CA2232048A1 - Treatment or prevention of neoplastic and infectious diseases with heat shock/stress proteins - Google Patents
Treatment or prevention of neoplastic and infectious diseases with heat shock/stress proteinsInfo
- Publication number
- CA2232048A1 CA2232048A1 CA002232048A CA2232048A CA2232048A1 CA 2232048 A1 CA2232048 A1 CA 2232048A1 CA 002232048 A CA002232048 A CA 002232048A CA 2232048 A CA2232048 A CA 2232048A CA 2232048 A1 CA2232048 A1 CA 2232048A1
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The present invention relates to methods and compositions for eliciting an immune response and the prevention and treatment of primary and metastatic neoplastic diseases and infectious diseases. The methods of the invention comprise administering a composition comprising an effective amount of a complex, in which the complex consists essentially of a heat shock protein (hsp) noncovalently bound to an antigenic molecule. "Antigenic molecule" as used herein refers to the peptides with which the hsps are endogenously associated in vivo as well as exogenous antigens/immunogens (i.e., with which the hsps are not complexed in vivo or antigenic/immunogenic fragments and derivatives thereof. In a preferred embodiment, the complex is autologous to the individual. The effective amounts of the complex are in the range of 100-600 micrograms for complexes comprising hsp70, 50-1000 micrograms for hsp90, and 10-600 micrograms for gp96. The invention also provides a method for measuring tumor rejection in vivo in an individual, preferably a human, comprising measuring the generation by the individual of MHC Class I-restricted CD8+ cytotoxic T lymphocytes specific to the tumor. Methods of purifying hsp70-peptide complexes are also provided.
Description
W O 97/10001 PCT~US96/14557 "TREATMENT OR PREVENTION OF NEOPLASTIC AND INFECTIOUS DISEASES WITH
HEAT SHOCK/STRESS PROTEINS"
This invention was made with government support under grant numbers CA44786 and CA64394 awarded by the National Institutes of Health. The government has certain rights in the invention.
1. INTRODUCTION
The present invention relates to methods and 10 compositions for the prevention and treatment of infectious diseases, primary and metastatic neoplastic diseases, including, but not limited to human sarcomas and carcinomas.
In the practice of the prevention and treatment of infectious diseases and cancer, compositions of complexes of heat 15 shock/stress proteins (hsps) including, but not limited to, hsp70, hsp90, gp96 alone or in combination with each other, noncovalently bound to antigenic molecules, are used to augment the immune response to genotoxic and nongenotoxic factors, tumors and infectious agents.
HEAT SHOCK/STRESS PROTEINS"
This invention was made with government support under grant numbers CA44786 and CA64394 awarded by the National Institutes of Health. The government has certain rights in the invention.
1. INTRODUCTION
The present invention relates to methods and 10 compositions for the prevention and treatment of infectious diseases, primary and metastatic neoplastic diseases, including, but not limited to human sarcomas and carcinomas.
In the practice of the prevention and treatment of infectious diseases and cancer, compositions of complexes of heat 15 shock/stress proteins (hsps) including, but not limited to, hsp70, hsp90, gp96 alone or in combination with each other, noncovalently bound to antigenic molecules, are used to augment the immune response to genotoxic and nongenotoxic factors, tumors and infectious agents.
2. BACRGROUND OF TRE lNv~NllON
The era of tumor immunology began with experiments by Prehn and Main, who showed that antigens on the methylcholanthrene (MCA)-induced sarcomas were tumor specific 25 in that transplantation assays could not detect these antigens in normal tissue of the mice (Prehn, R.T., et al., 1957, J. Natl. Cancer Inst. 18:769-778). This notion was confirmed by further experiments demonstrating that tumor specific resistance against MCA-induced tumors can be 30 elicited in the autochthonous host, that is, the mouse in which the tumor originated (Klein, G., et al., 1960, Cancer Res. 20: 1561-1572).
In subsequent studies, tumor specific antigens were also found on tumors induced with other chemical or physical ~ 5 carcinogens or on spontaneous tumors (Kripke, M.L., 1974, J.
Natl. Cancer Inst. 53:1333-1336; Vaage, J., 1968, Cancer Res.
W O97/lO001 PCTAUS96/14557 28:2477-2483; Carswell, E.A., et al., 1970, J. Natl. Cancer Inst. 44:1281-1288). Since these studies used protective immunity against the growth of transplanted tumors as the criterion for tumor specific antigens, these antigens are 5 also commonly referred to as "tumor specific transplantation antigens" or "tumor specific rejection antigens." Several factors can greatly influence the immunogenicity of the tumor induced, including, for example, the specific type of carcinogen involved, immunocompetence of the host and latency 10 period (Old, L.J., et al., 1962, Ann. N.Y. Acad. Sci. 101:80-106; Bartlett, G.L., 1972, J. Natl. Cancer Inst. 49:493-504).
Most, if not all, carcinogens are mutagens which may cause mutation, leading to the expression of tumor specific antigens (Ames, B.N., 1979, Science 204: 587-593; Weisburger, 15 J.H., et al., 1981, Science 214:401-407). Some carcinogens are immunosuppressive (Malmgren, R.A., et al., 1952, Proc.
Soc. Exp. Biol. Med. 79: 484-488). Experimental evidence suggests that there is a constant inverse correlation between immunogenicity of a tumor and latency period (time between 20 exposure to carcinogen and tumor appearance) (Old, L.J., et al., 1962, Ann. N.Y. Acad. sci. 101:80-106; and Bartlett, G.L., 1972, ~. Natl. Cancer Inst. 49: 493-504). Other studies have revealed the existence of tumor specific antigens that do not lead to rejection, but, nevertheless, can potentially 25 stimulate specific immune responses (Roitt, I., Brostoff, J
and Male, D., 1993, Immunology, 3rd ed., Mosby, St. Louis, pps. 17.1-17.12).
2.1. Tumor-8pecific Immunogenicities of ~eat 8hock/8tress Proteins hsP7o~ hsP9o and gP96 Srivastava et al. demonstrated immune response to methylcholanthrene-induced sarcomas o~ inbred mice (1988, Immunol. Today 9:78-83). In these studies it was found that 35 the molecules responsible for the individually distinct immunogenicity of these tumors were identified as cell-surface glycoproteins of 96kDa (gp96) and intracellular W O 97/10001 PCT~US96/14557 proteins of 84 to 86kDa (Srivastava, P.K., et al., 1986, Proc. Natl. Acad. sci. USA 83:3407-3411; Ullrich, S.J., et al., 1986, Proc. Natl . Acad . sci . USA 83:3121-3125.
Immunization of mice with gp96 or p84/86 isolated from a 5 particular tumor rendered the mice immune to that particular ~ tumor, but not to antigenically distinct tumors. Isolation and characterization of genes encoding gp96 and p84/86 revealed significant homology between them, and showed that gp96 and p84/86 were, respectively, the endoplasmic reticular 10 and cytosolic counterparts of the same heat shock proteins (Srivastava, P.K., et al., 1988, Immunogenetics 28:205-207;
Srivastava, P.K., et al., 1991, Curr. Top. Microbiol.
Immunol. 167:109-123). Further, hsp70 was shown to elicit immunity to the tumor from which it was isolated but not to 15 antigenically distinct tumors. However, hsp70 depleted of peptides was found to lose its immunogenic activity (Udono, M., and Srivastava, P.K., 1993, ~. Exp. Med. 178:1391-1396).
These observations suggested that the heat shock proteins are not immunogenic per se, but are carriers of antigenic 20 peptides that elicit specific immunity to cancers (Srivastava, P.K., 1993, Adv. Cancer Res. 62:153-177).
2.2. PathobioloqY of Cancer Cancer is characterized primarily by an increase in the 25 number of abnormal cells derived from a given normal tissue, invasion of adjacent tissues by these abnormal cells, and lymphatic or blood-borne spread of malignant cells to regional lymph nodes and to distant sites (metastasis).
Clinical data and molecular biologic studies indicate that 30 cancer is a multistep process that begins with minor preneoplastic changes, which may under certain conditions progress to neoplasia.
~ Pre-malignant abnormal cell growth is exemplified by hyperplasia, metaplasia, or most particularly, dysplasia (for ~ 35 review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-79.) Hyperplasia is a form of WO 97/lOOOI PCT~US96/14557 controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. As but one example, endometrial hyperplasia often precedes endometrial cancer. Metaplasia is 5 a form of controlled cell growth in which one type of adult or fully differentiated cell su~stitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic epithelium. Dysplasia is fre~uently a 10 forerunner of cancer, and is found mainly in the epithelia;
it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit 15 pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder.
The neoplastic lesion may evolve clonally and develop an 20 increasing capacity for invasion, growth, metastasis, and heterogeneity, especially under conditions in which the neoplastic cells escape the host's immune surveillance (Roitt, I., Brostoff, J and Kale, D., 1993, Immunology, 3rd ed., Mosby, St. Louis, pps. 17.1-17.12).
2.3. Immunother~py Four basic cell types whose function has been associated with antitumor cell immunity and the elimination of tumor cells from the body are: i) B-lymphocytes which secrete 30 immunoglobulins into the blood plasma for identifying and labeling the nonself invader cells; ii) monocytes which secrete the complement proteins which are responsible for lysing and processing the immunoglobulin-coated target invader cells; iii) natural killer lymphocytes having two 35 mech~ni~c for the destruction of tumor cells-antibody-dependent cellular cytotoxicity and natural killing; and iv) T-lymphocytes possessing antigen-specific receptors and each , T-lymphocyte clone having the capacity to recognize a tumor cell carrying complementary marker molecules (Schreiber, H., 1989, in Fundamental Immunology (ed). W.E. Paul, pp. 923-955).
Several factors can influence the immunogenicity of tumors induced. These factors include dose of carcinogen, immunocompetence of the host, and latency period.
Immunocompetence of the host during the period of cancer induction and development can allow the host to respond to 10 immunogenic tumor cells. This may prevent the outgrowth of these cells or select far less immunogenic escape variants that have lost their respective rejection antigen.
Conversely, immunosuppression or immune deficiency of the host during carcinogenesis or tumorigenesis may allow growth 15 of highly immunogenic tumors (Schreiber, ~., 1989, in Fundamental Immunology ( ed). W.E. Paul, pp. 923-955).
Three major types of cancer immunotherapy are currently being explored: i) adoptive cellular immunotherapy, ii) in vivo manipulation of patient plasma to remove blocking 20 factors or add tumoricidal factors, and iii) in vivo administration of biological response modifiers (e.g., interferons (IFN; IFN-alpha and IFN-gamma), interleukins (IL;
IL-2, IL-4 and IL-6), colony-stimulating factors, tumor necrosis factor (TNF), monoclonal antibodies and other 25 immunopotentiating agents, such as corynebacterium parvum (C.
parvum) (Kopp, W.C., et al., 1994, Cancer Chemotherapy and Biol. Response Modifiers 15: 226-286). There is little doubt that immunotherapy of cancer as it stands is falling short of the hopes invested in it. Although numerous 30 immunotherapeutic approaches have been tested, few of these procedures have proved to be effective as the sole or even as an adjunct form of cancer prevention and treatment.
2.3.1. I~terleu~ins (IL-2, IL-~ and IL-6) IL-2 has significant antitumor activity in a small percentage of patients with renal cell carcinoma and melanoma. Investigators continue to search for IL-2 based 5 _ W O 97/lO001 PCTAJS96/14557 regimens that will increase the response rates in IL-2 responsive tumors, but, for the most part, have neither defined new indications nor settled fundamental issues, such as whether dose intensity is important in IL-2 therapy (Kopp, s W.C., et al., 1994, Cancer Chemotherapy and Biol . Response Modifiers 15:226-286). Numerous reports have documented IL-2 associated toxicity involving increased nitrate levels and the syndrome of vascular leak and hypotension, analogous to septic shock. In addition, an increased incidence of 10 nonopportunistic bacterial infections and autoimmune complications are frequently accompanied by the antitumor response of IL-2 (Kopp, W.C., et al., 1994, Cancer Chemotherapy and Biol. Response Modifiers 15:226-286).
IL-4 and IL-6 are also being tested as antitumor agents 15 either directly or through immunomodulating mechanisms.
Dose-limiting toxicities have been observed with both agents in Phase I clinical trials (Gilleece, M.H., et al., 1992, Br.
J. Cancer 66:204-210, Weber, J., et al., 1993, J. Clin.
oncol. 11:499-506).
2.3.2. Tumor Necrosis Factor The toxicity of systemically administered TNF seriously limits its use for the treatment of cancer. TNF has been most effective when used for regional therapy, in which 25 measures, such as limb isolation for perfusion, are taken to limit the systemic dose and hence the toxicity of TNF. Dose-limiting toxicity of TNF consist of thrombocytopenia, headache, confusion and hypotension (Mittleman, A., et al., 1992, Inv. New Drugs 10:183-190).
2.3.3. Interferon~
The activity of IFN-~ has been described as being modest in a number of malignancies, including renal cell carcinoma, melanoma, hairy cell leukemia low-grade non-Hodgkin's 35 lymphoma, and others. Higher doses of IFN-~ are usually associated with higher response rates in some malignancies, but also cause more toxicity. In addition, more and more W O 97/lOOOI PCTAJS96/14S57 reports indicate that relapses after successful interferon therapy coincide with formation of neutralizing antibodies against interferon (Ouesada, J.R., et al., 1987, ~.
Interferon Res . 67: 678.
2.~. Pharmsco~inetic Models for Antic~ncer Chemother~peutic and Immunotherapeutic Drugs: Extrapolation and 8caling of Animal Data to ~, ~n~
The ethical and fiscal constraints which require the use 10 of animal models for most toxicology research also impose the acceptance of certain fundamental assumptions in order to estimate dose potency in humans from dose-response data in animals. Interspecies dose-response equivalence is most frequently estimated as the product of a reference species 15 dose and a single scaling ratio based on a physiological parameter such as body weight, body surface area, maximum lifespan potential, etc. Most frequently, exposure is expressed as milligrams of dose administered in proportion to body mass in kilograms (mg kg~~). Body mass is a surrogate 20 for body volume, and therefore, the ratio milligrams per kilogram is actually concentrations in milligrams per liter (Hirshaut, Y., et al., 1969, Cancer Res. 29:1732-1740). The key assumptions which accompany this practice and contribute to its failure to accurately estimate equipotent exposure 25 among various species are: i) that the biological systems involved are homogeneous, "well-stirred volumes" with specific gravity e~ual to 1.0; ii) that the administered compounds are instantly and homogeneously distributed throughout the total body mass; and iii) that the response of 30 the biological systems is directly proportional only to the initial concentration of the test material in the system. As actual pharmacokinetic conditions depart from these - assumptions, the utility of initial concentration scaling between species declines.
Through pharmacokinetics, one can study the time course of a drug and its metabolite levels in different fluids, tissues, and excreta of the body, and the mathematical WO 97/lOO01 PCT~US96/14557 relationships required to develop models to interpret such data. It, therefore, provides the basic information regarding drug distribution, availability, and the resulting toxicity in the tissues and hence, specifies the limitation 5 in the drug dosage for different treatment schedules and di~ferent routes of drug administration. The ultimate goal of the pharmacokinetic studies of anticancer drugs is thus to offer a framework for the design of optimal therapeutic dosage regimens and treatment schedules for individual 10 patients.
The currently utilized guidelines for prescription have evolved gradually without always having a complete and explicit justification. In 1966, Freireich and co-workers proposed the use of surface area proportions for interspecies 15 extrapolation of the acute toxicity of anticancer drugs.
This procedure has become the method of choice for many risk assessment applications (Freireich, E.J., et al., 1966, Cancer ChemotAerapy Rep. 50:219-244). For example, surface area scaling is the basis of the National Cancer Institute's 20 interspecies extrapolation procedure for anti-cancer drugs (Schein, P.S., et al., 1970, Clin. Pharmacol. Therap. 11:3-40; Goldsmith, M.A., et al., 1975, Cancer ~es. 35:1354-1364).
In accepting surface area extrapolation, the tenuous basis for initial concentration scaling has been replaced by an 25 empirical approach. The basic formula used for estimating prescription of cancer chemotherapy per body surface area (BSA) is BSA = k x kg~3, in which k is a constant that differs for each age group and species. For example, the k value for adult humans is 11, while for mice it is 9 (See Quiring, P., 30 1955, Surface area determination, in Glasser E. (ed.) Medical Physics I Chicago: Medical Year Book, p. 1490 and Vriesendorp, H.M., 1985, Hematol. (Supplm. 16) 13:57-63).
The major attraction of expressing cancer chemotherapy per m2 BSA appears to be that it offers an easily remembered ~5 simplification, i.e., equal doses of drug per m2 BSA will produce approximately the same effect in comparing different species and age groups. However, simplicity is not proof and W O 97/10001 PCT~US96/145~7 alternative methods for estimating prescription of anticancer drugs appear to have a better scientific foundation, with the added potential for a more effective use of anticancer agents (Hill, J.A., et al., 1989, ~ealth Physics 57:395-401).
The effectiveness of an optimal dose of a drug used in chemotherapy and/or immunotherapy can be altered by various factors, including tumor growth kinetics, drug resistance of tumor cells, total-body tumor cell burden, toxic effects of chemotherapy and/or immunotherapy on cells and tissues other 10 than the tumor, and distribution of chemotherapeutic agents and/or immunotherapeutic agents within the tissues of the patient. The greater the size of the primary tumor, the greater the probability that a large number of cells (drug resistant and drug sensitive) have metastasized before 15 diagnosis and that the patient will relapse after the primary.
Some metastases arise in certain sites in the body where resistance to chemotherapy is based on the limited tissue distribution of chemotherapeutic drugs administered in 20 standard doses. Such sites act as sanctuaries that shield the cancer cells from drugs that are circulating in the blood; for example, there are barriers in the brain and tests that impede drug diffusion from the capillaries into the tissue. Thus, these sites may require special forms of 25 treatment such as immunotherapy, especially since immunosuppression is characteristic of several types of neoplastic diseases.
The era of tumor immunology began with experiments by Prehn and Main, who showed that antigens on the methylcholanthrene (MCA)-induced sarcomas were tumor specific 25 in that transplantation assays could not detect these antigens in normal tissue of the mice (Prehn, R.T., et al., 1957, J. Natl. Cancer Inst. 18:769-778). This notion was confirmed by further experiments demonstrating that tumor specific resistance against MCA-induced tumors can be 30 elicited in the autochthonous host, that is, the mouse in which the tumor originated (Klein, G., et al., 1960, Cancer Res. 20: 1561-1572).
In subsequent studies, tumor specific antigens were also found on tumors induced with other chemical or physical ~ 5 carcinogens or on spontaneous tumors (Kripke, M.L., 1974, J.
Natl. Cancer Inst. 53:1333-1336; Vaage, J., 1968, Cancer Res.
W O97/lO001 PCTAUS96/14557 28:2477-2483; Carswell, E.A., et al., 1970, J. Natl. Cancer Inst. 44:1281-1288). Since these studies used protective immunity against the growth of transplanted tumors as the criterion for tumor specific antigens, these antigens are 5 also commonly referred to as "tumor specific transplantation antigens" or "tumor specific rejection antigens." Several factors can greatly influence the immunogenicity of the tumor induced, including, for example, the specific type of carcinogen involved, immunocompetence of the host and latency 10 period (Old, L.J., et al., 1962, Ann. N.Y. Acad. Sci. 101:80-106; Bartlett, G.L., 1972, J. Natl. Cancer Inst. 49:493-504).
Most, if not all, carcinogens are mutagens which may cause mutation, leading to the expression of tumor specific antigens (Ames, B.N., 1979, Science 204: 587-593; Weisburger, 15 J.H., et al., 1981, Science 214:401-407). Some carcinogens are immunosuppressive (Malmgren, R.A., et al., 1952, Proc.
Soc. Exp. Biol. Med. 79: 484-488). Experimental evidence suggests that there is a constant inverse correlation between immunogenicity of a tumor and latency period (time between 20 exposure to carcinogen and tumor appearance) (Old, L.J., et al., 1962, Ann. N.Y. Acad. sci. 101:80-106; and Bartlett, G.L., 1972, ~. Natl. Cancer Inst. 49: 493-504). Other studies have revealed the existence of tumor specific antigens that do not lead to rejection, but, nevertheless, can potentially 25 stimulate specific immune responses (Roitt, I., Brostoff, J
and Male, D., 1993, Immunology, 3rd ed., Mosby, St. Louis, pps. 17.1-17.12).
2.1. Tumor-8pecific Immunogenicities of ~eat 8hock/8tress Proteins hsP7o~ hsP9o and gP96 Srivastava et al. demonstrated immune response to methylcholanthrene-induced sarcomas o~ inbred mice (1988, Immunol. Today 9:78-83). In these studies it was found that 35 the molecules responsible for the individually distinct immunogenicity of these tumors were identified as cell-surface glycoproteins of 96kDa (gp96) and intracellular W O 97/10001 PCT~US96/14557 proteins of 84 to 86kDa (Srivastava, P.K., et al., 1986, Proc. Natl. Acad. sci. USA 83:3407-3411; Ullrich, S.J., et al., 1986, Proc. Natl . Acad . sci . USA 83:3121-3125.
Immunization of mice with gp96 or p84/86 isolated from a 5 particular tumor rendered the mice immune to that particular ~ tumor, but not to antigenically distinct tumors. Isolation and characterization of genes encoding gp96 and p84/86 revealed significant homology between them, and showed that gp96 and p84/86 were, respectively, the endoplasmic reticular 10 and cytosolic counterparts of the same heat shock proteins (Srivastava, P.K., et al., 1988, Immunogenetics 28:205-207;
Srivastava, P.K., et al., 1991, Curr. Top. Microbiol.
Immunol. 167:109-123). Further, hsp70 was shown to elicit immunity to the tumor from which it was isolated but not to 15 antigenically distinct tumors. However, hsp70 depleted of peptides was found to lose its immunogenic activity (Udono, M., and Srivastava, P.K., 1993, ~. Exp. Med. 178:1391-1396).
These observations suggested that the heat shock proteins are not immunogenic per se, but are carriers of antigenic 20 peptides that elicit specific immunity to cancers (Srivastava, P.K., 1993, Adv. Cancer Res. 62:153-177).
2.2. PathobioloqY of Cancer Cancer is characterized primarily by an increase in the 25 number of abnormal cells derived from a given normal tissue, invasion of adjacent tissues by these abnormal cells, and lymphatic or blood-borne spread of malignant cells to regional lymph nodes and to distant sites (metastasis).
Clinical data and molecular biologic studies indicate that 30 cancer is a multistep process that begins with minor preneoplastic changes, which may under certain conditions progress to neoplasia.
~ Pre-malignant abnormal cell growth is exemplified by hyperplasia, metaplasia, or most particularly, dysplasia (for ~ 35 review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-79.) Hyperplasia is a form of WO 97/lOOOI PCT~US96/14557 controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. As but one example, endometrial hyperplasia often precedes endometrial cancer. Metaplasia is 5 a form of controlled cell growth in which one type of adult or fully differentiated cell su~stitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic epithelium. Dysplasia is fre~uently a 10 forerunner of cancer, and is found mainly in the epithelia;
it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit 15 pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder.
The neoplastic lesion may evolve clonally and develop an 20 increasing capacity for invasion, growth, metastasis, and heterogeneity, especially under conditions in which the neoplastic cells escape the host's immune surveillance (Roitt, I., Brostoff, J and Kale, D., 1993, Immunology, 3rd ed., Mosby, St. Louis, pps. 17.1-17.12).
2.3. Immunother~py Four basic cell types whose function has been associated with antitumor cell immunity and the elimination of tumor cells from the body are: i) B-lymphocytes which secrete 30 immunoglobulins into the blood plasma for identifying and labeling the nonself invader cells; ii) monocytes which secrete the complement proteins which are responsible for lysing and processing the immunoglobulin-coated target invader cells; iii) natural killer lymphocytes having two 35 mech~ni~c for the destruction of tumor cells-antibody-dependent cellular cytotoxicity and natural killing; and iv) T-lymphocytes possessing antigen-specific receptors and each , T-lymphocyte clone having the capacity to recognize a tumor cell carrying complementary marker molecules (Schreiber, H., 1989, in Fundamental Immunology (ed). W.E. Paul, pp. 923-955).
Several factors can influence the immunogenicity of tumors induced. These factors include dose of carcinogen, immunocompetence of the host, and latency period.
Immunocompetence of the host during the period of cancer induction and development can allow the host to respond to 10 immunogenic tumor cells. This may prevent the outgrowth of these cells or select far less immunogenic escape variants that have lost their respective rejection antigen.
Conversely, immunosuppression or immune deficiency of the host during carcinogenesis or tumorigenesis may allow growth 15 of highly immunogenic tumors (Schreiber, ~., 1989, in Fundamental Immunology ( ed). W.E. Paul, pp. 923-955).
Three major types of cancer immunotherapy are currently being explored: i) adoptive cellular immunotherapy, ii) in vivo manipulation of patient plasma to remove blocking 20 factors or add tumoricidal factors, and iii) in vivo administration of biological response modifiers (e.g., interferons (IFN; IFN-alpha and IFN-gamma), interleukins (IL;
IL-2, IL-4 and IL-6), colony-stimulating factors, tumor necrosis factor (TNF), monoclonal antibodies and other 25 immunopotentiating agents, such as corynebacterium parvum (C.
parvum) (Kopp, W.C., et al., 1994, Cancer Chemotherapy and Biol. Response Modifiers 15: 226-286). There is little doubt that immunotherapy of cancer as it stands is falling short of the hopes invested in it. Although numerous 30 immunotherapeutic approaches have been tested, few of these procedures have proved to be effective as the sole or even as an adjunct form of cancer prevention and treatment.
2.3.1. I~terleu~ins (IL-2, IL-~ and IL-6) IL-2 has significant antitumor activity in a small percentage of patients with renal cell carcinoma and melanoma. Investigators continue to search for IL-2 based 5 _ W O 97/lO001 PCTAJS96/14557 regimens that will increase the response rates in IL-2 responsive tumors, but, for the most part, have neither defined new indications nor settled fundamental issues, such as whether dose intensity is important in IL-2 therapy (Kopp, s W.C., et al., 1994, Cancer Chemotherapy and Biol . Response Modifiers 15:226-286). Numerous reports have documented IL-2 associated toxicity involving increased nitrate levels and the syndrome of vascular leak and hypotension, analogous to septic shock. In addition, an increased incidence of 10 nonopportunistic bacterial infections and autoimmune complications are frequently accompanied by the antitumor response of IL-2 (Kopp, W.C., et al., 1994, Cancer Chemotherapy and Biol. Response Modifiers 15:226-286).
