MXPA99003341A - Cancer immunotherapy using tumor cells combined with mixed lymphocytes - Google Patents

Abstract

This invention comprises cellular vaccines and methods of using them in cancer immunotherapy, particularly in humans. The vaccines comprise stimulated lymphocytes allogeneic to the subject being treated, along with a source of tumor-associated antigen. The allogeneic lymphocytes can be stimulated by combining or coculturing them with leukocytes obtained from the subject to be treated or form another third-party donor. Tumor antigen may be provided in the form of primary tumor cells, tumor cell lines or tumor extracts prepared from the subject. Stimulated allogeneic lymphocytes and tumor antigen are combined and administered at a site distant from the primary tumor, in order to prime or boost a systemic cellular anti-tumor immune response. This approach overcomes the natural refractory nature of the immune system to stimulation by tumor antigens, generating a host response and potentially improving the clinical outcome.

Description

IMMUNOGENIC COMPOSITION THAT INCLUDES COMBINED TUMOR CELLS WITH MIXED LYMPHOCYTES AND METHOD FOR THE PRODUCTION OF THE SAME FIELD OF THE INVENTION The present invention relates generally to the fields of cellular immunology and cancer therapy. More specifically, it relates to the generation of an antitumor immune response in a subject (particularly a human) by administering a cellular vaccine, comprising inactivated tumor cells and stimulated immune cells, such as can be generated in a culture of mixed lymphocytes. - BACKGROUND OF THE INVENTION Despite numerous advances in medical research, cancer remains a leading cause of death throughout the developed world. Specific procedures for cancer management, such as surgery, radiotherapy and generalized chemotherapy, have been successful in the management of a selective group of circulating cancers and slow-growing solids. However, many REF .: 30053 solid tumors are considerably resistant to such procedures, and the prognosis in such cases is correspondingly serious. An example is brain cancer. Each year, approximately 15,000 cases of high-grade astrocytomas are diagnosed in the United States. The number is growing in pediatric and adult populations. Standard treatments include cytoreductive surgery followed by radiation or chemotherapy. There is no cure, and virtually all patients ultimately succumb to recurrent or progressive disease. Complete survival for grade IV astrocytomas (glioblase multiform) is poor, with less than ~ 50% of patients dying in the first year after diagnosis. Because these tumors are aggressive and highly resistant to standard treatments, new therapies are needed. An emerging area of cancer treatment is immunotherapy. The general principle is to confer on the subject being treated, an ability to mount what is in effect a response to rejection, specifically against malignant cells. There are a number of immune strategies under development, including: 1. adoptive immunotherapy using autologous stimulated cells of various types; 2. Systemic transfer of allogeneic lymphocytes; 3. Intratumoral implant of immunologically reactive cells; and 4. vaccination at a distant site to generate a systemic immune response specific to the tumor. The first of the strategies listed above, the adopted immunological method, is aimed at providing the patient with a level of immunity enhanced by ex vivo stimulatory cells, and then readminis bringing them to the patient. The cells are histocopable with the subject, and are generally obtained from a previous autologous donation. A procedure is to stimulate the autologous lymphocytes ex vivo, with the antigen associated with the tumor to make them specific to fear. Zarling et al. (1978) Nature 274: 269-271 generated cytotoxic lymphocytes in vitro against autologous human leukemia cells. Lee et al. (1996) extract * Gastroenterology, conducted a culture of mixed lymphocytes in vitro with inactivated leukemic blasts and autologous lymphocytes, and generated cytotoxic effector T lymphocytes for a tumor antigen on autologous blasts. It was thought that a D-locus MHC incompatibility was necessary to provide adequate support in the culture of lymphocytes. Lesham et al. (1984) Cancer Immuno1. Immunother. 17: 117-23 developed in vitro cytotoxic responses against murine thymoma cells by alosensitization. Gate and collaborators (1982) J. Nati. Cancer Inst. 69: 1245-54 found that 5 out of 9 human glioma cell lines do not elicit allogenic cytolytic lymphocyte responses in ex vivo cultures. However, if inactivated allogeneic lymphocytes were provided as stimulator cells in the cultures, tumor-specific cytolytic T lymphocytes and non-T, non-specific effectors would be generated for 4 of the non-stimulatory lines. In U.S. Patent No. 5,192,537, Osband suggests activation of mononuclear cells from a patient with tumor by culturing them ex vivo in the presence of the tumor cell extract and a non-specific activator such as phytohemagglutinin or IL-1, and then treating the culture for decrease the activity of suppressor cells. Despite these experimental observations, the systemic administration of autologous tumor-specific lymphocytes, stimulated ex vivo, has not become part of the standard therapy against cancer. Autologous lymphocytes and killer cells can also be stimulated not specifically. In one example, leukocytes expressing the Fe receptor that can mediate an antibody-mediated, cell-mediated cytotoxicity reaction are generated by culture with a combination of IL-2 and IFN-α. (North American Patent No. 5,308,626). In yet another example, lymphocytes derived from peripheral blood cultured in IL-2 form lymphokine-activated killer cells (LAK), which are cytolytic to a range of neoplastic cells, but not to normal cells. LAKs are mainly derived from natural killer cells that express the CD56 antigen but not CD3. Such cells can be purified from peripheral cell leukocytes by adhesion induced by IL-2 to the plastic (A-LAK cells; U.S. Patent No. 5,057,423). In combination with high doses of IL-2, LAK cells have had some success in the treatment of metastatic human melanoma and renal cell carcinoma. Rosenberg (1987) New Engl. J. Med. 316: 889-897. This strategy is labor intensive, expensive, and not suitable for all patients. Schwartz et al. (1989) Cancer Res. 49: 1441-1446 showed that A-LAK cells are superior to LAK cells in reducing metastases in lung and liver from breast cancer in experimental animal models, but this was not healing and there were no long-term survivors. For examples of tests conducted using LAK in the treatment of brain tumors, see Merchant et al. (1988) Cancer 62: 665-671 and (1990) J. Neuro-Oncol. 8: 173-198; Yoshida et al (1988) Cancer Res. 48: 5011-5016; Barba et al. (1989) J. Neurosurg. 70: 175-182; Hayes et al. (1988) Lymphokine Res. 7: 337-345; and Naganuma et al. (1989) Acta Neurochir. (Wien) 99: 157-160. Another study proposes therapy for high-grade recurrent glioma using murine lymphocytes activated by mitogen and stimulated with autologous IL-2 (MAK), in combination with IL-2. Jeffes et al. (1991) Lymphokine Res. 10: 89-94. While none of these tests was associated with serious clinical complications, the efficacy was only anecdotal or transient. The induction of tumor-specific immunity in patients receiving such treatments has not been shown. Another form of adoptive therapy using autologous cells has been proposed based on observations with tumor infiltration lymphocytes (TIL). The TILs are obtained by collecting lymphocyte populations that infiltrate inside the tumors, and cultivating them ex vivo with IL-2. Finke et al (199Q) Cancer Res. 50: 2363-2370 have characterized the cytolytic activity of CD4 + and CD6 + TILs in human renal cell carcinomas. The TILs have activity and tumor specificity superior to the LAK cells, and have been experimentally administered, for example, to humans with advanced melanoma. Rosenberg et al. (1990) New Engl. J. Med. 323: 570-578. The effector population within the TILs can be cytotoxic T lymphocytes (CTL) which are primed to be tumor-specific in the host, and are devoid of lytic granules, and become transformed into the cytolytic lymphoblasts when stimulated in culture. . Berke et al. (1988) J. Immunol. 129: 303 ff. Unfortunately, TILs can only be prepared in sufficient quantity to be clinically relevant in a limited number of tumor types. These strategies remain experimental, especially in human therapy. The second of the strategies for immunotherapy for cancer listed at the beginning is the adoptive transfer of lymphocytes to the gene. The reason for this experimental strategy is to create a general level of immune stimulation and thereby overcome the energy that prevents the host's immune system from rejecting the tumor. Strausser et al. (1981) J. Immunol. Vol. 127, No. 1 describe lysis of human solid tumors by autologous allergen-sensitized in vitro cells. Zarling et al (1978) Nature 274: 269-71 demonstrated human anti-lymphoma responses in vivo, after sensitization with allogeneic leukocytes. Kondo et al. (1984) Med Hypotheses 15: 241-77 observed the objective responses of this strategy in 20-30% of patients, and attributed the effect to the decrease of suppressor T cells. The studies were conducted on patients with disseminated or circulating disease. Even though these initial experiments were conducted a decade ago, the strategy has not gained general acceptance, especially for the treatment of solid tumors. The third of the immunotherapy strategies listed at the beginning is the intrathecal implants. This is a strategy aimed at the distribution of effector cells directly to the site of action. Since the transplanted cells do not circulate, these do not need to be histocompatible with the host. The intratumoral allogeneic cell implant can promote the ability of transplanted cells to react with the tumor and initiate a potent host versus tumor response. Kruse et al. (1990) Proc. Nati Acad. Sci. USA 87: 9577-9581 demonstrated that direct intratumoral implantation of allogeneic cytotoxic T lymphocytes (CTL) within brain tumors that develop in Fischer rats, resulted in a significant survival advantage over other lymphocyte populations, including CTL. syngenic, LAK cells, adherent LAK cells or IL-2 alone. Redd et al. (1992) Cancer Immunol. Immunother. 34: 349-354 developed cytotoxic T lymphocytes specific for an allogeneic brain tumor in rats. The lymphocytes were specific for a determinant expressed only by the tumor, and they were predicted to be useful for therapeutic purposes in vivo, Kruse et al. (1994), J. Neurooncol. 19: 161-168 prepared LTLs from four incompatible MHC rat strains, and used them to treat Fischer rats that have established 9L brain tumors. The CTL were administered in a biweekly scheme, each time a different CTL preparation incompatible with MHC was administered. The animals without tumor showed minimal localized brain damage. Those with tumors showed either: a) mononuclear cell infiltration, massive tumor necrosis beginning 2-4 days after treatment and total tumor destruction in 15 days; or b) cellular infiltration, early tumor destruction, and then redevelopment of the tumor progressing until the animal dies. The animals with tumor regression were resistant to the new intracranial challenge with viable tumor cells. Kruse and collaborators (1994). Intratumoral CTL implants can be optionally combined with chemotherapy using cyclophosphamide. Kruse et al. (1993) J. Neurooncol. 15: 97-112. Despite the promise of intratumoral implantation techniques, several warnings remain. For some reason, the implant is frequently performed by surgical techniques, which may be too invasive for routine maintenance. In addition, the strategy is aimed at generating a local response, not at generating a systemic response that is generally necessary for protection against metastasis. The fourth of the immunotherapy strategies listed at the beginning is the generation of a specific immune response of the tumor, if it is temporary, to the point of origin of the host. The response is elicited from the subject's own immune system by administering a vaccine composition at a site distant from the tumor. The specific antibodies or immune cells produced in the host will as a result optically migrate towards the tumor, and then eradicate the cancer cells, provided they are in the body. Various types of vaccine have been proposed, including isolated tumor antigen vaccines and anti-idiotypic vaccines. Mitchell and collaborators (1993) Ann. N. Y . Acad. Sci. 690: 153-166 have treated cancer patients with used mechanics from a plurality of allogeneic melanoma cell lines, combined with the DETOXMR adjuvant. These procedures are all based on the premise that tumors of a related tissue type all share a common antigen associated with the tumor. For patients with tumors that did not acquire antigen expression during malignant transformation, or that were subsequently differentiated to not express it, none of these vaccines will be successful. An alternative procedure for an anti-tumor vaccine is to use tumor cells from the subject to be treated, or a derivative of such cells. For a review see, Schirm Acher and collaborators (1995) J, Cancer Res. Clin ^ Oncol ,. 121: 487-489. In U.S. Patent No. 5,484,596, Hanna Jr. and collaborators, claim a method to treat a resectable carcinoma to prevent recurrence or metastasis, which comprises the surgical removal of the tumor, the dispersion of the cells with collagenase, the irradiation of the cells, and the vaccination of the patient with at least three doses consecutive of approximately 107 cells. The cells can optionally be cryopreserved, and the immune system can be checked periodically by skin test. This procedure does not resolve the well-established observations that many tumors are not naturally immunogenic. Many patients from whom the tumors have been resected are either tolerant or unable to respond to their own tumor antigen, even when included in a vaccine preparation. Various ways have arisen to prepare autologous or syngeneic tumor cells that potentially enhance immunogenicity. The tumor cells can be combined with extracts of the bacillus Calmette-Guerin (BCG) or the anticoagulant complex uicobacterial A6Q, or mixed with vaccinia virus or the Newcastle Disease Virus (NDV). Guo et al. (WO 95/16775) suggested that the tumor cells be fused with membrane components of a second cell such as a B cell that has a higher immunogenic potential. In a further procedure, the autologous or syngeneic tumor cells are genetically altered to produce a costimulatory molecule. Examples of costimulatory molecules include cell surface receptors, such as the costimulatory molecule B7-1 or the allogeneic histocompatibility antigens. Other examples are secreted activators, including cytokines. For a review see, Pardoll et al. (1992) Curr. Opin Immunol. 4: 619-23; Salto et al. (1994) Cancer Res. 54: 3516-3520; Vieweg et al. (1994) Cancer Res, 54: 1760-1765; Gastl et al. (1992) Cancer Res, 52: 6229-6236; and WO 96/07433). Tumor cells have been genetically altered to produce TNF-α, IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IFN-α, INF-? and GM-CSF. See, for example, Santin and collaborators, Gynecol. Oncol. 56: 230-239, 1995b; Int. J. Gynecol. Cancer 5: 401-410, 1995c; "Am. J. Obst. Gynecol. 174: 633-639, 1996- Golumbek et al. (1989) reported that mouse renal carcinoma cells inserted with a gene for IL-4 were strongly immunogenic for systemic T cell immunity. and protected the mice against a subsequent lethal challenge with unmodified progenitor tumor cells, Cavallo et al 1991 and 1992. Antitumor immunity is enhanced by a cancer vaccine that produces GM-CSF and IL-4, Wakimoto et al. 1996) Cancer Res. 56: 1828-33 The cytokine or cytokine combination can recruit or stimulate the cells of the immune system, thereby overcoming the normal barrier to immunity Certain cytokines also affect the expression of major histocompatibility molecules and intracellular adhesion molecules by cancer cells (Santin et al., 1995, Int. J. Cancer 65: 688-694; Santin (1996) Am. J. Obst. Gynecol), potentially improving the immunogenicity Experiments with histocompatible tumor cells that secrete cytokine have been carried out mainly in genetically restricted animal models, which are not directly equivalent to a heterogeneous patient population. Colombo et al. (1995) Cancer Immunoi. Immunother, 41; 265-270. Not all cancers respond to the same cytokines. There are problems regarding the injection of human patients with tumor cells competent to replication, particularly after genetic alteration. In addition, there usually is not enough time to genetically alter and develop sufficient cells of the patient to be treated for use in a vaccine, Blumbach (WO 96/05866) has suggested live tumor cell vaccines transduced with: a) a gene encoding for an immunostimulatory protein; b) a cytokine; and c) a thymidine kinase gene. The composition is provided as living cells which can develop in vivo and stimulate a response, and then be selectively killed via the thymidine kinase. The possibility of escape mutants is likely to be a matter of regulatory concern for this procedure in human therapy. Golumbek et al. (1992) J. Immunother, 12: 224-230 have shown that tumor cells proliferating with suicide genes can also survive toxin treatment when they exit the cell cycle temporarily or are pharmacologically sequestered. As an alternative, Cohen (WO 95/31107) suggested that the neoplastic disease can be treated with a cellular immunogen comprising allogeneic cells genetically altered to express one or more cytokines, and also to express tumor-associated antigens encoded by genomic tumor DNA, autologous In this procedure, an allogeneic cell (exemplified as a mouse LM cell) is genetically altered to express: a) a cytokine; and b) an antigen associated with the tumor, autologous to the subject to be treated. Consequently, the vaccine does not need to comprise living tumor cells. However, the application of the Cohen invention to human subjects could require prior knowledge for each patient of a particular antigen associated with the tumor, expressed by the particular tumor. Many human cancers of very widespread clinical interest do not have reliable, commonly shared markers. Once a relevant marker is identified for a particular patient, a cell line must be manipulated by genetic engineering accordingly, and grown to the required density before treatment. In this way, each patient could become their own research and development project. Since the immune response could be focused only on the particular antigen associated with the tumor used, it may be less effective than one directed against the spectrum of antigens expressed by a whole tumor cell. In addition, the vaccine comprises a genetically altered cell line, raising the problems described at the beginning. Cohen showed only a modest improvement in survival in animal studies, and failed to provide any evidence that this information could be effective in human patients with cancer. A suitable strategy for a human antitumor cell vaccine has to contend with the following problems: a) the heterogeneity between tumors (including tumors of the same type) when showing antigens associated with the tumor, b) the heterogeneity in the immune response between individuals with respect to antigens and cytokines; c) ethical and regulatory issues regarding compositions that can be used in humans; and d) the lack of development time in most clinical trials, limiting the ability to engineer the new cell lines or otherwise design the vaccine for each patient.

