat shock protein and the antigenic complex of at least a series connection heterozygosis excite immunne response or prevention or treatment disease.
Following embodiment has contrasted two kinds of antigenic mixture of heterozygosis and has contained the series connection heterozygosis immunogenicity of antigens and the dose response studies of same antigen.In an experiment, comprise the peptide that contains two connectors and epi-position but do not have the heat shock protein binding structural domain.
(contrast-200-72-01)
Immunogen The speckle number of per 300,000 cells
SIINFEKL The culture medium contrast The no pulse contrast RGYVYQGL
19.2μg NLLRLTGWFF RKSIINFEKLFF RKRGYVYGL 390±56 1.7±1.1 3.0±1.9 146±13
19.2μg NLLRLTGWFF RKRGYVYQGL FFRKSIINFEKL 180±11 1.3±1.1 2.7±1.1 321±5.8
(S200-72-02)
Immunogen The speckle number of per 300,000 cells
SIINFEKL The culture medium contrast RGYVYQGL
7.3μg FFRKSIINFEKLFFRKRGY VYQGL 8.3±1.1 1.7±0.4 31±5.5
9.6μg NLLRLTGWFFRKSIINFEK LFFRKRGYVYQGL 713±13 9.0±1.2 207±8.2
9.6μg NLLRLTGWFFRKRGYVY QGLFFRKSIINFEKL 69±12 0.7±0.4 460±14
(S200-72-12)
Immunogen The speckle number of per 300,000 cells
SIINFEKL The culture medium contrast RGYVYQGL
20μg NLLRLTGWFFRKSIINFEK LFFRKRGYVYQGL 410±49 0.3±0.4 250±11
10μg NLLRLTGWFFRKSIINFEK LFFRKRGYVYQGL 360±13 0.3±0.4 100±10
5μg NLLRLTGWFFRKSIINFEK LFFRKRGYVYQGL 130±3.3 0±0 35±6.6
20μg NLLRLTGWFFRKRGYVY QGLFFRKSIINFEKL 150±6 0±0 380±12
10μg NLLRLTGWFFRKRGYVY QGLFFRKSIINFEKL 30±3 0±0 83±5
In present embodiment and other embodiment, the epi-position of proximity thermal shock protein binding structural domain shows the strongest immunne response, therefore the selected epi-position of selecting to be used for bacterin preparation of the present invention can be positioned the whole immunogenicity of preparation is had the position of maximum contribution, no matter is to be not always the case under the situation of heat shock protein or as the complex with heat shock protein not having.
Embodiment 22
In following experiment, estimate the immunogenicity of series connection heterozygosis antigen mixture.Except from ovalbumin (SIINFEKL) with from the H2-K of VSV (RGYVYQGL) bOutside the I class peptide, also use H2-K bBeta-casein peptide IAYFYPEL and Sendai virus peptide FAPGNYPAL.In another experiment, mix two kinds of series connection heterozygosis antigens with the same antigen peptide that replaces configuration.Inspire strong immune response at 4 kinds of epi-positions.
The all preparations of this paper all contain 1mM ADP.In a following experiment, ADP is removed.