IL-4 and IL-6 are also being tested as antitumor agents 15 either directly or through immunomodulating mechanisms.
Dose-limiting toxicities have been observed with both agents in Phase I clinical trials (Gilleece, M.H., et al., 1992, Br.
J. Cancer 66:204-210, Weber, J., et al., 1993, J. Clin.
oncol. 11:499-506).
2.3.2. Tumor Necrosis Factor The toxicity of systemically administered TNF seriously limits its use for the treatment of cancer. TNF has been most effective when used for regional therapy, in which 25 measures, such as limb isolation for perfusion, are taken to limit the systemic dose and hence the toxicity of TNF. Dose-limiting toxicity of TNF consist of thrombocytopenia, headache, confusion and hypotension (Mittleman, A., et al., 1992, Inv. New Drugs 10:183-190).
2.3.3. Interferon~
The activity of IFN-~ has been described as being modest in a number of malignancies, including renal cell carcinoma, melanoma, hairy cell leukemia low-grade non-Hodgkin's 35 lymphoma, and others. Higher doses of IFN-~ are usually associated with higher response rates in some malignancies, but also cause more toxicity. In addition, more and more W O 97/lOOOI PCTAJS96/14S57 reports indicate that relapses after successful interferon therapy coincide with formation of neutralizing antibodies against interferon (Ouesada, J.R., et al., 1987, ~.
Interferon Res . 67: 678.
2.~. Pharmsco~inetic Models for Antic~ncer Chemother~peutic and Immunotherapeutic Drugs: Extrapolation and 8caling of Animal Data to ~, ~n~
The ethical and fiscal constraints which require the use 10 of animal models for most toxicology research also impose the acceptance of certain fundamental assumptions in order to estimate dose potency in humans from dose-response data in animals. Interspecies dose-response equivalence is most frequently estimated as the product of a reference species 15 dose and a single scaling ratio based on a physiological parameter such as body weight, body surface area, maximum lifespan potential, etc. Most frequently, exposure is expressed as milligrams of dose administered in proportion to body mass in kilograms (mg kg~~). Body mass is a surrogate 20 for body volume, and therefore, the ratio milligrams per kilogram is actually concentrations in milligrams per liter (Hirshaut, Y., et al., 1969, Cancer Res. 29:1732-1740). The key assumptions which accompany this practice and contribute to its failure to accurately estimate equipotent exposure 25 among various species are: i) that the biological systems involved are homogeneous, "well-stirred volumes" with specific gravity e~ual to 1.0; ii) that the administered compounds are instantly and homogeneously distributed throughout the total body mass; and iii) that the response of 30 the biological systems is directly proportional only to the initial concentration of the test material in the system. As actual pharmacokinetic conditions depart from these - assumptions, the utility of initial concentration scaling between species declines.
Through pharmacokinetics, one can study the time course of a drug and its metabolite levels in different fluids, tissues, and excreta of the body, and the mathematical WO 97/lOO01 PCT~US96/14557 relationships required to develop models to interpret such data. It, therefore, provides the basic information regarding drug distribution, availability, and the resulting toxicity in the tissues and hence, specifies the limitation 5 in the drug dosage for different treatment schedules and di~ferent routes of drug administration. The ultimate goal of the pharmacokinetic studies of anticancer drugs is thus to offer a framework for the design of optimal therapeutic dosage regimens and treatment schedules for individual 10 patients.
The currently utilized guidelines for prescription have evolved gradually without always having a complete and explicit justification. In 1966, Freireich and co-workers proposed the use of surface area proportions for interspecies 15 extrapolation of the acute toxicity of anticancer drugs.
This procedure has become the method of choice for many risk assessment applications (Freireich, E.J., et al., 1966, Cancer ChemotAerapy Rep. 50:219-244). For example, surface area scaling is the basis of the National Cancer Institute's 20 interspecies extrapolation procedure for anti-cancer drugs (Schein, P.S., et al., 1970, Clin. Pharmacol. Therap. 11:3-40; Goldsmith, M.A., et al., 1975, Cancer ~es. 35:1354-1364).
In accepting surface area extrapolation, the tenuous basis for initial concentration scaling has been replaced by an 25 empirical approach. The basic formula used for estimating prescription of cancer chemotherapy per body surface area (BSA) is BSA = k x kg~3, in which k is a constant that differs for each age group and species. For example, the k value for adult humans is 11, while for mice it is 9 (See Quiring, P., 30 1955, Surface area determination, in Glasser E. (ed.) Medical Physics I Chicago: Medical Year Book, p. 1490 and Vriesendorp, H.M., 1985, Hematol. (Supplm. 16) 13:57-63).
The major attraction of expressing cancer chemotherapy per m2 BSA appears to be that it offers an easily remembered ~5 simplification, i.e., equal doses of drug per m2 BSA will produce approximately the same effect in comparing different species and age groups. However, simplicity is not proof and W O 97/10001 PCT~US96/145~7 alternative methods for estimating prescription of anticancer drugs appear to have a better scientific foundation, with the added potential for a more effective use of anticancer agents (Hill, J.A., et al., 1989, ~ealth Physics 57:395-401).
The effectiveness of an optimal dose of a drug used in chemotherapy and/or immunotherapy can be altered by various factors, including tumor growth kinetics, drug resistance of tumor cells, total-body tumor cell burden, toxic effects of chemotherapy and/or immunotherapy on cells and tissues other 10 than the tumor, and distribution of chemotherapeutic agents and/or immunotherapeutic agents within the tissues of the patient. The greater the size of the primary tumor, the greater the probability that a large number of cells (drug resistant and drug sensitive) have metastasized before 15 diagnosis and that the patient will relapse after the primary.
Some metastases arise in certain sites in the body where resistance to chemotherapy is based on the limited tissue distribution of chemotherapeutic drugs administered in 20 standard doses. Such sites act as sanctuaries that shield the cancer cells from drugs that are circulating in the blood; for example, there are barriers in the brain and tests that impede drug diffusion from the capillaries into the tissue. Thus, these sites may require special forms of 25 treatment such as immunotherapy, especially since immunosuppression is characteristic of several types of neoplastic diseases.
3. 8U~MARY OF T~E lNv~-llON
The methods of the invention comprise methods of eliciting an immune response in an individual in whom the treatment or prevention of cancer is desired by administering a composition comprising an effective amount of a complex in which the complex consists essentially of a heat shock 35 protein (hsp) noncovalently bound to an antigenic molecule.
The amounts of the complex are within ranges of effective dosages, discovered by the present inventor to be effective, W O 97/lOO01 PCTAUS96/14~57 and which are surprisingly smaller than those amounts predicted to be effective by extrapolation by prior art methods from dosages used in animal studies. In a preferred embodiment, the complex is autologous to the individual; that 5 i5, the complex is isolated from the cancer cells of the individual himself (e.q., preferably prepared from tumor biopsies of the patient). Alternatively, the hsp and or the antigenic molecule can be isolated from the individual or ~rom others or by recombinant production methods using a 10 closed hsp originally derived from the individual or from others. "Antigenic molecule" as used herein refers to the peptides ~ith which the hsps are endogenously associated in vivo (e.g., in precancerous or cancerous tissue), as well as exogenous antigens/immunogens (i.e., with which the hsps are 15 not complexed in vivo) or antigenic/immunogenic fragments and derivatives thereof. Such exogenous antigens and fragments and derivatives (both peptide and non-peptide) thereof for use in complexing with hsps, can be selected from among those known in the art, as well as those readily identified by 20 standard immunoassays known in the art by detecting the ability to bind antibody or MHC molecules (antigenicity) or generate immune response (immunogenicity).
The invention also provides a method for measuring tumor rejection in vivo in an individual, preferably a human, 25 comprising measuring the generation by the individual of MHC
Class I-restricted CD8+ cytotoxic T lymphocytes specific to the tumor.
The invention provides methods for determining doses for treating infectious diseases or for human cancer 30 immunotherapy by evaluating the optimal dose of complexes of hsps noncovalently bound to antigenic molecules in experimental tumor models and extrapolating the data. The present invention relates to methods and compositions for prevention and treatment of primary and metastatic neoplastic 35 diseases.
Specific therapeutic regimens, pharmaceutical compositions, and kits are provided by the invention. In contrast to the prior art, the therapeutic regimen, and corresponding pharmaceutical compounds of the present invention are not based on body weight or surface area of the patient. The present inventor has discovered that a dosage 5 substantially equivalent to that seen to be effective in ~ smaller non-human mammals (e.g., mice) is effective for human administration, optionally subject to a correction factor not exceeding a fifty fold increase, based on the relative lymph node sizes in such mammals and in humans. Pharmaceutical 10 formulations are provided, based on a newly-discovered extrapolation and scaling of animal dosage to human, comprising compositions of complexes of antigenic molecules and heat shock/stress proteins, including but not limited to hsp70, hsp90, gp96 either alone or in combination.
15 Specifically, interspecies dose-response equivalence for hsp noncovalently bound to antigenic molecules for a human dose is estimated as the product of the therapeutic dosage observed in mice and a single scaling ratio, not exceeding a fifty fold increase.
The present invention encompasses methods for prevention and treatment of cancer by enhancing the host's immune competence and activity of immune effector cells.
Furthermore, the invention provides methods for evaluating the efficacy of drugs in enhancing immune responses for 25 treatment and monitoring the progress of patients participating in clinical trials for the treatment of primary and metastatic neoplastic diseases.
Immunotherapy using the therapeutic regimens of the invention, by administering such complexes of heat 30 shock/stress proteins noncovalently bound to antigenic molecules, can induce specific immunity to tumor cells, and leads to regression of the tumor mass. Cancers which are responsive to specific immunotherapy by the heat shock/stress proteins of the invention include but are not limited to - 35 human sarcomas and carcinomas. In a specific embodiment, hsp-antigenic molecule complexes are allogeneic to the W O 97/lOOOl PCT~US96/145~7 patient; in a preferred embodiment, the hsp are autologous to (derived from) the patient to whom they are administered.
Particular compositions of the invention and their properties are described in the sections and subsections s which follow. A preferred composition comprises hsp-peptide complexes isolated from the tumor biopsy of the patient to whom the composition is to be administered. Such a composition which comprises hsp70, hsp90 and/or gp96 demonstrates strong inhibition of a variety of tumors in 10 mammals. Moreover, the therapeutic doses that are effective in the corresponding experimental model in rodents as described infra, in Section 6 can be used to inhibit the in vivo growth of colon and liver cancers in human cancer patients as described in Section 7, infra. Preferred 15 compositions comprising hsp70, hsp90 and/or gp96 which preferably exhibit no toxicity when administered to human subjects are also described.
In another embodiment, the methods further optionally comprise administering biological response modifiers, e.g., 20 IFN-~, IFN-~, IL-2, IL-4, IL-6, TNF, or other cytokine growth factors affecting the immune cells, in combination with the hsp complexes.
In addition to cancer therapy, the complexes of hsps noncovalently bound to antigenic molecules can be utilized 25 for the prevention of a variety of cancers, e.g., in individuals who are predisposed as a result of familial history or in individuals with an enhanced risk to cancer due to environmental factors.
An improved method for purification of hsp70-peptide 30 complexes from cells is also provided.
The Examples presented in Sections 6 and 7 below, detail the use according to the methods of the invention of hsp-peptide complexes in cancer immunotherapy in experimental tumor models and in human patients suffering from advanced 35 colon and liver cancer.
, 4 . BRIEF DE8CRIPTION OF FIG~JRES
Figure 1. Effect of Al i n; stration of gp96 derived from W6138 or W 6139SJ carcinomas on tumor growth measured as average tumor diameter (mm).
Panel A: Lane 1, SDS-PAGE profile of gp96 preparation;
Lane 2, Immunoblot of lane 1 with antibody specific for gp96.
Panel B (top): All mice were challenged with W 6138 cells. The first group of mice (open circles) received PBS;
the second group of mice (solid circles) received gp96 10 derived from W 6138 cells; and the third group of mice received gp96 derived from W6139SJ cells.
Panel B (bottom): All mice were challenged with W6139SJ
cells. The first group of mice (open circles) received PBS;
the second group of mice (solid circles) received gp96 15 derived from W 6138 cells; and the third group of mice received gp96 derived from W6139SJ cells.
Figure 2. Effect in tumor-bearing mice of therapeutic administration of gp96 derived from W6139SJ cells on tumor growth measured as tumor volume (mm3). All mice were 20 challenged with W6139SJ cells. The first group of mice received no treatment, the second group received gpg6 derived form W6139SJ cells, and the third group received gpg6 derived from the liver.
Figure 3. Vaccination with cognate gp96 preparations 25 elicits MHC class I - restricted CTLs. Mice were immunized with gp96 derived from W 6138 (triangles) or W 6139SJ
(rectangles) or with intact tumor cells (circles), as described in legend to Fig. 1. Ten days after second immunization, spleens were removed and spleen cells were 30 cocultered in a mixed lymphocyte tumor culture (MLTC) with irradiated tumor cells used for immunization or gp96 preparation. MLTCs were tested for cytotoxicity in a chromium release assay. Open symbols refer to the cytotoxicity in presence of anti-MHC class I specific ~ 35 antibody K44. (A) In vitro cytotoxicity of non-immunized mice (triangle) or mice immunized with 20 microgram of gp96 derived from W 6138 (circle) or W6139SJ (rectangle) against WO 97/10001 PCTrUS96/14557 targets as indicated. (B) In vitro cytotoxicity of non-immunized mice (triangle) or mice immunized twice with 107 irradiated W 6138 cells (circles) or W 6139SJ cells (rectangles) against ta~rgets as indicated.
Figure 4. Vaccination with cognate gp96 preparations elicits radiation-resistant T cell response. Mice were immunized with gp96 derived from W6138 (triangles) or W 6139SJ (rectangles) and MLTCs set up as described in legend to Fig. 2, except that mice had been irradiated at 400 rad 10 twelve days after the last vaccination and MLTCs were set up three days after irradiation. Open symbols refer to the cytotoxicity in presence of anti-MHC class I specific antibody K44.
Figure 5. A. ADP-bound and ADP eluted hsp70 preparation 15 was found to be associated with peptides. B. ATP-bound and ATP eluted hsp70 preparation was found not to be associated with peptides.
The methods of the invention comprise methods of eliciting an immune response in an individual in whom the treatment or prevention of cancer is desired by administering a composition comprising an effective amount of a complex in which the complex consists essentially of a heat shock 35 protein (hsp) noncovalently bound to an antigenic molecule.
The amounts of the complex are within ranges of effective dosages, discovered by the present inventor to be effective, W O 97/lOO01 PCTAUS96/14~57 and which are surprisingly smaller than those amounts predicted to be effective by extrapolation by prior art methods from dosages used in animal studies. In a preferred embodiment, the complex is autologous to the individual; that 5 i5, the complex is isolated from the cancer cells of the individual himself (e.q., preferably prepared from tumor biopsies of the patient). Alternatively, the hsp and or the antigenic molecule can be isolated from the individual or ~rom others or by recombinant production methods using a 10 closed hsp originally derived from the individual or from others. "Antigenic molecule" as used herein refers to the peptides ~ith which the hsps are endogenously associated in vivo (e.g., in precancerous or cancerous tissue), as well as exogenous antigens/immunogens (i.e., with which the hsps are 15 not complexed in vivo) or antigenic/immunogenic fragments and derivatives thereof. Such exogenous antigens and fragments and derivatives (both peptide and non-peptide) thereof for use in complexing with hsps, can be selected from among those known in the art, as well as those readily identified by 20 standard immunoassays known in the art by detecting the ability to bind antibody or MHC molecules (antigenicity) or generate immune response (immunogenicity).
The invention also provides a method for measuring tumor rejection in vivo in an individual, preferably a human, 25 comprising measuring the generation by the individual of MHC
Class I-restricted CD8+ cytotoxic T lymphocytes specific to the tumor.
The invention provides methods for determining doses for treating infectious diseases or for human cancer 30 immunotherapy by evaluating the optimal dose of complexes of hsps noncovalently bound to antigenic molecules in experimental tumor models and extrapolating the data. The present invention relates to methods and compositions for prevention and treatment of primary and metastatic neoplastic 35 diseases.
Specific therapeutic regimens, pharmaceutical compositions, and kits are provided by the invention. In contrast to the prior art, the therapeutic regimen, and corresponding pharmaceutical compounds of the present invention are not based on body weight or surface area of the patient. The present inventor has discovered that a dosage 5 substantially equivalent to that seen to be effective in ~ smaller non-human mammals (e.g., mice) is effective for human administration, optionally subject to a correction factor not exceeding a fifty fold increase, based on the relative lymph node sizes in such mammals and in humans. Pharmaceutical 10 formulations are provided, based on a newly-discovered extrapolation and scaling of animal dosage to human, comprising compositions of complexes of antigenic molecules and heat shock/stress proteins, including but not limited to hsp70, hsp90, gp96 either alone or in combination.
15 Specifically, interspecies dose-response equivalence for hsp noncovalently bound to antigenic molecules for a human dose is estimated as the product of the therapeutic dosage observed in mice and a single scaling ratio, not exceeding a fifty fold increase.
The present invention encompasses methods for prevention and treatment of cancer by enhancing the host's immune competence and activity of immune effector cells.
Furthermore, the invention provides methods for evaluating the efficacy of drugs in enhancing immune responses for 25 treatment and monitoring the progress of patients participating in clinical trials for the treatment of primary and metastatic neoplastic diseases.
Immunotherapy using the therapeutic regimens of the invention, by administering such complexes of heat 30 shock/stress proteins noncovalently bound to antigenic molecules, can induce specific immunity to tumor cells, and leads to regression of the tumor mass. Cancers which are responsive to specific immunotherapy by the heat shock/stress proteins of the invention include but are not limited to - 35 human sarcomas and carcinomas. In a specific embodiment, hsp-antigenic molecule complexes are allogeneic to the W O 97/lOOOl PCT~US96/145~7 patient; in a preferred embodiment, the hsp are autologous to (derived from) the patient to whom they are administered.
Particular compositions of the invention and their properties are described in the sections and subsections s which follow. A preferred composition comprises hsp-peptide complexes isolated from the tumor biopsy of the patient to whom the composition is to be administered. Such a composition which comprises hsp70, hsp90 and/or gp96 demonstrates strong inhibition of a variety of tumors in 10 mammals. Moreover, the therapeutic doses that are effective in the corresponding experimental model in rodents as described infra, in Section 6 can be used to inhibit the in vivo growth of colon and liver cancers in human cancer patients as described in Section 7, infra. Preferred 15 compositions comprising hsp70, hsp90 and/or gp96 which preferably exhibit no toxicity when administered to human subjects are also described.
In another embodiment, the methods further optionally comprise administering biological response modifiers, e.g., 20 IFN-~, IFN-~, IL-2, IL-4, IL-6, TNF, or other cytokine growth factors affecting the immune cells, in combination with the hsp complexes.
In addition to cancer therapy, the complexes of hsps noncovalently bound to antigenic molecules can be utilized 25 for the prevention of a variety of cancers, e.g., in individuals who are predisposed as a result of familial history or in individuals with an enhanced risk to cancer due to environmental factors.
An improved method for purification of hsp70-peptide 30 complexes from cells is also provided.
The Examples presented in Sections 6 and 7 below, detail the use according to the methods of the invention of hsp-peptide complexes in cancer immunotherapy in experimental tumor models and in human patients suffering from advanced 35 colon and liver cancer.
, 4 . BRIEF DE8CRIPTION OF FIG~JRES
Figure 1. Effect of Al i n; stration of gp96 derived from W6138 or W 6139SJ carcinomas on tumor growth measured as average tumor diameter (mm).
Panel A: Lane 1, SDS-PAGE profile of gp96 preparation;
Lane 2, Immunoblot of lane 1 with antibody specific for gp96.
Panel B (top): All mice were challenged with W 6138 cells. The first group of mice (open circles) received PBS;
the second group of mice (solid circles) received gp96 10 derived from W 6138 cells; and the third group of mice received gp96 derived from W6139SJ cells.
Panel B (bottom): All mice were challenged with W6139SJ
cells. The first group of mice (open circles) received PBS;
the second group of mice (solid circles) received gp96 15 derived from W 6138 cells; and the third group of mice received gp96 derived from W6139SJ cells.
Figure 2. Effect in tumor-bearing mice of therapeutic administration of gp96 derived from W6139SJ cells on tumor growth measured as tumor volume (mm3). All mice were 20 challenged with W6139SJ cells. The first group of mice received no treatment, the second group received gpg6 derived form W6139SJ cells, and the third group received gpg6 derived from the liver.
Figure 3. Vaccination with cognate gp96 preparations 25 elicits MHC class I - restricted CTLs. Mice were immunized with gp96 derived from W 6138 (triangles) or W 6139SJ
(rectangles) or with intact tumor cells (circles), as described in legend to Fig. 1. Ten days after second immunization, spleens were removed and spleen cells were 30 cocultered in a mixed lymphocyte tumor culture (MLTC) with irradiated tumor cells used for immunization or gp96 preparation. MLTCs were tested for cytotoxicity in a chromium release assay. Open symbols refer to the cytotoxicity in presence of anti-MHC class I specific ~ 35 antibody K44. (A) In vitro cytotoxicity of non-immunized mice (triangle) or mice immunized with 20 microgram of gp96 derived from W 6138 (circle) or W6139SJ (rectangle) against WO 97/10001 PCTrUS96/14557 targets as indicated. (B) In vitro cytotoxicity of non-immunized mice (triangle) or mice immunized twice with 107 irradiated W 6138 cells (circles) or W 6139SJ cells (rectangles) against ta~rgets as indicated.
Figure 4. Vaccination with cognate gp96 preparations elicits radiation-resistant T cell response. Mice were immunized with gp96 derived from W6138 (triangles) or W 6139SJ (rectangles) and MLTCs set up as described in legend to Fig. 2, except that mice had been irradiated at 400 rad 10 twelve days after the last vaccination and MLTCs were set up three days after irradiation. Open symbols refer to the cytotoxicity in presence of anti-MHC class I specific antibody K44.
Figure 5. A. ADP-bound and ADP eluted hsp70 preparation 15 was found to be associated with peptides. B. ATP-bound and ATP eluted hsp70 preparation was found not to be associated with peptides.
5. DETAILED DE8CRIPTION OF THE lNV~lION
Methods and compositions for the prevention and treatment of primary and metastatic neoplastic diseases and infectious diseases and for eliciting an immune response in a human individual, are described. The invention is based, in part, on a newly discovered dosage regimen for administration 25 of compositions comprising complexes of hsps noncovalently bound to antigenic molecules.
"Antigenic molecule" as used herein refers to the peptides with which the hsps are endogenously associated in vivo (e.g., in infected cells or precancerous or cancerous 30 tissue) as well as exogenous antigens/immunogens (i.e., with which the hsps are not complexed in vivo) or antigenic/immunogenic fragments and derivatives thereof.
The methods of the invention comprise methods of eliciting an immune response in an individual in whom the 35 treatment or prevention of infectious diseases or cancer is desired by administering a composition comprising an effective amount of a complex, in which the complex consists W O 97/lOOOI PCT~US96tl4557 essentially of a hsp noncovalently bound to an antigenic molecule. In a preferred embodiment, the complex is autologous to the individual; that is, the complex is isolated from either from the infected cells or the cancer S cells for precancerous cells of the individual himself (e.g., preferably prepared from infected tissues or tumor biopsies of the patient). Alternatively, the complex is produced in vitro (e . g ., wherein a complex with an exogenous antigenic molecule is desired). Alternatively, the hsp and/or the 10 antigenic molecule can be isolated from the individual or from others or by recombinant production methods using a cloned hsp originally derived from the individual or from others. Exogenous antigens and fragments and derivatives (both peptide and non-peptide) thereof for use in complexing 15 with hsps, can be selected from among those known in the art, as well as those readily identified by standard immunoassays know in the art by the ability to bind antibody or MHC
molecules (antigenicity) or generate immune response (immunogenicity). Complexes of hsps and antigenic molecules 20 can be isolated from cancer or precancerous tissue of a patient, or from a cancer cell line, or can be produced in vitro (as is necessary in the embodiment in which an exogenous antigen is used as the antigenic molecule).
The invention also provides a method for measuring tumor 25 rejection in vivo in an individual, preferably a human comprising measuring the generation by the individual of MHC
Class I-restricted CD8+ cytotoxic T lymphocytes specific to the tumor.
The hsps of the present invention that can be used 30 include but are not limited to, hsp70, hsp90, gp96 alone or in combination. Preferably, the hsps are human hsps.
Heat shock proteins, which are also referred to interchangeably herein as stress proteins, useful in the practice of the instant invention can be selected from among 35 any cellular protein that satisfies any one of the following criteria. It is a protein whose intracellular concentration increases when a cell is exposed to a stressful stimuli, it W O 97/lOOOl PCT~US96/14557 is capable of binding other proteins or peptides, it is capable of releasing the bound proteins or peptides in the presence of adenosine triphosphate ~ATP) or low pH, or it is a protein showing at least 35% homology with any cellular 5 protein having any of the above properties.
The first stress proteins to be identified were the heat shock proteins (hsps). As their name implies, hsps are synthesized by a cell in response to heat shock. To date, three major families of hsp have been identified based on 10 molecular weight. 'rhe families have been called hsp60, hsp70 and hsp90 where the numbers reflect the approximate molecular weight of the stress proteins in kilodaltons. Many members of these families were found subsequently to be induced in response to other stressful stimuli including, but not 15 limited to, nutrient deprivation, metabolic disruption, oxygen radicals, and infection with intracellular pathogens.
tSee Welch, May 1993, sci enti fic American 56--64; Young, 1990, Annu. ~ev. Immunol. 8:401-420; Craig, 1993, Science 260:1902-1903; Gething, et al., 1992, Nature 355:33-45; and Lindquist, 20 et al., 1988, Annu. Rev. Genetics 22:631-677), the disclosures of which are incorporated herein by reference.
It is contemplated that hsps/stress proteins belonging to all of these three ~amilies can be used in the practice of the instant invention.
The major hsps can accumulate to very high levels in stressed cells, but they occur at low to moderate levels in cells that have been stressed. For example, the highly inducible m~ ~ lian hsp70 is hardly detectable at normal temperatures but becomes one Of the most actively synthesized 30 proteins in the cell upon heat shock (Welch, et al., 1985, J.
Cell. Biol. 101:1198-1211). In contrast, hsp90 and hsp60 proteins are abundant at normal temperatures in most, but not all, ~ ~lian cells and are further induced by heat (Lai, et al., 1984, Mol . Cell . Biol . 4:2802-10; van Bergen en 35 Henegouwen, et al., 1987, Genes Dev. 1:525-31).
