MXPA97008989A - Therapy of cellulas alogeneicas for the cancer after the transplant of cells mother alogenei - Google Patents

Abstract

Methods and materials are described for treating cancer patients with solid tumors who have undergone a cancer therapy regimen that includes allogeneic bone marrow transplantation. Allogeneic lymphocytes are administered to these patients. Patients with solid tumors and hematopoietic patients are also treated with "pre-activated" allogeneic lymphocytes by in vitro exposure to a T-cell activator. The same activator or a different T-cell activator can be further administered to the patient in vi

Description

THERAPY OF ALLOGENETIC CELLS FOR CANCER AFTER TRANSPLANTATION OF ALOGENEIC STEM CELLS FIELD OF THE INVENTION This invention relates to methods for eradicating tumor cells that remain viable in a patient after transplantation of allogeneic stem cells. More particularly, this invention relates to the use of allogeneic lymphocytes for the eradication of solid tumor cells after transplantation of allogeneic stem cells. The invention also specifically relates to the use of activated, allogeneic, donor lymphocytes for the treatment of cancer patients, including relapsing patients.

BACKGROUND OF THE INVENTION Patients suffering from malignant hematological disorders such as leukemia or lymphoma, under appropriate circumstances, may be treated with autologous or allogeneic bone marrow transplants, as part of a therapeutic regimen.

REP: 026084 These transplants may also be useful in conjunction with the therapy of non-hematological malignancies such as breast carcinomas or other solid tumors. Bone marrow transplantation makes it possible to treat patients with superior "supra-lethal" combinations, resistant to chemotherapy and / or radiation disease, ignoring the irreversible toxicity of these therapeutic combinations in the normal compartment of the bone marrow. However, this "deflation" of the patient can leave a fraction of residual malignant cells that can lead to the relapse of the disease. Several lines of evidence suggest that a significant proportion of the beneficial effect of allogeneic bone marrow transplantation (ie, bone marrow transplantation from an individual not genetically identical to the host patient) comes from the cell-mediated interactions of the immune cells of the donor origin , against residual tumor cells in the host that have escaped the chemoradiotherapy deflation regimen. After transplantation of the allogeneic bone marrow (Alo-BMT), the incidence of relapse is significantly lower in patients with leukemia with clinical manifestations, than host disease against graft (GVHD), acute or Chronic, in comparison with patients without GVHD, indicating that immunologically mediated allogeneic interactions of the immunocompetent cells of the donor origin against the host can be achieved by the effects of the graft against leukemia (GVL, for its acronym in English). The highest relapse rates seem to occur in patients who undergo Alo-BMT with T lymphocyte reduction for the prevention of GVHD, compared to non-reduced marrow allograft recipients in cells, despite the severity of GVHD . Likewise, the relapse rates in patients with acute leukemia or chronic myelogenous leukemia reconstituted by bone marrow grafts obtained from an identical twin (syngeneic grafts) are significantly higher than those reconstituted by bone marrow cells obtained from an HLA-identical but not syngeneic brother. Similarly, relapse rates after transplantation of the patient's own marrow (autologous), even after adequate depuration in vitro for the elimination of residual leukemia cells, are significantly higher than after Alo-BMT. Recent studies by several groups have shown that patients with chronic myelogenous leukemia (CML) who relapse after Alo-BMT can be successfully treated by infusion of HLA-matched leukocytes at rest (ie, inactivated by in vitro treatment with activators). of T cells such as cytokines), from the donor of the Alo-BMT in order to achieve a second revision. Slavin et al, Blood 72 (Suppl 1): 407a (1988); Kolb and collaborators, Blood 76: 2462 (1990); Baer et al., J. Clin.

Oncology 11: 513 (1993); Jiang et al., Bone Marrow Transpl. 11: 133 (1993); Drobyski et al., Blood 82: 2310 (1993); Antin, Blood 82: 2273 (1993); Porter et al., N. Engl. J. Med. 330: 100-06 (1994). The therapeutic effects of the infused leukocytes are mediated by the potentiation of the effects of GVL, induced after Alo-BMT, by immunocompetent donor T cells that are not tolerant to malignant hematopoietic cells. Slavin et al., Blood 72 (Suppl.l): .407a (1988); Slavin et al., Bone Marrow Transpl. 6: 155-61 (1990); Kolb et al., Blood 76: 2462 (1990); Baer et al., J. Clin. Oncology 11: 513 (1993); Jiang et al., Bone Marrow Transpl. 11: 133 (1993); Drobyski et al., Blood 82: 2310 (1993); Antin, Blood 82: 2273 (1993); Porter et al., N. Engl. J. Med. 330: 100-06 (1994). Unfortunately, only approximately 50-70% of patients with CML who relapse after Alo-BMT respond favorably to Alo-CT. Kolb et al., Clin. Blood 82 (Suppl 1): 840 (1993). In addition, long-term disease-free survival is far from optimal due to failure in response, subsequent relapse, and complications that arise from GVHD and spinal cord aplasia. Finally, the possible anti-solid tumor effects of allogeneic lymphocytes after Alo-BMT have been relatively unknown in comparison to the documented effects of allogeneic lymphocytes on malignant hematopoietic cells.

BRIEF DESCRIPTION OF THE INVENTION The present invention includes a method for treating a human patient with cancer who has undergone a cancer therapy regimen that includes the transplantation of allogeneic stem cells. The term "stem cell transplantation" as used herein includes infusing a patient with hematopoietic stem cells derived from any appropriate source of stem cells in the body. The stem cells can be derived, for example, from the bone marrow, from the peripheral circulation after mobilization of the bone marrow, or from fetal sources such as fetal tissue, fetal circulation and umbilical cord blood. The "bone marrow transplant" is considered in the present to be simply a form of stem cell transplantation. Mobilization of the stem cells from the bone marrow can be achieved, for example, by treatment of the donor with the granulocyte colony stimulating factor (G-CSF) or other appropriate factors (eg IL). -8) that induces the movement of stem cells from the bone marrow to the peripheral circulation.

After mobilization, stem cells can be harvested from the peripheral blood by any appropriate cellular pheresis technique, for example, through the use of a commercially available blood collection device, as exemplified by the delivery device. CS 3000R Plus Blood Cell Collection sold by Baxter Healthcare Coporation. Methods for performing apheresis with the CS 3000 Plus machine are described in Williams et al., Bone Marrow Transplantation 5: 129-33 (1990) and Hillyer et al., Transfusion 33: 316-21 (1993), both publications incorporated herein. in the present by reference Infusion of the hematopoietic stem cells can result in complete and permanent grafting (ie, 100% of the donor's hematopoietic cells), or it can result in partial and momentary grafting, provided the donor cells persist sufficiently to allow the performance of allogeneic cell therapy as described herein. In this way, the term "stem cell transplantation" covers the infusion of stem cells in a patient that results in either partial complete grafting as described above. As used herein, the term "Alo-CT" (allogeneic cell therapy) refers to the infusion of resting allogeneic lymphocytes, i.e., lymphocytes that have not previously been exposed to the T cell activator in vitro; the term "Alo-ACT" (activated cell therapy, allogeneic) refers to the infusion of allogeneic lymphocytes preactivated in vitro with a T-cell activator such as recombinant human interleukin-2 (rhlL-2). These activated donor lymphocytes are called in the present "ADL". It is to be understood that allogeneic lymphocytes infused in a patient need not be infused as a purified preparation of T cells. Although it is possible to infuse a relatively pure T cell preparation, the cells can be infused in the form of a mononuclear cell preparation. , peripheral blood (PBMC). For example, the preparation of PBMC obtained as a result of pheresis with the blood collection device CS 3000R Plus is appropriate for the present invention. This cell preparation is approximately 95% mononuclear cells, most of which are T cells. Under appropriate circumstances, it is still possible to administer allogeneic lymphocytes to the patient by simply supplying whole blood. Both Alo-CT and Alo-ACT can be performed with or without the accompanying in vivo administration of a T-cell activator. Typically, the allogeneic lymphocytes infused are derived from the same donor who provided the stem cells for stem cell transplantation Allogeneic However, infused lymphocytes can be derived from other donors in appropriate circumstances. For example, if the infused lymphocytes are from a limited living space as described below, the same donor or a different donor can provide the cells, depending on the clinical situation. The term "cancer" as used herein, includes all pathological conditions comprising the malignant cells; this may include "solid" tumors that appear in solid tissues or organs (ie, tumor cells that grow as multicellular masses supported by blood spleens), as well as tumor cells that originate from hematopoietic stem cells.

