JPH11513369A - Treatment and prevention methods using heat shock proteins - Google Patents

Abstract

  (57) [Summary] The present invention relates to an immunogenic complex of a heat shock protein (hsp) non-covalently linked to an exogenous antigen molecule that elicits a specific immune response in a host when administered to an individual. Methods for treating or preventing cancer and infectious diseases are provided.

Description

DETAILED DESCRIPTION OF THE INVENTION               Treatment and prevention methods using heat shock proteins   This invention is a governmental application under grant number CA44786 approved by the National Institutes of Health. Made with support. The government has certain rights in the invention. 1.Introduction   The present invention relates to a set for the prevention and treatment of primary and metastatic cancers and / or infectious diseases. About adult. The practice of the prophylactic and therapeutic methods of the present invention is not limited to these. Hsp70, hsp90, gp96 alone or in combination with each other, and A composition of a non-covalent complex of heat shock / stress protein (hsp), comprising: Immunity to genotoxic and non-genotoxic agents, tumors, pathogens and infectious agents Used to enhance epidemic response. 2.Background of the Invention   Research on cellular responses to heat shock and other physiological stresses Stress-free cells, as well as cellular defenses against those attacks Key proteins involved in essential biochemical and immunological processes in The family has been identified. As their heat shock proteins, But hsp70, hsp90, gp96, and hsp100; these hsp families Perform different types of chaperoning functions. For example, hsp70 is cytoplasmic, nuclear, mito Localized in chondria or the endoplasmic reticulum, (Lindquist, S. et al., 1988, Ann. Rev. Geneti cstwenty two: 631-677) are involved in the presentation of antigens to cells of the immune system and are also It is also involved in the movement, folding and assembly of proteins in the vesicle. Likewise Hsp90 localized in the cytosol is involved in chaperoning and is present in the endoplasmic reticulum Gp96 is involved in antigen presentation (Strivastava, PK et al., 1991, Curr. Topics in M. icrobiology & Immun.167: 109-123). 2.1.Immunotherapy   In modern medicine, immunotherapy or vaccination can be used to control polio, tetanus, tuberculosis, Diseases such as pox, measles, hepatitis, etc. have been virtually eliminated. Using vaccinations The approach has exploited the ability of the immune system to prevent infections. Like protein Immunizations with various non-biological substances generally involve antibody response or CD4 + helper T cell response. Deriving answers (Raychaudhuri, S. and Morrow, WJW, 1993, Immunology Today,14 : 344-348). On the other hand, vaccination with biological substances such as live cells or infectious viruses Or, the infection generally leads to a CD8 + cytotoxic T lymphocyte (CTL) response. The CTL response is Important for protection against cancer, infectious viruses and bacteria. This reaches the CTL response The only way to achieve this is to use a live pathogen that is itself pathogenic. Therefore, there is a practical problem. This problem is generally associated with attenuated viruses and bacteria. By using strains or by killing all cells that can be used for vaccination. Is avoided. These strategies have been successful, but the use of attenuated strains is not. First, the attenuated pathogen is genetically recombined with the host DNA and transformed into a virulent strain. Risk. Therefore, non-living substances such as proteins in a special way There is a need for a method that can elicit a CD8 + CTL response by vaccination with.   In the age of tumor immunity, antigens on methylcholanthrene (MCA) -induced sarcoma The tumors do not detect these antigens in normal tissues of mice An experiment by Prehn and Main, which was shown to be unusual (Prehn, RT et al., 195 7, J. Natl. Cancer Inst.18: 769-778). This view suggests that MCA induction Tumor-specific resistance to the tumor is in its own host (i.e., the origin of the tumor (Klein). G. et al., 1960, CancerRes.20: 1561-1572).   In subsequent experiments, tumor-specific antigens may also have other chemical or physical carcinogens. It was also found in tumors induced by substances or spontaneous tumors (Klipke, M.L., 197 4, J. Natl. Cancer Inst. 53: 1333-1336; Vaage, J., 1968, Cancer Res.28: 2477-2483; Carswell, E.A. et al., 1970, J. Am. Natl. Cacer Inst.44: 1281-1288). In these studies, protective immunity against the growth of transplanted tumors was These antigens were also commonly referred to as "tumor-specific Target antigen "or" tumor-specific rejection antigen ". Several factors are induced Can have a significant effect on the immunogenicity of a given tumor, for example, the specificity of the carcinogen involved Sex, host immunocompetence and latency (Old, LJ et al., 1962, Ann. NY Acad. . Sci. 101: 80-106; Bartlett, G .; L., 1972, J.A. Natl, CancerInst.49: 493- 504).   Most, if not all, carcinogens are mutagens that can cause mutations Which leads to the expression of tumor-specific antigens (Ames, B.N., 1979, Science 204: 5). 87-593; Weisburger, J.H. et al., 1981, Science214: 401-407). Immunosuppressive development There are also cancer substances (Malmgren, R.A., et al., 1952, Proc. Soc. Exp. Biol. Med.79: 484- 488). Tumor immunogenicity and latency (time from tumor exposure to carcinogen exposure) Experimental evidence suggests that there is a certain opposite correlation between (Old, L.J. et al., 1962, Ann. N.Y. Acad. Sci.101: 80-106; and Bartlett, G.L., 1972. J. Natl. Cancer Inst.49: 493-504). Other studies report tumors that do not lead to rejection Despite revealing the presence of specific antigens, nevertheless, (Roitt, I., Brostoff, J. and Male, D., 1993, I mmunology, 3rd ed., Mosby, St. Louis, pp. 17.1-1-17.12). 2.2.Tumor-specific immunization of heat shock / stress proteins hsp70, hsp90 and gp96 Epidemiological   Srivastava and colleagues respond to methylcholanthrene-induced sarcomas in inbred mice. (1988, Immunol. Today9: 78-83). In these studies The molecule that causes each of these tumors to have different immunogenicity is a 96kDa cell surface. To be identified as surface glycoprotein (gp96) and intracellular protein of 84-86 kDa (Srivastava, PK et al., 1986, Proc. Natl. Acad. Sci. USA83: 3407- 3411; Ullrich, S.J. et al., 1986, Proc. Natl. Acad. Sci. USA83: 3121-3125). Immunization of mice with gp96 or p84 / 86 isolated from a particular tumor Was immunized against that particular tumor, but against an antigenically different tumor Was not immunized. of the genes encoding gp96 and p84 / 86 Isolation and characterization showed significant homology between them, gp96 and p84 / 86 is the ER and cytosolic equivalent of the same heat shock protein, respectively (Srivastava, PK et al., 1988, Immunogenetics).28: 205-207 Srivastava, P.K. et al., 1991, Curr. Top. Microbiol. Immunol.167: 109-123) . In addition, hsp70 elicits immunity against the tumor from which it was Do not elicit immunity to different tumors. However, Hsp70 depleted of peptide was found to lose its immunogenic activity (Udono, M. et al. And Srivastava, P.K., 1993, J.M. Exp. Med.178: 1391-1396). These observations However, these heat shock proteins are not immunogenic themselves, but Suggests that it is a carrier of antigenic peptide that elicits specific immunity against (Srivastava, PK, 1993, Adv. Cancer Res.62: 153-177). 2.3.Pathological biology of cancer   Cancer is primarily an increase in the number of abnormal cells from a given normal tissue. Invasion of adjacent tissues by these abnormal cells, and to local lymph glands and other parts Characterized by lymphatic or hematogenous spread (metastasis) of malignant cells in . From clinical data and molecular biology studies, cancer has progressed to neoplasia under certain conditions. It is shown to be a multi-step process starting from a small pre-tumor change that can be performed I have.   Precancerous abnormal cell growth is exemplified by hyperplasia, dysplasia, or, most strictly, dysplasia. (For a review of such abnormal growth states, see Robbins and Angell, 197. 6, Basic Pathology, 2nd edition, W.B. See Saunders Co., Philadelphia, pp.68-79 Illuminated). Hyperplasia occurs in tissues or without significant structural or functional changes. Is a form of controlled cell proliferation involving an increase in the number of cells in an organ. Just one example Thus, endometrial hyperplasia often precedes endometrial cancer. There is a metamorphosis 1 A system in which one type of adult cell or a fully differentiated cell is replaced by another type of adult cell Controlled form of cell growth. Metaplasia affects epithelial or connective tissue cells Can occur. Exceptional metaplasia involves a somewhat disorderly metaplastic epithelium. Dysplasia , Often a precursor to cancer, found primarily in the epithelium, The most disorderly form of non-neoplastic cell growth, loss of individual cell homogeneity and With loss of structural orientation of the cells. Dysplastic cells are often abnormally large and strongly stained It has a colored core and exhibits polymorphism. Dysplasia is present with chronic irritation or inflammation Characteristically in the anatomy, often found in the neck, respiratory tract, oral cavity and gall bladder .   Neoplastic lesions are clonally generated, especially if the tumor cells Escalate the ability to invade, proliferate, metastasize, and heterogeneize (Roitt, I., Brostoff, J and Kale, D., 1993, Immunology, No. 3. Edition, Mosby, St. Louis, pps. 17.1-1-17.12). 3.Summary of the Invention   The present invention relates to enhancing the immune capacity of a host and the activity of immune effector cells. , Pharmaceutical compositions and methods for preventing and treating cancer and / or infectious diseases And a kit. The pharmaceutical composition of the present invention binds non-covalently to an exogenous antigen molecule. It contains a complex of hsps that are joined together. Exogenous antigen molecules are associated with hsps in vivo. This is different from a peptide that forms a complex and is co-purified with hsp. Extrinsic An antigenic molecule is an antigen / immunogen or an antigenic / immunogenic fragment or derivative thereof. You. Such antigenic molecules can be selected from those known in the art. Antibodies or MHC can also be obtained by standard immunoassays known in the art. Depending on the ability to bind to a molecule (antigenic) or generate an immune response (immunogenicity) Assay. This antigen molecule can be used in vitro before administration to patients. Non-covalently bound to p. In the treatment or prevention of cancer, antigen molecules Molecules that elicit an immune response against them, such as tumor-specific antigens or tumor-associated Antigen, preferably human. Antigen molecules for treating or preventing infectious diseases Are molecules that induce an immune response against infectious agents, such as viruses, It is preferably an antigen of a pathogen that infects humans, such as bacteria, fungi and parasites. Special In certain embodiments, a pharmaceutical composition of the present invention comprises a single antigen as well as one or more antigens. Hsp complexed with a whole cocktail of antigens or antigens (eg, cancer-specific) . Preferably, the patient is human and the hsp is Human hsp. The hsps in the complex can be autologous or allogeneic to the patient. You.   Certain compositions of the invention and their properties are described in the following sections and subsections. Preferably , Hsp and a complex of antigen molecules can be expressed by hsp70, hsp90, gp96, or a combination thereof. Including.   In another embodiment, the pharmaceutical composition further comprises an effective amount of a biological response modifier. No. Biological response modifiers include interferon-α (IFN-α), IFN-γ, Tarleukin-2 (IL-2), IL-4, IL-6, tumor necrosis factor (TNF), or other sites Include, but are not limited to, caine growth factor. 4.BRIEF DESCRIPTION OF THE FIGURES   FIG. Effect of administration of hsp70 complexed with ovalbumin, or EG7 cells Cell line (expressing ovalbumin antigen) or EL4 cell line (for ovalbumin antigen) T cell cytotoxicity against (negative).   