BRIEF DESCRIPTION OF THE INVENTION This invention provides compositions and methods for eliciting an anti-tumor immune response in a human patient in need thereof. The compositions of the invention are cellular mixtures in a physiologically compatible excipient, and are referred to herein as a vaccine or an immunogenic composition. These can be administered to patients either to treat or palliate a clinically detectable tumor, or for prophylaxis, particularly after surgical removal, chemotherapy or radiotherapy of a previously detectable tumor. The compositions are typically administered at a site distant from the original tumor, with the aim of stimulating a systemic reactivity against the primary tumor and metastasis. The reactivity can in turn eradicate or diminish the development of the tumor cells, either at the primary site, within the metastases (if they exist), or both. Minimally, the vaccines of this invention comprise two components. The first is a source of tumor antigen, preferably a plurality of antigens, which is associated for the cancer for which the patient is at risk. A convenient source of antigen associated with the tumor are the tumor cells previously obtained from the patient, such as during surgical resection. The second component is a population of stimulated lymphocytes that can participate in the stimulation of the patient's immune system to produce an antitumor response. In particular, the population of stimulated lymphocytes comprises lymphocytes that are allogeneic to the patient. These are preferably reactivated by ex vivo co-culture with stimulator cells such as leukocytes obtained from the patient or from a second third-party donor, allogeneic to the donor, which contributes to the responder cells. Included in the invention are compositions comprising a plurality of stimulator or responder cells, or both, wherein the stimulator cells are capable of alloactivating the responder cells in culture. The embodiments of the invention include compositions for the treatment of cancer. One embodiment is a cellular vaccine suitable for administration to a human or other subject being treated, comprising an effective combination of the following components in a pharmacologically or physiologically compatible excipient: a) leukocytes from the human or from a donor third party; b) allogeneic lymphocytes for leukocytes, and preferably alloactivated against this; and c) a population of inactivated tumor cells, which consists of either primary tumor cells obtained from the human, the progeny of such cells of cell lines, or a combination thereof. The first ingredient is optional where the allogeneic lymphocytes are otherwise stimulated before inclusion in the mixture. The inactivated tumor cell can be replaced by an alternative source of tumor-associated antigen, such as a tumor cell homogenate, detergent lysate, or a purified derivative thereof, such as an isolated protein. The population of inactivated tumor cells preferably consists essentially of primary tumor cells dispersed from a solid tumor resected from a human, and can be selected from the group consisting of glioma cells, glioblastoma cells, gliosarcoma cells, astrocytoma cells and ovarian cancer cells. In other embodiments, non-solid tumors may be used, including but not limited to leukemias and lymphomas. Allogeneic lymphocytes are typically isolated from peripheral blood of a suitable donor, and may optionally be genetically altered to express a cytokine at a high level, particularly IL-2, 11-4, GM-CSF, TNF-a or M-CSF, or any combination thereof. In a preferred embodiment, leukocytes and lymphocytes are co-cultured for a duration and under sufficient conditions for allogeneic stimulation or lymphocyte proliferation, before combining with the population of tumor cells. The invention also exemplifies the vaccines and immunogenic compositions of this invention in unit dose or in the form of equipment. Also exemplified are methods for producing any of the compositions of this invention, by preparing or mixing the various components of the compositions, including co-culturing the leukocytes from a subject to be treated with allogeneic lymphocytes. and / or combining the stimulated allogeneic lymphocytes with primary tumor cells, the progeny, or the tumor antigen from the subject. Also exemplified are methods for the induction, firing or otherwise stimulation of an antitumor immune response, especially a cellular response, or for the treatment of a neoplastic disease such as cancer, comprising the administration of any of the compositions or vaccines. of this invention. Further embodiments are methods for inducing, triggering, or otherwise stimulating an anti-tumor immune response or treating a neuroplastic disease in a human in need of such treatment, comprising the steps of; a) mixing together in vi tro an antigen associated with the tumor (particularly a population of tumor cells) with a second cell population comprising human allogenic stimulated lymphocytes; and b) the administration of an immunogenic amount of the cellular mixture to the human. An immune response stimulated by one of the exemplified methods may be a primary response, or a secondary response, and the human may have been optionally previously treated by administration of the allogeneic lymphocytes within a solid tumor in the human or within a cavity in the human formed by the removal of a solid tumor or a portion of it. Preferably, the population of tumor cells comprises primary tumor cells obtained from said human, or a tumor cell line derived from the primary tumor cells obtained from the human, and is inactivated. In particular embodiments, the second cell population also comprises leukocytes from the human, which are preferably inactivated, and typically isolated from peripheral blood. Preferably, the leukocytes and lymphocytes are co-cultured before addition to the tumor cells, for a duration and under conditions sufficient for the allogeneic stimulation of the lymphocytes, or for the proliferation of the lymphocytes, before the combination with the population of tumor cells. In particular embodiments, the lymphocytes from the donor alternatively or in addition have been genetically altered to express a cytokine at a high level.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a three-panel graph showing the relative size of the tumor measured by magnetic resonance imaging (MRI) in nine different patients at various times after intratumoral implantation with 2, 4, or 6 x 109 cells (panels A, B, and C respectively). The implant cells were obtained from a culture of mixed lymphocytes of autologous and allogeneic leukocytes. The lines with downward slope are indicative of progressive reduction in the tumor mass attributed in part to a local immunological reaction resulting from the implant.

Figure 2 is a three-panel graph showing the effects of irradiation on a line of established tumor cells secreting IL-4. Panel A shows the pattern of development of the cells to which they were administered 5,000 (Q) or 10,000 (ß) rads. Panels B and C show IL-4 detected by ELISA in the culture medium, expressed as the total concentration (Panel B) or per cell (Panel C) at various times after irradiation.

Figure 3 is a bar chart showing the effect of different alloactivated lymphocyte preparations on the provision of resistance to a secondary challenge with J588L lymphoma cells in Balb / c mice. Allogeneic cells stimulated either with syngeneic splenocytes or certain third-party splenocytes are both effective.

Figure 4 is a bar chart showing the effect of different cell culture ratios on survival time in the mouse lymphoma model.

Figure 5 is a diagram showing the degree of functional activity in different preparations of alloactivated, human cells, as determined in four different assays.

Figure 6 is a bar chart showing the level of secretion of the cytokines IL-2 and IFN-α. by alloactivated, human cellular preparations.

Figure 7 is a bar chart showing the improvement of human lymphocyte alloactivation by "the use of a plurality of different stimulator cells.

Figure 8 is a bar chart showing the degree of functional activity of different preparations of human alloactivated cells, depending on the proportion of responder cells: stimulators.

Figure 9 is a bar graph showing the effect of inclusion of 20 μg / ml histidine (dark shading) or cimetidine (light shading) on human cell cultures; either responders alone, stimulators alone, or mixed cultures at a ratio of responders: stimulators of 10: 1.

Figure 10 is a half-tone reproduction of a photograph in a human patient vaccinated with a combination of mixed lymphocyte culture cells and autologous tumor cells, showing the immediate hypersensitivity reaction at the four injection sites. The doses were: 100 x 10 ° cells (upper right quadrant); 50 x 10 cells (upper left quadrant); 25 x 106 cells (lower right quadrant); and 10 x 10 cells (lower left quadrant).

Figure 11 is a half-tone reproduction of a photograph taken of the same human patient two days later, showing the late hypersensitivity reaction at the four injection sites. The reaction confirms that the patient is responding to the components of the vaccine composition.

DETAILED DESCRIPTION A central feature of the cellular vaccines of this invention is the use of multiple components that act in synchrony once inside the host, to produce the desired effect. In other words, the strategy is more than just an adoptive immune transfer. One component of the vaccine is the tumor antigen, preferably provided in the form of a cancer cell expressing multiple antigens associated with the tumor, shared by the tumor of the patient to be treated. The previously established tumor cell lines can be used for this purpose, but it is particularly convenient to use cells obtained from the patient to be treated, either by surgical resection, biopsy, blood sample collection, or other suitable technique. The other component is a mixture of cells that include lymphocytes that are activated (or capable of being activated) and as a result are capable of stimulating an increased immune response in the host. In certain preferred embodiments, the cell mixture is a culture of mixed lymphocytes of allogeneic cells stimulated using cells obtained from the patient to be treated. The strategy is a significant departure from previous procedures for immunotherapy against cancer in humans. Stimulated lymphocytes have been used in experimental human therapy, but as part of adoptive therapy-lymphocytes were originally obtained from the subject or a closely related donor. In this invention, the stimulated lymphocytes are allogeneic to the subject. Stimulated lymphocytes provide a potent immunostimulation that elicits a response against the tumor-associated antigen, simultaneously injected. As a result, a cellular immune response arises that is tumor specific, and much stronger than what can be achieved simply by administering the tumor cells to the patient, or a derivative thereof. The present invention was developed in conjunction with the observation that mixed lymphocytes implanted directly into a tumor bed limit or even reverse tumor growth. These experiments and the observations obtained therefrom are described in Example 1 below, and in the co-pending, co-pending US patent application Serial No. 08 / 616,880, a continuation in part of serial number 08 / 406,388, which they are incorporated by reference herein. The effect on the tumor mass seems to be at least partly due to an active immunological reaction of host origin. In one patient, there was no evidence of residual allogeneic lymphocytes, even when there was histological evidence for extensive tumor necrosis. In addition, the effect seems to be long-lasting. Some patients managed with a simple implant experienced a resolution in their condition that lasted for years. The hypothesis was hypothesized that increased expression of transplant antigens stimulated by allogeneic lymphocytes in the implant resulted in the massive recruitment of lymphoid cells near the tumor site. Were generated either the graft versus local tumor, graft versus host, host versus graft, or some other combination of reactions of this type, and opened an immunological window for the otherwise poorly immunogenic tumor antigens to initiate an antitumor immune response , mediated by the cells. An animal model has been developed for the implant protocol and a different type of cancer.

As a metastatic cancer model, the MADB 106 L_1 breast carcinoma cell line was used to infiltrate primary tumors in the middle lymphatic lobe of Fisher 344 rats. Once the tumors were established, they were implanted with syngeneic stimulating lymphocytes and allogeneic responder lymphocytes pre-cultured at a 1: 1 ratio. The treated animals survived an average of almost twice as much as those who were given live tumor cells but not the implant. The long-term survivors were immune to the new challenge with normally fatal doses of progenitor breast cancer cells. The preculture of mixed lymphocytes together was found to be important in obtaining the full effect (see Example 3 below). The combined results of these studies inspired the following conclusions: a) the strategy of implanting mixed lymphocytes is effective in improving survival in at least two different cancers (glioma and the metastatic breast cancer model) and at least two sites different (brain and liver); b) the survivors are resistant to the new challenge with the progenitor tumor cells; and c) the effect can be mediated through immune activation of the antitumor immunity of the host. A patient with a particular glioblastoma, treated with two successive implants did not respond adequately, and the implants were surgically removed. The patients were then treated with cryopreserved tumor cells recovered from the surgical procedure, mixed with another culture prepared from allogeneic and autologous lymphocytes. The mixture was administered not within the tumor bed, but at a subcutaneous site distant from the tumor (Examples 4 and 8). This "experimental procedure worked surprisingly well in the generation of a systemic antitumor response." Even though this patient did not have an intratumoral implant, he responded as well as he did.The conclusion is that the distal administration of the autologous tumor cells m S lymphocytes Mixed is an effective method of treatment against cancer, probably due to a potent ability to increase the immunogenicity of the tumor. A remarkable mark of the cellular vaccines of the present invention is that the effect is substantially greater than that obtained using tumor cells alone, or the tumor cells mixed with the adjuvants or co-factors previously used.The interaction between the tumor cells and the stimulated lymphocytes of the vaccines is probably complex.While not wishing to be bound by any theory, it seems possible that the tumor cells (or an antigen associated with the tumor) in fact a spectator in an immunological reaction generated in the host, stimulated by the activated lymphocytes. Activated lymphocytes can participate in one or more of the following forms. Firstly, these probably provide cytokines that are effective in the recruitment, activation or stimulation of the interaction of host immune cells. The cytokine mixture produced is superior to a cytokine provided in isolated form or via a transduced cell, in part, because it is a combination or cocktail of factors. Individual cocktail cytokines can work in concert or even synergistically to stimulate a variety of activities that go beyond those that can be reliably stimulated by a simple cytokine. The cocktail or cocktail may also comprise cytokines that have not been identified or isolated yet. Second, activated lymphocytes can also play a direct role in the presentation of the tumor antigen. Third, activated lymphocytes (particularly allogeneic lymphocytes) can play a contact role in the stimulation of host immune cells., during which the tumor antigen can participate as a spectator. An immune response resulting from the administration of the vaccine may comprise humoral and cellular components, but a cellular response is especially preferred. Cellular immunity (either cytotoxic lymphocytes, or helper-inducing cells that recruit other effector mechanisms) is believed to be important in the provision of a specific effect against the cells of the target neoplasm. The presence of an immune response can be verified periodically by standard immunological techniques. However, in human therapy, a major objective is an improvement in the patient's clinical condition. Clinical performance is therefore a superior test for the effectiveness of the compositions and methods of this invention, when directed towards the treatment of cancer. The present invention is superior to the strategies used or previously suggested. The advantages of the vaccine compositions of this invention include the following: • Vaccines improve the clinical condition or prognosis of human patients with cancer. This is true even when the tumor cells that reside in cancer patients are apparently poorly in themselves univocal. • Although the response is presumably mediated by an antigen associated with the tumor, there is no need to confirm the presence of any particular antigen on the tumor of a treated subject. The use of the patient's own tumor cells ensures a spectrum of relevant antigens. • There is no need to genetically alter patients 'cells, or use patients' DNA to genetically alter the cells of the vaccine. In fact, genetically altered cells are not required to obtain a response (although these can be included in particular embodiments of the invention, as described below). • The strategy is aimed at the generation of a systemic immune response, and can therefore be effective not only against the primary tumor, but also against the metastatic cells that share the 'tumor antigen with the primary tumor. • With the possible exception of the initial sampling of the tumor cells, the protocol can be performed with minimally invasive procedures. The vaccine compositions are preferably administered n site distant from the tumor. Subcutaneous routes of administration are preferred. • The effect is long lasting, persistent for at least approximately two months. Since the vaccination procedure is designed to stimulate the host's immune system, it should require at most occasional supplementation. This is a considerable advance on adoptive transfer methods. A further description of the preferred methods for preparing and using the vaccine compositions of this invention are provided in the sections that follow.

DEFINITIONS The terms "vaccine", "immunogen" or "immunogenic composition" are used herein to refer to a compound or composition, as appropriate, that is capable of conferring a specific degree of immunity when administered to a human or animal subject. . As used in this description, a "cellular vaccine" or a "cellular immunogen" refers to a composition comprising at least one cell population, which is optionally inactivated, as an active ingredient. The vaccines, immunogens and immunogenic compositions of this invention are active vaccines, which means that they are capable of stimulating a specific immune response (such as an antitumor antigen or anti-cancer cell response) mediated at least in part by the immune system of the immune system. Guest. The immune response may comprise antibodies, immunoreactive cells (such as helper / inducer or cytotoxic cells), or any combination thereof, and is preferably directed to an antigen that is present on a tumor to which the treatment is directed. The response can be provoked or restimulated in a subject by the administration of either single or multiple doses. No additional composition is required in order for it to qualify as a vaccine, unless otherwise specified. A compound or composition is "immunogenic" if it is capable of either: a) generating an immune response against an antigen (such as a tumor antigen) in a healthy individual; or b) the reconstitution, firing, or maintenance of an immune response in an individual, beyond what could occur if the compound or composition were not administered. A composition is immunogenic if it is capable of "reaching any of these criteria when administered in single or multiple doses." "Stimulation" of an immune or immunological response refers to the administration of a compound or composition that initiates, triggers, or maintains the ability of the host immune system to react against a target substance, such as a foreign molecule, an allogeneic cell, or a tumor cell, at a higher level than would otherwise occur- The stimulation of a primary immune response "refers herein to the production of specific immune reactivity in a subject in which no prior reactivity was detected, for example, due to lack of exposure to the target antigen, refractoriness to the target, or immunosuppression. of a "secondary" response refers to the reboot, trigger, or maintenance of reactivity in a subject in which the response was detected. prior activity, for example, due to natural immunity, spontaneous immunization, or treatment using one or more compositions or procedures. A "cell line" or "cell culture" denotes higher eukaryotic cells developed or maintained in vi tro. It is understood that the descendants of a cell may not be completely identical (either morphologically, genotypically, or phenotypically) to the progenitor cell. The "inactivation" of a cell is used in the present to indicate that the cell has been rendered incapable of performing cell division to form progeny. The cell can nevertheless be able to respond to the stimulus, or the biosynthesis and / or secretion of cellular products such as cytokines. Nactivation methods are known in the art. Preferred methods of inactivation are treatment with toxins such as mitomycin C, or irradiation. Cells that have been fixed or permeabilized and are unable to divide are also examples of inactivated cells. "Mixed lymphocyte reaction", "mixed lymphocyte culture", "MLR", and "MLC" are used interchangeably to refer to a mixture comprising a minimum of two different cell populations that are allotypically different. At least one of the alotypically different cells is a lymphocyte. The cells are cultured together for a time and under suitable conditions to result in stimulation of the lymphocytes. A frequent goal of an MLC is to provide allogeneic stimulation such as can initiate lymphocyte proliferation; but unless indicated, proliferation is not required during cultivation. In the proper context, these terms may alternatively refer to a mixture of cells derived from such a culture. When the cells from an MLC are administered as a bolus to a human, especially in a tumor bed, it refers to a "cytoimplant". "Genetic alteration" refers to a process in which a genetic element is introduced into a cell differently from mitosis or meiosis. The element can be heterologous to the cell, or this can be an additional copy or improved version of an element already present in the cell. The genetic alteration can be effected, for example, by transducing a cell with a recombinant plasmid or other polynucleotide, through any process known in the art, such as electroporation, calcium phosphate precipitation, or contacting a polynucleotide-liposome complex. The genetic alteration can also be effected, for example, by transduction or infection with DNA- or RNA-virus or viral vector. It is preferable that the genetic alteration be inheritable by the progeny of the cell, but in general this is not required for the practice of this invention, particularly when the altered cells are used in a pharmaceutical composition without further proliferation. The terms "tumor cell" or "cancer cell" used either singly or plurally, refer to cells that have undergone a malignant transformation that makes them pathological for the host organism. Primary cancer cells (ie, cells obtained near the site of malignant transformation) can be easily distinguished from non-cancerous cells by well-established techniques, particularly histological examination.

The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but any cell derived from an ancestor of the cancer cell. This includes The term "tumor-associated antigen" or "TAA" is used herein to refer to a molecule or complex that is expressed at a higher frequency or density by tumor cells than by non-tumor cells of the same tissue type. The antigens associated with the tumor may be antigens not normally expressed by the host; these can be mutated, truncated, misfolded, or otherwise abnormal manifestations of the molecules normally expressed by the host; these may be identical to molecules normally expressed, but expressed at abnormally high levels; or they can be expressed in a context or environment that is abnormal. Tumor-associated antigens may be, for example, proteins or protein fragments, complex carbohydrates, gangliosides, haptens, nucleic acids, or any combination of these or other biological molecules. Knowledge of the existence or characteristics of a particular antigen associated with the tumor is not necessary for the practice of the invention. As used herein, "treatment" refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, or may be performed either for prophylaxis or during the course of clinical pathology . Desirable effects include the prevention of the occurrence or recurrence of the disease, the alleviation of symptoms, the reduction of any direct or indirect pathological consequences of the disease, the prevention of metastasis, the decrease in the rate of progression of the disease, the improvement or attenuation of the state of the disease, and the improved remission or prognosis. The "pathology" associated with a disease condition is anything that compromises the welfare, normal physiology, or normal quality of life of the affected individual. This may involve (but is not limited to) destructive invasion of affected tissues in previously unaffected areas, developed at the expense of normal tissue function, irregular or suppressed biological activity, aggravation or suppression of an immunological inflammatory response, increased susceptibility to other organisms or pathogens, and undesirable clinical symptoms such as pain, fever, nausea, fatigue, mood swings, and other features such as can be determined by a attending physician. An "effective amount" is an amount sufficient to effect a beneficial or desired clinical result, particularly the generation of an immune response, the remarkable improvement in the clinical condition. An immunogenic amount is a sufficient amount in the subject group (either sick or not) sufficient to elicit an immune response, which "may comprise either a humoral response, a cellular response, or both. clinical response for subjects who have a neoplastic disease, an effective amount is an amount sufficient to alleviate, improve, stabilize, reverse or retard the progression of the disease, or otherwise reduce the pathological consequences of the disease. The preferred amounts and cellular proportions for use in an effective amount are given below in this description: An "individual" or "subject" is a vertebrate, preferably a mammal, more preferably a Human mammals include, but are not limited to, farm animals, sport animals, and pets.