Heat .shock proteins are among the most highly conserved proteins in existence. For example, DnaK, the hsp70 from E.
.
W O 97/10001 PCT~US96/14557 coli has about 50% amino acid sequence identity with hsp70 proteins from excoriates (Bardwell, et al., 1984, Proc. Natl .
Acad. Sci. 81:848-852). The hsp60 and hsp90 families also show similarly high levels of intra families conservation 5 (Hickey, et al., 1989, Mol . Cell . Biol . 9: 2615-2626; Jindal, 1989, Mol. Cell. Biol. 9:2279-2283). In addition, it has been discovered that the hsp60, hsp70 and hsp90 families are composed of proteins that are related to the stress proteins in sequence, for example, having greater than 35% amino acid 10 identity, but whose expression levels are not altered by stress. Therefore it is contemplated that the definition of stress protein, as used herein, embraces other proteins, muteins, analogs, and variants thereof having at least 35% to 55%, preferably 55% to 75%, and most preferably 75~ to 85%
15 amino acid identity with members of the three families whose expression levels in a cell are enhanced in response to a stressful stimulus. The purification of stress proteins belonging to these three families is described below.
The immunogenic hsp-peptide complexes of the invention 20 may include any complex containing an hsp and a peptide that is capable of inducing an immune response in a mammal. The peptides are preferably non covalently associated with the hsp. Preferred complexes may include, but are not limited to, hsp60-peptide, hsp70-peptide and hsp90-peptide complexes.
25 For example, an hsp called gp96 which is present in the endoplasmic reticulum of eukaryotic cells and is related to the cytoplasmic hsp90's can be used to generate an effective vaccine containing a gp96-peptide complex.
Although the hsps can be allogeneic to the patient, in a 30 preferred embodiment, the hsps are autologous to (derived from) the patient to whom they are administered. The hsps and/or antigenic molecules can be purified from natural sources, chemically synthesized, or recombinantly produced.
The invention provides methods for determining doses for 35 human cancer immunotherapy by evaluating the optimal dose of hsp noncovalently bound to peptide complexes in experimental tumor models and extrapolating the data. Specifically, a W O 97/10001 PCT~US96/14557 scaling factor not exceeding a fifty fold increase over the effective dose estimated in animals, is used as the optimal prescription method for cancer immunotherapy or vaccination in human subjects.
s The invention provides combinations of compositions which enhance the immunocompetence of the host individual and elicit specific immunity against infectious agents or specific immunity against preneoplastic and neoplastic cells.
The therapeutic regimens and pharmaceutical compositions of 10 the invention are described below. These compositions have the capacity to prevent the onset and progression of infectious diseases and prevent the development of tumor cells and to inhibit the growth and progression of tumor cells indicating that such compositions can induce specific 15 immunity in infectious diseases and cancer immunotherapy.
Hsps appear to induce an inflammatory reaction at the tumor site and ultimately cause a regression of the tumor burden in the cancer patients treated. Cancers which can be treated with complexes of hsps noncovalently bound to 20 antigenic molecules include, but are not limited to, human sarcomas and carcinomas.
Accordingly, the invention provides methods of preventing and treating cancer in an individual comprising administering a composition which stimulates the 25 immunocompetence of the host individual and elicits specific immunity against the preneoplastic and/or neoplastic cells.
As used herein, "preneoplastic" cell refers to a cell which is in transition from a normal to a neoplastic form; and morphological evidence, increasingly supported by molecular 30 biologic studies, indicates that preneoplasia progresses through multiple steps. Non-neoplastic cell growth commonly consists of hyperplasia, metaplasia, or most particularly, dysplasia (for review of such abnormal growth conditions (See Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B.
35 Saunders Co., Philadelphia, pp. 68-79~. Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant , W O 97/10001 PCT~US96/14557 alteration in structure or function. As but one example, endometrial hyperplasia often precedes endometrial cancer.
Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for 5 another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic epithelium.
Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of 10 non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic 15 irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder.
Although preneoplastic lesions may progress to neoplasia, they may also remain stable for long periods and may even regress, particularly if the inciting agent is removed or if 20 the lesion succumbs to an immunological attack by its host.
The therapeutic regimens and pharmaceutical compositions of the invention may be used with additional immune response enhancers or biological response modifiers including, but not limited to, the cytokines IFN-~, IFN-~, IL-2, IL-4, IL-6, 25 TNF, or other cytokine affecting immune cells. In accordance with this aspect of the invention, the complexes of the hsp and antigenic molecule are administered in combination therapy with one or more of these cytokines.
The invention further relates to administration of 30 complexes of hsp-antigenic molecules to individuals at enhanced risk of cancer due to familial history or environmental risk factors.
5.1. Dos~qe Reqimens It was established in experimental tumor models (Blachere et al., 1993, J. Immunotherapy 14:352-356) that the lowest dose of hsp noncovalently bound to peptide complexes CA 02232048 l99X-03-13 which produced tumor regression in mice was between 10 and 25 microgram/mouse weighing 20-25g which is equal to 25mg/25g =
lmg/kg. Prior art methods extrapolate to human dosages based on body weight and surface area. For example, prior art s methods o~ extrapolating human dosage based on body weight can be carried out as follows: since the conversion factor for converting the mouse dosage to human dosage is Dose Human per kg = Dose Mouse per kg x 12 (See Freireich, E.J., et al., 1966, Cancer Chemotherap. ~ep. 50:219-244), the effective 10 dose of hsp-peptide complexes in humans weighing 7Okg should be lmg/kg - 12 x 70, i.e., about 6mg (5.8mg).
Drug doses are also given in milligrams per square meter of body surface area because this method rather than body weight achieves a good correlation to certain metabolic and 15 excretionary functions (Shirkey, H.C., 1965, ~AMA 193:443).
Moreover, body surface area can be used as a common denominator for drug dosage in adults and children as well as in differemt animal species as indicated below in Table 1 (Freireich, E.J., et al., 1966, Cancer Chemotherap. Rep.
20 50:219-244~.
~ABLE 1 REPRE8ENTATIVE 8~RFACE AREA To WEIG~T
R~TIOS (km) FOR VARIOUg SPECIESI
Species Body Weight Surface Area km Factor (kg) (Sqm) Mouse 0.02 0.0066 3.0 Rat 0.15 0.025 5.9 Monkey 3.0 0.24 12 30Dog 8.0 0.40 20 Human, Child 20 0.80 25 Adult 60 1.6 37 Example: To express a mg/kg dose in any given species as the equivalent mg/sq m dose, multiply the Freireich, et al., 1966, Cancer Chemotherap. Rep. 50:
219-244.
W O97/lOOOI PCTAJS96/14557 dose by the appropriate km factor. In adult human, lOOmg/kg is equivalent to 100 mg/kg x 37 kg/sq m = 3700 mg/sq m.
In contrast to both of the above-described prior art 5 methods of determining dosage levels, the present invention provides dosages of the purified complexes of hsps and antigenic molecules that are much smaller than the dosages estimated by the prior art. For example, according to the invention, an amount of hsp70- and/or gp96-antigenic molecule 10 complexes is administered that is in the range of about lO
microgram to about 600 micrograms for a human patient, the preferred human dosage being the same as used in a 25g mouse, i.e., in the range of lO-100 micrograms. The dosage for hsp-90 peptide complexes in a human patient provided by the 15 present invention is in the range of about 50 to 5,000 micrograms, the preferred dosage being lO0 micrograms.
The doses recited above are preferably given once weekly for a period of about 4-6 weeks, and the mode or site of administration is preferably varied with each administration.
20 In a preferred example, subcutaneous administrations are given, with each site of administration varied sequentially.
Thus, by way of example and not limitation, the first injection may be given subcutaneously on the left arm, the second on the right arm, the third on the left belly, the 25 fourth on the right belly, the fifth on the left thigh, the sixth on the right thigh, etc. The same site may be repeated after a gap of one or more injections. Also, split injections may be given. Thus, for example, half the dose may be given in one site and the other half on an other site 30 on the same day.
Alternatively, the mode of administration is sequentially varied, e.g., weekly injections are given in sequence subcutaneously, intramuscularly, intravenously or intraperitoneally.
~ 35 After 4-6 weeks, further injections are preferably given at two-week intervals over a period of time of one month.
Later injections may be given monthly. The pace of later W O 97/10001 PCT~US96/14557 injections may be modified, depending upon the patient's clinical progress and responsiveness to the immunotherapy.
The invention is illustrated by non-limiting examples in Sections 6 and 7.
5.2. Therapeutic C _~sitions for Immune ResPonses to C~nc~
The compositions comprising hsp noncovalently bound to antigenic molecules are administered to elicit an effective 10 specific immune response to the complexed antigenic molecules (and not to the hsp).
In a preferred embodiment, non-covalent complexes of hsp70, hsp90 and gp96 with peptides are prepared and purified postoperatively from tumor cells obtained from the cancer 15 patient.
In accordance with the methods described herein, immunogenic or antigenic peptides that are endogenously complexed to hsps or MHC antigens can be used as antigenic molecules. For example, such peptides may be prepared that 20 stimulate cytotoxic T cell responses against different tumor antigens (e.g., tyrosinase, gplOO, melan-A, gp75, mucins, etc.) and viral proteins including, but not limited to, proteins of immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-II), hepatitis type A, 25 hepatitis type B, hepatitis type C, influenza, Varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, 30 huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus and polio virus. In the embodiment wherein the antigenic molecules are peptides noncovalently complexed to hsps in vivo, the complexes can be isolated from cells, or alternatively, produced in vitro from purified preparations 35 each of hsps and antigenic molecules.
In another specific embodiment, antigens of cancers (e.g., tumors) or infectious agents (e.g., viral antigen, bacterial antigens, etc.) can be obtained by purification from natural sources, by chemical synthesis, or recombinantly, and, through in vitro procedures such as that described below, noncovalently complexed to hsps.
In an embodiment wherein the hsp-antigenic molecule complex to be used is a complex that is produced in vivo in cells, exemplary purification procedures such as described in Sections 5.2.1-5.2.3 below can be employed. Alternatively, in an embodiment wherein one wishes to use antigenic 10 molecules by complexing to hsps in vitro, hsps can be purified for such use from the endogenous hsp-peptide complexes in the presence of ATP or low pH (or chemically synthesized or recombinantly produced). The protocols described herein may be used to isolate hsp-peptide 15 complexes, or the hsps alone, from any eukaryotic cells for example, tissues, isolated cells, or immortalized eukaryote cell lines infected with a preselected intracellular pathogen, tumor cells or tumor cell lines.
5.2.1. Prepar~tion and Purification of HsP 70-DeDtide Complexes The purification of hsp70-peptide complexes has been described previously, see, for example, Udono et al., 1993, J. Exp. Med. 178 :1391-1396. A procedure that may be used, 25 presented by way of example but not limitation, is as follows:
Initially, tumor cells are suspended in 3 volumes of lX
Lysis buffer consisting of 5mM sodium phosphate buffer, pH 7, lSOmM NaCl, 2mM CaCl2, 2mM MgCl2 and lmM phenyl methyl 30 sulfonyl fluoride (PMSF). Then, the pellet is sonicated, on ice, until >99% cells are lysed as determined by microscopic ~Y~ tion. As an alternative to sonication, the cells may be lysed by mechanical shearing and in this approach the cells typically are resuspended in 30mM sodium bicarbonate - 35 pH 7.5, lmM PMSF, incubated on ice for 20 minutes and then homogenized in a dounce homogenizer until >95% cells are lysed.
PCT~S96/14557 Then the lysate is centrifuged at 1,OOOg for 10 minutes to remove unbroken cells, nuclei and other cellular debris.
The resulting supernatant is recentrifuged at lOO,OOOg for 90 minutes, the supernatant harvested and then mixed with Con 5 A Sepharose equilibrated with phosphate buffered saline (PBS) containing 2mM Ca2+ and 2mM Mg~+. When the cells are lysed by mechanical shearing the supernatant is diluted with an equal volume of 2X lysis buffer prior to mixing with Con A
Sepharose. The supernatant is then allowed to bind to the 10 Con A Sepharose for 2-3 hours at 4~C. The material that fails to bind is harvested and dialyzed for 36 hours (three times, 100 volumes each time) against lOmM Tris-Acetate pH 7.5, O.lmM EDTA, lOmM NaCl, lmM PMSF. Then the dialyzate is centrifuged at 17,000 rpm (Sorvall SS3~ rotor) for 20 15 minutes. Then the resulting supernatant is harvested and applied to a Mono Q FPLC column equilibrated in 2OmM Tris-Acetate pH 7.5, 20mM NaCl, O.lmM EDTA and 15mM 2-mercaptoethanol. The column is then developed with a 2OmM to SOOmM NaCl gradient and then eluted fractions fractionated by 20 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and characterized by immunoblotting using an appropriate anti-hsp70 antibody (such as from clone N27F3-4, from StressGen).
Fractions strongly immunoreactive with the anti-hsp70 25 antibody are pooled and the hsp70-peptide complexes precipitated with ammonium sulfate; specifically with a 50%-70% ammonium sulfate cut. The resulting precipitate is then harvested by centrifugation at 17,000 rpm (SS34 Sorvall rotor) and washed with 70~ ammonium sulfate. The washed 30 precipitate is then solubilized and any residual ammonium sulfate removed by gel filtration on a SephadexR G25 column (Pharmacia). If necessary the hsp70 preparation thus obtained can be repurified through the Mono Q FPCL Column as described above.
The hsp70-peptide complex can be purified to apparent homogeneity using this method. Typically lmg of hsp70-peptide complex can be purified from lg of cells/tissue.
W O 97/lOOOI PCT~US96/14557 The present invention further describes a new and rapid method for purification of hsp70-peptide complexes. This improved method comprises contacting cellular proteins with ADP or a nonhydrolyzable analog of ATP affixed to a solid 5 substrate, such that hsp70 in the lysate can bind to the ADP
or nonhydrolyzable ATP analog, and eluting the bound hsp70.
A preferred method uses column chromatography with ADP
affixed to a solid substratum (e.g., ADP-agarose). The resulting hsp70 preparations are higher in purity and devoid 10 of contaminating peptides. The hsp70 yields are also increased significantly by about more than 10 fold.
Alternatively, chromatography with nonhydrolyzable analogs of ATP, instead of ADP, can be used for purification of hsp70-peptide complexes. By way of example but not limitation, 15 purification of hsp70-peptide complexes by ADP-agarose chromatography was carried out as described in Example Section 9.
5.2.2. Preparation and Purification of Hsp 90-Peptide Complexes A procedure that can be used, presented by way of example and not limitation, is as follows:
Initially, tumor cells are suspended in 3 volumes of lX
Lysis buffer consisting of 5mM sodium phosphate buffer (pH7), 25 150mM NaCl, 2mM CaCl2, 2mM MgCl2 and lmM phenyl methyl sulfonyl fluoride (PMSF). Then, the pellet is sonicated, on ice, until >99~ cells are lysed as determined by microscopic ~;nation. As an alternative to sonication, the cells may be lysed by mechanical shearing and in this approach the 30 cells typically are resuspended in 30mM sodium bicarbonate pH 7.5, lm~ PMSF, incubated on ice for 20 minutes and then homogenized in a dounce homogenizer until >95% cells are lysed.
Then the lysate is centrifuged at l,OOOg for 10 minutes 35 to remove unbroken cells, nuclei and other cellular debris.
The resulting supernatant is recentrifuged at lOO,OOOg for 90 minutes, the supernatant harvested and then mixed with Con A
W O 97/lOOOI PCTnUS96/14557 Sepharose equilibrated with PBS containing 2mM Ca2+ and 2mM
Mg2+. When the cells are lysed by mechanical shearing the supernatant is diluted with an equal volume of 2X Lysis buffer prior to mixing with Con A Sepharose. The supernatant 5 is then allowed to bind to the Con A Sepharose for 2-3 hours at 4~C. The material that fails to bind is harvested and dialyzed for 36 hours (three times, 100 volumes each time) against lOmM Tris-Acetate pH 7.5, 0.lmM EDTA, lOmM NaCl, lmM
PMSF. Then the dialyzate is centrifuged at 17,000 rpm lo (Sorvall SS34 rotor) for 20 minutes. Then the resulting supernatant is harvested and applied to a Mono Q FPLC column equilibrated with lysis buffer. The proteins are then eluted with a salt gradient of 200mM to 600mM NaCl.
The eluted fractions are fractionated by SDS-PAGE and 15 fractions containing the hsp90-peptide complexes identified by immunoblotting using an anti-hsp90 antibody such as 3G3 (Affinity Bioreagents). Hsp90-peptide complexes can be purified to apparent homogeneity using this procedure.
Typically, 150-200 ~g of hsp90-peptide complex can be 20 purified from lg of cells/tissue.
5.2.3. Preparation ~nd Purification o~ qD96-DeDtide Complexes A procedure that can be used, presented by way of 25 example and not limitation, is as follows:
A pellet of tumors is resuspended in 3 volumes of buffer consisting of 30mM sodium bicarbonate buffer (pH 7.5) and lmM
PMSF and the cells allowed to swell on ice 20 minutes. The cell pellet then is homogenized in a Dounce homogenizer (the 30 appropriate clearance of the homogenizer will vary according to each cells type) on ice until >95% cells are lysed.
The lysate is centrifuged at 1,OOOg for 10 minutes to remove unbroken cells, nuclei and other debris. The supernatant from this centrifugation step then is 35 recentrifuged at lOO,OOOg for 90 minutes. The gp96-peptide complex can be purified either from the 100,000 pellet or from the supernatant.
W O 97/lOOOl When purified from the supernatant, the supernatant is diluted with equal volume of 2X lysis buffer and the supernatant mixed for 2-3 hours at 4~C with con a sepharose equilibrated with PBS containing 2mM Ca2+ and 2mM Mg2+ Then, 5 the slurry is packed into a column and washed with lX lysis buffer until the OD280 drops to baseline. Then, the column is washed with 1/3 column bed volume of 10% ~-methyl mannoside (~-MM) dissolved in PBS contA;~ing 2mM Ca2+ and 2mM Mg2+, the column sealed with a piece of parafilm, and incubated at 37~C
10 for 15 minutes. Then the column is cooled to room temperature and the parafilm removed from the bottom of the column. ~ive column volumes of the ~-MM buffer are applied to the column and the eluate analyzed by SDS-PAGE. Typically the resulting material is about 60-95% pure, however this 15 depends upon the cell type and the tissue-to-lysis buffer ratio used. Then the sample is applied to a Mono Q FPLC
column (Pharmacia) e~uilibrated with a buffer containing 5mM
sodium phosphate, pH 7. The proteins then are eluted from the column with a O-lM NaCl gradient and the gp96 fraction 20 elutes between 400mM and 550mM NaCl.
The procedure, however, may be modified by two additional steps, used either alone or in combination, to consistently produce apparently homogeneous gp96-peptide complexes. One optional step involves an ammonium sulfate 25 precipitation prior to the Con A purification step and the other optional step involves DEAE-Sepharose purification after the Con A purification step but before the Mono Q FPLC
step.
In the first optional step, the supernatant resulting 30 from the lOO,OOOg centrifugation step is brought to a final concentration of 50% ammonium sulfate by the addition of ammonium sulfate. The ammonium sulfate is added slowly while gently stirring the solution in a beaker placed in a tray of ice water. The solution is stirred from about 1/2 to 12 35 hours at 4~C and the resulting solution centrifuged at 6,000 rpm (Sorvall SS34 rotor). The supernatant resulting from this step is removed, brought to 70~ ammonium sulfate _ _ _ ~ _ WO 97/iOOOl PCTAJS96/14557 saturation by the addition of ammonium sulfate solution, and centrifuged at 6,000 rpm (Sorvall SS34 rotor). The resulting pellet from this step is harvested and suspended in PBS
containing 70% ammonium sulfate in order to rinse the pellet.
5 This mixture is centrifuged at 6,000 rpm (Sorvall SS34 rotor) and the pellet dissolved in PBS containing 2mM Ca2+ and Mg2+.
Undissolved material is removed by a brief centrifugation at 15,000 rpm (Sorvall SS34 rotor). Then, the solution is mixed with Con A Sepharose and the procedure followed as before.
In the second optional step, the gp96 containing fractions eluted from the Con A column are pooled and the buffer exchanged for 5mM sodium phosphate buffer, pH 7, 300mM
NaCl by dialysis, or preferably by buffer exchange on a Sephadex G25 column. After buffer exchange, the solution is 15 mixed with DEAE-Sepharose previously equilibrated with 5mM
sodium phosphate buffer, pH 7, 300mM NaCl. The protein solution and the beads are mixed gently for 1 hour and poured into a column. Then, the column is washed with 5mM sodium phosphate buffer, pH 7, 300mM NaCl, until the absorbance at 20 280nM drops to baseline. Then, the bound protein is eluted from the column with five volumes of 5mM sodium phosphate buffer, pH 7, 700mM NaCl. Protein containing fractions are pooled and diluted with 5mM sodium phosphate buffer, pH 7 in order to lower the salt concentration to 175mM. The 25 resulting material then is applied to the Mono Q FPLC column (Pharmacia) equilibrated with 5mM sodium phosphate buffer, pH 7 and the protein that binds to the Mono Q FPLC column (Pharmacia) is eluted as described before.
It is appreciated, however, that one skilled in the art 30 may assess, by routine experimentation, the benefit of incorporating the second optional step into the purification protocol. In addition, it is appreciated also that the benefit of adding each of the optional steps will depend upon the source of the starting material.
When the gp96 fraction is isolated from the lOO,OOOg pellet, the pellet is suspended in 5 volumes of PBS
containing either 1% sodium deoxycholate or 1% oxtyl WO97/l0001 PCT~S96/14557 glucopyranoside (but without the Mg2+ and Ca2+) and incubated on ice for 1 hour. The suspension is centrifuged at 20,000g for 30 minutes and the resulting supernatant dialyzed against several changes of PBS (also without the Mg2+ and Ca2~) to 5 remove the detergent. The dialysate is centrifuged at 100,000g for 90 minutes, the supernatant harvested, and calcium and magnesium are added to the supernatant to give final concentrations of 2mM, respectively. Then the sample is purified by either the unmodified or the modi~ied method 10 for isolating gp96-peptide complex from the 100,000g supernatant, see above.
The gp96-peptide complexes can be purified to apparent homogeneity using this procedure. About 10-20~g of gp96 can be isolated from lg cells/tissue.
Infectious Disease In an alternative embodiment wherein it is desired to treat a patient having an infectious disease the above-described methods in Sections 5.2.1 - 5.2.3 are used to 20 isolate hsp-peptide complexes from cells infected with an infectious organism, e.g., of a cell line or from a patient.
Such infectious organisms include but are not limited to, viruses, bacterial, protozoa, fungi, and parasites as described in detail in Section 5.2.4 below.
5.2.~. Isolation of Antiqenic/I~munoqenic Components It has been found that antigenic peptides and/or components can be eluted from hsp-complexes either in the 30 presence of ATP or low pH. These experimental conditions may be used to isolate peptides and/or antigenic components ~rom cells which may contain potentially useful antigenic determinants. Once isolated, the amino acid sequence of each antigenic peptide may be determined using conventional amino 35 acid sequencing methodologies. Such antigenic molecules can then be produced by chemical synthesis~or recombinant methods, purified, and complexed to hsps in vitro.
W O 97/10001 PCTrUS96/14557 Similarly, it has been found that potentially immunogenic peptides may be eluted from MHC-peptide complexes using techniques well know in the art (Falk, K. et al., 1990 Nature 348:248-251; Elliott, T., et al., 1990, Nature 5 348:195-197; Falk, K., et al., 1991, Nature 351:290-296).
Thus, potentially immunogenic or antigenic peptides may be isolated from either endogenous stress protein-peptide complexes or endogenous MHC-peptide complexes for use subseguently as antigenic molecules, by complexing in vi tro 10 to hsps. Exemplary protocols for isolating peptides and/or antigenic components from either of the these complexes are set forth below in Sections 5.2.4.1 and 5.2.4.2.
5.2.4.1 Peptide~ From 8tres~ Protein-Peptide Complexes Two methods may be used to elute the peptide from a stress protein-peptide complex. One approach involves incubating the stress protein-peptide complex in the presence of ATP. The other approach involves incubating the complexes in a low pH buffer.
Briefly the complex of interest is centrifuged through a Centricon lo assembly (Millipore) to remove any low molecular weight material loosely associated with the complex. The large molecular weight fraction may be removed and analyzed by SDS-PAGE while the low molecular weight may be analyzed by 25 HPLC as described below. In the ATP incubation protocol, the stress protein-peptide complex in the large molecular weight fraction is incubated with 10mM ATP for 30 minutes at room temperature. In the low pH protocol, acetic acid or trifluoro acetic acid is added to the stress protein-peptide 30 complex to give a final concentration of 10~ (vol/vol) and the mixture incubated at room temperature or in a boiling water bath or any temperature in between, for 10 minutes (See, Van Bleek, et al., 1990, Nature 348:213-216; and Li, et al., 1993, EMBO Journal 12: 3143-3151).
The resulting samples are centrifuged through an Centricon 10 assembly as mentioned previously. The high and low molecular weight fractions are recovered. The remaining W O 97/lO001 PCT~US96/14557 large molecular weight stress protein-peptide complexes can be reincubated with ATP or low pH to remove any remaining peptides.
The resulting lower molecular weight fractions are 5 pooled, concentrated by evaporation and dissolved in 0.1%
trifluoroacetic acid (TFA). The dissolved material is then fractionated by reverse phase high pressure liquid chromatography (HPLC) T using for example a VYDAC CIB reverse phase column equilibrated with 0.1% TFA. The bound material 10 is then eluted at a flow rate of about 0.8 ml/min by developing the column with a linear gradient of 0 to 80%
acetonitrile in 0.1% TFA. The elution of the peptides can be monitored by OD2l0 and the fractions containing the peptides collected.
5.2.4.2 PePtide~ from M~C-peptid~ Complexes.
The isolation of potentially immunogenic peptides from MHC molecules is well known in the art and so is not described in detail herein (See, Falk, et al., 1990, Nature 20 348:248-251; Rotzsche, at al., 1990, Na~ure 348:252-254;
~lliott, et al., 1990, Nature 348:191-197; Falk, et al., 1991, Nature 351:290--296; Demotz, et al., 1989, Nature 343:682-684; Rotzsche, et al., 1990, Science 249:283-287), the disclosures of which are incorporated herein by 25 reference.
Briefly, MHC-peptide complexes may be isolated by a conventional immunoaffinity procedure. The peptides then may be eluted from the MHC-peptide complex by incubating the complexes in the presence of about 0.1% TFA in acetonitrile.