Figure 1A: Two mice from each group were tested for a) control vehicle (squares); b) ovalbumin. Only (plus sign); c) hsp70 only (triangle); or d) exempt from hsp70-ovalbumin complex Quarreled. T cells recovered from immunized mice were EG7 cells (FIG. 1A) or EL4 cells (FIG. 1B) Were tested for cytotoxicity. The result is the hsp70-ovalbumin complex The body has more cytotoxic T lymphocytes than ovalbumin alone or hsp70 alone (Figure 1A). It is a much better reagent in inducing a response. The T cells There was no response in the presence of EL4 cells lacking ovalbumin antigen. 5.Detailed description of the invention   Compositions for prevention and treatment of primary and metastatic cancers and / or infectious diseases And method. The present invention relates to hsps non-covalently linked to an exogenous antigen molecule. A pharmaceutical composition is provided.   This exogenous antigen molecule is endogenously complexed with hsp in vivo and purified with hsp Different from the peptide to be used. This exogenous antigen molecule may be an antigen / immunogen or antigenic / immune Epidemiological fragments or derivatives thereof. Such antigenic molecules are publicly available in the art. Can be selected from those known or have the ability to bind to an antibody or MHC molecule (anti- (Immunogenicity) or the ability to generate an immune response (immunogenicity). It can be measured by an immunoassay. The antigen molecule is administered in v It is non-covalently complexed with hsp by itro. Antigen molecules for treating or preventing cancer , A molecule that induces an immune response to cancer, such as a tumor-specific antigen, or a tumor. Tumor binding antigens, preferably antigens of human tumors. Antigen for the treatment or prevention of infectious diseases A molecule is a molecule that elicits an immune response against an infectious agent, for example, a virus. , Bacteria, fungi, parasites, etc., preferably pathogens that infect humans . In certain embodiments, the pharmaceutical compositions of the present invention comprise a single antigen as well as 1 Hsps complexed with the above antigens or with a cocktail of all antigens (e.g., cancer specific). No. Preferably, the loyal is human and the hsp is a human hsp. Hsp in the complex Can be self- or allogeneic to the patient.   The method of the invention provides for an effective amount of the conjugate (the conjugate is essentially non-covalent to the exogenous antigen molecule). Comprising a covalently bound hsp). Include methods of enhancing an immune response in an individual in whom treatment or prevention of cancer is desired. hsps and / or antigenic molecules may be from individuals or others, or from individuals or other sources. It can be isolated by recombinant production methods using a loaned hsp. Used for complexing with hsp Exogenous antigens and their fragments and their derivatives (peptides and non-peptides) Both) are those known in the art, as well as the ability to bind to an antibody or MHC molecule (antigen ) Or the ability to generate an immune response (immunogenicity) by standard immunoassays known in the art. Can be selected from those easily identified by the method.   The hsp of the present invention that can be used includes only hsp70, hsp90, gp96 or a combination thereof. But not limited thereto. Preferably, the hsp is a human hsp You.   Heat shock proteins (also known as stress proteins) useful in the practice of the present invention. Select) from any cellular protein that meets any of the following criteria: be able to. This is because when cells are exposed to stressful stimuli, An increasing concentration of a protein that binds to other proteins or peptides Can be adenosine triphosphate (ATP) or low pH Can release the bound protein or peptide in the presence of It can be combined with any cellular protein having any of the above properties with at least 3 It is a protein showing 5% homology.   The first stress proteins identified were heat shock proteins (hsp) is there. As the name implies, hsp is synthesized by cells in response to heat shock. Is done. To date, three large families of hsp have been identified based on molecular weight. Have been. This family is called hsp60, hsp70, hsp90, The numbers reflect the approximate molecular weight (kilodalton) of the stress protein. This Many members of these families respond to other stressful stimuli (eg, nutrition Deficiency, metabolic destruction, oxygen radicals, and infection by intracellular pathogens. (Welch, May 1993, Scientific American 56-64; Young, 1990, Annu. Rev. Immunol. 8: 401-420; Craig, 1993, Science 260: 1902-1903; Gething et al., 1992, Nature 355: 33-45; And Lindquist et al., 1988, Annu. Rev. See Genetics 22: 631-677, These disclosures are incorporated herein by reference). These three files Hsp / stress proteins belonging to all of Millies can be used in the practice of the present invention. It is thought that it can come.   Major hsps accumulate at high levels in stressed cells, It is present at low to moderate levels in cells that have not undergone radiation. For example, inductive Hsp70 in mammals with high In recipient cells, it becomes one of the most actively synthesized proteins (Welch et al., 1985). , J. et al. Cell. Biol. 101: 1198-1211). In contrast, hsp90 and hsp90 60 proteins are normal in most, but not all, mammalian cells. And is further induced by heat (Lai et al., 1984, Mol. Cell. Biol. 4: 2802-10; van Bergen en Henegouwen et al., 1987, Genes Dev. 1: 525-31).   Heat shock proteins are among the most highly conserved proteins in existence . For example, hsp70 from DnaK, E. coli is the hsp70 protein from excoriate Has about 50% amino acid sequence identity with protein (Bardwell et al., 1984, p. roc.Natl.Acad.Sci. 81: 848-852). Hsp60 family and hsp90 family Indicates similarly high levels of intra-family conservation (Hickey et al., 1989, Mol. Cell. Biol. . 9: 2615-2626; Jindal, 1989, Mol. Cell. Biol. 9: 2279-2283). In addition, hsp60 The family, hsp70 family and hsp90 family are, for example, greater than 35% Proteins related to stress proteins in sequences with different amino acid identity However, it was found that its expression level was not changed by stress. Therefore As used herein, the definition of stress protein is that expression levels in cells At least three members of the family that are enhanced in response to tress-filled stimuli 35% to 55%, preferably 55% to 75%, and most preferably 75% to 85% amino acids. Other proteins, mutant proteins, their analogs, And variants. Members belonging to these three families Purification of the tress protein is described below.   As an immunogenic complex of the hsp of the present invention and an exogenous antigen molecule, hsp and a mammal Complex containing exogenous antigen molecules that can induce an immune response in . This antigen molecule associates non-covalently with hsp. Preferred complexes are tamper Hsp60, hsp70, or hsp90 non-covalently bound to a cytoplasmic antigen. specific In embodiments, the complex is in the endoplasmic reticulum of a eukaryotic cell and Includes an hsp called gp96, which is related to hsp90.   Although the hsps may be allogeneic to the patient, in a preferred embodiment, the hsps are Be (self-derived) to the patient given. hsp and / or antigen molecule is a natural source Can be purified, chemically synthesized, or recombinantly produced. The present invention relates to an experimental tumor model. The optimal dosage of hsp non-covalently attached to the peptide conjugate with Provides a method for measuring dosages for human cancer immunotherapy by extrapolating . Specifically, scaling factors that do not exceed 50 times the estimated effective dose in animals It is used as an optimal formulation for cancer immunotherapy or vaccination of human subjects.   The present invention enhances the immune competence of a host individual and provides specificity against infectious pathogens. Hsp-antigenicity elicits immunity or an immune response against pre-neoplastic and neoplastic cells A composition comprising a molecular complex is provided. Therapeutic regimens and pharmaceutical compositions of the invention The objects are described below. These compositions prevent the onset and progression of infections and Prevents the development of tumor cells, and such compositions are useful in immunotherapy of infectious diseases and cancer. To inhibit tumor cell growth and progression, indicating that specific immunity can be induced in available.   The conjugates of the present invention may be used to induce an inflammatory response at the tumor site, and ultimately Can be used to reduce tumor burden in cancer patients. Exogenous antigen molecule Human sarcomas and cancers that can be treated with a complex of hsps non-covalently attached to Carcinomas, but are not limited to these.   Accordingly, the present invention stimulates the immune competence of a host individual and enhances pre-neoplastic cells and / or Administering to the individual a composition that elicits specific immunity against the tumor cells. Provided are methods for preventing and treating cancer. "Preneoplastic" as used herein Cells refer to cells that are in the transition from normal morphology to tumor morphology; Evidence is increasingly supported by molecular biology studies, suggesting that pre-cancerous It shows that it progresses. Non-tumor cell growth is usually hyperplastic, metaplastic, or minimal. And especially consists of dysplasia (for a review of such abnormal growth states, see Robbi ns and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philade lphia, pp. 68-79).   The therapeutic regimen and pharmaceutical composition of the invention may further comprise an immune response enhancer or Without limitation, cytokines IFN-α, IFN-γ, IL-2, IL-4, IL-6, TNF, Or with cytokines, including other cytokines that affect immune cells Can be used. According to this aspect of the invention, the complex between the hsp and the antigen molecule comprises 1 These may be administered in combination therapy with these cytokines.   The present invention further increases the risk of cancer due to family history or environmental risk factors For administration of hsp-antigen molecules to an individual.   A composition comprising a hsp non-covalently bound to an exogenous antigen molecule is provided by a complexed antigen. Trigger an effective specific immune response against the molecule (and not against the hsp) To administer.   In a preferred embodiment, hsp70, hsp90, and / or gp96 are exogenous antigens. Non-covalently complexed with the molecule.   According to the methods described herein, the exogenous antigen molecule can be immunogenic or antigenic. Protein, or other molecule, or immunogenic / antigenic fragment or derivative thereof Body. For example, exogenous immunogenic molecules include but are not limited to different tumor characteristics Differentially translatable antigens (e.g., tyrosinase, gp100, melan-A, gp75, mucin, etc.) And, but not limited to, type I immunodeficiency virus (HIV-I), type II human immunity Deficiency virus (HIV-II), hepatitis A virus, hepatitis B virus, hepatitis C virus Virus, influenza virus, varicella-zoster virus, adenovirus, type I Herpes simplex virus (HSV-I), herpes simplex virus type II (HSV-II), cowpox virus Rus, Rhinovirus, Econovirus, Rotavirus, Respiratory syncytial virus, Papiro Virus, papovavirus, cytomegalovirus, echinovirus, Voyurus, Hantavirus, Coxsackievirus, Mumpsvirus, Measles Viral antigens, including viral, rubella and poliovirus proteins Is mentioned.   In certain embodiments, the cancer (e.g., tumor) or infectious pathogen (e.g., Antigens (e.g., irs antigens, bacterial antigens) are chemically synthesized by purification from natural sources. Alternatively, it can be obtained by recombination and in vitro procedures as described below. Can be non-covalently complexed to hsp. 5.1.Hsp Purification   In certain embodiments, the hsp portion of the hsp-antigen molecule complex is purified from the cell It is desired that an exemplary purification procedure as described in Sections 5.1.1. can be used to purify the hsp-peptide complex, where hsp is used in the presence of ATP or Can be purified from the endogenous hsp-peptide complex at low pH, followed by It can be conjugated to a causative antigen molecule in vitro. Description of tumor cells However, the protocols described herein below are compatible with any eukaryotic cell, for example, Immortalized eukaryote infected with tissue, isolated cells, or preselected intracellular pathogen It can be used to isolate hsps from cell lines, tumor cells or tumor lines.   