MXPA97007077A - Method for treating tumor - Google Patents

Abstract

A method is provided for the treatment of a mammalian patient having a tumor, by administering to this patient lymphocytes from an allogeneic donor, which have been cultured together in the presence of lymphocytes derived from the patient, under conditions sufficient to alloactivate the donor lymphocytes. It is preferred that the donor lymphocytes be introduced intralesionally. This method is preferred for the treatment of glioblastoma in human beings

Description

METHOD TO TREAT TUMORS BACKGROUND OF THE INVENTION This application is a partial continuation of the patent application of the United States of America, Serial No. 08 / 406,388, filed March 17, 1995. 1. Field of the Invention This invention relates to methods to inhibit the proliferation of tumor cells. More particularly, this invention relates to a method for inhibiting the proliferation of tumor cells, increasing the ability of the patient to immunologically respond to the tumor. 2. Description of Related Art Traditional therapies have done little to alter the effect of patients high-grade brain tumors, such as glioblastomas, and many other types of tumors, such as systemic melanoma, and cancers of the head and By the neck. Patients primary tumors, which may undergo resection, generally experience recurrence of the tumor in one year after surgery, chemotherapy or radiation. Often these tumors progress rapidly, or out the subsequent conventional therapy. Thus, there is a need to develop new modes of therapy for these deadly tumors.