GENERAL TECHNIQUES The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry and immunology, which are within the skill in the art. Such techniques are fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual". Second edition (Sambrook et al., 1989); "Oligonucleot ide Synthesis" (M.J. Gait, ed., 1984); "Animal Cell Culture" (R. Freshney ed., 1987) "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D.M. Weir &C.C. Blakwell, eds.); "Gene Transfer Vector for Mammalian Cells" (J.M. Miller &M.P. Calos, eds., 1987); "Current Protocols in Molecular Biology "(F.M. Ausubel et al., Eds, 1987);" PCR: The Polymerase Chain Reaction "(Mullis et al, eds., 1994);" Current Protocols in Immunology "(JE Coligan et al., Eds., 1991) See also Gately et al., Lee et al. And Zaling et al. Examples of techniques in mixed lymphocyte cultures The general procedures for the preparation and administration of pharmaceutical compositions are described in Remington's Pharmaceutical Sciences 18"Edition (1990), EW Martin ed., Magk Publishing Co. PA. There are a number of animal models for cancer that can be used to test and adjust the compositions and methods of this invention. Certain models involve injection into offspring of consanguineous parents with established syngeneic tumor lines. Tumors can be co-injected with a potentially therapeutic composition, allowed to be established before therapy is started, or are administered as a challenge at some time after vaccination of a healthy animal. Illustrations are provided in the Examples section. Also useful are the chimeric animal models described in U.S. Patent Nos. 5,663,481, 5,602,305 and 5,476,993; European application 379,554; and the international application WO 91/01760.

All patents, patent applications, articles and publications mentioned herein, above and below, are incorporated by reference herein.

PREPARATION OF CELLULAR VACCINES The cellular vaccines of this invention are typically assembled by preparing each cell population or equivalent thereof in an appropriate manner, combining the components, and optionally co-culturing or storing the cell mixtures prior to administration to a subject.

Tumor associated antigen: the source of tumor-associated antigen is more usually a tumor cell or cell line that is phenotypically similar to that for which the patient is being treated. Tumors from the same type of tissue and with similar histological characteristics tend to share antigens associated with the tumor. While the full spectrum of antigens may vary among individual tumors, there is a substantial likelihood that at least one will be shared.

Preferably, the tumor cells are histocompatible with the subject to be treated. In general, when it is possible to obtain tumor cells of patient origin, these cells are preferred since they most likely share a complete complement of the relevant antigens associated with the tumor. Circulating tumors such as leukemias and lymphomas can be easily taken from the peripheral blood. Otherwise, tumor cells are generally taken by a surgical procedure, including but not limited to biopsy, or surgical resection or volume reduction. Tumor cells can also be harvested from metastatic sites. Solid tumors can be dissociated into separate cells by physical manipulation optionally combined with enzymatic treatment with proteases such as collagenase and the like. The cells are then transferred to fresh physiological medium. The cells can be stored until later use, for example, by freezing in liquid nitrogen. Optionally, and especially when the original tumor mass is small, it is permissible to expand the population of tumor cells to ensure an adequate supply. The cells are cultured in a growth medium suitable for propagation, optionally supplemented with growth factors. Preferably, a population of stable cells comprising the characteristics of the tumor cells is obtained without further transformation, although this is permissible where required. The cell population can optionally be cloned to increase its stability or refine its characteristics although this is not generally necessary. The conditions for reliably establishing short-term cultures and obtaining at least 10b cells from a variety of tumor types are described by Dillman et al. (1993) J. Immunother; 14: 65-69. If possible, the preparation of original tumor cells is used without proliferation, since it is possible that a critical tumor antigen will be lost through the proliferative process. Cancer cells or cancer cell lines obtained as described can be combined as described can be combined directly with the other components of the vaccine. However, it is preferable to inactivate the cancer cells to prevent further proliferation once it is administered to the subject. Any physical, chemical or biological inactivation medium can be used, including but not limited to irradiation (preferably at least about 5,000 cGy, more preferably at least about ,000 cGy, more preferably at least about 20,000 cGy); or treatment with mitomycin C (preferably at least 10 μg / ml, more preferably at least about 50 μg / ml). Cancer cells for use as a source and tumor antigen can be alternatively fixed with agents such as glutaraldehyde, paraformaldehyde, or formalin. These can also be solubilized in an ionic or non-ionic detergent such as deoxycholate or octyl glucoside, or used, for example *, using the vaccinia virus. If desired, suspensions of solubilized cells can be clarified or subjected to any of a number of standard biochemical separation procedures to enrich or isolate the particular antigens associated with the tumor. Before the combination with other components of the vaccine, the preparation is depleted of the agent used to treat it; for example, by centrifugation and washing the fixed cells, or dialysis of the solubilized suspension. Such treatment of the tumor cell population particularly beyond inactivation may be considered as optional and unnecessary for the practice of the embodiments of the invention, unless specifically required.

Allogeneic cells: The cellular vaccines of this invention also comprise a second cell population, of which at least a portion are cells allogenic for the subject to be treated, and capable of reacting specifically to an allogeneic stimulus. This generally means lymphocytic cells or cells of the lymphocytic line, particularly T cells, lymphocytes that express CD4 antigens (CD4 + cells), and cells that express the DC8 antigen (CD8 + cells) are both included in the definition of T lymphocytes, and either or both of them can be included in the vaccine. Preferably, the allogeneic cells in the second cell population are at least 10% CD4 + cells or 10% helper / inducer cells; more preferably, these are at least about 20% CD4 + cells or helper / inducer cells; still more preferably the portion is at least about 30%; still more preferably the portion is at least 50%. CD4 + cells can be conveniently quantified with a commercially available specific antibody such as 0KT4 in conjunction with fluorescence-activated counting. Cells are generally described as allogeneic if they have a sufficient phenotypic difference to stimulate an alloreaction. In the context of this description, the use of the term "allogeneic" is restricted to a difference in the phenotype of the major histocompatibility complex (MHC) antigens. Any qualitative difference in the identity of MHC allotypes between cells of the same species means that these are allogeneic cells. In humans, differences in any of the HLA-A, B, C, D, DP, DQ and DR loci constitute allotypic differences relevant to this invention. The identity of HLA-A, B, C, DP, DQ, and DR are typically determined using allotype-specific antibodies in a cytotoxicity or immunofluorescence technique. Preferred alotypic differences for purposes of the present invention relate to HLA antigens of class II. The comparison of the class II antigens of the DP, DQ and DR loci between the putative allogenic cells and the cells of the subject to be treated, preferably at least 1, and increasingly, preferably 2, 3, 4, 5 or even 6 loci, are different between allogeneic cells. Class II antigens can also be determined at the D locus by reaction of mixed lymphocytes using typed cells. Allogenic cell donors are generally unrelated to the subject being treated, to maximize the number of MHC differences. The number of differences in the region of class II is related but is secondary to a functional determination of halogenicity. Allogenic cells are particularly suitable for use in the present invention if they demonstrate a strong proliferative response in a single-path MLR, using inactivated mononuclear cells of the subject to be treated as stimulator cells. Cell donors known to produce a particularly strong response, particularly for stimulator cells of the same genotype as the subject, are especially suitable. A panel of different allogeneic cells can be tested against the patient's cells to determine those that elicit the strongest response. The population of allogeneic cells is not necessarily restricted to those obtained from a simple donor. Two, three or a higher plurality of donors can optionally be used to facilitate the collection of allogeneic cells, to increase the stimulation of allogenic cells, to minimize the production of an anti-allotype response, or to improve another the efficiency of the cellular vaccine. Allogenic cells are preferably activated or stimulated prior to administration to the subject, or are capable of performing activation or stimulation once administered. "Activation" in this context means induced to proliferate. Proliferation can be measured by counting the cells, or by a standard assay of ["H] -thymidine uptake during the last approximately 18 hours of culture.The stimulation of the cells with a mitogen such as PHA shows the maximum proliferation , and the culture without the cell-inducing agent measures the proliferation of baseline.The substantially proliferating cells will show uptake substantially above the baseline levels.The "stimulation" in this context means induced by an external agent to suffer a metabolic alteration, particularly an increase in the synthesis and / or secretion of biologically relevant molecules such as cytokines or other soluble mediators, a morphological change from a lymphocyte at rest to a lymphoblast, or an increase in biological reactivity in the cell including but not limited to an increase in endocytosis, exocytosis, phagocytosis, or intracellular transport and processing of molecules from the external environment. A number of biological assays are relevant for the measurement of activation and stimulation, including proliferation and cytoxicity assays, histological examination, measurement of the density of cell surface markers, measurement of mediators (particularly cytokines) synthesized and / or excreted by the cell, or an increased ability to recruit effector cells, such as neutrophils, basophils, mast cells, monocytes, macrophages, eosinophils, and other lymphocytes. The stimulation takes place when the test reveals a change in the characteristic that is measured after the induction, which exceeds those observed in the absence of induction. Particularly relevant forms of stimulation are those correlated with the increased frequency of participation of cells that possess such characteristics, in an immunological response. Such characteristics include blastogenesis, proliferation or cytoxicity in response to specific antigen, and increased secretion of mediators IL-2, IL-4, IL-6, TNF-α, IFN- ?, GtCSF, M-CSF, or GM-CSF. Certain assays for the measurement of stimulation are provided in Example 5. Lymphocytes can be collected for use with this invention from any suitable tissue source, but are more conveniently obtained from the blood of an allogeneic donor. Donors are preferably pre-selected to identify those with sufficient leukocyte counts, and exclude those with neoplastic conditions or transmissible infections. Collection can be done by donation of whole blood followed by separation of blood cell populations, and more conveniently by leukapheresis. Sufficient blood is processed to obtain approximately 100-500 ml of leukapheresis suspension, preferably at least 200 ml. The blood is collected in anticoagulant, such as citrate or EDTA. For example, leukapheresis can be performed using a Cobe 2997 blood cell separator (COBE SPECTRA®, Lakewood CO); Fenwall Cs 300 (Fenwall, Deerfield IL); or Haemonetrics (Baintree MA). Flow rates of approximately 40-50 ml / min for 2 to 4 hours produce approximately 200 to 250 ml of leukapheresis suspension having less than 1 ml of red cells, with variations between individual donors and the equipment used. Lymphocytes prepared by any suitable method are generally washed to remove the platelets, and are resuspended in a suitable medium, such as AIM V supplemented with 2% inactivated fetal calf serum. If desired, the cells can be further separated into subpopulations in order to enrich lymphocytes, particularly T cells. Positive and negative selection methods can be used. Suitable methods include centrifugation on a suitable medium such as FIC0LLMR or HISTOPAQUE® the pass on a nylon-wool column, affinity separation methods such as panning, or selection on a fluorescent cell sorter using an antibody against a marker relevant of the cell surface. For convenience, it may be preferable to decrease the number of handling steps. For example, the best separation of leukapheresis can avoid the need for subsequent separation over FICOLLMR. The allogenic cells can be stimulated in any way that enhances the immune response to the relevant antigens associated with the tumor. LAK cells and TIL cells are included in the definition of stimulated allogeneic cells. The LAK and TIL cells can be generated and separated according to the techniques known in the art; a major difference for use in the present invention is that they are allogeneic to the intended container. This invention encompasses any means of activating or stimulating allogeneic cells, including but not limited to preculture with cells of either donor or patient origin, precultivating with living tumor cells (not inactivated), preculturing with feeder cells of donor origin, precultivating with isolated or recombinant cytokines or mixtures thereof preculting with mitogens such as ConA, or any combination of the techniques listed. Methods for the stimulation of immune cells and assays for the determination of stimulation can be found among others in U.S. Patent No. 5,569,585.

A particularly preferred method of stimulation is by the culture of mixed lymphocytes, as is known in the art and as is further described herein.

Stimulating Cells: A preferred method to stimulate allogeneic lymphocytes is to combine them with cells that are capable of providing adequate stimulation. Such cells are referred to herein as "stimulatory" cells. Allogeneic lymphocytes, when stimulated in this way, are referred to as "responder" cells. Preferred stimulator cells are cells that are allogeneic to responder cells. In certain embodiments of this invention, the source of stimulator cells is an individual genetically similar to the subject being treated; even more preferably, the stimulator cells are from an individual who is similar or identical to the subject in terms of MHC antigens, even more preferably the cells are autologous to the subject. In some cases, the subject may have had a previous immune response against the antigen associated with the tumor, due to stimulation by the tumor or previous treatment.

In other embodiments of this invention, the stimulator cells are not from the subject to be treated, but from another donor or plurality of donors. The advantage of using the subject's cells is that the responders will continue to receive the stimulation of the alloantigens of the subject after administration. However, the use of third-party stimulators has its own group of advantages. For example, batches of stimulated cells can be prepared for use in different and different patients. By way of illustration, leukapheresis cells from various donors other than the subjects to be treated are combined and cultured together under conditions sufficient for stimulation. Aliquots of the cultured cells are then combined from the tumor cells from each individual patient, in order to form a cellular vaccine designed for the tumor-associated antigens of each patient. In additional embodiments of the invention, a plurality of stimulator cells from different individuals is used, which may or may not include those of the subject to be treated. Suitable cell types for use as stimulator cells are those that possess a high density of histocompatibility antigens, particularly class II antigens. The cells of the lymph nodes are adequate, but a more usual source is the peripheral blood. Leukocytes or white cells can. be collected from the subject's circulation by leukapheresis; however, collections of whole blood are more usual, and usually more convenient since the number of stimulatory cells required is much lower than the number of responding cells. 200 to 400 ml of peripheral blood collected via a vein puncture in a suitable anticoagulant typically provides enough cells to prepare the vaccine. The separation methods described above can in general be employed to rescue the stimulator cells from the entire blood sample of the subject. It is desirable to enrich, or at least not decrease, the cells expressing the histocompatibility antigens of class II of the population, such as B cells and monocytes. Extensive subfractionation of cells is not usually required, and a population of simple peripheral blood mononuclear cells (PBMC) is adequate for most purposes.

Mixed lymphocyte cultures: Allogeneic donor lymphocytes and stimulator cells, when used, can be combined just before administration to the patient, with the expectation that allogeneic stimulation will take place in vi ve. However, the data provided in example 3 have shown that the co-culture of the cells before administration, improves the effect of the composition. This invention encompasses the use of mixed two-way lymphocyte cultures as a way to stimulate allogeneic cells. However, when the leukocytes of the tumor patient are used as stimulator cells, the one-way MLCs are preferred. To drive a single-pathway MLC, the subject's stimulator cells are inactivated, for example, by treating approximately 10 7 cells / ml with 50 μm / ml mitomycin C, followed by washing. The allogeneic lymphocytes are combined with the stimulator cells in a suitable medium, generally supplemented with fetal calf serum or a substitute thereof, and optionally including other growth factors. The ratio of the responder: stimulator cells is preferably between about 100: 1 to 1:10, more preferably about 50: 1 to 1: 1.; still more preferably from about 20: 1 to 5: 1, and even more preferably about 10: 1. Where there are a plurality of stimulator or responder cells in a single-path MLC, the same approximate ratio of responders: stimulators is maintained. Thus, when two inactivated stimulators are used, the ratio can be about 9: (1: 1); When three inactivated stimulators are used, the ratio can be approximately 8: (1: 1: 1). Similarly, when multiple responders are used, the ratio can be (5: 5): 1 or (3: 3: 3): 1. If it is cultivated jointly, the composition of multiple responders becomes a multipath MLC. A single pathway activation of multiple responders can be achieved by conducting a separate culture for each responding population at a ratio of 10: 1, and then combining the alloactivated cells just before use. Once combined in the desired ratio, cells cultured at an appropriate density in a suitable atmosphere (such as 95% 02.5% C02 at approximately 37 ° C). The culture period is preferably at least about 12 hours, more preferably between about 24 hours and 72 hours. Additional stimulation can be obtained by culture for 3 to 5 days, although this is generally not preferred, since cytokine levels are normally higher during the first 48 to 72 hours of culture. The practitioner can determine if the conditions are sufficient for the proliferation or stimulation of the responding cells, by performing a bioassay for these properties as described at the beginning or in example 5. In another method, the levels of TNF-a , LT and / or IFN-? they are measured in the culture supernatant. Stimulation is indicated by the levels of the biological activity of TNF-a or LT at 50-150 U / ml, or 500-3500 pg / ml. The preferred cultures are those that show an activation level greater than or equal to 10. % above the control value of the unstimulated donor, within the first 3 days of culture, as measured by the Tetrazolium Reduction Assay (XTT), or by Flow Cytometry (CD69 or intracellular esterase), or both. The citation within that description of preferred cellular sources, cell proportions, culture conditions, and other characteristics, is intended as an aid to the practitioner, and is not to be understood as limiting the scope of the invention, unless explicitly required. . No limitation is implied with respect to any of the individual parameters, since other various combinations of parameters will generate a cell population with the desired functional effect. The culture of mixed lymphocytes is generally conducted with the allogeneic responder cells and the stimulator cells of the subject before the addition of the tumor cells. Usually, the stimulator cells, although derived from a patient with cancer, are themselves noncancerous. However, it is permissible for the tumor cells to be present during the MLC. Occasionally, the stimulator cells and tumor cells may be the same, such as in the treatment of a leukemia.

Functional effect optimization: Experience in animal model experiments shows that not all third-party donors provide the same degree of alloactivation, particularly when using a different donor third party as the stimulator cell. To the extent that viability is dependent on the donor cell, donors can be chosen according to experience, both in terms of the degree of alloactivation observed in the culture, and the clinical outcome. Functional criteria indicate a particular level of activation, such as the Tetrazolium Reduction Assay (XTT), the Flow Cytometry Assay, or the secretion level of certain lymphokines determined by ELISA, may be sufficiently predictive of the results, depending of clinical experience. Once successful donors are identified, they can be constituted in a panel of regular donors provided by the service laboratory that provides the immunogenic compositions. To the extent that the variability depends on the similarity between donors and patients, various other selection criteria may also be used. Since the efficacy of certain donor-patient combinations can migrate according to histocompatibility, the donors can be selected, if desired, based on tissue similarity. Donors of particular human histocompatibility types can be tested for efficacy with particular tumors, if desired, using one of the chimeric animal models listed at the beginning. A more immediate donor identification test can be conducted using the PBL from the patient and PBL from a selection of potential donors in an in vi tro trial. An assay of this type is a reverse functional test. In this assay, patient cells are established in a culture of mixed leukocytes as the responder, using the potential donor of the alloactivated cells as the inactivated stimulator. Since the response is thought to involve cytokine secretion by the alloactivated cells, an alternative predictor may be a two-step culture. In this procedure, a responder culture is established: stimulator using the same replenishing and stimulating cells that are tested for use in the preparative culture. After three days, the culture is inactivated with mitomycin or sublethal irradiation, so that the cells can still produce cytokines but not replicate. The leukocytes from the patient are then added, and their response is followed by a functional assay, by cytokine secretion, or by the proliferation of T cells. In a variation of this procedure, the inactivated tumor cells are also provided in the second stage. of the culture, and the reading is determined at the end of the second stage between the measurement of the cytolysis of the tumor cells marked with 51 Cr. These assays are described for the benefit of the reader who may wish to optimize the compositions of this invention in various ways, particularly in the establishment of a panel of enriched donors for high responders. It should be emphasized that the invention can be practiced without employing the additional refinements described in this section. The degree of alloactivation or the potential therapeutic result can also be improved by employing one or more of the following strategies, where available and appropriate a) using a plurality of donor cells as the responders or stimulators in the MLC; b) using cells from the patient to be treated, as stimulators in the MCL; c) the addition of an H2 receptor antagonist to the culture medium of the MLC. A preferred H2 receptor antagonist is cimetidine, added to the culture medium between 5 μg / ml and 100 μg / ml, typically 20 μg / ml. The benefits of using cimetidine or a plurality of donor cells are illustrated in Examples 6 and 7, respectively.

