WO2002030434A1 - Compositions et procedes pour la prevention et le traitement de maladies neoplasiques primaires, de maladies neoplasiques metastatiques, et de maladies infectieuses, a l'aide de compositions comprenant des proteines cellulaires non fractionnees - Google Patents

Compositions et procedes pour la prevention et le traitement de maladies neoplasiques primaires, de maladies neoplasiques metastatiques, et de maladies infectieuses, a l'aide de compositions comprenant des proteines cellulaires non fractionnees Download PDF

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WO2002030434A1
WO2002030434A1 PCT/US2001/028841 US0128841W WO0230434A1 WO 2002030434 A1 WO2002030434 A1 WO 2002030434A1 US 0128841 W US0128841 W US 0128841W WO 0230434 A1 WO0230434 A1 WO 0230434A1
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cells
proteins
cancer
cellular proteins
cell
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PCT/US2001/028841
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Pramod K. Srivastava
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University Of Connecticut Health Center
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Priority to US10/380,442 priority Critical patent/US20030211971A1/en
Priority to EP01975212A priority patent/EP1322317A4/fr
Priority to CA002422718A priority patent/CA2422718A1/fr
Priority to AU2001294560A priority patent/AU2001294560B2/en
Priority to JP2002533874A priority patent/JP2005516884A/ja
Priority to AU9456001A priority patent/AU9456001A/xx
Publication of WO2002030434A1 publication Critical patent/WO2002030434A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/02—Local antiseptics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to methods and compositions for the prevention and treatment of infectious diseases, and primary and metastatic neoplastic diseases, including, but not limited to human sarcomas and carcinomas.
  • compositions comprising unfractionated cellular proteins are used to augment the immune response to genotoxic and nongenotoxic factors, tumors and infectious agents .
  • tumor specific transplantation antigens also commonly referred to as "tumor specific rejection antigens.”
  • 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 period (Old, L.J., et al . , 1962, Ann . N. Y. Acad. Sci . 101:80- 106; Bartlett, G.L., 1972, J “ . Na tl . Cancer Inst . 49:493-504).
  • carcinogens are mutagens which may cause mutation, leading to the expression of tumor specific antigens (Ames, B. ⁇ ., 1979, Science 204:587-593; eisburger, J.H., et al . , 1981, Science 214:401-407) .
  • Some carcinogens are immunosuppressive (Malmgren, R.A., et al . , 1952, Proc . Soc . Exp . Biol . Med . J_9_ : 484 - 488 ) .
  • chemoimmunotherapy program for the treatment of breast cancer comprising the administration of cyclophosphamide, methotrexate, fluorouracil and BCG vaccine either with or without a second vaccine consisting of irradiated, allogeneic breast cancer cells (Sparks, F.C., et al . , 1976, Arch Surg, 35 111, 1057-62) .
  • the tumor cell vaccine was made up of 3.5 x 10 7 irradiated cells from each of three different breast carcinoma cell lines: MDA-MB-231, MDA-MB-157, and NBL-374B.
  • Hughes et al . reported the use of homogenized, fractionated tumor tissue as a vaccine for clinical cancer immunotherapy (Hughes, L. E. et al . , 1970, Cancer,
  • Subcellular extracts were prepared by homogenizing autologous tumor samples and then breaking the isolated cells by sonication. Lysed material was subjected to low speed centrifugation (600x g, 10 min.), to provide a first supernatant that was recentrifuged at 8500 x g for 10
  • composition comprising cell sap combined with the microsomal pellet while five patients were treated with a composition comprising a combination of the microsomal and mitochondrial fractions.
  • the clinical results obtained in these studies were deemed to fall within the limits of the natural course 25 of this disease, indicating that no apparent benefit had been provided by this treatment (Hughes, L. E. et al . , 1970, Cancer, 26(2) :269-78) .
  • Humphrey et al . administered a tumor extract referred to as a "tumor associated antigen preparation, " for
  • Tumor tissue was homogenized as a 20% w/v suspension in a 0.25M sucrose buffer and centrifuged (102,000 x g, 74 min.) . The supernatant obtained was 35 concentrated two-fold against a UM-10 Amicon filter and then frozen. One milliliter aliquots were administered weekly for eight weeks and then quarterly for two years . Since there were no control patients involved in this study, patient survival was compared to historical data. As measured by this standard, the authors suggested that the immunotherapy had modified the host's response to the melanoma and warranted further exploration (Humphrey, L. J. , et al . , 1984, Journal of Surgical Oncology, 25 . : 303 -05) .
  • Cassel et al . have described a cell extract prepared by infecting cultured malignant melanoma tumor cells with Newcastle disease virus and collecting the resulting lysate.
  • the "viral oncolysate” was clarified by centrifugation at 700 x g for 10 minutes and concentrated ten-fold against a PM-10 membrane in a Diaflo Cell (Amicon Corp.), providing an extract in which one milliliter of this ten-fold concentrate comprises approximately 8 x 10 6 cell equivalents (Cassel, W. A. et al . , 1977, Cancer 40 : 672-79).
  • Each dose of lysate represented a mixture of three components: (1) 0.5 ml of lysate prepared from cell line MRD; (2) one ml of lysate prepared from either cell line BMCL or M40; and (3) 1 ml of one lysate prepared from a cell line selected from the group consisting of seven specified cell lines (the choice was rotated with each patient visit) .
  • autologous viral oncolysates were prepared from tissue melanoma tissue taken from the patient, and in those instances, the autologous material was used in place of the third component of the mixture administered.
  • Plasma membrane fractions were isolated from clarified cell extracts by ultracentrifugation in a discontinuous sucrose-dextran T40 gradient. Treatment of mice with 1-2 x 10 3 cell equivalents of material prepared from the plasma membrane fraction or from the 100,000 x g pellet of Meth A cells provided substantial protection against the subsequent challenge of those mice with Meth A ascites cells. In a similar manner, subcellular fractions prepared from CI-4 cells protected inoculated mice against the subsequent challenge with CI-4 cells.
  • DuBois et al demonstrated the presence of a soluble tumor-associated tumor antigen in Meth A murine sarcoma cells in a fractionated cell lysate (DuBois, G. C, et al . , 1980, Cancer Research, 40.4204 -08) .
  • Meth A cells were separated from ascites fluid by low-speed centrifugation, washed, and mechanically disrupted to provide a crude lysate, which was centrifuged at 100,000 x g for fifty minutes. The high-speed supernatant was fractionated using selective precipitation with ammonium sulfate (0-55% cut) .
  • Precipitated proteins were resuspended in buffer and the resulting clarified solution concentrated against a Diaflo PM-30 membrane (Amicon Corporation, Lexington, Mass.). The concentrated material was size-fractionated on a Sephacryl S-200 column (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) . In vivo tumor rejection assays performed on individual fractions of the eluate of that column indicated that Meth A tumor-specific tumor associated transplantation antigen eluted from the Sephacryl S-200 column in a molecular weight range of 43,000 to 67,000.
  • Srivastava et al demonstrated the presence of an additional tumor-specific tumor rejection antigen, a 96,000 molecular weight glycoprotein, that could be isolated from chemically-induced murine sarcoma cell lines, including Meth A and CMS5 (Srivastava, P. K. , 1986, Proc . Natl . Acad . Sci . . 83 . : 3407-11) .
  • Srivastava et al also demonstrated that immunization of mice with unfractionated cytosol corresponding 5 x 10 7 cell equivalents enhanced tumor growth when the treated animals were subsequently challenged with Meth A cells (Srivastava, P. K. , 1986, Proc . Natl . Acad . Sci . 83 .
  • hsp70 was shown to elicit immunity to the tumor from which it was isolated but not to antigenically distinct tumors. However, hsp70 depleted of peptides was found to lose its immunogenic activity (Udono, M., and Srivastava, P.K., 1993, J " . Exp . Med . 178 . : 1391-1396) . These observations suggested that the heat shock proteins are not immunogenic per se, but are carriers of antigenic peptides that elicit specific immunity to cancers (Srivastava, P.K., 1993, Adv. Cancer Res . 62:153-177).