(200-72-04)
Immunogen The speckle number of per 300,000 cells
SIINFEKL RGYVYQGL IAYFYPEL FAPGNYPAL
9.6μg NLLRLTGWFFRKSIINF EKLFFRKRGYVYQGL 537±16 150±10 4.7±0.8 5.7±2.5
9.7μg NLLRLTGWFFR- KIAYFYPELFFRKFAPG NYPAL 1.7±1.1 1.7±0.8 128±9.2 136±6.6
9.6μg NLLRLTGW- FFRKSIINFEKLFFRKRG YVYQGL +9.7μg NLLRLTGW- FFRKIAYFYPELFFRKF APGNYPAL 363±31 256±5.3 127±7.9 155±28
5 S200-72-13
Immunogen The speckle number of per 300,000 cells
SIINFEKL RGYVYQGL IAYFYPEL FAPGNYPAL
9.6μg NLLRLTGW- FFRKIAYFYPELFFRKF APGNYPAL +9.6μg NLLRLTGW- FFRKSIINFEKLFFRKR GYVYQGL 388±6.8 72±5.0 402±17 379±30
9.6μg NLLRLTGW- FFRKRGYVYQGLFFR KSIINFEKL +9.6μg NLLRLTGW- FFRKIAYFYPELFFRKF APGNYPAL 76±1.9 159±8.3 115±20 172±5.9
S200-72-13
Immunogen The speckle number of per 300,000 cells
SIINFEKL RGYVYQGL IAYFYPEL Culture medium
9.6μg NLLRLTGWFFRKSIINFE KLFFRKRGYVYQGL 450±10 273±12 3.0±1.4 0.33±0.41
9.6μg NLLRLTGWFFRKRGYV YQGLFFRKSIINFEKL 82±4 445±30 1.3±0.41 0±0
9.6μg NLLRLTGWFFRKSIINFE KLFFRKRGYVYQGL+9.6 μg NLLRLTGWFFRKRGYV YQGLFFRKSIINFEKL 202±7.6 188±24 1.0±0.7 0.67±0.41
S200-72-13, no ADP
Immunogen The speckle number of per 300,000 cells
SIINFEKL RGYVYQGL IAYFYPEL Culture medium
9.6μg NLLRLTGWFFRKSIINFE KLFFRKRGYVYQGL 228±2.5 126±2.9 1.7±0.4 0±0
9.6μg NLLRLTGWFFRKRGYV YQGLFFRKSIINFEKL 83±9 189±19 13±15 0.33±0.41
9.6μg NLLRLTGWFFRKSIINFE KLFFRKRGYVYQGL +9.6μg NLLRLTGWFFRKRGYV YQGLFFRKSIINFEKL 115±7.8 86±11 0.33±0.41 0±0
Embodiment 23
In following experiment, when giving the B6 mice, import nearly 5 kinds of antigenic peptides into two kinds of series connection heterozygosis antigens and the antigenic mixture of single heterozygosis, and under the situation that does not give HSP70 altogether induction of immunity originality.Series connection heterozygosis antigen contains VSV and with ovalbumin peptide in an antigen, contain beta-casein and Sendai virus peptide in another antigen.Single heterozygosis antigen contains NS2-114 influenza virus peptide (RTFSFQLI).
S200-72-15
Immunogen The speckle number of per 300,000 cells
SIINFEKL RGYVYQGL LAYFYPEL FAPGNYPAL RTFSFQLI
9.6μg NLLRLTGW- FFRKRGYVYQGLFFRKSIIN FEKL+ 19μg NLLRLTGW- FFRKIAYFYPELFFRKFAPG NYPAL 67±6.1 205±20 229±28 266±33 0±0
9.6μg NLLRLTGW- FFRKRGYVYQGLFFRKSIIN FEKL+ 19μg NLLRLTGW- FFRKIAYFYPELFFRKFAPG NYPAL+ 12.2μg NLLRLTGW- FFRKRTFSFQLI 156±3.3 299±18 175±12 125±3.3 33±4.7
Embodiment 24
Do not having to give aforesaid single heterozygosis antigen under the situation of heat shock protein, estimating the immunogenicity of single heterozygosis antigen when having helper T cell epitope in the heterozygosis antigen.In the great majority experiment, use ovalbumin H2-K bThe aminoacid 323-339TEWTSSNVMEERKIKV of II class epi-position (being that the heterozygosis antigen sequence is NLLRLTGWFFRKTEWTSSNVMEERKIKV).The heterozygosis antigen that contains II class peptide will resist replying of I class epi-position on average to increase to about 7 times.