The invention offers a method for treating human patients with cancer with solid tumors, including without limitation breast carcinomas, composed of malignant cells. The patients have undergone allogeneic stem cell transplantation. After transplantation, patients are infused with allogeneic lymphocytes in order to induce a graft response against the tumor in the patient. The infused allogeneic lymphocytes can be activated, prior to infusion, by in vitro exposure to a T cell activator. Whether lymphocytes are activated or not before infusion, the patient can also be provided with a T cell activator in I live in order to provide a continuous stimulus of activation to the lymphocytes after the infusion. The T cell activator comprises at least one activator of the T cell signal transduction pathway. The T cell activator may include, without limitation, one or more of the following activators of the signal transduction pathway: interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7) , interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interferon-alpha (IFNa), interferon-gam a (IFNY), tumor necrosis factors such as TNFa, anti-CD3 antibodies (anti-CD3), anti-CD28 antibodies (anti-CD28), phytohemagglutinin, concanavalin-A and phorbol esters. T cell activators may be native factors obtained from natural sources, factors produced by recombinant DNA technology, chemically synthesized polypeptides or other molecules, or any derivative having the functional activity of the native factor. More preferably, the T cell activator is IL-2, for example human IL-2, recombinant (rhIL-2). The T cell activator used to activate the donor lymphocytes in vitro, and the T cell activator used for in vivo administration, may be the same or different activators of the T cell signal transduction pathway. The allogeneic lymphocytes are they can provide the patient in a series of incrementally increasing amounts, with the patient being inspected for the GVHD signals between increments. If GVHD is not manifested, if the GVHD is not severe and is controllable with normal anti-GVHD prophylaxis, then the patient can be treated with an incrementally higher dose of allogeneic lymphocytes than was provided in the previous infusion. Typically, although not necessarily, the doses are adjusted by logarithmic increments, for example, 105, 106, 107 lymphocytes / kg and so on. Preferably, the allogeneic lymphocytes are HLA-compatible (see below) with the patient, although this is not necessary in all cases, particularly if the infused lymphocytes are from a limited space of life. For example, allogeneic lymphocytes can carry a "suicide gene", which allows cells to be removed after infusion in the patient, by the use of a chemotherapeutic agent. After the infusion of the allogeneic lymphocytes, the patient is inspected for the levels of malignant cells. The invention also includes the treatment of human patients with cancer who have malignant hematopoietic cells, for example patients with chronic myelogenous leukemia or acute lymphocytic leukemia. As in the case of patients with solid tumors, these patients have undergone allogeneic stem cell transplantation as part of a regimen to treat malignancy. After transplantation of allogeneic stem cells, the patient is infused with allogeneic lymphocytes that have been activated in vitro by exposure to a T cell activator before administration to the patient. After the infusion, the patient is inspected for the levels of malignant hematopoietic cells. The patient can also be provided with a T cell activator in vivo. The T cell activator, if used for in vivo activation or administered in vivo, may be as described above for solid tumor modalities. The present invention is particularly useful for those unfortunate patients who, despite a transplant of allogeneic stem cells, continue to exhibit malignant cells as evidenced by open relapse or other indication that the malignant cells have not been completely eradicated. The methods are further applicable to patients who not only fail the response to an allogeneic stem cell transplant, but also fail a response to a cell therapy regimen after transplantation that includes the infusion of allogeneic resting donor lymphocytes. (Alo-CT). In one embodiment, the patient is treated with approximately 10 5 cells / kg to approximately 10 cells / kg of allogeneic ADL and then inspected for malignant cell levels. In an alternative embodiment, the patient can also be treated with the T cell activator in vivo, for example by injection according to a pharmaceutically acceptable carrier. Preferably, the T cell activator is given to the patient over a period of time of two to seven days, more preferably two to five days, and most preferably two to four days. The T cell activator can be administered starting on the same day as the infusion of activated, allogeneic, donor lymphocytes. More preferably, allogeneic donor lymphocytes are HLA-compatible with the patient. HLA-compatible lymphocytes include cells that are completely HLA-matched with the patient. Alternatively, HLA-compatible cells must be at least haploidentical with the patient. If the HLA-compatible lymphocytes are derived from a sibling of the patient, the cells are preferably HLA-fully matched with the patient, although some inequality can be tolerated. For example, HLA-compatible lymphocytes of a sibling may, in some cases, be unequal individual HLA loci. If the HLA-compatible lymphocytes are derived from an unrelated individual, preferably the cells are completely HLA-matched with the patient. The present invention also includes the use of donor lymphocytes, allogeneic, inactivated or activated in vitro, as well as T cell activators in the manufacture of a medicament for the treatment of human patients with cancer as described above. The invention further includes a manufacturing article comprising a packaging material and a container within the packaging material. The packaging material contains a label or an insert or instruction sheet of the package indicating that the contents of the container can be used for the treatment of human patients with cancer, as described above. The container can be a collapsible container comprising opposite walls of flexible material and a flexible tube projecting from the container. The contents of the container may include non-activated lymphocytes or ADL that are allogeneic with respect to the patient to be treated. Alternatively, the container may include a T cell activator.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Survival in percent as a function of the days after intradermal inoculation of 4T1 tumor cells (104) in 13 BALB / c or Fl mice in 3 separate experiments for each mouse race.

Figure 2. Percent survival as a function of the days after a dose of intradermal stimulation of (104) 4T1 cells given to individual BALB / c mice (n = 20) or BALB / c mice (n = 20) immunized intradermally 3 times with 107 irradiated 4T1 cells, given at intervals of 7-10 days. The dose of stimulation was injected 7 days after the 3rd. immunization dose. Figure 3. Chromosome detection and by PCR analysis. Peripheral blood samples were taken from female BALB / c recipient mice, 1, 3, 6 and 9 months after hematopoietic reconstitution with DBA / 2 male donor cells (Division: 1-4, respectively). Division 5: Direct PCR of positive control males. Division 6: positive DNA control. Division 7: no DNA control. Division 8: markers of size Hae III. The intensity of the signal depends on the number of cells, which was different in each sample. Figure 4. Size of the 4T1 tumor as a function of the days after tumor inoculation in 13 single BALB / c mice and 15 chimeric BALB / c mice reconstituted with DBA / 2 hematopoietic cells (DBA-BALB / c). In the tumor cells they were inoculated in chimeric mice 60-90 days after reconstitution with bone marrow cells. The results represent 3 separate experiments.

Figure 5. Tumor size 4T1 as a function of the days after inoculation of the tumor cells in 18 simple Fl (BALB / cx C57B1 / 6) mice and 11 chimeric Fl mice reconstituted with C57B1 / 6 hematopoietic cells (C57-Fl ). Tumor cells were clouded in chimeric mice 60-90 days after reconstitution of the bone marrow cells. The results represent 3 separate experiments.

Figure 6. Development of leukemia in normal BALB / C control mice (group A, n = 10) and in B6 chimeric mice? BALB / c (group B, n = 12) inoculated intravenously with 106 BCL1 cells.

Figure 7. Time intervals necessary for the effective effects of GVL: development of leukemia in BALB / c recipient, adoptive, secondary mice after receiving 105 spleen cells obtained from B6 chimeras? BALB / c inoculated with 106 BCL1 cells in 7 days (group A), 14 days (group B) and 21 days (group C) before adoptive transfer; and from normal BALB / c mice inoculated with 106 BCL1 cells: 7 days (group D), 14 days (group E) and 21 days (group F) before adoptive transfer. Each group consisted of 10 mice.

Figure 8. Amplification of the effects of GVL by allogeneic spleen cells and rhIL-2: development of leukemia in BALB / c receptors, adoptive, secondary mice after receiving 105 spleen cells obtained from B6 chimeras? BALB / c or normal BALB / c mice 7 days after inoculation with 10 6 BLI cells. Group A: untreated chimeras: group B: chimeras injected with rhIL-2; Group C: chimeras infused with spleen cells; group D: chimeras infused with spleen cells and with rhIL-2; Group E: normal BALB / c controls without additional treatment; Group F: normal BALB / c controls injected with rhIL-2; Group G: normal BALB / c controls infused with spleen cells; Group H: normal BALB / c controls infused with spleen cells and with rhIL-2. Each experimental group consisted of 10 mice.