Genetically altered cells: Allogenic cells used in the vaccine can be genetically altered in a way that enhances the immune response to the vaccine. Particularly preferred are allogeneic cells genetically altered to express cytokines at high levels. It is recognized that lymphocytes, particularly those in an allogeneic mixture, can already produce detectable levels of certain cytokines. "Elevated levels" of expression that occur as a result of genetic alteration, exceed the levels observed in non-genetically altered cells in the same way, but otherwise similar. Any cytokine can be used for this purpose, especially those that have the ability to recruit or stimulate cells of the lymphocyte line or antigen presentation, or otherwise participate in the enhancement of the immune response. Preferred cytokines include, but are not limited to, tumor necrosis factors, exemplified in TNF-α; interleukins, as exemplified in IL-2, IL-4, IL-6, IL-7 and IL-10; Interferons, exemplified by IFN-a and IFN- ?; hematopoietic factors and colony-stimulating factors, exemplified in GM-CSF and M-CSF.

Among the possible cytokines that can be used with this invention, GM-CSF is especially preferred because of its important role in the maturation and function of specialized cells that present antigens. This is believed to be important because many tumor cells, such as those of epithelial origin, do not express detectable MHC class II molecules. IL-4 is also especially preferred, as a pluripontencial cytokine endowed with a wide range of stimulatory activities in B and T lymphocytes, as well as in the hematopoietic cells. Their roles include the recruitment and activation of antigen presenting CD4 + cells, as well as the induction of cytotoxic T lymphocytes. TNF-a is a third cytokine that is especially preferred, in part because of its wide range of effects on the immune and inflammatory response. The sequences encoding the protein and DNA of human IL-4 and TNF-α are known, and the vectors comprising the coding sequences are available. For sequences of IL-4 and vectors, see U.S. Patent No. 5,017,691 and European Patent No. 230107. Genetically altered CHO cells are described in U.S. Patent No. 5,034,133. The use of IL-4 (either as the isolated recombinant or in a genetically altered cell) in the treatment of solid tumors is described in Note American Patent No. 5,382,427. TNF polypeptides, coding sequences, vectors, and genetically altered host cells are described in U.S. Patent No. 5,288,852 and European Patent 4,879,226. TNF variants, which may also be used in this invention, are described in U.S. Patent No. 4,677,063. The compositions comprising TNF-a and interferon are shown in European patent EP 131789. The synergism of TNF and IL-4 in the inhibition of the development of cancer cells is described in International Patent WO 92/05805. The genetic alteration can be effected by any method known in the art. Typically, a coding sequence for the desired cytokine is operably linked to a heterologous promoter that will be constitutively or inducibly active in the target cell, along with other control elements and a poly-A sequence necessary for the transcription and translation of the protein . The expression cassette thus compounded is introduced into the cell by any method known in the art, such as calcium phosphate precipitation, insertion using cationic liposomes, or using a tropic viral vector for the cells. Methods of alteration, genetics are described in the patent publications cited in the preceding paragraph. A preferred method of genetic alteration is the use of the retroviral vector LXSN comprising a suitable expression cassette, as illustrated in Example 2. Another preferred method is the use of adenoviral vectors (M. Graf et al., extract 1994). In summary, recombinant adenoviral expression vectors are engineered from commercially available plasmids such as those provided by Microbix, Canada. The conditions and multiplicities of infection infection (MOI) can be determined in premilinary experiments using a reporter gene such as β-galactosidase, and then used for cytokine transfer (Kammersheidt et al.). An advantage of using a viral vector is that the vector can first be replicated, and then a whole population of cells can be infected and altered. Accordingly, genetically altered cytokine secretion cells can be established as a cell line, or a fresh leukapheresis preparation is altered de novo just before use in a vaccine of this invention. In the latter case, the preparation of the vaccine could further comprise the step of infecting a population of allogeneic cells to the intended recipient, with a viral vector comprising a coding region for a particular cytokine of interest. The genetic alteration of allogeneic lymphocytes can be conducted as an alternative to or in addition to co-culture with leukocytes from the subject to be treated. Conferring the ability to produce an adequate cytokine or cytokine mixture can allow allogeneic lymphocytes to be self-stimulatory, avoiding the requirement of co-culture with leukocytes of the subject. More often, the two effects will be complementary or even synergistic, and it may be preferable that they be both. For example, lymphocytes altered to produce a major cytokine are then co-cultured with leukocytes from the subject which can then activate the production of other cytokines in minor but important amounts. Similar methods can be used to genetically alter primary tumor cells or tumor cell lines for use in vaccine compositions, so that they produce cytokines. The description of such altered tumor cells is provided in Example 2. In general, it is preferable to genetically alter the allogeneic lymphocytes instead of the tumor cells; in part, because the tumor cells are typically irradiated before administration to the subject. However, the alteration of the cells] 8_t_ £ a _._! Tr, putede fo? Fit &naj? &Cilme i © stable cell lines. Such lines (particularly those that possess a spectrum of common antigens associated with the tumor) can be used to create a standard cytokine secreting cell line for use in vaccines to treat a plurality of subjects. As shown in Example 2, the tumor lines can be created, which contribute to produce cytokine from the genetic alteration for some time after a dose of radiation that promotes proliferation. For use in the present invention, lines with these properties are developed, and propagated or maintained in culture right up to the assembly of the vaccine. The required number of tumor cells are irradiated at the correct dose and mixed with the allogeneic lymphocytes; meanwhile a reserve of the altered, living tumor cells is kept in reserve if they are needed for the reinforcement injections.

Montage of the vaccine: to maximize the viability of the various cells in the population or to maintain their intended function, it is generally preferable to assemble the vaccine near the time of administration. Various cell populations can be racoleched in advance, and cultured or cryopreserved to the degree consistent with the cell type and with the function in the vaccine. The newly obtained cells are preferred. Cells from mixed lymphocyte culture, or the whole vaccine, can also be cryopreserved as a mixture. However, it is preferable to conduct the MLC and then add the tumor cells shortly before administration to the patient. When the allogeneic cells are stimulated by an MLC, there will usually be three cell populations in the vaccine: tumor cells of the subject, stimulatory cells, and allogeneic responsive cells. The role of the stimulator cells is primarily to stimulate the allogenic cells in vi tro, and it may not be necessary that they be present in the final vaccine composition. In this way, they can be finally removed, although this is not usually necessary. It is important to remove any additional components used in the preparation of the cells, particularly in the MLR, which may have an undesired effect on the subject. In particular, fetal calf serum, bovine albumin, or other biological supplements in the culture medium are typically removed to avoid a collateral immune reaction against them. Typically, the cellular components of the vaccine are washed, such as by repeated gentle centrifugation, in a suitable pharmacologically compatible excipient. Compatible excipients include isotonic saline with or without a physiologically compatible buffer such as phosphate or Hepes and nutrients such as dextrose, physiologically compatible ions, or amino acids, and various culture media suitable for use with lymphocyte populations, particularly those devoid of of other immunogenic components. Carrier reagents, such as albumin and blood plasma fractions and non-active thickening agents, can also be used. The non-active biological components, to the extent that they are present in the pharmacological preparation, are preferably derived from the same species as that to be treated, and are even more preferably previously obtained from the subject. The vaccine compositions of this invention may optionally include additional active components that function independently or in synchrony with the tumor associated antigen and the activated allogeneic cells. Such optional components include, but are not limited to, isolated or recombinant cytokines, particularly those explicitly referred to in this disclosure, adjuvants and other cell types. A vaccine composition of this invention is considered "adequate" for administration to a human if reasonable and acceptable standards have been taken to ensure that the vaccine itself will not confer additional major pathology to the recipient. Side effects such as local inflammation, induration, or pain, or a febrile response, may be unavoidable and are generally acceptable and treatment is otherwise successful in a substantial proportion of patients. However, the composition should be reasonably free from: a) infectious or chemical unrelated and pathological agents, particularly from the donor of allogeneic lymphocytes; b) undesirable growths such as can be generated or propagated in tissue culture, such as bacteria or bacterial toxins, mycobacteria, and viruses; c) unacceptable levels of oncogenic agents or cancer cells that develop aggressively, which do not originate from the subject being treated; and d) components committed to initiate or effect an undesirable immune reaction, particularly anaphylactic shock. The particular tests that can be used are listed in the examples section of this description. The compositions of the present invention, and the subcomponents thereof can be supplied, in unit dose or in the form of equipment. The kits of this invention which may comprise various components of a cellular vaccine or the pharmaceutical composition, are provided in separate containers. The containers may separately contain cells or antigens such that when mixed together they constitute a vaccine of this invention in unit dose or multiple dose form. Preferred kits comprise in separate containers: stimulated lymphocytes allogeneic to the human being, particularly cells obtained from a co-culture of allogeneic lymphocytes and autologous leukocytes; and antigen associated to the tumor coming from the human, particularly primary tumor cells coming from the human, or the progeny thereof. Alternatively, the kits may comprise a cellular mixture in one container and a pharmaceutical excipient in another container. A preferred device in this category comprises, in a first container: stimulated lymphocytes allogeneic to the subject to be treated, particularly cells from a mixed lymphocyte culture; and in a second container, a pharmaceutical excipient. The user can use the excipient to prepare his own tumor cells of the subject; the cells are then combined with the allogeneic lymphocytes stimulated for administration to the subject. The packaged compositions and equipment of this invention typically include instructions for the storage, preparation, and administration of the composition.

USE OF CELL ULTRA VACCINES IN THE TREATMENT OF CANCER The compositions of this invention can be administered to subjects, especially but not limited to human subjects. These are particularly useful for eliciting an immune response against an antigen associated with the tumor, or for the treatment of cancer.

Treatment objectives: One purpose of administering the vaccine is to elicit an immune response. The immune response may include humoral or cellular components, or both. The humoral immunity can be determined by standard immunoassay for antibody levels in a serum sample from the treated individual. Since it is thought that cellular immunity plays an important role in the immune survival of cancer, the generation of a cellular immune response is often a particular goal of the treatment. As used herein, a "cellular immune response" is a response that involves T cells, and can be observed in vi tro or in vi vo.

A general cellular immune response can be measured as the proliferative activity of T cells in cells (particularly PLB) taken from the subject after administration of the vaccine. The inactivated tumor cells, preferably derived from the subject, are used as stimulators. A non-specific mitogen such as PHA serves as a positive control; Incubation with an unrelated stimulatory cell serves as a negative control. After incubation of the PBMCs with the stimulators for an appropriate period (typically 5 days), the incorporation of tritiated thymidine is measured. If desired, the determination of the subgroup of proliferating T cells can be performed using flow cytometry. The cytotoxicity of T cells (CTL) can also be measured. In this test, a population of enriched T cells from the subject are used as effectors in a standard 1Cr release assay. The tumor cells are radiolabeled as targets with approximately 200 μCi of Na2 5lCr0 for 60 minutes at 37 ° C, followed by washing. T cells and target cells (~ 1 x loVpozo) are then combined at various effector-to-target ratios in 96 well plates with U-bottom. Plates are centrifuged at 100 xg for 5 minutes to initiate cell contact, and they are incubated for 4-16 hours at 37 ° C with 5% C02. The release of 51 Cr is determined in the supernatant, and compared, with the targets incubated in the absence of T cells (negative control) or with OA% of TRITONMR X-100 (positive control). Another purpose of the administration of the vaccine is for the treatment of a neoplastic disease, particularly cancer. The beneficial effect of the vaccine will generally be at least in part mediated by the immune system, although an immune response need not be positively demonstrated in order that the compositions and methods of treatment fall within the scope of this invention, not to be required in another way.

Suitable Subjects: The compositions of this invention can be used for administration to human and non-human vertebrates. These provide advantages over the previously available compositions, particularly in populations descended from consanguineous parents, and particularly in spontaneous tumors. Veterinary applications are contemplated within the scope of the invention.

Cellular vaccines have been tested in human subjects, and are especially suitable for human treatment. Vaccines can be administered to any human subject at the discretion of the attending physician. Typically, the subject will either have cancer, or be at substantial risk of developing cancer. Typical human subjects for therapy comprise two groups, which can be distinguished by clinical criteria. Patients with "advanced disease" or "high tumor burden" are those who have a clinically measurable tumor. A clinically measurable tumor is one that can be detected based on the tumor mass (for example, by palpation, MRI, CAT scan, X-ray, or radio-syn- chrongraphy, positive biochemical or histopathological markers by themselves, are insufficient to identify this population). A vaccine composition exemplified in this invention is administered to patients with advanced disease in order to alleviate their condition. Ideally, the reduction in tumor mass occurs as a result, but any clinical improvement is a benefit. Clinical improvement includes the decreased risk or decreased proportion of progression or reduction in the pathological consequences of the tumor. A second group of suitable subjects is known in the Xécnica as the "adjuvant group". These are individuals who have had a history of cancer, but have responded to another mode of therapy. A previous therapy may have influenced (but is not restricted to) surgical resection, radiotherapy, traditional chemotherapy, and other forms of immunotherapy. As a result, these individuals do not have tumor clinically measurable by the definition given above. However, they are suspected of being at risk for the recurrence or progression of the disease, either near the site of the original tumor or due to metastasis. The adjuvant group can be further divided into high risk and low risk individuals. The subdivision is made based on the characteristics observed before or after the initial treatment. These characteristics are known in clinical techniques, and are properly defined for each different cancer. The typical characteristics of subgroups of high risk are those in which the tumor has invaded neighboring tissues, or which show involvement of the lymph nodes. A vaccine composition exemplified in this invention is administered to patients in the adjuvant group, in order to elicit an anticancer response primarily as a prophylactic measure against recurrence. Ideally, the composition delays the recurrence of cancer, or more preferably, reduces the risk of recurrence (e.g. improves the cure rate). Such parameters can be determined in comparison with other patient populations and other modes of therapy. Of course, crossings occur between these two groups of patients, and the vaccine compositions of this invention can be administered at any time that is appropriate. For example, therapy may be conducted before or during therapy, traditionally of a patient with high tumor burden, and continued after the tumor becomes clinically undetectable. Therapy can be continued in a patient who initially fell into the adjuvant group, but who is showing signs of recurrence.

Examples of tumors that can be treated with the compositions and methods of this invention include the following: pancreatic tumors, such as adenocarcinomas of the pancreatic ducts; lung tumors, such as adenocarcinomas of small and large cells, squamous cell carcinoma, and bronchialveolar carcinoma; colon tumors, such as epithelial adenocarcinoma and its metastases; and liver tumors, such as hepatoma and cholangiocarcinoma. Also included are breast tumors, such as ductal and lobular adenocarcinoma, gynecological tumors, such as squamous and adenocarcinoma of the uterine cervix, and uterine and ovarian epithelial adenocarcinoma; prosthetic tumors, such as prosthetic adenocarcinoma; bladder tumors, such as transitional squamous cell carcinoma; RES system tumors, such as nodular or diffuse B-cell or T-cell lymphoma, plasmacytoma, and acute or chronic leukemia; skin tumors, such as malignant melanoma; and soft tissue tumors, such as sarcoma and soft tissue leiomyosarcoma. Of special interest are brain tumors, such as astrocytoma, oligodendroglioma, ependymoma, medulloblastomas, and primitive neural ectodermal tumor. Included in this category are gliomas, glioblastomas, and gliosarcomas. The immune status of the individual can be any of the following. The individual may be immunologically intact with respect to certain antigens associated with the tumor, present in the composition, in which case the compositions may be administered to initiate or promote the maturation of an antitumor response. The individual may not currently be expressing antitumor immunity, but may have immunological memory, particularly memory in T cells related to a tumor associated antigen, comprised in the vaccine, in which case the compositions may be administered to stimulate a memory response. The individual may also have active immunity (either tumor or cellular immunity, or both) to a tumor-associated antigen, comprised in the vaccine, in which case the compositions may be administered to maintain, trigger, or mature the response, or recruit other arms of the immune system. The subject must be at least partially immunocompetent, to minimize a graft versus host reaction of pathological extent. However, it is recognized that cancer patients frequently show a degree of immunosuppression, and this does not necessarily prevent the use of the compositions of the invention, as long as the compositions can be administered safely and effectively. The immunocompetence of the subject may be of the host's origin, or may be provided by means of an adoptive, concurrent transfer treatment.

Modes of administration and dosage: The compositions of this invention can be administered to the subject at any site, particularly a site that is "distal" to or "distant" from the primary tumor. The route of administration of a pharmaceutical composition can be parenteral, intramuscular, subcutaneous, intradermal, intraperitoneal, intranasal, intravenous (including via an internal residence catheter), via an afferent lymphatic vessel, or by any other route that is suitable in view of the tumor that is treated and the condition of the subject. Due to the low level inflammation or induration that can occur for a few days after administration, relatively non-invasive methods are preferred, particularly subcutaneous routes. The dose administered is an "effective" amount to give rise to a desired therapeutic response, be it the situation of an immune response, or the treatment of cancer as defined elsewhere in this description. For the pharmaceutical compositions of this invention, effective doses typically fall within the range of about 10 5 to 10 11 cells, including allogeneic lymphocytes, and tumor cells and other cells from the subject being treated, if present. Preferably, between 10 to 10 10 cells are used; more preferably between about 1 x 107 and 2 x 109 cells are used; more preferably between about 5 x 10 7 and 2 x 10 9 cells are used; even more preferably between about 1 x 10"and 1 x 109 cells are used.Multiple doses, when used in combination to achieve a desired effect, fall within the definition of an effective amount.The various components of the cellular vaccine they are present in an "effective combination", which means that there are sufficient amounts of each of the components for the vaccine to be effective.Preferably, at least about 10b, more preferably at least about 107 but not more than 1010 lymphocytes are present Preferably, at least 105, more preferably at least IO '' ', and still more preferably approximately 10 but in general less than 108 and typically less than 5 x 10 tumor cells, the progeny of the tumor cells, or the equivalents of them If autologous or third-party stimulatory leukocytes are present, preferential between about 10"and 10A The proportions of allogeneic lymphocytes to stimulatory leukocytes are generally between 1: 1 and 100: 1, usually between about 5: 1 and about 25: 1, and typically about 10: 1. However, any number of component cells or other constituents can be used, as long as the vaccine is effective as a whole. This will also depend on the method used to prepare the vaccine, such as whether the allogeneic lymphocytes and autologous leukocytes are co-cultured before administration. The effectiveness of the composition is probably related to the proximity of the stimulated lymphocytes and the tumor antigens. once administered to the subject. While it is more convenient to pre-mix the components prior to administration, it is recognized since a similar effect is potentially achievable by administration separated to approximately the same neighborhood in the subject. Accordingly, the embodiments of this invention include not only the compositions in which the components are prebleached, but also the combined preparations containing stimulated lymphocytes. (such as allogeneic lymphocytes allogeneic to a human patient) and tumor antigen (such as primary tumor cells from the human patient or the progeny thereof) for simultaneous, separate or sequential use in a method for the treatment of a human by surgery or therapy, particularly for the treatment of a tumor in the subject or to elicit an antitumor response.