(250-72-08)
The immunogen that contains I class T1249 The speckle number of per 300,000 cells
Replying when giving I class heterozygosis antigen to I class epi-position Replying when giving I class and the antigenic mixture of II class heterozygosis to I class epi-position Culture medium Splenocyte
24.2μg NLLRLTGW- FFRKDAPIYTNV 2±1.9 13±3.9 0.7±0.4 0±0
24.9μg NLLRLTGW- FFRKSSWDFITV 18±0.7 98±5.8 0.7±0.8 0.7±0.4
25.4μg NLLRLTGW- FFRKRTFSFQLI 5.3±1.5 43±7.6 0.3±0.4 0±0
25.5μg NLLRLTGW- FFRKIAYFYPEL 11±3 73±9.8 0±0 0±0
Embodiment 25
Do not having under the situation of heat shock protein, the heterozygosis antigen of estimating heterozygosis antigen and the various H2-Kb of containing II class peptides gives altogether to immunogenic influence.I class peptide is SSWDFITV or DAPIYTNV; II class peptide comprises with ovalbumin peptide mentioned above, derive from the II class peptide NNFTVSFWLRVPKVSASHL of tetanus toxoid (being that the heterozygosis antigen sequence is NLLRLTGWFFRKNNFTVSFWLRVPKVSASHL) or HBVc (amino acid/11 28-140) peptide TPPAYRPPNAPIL.
250-72-13
Immunogen The speckle number of per 300,000 cells
Culture medium SSWDFITV
24.9μg NLLRLTGWFFRKSSWDFITV 3.0±0.7 78±3.9
24.9μg NLLRLTGWFFRKSSWDFITV+27.4μg NLLRLTGWFFRKTPPAYRPPNAPIL 8.0±3.1 84±7.1
24.9μg NLLRLTGWFFRKSSWDFITV+33.6μg NNFTVSFWLRVPKVSASHLGSGNLLRLTGW 3.7±1.1 315±15
24.9μg NLLRLTGWFFRKSSWDFITV+36.4μg HWDFAWPWNGSGNNFTVSFWLRVPKVSASHL 2.7±2.0 135±5.7
24.9μg NLLRLTGWFFRKSSWDFITV+34.7μg NLLRLTGWFFRKTEWTSSNVMEERKIKV 1.7±0.4 229±12
Therefore, helper T cell epitope can be included in the heterozygosis antigen as unique epi-position, gives with mixture with other heterozygosis antigen that contains I class epi-position, and perhaps helper T cell epitope can be included in the series connection heterozygosis antigen as one of epi-position.These only are the examples about the numerous variations of heterozygosis antigen composition of the present invention.
Embodiment 26
In the mode similar, estimate the heterozygosis immunogenicity of antigens of when having and do not have to give altogether, connecting with the heterozygosis antigen that contains ovalbumin II class peptide with previous embodiment.
S250-72-12
Immunogen The speckle number of per 300,000 cells
IAYFYPEL FAPGNYPAL Culture medium
19μg NLLRLTGWFFRKIAYFYPEL- FFRKFAPGNYPAL 9.3±4.7 17±9 0.7±0.6
19μg NLLRLTGWFFRKIAYFYPEL- FFRKFAPGNYPAL+20.8μg NLLRLTGWFFRKTEWTSSNVMEERKIKV 44±5.1 58±5.2 0.7±0.6
250-72-15
Immunogen The speckle of per 300,000 cells is counted IAYFYPEL
25.5μg NLLRLTGWFFRKIAYFYPEL 3.7±3.1
25.5μg NLLRLTGWFFRKIAYFYPEL+34.7μg NLLRLTGWFFRKTEWTSSNVMEERKIKV 133±11
25.5μg NLLRLTGWFFRKIAYFYPEL+25Mg NLLRLTGWFFRKSIINFEKL 88±9.9
Embodiment 27
In addition, not having to contain the heterozygosis antigen and at least a heterozygosis antigen of connecting of helper T cell epitope altogether under the situation about giving altogether, similarly test with heat shock protein.