30 The eluted peptides may be fractionated and purified by reverse phase HPLC, as before.
The amino acid sequences of the eluted peptides may be determined either by manual or automated amino acid sequencing techniques well known in the art. Once the amino 35 acid sequence of a potentially protective peptide has been determined the peptide may be synthesized in any desired W O 97/lOOOI PCTrUS96/145~7 amount using conventional peptide synthesis or other protocols well known in the art.
Peptides having the same amino acid sequence as those isolated above may be synthesized by solid-phase peptide 5 synthesis using procedures similar to those described by Merrifield, 1963, J. Am. Chem. Soc., 85:2149. During synthesis, N-~-protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C-terminal and to an insoluble polymeric 10 support i e., polystyrene beads. The peptides are synthesized by linking an amino group of an N-~-deprotected amino acid to an ~-car~oxy group of an N-~-protected amino acid that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide. The attachment of a free 15 amino group to the activated carboxyl leads to peptide bond formation. The most commonly used N-~-protecting groups include Boc which is acid labile and Fmoc which is base labile.
Briefly, the C-terminal N-~-protected amino acid is 20 first attached to the polystyrene beads. The N-~-protecting group is then removed. The deprotected ~-amino group is coupled to the activated ~-carboxylate group of the next N-~-protected amino acid. The process is repeated until the desired peptide is synthesized. The resulting peptides are 25 then cleaved from the insoluble polymer support and the amino acid side chains deprotected. Longer peptides can be derived by condensation of protected peptide fragments. Details of appropriate chemistries, resins, protecting groups, protected amino acids and reagents are well known in the art and so are 30 not discussed in detail herein (See, Atherton, et al., 1989, Solid Phase Peptide Synthesis: A Practical Approach, IRL
Press, and Bodanszky, 1993, Peptide Chemistry, A Practical Textbook, 2nd Ed., Springer-Verlag).
Purification of the resulting peptides is accomplished 35 using conventional procedures, such as preparative HPLC using gel permeation, partition and/or ion exchange chromatography.
W O 97/10001 PCT~US96/14557 The choice of appropriate matrices and buffers are well known in the art and so are not described in detail herein.
5.2.5 Exoqenous Antiqenic Molecules Antigens or antigenic portions thereof can be selected for use as antigenic molecules, for complexing to hsps, from among those known in the art or determined by immunoassay to be able to bind to antibody or MHC molecules (antigenicity) or generate immune response (immunogenicity). To determine 10 immunogenicity or antigenicity by detecting binding to antibody, various immunoassays known in the art can be used, including but not limited to competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich"
15 immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in vivo immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, immunoprecipitation reactions, agglutination assays (e.g., gel agglutination 20 assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary 25 antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labelled. Many means are known in the art for detecting binding in an immunoassay and are envisioned for use. In one embodiment for detecting 30 immunogenicity, T cell-mediated responses can be assayed by stAn~d methods, e.g., in vitro cytoxicity assays or in vivo delayed-type hypersensitivity assays.
Potentially useful antigens or derivatives thereof for use as antigenic molecules can also be identified by various 35 criteria, such as the antigen's involvement in neutralization of a pathogen's infectivity (wherein it is desired to treat or prevent infection by such a pathogen) (Norrby, 1985, Summary, in Vaccines 85, ~erner, et al. (eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp.
388-389), type or group specificity, recognition by patients' antisera or immune cells, and/or the demonstration of 5 protective effects of antisera or immune cells specific for the antigen. In addition, where it is desired to treat or prevent a disease caused by pathogen, the antigen's encoded epitope should preferably display a small or no degree of antigenic variation in time or amongst different isolates of 10 the same pathogen.
Preferably, where it is desired to treat or prevent cancer, known tumor-specific antigens or fragments or derivatives thereof are used. For example, such tumor specific or tumor-associated antigens include but are not 15 limited to KS 1/4 pan-carcinoma antigen (Perez and Walker, 1990, J. Inununol. 142:3662-3667; Bumal, 1988, Hybridoma 7(4):407-415); ovarian carcinoma antigen (CA125) (Yu, et al., 1991, Cancer Res. 51(2):468-475); prostatic acid phosphate (Tailer, et al., 1990, Nucl. Acids Res. 18(16):4928);
20 prostate specific antigen (Henttu and Vihko, 1989, Biochem.
Biophys. Res. Comm. 160(2):903-910; Israeli, et al., 1993, Cancer Res. 53:227-230); melanoma-associated antigen p97 (Estin, et al., 1989, J. Natl. Cancer Inst. 81(6):445-446);
melanoma antigen gp75 (Vijayasardahl, et al., 1990, J. Exp.
25 Med. 171(4):1375-1380); high molecular weight melanoma antigen (Natali, et al., 1987, Cancer 59:55-63) and prostate specific m~embrane antigen.
In a specific embodiment, an antigen or fragment or derivative thereof specific to a certain tumor is selected 30 for complexing to hsp and subsequent administration to a patient having that tumor.
Preferably, where it is desired to treat or prevent viral diseases, molecules comprising epitopes of known viruse~ are used. For example, such antigenic epitopes may 35 be prepared from viruses including, but not limited to, hepatitis ~ype A hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, herpes simplex type I (HSV-W O97/lOO01 PCTAJS96/14557 I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsachie virus, mumps virus, measles virus, 5 rubella virus, polio virus, human immunodeficiency virus type I (HIV-I), and human immunodeficiency virus type II (HIV-II).
Preferably, where it is desired to treat or prevent bacterial infections, molecules comprising epitopes of known bacteria are used. For example, such antigenic epitopes may 10 be prepared from bacteria including, but not limited to, mycobacteria rickettsia, mycoplasma, neisseria and legionella.
Preferably, where it is desired to treat or prevent protozoal infectious, molecules comprising epitopes of known 15 protozoa are used. For example, such antigenic epitopes may be prepared from protozoa including, but not limited to, leishmania, kokzidioa, and trypanosoma.
Preferably, where it is desired to treat or prevent parasitic infectious, molecules comprising epitopes of known 20 parasites are used. For example, such antigenic epitopes may be from parasites including, but not limited to, chlamydia and rickettsia.
5 . 2 . 6 In Vitro Production of ~tress Protein-Anti~enic Molecule Complexes In an embodiment in which complexes of hsps and the peptides with which they are endogenously associated in vivo are not employed, complexes of hsps to antigenic molecules are produced in vitro. As will be appreciated by those 30 skilled in the art, the peptides either isolated by the aforementioned procedures or chemically synthesized or recombinantly produced may be reconstituted with a variety of naturally purified or recombinant stress proteins in vitro to generate immunogenic non-covalent stress protein-antigenic 35 molecule complexes. Alternatively, exogenous antigens or antigenic/immunogenic fragments or derivatives thereof can be noncovalently complexed to stress proteins for use in the W O 97/10001 PCT~US96/14557 immunotherapeutic or prophylactic vaccines of the invention.
A preferred, exemplary protocol for noncovalently complexing a stress protein and an antigenic molecule in vitro is discussed below.
Prior to complexing, the hsps are pretreated with ATP or low pH to remove any peptides that may be associated with the hsp of interest. When the ATP procedure is used, excess ATP
is removed from the preparation by the addition of apyranase as described by Levy, et al., 1991, Cell 67: 265-274 . When 10 the low pH procedure is used, the buffer is readjusted to neutral pH by the addition of pH modifying reagents.
The antigenic molecules (l~g) and the pretreated hsp (9~g) are admixed to give an approximately 5 antigenic molecule: 1 stress protein molar ratio. Then, the mixture is 15 incubated for 15 minutes to 3 hours at 4~ to 45~C in a suitable binding buffer such as one containing 20mM sodium phosphate, pH 7.2, 350mM NaCl, 3mM MgCl2 and lmM phenyl methyl sulfonyl fluoride (PMSF). The preparations are centrifuged through Centricon 10 assembly (Millipore) to remove any 20 unbound peptide. The association of the peptides with the stress proteins can be assayed by SDS-PAGE. This is the preferred method for in vitro complexing of peptides isolated from MHC-peptide complexes of peptides disassociated from endogenous hsp-peptide complexes.
In an alternative embodiment of the invention, preferred for producing complexes of hsp70 to exogenous antigenic molecules such as proteins, 5-10 micrograms of purified hsp is incubated with equimolar quantities of the antigenic molecule in 20mM sodium phosphate buffer pH 7.5, 0.5M NaCl, 30 3mM MgCl2 and lmM ADP in a volume of 100 microliter at 37~C
for 1 hr. This incubation mixture is further diluted to lml in phosphate-buffered saline.
In an alternative embodiment of the invention, preferred for producing complexes of gp96 or hsp90 to peptides, 5 -10 35 mi~loy~ams of purified gp96 or hsp90 is incubated with equimolar or excess quantities of the antigenic peptide in a suitable buffer such as one containing 20mM sodium phosphate W O 97/lO001 PCTrUS96/14557 buffer pH 7.5, 0.5M NaCl, 3nM MgC12 at 60-65OC ~or 5-20 min.
This incubation mixture is allowed to cool to room temperature and centrifuged one or more times if necessary, through Centricon 10 assembly (Millipore) to remove any 5 unbound peptide.
Following complexing, the immunogenic stress protein-antigenic molecule complexes can optionally be assayed in vitro using for example the mixed lymphocyte target cell assay (MLTC) described below. Once immunogenic complexes 10 have been isolated they can be optionally characterized further in animal models using the preferred administration protocols and excipients discussed below.
5.2.7 Determination of Immunogenicity of 8tre~ Protein-Peptide Complexe~
The purified stress protein-antigenic molecule complexes can be assayed for immunogenicity using the mixed lymphocyte target culture assay (MLTC) well known in the art.
By way of example but not limitation, the following 20 procedure can be used. Briefly, mice are injected subcutaneously with the candidate stress protein-antigenic molecule complexes. Other mice are injected with either other stress protein peptide complexes or whole infected cells which act as positive controls for the assay. The mice 25 are injected twice, 7-10 days apart. Ten days after the last i -lnization, the spleens are removed and the lymphocytes released. The released lymphocytes may be restimulated subsequently in vitro by the addition of dead cells that expressed the complex of interest.
For example, 8X106 immune spleen cells may be stimulated with 4x104 mitomycin C treated or ~-irradiated (5-10,000 rads) infected cells (or cells transfected with an appropriate gene, as the case may be) in 3ml RPMI medium containing 10%
fetal calf serum. In certain cases 33% secondary mixed 35 lymphocyte culture supernatant may be included in the culture medium as a source of T cell growth factors (See, Glasebrook, et al., 1980, J. Exp. Med. 151:876). To test the primary W O97/lOO01 PCT~US96/1~57 cytotoxic T cell response after immunization, spleen cells may be cultured without stimulation. In some experiments spleen cells of the immunized mice may also be restimulated with antigenically distinct cells, to determine the 5 speci~icity of the cytotoxic T cell response.
Six days later the cultures are tested for cytotoxicity in a 4 hour 5~Cr-release assay (See, Palladino, et al., 1987, Cancer ~es . 47:5074-5079 and Blachere, at al., 1993, J.
Immunotherapy 14:352-356). In this assay, the mixed 10 lymphocyte culture is added to a target cell suspension to give different effector:target (E:T) ratios (usually 1:1 to 40:1). The target cells are prelabelled by incubating lx1O6 target cells in culture medium containing 200 mCi s~Cr/ml for one hour at 37~C. The cells are washed three times following 15 labeling. Each assay point (E:T ratio) is performed in triplicate and the appropriate controls incorporated to measure spontaneous 5~Cr release (no lymphocytes added to assay) and 100% release (cells lysed with detergent). After incubating the cell mixtures ~or 4 hours, the cells are 20 paletted by centrifugation at 200g for 5 minutes. The amount of 5ICr released into the supernatant is measured by a gamma counter. The percent cytotoxicity is measured as cpm in the test sample minus spontaneously released cpm divided'by the total detergent released cpm minus spontaneously released 25 cpm.
In order to block the MHC class I cascade a concentrated hybridoma supernatant derived from K-44 hybridoma cells (an anti-MHC class I hybridoma) is added to the test samples to a final concentration of 12.5~.
5.3. Formul~tio~
Hsp-antigenic molecule complexes of the invention may be formulated into pharmaceutical preparations for administration to mammals for treatment or prevention of 35 cancer or infectious diseases. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may be prepared, packaged, and W O 97/lO001 PCT~US96114557 labelled for treatment of the indicated tumor, such as human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, S lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland 10 carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung 15 carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute 20 lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia);
and polycythemia vera, lymphoma (Hodgkin's disease and non-25 Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. Alternatively, it can be labeled for treatment of the appropriate infectious disease. Alternatively, pharmaceutical compositions may be formulated for treatment of appropriate infectious diseases.
If the complex is water-soluble, then it may be formulated in an appropriate buffer, for example, phosphate buffered saline or other physiologically compatible solutions. Alternatively, if the resulting complex has poor solubility in aqueous solvents, then it may be formulated 35 with a non-ionic surfactant such as Tween, or polyethylene glycol. Thus, the compounds and'their physiologically acceptable solvates may be formulated for administration by W O 97/lO001 PCTAJS96/14557 inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, rectal administration or, in the case of tumors, directly injected into a solid tumor.
For oral administration, the pharmaceutical preparation 5 may be in liquid form, for example, solutions, syrups or suspensions, or may be presented as a drug product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives 10 such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or 15 sorbic acid). The pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl 20 methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by 25 methods well-known in the art.
Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional 30 manner.
For administration by inhalation, the compounds for use according ~o the present invention are conveniently delivered in the for~ of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, 35 e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit W O 97/lOOOl PCTAUS96/145S7 may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as 5 lactose or starch.
The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-10 dose containers, with an added preservative. Thecompositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may 15 be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa 20 butter or other glycerides.
In addition to the formulations described previously, the compounds may also be formulated as a depot preparation.
Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) 25 or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble 30 salt. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophilic drugs.
The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for 35 example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.
WO 97/lO001 PCT~US96/14557 The invention also provides kits for carrying out the therapeutic regimens of the invention. Such kits comprise in one or more containers therapeutically or prophylactically effective amounts of the hsp-antigenic molecule complexes in S pharmaceutically acceptable form. The hsp-antigenic molecule complex in a vial of a kit o~ the invention may be in the form of a pharmaceutically acceptable solution, e.g., in combination with sterile saline, dextrose solution, or buffered solution, or other pharmaceutically acceptable 10 sterile ~luid. Alternatively, the complex may be lyophilized or desiccated; in this instance, the kit optionally further comprises in a container a pharmaceutically acceptable solution (e.g., saline, dextrose solution, etc.), preferably sterile, to reconstitute the complex to form a solution for 15 injection purposes.
In another embodiment, a kit of the invention further comprises a needle or syringe, preferably packaged in sterile form, for injecting the complex, and/or a packaged alcohol pad. Instructions are optionally included for administration 20 of hsp-antigenic molecule complexes by a clinician or by the patient.
5.4 Tarqet Infectious Diseases Infectious diseases that can be treated or prevented by 25 the methods of the present invention are caused by infectious agents including, but not limited to, viruses, bacteria, fungi protozoa and parasites.
Viral diseases that can be treated or prevented by the methods of the present invention include, but are not limited 30 to, those caused by hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, 35 cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsachie virus, mumps virus, measles virus, rubella virus, W O 97/10001 PCT~US96/14557 polio virus, human immunodeficiency virus type I (HIV-I), and human immunodeficiency virus type II (HIV-II).
Bacterial diseases that can be treated or prevented by - the methods of the present invention are caused by bacteria 5 including, but not limited to, mycobacteria rickettsia, mycoplasma, neisseria and legionella.
Protozoal diseases that can be treated or prevented by the methods of the present invention are caused by protozoa including, but not limited to, leishmania, kokzidioa, and 10 trypanosoma.
Parasitic diseases that can be treated or prevented by the methods of the present invention are caused by parasites including, but not limited to, chlamydia and rickettsia.
5.5. Tarqet Cancers Cancers that can be treated or prevented by the methods of the present invention include, but not limited to human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, 20 angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, 25 adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, 30 Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, ~ craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuL~ ~, oligodendroglioma, meningioma, melanoma, 35 neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and WO 97/lOOOI PCT~US96/14557 erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia);
and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's 5 macroglo~ulinemia, and heavy chain disease. Specific examples of such cancers are described in the sections ~elow.
In a specific embodiment the cancer is metastatic. In another specific embodiment, the patient having a cancer is immunosuppressed by reason of having undergone anti-cancer 10 therapy (e.g., chemotherapy radiation) prior to administration of the hsp-antigenic molecule complexes of the invention.
5.5.1. Colorectal CancQr Metastatic to the Liver In 1992, approximately 150,000 Americans were diagnosed with colorectal cancer and more than 60,000 died as a result of colorectal metastases. At the time of their deaths, 80 percent of patients with colorectal cancer have metastatic 20 disease involving the liver, and one-half of these patients have no evidence of other (extrahepatic) metastases. Most metastatic tumors of the liver are from gastrointestinal primaries. Unfortunately, the natural history of metastatic liver lesions carries a grave prognosis and Systemic 25 chemotherapy regimens have been unable to induce significant response rates or alter length of survival (Drebin, J.A., et al., in Cu~rent Therapy In Oncology, ed. J.E. Niederhuber, B.C. Decker, Mosby, 1993, p.426).
Colorectal cancer initially spreads to regional lymph 30 nodes and then through the portal venous circulation to the liver, which represents the most common visceral site of metastasis. The symptoms that lead patients with colorectal c~n~r to seek medical care vary with the anatomical location of the lesion. For example, lesions in the ascending colon 35 frequency ulcerate, which leads to chronic blood loss in the stool.
W O 97/10001 PCT~US96/14557 Radical resection offers the greatest potential for cure in patients with invasive colorectal cancer. Before surgery, the CEA titer is determined. Radiation therapy and chemotherapy are used in patients with advanced colorectal S cancer. Results with chemotherapeutic agents (e.g., 5-fluorouracil) are mixed and fewer than 25 percent of patients experience a greater than 50 percent reduction in tumor mass (Richards, 2d., F., et al., 1986, ~. Clin. Oncol. 4:565).
Patients with widespread metastases have limited ~o survival and systemic chemotherapy has little impact in this group of patients. In addition, systemically administered chemotherapy is often limited by the severity of toxicities associated with the various agents, such as severe diarrhea, mucositis and/or myelosuppression. Other techniques, 15 including hepatic radiation, systemic chemotherapy, hepatic arterial ligation, tumor embolization and immunotherapy have all been explored, but, for the most part, have proven ineffectual in prolonging patient survival.
In a specific embodiment, the present invention provides 20 compositions and methods for enhancing tumor specific immunity in individuals suffering from colorectal cancer metastasized to the liver, in order to inhibit the progression of the neoplastic disease. Preferred methods of treating these neoplastic diseases comprise administering a 25 c~ ~ocition of autologous hsp noncovalently bound to peptide complexes, which elicits tumor-specific immunity against the tumor cells. Most specifically, the use of a composition of the invention, comprising gp96, can result in nearly complete inhibition of liver cancer growth in cancer patients, without 30 inducing toxicity and thus providing a dramatic therapeutic effect.
Accordingly, as an example of the method of the invention, gp96 is administered to a patient diagnosed with colorectal cancer, with or without liver metastasis, via one 35 of many different routes of administration, the preferred routes being subcutaneous at different anatomical sites, e.g., left arm, right arm, left belly, right belly, left W O 97/10001 PCT~US96/14557 thigh, right thigh, etc. These routes of administration are used in sequence and the site of injection i5 varied for each weekly injection as described in Section 7. The preparations and use of therapeutically effective compositions for the S prevention and treatment of primary and metastatic cancers are described in detail in the sections which follow and by way of example, infra.
5.5.2. ~ePatocQllular Carcinoma Hepatocellular carcinoma is generally a disease of the elderly in the United States. Although many factors may lead to hepatocellular carcinoma, the disease is usually limited to those persons with preexisting liver disease.
Approximately 60 to 80 percent of patients in the United 15 States with hepatocellular carcinoma have a cirrhotic liver and about four percent of individuals with a cirrhotic liver eventually develop hepatocellular carcinoma (Niederhuber, J.E., (ed.), 1993, Current Therapy in Oncology, B.C. Decker, Mosby). The risk is highest in patients whose liver disease 20 is caused by inherited hemochromatosis or hepatic B viral infection (Bradbear, R.A., et al., 1985, ~. Natl. Cancer Inst. 75:81; Beasley, R.P., et al., 1981, Lancet 2:1129).
Other causes of cirrhosis that can lead to hepatocellular carcinoma include alcohol abuse and hepatic fibrosis caused 25 by chronic administration of methotrexate. The most frequent symptoms of hepatocellular carcinoma are the development of a painful mass in the right upper quadrant or epigastrium, a~. __~nied by weight loss. In patients with cirrhosis, the development of hepatocellular carcinoma is preceded by 30 ascites, portal hypertension and relatively abrupt clinical deterioration. In most cases, abnormal values in standard liver function tests such as serum aminotransferase and alkaline phosp~atase are observed.
CT scans of the liver are used to determine the anatomic 35 distribution of hepatocellular carcinoma and also provide orientation for percutaneous needle biopsy. Approximately 70 percent of patients with hepatocellular carcinoma have an W O 97/lOOOl PCTAUS96/14557 elevated serum alpha-fetoprotein concentration (McIntire, R.R., et al., 1975, Cancer Res. 35:991) and its concentration correlates with the extent of the disease.
~ Radical resection offers the only hope for cure in 5 patients with hepatocellular carcinoma. Such operative procedures are associated with five-year survival rates of 12 to 30 percent. Liver transplantation may improve survival of some younger individuals. However, most patients are not surgical candidates because of extensive cirrhosis multifocal 10 tumor pattern or scarcity of compatible donor organs.
Chemotherapeutic agents have been administered either by intravenous route or through an intrahepatic arterial catheter. Such therapy has sometimes been combined with irradiation to the liver. Reductions in the size of 15 measurable tumors of 50~ or more have been reported in some patients treated with either systemic doxorubicin or 5-fluorouracil. However, chemotherapy often induces immunosuppression and rarely causes the tumor to disappear completely and the duration of response is short. The 20 prognosis for patients with hepatocellular carcinoma is negatively correlated with cirrhosis and metastases to the lungs or bone. Median survival for patients is only four to six months. In another specific embodiment, the present invention provides compositions and methods for enhancing 25 specific immunity in individuals suffering from hepatocellular carcinoma in order to inhibit the progression of the neoplastic disease and ultimately irradiate all preneoplastic and neoplastic cells.
5.5.3. Breast C~ncer Another specific aspect of the invention relates to the treatment of breast cancer. The American Cancer Society estimated that in 1992 180,000 American women were diagnosed with breast cancer and 46,000 succumbed to the disease 35 (Niederhuber, J.E.ed. Current Therapy in Oncology B.C.
Decker, Mosby, 1993). This makes breast cancer the second major cause of cancer death in women, ranking just behind W O 97/lO001 PCT~US96/14557 lung cancer. A disturbing fact is the observation that breast cancer has been increasing at a rate of 3 percent per year since 1980 (Niederhuber, J.E., ed. Current TheraPY in Oncoloq~, B.C. Decker, Mosby, (1993)). The treatment of 5 breast cancer presently involves surgery, radiation, hormonal therapy and/or chemotherapy. Consideration of two breast cancer characteristics, hormone receptors and disease extent, has governed how hormonal therapies and standard-dose chemotherapy are sequenced to improve survival and maintain lo or improve quality of life. A wide range of multidrug regimens have been used as adjuvant therapy in breast cancer patients, including, but not limited to combinations of 2 cyclophosphamide, doxorubicin, vincristine methotrexate, 5-fluorouracil and/or leucovorin. In a specific embodiment, 15 the present invention provides hsp compositions and methods for enhancing specific immunity to preneoplastic and neoplastic mA ~ry cells in women. The present invention also provides compositions and methods for preventing the development of neoplastic cells in women at enhanced risk for 20 breast cancer, and for inhibiting cancer cell proliferation and metastasis. These compositions can be applied alone or in combination with each other or with biological response modifiers 5.6. Autoloqous Embodime~t The specific immunogenicity of hsps derives not from hsps per se, but from the peptides bound to them. In a preferred embodiment of the invention directed to the use of autologous complexes of hsp-peptides as cancer vaccines, two 30 of the most intractable hurdles to cancer immunotherapy are circumvented. First is the possibility that human cancers, like cancers of experimental animals, are antigenically distinct. In an embodiment of the present invention, hsps chaperone antigenic peptides of the cancer cells from which 35 they are ~erived and circumvent this hurdle. Second, most current approaches to cancer immunotherapy focus on determining the CTL-recognized epitopes of cancer cell lines.
W O 97/lOOOl PCTAJS96/145S7 This approach requires the availability of cell lines and CTLs against cancers. These reagents are unavailable for an overwhelming proportion of human cancers. In an embodiment - of the present invention directed to autologous complexes of 5 hsp peptides, cancer immunotherapy does not depend on the ~ availability of cell lines or CTLs nor does it require definition of the antigenic epitopes of cancer cells. These advantages make autologous hsps noncovalently bound to peptide complexes attractive and novel immunogens against 10 cancer.
5.7. Prevention and ~reatment of Primary ~nd Metastatic Neoplastic Diseases There are many reasons why immunotherapy as provided by 15 the present invention is desired for use in cancer patients.
First, if cancer patients are immunosuppressed and surgery, with anesthesia, and subsequent chemotherapy, may worsen the immunosuppression, then with appropriate immunotherapy in the preoperative period, this immunosuppression may be prevented 20 or reversed. This could lead to fewer infectious complications and to accelerated wound healing. Second, tumor bulk is minimal ~ollowing surgery and immunotherapy is most likely to be effective in this situation. A third reason is the possibility that tumor cells are shed into the 25 circulation at surgery and effective immunotherapy applied at this time can eliminate these cells.
The preventive and therapeutic methods of the invention are directed at enhancing the immunocompetence of the cancer patient either before surgery, at or after surgery, and to 30 induce tumor-specific immunity to cancer cells, with the objective being inhibition of cancer, and with the ultimate clinical objective being total cancer regression and eradication.