  • Cancer is characterized primarily by an increase in the 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 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
  • Hyperplasia is a form of 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 a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult ceil . 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 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 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 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) .
  • B-lymphocytes which secrete immunoglobulins into the blood plasma for identifying and labeling the nonself invader cells
  • monocytes which secrete the complement proteins which are responsible for lysing and processing the immunoglobulin-coated target invader cells
  • natural killer lymphocytes having two mechanisms for the destruction of tumor cells-antibody- dependent cellular cytotoxicity and natural killing
  • T-lymphocytes possessing antigen-specific receptors wherein 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) .
  • Immunocompetence of the host during the period of cancer induction and development can allow the host to respond to 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.
  • immunosuppression or immune deficiency of the host during carcinogenesis or tumorigenesis may allow growth of highly immunogenic tumors (Schreiber, H., 1989, in Fundamental Immunology (ed) . W.E. Paul, pp. 923-955) .
  • active immunotherapy e . g. , administration of whole attenuated tumor cells or purified tumor antigens
  • adoptive cellular immunotherapy iii) in vivo manipulation of patient plasma to remove blocking factors or add tumoricidal factors
  • 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)
  • TNF tumor necrosis factor
  • monoclonal antibodies and other immunopotentiating agents such as Corynebacterium parvum (C. parvum) (Rosenberg, 2001, In Cancer Principles and Practice of Oncology, 6 th Edition, edited by DeVita et al . , Chapter 18, Lippincott, Williams and Wilkins, Philadephia; Kopp, W.C., et al . , 1994, Cancer Chemotherapy and Biol . Response Modifiers 15:226-286) .
  • 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 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, W.C., et al . , 1994, Cancer Chemotherapy and Biol . Response Modifi ers 1_5: 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.
  • TNF systemically administered TNF seriously limits its use for the treatment of cancer.
  • TNF has been most effective when used for regional therapy, in which 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) .
  • IFN-c. The activity of IFN-c. has been described as being modest in a number of malignancies, including renal cell carcinoma, melanoma, hairy cell leukemia low-grade non- Hodgkin's lymphoma, and others. Higher doses of IFN-c. are usually associated with higher response rates in some malignancies, but also cause more toxicity.
  • Chemotherapeutic and Immunotherapeutic Drugs Extrapolation and Scaling of Animal Data to Humans
  • BSA k x kg 2/3 , 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., 1955, Surface area determina tion , in Glasser E. (ed.) Medical Physics I Chicago: Medical Year Book, p. 1490 and Vriesendorp, H.M., 1985, Hema tol . (Supplm. 16) 13 .
  • 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 or infectious agents, total-body tumor cell burden, toxic effects of chemotherapy and/or immunotherapy on cells and tissues other than the tumor, and distribution of anti-infective, chemotherapeutic, and/or immunotherapeutic agents within the tissues of the patient.
  • tumor growth kinetics drug resistance of tumor cells or infectious agents
  • total-body tumor cell burden toxic effects of chemotherapy and/or immunotherapy on cells and tissues other than the tumor
  • anti-infective, chemotherapeutic, 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 diagnosis and that the patient will relapse after the primary.
  • metastases arise in certain sites in the body where resistance to chemotherapy is based on the limited tissue distribution of chemotherapeutic drugs administered in 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 testes that impede drug diffusion from the capillaries into the tissue. Thus, these sites may require special forms of treatment such as immunotherapy, especially since immunosuppression is characteristic of several types of neoplastic diseases. Similarly, infectious agents, especially intracellular pathogens, may be insulated within environments protected from anti-infective compounds circulating in the blood and, therefore may also required special forms of treatment, including immunotherapy. 3. SUMMARY OF THE INVENTION
  • the methods of the invention comprise methods of eliciting an immune response in an individual in whom the treatment or prevention of cancer or infectious disease is desired by administering a composition comprising an immunogenic amount of unfractionated cellular proteins.
  • the composition is autologous to the individual; that is, the unf actionated cellular proteins are isolated from the cancer cells of the individual himself ⁇ e . g. , preferably prepared from tumor biopsies of the patient), or from cells isolated from a metastasis thereof.
  • the composition is allogeneic to the individual, that is, the unf actionated cellular proteins of the composition are prepared from other individuals or from recombinant or non-recombinant cell lines that express one or more antigens of interest .
  • the present invention encompasses methods for prevention and treatment of cancer by enhancing the host's immune competence and activity of immune effector cells.
  • the amounts of the composition as discovered by the present inventor to be effective are surprisingly smaller than those amounts predicted to be effective by extrapolation by prior art methods or from dosages used in animal studies.
  • Immunotherapy using the therapeutic regimens of the invention by administering an immunogenic amount of such compositions comprising unfractionated cellular proteins that is effective for such treatment, can induce specific immunity to tumor cells, and leads to regression of the tumor mass.
  • compositions comprising unfractionated cellular proteins of the invention include but are not limited to human sarcomas and carcinomas.
  • compositions comprising unf actionated cellular proteins are allogeneic to the patient; in a preferred embodiment, the compositions comprising unfractionated cellular proteins are autologous to (derived from) the patient to whom they are administered.
  • compositions of the invention and their properties are described in the sections and subsections which follow.
  • Preferred compositions comprising unfractionated cellular proteins or unfractionated cytosolic soluble proteins, are isolated from tumor biopsy of the patient to whom the composition is to be administered.
  • Such compositions may be combined with hsp70, hsp90 and/or gp96 complexes, and it would be expected that these compositions would demonstrate strong inhibition of a variety of tumors in mammals.
  • compositions comprising unfractionated cellular proteins that are effective in the corresponding experimental model in rodents as described infra , in Section 8 can be used to inhibit the in vivo growth of colon and liver cancers in human cancer patients as described in Sections 6 and 7, infra .
  • Preferred compositions which preferably exhibit no toxicity when administered to human subjects are also described.
  • the methods further optionally comprise administering biological response modifiers, e . g. , IFN-c., IFN- ⁇ , IL-2, IL-4, IL-6, TNF, or other cytokine growth factors affecting the immune cells, in combination with compositions comprising unfractionated cellular proteins .
  • biological response modifiers e . g. , IFN-c., IFN- ⁇ , IL-2, IL-4, IL-6, TNF, or other cytokine growth factors affecting the immune cells.
  • compositions comprising unfractionated cellular proteins can be utilized 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 .
  • the invention provides a method of inducing an immune response in a subject against a type of cancer which comprises administering to the subject a composition comprising an immunogenic amount of unfractionated cellular proteins obtained from cells of said type of cancer or a metastasis thereof effective to induce said immune response.
  • the invention also provides a method of treating or preventing a type of cancer, comprising administering to a subject in need of such treatment or prevention a composition comprising an amount, effective for treatment or prevention, of unfractionated cellular proteins obtained from cells of the type of cancer or a metastasis thereof.
  • the disclosed methods for inducing an immune response and for preventing or treating a type of cancer are carried out by administering un ractionated cellular proteins from 10 2 to 10 9 cell equivalents of cells from the target type of cancer or metastasis thereof, from 10 2 to 10 7 cell equivalents of cells from the target type of cancer or metastasis thereof, and, preferably, proteins from less than 10 6 cell equivalents, from 10 2 to 5 x 10 5 cell equivalents of cells.
  • the amount of unf actionated cellular proteins administered are in the range of proteins of 10 3 cell equivalents or less of the cells, in a more preferred embodiment from 10 4 cell equivalents or less, and in a even more preferred embodiment, proteins of 5 x 10 5 cell equivalents or less.
  • the unfractionated cellular proteins used in the disclosed methods for inducing an immune response and for preventing or treating a type of cancer are preferably prepared by a method comprising subjecting a lysed sample of said cells to centrifugation one or more times with the highest force being about 100,000 x g, and substantially not subjecting the proteins within the lysed sample to any method that selectively removes particular soluble proteins.
  • the unfractionated cellular proteins so prepared are contained in a solution substantially free of plasma membrane, cell organelles or particles thereof, and viral particles.
