WO2000038710A2 - Dual hapten modified tumor cells and tumor polypeptides and methods of treating cancer - Google Patents

Abstract

This invention relates to compositions comprising dual haptenized tumor cells and extracts thereof, methods for preparing the compositions, vaccines comprising such double haptenized tumor cells, and methods for treating cancer with such vaccines. In a specific embodiment, melanoma and ovarian adenocarcinoma cells are dual-haptenized with a dinitrophenyl group coupled to epsilon -amino groups, and with a sulfanilic acid group coupled to aromatic side chains of histidine and tyrosine. The dual-haptenized tumor cells can be used in vaccine preparations at a lower dosage, and are more effective for eliciting favorable immune responses.

Description

DUAL HAPTEN MODIFIED TUMOR CELLS AND TUMOR POLYPEPΗDES AND METHODS OF TREATING CANCER

5

FIELD OF THE INVENTION

The invention relates to compositions comprising dual haptenized tumor cells and extracts thereof, methods for preparing the compositions, vaccines comprising such dual haptenized tumor cells, and methods for treating cancer with 10 such vaccines.

BACKGROUND OF THE INVENTION Haptenized Tumor Cell Vaccines

An autologous whole-cell vaccine modified with the hapten

15 dinitrophenyl (DNP) has been shown to produce inflammatory responses in metastatic sites of melanoma patients. The survival rates of patients receiving post-surgical adjuvant therapy with DNP -modified vaccine are markedly higher than those reported for patients treated with surgery alone. Intact or viable cells are preferred for the vaccine.

20 U.S. Patent No. 5,290,551 , to David Berd, discloses and claims vaccine compositions comprising haptenized melanoma cells. Melanoma patients who were treated with these cells developed a strong immune response. This response was detected, e.g., in a delayed-type hypersensitivity (DTH) response to haptenized and non-haptenized tumor cells. More importantly, the immune response increased

25 the survival rates of melanoma patients.

Haptenized tumor cell vaccines have also been described for other types of cancers, including lung cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, and leukemia (see U.S. Patent Application No. 08/203,004, filed February 28, 1994; International Patent Application No. PCT/US96/09511; U.S.

30 Patent Application No. 08/899,905, filed July 24, 1997). Generally, the immune response to haptenized cells has been found to be independent of the choice of hapten, but dependent on the functional group to which the hapten is attached. In particular, it has been reported that haptenization of e-amino groups of lysine and -COOH groups of aspartic acid and glutamic acid is effective for a robust immune response, and that haptenization of aromatic groups (such as tryptophan) potentially results in a less effective or ineffective immune response (Nahas and Leskowitz, Cellular Immunol., 1980;54:241).

It is known from animal studies that immunization of mice with syngeneic lymphocytes modified with arsanilic acid induces strong T cell responses against those modified cells, including DTH (Bach et al. , J. Immunol.,

1978;121 :1460) and cytotoxic T cells (Sherman et a!., J. Immunol., 1978;121 :1432). Injection of arsanilic acid into the rat kidney induced a brisk autoimmune nephritis (Rennke et al., Kidney International, 1994;45:1044). Obviously, the administration of even minute amounts of arsanilic acid into human is unacceptable, but sulfanilic acid, a non-toxic compound in small amounts, should induce a similar immunological effect (Nahas and Leskowitz, supra, 1980). Both compounds can be coupled to tyrosine and histidine after being diazotized by treatment with sodium nitrite. Moreover, immunization of animals with sulfanilic acid-modified protein can induce autoimmunity (Weigle, J. Exp. Med., 1965; 122: 1049). A third potentially interesting hapten in this category is phosphorylcholine (PC), in light of the work of Kim et al. (Eur. J. Immunol., 1992;22:775). However, it has not been established that these haptens will be effective in humans; on the contrary, Nahas and Leskowitz, supra, suggest otherwise.

These discoveries have led to rapid advances in the treatment of cancer, particularly melanoma, by immunotherapy. Nevertheless, there remains a need in the art for even more effective therapies, since the response rates achieved with the haptenized tumor cell vaccine technologies mentioned above, while impressive, have not reached 100%. There is also a need in the art for effective vaccines using fewer cells, e.g., fewer than about 10⁷ cells per dose. This is especially critical for the treatment of an early stage cancer, when the number of cells obtainable from a resected tumor may be fewer than necessary for vaccine preparation as described above.

The present invention addresses these and other needs in the art in a surprisingly effective way.

The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

SUMMARY OF THE INVENTION

The present invention advantageously provides a composition comprising dual-haptenized tumor cell polypeptides. The polypeptides are conjugated with two haptens by derivitization of two different functional groups found on the polypeptides, wherein the functional groups are selected from the group consisting of a primary amine group, a carboxylic acid group, an aromatic group, and a sulfhydryl group. In addition, the polypeptides originate from the same tumor type as that of a cancer patient intended for treatment with the composition of the invention. In various embodiments, the polypeptides are separated from non-polypeptide components of haptenized tumor cells; the polypeptides are associated with tumor cell membrane components separated from other cellular components and from whole tumor cells; or the polypeptides are associated with whole autologous tumor cells, wherein the tumor cells have been rendered incapable of growth before administration to a patient. In particular, the invention provides a composition comprising dual-haptenized tumor cells.

The invention also provides a vaccine for treating cancer comprising a dual-haptenized tumor cell polypeptide composition and an adjuvant.

The invention also provides a method for preparing a composition for use in a cancer vaccine. This method comprises haptenizing tumor cells, originating from same tumor type as that of a cancer patient intended for treatment with the vaccine, with two haptens attached to two different functional groups on the polypeptides, wherein the functional groups are selected from the group consisting of a primary amine group, a carboxylic group, an aromatic group, and a sulfhydryl group.

In addition, a method for treating cancer in a subject comprising administering a vaccine of the invention to the subject is provided.

Thus, one object of the invention is to provide more effective cancer vaccines by modifying cells, and particularly cell-associated peptides by dual-haptenization.

This and other aspects of the invention are further elaborated in the Detailed Description of the Invention and Examples, infra.