PCT~S96/14557 W097tlO001 5.8. ~onitoring of BffeGts During ~n~ - Prevention And Immunotherapy with Hs~-~eptide ComPlexe~
The effect of immunotherapy with hsp-antigenic molecule complexes on development and progression of neoplastic 5 diseases can be monitored by any methods known to one skilled in the art, including but not limited to measuring: a) delayed hypersensitivity as an assessment of cellular immunity; b) activity of cytolytic T-lymphocytes in vitro; c) levels of tumor specific antigens, e.g., carcinoembryonic 10 (CEA) antigens; d) changes in the morphology of tumors using techniques such as a computed tomographic (CT) scan; and e) changes in levels of putative biomarkers of risk for a particular cancer in individuals at high risk, and f) changes in the morphology of tumors using a sonogram.
5.8.1. DelaYed HypersensitivitY 8kin Test Delayed hypersensitivity skin tests are of great value in the overall immunocompetence and cellular immunity to an antigen. Inability to react to a battery of common skin 20 antigens is termed anergy (Sato, T., et al, 1995, Clin.
Immunol . Pathol . 74:35-43).
Proper technique of skin testing requires that the antigens be stored sterile at 4~C, protected from light and reconstituted shorted before use. A 25- or 27-gauge need 25 ensures intradermal, rather than subcutaneous, administration of antigen. Twenty-four and 48 hours after intradermal administration of the antigen, the largest dimensions of both erythema and induration are measured with a ruler.
Hypoactivity to any given antigen or group of antigens is 30 confirmed by testing with higher concentrations of antigen or, in ambiguous circumstances, by a repeat test with an intermediate test.
-- ~0 --WO97/10001 PCT~S96/14557 5.8.2. Activity of Cytolytic ~-lymphocytes In Vitro 8xlO6 Peripheral blood derived T lymphocytes isolated by the Ficoll-Hypaque centrifigation gradient technique, are 5 restimulated with 4x104 mitomycin C treated tumor cells in 3ml RPMI medium containing 10% fetal calf serum. In some experiments, 33% secondary mixed lymphocyte culture supernatant or IL-2, is included in the culture medium as a source of T cell growth factors.
In order to measure the primary response of cytolytic T-lymphocytes after immunization, T cells are cultured without the stimulator tumor cells. In other experiments, T cells are restimulated with antigenically distinct cells. After six days, the cultures are tested for cytotoxity in a 4 hour lS 51Cr-release assay. The spontaneous 5~Cr-release of the targets should reach a level less than 20%. For the anti-MHC
class I blocking activity, a tenfold concentrated supernatant of W6/32 hybridoma is added to the test at a final concentration of 12.5% (Heike M., et al., J. Im~unotherapy 20 l5:l65-l74~
5.8.3. Levels of Tumor Specific Antiqens Although it may not be possible to detect unique tumor antigens on all tumors, many tumors display antigens that 25 distinguish them from normal cells. The monoclonal antibody reagents have permitted the isolation and biochemical characterization of the antigens and have been invaluable diagno~tically for distinction of transformed from nontransformed cells and for definition of the cell lineage 30 of transformed cells. The best-characterized human tumor-a~ociated antigens are the oncofetal antigens. These antigens are expressed during embryogenesis, but are absent or very difficult to detect in normal adult tissue. The prototype antigen is carcinoembryonic antigen (CEA), a 35 glycoprotein found on fetal gut an human colon cancer cells, but not on normal adult colon cells. Since CEA is shed from colon carcinoma cells and found in the serum, it was - Sl -W O 97/lO001 PCT~US96/14557 originally thought that the presence of this antigen in the serum could be used to screen patients for colon cancer.
However, patients with other tumors, such as pancreatic and breast cancer, also have elevated serum levels of CEA.
5 Therefore, monitoring the fall and rise of CEA levels in cancer patients undergoing therapy has proven useful for predicting tumor progression and responses to treatment.
Several other oncofetal antigens have been useful for diagnosing and monitoring human tumors, e.g., alpha-lO fetoprotein, an alpha-globulin normally secreted by fetal liver and yolk sac cells, is found in the serum of patients with liver and germinal cell tumors and can be used as a matter of disease status.
5.8.~. Computed Tomo~raphic (CT) Scan CT remains the choice of techniques for the accurate staging of cancers. CT has proved more sensitive and specific than any other imaging techniques for the detection of metastases.
5.8.5. Measurement of Putative Biomarkers The levels of a putative biomarker for risk of a specific cancer are measured to monitor the effect of hsp noncovalently bound to peptide complexes. For example, in 25 individuals at enhanced risk for prostate cancer, serum prostate-specific antigen (PSA) is measured by the procedure described by Brawer, M.K., et. al., 1992, J. Urol. 147:841-845, and Catalona, W.J., et al., 1993, JAMA 270:948-958; or in individuals at risk for colorectal cancer CEA is measured 30 as described above in Section 4.5.3; and in individuals at enhanced risk for breast cancer, 16-~-hydroxylation of estradiol is measured by the procedure described by Schneider, J. et al., 1982, Proc. Natl. Acad. Sci. ISA
79:3047-3051. The references cited above are incorporated by 35 reference herein in their entirety.
-W O 97/10001 PCT~US96/14557 5.8.6. 80noqr~m A Sonogram remains an alternative choice of technique for the accurate staging of cancers.
s 6. E~AMPLE: ~nMTNIgTRATION OF H8P-PEPTIDE
a) Tumor models:
Two W -induced carcinomas were studied in the C3H/HeN
mice (Ward, et al., 1989, J. Exp. Med. 170:217): (i) the 10 highly immunogenic 6138 carcinoma, and (ii) the less immunogenic 6139ST carcinoma.
b) Gp96 preparations were prepared from the 6138 and 6139SJ carcinomas by the procedures described above in Section 5.2.3. The gp96 preparations were administered 15 without adjuvants.
Methods and compositions for the prevention and treatment of primary and metastatic neoplastic diseases and infectious diseases and for eliciting an immune response in a human individual, are described. The invention is based, in part, on a newly discovered dosage regimen for administration 25 of compositions comprising complexes of hsps noncovalently bound to antigenic molecules.
"Antigenic molecule" as used herein refers to the peptides with which the hsps are endogenously associated in vivo (e.g., in infected cells or precancerous or cancerous 30 tissue) as well as exogenous antigens/immunogens (i.e., with which the hsps are not complexed in vivo) or antigenic/immunogenic fragments and derivatives thereof.
The methods of the invention comprise methods of eliciting an immune response in an individual in whom the 35 treatment or prevention of infectious diseases or cancer is desired by administering a composition comprising an effective amount of a complex, in which the complex consists W O 97/lOOOI PCT~US96tl4557 essentially of a hsp noncovalently bound to an antigenic molecule. In a preferred embodiment, the complex is autologous to the individual; that is, the complex is isolated from either from the infected cells or the cancer S cells for precancerous cells of the individual himself (e.g., preferably prepared from infected tissues or tumor biopsies of the patient). Alternatively, the complex is produced in vitro (e . g ., wherein a complex with an exogenous antigenic molecule is desired). Alternatively, the hsp and/or the 10 antigenic molecule can be isolated from the individual or from others or by recombinant production methods using a cloned hsp originally derived from the individual or from others. Exogenous antigens and fragments and derivatives (both peptide and non-peptide) thereof for use in complexing 15 with hsps, can be selected from among those known in the art, as well as those readily identified by standard immunoassays know in the art by the ability to bind antibody or MHC
molecules (antigenicity) or generate immune response (immunogenicity). Complexes of hsps and antigenic molecules 20 can be isolated from cancer or precancerous tissue of a patient, or from a cancer cell line, or can be produced in vitro (as is necessary in the embodiment in which an exogenous antigen is used as the antigenic molecule).
The invention also provides a method for measuring tumor 25 rejection in vivo in an individual, preferably a human comprising measuring the generation by the individual of MHC
Class I-restricted CD8+ cytotoxic T lymphocytes specific to the tumor.
The hsps of the present invention that can be used 30 include but are not limited to, hsp70, hsp90, gp96 alone or in combination. Preferably, the hsps are human hsps.
Heat shock proteins, which are also referred to interchangeably herein as stress proteins, useful in the practice of the instant invention can be selected from among 35 any cellular protein that satisfies any one of the following criteria. It is a protein whose intracellular concentration increases when a cell is exposed to a stressful stimuli, it W O 97/lOOOl PCT~US96/14557 is capable of binding other proteins or peptides, it is capable of releasing the bound proteins or peptides in the presence of adenosine triphosphate ~ATP) or low pH, or it is a protein showing at least 35% homology with any cellular 5 protein having any of the above properties.
The first stress proteins to be identified were the heat shock proteins (hsps). As their name implies, hsps are synthesized by a cell in response to heat shock. To date, three major families of hsp have been identified based on 10 molecular weight. 'rhe families have been called hsp60, hsp70 and hsp90 where the numbers reflect the approximate molecular weight of the stress proteins in kilodaltons. Many members of these families were found subsequently to be induced in response to other stressful stimuli including, but not 15 limited to, nutrient deprivation, metabolic disruption, oxygen radicals, and infection with intracellular pathogens.
tSee Welch, May 1993, sci enti fic American 56--64; Young, 1990, Annu. ~ev. Immunol. 8:401-420; Craig, 1993, Science 260:1902-1903; Gething, et al., 1992, Nature 355:33-45; and Lindquist, 20 et al., 1988, Annu. Rev. Genetics 22:631-677), the disclosures of which are incorporated herein by reference.
It is contemplated that hsps/stress proteins belonging to all of these three ~amilies can be used in the practice of the instant invention.
The major hsps can accumulate to very high levels in stressed cells, but they occur at low to moderate levels in cells that have been stressed. For example, the highly inducible m~ ~ lian hsp70 is hardly detectable at normal temperatures but becomes one Of the most actively synthesized 30 proteins in the cell upon heat shock (Welch, et al., 1985, J.
Cell. Biol. 101:1198-1211). In contrast, hsp90 and hsp60 proteins are abundant at normal temperatures in most, but not all, ~ ~lian cells and are further induced by heat (Lai, et al., 1984, Mol . Cell . Biol . 4:2802-10; van Bergen en 35 Henegouwen, et al., 1987, Genes Dev. 1:525-31).
Heat .shock proteins are among the most highly conserved proteins in existence. For example, DnaK, the hsp70 from E.
.
W O 97/10001 PCT~US96/14557 coli has about 50% amino acid sequence identity with hsp70 proteins from excoriates (Bardwell, et al., 1984, Proc. Natl .
Acad. Sci. 81:848-852). The hsp60 and hsp90 families also show similarly high levels of intra families conservation 5 (Hickey, et al., 1989, Mol . Cell . Biol . 9: 2615-2626; Jindal, 1989, Mol. Cell. Biol. 9:2279-2283). In addition, it has been discovered that the hsp60, hsp70 and hsp90 families are composed of proteins that are related to the stress proteins in sequence, for example, having greater than 35% amino acid 10 identity, but whose expression levels are not altered by stress. Therefore it is contemplated that the definition of stress protein, as used herein, embraces other proteins, muteins, analogs, and variants thereof having at least 35% to 55%, preferably 55% to 75%, and most preferably 75~ to 85%
15 amino acid identity with members of the three families whose expression levels in a cell are enhanced in response to a stressful stimulus. The purification of stress proteins belonging to these three families is described below.
The immunogenic hsp-peptide complexes of the invention 20 may include any complex containing an hsp and a peptide that is capable of inducing an immune response in a mammal. The peptides are preferably non covalently associated with the hsp. Preferred complexes may include, but are not limited to, hsp60-peptide, hsp70-peptide and hsp90-peptide complexes.
25 For example, an hsp called gp96 which is present in the endoplasmic reticulum of eukaryotic cells and is related to the cytoplasmic hsp90's can be used to generate an effective vaccine containing a gp96-peptide complex.
Although the hsps can be allogeneic to the patient, in a 30 preferred embodiment, the hsps are autologous to (derived from) the patient to whom they are administered. The hsps and/or antigenic molecules can be purified from natural sources, chemically synthesized, or recombinantly produced.
The invention provides methods for determining doses for 35 human cancer immunotherapy by evaluating the optimal dose of hsp noncovalently bound to peptide complexes in experimental tumor models and extrapolating the data. Specifically, a W O 97/10001 PCT~US96/14557 scaling factor not exceeding a fifty fold increase over the effective dose estimated in animals, is used as the optimal prescription method for cancer immunotherapy or vaccination in human subjects.
s The invention provides combinations of compositions which enhance the immunocompetence of the host individual and elicit specific immunity against infectious agents or specific immunity against preneoplastic and neoplastic cells.
The therapeutic regimens and pharmaceutical compositions of 10 the invention are described below. These compositions have the capacity to prevent the onset and progression of infectious diseases and prevent the development of tumor cells and to inhibit the growth and progression of tumor cells indicating that such compositions can induce specific 15 immunity in infectious diseases and cancer immunotherapy.
Hsps appear to induce an inflammatory reaction at the tumor site and ultimately cause a regression of the tumor burden in the cancer patients treated. Cancers which can be treated with complexes of hsps noncovalently bound to 20 antigenic molecules include, but are not limited to, human sarcomas and carcinomas.
Accordingly, the invention provides methods of preventing and treating cancer in an individual comprising administering a composition which stimulates the 25 immunocompetence of the host individual and elicits specific immunity against the preneoplastic and/or neoplastic cells.
As used herein, "preneoplastic" cell refers to a cell which is in transition from a normal to a neoplastic form; and morphological evidence, increasingly supported by molecular 30 biologic studies, indicates that preneoplasia progresses through multiple steps. Non-neoplastic cell growth commonly consists of hyperplasia, metaplasia, or most particularly, dysplasia (for review of such abnormal growth conditions (See Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B.
35 Saunders Co., Philadelphia, pp. 68-79~. Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant , W O 97/10001 PCT~US96/14557 alteration in structure or function. As but one example, endometrial hyperplasia often precedes endometrial cancer.
Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for 5 another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic epithelium.
Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of 10 non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic 15 irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder.
Although preneoplastic lesions may progress to neoplasia, they may also remain stable for long periods and may even regress, particularly if the inciting agent is removed or if 20 the lesion succumbs to an immunological attack by its host.
The therapeutic regimens and pharmaceutical compositions of the invention may be used with additional immune response enhancers or biological response modifiers including, but not limited to, the cytokines IFN-~, IFN-~, IL-2, IL-4, IL-6, 25 TNF, or other cytokine affecting immune cells. In accordance with this aspect of the invention, the complexes of the hsp and antigenic molecule are administered in combination therapy with one or more of these cytokines.
The invention further relates to administration of 30 complexes of hsp-antigenic molecules to individuals at enhanced risk of cancer due to familial history or environmental risk factors.
5.1. Dos~qe Reqimens It was established in experimental tumor models (Blachere et al., 1993, J. Immunotherapy 14:352-356) that the lowest dose of hsp noncovalently bound to peptide complexes CA 02232048 l99X-03-13 which produced tumor regression in mice was between 10 and 25 microgram/mouse weighing 20-25g which is equal to 25mg/25g =
lmg/kg. Prior art methods extrapolate to human dosages based on body weight and surface area. For example, prior art s methods o~ extrapolating human dosage based on body weight can be carried out as follows: since the conversion factor for converting the mouse dosage to human dosage is Dose Human per kg = Dose Mouse per kg x 12 (See Freireich, E.J., et al., 1966, Cancer Chemotherap. ~ep. 50:219-244), the effective 10 dose of hsp-peptide complexes in humans weighing 7Okg should be lmg/kg - 12 x 70, i.e., about 6mg (5.8mg).
Drug doses are also given in milligrams per square meter of body surface area because this method rather than body weight achieves a good correlation to certain metabolic and 15 excretionary functions (Shirkey, H.C., 1965, ~AMA 193:443).
Moreover, body surface area can be used as a common denominator for drug dosage in adults and children as well as in differemt animal species as indicated below in Table 1 (Freireich, E.J., et al., 1966, Cancer Chemotherap. Rep.
20 50:219-244~.
~ABLE 1 REPRE8ENTATIVE 8~RFACE AREA To WEIG~T
R~TIOS (km) FOR VARIOUg SPECIESI
Species Body Weight Surface Area km Factor (kg) (Sqm) Mouse 0.02 0.0066 3.0 Rat 0.15 0.025 5.9 Monkey 3.0 0.24 12 30Dog 8.0 0.40 20 Human, Child 20 0.80 25 Adult 60 1.6 37 Example: To express a mg/kg dose in any given species as the equivalent mg/sq m dose, multiply the Freireich, et al., 1966, Cancer Chemotherap. Rep. 50:
219-244.
W O97/lOOOI PCTAJS96/14557 dose by the appropriate km factor. In adult human, lOOmg/kg is equivalent to 100 mg/kg x 37 kg/sq m = 3700 mg/sq m.
In contrast to both of the above-described prior art 5 methods of determining dosage levels, the present invention provides dosages of the purified complexes of hsps and antigenic molecules that are much smaller than the dosages estimated by the prior art. For example, according to the invention, an amount of hsp70- and/or gp96-antigenic molecule 10 complexes is administered that is in the range of about lO
microgram to about 600 micrograms for a human patient, the preferred human dosage being the same as used in a 25g mouse, i.e., in the range of lO-100 micrograms. The dosage for hsp-90 peptide complexes in a human patient provided by the 15 present invention is in the range of about 50 to 5,000 micrograms, the preferred dosage being lO0 micrograms.
The doses recited above are preferably given once weekly for a period of about 4-6 weeks, and the mode or site of administration is preferably varied with each administration.
20 In a preferred example, subcutaneous administrations are given, with each site of administration varied sequentially.
Thus, by way of example and not limitation, the first injection may be given subcutaneously on the left arm, the second on the right arm, the third on the left belly, the 25 fourth on the right belly, the fifth on the left thigh, the sixth on the right thigh, etc. The same site may be repeated after a gap of one or more injections. Also, split injections may be given. Thus, for example, half the dose may be given in one site and the other half on an other site 30 on the same day.
Alternatively, the mode of administration is sequentially varied, e.g., weekly injections are given in sequence subcutaneously, intramuscularly, intravenously or intraperitoneally.
~ 35 After 4-6 weeks, further injections are preferably given at two-week intervals over a period of time of one month.
Later injections may be given monthly. The pace of later W O 97/10001 PCT~US96/14557 injections may be modified, depending upon the patient's clinical progress and responsiveness to the immunotherapy.
The invention is illustrated by non-limiting examples in Sections 6 and 7.
5.2. Therapeutic C _~sitions for Immune ResPonses to C~nc~
The compositions comprising hsp noncovalently bound to antigenic molecules are administered to elicit an effective 10 specific immune response to the complexed antigenic molecules (and not to the hsp).
In a preferred embodiment, non-covalent complexes of hsp70, hsp90 and gp96 with peptides are prepared and purified postoperatively from tumor cells obtained from the cancer 15 patient.
In accordance with the methods described herein, immunogenic or antigenic peptides that are endogenously complexed to hsps or MHC antigens can be used as antigenic molecules. For example, such peptides may be prepared that 20 stimulate cytotoxic T cell responses against different tumor antigens (e.g., tyrosinase, gplOO, melan-A, gp75, mucins, etc.) and viral proteins including, but not limited to, proteins of immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-II), hepatitis type A, 25 hepatitis type B, hepatitis type C, influenza, Varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, 30 huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus and polio virus. In the embodiment wherein the antigenic molecules are peptides noncovalently complexed to hsps in vivo, the complexes can be isolated from cells, or alternatively, produced in vitro from purified preparations 35 each of hsps and antigenic molecules.
In another specific embodiment, antigens of cancers (e.g., tumors) or infectious agents (e.g., viral antigen, bacterial antigens, etc.) can be obtained by purification from natural sources, by chemical synthesis, or recombinantly, and, through in vitro procedures such as that described below, noncovalently complexed to hsps.
In an embodiment wherein the hsp-antigenic molecule complex to be used is a complex that is produced in vivo in cells, exemplary purification procedures such as described in Sections 5.2.1-5.2.3 below can be employed. Alternatively, in an embodiment wherein one wishes to use antigenic 10 molecules by complexing to hsps in vitro, hsps can be purified for such use from the endogenous hsp-peptide complexes in the presence of ATP or low pH (or chemically synthesized or recombinantly produced). The protocols described herein may be used to isolate hsp-peptide 15 complexes, or the hsps alone, from any eukaryotic cells for example, tissues, isolated cells, or immortalized eukaryote cell lines infected with a preselected intracellular pathogen, tumor cells or tumor cell lines.
5.2.1. Prepar~tion and Purification of HsP 70-DeDtide Complexes The purification of hsp70-peptide complexes has been described previously, see, for example, Udono et al., 1993, J. Exp. Med. 178 :1391-1396. A procedure that may be used, 25 presented by way of example but not limitation, is as follows:
Initially, tumor cells are suspended in 3 volumes of lX
Lysis buffer consisting of 5mM sodium phosphate buffer, pH 7, lSOmM NaCl, 2mM CaCl2, 2mM MgCl2 and lmM phenyl methyl 30 sulfonyl fluoride (PMSF). Then, the pellet is sonicated, on ice, until >99% cells are lysed as determined by microscopic ~Y~ tion. As an alternative to sonication, the cells may be lysed by mechanical shearing and in this approach the cells typically are resuspended in 30mM sodium bicarbonate - 35 pH 7.5, lmM PMSF, incubated on ice for 20 minutes and then homogenized in a dounce homogenizer until >95% cells are lysed.
PCT~S96/14557 Then the lysate is centrifuged at 1,OOOg for 10 minutes to remove unbroken cells, nuclei and other cellular debris.
The resulting supernatant is recentrifuged at lOO,OOOg for 90 minutes, the supernatant harvested and then mixed with Con 5 A Sepharose equilibrated with phosphate buffered saline (PBS) containing 2mM Ca2+ and 2mM Mg~+. When the cells are lysed by mechanical shearing the supernatant is diluted with an equal volume of 2X lysis buffer prior to mixing with Con A
Sepharose. The supernatant is then allowed to bind to the 10 Con A Sepharose for 2-3 hours at 4~C. The material that fails to bind is harvested and dialyzed for 36 hours (three times, 100 volumes each time) against lOmM Tris-Acetate pH 7.5, O.lmM EDTA, lOmM NaCl, lmM PMSF. Then the dialyzate is centrifuged at 17,000 rpm (Sorvall SS3~ rotor) for 20 15 minutes. Then the resulting supernatant is harvested and applied to a Mono Q FPLC column equilibrated in 2OmM Tris-Acetate pH 7.5, 20mM NaCl, O.lmM EDTA and 15mM 2-mercaptoethanol. The column is then developed with a 2OmM to SOOmM NaCl gradient and then eluted fractions fractionated by 20 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and characterized by immunoblotting using an appropriate anti-hsp70 antibody (such as from clone N27F3-4, from StressGen).
Fractions strongly immunoreactive with the anti-hsp70 25 antibody are pooled and the hsp70-peptide complexes precipitated with ammonium sulfate; specifically with a 50%-70% ammonium sulfate cut. The resulting precipitate is then harvested by centrifugation at 17,000 rpm (SS34 Sorvall rotor) and washed with 70~ ammonium sulfate. The washed 30 precipitate is then solubilized and any residual ammonium sulfate removed by gel filtration on a SephadexR G25 column (Pharmacia). If necessary the hsp70 preparation thus obtained can be repurified through the Mono Q FPCL Column as described above.
The hsp70-peptide complex can be purified to apparent homogeneity using this method. Typically lmg of hsp70-peptide complex can be purified from lg of cells/tissue.
W O 97/lOOOI PCT~US96/14557 The present invention further describes a new and rapid method for purification of hsp70-peptide complexes. This improved method comprises contacting cellular proteins with ADP or a nonhydrolyzable analog of ATP affixed to a solid 5 substrate, such that hsp70 in the lysate can bind to the ADP
or nonhydrolyzable ATP analog, and eluting the bound hsp70.
A preferred method uses column chromatography with ADP
affixed to a solid substratum (e.g., ADP-agarose). The resulting hsp70 preparations are higher in purity and devoid 10 of contaminating peptides. The hsp70 yields are also increased significantly by about more than 10 fold.
Alternatively, chromatography with nonhydrolyzable analogs of ATP, instead of ADP, can be used for purification of hsp70-peptide complexes. By way of example but not limitation, 15 purification of hsp70-peptide complexes by ADP-agarose chromatography was carried out as described in Example Section 9.
5.2.2. Preparation and Purification of Hsp 90-Peptide Complexes A procedure that can be used, presented by way of example and not limitation, is as follows:
Initially, tumor cells are suspended in 3 volumes of lX
Lysis buffer consisting of 5mM sodium phosphate buffer (pH7), 25 150mM NaCl, 2mM CaCl2, 2mM MgCl2 and lmM phenyl methyl sulfonyl fluoride (PMSF). Then, the pellet is sonicated, on ice, until >99~ cells are lysed as determined by microscopic ~;nation. As an alternative to sonication, the cells may be lysed by mechanical shearing and in this approach the 30 cells typically are resuspended in 30mM sodium bicarbonate pH 7.5, lm~ PMSF, incubated on ice for 20 minutes and then homogenized in a dounce homogenizer until >95% cells are lysed.
Then the lysate is centrifuged at l,OOOg for 10 minutes 35 to remove unbroken cells, nuclei and other cellular debris.
The resulting supernatant is recentrifuged at lOO,OOOg for 90 minutes, the supernatant harvested and then mixed with Con A
W O 97/lOOOI PCTnUS96/14557 Sepharose equilibrated with PBS containing 2mM Ca2+ and 2mM
Mg2+. When the cells are lysed by mechanical shearing the supernatant is diluted with an equal volume of 2X Lysis buffer prior to mixing with Con A Sepharose. The supernatant 5 is then allowed to bind to the Con A Sepharose for 2-3 hours at 4~C. The material that fails to bind is harvested and dialyzed for 36 hours (three times, 100 volumes each time) against lOmM Tris-Acetate pH 7.5, 0.lmM EDTA, lOmM NaCl, lmM
PMSF. Then the dialyzate is centrifuged at 17,000 rpm lo (Sorvall SS34 rotor) for 20 minutes. Then the resulting supernatant is harvested and applied to a Mono Q FPLC column equilibrated with lysis buffer. The proteins are then eluted with a salt gradient of 200mM to 600mM NaCl.
The eluted fractions are fractionated by SDS-PAGE and 15 fractions containing the hsp90-peptide complexes identified by immunoblotting using an anti-hsp90 antibody such as 3G3 (Affinity Bioreagents). Hsp90-peptide complexes can be purified to apparent homogeneity using this procedure.
Typically, 150-200 ~g of hsp90-peptide complex can be 20 purified from lg of cells/tissue.