The pharmaceutical compositions of this invention can be administered after, before or in place of, or in combination with other therapies related to the generation of an immune response or for the treatment of cancer in the subject. For example, the subject may be previously or concurrently treated by chemotherapy, radiation therapy, and other forms of immunotherapy and adoptive transfer. Where such embodiments are used, they are preferably employed in a manner or at a time that does not interfere with the immunogenicity of the compositions of this invention. The subject may also have been administered with another vaccine or other composition in order to stimulate an immune response. Such alternative compositions may include tumor antigen vaccines, nucleic acid vaccines encoding tumor antigens, anti-idio type vaccines, and other types of cellular vaccines, including tumor cell lines expressing cytokine. Certain embodiments of this invention relate to combination therapies, which comprise the administration of a combination of cellular vaccine described herein in conjunction with another strategy directed toward providing an anti-tumor immune response. In a preferred combination therapy, the subject is administered an intratumoral implant of stimulated allogeneic lymphocytes, either before, during or after treatment at a site distant from the tumor with a composition comprising stimulated allogeneic lymphocytes and autologous tumor cells. In another preferred combination therapy, the subject is treated at sites distant from the tumor with an alternative cellular vaccine composition, either before, during or after treatment with a composition comprising stimulated allogeneic lymphocytes and autologous tumor cells. A preferred alternative composition for this purpose comprises autologous tumor cells mixed with allogeneic cells (particularly tumor cells) that have been genetically altered to express a cytokine at a high level. Where a plurality of different compositions or different administration modalities are employed throughout the course of therapy, the order and timing of each treatment element is chosen to optimize the immune or antitumor effect.

The timing of the administration of the compositions of this invention is within the judgment of the attending physician, and depends on the patient's clinical condition, the objectives of the treatment and the concurrent therapies that are also administered. Typically, at an appropriate time in patient management, a first dose is given, and the patient is periodically checked for either an immunological or clinical response, often both. Suitable means of immunological monitoring include a one-way MLR using the patient's PBL as responders, and primary tumor cells as stimulators. An immunological reaction may also be manifested by a delayed inflammatory response at the site of the injection. Suitable means for periodically verifying the tumor are selected depending on the type of tumor and the characteristics, and may include CT scanning, magnetic resonance imaging (MRI), radiography with a suitable imaging agent, radiography. monitoring of circulating tumor marker antigens, and the subject's clinical response. Additional doses can be given, such as on a monthly or weekly basis, until the desired effect is achieved. After this, and particularly when the immunological or clinical benefit seems to decrease, booster or maintenance doses may be administered, as required. When multiple doses of a cellular vaccine are administered to the same patient, some attention should be paid to the possibility that allogeneic lymphocytes in the vaccine may generate an anti-allotype response. The use of a mixture of allogeneic cells from a plurality of donors, and the use of different populations of allogeneic cells in each dose, are both strategies that can help to minimize the appearance of an anti-allotype response. During the course of therapy, the subject is evaluated on a regular basis for side effects at the site of injection, or general side effects such as a feverish response. Side effects are managed with appropriate clinical supportive care. The examples presented below are provided as an additional guidance to a practitioner of ordinary skill in the art, and are not understood to be limiting in any way.

EXAMPLES EXAMPLE 1: TREATMENT AGAINST CANCER WITH MIXED LYMPHOCYTE IMPLANTS This example describes a study in humans in which allogeneic lymphocytes stimulated in a culture of mixed lymphocytes were implanted in the tumor bed of advanced brain cancer. As a result, the local environment compd stimulated lymphocytes and any residual autologous tumor cells. This treatment was effective in limiting or reversing tumor progression and improving survival in some of the treated subjects. The coincidental observations support the present invention; particularly the apparent active involvement of the antitumor response characteic of the subjects. A clinical trial was performed in patients with high-grade recurrent astrocytomas to evaluate the feasibility, tolerance capacity and toxicities associated with the direct intratumoral implant of the allogeneic lymphocytes activated against the alloantigens of the patient by culture of mixed lymphocytes. The results indicate that the direct intratumoral implant of allogeneic lymphocytes activated by MLC, in patients with recurrent high-grade gliomas, is feasible and safe and seems to provide a clinical benefit. Nine biopsy patients who had high-grade astrocytomas (grade III or IV of Daumas-Duport) were randomly selected for the intratumoral implant of allogeneic lymphocytes activated by MLC after the recurrence or progression of their astrocytomas, after the therapies standards The test was approved by the Institutional Review Board of The Good Samaritan Hospital, Los Angeles, California. All patients were enrolled with consent and informed. The ages of the patients were in the range of 24 to 67 years (average of 50 years) and there were 4 men and 5 women. Eight patients had grade IV astrocytoma (glioblastoma multiforme, GBM), and one patient had grade III astrocytoma (anaplastic astrocytoma).

All patients had failed previous surgery, radiotherapy, chemotherapy and immunotherapy (autologous LAK cells plus IL-2), and had the tumor progressively developing. Kamofsky's performance ratings were in the range of 60 to 80 (mean of 72.6) at the time of immunotherapy. The characteics of the patients are listed in Table 1.

= Right Parietal Lobe; LTL = Left Temporary Lobe; ROL = Right Occipital Lobe; LOL = Left Occipital Lobe. *** DBS = Discharge Surgery; RT = Beam Radiotherapy External; CT = Chemotherapy; Itx = Previous immunotherapy (LAK cells) + IL-2); GK = Gamma Blade Therapy (months before alloimplant). **** KPS = Performance Rating of Kamofsky (at the time of immunotherapy).

The cultivation of mixed lymphocytes on a preparative scale was conducted as follows. Three days before implantation, a genetically unrelated donor was identified and subjected to leukapheresis to obtain the desired number of leukocytes. Leukapheresis for approximately 2.5 hours routinely provides up to 10 x 108 mononuclear cells. At the same time, one unit of the patient's blood was obtained, and the yellow layer was obtained by centrifugation. The mononuclear cells of the donor and the patient were then obtained by centrifugation on gradients of FICOLLMR-Hypaque (density = 1.077). The patient's mononuclear cells were inactivated by treatment with mitomycin-C (MC, Mutamycin) at 10 μg / ml for 1 hour at 37 ° C, and washed to remove excess drug. The mononuclear cells of the donor were then mixed with the mononuclear cells of the patient treated with MC at a ratio of 10: 1 to 20: 1 in AIM V medium (total cell density = 2 x 10 6 cells / ml). The cells were dispersed in plastic culture bags (Baxter), and placed at 37 ° C in a humidified air incubator with 5% C02 / 95% air. After a three-day incubation period, the viable cells were recovered by centrifugation, counted, suspended in 4-5 ml of the patient's sterile plasma, and transported to the operating room. At the time of implantation, calcium gluconate was added to start a clot. The clot was then thinned on a sterile metal plate. The tumor was resected where possible, forming a cavity circumscribed by the tumor bed. The thinned clot containing the stimulated allogeneic lymphocytes was placed inside the cavity, and in or near any remaining tumor. The clinical toxicities associated with the intratumoral implants of the MLC-activated allogenic lymphocytes are documented in Table 2. At each dose level, some patients experienced grade 1 and grade 2 toxicities. However, these were transient effects, and it is not clear if these were effects of immunotherapy or surgical reaction. The degree of cerebral edema at each dose level was controlled by administration of moderate doses of dexamethasone (between 8 and 24 mg / day), which was maintained for up to several months.

The toxicities did not appear to be increased at higher doses of cytoimplant up to 6 x 108 cells. Due to the physical limitations in obtaining the lymphocytes from the donor, the maximum dose implanted did not exceed 6 x 109 cells. Clinical responses were evaluated using three criteria: a) serial MRI scans, using contrast enhancement with triaxial measurements of maximum magnification diameter; b) Kamofsky performance ratings; and c) survival. The tumor volumes of the serial MRI-scans for the 9 patients enrolled in the test are shown in Figure 1. The MRI evidence of the tumor response to the alloimplant (as assessed by TI-weighted MRI images, augmented with gadolinium) was observed in 3 of 9 patients. There was complete regression of the tumor in two patients and partial regression of the tumor (> 50% shrinkage) in a patient in an observation period of 10 to 130 weeks. In five patients, serial MRI scans showed stabilization of tumor size, essentially without tumor growth in an observation period of 8 to 20 weeks.

Only one patient showed progressive tumor growth after alloimplantation. The average complete survival for patients at each dose level measured from the time of immunotherapy was 24 weeks at 2 x 109 cells (range of 18 to 24 weeks), 64 weeks at 4 x 109 cells (range of 10 to 135 weeks), and 72 weeks at 6 x 109 cells (20 to 140 weeks). Importantly, there were two long-term survivors; one at a dose of 4 x 109 cells (BTP-006,> 125 weeks), and one at a dose of 6 x 109 cells (BTP-008,> 135 weeks). The clinical toxicities associated with the intratumoral cytoimplantation are listed in Table 3. The toxicities were graded according to the following criteria: Grade 0 = No headache, no fever, no attacks; Grade 1 = Mild headache; Grade 2 = Headache, mild edema (MRI); Grade 3 = Severe headache, moderate edema (MRI); Grade 4 = severe headache, severe edema (MRI), neurological changes. Grade 3 or Grade 4 irreversible toxicity was dose-limiting.

Each of these patients were classified in their Karmofsky performance ratings from 80 (preimplantation) to 100. Two patients are currently alive and enjoying a good quality of life. Serial MRI scans of patient BTP-006 indicated continuous regression of the tumor over a period of 24 months. Serial MRI scans of patient BTP-008 also indicated a persistent and slow reduction in tumor size over a 24-month observation period. Histology of the implant site was determined after an autopsy performed on a patient who died 60 days after implantation. Immunohistochemistry was performed on tissue sections fixed in 10% neutral buffered formalin. Sections of five μm were prepared on siliconized glass slides and stained with primary antibodies against different cellular antigens using an automated immunotherapy system TECHMATEMR "(Biotech solutions, Inc., Santa Barbara, California) composing the avidin-biotin complex method with DAB As the chromogen, the primary antigens used included anti-CD68 (HAM 56, macrophages), L26 (CD20, B cells), UCHL-1 (T cells) and GFAP (dual cells) After immunostaining, the tissue sections were counterstained with haematoxylin, and examined microscopically for immunopositive cells.The identification of the other inflammatory cell types and the degree of tissue necrosis were determined in parallel by histological criteria using stained sections stained with standard haematoxylin / eosin. the alloimplant was placed, a cystic cavity was found, filled only with fibrin and blood clot organization. Microscopically, there was no evidence of implanted, residual lymphoid cells. However, sections taken from the periphery of the tumor, 1.5 cm from the site of the implant showed massive infiltration of the tumor tissue by CD68 + macrophages and scattered lymphocytes, and evidence of extensive tumor necrosis. Interestingly, numerous CD68 + macrophages (microglia) were identified, apparently migrating from adjacent vessels in the normal brain parenchyma to areas of dead tumor tissue. This study demonstrates that the intratumoral implant of allogeneic lymphocytes activated by MLC is feasible and well tolerated by patients. The toxicities associated with alloimplantation included occasional headache, low grade fever, and cerebral edema, which was controlled by administration of glucocorticoids. Patients who survive long-term remained on steroids for many months after implantation, but were eventually reduced to very low maintenance doses. The procedure was associated with a significant number of responses as determined by the tumor responses noted in serial MRI scans and, more importantly, the patient's prolonged survival.

Example 2: GENETIC ALTERATION OF CELLS TO EXPRESS CYTOKINES In certain embodiments of this invention, a cell or other population is genetically altered to express a cytokine at a high level. This level provides two non-limiting illustrations of how cell populations can be genetically altered to express a cytokine. The illustrations make use of the pLXSN plasmid derived from a Maloney murine leukemia virus, or the LNCX retroviral expression vector. Genetically altered cells are produced, which express the desired product in a stable manner, even after cell division or irradiation.

Cell line that secretes IL-4: The line of human ovarian cancer cells was genetically altered to secrete IL-4, using a retroviral vector comprising a construct encoding IL-4. The cell line was stable, and capable of biosynthesizing IL-4 even after a radiation dose of inactivation. The cell line expresses Class I and Her-2 / neu MHC antigens, but not Class II MHC antigens, ICAM-1, CA-125 or IL-4 receptors. The details of the techniques useful for the production of such cell lines are described elsewhere (Santin et al 1995b and c). In summary, the human ovarian cell line UCI-107 was established from a previously untreated patient with a stage III primary papillary ovarian adenocarcinoma. The cell lines UCI-101 and UCI-107 have previously been characterized by Gamboa-Vuj icic and collaborators, and were kindly provided by Dr. Alberto Manetta (University of California, Irvine Medical Center). The cells were maintained at 37 ° C, at 5% C02 in complete medium (CM) containing RPMl 1640 (Gibco Life Technologies), 10% fetal bovine serum (FBS, Ge ini Bioproducts, Calabassas, CA), and 1 percent of penicillin / streptomycin sulfate (Irvine Scientific, Santa Ana, CA). Retroviral vectors were constructed as follows: Plasmid pLXSN was kindly provided by Dr. A. Dusty Miller (Fred Hutchinson Cancer Center, Seattle, WA). This plasmid, derived from a Moloney murine leukemia virus (MMLV) contains the neofosphotransferase gene whose constitutive expression is driven by the SV40 enhancer / promoter, the 5 'retroviral LTR of the integrated vector drives the expression of an inserted gene. The human IL-4 cDNA was obtained from the ATCC in the cloning vector Okaiama and Berg pCD, and was excised using the restriction enzyme BamHI. The cDNA was then cloned into the "restriction site." BamHI in the multiple cloning region of pLSXN The proper orientation of the cDNA was determined by diagnostic restriction endonuclease digestions.Once constructed, the retroviral plasmid DNA was then purified by cesium chloride gradient density centrifugation The purified retroviral plasmid DNA (LXSN / IL-4) was used to transduce the murine esotropic packaging cell line GP-E86 by the calcium phosphate method, the supernatant of forty-eight hours of these cells was then used to infect the murine amphotropic packaging cell line, PA317. The packaging cell line PA-317 was obtained from the ATCC and was maintained in CM.The transduced PA317 cells were selected for resistance to G418. isolated clones were expanded, aliquots were taken and frozen under liquid nitrogen in a master cell bank. The supernatant of a transduced PA317 clone, containing infectious replication incompetent retroviruses, was used to infect the human carcinoma cell lines. Briefly, the human ovarian carcinoma cell lines were seeded in 100 mm tissue culture plates at densities of 1 x 10c cells in 10 ml of CM and incubated for 4 hours at 37 ° C., with 5% C02 to allow adhesion. After incubation, the medium was aspirated and replaced with 5 ml of 2% polybrene in phosphate buffered saline (PBS), (Aldrich Chemical Co., Inc., Milwaukee, Wl). After 30 minutes at 37 ° C, 5% C02, 10 ml of the retroviral supernatant was added, and the retrovirus-mediated gene transfer was achieved by overnight incubation. The supernatants were then aspirated and replaced with CM. After an additional incubation of 48 hours in CM at 37 ° C, 5% C02, the selection of the transduced clones was achieved by the CM culture containing 0.075% of G418 (geneticin, Gibco Life Technologies). The clones were isolated after 14 days using sterile cloning cylinders of 8 x 8.8 mm (Belco Glass, Inc., Vineland NJ) and expanded for 3 weeks in CM containing G418. The progenitor cell lines were used as positive controls for resistance to G418. After the clonal selection in G418, the transduced cell lines were returned to CM for expansion and study. Cells were established in CM at a density of 0.5 x 10 cells / 10 ml in 100 mm tissue culture plates. Cell counts were conducted every 12, 24, 48, 72 and 96 hours, and the number of viable cells was determined using the trypan blue exclusion. The experiments were conducted to compare the growth of untransduced tumor cell lines (progenitors) and transduced, and to evaluate the level of cytokine production over time. Supernatants were collected and frozen at -20 ° C (for subsequent evaluation by ELISA of cytokine levels) and culture plates were trypsinized to determine cell counts and viability. The progenitor cells, transduced with IL-4 and vector control, were seeded in 100 mm tissue culture plates (Corning) at a density of 1 x lOV cells / ml in 10 ml of CM. After 48 hours of incubation at 37 ° C, and 5% C02, the supernatant was aspirated, freed from the cells by centrifugation at 1,500 rpm for 10 minutes, and then stored at -20 ° C. The concentration of IL-4 was then determined by ELISA, using commercially available equipment (Research &Diagnostic Systems, Minneapolis, Minnesota). Table 4 shows the secretion level of interleukin 4 from individual clones of genetically altered human serous papillary ovarian cancer cells.

As expected, each progenitor line and cells transduced with the vector alone did not produce detectable levels of IL-4. The best IL-4 producing clone, named UCI 107E IL-4 GS, was expanded and used to form a master cell bank for subsequent testing and extensive characterization. The UCI 107 progenitor cell line has the characteristic morphology of ovarian eelial cells developed in vi tro. The morphology of the UCI 107 cells transduced with the LXSN vector alone or LXSN containing the IL-4 gene, was indistinguishable from that of the progenitor cells. It was determined that the doubling time of the control vector, progenitor and the UCI 107E IL-4 GS cells was 15.3, 15.7 and 18.6 hours, respectively. No changes in the rate of development of these cells have been observed in 35 passages and 6 months of culture. The production levels of IL-4 were consistently in the range of 900 to 1300 pg / ml / 10 5 cells / 48 hours during the 6 months of passage. Extensive tests performed on the UCI 107E IL-4 GS cell master bank (MCB) revealed that this line is free of the presence of mycoplasma, bacteria and infectious viruses.The southern analysis was conducted using the NeoR gene to probe the UCI line 107E IL-4 and progenitor line UCI 107. In summary, the concentrated suspensions of tissue culture cells were used in TNE buffer (10 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 7.5) containing 0.5% of SDS, treated with 50 μg / ml proteinase K overnight at 37 ° C, then extracted with phenol and chloroform.The DNA solution was precited in 100% ethanol, wound and resuspended in 10 mm Tris, 0.1 M EDTA (pH 8): 10 Ug of the high molecular weight DNA were digested with Sstl (GIBCO / BRL, Grand Island, New York), separated by electrophoresis on a 0.8% agarose gel and transferred to Gene Screen Plus (Dupont NEN, Boston, Massachusetts) Transfer, hybridization and The washing was performed according to the manufacturer's specifications. The random primer IL-4 probe was prepared by the method of Tabor and Struhl (1988) in Current Protocols in Molecular Biology Vol. 2.2.1-2.2.3. The results confirmed that after 20 passes, ICU 107E IL-4 still contained the vector DNA. The stability of IL-4 secretion after irradiation was tested as follows: cells were irradiated in a 15 ml conical tube in CM at room temperature with gamma rays (137Cs) at a dose rate of 200 rads / minute. Immediately after irradiation, the cells were seeded in a Petri dish culture plate at a density of 1 x 10 cells in 10 ml of CM Test doses of 1,000 to 10,000 rads were applied Irradiated cells were cultured at 37 ° C in an atmosphere with 5% C02 and the medium was completely changed every four days in all boxes.