DETAILED DESCRIPTION OF THE INVENTION A series of animal experiments was attempted to evaluate 1) the ability of allogeneic lymphocytes to effect a response to the solid tumor after transplantation of allogeneic stem cells, and 2) the feasibility and efficiency of Alo-ACT with and without the in vivo administration of the T cell activator, after the transplantation of allogeneic stem cells. The concepts developed in animal experiments also extend to the clinical situation to demonstrate efficacy in human patients with breast cancer as well as human patients with cancer not sensitive to Alo-BMT and Alo-CT. The animal experiments discussed below demonstrate the feasibility for the induction of an immune-mediated tumor-graft defect (GVT) in solid tumors., using a murine model of mammary adenocarcinoma derived from BALB / c (H-2) mice. A murine breast cancer cell line (4T1) that is highly tumorigenic in Fl haploidentical Fl (BALB / cx C57B1 / 6) or syngeneic (BALB / c) mice was used, it is only partially tumorigenic in a H-2d congenital race. mice (DBA / 2) and is not tumorigenic in an unrelated MHC strain (H-2b) of mice (C57B1 / 6). 4T1 cells express on their surfaces the major histocompatibility antigens (MHC, for its acronym in English) of class I, addition molecules and addition molecules associated with the return of CD44, but the MHC class II antigens or co-stimulatory molecules such as B7.

Female BALB / c (H-2d) or Fl (H-Dd b) mice were reconstituted with bone marrow cells derived from DBA (H-2d), unequal, minor, from male or with bone marrow cells derived from C57 (H-2b), uneven, major, respectively, 24 hours after lethal, total body irradiation. Receiving mice having donor cells, inserted, unequal, minor or major were inoculated with 4T1 tumor cells, 2-3 months after reconstitution with the bone marrow. Allogeneic donor cells, if differentiated from tumor cells in minor antigens or MHC, were able to affect the development of the primary tumor, which expressed host-like MHC alloantigens. The size of the tumor in the bone marrow chimeras through the MHC antigens or lower was significantly (p <0.05) lower than the tumor size observed in the non-grafted control mice, BALB / c or Fl. All the results show that it is possible to induce a GVT effect by alloreactive cells in a murine model and mammary carcinoma. Previous studies (Cohen et al., J. Immunol., 151: 1803-10 (1993), incorporated herein by reference) have shown that the use of donor lymphocytes, activated, in vitro (ADL) with or without rhIL-2 in vivo (Alo-ACT-rhIL-2) provides significant GVL effects in the mice. However, since 100% survival was observed in all mice given the allogeneic lymphocytes, it was not possible to identify particular improvements in the effects of GVL through the use of ADL or rhIL-2 in vivo. This study also provided confirmation that the effects of GVL are predominantly caused by allogeneic T cells and not by natural killer (NK) cells that were considered until recently to be unrestricted by the MHC. Non-reactive NK cells, B cells and macrophage cells may, however, play a role in the effects of GVL or GVT induced by allogeneic T cells. The results further indicated that the effects of GVL are not due to the cascade of allogeneic responses, inflammatory reactions and cytokine release in vivo that result from GVHD per se. In a further set of experiments reported subsequently, BALB / c, C57B1 / 6 (B6) and (BALB / cx B6) Fl (Fi) mice were used to evaluate various allogeneic cell therapy protocols accompanied or unaccompanied by live from a T-cell activator. BCL1 cells, representing a spontaneous B-cell / B-cell leukemia / lymphoma of origin BALB / c, originally described by Slavin and Strober, Nature 272: 624 (1978), were used as a model of tumor. Infusion of 10 to 100 BCL1 cells in BALB / c mice resulted in a typical B-cell leukemia / lymphoma characterized by splenomegaly of peripheral blood lymphocytosis, subsequent and death in 100% of the recipients. BCL1 causes leukemia in the Fl receivers but also takes more time to develop as compared by the BALB / c receivers. The present inventor investigated the susceptibility of B6 chimeras? BALB / c, well established and completely reconstituted, tolerant, to BCL1 cells. Chimeras were generated by lethally irradiating BALB / c mice and reconstituted 24 hours later with bone marrow cells, B6, depleted of T cells. None of the chimeras showed any clinical evidence of GVHD. Normal BALB / c mice and B6 chimeras - > • BALB / c were injected intravenously with 104, 105, or 106 BCL1 cells.