250-72-12
Immunogen The speckle number of per 300,000 cells
Culture medium SIINFEKL RGYV YQGL IAYF YPEL FAPGN YPAL
24μg NLLRLTGWFFRKIAYFYP BLFFRKFAPGNYPAL 0.7±0.6 NT NT 9.3± 4.7 17±8.7
24μg NLLRLTGWFFRKIAYFYP ELFFRKFAPGNYPAL+ 21μg NLLRLTGWFFRK- TEWTSSNVMEERKIKV 0.7±0.6 NT NT 44± 5.1 67±5.5
15μg NLLRLTGWFFRK- FAPGNYAL 0±0 NT NT 0.3± 0.6 4.3±3.2
15μg NLLRLTGWFFRKF- APGNYPA+ 21μg NLLRLTGWFFRK- TEWTSSNVMEERKIKV 0±0 NT NT 2.3± 2.1 58±5.2
Embodiment 28
Use the heterozygosis antigen that contains people I class (HLA-A2) epi-position in HHD II mice, to carry out immune Research as mentioned above.Use complex inoculation mice by 5 μ g hsp70 and 33 μ gNLLRLTGWFFRKYMDGTMSQV preparation.ELISPOT result in per 300,000 cells is: culture medium, 1.33 ± 0.58; Splenocyte 1 ± 0; Splenocyte+YMDGTMSQV 123 ± 13; Splenocyte+IMDQVPFSV 4 ± 1.
Embodiment 29
Use in the experiment of HHDII mice at another, use the immunogenicity HLA-A2 epi-position (SVYDFFVWL) of Trp-2.Because this epi-position also is the H2-Kb epi-position, and the HHDII mice is based upon on B6 mice (H2-Kb) basis, show the tolerance of in mouse model, having broken to self epi-position so induce the immunne response of anti-Trp-2 peptide.This result of experiment confirms the tolerance of self epi-position is broken, and the invention further relates to by giving heterozygosis antigen of the present invention and complex and breaks the method for tolerance.
HHDII-200-72-03
Immunogen The speckle number of per 300,000 cells
Culture medium SVYDFFVW L IMDQVPFSV YMDGTMSQ V
4.33μg NLLRLTGWFFRKSVYD FFVWL+25μg hsp70 0.5±0.71 166±25 2.0±1.4 35±0.71
8.66μg NLLRLTGW- FFRKSVYDFFVWL+25 μg hsp70 3.5±0.71 114±11 7.7±2.1 11±3.1
4.1μg NLLRLTGW- FFRKYMDGTMSQV+ 25μg hsp70 3.0 1.0 2.0±1.4 74±2.8
4.1μg NLLRLTGW- FFRKIMDQVPQV+ 25μg hsp70 1.0±1.4 2.0±2.0 984±26 2.3±1.5
Embodiment 30
Use the HHDII mice to estimate the immunogenicity of hsp70 and three kinds of antigenic complex of heterozygosis, these three kinds of heterozygosis antigens contain some HIV virus component epi-position that embodiment 27 proposes.
HDDII-200-72-07
Immunogen The speckle number of per 300,000 cells
Culture medium ILKEPVHGV VIYQYMDDL SLYNTVATL
36μg NLLRLTGW- FFRKILKEPVHGV+25μg hsp70 1.0±1.0 34±12 0±0 NT
36μg NLLRLTGW- FFRKVIYQYMDDL+25μg hsp70 0±0 0.67±0.58 24±6.1 NT
36μg NLLRLTGW- FFRKSLYNTVATL+25μg hsp70 0.67±0.58 NT NT 140±6.7
NT=does not test
Embodiment 31
As mentioned above, estimate and the compound immunogenicity of heterozygosis antigen mixture in the B6 mice that contains the H2-Kb epi-position of hsp70.
OBS-72-01
Immunogen The speckle number of per 300,000 cells
Culture medium Splenocyte SIINFEKL RAPGNYPAL IAYFYPEL
2μg NLLRLTGWFFRKSIINFEKL +13.7μg hsp70 1 2±1 148±11 7±2 3±2
10μg NLLRLTGWFFRKIAYFYPEL +13.7μg hsp70 0 2±2 3±1 8±2 47±13
10μg NLLRLTGWFFRKFAPGNYPAL +13.7μg hsp70 3 3±3 3±2 83±6 6±1
2μg NLLRLTGWFFRKSIINFEKL+ 10μg NLLRLTGWFFRKIAYFYPEL + 27.4μg hsp70 2 4±2 94±4 9±3 29±4
2μg NLLRLTGWFFRKSIINFEKL+ 10μg NLLRLTGWFFRKFAPGNYPAL + 27.4μg hsp70 3 3±0 169±7 157±27 4±2
10μg NLLRLTGWFFRKIAYFYPEL+ 10μg NLLRLTGWFFRKFAPGNYPAL+ 27.4μg hsp70 3 3±3 4±3 46±8 39±2
2μg NLLRLTGWFFRKSIINFEKL+ 10μg NLLRLTGWFFRKIAYFYPEL+ 10μg NLLRLTGWFFRKFAPGNYPAL+ 41μg hsp70 1 5±2 149±19 61±5 60±7
Embodiment 32
With B6 mice study and the compound heterozygosis immunogenicity of antigens of connecting of hsp7.