Every 48 hours, the culture supernatants were collected from the box for cytokine production and the number of viable cells was evaluated by light microscopy by means of trypan blue exclusion. The results of this experiment are shown in Figure 2. Irradiated cells with between 2,500 and 10,000 rads remained viable for approximately 8 days, but all cells died at 3 weeks. The cells irradiated with 1,000 rads recovered and continued to proliferate. The levels of cytokine production were detectable for 8 days at all doses and were closely parallel to the number of viable cells. Panel B shows the production of IL-4 after irradiation at 5,000 rad (D) or 10,000 rad (_-) in three separate experiments. Panel C shows the production of IL-4 standardized in pg / ml / 10 5 cells / 48 hours by UCI 107E IL-4 GS cells after irradiation at 5,000 or 10,000 rads in two separate experiments. There were no statistically significant differences in survival between irradiated cells with 2,500, 5,000 and 10,000 rads on days 2 (p = 0.72), 4 (p = 0.14), 6 (p = 0.10), and 8 (p = 0.3). ).

Collectively, these results indicate that the UCI 107E IL-4 GS cells constitute a stable cell line of IL-4 secretion. The cells can be irradiated to stop replication effectively, maintaining a production of IL-4 for up to 1 week.

Cell line that secretes TNF-a: A human TNF-a coding sequence was used to genetically alter a line of rat glioblastoma, and it is shown to confer protection against several different glioma lines. Graf et al. (1994) Soc. "Neuroscience (extract) In summary, the TNF-a coding sequence was inserted into a T9 glioblastoma cell line of Fischer rat, insensitive to TNF by retrovirus-mediated gene transduction, using a LNCX retroviral expression vector A clone designated T9 / LNCT2 secreting biologically active TNF was isolated at a level of 2,000 pg / 10 6 cells / 48 hours The rates or rates of growth of the transduced cells, the T9 progenitor cells, and cells transduced with the vector alone (designated T9 / LNCX) were identical.The T9 / LNCT2 line has been maintained for a full year without loss of TNF secretion capacity.When the progenitor cells and T9 control vector were injected subcutaneously in On the flank of Fischer rats, tumors were established and developed to kill animals, In contrast, T9 / LNCT2 cells injected subcutaneously. they developed initially, but returned in 40-50% of the animals in 3 to 4 weeks. TNF was secreted by subcutaneous T9 / LNCT2 cells during this period. The survivors were totally resistant to the new intracranial challenge with T9 progenitor, even after 1 year. In addition, these animals were also resistant to challenge with the syngeneic glioma cell line, 9L.

EXAMPLE 3: RESISTANCE TO THE CHALLENGE AFTER TREATMENT WITH A CYTOIMPLANT A rat model for the treatment of cancer was developed by injecting the metastatic breast carcinoma cell line MADB 106 L_1 into the middle lobe of the liver of the Fischer 344 rat strain born to consanguineous parents. The tumors were measured regularly, and they were established for 14 days before treatment. Mixed lymphocyte cultures were prepared using Fischer 344 inactivated rat stimulatory lymphocytes and Wistar-Furth rat (W / F) allogeneic responder lymphocytes at a ratio of 1: 1. Three days after the start of the culture, 80 x 106 cells were injected directly into the established tumors, progressively in development. Groups of 5 rats were treated as follows: Group 1 did not receive injection; Group 2 was injected with non-sensitized W / F allogeneic lymphocytes directly into the tumor; Group 3 was similarly injected with the cells obtained from the reaction of mixed lymphocytes. The effect of treatment with MLC on survival is shown in Table 5: The group treated with MLC was the only group with long-term survivors. The survivors were resistant to the new challenge with the progenitor tumor cells. It was concluded that the direct intratumoral implant of the allogeneic lymphocytes stimulated via a culture of mixed lymphocytes confers a significant survival advantage. The effect can be mediated through the immune activation of the antitumor immunity of the host to the activated lymphocytes and the production of cytokine in the local microenvironment of the tumor.

EXAMPLE 4: CELL VACCINATION IN TUMOR DISTANT SITES This example establishes the use of a cellular vaccine comprising stimulated allogeneic lymphocytes blended with syngeneic tumor cells in a mouse model, and identifies an effective amount in the generation of an immune response and confers resistance to the tumor. The general materials and procedures are as follows. Approximately 1 to 2 x 108 splenic spleen cells from a single mouse are typically recovered. Splenic responsive and stimulating cells are mixed and cultured in 1 ml of RPMI medium supplemented with 10% fetal calf serum and 1 mM BME in a C02 incubator at 37 ° C for 3 days. Culture supernatants are analyzed by ELISA for IL-2, TNF, IFN-α. and IL-4. CD cell surface markers in cultured cells are determined by flow cytometric analysis. The cells are also checked periodically by morphological criteria to determine the number of small lymphocytes, blasts, and apoptotic bodies. The required number of cells from the mixed lymphocyte culture are combined with tumor cells in phosphate buffered saline at a final injection volume of approximately 100 μl. the composition is administered by subcutaneous injection in the right flank. The site of the injection is examined for signs of inflammation, and the spleen cells are collected periodically for the determination of immunological criteria. The biopsy and autopsy samples are examined by standard morphological criteria and by immunohistology. J588L is a line of plasmacytoma cells derived from a spontaneous tumor in a Balb / c mouse. Subcutaneous injections of 106 viable J588L cells form palpable tumors greater than 5 mm in diameter in 100% of the histocompatible mice treated within approximately 12 days, accompanied by cell necrosis. In experiments of this type, the mice are sacrificed after the tumors reach approximately 10 mm in diameter. In one experiment, Balb / c mice were injected subcutaneously with 106 J588L plasmacytoma cells mixed with 106 cytoimplant cells. Cells for cytoimplantation were generated by co-culturing C57BL / 6 splenocytes with Balb / c splenocytes at a ratio of 10: 1 for 3 days. Tumor growth at the site of injection was measured in mice treated with the cell mixture, and compared to that of mice injected with 10"J588L cells mixed with 106 C57BL / 6 splenocytes alone, as a control. The control group had tumors at least 1 cm in diameter after 14 days, however, 11 of 15 mice in the first group had no tumor growth, the other 4 mice had tumors that developed slower than in the controls. surviving mice in this group were subsequently challenged with an additional bolus of J588L cells on the opposite flank to determine if there was a progressive systemic immune response against the tumor, Seven of the 10 mice were resistant to the new challenge, showing no tumor growth, or limited growth followed by regression The additional characterization of the interrelation between the cells of the cytoimplant and the response obtained da, is performed by testing the combinations shown in Table 6. cultured cells are mixed with 106 J588L cells and injected into Balb / c mice, and their response is checked periodically as described above. Surviving mice are tested for progressive immunity and tumor resistance by a subsequent challenge with 106 J588L cells alone.

A second group of experiments is directed toward determining the benefit of including modulators in the vaccine composition. The helper T cells can be functionally divided into two subgroups. 'TH1 cells that can be elaborated in the presence of IL-2, IFN or IL-12, and that are believed to favor cytotoxicity in vi vo. TH2 cells can be elaborated in the presence of IL-4, IL-5 or IL-10, and are believed to favor the secretion of B cells. TH1 cells may predominate during typical mixed lymphocyte cultures in vi tro, due to in the presence of IL-2. TH2 cells may play a role in strong antitumor immunity through the production of IgE and the involvement of tumor cytolysis mediated by eosinophils. Experiments are conducted in which the culture of mixed lymphocytes used to provide the stimulated allogeneic lymphocytes of the vaccine composition is supplemented with relevant modulators, particularly IL-2, IL-4 or prednisone. The level of modulators is first tested in the ranges used by Piccinni et al. (1995) J. Immunol. 155: 128 ff. And Spits et al. (1988) J. Immun ol. 141: 29-36. Splenocytes C57BL / 6 with Balb / c splenocytes are co-cultured at a 10: 1 ratio in medium supplemented with each test mediator, starting on day 0 of the culture. IL-2 is expected to improve the proportion of TH1 cells, while IL-4 or prednisone is expected to increase the proportion of TH2 cells, compared to unsupplemented cultures. The characteristics of each culture are determined by measuring cytokine levels in the supernatant by immunoassay or bioassay; for example, IL-2 (secreted by TH1), IL-4, IL-5 (secreted by TH2), IFN-? or TNF-a. The characteristics of each culture are also determined by the cell surface markers by flow cytometry: for example, CD45RB (higher on TH1 than TH2); CD69, and the IL-2 receptor (elevated in activated cells). The protection experiments are conducted as follows: 10"cultured cells are mixed with 10 6 J588L cells and injected into Balb / c mice, and their response is checked periodically as described above.The surviving mice are tested for progressive immunity and Tumor resistance by subsequent challenge with 10"J588L cells alone. The specificity of the immune response is tested by challenging the immune mice with unrelated tumor lines. The effect of vaccines made from cultures enriched for TH1 cells is compared to the vaccine made from cultures enriched with TH2 cells. Further experiments are conducted in which the animals are treated with a composition comprising combined cells from both types of cultures, together with the plasmacytoma cells J588L.

EXAMPLE 5: MEASUREMENT OF THE DEGREE OF ALOACTIVATION In order to ensure the production of high quality, effective MLC cells, a method for measuring the potency of the alloactivated cells can be employed. Only cell cultures with activity above and above the unstimulated control cells should be used clinically. It is beneficial to compare the activity to the unstimulated control, since the baseline activity of mononuclear cells from different individuals varies widely. Various methods are available to measure the activation of lymphocytes. Compared to unstimulated mononuclear cells, the alloactivated cells reduce more formazan dye and have more esterase activity. The production of XTT (a formazan dye) can be easily demonstrated in a 96-well plate by colorimetric spectrophotometry at 470 nm (reference 650 nm). The activated cells typically show higher absorbance than the controls. Activation of lymphocytes can also be demonstrated by the flow cytometric determination of esterase activity using the esterase substrate, fluorescein diacetate (FDA). T cells with high esterase grade are not determined using FDA and a CD3 antibody labeled with phycoerythrin. Esterase activity can be accurately measured in a plate assay by using higher concentrations of ADF and determination of esterase activity by spectrophotometry at 494 nm (reference 650 nm) in a 96-well plate format . The antecedent esterase activity, inherent in serum-containing media, is inhibited by the addition of a competitive esterase inhibitor (approximately 10 mM), the arginine methyl ester. For the most part, these measurements show good correlation with one another and with blastogenesis.

I: Formazan MTT Reduction Test This assay is used to enumerate living cells by the ability for the culture sample to reduce MTT to the blue-green formazan dye, and is also helpful in distinguishing activated from inactive cells. This can be used for virtually any cell in virtually any medium. The useful cell range is between 105 and 5 x 10c per ml.

Rea c ti vos: • 96 well plates, flat bottomed (not ELISA plates) • 5 mg / ml MTT (Sigma) in PBS (frozen) • 20% SDS in 45% DMF, 0.2 N HCl preheated to 37 ° C) Procedure: Place 100 μl of culture medium with cells in the 96-well plate in duplicate or in triplicate. Use 100 μl of medium only for the controls. Leave the first column for the target.

Add 10 μl of MTT to each well. Hit to mix. Cover the plate and incubate at 37 ° C for 4 hours. Add 50 μl of the SDS solution, avoiding bubbles. Hit to mix. If bubbles are present, blow on the surface. Count the plate at 570 nm (reference 650 nm).

II: Formazan XTT Reduction Test This assay is used to enumerate living cells by the ability of the sample culture to reduce XTT to the red-orange formazan dye, and is also helpful in distinguishing activated from inactive cells. This can be used for virtually any cell in virtually any medium. The useful cell interval is between 105 and 5 x 10 * per ml.

Reagents: • 96 well, flat bottom plates (not ELISA plates) • 1 mg / ml MTT (2,3-bis- (2-methoxy-4-nitro-5-sulfo-phenyl-2H- salt) tetrasolium-5-carboxanilide), Sigma) in PBS (fresh) • 1.53 mg / ml PMS (phenylmetanesulfonyl fluoride, Sigma) in PBS (frozen, protected from light) Procedure: Place 100 μl of culture medium with cells in the 96-well plate in duplicate or triplicate. Use 100 μl of medium only for the controls. Leave the first column for the target. Premix the PMS with XTT immediately before use (5 μl per ml of XTT). Add 50 μl of XTT to each well. Hit the plate to mix. Cover the plate and incubate at 37 ° C for 4 hours. Count the plate at 470 nm (reference 650 nm).

III: Flow Cytometry for CD3 / CD69 or CD3 / FDA This is a measurement of the activation of the T lymphocytes after the alloactivation of mixed lymphocytes. Activities such as CD69 expression or esterase activity correlate with cytokine secretion and can be used as surrogate measures of lymphocyte activity. Unstimulated lymphocytes do not express surface CD69 and have only low levels of non-specific esterases. Once activated by allo-antigen or non-specific mitogens, the expression of CD69 appears within 48 hours (peak at 24). Esterase activity increases shortly after stimulation, and continues for several days. Not all alloestimulated lymphocyte reactions proceed with the same kinetics, and it is preferable to measure activation on day 1, 2 and 3 of the culture.

Sample: Test samples from donor and patient cells are mixed in small cultures of 0.5 x 10 cells / ml in 2% FCS-RPMI. These cultures are maintained at 37 ° C in an incubator with 5% C02 until the test.

Reactives: • Monoclonal antibodies • CD3-PE (Coulter) • CD69-FITC (Becton-Dickinson). Keep refrigerated when not in use and protect it from light. • Fluorescein diacetate (Sigma): a stock solution is prepared at lOmg / ml in DMSO, protected from light, and stored in batch frozen test aliquots. Prepare the working solution weekly by diluting the reserve solution 1: 100 in DMSO, keep the working solution refrigerated and protect it from light. • D-PBS, 0.5% paraformaldehyde-0.05% TRIT0NMR X-100 in PBS Procedure: Samples of unstimulated and activated mononuclear cells, of internal control, are produced on a basis as necessary. The large batches of test by lot will be frozen in aliquots of 250 μl in 10% DMSO freezing medium. Mononuclear cells from a normal donor can be used to produce activated control specimens. These cells are placed in two portions of FCS-RPMI at 0.5 x 10 b cells / ml up to 100 ml. The cells were cultured for 2 days at 37 ° C in the presence or absence of 2 μg / ml of PHA lectin, or mixed at a ratio of 10: 1 with a second population of the donor. The cells are harvested by centrifugation at 350 X g for 5 minutes. The medium is removed and replaced with 1/10 of the volume of freezing medium DMSO, and frozen. When necessary, the unstimulated and stimulated control cells can be rapidly frozen and resuspended to the original volume by the addition of 9 volumes of PBS. The control cells are analyzed according to the protocol described below, together with the samples from the test culture. The duplicate use of the control specimens is an addition to the measure of quality assurance. The percentage of CD69 or esterase-positive lymphocytes should be between a variance of 5%. Dilute 5 μl of the CD3-PE antibody (per sample) in 0.5 ml of PBS (per sample). Add either 10 μl of CD69 (per sample) or 1 μl of FDA working solution (per sample). To labeled polystyrene tubes, 12 x 75 mm, 0.5 ml of the diluted antibody is added. Add 100 μl of the perfectly mixed sample to each tube, including the reference controls, the unstimulated donor cells and the alloactivated cells. Swirl gently and incubate for 30 minutes at room temperature. Add 0.5 ml of 0.5% paraformaldehyde-0.05% TRITONMR X-100 PBS and mix. Counting is performed on an appropriately equipped flow cytometer, such as the EPICS XL flow cytometer from Coulter. Histogram 1 (forward scatter versus CD3) of any protocol must have a generous entry around the CD3 + mononuclear cells. Region A should approximate the% of T lymphocytes and should be passed to Histogram 2. In Histogram 2, the use of lateral scatter versus CD3 allows for the discrimination of lymphocytes (low lateral scattering level) from unused RBSs ,. RBC ghosts, platelet aggregates, residual granulocytes and / or other debris. An entry is drawn around the lymphocytes (see Histogram 2 for example). This second entry is passed to Histogram 3, where the CD3 + CE69 + cells or the CD3 + FDA + cells are shown. Control values are run first to adjust the inputs (unstimulated controls). The statistical cursor of the Histogram 3 quadrant is placed, so that the high values of CD69 or FDA (Quadrant 2) are 2%. Leave this entry adjusted when analyzing stimulated samples. At least 5,000 cells are counted with entry for each sample, to obtain a confidence interval of 97%.

IV: FDA Plate Test This assay is used to enumerate living cells by the ability for the culture sample to produce the esterase substrate, fluorescein diacetate, and is also helpful in distinguishing activated from non-activated cells. This test can be used for virtually any medium. The useful cell range is between 105 and 5 x 106 per ml.

Reactives: • 96-well, flat-bottom plates (not ELISA plates) • 10 mg / ml of FDA (Sigma) in DMSO (store, protect from light) • 10 mg / ml arginine methyl ester (Sigma) ) at DMSO Procedure: Place 100 μi of culture medium with cells in the 96-well plate in duplicate or triplicate. Use 100 μl of medium alone, for the controls.

Prepare a fresh working solution of FDA by adding 10 μl per ml of PBS from the reserve FDA plus 50 μl of AME reserve per ml. Add 20 μl of FDA working solution to each well. Hit the plate to mix. Cover the plate and incubate at 37 ° C for 1 hour. Count the plate at 494 nm (reference 650 nm).

V: Acid Production Test This test is used to quantify the relative production of organic acid in crops. This correlates with the activation state of the cells. This assay requires the use of medium containing no more than 2% serum. The practical cell interval is 1-5 x 10 cells / ml incubated 24 to 48 hours.

