MXPA00007995A - Hapten-modified tumor cell membranes, and methods of making and using hapten-modified tumor cell membranes - Google Patents

Abstract

The present invention is directed to isolated tumor cell membranes, compositions thereof, methods of making the membranes and compositions, and methods of treating cancer. The compositions of the present invention include a composition prepared from a tumor cell which is hapten modified. The tumor cell membranes and compositions of the invention have the properties, when administered to a mammal suffering from a malignant tumor of the same type as said tumor cell, of eliciting T lymphocytes that infiltrate the tumor of the mammal, of eliciting an inflammatory immune response against the tumor of the mammal, and of eliciting a delayed-type hypersensitivity response to the tumor of the mammal. The membranes and compositions of the invention also stimulate T cells in vitro. The methods of the present invention are directed to treating cancer comprising administering a therapeutically effective amount of a tumor cell membrane. The presentinvention is also directed to a method of making a hapten-modified tumor cell membrane and dosage forms containing the membranes.

Description

MEMBRANES OF TUMOR CELLS MODIFIED WITH HAPTEN AND ITS USE Reference to Government Guarantees The invention described herein was made in the course of work under a guarantee or grant from the National Institutes of Health - National Cancer Institute, grant No. CA-39248. The Government of the United States has certain rights in this invention.

BACKGROUND OF THE INVENTION Theories were made in the 1960s that specific antigens carrying tumor cells (TSA), which are not present in normal cells and that the immune response of these antigens can enable an individual to reject a tumor. It has been strongly suggested that the immune response has specific antigens that carry tumor cells can be increased by introducing new immunological determinants in the cell. Mitchison, Transplant. Proc., 1970, 2, 92. Said "auxiliary determinant" which may be a hapten, a protein, a viral coat antigen, a transplant antigen, or a xenogenic cell antigen, may be introduced into a population of cells tumor The cells can then be injected to an individual who can be expected to be tolerant to the development of tumor or modified cells. Clinically, the hope was that an immunological reaction against the auxiliary determinants could occur, as a consequence of which the reaction to the accompanying TSA is increased, and the tumor cells that can otherwise be tolerated, are destroyed. Mitchison, supra, also suggests various modes of action of the auxiliary determinants including, 1) that the unmodified cells are merely attenuated, in the sense that their growth rate is reduced or their susceptibility to immune attack is increased; 2) that the auxiliary determinants merely provide points of attack and thus enable the modified cells to be annihilated by a non-directed immune response against TSA; 3) that the auxiliary determinants have an auxiliary action such as binding to an antibody or promotion of localization of the cells in the correct part of the body for immunization, in particular, in lymph nodes. Fujiwara et al., J. Immunol., 1984, 132, 1571 showed that certain haptenized tumor cells, i.e., tumor cells conjugated with hapten-trinitrophenyl (TNP), can induce systematic immunity against unmodified tumor cells in a murine system. , provided that the mice were first sensitized to the hapten in the absence of hapten-specific suppressor T cells. The passage cells of the treated mice completely and specifically prevented the growth of tumors in untreated recipient animals. Flood et al., J. Immunol., 1987, 138, 3573 showed that mice immunized with a "regressor" tumor induced with ultraviolet light, conjugated to TNP were able to reject a "progressor" tumor conjugated with TNP that is otherwise not immune In addition, these mice were subsequently resistant to attack with the unconjugated "progressor" tumor. In another experimental system, Fujiwara et al., J. Immunol., 1984, 133,510 demonstrated that mice sensitized to trinitrochlorobenzene (TNCB) after pretreatment with cyclophosphamide could be cured of large tumors (10 mm) through in situ haptenization of cells. tumor Subsequently, these animals were specifically resistant to attack with unconjugated tumor cells. The teachings of Fujiwara et al. Differ from the present invention for several reasons, including the following: A. The cells used in the Fujiwara composition are derived from murine tumors that can be transplanted, induced, not from spontaneous human tumors; B. The Fujiwara composition is used in immunoprophylaxis, the present invention uses immunotherapy; C. The Fujiwara composition is administered as a local therapy, the composition of the present invention is administered through systemic inoculation; D. The Fujiwara composition did not result in tumor regression, the composition of the present invention results in regression and / or prolonged survival for at least a substantial portion of the treated patients; and E. Fujiwara administers tumor cells, the present invention teaches the administration of tumor cell membranes. The existence of T cells, which cross-react with unmodified tissues, has recently been demonstrated. Weltzien et al. Have shown that class I MHC-restricted T-cell clones generated from mice immunized with syngeneic lymphocytes modified with TNP responded to "own" peptides modified with TNP, aiated with MHC. Ortmann, B. et al., J. Immunol., 1992, 148, 1445. In addition, it has been established that immunization of mice with lymphocytes modified with TNP results in the development of splenic T cells that exhibit secondary proliferative and cytotoxic responses to cells modified with TNP in vitro. Shearer, GM Eur. J. Immunol., 1974, 4, 527. The potential of lymphocytes produced by immunization with autologous cells modified with DNP or TNP to respond to unmodified autologous cells is of important interest, since it may be important for two clinical problems: 1) drug-induced autoimmune disease, and 2) cancer immunotherapy. With respect to the former, it has been suggested that the ingested drugs act as haptens, which are combined with immunogenic protein-forming complexes of normal tissue that are recognized by T cells. Tsutsui, H., et al., J. Immunol., 1992 , 149, 706. Subsequently, the autoimmune disease, for example, systemic lupus erythematosus, may develop and continue even after the withdrawal or absence of the offending drug. This could involve the final generation of T lymphocytes that cross-react with unmodified tissues. The common denominator of these experiments is hapten sensitization in a medium where the suppre cells are not induced. Spleen cells from mice sensitized with TNCB, pretreated with cyclophosphamide exhibited an "amplified auxiliary function" radio resistant, that is, they specifically increased the generation of anti-TNP cytotoxicity in vitro. Furthermore, once these amplified aids were activated through in vitro exposure to autologous lymphocytes conjugated with TNP, they could increase cytotoxicity in unrelated antigens as well, including tumor antigens (Fujiwara et al., 1984). Flood et al., (1987), supra, showed that this amplified helper activity was mediated by T cells with the Lyt "1+, Lyt" 2", L3T4 +, l" J + phenotype and suggests that these cells were contrasuppre cells, a new class of immunoregulatory T cell. Immunotherapy of patients with melanoma has shown that the administration of cyclophosphamide, at a high dose (1000 mg / M2) or at a low dose (300 mg / M2), three days before sensitization with primary antigen hole limpet hemocyanin markedly increases the acquisition of delayed-type hypersensitivity to that of the antigen (Berd et al., Cancer Res., 1982, 42, 4862; Cancer Res., 1984, 1275). Pre-treatment with a low cyclophosphamide allows patients with metastatic melanoma to develop delayed-type hypersensitivity to autologous melanoma cells in response to injection with the autologous melanoma vaccine (Berd et al., Cancer Res., 1986, 46 , 2572; Cancer Invest., 1988, 6, 335). Administration of cyclophosphamide results in the reduction of non-specific lymphocyte T suppre function in the peripheral blood (Berd et al., Cancer Res., 1984, 44, 5439; Cancer Res., 1987, 47, 3317), possibly lacking of T cell induction of suppre CD4 +, CD45R + (Berd et al., Cancer Res., 1988, 48, 1671). The antitumor effects of this immunotherapy regimen appear to be limited by the excessively long interval between the initiation of vaccine administration and the development of delayed-type hypersensitivity to tumor cells (Berd et al., Proc. Amer. Assoc. Cancer Res., 1988, 29, 408 (# 1626)). Therefore, there is a need to increase the therapeutic efficacy of said vaccine to make it more immunogenic. Most tumor immunologists now agree that the infiltration of T lymphocytes, white blood cells responsible for tumor immunity, in the tumor mass is a prerequisite for the destruction of tumors through the immune system. Consequently, much attention has been focused on what has become known as "TIL" therapy, pioneered by Dr. Stephen Rosenberg at NCI. Dr. Rosenberg and others extracted from the metastasis of human cancer, some T lymphocytes that are naturally present and greatly expand their numbers by growing them in vitro with interleukin 2 (IL2), a growth factor for T lymphocytes. Topalian et al., J. Clin. Oncol., 1988, 6, 839. However, this therapy has not been very effective, since the injected T cells are limited in their ability to "receive" at the tumor site. The ability of high concentrations of IL2 to induce lymphocytes to become non-specifically cytotoxic killing cells has been exploited therapeutically in a number of studies (Lotze et al., J. Biol. Response, 1982, 3.475; West and others, New Engl. J. Med., 1987, 316,898). However, this aspect has limitations due to the severe toxicity of the high intravenous dose of IL2. Less attention has been given to the observation that much lower concentrations of IL2 can act as an immunological aid by inducing the expansion of antigen-activated T cells (Talmadge et al., Cancer Res., 1987, 47, 5725; Meuer et al. Lancet, 1989, 1, 15). Therefore, there is a need to understand and try to exploit the use of IL2 as an immunological aid. It is believed that human melanomas express unique surface antigens recognizable by T lymphocytes. Oíd, L. J., Cancer Res., 1981, 41, 361; Van der Bruggen, P. et al., Science, 1991, 254, 1643; Mukherji, B. et al., J. Immunol., 1986, 136, 1888; and Anichini, A., et al., J. Immunol., 1989, 142, 3692. However, the immunotherapeutic aspects prior to the work performed by the inventor of the present have been limited by the difficulty of inducing a T cell-mediated response. , effective to said antigens, in vivo. There are several proposed models to explain what appears to be a tolerance to antigens associated with human tumors. These include: 1) Tumor-specific antigen suppressor cells that underestimate the incipient antitumor responses. Mukherji, et al., Supra: Berendt, MJ and RJ North, J. Exp. Med., 1980, 151, 69. 2) The failure of human tumor cells to produce T helper cells or to provide costimulatory signals for those T cells Fearon, RT et al., Cell, 1990, 60, 397; Townsend, S. E. and J. P. Allison, Science, 1993, 259, 368; and 3) Expression of reduced surface area of major histocompatibility products in tumor cells, which limits their recognition by T. Ruiter, DJ, Seminars in Cancer Biology, 1991, 2, 35. None of these hypotheses has been corroborated in the system. clinical. Regardless of whether these explanations are true or not, there is a continuing need for more effective treatment of various diseases.