A new family of cancer therapies, developed in recent years, are based on immunotherapy. In general, immunotherapies take one of two approaches: 1) several techniques are used to activate the patient's immune system to attack the tumor or 2) the patient's lymphoid cells are removed and activated by in vi tro techniques, to produce the activity against cancer; and the activated cells are then reintroduced systemically into the patient. The clinical effectiveness of these various types of immunotherapy is being evaluated in patients different types of cancers. However, one of the major problems, associated both of these types of immunotherapy, is the toxicity observed when the immunotherapeutic agents are administered systemically. One method developed to avoid this toxicity is the intralesional administration of immunotherapy, for example by direct injection into the tumor. The intralesional administration of various forms of immunotherapy to cancer patients does not cause the toxicity seen the systemic administration of the immunological agents (M. Fletcher, et al., Lymphokine Res. 6:45, 1987, H. Rabinowich, et al. ., Cancer Res. 41_: 1987; SA Rosenberg, et al., Science 233: 1318, 1989, and G. Pizz., Et al., Int. J. Cancer 34: 359, 1984). Recent studies indicated that the immunization of animals tumor cells, which were treated by genetic engineering, to secrete different cytokines, increases the induction of a therapeutic immune response. Cytokines are believed to induce a complete set of reactions, including: a) an increased expression of tumor antigens; b) inflammation and infiltration of the tumors host lymphoid cells; c) induction of tumor-specific immunity; and d) activation of effector mechanisms against specific host tumors, which destroy the tumor. However, although this technique can finally prove useful, because it is extremely expensive and time consuming, its application may be limited. Studies in experimental animals (mainly monkeys), showed that the chronic release of cytokines in a tumor, can induce a response against the tumor and the regression of this tumor. Repeated intralesional injection of cytokines, such as Interleukin-2 (IL-2), Tumor Necrosis Factor (TNF), and Interferon-? (INF-) has been shown to cause regression of cutaneous sarcomas (SP Creekmore, et al., Resident and Staff Physician 34: 23-31, 1988, P. Greenberg et al., Basic and Tumor Immunology (R. Herberman, Ed.) P.302, 1983); E. Grimm, et al. , Lymphokines, 9: 279-311, 1984; G. Forni, et al. , Lymphokines L4: 335-360, 1987). It has also been shown that the injection into a tumor of the tumor cells of the animal, which have been engineered to secrete cytokines, such as IL-2, IL-4, TNF and the Colony Stimulation Factor of Granulocyte monocytes (GM-CSF), will induce immunity against host tumors (E. Feron, et al., Cell 60, 397-403; P. Galumbek, et al. , Science 254: 713-716, 1991; A. Ascher, et al. , J. of Immunol. 146: 3227-3234, 1991). These latter results have been obtained even in the treatment of tumors that were previously thought to be non-immunogenic. J. M. Redd, et al. (Cancer Immunology and Imm a therapy 34 (5): 349, 1992) have shown in rats that the allogeneic lymphocytes sensitized against the alloantigens of the donor, can inhibit the formation of tumors when they are injected together into the brain of a rat with 9L glioblastoma. In a separate study, spleen cells, both normal and alloimmune, from Wistar rats were injected into T9 brain tumors established at 6 days in Fischer rats. Intralesional injection of normal spleen cells from Wistar rats, previously immunized against Fischer alloantigens, cured tumors in 50% of Fischer rats. In contrast, untreated and unresponsive animals died within 30 days. The survivors appeared completely normal and the intracranial injections of the allogenic cells in the normal rats did not cause detectable changes in behavior or survival in a period of three months. The histopathological examination of the brains of the treated animals bearing tumors revealed: 1) infiltration of mononuclear cells, massive necrosis of tumors, which start from 2 to 4 days and the total destruction of the tumor by 15 days; or 2) cellular infiltration, early destruction of the tumor and then regrowth of the tumor, which progresses until the animal dies. No damage to normal brain tissue was evident at any time in these animals. The animals with tumor regression developed systemic immunity and proved to be totally resistant to the repeated challenge with the viable tumor. Although these results in rats are of interest, their value in reasonably forecasting what will be observed in highly unrelated species, such as a human being, is very questionable, in view of the considerable diversity of species that exist, especially with respect to to the immunological response to tumors. Human glioma patients with surgically accessible and localized tumors are logical candidates for intralesional immunotherapy. Multiple Phase I studies in adult patients with gliomas have been reported, employing intratumor implants of peripheral blood lymphocytes analogues, activated in vitro with IL-2. While little clinical effect was noted, side effects were few and occurred only when excessive levels of IL-2 were administered in conjunction with the cells (KS Jacobs, et al., Cancer Res., 47: 2101, 1986; Merchant, et al., Neurosurgery 23: 725, 1988). Studies of these patients revealed that survival directly correlated with the ability of the implanted cells to secrete the cytokine TNF. The discovery of the inhibitors for both TNF and IL-1 in the serum or cystic fluid of the tumor, and in primary cultures of the tumors of these patients, suggests that the tumor cells surround themselves with blocking agents. the response against the host tumor. These inhibitors can prevent the activated cytokine implanted cells from remaining active in the tumor for a long enough time to cause their destruction. This concept is supported by the findings in studies of brain tumors in rats. When active IL-2 lymphoid cells are implanted in brain tumors of C6 and T9 glioma, in Wistar and Fischer rats, respectively, the histopathological examination revealed that lymphoid cells activated with IL-2, implanted, only remain in the brain. tumor site for 4 to 6 days (W. Carson et al., J. of Immunotherapy 10 (2): 131-140, 1991). The operative mechanisms in causing tumor regression in animals treated with allogenic lymphoid cells include a graft reaction vs. guest and guest vs. graft at the site of the tumor. These potent immunological reactions can stimulate high levels of endogenous cytokine production in the tumor, overcome local levels of cytokine inhibitors and presumably stimulate the infiltration, enlistment and activation of the anti-tumor activity of the host, both specific and non-specific. Animals with regression of the tumor were found to be resistant to the neural network of the tumor. However, until now it is unknown whether any treatment based on similar methods will achieve similar results in humans, sufficient to be considered effective in the treatment of human tumors. Accordingly, in view of the limitations of the prior art,. New and better methods are needed to treat mammalian tumors. In particular, new intra-tumor immunotherapy methods are necessary for human cancer patients, for whom the regression of an individual solid tumor can be demonstrated to save life. COMPENDIUM OF THE INVENTION The present invention provides a