Instead of isolating the native hsp from the cells described in sections 5.1.1. It can be produced chemically or recombinantly. 5.1.1.hsp70- Preparation and purification of peptide conjugate   Purification of the hsp70-peptide complex has been described previously. See, for example, Udono et al., 1993. J. Exp. Med. See 178: 1391-1396. Uses that are given as examples rather than limitations The operations that can be used are as follows.   First, tumor cells were treated with 5 mM sodium phosphate buffer, pH 7, 150 mM NaCl, 2 mM Ca. Cl_Two, 2mM MgCl_TwoAnd 1 mM phenylmethylsulfonyl fluoride (PMSF) Resuspend in 3 volumes of 1 × lysis buffer. Second,> 99% of the cells were Sonicate the pellet on ice until measured and melted. Alternative methods of sonication The cells may then be lysed by mechanical shearing, which approach Resuspend in 30 mM sodium bicarbonate pH 7.5, 1 mM PMSF and incubate on ice for 20 minutes. Incubate and then in a dounce homogenizer until> 95% of cells are lysed And make it uniform.   The lysate is then centrifuged at 1,000 g for 10 minutes to remove unbroken cells, nuclei and Remove other cell debris. The resulting supernatant is weighed at 100,000 g for 90 minutes. Centrifuge again and collect the supernatant, then 2 mM Ca²⁺And 2 mM Mg²⁺Phosphoric acid containing Mix with Con A Sepharose equilibrated in buffered saline (PBS). Cells are mechanically sheared When lysed, remove the supernatant to an equal volume of 2x lysis buffer before mixing with Con A Sepharose. Dilute with liquid. The supernatant is then ligated to Con A Sepharose at 4 ° C for 2-3 hours. Combine. Collect the unbound material, 10 mM Tris-acetic acid pH 7.5, 0.1 mM EDTA, 10 mM Dialysis against M NaCl, 1 mM PMSF for 36 hours (3 times, 100 volumes each time). Next Centrifuge dialysate at 17,000 rpm (Sorvall SS34 rotor) for 20 minutes You. Next, the obtained supernatant was collected and 20 mM Tris-acetic acid pH 7.5, 20 mM NaCl, 0.1 mM ED Negative load on Mono Q FPLC column equilibrated in TA and 15 mM 2-mercaptoethanol Load. The column was then developed with a 20 mM to 500 mM NaCl gradient and the eluted fractions were Fractionation by sodium sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Anti-hsp70 antibody (eg, from clone N27F3-4 from StressGen) And characterized by immunoblotting.   The fraction that is strongly immunoreactive with the anti-hsp70 antibody is pooled, and the hsp70-peptide complex is sulfated. Precipitate with ammonium, specifically 50% -70% ammonium sulfate cut. next The resulting precipitate was collected by centrifugation at 17,000 rpm (SS34 Sorvall rotor), Wash with 0% ammonium sulfate. Next, the washed precipitate is solubilized and residual sulfuric acid is added. Ammonium by gel filtration through a Sephadex® G25 column (Pharmacia). Removed. If necessary, the hsp70 preparation thus obtained may be combined with Mono Q as described above. It can be purified again by FPLC column.   Using this method, the hsp70-peptide complex was purified to apparent homogeneity. obtain. Typically, 1 mg of the hsp70-peptide conjugate can be purified from 1 g of cells / tissue.   The present invention further discloses a novel and rapid method for purifying hsp70. This improved method In this case, a method using ATP (for example, ATP-agarose) attached to a solid base layer is used. Use column chromatography. The yield of hsp70 is significantly increased and the purity is high. I think it is. For example, but not limited to, ATP-agarose chromatography Purification of hsp70 by chromatography is performed as follows. Meth A sarcoma cells ( (500 million cells) in a hypotonic buffer, and the resulting lysate is centrifuged. (100,000 × g, 4 ° C., 90 minutes). The supernatant is divided into two parts, and ADP-A The solution was applied to a galose column or an ATP-agarose column. Loosen these columns Washed with buffer and eluted with 3 mM ADP or 3 mM ATP, respectively. Elution The fractions thus analyzed were analyzed by SDS-PAGE. In both cases, h looks uniform A preparation of sp70 was obtained. However, each preparation was tested for the presence or absence of the peptide. ADP-binding / eluting hsp70 preparation was accompanied by peptide However, the ATP-binding / eluting hsp70 preparation does not I understood. 5.1.2.hsp90- Preparation and purification of peptide conjugate   The operations that may be used, given by way of example and not limitation, are as follows.   First, tumor cells were treated with 5 mM sodium phosphate buffer (pH 7), 150 mM NaCl, 2 mM C aCl_Two, 2mM MgCl_TwoAnd 1 mM phenylmethylsulfonyl fluoride (PMSF) Resuspend in 3 volumes of 1 × lysis buffer. Next,> 99% of the cells are examined by microscopy Sonicate the pellet on ice until melted as determined by. Sonication Alternatively, the cells may be lysed by mechanical shear, an approach that Typically resuspended in 30 mM sodium bicarbonate pH 7.5, 1 mM PMSF and placed on ice for 20 minutes. Incubate for 1 minute, then dounce homogenize until> 95% of cells are lysed In the oven.   The lysate is then centrifuged at 1,000 g for 10 minutes to remove non-destructed cells, nuclei and And other cell debris. The resulting supernatant was weighed at 100,000 g for 90 minutes. And centrifuged again to collect the supernatant, then 2 mM Ca²⁺And 2 mM Mg²⁺PBS containing Mix with Con A Sepharose equilibrated in. When cells are lysed by mechanical shear Dilute supernatant with an equal volume of 2x lysis buffer before mixing with Con A Sepharose . The supernatant is then bound to Con A Sepharose at 4 ° C. for 2-3 hours. Conclusion Collect substances that cannot be combined, 10 mM Tris-acetic acid pH 7.5, 0.1 mM EDTA, 10 mM NaCl, 1 mM Dialyze PMSF for 36 hours (3 times, 100 volumes each time). Next, the dialysate is Centrifuge at 000 rpm (Sorvall SS34 rotor) for 20 minutes. Then got Collect the supernatant and load onto a Mono Q FPLC column equilibrated with lysis buffer. Next The protein is eluted with a salt gradient from 200 mM to 600 mM NaCl.   Next, the eluted fraction was fractionated by SDS-PAGE, and an anti-hsp90 antibody such as 3G3 (Af finity Bioreagents) using immunoblotting of the fraction containing the hsp90-peptide complex. Identify by lotting. Using this procedure, the hsp90-peptide complex is It can be purified until it is clearly homogeneous. Typically, the hsp90-peptide complex 150-20 0 μg can be purified from 1 g of cells / tissue. 5.1.3.gp96- Preparation and purification of peptide conjugate   The operations that may be used, given by way of example and not limitation, are as follows.   Tumor pellets were prepared from 30 mM sodium bicarbonate buffer (pH 7.5) and 1 mM PMSF. Resuspend in 3 volumes of buffer and swell cells on ice for 20 minutes. Then> 95% Cell pellet on ice in dounce homogenizer until cells are lysed Make uniform (appropriate clearance of homogenizer depends on each cell type) Will change).   The lysate is centrifuged at 1,000 g for 10 minutes to remove non-destructed cells, nuclei, and Remove other debris. Next, the supernatant from this rapid centrifugal separation process is 90 minutes at 100,000 g. Separate again quickly over the interval. gp96-peptide complex containing 100,000 pellets or Can be purified from the supernatant.   If purifying from the supernatant, dilute the supernatant with the same volume of 2X cell lysis buffer, then At 2 ° C., the supernatant is²⁺And 2 mM Mg²⁺In equilibrium with PBS containing Mix with some Con A Sepharose for 2-3 hours. After this, the slurry is applied to the column. Fill and wash with 1X cell lysis buffer until OD280 drops to baseline . Next, 2mM Ca²⁺And 2 mM Mg²⁺1/3 of the column bed volume dissolved in PBS containing Wash the mosquito / ram with a volume of 10% α-methyl mannoside (α-MM) and use a piece of parafilm Seal the column with and incubate at 37 ° C for 15 minutes. Next, let the column cool to room temperature. And remove the parafilm from the bottom of the column. 5 times the column volume of α- MM buffer is supplied to the column and the eluate is analyzed by SDS-PAGE. Typically obtained The purity of the substance is about 60% -95%, which depends on the cell type and the Depends on tissue to lysis buffer ratio. Next, the sample was diluted with 5 mM sodium phosphate. Supply to Mono Q FPLC column (Pharmacia) in equilibrium with buffer solution (pH 7) containing I do. The protein is then eluted from the column using a 0M to 1M NaCl gradient. Elutes the gp96 fraction in the range of 400 mM to 550 mM NaCl.   However, by utilizing the two additional steps alone or in combination, Modify this procedure to ensure that an apparently uniform gp96 peptide complex is formed It may be. In one optional step, ammonium sulfate is added before the Con A purification step. Precipitate and, optionally, after the Con A purification step and before the Mono Q FPLC step Perform DEAE-Sepharose purification.   In the first optional step, the supernatant obtained from the 100,000 g centrifugation step Add ammonium to a final concentration of 50% ammonium sulfate. To Gently agitate the solution in a beaker placed in a tray with ice water. While slowly adding ammonium sulfate. Approximately 1/2 hour at 4 ° C Stir for ~ 12 hours and centrifuge the resulting solution at 6,000 rpm (Sor vall SS34 rotor). Take out the supernatant obtained in this step, and remove the aluminum sulfate. To a 70% saturated solution of aluminum sulfate, and centrifuged at 6,000 rpm (Sorval l Use SS34 rotor). The pellets obtained in this step are collected and 70% ammonium sulfate The pellet is washed by suspending in PBS containing PBS. 6,000 rpm Centrifuge (using a Sorvall SS34 rotor) at 2 mM Ca²⁺And Mg²⁺PBS containing Dissolve the pellet in. Perform simple centrifugation at 15,000 rpm to remove undissolved material. Remove (using Sorvall SS34 rotor). Next, mix this solution with Con A Sepharose. If so, repeat the procedure described above.   In a second optional step, the gp96-containing fractions eluted from the Con A column were pooled. By dialysis, or preferably by buffer exchange using a Sephadex G25 column. The buffer is exchanged for 5 mM sodium phosphate buffer (pH 7, 300 mM NaCl). Buffer After exchange, the solution is pre-equilibrated with 5 mM sodium phosphate buffer (pH 7, 300 mM NaCl). Mix with DEAE-Sepharose in state. Add the protein solution and beads to Mix gently while pouring onto the column. Next, the absorbance at 280 nM was Using a 5 mM sodium phosphate buffer (pH 7, 300 mM NaCl) until the Wash the ram. Next, use 5 volumes of 5 mM sodium phosphate (pH 7, 700 mM NaCl). To elute the bound protein from the column. Pool protein containing fractions , Diluted with 5 mM sodium phosphate buffer (pH 7) to reduce salt concentration to 175 mM. Next, the obtained substance was mixed with Mono mM in equilibrium with 5 mM sodium phosphate buffer (pH 7).  Apply to a Q FPLC column (Pharmacia) and apply a Mono Q FPLC column (Ph armacia).   However, those skilled in the art will appreciate that the optional second step can be incorporated into the purification protocol. It is believed that the benefits can be assessed by routine experimentation. In addition, any of the optional steps It is believed that the benefits of the addition will depend on the source of the starting materials.   When isolating the gp96 fraction from a 100,000 g pellet, 1% deoxycholate or 1% 5 volumes of PBS containing any of octylglucopyranoside (but Mg²⁺You And Ca²⁺(Not included) and incubate on ice for 1 hour I do. The suspension was centrifuged at 20,000 g for 30 minutes, and the resulting supernatant was Several variants of PBS (again, Mg²⁺And Ca²⁺(Not included) And remove the cleaning agent. Centrifuge the dialysate at 100,000g for 90 minutes and collect the supernatant Then, calcium and magnesium were added to the supernatant to a final concentration of 2 mM each. Degree. Next, the sample is collected using either the unmodified or improved methods. Purify and isolate gp96-peptide from 100,000 g supernatant.   