  • the proteins used in the disclosed methods for inducing an immune response and for preventing or treating a type of cancer are prepared by a method comprising subjecting a lysed sample of said cells to centrifugation one or more times with the highest force being 1,000 x g, and substantially not subjecting the proteins within the lysed sample to any method that selectively removes particular proteins.
  • the unfractionated cellular proteins so prepared are contained in a solution substantially free of intact cells.
  • the proteins are autologous to the subject. In another embodiment, the proteins are allogeneic to the subject.
  • the unfractionated cellular proteins are isolated from cells obtained from a tumor or from cells of a tumor cell line.
  • the composition administered further comprises an adjuvant.
  • the composition administered is substantially free of adjuvant.
  • the composition is administered at weekly intervals.
  • This administration in one embodiment, is repeated at the same site of the subject. Alternatively, the administration is repeated at different sites.
  • the composition may be administered intradermally, or subcutaneously .
  • the type of cancer is 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,
  • Also disclosed herein is a method of inducing an immune response in a subject against a pathogen, which comprises administering to the subject a composition comprising an immunogenic amount of unfractionated cellular proteins obtained from cells having an antigenicity (antigenic determinant) of said pathogen.
  • a further method disclosed herein is for treating or preventing an infection by a pathogen in a subject comprising administering to a subject in need of such treatment or prevention a composition comprising an amount, effective for such treatment or prevention, of unfractionated cellular proteins obtained from cells having an antigenicity of said pathogen.
  • the proteins are obtained from cells infected with an agent displaying the antigenicity of said pathogen or from cells transformed with and expressing a nucleic acid displaying the antigenicity of said pathogen.
  • the disclosed methods for inducing an immune response against, and for preventing or treating an infection by a pathogen in a subject are carried out by administering unfractionated cellular proteins from 10 2 to 10 7 cell equivalents of cells having an antigenicity of the pathogen, from 10 2 to 10 6 cell equivalents of cells having an antigenicity of the pathogen, and, preferably, proteins from less than 10 s cell equivalents, or from 10 2 to 5 x 10 5 cell equivalents of cells.
  • the proteins are isolated from 10 3 cell equivalents or less of the cells, in a more preferred embodiment from 10 4 cell equivalents or less, and in a even more preferred embodiment, the proteins are isolated from 5 x 10 s cell equivalents or less .
  • the proteins used in the disclosed methods for inducing an immune response against, and for preventing or treating an infection by a pathogen in a subject are prepared by a method comprising subjecting a lysed sample of said cells to centrifugation one or more times with the highest force being 100,000 x g, and substantially not subjecting cytosolic, soluble proteins within the lysed sample to any method that selectively removes particular soluble proteins .
  • the unfractionated cellular proteins so prepared are contained in a solution substantially free of plasma membrane, and cell organelles or particles thereof.
  • the proteins used in the disclosed methods for inducing an immune response against, and for preventing or treating an infection by a pathogen in a subject are prepared by a method comprising subjecting a lysed sample of said cells to centrifugation one or more times with the highest force being 1,000 x g, and substantially not subjecting the proteins within the lysed sample to any method that selectively removes particular soluble proteins.
  • the unfractionated cellular proteins so prepared are contained in a solution substantially free of intact cells.
  • the proteins are autologous to the subject.
  • the proteins are allogeneic to the subject.
  • the unfractionated cellular proteins are isolated from cells infected with the pathogen, from cells infected with replication-defective or other impaired or attenuated derivatives of the pathogen, or from cells transformed with recombinant molecules expressing an antigenicity of the pathogen.
  • the composition administered further comprises an adjuvant.
  • the composition administered is substantially free of adjuvant.
  • the composition is administered at weekly intervals. This administration, in one embodiment, is repeated at the same site of the subject. Alternatively, the administration is repeated at different sites.
  • the composition may be administered intradermally, or subcutaneously.
  • the disclosed methods are used, in a further embodiment, for inducing an immune response against, and for preventing or treating an infection by a pathogen in a subject, where that pathogen is a virus, bacterium, or a parasite; that is, the disclosed methods may be used where the pathogen is selected from the group consisting of hepatitis virus type A, hepatitis virus type B, hepatitis virus type C, influenza virus, varicella virus, adenovirus, herpes simplex virus type I (HSV-I) , herpes simplex virus type II (HSV-II), rinderpest virus, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus , echinovirus, arbovirus , hantavirus, coxsackie virus, mumps virus, measles virus, rubella virus, polio virus, human immunodeficiency virus type I (HI
  • the administered composition further comprises at least one biological response modifier selected from the group comprising heat shock proteins, interferons, interleukins , colony stimulating factors, monoclonal antibodies, and tumor necrosis factor.
  • the invention further provides a method for preparing a vaccine for treatment or prevention of cancer comprising the steps of lysing cancer cells to produce a crude cell lysate; centrifuging the crude cell lysate or supernatant derived therefrom one or more times, to remove intact cells, cell membranes, and organelles, whereby there is substantially no subjecting of cellular proteins within said lysate to any method that selectively removes particular soluble proteins.
  • the lysing step is done by using hypotonic shock combined with mechanical disruption.
  • the lysing step is carried out by suspending cells in a buffer that can, but need not be a hypotonic buffer, and subjecting the suspension to repeated cycles of freezing and thawing.
  • resuspended cells are lysed by sonication or Dounce homogenization .
  • the centrifuging step comprises a first centrifuging at 1,000 x g to produce a first supernatant, and a second centrifuging at 100,000 x g of said first supernatant to produce a second supernatant.
  • the second supernatant is dialyzed against a suitable buffer.
  • the centrifuging step comprises centrifuging the cell lysate only at 1,000 x g to produce a supernatant that is then dialyzed against a suitable buffer.
  • the present invention also encompasses a method of treating or preventing a type of cancer, comprising administering to a subject in need of such treatment or prevention a composition comprising an amount, effective for the treatment or prevention, of unfractionated cellular proteins obtained from cells transformed with and expressing a nucleic acid encoding a molecule displaying antigenicity of a tumor-associated antigen or tumor-specific antigen of said type of cancer.
  • kits comprising in one or more containers an amount, effective for treatment or prevention of a type of cancer, of unfractionated cellular proteins obtained from cells of said type of cancer or a metastasis thereof or from cells transformed with and expressing a nucleic acid encoding a molecule displaying antigenicity of a tumor-associated antigen or tumor-specific antigen of said type of cancer.
  • a further embodiment of the present invention includes a kit comprising in one or more containers an amount, effective for treatment or prevention of an infectious disease, of unfractionated cellular proteins obtained from cells having an antigenicity of a pathogen that causes the infectious disease.
  • the proteins are obtained from cells infected with an agent displaying the antigenicity of said pathogen or from cells transformed with and expressing a nucleic acid displaying the antigenicity of said pathogen.
  • compositions comprising unfractionated cellular proteins in cancer immunotherapy in experimental tumor models and in human patients suffering from advanced colon and liver cancer.
  • 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 involves administration of compositions comprising unfractionated cellular proteins.
  • unfractionated cellular proteins means a collection of proteins contained within a clarified extract of lysed cells, wherein the clarified extract is not subjected to any methods that selectively remove particular proteins, and substantially lacks intact cells .
  • unfractionated cytosolic soluble proteins refers to the collection of proteins contained within a clarified extract of lysed cells, wherein the clarified extract is not subjected to any methods that selectively remove particular soluble proteins, and substantially lacks not only intact cells, but also cell debris, nuclei, organelles and membranes.
  • a clarified cell extract can be prepared by cell lysis and low-speed centrifugation.
  • the low-speed centrifugation is designed to remove intact cells.
  • the clarified cell extract comprises unfractionated cellular proteins, including proteins associated with organelles, and cellular membranes, such as plasma membranes and membranes of the endoplasmic reticulum.
  • the clarified cell extract may be dialyzed against a suitable buffer solution before use.
  • the clarified cell extract is subjected to a high-speed centrifugation.
  • the high-speed centrifugation is generally at 100,000 x g for one hour, which is sufficient to remove most of the remaining cell debris, nuclei, organelles and cellular membranes.