method for inhibiting tumors in mammals. In the general practice of this invention, peripheral blood mononuclear cells from a tumor patient (PBMC) are cultured together in vi tro to induce a mixed lymphocyte cell reaction with healthy lymphocytes derived from a normal donor, preferably a allogeneic donor Preferably, normal lymphocytes are from a donor unrelated to the tumor patient and preferably normal lymphocytes are obtained by leukapheresis of whole blood. During co-culture, the lymphocytes of the allogeneic donor are specifically activated against the alloantigens of the patient. The mixture of the alloactivated donor and the lymphocytes of the patient, produced by the mixed culture of lymphocytes, is referred to herein as the "MLC." Activated MLC cells produce a mixture of cytokines, which has been shown to induce a primary immune response in vitro. In the treatment of glioblastoma, for example, the patient is a human and the alloactivated lymphocytes are surgically implanted into the patient's brain at the primary site of the tumor, optionally together with the PBMCs of the co-culture patient as a mixture of MLC. , to induce the patient's immune system to attack the tumor cells derived from it. In the treatment of other types of tumors, MLCs are injected into a tumor site, the site of metastasis or body cavity, such as the peritoneum. Optionally, MLCs are administered peripherally, such as in a non-primary tumor site, in the treatment of tumors in addition to glioblastoma. In one embodiment of the invention, the lymphocytes of the alloactivated donor, obtained by co-culture with lymphocytes derived from the patient, are isolated from the MLC mixture and administered intralesionally as an implantation site directly into the tumor of a patient who desires protection. against the recurrence of a tumor, for example in the vicinity of a tumor that is not very large surgically, or wishes to treat an inoperable tumor. Alternatively, alloactivated donor lymphocytes may be peripherally administered in the treatment of a tumor, such as a secondary or metastatic tumor site. In another embodiment, the allogeneic activated lymphocytes are co-cultured with lymphocytes derived from the patient, and the mixture of the MLCs are implanted or introduced peripherally as a mixture. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the survival times of patients with glioblastoma treated with the intra-tumor implant of MLC cells. Figure 2 is a graph showing the magnetic resonance imaging (MRI) scans, which indicate the reduction in tumor size in nine glioblastoma patients, two patients, one who received a simple implant dose of 4 x 10 ^ MLC and another which received a simple implant dose of 6 x 10 ^ MLC, have continued to date to show a progressive reduction of the tumor mass in periods of 58 and 74 weeks, respectively.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for treating tumors in mammals, with the use of an allograft of donor lymphocytes and which have been co-cultured in vitro in a mixed lymphocyte reaction (MLC) with the mononuclear cells of the patient, preferably peripheral blood, to activate the donor lymphocytes against the antigens associated with the patient's tumor. It is believed that the graft response vs. guest and the host vs. guest response Allograft grafting induces a potent immunological reaction at the tumor site, resulting in cytokine production and tissue destruction. During this reaction, the host lymphoid cells identify both the graft and tissue lymphoid cells as foreign cells and both are inhibited or destroyed by the local and systemic immune response. This host reaction occurs not only at the implant site of the lymphoid cells of the graft, but may also occur peripherally, such as at a secondary or metastatic tumor site. In addition, the implanted donor lymphocytes immunologically attack the tumor in a graft response. Guest. Thus, the strong immunological reaction in the center of the tumor produces an environment that overcomes the cytokine inhibitors that may be present at the tumor site. The increase in the immune reaction will inhibit the growth, decrease the size and / or eradicate established or metastatic tumors, as well as inhibit the recurrence at the site of the tumors made less bulky surgically. Thus, the present invention provides a method for enhancing the systemic immune response to the patient's tumor by introducing into the patient a viable preparation of the alloactivated cells described herein. Numerous studies have shown that the in vitro environment in a mixed lymphocyte reaction facilitates an active primary immune response to allogeneic and tumor-associated antigens (M. Gately, et al., JNCI 69: 1245, 1982; S. Lee, et al. al., J. Experimental Medicine 147: 912, 1978; J. Zarling, et al., Nature 262: 691, 1976). In the present invention, the MLC reaction occurs within the patient's own tumor tissue, thereby stimulating the patient to respond against his own tumor. Preferably, the co-culture is performed, generally in 1 to 5 days. Since the release of key cytokines takes place during the early stages of co-culture, it is preferred that the co-cultured cells be implanted in the early stages of the co-culture reaction, usually during the first 48 hours of the reaction. , when the levels of the cytokine are normally higher. This method results in the release of the key cytokines directly into the tumor tissue, so that an environment is created that is conducive to the recognition of the antigen and development of the cell-mediated immunity directed against the antigen. Standard techniques, such as those based on the immunoassay, can be used to measure the level of various cytokines, which include TNF, LT and interferon gamma, present in the supernatants of the MLC culture. Levels of TNF and LT vary from 50 to 150 units of biological activity / ml, or 500 to 3500 pg / ml of supernatant. As a result of in vitro co-culture, healthy allogeneic lymphoid cells from a donor are specifically activated against the patient's alloantigens. The implanted cells are generally allowed to come into direct contact with the tumor cells. Thus, as used herein, the term "implant" and "implanted" mean that the cells are placed in the patient's body as a group, optionally within a thick matrix of coagulated serum or other suspension of a gel-like substance. Examples of tumors that can be treated by the method of this invention include the following: Tumors of the brain, such as astrocytoma, oligodendroglioma, ependymoma, medulloblastomas and PNET (Neural Primitive Ectodermal Tumor); Pancreatic tumors, such as pancreatic ductal adenocarcinomas; Tumors of the lung, such as small and large adenocarcinomas, squamous cell carcinoma, and bronchoalveolar carcinoma; Tumors of the colon, such as epithelial adenocarcinoma and liver metastasis of these tumors; Tumors of the liver, such as hepatoma and cholangio-carcinoma; Tumors of the breast, such as ductal and lobular adenocarcinoma; Gynecological tumors, such as squamous and adenocarcinoma of the uterine cervix, and epithelial adenocarcinoma of the uterus and ovary; Prostate tumors, such as prosthetic adenocarcinoma; Tumors of the bladder, such as transitional squamous cell carcinoma; Tumors of the RES system, such as B and T cell lymphoma (nodular and diffuse), plasmacytoma, and acute and chronic leukemia; Tumors of the skin, such as malignant melanoma; Y Tumors of soft tissues, such as soft tissue sarcoma and leiomyosarcoma.