With respect to acute myelogenous leukemia (AML), treatment for acute myelogenous leukemia is divided into one or more initial induction phases and several subsequent courses of remission, also known as consolidation, chemotherapy. Initial induction chemotherapy can induce a complete response in 55 to 88% of patients, depending on the protocol used. However, the vast majority of these patients relapsed, and the long-term survival (5 years +) of patients with acute myelogenous leukemia is only 20-30%. The addition of high-dose chemotherapy and spinal cord transplantation (BMT) for this therapeutic regimen during the first remission may produce modest results at the end. For example, patients undergoing allogeneic spinal cord transplantation are offered an increase of 5 to 10% in a 5-year survival. However, strict acceptability criteria for spinal cord transplantation (eg, age, availability of a donor with HLA to match) severely limit the number of patients who can be treated. Once patients with acute myelogenous leukemia relapse, there is only 30% chance for a second remission, and very few of these patients remain free of the long-term disease. Treatment modalities in relapse include protocols similar to those used to achieve first remission (induction therapy followed by several courses of consolidation chemotherapy), although a high dose of an individual agent and spinal cord transplant may also be used ( Keating and others). The experience with spinal cord transplantation has suggested that immunological rejection plays an important role in the control of the disease. Graft-versus-host disease (GVHD) and relapse are the two leading causes of death in patients treated with spinal cord transplantation. The risk of relapse is reduced if moderate graft-versus-host disease occurs (Horowitz et al.). Therefore, it has been hypothesized that lymphocytes grafted with capable of immunologically rejecting leukemia host cells (graft versus leukemia reaction, GVL). This graft reaction against leukemia can be mediated by a T cell response against specific leukemia cell antigens, although the immunogenic human leukemia antigens have not yet been demonstrated, (the same applies true to the melanoma). It is known that human acute myelogenous leukemia cells strongly express major histocompatibility complex antigens of both class I and class II (MHC) (Ashman et al., Andreasen et al.), Which are prerequisites for the induction of cell responses. T mediated by CD8 and CD4, respectively. However, an induction of a T cell response directed to leukemia cells has not been successful. Several immunological aspects have been used for the treatment of acute leukemia (Foon et al, Caron and Scheinberg). These aspects are divided into non-specific, such as Bacillus Calmette Guerin (BCG), interleukin-2, levamisole, methanol extraction residue from tuberculous bacillus, and specific, such as monoclonal antibodies and vaccines (harvested leukemia cells, free extracts of cell and cultured cells). Most of these studies have been performed in patients already in remission, where immunotherapy may have been successful in controlling residual disease. In the late 1960s and early 1970s, R. Powles' research group at St. Barthlomew's Hospital, in England, led to a series of vaccine treatment studies of patients with acute myelogenous leukemia after induced remission by chemotherapy (Powles, 1974; Powles et al., 1977). These used allogenic acute myelogenous leukemia cells with BCG as an adjuvant. Several experiments were carried out, all with small sample sizes. There was some prolongation of survival using a combination of chemotherapy and immunotherapy compared to chemotherapy alone, but there was no prolongation of relapse-free survival. No serious toxicity was observed; nor did they observe autoimmunity (for example toxicity to the normal bone marrow.) In retrospect, there are a number of technical problems with these experiments: 1) allogenic rather than autologous leukemia cells were used; 2) the dose of leukemia cells in the vaccine was excessive (up to 109 cells / dose); 3) the dose of BCG was very high and administration of BCG was separated by time and location from the leukemia cell vaccine; and 4) the vaccine was administered while patients received pharmacocytotoxic agents (maintenance or consolidation chemotherapy). The immunochemical basis of the limited success of the aforementioned treatments remains speculative, but several hypotheses are being tested. Kim and Jang (1992) have suggested that the lack of T cell response to a particular epitope may not be due to the absence of a T cell repertoire, but rather to the difficulty in generating the particular epitope. Martin and others (1993) have explained their results by hypothesizing the existence of autoreactive T cells that escape thymic selection due to the low affinity of "own" peptides. Conventional attempts to treat human cancer have not been successful. The administration of compositions, illustrated by those set forth above, failed to reliably induce the development of cell-mediated immunity as indicated by delayed-type hypersensitivity (DTH), T cell infiltration and inflammatory immune response. Accordingly, despite numerous attempts based on various theories proposed for the immunological effects reported in cancer treatments, there is a need for a composition that, after being administered to a mammal, is capable of producing T lymphocytes that infiltrate a tumor, producing an inflammatory immune response to a tumor, and producing a delayed-type hypersensitivity response to a tumor. Applicants have now surprisingly discovered that using isolated membranes from either syngeneic or allogeneic tumor cells have these desired properties.

COMPENDIUM OF THE INVENTION The present invention is directed to an isolated tumor cell membrane, a composition containing said membrane methods for isolating and preparing the tumor cell membrane and compositions containing said membrane, and its use in vitro and for the treatment of cancer. The tumor cell membrane, which may be modified hapten, is preferably a tumor cell plasma membrane which may be syngeneic and allogeneic. The syngeneic tumor cell membrane can be autologous. The cancer that will be treated includes carcinomas and non-solid tumors, including leukemia (such as acute myelogenous leukemia), lymphoma, multiple myeloma, ovarian, colon, rectal, colorectal, melanoma, breast, lung, kidney and prostate.

In one aspect, the present invention relates to a mammalian tumor cell membrane, preferably human being, isolated, modified with hapten. The hapten may be selected from the group consisting of dinitrophenyl, trinitrophenyl, N-iodoacetyl-N '- (5-sulfonic 1-naphthyl) ethylene diamine, trinitrobenzenesulfonic acid, fluorescein isothiocyanate, arsenic acid-benzene isothiocyanate, trinitrobenzenesulfonic acid, osf orilcolina, sulphanilic acid, arsanílico acid and dinitrobencen-S-mustard and their combinations. In another aspect, the present invention is directed to a composition comprising a mammalian tumor cell membrane modified with hapten, alone or in combination with a mammalian tumor cell modified with hapten. In still another aspect, the invention provides a vaccine composition comprising a therapeutically effective amount of a mammalian tumor cell membrane, preferably a human, to be administered to a mammal suffering from a malignant tumor of the same type as the cell membrane. tumor. In another aspect of the invention, the composition contains an auxiliary, such as, for example, Bacille Calmette-Guerin, QS-21, detoxified endotoxin and cytokines such as interleukin-2, interleukin-4, gamma interferon (IFN-γ), interleukin-12, interleukin-15 and GM-CSF. The membrane and composition of the present invention have (when administered to a mammal, preferably a human being, who suffers from a malignant tumor of the same type as the tumor cell from which the membrane was made) at least one of the following properties: (i) producing T lymphocytes that infiltrate the tumor of a treated mammal, (ii) producing an inflammatory immune response against the mammalian tumor, (iii) producing a delayed-type hypersensitivity response in the mammalian tumor . The membrane and the composition of the invention also have the property of stimulating T cells, in vitro. In yet another aspect, the present invention is directed to a method of treating cancer, which comprises administering to a mammal, preferably a human, a composition comprising a therapeutically effective amount of a human tumor cell membrane modified with hapten, wherein said mammal suffers from a malignant tumor of the same type as the tumor cell membrane. Still further, the present invention relates to a method for producing T lymphocytes that have the property of infiltrating said tumor from the mammal, preferably a human, and, optionally, measuring said T lymphocytes that infiltrate the tumor of said mammal. The invention further relates to a method for producing an inflammatory immune response in the tumor of the mammal and, optionally, measuring said inflammatory immune response, or to a method for producing a delayed-type hypersensitivity response in the tumor of said mammal and, optionally, , measure said delayed-type hypersensitivity response. The invention also relates to a method for stimulating T cells in vitro. In still another aspect of the invention, this relates to a method for making a tumor cell membrane modified with hapten.