  • the supernatant collected after this step comprises unfractionated cytosolic soluble proteins.
  • the supernatant may be dialyzed against a suitable buffer solution, but it is not subject to any additional chromatographic separations.
  • compositions comprising unfractionated cellular proteins or unfractionated cytosolic soluble proteins are administered to elicit an effective specific immune response.
  • Unfractionated Cellular Proteins comprise methods of eliciting an immune response in an individual or in whom the treatment or prevention of infectious diseases or cancer is desired by administering a composition comprising unfractionated cellular proteins.
  • the unfractionated cellular proteins which include unfractionated cytosolic soluble proteins can be obtained from non-recombinant cells (containing endogenous heat shock protein-peptide complexes) or recombinant cells containing non-endogenous heat shock protein-peptide complexes.
  • the unfractionated cellular proteins are prepared, postoperatively, from tumor cells obtained from the cancer patient.
  • proteins, protein fragments, and peptide antigens of interest are synthesized in cell lines modified by the introduction of recombinant expression systems that encode such antigens, and such cells are used to prepare the unfractionated cellular proteins.
  • Suitable proteins and peptides that may be expressed in such cells include, but are not limited to those displaying the antigenicity of (for the treatment or prevention of cancer) : tumor antigens including tyrosinase, gplOO, melan-A, gp75, mucins; and (for the treatment or prevention of infectious disease) : viral proteins including proteins of immunodeficiency virus type I (HIV-I) , human immunodeficiency virus type II (HIV-II) , 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, cytomegalovirus, echinovirus, arbovirus, hantavirus, co
  • the unfractionated cellular proteins including unfractionated cytosolic soluble proteins are autologous to the individual; that is, the un ractionated cytosolic soluble proteins are isolated from either from the infected cells or the cancer cells or precancerous cells of the individual himself ⁇ e . g. , preferably prepared from infected tissues or tumor biopsies of the patient) .
  • the unfractionated cellular proteins are allogeneic to the treated individual, that is, the proteins are prepared from an individual other than the patient to whom they are administered.
  • Unfractionated cellular proteins are also prepared from cell lines produced in vi tro by recombinant methods whereby the modified cells express an exogenous antigenic molecule of interest, or an increased level of an endogenous antigenic molecule of interest .
  • exogenous antigens, and fragments or derivatives thereof can be selected from among those known in the art, as well as those readily identified by standard immunoassays known in the art by the ability to bind antibody or MHC molecules (antigenicity) or generate immune response (immunogenicity) .
  • Unfractionated cytosolic soluble proteins can be prepared from cancerous or precancerous tissue of a patient, or from a cancer cell line, or can be produced from cell lines constructed by recombinant methods, in vi tro, and which express one or more antigenic molecules of interest.
  • the unfractionated cellular proteins of the present invention can be used alone or may be combined with heat shock proteins, including but not limited to, hsp70, hsp90, gp96 alone or combinations thereof, preferably complexed noncovalently or covalently with an antigenic molecule.
  • the heat shock proteins are human heat shock proteins .
  • 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 any cellular protein that satisfies the following criteria. It is a protein whose intracellular concentration increases when a cell is exposed to a stressful stimulus, it 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 protein having any of the above properties.
  • ATP adenosine triphosphate
  • the first stress proteins to be identified were the heat shock proteins (hsps) .
  • hsps heat shock proteins
  • hsps are synthesized by a cell in response to heat shock.
  • three major families of hsp have been identified based on molecular weight. The 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 limited to, nutrient deprivation, metabolic disruption, oxygen radicals, and infection with intracellular pathogens. (See Welch, May 1993, Scientific American 56-64; Young, 1990, ⁇ i.-.-!.
  • the major hsps can accumulate to very high levels in stressed cells, but they occur at low to moderate levels in cells that have not been stressed.
  • the highly inducible mammalian hsp70 is hardly detectable at normal temperatures but becomes one of the most actively synthesized proteins in the cell upon heat shock (Welch, et al . , 1985, J. Cell . Biol . 101:1198-1211).
  • hsp90 and hsp60 proteins are abundant at normal temperatures in most, but not all, mammalian cells and are further induced by heat (Lai, et al . , 1984, Mol . Cell . Biol .
  • Heat shock proteins are among the most highly conserved proteins in existence.
  • DnaK the hsp70 from E. 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 (Hickey, et al . , 1989, Mol . Cell . Biol . 9:2615-
  • 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 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% amino acid identity with members of the three families whose expression levels in a cell are enhanced in response to a stressful stimulus.
  • the immunogenic hsp-peptide complexes may include any complex containing an hsp and a peptide that is capable of inducing an immune response in a mammal .
  • the peptides are pre erably non covalently associated with the hsp.
  • Preferred complexes may include, but are not limited to, hsp60-peptide, hsp70-peptide and hsp90 -peptide complexes.
  • a complex of an antigenic molecule and an hsp called gp96 which is present in the endoplasmic reticulum of eukaryotic cells and is related to the cytoplasmic hsp90's can be combined with the compositions of the present invention comprising unfractionated cellular proteins, to generate an effective vaccine .
  • compositions comprising unfractionated cellular proteins can be allogeneic to the patient, in a preferred embodiment they are autologous to (derived from) the patient to whom they are administered.
  • the invention provides methods for determining doses for human cancer immunotherapy by evaluating the optimal dose of compositions comprising un ractionated cellular proteins or unfractionated cytosolic soluble proteins m experimental tumor models.
  • the invention provides 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 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 immunity m infectious diseases and cancer immunotherapy.
  • compositions comprising unfractionated cellular proteins appear to induce an inflammatory reaction at the tumor site and ultimately may cause a regression of the tumor burden m the cancer patients treated.
  • Cancers which can be treated with compositions comprising unfractionated cellular proteins include, but are not limited to, human sarcomas and carcinomas.
  • the unfractionated cellular proteins are unfractionated cytosolic soluble proteins.
  • compositions of the present invention comprising an immunogenic, effective amount of unfractionated cellular proteins obtained from cancer cells or a metastasis thereof, are administered to a subject m need of treatment against cancer, as a method of inducing an immune response against that cancer.
  • the proteins administered are preferably from 10 7 cell equivalents or less, from 10 6 cell equivalents or less, or from 10 2 to 5 x 10 5 cell equivalents, or from 10 3 cell equivalents or less, more preferably from 10 4 cell equivalents or less, and most preferably from 5 x 10 5 cell equivalents or less.
  • the invention provides methods of preventing and treating cancer m an individual comprising administering a composition which stimulates the immunocompetence of the host individual and elicits specific immunity against the preneoplastic and/or neoplastic cells.
  • preneoplastic refers to a cell which is in transition from a normal to a neoplastic form; and morphological evidence, increasingly supported by molecular 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.
  • Hyperplasia is a form of 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 a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes 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 frequently a 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 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.
  • 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 the lesion succumbs to an immunological attack by its host.
  • compositions of the invention that comprise unfractionated cellular proteins, may be used in conjunction with additional immune response enhancers or biological response modifiers including, but not limited to, the cytokines IFN- ⁇ , IFN- ⁇ , IL- 2, IL-4 , IL-6, TNF, or other cytokine affecting immune cells, as well as complexes of heat shock proteins and antigenic molecules.
  • additional immune response enhancers or biological response modifiers including, but not limited to, the cytokines IFN- ⁇ , IFN- ⁇ , IL- 2, IL-4 , IL-6, TNF, or other cytokine affecting immune cells, as well as complexes of heat shock proteins and antigenic molecules.
  • the compositions of the present invention may be administered either with, or in a preferred embodiment, without an adjuvant.
  • the invention further relates to administration of compositions comprising unfractionated cellular proteins to individuals at enhanced risk of cancer, e . g . , due to familial history or environmental risk factors.
  • Cells which may be tumor cells derived from a biopsy of the patient or tumor cells cultivated in vi tro, or cells lines infected with a pathogenic agent, are suspended in 3 volumes of IX Lysis buffer consisting of 30mM sodium bicarbonate pH 7.5, and ImM phenyl methyl sulfonyl fluoride (PMSF) .