DETAILED DESCRIPTION OF THE INVENTION

The invention advantageously provides for modification of tumor cells with two haptens, particularly modifications in which haptens are coupled to hydrophobic residues of major histocompatibility complex (MHC)-bound peptides, which augments the immunogenicity of autologous, hapten-modified cells. The work described herein has provided strong support for the idea that immunizing patients with hapten-modified tumor cells can induce immunity to unmodified tumor cells. Animal data suggest that it should be possible to increase the immunogenicity of hapten-modified cells by the addition of a second hapten. The present invention provides a rationale for achieving improved results in humans. More particularly, the invention permits the use of a decreased number of tumor cells in a vaccine, or a more effective immune response, or both. Furthermore, the immune response is, in a specific embodiment, enhanced by a method of haptenization previously believed to be ineffective. The various aspects of the invention will be set forth in greater detail in the following sections. This organization into various sections is intended to facilitate understanding the invention, and is in no way intended to be limiting thereof.

Definitions The following defined terms are used throughout the present specification, and should be helpful in understanding the scope and practice of the present invention.

In a specific embodiment, the term "about" or "approximately" means within 20%, preferably within 10%), and more preferably within 5% of a given value or range.

A "formulation" refers to an aqueous medium or solution for the preservation of haptenized tumor cells, which is preferably directly injectable into an organism. The aqueous medium will include salts or sugars, or both, at about an isotonic concentration. The phrase "pharmaceutically acceptable" refers to molecular entities, at particular concentrations, and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, fever, dizziness and the like, when administered to a human or non-human animal. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in humans or non-human animals.

As used herein, the term "isolated" means that the referenced material is removed from the natural environment in which it is normally found. In particular, isolated biological material if free of cellular components. An isolated peptide may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is membrane-associated. An isolated organelle, cell, or tissue is removed from the anatomical site in which it is found in an organism. An isolated material may be, but need not be, purified.

The term "purified" as used herein refers to material that has been isolated under conditions that reduce or eliminate unrelated materials, i.e., contaminants. For example, a purified protein is preferably free of other proteins or nucleic acids with which it is associated in a cell; a purified cell is free of unrelated cells and tissue matrix components.

A "subject" is a human or a non-human animal who may receive haptenized tumor cells formulated in a composition of the invention. Preferably the subject is a human. However, the invention is also contemplated for veterinary medicine, particularly for treatment of domestic pets (dogs, cats), and livestock (horses, cows, pigs, etc.)

Dual-Haptenized Tumor Cells

The present invention is directed for use in the preparation of haptenized tumor cell vaccines for treating cancer, including metastatic and primary cancers. Cancers treatable with the present invention include solid tumors, including carcinomas, and non-solid tumors, including hematologic malignancies. Examples of solid tumors that can be treated according to the invention include sarcomas and carcinomas such as, but not limited to: 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 adenocarcmomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. Hematologic malignancies include leukemias, lymphomas, and multiple myelomas. The following are non- limiting preferred examples of the cancers treatable with the composition and methods of the present invention: melanoma, including stage-4 melanoma; ovarian, including advanced ovarian; leukemia, including but not limited to acute myelogenous leukemia; colon, including colon metastasized to liver; rectal, colorectal, breast, lung, kidney, and prostate cancers. Tumor Cells

The compositions of the present invention are prepared from tumor cells, e.g., cells obtained from tumors surgically resected in the course of a cancer treatment regimen as described above. Tumor cells to be used in the present invention may be prepared as follows. Tumors are processed as described by Berd et al, Cancer Res., 1986;46:2572, Sato, et al, Cancer Invest., 1997;15:98, U.S. Patent No. 5,290,551, and applications U.S. Serial Nos. 08/203,004, 08/479,016, 08/899,905, 08/942,794, or corresponding PCT application PCT/US96/09511, each of which is incorporated herein by reference in its entirety. Briefly, the cells are extracted by dissociation, such as by enzymatic dissociation with collagenase and DNase, by mechanical dissociation in a blender, by teasing with tweezers, using mortar and pestle, cutting into small pieces using a scalpel blade, and the like. With respect to liquid tumors, blood or bone marrow samples may be collected and tumor cells isolated by density gradient centrifugation.

The tumor cells of the present invention may be live, attenuated, or killed cells. Tumor cells incapable of growth and division after administration into the subject, such that they are substantially in a state of no growth, are preferred for use in the present invention. It is to be understood that "cells in a state of no growth" means live cells that will not divide in vivo. Conventional methods of rendering cells incapable of division are known to skilled artisans and may be useful in the present invention. For example, cells may be irradiated prior to use. Tumor cells may be irradiated to receive a dose of about 2500 cGy to prevent the cells from multiplying after administration. Alternatively, haptenization, and particularly dual haptenization, can render the cells incapable of growth.

The tumor cells should preferably originate from the same type of cancer as that to be treated, and are even more preferably syngeneic (e.g., autologous or tissue-type matched). For purposes of the present invention, syngeneic refers to tumor cells that are closely enough related genetically that the immune system of the intended recipient will recognize the cells as "self, e.g., the cells express the same or almost the same complement of MHC molecules. Another term for this is "tissue- type matched." For example, genetic identity may be determined with respect to antigens or immunological reactions, and any other methods known in the art. A syngeneic tumor cell can be created by genetically engineering a tumor cell to express the required MHC molecules.

Preferably the cells originate from the type of cancer which is to be treated, and, more preferably, from the same patient who is to be treated. The tumor cells may be, but are not limited to, autologous cells dissociated from biopsy or surgical resection specimens, or from tissue culture of such cells. Nonetheless, allogeneic cells and stem cells are also within the scope of the present invention.

Tumor Cell Membranes The isolated, modified tumor cell membranes of the present invention are prepared from mammalian, preferably human, tumor cells. In one embodiment of the invention, tumor cell membrane are isolated from a tumor of an animal, e.g., from a feline, canine, equine, bovine, or porcine family. Isolation and preparation of haptenized tumor cell membranes is described in US Patent Application No. 08/479,016, filed June 7, 1995 and US Application No. 90/025,012, filed February 17, The tumor cells from which membranes are isolated may be live, attenuated, or killed cells. Tumor cells rendered incapable of growth and division prior to administration into the patient, such that the cells are substantially in a state of no growth, can be used in the present invention. Alternatively, tumor cell membranes may also be isolated from tumor cells capable of in vivo growth and division, since the membranes by themselves cannot multiply. Preferably, in such a case, the tumor cell membrane preparation is not contaminated with tumor cells capable of multiplying in vivo.

As with tumor cells, tumor cell membranes are preferably isolated from the tumor cells of the same type of cancer as that to be treated. For example, membranes to be used for treating ovarian cancer are isolated from ovarian cancer cells. Preferably, the tumor cells originate from the same subject who is to be treated. The tumor cells are preferably syngeneic (e.g. autologous), but may also be allogeneic to that subject. There may be genetic identity between a particular antigen on the tumor cell used as a membrane source and an antigen present on the patient's tumor cells. The tumor cells may be, but are not limited to, cells dissociated from biopsy specimens or from tissue culture. Membranes isolated from allogeneic cells and stem cells are also within the scope of the present invention.