In one embodiment of the method of this invention, the alloactivated lymphocytes of the donor are implanted at the site of the tumor, optionally together with the PBMCs of the co-culture, to supplement and increase the attack on the patient's immune system on tumor cells derived from the same. For example, in one embodiment of the invention, alloactivated human donor m lymphocytes, obtained by co-culture with the PBMCs derived from the patient or PBMC cell lysates, are administered intralesionally in a human, for example as a placed implant. directly inside the brain of a patient who desires protection against the recurrence of a glioblastoma tumor. The alloactivated lymphocytes of the donor can be implanted in the vicinity of a tumor made less bulky surgically, or a tumor treated by irradiation, chemotherapy or other appropriate techniques. In another preferred embodiment, the allogeneic activated lymphocytes are co-cultured with lymphocytes derived from the patient and the mixture of the co-cultured and allogeneic cells derived therefrom, known herein as "the MLCs", are implanted intralesionally. The typical implant comprises a therapeutic amount of the alloestimulated donor lymphocytes. As used herein, the term "a therapeutic amount" means a sufficient amount of the MLC or allo-stimulated lymphoid cells of the donor, obtained the culture of mixed lymphocytes to inhibit growth, decrease the size of and / or eradicate established tumors and / or preventing recurrence of the tumor at the site of a tumor, which has become less bulky surgically or treated with chemotherapy or irradiation. More generally, a therapeutic amount may vary with the potency of each batch of the alloactivated donor cells; the amount required for the desired therapeutic or other effect, the mode of administration, i.e. whether by direct implantation within a tumor or body cavity or by peripheral administration, such as intravenously; and the regime of elimination or interruption of MLC by the body, once implanted or administered. In accordance with conventional prudent formulation practices, a dose near the lower end of the useful range can be used initially and the dose increased or decreased as indicated by the observed response, as in the routine procedure of the physician. However, in general a unit dose for the direct implant comprises approximately 2 x 10 ^ to 6 x 10 ^ MLC. For example, it has been found that in the treatment of brain tumors, the upper limit of the cells that can be implanted is approximately 6 x 10 ^. Alternatively, a unit dose for peripheral administration usually comprises about 2 x 10 ^ to 2 x 1010 MLC. The invention further comprises a sterile bottle or other container for retaining a composition, comprising a unit dose of the MLC. Typically, the vial or container will bear a label indicating information regarding the pharmaceutical use of the composition in the treatment of a tumor in a human, as approved by the FDA for the use of the composition in the treatment of a human having one or more of the tumors, in which the method of treatment of the invention is effective, as described herein. Although any known method for obtaining PBMC from a donor can be used, it is preferred to obtain about 150 to 300 ml of leukapheresis suspension containing the donor PBMC, which uses leukapheresis techniques that are well known in the art. for support apheresis, according to the manufacturer's instructions for leukapheresis equipment. For example, leukapheresis can be performed with the use of a Cobe 2997 blood cell separator, Cobe Spectra® (Lakewood, CO), Fen all CS 3000 (Deerfield, II) or Haemonetics (Braintree, MA). Generally, a flow regime of 40 to 50 ml / min for 2 to 4 hours with lymphocyte yield of 2-4 x IO ^ can be used to process a total donor blood volume of 7,000 to 12,000 ml and deliver 200 to 250 ml of leukapheresis suspension having less than 1 ml of red blood cells. For example, if a Cobe 2997 blood cell separator is used, the rate of the centrifuge is generally around 5 x G, the flow rate is up to about 45 ml / min and the collection rate is not higher of or equal to 2.5 ml / min. One skilled in the art will appreciate that the performance of the lymphocytes will vary with the donor and the leukapheresis machine used. For example, if the donor's pre-absolute lymphocyte count is at the level of 0.6 x 10 ^ to 1.0 x 10 ^, as few as 150 ml of the leukapheresis suspension of the donor can be removed. If desired, the donor cells can make contact with a stimulatory cytokine, such as IL-2, to initiate the activation of the cells to the antigen derived from the patient, during co-culture and further stimulate the proliferation of the lymphocytes. The PBMCs of the donor are obtained from the blood fraction of the donor, taking care to avoid the rupture of the mononuclear cells, for example, by centrifugation of the fraction of blood containing the mononuclear cells through a cell separation medium, such as Histopaque® 1.077 at 350 x G for 7 to 10 minutes. Those skilled in the art will know other techniques for separating PBMC cells that can be used easily. Donor blood is typically pre-examined 3 to 7 days before surgery, to detect HIV, Hepatitis A, B and C, and VDRL. Sufficient anticoagulant, such as 2% citrate, is added to the donor and the patient's blood or blood fraction to prevent coagulation when removing blood. Alternative anticoagulants and their mixtures are known to those skilled in the art, and can also be used, such as formula A dextrose citrate anticoagulant (ACDA) 15 ml / citrate 100 ml; formula B dextrose anticoagulant citrate (ACDB) 25 ml / citrate 100 ml; or citrate phosphate dextrose (CPD) 14 ml / citrate 100 ml. Typically, whole blood is removed from the patient to be treated, according to the invention, using methods known in the art, such as vein puncture. The PBMCs are isolated from the patient's whole blood, usually by centrifugation through a cell separation medium, such as Histopaque 1.077 (Sigma, St. Louis, MO) and washed thoroughly to release the cells from the blood coagulant factor. of the patient. Samples of both donor blood and patient or blood fraction must be fully tested to ensure sterility before co-culturing the cells. Typical of tests for the sterility of blood components that can be conducted by a person skilled in the art, are those that use such growth medium as thioglycolate broth, tryptic soy broth and Tissue Culture Medium. Rosewell Park Memorial Institute (RPMI) with 10% heat inactivated fetal bovine serum 10% (BS) (RPMI - 10%) and 1% L-glutamine without adding antibiotics. Sterility tests using the blood cell culture in such growth medium are illustrated in Example I of this application. Alternative sterility tests are known to those skilled in the art. Before the mixed culture of lymphocytes can be performed, PBMCs are typically isolated from whole blood and analyzed to determine the number of living cells per unit volume. This can be done, for example, using a dye that differentiates between living and dead cells and counts the cells in a Neubauer chamber. Typical dyes for this use are Tryptan Blue and Eosin Y dyes, both of which can be used in wet preparations. Alternative dyes will be known to the experts in the material. Generally, the concentration of living cells is standardized by diluting the PBMC preparation to achieve a predetermined concentration of living cells per volume unit. Although a person skilled in the art can select a somewhat larger or smaller number, it is generally preferred that the number of beams cells be set at an approximate concentration of 10 *? cells / ml, for the purposes of conducting the mixed culture of lymphocytes. The donor cells are typically grown in a ratio of 10: 1 to 20: 1 compared to the patient's cells. Standard techniques for conducting the mixed culture of lymphocytes with the use of mammalian cells (for example a human) are well known in the art and are illustrated in Example 1 of this application. See, for example, Current Protocols in Immunology, Ed. J. E. Coligan, et al. , John Wiley & Sons, Inc., 1994, Sec. 7.10 and M. Gately, et al. , supra; S. Lee, et al. , supra; and J. Zarling, et al. , supra, that are incorporated here as a reference in its entirety. To block the response of the patient's stimulator cells to the donor response cells (stimulation again) it is preferred that the patient's cells be irradiated or treated with a DNA binding agent, such as mitomycin C, before the mixing the cells during co-culture, to reduce or eliminate the proliferative potential of the patient's cells, as is well known in the art. In the present invention, the lymphocyte cells of the donor are typically co-cultured in a short period of the mixed culture of lymphocytes with the patient's PBMC for a period of at least 48 hours and preferably from 1 to 5 days. As a result of co-culture in vi tro, healthy lymphoid cells, from the unrelated donor, are specifically activated against the antigens associated with the patient's tumor. Preparations for parenteral or intravenous administration are typically contained in a "physiologically compatible carrier". Since the cells used in the practice of this invention are alive, a physiologically compatible carrier is one that does not impart viability of the cells, that is, it is hypotonic and with a physiological pH. Such carriers include sterile aqueous solutions of salts, suspensions and emulsions, including saline and regulated media. Ringer dextrose, dextrose and sodium chloride and lactated Ringer's solution. Intravenous vehicles include fluid and nutrient fillers, electrolyte fillers, such as those based on Ringer's dextrose, and the like. For administration by the non-intravenous routes, the carrier may be in the form of coagulated plasma, preferably coagulated plasma of the patient. Alternatively, the carrier can be a biodegradable, physiologically compatible, plasma-free solid or semi-solid, such as a gel, suspension or water-soluble jelly. Acacia, methylcellulose and other cellulose derivatives, sodium alginate and tragacanth suspensions or gels are suitable for use as carriers in the practice of this invention, for example, sodium carboxymethylcellulose 2.5%, tragacanth 1.25% and gum Guar 0.5%. In the preferred method of implanting into a tumor site the co-cultured lymphocytes are harvested from the co-culture supernatant (after at least 48 hours of co-culture) by centrifugation at the time of surgery. The collected cells are washed twice with an injectable saline solution and resuspended in the decalcified plasma obtained from the patient on the previous day, free of platelets. The cells in the plasma are transported to surgery, the plasma is re-calcified by the addition of calcium, preferably in the form of calcium gluconate, so that the plasma clots, embed the cells in a plasma clot derived from the same. The patient's tumor is then made less voluminous surgically, and the clot crumbles aseptically and is implanted at the site of the tumor. The following examples illustrate the manner in which the invention can be practiced. However, it will be understood that the examples are for purposes of illustrating the invention and should not be construed as limiting any of the materials or specific conditions therein.