Using this procedure, it is possible to purify the gp96-peptide until it is apparently homogeneous. it can. About 10-20 μg of gp96 is isolated from 1 g cells / tissue. 5.2Exogenous antigen molecule 5.2.1.Peptides from the MHC complex   Potentially immunogenic peptides are derived from MHC-peptide conjugates in the art. It has been found that it may be eluted using well-known techniques (Falk, K. et al., 19 90 Nature 348: 248-251; Elliott, T. et al., 1990 Nature 348: 195-197; Falk, K Et al., 1991, Nature 351: 290-296). Once isolated, each antigen pair The amino acid sequence of the peptide can be determined using conventional amino acid sequencing techniques. Wear. Subsequently, such antigen molecules are produced by chemical synthesis or recombinant methods. Can be purified and purified to form a complex with hsp in vitro. You.   Thus, potentially immunogenic or antigenic peptides can be converted to exogenous MHC-pepti From the complex and then form a complex with hsp in vitro Thus, it can be used as an exogenous antigen molecule. Peptide and / or Specific examples of a protocol for isolating an antigen component from an MHC complex are described below. Section 5.2.1.1. 5.2.1.1.MHC- Peptides derived from peptide complexes   The isolation of promising immune peptides from MHC molecules is well known in the art and , Not detailed herein (Falk, et al., 1990, Nature 348: 248-251; Rotzsc he, et al., 1990, Nature 348: 252-254; Elliott, et al., 1990, Nature 348: 1. 91-197; Falk, et al., 1991, Nature 351: 290-296; Demotz, et al., 1989, Na ture 343: 682-684; Rotzsche, et al., 1990, Science 249: 2. 83-287). These disclosures are incorporated herein by reference. Shall be.   Briefly, MHC-peptide conjugates can be isolated by conventional immunoaffinity methods. Can be isolated. In this case, about 0.1% TFA is contained in acetonitrile. Incubating the complex in the presence of Can be eluted from As described above, the eluted peptide was in reversed phase. Separation and purification can be performed by HPLC.   The determination of the amino acid sequence of the eluted peptide can be performed manually or by well-known techniques in the art. This can be done by any of the automated amino acid sequencing methods. Promising defense pepti After the amino acid sequence of the peptide has been determined, conventional peptide synthesis or The peptide can be synthesized in the desired amount using other known protocols. .   A peptide having the same amino acid sequence as the peptide isolated as described above, Merrifield, 1963, J.M. Am. Chem. Soc., 85: 2149. It can be synthesized by peptide synthesis. N-α-protection with protected side chains during synthesis Amino acids are used to grow C-terminally linked polypeptide chains and insoluble polymer chains. It is added little by little to the carrier (ie, polystyrene beads). N-α-deprotected amine Reaction of the amino group of carboxylic acid with a reagent such as dicyclohexylcarbodiimide. And the α-carboxy group of the N-α-protected amino acid activated by To synthesize a peptide. Free amino group bound to activated carboxyl group This produces a peptide bond. The most commonly used N-α-protecting groups are , Boc having an acid reaction activity and Fmoc having a base reaction activity.   Briefly, first, a C-terminal N-α-protected amino acid was To join. Next, the N-α-protecting group is removed. The deprotected α-amino group is Link to the activated α-carboxylate group of the N-α-protected amino acid. Desired pep This process is repeated until the tide is synthesized. Next, make the resulting peptide insoluble Cleavage from the polymer support and deprotection of the amino acid side chains. Protected peptide fragment By condensation, a longer peptide can be derived. For more information on appropriate chemistries, resins, protecting groups, protected amino acids, and reagents, see Since they are well known in the art, they will not be described in detail herein (Atherton, et al., 198). 9, Solid Phase Peptide Synthesis: A Practical Approach, IRL Press and Bo danszky, 1993, Peptide Chemistry, A Practical Textbook, 2nd Ed., Springer -See Verlag).   Purification of the resulting peptide was performed by gel permeation preparative chromatography, partition chromatography. Conventional methods such as chromatography and / or ion exchange chromatography It is performed using. Selection of the appropriate matrix and buffer is well known in the art. Since it is known, it will not be described in detail in this specification. 5.2.2Other exogenous antigen molecules   Antigen molecules that are not isolated from the MHC-peptide complex are also referred to as exogenous antigen molecules. Can be used. Antigen or its antigen for use as an antigen molecule The sex moiety is selected from those known in the art to form a complex with hsp. That can bind to the antibody by immunoassay (antigen Sex) or those that can produce an immune response (immunogenicity) it can.   To determine immunogenicity or antigenicity by detecting antibody binding, Various immunoassays well known in the art can be utilized. For example, LA Geoimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" Muno assay, immunoradiometric assay, gel diffusion sedimentation reaction, immunodiffusion Assays, in vivo immunoassays (e.g., colloidal gold, enzymes, or radio Isotope labeling), Western blot, immunoprecipitation, agglutination Assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, Competitive assays such as epifluorescence assays, protein A assays, and immunoelectrophoresis assays Assay systems and non-competitive assay systems, including but not limited to It is not something to be done. In one embodiment, detection of antibody binding comprises labeling on the primary antibody This is performed by detecting In another embodiment, a connection with a secondary antibody is provided. Detection of binding to reagents for binding or primary antibodies Thus, detection of the primary antibody is performed. In a further embodiment, the secondary antibody is labeled I do. Many means for detecting binding in immunoassays are known in the art. And it is considered that these can be used. To detect immunogenicity In one embodiment of the invention, standard methods (eg, in vitro cytotoxicity assays or Can be used to assay T cell-mediated responses in an in vivo delayed-type hypersensitivity assay.   Also, promising antigens or derivatives thereof for use as antigen molecules are Antigen contribution to neutralizing infectivity (the treatment or prevention of infection by these pathogens) Is desired) (Norrby, 1985, Summary, in Vaccines 85, Lerner, et al. (E ds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 38 8-389), type or group specificity, recognition by patient antisera or immune cells, And / or the appearance of protective effects of antisera or immune cells specific to the antibody. It can be recognized according to various criteria. Antigens are derived from any pathogen Pathogens include, but are not limited to, viruses, bacteria, fungi, Live animals and parasites are included. The term "pathogen" is not limiting None, but also includes intracellular pathogens. Intracellular pathogens that infect mammals are Can be present in the vesicles and cause disease in the body of mammals. Other than intracellular pathogen May also be used to form a complex with hsp . If you want to treat or prevent a disease caused by a pathogen, code for that antigen Epitopes are preferably between or between different isolates of the same pathogen. Occasionally, antigenic mutations may or may not be represented.   Preferably, where treatment or prevention of cancer is desired, a known cancer-specific antigen or Used are fragments or derivatives thereof. For example, such tumor-specific antigens Or KS1 / 4 whole carcinoma antigen (Perez and Walker, 1990, J. Imm unol. 142: 3662-3667; Bumal, 1988, Hybridoma 7 (4): 407-415); ovarian cancer antigen (CA1 25) (Yu, et al., 1991, Cancer Res. 51 (2): 466-475); (Tailer, et al., 1990, Nucl. Acids Res. 18 (16): 4928); prostate specific antigen ( Henttu and Vihko, 1989, Biochem. Biophys. Res. Comm. 160 (2): 903-910; Isra eli, et al., 1993, Cancer Res. 53: 227-2300); melanoma-associated antigen p97 (Estin, et al., 1989, 1J. Natl. Cancer In st. 81 (6): 445-446); melanoma antigen gp75 (Vijayasardahl, et al., 1990, JE). xp. Med. 17184): 1375-1380); High molecular weight melanoma antigen (Natali, et al., 1987) , Cancer 59: 55-63); and prostate-specific membrane antigens. It is not something to be done.   In certain embodiments, an antigen specific to a tumor or a fragment or derivative thereof is selected. To form a complex with hsp and then administer to the patient with the tumor.   Preferably, if treatment or prevention of a viral disease is desired, the known virus Molecules containing the epitope of the protein are used. For example, such antigenic epitopes Means hepatitis A virus, hepatitis B virus, hepatitis C virus, influenza Virus, varicella virus, adenovirus, herpes simplex virus type I (HSV-I) , Herpes simplex virus type II (HSV-II), rinderpest virus, rhinovirus, Irs, rotavirus, respiratory syncytial virus, papillomavirus, papovau Irus, cytomegalovirus, echinovirus, arbovirus, Hantawi Lus, coxsackie virus, mumps virus, measles virus, rubella virus Virus, poliovirus, human immunodeficiency virus type I (HIV-I), and human immunodeficiency virus It can be prepared from viruses such as whole virus type II (HIV-II). It is not limited.   Preferably, if the treatment or prevention of a bacterial infection is desired, the known bacterial Molecules containing pitopes are used. For example, such antigenic epitopes Such as Icobacteria, Rickettsia, Mycoplasma, Neisseria, Legionella, etc. It can be prepared from bacteria, but is not limited to these. If it is desired to treat or prevent a protozoan infection, preferably a known protozoan A molecule containing the epitope is used. For example, such protozoa are limited It doesn't mean that leishmania, kokzidio (kokzidio) a) and trypanosoma.   Preferably, if treatment or prevention of a parasitic infection is desired, the known parasitic Molecules containing the epitope are used. For example, such antigenic epitopes It may be derived from parasites such as chlamydia and rickettsia, It is not limited to them. 5.3.In vitro production of stress protein-antigen molecule complex   Utilizing a complex of hsp and a peptide that causes endogenous association with hsp in vivo In one embodiment, the complex of the hsp and the antigen molecule is produced in vitro. This As will be appreciated by those of skill in the art, either isolated by the procedure described above or chemically synthesized The synthesized or recombinantly produced peptides can be used in various naturally purified forms or Reconstituted in vitro with recombinant stress proteins, A combined stress protein-antigen molecule complex can be formed. In addition, external factors Sex antigens or antigen / immune fragments or derivatives thereof and stress proteins To form a non-covalent complex with the immunotherapeutic or prophylactic vaccine of the present invention. Can be used for chin. Non-covalent form of stress protein and antigen molecule Preferred specific protocols for forming the complex in vitro are described below. It is described in.   Prior to complex formation, hsps are pretreated with ATP or low pH to Eliminate peptides that may cause association. When using the ATP method, Levy, et Add apyranase as described in al., 1991, Cell 67: 265-274. This removes excess ATP from the preparation. If using low pH method, adjust pH Reagents are added and buffers are readjusted to neutral pH.   Antigen molecule (1 μg) and pretreated hsp (9 μg) were Mix in a molar ratio of about 5: 1. Next, at 4 ° C to 45 ° C, a suitable Combination buffer [eg 20 mM sodium phosphate (pH 7.2), 350 mM NaCl, 3 mM MgCl 2 Containing 1 mM and 1 mM phenylmethylsulfonyl fluoride (PMSF) Incubate for 15 minutes to 3 hours. Prepare the preparation with Centricon 10 assembly (Millipor e) Centrifuge to remove unbound peptide. Peptides and stress tampers Association with the protein can be assayed by SDS-PAGE. This method uses endogenous hs The peptide obtained by disassociation of the p-peptide complex is simply It is a preferred method for performing in vitro complex formation of the released peptides.   