Tumor cell membranes may include all cellular membranes, such as outer membrane, nuclear membranes, mitochondrial membranes, vacuole membranes, endoplasmic reticular membranes, golgi complex membranes, and lysosome membranes. In one embodiment of the invention, more than about 50%> of the membranes are tumor cell plasma membranes. Preferably, more than about 60%> of the membranes consist of tumor cell plasma membranes, with more than about 70% being more preferred, 80% being even more preferred, 90% being even more preferred, 95% being even more preferred, and 99% being most preferred.

Preferably, the isolated membranes are substantially free of nuclei and intact cells. For example, a membrane preparation is substantially free of nuclei or intact cells if it contains less than about 100 cells and/or nuclei in about 2 x 10⁸ cell equivalents (c.e.) of membrane material. A cell equivalent is that amount of membrane isolated from the indicated number of cells. An isolated tumor cell membrane which is substantially free of cells and/or nuclei may contain lymphocytes and/or lymphocyte membranes.

Preferably, the isolated tumor cell membranes are the outer cell membranes, i.e., tumor cell plasma membranes. The membrane preparation of the invention may contain the entire outer membrane or a fraction thereof. An isolated membrane of the invention, preferably including a fraction of the outer membrane, contains an MHC molecule fraction and/or a heat shock protein fraction. The size of the membrane fragments is not critical.

Allogeneic tumor cell membranes may also be used in the methods of the present invention with syngeneic (e.g. autologous) antigen presenting cells. This approach permits immunization of a patient with tumor cell membranes originating from a source other than the patient's own tumor. Syngeneic antigen-presenting cells process allogeneic membranes such that the patient's cell-mediated immune system may respond to them.

A tumor cell membrane (modified or un-modified) as referred to in this specification includes any form in which such a membrane preparation may be stored or administered, such as, for example, a membrane resuspended in a diluent, a membrane pellet, or a frozen or a lyophilized membrane.

The tumor cell membranes can be obtained from haptenized cells, or may be haptenized after extraction from the cells using the techniques described infra.

Tumor cell membranes are prepared from tumor cells, e.g., obtained as described above, by disrupting the cells using, for example, hypotonic shock, mechanical dissociation and enzymatic dissociation, and separating various cell components by centrifugation. Briefly, the following steps may be used: lysing tumor cells, removing nuclei from the lysed tumor cells to obtain nuclei-free tumor cells, obtaining substantially pure membranes free from cells and nuclei, and coupling the tumor cell membranes to a hapten to obtain hapten-modified tumor cell membranes. Membrane isolation may be conducted in accordance with the methods of Heike et al. In one embodiment of the invention, intact cells and nuclei may be removed by consecutive centrifugation until membranes are substantially free of nuclei and cells, as determined microscopically. For example, lysed cells may be centrifuged at low speed, such as for example, at about 500-2,000 g for about five minutes. The separation procedure is such that less than about 100 cells or nuclei remain in about 2 x 10⁸ cell equivalents (c.e.) of membrane material. The retrieved supernatant contains membranes which, for example, may be pelleted by ultracentrifugation at about 100,000 g for about 90 minutes. The pellet contains mainly membranes. Membranes may be resuspended, for example, in about 8% sucrose, 5 mM Tris, pH 7.6 and frozen at about -80°C until use. Any diluent may be used, preferably one that acts as a stabilizer. To determine the quality of membrane preparation, a fraction (about 6 x 10⁷ c.e. membranes) may be cultured regularly. Cell colonies should not develop and cells or nuclei should not be detected by light microscopy.

Modification of the prepared cells or membranes with DNP or another hapten may be performed by known methods, e.g. by the method of Miller and Claman (J. Immunol., 117:1519;1976) which involves a 30 minute incubation of tumor cells or membranes with a hapten under sterile conditions, followed by washing with sterile saline. Hapten-modification may be confirmed by flow cytometry using a monoclonal anti-hapten antibody. The dissociated cells or isolated membranes may be used fresh or stored frozen, such as in a controlled rate freezer or in liquid nitrogen until needed. The cells and membranes are ready for use upon thawing. Preferably, the cells or membranes are thawed shortly before they are to be administered to a patient. For example, the cells or membranes may be thawed on the day that a patient is to be skin tested or treated.

Allogeneic tumor cell membranes may be prepared as described above. However, prior to administration to a subject the preparation may be co-incubated with syngeneic (e.g. autologous) antigen presenting cells. Syngeneic antigen- presenting cells process allogeneic membranes such that the patient's cell-mediated immune system may respond to them. This approach permits immunization of a patient with tumor cell membranes originating from a source other than the patient's own tumor. Allogeneic tumor cell membranes are incubated with antigen-presenting cells for a time period varying from about a couple of hours to about several days. The membrane-pulsed antigen presenting cells are then washed and injected into the patient.

Antigen-presenting cells may be prepared in a number of ways including for example the methods of Grabbe et al. (Immunol. Today, 1995; 16:117- 121) and Siena et al. (Exp. Hematol., 1995;23:1463-1471). Briefly, blood is obtained, for example by venipuncture, from the patient to be immunized. Alternatively, a sample of bone marrow may be collected. Alternatively, blood leukocytes may be obtained by leukapheresis. From any of these sources, mononuclear leukocytes are isolated by gradient centrifugation. The leukocytes may be further purified by positive selection with a monoclonal antibody to the antigen, CD34. The purified leukocytes are cultured and expanded in tissue culture medium (for example, RPMI- 1640 supplemented with serum, such as fetal calf serum, pooled human serum, or autologous serum). Alternatively, serum-free medium may be used. To stimulate the growth of antigen-presenting cells, cytokines may be added to the culture medium. Cytokines include but are not limited to granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin 4 (IL4), TNF (tumor necrosis factor), interleukin 3 (IL3), FLT3 ligand and granulocyte colony stimulating factor (G-CSF).

The antigen-presenting cells isolated and expanded in culture, for example, may be characterized as dendritic cells, monocytes, macrophages, and Langerhans cells.

Tumor Cell Peptides The isolation of peptides to be used in hapten-modified anti-cancer vaccines is described in US Patent Application 08/479,016, filed June 7, 1995 and provisional Patent Application 60/109,622, filed November 24, 1998. Both applications disclose extraction and isolation of hapten-modified peptides, which can be adapted for the present invention. Peptides can also be synthesized based on known sequences, or isolated prior to haptenization. The isolated peptides can then be modified by dual-haptenization.