DETAILED DESCRIPTION OF THE INVENTION All patents, patent applications and references cited herein are incorporated herein by reference. In case of inconsistencies, the present description governs. The present invention is directed to an isolated modified unmodified tumor cell membrane, a novel composition containing said membrane, methods for isolating and making the membrane and compositions containing said membrane, and methods for using the membrane and the compositions of said membrane. the invention. The membranes and compositions of the present invention can be used to treat cancer in a mammal, preferably a human being, including metastatic and primary cancers, solid and non-solid cancers such as, for example, rectal, colorectal, melanoma, breast cancers , of lung, of kidney, and of prostate. Stage I, II, III or IV cancer can be treated with the isolated modified membranes, compositions and methods of the present invention, preferably stages III and IV, even more preferably stage III. Mammals, particularly humans, that have metastatic cancer of the above type can be treated with the membranes, compositions and methods of the present invention. In one embodiment, the present invention is used to treat domestic animals, such as, for example, members of the feline, canine, equine and bovine families. The membranes and compositions of the invention can also be used to produce T lymphocytes and have the property of infiltrating a mammalian tumor, producing an inflammatory immune response in the mammalian tumor, producing a delayed-type hypersensitivity response in the tumor of a mammal and / or stimulate T lymphocytes in vitro. It will be understood that any description in this specification with respect to the use of isolated tumor cells equally applies to the use of tumor cell membranes, or to a combination of tumor cells and tumor cell membranes.

MEMBRANES OF TUMOR CELLS AND THEIR COMPOSITIONS The membranes of modified tumor cells isolated from the present invention are prepared from mammalian tumor cells, preferably human. In one embodiment of the invention, the tumor cell membrane is isolated from a tumor of an animal of a family of phenyls, canines, equines or cattle. Included within the definition of a tumor cell for the purposes of the present invention are whole and separate tumor cells. The tumor cells from which the membranes are isolated can be living, attenuated or annihilated cells. Tumor cells that do not grow and divide after administration to the subject, so that they are substantially in the non-growth state, can be used in the present invention. Said cells are preferred if they are administered to the patient alone or in combination with membranes of isolated tumor cells. It should be understood that "cells in a non-growth state" means whole or divided, living, attenuated or annihilated cells (both whole and divided) that do not divide in vivo. Conventional methods for suspending cells in a growth state are known to those skilled in the art and may be useful in the present invention. For example, the cells can be irradiated before being used, so that they do not grow and do not divide. The tumor cells can be irradiated, for example, at 2,500 R to prevent the cells from growing after administration. Alternatively, the tumor cell membranes can also be isolated from tumor cells that can grow and divide in vivo. Preferably, in such a case, the preparation of the tumor cell membrane is not contaminated with tumor cells that are capable of dividing in vivo. Tumor cell membranes are isolated from tumor cells of the same type as the cancer that will be treated. For example, the membranes that will be used for the treatment of ovarian cancer are isolated from ovarian cancer cells. Preferably, the tumor cells originate from the same subject to be treated. The tumor cells are preferably syngeneic (eg, autologous), but may also be allogenic for that subject. To be defined as "syngeneic", the tumor cell does not need to be complete (ie 100%) and genetically identical to any tumor cell or somatic, non-tumor cell of the treated patient. The genetic identity of the MHC molecules between the tumor cell (from which the membranes are isolated) and the patient is generally sufficient. In addition, there may be a genetic identity between a particular antigen and the tumor cell used as a membrane source and an antigen present in tumor cells of the patient. The genetic identity can be determined according to methods known in the art. A syngeneic tumor cell also means a cell that has been genetically altered (using, for example, recombinant DNA technology) to become genetically identical with respect to, for example, the particular MHC molecules of the patient and / or the particular antigen of the cells cancerous of the patient. Tumor cells from animals of the same species that differ genetically, such as allogeneic cells, can also be used for the preparation of tumor cell membranes of the invention. Tumor cells can be, and are not limited to, cells disassociated from biopsy specimens or from tissue culture. Membranes isolated from allogenic cells and mast cells are also within the scope of the present invention. The membranes of tumor cells can include all cell membranes, such as outer membrane, nuclear membranes, mitochondrial membranes, vacuoles membranes, endoplasmic reticular membranes, Golgi complex membranes, and lysosome membranes. In one embodiment of the present invention, more than about 50% of the membranes are plasma membranes of tumor cells. Preferably, more than about 60% of the membranes consist of plasma membranes of tumor cells, with more than about 70% being preferred, 80% being very preferred, 90% still very preferred, 95% being highly preferred, and 99% being very preferred. Preferably, the isolated membranes are substantially free of nuclei and cells. For example, a membrane preparation is substantially free of nuclei or cells if it contains less than about 100 cells and / or nuclei in about 2 x 108 cell equivalents (c. E.) Of the 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 membranes of isolated tumor cells are the outer cell membranes, i.e., plasma membranes of tumor cells. The membrane preparation of the invention may contain the entire outer membrane or a fraction thereof. An isolated membrane of the invention containing a fraction of the outer membrane, contains at least a fraction of the MHC molecule and / or a thermal shock protein fraction of the outer membrane. The size of the membrane fragments is not critical.

Allogenic tumor cell membranes can also be used in the methods of the present invention with antigen-presenting, syngeneic (eg, autologous) cells. This aspect allows immunization of a patient with tumor cell membranes originating from a source other than the patient's own tumor. The syngeneic antigen presentation cells process allogeneic membranes so that the immune system mediated by the patient's cell can respond to them. The membranes of isolated tumor cells as well as such cells can be modified, for example, with a hapten. Said membranes of modified tumor cells (and tumor cells) have at least one of the following properties: i) producing T lymphocytes that infiltrate the tumor of a treated mammal, ii) producing an inflammatory immune response against the tumor of the mammal, and iii) producing a delayed-type hypersensitivity response to the mammalian tumor. The membranes of modified tumor cells and cells also have the property of stimulating T cells in vitro. A tumor cell membrane (modified or unmodified) as presented in this specification includes any form in which said membrane preparation can be stored or administered, such as, for example, a membrane resuspended in a diluent, a membrane pellet. , or a frozen or lyophilized membrane.

The membranes of the invention can be used in the methods of the invention individually or in combination with other compounds, including and not limited to other compositions of the invention. Accordingly, tumor cells and tumor cell membranes can be used alone or co-administered. For the purposes of the present invention, co-administration includes administration together and consecutively. In addition, tumor cells and tumor cell membranes can be co-administered with other compounds, including and not limited to cytokines such as interleukin-2, interleukin-4, gamma-interferon (IFN-α), interleukin-12, interleukin -15 and GM-CSF. The tumor cells and membranes of tumor cells of the invention can also be used in conjunction with other cancer treatments including, but not limited to chemotherapy, radiation, antibodies and antisense oligonucleotides. However, it is an advantage of the present invention that it may be useful only as a treatment for cancer, so that the need for additional therapies is unnecessary. A composition of the present invention may contain the isolated tumor cell membrane of the invention (modified or unmodified) and a pharmaceutically acceptable carrier or diluent, such as, and not limited to, Hanks' solution, saline, saline regulated at its pH with phosphate, sucrose solution, and water. In general, the pharmaceutically acceptable carrier is selected with respect to the intended route of administration and standard pharmaceutical practice. The proportional ratio of the active ingredient to vehicle naturally depends on the chemical nature, solubility and stability of the compositions, as well as the contemplated dose and can be optimized using common knowledge of the art. In a preferred embodiment of the invention, a composition of the invention is a vaccine composition containing an effective amount of the modified, isolated tumor cell membrane. For the purposes of this description, "an effective amount" is the amount necessary to achieve a desired result. For example, in a method for treating cancer "an effective amount" represents that number of membranes of isolated, modified tumor cells that has the property of causing at least one of the following points: (i) producing T lymphocytes that infiltrate the tumor , (ii) produce an inflammatory response against the tumor, (iii) produce a delayed-type hypersensitivity response to a tumor, and (iv) regression of the tumor. Similarly, in a method for stimulating T cells in vitro, "an effective amount" is that amount of membranes that results in T cell stimulation. The vaccine composition may contain, for example, at least 104 c. and. of membranes isolated per dose, preferably at least 105 c. e., and most preferably at least 106 c. and. A dose is that amount of the vaccine composition that is administered in a single administration. In one embodiment, the vaccine composition contains from about 105 to about 2.5 x 107 c. e., membranes per dose, most preferably around 5 x 106 c. and. The amount of the tumor cells and the membranes of tumor cells of the invention that will be used, generally depends on factors such as the affinity of the compound for cancer cells, the amount of cancer cells present and the solubility of the composition. The doses can be fixed with respect to the weight, and clinical condition of the patient. A vaccine composition of the invention can be packaged in a dosage form suitable for intradermal, intravenous, intraperitoneal, intramuscular and subcutaneous administration. Alternatively, the dosage form may contain membranes of isolated tumor cells that will be reconstituted at the time of administration with, for example, a suitable diluent.