All normal BALB / c mice developed leukemia within 21-58 days and died, while all well-established chimeras (ie, remaining chimeras 2-3 months of induction of chimerism) survived without evidence of disease for more than 6 months. On the contrary, previous studies showed that early inoculation of BCLl in B6 receptors? BALB / c or B6? Fl resulted in leukemia in all recipients with MRD. Weiss et al, Cancer. Immunol. Immunother. 31: 236 (1990). Adoptive transfer experiments were performed with both normal BALB / c and B6 chimeras - >; BALB / c that have been injected with 106 BCLl cells. Spleen cells (105) were transferred to 10 simple, secondary BALB / c mice on day 7, 14 and 21 after inoculation of BCLl cells. Seven out of 10 secondary recipients who received cells from chimeras removed 7 days after inoculation with BCLl developed leukemia in the space of 44 days. In contrast, none of the secondary receptors that received cells obtained from chimeras inoculated with BCLl cells 14 and 21 days before the cell transfer, developed leukemia. The data suggest that a period of at least 14 days is required for the complete eradication of 106 BLC1 cells, while at 7 days, the eradication of leukemic cells is still incomplete. Additional experiments were carried out on the chimeras to determine the effects of in vivo administration of the T cell activator (rhIL-2). Chimeras were injected with 10 6 Bcll cells and then treated in a variety of manner with rhIL-2 in vivo, lymphocytes or combinations of rhIL-2 and lymphocytes. After seven days, all mice were sacrificed and spleen cells were used for adoptive transfer in secondary BALB / c receptors, as before. All of the secondary BALB / c recipients who received spleen cells from normal, control BALB / c mice developed leukemia. Additionally, no antileukemic effects were detected in normal BALB / c mice, control with rhIL-2, allogeneic splenocytes or both. In contrast, 70% of the chimeras in the group without any additional treatment did not develop leukemia for a period greater than 6 months. Of the 30% who developed leukemia, the onset was delayed to 44-52 days. Of the chimeras that received only B6 lymphocytes, 80% remained free of disease and the remaining 20% showed delayed onset of leukemia. Of the chimeras that were treated with rhIL-2 or that were treated with both rhIL-2 and B6 lymphocytes, 100% were free of disease for more than 6 months. Taken together, these results indicate that chimeras generated by irradiation of BALB / c mice and reconstitution with bone marrow cells, B6, depleted of T cells are able to resist the leukemogenic potential of BLC1 cells (which are of BALB / origin). c), assuming that the chimerism was established and the recipients are immunocompetent. This is despite the fact that chimeras are completely intolerant to BALB / c alloantigens, since these chimeras have been shown to be completely tolerant to host alloantigens (BALB / c) and accept indefinitely allografts of skin type donor Levite and Reisner, Transplantation 55: 3 (1993). In addition, the chimeras are resistant to BCLl cells in the absence of GVHD. Thus, antitumor effects in tolerant chimeras may include recognition of tumor-specific or tumor-associated cell surface determinants different from other determinants of the host-type major histocompatibility complex (MHC), independent of GVHD. Significantly, improvement of the effects of GVL can be achieved, without GVHD or alternatively, with controllable GVHD, by administration after ADL transplantation with or without a short course of rhIL-2 with a relatively low dose. It is especially advantageous to use graduated increments of allogeneic cells while controlling the GVHD. The longer the time intervals from BMT to cell therapy, the less likely it is to develop uncontrolled GVHD and the greater the number of donor T cells that may be allogeneic. See, Slavin et al., J. Exp. Med. 147: 963 (1978); Slavin et al., Cancer Invest. 10: 221-7 (1992). This can be contrasted to mice with residual tumor cells giving allogeneic T cells during the early post-BMT period. The allogeneic T cells infused in these cases may become tolerant to the host, resulting in the GVHD. In this way, the infusion of allogeneic lymphocytes, especially after in vitro and in vivo activation of donor T cells, by T cell activators such as rhIL-2, makes it possible to infuse donor T cells, not tumor tolerant, post-BMT, relatively late that are accepted by the recipient but that engender potent GVL effects. T cells can be given in graduated increments, with proportionally more cells administered as the time from BMT increases. The results and indications derived from the experiments with mice were extended to the clinical situation with human patients suffering from breast cancer and malignant hematological disorders, including acute and chronic leukemias. Specifically, the present inventor has discovered that a therapeutic regimen of Alo-CT may be effective in the treatment of breast cancer after allogeneic BMT. The present inventor has also discovered that activated donor lymphocytes can provide antitumor effects even beyond those obtained with non-activated allogeneic lymphocytes. For example, Alo-ACT and the T cell activator in vivo can be successfully used in a clinical situation to treat relapse after Alo-BMT. In this way, the results in human patients provide the important confirmation and extension of the animal data reported previously. More particularly, the present inventor has discovered that in vitro activation of the PBLs of the donor prior to infusion in the patient provides a means to induce remission following an unsuccessful bone marrow transplant and cellular immunotherapy regimen. Surprisingly, the PBL of the in vitro activated donor provided a measurable GVL effect when the same cells, absent from the in vitro treatment, were found to eradicate the tumor cells. It is noteworthy that, in some cases, these non-activated cells, although not exposed to the T-cell activator before the infusion, were, however, placed on the activation T cell activator in vivo after the infusion. In contrast, PBLs from the same donor were effective when preactivated before the infusion (Alo-ACT approach) and were accompanied by the T cell activator in vivo. Furthermore, it is demonstrated here that an Alo-ACT regimen can be attempted in this situation without necessarily inducing clinically significant GVHD. To document the ability of allogeneic lymphocytes to provide a therapeutic effect in patients with solid tumor, a human patient with water myeloid leukemia (AML) and recurrent breast cancer was treated with induction chemotherapy, allogeneic stem cell transplantation and allogeneic cells after transplantation (see Example 3 below). The approach in the treatment of this patient was oriented towards AML, although most of the components used for induction chemotherapy are known to be active against breast cancer as well. However, the intensity of the dose was less than optimal for the treatment of recurrent breast cancer, and it would not be expected that a patient with a recurrent, aggressive breast cancer would respond to this "sub-optimal" chemotherapy. Therefore, the response of breast cancer can be attributed to immunotherapy mediated by allogeneic cells, received by this patient. As an illustrative example to demonstrate the clinical efficacy of Alo-ACT plus the treatment regimen with T cell activator in vivo, a human patient with chronic myelogenous leukemia (CML) who has a very poor prognosis was pretreated (see Patient No. 1 in Example 3, below). Chronic myeloid leukemia (CML) is a hematological disorder that is the result of the neoplastic transformation of pluripotent stem cells. The chromosome Philadelphia (Ph) was first described in 1960 as an abbreviated chromosome found in the bone marrow of patients with CML. The Ph chromosome is the result of a reciprocal translocation between the long arms of chromosomes 9 and 22. Potential breakpoints on chromosome 22 occur in a first region of 5.8 kb called the breakpoint group region (bcr) . The breakpoint group region is part of a large bcr gene that contains four exons. Potential breakpoints on chromosome 9 are scattered over a distance of at least 100 kb, but all are located 5 'to the c-abl proto-oncogene. The Ph translocation transfers the c-abl gene from its position on chromosome 9 to the Ph chromosome. Because 90% of CML patients carry the Ph chromosome, it is the hallmark of CML, and is diagnostic of the disease. Approximately 5% of acute lymphocytic leukemia (ALL) in childhood and 30% of adults also carry the Ph chromosome. The Ph chromosome in CML and ALL result from the same translocation of c-abl to different introns of the bcr gene . The removal of cells that exhibit the Ph-karyotype is an indication of remission. An alternative method of assessing the presence of the Ph chromosome is through the use of the polymerase chain reaction (PCR) to detect the bcr / abl transcript. The elimination of the bcr / abl transcript in the PCR analysis is indicative of the successful elimination of the cells that lead to the CML. Prior to treatment, the patient (Patient No. 1 of Example 3, below) had relapsed after Alo-BMT and remained positive for CML markers after an Alo-CT course accompanied by in vivo treatment with rhIL-2. . The patient had not experienced GVHD as a result of bone marrow transplantation or the Alo-CT / rhIL-2 regimen. Peripheral blood leukocytes (PBL), taken from the same HLA-matched brother who donated cells for Alo-BMT, were preactivated in vitro by incubation with rhIL-2. The activated lymphocytes of the donor were administered at a dose between 107 and 108 cells per kilogram of body weight. This was followed immediately by a three-day course of rhIL-2 administered in vivo in order to provide an additional stimulus for activation after the infusion of the cells into the patient. The patient treated thus has not experienced any clinical laboratory sign of GVHD, and has a completely normal morphology of the bone marrow. Significantly, after the Alo-ACT / rhIL-2 regimen, the PCR test for the presence of the bcr / abl fusion product became negative. After 28 months after treatment, the patient did not show evidence for the Ph chromosome, either by cytogenetic analysis or PCR. Additional patients treated with allogeneic cell therapy are provided in Example 3, below. In a preferred embodiment for the treatment of human patients with solid tumors, donor PBLs, inactivated or activated in vitro, are infused into the patient after transplantation of allogeneic stem cells.

In general, donor PBLs were infused after the patient had at least achieved partial hematopoietic recovery from stem cell transplantation; in many cases, the longer the time interval from stem cell transplantation to the administration of donor PBLs, the less lymphocytes can be provided since the risk of uncontrollable GVHD is proportionally less at later times after transplantation. The patient can be attended with graduated increments of the donor PBLs, typically starting with 105 or 106 T cells / kg and progressively in logarithmic increments, for example, 107, 108, 109 T cells / kg depending on the minimum GVHD (controllable) or not, after the previous infusion. If used, fewer activated lymphocytes from the donor are proportionally administered compared to the donor inactivated, corresponding PBLs. This is because activated lymphocytes, although they may generate a higher antitumor effect compared to inactivated lymphocytes, may also put the patient at some high risk of GVHD. However, it should be noted that if ADL are used with a limited life span, then issue the risk of the GVHD and large ADL numbers can be used. For example, as discussed below, allogeneic lymphocytes can be transduced with a "suicide" gene construct that allows the infused cells to be selectively removed after they have exerted the effect of antitumor cells in the patient. For the activation of the donor PBLs, the cells are incubated in rhIL-2 at a concentration of 60 IU / ml at 12,000 IU / ml, preferably at 600 IU / ml up to 8,000 IU / ml, and in the most preferred at 6,000 IU / ml. It will be apparent that these concentrations can be varied to suit the particular incubation media, different batches and rhIL-2 preparations, and other routine variations of clinical and laboratory procedures. For example, if the T cell activator comprises monoclonal antibodies such as anti-CD3 and / or anti-CD28 used in conjunction with rhlL-2, then a correspondingly lower concentration of rhIL-2 may be required. Activators of T cells, other than IL-2, may be employed in the present methods, while the PBLs of the donor are activated appropriately. These alternative T cell activators may include, without limitation, interleukin-1 (IL-1), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin- 7 (IL-7), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interferon-alpha (IFNa), interferon-gamma (IFN?), Factors of tumor necrosis such as TNFa, anti-CD3 antibodies including antigen binding fragments thereof (anti-CD3), anti-CD28 antibodies including the antigen binding fragments thereof (anti-CD28), phytohemagglutinin, concanavalina-A and phorbol esters. The donor PBLs are incubated in the T cell activator until a sufficient level of activation is achieved. For example, the cells can be incubated in the T cell activator such as rhIL-2 for 2 to 14 days, preferably for 4 or 5 days. The duration of the incubation can be varied to adjust to routine variations in temperature, media formulations, normal swings in the sensitivity of the PBL, the use of additional cytokines required to optimize cell growth and activation, and other routine variables, with the condition that the PBL achieve an appropriate state of activation.