EXAMPLE 1 A. Mixed Lymphocyte Cell Cultivation Procedure (MLC) 1. Collection of the response PBMC from the unrelated donor. Peripheral blood mononuclear cells (PBMC) were collected by leukapheresis from normal healthy donors unrelated to the patient. The donors were previously examined to test the complete blood count (CBC) with hepatitis A, B and C, VDRL and differential HIV-I. Approximately 150 to 300 ml of the leukapheresis suspension containing the PBMC were collected from each donor, using standard blood donation procedures for apheresis support, according to the manufacturer's instructions. Leukapheresis was performed using a Fen all CS 3000 blood cell separator (Deerfield, IL). A flow rate of 40 to 50 ml / min for 2 to 4 hours, with lymphocyte yield of 2-4 x 10 ^ processed a total donor blood volume of 7,000 to 12,000 ml, to deliver 200 to 250 ml of suspension of leukapheresis that has less than 1 ml of red blood cells. When a Cobe 2997 blood cell separator was used, the centrifugation rate was 5xG, the flow rate up to 45 ml / min and the collection rate was equal to or greater than 2.5 ml / min. However, if the donor's pre-absolute lymphocyte count is 0.6 x 10 ^ to 1.0 x 10 * 9, as little as 150 ml of the leukapheresis product were removed. The hematocrit for the final product was 3.5%. At least one total volume of blood was processed for 80% efficiency of lymphocyte collection. The anticoagulant used was 2% citrate or a citrate / anticoagulant ratio of ACDA-15 ml / citrate -100 ml; ACDB - 25 ml / citrate - 100 ml; or CPD 14 ml / citrate -100 ml. To obtain the maximum purity of the product, the actual and final product of the cell separator was transported as a pure concentrate of cells in the plasma derived therefrom. The cells were not washed and no albumin was added. 1. Preparation of donor cells The leukapheresis product was transported to the MC Oncology Research Laboratory for the production of mixed allogeneic lymphocyte cells (MLC) for immunotherapy. The cells were drained from the leukapheresis package into two or three 250 ml centrifuge tubes, stirring and placing 3 ml apart for the sterility tests to be done during centrifugation. The cell concentrate was diluted with phosphate buffered saline (PBS) and centrifuged for 7 minutes at 2,000 rpm. The centrifugation was repeated twice for a total of three times, to wash the cells and release them from the coagulation factor in the donor serum. Three 1 ml aliquots of the 3 ml removed from the leukocyte suspension were placed in sterile tubes with a lid, for the sterility test. The first 1 ml aliquot was added to the thioglycolate medium (Difco, Detroit, MI) (30-35 ° C, 48 h); a second part of 1 ml was added to the tryptic soy broth (Difco, Detroit, MI) (25-30 ° C, 48 h); and a third of 1 ml added to RPMI 1640 (GIBCO, Gaithersburg, MD) with 10% heat-inactivated FBS (RPMI-10%) and 1% L-glutamine, but without antibiotics. The cells were washed by rotation twice at 150g for 10 minutes in PBS, to remove the platelets. The supernatant was discarded very carefully as the cells were in an aqueous paste and not in a pellet. The cells were resuspended in AIM V (GIBCO, Gaithersburg, MD) supplemented with 2% heat-inactivated FBS (2% AIM V) at 420 ml and placed in a 175 cm3 T-flask. The blood of the patient or donor was diluted 1: 1 with a sterile saline solution. For cell separation, 35 ml of the cell suspension was carefully layered onto 15 ml of the Histopaque® 1.077 suspension medium (Sigma, St. Louis, MO) in each 50 ml tube and centrifuged at 250 G for 45 minutes. minutes The centrifugation started slowly and was gradually increased at full speed. After centrifugation, the mononuclear cells containing the interface, between the Histopaque® suspension medium and the plasma layer, were carefully collected with a sterile 25 ml pipette, placed in clean 50 ml centrifuge tubes, diluted with 2% of the medium AIM VI: 1 and centrifuged at 550G for 7 to 10 minutes, to form a pellet of cells. The cells remained a minimum of time in the Histopaque® suspension medium, because it is toxic to the cells. The supernatant was discarded, the pellet was resuspended in 2% of AIM V and divided into two centrifuge tubes of 50 ml at a total volume of 40 ml and centrifuged at 550G for 5 minutes. After washing, the supernatant was discarded. The washing step was repeated twice for a total of three minutes. After the last wash, the cells in each tube were resuspended in 50 ml of 2% AIMV. Aliquots of 1 ml of the resuspended cells were diluted at a ratio of 1:10 in 2% AIM V per tube, then further diluted 1: 1 in the Tryptan Blue dye (Sigma, St. Louis, MO) to distinguish The dead cells of living and living cells are counted in a hemocytometer. Cells were adjusted to 2 x 106 / ml with 2 & amp;; of AIM V. 3. Collection of PBMC stimulator for patients with tumor. From 200 to 400 ml of the peripheral blood cells were removed from each patient of glioblastoma by puncture in the vein and placed in 250 ml centrifuge tubes, stirring and placing 3 ml apart for the sterility tests that are done during the rotation. The blood cells in the centrifuge tubes were diluted with saline and centrifuged for 7 minutes at 550G. The centrifugation was repeated twice for a total of three times to wash the free cells of the coagulation factor in the patient's serum. The sterility tests were conducted as described above. The cells were washed twice by centrifugation at 150G for 10 minutes in saline to remove the platelets, the supernatant was discarded very carefully and 420 ml of cells were resuspended in a 175 cm3 T-flask in saline. 15 ml of the Histopaque® 1.077 cell separation medium was added to twelve 50 ml centrifuge tubes, and 35 ml of cells suspended in the saline were layered on the Histopaque® 1.077 medium in each 50 ml tube. Cell suspensions were rotated at 250G for 45 minutes, initiating centrifugation slowly and gradually increasing speed.