5.2.3. Preparation ~nd Purification o~ qD96-DeDtide Complexes A procedure that can be used, presented by way of 25 example and not limitation, is as follows:
A pellet of tumors is resuspended in 3 volumes of buffer consisting of 30mM sodium bicarbonate buffer (pH 7.5) and lmM
PMSF and the cells allowed to swell on ice 20 minutes. The cell pellet then is homogenized in a Dounce homogenizer (the 30 appropriate clearance of the homogenizer will vary according to each cells type) on ice until >95% cells are lysed.
The lysate is centrifuged at 1,OOOg for 10 minutes to remove unbroken cells, nuclei and other debris. The supernatant from this centrifugation step then is 35 recentrifuged at lOO,OOOg for 90 minutes. The gp96-peptide complex can be purified either from the 100,000 pellet or from the supernatant.
W O 97/lOOOl When purified from the supernatant, the supernatant is diluted with equal volume of 2X lysis buffer and the supernatant mixed for 2-3 hours at 4~C with con a sepharose equilibrated with PBS containing 2mM Ca2+ and 2mM Mg2+ Then, 5 the slurry is packed into a column and washed with lX lysis buffer until the OD280 drops to baseline. Then, the column is washed with 1/3 column bed volume of 10% ~-methyl mannoside (~-MM) dissolved in PBS contA;~ing 2mM Ca2+ and 2mM Mg2+, the column sealed with a piece of parafilm, and incubated at 37~C
10 for 15 minutes. Then the column is cooled to room temperature and the parafilm removed from the bottom of the column. ~ive column volumes of the ~-MM buffer are applied to the column and the eluate analyzed by SDS-PAGE. Typically the resulting material is about 60-95% pure, however this 15 depends upon the cell type and the tissue-to-lysis buffer ratio used. Then the sample is applied to a Mono Q FPLC
column (Pharmacia) e~uilibrated with a buffer containing 5mM
sodium phosphate, pH 7. The proteins then are eluted from the column with a O-lM NaCl gradient and the gp96 fraction 20 elutes between 400mM and 550mM NaCl.
The procedure, however, may be modified by two additional steps, used either alone or in combination, to consistently produce apparently homogeneous gp96-peptide complexes. One optional step involves an ammonium sulfate 25 precipitation prior to the Con A purification step and the other optional step involves DEAE-Sepharose purification after the Con A purification step but before the Mono Q FPLC
step.
In the first optional step, the supernatant resulting 30 from the lOO,OOOg centrifugation step is brought to a final concentration of 50% ammonium sulfate by the addition of ammonium sulfate. The ammonium sulfate is added slowly while gently stirring the solution in a beaker placed in a tray of ice water. The solution is stirred from about 1/2 to 12 35 hours at 4~C and the resulting solution centrifuged at 6,000 rpm (Sorvall SS34 rotor). The supernatant resulting from this step is removed, brought to 70~ ammonium sulfate _ _ _ ~ _ WO 97/iOOOl PCTAJS96/14557 saturation by the addition of ammonium sulfate solution, and centrifuged at 6,000 rpm (Sorvall SS34 rotor). The resulting pellet from this step is harvested and suspended in PBS
containing 70% ammonium sulfate in order to rinse the pellet.
5 This mixture is centrifuged at 6,000 rpm (Sorvall SS34 rotor) and the pellet dissolved in PBS containing 2mM Ca2+ and Mg2+.
Undissolved material is removed by a brief centrifugation at 15,000 rpm (Sorvall SS34 rotor). Then, the solution is mixed with Con A Sepharose and the procedure followed as before.
In the second optional step, the gp96 containing fractions eluted from the Con A column are pooled and the buffer exchanged for 5mM sodium phosphate buffer, pH 7, 300mM
NaCl by dialysis, or preferably by buffer exchange on a Sephadex G25 column. After buffer exchange, the solution is 15 mixed with DEAE-Sepharose previously equilibrated with 5mM
sodium phosphate buffer, pH 7, 300mM NaCl. The protein solution and the beads are mixed gently for 1 hour and poured into a column. Then, the column is washed with 5mM sodium phosphate buffer, pH 7, 300mM NaCl, until the absorbance at 20 280nM drops to baseline. Then, the bound protein is eluted from the column with five volumes of 5mM sodium phosphate buffer, pH 7, 700mM NaCl. Protein containing fractions are pooled and diluted with 5mM sodium phosphate buffer, pH 7 in order to lower the salt concentration to 175mM. The 25 resulting material then is applied to the Mono Q FPLC column (Pharmacia) equilibrated with 5mM sodium phosphate buffer, pH 7 and the protein that binds to the Mono Q FPLC column (Pharmacia) is eluted as described before.
It is appreciated, however, that one skilled in the art 30 may assess, by routine experimentation, the benefit of incorporating the second optional step into the purification protocol. In addition, it is appreciated also that the benefit of adding each of the optional steps will depend upon the source of the starting material.
When the gp96 fraction is isolated from the lOO,OOOg pellet, the pellet is suspended in 5 volumes of PBS
containing either 1% sodium deoxycholate or 1% oxtyl WO97/l0001 PCT~S96/14557 glucopyranoside (but without the Mg2+ and Ca2+) and incubated on ice for 1 hour. The suspension is centrifuged at 20,000g for 30 minutes and the resulting supernatant dialyzed against several changes of PBS (also without the Mg2+ and Ca2~) to 5 remove the detergent. The dialysate is centrifuged at 100,000g for 90 minutes, the supernatant harvested, and calcium and magnesium are added to the supernatant to give final concentrations of 2mM, respectively. Then the sample is purified by either the unmodified or the modi~ied method 10 for isolating gp96-peptide complex from the 100,000g supernatant, see above.
The gp96-peptide complexes can be purified to apparent homogeneity using this procedure. About 10-20~g of gp96 can be isolated from lg cells/tissue.
Infectious Disease In an alternative embodiment wherein it is desired to treat a patient having an infectious disease the above-described methods in Sections 5.2.1 - 5.2.3 are used to 20 isolate hsp-peptide complexes from cells infected with an infectious organism, e.g., of a cell line or from a patient.
Such infectious organisms include but are not limited to, viruses, bacterial, protozoa, fungi, and parasites as described in detail in Section 5.2.4 below.
5.2.~. Isolation of Antiqenic/I~munoqenic Components It has been found that antigenic peptides and/or components can be eluted from hsp-complexes either in the 30 presence of ATP or low pH. These experimental conditions may be used to isolate peptides and/or antigenic components ~rom cells which may contain potentially useful antigenic determinants. Once isolated, the amino acid sequence of each antigenic peptide may be determined using conventional amino 35 acid sequencing methodologies. Such antigenic molecules can then be produced by chemical synthesis~or recombinant methods, purified, and complexed to hsps in vitro.
W O 97/10001 PCTrUS96/14557 Similarly, it has been found that potentially immunogenic peptides may be eluted from MHC-peptide complexes using techniques well know in the art (Falk, K. et al., 1990 Nature 348:248-251; Elliott, T., et al., 1990, Nature 5 348:195-197; Falk, K., et al., 1991, Nature 351:290-296).
Thus, potentially immunogenic or antigenic peptides may be isolated from either endogenous stress protein-peptide complexes or endogenous MHC-peptide complexes for use subseguently as antigenic molecules, by complexing in vi tro 10 to hsps. Exemplary protocols for isolating peptides and/or antigenic components from either of the these complexes are set forth below in Sections 5.2.4.1 and 5.2.4.2.
5.2.4.1 Peptide~ From 8tres~ Protein-Peptide Complexes Two methods may be used to elute the peptide from a stress protein-peptide complex. One approach involves incubating the stress protein-peptide complex in the presence of ATP. The other approach involves incubating the complexes in a low pH buffer.
Briefly the complex of interest is centrifuged through a Centricon lo assembly (Millipore) to remove any low molecular weight material loosely associated with the complex. The large molecular weight fraction may be removed and analyzed by SDS-PAGE while the low molecular weight may be analyzed by 25 HPLC as described below. In the ATP incubation protocol, the stress protein-peptide complex in the large molecular weight fraction is incubated with 10mM ATP for 30 minutes at room temperature. In the low pH protocol, acetic acid or trifluoro acetic acid is added to the stress protein-peptide 30 complex to give a final concentration of 10~ (vol/vol) and the mixture incubated at room temperature or in a boiling water bath or any temperature in between, for 10 minutes (See, Van Bleek, et al., 1990, Nature 348:213-216; and Li, et al., 1993, EMBO Journal 12: 3143-3151).
The resulting samples are centrifuged through an Centricon 10 assembly as mentioned previously. The high and low molecular weight fractions are recovered. The remaining W O 97/lO001 PCT~US96/14557 large molecular weight stress protein-peptide complexes can be reincubated with ATP or low pH to remove any remaining peptides.
The resulting lower molecular weight fractions are 5 pooled, concentrated by evaporation and dissolved in 0.1%
trifluoroacetic acid (TFA). The dissolved material is then fractionated by reverse phase high pressure liquid chromatography (HPLC) T using for example a VYDAC CIB reverse phase column equilibrated with 0.1% TFA. The bound material 10 is then eluted at a flow rate of about 0.8 ml/min by developing the column with a linear gradient of 0 to 80%
acetonitrile in 0.1% TFA. The elution of the peptides can be monitored by OD2l0 and the fractions containing the peptides collected.
5.2.4.2 PePtide~ from M~C-peptid~ Complexes.
The isolation of potentially immunogenic peptides from MHC molecules is well known in the art and so is not described in detail herein (See, Falk, et al., 1990, Nature 20 348:248-251; Rotzsche, at al., 1990, Na~ure 348:252-254;
~lliott, et al., 1990, Nature 348:191-197; Falk, et al., 1991, Nature 351:290--296; Demotz, et al., 1989, Nature 343:682-684; Rotzsche, et al., 1990, Science 249:283-287), the disclosures of which are incorporated herein by 25 reference.
Briefly, MHC-peptide complexes may be isolated by a conventional immunoaffinity procedure. The peptides then may be eluted from the MHC-peptide complex by incubating the complexes in the presence of about 0.1% TFA in acetonitrile.
30 The eluted peptides may be fractionated and purified by reverse phase HPLC, as before.
The amino acid sequences of the eluted peptides may be determined either by manual or automated amino acid sequencing techniques well known in the art. Once the amino 35 acid sequence of a potentially protective peptide has been determined the peptide may be synthesized in any desired W O 97/lOOOI PCTrUS96/145~7 amount using conventional peptide synthesis or other protocols well known in the art.
Peptides having the same amino acid sequence as those isolated above may be synthesized by solid-phase peptide 5 synthesis using procedures similar to those described by Merrifield, 1963, J. Am. Chem. Soc., 85:2149. During synthesis, N-~-protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C-terminal and to an insoluble polymeric 10 support i e., polystyrene beads. The peptides are synthesized by linking an amino group of an N-~-deprotected amino acid to an ~-car~oxy group of an N-~-protected amino acid that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide. The attachment of a free 15 amino group to the activated carboxyl leads to peptide bond formation. The most commonly used N-~-protecting groups include Boc which is acid labile and Fmoc which is base labile.
Briefly, the C-terminal N-~-protected amino acid is 20 first attached to the polystyrene beads. The N-~-protecting group is then removed. The deprotected ~-amino group is coupled to the activated ~-carboxylate group of the next N-~-protected amino acid. The process is repeated until the desired peptide is synthesized. The resulting peptides are 25 then cleaved from the insoluble polymer support and the amino acid side chains deprotected. Longer peptides can be derived by condensation of protected peptide fragments. Details of appropriate chemistries, resins, protecting groups, protected amino acids and reagents are well known in the art and so are 30 not discussed in detail herein (See, Atherton, et al., 1989, Solid Phase Peptide Synthesis: A Practical Approach, IRL
Press, and Bodanszky, 1993, Peptide Chemistry, A Practical Textbook, 2nd Ed., Springer-Verlag).
Purification of the resulting peptides is accomplished 35 using conventional procedures, such as preparative HPLC using gel permeation, partition and/or ion exchange chromatography.
W O 97/10001 PCT~US96/14557 The choice of appropriate matrices and buffers are well known in the art and so are not described in detail herein.
5.2.5 Exoqenous Antiqenic Molecules Antigens or antigenic portions thereof can be selected for use as antigenic molecules, for complexing to hsps, from among those known in the art or determined by immunoassay to be able to bind to antibody or MHC molecules (antigenicity) or generate immune response (immunogenicity). To determine 10 immunogenicity or antigenicity by detecting binding to antibody, various immunoassays known in the art can be used, including but not limited to competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich"
15 immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in vivo immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, immunoprecipitation reactions, agglutination assays (e.g., gel agglutination 20 assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary 25 antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labelled. Many means are known in the art for detecting binding in an immunoassay and are envisioned for use. In one embodiment for detecting 30 immunogenicity, T cell-mediated responses can be assayed by stAn~d methods, e.g., in vitro cytoxicity assays or in vivo delayed-type hypersensitivity assays.
Potentially useful antigens or derivatives thereof for use as antigenic molecules can also be identified by various 35 criteria, such as the antigen's involvement in neutralization of a pathogen's infectivity (wherein it is desired to treat or prevent infection by such a pathogen) (Norrby, 1985, Summary, in Vaccines 85, ~erner, et al. (eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp.
388-389), type or group specificity, recognition by patients' antisera or immune cells, and/or the demonstration of 5 protective effects of antisera or immune cells specific for the antigen. In addition, where it is desired to treat or prevent a disease caused by pathogen, the antigen's encoded epitope should preferably display a small or no degree of antigenic variation in time or amongst different isolates of 10 the same pathogen.
Preferably, where it is desired to treat or prevent cancer, known tumor-specific antigens or fragments or derivatives thereof are used. For example, such tumor specific or tumor-associated antigens include but are not 15 limited to KS 1/4 pan-carcinoma antigen (Perez and Walker, 1990, J. Inununol. 142:3662-3667; Bumal, 1988, Hybridoma 7(4):407-415); ovarian carcinoma antigen (CA125) (Yu, et al., 1991, Cancer Res. 51(2):468-475); prostatic acid phosphate (Tailer, et al., 1990, Nucl. Acids Res. 18(16):4928);
20 prostate specific antigen (Henttu and Vihko, 1989, Biochem.
Biophys. Res. Comm. 160(2):903-910; Israeli, et al., 1993, Cancer Res. 53:227-230); melanoma-associated antigen p97 (Estin, et al., 1989, J. Natl. Cancer Inst. 81(6):445-446);
melanoma antigen gp75 (Vijayasardahl, et al., 1990, J. Exp.
25 Med. 171(4):1375-1380); high molecular weight melanoma antigen (Natali, et al., 1987, Cancer 59:55-63) and prostate specific m~embrane antigen.
In a specific embodiment, an antigen or fragment or derivative thereof specific to a certain tumor is selected 30 for complexing to hsp and subsequent administration to a patient having that tumor.
Preferably, where it is desired to treat or prevent viral diseases, molecules comprising epitopes of known viruse~ are used. For example, such antigenic epitopes may 35 be prepared from viruses including, but not limited to, hepatitis ~ype A hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, herpes simplex type I (HSV-W O97/lOO01 PCTAJS96/14557 I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsachie virus, mumps virus, measles virus, 5 rubella virus, polio virus, human immunodeficiency virus type I (HIV-I), and human immunodeficiency virus type II (HIV-II).
Preferably, where it is desired to treat or prevent bacterial infections, molecules comprising epitopes of known bacteria are used. For example, such antigenic epitopes may 10 be prepared from bacteria including, but not limited to, mycobacteria rickettsia, mycoplasma, neisseria and legionella.
Preferably, where it is desired to treat or prevent protozoal infectious, molecules comprising epitopes of known 15 protozoa are used. For example, such antigenic epitopes may be prepared from protozoa including, but not limited to, leishmania, kokzidioa, and trypanosoma.
Preferably, where it is desired to treat or prevent parasitic infectious, molecules comprising epitopes of known 20 parasites are used. For example, such antigenic epitopes may be from parasites including, but not limited to, chlamydia and rickettsia.
5 . 2 . 6 In Vitro Production of ~tress Protein-Anti~enic Molecule Complexes In an embodiment in which complexes of hsps and the peptides with which they are endogenously associated in vivo are not employed, complexes of hsps to antigenic molecules are produced in vitro. As will be appreciated by those 30 skilled in the art, the peptides either isolated by the aforementioned procedures or chemically synthesized or recombinantly produced may be reconstituted with a variety of naturally purified or recombinant stress proteins in vitro to generate immunogenic non-covalent stress protein-antigenic 35 molecule complexes. Alternatively, exogenous antigens or antigenic/immunogenic fragments or derivatives thereof can be noncovalently complexed to stress proteins for use in the W O 97/10001 PCT~US96/14557 immunotherapeutic or prophylactic vaccines of the invention.
A preferred, exemplary protocol for noncovalently complexing a stress protein and an antigenic molecule in vitro is discussed below.
Prior to complexing, the hsps are pretreated with ATP or low pH to remove any peptides that may be associated with the hsp of interest. When the ATP procedure is used, excess ATP
is removed from the preparation by the addition of apyranase as described by Levy, et al., 1991, Cell 67: 265-274 . When 10 the low pH procedure is used, the buffer is readjusted to neutral pH by the addition of pH modifying reagents.
The antigenic molecules (l~g) and the pretreated hsp (9~g) are admixed to give an approximately 5 antigenic molecule: 1 stress protein molar ratio. Then, the mixture is 15 incubated for 15 minutes to 3 hours at 4~ to 45~C in a suitable binding buffer such as one containing 20mM sodium phosphate, pH 7.2, 350mM NaCl, 3mM MgCl2 and lmM phenyl methyl sulfonyl fluoride (PMSF). The preparations are centrifuged through Centricon 10 assembly (Millipore) to remove any 20 unbound peptide. The association of the peptides with the stress proteins can be assayed by SDS-PAGE. This is the preferred method for in vitro complexing of peptides isolated from MHC-peptide complexes of peptides disassociated from endogenous hsp-peptide complexes.
In an alternative embodiment of the invention, preferred for producing complexes of hsp70 to exogenous antigenic molecules such as proteins, 5-10 micrograms of purified hsp is incubated with equimolar quantities of the antigenic molecule in 20mM sodium phosphate buffer pH 7.5, 0.5M NaCl, 30 3mM MgCl2 and lmM ADP in a volume of 100 microliter at 37~C
for 1 hr. This incubation mixture is further diluted to lml in phosphate-buffered saline.
In an alternative embodiment of the invention, preferred for producing complexes of gp96 or hsp90 to peptides, 5 -10 35 mi~loy~ams of purified gp96 or hsp90 is incubated with equimolar or excess quantities of the antigenic peptide in a suitable buffer such as one containing 20mM sodium phosphate W O 97/lO001 PCTrUS96/14557 buffer pH 7.5, 0.5M NaCl, 3nM MgC12 at 60-65OC ~or 5-20 min.
This incubation mixture is allowed to cool to room temperature and centrifuged one or more times if necessary, through Centricon 10 assembly (Millipore) to remove any 5 unbound peptide.
Following complexing, the immunogenic stress protein-antigenic molecule complexes can optionally be assayed in vitro using for example the mixed lymphocyte target cell assay (MLTC) described below. Once immunogenic complexes 10 have been isolated they can be optionally characterized further in animal models using the preferred administration protocols and excipients discussed below.
5.2.7 Determination of Immunogenicity of 8tre~ Protein-Peptide Complexe~
The purified stress protein-antigenic molecule complexes can be assayed for immunogenicity using the mixed lymphocyte target culture assay (MLTC) well known in the art.
By way of example but not limitation, the following 20 procedure can be used. Briefly, mice are injected subcutaneously with the candidate stress protein-antigenic molecule complexes. Other mice are injected with either other stress protein peptide complexes or whole infected cells which act as positive controls for the assay. The mice 25 are injected twice, 7-10 days apart. Ten days after the last i -lnization, the spleens are removed and the lymphocytes released. The released lymphocytes may be restimulated subsequently in vitro by the addition of dead cells that expressed the complex of interest.
For example, 8X106 immune spleen cells may be stimulated with 4x104 mitomycin C treated or ~-irradiated (5-10,000 rads) infected cells (or cells transfected with an appropriate gene, as the case may be) in 3ml RPMI medium containing 10%
fetal calf serum. In certain cases 33% secondary mixed 35 lymphocyte culture supernatant may be included in the culture medium as a source of T cell growth factors (See, Glasebrook, et al., 1980, J. Exp. Med. 151:876). To test the primary W O97/lOO01 PCT~US96/1~57 cytotoxic T cell response after immunization, spleen cells may be cultured without stimulation. In some experiments spleen cells of the immunized mice may also be restimulated with antigenically distinct cells, to determine the 5 speci~icity of the cytotoxic T cell response.
Six days later the cultures are tested for cytotoxicity in a 4 hour 5~Cr-release assay (See, Palladino, et al., 1987, Cancer ~es . 47:5074-5079 and Blachere, at al., 1993, J.
Immunotherapy 14:352-356). In this assay, the mixed 10 lymphocyte culture is added to a target cell suspension to give different effector:target (E:T) ratios (usually 1:1 to 40:1). The target cells are prelabelled by incubating lx1O6 target cells in culture medium containing 200 mCi s~Cr/ml for one hour at 37~C. The cells are washed three times following 15 labeling. Each assay point (E:T ratio) is performed in triplicate and the appropriate controls incorporated to measure spontaneous 5~Cr release (no lymphocytes added to assay) and 100% release (cells lysed with detergent). After incubating the cell mixtures ~or 4 hours, the cells are 20 paletted by centrifugation at 200g for 5 minutes. The amount of 5ICr released into the supernatant is measured by a gamma counter. The percent cytotoxicity is measured as cpm in the test sample minus spontaneously released cpm divided'by the total detergent released cpm minus spontaneously released 25 cpm.
In order to block the MHC class I cascade a concentrated hybridoma supernatant derived from K-44 hybridoma cells (an anti-MHC class I hybridoma) is added to the test samples to a final concentration of 12.5~.
5.3. Formul~tio~
Hsp-antigenic molecule complexes of the invention may be formulated into pharmaceutical preparations for administration to mammals for treatment or prevention of 35 cancer or infectious diseases. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may be prepared, packaged, and W O 97/lO001 PCT~US96114557 labelled for treatment of the indicated tumor, such as human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, S lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland 10 carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung 15 carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute 20 lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia);
and polycythemia vera, lymphoma (Hodgkin's disease and non-25 Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. Alternatively, it can be labeled for treatment of the appropriate infectious disease. Alternatively, pharmaceutical compositions may be formulated for treatment of appropriate infectious diseases.
If the complex is water-soluble, then it may be formulated in an appropriate buffer, for example, phosphate buffered saline or other physiologically compatible solutions. Alternatively, if the resulting complex has poor solubility in aqueous solvents, then it may be formulated 35 with a non-ionic surfactant such as Tween, or polyethylene glycol. Thus, the compounds and'their physiologically acceptable solvates may be formulated for administration by W O 97/lO001 PCTAJS96/14557 inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, rectal administration or, in the case of tumors, directly injected into a solid tumor.
For oral administration, the pharmaceutical preparation 5 may be in liquid form, for example, solutions, syrups or suspensions, or may be presented as a drug product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives 10 such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or 15 sorbic acid). The pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl 20 methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by 25 methods well-known in the art.
Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional 30 manner.
For administration by inhalation, the compounds for use according ~o the present invention are conveniently delivered in the for~ of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, 35 e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit W O 97/lOOOl PCTAUS96/145S7 may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as 5 lactose or starch.
The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-10 dose containers, with an added preservative. Thecompositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may 15 be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa 20 butter or other glycerides.
In addition to the formulations described previously, the compounds may also be formulated as a depot preparation.
Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) 25 or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble 30 salt. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophilic drugs.
The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for 35 example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.
WO 97/lO001 PCT~US96/14557 The invention also provides kits for carrying out the therapeutic regimens of the invention. Such kits comprise in one or more containers therapeutically or prophylactically effective amounts of the hsp-antigenic molecule complexes in S pharmaceutically acceptable form. The hsp-antigenic molecule complex in a vial of a kit o~ the invention may be in the form of a pharmaceutically acceptable solution, e.g., in combination with sterile saline, dextrose solution, or buffered solution, or other pharmaceutically acceptable 10 sterile ~luid. Alternatively, the complex may be lyophilized or desiccated; in this instance, the kit optionally further comprises in a container a pharmaceutically acceptable solution (e.g., saline, dextrose solution, etc.), preferably sterile, to reconstitute the complex to form a solution for 15 injection purposes.
In another embodiment, a kit of the invention further comprises a needle or syringe, preferably packaged in sterile form, for injecting the complex, and/or a packaged alcohol pad. Instructions are optionally included for administration 20 of hsp-antigenic molecule complexes by a clinician or by the patient.
5.4 Tarqet Infectious Diseases Infectious diseases that can be treated or prevented by 25 the methods of the present invention are caused by infectious agents including, but not limited to, viruses, bacteria, fungi protozoa and parasites.
Viral diseases that can be treated or prevented by the methods of the present invention include, but are not limited 30 to, those caused by hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, 35 cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsachie virus, mumps virus, measles virus, rubella virus, W O 97/10001 PCT~US96/14557 polio virus, human immunodeficiency virus type I (HIV-I), and human immunodeficiency virus type II (HIV-II).
Bacterial diseases that can be treated or prevented by - the methods of the present invention are caused by bacteria 5 including, but not limited to, mycobacteria rickettsia, mycoplasma, neisseria and legionella.
Protozoal diseases that can be treated or prevented by the methods of the present invention are caused by protozoa including, but not limited to, leishmania, kokzidioa, and 10 trypanosoma.
Parasitic diseases that can be treated or prevented by the methods of the present invention are caused by parasites including, but not limited to, chlamydia and rickettsia.
5.5. Tarqet Cancers Cancers that can be treated or prevented by the methods of the present invention include, but not limited to human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, 20 angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, 25 adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, 30 Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, ~ craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuL~ ~, oligodendroglioma, meningioma, melanoma, 35 neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and WO 97/lOOOI PCT~US96/14557 erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia);
and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's 5 macroglo~ulinemia, and heavy chain disease. Specific examples of such cancers are described in the sections ~elow.
In a specific embodiment the cancer is metastatic. In another specific embodiment, the patient having a cancer is immunosuppressed by reason of having undergone anti-cancer 10 therapy (e.g., chemotherapy radiation) prior to administration of the hsp-antigenic molecule complexes of the invention.