The present invention for forming a complex between hsp70 and an exogenous antigen molecule such as a protein In another preferred embodiment of the purification of 5 micrograms to 10 micrograms hsp together with equimolar amounts of antigen molecules at 37 ° C, 20 mM sodium phosphate pH 7.5. Volume consisting of 0.5 M NaCl, 3 mM MgCl2, and 1 mM ADP. Incubate for 1 hour in the solution of the bottle. This incubation mixture Further dilute with phosphate buffered saline to 1 ml.   In yet another embodiment of the present invention, a gp96 or hsp90 complex to a peptide A preferred method for producing 0.5 M NaCl and 3 nM MgCl_Two20mM phosphorus containing Sodium acid buffer, 5-20 minutes in a suitable buffer, such as a buffer containing pH 7.5, At 60-65 ° C, 5-10 micrograms of purified gp96 or hsp90 is equimolar or excess. Incubate with excess antigenic peptide. This incubation mix Allow the material to cool to room temperature and use one or more Centricon 10 assemblies ( The unbound peptide is removed by centrifugation using Lipore).   After complex formation, the resulting immunogenic stress protein-antigen molecule complex is In some cases, for example, using the mixed lymphocyte target cell assay (MLTC) described below, Can be measured with o. Once the immunogenic complexes are isolated, they Advantageously, in animal models using the preferred dosing protocols and excipients described below. Can be further characterized. 5.4.Determination of immunogenicity of stress protein-peptide complex   Purification of the purified stress protein-antigen molecule complex by any procedure Immunogenicity using a mixed lymphocyte target culture assay (MLTC) well-known in Assay.   As an example, the following method can be used, but the present invention It is not limited. Briefly, mice have stress protein-antigen components The child conjugate is injected subcutaneously. Other mice have other stress protein-peptide complexes Inject either the coalesced or all infected cells that serve as positive controls for this assay. Mau Inject 2 doses at 7-10 days. 10 days after the last immunization, remove the spleen Collect lymphocytes. Next, the phosphorus obtained from the dead cells expressing the complex of interest was obtained. It may be added to the papule and stimulated again in vitro.   For example, 8x10⁶  The immune spleen cells were 4x1 in 3 ml of RPMI medium containing 10% fetal bovine serum. 0^Four  Mitomycin C-treated or gamma-irradiated (5-10,000 rads) infected cells ( Or, if necessary, cells transfected with the appropriate gene). In some cases, a 33% secondary mixed lymphocyte culture supernatant was used as a source of T cell growth factor. May be contained in the medium (Glasebrook, et al., 1980, J. Exp. Med. 151: 876). To test the primary cytotoxic T cell response after immunization, spleen cells were You may culture without giving a stimulus. In one experiment, spleen cells from immunized mice were Vesicles are restimulated with cells of different antigenicity to determine the specificity of the cytotoxic T cell response You can also.   6 days later, 4 hours⁵¹Test cultures for cytotoxicity in a Cr-release assay ( Palladino, et al., 1987, Cancer Res. 47: 5074-5079, Blachere, et al., 19 93, J.S. Immunotherapy 14: 352-356). This assay uses mixed lymphocyte cultures. Adding nutrients to the target cell suspension results in different effector-to-target (E: T) ratios. (Usually 1: 1 to 40: 1). 200 mCi^5l1x10 for 1 hour at 37 ° C in medium containing Cr / ml⁶ The target cells are pre-labeled by incubating You. After labeling, the cells are washed three times. Perform each assay point (E: T ratio) three times, Spontaneous with control⁵¹Cr release (assay without added lymphocytes) and 100% release The outflow (cells lysed with detergent) is measured. Incubate the cell mixture for 4 hours After centrifugation, centrifuge at 200 xg for 5 minutes to pellet the cells. Release to supernatant Was done⁵¹The amount of Cr is measured with a gamma counter. The% cytotoxicity is determined by the following equation: Measure as cpm. (Test sample-spontaneous release cpm) ÷ (total surfactant release cpm-spontaneous release cpm)   To block the MHC class I cascade, K-44 hybridoma cells (anti-MH The concentrated hybridoma supernatant obtained from C class I hybridoma) was To a final concentration of 12.5%. 5.5.Formulation   The complex of the present invention (hsp non-covalently bound to an exogenous antigen molecule) is Pharmaceutical preparation for administration to mammals for the treatment of tumors and infectious diseases Things. About preferred doses, routes of administration and therapeutic regimens Is P. Srivastava's co-pending application “Heat Shock / Stress Tan Prevention and treatment of primary and metastatic neoplastic diseases and infectious diseases using proteins Compositions and Methods for the Prevention and Treatment of Primary and Metastatic Neoplastic Diseases and Infectio us Diseases with Heat Shock / Stress Proteins) (filed on the same day as the present application). And the specification of which is incorporated herein by reference in its entirety. For example, Pharmaceutical formulations scale from newly discovered estimation methods and animal doses to humans Of the complex between the antigen molecule and the heat shock / stress protein. The composition. Why are heat shock / stress proteins limited? But not hsp70, hsp90 and gp96, which alone or Used in combination. Specifically, h bound non-covalently to an antigen molecule Cross-species dose-response equivalents of human doses for sp were observed in mice. As a product of a single therapeutic ratio and a single scaling ratio not exceeding 50-fold increase Is done. In a preferred embodiment, hsp70- and / or gp96- administered to a human. The amount of the antigen molecule complex is about 10-600 μg, preferably 10-100 μg, most preferably Is in the range of about 25 μg, and is administered once a week for 4 to 6 weeks. This is a subcutaneous injection with a different application site. hsp90-antigen molecule complex Preferred amounts are in the range of 50-5,000 μg, preferably 100 μg.   A composition comprising a compound of the present invention, formulated in a compatible pharmaceutical carrier, may comprise a human sarcoma. And packaged and labeled for the treatment of adaptive tumors such as cancer Is also good. Human sarcomas and cancers include, for example, fibrosarcomas, myxosarcomas, liposarcomas, Chondrosarcoma, osteogenic sarcoma, chordoma, hemangiosarcoma, endothelial sarcoma, lymphatic sarcoma, lymphatic vessels Endothelial sarcoma, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, Pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma , Sebaceous carcinoma, papillary carcinoma, papillary carcinoma, cystoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma , Liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Bilums tumor, cervical cancer, sperm cancer Nest cancer, lung cancer, small cell lung cancer, bladder cancer, Epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, Pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblast Tumors, retinoblastoma; leukemias such as acute lymphocytic leukemia and acute myeloid leukemia Disease (myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia); chronic leukemia ( Chronic myeloid (granular cell) leukemia and chronic lymphocytic leukemia); and polycythemia vera Coccyemia, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Walden Strom macroglobulin, as well as heavy chain disease. Or appropriate It can be formulated and labeled for treatment of an infection.   If the conjugate is water-soluble, the conjugate is combined with a suitable buffer, for example, phosphate buffered saline. It may be formulated in saline or other physiologically compatible solutions. There If the obtained complex is poorly soluble in an aqueous solvent, the complex It may be formulated with an ionic surfactant or polyethylene glycol. Obedience Thus, the compound and its physiologically acceptable solvate can be Inhalation or spraying or oral, buccal, parenteral, rectal administration Alternatively, in the case of tumors, they may be formulated for administration by direct injection into solid tumors. No.   For oral administration, the pharmaceutical preparation is in liquid form, for example, as a solution, syrup or suspension. Liquid or reconstituted with water or other suitable vehicle before use It may be a drug product for Such liquid preparations are pharmaceutically acceptable. It can be prepared by conventional methods using the additives used. Such an addition As a substance, a suspending agent (for example, sorbitol syrup, cellulose derivative or Hydrogenated edible fats), emulsifiers (eg, lecithin or acacia), non-aqueous vehicles (Eg, almond oil, oily esters or fractionated vegetable oils) and preservatives (eg, For example, methyl or propyl p-hydroxybenzoate or sorbic acid) No. The pharmaceutical composition can be in the form of, for example, a tablet or capsule. And can be prepared by conventional methods using pharmaceutically acceptable excipients. . Such excipients include binders (eg, pregelatinized maize starch). , Polyvinylpyrrolidone or hydroxypropylmethylcellulose), Fillers (eg lactose, microcrystalline cellulose or calcium hydrogen phosphate), lubrication Agent (Eg, magnesium stearate, talc or silica), disintegrants (eg, , Potato starch or sodium starch glycolat e)) or wetting agents (eg sodium lauryl sulfonate). Tablets may be coated by methods well known in the art.   Preparations for oral administration should be suitably formulated to give controlled release of the active compound It may be.   For oral mucosal administration, the compositions may be formulated in conventional manner as tablets or drops. May be used.   For administration by inhalation, the compounds used according to the invention are suitable propellants Suitable in the form of an aerosol spray from a compression pack or nebulizer, with the use of Delivered to Suitable propellants include, for example, dichlorodifluoromethane, Lichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other Suitable gases. In case of compressed aerosol, deliver a metered amount The dosage unit can be determined by installing a valve for use. For example, Gelatin capsules or cartridges used in inhalers or sprayers Contains a powdered mixture of the compounds and a suitable powder base such as lactose or starch. May be formulated.   The compound may be administered parenterally by injection, for example, by bolus injection or continuous infusion. Can be formulated for parenteral administration. Injectable preparations may be presented in unit dosage form, e.g. For example, it can be placed in an ampoule or multi-dose container and provided with a preservative. Wear. Compositions include suspensions, solutions or emulsions in oily or aqueous vehicles, etc. Formulation agents such as suspending, stabilizing and / or dispersing agents May be included. Alternatively, the active ingredient may be prepared prior to use, for example, aseptically and pyrogenically. It may be in powder form for reconstitution with a suitable vehicle such as water-free.   The compound may also be a conventional suppository such as, for example, cocoa butter or other glycerides. Formulation as a rectal composition such as a suppository or retention enema containing a base Can also.   In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. This Such long-acting formulations may be implanted (eg, subcutaneously or intramuscularly) or injected intramuscularly. May be administered. Thus, for example, compounds can be converted to suitable macromolecules or hydrophobic Using a substance (eg, as an emulsion in an acceptable oil) or an ion exchange resin It may be formulated or as a slowly soluble derivative, for example, loosely It may be formulated as a salt dissolving in crab. Liposomes and emulsions are hydrophilic drugs It is a well-known example of a delivery vehicle or carrier for a product.   The composition may, if desired, be packaged or one or more units containing the active ingredient. It may be provided in a dispenser device containing the dosage form. The pack is an example For example, including metal or plastic foil, such as blister packs Good. The pack or dispenser device should be accompanied by instructions for administration. Is also good.   