For example, if relatively large numbers of cells are desired for infusion in the patient, then a correspondingly enlarged incubation time may be required. Several laboratory tests can be used to determine an appropriate endpoint for the in vitro activation period. These could include classification of fluorescent activated cells (FACS, for its acronym in English), to detect several phenotypes of T cells, relevant, and the measurement of the precursor activity of cytotoxic T lymphocytes (CTLp). To diminish or eliminate the possibility of GVHD, allogeneic lymphocytes of the donor which have a limited period of life can be used. For example, donor lymphocytes can be transduced with a susceptibility factor, or "suicide gene" that makes the lymphocytes susceptible to a chemotherapeutic agent. See, for example, Tiberghien, J. Leukocyte Biol. 56: 203-09 (1994). In one embodiment, it is used as the suicide gene thymidine kinase for the simple herpes virus (HS-tk). Cells expressing HS-tk are sensitive to elimination by exposure to acyclovir or ganciclovir. The HS-tk gene can be transferred to T cells via a retroviral vector containing appropriate promoters, selectable markers and / or other flanking elements. Tiberghien et al., Blood 84: 1333-41 (1994); Mavilio et al., Blood 83: 1988-97 (1994). After infusion in the patient, the lymphocytes can be removed selectively, after an antitumor effect has been generated, but before the onset of severe GVHD. Other methods for limiting the life span of the allogeneic lymphocytes infused may include without limitation, irradiation, photosensitization and the use of anti-lymphocyte antibodies. The exact number of allogeneic lymphocytes infused may depend on the availability and previously identified risk factors of the patient for GVHD. For example, the patient can be started with 105 cells / kg, with increase by a logarithmic increase (1) every 1-4 weeks if the GVHD is not developed after the previous administration. To allow continued activation of the allogeneic lymphocytes after infusion in the patient, the T-cell activator such as IL-2 can be administered to the patient by subcutaneous injection or any other method appropriate for the routine distribution of drugs. This in vivo administration of the T cell activator is preferably initiated on the same day as the infusion of the allogeneic lymphocytes, or it can be started at any time up to about 7 days after the infusion. The T cell activator administered in vivo can be given for a period of time of 1 to 14 days, preferably for a period of time of 2 to 7 days, more preferably for a period of time of 2 to 4 days. , and most preferably for 3 days. In a preferred embodiment, rhIL-2 is infused into the patient for 3 days at a concentration of 106, to 107, preferably 6 x 106, IU / m2 of the body surface area. The time course and concentrations may be varied to conform to clinical indications such as the trend for GVHD or the patient's ability to tolerate the chosen T-cell activator. After the treatment of the Alo-CT and / or Alo-ACT, and if desired, the in vivo treatment with the T cell activator, the patient is inspected for the signs and symptoms of the GVHD, and where appropriate, for the levels of malignant residual cells. Inspection of residual malignant cell levels may include clinical inspection of the patient for physical symptoms of relapse. Preferably, the inspection includes the evaluation of diagnostic criteria that allow the detection of malignant cells before the manifestation of physical symptoms. For example, cytogenetic studies can be performed in which the macroscopic morphology of the chromosome is examined. Alternatively, the inspection may include the use of molecular probes to detect, for example, aberrant nucleic acid sequences, characteristics of malignant cells. In the case of CML, the patient can be inspected for the Ph chromosome evidence as revealed by the cytogenetic examination or as revealed by the PCR analysis of the bcr / abl transcript in the nucleic acid preparations taken from the peripheral circulation. Other specific disease markers may be equally useful, such as alpha-RAR labeling for AML-M3 as well as a variety of markers developed for solid tumors of varying origin. The disappearance of the selected markers is an indication that the patient has gone into remission as a result of the Alo-CT or Alo-ACT regimen and treatment. In the absence of specific disease markers, other markers can provide equally useful information about the state of the cells derived from the host against cells derived from the donor. For example, the presence or absence of specific markers of the sex chromosome in the host circulation can be used to inspect the female-to-male or male-female combinations of the host / donor. Likewise, the presence or absence of specific host bands after the search for VNTR (tandem, nuclear, variable repeat) is also indicative of the efficacy of cell therapy. The invention will be further understood with reference to the following illustrative embodiments, which are exemplary only, and should not be taken as limiting the true scope of the present invention as described in the claims.

EXAMPLE 1 Induction of an Graft Effect Against Tumor in a Murine Model of Breast Carcinoma Materials and methods Mice BALB / c (H-2d) and Fl (BALB / cXC57Bl / 6) (H-2d b) mice of 10-12 weeks, DAB / 2 (H-2d) and C57B1 / 6 (H-2b) mice 7-9 weeks were obtained from Harlan Sprague Dawley, USA, and kept in a specific pathogen-free animal housing at Hebrew University Hadassah Medical School, in accordance with Israel's specific national laws.

Tumor 4T1 is one of a series of subpopulations isolated from a spontaneous, individual breast tumor of a BALB / cfC3H mouse.

Dexter et al., Cancer Res. 38: 3174-81 (1978). It is maintained by passage in vitro in RPMI 1640 medium containing 10% of Bovine Serum Fetal heat inactivated (FBS) (Grand Island Biological CO., Grand Island, NY), 2 mM glutamine, 100 mg / ml streptomycin, 100 U / ml penicillin and 1% non-essential amino acids. The preparation of cells for injection includes collection by 0.25% trypsin in 0.05% EDTA, washing with RPMI 1640 and resuspension in Hank's medium for intradermal injection (ID) in mice in a volume of 0.1 ml. All tissue culture media and reagents were purchased from Biological Industry, Beit Ha'emek, Israel. Cells were maintained at 37 ° C in a C02 / 5% / air incubator, humidified.

Measurement of the growth of the main tumor in vivo The tumor size was measured once a week in two perpendicular dimensions in a calibrator. The size of the tumor in cm3 was calculated by the formula (a x b2) / 2 where a is the largest dimension and b is the smallest dimension of the tumor.

Flow cytometric analysis An amount of 5 x 106 cells was stained directly with the following monoclonal antibodies: Fluorescein-Isothiocyanate (FITC) anti-H-2K and R-phycoerythrin (PE) anti-I-Ad (Pharmigen, CA, USA). Indirect dyeing was carried out using the ICAM-1 antibodies (Takei, J. Immunol., 134: 1403-07 (1985)), VCAM-1 (Miyake et al., J. Exp. Med. 173. 599-607 ( 1991)), CD44 (Trowbridge et al., Immunogenetics 15: 299-312 (1982) and B7-1 (Razi-Wolf et al., Proc. Nati, Acad. Sci. USA 89: 4210-14 (1992)), anti-mouse A fragment (Fab) 2 purified by affinity, conjugated to FITC of mouse anti-rat IgG, was used as a secondary antibody (Jackson ImmunoResearch Laboratories Inc., PA., USA). of dyeing on ice for 30 minutes, followed by washing with phosphate buffered saline containing 1% bovine serum albumin and 0.03% sodium azide.The cells were fixed in 1% paraformaldehyde and analyzed by FACScan cytometry using the Lysys II program (Becton Dickinson, Santa Clara USA).

Immunization Protocol Cultured 4T1 cells (107) were irradiated (120 Gy) to measure the absence of cell proliferation of the immunization dose, then injected intradermally (ID) into BALB / c mice, single 3 times at 7-10 intervals days. Seven to 10 days after the last immunization dose, a stimulus of 104 fresh, non-irradiated 4T1 cells was given ID. The parallel was inoculated with a control group of unimmunized, simple BALB / c mice with 104 fresh 4T1 cells.

Induction of bone marrow chimeras Female BALB / c mice were exposed to a lethal dose of total body irradiation (TBI) of 9 Gy, at 24 hours prior to intravenous injection with 107 bone marrow cells derived from DBA / 2 male mice. Female mice Fl (BALB / C X C57B1 / 6) were exposed to a lethal dose of TBI of 11 Gy 24 hours before intravenous injection with 107 bone marrow cells derived from male C57B1 / 6 mice. The TBI was distributed by linear accelerator to an energy of 6 mev. with a dose rate of 1.9 Gy / min. The bone marrow cells were prepared by flooding the RPMI 1640 medium through the reeds of the bone marrow.

Femora and the tibia of the donors with a 25 gauge needle.