5.5.1. Colorectal CancQr Metastatic to the Liver In 1992, approximately 150,000 Americans were diagnosed with colorectal cancer and more than 60,000 died as a result of colorectal metastases. At the time of their deaths, 80 percent of patients with colorectal cancer have metastatic 20 disease involving the liver, and one-half of these patients have no evidence of other (extrahepatic) metastases. Most metastatic tumors of the liver are from gastrointestinal primaries. Unfortunately, the natural history of metastatic liver lesions carries a grave prognosis and Systemic 25 chemotherapy regimens have been unable to induce significant response rates or alter length of survival (Drebin, J.A., et al., in Cu~rent Therapy In Oncology, ed. J.E. Niederhuber, B.C. Decker, Mosby, 1993, p.426).
Colorectal cancer initially spreads to regional lymph 30 nodes and then through the portal venous circulation to the liver, which represents the most common visceral site of metastasis. The symptoms that lead patients with colorectal c~n~r to seek medical care vary with the anatomical location of the lesion. For example, lesions in the ascending colon 35 frequency ulcerate, which leads to chronic blood loss in the stool.
W O 97/10001 PCT~US96/14557 Radical resection offers the greatest potential for cure in patients with invasive colorectal cancer. Before surgery, the CEA titer is determined. Radiation therapy and chemotherapy are used in patients with advanced colorectal S cancer. Results with chemotherapeutic agents (e.g., 5-fluorouracil) are mixed and fewer than 25 percent of patients experience a greater than 50 percent reduction in tumor mass (Richards, 2d., F., et al., 1986, ~. Clin. Oncol. 4:565).
Patients with widespread metastases have limited ~o survival and systemic chemotherapy has little impact in this group of patients. In addition, systemically administered chemotherapy is often limited by the severity of toxicities associated with the various agents, such as severe diarrhea, mucositis and/or myelosuppression. Other techniques, 15 including hepatic radiation, systemic chemotherapy, hepatic arterial ligation, tumor embolization and immunotherapy have all been explored, but, for the most part, have proven ineffectual in prolonging patient survival.
In a specific embodiment, the present invention provides 20 compositions and methods for enhancing tumor specific immunity in individuals suffering from colorectal cancer metastasized to the liver, in order to inhibit the progression of the neoplastic disease. Preferred methods of treating these neoplastic diseases comprise administering a 25 c~ ~ocition of autologous hsp noncovalently bound to peptide complexes, which elicits tumor-specific immunity against the tumor cells. Most specifically, the use of a composition of the invention, comprising gp96, can result in nearly complete inhibition of liver cancer growth in cancer patients, without 30 inducing toxicity and thus providing a dramatic therapeutic effect.
Accordingly, as an example of the method of the invention, gp96 is administered to a patient diagnosed with colorectal cancer, with or without liver metastasis, via one 35 of many different routes of administration, the preferred routes being subcutaneous at different anatomical sites, e.g., left arm, right arm, left belly, right belly, left W O 97/10001 PCT~US96/14557 thigh, right thigh, etc. These routes of administration are used in sequence and the site of injection i5 varied for each weekly injection as described in Section 7. The preparations and use of therapeutically effective compositions for the S prevention and treatment of primary and metastatic cancers are described in detail in the sections which follow and by way of example, infra.
5.5.2. ~ePatocQllular Carcinoma Hepatocellular carcinoma is generally a disease of the elderly in the United States. Although many factors may lead to hepatocellular carcinoma, the disease is usually limited to those persons with preexisting liver disease.
Approximately 60 to 80 percent of patients in the United 15 States with hepatocellular carcinoma have a cirrhotic liver and about four percent of individuals with a cirrhotic liver eventually develop hepatocellular carcinoma (Niederhuber, J.E., (ed.), 1993, Current Therapy in Oncology, B.C. Decker, Mosby). The risk is highest in patients whose liver disease 20 is caused by inherited hemochromatosis or hepatic B viral infection (Bradbear, R.A., et al., 1985, ~. Natl. Cancer Inst. 75:81; Beasley, R.P., et al., 1981, Lancet 2:1129).
Other causes of cirrhosis that can lead to hepatocellular carcinoma include alcohol abuse and hepatic fibrosis caused 25 by chronic administration of methotrexate. The most frequent symptoms of hepatocellular carcinoma are the development of a painful mass in the right upper quadrant or epigastrium, a~. __~nied by weight loss. In patients with cirrhosis, the development of hepatocellular carcinoma is preceded by 30 ascites, portal hypertension and relatively abrupt clinical deterioration. In most cases, abnormal values in standard liver function tests such as serum aminotransferase and alkaline phosp~atase are observed.
CT scans of the liver are used to determine the anatomic 35 distribution of hepatocellular carcinoma and also provide orientation for percutaneous needle biopsy. Approximately 70 percent of patients with hepatocellular carcinoma have an W O 97/lOOOl PCTAUS96/14557 elevated serum alpha-fetoprotein concentration (McIntire, R.R., et al., 1975, Cancer Res. 35:991) and its concentration correlates with the extent of the disease.
~ Radical resection offers the only hope for cure in 5 patients with hepatocellular carcinoma. Such operative procedures are associated with five-year survival rates of 12 to 30 percent. Liver transplantation may improve survival of some younger individuals. However, most patients are not surgical candidates because of extensive cirrhosis multifocal 10 tumor pattern or scarcity of compatible donor organs.
Chemotherapeutic agents have been administered either by intravenous route or through an intrahepatic arterial catheter. Such therapy has sometimes been combined with irradiation to the liver. Reductions in the size of 15 measurable tumors of 50~ or more have been reported in some patients treated with either systemic doxorubicin or 5-fluorouracil. However, chemotherapy often induces immunosuppression and rarely causes the tumor to disappear completely and the duration of response is short. The 20 prognosis for patients with hepatocellular carcinoma is negatively correlated with cirrhosis and metastases to the lungs or bone. Median survival for patients is only four to six months. In another specific embodiment, the present invention provides compositions and methods for enhancing 25 specific immunity in individuals suffering from hepatocellular carcinoma in order to inhibit the progression of the neoplastic disease and ultimately irradiate all preneoplastic and neoplastic cells.
5.5.3. Breast C~ncer Another specific aspect of the invention relates to the treatment of breast cancer. The American Cancer Society estimated that in 1992 180,000 American women were diagnosed with breast cancer and 46,000 succumbed to the disease 35 (Niederhuber, J.E.ed. Current Therapy in Oncology B.C.
Decker, Mosby, 1993). This makes breast cancer the second major cause of cancer death in women, ranking just behind W O 97/lO001 PCT~US96/14557 lung cancer. A disturbing fact is the observation that breast cancer has been increasing at a rate of 3 percent per year since 1980 (Niederhuber, J.E., ed. Current TheraPY in Oncoloq~, B.C. Decker, Mosby, (1993)). The treatment of 5 breast cancer presently involves surgery, radiation, hormonal therapy and/or chemotherapy. Consideration of two breast cancer characteristics, hormone receptors and disease extent, has governed how hormonal therapies and standard-dose chemotherapy are sequenced to improve survival and maintain lo or improve quality of life. A wide range of multidrug regimens have been used as adjuvant therapy in breast cancer patients, including, but not limited to combinations of 2 cyclophosphamide, doxorubicin, vincristine methotrexate, 5-fluorouracil and/or leucovorin. In a specific embodiment, 15 the present invention provides hsp compositions and methods for enhancing specific immunity to preneoplastic and neoplastic mA ~ry cells in women. The present invention also provides compositions and methods for preventing the development of neoplastic cells in women at enhanced risk for 20 breast cancer, and for inhibiting cancer cell proliferation and metastasis. These compositions can be applied alone or in combination with each other or with biological response modifiers 5.6. Autoloqous Embodime~t The specific immunogenicity of hsps derives not from hsps per se, but from the peptides bound to them. In a preferred embodiment of the invention directed to the use of autologous complexes of hsp-peptides as cancer vaccines, two 30 of the most intractable hurdles to cancer immunotherapy are circumvented. First is the possibility that human cancers, like cancers of experimental animals, are antigenically distinct. In an embodiment of the present invention, hsps chaperone antigenic peptides of the cancer cells from which 35 they are ~erived and circumvent this hurdle. Second, most current approaches to cancer immunotherapy focus on determining the CTL-recognized epitopes of cancer cell lines.
W O 97/lOOOl PCTAJS96/145S7 This approach requires the availability of cell lines and CTLs against cancers. These reagents are unavailable for an overwhelming proportion of human cancers. In an embodiment - of the present invention directed to autologous complexes of 5 hsp peptides, cancer immunotherapy does not depend on the ~ availability of cell lines or CTLs nor does it require definition of the antigenic epitopes of cancer cells. These advantages make autologous hsps noncovalently bound to peptide complexes attractive and novel immunogens against 10 cancer.
5.7. Prevention and ~reatment of Primary ~nd Metastatic Neoplastic Diseases There are many reasons why immunotherapy as provided by 15 the present invention is desired for use in cancer patients.
First, if cancer patients are immunosuppressed and surgery, with anesthesia, and subsequent chemotherapy, may worsen the immunosuppression, then with appropriate immunotherapy in the preoperative period, this immunosuppression may be prevented 20 or reversed. This could lead to fewer infectious complications and to accelerated wound healing. Second, tumor bulk is minimal ~ollowing surgery and immunotherapy is most likely to be effective in this situation. A third reason is the possibility that tumor cells are shed into the 25 circulation at surgery and effective immunotherapy applied at this time can eliminate these cells.
The preventive and therapeutic methods of the invention are directed at enhancing the immunocompetence of the cancer patient either before surgery, at or after surgery, and to 30 induce tumor-specific immunity to cancer cells, with the objective being inhibition of cancer, and with the ultimate clinical objective being total cancer regression and eradication.
PCT~S96/14557 W097tlO001 5.8. ~onitoring of BffeGts During ~n~ - Prevention And Immunotherapy with Hs~-~eptide ComPlexe~
The effect of immunotherapy with hsp-antigenic molecule complexes on development and progression of neoplastic 5 diseases can be monitored by any methods known to one skilled in the art, including but not limited to measuring: a) delayed hypersensitivity as an assessment of cellular immunity; b) activity of cytolytic T-lymphocytes in vitro; c) levels of tumor specific antigens, e.g., carcinoembryonic 10 (CEA) antigens; d) changes in the morphology of tumors using techniques such as a computed tomographic (CT) scan; and e) changes in levels of putative biomarkers of risk for a particular cancer in individuals at high risk, and f) changes in the morphology of tumors using a sonogram.
5.8.1. DelaYed HypersensitivitY 8kin Test Delayed hypersensitivity skin tests are of great value in the overall immunocompetence and cellular immunity to an antigen. Inability to react to a battery of common skin 20 antigens is termed anergy (Sato, T., et al, 1995, Clin.
Immunol . Pathol . 74:35-43).
Proper technique of skin testing requires that the antigens be stored sterile at 4~C, protected from light and reconstituted shorted before use. A 25- or 27-gauge need 25 ensures intradermal, rather than subcutaneous, administration of antigen. Twenty-four and 48 hours after intradermal administration of the antigen, the largest dimensions of both erythema and induration are measured with a ruler.
Hypoactivity to any given antigen or group of antigens is 30 confirmed by testing with higher concentrations of antigen or, in ambiguous circumstances, by a repeat test with an intermediate test.
-- ~0 --WO97/10001 PCT~S96/14557 5.8.2. Activity of Cytolytic ~-lymphocytes In Vitro 8xlO6 Peripheral blood derived T lymphocytes isolated by the Ficoll-Hypaque centrifigation gradient technique, are 5 restimulated with 4x104 mitomycin C treated tumor cells in 3ml RPMI medium containing 10% fetal calf serum. In some experiments, 33% secondary mixed lymphocyte culture supernatant or IL-2, is included in the culture medium as a source of T cell growth factors.
In order to measure the primary response of cytolytic T-lymphocytes after immunization, T cells are cultured without the stimulator tumor cells. In other experiments, T cells are restimulated with antigenically distinct cells. After six days, the cultures are tested for cytotoxity in a 4 hour lS 51Cr-release assay. The spontaneous 5~Cr-release of the targets should reach a level less than 20%. For the anti-MHC
class I blocking activity, a tenfold concentrated supernatant of W6/32 hybridoma is added to the test at a final concentration of 12.5% (Heike M., et al., J. Im~unotherapy 20 l5:l65-l74~
5.8.3. Levels of Tumor Specific Antiqens Although it may not be possible to detect unique tumor antigens on all tumors, many tumors display antigens that 25 distinguish them from normal cells. The monoclonal antibody reagents have permitted the isolation and biochemical characterization of the antigens and have been invaluable diagno~tically for distinction of transformed from nontransformed cells and for definition of the cell lineage 30 of transformed cells. The best-characterized human tumor-a~ociated antigens are the oncofetal antigens. These antigens are expressed during embryogenesis, but are absent or very difficult to detect in normal adult tissue. The prototype antigen is carcinoembryonic antigen (CEA), a 35 glycoprotein found on fetal gut an human colon cancer cells, but not on normal adult colon cells. Since CEA is shed from colon carcinoma cells and found in the serum, it was - Sl -W O 97/lO001 PCT~US96/14557 originally thought that the presence of this antigen in the serum could be used to screen patients for colon cancer.
However, patients with other tumors, such as pancreatic and breast cancer, also have elevated serum levels of CEA.
5 Therefore, monitoring the fall and rise of CEA levels in cancer patients undergoing therapy has proven useful for predicting tumor progression and responses to treatment.
Several other oncofetal antigens have been useful for diagnosing and monitoring human tumors, e.g., alpha-lO fetoprotein, an alpha-globulin normally secreted by fetal liver and yolk sac cells, is found in the serum of patients with liver and germinal cell tumors and can be used as a matter of disease status.
5.8.~. Computed Tomo~raphic (CT) Scan CT remains the choice of techniques for the accurate staging of cancers. CT has proved more sensitive and specific than any other imaging techniques for the detection of metastases.
5.8.5. Measurement of Putative Biomarkers The levels of a putative biomarker for risk of a specific cancer are measured to monitor the effect of hsp noncovalently bound to peptide complexes. For example, in 25 individuals at enhanced risk for prostate cancer, serum prostate-specific antigen (PSA) is measured by the procedure described by Brawer, M.K., et. al., 1992, J. Urol. 147:841-845, and Catalona, W.J., et al., 1993, JAMA 270:948-958; or in individuals at risk for colorectal cancer CEA is measured 30 as described above in Section 4.5.3; and in individuals at enhanced risk for breast cancer, 16-~-hydroxylation of estradiol is measured by the procedure described by Schneider, J. et al., 1982, Proc. Natl. Acad. Sci. ISA
79:3047-3051. The references cited above are incorporated by 35 reference herein in their entirety.
-W O 97/10001 PCT~US96/14557 5.8.6. 80noqr~m A Sonogram remains an alternative choice of technique for the accurate staging of cancers.
s 6. E~AMPLE: ~nMTNIgTRATION OF H8P-PEPTIDE
a) Tumor models:
Two W -induced carcinomas were studied in the C3H/HeN
mice (Ward, et al., 1989, J. Exp. Med. 170:217): (i) the 10 highly immunogenic 6138 carcinoma, and (ii) the less immunogenic 6139ST carcinoma.
b) Gp96 preparations were prepared from the 6138 and 6139SJ carcinomas by the procedures described above in Section 5.2.3. The gp96 preparations were administered 15 without adjuvants.
6.1 Prevention Modality (a) Materials and Method:
The ability of gp96 preparations to prevent development 20 of W-induced carcinoma was tested. A total of six groups of female C3H/HeN mice (obtained from the National Cancer Institute, Frederick, MD), weighing approximately 25g each, were used. Two groups of mice were given twice at a ten day interval, either (i) phosphate buffer saline (PBS), (ii) 25 microgram/mouse of gp96 derived from W6138 carcinomas, or (iii) 25 microgram/mouse of gp96 derived from W 3169SJ
carcinoma.
In each set, mice were challenged with 107 cells from either the W6138 carcinoma or the W6139SJ carcinoma 15 days 3 after the second injection with PBS or gp96. Tumors were measured at 2 day intervals. Since the W6138 tumor is a regre~sor tumor, mice were irradiated at 400 rad 10 days after the second injection with PBS or gp96 in order to permit growth of the tumor. The W6139SJ challenged mice 5 were not irradiated.
b) Results W O 97JlO001 PCTAJS96/14557 Administration of gp96 isolated from the W 6138 carcinoma rendered the mice immune to the W6138 challenge but not the W6139SJ challenge (Figure 1). Conversely, administration of gp96 isolated from the W6139SJ conferred 5 resistance to the W6139SJ cells but not to the W 6138 cells.
The resistance rendered by the gp96 derived from the W6138 against the W6138 cells was much greater (6 out of 7 mice) than the resistance rendered by the gp96 derived ~rom the W 6139 against the W6139 SJ cells (2 out of 4 mice) 10 (Figure 1). These results indicate that administration of gp96 preparations derived from the two W-induced carcinomas immunized syngeneic mice from the respective cancer cell type and that the resistance rendered was greater and more uniform against the more immunogenic carcinoma cells.
6.2 ~reatment Modality a) Materials and Methods The ability of gp96 preparations to mediate therapy of pre-existing cancers was tested. Three groups of mice were 20 injected intradermally with 107 cells of the W6139SJ
carcinoma. The mice were kept under observation until the tumors became visible and palpable at day 4. Thereafter, the mice in the first group received no treatment, each mouse in the second group received every other day for a total of 5 25 injections of 6 micrograms each of gp96 derived from the W6139SJ carcinoma cells, and each mouse in the third group received in a similar manner a total of 5 injections of gp96 derived from the normal liver.
b) Results Tumor growth monitored as diameter width, was significantly retarded in mice treated with tumor-derived gp96 but not in mice treated with the liver-derived gp96 or in the untreated mice (Figure 2). These results indicated a therapeutic effect of gp96-complexes in the W613ssJ
35 carcinoma model. All mice eventually succumbed to tumor growth. A scrutiny of the kinetics of tumor growth in treated and controlled mice shows that administration of W O 97/lOOOI PCTAUS96/14557 tumor-derived gp96 had an immediate inhibitory effect on tumor growth and that the effect appears to have diminished after treatment with gp96 was terminated.
6.3 Measuring Generation of M~C Class I
Restricted CD8+ CTLs Provides An AssaY For In Vivo Tumor Re~ection The effect of vaccination with hsps has been measured thus far in the prior art by tumor rejection assays in vivo.
While this assay is clearly the most demanding and rigorous 10 evidence for immunogenicity, it is impractical for the purpose of monitoring immune response in humans. We tested the ability of tumor-derived gp96 preparations to elicit a CD8+ T cell response in order to define an in vitro correlate for in vivo tumor rejection. Mice were immunized twice with 15 20 micrograms gp96 derived from 6138 or 6139SJ cells. Mixed lymphocyte-tumor cultures (MLTCs) generated from immunized mice were tested in a 5IChromium release assay and showed tumor-specific cytotoxicity for the tumor used as the source of gp96. This cytotoxic activity could be blocked by anti-MHC class I antibody K44 (Ozato, K., et al., 1985, Proc.
Natl . Acad . Sci . USA 82:2427) (Fig. 3A) and by anti-CD8 antibody YTS169.4 (Cobbold, S.P., et al., 1984, Na ture 312:548) (not shown). No corresponding activity was detected in MLTCs generated from spleens of naive mice. These results demonstrate that vaccination with gp96 elicits effective tumor-specific CTL response, which may be measured in vitro, and independently of the tumor regression responses shown in Figs. 1 and 2. In light of the general paradigm that exogenous antigens are usually presented through MHC class II
molecules and elicit a helper T cell response (Townsend, A.
et al., 1989, Ann. Rev. Immunol . 7:601), the ability of exogenous HSP preparations to elicit MHC class I-restricted CTLs is unusual.
While testing the ability of tumor-derived gpg6 preparations to elicit CTL responses, vaccination with irradiated whole tumor cells was carried out as a positive W O 97/lOO01 PCTAUS96/14557 control. As expected, vaccination with intact irradiated 6138 cells led to vigorous tumor--specific CTL response.
However, vaccination with intact irradiated 6139SJ cells did not lead to a corresponding CTL response (Fig. 3B). This 5 result was surprising as W--inducedcancers of C3H mice are generally highly immunogenic (Kripke, M.L., 1977, Cancer ~es.
37:1395. In view of the observation that 6139SJ cells are suitable targets for cytoto~cic T cells (as seen in Fig. 3A), we deduce that they are not defective in antigen 10 pre~:entation; instead, their inability to elicit CTL response suggests that they are deficient in a crucial, as yet undefined step necessary specifically for priming a CTL
response in vivo . It is most significant in this regard that although 6139SJ cells do not elicit a CTL response, gp96 15 preparations derived from them do so efficiently. This suggest that intact tumor cells and HSPs derived from them elicit immunity through distinct immunological pathways. The ability of gp96 preparations derived from a tumor to elicit a potent CTL response even when the tumor from which gp96 is 20 derived is unable to do so, makes hsp preparations attractive as therapeutic vaccines.
6.4 GP96--PeptidQComplexos Elicit A MemorY T Cell ResPon~3e The ability to elicit a memory response is crucial for any vaccine and the ability of gp96 to elicit a memory T cell population was tested. A number of criteria, i.e., radiation resistance, kinetics of appearance, loss of CD45RB and L--selectin ly}nphocyte surface antigens, were used to identify 30 memory T response. In contrast to naive T cells (Schrek, R., 1961, Ann. N.Y. Acad. sci 95:839), memory T cells are cycling cells (Mac3cay, C.R., et al, 1992, Nature 360:264~ and like other cycling lymphocytes, are resistant to sub-lethal irradiation (Lowenthal, J.W., et al., 1991, Leuc. Biol.
35 49:388). Thus radiation-resistance can be used to distinguish naive resting T cells from activated e~fector and memory T cells. However, no known surface markers W O 97/10001 PCT~US96/14557 distinguish activated effector T cells from memory T cells and the two are distinguishable only by the kinetics of their appearance. Activated effector T cells disappear from circulation within seven to ten days of depletion of 5 significant quantities of antigen (Sprent, J., 1994, Cell 76:315); in contrast, memory T cells continue to circulate well beyond this window of time. In order to test, if vaccination with tumor-derived gp96 elicits a memory T cell response in addition to the effector response shown in 10 Fig. 3, mice were vaccinated twice at ten day intervals, with tumor-derived gp96 and were irradiated (400 rad) twelve days after the last vaccination. Three days after irradiation, MLTCs were generated from spleens of mice and tested for tumor-specific CTL response. It was observed (Fig. 4) that 15 similar to the response in unirradiated mice (Fig. 3A~, the irradiated, gp96-vaccinated mice generated powerful, MHC
class I - restricted and tumor-specific CTL responses. Under this regimen of vaccination and irradiation, the irradiation eliminates the non-memory resting T cells, while the delay 20 between the last vaccination and generation of MCTCs eliminates activated T lymphocytes (Sprent, J., 1994, Cell 76:315). Thus, the observed CTL response derives from radiation-resistant memory T cells elicited by gp96 preparations. This phenomenon was also tested in tumor 25 rejection assays in vivo and mice vaccinated with pg96 and irradiated were observed to resist tumor challenges up to 17 days after vaccination, even though they had bene irradiated (data not shown). These observations indicate that vaccination with gp96 elicits a long-lived, radiation-30 resistant T cell population.
As an independent parameter for memory response,expression of CD45RB (Birkeland, M.L., et al., 1989, Proc .
Natl. Acad. sci. USA 86:6734) on CD8+ lymphocytes from irradiated and non-irradiated, naive and gp96-vaccinated mice 35 was tested (Fig. 4). In each case, lymphocytes were obtained under the same regimen as described in the preceding paragraph, i.e., fifteen days after the last vaccination WO97/10001 PCT~S96/14557 including three days after irradiation, in order to allow the activated effector cells to be depleted. It was observed that vaccination with gp96 led to relative loss of expression of CD45RB on CD8+ T lymphocytes in irradiated as well as non-5 irradiated, immunized mice. Similar results were observedwith L-selectin (data not shown). These results indicated that as judged from two independent sets of criteria, vaccination with gp96 elicits a memory T cell response. To the best of my knowledge, this is the first demonstration of l0 generation of a memory CTL response by vaccination with a biochemically defined, purified cancer vaccine.
The ability of gp96 preparations to prevent development 20 of W-induced carcinoma was tested. A total of six groups of female C3H/HeN mice (obtained from the National Cancer Institute, Frederick, MD), weighing approximately 25g each, were used. Two groups of mice were given twice at a ten day interval, either (i) phosphate buffer saline (PBS), (ii) 25 microgram/mouse of gp96 derived from W6138 carcinomas, or (iii) 25 microgram/mouse of gp96 derived from W 3169SJ
carcinoma.
In each set, mice were challenged with 107 cells from either the W6138 carcinoma or the W6139SJ carcinoma 15 days 3 after the second injection with PBS or gp96. Tumors were measured at 2 day intervals. Since the W6138 tumor is a regre~sor tumor, mice were irradiated at 400 rad 10 days after the second injection with PBS or gp96 in order to permit growth of the tumor. The W6139SJ challenged mice 5 were not irradiated.
b) Results W O 97JlO001 PCTAJS96/14557 Administration of gp96 isolated from the W 6138 carcinoma rendered the mice immune to the W6138 challenge but not the W6139SJ challenge (Figure 1). Conversely, administration of gp96 isolated from the W6139SJ conferred 5 resistance to the W6139SJ cells but not to the W 6138 cells.
The resistance rendered by the gp96 derived from the W6138 against the W6138 cells was much greater (6 out of 7 mice) than the resistance rendered by the gp96 derived ~rom the W 6139 against the W6139 SJ cells (2 out of 4 mice) 10 (Figure 1). These results indicate that administration of gp96 preparations derived from the two W-induced carcinomas immunized syngeneic mice from the respective cancer cell type and that the resistance rendered was greater and more uniform against the more immunogenic carcinoma cells.
6.2 ~reatment Modality a) Materials and Methods The ability of gp96 preparations to mediate therapy of pre-existing cancers was tested. Three groups of mice were 20 injected intradermally with 107 cells of the W6139SJ
carcinoma. The mice were kept under observation until the tumors became visible and palpable at day 4. Thereafter, the mice in the first group received no treatment, each mouse in the second group received every other day for a total of 5 25 injections of 6 micrograms each of gp96 derived from the W6139SJ carcinoma cells, and each mouse in the third group received in a similar manner a total of 5 injections of gp96 derived from the normal liver.
b) Results Tumor growth monitored as diameter width, was significantly retarded in mice treated with tumor-derived gp96 but not in mice treated with the liver-derived gp96 or in the untreated mice (Figure 2). These results indicated a therapeutic effect of gp96-complexes in the W613ssJ
35 carcinoma model. All mice eventually succumbed to tumor growth. A scrutiny of the kinetics of tumor growth in treated and controlled mice shows that administration of W O 97/lOOOI PCTAUS96/14557 tumor-derived gp96 had an immediate inhibitory effect on tumor growth and that the effect appears to have diminished after treatment with gp96 was terminated.