HAPTEN The tumor cells and membranes of tumor cells of the present invention can be used as tumor cells and membranes of modified tumor cells, unmodified, or a combination of modified and unmodified. For the purposes of the present invention, the modified term includes and is not limited to modification with a hapten. Any small molecule that not only induces an immune response (but improves the immune response against another molecule to which it is conjugated or otherwise binds) can function as a hapten. Generally, the molecule used must have a molecular weight of less than about 1000. A variety of haptens are known in the art such as, for example: TNP (Kempkes et al., J. Immunol, 1991 1472: 2467); phosphorylcholine (Jang et al., Eur. J. Immunol., 1991 21: 1303); nickel (Pistoor et al., J. Invest, Dermatol, 1995 105: 92); arsenate (Nalefski and Rao, J. Immunol., 1993, 150: 3806). Generally, haptens suitable for use in the present invention have the property of binding to a hydrophilic amino acid (such as, for example, lysine). The hapten can be conjugated to a cell through e-amino groups of lysine or -COOH groups. In addition, the hapten that can bind to hydrophobic amino acids such as tyrosine and histidine, through diaza coupling can also be used. Examples of haptens suitable for use in the present invention are: dinitrophenyl, trinitrophenyl, N-iodoacetyl-N '- (5-sulfonic 1-naphthyl) ethylene diamine, trinitrobenzenesulfonic acid fluorescein isothiocyanate, arsenic acid-benzene isothiocyanate, trinitrobenzenesulfonic acid, phosphorylcholine, sulfanilic acid, arsanilic acid, dinitrobenzene-S-mustard (Nahas and Leskowitz, Cellular Immunol., 1980, 54: 241), and mixtures thereof. Once armed with the present disclosure, those skilled in the art may be able to select haptens for use in the present invention. For example, haptens can be tested routinely using a delayed-type hypersensitivity (DTH) test.

AUXILIARY In a preferred embodiment, the tumor cell or tumor cell membrane is administered with an immunological aid. The helper has the property of increasing the immune response of tumor cells and membranes modified with hapten. Representative auxiliary examples are Bacille Calmette-Guerin, BCG, or the synthetic auxiliary, QS-21 comprising a homogenous purified saponin from the bark of Quillaja saponaria, Corynebacteriutn parvum, McCune et al., Cancer 1989, 43-1619, saponins in general , detoxified endotoxin and cytokine such as interleukin-2, interleukin-4, gamma-interferon (IFN-γ), interleukin-12, interleukin-15, GM-CSF and combinations thereof. It must be understood that the auxiliary can be subjected to optimization. In other words, those skilled in the art can use routine experimentation to determine the most optimal aid to use.

METHODS FOR MAKING TUMOR CELL MEMBRANES OF THE INVENTION Tumor cells for use in the present invention can be prepared as follows. Tumors are processed as described by Berd et al., (1986), supra, Sato et al. (1997), US Patent No. 5,290,551, and US applications series Nos. 08 / 203,004, 08 / 479,016, 08 / 899,905, 08 / 942,794, or the corresponding PCT application PCT / US96 / 09511, each of which is hereby incorporated by reference in its entirety. In summary, the cells are extracted through dissociation, such as through enzymatic dissociation with collagenase and DNase, through mechanical dissociation in a mixer, shredding with tweezers, using a mortar, counting in small pieces using a scalpel, and Similar. With respect to liquid tumors, blood or bone marrow samples can be collected and the tumor cells are isolated through density gradient centrifugation. Tumor cell membranes are prepared from tumor cells by dividing the cells using, for example, hypotonic shock, mechanical dissociation and enzymatic dissociation, and separating several cellular components through centrifugation. In summary, the following steps can be used: lysing tumor cells, removing the nuclei of the tumor cells Used to obtain nucleus-free tumor cells, obtaining substantially pure membranes free of cells and nuclei, and subjecting the tumor cell membranes to a hapten to obtain membranes of tumor cells modified with hapten. The isolation of the membrane can be conducted according to the methods of Heike et al. In one embodiment of the invention, intact cells and nuclei can be removed through consecutive centrifugation until the membranes are substantially free of nuclei and cells, as determined microscopically. For example, the Used cells can be centrifuged at low speed, such as, for example, about 500-2000 g for about 5 minutes. The separation process is such that less than about 100 cells and / or nuclei remain at about 2 x 108 cell equivalents (c. E.) Of the membrane material. The post-nuclear supernatant containing the membranes can be formed into pellets through ultra centrifugation at approximately 100,000 g for about 90 minutes, for example. The pellet contains total membranes. The membranes may be resuspended, for example, in about 8% sucrose, 5 mm tris, pH 7.6 and frozen at about -80 ° C until used. Any diluent may be used, preferably one that acts as a stabilizer. To determine the quality of the membrane preparation, approximately 6 x 107 of membrane c. and. of membranes, can be regularly cultivated. The cell colonies should not develop and the cells or nuclei should not be detected through a light microscope. Modification of the prepared cells or membranes with DNP or other hapten can be carried out by known methods, for example the method of Miller and Claman, J. Immunol., 1976, 117, 1519, incorporated herein by reference in its entirety, which involves a 30 minute incubation of tumor cells or membranes with a hapten under sterile conditions, followed by washing with sterile saline. The hapten modification can be confirmed by flow cytometry using a monoclonal anti-hapten antibody. 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 to be used after thawing. Preferably, the cells or membranes are thawed just before they are to be delivered to a patient. For example, on the day the patient is going to be tested or treated on the skin, the cells or membranes can be thawed. Optionally, the cells or membranes can be washed, and optionally irradiated at 2,500 R. These can be washed again and then suspended in a balanced salt solution of Hanks without phenol red. Membranes of allogeneic tumor cells can be prepared as described above. However, prior to administration to a subject, they are co-incubated with syngeneic antigen presenting cells (eg, autologous). Syngeneic antigen presenting cells process allogeneic membranes, so that the patient's cell-mediated immune system can respond to these. This aspect allows the immunization of a patient with tumor cell membranes originating from a source other than the patient's own tumor. The membranes of allogeneic tumor cells are incubated with antigen presenting cells for a period ranging from about several hours to about several days. The membrane-pulsed antigen display cells are then washed and injected into the patient. Antigen presenting cells can be prepared in a number of ways, including, for example, the methods of Grabbe et al., 1995 and Siena et al., 1995. In summary, blood is obtained, for example, by puncture in the vein, of the patient to be immunized. Alternatively, bone marrow can be obtained. Alternatively, leukocytes can be obtained from the blood through leukapheresis. From any of these sources, mononuclear leukocytes can be isolated through gradient centrifugation. The leukocytes can also be purified through positive selection with a monoclonal antibody to the antigen, CD34. The purified leukocytes are cultured and expanded in the tissue culture medium (e.g., RPMI-1640 supplemented with serum, such as fetal bovine serum, combined human serum, or autologous serum). Alternatively, a serum-free medium can be used. To stimulate the growth of antigen presenting cells, cytokines can be added to the culture medium. Cytokines include and are not limited to, macrophage-granulocyte colony stimulation factor (GM-CSF), interleukin-4 (IL4), TNF (tumor necrosis factor), interleukin-3 (IL3), FLT3 ligand and granulocyte colony stimulation factor (G-CSF). The antigen presentation cells isolated and expanded in the culture can be characterized as dendritic cells, monocytes, macrophages and Langerhans cells, for example.

METHODS FOR USING TUMOR CELL MEMBRANES AND COMPOSITIONS. Method for Cancer Treatment. The present invention relates to a method for treating a mammal, preferably a human being, diagnosed with or suspected of having cancer, by administering a pharmaceutically acceptable amount of a hapten-modified tumor cell membrane, a hapten-modified tumor cell, or a combination thereof. The membranes and / or cells can be mixed with an immunological aid and / or a pharmaceutically acceptable carrier. A pharmaceutically acceptable amount of a low dose of cyclophosphamide or other low dose chemotherapy may be administered in a manner preceding the administration of the composition. The haptenized composition may optionally be followed by the administration of a pharmaceutically acceptable amount of a non-haptenized tumor cell or a tumor cell membrane. A non-haptenized composition can also be administered according to the methods of the present invention.