After centrifugation, the mono-nuclear cells at the interface between the Histopaque® cell separation medium and the plasma layer were collected with a sterile 25 ml pipette in 2 sterile 250 ml centrifuge tubes and diluted with 2 ml. % of AIM-V to a final volume of 250 ml. The diluted mononuclear cells were subjected to centrifugation at 550G for 7 to 10 minutes. For washing, the supernatant was discarded, then the cell pellet was resuspended with 2% of AIM V and centrifuged at 550G for 5 minutes. The washing step was repeated for a total of three times. After the last washing step, the cells were resuspended in 50 ml of 2% AIM V. 1 ml of the cell suspension was diluted 1:10 in 2% of AIM-V per tube and the number of viable cells was determined by 1: 1 enumeration in the Trypan Blue dye, as described above. The procedures, described above, for cell collection and sterility testing, were approved by the State of California for GMP for the control of sterility and quality. 4. Alloactivation of the patient's mononuclear cells (PBMC) with donor leukocytes The PBMC isolated from the patient were resuspended in ^ cells / ml in AIM-V, 50 μg of Mitomycin C (Bristol-Mayer Squibb, Princeton, NJ) was added per ml of the patient's cell suspension, and the PBMC suspension was incubated at 37 ° C for one hour to block the response of the stimulus cells to the response cells (stimulation again). After one hour of incubation, the excess mitomycin C was washed from the cells by alternative centrifugation (250G for 5 minutes) and the cells were resuspended in AIM-V. After treatment of the mitomycin of the patient PBMC, the cells were added in a ratio of 20: 1 to 10: 1 to the donor culture (obtained as described above). For co-culture, the PBMC suspension of the patient treated with mitomycin C and the donor was placed in a sterile, sealed Fenwal tissue culture system, specially designed for the cultivation of PBMCs for implantation in patients. The cells were passed in sealing systems by means of Fenwal cell transfer units and pumps, according to the instructions of the manufacturers, and cultured in an incubator at 37 ° C for 48 hours. 5. Tests of sterility of alloactivated cells Two days before implantation of the cell suspension, the following three sterility tests were performed. Sterile 10 ml aliquots were removed from each tissue culture bag, placed in sterile, capped 15 ml centrifuge tubes, and subjected to centrifugation for 10 minutes at 450G. In each tube, the pellet was resuspended in 3.0 ml of PBS. An aliquot part of 1 ml of the cell suspension was added to each of the three sterile cap tubes, containing 2 ml of thioglycolate broth, tryptic soy broth or RPMI-10% and incubated for 48 hours. Each cell suspension was examined microscopically before implantation, to detect signs of microbial growth. On the day of surgery, the cells were centrifuged and separated from their medium, washed twice with a saline solution and resuspended in the decalcified plasma, free of platelets, obtained from the patient on the previous day. The cells were transported to the operating room in the plasma, then the plasma was recalcified by the addition of calcium gluconate, so that it coagulated just before implantation in the tumor bed. On the day of surgery, a drop of the collected cell pellet was examined again in sterility under the microscope. Just before coagulation, an aliquot of 100 μl of the cell suspension was added to each 2 ml of RPMI-10% without antibiotics, thioglycolate broth and tryptic soy in a sterile tube with lid. The samples were incubated for four days after surgery and the operation record was maintained for this last sterility test.