6.3 Measuring Generation of M~C Class I
Restricted CD8+ CTLs Provides An AssaY For In Vivo Tumor Re~ection The effect of vaccination with hsps has been measured thus far in the prior art by tumor rejection assays in vivo.
While this assay is clearly the most demanding and rigorous 10 evidence for immunogenicity, it is impractical for the purpose of monitoring immune response in humans. We tested the ability of tumor-derived gp96 preparations to elicit a CD8+ T cell response in order to define an in vitro correlate for in vivo tumor rejection. Mice were immunized twice with 15 20 micrograms gp96 derived from 6138 or 6139SJ cells. Mixed lymphocyte-tumor cultures (MLTCs) generated from immunized mice were tested in a 5IChromium release assay and showed tumor-specific cytotoxicity for the tumor used as the source of gp96. This cytotoxic activity could be blocked by anti-MHC class I antibody K44 (Ozato, K., et al., 1985, Proc.
Natl . Acad . Sci . USA 82:2427) (Fig. 3A) and by anti-CD8 antibody YTS169.4 (Cobbold, S.P., et al., 1984, Na ture 312:548) (not shown). No corresponding activity was detected in MLTCs generated from spleens of naive mice. These results demonstrate that vaccination with gp96 elicits effective tumor-specific CTL response, which may be measured in vitro, and independently of the tumor regression responses shown in Figs. 1 and 2. In light of the general paradigm that exogenous antigens are usually presented through MHC class II
molecules and elicit a helper T cell response (Townsend, A.
et al., 1989, Ann. Rev. Immunol . 7:601), the ability of exogenous HSP preparations to elicit MHC class I-restricted CTLs is unusual.
While testing the ability of tumor-derived gpg6 preparations to elicit CTL responses, vaccination with irradiated whole tumor cells was carried out as a positive W O 97/lOO01 PCTAUS96/14557 control. As expected, vaccination with intact irradiated 6138 cells led to vigorous tumor--specific CTL response.
However, vaccination with intact irradiated 6139SJ cells did not lead to a corresponding CTL response (Fig. 3B). This 5 result was surprising as W--inducedcancers of C3H mice are generally highly immunogenic (Kripke, M.L., 1977, Cancer ~es.
37:1395. In view of the observation that 6139SJ cells are suitable targets for cytoto~cic T cells (as seen in Fig. 3A), we deduce that they are not defective in antigen 10 pre~:entation; instead, their inability to elicit CTL response suggests that they are deficient in a crucial, as yet undefined step necessary specifically for priming a CTL
response in vivo . It is most significant in this regard that although 6139SJ cells do not elicit a CTL response, gp96 15 preparations derived from them do so efficiently. This suggest that intact tumor cells and HSPs derived from them elicit immunity through distinct immunological pathways. The ability of gp96 preparations derived from a tumor to elicit a potent CTL response even when the tumor from which gp96 is 20 derived is unable to do so, makes hsp preparations attractive as therapeutic vaccines.
6.4 GP96--PeptidQComplexos Elicit A MemorY T Cell ResPon~3e The ability to elicit a memory response is crucial for any vaccine and the ability of gp96 to elicit a memory T cell population was tested. A number of criteria, i.e., radiation resistance, kinetics of appearance, loss of CD45RB and L--selectin ly}nphocyte surface antigens, were used to identify 30 memory T response. In contrast to naive T cells (Schrek, R., 1961, Ann. N.Y. Acad. sci 95:839), memory T cells are cycling cells (Mac3cay, C.R., et al, 1992, Nature 360:264~ and like other cycling lymphocytes, are resistant to sub-lethal irradiation (Lowenthal, J.W., et al., 1991, Leuc. Biol.
35 49:388). Thus radiation-resistance can be used to distinguish naive resting T cells from activated e~fector and memory T cells. However, no known surface markers W O 97/10001 PCT~US96/14557 distinguish activated effector T cells from memory T cells and the two are distinguishable only by the kinetics of their appearance. Activated effector T cells disappear from circulation within seven to ten days of depletion of 5 significant quantities of antigen (Sprent, J., 1994, Cell 76:315); in contrast, memory T cells continue to circulate well beyond this window of time. In order to test, if vaccination with tumor-derived gp96 elicits a memory T cell response in addition to the effector response shown in 10 Fig. 3, mice were vaccinated twice at ten day intervals, with tumor-derived gp96 and were irradiated (400 rad) twelve days after the last vaccination. Three days after irradiation, MLTCs were generated from spleens of mice and tested for tumor-specific CTL response. It was observed (Fig. 4) that 15 similar to the response in unirradiated mice (Fig. 3A~, the irradiated, gp96-vaccinated mice generated powerful, MHC
class I - restricted and tumor-specific CTL responses. Under this regimen of vaccination and irradiation, the irradiation eliminates the non-memory resting T cells, while the delay 20 between the last vaccination and generation of MCTCs eliminates activated T lymphocytes (Sprent, J., 1994, Cell 76:315). Thus, the observed CTL response derives from radiation-resistant memory T cells elicited by gp96 preparations. This phenomenon was also tested in tumor 25 rejection assays in vivo and mice vaccinated with pg96 and irradiated were observed to resist tumor challenges up to 17 days after vaccination, even though they had bene irradiated (data not shown). These observations indicate that vaccination with gp96 elicits a long-lived, radiation-30 resistant T cell population.
As an independent parameter for memory response,expression of CD45RB (Birkeland, M.L., et al., 1989, Proc .
Natl. Acad. sci. USA 86:6734) on CD8+ lymphocytes from irradiated and non-irradiated, naive and gp96-vaccinated mice 35 was tested (Fig. 4). In each case, lymphocytes were obtained under the same regimen as described in the preceding paragraph, i.e., fifteen days after the last vaccination WO97/10001 PCT~S96/14557 including three days after irradiation, in order to allow the activated effector cells to be depleted. It was observed that vaccination with gp96 led to relative loss of expression of CD45RB on CD8+ T lymphocytes in irradiated as well as non-5 irradiated, immunized mice. Similar results were observedwith L-selectin (data not shown). These results indicated that as judged from two independent sets of criteria, vaccination with gp96 elicits a memory T cell response. To the best of my knowledge, this is the first demonstration of l0 generation of a memory CTL response by vaccination with a biochemically defined, purified cancer vaccine.
7. EXAHPLE: ~nMTNISTRATION OF ~SP-PEPTIDE COMPLEXE8 IN T~E T~M~T OF EEPATOCELLULAR
CARCINOMA
Patients with hepatocellular carcinoma are injected with hsp-peptide complexes (derived from their own tumors or from other tumors) post surgery. Treatment with hsp-peptide complexes is started any time after surgery. However, if the patient has received chemotherapy, hsp-peptide complexes are 20 usually administered after an interval of four weeks or more so as to allow the immune system to recover. The immunocompetence of the patient is tested by procedures described in sections 5.7 above.
The therapeutic regiment of hsp-peptide complexes, for 25 example, gp96, hsp90, hsp70 or a combination thereof, includes weekly injections of the hsp-peptide complex, dissolved in saline or other physiologically compatible solution.
The dosage used for hsp70 or gp96 is in the range of l0-30 600 micrograms, with the preferred dosage being lO-lO0 micrograms. The dosage used for hsp90 is in the range of 50 to 5,000 micrograms, with the preferred dosage being about l00 micrograms.
The route and site of injection is varied each time, for 35 example, the first injection is given subcutaneously on the left arm, the second injection on the right arm, the third W O 97/lOOOl PCT~US96/14557 injection on the left abdominal region, the fourth injection on the right abdominal region, the fifth injection on the left thigh, the sixth injection on the right thigh, etc. The same site is repeated after a gap of one or more injections.
5 In addition, injections are split and each half of the dose is administered at a different site on the same day.
overall, the first four to six injections are given at weekly intervals. Subsequently, two injections are given at two-week intervals; followed by a regimen of injections at 10 monthly intervals. The effect of hsp-peptide complexes therapy is monitored by measuring: a) delayed hypersensitivity as an assessment of cellular immunity; b) activity of cytolytic T-lymphocytes in vitro; c) levels of tumor specific antigens, e.g., carcinoembryonic (CEA) 15 antigens; d) changes in the morphology of tumors using techni~ues such as a computed tomographic (CT) scan; and eO
changes in putative biomarkers of risk for a particular cancer in individuals at high risk.
Depending on the results obtained, as described above 20 Section 5.7, the therapeutic regimen is developed to maintain and/or boost the immunological responses of the patient, with the ultimate goal of achieving tumor regression and complete eradication of cancer cells.
CARCINOMA
Patients with hepatocellular carcinoma are injected with hsp-peptide complexes (derived from their own tumors or from other tumors) post surgery. Treatment with hsp-peptide complexes is started any time after surgery. However, if the patient has received chemotherapy, hsp-peptide complexes are 20 usually administered after an interval of four weeks or more so as to allow the immune system to recover. The immunocompetence of the patient is tested by procedures described in sections 5.7 above.
The therapeutic regiment of hsp-peptide complexes, for 25 example, gp96, hsp90, hsp70 or a combination thereof, includes weekly injections of the hsp-peptide complex, dissolved in saline or other physiologically compatible solution.
The dosage used for hsp70 or gp96 is in the range of l0-30 600 micrograms, with the preferred dosage being lO-lO0 micrograms. The dosage used for hsp90 is in the range of 50 to 5,000 micrograms, with the preferred dosage being about l00 micrograms.
The route and site of injection is varied each time, for 35 example, the first injection is given subcutaneously on the left arm, the second injection on the right arm, the third W O 97/lOOOl PCT~US96/14557 injection on the left abdominal region, the fourth injection on the right abdominal region, the fifth injection on the left thigh, the sixth injection on the right thigh, etc. The same site is repeated after a gap of one or more injections.
5 In addition, injections are split and each half of the dose is administered at a different site on the same day.
overall, the first four to six injections are given at weekly intervals. Subsequently, two injections are given at two-week intervals; followed by a regimen of injections at 10 monthly intervals. The effect of hsp-peptide complexes therapy is monitored by measuring: a) delayed hypersensitivity as an assessment of cellular immunity; b) activity of cytolytic T-lymphocytes in vitro; c) levels of tumor specific antigens, e.g., carcinoembryonic (CEA) 15 antigens; d) changes in the morphology of tumors using techni~ues such as a computed tomographic (CT) scan; and eO
changes in putative biomarkers of risk for a particular cancer in individuals at high risk.
Depending on the results obtained, as described above 20 Section 5.7, the therapeutic regimen is developed to maintain and/or boost the immunological responses of the patient, with the ultimate goal of achieving tumor regression and complete eradication of cancer cells.
8. EXAMPLE: ~n~TNI8TRATION OF ~SP-PEPTIDE COMPLEXES
IN T~E TREATMENT OF COLORECTAL CANCER
Hsp-peptide complexes (gp96, hsp70, hsp90 or a combination thereof) are administered as adjuvant therapy and as prophylactic adjuvant therapy in patients after complete 30 reduction of colorectal cancer to eliminate undetectable micrometastases and to improve survival.
The therapeutic and prophylactic regimens used in patients suffering from colorectal cancer are the same as those described in Section 7 above for patients recovering 35 with hepatocellular carcinoma. The methods of monitoring of patients under clinical evaluation for prevention and treatment of colorectal cancer is done by procedures W O 97/10001 PCT~US96/14557 described in Section 5.7. Specifically, CEA levels are measured as a useful monitor of tumor regression and/or recurrence (Mayer, R.J., et al., 1978, Cancer 42:1428).
IN T~E TREATMENT OF COLORECTAL CANCER
Hsp-peptide complexes (gp96, hsp70, hsp90 or a combination thereof) are administered as adjuvant therapy and as prophylactic adjuvant therapy in patients after complete 30 reduction of colorectal cancer to eliminate undetectable micrometastases and to improve survival.
The therapeutic and prophylactic regimens used in patients suffering from colorectal cancer are the same as those described in Section 7 above for patients recovering 35 with hepatocellular carcinoma. The methods of monitoring of patients under clinical evaluation for prevention and treatment of colorectal cancer is done by procedures W O 97/10001 PCT~US96/14557 described in Section 5.7. Specifically, CEA levels are measured as a useful monitor of tumor regression and/or recurrence (Mayer, R.J., et al., 1978, Cancer 42:1428).
9. E~AXPL~8: ME~HOD FOR RAPID P~RIF~CATION OF PEPTIDE-A8SQCIATED ~8P70 Hsp70-peptide complexes can be readily obtained from ~-Anc~ cells or cells infected by an infectious agent or other cells by a rapid, one-step ADP-agarose chromatography, 10 described below.
9.1 Method and Re~ult~
Meth A sarcoma cells (500 million cells) were homogenized in hypotonic buffer and the lysate was 15 centrifuged at 100,000 g for 90 minutes at 4~C. The supernatant was divided into two and was applied to an ADP-agarose or an ATP-agarose column. The columns were washed in buffer and were eluted with 3 mM ADP or 3 mM ATP, respectively. The eluted fractions were analyzed by 20 SDS-PAGE: in both cases, apparently homogeneous preparations of hsp70 were obtained. However, when each of the preparations was tested for presence of peptides, the ADP-bound/eluted hsp70 preparation was found to be associated with peptides, while the ATP-bound/eluted hsp70 preparation 25 was not. (Figures. 5A and SB) The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described 30 herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
Various publications are cited herein, the disclosures 35 of which are incorporated by reference in their entireties.
9.1 Method and Re~ult~
Meth A sarcoma cells (500 million cells) were homogenized in hypotonic buffer and the lysate was 15 centrifuged at 100,000 g for 90 minutes at 4~C. The supernatant was divided into two and was applied to an ADP-agarose or an ATP-agarose column. The columns were washed in buffer and were eluted with 3 mM ADP or 3 mM ATP, respectively. The eluted fractions were analyzed by 20 SDS-PAGE: in both cases, apparently homogeneous preparations of hsp70 were obtained. However, when each of the preparations was tested for presence of peptides, the ADP-bound/eluted hsp70 preparation was found to be associated with peptides, while the ATP-bound/eluted hsp70 preparation 25 was not. (Figures. 5A and SB) The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described 30 herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
Various publications are cited herein, the disclosures 35 of which are incorporated by reference in their entireties.
Claims (61)
1. A method of eliciting an immune response in a human individual comprising administering to the individual a composition comprising an amount of a complex in the range of 10 to 600 micrograms, said complex consisting essentially of a heat shock protein 70 noncovalently bound to an antigenic molecule.
2. A method of eliciting an immune response in a human individual comprising administering to the individual a composition comprising an amount of a complex in the range of 50 to 5,000 micrograms, said complex consisting essentially of a heat shock protein 90 noncovalently bound to an antigenic molecule.
3. A method of eliciting an immune response in a human individual comprising administering to the individual a composition comprising an amount of a complex in the range of 10 to 600 micrograms, said complex consisting essentially of a heat shock protein gP96 noncovalently bound to an antigenic molecule.
4. The method according to claim 1, 2 or 3 in which the individual has liver cancer, colon cancer, or breast cancer.
5. The method according to claim 1 in which the amount of the complex is in the range of 10 to 100 micrograms.
6. The method according to claim 2 in which the amount of the complex is in the range of about 100 micrograms.
7. The method according to claim 3 in which the amount of the complex is in the range of 10 to 100 micrograms.
8. The method according to claim 1, 2 or 3, further comprising administering to the individual an effective amount of a biological response modifier selected from the group consisting of interferon-.alpha., interferon-.gamma., interleukin-2, interleukin-4, interleukin-6, and tumor necrosis factor.
9. The method according to claim 1, 2 or 3 in which said administering step is repeated at weekly intervals.
10. The method according to claim 1, 2 or 3 in which said complex is administered intramuscularly, subcutaneously, intraperitoneally or intravenously.
11. The method according to claim 1, 2 or 3 in which said administering step is repeated five times, the first administration being on the left arm, the second administration being on the right arm, the third administration being on the left belly, the fourth administration being on the right belly, the fifth administration being on the left thigh, and the sixth administration being on the right thigh; said first through sixth administration being subcutaneously.
12. A method of treating a human individual having cancer, comprising administering to the individual a composition comprising an amount of a complex in the range of 10 to 600 micrograms, said complex consisting essentially of a heat shock protein 70 noncovalently bound to an antigenic molecule.
13. A method of treating a human individual having cancer, comprising administering to the individual a composition comprising an amount of a complex in the range of 50 to 5,000 micrograms, said complex consisting essentially of a heat shock protein 90 noncovalently bound to an antigenic molecule.
14. A method of treating a human individual having cancer, comprising administering to the individual a composition comprising an amount of a complex in the range of 10 to 600 micrograms, said complex consisting essentially of a heat shock protein gp96 noncovalently bound to an antigenic molecule.
15. The method according to claim 12, 13 or 14 in which the cancer comprises a sarcoma or carcinoma, selected from the group consisting of fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease.
16. The method according to claim 12, in which the amount of the complex is in the range of 10 to 100 micrograms.
17. The method according to claim 13 in which the amount of the complex is in the range of about 100 micrograms.
18. The method according to claim 14 in which the amount of the complex is in the range of 10 to 100 micrograms.
19. The method according to claim 12, 13 or 14 in which the complex is prepared from cancerous tissue autologous to the individual.
20. The method according to claim 12, 13 or 14 in which the complex is prepared from cancerous tissue allogeneic to the individual.
21. The method according to claim 12, 13 or 14, further comprising administering to the individual an effective amount of a biological response modifier selected from the group consisting of interferon-.alpha., interferon-.gamma., interleukin-2, interleukin-4, interleukin-6, and tumor necrosis factor.
22. The method according to claim 12, 13 or 14 in which said administering step is repeated at weekly intervals.
23. The method according to claim 16, 17 or 18 in which said administering step is repeated five times, the first administration being on the left arm, the second administration being on the right arm, the third administration being on the left belly, the fourth administration being on the right belly, the fifth administration being on the left thigh, and the sixth administration being on the right thigh; said first through sixth administration being subcutaneously.
24. A method of treating a human individual having cancer comprising:
(a) administering to the individual a composition comprising about 25 micrograms of a complex, said complex consisting essentially of a heat shock protein gp96 noncovalently bound to a peptide, said complex having been isolated from cancerous tissue of said individual; and (b) repeating said administering of step (a) at weekly intervals for five weeks, the first administration being on the left arm, the second administration being on the right arm, the third administration being on the left belly, the fourth administration being on the right belly, the fifth administration being on the left thigh, and the sixth administration being on the right thigh; said first through sixth administration being subcutaneously.
(a) administering to the individual a composition comprising about 25 micrograms of a complex, said complex consisting essentially of a heat shock protein gp96 noncovalently bound to a peptide, said complex having been isolated from cancerous tissue of said individual; and (b) repeating said administering of step (a) at weekly intervals for five weeks, the first administration being on the left arm, the second administration being on the right arm, the third administration being on the left belly, the fourth administration being on the right belly, the fifth administration being on the left thigh, and the sixth administration being on the right thigh; said first through sixth administration being subcutaneously.
25. A method of preventing cancer in a human individual in whom prevention of cancer is desired comprising administering to the individual a composition comprising an amount of a complex in the range of 10 to 600 micrograms, said complex consisting essentially of a heat shock protein 70 noncovalently bound to an antigenic molecule.
26. A method of preventing cancer in a human individual in whom prevention of cancer is desired, comprising administering to the individual a composition comprising an amount of a complex in the range of 50 to 5,000 micrograms, said complex consisting essentially of a heat shock protein 90 noncovalently bound to an antigenic molecule.
27. A method of preventing cancer in a human individual in whom prevention of cancer is desired, comprising administering to the individual a composition comprising an amount of a complex in the range of 10 to 600 micrograms, said complex consisting essentially of a heat shock protein gp96 noncovalently bound to an antigenic molecule.
28. The method according to claim 25, in which the amount of the complex is in the range of 10 to 100 micrograms.
29. The method according to claim 26, in which the amount of the complex is in the range of 100 micrograms.
30. The method according to claim 27, in which the amount of the complex is in the range of 10 to 100 micrograms.
31. The method according to claim 12, 13, or 14 in which the antigenic molecule is a peptide with which the heat shock protein is endogenously associated in vivo, and the complex is isolated from cancerous tissue.
32. The method according to claim 31 in which the cancerous tissue is from the individual.
33. The method according to claim 12, 13, or 14 in which the noncovalent complex of the heat shock protein and antigenic molecule is produced in vitro.
34. The method according to claim 33 in which the antigenic molecule is a tumor-specific antigen.
35. The method according to claim 25, 26, or 27 in which the antigenic molecule is a peptide with which the heat shock protein is endogenously associated in vivo.
36. A method of treating or preventing an infectious disease in a human individual in whom such treatment or prevention is desired comprising administering to the individual a composition comprising an amount of a complex in the range of 10 to 600 micrograms, said complex consisting essentially of a heat shock protein 70 noncovalently bound to an antigenic molecule.
37. A method of treating or preventing an infectious disease in a human individual in whom such treatment or prevention is desired comprising administering to the individual a composition comprising an amount of a complex in the range of 50 to 5,000 micrograms, said complex consisting essentially of a heat shock protein 90 noncovalently bound to an antigenic molecule.
38. A method of treating or preventing an infectious disease in a human individual in whom such treatment or prevention is desired comprising administering to the individual a composition comprising an amount of a complex in the range of 10 to 600 micrograms, said complex consisting essentially of a heat shock protein gp96 noncovalently bound to an antigenic molecule.
39. The method according to claim 36 in which the amount of the complex is in the range of 10 to 100 micrograms.
40. The method according to claim 37 in which the amount of the complex is in the range of about 100 micrograms.
41. The method according to claim 38 in which the amount of the complex is in the range of 10 to 100 micrograms.
42. The method according to claim 36, 37, or 38 in which the antigenic molecule is a peptide with which the heat shock protein is endogenously associated in cells infected with an infectious agent that causes the infectious disease.
43. The method according to claim 36, 37, or 38 in which the antigenic molecule is an antigen of an infectious agent that causes the infectious disease.
44. The method according to claim 43 in which the infectious agent is a virus, bacterium, protozoa, fungus, or parasite.
45. A method for measuring tumor rejection in vivo in an individual having a tumor comprising measuring the generation by the individual of MHC Class I-restricted CD8+
cytotoxic T lymphocytes specific to the tumor.
cytotoxic T lymphocytes specific to the tumor.
46. A kit comprising in a container a composition comprising an amount of a complex in the range of 10 to 600 micrograms, said complex consisting essentially of a heat shock protein 70 noncovalently bound to an antigenic molecule.
47. A kit comprising in a container a composition comprising an amount of a complex in the range of 50 to 5,000 micrograms, said complex consisting essentially of a heat shock protein 90 noncovalently bound to an antigenic molecule.
48. A kit comprising in a container a composition comprising an amount of a complex in the range of 10 to 600 micrograms, said complex consisting essentially of a heat shock protein gp96 noncovalently bound to an antigenic molecule.
49. A kit comprising a plurality of containers, each container having a composition comprising an amount of a complex in the range of 10 to 600 micrograms, said complex consisting essentially of a heat shock protein 70 noncovalently bound to an antigenic molecule.
50. The kit of claim 46 in which the amount of the complex is in the range of 10 to 100 micrograms.
51. The kit of claim 47 in which the amount of the complex is in the range of about 100 micrograms.
52. The kit of claim 48 in which the amount of the complex is in the range of 10 to 100 micrograms.
53. A method of purifying hsp70-peptide complexes comprising:
(a) contacting a sample containing cellular proteins with ADP affixed to a solid substrate under conditions such that hsp70 in the sample can bind to the ADP; and (b) eluting the hsp70 bound to the ADP in step (a).
(a) contacting a sample containing cellular proteins with ADP affixed to a solid substrate under conditions such that hsp70 in the sample can bind to the ADP; and (b) eluting the hsp70 bound to the ADP in step (a).
54. The method according to claim 53 wherein the contacting is carried out by column chromatography over ADP-agarose.
55. The method according to claim 53 wherein the cell is a tumor cell.
56. The method according to claim 53 wherein the cell is infected with a virus.
57. The method according to claim 53 wherein the cell is infected with a bacterium.
58. The method according to claim 53 wherein the cell is infected with a protozoa.
59. The method according to claim 53 wherein the cell is infected with a parasite.
60. A method of purifying hsp70-peptide complexes from a cell comprising:
(a) homogenizing the cell with a hypotonic buffer solution to produce a cell lysate;
(b) centrifuging the cell lysate to obtain a supernatant;
(c) running the supernatant over an ADP-agarose column;
(d) washing the ADP-agarose column with a buffer containing ADP; and (e) collecting the hsp70-peptide complex.
(a) homogenizing the cell with a hypotonic buffer solution to produce a cell lysate;
(b) centrifuging the cell lysate to obtain a supernatant;
(c) running the supernatant over an ADP-agarose column;
(d) washing the ADP-agarose column with a buffer containing ADP; and (e) collecting the hsp70-peptide complex.
61. A method of purifying hsp70-peptide complexes comprising:
(a) contacting a sample containing cellular proteins with a nonhydrolyzable analog of ATP
affixed to a solid substrate under conditions such that hsp70 in the sample can bind to the nonhydrolyzable analog of ATP; and (b) eluting the hsp70 bound to the nonhydrolyzable analog of ATP in step (a).
(a) contacting a sample containing cellular proteins with a nonhydrolyzable analog of ATP
affixed to a solid substrate under conditions such that hsp70 in the sample can bind to the nonhydrolyzable analog of ATP; and (b) eluting the hsp70 bound to the nonhydrolyzable analog of ATP in step (a).
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PCT/US1996/014557 WO1997010001A1 (en) | 1995-09-13 | 1996-09-11 | Treatment or prevention of neoplastic and infectious diseases with heat shock/stress proteins |
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US5750119A (en) | 1994-01-13 | 1998-05-12 | Mount Sinai School Of Medicine Of The City University Of New York | Immunotherapeutic stress protein-peptide complexes against cancer |
US5997873A (en) * | 1994-01-13 | 1999-12-07 | Mount Sinai School Of Medicine Of The City University Of New York | Method of preparation of heat shock protein 70-peptide complexes |
US5961979A (en) * | 1994-03-16 | 1999-10-05 | Mount Sinai School Of Medicine Of The City University Of New York | Stress protein-peptide complexes as prophylactic and therapeutic vaccines against intracellular pathogens |
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|
MKLA | Lapsed |
Effective date: 20160912 |