  • the cells may be lysed by mechanical shearing in the same Lysis buffer, which are incubated on ice for about 20 minutes to allow the cells to become hypotonically- swollen, and which are then homogenized in a dounce homogenizer until >95% cells are lysed.
  • cells resuspended in a non-hypotonic buffer are lysed by cycles of freezing and thawing, or sonication. For example, two to five, and preferably three, such cycles of freezing and thawing are used, as necessary, generally until at least 90% of the cells have been lysed.
  • a non-hypotonic buffer such as PBS
  • sonication cells in PBS and on ice can be sonicated using a Ultrasonic Processor GE130 for 5 cycles ,- each cycle consisting of 10 seconds of exposure to ultrasound and thirty seconds of rest before the next cycle of sonication.
  • the lysate can be centrifuged one or more times at a maximum of about 1,000 x g for a period of time, e.g., 10 minutes, to remove unbroken cells, or until the lysate becomes classified.
  • the clarified cell extract which comprises unfractionated cellular proteins can be dialyzed, generally for 36 hours at 4°C (three times, 100 volumes each time) against PBS (phosphate buffered saline) or other suitable buffer, to provide the unfractionated cellular proteins of the present invention. If necessary, insoluble material in the cell extract may be removed by filtration or further low-speed centrifugation .
  • Cytosolic Soluble Proteins An exemplary, but not limiting, method that may be used to prepare unfractionated cytosolic soluble proteins is as follows :
  • the clarified cell extract which comprises unfractionated cellular proteins prepared as described in Section 4.1.1 is recentrifuged for one or more times at about 100,000 x g for a period of time, for example, about one hour, or until the supernatant is substantially free of plasma membranes, cell organelles or particles there of, and viral particles.
  • a higher speed centrifugation can also be used, e.g., greater than or about 125,000xg, 150,000xg, 175,000xg or 200,000xg.
  • the recovered supernatant comprises unfractionated cytosolic soluble proteins of the present invention and may be dialyzed for 36 hours at 4° (three times, 100 volumes each time) against PBS (phosphate buffered saline) or other suitable buffer. If necessary, any remaining insoluble material in the preparation may be removed by filtration or further centrifugation .
  • unfractionated cellular proteins are isolated from cells transformed with nucleic acids encoding and expressing one or more tumor-associated antigens or tumor-specific antigens (for the treatment or prevention of cancer) , or expressing one or more molecules that display the antigenicity of a pathogen (for the treatment or prevention of infectious disease) .
  • Specific antigens or antigenic portions thereof that can be used as antigenic molecules to be expressed in cell lines from which the unfractionated cytosolic soluble proteins of the present invention may be isolated are selected from among those known in the art or determined by immunoassay to be able to bind to antibody or MHC molecules (antigenicity) or generate an immune response (immunogenicity) .
  • 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” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in vivo immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, immunopreci itation reactions, agglutination assays (e.gr., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
  • ELISA enzyme linked immunosorbent assay
  • sandwich immunoassays immunoradiometric assays
  • gel diffusion precipitin reactions immunodiffusion assays
  • immunodiffusion assays
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled.
  • Many means are known in the art for detecting binding in an immunoassay and are envisioned for use.
  • T cell-mediated responses can be assayed by standard methods, e . g . , in vi tro cytotoxicity assays or in vivo delayed-type hypersensitivity assays.
  • Potentially useful antigens or derivatives thereof for use as antigenic molecules can also be identified by 5 various 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, Lerner, et al . (eds.),
  • the antigen's encoded epitope 15 should preferably display a small or no degree of antigenic variation in time or amongst different isolates of the same pathogen.
  • tumor-specific antigens or fragments or 20 derivatives thereof are used.
  • tumor specific or tumor-associated antigens include but are not limited to KS l/4 pan-carcinoma antigen (Perez and Walker, 1990, J. Immunol .
  • an antigen or fragment or 35 derivative thereof specific to a certain tumor is selected for expression in a cell line to be used for the preparation of unfractionated cellular proteins that are subsequently administered to a patient having that tumor.
  • molecules comprising epitopes of known viruses are expressed in a cell line to be used for the preparation unfractionated cytosolic soluble proteins.
  • antigenic epitopes may be prepared from viruses including, but not limited to, 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, cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus, polio virus, human immunodeficiency virus type I (HIV-I) , and human immunodeficiency virus type II (HIV- II) .
  • viruses including, but not limited to, hepatitis type
  • molecules comprising epitopes of known bacteria are used.
  • antigenic epitopes may be prepared from bacteria including, but not limited to, mycobacteria rickettsia, mycoplasma, neisseria and legionella.
  • molecules comprising epitopes of known protozoa are expressed in recombinant cells lines from which unfractionated cytosolic soluble proteins are prepared.
  • antigenic epitopes may be prepared from protozoa including, but not limited to, leishmania, kokzidioa, and trypanosoma.
  • molecules comprising epitopes of known parasites are expressed in recombinant cells lines from which unfractionated cellular proteins are prepared.
  • antigenic epitopes may be from parasites including, but not limited to, chlamydia and rickettsia.
  • Unfractionated cellular Proteins The unfractionated cellular proteins prepared as described herein can be assayed for immunogenicity using the mixed lymphocyte target culture assay (MLTC) well known in the art .
  • MLTC mixed lymphocyte target culture assay
  • mice are injected subcutaneously with the candidate unfractionated cytosolic soluble proteins, while other mice are injected with either another composition comprising unfractionated cytosolic soluble proteins, whole tumor cells or whole infected cells which act as positive controls for the assay.
  • the mice are injected twice, 7-10 days apart. Ten days after the last immunization, spleens are removed and lymphocytes released. The released lymphocytes may be restimulated subsequently in vi tro by the addition of dead cells that expressed the complex of interest.
  • 8x10 s immune spleen cells may be stimulated with 4xl0 4 mitomycin C treated or ⁇ -irradiated (5-
  • infected cells or tumor cells, or cells transfected with an appropriate gene, as the case may be
  • 3ml RPMI medium containing 10% fetal calf serum.
  • 33% secondary mixed 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).
  • spleen cells may be cultured without stimulation.
  • spleen cells of the immunized mice may also be restimulated with antigenically distinct cells, to determine the specificity of the cytotoxic T cell response.
  • the mixed lymphocyte culture is added to a target cell suspension to give different effector : target (E:T) ratios (usually 1:1 to 40:1).
  • E:T effector : target
  • the target cells are prelabeled by incubating 1x10 s target cells in culture medium containing 200 mCi 51 Cr/ml for one hour at 37°C. The cells are washed three times following labeling.
  • Each assay point (E:T ratio) is performed in triplicate and the appropriate controls incorporated to measure spontaneous 51 Cr release (no lymphocytes added to assay) and 100% release (cells lysed with detergent) . After incubating the cell mixtures for
  • the amount of 51 Cr 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 cpm.
  • 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%.
  • Unfractionated cellular proteins including unfractionated cytosolic soluble proteins of the invention may be formulated into pharmaceutical preparations for administration to animals for inducing an immune response or for treatment or prevention of cancer or infectious diseases.
  • the subject to which compositions comprising unfractionated cytosolic soluble proteins effective in such treatment or prevention may be administered can be an animal. More specifically, the subject may be a domestic animal, such as a cat, dog, horse, cow, chicken, mouse, rat, etc., or, preferably, a mammal or primate, and, most preferably, a human.
  • Compositions comprising unfractionated cytosolic soluble proteins of the invention formulated in a compatible pharmaceutical carrier may be prepared, packaged, and labeled for treatment of the indicated tumor, such as human sarcomas and carcinomas, e . g. , fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothel iosarcoma , lymphangiosarcoma , lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing ' s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate 5 cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary
  • choriocarcinoma 10 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,
  • leukemias e . g. , acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia) ; chronic leukemia (chronic myelocytic (granulocytic) leukemia and
  • compositions may be formulated for treatment of appropriate infectious diseases.