For purposes of the present invention, peptides are compounds of two or more amino acids and include proteins. Peptides will preferably be of low molecular weight, of about 1,000 kD to about 10,000 kD, more preferably about 1,000 kD to about 5,000 kD, which are isolated from a haptenized tumor cell and which stimulate T cell lymphocytes to produce gamma interferon. The peptide of the invention may be from about 8 to about 20 amino acids, preferably from about 8 to about 12 amino acids. In addition, the peptide is preferably haptenized. Peptides may be isolated from the cell surface, cell interior, or any combination of the two locations. The extract may be particular to type of cancer cell (versus normal cell). The peptides of the present invention include but are not limited to peptides which bind to MHC molecules, a cell surface-associated protein, a peptide associated with a heat shock protein chaperonin, a protein encoded by cancer oncogenes, or mutated anti- oncogenes. In one preferred embodiment of the invention, peptides are bound to the MHC molecules. For purposes of the present invention "a peptide equivalent" is the peptide having the same amino acid sequence as the peptide isolated from an MHC molecule, although prepared either by degradation of a protein comprising the peptide, synthesized in vitro or recombinant DNA technology. Preferably, the peptides are derived from tumor specific antigens. There is substantial evidence that the same T-cell-defined tumor antigens are expressed by different human melanoma tumors, suggesting that transformation-associated events may give rise to recurrent expression of the same tumor antigen in tumors of related tissue and/or cellular origin (Sahasrabudhe et al, J. Immunol, 1993;151:6302-6310; Shamamian et al., Cancer Immunol. Immunother., 1994;39:73-83; Cox et al., Science, 1994;264:716; Peoples et al., J. Immunol., 1993;151 :5481-5491; Jerome et al., Cancer Res., 1991 ;51 :2908-2916; Morioke et al, J. Immunol., 1994;153:5650-5658). Examples of such antigens include, but are not limited to, MART 1/Melan A, gp-100, and tyrosinase (melanoma); MAGE-1 and MAGE-3 (bladder, head and neck, non-small cell carcinoma); HPV E6 and E7 proteins (cervical cancer); HER2/neu/c-erbB-2 (breast cancer); HER3, HER4, Mucin (MUC-1) (breast, pancreas, colon, prostate); prostate specific antigen (PSA) (prostate); and CEA (colon, breast, GI). The cell extracts of the invention, including peptides originally isolated from MHC molecules located on tumor cell plasma membranes, have the property of stimulating T cells. For purposes of the present invention, stimulation refers to proliferation of T cells as well as production of cytokines by T cells in response to the cell extract. Proliferation of T cells may be observed by uptake by T cells of modified nucleic acids, such as but not limited to ³H thymidine, ¹²⁵IUDR (iododeoxyuridine); and dyes such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) which stains live cells. In addition, production of cytokines such as but not limited to γ-interferon (INFγ), tumor necrosis factor (TNF), and interleukin-2 (IL-2) may be tested. Production of cytokines is preferably in an amount greater than 15 picograms/ml, more preferably about 20 to about 30 picograms/ml, even more preferably about 50 picograms/ml. Alternatively, cytotoxicity assays can be used to evaluate T cell stimulation.

From the hapten-modified cells, peptides may be extracted, some of which are hapten-modified as a result of modifying the cells. Alternatively, extracted or synthetic peptides can be reacted with a hapten after isolation or synthesis. Protein extraction techniques known to those of skill in the art may be followed by antigen assays to isolate proteins or peptides effective for patient treatment. The methods of isolating cell extracts are readily known to those skilled in the art. Briefly, cancer cells are isolated from a tumor and cultured in vitro. A hapten preparation is added to the cultured cells in accordance with the method set forth above. Peptides are isolated from cells according to an established technique, e.g., the technique of Rotzschke et al, Nature. 1990;348:252, the disclosure of which is hereby incorporated by reference in its entirety. The cells are treated with a weak acid such as but not limited to trifluoroacetic acid (TFA). The cells are thereafter centrifuged and the supernatant is saved. Compounds having a molecular weight greater than 5,000 are removed from the supernatant by gel filtration (G25 Sepharose, Pharmacia). The remainder of the supernatant is separated on a reversed-phase HPLC column (Superpac Pep S, Pharmacia LKB) in 0.1 % TFA using a gradient of increasing acetonitrile concentration; flow rate = 1 ml/min, fraction size = 1 ml. Fractions containing small peptides are collected by HPLC according to the method of Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), concentrated, and frozen. The HPLC fractions containing small peptides are screened for immunological activity, e.g., by allowing them to bind to autologous B lymphoblastoid cells which are then tested for their ability to stimulate tumor-specific T lymphocytes. T cells used for this testing are isolated from a human patient and propagated in vitro as described in International Application No. PCT/US97/15741, published on April 9, 1998 (WO98/14206). The peptides that stimulate T cells are then analyzed for their structure. For example, the peptides are sequenced using methods known in the art to determine their amino acid sequence. In one embodiment of the invention, the peptides are sequenced as a pool as described by Burrows et al. (J. NeuroSci. Res., 1997;49:107-116) and Gavin et al. (Eur. J. Immunol., 1994;24:2124-33) to determine prevailing motifs. In another embodiment of the invention, the peptides are further separated using methods known in the art, such as HPLC, as described in U.S. Patent Nos. 5,747,269; 5,487,982; 5,827,516 and 5,820,862 and sequenced. Sequencing is performed by using Edman degradation as described in Edman and Berg, Eur. J. Biochem., 1967;80:116-132, or any modification thereof known in the art. One powerful technique for characterizing isolated peptides is mass spectrometry.

Once the sequence of the peptides isolated from the MHC molecules is known, synthetic peptides having the same sequence are synthesized and used as a vaccine alone, presented on an antigen presenting cell and/or in combination with other extracts or whole cells using the methods described above. The equivalent peptides may also be produced recombinantly or by chemical degradation of proteins containing the isolated peptides. In another embodiment, the structure of known peptides is altered by changing at least one amino acid and the so altered peptides are tested for their ability to stimulate T cells.