The present invention also provides kits for performing the therapeutic regimen of the present invention. Such kits contain a therapeutically or prophylactically effective amount of a pharmaceutical in one or more containers. Hsp-antigen molecule complex in an acceptable form. For vials of the kit of the present invention The contained hsp-antigen molecule conjugate is a pharmaceutically acceptable solution, for example, sterile physiology. Saline, dextrose or buffered solutions, or other pharmaceutically acceptable It can be combined with a sterile liquid. Alternatively, the complex is freeze-dried. Or dried in a desiccator. In this case, The kit may optionally include a pharmaceutically acceptable solution (eg, saline, A sterile solution), preferably sterile, to give a solution for injection. It may be included for the purpose of reconstructing the complex.   In other embodiments, the kit of the invention further comprises a needle or a needle for injecting the conjugate. Syringe, preferably packaged in sterile form, and / or package Includes an assembled alcohol pad. Instructions are provided by the clinician or patient for the hsp Optionally included for administration of the antigen molecule conjugate. 5.6.Target infection   Infections that can be treated or prevented by the methods of the present invention are induced by infectious agents. woken up. Examples of infectious agents include viruses, bacteria, molds, protozoa, and parasites. Although the present invention can be mentioned, the present invention is not limited to this.   Examples of viral diseases that can be treated or prevented by the method of the present invention include hepatitis A Type, hepatitis B, hepatitis C, influenza, varicella, adenovirus, herpes simplex Type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echo Virus, rotavirus, respiratory syncytial virus, papillomavirus, papova Virus, cytomegalovirus, echinovirus, arbovirus, hantau Huntaviras, coxsackie virus, mumps virus, measles Ruth, rubella virus, poliovirus, human immunodeficiency virus type I (HIV-I) And human immunodeficiency virus type II (HIV-II). However, it is not limited to these.   Bacterial diseases that can be treated or prevented by the method of the present invention are caused by bacteria. Is done. Examples of bacteria include mycobacterium, rickettsia, mycoplasma , Neisseria and Legionella, but the invention is not limited thereto. Not something.   Protozoal diseases that can be treated or prevented by the method of the present invention include, for example, leishma Nia, caused by kokzidioa, a protozoan of trypanosoma However, the present invention is not limited to these.   Parasitic diseases that can be treated or prevented by the method of the present invention include, for example, Chlamydia A) Caused by Ricketia parasites, but the invention is not limited to these. It is not something to be done. 5.7. Target cancer   Examples of cancers that can be treated or prevented by the method of the present invention include the following. , But is not limited to tumors such as human sarcomas and carcinomas. For example, Fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, hemangiosarcoma, within Sarcoma, lymphatic sarcoma, lymphatic endothelial sarcoma, synovial tumor, mesothelioma, Ewing's tumor, flat Synovial sarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell Squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous carcinoma, papillary carcinoma, papillary carcinoma, cystoma Medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, fetal stage Cancer, Birms tumor, cervical cancer, testis cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, God Gliomas, astrocytomas, marrow Blastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendrolymphoma Glioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemia, eg acute Lymphocytic leukemia and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic) , Monocytic, erythroleukemia); chronic leukemia (chronic myeloid (granular cell) leukemia and chronic Lymphocytic leukemia); polycythemia vera, lymphomas (Hodgkin's disease and Zykin's disease), multiple myeloma, Waldenstrom macroglobulin, and H Chain disease can be exemplified. Specific examples of such cancers are described in the following sections. I have.   In certain aspects, the cancer is metastatic. In other specific aspects, the patient with cancer has Before administering the hsp-antigen molecule conjugate of the present invention, an anti-cancer treatment (eg, chemotherapy) , Radiation), and is immunosuppressed. In other specific aspects , A cancer is a tumor. 5.7.1 Colorectal cancer that has spread to the liver   In 1992, approximately 150,000 Americans were diagnosed with colorectal cancer. And More than 60,000 people have died as a result of colorectal metastasis. Colorectal cancer patients when they die 80% of patients have metastatic disease involving the liver, and half of those patients have other (extrahepatic) No evidence of metastases was found. The most metastatic tumor in the liver is the gastrointestinal It starts from the nuclear power plant. Unfortunately, the history of metastatic liver disease has a severe prognosis, Chemotherapy does not provide a significant response rate and alters survival (Drebin, J.A. et al., Ed., Current Therapy In Oncology, J.E. Nieder huber, B.C. Decker, Mosby, 1993, p.426).   Colorectal cancer first spreads from local lymph nodes to the liver through the portal venous circulation . The liver represents the most common visceral site of metastasis. Colorectal cancer patients need treatment The symptoms sought depend on the anatomical location of the lesion. For example, in the ascending colon Certain lesions often cause ulcers. This ulcer causes chronic blood loss during bowel movement. You.   Radical resection has the greatest effect in treating patients with invasive colorectal cancer. Surgical Before CEA titers are measured. Radiation therapy and chemotherapy are advanced colorectal cancer Applies to patients. The result of administering a chemotherapeutic agent (for example, 5-fluorouracil) Variability, with less than 25% of patients experiencing more than 50% regression of tumor mass (Rchards Ed., 2nd edition, F. et al., 1986, J. Mol. Clin. Oncol. 4: 565).   Patients with extensive metastases have limited survival, and patients in this group Systemic chemotherapy has little effect. In addition, systemic chemotherapy is Severe toxicity associated with, for example, severe diarrhea, mucositis and / or spinal cord depression Therefore, it is often limited. Liver irradiation, systemic chemotherapy, hepatic artery cording, tumor Other treatments, such as embolization and immunotherapy, were all considered, but for the majority Has not been proven to be effective in patient survival.   In certain aspects, the invention relates to an individual having colorectal cancer that has spread to the liver. Compositions for enhancing tumor-specific immunity and inhibiting the progression of neoplastic disease in And methods.   Thus, as an example of the method of the present invention, gp96 is used in the colon with or without liver metastases. Administered to patients diagnosed with rectal cancer. Administration can be by one of many different routes of administration. But preferably by subcutaneous administration at different anatomical sites. This Examples of the anatomy include the left arm, right arm, left abdomen, right abdomen, left thigh, and right thigh. I can do it. These administration routes are used sequentially, and the injection site is once a week as described in Section 7. Depends on each injection. Cures for the prevention and treatment of primary and metastatic cancer The preparation and use of therapeutically effective compositions is described in detail in the sections below and is exemplified by Will be described later. 5.7.2 Hepatocellular carcinoma   Hepatocellular carcinoma is a common disease in the elderly in the United States. Many factors can cause hepatocellular carcinoma Although it does, the disease is usually limited to people with a history of liver disease. United States Approximately 60-80% of patients with hepatocellular carcinoma have cirrhosis, and about 4% of patients with cirrhosis Eventually results in hepatocellular carcinoma (Niederhuber, edited by J.E., 1994, Current Therapy i n Oncology, B.C. Decker, Mosby). If the liver disease is hereditary hemochromatosis or hepatitis B This risk is high in patients caused by ills infection (Bradbear, R. et al. A. et al., 1985, J.A. Natl. Cancer Inst. 75:81; Beasley, R.P. et al., 198. 1, Lancet 2: 1129). Other causes of cirrhosis that can lead to hepatocellular carcinoma Liver abuse caused by alcohol abuse and chronic administration of methotrexate If you have fibrosis. The most frequent symptom of hepatocellular carcinoma is weight loss in the upper right abdomen The appearance of a nociceptive mass in the quadrant or upper abdomen. In patients with cirrhosis, belly Hydrolysis, portal hypertension, and relatively rapid clinical deterioration are precursors to the development of hepatocellular carcinoma. It becomes. In most cases, serum aminotransferase or alkaline phosphatase In the standard liver function test for vatase, abnormal values are observed.   A liver CT scan can be used to determine the anatomical distribution of hepatocellular carcinoma. And provide orientation for percutaneous puncture biopsy. About 70% of hepatocellular carcinoma patients also have elevated serum alpha-fetoprotein levels. (McIntire, K.R. et al., 1975, Cancer Res. 35: 991). Correlates with degree.   Radical resection has little hope for treating hepatocellular carcinoma patients. So According to such a surgical procedure, the survival rate for 5 years is 12-30%. Liver transplant is more It can improve the survival rate of young patients. However, enlarged cirrhosis Most patients undergo this surgery because of nest tumor patterns or lack of matched donor organs I can not receive. Chemotherapy can be administered via intravenous routes or intrahepatic artery catheters Have been administered. Such treatment is combined with radiation therapy to the liver There is. Receiving systemic doxorubicin or 5-fluorouracil Some patients reported regression of more than 50% of measurable tumor size I have. However, chemotherapy often induces immunosuppression and completely It rarely disappears, and its response duration is short. Prognosis of patients with hepatocellular carcinoma Later, there is no correlation with cirrhosis or metastasis to lungs or bone. The average patient survival rate is Only 4 to 6 months. In other embodiments, the progression of neoplastic disease is prevented and In order to finally treat all pre-neoplastic and neoplastic cells with radiation, Ming provides compositions and methods for enhancing specific immunity in colorectal cancer patients. 5.7.3. Breast cancer   Another embodiment of the present invention relates to the treatment of breast cancer. American Cancer Society, 1992 Estimated that 180,000 American women were diagnosed with breast cancer and 46,000 had fallen for the disease. (Niederhuber, edited by J.E., Current Therapy in Oncology B.C. Decker, Mo. sby, 1993). This makes breast cancer the second leading cause of cancer death in women, after lung cancer Cause. What is disturbing is that breast cancer has been increasing at a rate of 3% since 1980 (Niederhuber, J.E., Current Therapy in Oncology BC Decker) , Mosby, 1993). Current treatments for breast cancer include surgery, radiation therapy There are laws, hormonal therapy and / or chemotherapy. Hormone receptors and disease Considering the two major characteristics of breast cancer, the degree of Improve survival and maintain or improve quality of life with sub-dose chemotherapy It is determined whether to be good. As adjunctive therapy in breast cancer patients, 2-cyclophosph Amide, doxorubicin, vincristine methotrexate, 5-fluoroura Extensive combined with (but not limited to) sill and / or leucovorin Of multi-drug therapies are used. In one specific aspect, the invention relates to a female Compositions and methods for enhancing specific immunity against pre-neoplastic and neoplastic mammalian cells provide. The invention also relates to neoplastic cells in women at high risk of developing breast cancer. Provided are compositions and methods for preventing the occurrence of cancer, and further inhibiting the growth and metastasis of cancer cells Compositions and methods are provided. These compositions can be used alone or Alternatively, it can be applied in combination with a biological response modifier. 