  • the administered dosage of unfractionated cellular proteins or unfractionated cytosolic, soluble proteins is 10 7 , 5 x 10 6 , 10 s , 5 x 10 s , 10 s , 5 x 10 4 ,
  • Cell equivalents can be determined by comparing the protein content of a sample comprising unfractionated cellular proteins or unfractionated cytosolic soluble proteins to the total protein recovered in the low- speed, or high-speed supernatant fraction, depending upon the method used, prepared from a known number of cells.
  • composition comprising the unfractionated cellular proteins of the present invention is water-soluble, then it may be formulated in an appropriate buffer, for example, phosphate buffered saline or other physiologically compatible solutions. Alternatively, it may be formulated with a non-ionic surfactant such as Tween, or polyethylene glycol .
  • a non-ionic surfactant such as Tween, or polyethylene glycol .
  • the compositions and their physiologically acceptable solvates may be formulated for administration by 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.
  • the pharmaceutical preparation 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 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 sorbic acid) .
  • suspending agents ⁇ e . g. , sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e . g.
  • 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 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 methods well-known in the art. Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • compositions for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e . g . , d i c h 1 o r o d i f 1 u o r ome t h a n e , trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a pressurized aerosol the dosage unit 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 composition and a suitable powder base such as lactose or starch.
  • compositions 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- dose containers, with an added preservative.
  • the compositions 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.
  • the active ingredient may 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 butter or other glycerides .
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
  • 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 salt.
  • suitable polymeric or hydrophobic materials for example, as an emulsion in an acceptable oil
  • ion exchange resins for example, as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophilic drugs.
  • compositions of the invention may further comprise one or more cytokines, complexes of heat shock proteins and antigenic molecules, and/or adjuvants.
  • 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 example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • kits for carrying out the therapeutic regimens of the invention comprise in one or more containers therapeutically or prophylactically effective amounts of compositions comprising unfractionated cellular proteins, in pharmaceutically acceptable form.
  • the compositions comprising unfractionated cellular proteins in a vial of a kit of 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 sterile fluid.
  • the composition comprising unfractionated cytosolic soluble proteins 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 composition to form a solution for injection purposes.
  • the unfractionated cellular proteins complexes in the kits are unfractionated cytosolic soluble proteins .
  • kits 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 of hsp- antigenic molecule complexes by a clinician or by the patient .
  • the unfractionated cellular proteins can be administered in combination with complexes of heat shock proteins and antigenic molecules, for eliciting a specific immune response or for treatment or prevention of cancer or infectious disease (e . g. , where the antigenic molecule displays the antigenicity of a cancer cell or infectious agent, respectively) .
  • An "antigenic molecule" that is complexed to a heat shock protein refers to the peptides with which heat shock proteins are endogenously associated in vivo ( e . g. , in infected cells or precancerous or cancerous tissue), as well as exogenous antigens/immunogens (i.e. with which the hsps are not naturally complexed in vivo) or antigenic/immunogenic fragments and derivatives thereof.
  • the peptides are noncovalently complexed to hsps in vivo, and the complexes can be isolated from cells; or alternatively, complexes of hsps and antigenic molecules can be produced in vi tro from purified preparations each of hsps and antigenic molecules, and then combined with the unfractionated cellular proteins .
  • compositions of the present invention may also comprise immunogenic or antigenic peptides that are endogenously complexed to hsps or MHC antigens.
  • immunogenic or antigenic peptides that are endogenously complexed to hsps or MHC antigens.
  • such peptides may be prepared that stimulate cytotoxic T cell responses against by displaying the antigenicity of a tumor antigen (e.g., tyrosinase, gplOO, melan-A, gp75, mucins, etc.) .
  • a tumor antigen e.g., tyrosinase, gplOO, melan-A, gp75, mucins, etc.
  • a protein of an infectious agent e . g.
  • a viral protein including, but not limited to, a protein of immunodeficiency virus type I (HIV-I) , human immunodeficiency virus type II (HIV-II) , 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, cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsackie virus, mumps virus, measles virus, rubella virus, or polio virus .
  • HSV-I protein of immunodeficiency virus type I
  • HV-III human immunodeficiency virus type II
  • hepatitis type A hepatitis type B
  • antigens of cancers e.g., tumors
  • infectious agents e.g., viral antigen, bacterial antigens, etc.
  • hsp-antigenic molecule complex combined with the unfractionated cellular proteins of the invention is a complex produced in vivo in cells
  • exemplary purification procedures for those complexes are described in U.S. Patent No. 5,837,251.
  • 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 known in the art may be used to isolate hsp-peptide 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.
  • the unfractionated cellular proteins used in combination with hsp-antigenic molecule complexes are unfractionated cytosolic soluble proteins.
  • unfractionated cytosolic soluble proteins are prepared from cells infected with an infectious agent, e.g., of a cell line or from a patient.
  • infectious agents include but are not limited to, viruses, bacterial, protozoa, fungi, and parasites as described in detail hereinbelow.
  • unfractionated cytosolic soluble proteins may be combined with hsp-peptide complexes that may either may be isolated from cells infected with the pathogenic agent of interest, or prepared in vi tro by forming complexes of a heat shock protein and an antigenic peptide.
  • the unfractionated cytosolic soluble proteins may also be prepared from recombinant cells expressing one or more protein or peptide antigens against which it is desired to generate an immune response.
  • the unfractionated cellular proteins used are unfractionated cytosolic soluble proteins.
  • Infectious diseases that can be treated or prevented by 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 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, cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus, polio virus, human immunodeficiency virus type I (HIV-I) , and human immunodeficiency virus
  • Bacterial diseases that can be treated or prevented by the methods of the present invention are caused by bacteria 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 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.
  • Target 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, angiosarcoma , endotheliosarcoma , lymphangiosarcoma , lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewin ' 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,
  • the cancer is metastatic.
  • the patient having a cancer is immunosuppressed by reason of having undergone anti-cancer therapy (e.g., chemotherapy radiation) prior to administration of the compositions of the invention that comprise unfractionated cellular proteins. 4.4.1. Colorectal Cancer
  • Colorectal cancer initially spreads to regional lymph 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 cancer to seek medical care vary with the anatomical location of the lesion. For example, lesions in the ascending colon frequency ulcerate, which leads to chronic blood loss in the stool .
  • Radical resection offers the greatest potential for cure in patients with invasive colorectal cancer.
  • the CBA titer is determined. Radiation therapy and chemotherapy are used in patients with advanced colorectal 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, J. Clin . Oncol . 4:565). Patients with widespread metastases have limited survival and systemic chemotherapy has little impact in this group of patients.
  • chemotherapeutic agents e.g., 5- fluorouracil
  • 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 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 .
  • the present invention provides 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 an autologous composition comprising unfractionated cellular proteins, which elicits tumor-specific immunity against the tumor cells.
  • an autologous composition comprising unfractionated cellular proteins, which elicits tumor-specific immunity against the tumor cells.
  • the use of a composition of the invention, comprising unfractionated cellular proteins could result in nearly complete inhibition of liver cancer growth in cancer patients, without inducing toxicity and thus providing a dramatic therapeutic effect.
  • a composition comprising unfractionated cellular proteins is administered to a patient diagnosed with colorectal cancer, with or without liver metastasis, via one of many different routes of administration, the preferred routes being intradermal at different anatomical sites, e.g., left arm, right arm, left belly, right belly, left thigh, right thigh, etc.
  • routes of administration are used in sequence and the site of injection is varied for each weekly injection as described in Section 7.
  • the preparations and use of therapeutically effective compositions for the prevention and treatment of primary and metastatic cancers are described in detail in the sections which follow and by way of example, infra .
  • 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 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 is caused by inherited hemochromatosis or hepatic B viral infection (Bradbear, R.A. , et al .
  • cirrhosis causes of cirrhosis that can lead to hepatocellular carcinoma include alcohol abuse and hepatic fibrosis caused 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, accompanied by weight loss.
  • the development of hepatocellular carcinoma is preceded by ascites, portal hypertension and relatively abrupt clinical deterioration.