S200-72-01
Immunogen The speckle number of per 300,000 cells
The culture medium contrast The no pulse contrast SIINFEKL RGYVYQG L
5.6μg hsp70+ 3μg NLLRLTGWFFRKSIINFEKL 0.33±0.41 0.67± 0.41 43±9.2 1±0
11.2μg hsp70+ 5.9μg NLLRLTGWFFRKRGYVYQGL 0.33±0.41 0.33± 0.41 1±0.71 102±16
11.2μg hsp70+ 3μg NLLRLTGWFFRKSIINFEKL+ 5.9μg NLLRLTGWFFRKRGYVYQGL 0.67±0.82 1.7±0.41 182±11 113±10
5.6μg hsp70+ 4.8μg NLLRLTGWFFRKSIINFBKLFFRKRGYVYQGL 0±0 4±1.4 456±19 113±1.1
11.2μg hsp70+ 9.6μg NLLRLTGWFFRKSIINFEKLFFRKRGYVYQGL 0±0 10±3.3 505±57 90±11
22.4μg hsp70+ 19.2μg NLLRLTGWFFRKSIINFEKLFFRKRGYVYQGL 0.67±0.82 1.7±0.41 289±26 130±12
5.6μg hsp70+ 4.8μg NLLRLTGWFFRKRGYVYQGLFFKSIINFEKL 0.33±0.41 2.3±0.41 72±9.5 98±9.2
11.2μg hsp70+ 9.6μg NLLRLTGWFFRKRGYVYQGLFFRKSIINFEKL 2±0 2.3±1.5 370±16 617±23
22.4μg hsp70+ 19.3μg NLLRLTGWFFRKRGYVYQGLFFRKSIINFEKL 0.67±0.41 4.0±2.1 336±7.8 728±12
The invention is not restricted to the scope of specific embodiments described herein.In fact,, describe and accompanying drawing according to preamble these except described herein, various modifications of the present invention are apparent to those skilled in the art.This modification belongs to the scope of appended claims.
Its content of various publications that this paper quotes all by reference integral body be attached to herein.

Claims (21)

1. heterozygosis antigen, described antigen comprises: the antigenic structure territory of at least one infectious agent or tumor antigen, at least one is non-covalent in conjunction with the binding structural domain of heat shock protein and at least one the peptide connector between them, and described peptide connector 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 LeuGlu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO:1001); Lys Asn (KN); Arg Lys (RK); And the AA1-AA2-AA3-leucine, wherein AA1 is A, S, V, E, G, L or K, and AA2 is K, V or E, and AA3 is V, S, F, K, A, E or T.
2. compositions of inducing the immunne response of infectious agents or tumor antigen, described compositions comprises the heterozygosis antigen of at least a claim 1.
3. compositions of inducing the immunne response of infectious agents or tumor antigen, described compositions comprises the antigenic complex of heterozygosis of at least a heat shock protein and at least a claim 1.
4. the compositions of claim 3, wherein said heat shock protein is hsp70.
5. method of inducing the immunne response of infectious agents or tumor antigen, described method comprises the heterozygosis antigen that gives at least a claim 1 of patient.
6. method of inducing the immunne response of infectious agents or tumor antigen, described method comprise and give the patient following (a) and complex (b):
(a) the heterozygosis antigen of claim 1; With
(b) heat shock protein;
Wherein said heterozygosis antigen and the non-covalent combination of heat shock protein.
7. the method for claim 6, wherein said heat shock protein is hsp70.