5.8.Prevention and treatment of primary and metastatic neoplastic disease   The reasons why the immunotherapy provided by the present invention is desirable for use in cancer patients include: Many things can be mentioned. First, if a cancer patient is immunosuppressed, Surgery with anesthesia, followed by chemotherapy, exacerbates immunosuppression. Also appropriate before surgery With appropriate immunotherapy, this immunosuppression can be prevented or reversed. You. This reduces infectious complications and promotes wound healing. Second, postoperative tumor In this situation, immunotherapy may be most effective in minimizing tumor volume. No. The third reason is that tumor cells are flushed into the circulatory system during surgery and are applied at this time effective It is possible that proper immunotherapy can eliminate these cells.   In certain embodiments, the method of preventing and treating according to the present invention comprises treating cancer before, during or after surgery. Is directed at enhancing the patient's immune capacity and is tumor specific for cancer cells It is directed at inducing immunity. The purpose of the present invention is the inhibition of cancer and ultimate clinical The goal is comprehensive remission and eradication of cancer. 5.9.Monitoring effects during cancer prevention and immunotherapy using Hsp-peptide conjugates       ring   Hsp-antigen molecule conjugate immunotherapy for the development and progression of neoplastic diseases The effect can be monitored by any method known to those skilled in the art, For example, but not limited to, a) delayed hypersensitivity reactions as an assessment of cellular immunity B) measurement of the activity of cytotoxic T lymphocytes in vitro, c) tumor characteristics Measurement of levels of foreign antigens, eg, carcinoembryonic antigen (CEA), d) Computer Measurement of morphological changes in tumors using techniques such as linked tomography (CT) scans, e) Putative biomers that indicate the risk of a particular cancer in individuals at high risk Measurement of changes in Kerr level, and f) morphological changes of tumors using sonograms Measurement. 5.9.1.Delayed hypersensitivity skin test   The delayed-type hypersensitivity skin test measures overall immunocompetence and cellular immunity to antigens. Very worthwhile. The inability to react to a range of common skin antigens depends on anergy and (Sato, T., et al., 1995, Clin. Immunol. Pathol. 74: 35-43).   For proper skin testing, store the antigen sterile at 4 ° C, protect it from light, and Constructing technology is needed. How to ensure intradermal rather than subcutaneous administration of antigen , 25 or 27 gauge required. 24 and 48 hours after intradermal administration of antigen Both erythema and induration are measured with a ruler. Activity against the administered antigen or antigens For reduced sex by testing with higher concentrations of antigen or when the situation is ambiguous If this is the case, confirm by a repeated test with an intermediate test. 5.9.2In vitro activity of cytolytic T-lymphocytes   8 × 10 5 isolated by Ficoll Hypaque centrifugation gradient method⁶Peripheral Blood-derived T lymphocytes were added to 3 ml of RPMI medium containing 10% fetal bovine serum at 4 × 10 4^FourPieces Restimulate with tumor cells treated with C. In some experiments, T cell expansion 33% secondary mixed lymphocyte culture supernatant or IL-2 is added to the medium as a factor source .   To measure the primary response of cytolytic T-lymphocytes after immunization, the tumor Culture T cells without cells. In other experiments, cells with different antigenicity were Restimulate the vesicles. After 6 days, the cytotoxicity of the culture was⁵¹Cr release assay Test. Spontaneous target⁵¹Cr release should be below 20% level. Anti-MHC For Las I blocking activity, 10-fold concentrated supernatant of W6 / 32 hybridoma Add to the test to a final concentration of 12.5% (Heike, M., et al., J. Immun otherapy 15: 165-174). 5.9.3Tumor-specific antigen levels   While it is not possible to detect unique tumor antigens for all tumors, many Tumors develop antigens that can be distinguished from normal cells. Depending on the monoclonal antibody reagent Enables the biochemical characterization and isolation of antigens, and The identification of cells and the identification of the cell line of the transformed cells were of great diagnostic value. The most well characterized human tumor associated antigens are fetal neoplastic antigens. This These antigens are expressed during embryogenesis, but are not present or present in normal adult tissues. However, detection is extremely difficult. The prototype antigens are carcinoembryonic antigen (CEA), It is a glycoprotein found in the infant digestive tract. CEA is present in human colon cancer cells, Not found in normal adult colon cells. CEA is found in serum flowing out of colon cancer cells As such, the presence of antigen in this serum originally screened colon cancer patients It was thought that it could be used to do so. However, the pancreas Patients with other tumors, such as cancer and breast cancer, also had elevated levels of serum CEA. Therefore, Monitoring Decrease and Elevation of CEA Levels in Cancer Patients During Treatment Is Tumor Progression And was useful in predicting the outcome of treatment.   Several other fetal neoplastic antigens are also useful for diagnosing and monitoring human tumors. It turned out to be useful. For example, α-fetoprotein is found in fetal liver and yolk sac Α-globulin normally secreted by the vesicle It is present in the patient's serum and can be used as an indicator of the current state of the disease. You. 5.9.4.Computerized tomography (CT) scan   CT leaves the opportunity to choose a technique for accurate disease classification of cancer. CT is Is more sensitive and specific than all other imaging techniques for detecting metastases I know. 5.9.5.Measurement of putative biological markers   The level of a putative biological marker for the risk of a particular cancer It is measured to monitor the effect of hsp non-covalently bound to the complex. example For example, in individuals at increased risk for prostate cancer, serum prostate specific antigen (P SA) is described in Brawer, M.K., et al., 1992, J. Am. Urol. 147: 841-845 and Catalona, W.J., Et al., 1993, JAMA 270: 948-958; or colorectal cancer In individuals at risk for CEA, as described in Section 4.5.3, Estradio in individuals at increased risk for breast cancer 16-α-hydroxylation of Schleider, Schneider, J. et al. Et al., 1982, Proc. Natl. Ac ad. Sci. It is measured by the method described in ISA 79: 3047-3051. Quoted above The referenced references are hereby incorporated by reference. 5.9.6.Sonogram   Sonograms leave the opportunity for another choice of technology for accurate disease classification of cancer. doing. 6. Example: Administration of HSP-exogenous antigen in the treatment of hepatocellular carcinoma   For patients with hepatocellular carcinoma, h prepared in vivo from purified hsp and purified antigen The sp-antigen molecule peptide complex is administered. The antigen used was carcinoembryonic antigen (CEA) It is. Treatment with the hsp-antigen complex is initiated at any time after surgery, If the individual is receiving chemotherapy, usually 4 weeks or more to restore the immune system Hsp-antigen complex is administered after an interval of The immunity of the loyal Test as described in Section 5.9.   The treatment regimen consists of hsp-antigen dissolved in saline or other physiologically compatible solution. Administering the conjugate weekly.   The dose of hsp70 or gp96 used is 10-600 μg, preferably 10-600 μg. 〜100 μg. The dose of hsp90 used is 50-5,000 μg , Preferably about 100 μg.   The route and site of injection will vary at each point in time, eg, The second injection was subcutaneously administered to the right arm, and the third injection was left abdomen Subcutaneous administration to the right flank region, and the fifth injection The second injection is subcutaneous to the left thigh and the sixth injection is subcutaneous to the right thigh. And so on. Repeat the injection at the same site after one or more injection gaps. In addition, the injections are split and each half of the dose is administered to different sites on the same day.   Overall, make 4-6 injections every other week, then twice every two weeks After the injection, the injection plan is performed every other month. Effect of hsp-antigen complex therapy A) delayed-type hypersensitivity to evaluate cell-mediated immunity, b) inviting cytotoxic T cells ro activity, c) the level of a tumor-specific antigen such as, for example, carcinoembryonic antigen (CEA), d) Tumor morphology using techniques such as computed tomography (CT) scans And e) a putative indication of a particular cancer risk at high risk in an individual Monitor by measuring changes in biomarkers.   Depending on the results obtained, it may be necessary to maintain and / or enhance the patient's immunological response. Review treatment plan to achieve final goal of tumor regression and complete eradication of cancer cells To achieve. 7. Example: Administration of hsp-exogenous antigen complex in the treatment of colorectal cancer   hsp-antigen complex (gp96, hsp70, hsp90 or a combination thereof) Adjuvant therapy after colorectal cancer has completely declined. Or as a prophylactic adjuvant therapy to eliminate unexpected micrometastases and prolong life Improve.   Treatment and prevention strategies for patients with colorectal cancer Is the same as described in Section 6 for recovery of the elderly. As an exogenous antigen molecule The antigen used was a carcinoembryonic antigen. Prospects for prevention and treatment of colorectal cancer The method of monitoring the patient in the floor assessment shall be as described in Section 5.9. It is. 8. Example: Induction of CTL response to hsp70-ovalbumin complex 8.1.Materials and methods   The hsp70-ovalbumin conjugate was prepared in vitro. Brief description Then, 5 to 10 μg of purified hsp70 was added to a 20 mM sodium phosphate buffer (p H7.5), 0.5 NaCl, 3 mM MgCl_Two, And isoforms in 1 mM ADP Incubate for 1 hour at 37 ° C in a volume of 100 μl with 1 volume of ovalbumin did. The incubation mixture is then made up to 1 ml in phosphate buffered saline. And subcutaneously administered to selected mammalian C57BL / 6 strain mice.   The injection was repeated once every other week. hsp70-ovalbumin complex Fresh was prepared for each injection. After administration of a total of two injections, the animals are killed. Was. Two mice in each group were a) control vehicle, b) ovalbumin only, c) h Immunization with sp70 alone or d) hsp70-ovalbumin complex .   T cells were isolated from the spleen of each mouse using the T cell gradient fast-heart method and^Five 4 × 10 T cells^FourEG7 cells (ovalbumin antigen positive) or EL Incubated with 4 cells (ovalbumin antigen negative). CTL response⁵¹ It was measured as% Cr release. 8.2.result   The hsp70-ovalbumin complex may be ovalbumin alone or hsp70 Induced a significantly greater CTL response compared to only (FIG. 1A). However, T cells It did not react against EL4 cells lacking ovalbumin antigen (FIG. 1B).   The scope of the invention is not limited by the specific embodiments described herein. Real In this regard, various modifications of the present invention, in addition to those described in the present specification, may be subject to the foregoing descriptions and appendices. The accompanying drawings will become apparent to those skilled in the art. Such changes are subject to the scope of the appended claims. It is intended to fall within the bounds.   Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties. Included in this specification.