  • abnormal values in standard liver function tests such as serum aminotransferase and alkaline phosphatase are observed.
  • CT scans of the liver are used to determine the anatomic distribution of hepatocellular carcinoma and also provide orientation for percutaneous needle biopsy.
  • Approximately 70 percent of patients with hepatocellular carcinoma have an elevated serum alpha-fetoprotein concentration (Mclntire, K.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 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 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 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 present invention provides compositions and methods for enhancing 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.
  • Another specific aspect of the invention relates to the treatment of breast cancer.
  • the American Cancer Society estimated that in 2000, 184,200 American women will be diagnosed with breast cancer and 41,200 will succumb to the disease (Cancer Facts & Figures 2000, American Cancer Society (ACS) , Atlanta, Georgia, 2000) . This makes breast cancer the second major cause of cancer death in women, ranking just behind lung cancer.
  • the treatment of 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 or improve quality of life.
  • the present invention provides compositions of unfractionated cellular proteins for enhancing specific immunity to preneoplastic and neoplastic mammary cells in women.
  • the present invention also provides compositions of unfractionated cellular proteins and methods for preventing the development of neoplastic cells in women at enhanced risk for 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.
  • 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.
  • immunotherapy as provided by the present invention is desired for use in cancer patients.
  • a third reason is the possibility that tumor cells are shed into the 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 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 . 4.6.1.
  • the effect of immunotherapy with compositions comprising unfractionated cellular proteins, on development and progression of neoplastic diseases can be monitored by any method 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 vi tro; c) levels of tumor specific antigens, e.g., carcinoembryonic (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.
  • a) delayed hypersensitivity as an assessment of cellular immunity b) activity of cytolytic T- lymphocytes in vi tro
  • c) levels of tumor specific antigens e.g., carcinoembryonic (CEA) antigens
  • CCA carcinoembr
  • Delayed Hypersensitivity Skin 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 antigens is termed anergy (Sato, T., et al . , 1995, Clin . Immunol . Pathol . 74:35-43) .
  • antigens 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 ensures intradermal, rather than subcutaneous, administration of antigen.
  • Hypoactivity to any given antigen or group of antigens is confirmed by testing with higher concentrations of antigen or, in ambiguous circumstances, by a repeat test with an intermediate tes .
  • Peripheral blood derived T lymphocytes isolated by the Ficoll-Hypaque centrifugation gradient technique are restimulated with 4xl0 4 mitomycin C treated tumor cells in 3ml RPMI medium containing 10% fetal calf serum.
  • 33% secondary mixed lymphocyte culture supernatant or IL-2 is included in the culture medium as a source of T cell growth factors.
  • 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 cytotoxicity in a 4 hour sl Cr- release assay. The spontaneous 51 Cr-release of the targets should reach a level less than 20%.
  • 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. Immunotherapy 15:165-174).
  • tumors Although it may not be possible to detect unique tumor antigens on all tumors, many tumors display antigens that distinguish them from normal cells.
  • the monoclonal antibody reagents have permitted the isolation and biochemical characterization of the antigens and have been invaluable diagnostically for distinction of transformed from nontransformed cells and for definition of the cell lineage of transformed cells.
  • the best-characterized human tumor- associated 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 glycoprotein found on fetal gut an human colon cancer cells, but not on normal adult colon cells.
  • CEA carcinoembryonic antigen
  • CEA is shed from colon carcinoma cells and found in the serum, it was originally thought that the presence of this antigen in the serum could be used to screen patients for colon cancer.
  • patients with other tumors, such as pancreatic and breast cancer also have elevated serum levels of CEA. 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.
  • alpha- fetoprotein an alpha-globulin normally secreted by fetal liver and yolk sac cells
  • alpha- fetoprotein an alpha-globulin normally secreted by fetal liver and yolk sac cells
  • Computed Tomographic (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 .
  • the levels of a putative biomarker for risk of a specific cancer are measured to monitor the effect of compositions comprising unfractionated cellular proteins.
  • serum prostate-specific antigen PSA
  • PSA serum prostate-specific antigen
  • et al . 1992, J. Urol . 147:841-845
  • Catalona W.J., et al . , 1993, JAMA 270:948- 958
  • individuals at risk for colorectal cancer CEA is measured as described above in Section 4.5.3
  • 16-o.- 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 reference herein in their entirety.
  • Sonogram A Sonogram remains an alternative choice of technique for the accurate staging of cancers .
  • compositions comprising unfractionated cellular proteins can be measured by tumor rejection assays in vivo . While this assay is clearly the most demanding and rigorous evidence for immunogenicity, it is impractical for the purpose of monitoring immune response in humans.
  • the ability of tumor-derived compositions comprising unfractionated cellular proteins, to elicit a CD8 + T cell response is evaluated in order to define an in vi tro correlate for in vivo tumor rejection.
  • Mice are immunized twice with a composition comprising 10 2 to 10 s cell equivalents of unfractionated cellular proteins derived from 6138 or 6139SJ cells (Ward et al., 1989, J. Exp. Med. 170:217).
  • MLTCs Mixed lymphocyte-tumor cultures
  • a composition comprising 10 2 to 10 6 cell equivalents of unfractionated cellular proteins, elicits an effective tumor-specific CTL response, which may be measured in vi tro .
  • compositions Comprising Unfractionated Cellular Proteins to Elicit a Memory
  • the ability to elicit a memory response is crucial for any vaccine and, therefore, the ability of a composition comprising unfractionated cellular proteins to elicit a memory T cell population is tested.
  • a number of criteria i.e., radiation resistance, kinetics of appearance, loss of CD45RB and L-selectin lymphocyte surface antigens, are used to identify memory T response.
  • memory T cells are cycling cells (Mackay, C.R., et al .
  • mice are vaccinated twice at ten day intervals, with a composition comprising unfractionated cellular proteins and the vaccinated mice are irradiated (400 rad) twelve days after the last vaccination.
  • MLTCs are generated from spleens of mice and tested for tumor- specific CTL response. It is expected that the irradiated, mice vaccinated with unfractionated cellular proteins generate powerful, MHC class I - restricted and tumor-specific CTL responses.
  • the irradiation eliminates the non-memory resting T cells, while the delay between the last vaccination and generation of MCTCs eliminates activated T lymphocytes (Sprent, J., 1994, Cell 76:315) . Therefore, under these conditions, detection of a desired CTL response indicates that the composition comprising unfractionated cellular proteins elicits a response derived from radiation-resistant memory T cells. This phenomenon is also tested in tumor rejection assays in vivo . Mice vaccinated with a composition comprising unfractionated cellular proteins are irradiated and then observed to evaluate their resistance to tumor challenges for up to 17 days after vaccination. Tumor rejection under these conditions indicates that vaccination with a composition comprising unfractionated cellular proteins elicits a long-lived, radiation-resistant T cell population.
  • CD45RB (Birkeland, M.L., et al . , 1989, Proc . Na tl . Acad . Sci . USA 86:6734) on CD8 + lymphocytes from irradiated and non-irradiated, naive mice and mice vaccinated with a composition comprising unfractionated cellular proteins are also tested.
  • lymphocytes are obtained under the same regimen as described in the preceding paragraph, i . e . , fifteen days after the last vaccination including three days after irradiation, in order to allow the activated effector cells to be depleted.
  • Patients with hepatocellular carcinoma are injected with a composition comprising unfractionated cellular proteins
  • compositions comprising unfractionated cellular proteins are 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 well known in the art or as described herein.
  • the therapeutic regimen of compositions comprising unfractionated cellular proteins includes weekly injections of the composition comprising unfractionated cellular proteins dissolved in saline or other physiologically compatible solution .
  • the dosage used is in the range of from about 10 2 to about 10 9 cell equivalents, preferably in the range of from about 10 2 to about 5 x 10 7 cell equivalents, and more preferably in the range of from about 10 3 to about 5 x 10 s cell equivalents of cell unfractionated cellular proteins.
  • the route and site of injection are varied each time, for example, the first injection is given subcutaneously on the left arm, the second injection on the right arm, the third 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.