Any malignant tumor can be treated according to the present invention, including metastatic and primary cancers and solid and non-solid tumors. Solid tumors include carcinomas, and non-solid tumors include hematologic diseases. Carcinomas include and are not limited to adenocarcinomas and epithelial carcinomas. The hematological evils include leukemias, lymphomas and multiple myelomas. The following are non-limiting examples of cancers that can be treated with membranes of modified tumor cells, isolated, according to the methods of the present invention: ovarian cancers, including advanced ovarian cancer, leukemia, including and not limited to myelogenous leukemia acute, colon cancer, including colon with metastasis, liver, rectal, colorectal, melanoma, breast, lung, kidney and prostate cancer. Ovarian cancers can be adenocarcinomas or epithelial carcinomas. Cancers of the colon and prostate are adenocarcinoma. Leukemias can originate from the myeloid spinal cord or lymph nodes. Leukemias can be acute, exhibited by a maturation stopped at a primitive stage of development, and chronic, exhibited by an excess increase of mature lymphoid or myeloid cells. Cancer of stage I, II, III or IV can be treated according to the present invention, preferably stages III and IV, and most preferably stage III. Mammals, particularly humans, that have metastatic cancer of the above type can be treated with the membranes, compositions and methods of the present invention. In one embodiment of the invention, domestic animals can be treated. Prior to administration of the vaccine composition of the invention, the subject can be immunized to the hapten, which is used to modify tumor cells and membranes by applying it to the skin. For example, dinitrofluorobenzene (DNFB) can be used. Subsequently (approximately 2 weeks later, for example), the subject can be injected with a tumor cell membrane composition. The composition can be administered (such as through a re-injection) for a total of at least three and preferably six treatments. In one modality, the total number of administrations (including initial administration) can be 8, and in another modality they can be 10. The vaccination schedule can be designed by the attending physician to adapt the particular condition of the subject. Vaccine injections can be administered, for example, every 2 weeks, and preferably weekly. A booster shot can be given. Preferably, one or two booster vaccines are administered. The booster vaccine can be administered, for example, after approximately 6 months or approximately 1 year after the initial administration. The immune response of the subject can be increased with the drugs. For example, cyclophosphamide (CY) can be administered before each administration. The present invention can be used following a conventional treatment for cancer, such as surgery. In the case of solid tumors such as ovarian cancer, optimal or suboptimal to the tumor volume may be removed. Optimally removing the volume refers to removing the tumor so that only small pieces remain in the treated subject. Suboptimally removing the volume refers to removing the tumor, although large pieces remain in the subject. In the case of non-solid tumors, an appropriate blood or spinal cord sample can be taken, and the cancer cells are isolated by known techniques. The excised tumors or the collected tumor cells can be used to prepare tumor cell membranes as described above. The tumor cell membranes can be administered through any suitable route, including inoculation and injection, for example, intradermal, intravenous, intraperitoneal, intramuscular and subcutaneous. Multiple administration sites may exist for each vaccine treatment, for example, the vaccine composition may be administered through intradermal injection in at least 2, and preferably 3 contiguous sites per administration. In one embodiment of the invention, the vaccine composition is administered in the arms or legs, in the upper part. The effectiveness of the vaccine can be improved by administering several biological response modifiers. These agents work ctly or inctly stimulating the immune response. The biological response modifiers of the present invention include and are not limited to interleukin-12, interleukin-15 and gamma interferon. In one modality, IL-12 is given following each vaccine injection. The administration of IL-12 to patients with inflammatory responses may cause the T lymphocytes within the tumor mass to proliferate and become more active. The high T cell numbers and functional capacity leads to the immunological destruction and regression of the tumors. Human cancer vaccines have been developed and tested through a number of workers. Although they can sometimes induce weak immunity to a patient's cancer, they rarely cause regression of tumors or prolong survival. Evidence of an inflammatory response was surprisingly found with the vaccine of the present invention. Microscopically, infiltration of T lymphocytes was observed. Therefore, this aspect, which increases the inflammatory response and the number of lymphocytes, is a significant advance in the technique. Therefore, the present invention also provides methods for producing T cells having at least one of the following properties: i) producing T lymphocytes that infiltrate the tumor of a treated mammal, ii) producing an inflammatory immune response against the tumor of the treated mammal; mammal, and iii) produce a delayed-type hypersensitivity response to the mammalian tumor when administered.

Method for Stimulating T Cells. Membranes of isolated tumor cells can be used to stimulate T cells in vitro. This assay can be used to determine, for example, whether a therapy using a particular tumor membrane is likely to be successful. The T cell for use in this assay can be obtained according to the following method, which is described in humans, but can be applied to any mammal. The T cells are generated by administering to a patient with cancer of a certain type, a pharmaceutically acceptable amount of a composition comprising hapten-modified tumor cells, tumor cell membranes, or a combination thereof. The composition may optionally contain an immunological aid and / or a pharmaceutically acceptable carrier. A pharmaceutically acceptable amount of a low dose of cyclophosphamide or other low dose chemotherapy, such as and not limited to melphalan, from about 5 to about 10 mg / m2, optionally can be administered prior to the administration of the first tumor cell composition. . The haptenized composition may optionally be followed by the administration of a pharmaceutically acceptable amount of a non-haptenized vaccine composition containing non-haptenized membranes, tumor cells or a combination thereof.

A non-haptenized composition can be administered according to the methods of the present invention. Peripheral blood lymphocytes (PBL) can be obtained from patients who develop a strong delayed-type hypersensitivity reaction (DTH) to autologous cells or membranes modified with hapten after administration. The DTH reaction preferably has a diameter of about 10 mm, still most preferably greater than 10 mm. A T cell line can be established from PBL through repeated stimulation with hapten-modified cancer cells. T cells can be isolated by known techniques, such as the preparation of single cell suspension, filtration, elimination of monocytes and isolation of a subgroup expressing a particular T cell receptor (TCR) type, causing the subgroup to expand in the presence of the TCR subtype-specific antibody and / or in the presence of IL-2 and / or in the presence of a superantigen. The T cells of interest can be expanded in vivo, since they are picked up from filtrations from or within the tumor, which are already rich in T cells of interest. Modified tumor cells and tumor cell membranes each have the property of stimulating T cells. "Stimulation", for purposes of the present invention, refers to inducing T cell proliferation, as well as the production of cytokines. through T cells, in vitro. The membranes and tumor cells each independently have the activity of stimulating T cells. The proliferation of T cells can be detected and measured through consumption by T cells of modified nucleotides, such as and not limited to 3H thymidine, 125 IUDR (iododeoxyuridine); and dyes such as 3-4,5-dimethylthiazol-2-yl) -2,5-diphenyltrazolium bromide (MTT), which dyes living cells. In addition, the production of cytokines such as and not limited to IFN ?, tumor necrosis factor (TNF), and IL-2 may be useful for exhibiting T-cell proliferation. Cytokine production can be detected and measured using well tests. known in the art. The cytokine production must be above the background level, which is generally above 25 picograms / ml, and preferably is above 100 picograms / ml. T cells are lymphocytes that mediate two types of immunological, effector and regulatory functions, secrete proteins (lymphokines), and annihilate other cells (cytotoxicity). Effector functions include reactivity such as delayed type hypersensitivity, allograft rejection, tumor immunity, and graft-versus-host reactivity. The production of lymphokine and cytotoxicity are demonstrated by the effector functions of the T cell. The regulatory functions of T cells are represented either by their ability to amplify cell-mediated cytotoxicity through other T cells such as the production of immunoglobulin through B cells. Regulatory functions also require the production of lymphokines. B cells produce gamma interferon (IFNα) in response to an induction stimulus including and not limited to mitogens, antigens, or lectins. In one embodiment of the invention, a T-cell line can be developed as follows. 1 x 106 of PBL are mixed with autologous DNP-conjugated B lymphoblastoid B cells (1 x 105) in 24-well flat bottom plates in a lymphocyte culture medium. After 7 days of culture, IL2 100U / ml was added (Cetus Oncology, Emeryville, CA) the expended T cell cultures were maintained in the medium containing IL2 and were divided as necessary to maintain a concentration of approximately 2 x 10 6 cells in a cavity with a diameter of 22 mm. Every 14 days, the cultures were further stimulated by adding B lymphoblastoid cells conjugated with autologous DNP. The phenotypes can be determined through flow cytometry with a panel of monoclonal antibodies (Becton-Dickinson, San José, CA). Separation of CD8 + and CD4 + T cells is achieved through indirect panning where T cells coated with anti-CD8 or anti-CD4 monoclonal antibodies adhere to anti-immunoglobulin coated dishes using standard techniques according to the methods of Wysocki, LJ and VL Sato, Proc. Nati Acad. Sci. USA, 1978, 75, 2844, will be incorporated here by reference in its entirety; the adherent cells are isolated and expanded with DNP-modified stimulants, including and not limited to those established below, melanoma cells and ß-lymphoblastoid cells; and IL2. Phenotypically homogeneous T cell subpopulations were obtained, for example, by culturing at a limiting dilution in round bottom microtiter cavities in a lymphocyte culture medium containing 2 x 10 5 irradiated allogeneic feeder cells, 200 U / ml IL2, and phytohemagglutinin. Cavities with developing lymphocyte colonies were classified for the ability to proliferate in response to DNP-modified B lymphoblastoid cells. Positive cells expanded IL2 and were further stimulated with autologous DNP-conjugated B lymphoblastoid cells every 14 days. Peripheral blood lymphocytes (PBL) can be tested as response cells. These are suspended in a lymphocyte culture medium (RPMI-1640, 10% combined human AB + serum, insulin-transferred-selenite medium supplement (Sigma Chemical Co.), 2 mm L-glutamine, 1% amino acids non-essential, 25 mM pH buffer of HEPES, penicillin + streptomycin) and were added to round bottom microtiter plates, from 96 wells to 1 x 105 cells / well. Stimulant cells were also added, including: 1) autologous or allogeneic PBL, 2) autologous or allogeneic B lymphoblastoid lines made through transfection with the Epstein-Barr virus, 3) autologous cultured melanoma cells; inactivated through radiation (5000 R). in most experiments, the response: stimulant ratio is preferably 1: 1. Plates were incubated in a CO2 incubator at 37 ° C for 5 days; then the cavities were pulsed with IUDR labeled with 125 I (ICN Radiochemical, Costa Mesa, CA) for 6 hours, harvested with an automatic harvesting device, and counted in a gamma counter. The average of cavities in triplicate was calculated. Combined T cells were also tested for a lymphoproliferative response according to the above methods. PBL, obtained and cryopreserved from patients at the time of maximum DTH reactivity to autologous cells modified with DNP, were thawed and tested for in vitro proliferative responses. The application of DNFB alone did not result in detectable numbers of response cells in circulation. It is expected that reactive PBL will be detected after 2 injections of the DNP vaccine (day 63) and will continue to be detected throughout the period of vaccine treatment, based on previous experience with DNP-modified melanoma cells. To test cytokine production, T cells can be added to round bottom microtiter plates at approximately 1 x 10 5 cells / well. An equal number of stimulants (autologous B lymphoblastoid cells modified with DNP) were added, and the supernatants were collected after 18 hours of incubation. Commercially available ELISA kits were used to measure gamma-interferon (Endogen, Boston, MA, sensitivity = 5 pg / ml). To determine the MHC dependence of the response, stimulating cells can be pre-incubated with monoclonal antibodies for class I MHC (W6 / 32) or class II MHC (L243) at a concentration of 10 μg / ml for 1 hour before adding the response cells. The non-specific mouse immunoglobulin can be tested at the same concentration as a negative control. DNP-reactive CD8 + T cells obtained by panning the bulk population are capable of being maintained in long-term culture (> 3 months) in a medium containing IL2 through repeated stimulation with autologous B lymphoblastoid cells modified with DNP , these retained the stable phenotype, CD3 +, CD8 +. Two lines of evidence suggest that their response will be class I MHC restricted: 1) production of interferon gamma will be blocked through pre-incubation of stimulating cells with the anti-class I framework antibody, but not by the anti-class II antibody, 2). T cells will be able to respond to allogeneic DNP-modified stimulants that match 1 or both HLA-A sites, but not with stimulants that are incapacitated HLA-A. To test the production of interferon gamma through T cells, lymphocytes can be obtained from a patient's blood. Approximately one million lymphocytes are mixed with autologous melanoma cell membranes modified with DNP to stimulate T cells. Every 7 days, 100 U / ml of interleukin-2 can be added. The T cells are expanded through the passage. The T cells are then re-stimulated through the autologous melanoma cell membranes modified with DNP.