Polymerase chain reaction (PCR) The PCR is carried out as previously described. Pugatsch et al., Leukemia Res. 17: 900-1002 (1993). Again, the blood samples were used in sterile distilled water, and centrifuged for 10 seconds at 12,000 g in an Eppendorf centrifuge. The supernatants were discarded and 50 ml of 0.05 M NaOH was added to the cell pellets. The samples were divided during 10 minutes, and 6 ml of 1 M Tris, pH 7.2 was added. The samples were centrifuged for 5 minutes at 12000 g, and care was taken to use only the supernatants for the assay. Oligonucleotide primers were chosen according to the published sequence of a chromosome-specific gene (Gubbay et al., Nature 346: 245-50 (1990)) from position 22-39 of the 5 'primer and from 342-359 of the 3 'primer, respectively. The DNA was amplified in an MJR-Mini cycler in a total volume of 50 ml. The primers are added to a final concentration of 100 p.sup.-ol and Taq DNA polymerase (Appligen, France) at 1U / sample. The following program was used: 94 ° C, 30 minutes; 50 ° C, 45 minutes; 72 ° C, 1 second; for a total of 35 cycles. The reaction products were visualized on 1.6% agarose gels (Sigma, St. Louis, USA) containing 10.05% ethidium bromide.

Results Phenotypic analysis of surface markers of 4T1 cells The phenotypic characterization of the surface markers of murine mammary cells 4T1 was carried out using the flow cytometry FACS analysis as described above. Cultured 4T1 cells express the class I H-2d antigens (93%) as well as addition molecules similar to ICAM, VCAM (64%, 59%, respectively) and the addition molecules associated with a CD44 return (76%) . 4T1 cells do not express the class II antigens of I-Ad, or costimulatory molecules similar to B7-1.

Tumorigenicity of 4T1 The ability of 4T1 H-2d cells of BALB / c origin to test tumors in hosts compatible with H-2 as well as incompatible was tested. Intradermal inoculation of 104 4T1 cells in syngeneic BALB / c H-2d mice resulted in a local tumor measurable in 100% of the mouse within 21 days. The main tumor eventually led to tumor metastasis and death of all mice in the space of a mean of 39 days (Figure 1). A delayed appearance of the local tumor was observed only in a fraction of the BALB / c hosts (44%) after the inoculation of 103 4T1 cells. Intradermal inoculation of 106 4T1 cells in congenital DBA / 2 H-2d mice elicited total tumor and death in only 20% of the mice, while a lower dose in cells (105) led to a local tumor that returned momentarily. days after the tumor inoculation. A measurable primary tumor and lung metastasis appeared in 84% of the host mice (H-2d semi-allogeneic in the space of 38 after inoculation of the 104 4T1 cells.All H-2d b hosts with tumor developed died in the space of an average of 50 days (Figure 1). Inoculation of 4T1 H-2d cells in C57B1 / 6 H-2b allogeneic mice failed to elicit the tumor in any of the hosts at a cell dose of 5 x 105. The results show that 4T1 mammary tumor cells having antigens H-2d can be highly tumorigenic in haploidentical and completely histocompatible H-2 hosts mainly (BALB / c and (BALB / cx C57B1 / 6) F1, respectively), weakly tumorigenic in host (DBA / 2) minor histoincompatible and are non-tumorigenic in main histoincompatible hosts (C57B1 / 6).

Immunogenicity of 4T1 cells 4T1 (107) cells irradiated 3 times in 7-10 day intervals in syngeneic BALB / c mice were inoculated prior to stimulation with a fresh tumorigenic dose of 104 4T1 cells. These multiple indications of irradiated 4T1 cells did not induce immunological protection against stimulation of unirradiated 4T1 tumor cells (Figure 2). All mice died with a major, local, large tumor as well as lung metastasis at a mean of 42 days after stimulation inoculation. The simple BALB / c mice inoculated with a dose of stimulation only measured in a mean of 45 days. Inoculation of either a lower dose of irradiated cells or the same cell dose given only once or twice failed to induce tumor immunity (data not shown).

Induction of BM chimeras through minor and major histocompatible antigens Female mice BALB / c (H-2d) and (BALB / cx C57B1 / 6) Fl (H-2d b), receptors were reconstituted with bone marrow cells (H-2d) derived from DBA / 2, minor histoincompatible, derived from males and (H-2b) derived from C57B1 / 6 major histoincompatible, respectively, after a lethal dose of TBI (data not shown) the induction of hematopoietic chimeras was tested using molecular analysis for the detection of chromosome sequences And of the male samples and peripheral blood cells taken 1, 3, 6 and 9 months after reconstitution with BM. The results presented in Figure 3 show the evidence for the presence of the Y chromosome marker as early as one month after the inoculation of bone marrow cells and the continuation of their dominant presence throughout a period > 280 days in DBA-BALB / c chimeras. A stable hematopoietic chimerism with mild symptoms of chronic GVHD (mild weight loss and skin loss) was established through minor histocompatible antigens and without overt symptoms of GVHD through major histocompatible antigens. Respectively, the survival time of 9 DBA-BALB / c chimeras was 261 (median) with an interval of 147-341 and > 300 days in 14 C57B1 / 6-F1 chimeras.

Tumorigenicity of T41 cells in hematopoietic chimeras 4T1 tumor cells having H-2d histocompatible antigens of BALB / c origin were inoculated intradermally into individual BALB / c (H-2d) mice and into BALB / c chimeras having minor histoincompatible hematopoietic cells of DBA / 2 origin (H -2d). The size of the tumor as a function of the days after the tumor inoculation is presented in Figure 4. A local tumor measurable on day 20 increased markedly with time up to 1.43 cm3 in the BALB / c single mice. A significantly smaller tumor (p <0.01) with limited growth of up to 0.3 cm3 was observed in the chimeric DBA-BALB / c mice. Inoculation of 4T1 cells in simple mice Fl (H-2d / b) and Fl chimeras having major histocompatible hematopoietic cells of C51B1 / 6 origin, showed a primary local primary tumor of 0.26 cm3 that was further increased to 2.40 cm3 in mice F simple and was significantly lower (p <0.05) with limited growth up to 0.31 cm3 in the chimeric C571B1 / 6-Fl mice (Figure 5).

EXAMPLE 2 Effects of Graft versus Tumor in a Murine Model of Leukemia 1. Procedures Mice BALB / c, C57B1 / 6 (B6) and (BALB / c x B6) Fl (Fl), males and females 8-12 weeks of age, inborn were found from Jackson Memorial Laboratory, BAR Harbor ME, USA. The mice were kept in small isolated cages (5 animals in each cage) and fed with sterile feed and acidic water (pH 3.0) by induction of chimerism. Inoculation of leukemia and post-transplant immunotherapy were carried out in a non-isolated, normal animal scaling. BALB / ca mice were exposed to a single dose of total body irradiation (TBI) of 10 Gy from a gamma source 150-A60CO (Atomic Energy Canada) with a skin focus, distance of 75 cm at a speed Dosage of 58 cGy / min. Twenty-four hours later, the lethally irradiated mice received 5 x 10 6 bone marrow cells depleted of T cells from B6 donors via the lateral vein of the tail. The marrow inocula were enriched for the stem cells and depleted of immunocompetent T cells by agglutination of soybean lectin, according to Reisner et al. Reisner et al., Proc. Nati Acad. Sci. USA 75: 2933 (1978), with minor modifications as reported in Schawarz et al., J. Immunol 138: 460 (1987). RhIL-2 was delivered by Dr. C.R. Franks, EuroCetus BV. Amsterdam, The Netherlands, as 1 mg of Proleucine (18 x 106 International Units = 3 x 106 Cetus Units). RhIL-2 was initially diluted with water for injection and subsequently re-diluted with 5% dextrose. BCL1 cells were maintained in vivo in BALB / c mice by intravenous passages of 106-107 peripheral blood lymphocytes (PBL) obtained from mice having tumor. All recipients of BCL1 cells developed splenomegaly and marked lymphocytosis in the blood at the time they were sacrificed to be used as donors for BCLl cells in experimental mice. Slavin et al., Cancer Res. 41: 4162 (1981). The PBL accounts of all the experimental groups were carried out weekly. The onset of leukemia was defined as PBL counts exceeding 20,000 / mm3. The maximum value of the PBL accounts for the disease usually changed >; 100,000 / mm3. The survival of the BCLl receivers was inspected daily. Chimerism (ie, the presence of hematopoietic cells, not auto, ie, donor, in a recipient) was determined 4-9 weeks after BMT from spleen or peripheral blood cells, as described previously. Lapidot et al., Blood 73: 2025 (1989). The chimerism was reconfirmed by titrating the PBLs using a microcitotoxicity-dependent, in vitro test, with specific alloantisera (BALB / c anti-B6 and anti-BALB / c B6) and rabbit complement, before inoculation with BCLl cells. The percentage of host or donor type cells was determined by the trypan blue ink exclusion test. Specific alloantiseres were prepared by cross-immunizing mice with a full thickness skin allograft followed by 6 intraperitoneal injections of 30-50 x 10 6 donor spleen cells, given with a 1-2 week separation. The mice were bled and the sera were stored at -70 ° C. Chimerism was tested by classifying each lymphocyte sample with both antisera: lymphocytes obtained from Fl-receivers were used 100% by both antisera BALB / c anti-B6 and B6 anti-BALB / c, while lymphocytes obtained from of the B6-BALB / c chimeras were used 100% only by the anti-B6 BALB / c antiserum; the anti-BALB / c B6 antiserum was used to confirm the elimination of the host cells. The net percentage of chimerism was calculated as follows: percentage of cells used after treatment with anti-B6 BALB / c antisera (average of assays involved) minus lysed cells after treatment with B6 antisera anti-BALB / c minus lysed cells with complement only. 2. Results Evidence of Chimerism in BALB / c Mice Transplanted with Bone Marrow B6 Exhausted from T Cells As described above, they were lethally irradiated and BALB / c mice were reconstituted with B6 bone marrow cells depleted of T cells. Chimerism was confirmed by assaying the PBL briefly after transplantation and again three months later, immediately before inoculation with BCLl cells. All mice were found to be chimeric. The percentages of the donor type cells in the blood varied between 74 to 100%. None of the chimeras showed any clinical evidence of GVHD and the body weight of the chimeras was comparable to the body weight of the normal controls (data not shown).