Haptenization The tumor cells, membranes, or peptides are haptenized. For purposes of the present invention, virtually any small molecule, including peptides, that fails to induce an immune response when administered alone, may function as a hapten. A variety of haptens of different chemical structure have been shown to induce similar types of immune responses: e.g., dinitrophenyl (DNP); trinitrophenyl (TNP) (Kempkes et al, J. Immunol., 1991;147:2467); phosphorylcholine (Jang et al, Eur. J. Immunol, 1991;21 :1303); nickel (Pistoor et al. , J. Invest. DermatoL, 1995;105:92); and arsenate (Nalefski and Rao, J. Immunol., 1993;150:3806). Conjugation of a hapten to a cell may preferably be accomplished by conjugation via e-amino groups of lysine or -COOH groups. This group of haptens include a number of chemically diverse compounds: halonitrobenzenes (including dinitrofluorobenzene, difluorodinitrobenzene, trinitrofluorobenzene),

N-iodoacetyl-N'-(5-sulfonic-l-naphthyl) ethylene diamine, nitrobenzene sulfonic acids (including trinitrobenzenesulfonic acid and dinitrobenzene sulfonic acid), fluorescein isothiocyanate, arsenic acid benzene isothiocyanate, and dinitrobenzene-S-mustard (Nahas and Leskowitz, Cellular Immunol., 1980;54:241). Once familiar with the present disclosure, skilled artisans would be able to choose haptens for use in the present invention. Haptens generally include a reactive group for conjugation to a substituent on an amino acid side chain of a protein or polypeptide (e.g., a free carboxylic acid group as in the case of aspartic acid or glutamic acid; the e-amino group of lysine; the thiol moiety of cysteine: the hydroxyl group of serine or tyrosine; the imidazole moiety of histidine; or the aryl groups of tryptophan, tyrosine, or phenylalanine). As used herein, the term "reactive group" refers to a functional group on the hapten that reacts with a functional group on a peptide or protein. The term "functional group" retains its standard meaning in organic chemistry. These reactive groups on a hapten are termed herein the "hapten reactive group". Numerous hapten reactive groups are known, which interact with the substituents present on the side chains of amino acids that comprise peptides and proteins. Preferred examples of such reactive groups for conjugation to specific polypeptide substituents are carboxylic acid or sulfonic acid derivatives (including acid chlorides, anhydrides, and reactive carboxylic esters such as N-hydroxysuccinimide esters), imidoesters, diazonium salts, isocyanates, isothiocyanates, halonitrobenzenes, α-halocarbonyl compounds, maleimides, sulfur mustards, nitrogen mustards, and aziridines.

Functional groups reactive with primary amines. Hapten reactive groups that would form a covalent bond with primary amines present on amino acid side chains would include, but not be limited to, acid chlorides, anhydrides, reactive esters, α,β-unsaturated ketones, imidoesters, and halonitrobenzenes. Various reactive esters with the capability of reacting with nucleophilic groups such as primary amines are available commercially, e.g., from Pierce (Rockford, Illinois).

Functional groups reactive with carboxylic acids. Carboxylic acids in the presence of carbodiimides, such as EDC, can be activated, allowing for interaction with various nucleophiles, including primary and secondary amines. Alkylation of carboxylic acids to form stable esters can be achieved by interaction with sulfur or nitrogen mustards, or haptens containing either an alkyl or aryl aziridine moiety.

Functional groups reactive with aromatic groups. Interaction of the aromatic moieties associated with certain amino acids can be accomplished by photoactivation of aryl diazonium compound in the presence of the protein or peptide. Thus, modification of the aryl side chains of histidine, tryptophan, tyrosine, and phenylalanine, particularly histidine and tryptophan, can be achieved by the use of such a reactive functionality. Functional groups reactive with sulfhydryl groups. There are several reactive groups that can be coupled to sulfhydryl groups present on the side chains of amino acids. Haptens containing an α,β-unsaturated ketone or ester moiety, such as maleimide, provide a reactive functionality that can interact with sulfhydryl as well as amino groups. In addition, a reactive disulfide group, such as 2-pyridyldithio group or a 5,5'-dithio-bis-(2-nitrobenzoic acid) group is also applicable. Some examples of reagents containing reactive disulfide bonds include N-succinimidyl 3-(2-pyridyl- dithio) propionate (Carlsson, et al, Biochem J., 1978;173:723-737), sodium S-4- succinimidyloxycarbonyl-alpha-methylbenzylthiosulfate, and 4-succinimidyloxycarbonyl-alpha-methyl-(2-pyridyldithio)toluene. Some examples of reagents comprising reactive groups having a double bond that reacts with a thiol group include succinimidyl 4-(N-maleimidomethyl)cyclohexahe-l-carboxylate and succinimidyl m-maleimidobenzoate.

Other functional molecules include succinimidyl 3- (maleimido)propionate, sulfosuccinimidyl 4-(p-maleimido-phenyl)butyrate, sulfosuccinimidyl 4-(N-maleimidomethyl- cyclohexane)- 1 -carboxylate, maleimidobenzoyl-N-hydroxy-succinimide ester. Many of the above-mentioned reagents and their sulfonate salts are available from Pierce. Haptens also include a hapten recognition group that interacts with antibody. The recognition group is irreversibly associated with the hapten reactive group. Thus, when the hapten reactive group is conjugated to a functional group on the target molecule, the hapten recognition group is available for binding with antibody. By selecting an appropriate hapten reactive group, antibody recognition of, and binding to, a hapten recognition group can be made independent of the functional group to which the hapten is conjugated. When this is the case, the haptens are functionally equivalent, and are said to share antibody binding features. Naturally, in cases where the recognition groups of two haptens differ chemically, the reactive groups may be the same or different, i.e., reactive with the same or different functional groups on the target molecule.

Examples of different hapten recognition groups include without limitation to dinitiophenyl, trinitrophenyl, fluorescein, other aromatics, phosphorylcholine, peptides, advanced glycosylation endproducts (AGE), carbohydrates, etc. In a specific embodiment, the same hapten recognition group can be coupled to different amino acids through different hapten reactive groups. For example, the reagents dinitrobenzene sulfonic acid, dinitro-phenyldiazonium, and dinitrobenzene-S-mustard, all form the dinitrophenyl hapten coupled to amino groups, aromatic groups, and carboxylic acid groups, respectively. Similarly, an arsonic acid hapten can be coupled by reacting arsonic acid benzene isothiocyanate to amino groups or azobenzenearsonate to aromatic groups.