As a result, a rich population of T cells is obtained, which are sensitive to autologous melanoma cells modified with DNP. Stimulation is determined through the amount of interferon gamma production through T cells. Generally, the production of gamma interferon at more than 15 picograms / ml is considered important. The invention is further illustrated by the following examples, which represent an illustration and are not intended to limit the present invention to these specific embodiments.

EXAMPLE 1 In vitro stimulation of T Cells through Melanoma Membranes Isolated Establishment of the T Cell Line. Peripheral blood lymphocytes (PBL) were obtained from a patient who developed a strong delayed-type hypersensitivity reaction (DTH) to autologous melanoma cells modified with DNP after administration of the DNP vaccine. according to the method of the present. The PBLs were separated from the blood by density gradient centrifugation, suspended in a freezing medium, such as RPMI-1640 with 2.5% human albumin and DMSO, frozen in a speed control freezer, and stored in liquid nitrogen until used (Sato et al., 1995).

A T cell line was established from these PBLs through repeated stimulation with autologous melanoma cells modified with DNP (DNP-Mel) and maintained with recombinant interleukin-2 (IL-2) (Sato et al., 1995). . Melanoma cells. Melanoma cells were enzymatically extracted as described above from metastatic masses surgically removed from the same patient and cryopreserved through a previously described method (Sato et al., 1997). An autologous melanoma cell line was established from the melanoma cell suspension. Briefly, the melanoma cells were enzymatically dissociated from metastatic masses and suspended in the tissue culture medium (RPMI-1640 with fetal bovine serum or human serum) and added to tissue culture plates. After several days, non-adherent tumor cells were removed and a fresh medium was added. After several weeks, the adherent melanoma cells began to proliferate rapidly. When the cells grew to confluence in the culture dish, they were divided by removing the cells with EDTA and adding to fresh tissue culture plates. Cells of the melanoma cell line were modified with DNP through the method of Miller and Claman. This involves a 30 minute incubation of the tumor cells with dinitrofluorobenzene (DNFB, Sigma Chemical Co.) under sterile conditions, followed by washing excess DNFB with the Hanks solution. Modification with DNP was confirmed by flow cytometry with a mouse monoclonal anti-DNP antibody (SPE-7, Sigma Immunochemicals, St. Louis, MO), (100% of the cells showed to be modified with DNP). As an alternative procedure, melanoma cells criconserved with DNP were modified as described above in the intervention step of establishing a cell line. Cell Membrane Extraction. The cell membranes were extracted from melanoma cells modified with DNP (DNP-Mel) through the methods of Heike et al. Briefly, cells modified with DNP were used through hypotonic shock in 5 volumes of 30 mm pH regulator in sodium bicarbonate, 1 mm of phenylmethylsulfonyl fluoride (in PMSF), and through homogenization of Dounce (10- 20 shocks). The residual intact cells and nuclei were removed by consecutive centrifugation at 1000 g for 5 minutes, until the supernatant was free of nuclei and cells. Then the membranes were pelleted through centrifugation at 100,000 g for 90 minutes. The total membranes in the pellet were resuspended in 8% sucrose, 5 mm of tris, pH 7.6 to 107 cell equivalent units (ie, membranes extracted from 107 cells / ml) and frozen at -80 ° C until used. As an alternative procedure, the cell membranes were isolated from unmodified melanoma cells in a manner identical to that described above. The membranes were suspended in a Hanks solution without albumin at various equivalent cell concentrations (from 105-109 cell equivalents / ml). Then DNFB was added as described above. Then, the membranes were pelleted through centrifugation at 100 g for 90 minutes and washed twice with saline. Cytokine Production in Response to Membrane Preparation. T-cell responses induced by melanoma membranes modified with DNP were measured through the production of IFN-gamma. The T cells obtained from PBL of patients were plated on a plate with a round bottom of 96 cavities at 105 / cavity in a culture medium of 100 μl (RPMI-1640 supplemented with 10% human AB serum, 2 mm L-glutamate, 100 mg / ml / 100 U / ml streptomycin / penicillin, 10 mM HEPES, 1% non-essential amino acids). Various amounts (approximately 105 to about 108 cell equivalents) of cell membranes were added to each well and an additional culture medium was added to make the total volume of each well in 250 μl. The supernatants were collected for the IFN-gamma assay after 18 hours of incubation. The concentration of IFN-gamma in supernatant was measured through a commercially available ELISA kit (Endrogen, Boston, MA; Sensitivity = 5 pg / ml). The production of significant IFN-gamma through T cells (750 pg / ml) was detected after incubation with autologous DNP-Mel membranes. The production of IFN-gamma through T cells was related to the number of co-incubated DNP-Mel membranes. There was no significant response in the unmodified Mel membranes. Two sub-lines of T-cell enrichment were developed for CD4 + and CD8 + T cells through a positive panning technique. Each subline responded to DNP-Mel membranes through the production of IFN-gamma. The response of the CD4 + T subline to the DNP-Mel membranes was blocked by the MHC class II antibody, and the response of the CD8 + subline was blocked by the MHC class I antibody (73% block and 80% block). %, respectively). These results show that the membranes of hapten-modified tumor cells can be successfully used to vaccinate patients in need of tumor treatment.