The cellular implants produced by the method described above have received the approval of the FDA administration for use in human cancer patients (IND-BB-6288). B. Intralesional Implantation of Lymphocyte Mixed Cell Culture A Phase I human trial was initiated to test the effects of intracranial injection of alloactivated allogeneic lymphoid cells in patients with recurrent glioblase-taking. A total of 9 patients with recurrent glioblastoma of Grade 3 and Grade 4, entered this trial. The MLC obtained from the mixed culture of lymphocytes in Stage 3 above was implanted intralesionally in the less bulky tumor at the time of surgery. These patients were tested in each of three doses of 2 x 10 ^ cells, and 4 x 10 ^ and 6 x 10 ^ of the MLC. The patients were clinically followed and screened by magnetic resonance imaging (MRI), performed at several monthly intervals in each patient, followed by implantation, to monitor the disease process. All patients complained of headache and nausea for 1 to 2 months after surgery. Patients who received the highest cell dose experienced the greatest number of symptoms. Four of the five patients, which included all those in the highest dose and one in each smaller dose, showed a 50% or more reduction in the area of tumor improvement in the MRI scans in a period of 2 to 3 months after the implant. The results of the MRI scans are shown in Figure 1, which measures the survival time of glioblastoma patients treated with intratumoral implants of MLC cells. Two patients, one who received a single implant dose of 4 x 109 MLC and another who received a single implant dose of 6 x 109 MLC, have continued to date showing a progressive reduction in the tumor mass in periods of 58 and 74 weeks, respectively. These two patients have also shown a marked improvement in Kamofsky's classification. The results of the MRI scans are shown in Figure 2, which measure the reduction of tumor volume as a function of time since the implantation of the MLC. EXAMPLE 2 The Phase I studies have been conducted in two patients with systemic melanoma. Mixed cultures of patient and donor lymphocytes were prepared, as described in Example 1. Patients were treated by intralesional injection of 2 x 10 9 MLC cells into skin tumors. No patient exhibited negative effects. The first patient experienced necrosis and destruction of the injected cutaneous tumor and inflammation in a distant metastasis.