Isolation and Haptenization of Tumor Cells The dissociated cells, cell membranes, or peptides may be stored frozen in a freezing medium (e.g., prepared from a sterile-filtered solution of 50 ml Human Serum Albumin (HSA) (American Red Cross) added to 450 ml of RPMI 1640 (Mediatech) supplemented with L-glutamine and adjusted to an appropriate pH with NaOH), such as in a controlled rate freezer or in liquid nitrogen until needed. The cells are ready for use upon thawing. Preferably, the cells are thawed shortly before haptenization. Optionally, the cells may be washed, and optionally irradiated to receive a dose of about 2500 cGy. They may then be washed again and suspended in Hanks Balanced Salt Solution (HBSS) without phenol red and without HSA.

Modification of the prepared cells with DNP or another hapten may be performed by known methods, e.g. by the method of Miller and Clanian (J. Immunol., 1976;117:151), incorporated herein by reference in its entirety, which involves a 30 minute incubation of tumor cells with DNFB under sterile conditions, followed by washing with sterile saline or HBSS/HSA. Since DNP modifies hydrophilic residues of MHC-bound peptides (mainly lysine e-amino groups) (Nahas and Leskowitz, Cellular Immunol, 1980;54:241), the second hapten could advantageously be conjugated to hydrophobic residues (such as tyrosine and histidine). Such haptens, binding proteins through an azo linkage, include sulfanilic acid, arsanilic acid, and phosphorylcholine.

Vaccine Preparations

The compositions of the invention may be administered in a mixture with a pharmaceutically-acceptable carrier, selected with regard to the intended route of administration and standard pharmaceutical practice. Dosages may be set with regards to weight, and the clinical condition of the patient. The proportional ratio of active ingredient to carrier naturally depend on the chemical nature, solubility, and stability of the compositions, as well as the dosage contemplated. The amount of the tumor cells of the invention to be used depend on such factors as the affinity of the compound for cancer cells, the amount of cancer cells present, and the solubility of the composition. The compounds of the present invention may be administered by any suitable route, including inoculation and injection via, for example, intradermal, intravenous, intraperitoneal, intramuscular, and subcutaneous routes.

In a preferred embodiment of the invention, the composition comprises a vaccine comprising about 1 x 10⁶ to about 25 x 10⁶, more preferably about 2.5 x 10⁶ to about 7.5 x 10⁶, live, irradiated, tumor cells suspended in a pharmaceutically acceptable carrier or diluent, such as but not limited to Hanks solution, saline, phosphate-buffered saline (PBS), and water. The composition may be administered by intradermal injection into 3 contiguous sites per administration on the upper arms or legs, excluding limbs ipsilateral to a lymph node dissection.

Formulations The formulations according to the invention may be prepared in various ways. The different components may be mixed together, and then added to haptenized tumor cells. It is also possible to mix one or several of the components with the haptenized tumor cells and then add the remaining component(s). The preparation of the formulation and its addition of the haptenized tumor cells are preferably performed under sterile conditions.

The respective proportions of the components of the media according to the invention may be adapted by persons skilled in the art. Generally, HSA will be added to an appropriate buffered cell culture medium. "Human serum albumin" or "HSA" refers to a non-glycosylated monomeric protein consisting of 585 amino acid residues, having a molecular weight of about 66 kD. Its globular structure is maintained by 17 disulfide bridges, which create a sequential series of 9 double loops (Brown, "Albumin structure, function and uses", Rosenoer, V.M. et al. (eds.), Pergamon Press:Oxford, pp. 27-51, 1977). The genes encoding for HSA are known to be highly polymorphic, and more than 30 apparently different genetic variants have been identified by electrophoretic analysis (Weitkamp, L.R. et al, Ann. Hum. Genet., 1973;37:219-226). The HSA gene comprises 15 exons and 14 introns corresponding to 16,961 nucleotides from the putative mRNA "capping" site up to the first site of addition of poly(A).

In its essence, a buffered cell culture medium is an isotonic buffered aqueous solution, such as phosphate buffered saline, Tris-buffered saline, or HEPES buffered saline. In a preferred embodiment, the medium is plain Hank's medium (no phenol red), e.g., as sold commercially by Sigma Chemical Co. (St. Louis, Missouri, USA). Other tissue culture media can also be used, including basal medium Eagle (with either Earle's or Hank's salts), Dulbecco's modified, Eagle's medium (DMEM), Iscove's modified Dulbecco's medium (IMDM), Medium 199, Minimal Essential Medium (MEM) Eagle (with Earle's or Hank's salts), RPMI, Dulbecco's phosphate buffered salts, Earle's balanced salts (EBSS), and Hank's Balanced Salts (HBSS). These media can be supplemented, e.g., with glucose, Ham's nutrients, or HEPES. Other components, such as sodium bicarbonate and L-glutamine, can be specifically included or omitted. Media, salts, and other reagents can be purchased from numerous sources, including Sigma, Gibco, BRL, Mediatech, and other companies. For use in humans, an appropriate medium is pharmaceutically acceptable.

Preferably, a formulation of whole, viable cells comprises an optimized HSA concentration in a buffered cultured medium, preferably HBSS. In a specific embodiment, the final concentration of HSA is about 1.0% in a HBSS. However, an unexpected improvement in cell viability can be achieved using at least about 0.25% HSA, a greater improvement in cell viability with 0.3%> HSA (as compared to 0.1 % HSA), and an even greater improvement is possible using at least about 0.5%o HSA. Upper limits to the concentration are determined by the need to avoid contaminants that may be present in naturally-derived HSA, or alternatively to avoid allergic reactions to recombinant HSA. Preferably, the concentration of HSA in a formulation of the invention is no more than about 10%. More preferably, the concentration is less than or equal to about 5% and, more preferably still, less than or equal to about 2%. Also, a composition or formulation of the invention may contain other components in addition to HSA to further stabilize the haptenized tumor cells. Examples of such components include, but are not limited to, carbohydrates and sugars, such as dextrose, sucrose, glucose, and the like, e.g., at a 5%> concentration; medium to long chain polyols, such as glycerol, polyethylene glycol, and the like, e.g., at 10%) concentration; other proteins; amino acids; nucleic acids; chelators; proteolysis inhibitors; preservatives; and other components. Preferably, any such constituent of a composition of the invention is pharmaceutically acceptable.

Adjuvant In preferred embodiment, a tumor cell composition may be administered with an immunological adjuvant. The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen (Hood et al, Immunology, Second Edition, 1984, Benjamin-Cummings: Menlo Park, California, p. 384). While commercially available pharmaceutically acceptable adjuvants are limited, representative examples of adjuvants include Bacille Calmette-Guerin (BCG) the synthetic adjuvant QS-21 comprising a homogeneous saponin purified from the bark of Quillaja saponaria and Corynebacterium parvum (McCune et al. , Cancer, 1979;43 : 1619). Other adjuvants include Complete and Incomplete Freund's Adjuvant, mineral oils, surface active substances such as lipolecithin, pluronic polyols, polyanions, peptides, and oil or hydrocarbon emulsions.