Resistance of the Chimeras to BCLl Normal BALB / c mice and B6-BALB / c chimeras were intravenously injected with 10 6 Bcll cells. All normal BALB / c mice developed leukemia, mostly in the space of less than 40 days (median 21 days), and died, while the 10 tested chimeras survived without evidence of disease during > 6 months (Figure 6). A total dose of 102 BCLl cells is sufficient to cause 100% death of leukemia in the normal BALB / c received (data not shown). Slavin et al., Cancer Res. 41: 4162 (1989).

Elimination of BCLl clonogenic cells in Chimeras B6? BALB / c without GVHD None of the B6-BALB / c chimeras exhibited any clinical evidence of GVHD. In order to track the fate of large numbers of clonogenic BCLl cells given to B6 chimeras - > BALB / c, the adoptive transfer experiments were carried out. Ten spleen cells (prepared from a pool of 3 chimeras) were transferred to 10 simple, secondary BALB / c mice 7, 14 and 21 days after inoculation with 10 6 BCL 1 cells (Figure 7). With the exception of an individual mouse (1/30), all adoptive receptors of control spleen cells, obtained from normal mice 1, 2, and 3 weeks after inoculation with BCLl cells developed leukemia in the space of 37 days and they died. Seven of 10 secondary recipients of cells obtained from inoculated chimeras 7 days after the transfer of the cells developed leukemia in the space of 44 days. In contrast, none of the adoptive receptors of spleen cells obtained from B6 chimeras - > BALB / c at 14 and 21 days after inoculation with BCLl developed leukemia when it was inspected for more than 6 months. The data suggest that a period of at least 14 days is required for complete eradication and / or inactivation of the 106 BCLl cells. While at 7 days the eradication of leukemic cells is still incomplete.

Amplification of the effects of GVL by cells de1 Bazo Al < Ogeneic, Immunocompetent and rhIL-2 Therapy in Chimeras Inoculated with BCLl Cells Twenty-four normal BALB / c mice and 24 well-established B6-BALB / C chimeras were injected with 106 BCLl cells. The injected chimeras were divided into 4 groups. (A) Chimeras B6 - > BALB / c that serve as controls without additional therapy; (B) Chimeras B6? BALB / c receiving rhIL-2 (10,000 IU x 3 / day intraperitoneally for 5 days) starting one day after inoculation with leukemic cells (C) Chimeras B6 - > BALB / c receiving 107 normal, immunocompetent B6 spleen cells; (D) Chimeras B6? BALB / c receiving both 107 normal B6 spleen cells and rhIL-2. For comparison, several controls were included: (E) a control group of BALB / c mice inoculated with 106 BCLl cells without additional therapy; Normal BALB / c mice inoculated with 106 BCLl cells received either rhIL-2 (F) or allogeneic spleen cells (G) or both (H). Seven days later all the mice were sacrificed and their spleen cells were used for the adoptive transfer experiments to assess the presence of clonogenic BCLl cells.

Secondary BALB / c recipients (5 in each group) received 105 spleen cells obtained from a pool of 3 control BALB / c mice or from 3 B6-BALB / c chimeras from each experimental group. Equalization results were obtained when the experiment doubled with the three remaining mice in each group. Therefore, the data were pooled and each experimental group shown in Figure 8 consists of 10 mice. All of the secondary BALB / c receptors that receive spleen cells obtained from normal BALB / c mice (E) developed leukemia in the space of 32-37 days. Seventy percent of secondary recipients who receive spleen cells obtained from B6 chimeras? BALB / c (group A) did not develop leukemia during > 6 months, while in 30% who did not develop leukemia the onset of the disease was delayed (start in the space of 44-52 days). B6 - »BALB / c chimeras treated with rhIL-2 (B) allogeneic immunocompetent donor splenocytes (C) or the combination treatment of both (D) exhibited marked resistance against leukemia, with no evidence of disease during > 6 months in all secondary recipients of spleen cells obtained from groups B and D and with delayed onset of leukemia only in 20% of mice that received spleen cells from group C. No effects were detected antileukemia in normal control BALB / c mice treated with rhIL-2, allogeneic splenocytes or both (F, G and H, respectively).

EXAMPLE 3 CLINICAL RESULTS 1. Breast Cancer A 40-year-old female patient presented who had developed a mass in the upper middle quadrant in the left breast at the age of 37 years. The physical examination revealed an indefinite mass in that region and a mass of 4 x 3 cm in the left axilla. The excisional biopsy was taken from the breast mass and the pathological examination revealed a grade III multifocal infiltration ductal carcinoma with three masses of 3 x 2 x 2, 1.5 x 1 x 1 and 1 x 1 x 1 cm in size . In addition, there was tumor invasion to the lymphatic spleens with a positive surgical margin. The patient was treated with 7 cycles of CAF (Cyclophosphamide, Adriamycin and 5-Fluoro-uracil). Chemotherapy was followed by upper left quadrantectomy and dissection of the axillary lymph node. The pathological report of this specimen rebelled three residual foci of infiltration ductal carcinoma of l x l x l, l x l x 1.5 and 0.5 x 0.5 x 0.5 cm in size. Three of the 17 nodules were comprised of cancer. The patient completed irradiation of the 56 Gy chest followed by a booster dose of 14 Gy to the tumor area using the irradiation of the 12 MeV electron beam. Twenty-three months later a mass of 1.5 cm was noted in the mid aspect of the scar of the quadrantectomy adherent to the chest wall. FNA aspiration was performed and cytological analysis revealed malignant cells that were consistent with breast cancer. The blood count at that time showed HgB DE 7.5% and WBC of 1.3 X 109 / L. Bone marrow biopsy was performed and the diagnosis was compatible with AML-M2. Analysis of the hemocytoblast phenotype by the fluorescence activated cell sorter showed HLA-DR of 76%, CD34, 65%, CD33 66%, CD13 56%, CD15 41%, CD11B 40% and CD11C 80%. The systemic evaluation included full body CT scan, abdominal ultrasound, liver scan, bone scan, CA-15-3 and CEA; all were within the normal range. The patient was treated with a cycle of amscarina and cytosar of high dose with the subsequent disappearance of the hemocitoblasts in the bone marrow. There was a slight decrease in the size of the mass of the chest wall. Four months after the diagnosis of AML, the patient underwent transplantation of allogeneic stem cells, depleted of T cells from a non-sensitive sibling of MLR completely HLA, A, B, C, DR and DRBl-equaled. The conditioning protocol included immunosuppression before transplantation with anti-thymocyte globulin (Fresenius) 10 mg / kg for 4 consecutive days and the subsequent administration of busulfan 4 mg / kg / day x 4, thiotepa 10 mg / kg / day x 1 , cytoxan 50 mg / kg / day x 4 and intrathecal ARA-C for the prophylaxis of CNS disease. Reduction of T cells was achieved by adding the monoclonal anti-human lymphocyte anti-lymphocyte antibody (CDw52) (Campath-IG provided by Dr. G. Hale, Oxford University, UK) at 0.3 ug / 106 nucleated cells to the bag that it contains the cells of the marrow as previously described (Naparstek et al, Exp. Hematol 17: 723 (abstr.) (1989)). The graft (ANC >; 0.5 X 109 / L, PLT > 25 x 109 / L) was documented on the 21st day after transplantation. After transplantation, cytogenetic studies revealed complete reconstitution with cells derived from the donor in the blood. Ten weeks after the transplant, there were no clinical signs of GVHD; therefore, the patient was treated with allogeneic cell-mediated immunotherapy (Alo-CT) consisting of the infusion of blood lymphocytes from the donor at an equivalent cellular dose of 1 x 10 5 T cells / kg.