EXAMPLE 2 Treatment of Stage III Ovarian Cancer with Modified Tumor Cell Membranes Patients can be initially pretreated according to standard medical practice (volume reduction surgery followed by chemotherapy). After completing the chemotherapy, a course of 6 weeks of treatment with a vaccine containing membranes of ovarian cancer cells modified with the hapten, dinitrophenyl (DNP), can be administered. A low dose of cyclophosphamide can be administered before the first injection. After the end of the course of treatment, patients can be tested for delayed-type hypersensitivity to the carcinoma cell membranes, both modified with DNP and unmodified. In vitro studies were performed with cryopreserved lymphocytes extracted from metastatic tumors and / or separated from peripheral blood. Patients who received surgical volume reduction or patients who exhibited tumor reduction through chemotherapy, for example, may be selected for treatment. The excised tumor mass of each patient may be sufficient to obtain at least 100 x 10 6 viable tumor cells. Said patient may receive chemotherapy, such as carboplatin and taxol, and preferably are clinically tumor free, after the end of chemotherapy (ie, normal physical examination and CT-studies and serum CA-125 <35 IU / L). Patients can be excluded from receiving the treatment of the present invention based on: insufficient amount of tumor cells to prepare a vaccine and skin test (<100 x 106 cells), Karnovsky's operating status less than 80, therapy of major field radiation with the preceding 6 months, administration of systemic corticosteroids, hematocrit < 30% or WBC < 3000, age < 18, active autoimmune disease, active, serious infection, other active malignancy, evidence of infection with hepatitis B virus (antigen in circulation) or with HIV (antibody in circulation), or inability to provide informed consent. The patients underwent surgical resection of the tumor and reduction of the volume of metastasis. Patients who experienced an optimal or suboptimal volume reduction may be acceptable. Tumor tissue can be supplied to the laboratory and processed to obtain membranes. The membranes can be cryopreserved and stored in liquid nitrogen. The membranes of both syngeneic and allogeneic tumor cells can be prepared and used as described in this specification. Beginning (6) weeks after surgery, patients can begin chemotherapy, such as with carboplatin or cisplatin plus taxol, according to the following dose schedule: carboplatin AUC 7.5 or cisplatin 75 mg / M2, every 3 weeks, taxol 175 mg / M2 i. v. for 3 hours, every 3 weeks. You can administer 6 cycles of chemotherapy. Any other chemotherapy can be administered. Approximately 4 weeks after the end of chemotherapy, patients may undergo a metastatic evaluation to include chest-abdomen-pelvis computer tomography (CT). Only patients who do not have evidence of recurrent carcinoma may be acceptable for vaccine treatment. Patients who raised serum CA125 levels may be acceptable as long as CT studies are negative for recurrences. Tumor cell membrane therapy can be started at least 4 weeks later, and no more than 12 weeks after the last chemotherapy administration. On day 7, patients can be tested on the skin, with: 1) cells or autologous ovarian cancer membranes modified with DNP, 2) diluent (balanced salt solution of (Hanks with 0.1% human albumin, and 3) intermediate PPD DTH reactions can be measured on day 5, on day 0, patients can receive cyclophosphamide, 300 mg / m2, as a rapid intravenous infusion. Three days later, they can be injected intradermally with a tumor cell membrane composition and can be repeated weekly for 6 weeks. The vaccines may consist of ovarian cancer cell membranes, modified with DNP mixed with BCG. Vaccines can be injected into the upper arm. If for some reason a dissection of the left auxiliary lymph node has been performed, the right arm can be used. Two and a half weeks after the sixth vaccine, patients may undergo a clinical evaluation, consisting of CBC, SMA-12, CA125, and chest X-rays. These can be tested for DTH with the following materials: autologous carcinoma cells, both modified with DNP and unmodified; autologous peripheral blood lymphocytes, both modified with DNP and unmodified; diluent; and PPD intermediary. Also, they can be tested for contact sensitivity to dinitrofluorobenzene (DNFB). Patients who remain free of relapse may be given a seventh (booster) vaccine in the sixth month (measured as of the beginning of the vaccine program). For each patient, at least one cryopreserved vial of tumor cell membranes can be saved for the sixth month booster injection. If the number of cells available is anticipated as insufficient for 6 weekly vaccinations plus booster at 6 months, then the initial course of weekly injections may be reduced to 5. Another booster vaccine may be administered per year, but only if one Sufficient number of cells is available. Just before reinforcement a year, patients can be tested on the skin with cells or tumor membranes to determine if their previous level of immunity has been maintained.

EXAMPLE 3 Treatment of Melanoma Cancer with Modified Tumor Cell Membranes Tumor masses can be processed as previously described. In summary, cells can be extracted through enzymatic cleavage with collagenase and DNase and through mechanical dissociation. Cell membranes can be isolated as described in this specification, and frozen in a controlled speed freezer and stored in liquid nitrogen until needed. On the day a patient is going to be treated, the membranes can be thawed, washed and resuspended in a balanced salt solution of Hanks without phenol red. Modification with DNP can be done through the method of Miller and Claman (1976). This involves a 30 minute incubation of the tumor cells with dinitrofluorobenzene (DNFB) under sterile conditions followed by washing with sterile saline. The vaccine composition may contain a minimum of 2.5 x 106 c. and. of trypan blue excluding membranes of melanoma cells, and a maximum of 7.5 x 106 membranes of melanoma cells, suspended in 0.2 ml of Hanks' solution. Each vaccine treatment can consist of 3 injections in contiguous sites. The freeze-dried material can be reconstituted with 1 ml of sterile water or saline regulated at pH with phosphate pH 7.2 (PBS). Appropriate dilutions can be made in regulated saline at its sterile pH. Then, 0.1 ml can be extracted and mixed with the vaccine just before the injection. The first and second vaccines can be mixed with 0.1 ml of a 1:10 dilution of Tice BCG ("Tice-1"). BCG is a strain Tice (subcepa of the strain of the Pasteur Institute) obtained from Organon Teknika Corporation (Durham, NC). The third and fourth vaccines can be mixed with 0.1 ml of a dilution of 1: 100 ("Tice-3"). The fifth and sixth and the booster vaccines can be mixed with 0.1 ml of a dilution of 1: 1000 ("Tice-5"). The ideal vaccine reaction is an inflammatory papule with a central ulceration more than small (<5 mm). The skin test can be performed through the intradermal injection of 0.1 ml of the test material into the forearm, and the DTH was analyzed at 48 hours by measuring the mean diameter of induration. The following materials can be tested: 1) 1 x 106 membrane cells of autologous melanoma cells not modified and modified with DNP; either enzymatically dissociated (TCE) or mechanically dissociated (TCM) tumor cells can be used; 2) 3 x 106 autologous peripheral blood lymphocytes not modified and modified with DNP; 3) Hanks solution; and 4) resistance to the PPD intermediate. Also, contact sensitivity for DNFB can be tested by applying 200 μg DNFB to the skin of the ventral surface of the upper arm by examining the area for an induration circle at 48 hours. The total battery of DTH tests can be made following the course of 6 weeks of vaccine administration. The pre-treatment DTH test can be limited to melanoma cell membranes modified with DNP, PPD and diluent. This strategy was designed to avoid: 1) the sensitivity of patients to lymphocytes modified with DNP and 2) the tolerance of patients to the injection of unmodified tumor cells.

All patients can be collected blood for separation and cryopreservation of lymphocytes and serum each time the skin test is performed. Periodically, these can be tested: response to autologous cancer cells, as measured by proliferation, cytokine release and cytotoxicity. Patients can be evaluated for metastatic disease before starting vaccine therapy. After completing the first 8 weeks of vaccine therapy, evaluations were conducted every 3 months. The evaluations can continue through 2 years, every 4 months for 3 years, and every 6 months thereafter. Physical examination and routine blood work (CBC), SMA-12, and CA125) can be performed with each evaluation. Breast-abdomen-pelvic CT can be performed before administration of the vaccine, at 6 months and 12 months (before booster shots), and then as clinically indicated. The free fall and total survival can be measured. All patients can be followed for at least 5 years or until the day of their death. Patients are expected to develop a local reaction to BCG, consisting of a drainage, soft pustule that heals in 2 3 months leaving a small-pox-like scar. As patients develop sensitivity to BCG, the intensity of these reactions may increase. Anaphylaxis, another allergic phenomenon, and auto immunity have never been observed in patients with haptenized vaccine.

Reactions at the vaccine sites were classified as follows: 0 no symptoms; 1- itching and discomfort, but without interference with arm movement or normal activity; 2-discomfort that causes interference with arm movement, but does not require modification of normal activity; 3-discomfort that causes a greater interference with the movement of the arm and requires modification of normal activity; and 4-discomfort that causes inability to use the limb for normal activity. Cyclophosphamide can be reconstituted with sterile water and the appropriate dose can be administered through a rapid intravenous infusion. Typically, about one third of patients may experience nausea and approximately 10% may have vomiting after the administration of a low dose of cyclophosphamide. Leukopenia, alopecia and cystitis do not occur at this dose. This protocol is expected to be associated with a lower incidence of nausea and vomiting, since cyclophosphamide can only be administered once in association with the final vaccine inoculation. Patients can be observed after the injection of the vaccine. Patients who experience unexpected symptoms or signs are extruded to make contact with the doctor to be evaluated immediately. Fever that causes discomfort can be treated with acetaminophen. Nausea caused by a low dose of cyclophosphamide can be treated with prochlorperazine (Compazine). If several local reactions (> 5 mm ulceration) occur at the vaccine site, subsequent doses of BCG should be reduced (see above). Patients who are free from relapse at the 1-year evaluation may receive a final booster injection. Afterwards, your condition can be followed without further treatment. Patients who develop metastases may be taken for study and treated as clinically indicated (usually surgery or chemotherapy). An effective study to determine if DNP vaccine prolongs relapse-free and / or total survival in these patients can also be conducted. Survival parameters can be measured (Kaplan-Meier method). All regulations of Thomas Jefferson University, NIH, and FDA regarding informed consent are followed with respect to informed consent. Previous studies of DNP-modified autologous melanoma vaccine using hapten-modified melanoma cells showed the following results: 100% of patients (N = 60) developed a positive DTH response (induration diameter> 5 mm) to cells autologous tumors modified with DNP after treatment, and 85% developed a large positive response (induration diameter> 10 mm). A similar success is expected with a DNP-modified autologous carcinoma membrane vaccine vaccine, i.e., patients are expected to develop DTH to the autologous carcinoma cell membranes modified with unmodified DNP. Various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description, said modifications also intend to fall within the scope of the appended claims.