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Claims (1)

[Claims] 1. An immunogenic amount of a heat shock protein non-covalently bound to an exogenous antigen molecule A composition comprising a heterogeneous complex, wherein the exogenous antigenic molecule is an MHC-peptide complex. The above composition, wherein the composition is not isolated from the union. 2. An immunogenic amount of a heat shock protein non-covalently bound to an exogenous antigen molecule A complex comprising the antigenic complex and the antigenic complex. The above composition, which is an antigenic fragment or an antigenic derivative. 3. The exogenous antigen molecule is a cancer antigen or an antigenic fragment or antigenic derivative thereof. The composition of claim 1, wherein 4. 2. The composition of claim 1, wherein the exogenous antigenic molecule is an antigen of a pathogen. 5. Exogenous antigenic molecule is a viral antigen or antigenic fragment or antigenic induction thereof The composition of claim 1, which is a body. 6. The exogenous antigen molecule is a bacterial antigen or an antigenic fragment or antigenic derivative thereof The composition of claim 1, wherein the composition comprises: 7. The exogenous antigen molecule is a fungal antigen or an antigenic fragment or derivative thereof The composition of claim 1, wherein the composition comprises: 8. The exogenous antigen molecule is an antigen of a parasite or an antigenic fragment or antigenic derivative thereof The composition of claim 1, wherein 9. Heat shock proteins are hsp70, hsp90, gp96 and these The composition according to claim 1, 2, 3, or 4, wherein the composition is selected from the group consisting of a combination. Ten. 3. The composition according to claim 1, wherein the heat shock protein is hsp70. Stuff. 11． 3. The composition according to claim 1, wherein the heat shock protein is hsp90. Stuff. 12． 3. The composition according to claim 1, wherein the heat shock protein is gp96. . 13. The virus is hepatitis B, hepatitis C, influenza, chickenpox, adenovirus, Herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, Virus, echovirus, rotavirus, respiratory syncytial virus, nipple Oncovirus, papovavirus, cytomegalovirus, echinovirus, arbovirus Virus, huntavirus, coxsackievirus, mumps Virus, measles virus, rubella virus, poliovirus, human immunodeficiency virus Virus type I (HIV-I) and human immunodeficiency virus type II (HIV-II). The composition according to claim 5, wherein the composition is selected from the group consisting of: 14. Bacteria are Mycobacterium, Rickettsia, Mycoplasma, Neisseria, 7. The composition of claim 6, wherein the composition is selected from the group consisting of and Legionella. 15． Exogenous antigenic molecule is a protozoan antigen or antigenic fragment or antigenic induction thereof The composition of claim 1, which is a body. 16． Protozoa are leishmania, kokzidioa, and trypano 16. The composition according to claim 15, wherein the composition is selected from the group consisting of soma. 17． 9. The parasite is selected from the group consisting of Chlamydia and Rickettsia. A composition according to claim 1. 18． Cancer is fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, blood vessel Sarcoma, endothelial sarcoma, lymphatic sarcoma, lymphatic endothelial sarcoma, synovial tumor, mesothelioma, Ein Gliomas, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, flat cancer Squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous carcinoma, papillary carcinoma, papillary carcinoma, sac Alveolar carcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver carcinoma, cholangiocarcinoma, choriocarcinoma, seminiferous carcinoma , Embryonic carcinoma, bilumus tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelium Cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal Somatomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, melanomas, neuroblastomas, Retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom macro 4. The method according to claim 2, which is selected from the group consisting of globulinemia and heavy chain disease. A composition as described. 19. The set according to claim 1 or 2, further comprising a pharmaceutically acceptable carrier. Adult. 20. The composition according to claim 1 or 2, further comprising a cytokine. twenty one. Cytokines are interferon-α, interferon-γ, interlo Ikin-2, interleukin-4, interleukin-6, and tumor necrosis cause 21. The composition of claim 20, wherein the composition is selected from the group consisting of: twenty two. Exogenous antigen molecules not isolated from the MHC-peptide complex Administering to the individual a covalently bound complex of heat shock proteins A method of eliciting an immune response in an individual. twenty three. The exogenous antigen molecule is a cancer antigen, a pathogen antigen, and a cancer antigen or pathogen. 23.Selected from the group consisting of antigenic fragments or antigenic derivatives of the antigen, according to claim 22 A composition as described. twenty four. Exogenous antigenic molecule which is a cancer antigen or an antigenic fragment or antigenic derivative thereof Administering a heat shock protein complex non-covalently bound to the individual to the individual A method of eliciting an immune response in an individual, comprising: twenty five. A complex of heat shock protein non-covalently bound to an exogenous antigen molecule A method of treating an individual with cancer comprising administering to the individual. 26. The exogenous antigen molecule is a cancer antigen or an antigenic fragment or antigenic derivative thereof. 26. The method of claim 25, wherein 27. Exogenous antigen molecule is tumor-specific antigen or antigenic fragment or antigenic induction thereof 26. The method of claim 25, which is a body. 28. Cancer is fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, blood vessel Sarcoma, endothelial sarcoma, lymphatic sarcoma, lymphatic endothelial sarcoma, synovial tumor, mesothelioma, Ein Gliomas, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, flat cancer Squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous carcinoma, papillary carcinoma, papillary carcinoma, sac Alveolar carcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver carcinoma, cholangiocarcinoma, choriocarcinoma, seminiferous carcinoma , Embryonic carcinoma, bilumus tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelium Cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal Somatomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, melanomas, neuroblastomas, Retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom macro 27. The method of claim 26, wherein the method is selected from the group consisting of globulinemia and heavy chain disease. Law. 29. Heat shock proteins are hsp70, hsp90, gp96 and these 26. The method according to claim 22, 24 or 25, wherein the method is selected from the group consisting of a combination. 30. The method according to claim 22, 24 or 25, wherein the heat shock protein is hsp70. Method. 31. 26. The heat shock protein of claim 22, 24 or 25, wherein the heat shock protein is hsp90. Method. 32. The method according to claim 22, 24 or 25, wherein the heat shock protein is gp96. Law. 33. A complex of heat shock protein non-covalently bound to an exogenous antigen molecule Preventing cancer in an individual wishing to prevent cancer, comprising administering to the individual. Way. 34. The exogenous antigen molecule is a cancer antigen or an antigenic fragment or antigenic derivative thereof. 34. The method of claim 33, wherein 35. Heat shock proteins are hsp70, hsp90, gp96 and these 34. The method of claim 33, wherein the method is selected from the group consisting of a combination. 36. Exogenous antigen molecules not isolated from the MHC-peptide complex Administering to the individual a covalently bound complex of heat shock proteins Treating or predicting an infection in an individual who wishes to treat or prevent the infection. How to prevent. 37. 37. The method of claim 36, wherein the exogenous antigenic molecule is an antigen of a pathogen. 38. 38. The pathogen is a virus, bacterium, fungus, parasite, or protozoan. The method described in. 39. Heat shock proteins are hsp70, hsp90, gp96 and these 38. The method of claim 37, wherein the method is selected from the group consisting of a combination. 40. 37. The method of claim 22, 24, 25, 33 or 36, wherein the individual is a human. 41. Heat shock protein non-covalently bound to an exogenous antigen molecule in a container Comprising a purified complex of high quality together with a pharmaceutically acceptable carrier . 42. 42. The kit of claim 41, wherein the exogenous antigen molecule is a cancer antigen. 43. 42. The kit of claim 41, wherein the exogenous antigen molecule is a pathogen antigen. 44. 44. The pathogen is a virus, bacterium, fungus, parasite, or protozoan. The kit according to 1. 45. An immunogenic amount of an exogenous antigen that is an antigen of a pathogen (the pathogen is not an intracellular pathogen) Contains a complex of non-covalently bound heat shock proteins to a neutral antigen molecule Composition. 46. Exogenous antigen molecules that are antigens of the pathogen (the pathogen is not an intracellular pathogen) Administering a non-covalently bound complex of heat shock proteins to the individual. A method of eliciting an immune response in an individual, comprising: 47. Exogenous antigen molecules that are antigens of the pathogen (the pathogen is not an intracellular pathogen) Administering a non-covalently bound complex of heat shock proteins to the individual. Treating or preventing an infection in an individual who wishes to treat or prevent the infection. Is a way to prevent. 48. Heat shock proteins are hsp70, hsp90, gp96 and these 48. The composition according to claim 45, 46 or 47, selected from the group consisting of a combination.