It will be understood that the adjuvant is subject to optimization. In other words, the skilled artisan can engage in experimentation that is no more than routine to determine the best adjuvant to use.

Immunostimulants and Combination Therapies The haptenized tumor cell compositions may be co-administered with other compounds including but not limited to cytokines such as IL-2, IL-4, LNFγ, IL- 12, and GM-CSF. The tumor cells and extracts of the invention may also be used in conj unction with other cancer treatments including but not limited to chemotherapy, radiation therapy, immunotherapy, and gene therapy.

EXAMPLES The following example is illustrative of the invention, but not limiting thereof.

Example 1 : Aromatic-Specific Hapten-Modified Autologous Tumor Cell Vaccine Three haptens disclosed in previous publications are sulfanilic acid, arsanilic acid, and phosphorylcholine. Arsanilic acid, an arsenic-containing compound, is not suitable for clinical studies. Sulfanilic acid (SA) is safe in small quantities and is preferable to phosphorylcholine because it does not require extensive chemical modification (see below). Materials and Methods

Haptenization. The technique for SA conjugation was developed via minor modifications of previously published methods (Bach et al, J. Immunol., 1978;121 :1460). SA (obtained from Sigma Chemical Co.) is first converted to a diazonium salt by adding a saturating amount of sodium nitrite. For example, ice-cold sodium nitrite may be added dropwise to 100 mg SA dissolved in 10 ml 0.1 HCl until saturation, corresponding approximately to a final concentration of a sulfanilic acid diazonium salt of about 40 mM. The diazotized SA is diluted 1 :4 (v/v) in borate- buffered saline at pH 8.2 and added to about 5xl0⁶ pelleted tumor cells to a final concentration of about 10 mM SA. After incubation for 7-30 minutes, the cells are washed in HBSS with 1% HSA. If the melanoma cells are not heavily pigmented, the cell pellet has a red-orange color.

The degree of cell haptenization is measured by spectrophotometric analysis of cell lysates at a wavelength of 360 nM, or, preferably, by enzyme-linked immunosorbent assays (ELISA) using anti-SA antibodies. Although polyclonal anti- SA antibodies are easily made (Weigle, J. Exp. Med., 1965;122:1049), a monoclonal antibody is preferable. For example, a custom-made anti-SA monoclonal antibody was prepared by Sigma and delivered in the form of partially purified ascites fluid from mice repeatedly immunized with sulfanilic acid-conjugated to KLH (keyhole limpet hemocyanin). In an ELISA assay, the binding of the anti-SA antibody bound to melanoma cells modified with SA is evaluated (See Table 1). Unmodified melanoma cells, as well as DNP -modified cells are used as controls.

Table 1 - Optical density (OD) of SA-haptenized melanoma cells.

Sample OD unmodified OD DNP-modified OD SA-modified

A .126 nd* 0.324

B .164 nd 0.547

C .068 nd 0.630

D .194 .176 0.366

E .226 .262 1.089

*nd = not done

Clinical Trials of SA-Modified Tumor Cell. Patients with surgically incurable melanoma or chemotherapy-refractory ovarian cancer, from which ample tumor tissue can be obtained, are included in the study. Except for the haptenization step, the tumor processing and vaccine preparation are identical to that of DNP- modified cells.

Cells are prepared and haptenized as described previously (U.S. Patent No. 5,290,551; U.S. Patent Applications No. 08/203,004; No. 08/475,016; and No. 08/899,905), except for the hapten being SA. After haptenization and washing, the cells are suspended in HBSS supplemented with 1% HSA and stored at 4°C.

The current established optimum dosage-schedule for DNP -vaccine is used (see, U.S. Patent Application No. 60/084,081, filed May 4, 1998). The vaccine (about 2.5-7.5xl0⁶ trypan-blue excluding (i.e., live) tumor cells mixed with BCG) is injected intradermally on the upper arm (excluding arms ipsilateral to a lymph node dissection). The schedule consists of weekly administrations for about 6 weeks. Cyclophosphamide 300 mg/M² is administered intravenously about 3 days before the first vaccine injection.

The study endpoints are the evaluations of immunological responses and toxicity. Toxicity is anticipated to be limited to local vaccine reactions as with the DNP-vaccine. The major immunological parameter is DTH to SA-modified autologous tumor cells; this may be tested pre-treatment and about 2V₂ weeks after the last vaccine administration. Establishing, with 95%> confidence interval, a positive DTH response (more than or equal to 5 mm mean diameter of induration) in at least 50% of the patients would require about 10 to about 20 patients (more than or equal to 9 out of 10 positive, or more than or equal to about 15 out of 20 positive). DTH to unmodified autologous tumor cells is measured a control. If equal to or more than 11 out of 20 patients develop a positive DTH to unmodified cells, it can be concluded, with 95%o confidence interval, that the response rate is at least 30%>, which is similar to what has been observed with DNP-modified vaccines. Negative controls include: diluent (HBSS balanced salt solution + 1% HSA), autologous peripheral blood lymphocytes (PBL), and autologous PBL coated with collagenase and DNAse (the enzymes used for tumor cell processing).

If positive DTH responses to SA modified tumor cells are observed, the T cell responses to PBL obtained post-treatment exhibit T cells will be evaluated by established methods using LNFγ production and proliferation as the primary indicators of T cell response (see, U.S. Patent Application 08/479,016, filed June 7, 1995).

In addition, a vaccine consisting of cells modified with phosphorylcholine (PC) can made and evaluated. A methodology for PC coupling has been developed (Jang et al, Eur. J. Immunol., 1991;21:1303), which involves the conversion of PC to p-nitrophenylphosphorylcohline (Chesbro and Metzger, Biochemistry, 1972; 11 :766). Example 2: Preparation and testing of a bihaptenized vaccine

Theoretical considerations and experimental data provide a strong rationale for immunizing patients with tumor cells modified with both DNP and SA.

Materials and Methods Dual-haptenization. In this example, melanoma cells are first modified with DNP (as referenced above in Example 1), washed, and then modified with SA (according to Example 1). The final product is analyzed for the degree of DNP- and SA-haptenization using anti-DNP (SIGMA) and anti-SA antibodies in an ELISA assay (Table 2). Unmodified melanoma cells, as well as mono-haptenized cells, are used as controls.