The second patient showed inflammation in the injected tumor, but not necrosis at the present dose level. The treatment of these patients is progressive. EXAMPLE 3 A human clinical study of phase I was conducted to test the effect of the intratumoral implant of MLC cells in patients with pancreatic cancer. Four patients with incurable, non-treatable pancreatic cancer received the implant intralesions of 4 x 109 MLC cells. Little or no toxicity was found. A reduction in the tumor mass occurred and an extension of life was observed. Three patients experienced a greater than 50 percent reduction in tumor mass. In patient No. 3, the serum levels of CA-19-9, a marker for this tumor, declined from the high level of 206 to 86 over a period of two months. Seven months after treatment, two of the treated patients returned to their jobs, have no clinical symptoms of the tumor and live a totally normal life. The above description of the invention is exemplary for purposes of illustration and explanation. It should be understood that various modifications can be made without departing from the spirit and scope of the invention. Therefore, the following claims are intended to be construed as encompassing all these modifications.

Claims (14)

1. A method for preparing a pharmaceutical composition containing lymphocytes from a human alloactivated donor to treat a tumor in a human patient, said method comprises the following steps: a) co-culturing lymphocytes from a human donor ex vivo with leukosites from the patient, in a manner that the lymphocytes of the donor are alloactive; and b) harvesting the cells and preparing them for human administration a moment after the initiation of co-culture, when the cultured cells, after the implantation in the bed of a solid tumor found in the patient, are effective in the treatment of the tumor.

2. The method according to claim 1, wherein the co-cultured cells are harvested at the time when the implantation of the co-cultured cells causes a response in the patient against the tumor.

3. The method according to claim 1, wherein the co-cultured cells are harvested at the time when a single implantation of the cells co-cultured in the bed of a solid tumor is effective in the treatment of cancer.

4. The method according to claim 1, wherein the tumor is a malignancy selected from the group consisting of melanoma, pancreatic cancer, cancer of the liver, cancer of the colon, cancer of the prostate and cancer of the chest. The method according to any of the preceding claims, wherein the culture in step a) is conducted for a period of 48 hours after the initiation of the culture. 6. A pharmaceutical composition containing lymphocytes of alloactivated human donor and is suitable for human administration, prepared according to any of the above methods. The pharmaceutical composition according to claim 6, having one or more of the following characteristics: i) it contains between about 2 x 10 9 and 2 x 10 10 of peripheral, cultured, blood mononuclear cells originating from the donor; ii) contains between about 1 x 10B and 2 x 109 of peripheral, cultured, blood mononuclear cells originating from the patient; iii) is substantially free of any exogenously added lymphocyte proliferation agent; iv) contains a physiologically compatible carrier selected from the group consisting of physiological saline, regulated medium and coagulated plasma. 8. A method for preparing a population of cultured cells containing alloactivated human donor lymphocytes effective in the treatment of a tumor in a human patient, said method comprising the following steps: a) obtaining leukocytes from the human patient; b) obtain lymphocytes from a human donor that is allogeneic to the human patient; c) co-cultivating the donor lymphocytes ex vivo with the leukocytes of the patient, so that the lymphocytes of the donor are alloactive; d) harvesting the cells of the culture at the moment in which the co-cultured cells, after the implantation in the bed of a solid tumor that is in the patient, are effective in the treatment of the tumor; e) washing the culture medium of the harvested cells; and f) verifying that the co-cultured cells are sufficiently sterile for human administration. The method according to claim 8, which incorporates one or more of the following characteristics: i) obtaining at least about 2 x 10a of leukocytes from the human patient in step a); ii) obtain leukocytes from a human patient having melanoma, pancreatic cancer, liver cancer, colon cancer, prostate cancer or breast cancer, in stage a); iii) obtain at least about 2 x 109 of the peripheral mononuclear cells from the blood of the human donor in step b); iv) blocking / proliferation of the patient's leukocytes before step c); v) co-culturing the donor lymphocytes with the patient's leukocytes in a ratio of approximately 10: 1 to 20: 1 in step c); vi) harvesting the cells in about 48 hours after the initiation of the culture in step d); or vii) producing between about 2 x 109 and 2 x 1010 of the co-cultured cells suitable for human administration after performing step e). 10. The use of a population of cells containing lymphocytes from a human donor that are alloactivated against the leukocytes of a human patient having a tumor, for the preparation of a medicament for the treatment of the tumor. 11. The use of a population of cells containing lymphocytes from a human donor that are alloactivated against the leukocytes of a human patient having a tumor, for the preparation of a medicament that elicits an anti-tumor immune response in the patient . The use according to claim 11 or 12, wherein the administration of the medicament comprises the implantation in or around the bed of a solid tumor that is found in a patient, with or without anterior resection or partial resection of the solid tumor . The use according to any of claims 11 to 13, which incorporates one or more of the following characteristics: i) a single implantation of the drug in or around the bed of the solid tumor is effective in the treatment of the tumor; ii) a single implantation of the drug in or around the bed of the solid tumor is effective in eliciting an anti-tumor immune response; or iii) a single implantation of the drug in or around the bed of the solid tumor is effective in extending the median life expectancy of the treated patients. 14. The use according to any of claims 11 to 14, wherein the tumor is a malignancy selected from the group consisting of melanoma, pancreatic cancer, liver cancer, cancer of the colon, cancer. - in the prostate and cancer of the chest.