Reacti vos: • 96 well plates, flat bottom (no plates ELISA) • Acid Analysis Reagent. It is prepared in bulk and stored at 4 ° C. Add 0.1 mg / ml bromophenol blue in distilled water. Add enough concentrated HCl until the appropriate titration point is reached. The titration is carried out until the yellow-green color is obtained after the addition of 75 μl of the reagent to 100 μl of RPMl with 2% FCS in a well of a 96-well plate.

Procedure: Place 100 μl of culture medium with cells in the 96-well plate in duplicate or triplicate. Use 100 μl of the medium alone, for the controls. Add 75 μl of reagent to each well.

Hit the plate to mix. Count the plate at 470 nm (reference 650 nm).

VI: Quantification of Blastogenesis This assay is used to quantify the absolute number of lymphoblasts produced in the cultures after 7 days. This assay can be used for peripheral blood mononuclear cells in virtually any medium. The useful cell interval is between 1 x 105 and 5 x 106 per ml.

Reactives: • Wright staining or Diff-Quick staining Procedure: Place 1 to 2 drops of a 7-day culture in a Cytospin chamber and perform Cytospin (cell centrifugation). Stain the glass slide dry with either Wright's Stain or Stain Diff-Quick. The number of lymphoblasts and the other cells are counted under the 100X lens of the microscope with immersion oil. Count above a total of 300 cells.

EXAMPLE 6: ADDITIONAL EXPERIMENTS IN ANIMAL MODEL Efficacy of Alloactivated Cells Prepared Using Third-Party Stimulators Cell compositions were prepared, composed either of non-stimulated allogeneic cells alone, alloactivated syngeneic cells, non-activated allogeneic cells or alloactivated allogeneic cells (two separate allogeneic cells) or allogeneic cells all activated (two separate allogeneic donors). Splenocytes from mice were used to produce the cells alloactivated by culture at a ratio of responder cells: stimulators 10: 1. Combinations of splenocytes were cultured in RPMl plus 10% fetal calf serum (FCS) supplemented with penicillin-streptomycin at 3 x 106 / ml at 37 ° C for 3 days. 1 x 10 6 live J588L lymphoma cells were then mixed with 10 x 10 6 cultured mouse splenocytes, and then injected into the subcutaneous tissue on the right flank of the Balb / c mice. The treated mice were observed for tumor growth for 3 weeks. Mice without tumor were challenged again 1 month later with 1 x 106 live lymphoma cells alone, by subcutaneous injections on the left flank, and were observed for tumor growth. Figure 3 shows the results of these experiments. The presence of activated allogenic cells correlates with an antitumour response of the host, in vi ve, subsequent. Cell populations prepared using two allogeneic donors to the treated animal could be used in place of syngeneic or autologous cells, in order to induce an antitumor response. However, not all combinations of donor cell populations: activated allogeneic donors were equally effective.

Effect of Proportion of Stimulating Responding Cells on Efficiency Cell populations composed of activated allogeneic cells were prepared by a variable number of syngeneic stimulating cells, using C57 splenocytes as the responder and Balb / c splenocytes as the stimulator. The cells were mixed with live lymphoma cells (J588L cells) and injected into the flanks of Balb / c mice. The treated mice were observed for tumor growth for 3 weeks. Figure 4 shows the percentage of mice without tumors after challenge with the primary tumor (6 mice per group). A lower cellular proportion may sometimes be better to induce an antitumor response in mice.

Impact of the Use of Splenosites from Tumor-bearing Mice on the Antitumor Effect Splenocytes were taken from healthy C57 or Balb / c mice or from a mouse that had a 1-cm lymphoma on the right flank. The cells were cultured for 3 days either alone or after mixing with Balb / c cells at a 10: 1 ratio at a concentration of 0.5 x 106 cells / ml in RPMI-10% FCS. The activation of the lymphocytes was judged by the analysis of the percentage of population high in CD3 + / esterase by Flow Cytometry. The percent of cells positive to FDA was approximately 3.5% using sterilizers from healthy Balb / c donors, but only approximately 2.5% using stimulators from donors that possess tumors. Cell populations alloactivated with stimulators from either healthy Balb / c mice or from mice bearing J588L tumors were mixed with live lymphoma cells (J588L cells), and injected into the flanks of healthy Balb / c mice (intact) The mice were checked periodically for tumor growth for 3 weeks. Mice without tumors were then challenged again with 1 × 10 6 live lymphoma cells alone on the left flank, and were observed for tumor growth. The percentage of mice without tumors after the secondary challenge with the tumor was between 30 and 40% in both groups.

Resistance of Immunized Mice with Aloactivated Lymphocytes and Irradiated Tumor Cells to the Subsequent Challenge with Tumor This experiment tested the immunogenic spectrum of a cellular vaccine containing alloactivated lymphocytes mixed with inactivated tumor cells. C57 / BL6 mice (3 per group) were injected subcutaneously with 10 irradiated B16 melanoma cells, alone, mixed with 10 7 alloactivated Balb / cx C57 lymphocytes, or mixed with 10 6 cells of J588L lymphoma secreting IL-4 (allogeneic to C57) . The alloactivated cells were prepared by culturing Balb / c splenocytes with C57 splenocytes at a ratio of 10: 1 to 3 x 10 ml in RPMl, 10% FCS for 3 days. The cells were washed in PBS, and injected subcutaneously into the flanks of healthy C57 mice. After 3 weeks, the mice were challenged again with 5 x 10"live B16 melanoma cells, subcutaneously on the opposite flank." The mice were observed for tumor formation and sacrificed after the tumors reached 1 cm in diameter.

Mice treated with the alloactivated cells survived significantly longer than the other groups. The two longest survival mice eventually developed cone-shaped tumors, both ulcerated. No other mouse develops ulcers. Two days after the ulcers appeared, both mice died. The necropsy of these mice revealed the presence of extremely necrotic tumor cells, with evidence of cellular lysis by recent tumor in the form of massive DNA deposition. This necrosis was accompanied by an inflammatory infiltrate, consisting mainly of lymphocytes. No other form of infection was observed anywhere else in the body. No pulmonary metastases were observed. This is in contrast to the large number of lung metastases in control mice harboring flank B16 melanoma tumors. The bilateral kidneys in both mice showed extensive erulonephritis, suggesting death from the tumor lysis syndrome. No other mice showed these changes. These results are consistent with the mice treated with the alloactivated cell vaccine, which develops a specific response that caused massive lysis of the live cancer cells administered in the subsequent challenge. In yet another experiment using a different tumor model, C57 / BL6 mice (3 per group) were injected subcutaneously with 10 6 Lewis lung carcinoma cells alone, mixed with 10 7 Balb / cx C57 alloactivated lymphocyte cells, or mixed with 10 cells of J588L lymphoma secreting IL-4 (allogeneic for C57). The alloactivated cells were prepared by culturing Balb / c splenocytes with C57 splenocytes at a ratio of 10: 1 to 3 x 106 / ml in RPMl, 10% FCS for 3 days. All cells were washed in PBS and injected subcutaneously into the flanks of healthy (intact) C57 mice. The mice were observed for tumor formation, and were sacrificed after the tumors reached 1 cm in diameter. Mice treated with cells that secrete IL-4 survived significantly longer than the other groups with 2 out of 3 long-term survivors. The group treated with alloactivated cells alone had no long-term survivors.

Correlation of Functional Markers with Antitumor Effect To determine the correlation between the results of the in vi tro functional assay and the potential therapeutic benefit, cultures showing varying degrees of activation are tested., in the mouse lymphoma treatment model. Mixed lymphocyte cultures are established using splenocytes from a variety of strains of mice born to consanguineous parents, to a proportion of responding cells: they are 10: 1 stimulators. Alternatively, cultures are established using a particular combination of responder strain: stimulator, but at different cell proportions. After 3 days of culture, the activity is measured in the Formazan XTT assay and the esterase assay. Just before injection, cultured cells are supplemented with additional splenocytes, as necessary, to normalize the cell ratio, and mixed with 1 x 10b live or irradiated J588L lymphoma cells. The preparation is injected into Balb / c mice, and the effect on survival is checked periodically. Mice can be challenged again with a subsequent dose of live lymphoma cells to test a persistent immune response. The survival data are then correlated with the functional activity measured during the culture period.

Effect of the Composition of Aloactivated Cells on the Antitumor Effect As described elsewhere in this description, histamine impairs alloactivation during lymphocyte culture, as measured in functional assays. Cimetidine, which is an H2 receptor antagonist, promotes alloactivation. In this study, alloactivation cultures are prepared in the presence or absence of 20 μg / ml of histidine or cimetidine, tested in Formazan XTT and esterase assays, and then injected into Balb / c mice with J588L lymphoma cells to correlate with effectiveness. In yet another study, the effect of having a plurality of different stimulating or responding cells is tested. Standard cultures containing C57: Balb / c splenocytes (10: 1) are compared for efficacy in the mouse lymphoma model with cultures containing: a) C57 splenocytes: Aj: Balb / c (9: 1: 1 or 5: 5: 1); b) C57 splenocytes: Aj: C3H (9: 1: 1 or 5: 5: 1); splenocytes C57: Aj: C3H: Balb / c (8: 1: 1: 1 or 3: 3: 3: 1).

EXAMPLE 7: EXPERIMENTS WITH CULTIVATED HUMAN CELLS Criteria for the Functionality of Aloactivated Cells The degree of alloactivation (a potential reflection of potency in therapy) can be measured according to the functional tests detailed in Example 5. This example illustrates the degree of activation revealed by the assays. Monocytes from human peripheral blood were isolated from samples taken from a number of unrelated human volunteers, and were established in mixed single-pathway lymphocyte cultures, at a responder: stimulator ratio of 10: 1, as further described in this description. The tests were run after 2 to 3 days in culture. The results are shown in Figures 5 and 6. Each of the individuals is indicated by a single letter, with the responding cells that are indicated before the stimulator cells. Thus, the designation A x B means that the cells from individual A were cultured with the inactivated cells from the individual B. Compared with the unstimulated mononuclear cells, the alloactivated cells have more esterase activity and further reduce the XTT (a formazan dye). The esterase activity can also be measured by flow cytometry using the esterase substrate, fluorescein diacetate (FDA). T cells with high esterase activity can be identified by the CD3 antibody labeled with phycoerythrin in conjunction with FDA. These measurements correlate well with blastogenesis (determined after culture for a week), or the level of IL-2 or IFN-? in the supernatant.

Impact of Using Multiple Allogenic Stimulatory Cells Alloactivated human lymphocyte cultures were produced using cells from one, two, three or four unrelated donors. 3 x 10 'cells / ml were cultured in 2% FCS-RPMI at 37 ° C for 2 days. Populations of two donors were produced by mixing the responder cells with the stimulator cells at a ratio of 10: 1. Populations containing cells of three or four donors were produced by mixing the responder cells with two or three different stimulator cells at ratios of 9: 1: 1 or 8: 1: 1: 1. Figure 7 shows the characteristics of the cells measured using flow cytometry. All values represent the percentage of brightly fluorescent cells after counting 4000 cells in a Coulter EPICS XL Citometer. The results show that cultures prepared with stimulators from a plurality of donors under certain conditions achieve higher levels of activation.

Impact of the Alteration of the Proportion of Responding Cells: Stimulatory Cultures of mixed lymphocytes composed of mononuclear cells of human peripheral blood, alloactivated, were produced using cells from the same two unrelated donors at proportions of 10: 1, 5: 1 or 1: 1. The cells were cultured at 0.5 x 10 6 cells / ml in 2% FCS-RPMI for 3 days. The strength of these cultures was measured using the Formazan XTT reduction assay. The results are shown in Figure 8.

Impact of Histamine or Cimetidine on Aloactivation It is known histamine induces the activity of suppressor T cells. Since suppressor T cells may play a role in the control of MLR activity, the effect of histamine and a potent drug receptor blocker (H2) of histamine type 2, the cimetidine. Cell populations composed of alloactivated human peripheral blood mononuclear cells were produced using cells from unrelated donors. All cultures contain a 10: 1 ratio of responder mononuclear cells: stimulators at 0.5 x 106 cells / ml. In some cultures, 20 μg / ml histamine or 20 μg / ml Cimetidine was added on day 0. Figure 9 shows the results measured using a formazan reduction medium (XTT). The suppression induced by histamine and the decreased strength of alloactivation. Cimetidine increased activity, possibly by blocking the development of suppression.