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Claims (52)

1. A composition comprising a maximum of 7.5 x 106 cell equivalents (ce.) Per dose of a hapten-modified human tumor cell membrane, isolated.

2. The composition according to claim 1, wherein the tumor cell membrane is isolated from a tumor cell selected from the group consisting of a carcinoma cell and a non-solid tumor cell.

3. The composition according to claim 1, wherein said tumor cell membrane originates from a tumor selected from the group consisting of leukemia, lymphoma, multiple myeloma, ovarian cancer, colon cancer, rectal cancer, colorectal cancer, melanoma. , chest, lung, kidney and prostate.

4. The composition according to claim 3, wherein the leukemia is acute myelogenous leukemia.

5. The composition according to claim 1, comprising at least 105 ce. per dose.

6. The composition according to claim 5, comprising at least 106 ce. per dose.

7. The composition according to claim 1, comprising about 2.5 x 106 to about 7.5 x 106 cell equivalents (ce.) Per dose.

8. The composition according to claim 1, wherein said hapten is selected from the group consisting of dinitrophenyl, trinitrophenyl, N-iodoacetyl-N '- (5-sulfonic 1-naphthyl) ethylenediamine, trinitrobenzenesulfonic acid, fluorescein isothiocyanate. , arsenic acid-benzene isothiocyanate, trinitrobenzenesulfonic acid, and dinitrobenzene-S-mustard.

9. The composition according to claim 1, further comprising an auxiliary.

10. The composition according to claim 9, wherein said auxiliary is selected from the group consisting of Bacille Calmette-Guerin, QS-21, detoxified endotoxin and a cytokine.

11. The composition according to claim 1, having at least one of the following properties: (i) producing T lymphocytes that infiltrate the tumor of a treated mammal, (ii) producing an inflammatory immune response against the tumor of the treated mammal; mammal, (iii) producing a delayed type hypersensitivity response in the mammalian tumor, and (iv) stimulating T cells in vitro.

12. The composition according to claim 1, further comprising an antigen presenting cell.

13. The composition according to claim 12, wherein said antigen presenting cell is allogeneic to the tumor cell.

14. The composition according to claim 12, wherein said antigen presenting cell is autologous to the tumor cell.

15. The composition according to claim 1, wherein said membrane is an outer cell membrane.

16. The composition according to claim 1, wherein the membrane comprises a membrane fraction comprising an MHC molecule, a heat shock protein or a combination thereof.

17. A method for treating cancer in a human being, comprising administering to a human being a composition comprising a therapeutically effective amount of the composition of claim 1, wherein said human being suffers from a malignant tumor of the same type as said tumor cell membrane.

18. The method according to claim 17, which comprises producing T lymphocytes that infiltrate said human tumor and measuring the T lymphocytes that infiltrate the tumor of the human being.

19. The method according to claim 17, which comprises producing an inflammatory immune response to said tumor of the human being and measuring said inflammatory immune response.

20. The method according to claim 17, which comprises producing a delayed-type hypersensitivity response to the tumor of said human and measuring the delayed-type hypersensitivity response.

21. The method according to claim 17, wherein the malignant tumor is selected from the group consisting of carcinomas and non-solid tumors.

22. - The method according to claim 17, wherein the malignant tumor is selected from the group consisting of leukemia, lymphoma, multiple myeloma, ovarian, colon, rectal, colorectal, melanoma, breast, lung, kidney and of prostate.

23. The method according to claim 22, wherein the leukemia is myelogenous or acute leukemia.

24. The method according to claim 17, wherein the tumor cell membrane is a tumor cell membrane selected from the group consisting of a syngeneic tumor cell membrane and an allogeneic tumor cell membrane.

25. The method according to claim 24, wherein the syngeneic tumor cell membrane is autologous.

26. The method according to claim 17, wherein said hapten is selected from the group consisting of dinitrophenyl, trinitrophenyl, N-iodoacetyl-N '- (5-sulfonic 1 -naphthyl) ethylenediamine, trinitrobenzenesulfonic acid, fluorescein isothiocyanate. , arsenic acid-benzene isothiocyanate, trinitrobenzenesulfonic acid, and dinitro benzene-S-mustard.

27. The method according to claim 17, further comprising an auxiliary.

28. The method according to claim 27, wherein said auxiliary is selected from the group consisting of Bacille Calmette-Guerin, QS-21, detoxified endotoxin and a cytokine.

29. The method according to claim 17, wherein the tumor cell membrane is allogeneic, the composition further comprising an antigen presenting cell.

30. The method according to claim 29, wherein said antigen presenting cell is syngeneic.

31. The method according to claim 29, wherein said antigen presenting cell is autologous.

32. A method for preparing the hapten-modified tumor cell membrane according to claim 1, comprising lysing a tumor cell to obtain a used tumor cell, removing the nuclei of the tumor cell used to obtain a tumor cell free of charge. nuclei, obtain a tumor cell membrane substantially free of cell from the tumor cell free of nuclei, and conjugate the tumor cell membrane with a hapten to obtain a tumor cell membrane modified with hapten.

33. The method according to claim 32, wherein the lysis is selected from the group consisting of hypotonic shock, mechanical dissociation and enzymatic dissociation.

34. The method according to claim 32, wherein said hapten is selected from the group consisting of dinitrophenyl, trinitrophenyl, N-iodoacetyl-N '- (5-sulfonic 1-naphthyl) ethylene diamine, trinitrobenzenesulfonic acid, isothiocyanate fluorescein, arsenic acid-benzene isothiocyanate, trinitrobenzenesulfonic acid, and dinitro benzene-S-mustard.

35.- A membrane of tumor cell of pet modified with hapten, isolated.

36. - The membrane according to claim 35, wherein said mammal is an animal selected from the families of canines, felines, bovines and equines.

37. The membrane according to claim 35, wherein said membrane is isolated from a tumor cell selected from the group consisting of carcinoma cell and non-solid tumor cell.

38.- The membrane according to claim 35, wherein said membrane originates from a tumor selected from the group consisting of leukemia, lymphoma, multiple myeloma, ovarian, colon, colorectal, melanoma, breast, lung cancer, kidney, and prostate.

39.- The membrane according to claim 38, wherein said leukemia is acute myelogenous leukemia.

40. The membrane according to claim 35, selected from the group consisting of a syngeneic tumor cell membrane and an allogeneic tumor cell membrane.

41. The membrane according to claim 40, wherein said syngeneic tumor cell membrane is autologous.

The membrane according to claim 35, wherein said hapten is selected from the group consisting of dinitrophenyl, trinitrophenyl, N-iodoacetyl-N '- (5-sulfonic 1-naphthyl) ethylenediamine, trinitrobenzenesulfonic acid, fluorescein isothiocyanate. , arsenic acid-benzene isothiocyanate, trinitrobenzenesulfonic acid, and dinitrobenzene-S-mustard.

43. The membrane according to claim 35, having at least one of the following properties: (i) producing T lymphocytes that infiltrate the tumor of a mammal, (ii) producing an inflammatory immune response against the tumor of the mammal, ( iii) producing a delayed-type hypersensitivity response in the mammalian tumor, and (iv) stimulating T cells in vitro.

44. The membrane according to claim 35, wherein said membrane is an outer cell membrane.

45.- The membrane according to claim 35, wherein said membrane comprises an MHC molecule.

46.- A composition comprising the membrane according to claim 35.

47.- A method for producing T lymphocytes that infiltrate a tumor of a mammal, comprising administering to said mammal a therapeutically effective amount of the composition of claim 1 or 46, wherein the mammal suffers from a malignant tumor of the same type as said tumor cell membrane.

48. The method according to claim 47, wherein said mammal is a human being, and an animal selected from the families of canines, felines, bovines and equines.

49. A method for producing an inflammatory immune response to a tumor of a mammal, comprising administering to said mammal a therapeutically effective amount of the composition of claim 1 or 46, wherein the mammal suffers from a malignant tumor of the same type as said tumor cell membrane.

50.- The method according to claim 49, wherein said mammal is a human being or an animal selected from the families of canines, felines, bovines and equines.

51. A method for producing a delayed-type hypersensitivity response to a tumor of a mammal, comprising administering to said mammal a therapeutically effective amount of the composition of claim 1 or 46, wherein said mammal suffers from a malignant tumor of the same type as the tumor cell.

52. The method according to claim 51, wherein said mammal is a human being or an animal selected from the families of canines, felines, bovines and equines.