8. treat infectious disease or method for cancer for one kind, described method comprises the heterozygosis antigen that gives at least a claim 1 of patient, and at least one antigenic structure territory derives from infectious disease or cancer in the described heterozygosis antigen.
9. a treatment infectious disease or method for cancer, described method comprise and give the patient following (a) and complex (b):
(a) the heterozygosis antigen of claim 1, wherein at least one antigenic structure territory derives from infectious disease or cancer; With
(b) heat shock protein;
Wherein said heterozygosis antigen and the non-covalent combination of heat shock protein.
10. the method for claim 9, wherein said heat shock protein is hsp70.
11. heterozygosis antigen, described antigen basic composition is: the antigenic structure territory of at least one infectious agent or tumor antigen, at least one is non-covalent in conjunction with the binding structural domain of heat shock protein and at least one the peptide connector between them, and wherein the peptide connector is selected from: Phe Phe ArgLys (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 LeuGlu (QLE), Ala Lys Val Leu (AKVL; SEQ ID NO:1001); Lys Asn (KN); Arg Lys (RK); And the AA1-AA2-AA3-leucine, wherein AA1 is A, S, V, E, G, L or K, and AA2 is K, V or E, and AA3 is V, S, F, K, A, E or T.
12. a compositions of inducing the immunne response of infectious agents or tumor antigen, described compositions comprise the heterozygosis antigen of at least a claim 11.
13. a compositions of inducing the immunne response of infectious agents or tumor antigen, described compositions comprise the antigenic complex of heterozygosis of at least a heat shock protein and at least a claim 11.
14. the compositions of claim 13, wherein said heat shock protein are hsp70.
15. a method of inducing the immunne response of infectious agents or tumor antigen, described method comprise the heterozygosis antigen that gives at least a claim 11 of patient.
16. comprising, a method of inducing the immunne response of infectious agents or tumor antigen, described method give the patient following (a) and complex (b):
(a) the heterozygosis antigen of claim 11; With
(b) heat shock protein;
Wherein said heterozygosis antigen and the non-covalent combination of heat shock protein.
17. the method for claim 16, wherein said heat shock protein are hsp70.
18. treat infectious disease or method for cancer for one kind, described method comprises the heterozygosis antigen that gives at least a claim 11 of patient, at least one antigenic structure territory derives from infectious disease or cancer in the described antigen.
19. comprising, a treatment infectious disease or method for cancer, described method give the patient following (a) and complex (b):
(a) the heterozygosis antigen of claim 1, wherein the antigenic structure territory derives from infectious disease or cancer; With
(b) heat shock protein;
Wherein said heterozygosis antigen and the non-covalent combination of heat shock protein.
20. the method for claim 19, wherein said heat shock protein are hsp70.
21. a peptide, described peptide are Phe Phe Arg Lys (FFRK; SEQ IDNO:1000); Phe Arg Lys (FRK); Phe Arg Lys Asn (FRKN, SEQ IDNO:1002); Arg Lys Asn (RKN); Phe Phe Arg Lys Asn (FFRKN, SEQ IDNO:1003); Phe Arg (FR), Gln Leu Lys (QLK), Gln Leu Glu (QLE), AlaLys Val Leu (AKVL; SEQ ID NO:1001); Lys Asn (KN); Arg Lys (RK); Or the AA1-AA2-AA3-leucine, wherein AA1 is A, S, V, E, G, L or K, and AA2 is K, V or E, and AA3 is V, S, F, K, A, E or T.
CN 200480015764 2003-04-11 2004-04-09 Improved heat shock protein-based vaccines and immunotherapies Pending CN1816348A (en)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US46246903P 2003-04-11 2003-04-11
US60/462,469 2003-04-11
US60/463,746 2003-04-18
US60/503,417 2003-09-16
US10/776,521 2004-02-12
USPCT/US04/04340 2004-02-13
US10/820,067 2004-04-08

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107847572A (en) * 2015-05-13 2018-03-27 艾吉纳斯公司 Vaccine for treatment of cancer and prevention

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107847572A (en) * 2015-05-13 2018-03-27 艾吉纳斯公司 Vaccine for treatment of cancer and prevention

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