EXAMPLE 8: CLINICAL APPLICATION OF THE CELLULAR VACCINE This section provides an example shows an immunological composition comprising activated lymphocytes and autologous cancer cells administered subcutaneously is successful in stimulating an immune response in a primed human container. The JT patient with cancer had an aggressive multiform glioblastoma which had progressed through traditional forms of cancer therapy. This was treated with intratumoral implants according to Example 1 in August and September 1995. Studies were conducted thereafter to determine whether JT had developed tumor-specific detectable immunity. First, a stable cell line was established from its tumor. Viable tumor cells from a surgically removed tumor were transferred to the laboratory and cultured in RPMI-1640 medium containing 10% fetal calf serum (FCS). The cell line was designated PGA-95. Viable tumor cells were also recovered from the subsequently removed occipital lobe, and used to generate a second cell line, designated PGA-96. The two cell lines have similar if not identical characteristics. The MLTC (mixed tumor-lymphocyte cell culture) was performed as follows: peripheral blood lymphocytes (PBL) had been collected and cryopreserved at the time of cytoimplantation in August were cultured with viable, developing PGA-95 cells. , to various proportions in RPMI-1640/10% FCS for up to 8 days. No antitumor activity was detected. However, when the PBL obtained from the patient in January 1996 were cultured with PGA-95 cells in an identical manner, a strong activity of antitumor cells was noted. Virtually 100% of the tumor cells were killed for 7 to 8 days of culture at a ratio of PB: PGA-95 of 100: 1, as determined by crystal violet staining of adherent tumor cells at the end of the assay. Similar results were obtained at a ratio of 50: 1, and a death of approximately 50% of the tumor cells was observed at 10: 1. Importantly, death of PGA-95 cells did not occur when unrelated PBLs (from third parties) were used in the co-culture. During the first 5 days of MLTC, high levels of IL-2 (1000-1500 pg / ml) were produced and then fell to much lower levels by day 8. No IL-4 was detected during the 8 days of culture. TNF was produced at significant levels only after 6 days, and continued to increase to 400-500 pg / ml by day 8. The phenotype of the responder cells was determined by flow cytometric analysis, and it was found to be a mixture of CD4 + and CD8 + cells. CD4 + cells proliferated early during culture, followed by proliferation of CD8 + cells. The CD8 + cells did not produce IL-2. In consecuense, the culture medium was supplemented with 200 U / ml of recombinant IL-2 (Hoffman-LaRoche), and approximately 1 x 109 CD8 + cytotoxic T lymphocytes (CTL) were generated in approximately 30 days. The specificity of the CTL generated was determined by standard 51Cr release assays. The CTL generated against the PGA-96 cells efficiently killed the PGA-95 and PGA-96 cells (30-50% lysis in 4 to 6 hours), or cryopreserved tumor cells that had not previously been cultured. CTLs did not kill autologous PBLs or autologous fibroblasts; nor did they kill the unrelated cancer cell lines; specifically, glioma cells (U373 or ACBT), prostatic adenocarcinoma cells (LNCAP), bladder cancer cells (BLT-2), ovarian carcinoma cells (ICU-107) or leukemia cells (K562). MLTC was used to periodically check the antitumor, systemic cell immunity in the JT patient as time passed. Beginning in January 1996, blood samples were collected every 2 to 3 weeks. In the MLTC assay, 100% of the tumor cells were killed at proportions of PBL: PGA of 100: 1 and 50: 1, with death proportionally lower at lower proportions. In April, activity dropped dramatically to the point where only 25% of the tumor cells were killed at 100: 1, and none at 50: 1. It was not long after this time that the patient developed a recurrence, requiring an occipital lobectomy. Subsequently, it was decided to try to trigger its systemic antitumor activity by administering a cutaneous immunization consisting of mixed mixed lymphocytes with tumor cells. The cryopreserved cells from the original tumor were inactivated with 10,000 cGy. The cytoimplant cells were prepared by culturing mixed lymphocytes from the patient's stimulator cells, with allogeneic responder cells from a donor, according to Example 1. The vaccine was prepared by mixing 100, 50, 25 or 10 x 108 cytoimplant cells with 1 x 107 irradiated tumor cells. The dose range was extrapolated from the animal experiments described in Example 5. The injections were administered at four different sites on the back. Skin reactions were noted at all four sites, and the patient developed a febrile response: Figure 10 is a reproduction of a photograph taken from the patient's back shortly after the administration of the four injections. Erythema is apparent at each injection site; probably a response to the soluble mediators already present in the cells stimulated in the MLC. Figure 11 is a reproduction of a photograph taken 2 days later, showing the evidence of induration. The marks indicate the measured size of the area involved. This evidence of delayed-type hypersensitivity is particularly important, because it suggests that the lymphocytes and / or cells presenting the antigen have been recruited to the injection site by the stimulated allogeneic cells. The allogeneic cells are expected to stimulate the recruited host cells, which in turn must react against the autologous tumor cells also present at the site. The history of the patient is shown in Table 7: TABLE 7 Date Observation Caucasian woman 7 years old, right-handed /93 glioblastoma multiforme tested by biopsy after recurrent attacks. Partial resection performed 11/93 • The tumor progressed, Partial resection performed 11/93 The tumor progressed. Total resection performed 11/93 Chemotherapy. 2 courses of cyclophosphamide 2/94 External beam radiation therapy 6/94 High dose chemotherapy: cyclophosphamide + melphalan, followed by autologous bone marrow transplantation 8/95 Recurrence of the tumor. Partial resection The MLTC trial performed 12 days after the cutaneous administration of the vaccine showed a dramatic return of the immune response to the levels previously noted in January-March. The MLTC results have continued to approximately this level through a determination made on July 14, 1996. The patient died approximately in September of 1996. Additional tests were conducted to verify the parameters of the vaccination protocol. Patients with astrocytoma III or IV are recruited in the studies conducted under the auspices of the appropriate Institutional Review Board as in Example 1. All patients are enrolled with informed consent, and randomly distributed in the various treatment groups. The tumor cells are cryopreserved from each patient at the time of surgery, and proliferated ex vi ve if necessary to obtain enough cells for the anticipated course of therapy. Thawed or cultured tumor cells are subjected to 10,000 rads of gamma radiation. Mixed lymphocyte cultures on a preparative scale using inactivated stimulator cells from the patient, and donor leukocytes are conducted essentially as described in Example 1. In a test, patients are administered two vaccines with two weeks of separation. The mononuclear cells used to prepare each cell vaccine are obtained from two unrelated healthy donors. The donors were pre-selected to minimize the risk of infectious diseases, and those with positive test were eliminated. The selection includes the test for the antibody specific for HIV-1, HIV-2, HTLV-I, HTLV-II, hepatitis C, or hepatitis B nucleus; HIV antigen, HBsAg, RPR, or elevation in liver markers such as ALT. The typing of HLA-A, -B, -C, and -DR is performed to select the allogeneic donors to the patient and to each other. By using genetically different donors, the likelihood of hyperacute rejection of the second administration is decreased. In addition, each injection is preceded by the major cross-comparison test (donor cells and patient's serum) for the presence of the preformed antibody. The coupling or comparison of blood types is not generally required, except that the administration of cells from an Rh-positive donor to an Rh-negative woman of reproductive age or younger is avoided where possible. The culture of mixed lymphocytes is conducted by mixing peripheral blood mononuclear cells from the donor and the patientand inactivated from the patient, at a ratio of 10: 1, and culturing at 3 x 106 cells / ml in AIMV supplemented with 2% fetal calf serum for 3 days at 37 ° C. The total number of mononuclear cells required for a single inoculum is no greater than 1 x 109. The stimulated cells are harvested and washed by centrifugation, then suspended in sterile, injectable saline. Quality control of activated cell production includes periodic verification of cell counts and viability, testing for mycoplasma and endotoxin, and verification for lymphocyte activation using early activation markers. Before use in the treatment, the preparation of alloactivated cells is also evaluated, according to the criteria of functional release. The Tetrazolium Reduction Assay (XTT) described in Example 5 is conducted on a cell sample. Flow cytometry is conducted to measure cell surface expression of CD69 using the fluorescent antibody; or the increased intracellular esterase activity using fluorescein diacetate. Cultured cells are considered to be sufficiently activated if the level measured in either (but preferably in both) of these assays is greater than or equal to 10% above the control value of the unstimulated donor, on any day of the culture period ( day 1, day 2 or day 3). Once the crop passes the criteria, no proof is needed in the subsequent days. The cells are harvested on day 3, mixed with the required number of primary or cultured tumor cells, and prepared for administration to the human. Patients recruited first in the study receive one of three doses graduated in two separate injections for two weeks (106 MLC cells: 108 tumor cells); Group 1 is administered 1 x 10r MLC cells; Group 2 is administered 5 x 10e MLC cells; Group 3 is administered 1 x 109 MLC cells. The MLC cells are mixed with 1 x 10b and 1 x 107 tumor cells derived from the patient for each inoculum, depending on the number available, and the tendency towards 1 x 107 where possible. Inoculations are administered subcutaneously using a 20-21 gauge needle, 10 cm inferior to the inguinal ligament on the anterior intermediate thigh, on opposite sides. The maximum tolerated dose (MTD) is determined as follows: if at least one of three patients receiving a given cell combination develops Reversible Grade 3 or irreversible Grade 2 toxicity, up to three additional patients are introduced at the same dose. If a second patient develops the same degree of toxicity or higher, the cell combination is defined as the MTD. Otherwise, the doses are raised in scale until the maximum level is reached. The clinical response is checked periodically by various criteria, including local induration, pruritus, or necrosis at the injection site, systemic effects such as fever, malaise, headache, and altered hematologic or renal parameters; Tumor volume detected by criteria such as RMI MRI results are interpreted carefully A growing tumor mass (a characteristic of progressive disease) or local induration by leukocytes (a possible feature of successful treatment) can appear both as an area However, a reduction in the area is consistent with shrinkage of the tumor mass and successful treatment.The presence of a cellular immune response in the treated patient is checked periodically by various criteria. obtained before and after each inoculation are cultured with alog cells irradiated nucleic acids of donor or third-party origin (for anti-allotype response), or irradiated patient tumor cells, or third-party tumor cells (for the specific antitumor response). The response of the lymphocytes of the patient in culture is determined by measurement of proliferation using the reduction of MTT or one of the other functional assays as a substitute marker for cell division. The expression of CD69 is determined by immunofluorocytometry using the antibody labeled with PE. Optionally, responding T cells are co-stained for CD4, CD8 or CD31, to identify subsets of helper or suppressor cells, or for CD45RF to distinguish TH_ cells from TH2 • Cytokines IL-2, IL-4, IFN -? and TNF-α secreted into the culture medium are quantified by ELISA. IL-2 and IFN-? correlate with the activity of TH ?, IL-4 correlates with the activity of TH2 and TNF-a correlates with the activity of both. The patient's PBLs are also tested for their ability to respond to autologous tumor cells, as described earlier in this example. CBLs are cultured in the presence or absence of tumor cells, and then measured for the degree of responsiveness. The general activation of T cells can be measured by the functional assays described in Example 5, the incorporation of tritiated thymidine, or blastogenesis. The activity of cytotoxic T cells can be measured as cytolysis of tumor cells labeled with 51 Cr. The antitumor response of hypersensitivity of delayed effective type (DTH) in the treated patient, is measured by comparing the response at 48 hours of the intradermal administration of 5 x 105 autologous tumor cells, antigens of mumps, tricofiton or PPD , with that observed for the same series before treatment. In a subsequent test, vaccine therapy is combined with implant therapy. At the time of surgical detachment, an implant of allogeneic lymphocytes stimulated with autologous stimulator cells is placed in the tumor bed, as described in Example 1. The removed tumor cells are frozen and / or cultured for the preparation of a cellular vaccine. Two donors are selected who are allogeneic to the patient, and preferably allogeneic to each other and to the cellular donor for the implant. The cells of each donor are used to prepare the MLC component of a vaccine, as described earlier in this example, and then mixed with the patient's tumor cells. A vaccine is given four weeks after the implant; the second at 6 weeks. The selected dose is at or below the MTD established in the preceding test. The clinical and immunological criteria are periodically verified as described above. The response of patients undergoing combination therapy is compared to that of patients receiving an intracranial implant alone, to determine the degree to which prior vaccination improves the effectiveness of the implant. Another study is conducted in patients with stage IV (metastatic) colon cancer. Patients are enrolled in the study under the terms of informed consent, and undergo a standard colectomy. Approximately 1 week later (around the time they are discharged from the hospital), they begin a course of four shots of vaccine. The vaccine composition consists essentially of a population of alloactivated cells, mixed with the tumor cells. Patients receive one of three different doses: 1 x 108 MLC cells; 3 x 108 MLC cells; or 1 x 109 MLC cells, mixed with up to 1 x 10 7 inactivated tumor cells, depending on availability. The same dose is administered four times in a weekly schedule. Initial studies are known mainly to determine the maximum tolerated dose (MTD). Undesirable collateral clinical effects at the site of injection include an unacceptable level of induration, inflammation or ulceration. Once BAT is determined, a comparison is made between the 4-week vaccination schedule alone, and the course of vaccination initiated by the direct implant within a tumor mass. The implant group is treated two days to a week after colectomy, using ultrasound to guide an injection needle into a metastatic tumor mass of appreciable size in the liver. The metastatic site is injected with a preparation of 10 x 109 MLC alloactivated cells alone, suspended in a minimal volume of saline. Beginning one week later, patients in this group also receive the 4-week course of the MLC tumor cell vaccine. Patients are checked periodically for the degree of clinical and immunological response for at least three months after therapy. The immunological criteria are followed as described above, the clinical criteria are followed, in part, by tracking the volume of the tumor metastasis present in the liver, performing a CT scan at regular intervals, calculating the volume of each metastatic site, and the volumes are compared with the measurements obtained before treatment.The progression of the disease is indicated by an increase in the volume of the metastasis, or an increase in the number of metastatic sites.A successful result is indicated by the reversal of the disease, or slower progression compared to the typical outcome for patients with colon cancer of the same degree.

EXAMPLE 9: COMMERCIAL PRODUCTION OF ALLOCATED CELLULAR COMPOSITIONS This protocol describes the complete procedure for the production of mixed lymphocyte culture. The design of this methodology takes into account the Good Manufacturing Practices (GMP) and Good Laboratory Practices (GLP) and complies with the requirements of Code 21 of the Federal Regulations of the United States. At least 2 x 10 9 peripheral blood mononuclear cells are collected from the patient by modified leukapheresis from the patient to be treated. Isolation of the cells is performed on a Baxter Fenwall apheresis machine or equivalent machine using the Procedure for Collecting Totipotential Cells. The cells are shipped in a bag of Baxter-type components on ice (4-10 ° C). The transit temperature is checked periodically using the MONITOR-MARKMR Time / Temperature Labels. At least 10 x 109 peripheral blood mononuclear cells from the donor are collected by modified leukapheresis from a healthy individual. The isolation of the cells is carried out in a Baxter Fenwall apheresis machine or equivalent, using the Procedure for the Collection of Totipotential Cells. The donors are unrelated, anonymous, and random individuals, chosen from a list of preselected potential donors. The preselection of donors should indicate negative risk factors for HIV, Hepatitis, Spongiform Encephalitis or Tuberculosis. Each cell component is tested to be negative for HIV 1/2 antibody (Ab), HIV Ag, CMV Ab, HTLV I / II Ab, HCV Ab, HBc Ab, Ag of HBs and RPR. The cells are shipped in a bag of Baxter-type components on ice (4-10 ° C). After the reception each component is tested for sterility, for the appropriate cellular accounts and for viability. The components are kept at 4-10 ° C until use, and are used or frozen within 72 hours after collection. The frozen material, after being thawed, is used within 2 hours and no longer refreezes. Pre-clinical studies indicate that components stored at 4 ° C in plasma anticoagulated with ACD or material frozen in media containing DMSO are suitable for the production of effective cellular compositions. The plasma is removed from the donor and patient components by centrifugation. The donor plasma can be collected and inactivated by heat for use as a supplement for the medium. The cellular components are suspended in small volumes in PBS and appropriate volumes of each suspension are mixed to produce a culture containing 3 x 10 mononuclear cells / ml in AIMV medium at a ratio of 10: 1 to 20: 1 (donor cells : patient). The donor plasma, inactivated by heat, is added to a final concentration of 2%. The mixed cells are pumped into 3-liter gas-permeable culture bags, Fenwall type, through the use of the Fenwall solution pump and the sterile equipment. Samples of the cellular components can also be established in small culture tubes to test the activation of the lymphocytes. The test of the functional activity is compared with the control cultures that contain the cells of the donor not stimulated, alone.

The cell mixtures are cultured in an incubator at 37 ° C of the humidified ISO-9000 form, filtered with HEPA, with 5% C02 for 3 days, and checked closely. The cells are harvested after cultivation by centrifugation. Samples are taken for quality assurance trials. Each preparation is tested for final sterility, for proper cell counts and for adequate viability and functional activity. Cell preparation is suspended in 25% sterile human albumin, and placed in sterile injectable vials for transport. Each preparation is labeled with an expiration date and time, which is 30 hours after packaging, and is accompanied by appropriate instructions, the results of the release specification, and a MONITOR-MARKMA Time / Temperature Label. Cell preparations are packaged and shipped via overnight mail service. If not used immediately, the cells are stored in a refrigerator at 4-10 ° C. Any preparation not implanted before the expiration date is discarded.

In-process tests that measure the consistency of the product include: • pre-selection infectious disease tests; • sterility tests of the final product and in process; • mycoplasma and endotoxin of the final product; • cellular accounts of the product in process and final; viability of the product in process and final (> 85%).

Cells must also meet satisfactory functional criteria. Preparations that do not meet any of these criteria are not used for the treatment of patients.

TABLE 8: Donor and Patient Selection (At the Time of the Leukapheresis Procedure) The patient can be positive for HBc Ab or CMV Ab, and the components are labeled as such. If the donor components negative for CMV are not available, a positive donor component for CMV Ab can be used, even for CMV negative patients.

TABLE 9: Pre-Process Test of Donor and Patient Mononuclear Cells (At the Time of Reception at the Facility, Prior to Irradiation of the Patient's Mononuclear Cells) TABLE 10: Test in Process of Aloactivated Cells TABLE 11: Final Product Test Although the above invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Therefore, the description and the examples should not be considered as limiting the scope of the invention, which is delineated by the following claims.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (42)

1. An immunogenic composition suitable for administration to a human, characterized in that it comprises an effective combination of: a) allogeneic stimulated lymphocytes for the human; and b) an antigen associated to the tumor, coming from the human.

2. The immunogenic composition according to claim 1, characterized in that the antigen associated to the tumor is comprised in a primary tumor cell of said human, or a progeny of such a tumor cell obtained during the culture of the ex vivo tumor cell.

3. The immunogenic composition according to claim 1, characterized in that the antigen associated to the tumor is comprised in an extract of a primary tumor cell from the human, a progeny of a primary tumor cell from the human, or a combination thereof.

4. The immunogenic composition according to claims 1 to 3, characterized in that the stimulated lymphocytes have been stimulated by culture with allogeneic leukocytes to the lymphocytes.

5. The immunogenic composition according to claims 1 to 3, characterized in that the stimulated lymphocytes have been stimulated by cultivation with a recombinantly produced cytokine, a mitogenic, or with a genetically altered cell to secrete a cytokine at a high level.

6. An immunogenic composition suitable for administration to a human, characterized in that it comprises an effective combination of: a) lymphocytes allogeneic to the human; b) allogeneic leukocytes to lymphocytes; and c) a population of inactivated tumor cells, consisting essentially of primary tumor cells obtained from the human, or the progeny of such cells.

7. The immunogenic composition according to claim 6, characterized in that the leukocytes are autologous to the human.

8. The immunogenic composition according to claim 6, characterized in that the leukocytes are allogeneic to the human.

9. The immunogenic composition according to claim 6, characterized in that it comprises leukocytes from at least three different human donors.

10. The immunogenic composition according to claim 6, characterized in that the population of inactivated tumor cells is selected from the group consisting of melanoma, pancreatic cancer, liver cancer, colon cancer, prostate cancer and cancer cells. of breast.

11. The immunogenic composition according to claim 6, characterized in that the leukocytes are inactivated.

12. The immunogenic composition according to claim 6, characterized in that the lymphocytes comprise a cell that has been genetically altered to express a cytokine at a high level.

13. The immunogenic composition according to claim 6, characterized in that the leukocytes and the lymphocytes are co-cultured for a duration and under conditions sufficient for the allogeneic stimulation of the lymphocytes, before the combination with the population of tumor cells.

14. The immunogenic composition according to claim 6, characterized in that the co-culture is for a duration and under conditions sufficient to stimulate the high secretion of the cytokine by the lymphocytes.

15. A unit dose of the immunogenic composition according to claims 6 to 14, characterized in that the number of allogeneic lymphocytes to the leukocytes in the dose is between approximately 1 x 108 and 2 x 109.

16. A unit dose of the immunogenic composition according to claims 6 to 14, characterized in that the population of inactivated tumor cells in the dose consists of between about 1 x 10 6 and 5 x 10 7 cells.

17. A method for producing the immunogenic composition according to claim 1, characterized in that it comprises the mixing of: a) human allogenic stimulated lymphocytes; with b) the antigen associated with the tumor, coming from the human.

18. A method for producing the immunogenic composition according to claim 1, characterized in that it comprises the mixing of: a) the cells obtained from a co-culture of allogeneic lymphocytes for the human and leukocytes allogeneic to the lymphocytes; with b) primary tumor cells from human, or the progeny thereof.

19. A kit for producing the immunogenic composition according to claim 1, characterized in that it comprises, in separate containers: a) allogeneic stimulated lymphocytes for the human; and b) the antigen associated to the tumor, coming from the human.

20. A kit for producing the immunogenic composition according to claim 1, characterized in that it comprises in separate containers: a) cells obtained from a co-culture of allogeneic lymphocytes "for the human and leukocytes allogeneic to the lymphocytes, and b) primary tumor cells from of the human, or the progeny of them.

21. A method for inducing an anti-tumor immune response in a human, characterized in that it comprises administering an immunogenic amount of the immunogenic composition according to any of claims 1 to 5, to the human.

22. A method for inducing an anti-tumor immune response in a human, characterized in that it comprises administering an immunogenic amount of the immunogenic composition according to any of claims 6 to 14, to the human.

23. A method for inducing an anti-tumor immune response in a human, characterized in that it comprises the steps of: a) ex vivo mixing of a first cell population comprising tumor cells, and a second cell population comprising lymphocytes allogeneic to lymphocytes , to produce a cellular mixture; and b) the administration of an immunogenic amount of the cellular mixture to the human.

24. The method according to claim 23, characterized in that the tumor cells comprise cells selected from the group consisting of melanoma, pancreatic cancer, liver cancer, colon cancer, prostate cancer, and breast cancer cells. .

25. The method according to claim 23, characterized in that the second cell population also comprises allogeneic leukocytes to the lymphocytes.

26. The method according to claim 23, characterized in that the second cell population contains leukocytes from at least three human donors.

27. The method according to claim 23, characterized in that the leukocytes are autologous to the human.

28. The method according to claim 23, characterized in that the leukocytes are allogeneic to the human.

29. The method according to claim 23, characterized in that the immune response is a primary response.

30. The method according to claim 23, characterized in that the immune response is a secondary response.

31. The method according to claim 23, characterized in that the human has been previously treated by the administration of alloactivated allogeneic lymphocytes, within a solid tumor in the human, or in or around a site where a solid tumor or a tumor has been removed. portion thereof.

32. A method for the treatment of a neuroplastic disease in a human, characterized in that it comprises administering an effective amount of the immunogenic composition according to any of claims 1 to 4, to the human.

33. A method for the treatment of a neoplastic disease in a human, characterized in that it comprises the steps of: a) ex vivo mixing of a first cell population comprising tumor cells, and a second cell population comprising lymphocytes allogeneic to lymphocytes, to produce a cellular mixture; and b) the administration of an effective amount of the cellular mixture to the human.

34. The method according to claim 23, characterized in that the second cell population also comprises allogeneic leukocytes to the lymphocytes.

35. A cell population, characterized in that it contains: a) lymphocytes "alloactivated, allogeneic to a human patient, and b) primary tumor cells from the human patient or the progeny thereof, for use in a method for the treatment of a human by surgery or therapy.

36. The cell population according to claim 35, characterized in that the lymphocytes have been alloactivated against leukocytes from the human patient.

37. The cell population according to claim 35, characterized in that the lymphocytes have been alloactivated against allogeneic leukocytes to the human patient.

38. The use of a cell population, which contains: a) alloactivated allogeneic lymphocytes to a human patient; and b) primary tumor cells from the human patient or the progeny thereof; for the manufacture of a medicament for the treatment of the tumor or for provoking an anti-tumor immune response in the human patient.

39. The use according to claim 38, wherein the lymphocytes have been alloactivated against leukocytes from the human patient.

40. The cell according to claim 38, characterized in that the lymphocytes have been alloactivated against the allogeneic leukocytes to the human patient.

41. A combined preparation, characterized in that it comprises: a) alloactivated lymphocytes, allogeneic to a human patient; b) primary tumor cells from the human patient, or the progeny thereof, for simultaneous, separate or substantial use in a method or treatment of a human by surgery or therapy.

42. A combined preparation, containing: a) alloactivated allogeneic lymphocytes to a human patient; b) primary tumor cells from the human patient or the progeny thereof, for simultaneous, separate or sequential use for the treatment of the tumor or to elicit an anti-tumor immune response in the human patient.