  • injections are split and each half of the dose is administered at a different site on the same day.
  • the effect of treatment with a composition comprising unfractionated cellular proteins is monitored by measuring: a) delayed hypersensitivity as an assessment of cellular immunity; b) activity of cytolytic T-lymphocytes in vi tro; c ) levels of tumor specific antigens, e.g., carcinoembryonic (CEA) antigens; d) changes in the morphology of tumors using techniques such as a computed tomographic (CT) scan; and e) changes in putative biomarkers of risk for a particular cancer in individuals at high risk.
  • a composition comprising unfractionated cellular proteins is monitored by measuring: a) delayed hypersensitivity as an assessment of cellular immunity; b) activity of cytolytic T-lymphocytes in vi tro; c ) levels of tumor specific antigens, e.g., carcinoembryonic (CEA) antigens; d) changes in the morphology of tumors using techniques such as a computed tomographic (CT) scan; and e) changes in put
  • 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.
  • compositions comprising unfractionated cellular proteins are administered as adjuvant therapy and as prophylactic adjuvant therapy in patients after complete reduction of colorectal cancer to eliminate undetectable icrometastases and to improve survival .
  • the therapeutic and prophylactic regimens used in patients suffering from colorectal cancer are the same as those described in Section 6 above for patients recovering with hepatocellular carcinoma.
  • the methods of monitoring of patients under clinical evaluation for prevention and treatment of colorectal cancer is done by procedures described in Section
  • CEA levels are measured as a useful monitor of tumor regression and/or recurrence (Mayer, R.J., et al . , 1978, Cancer 42 : 1428) .
  • Meth A tumor model is a methylcholanthrene-induced tumor that was originally isolated from a BALB/c mouse, and is now maintained by serial passage of ascites fluid in BALB/c mice (Old et al . Ann. N.Y. Acad. Sci. 101: 80-67 (1962) ) . Preparation of unfractionated cellular proteins:
  • compositions comprising unfractionated cellular proteins were prepared from Meth A tumors according to the procedures described above in Section 4.1.1.
  • the compositions comprising unfractionated cellular proteins, preparations are administered without adjuvants.
  • clarified Meth A tumor cell extracts were prepared by freezing and thawing cell suspensions that were then subjected centrifugation at only low speed, i.e., 1000 x g, to provide a supernatant comprising unfractionated cellular proteins, which were then administered to tumor-bearing mice for treatment.
  • compositions comprising unfractionated cellular proteins derived from Meth A tumor cells were tested.
  • mice were injected mtradermally with 10 s Meth A cells. Beginning five days after injection of the tumor cells (day 5) , each group of mice was administered PBS buffer, irradiated whole Meth A tumor cells, 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 s , or 1 x 10 7 cell equivalents of unfractionated cellular proteins prepared from Meth A tumor cells. These treatments were repeated for each group of mice at day 7, 9, 12, 14, and 16.
  • compositions comprising unfractionated cellular proteins derived from Meth A tumor cells to prevent development of Meth A tumors in vivo was tested.
  • mice were vaccinated intradermally at day 0 and day 7 with the indicated amount (in cell equivalents) of unfractionated cellular proteins, which were prepared by lysing Meth A tumor cells by freezing and thawing, Dounce homogenization, and sonication as indicated, and subjecting the lysate to centrifugation only at low-speed (1 , 000 x g) .
  • mice were challenged by intradermal injection of 10 s viable Meth A tumor cells.
  • composition Administered Number of Mice Without Tumor/

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Abstract

L'invention concerne des procédés et des compositions pour la prévention et le traitement de maladies infectieuses et de maladies néoplasiques primaires et métastatiques, notamment, mais pas uniquement, de sarcomes et carcinomes humains. Dans la pratique de la prévention et du traitement des maladies infectieuses et du cancer, des compositions comprenant des protéines cellulaires non fractionnées sont utilisées pour augmenter la réponse immunitaire à des facteurs génotoxiques et non génotoxiques, à des tumeurs et à des agents infectieux.
PCT/US2001/028841 2000-09-15 2001-09-17 Compositions et procedes pour la prevention et le traitement de maladies neoplasiques primaires, de maladies neoplasiques metastatiques, et de maladies infectieuses, a l'aide de compositions comprenant des proteines cellulaires non fractionnees WO2002030434A1 (fr)

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US10/380,442 US20030211971A1 (en) 2001-09-17 2001-09-17 Compositions and methods for prevention and treatment of primary and metastatic neoplastic diseases and infectious diseases with compositions comprising unfractionated cellular proteins
EP01975212A EP1322317A4 (fr) 2000-09-15 2001-09-17 Compositions et procedes pour la prevention et le traitement de maladies neoplasiques primaires, de maladies neoplasiques metastatiques, et de maladies infectieuses, a l'aide de compositions comprenant des proteines cellulaires non fractionnees
CA002422718A CA2422718A1 (fr) 2000-09-15 2001-09-17 Compositions et procedes pour la prevention et le traitement de maladies neoplasiques primaires, de maladies neoplasiques metastatiques, et de maladies infectieuses, a l'aide de compositions comprenant des proteines cellulaires non fractionnees
AU2001294560A AU2001294560B2 (en) 2000-09-15 2001-09-17 Compositions and methods for prevention and treatment of primary and metastatic neoplastic diseases and infectious diseases with compositions comprising unfractionated cellular proteins
JP2002533874A JP2005516884A (ja) 2000-09-15 2001-09-17 非分画細胞タンパク質を含有する組成物を用いて原発性および転移性新生物疾患並びに感染性疾患を予防および治療するための組成物および方法
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006123164A2 (fr) * 2005-05-19 2006-11-23 The Edward Jenner Institute For Vaccine Research Procedes de traitement et de prevention des infections
US7666581B2 (en) 2001-08-20 2010-02-23 University Of Connecticut Health Center Methods for preparing compositions comprising heat shock proteins useful for the treatment of cancer and infectious disease
US20110280911A1 (en) * 2008-11-17 2011-11-17 The Regents Of The University Of Michigan Cancer vaccine compositions and methods of using the same

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5098997A (en) * 1987-12-11 1992-03-24 Praxis Biologics, Inc. Vaccines for Haemophilus influenzae

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HOLLINSHEAD ARIEL, EDITED BY RICHARD LAFOND: "Immunotherapy", 1988, AMERICAN CHEMICAL SOCIETY, WASHINGTON, D.C., XP002908392 *
PAUL WILLIAM E.: "Fundamental immunology", 1993, RAVEN PRESS, NEW YORK, XP002908391 *
See also references of EP1322317A4 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7666581B2 (en) 2001-08-20 2010-02-23 University Of Connecticut Health Center Methods for preparing compositions comprising heat shock proteins useful for the treatment of cancer and infectious disease
WO2006123164A2 (fr) * 2005-05-19 2006-11-23 The Edward Jenner Institute For Vaccine Research Procedes de traitement et de prevention des infections
WO2006123164A3 (fr) * 2005-05-19 2007-05-10 Edward Jenner Inst For Vaccine Procedes de traitement et de prevention des infections
GB2441094A (en) * 2005-05-19 2008-02-20 Edward Jenner Inst For Vaccine Methods for treatment and prevention of infection
GB2441094B (en) * 2005-05-19 2010-11-03 Edward Jenner Inst For Vaccine Methods for treatment and prevention of infection
US20110280911A1 (en) * 2008-11-17 2011-11-17 The Regents Of The University Of Michigan Cancer vaccine compositions and methods of using the same
US9974844B2 (en) * 2008-11-17 2018-05-22 The Regents Of The University Of Michigan Cancer vaccine compositions and methods of using the same
US11116825B2 (en) 2008-11-17 2021-09-14 The Regents Of The University Of Michigan Cancer vaccine compositions and methods of using the same

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CA2422718A1 (fr) 2002-04-18
EP1322317A1 (fr) 2003-07-02
AU9456001A (en) 2002-04-22
JP2005516884A (ja) 2005-06-09
EP1322317A4 (fr) 2004-12-01

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