Table 2 - OD of dual haptenized melanoma cells

Treatment of Cells Antibodv Tested OD

None anttii--DDNNPP 0.172

DNP anttii--DDNNPP 0.458

S SAA a mnttii--DDNNPP 0.160

DNP + SA a innttii--DDNNPP 0.320

None aannttii--SSAA 0.603

DNP aannttii--SSAA 0.666 S SAA aannttii--SSAA 0.540

DNP + SA aannttii--SSAA 0.750

In addition, functional tests of hapten modification are performed to determine the autologous dual haptenized tumor cells elicit DTH in patients who have been immunized with mono-haptenized (either DNP or SA) vaccine, and dual haptenized cells are tested for their ability to stimulate T cell responses in PBL obtained from patients after immunization with mono-haptenized vaccine.

Clinical Trials. Provided are positive results are achieved for dual- haptenized cells in the above quality control tests, clinical trials are undertaken to determine their immunogenicity and toxicity. Study designs may be identical to that described above for the clinical trial of S A-modified tumor cells, except for a more extensive DTH testing: patients are skin-tested with both mono- and dual-haptenized autologous tumor cells. For this study, a positive result would require post-vaccine DTH responses of more than or equal to 5mm induration to DNP-modified, SA- modified, and dual-haptenized cells in more than or equal to about 15 out of 20 patients. More than 1 lout of 20 patients (lower end of 95%> confidence limit is about 30%>) may develop positive DTH to unmodified autologous tumor cells as well. Tests of in vitro T cell function (as described above) will be performed as well.

In another clinical study, patients with surgically incurable melanoma, from whom adequate amounts of tumor tissue can be harvested, would be included. Since preliminary data have indicated that small lung metastases are most likely to respond to DNP-vaccine, a separate trial including only this subset of patients may be performed. Additional studies in patients with e.g. chemotherapy-refractory ovarian cancer, or melanoma patients with bulky, resectable lymph node metastases, can be conducted. A phase LI trial may employ the standard two-stage design of Simon

(Controlled Clin. Trials, 1989; 10:1). Initially, 13 patients are treated and if at least one partial response (defined by standard clinical criteria) is observed, the number of patients would be expanded to 27. The dosage-schedule of the vaccine and the immunological endpoints would be similar to that used in the single hapten studies.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, are approximate and are provided for description only.

Patents, patent applications, and publications cited throughout this application are incorporated herein by reference in their entireties.

Claims

WHAT IS CLAIMED IS:

A composition comprising dual-haptenized tumor cell polypeptides, wherein the polypeptides: a) are conjugated with two haptens by derivitization of two different functional groups found on the polypeptides, wherein the functional groups are selected from the group consisting of an amine group, a carboxylic acid group, an aromatic group, a hydroxy group, an imidizole group, and a sulfhydryl group; and b) originate from the same tumor type as the tumor type of a subject intended for treatment with the composition.

2. The composition of claim 1, wherein a first hapten is conjugated to an aromatic group and a second hapten is conjugated to a primary amino group or a carboxylic acid group.

3. The composition of claim 2, wherein the second hapten is conjugated to a primary amino group.

4. The composition of claim 1, wherein the first and second haptens have different hapten recognition groups.

5. The composition of claim 3, wherein the first hapten is a sulfanilic acid (SA), and the second group is a dinitrophenyl (DNP).

6. The composition of claim 1 wherein the polypeptides are isolated.

7. The composition of claim 1 wherein the polypeptides are associated with tumor cell membrane components, wherein the tumor cell membrane components are isolated.

8. The composition of claim 1, wherein the polypeptides are associated with intact autologous tumor cells, which have been rendered incapable of growth.

9. A composition comprising dual-haptenized tumor cells, wherein the haptenized tumor cells: (a) are conjugated with two haptens by derivitization of two different functional groups found on peptides wherein the functional groups are selected from the group consisting of an amine group, a carboxylic acid group , an aromatic group, a hydroxy group, an imidizole group, and a sulfhydryl group; (b) originate from the same tumor type as the tumor of a subject intended for treatment with the composition; and (c) have been rendered incapable of growth.

10. The composition of claim 9, wherein a first hapten is conjugated to an aromatic group and a second hapten is conjugated to an amine group.

11. The composition of claim 10, wherein the first hapten is sulfanilic acid (SA), and the second group is dinitrophenyl (DNP).

12. A vaccine for treating cancer comprising the dual-haptenized tumor cell polypeptide composition of claim 1 and an adjuvant.

13. The vaccine of claim 12, wherein the adjuvant is BCG.

14. A vaccine for treating cancer comprising the dual-haptenized tumor cell membrane composition of claim 7 and an adjuvant.

15. The vaccine of claim 14, wherein the adjuvant is BCG.

16. A vaccine for treating cancer comprising the dual-haptenized tumor cells of claim 9 and an adjuvant.

17. The vaccine of claim 16, wherein the adjuvant is BCG.

18. A method for preparing a composition for use in a cancer vaccine, which method comprises haptenizing tumor cells originating from the same tumor type as the tumor of a human intended for treatment with the vaccine with two haptens by haptenization of two different functional groups found on polypeptides wherein the functional groups are selected from the group consisting of an amine group, a carboylic acid group, an aromatic group, a hydroxy group, an imidizole group, and a sulfhydryl group.

19. The method according to claim 18, wherein a first hapten is conjugated to an aromatic group and a second hapten is conjugated to a primary amino group or a carboxylic acid group.

20. The method according to claim 19, wherein the second hapten is conjugated to a primary amino group.

21. The method according to claim 18, wherein the first and second haptens have different hapten recognition groups.

22. The method according to claim 20, wherein the first hapten is sulfanilic acid (SA), and the second group is dinitrophenyl (DNP).

23. The method according to claim 18, wherein the tumor cells have been rendered incapable of growth.

24. The method according to claim 18, wherein membranes are extracted from the tumor cells to obtain a haptenized tumor cell membrane extract.

25. The method according to claim 18, wherein peptides are isolated from the tumor cells to obtain a haptenized tumor cell peptide preparation.

26. A method for treating cancer in a subject comprising administering a vaccine of claim 12 to the subject.

27. The method according to claim 26, wherein the subject is a human.

28. A method for treating cancer in a subject comprising administering a vaccine of claim 14 to the subject.

29. The method according to claim 28, wherein the subject is a human.

30. A method for treating cancer in a subject comprising administering a vaccine of claim 16 to the subject.

31. The method according to claim 30, wherein the subject is a human.