Four weeks later, a momentary deterioration of the liver function tests was observed (GGTP 712, ALT 301 and AST 258 Units). No other clinical findings indicative of GVHD were noted. Twenty weeks after transplantation, a higher dose of donor blood lymphocytes consisting of 0.6 x 10 6 T cells / kg was given. More than 8 months after the transplant, the patient was still free without evidence of breast cancer or AML. There is no evidence of acute or chronic graft-versus-host disease (GVHD), developed although the patient did not receive anti-GVHD prophylaxis. The present inventor is not aware of some cases in which a patient with this recurrent, aggressive breast cancer that appeared in the stable complete response after receiving only the "sub-optimal" chemotherapy as used in this patient.

II. Hematological malignancies Patient No. 1. A 17-year-old man with accelerated phase CML was admitted to the Hadassah University Hospital Department of Bone Marrow transplantation for the allogeneic BMT. The patient had a brother HLA-A, B, DR, DRB1 matched non-reactive in the culture of bilaterally mixed lymphocytes for allogeneic BMT. Cytogenetic analysis before transplantation of the patient described 100% positive pH 1 in the spontaneous metaphases of the bone marrow with three different clones of malignant translocation: 35% 46XY t (9:22); 35% 46XY t (9:22) and (15) (q26); 30% 46XY t (9:22) and (2) (q37). Additionally, the patient was classified as 100% positive for the bcr / abl fusion product, as detected by PCR in a peripheral blood sample. Conditioning before transplantation included cyclophosphamide (60 mg / kg x 2 days) and total body irradiation (200 cGy daily x 6 days).

On July 21, 1993, they were transplanted with 2.5 x 108 viable, nucleated cells / kg (not depleted of T cells) from their compatible sibling. It was treated with cyclosporin A (initiating day -l) and methotrexate (days 1, 3, 6 and 11) as anti-GVHD prophylaxis as previously described. Goldman, Leuk, and Lymph. 3: 159-64 (1990). The graft was normal with the white blood cell count (WBC) > 1 x 109 / L on day + 26, the neutrophil count > 0.5 x 109 / L on day + 25 and platelet count > 25 x 109 / L on day + 25. He was discharged 24 days after BMT in very good general condition without signs of GVHD. One month after BMT, PCR did not describe the bcr / abl fusion product. Cyclosporin A was decreased and discontinued 3 months after BMT. One month later, at 4 months after BMT, the PCR was converted to bcr / abl positivity and the cytogenetic analysis of the marrow revealed 100% Ph + with clonal selection. The morphology of the bone marrow was compatible with chronic phase CML. No significant increase in peripheral blood counts was observed.

In an attempt to re-induce the revision, it was treated with allogeneic cell-mediated immunotherapy (Alo-CT) using the peripheral blood lymphocytes (PBL) (8.9 x 107 infused cells / kg) of the compatible sibling. Two weeks later, in the absence of any sign of GVHD, the patient was given another infusion of PBL (5 x 107 cells / kg) with rhIL-2 in vivo (3 x 106 IU / ml2) given subsequently for 3 days consecutive on an outpatient basis. There are no signs of GVHD developed. There was a momentary decline in the WBC scores from 14.7 x 109 / L to 6.2 x 107 / L with no change in the hemoglobin and platelet counts. PCRs for the bcr / abl fusion product remained positive. With the continuous evidence of malignant cells after BMT and Alo-CT, the patient's prognosis was very poor, absent of additional therapeutic measures.

In an attempt to escalate the therapeutic regimen, the PBL of the compatible sibling were precultured in the RPMI medium (Beit Haemek, Israel) supplemented with 5% autologous AB serum., inactivated and supplemented additionally with 6,000 IU / ml of rhIL-2. The PBL were kept in this medium for 4 days in a unified 5% C02 incubator in air, at a concentration of 2.5 x 106 cells / ml. After 4 days of incubation of an initial cell dose of 17 x 108 viable cells, a total of 28 x 108 ADL were collected. In January 1994, the patient received 3.7 x 107 allogeneic ADL / kg, together with administration of subcutaneous rhIL-2, for 3 days (3 x 106 IU / m2), starting on the same day as ADL administration, for activation additional in vivo allogeneic ADL. The WBC fell from 11.3 x 109 / L to 1.3 x 109 / L. Hemoglobin fell from 11.5% of g to 8.3% of g and the platelet count fell from 346 x 109 / L to 23 x 109 / L. PCR became negative for the bcr / abl fusion product. The cytogenetic analysis of the bone marrow detected 100% normal male karyotype in all spontaneous metaphases. The morphology of the bone marrow was completely normal and there were no clinical laboratory signs of GVHD. The blood counts gradually improved without additional therapy. At 2 months after the Alo-ACT, at the time the WBC count was 2.8 x 109 / L, platelets 78 x 109 / L and hemoglobin 10.2% of g, the patient developed disseminated herpes zoster with function tests Abnormal liver that include bilirubin 17 (normal range 2.5-17) micromol / L, AST 444 (normal range 7-40) units, ALT 561 (normal range 6-53) units, GTP 346 (normal range 60-170) units . The patient is more than 28 months after the Alo-ACT without evidence of the Ph + clone (both by cytogenetic analysis and by PCR) and without signs of severe GVHD and good general condition.

Patient No. 2. A six-year-old boy was diagnosed with positive acute lymphocytic leukemia acalla (ALL) in 1988. The allogeneic BTM was performed in Barcelona, Spain on January 8, 1990. At the time of BMT , the patient was in a second complete remission. The conditioning regimen consisted of cyclophosphamide, 60 mg / kg on two consecutive days, plus total fractionated body irradiation (TBI), 200 cGY x 6 (a total of 1200 cGY). The bone marrow in the donor was from a fully matched sibling, and there was no decrease in T cells. The patient was given anti-GVHD prophylaxis after transplantation, normal with cyclosporin A. After BMT, GVHD Grade I was developed Hematological and cytogenetic relapse was diagnosed one month later with T (2; 3), (Q37; P14), DEL (13) (Q?), DEL (20) (Qll), clones. The patient received Alo-CT after the transplant which consisted of the PBLs of the donor at an equivalent dose of 1.4 x 107 T cells / kg given on October 8, 1991, followed on November 3, 1991 by 3.5 x 10 8 T cells / kg with concomitant administration of rhIL-2 subcutaneously (6 x 106 IU / m2) for 3 consecutive days starting on the day of cell infusion. Subsequently, in December 1991, the patient received 3 x 108 ADL / kg prepared by treating the PBLs of the in vitro donor with 6,000 IU / ml of rhIL-2 for 4 days. Five days later, the patient developed grade III cutaneous GVHD that responded during the course of one month to corticosteroid therapy. After the Alo-ACT + regimen of T-cell activators in vivo, the patient entered into a complete review, documented by a normal cytogenetic pattern observed in all metaphases investigated. The chronic GVHD of the skin has persisted, and the patient remains in full review for 53 months after the Alo-ACT.

Patient No. 3. A nine-year-old girl was initially diagnosed in September 1990 with adult-type CML, 100% Philadelphia chromosome-positive cells. He underwent allogeneic BMT on February 21, 1991, in Seattle, while he was in the Chronic Phase. Conditioning consisted of cyclophosphamide, 60 mg / kg, on two consecutive days, followed by fractionated TBI, 200 cGY x 6 (total dose of 1200 Cgy). A fully-fledged HLA AB DR-matched non-reactive MLR was the donor, and the donor cells were depleted of T cells. The patient received prophylaxis of the anti-GVHD with cyclosporin A, and did not develop GVHD. Nine months after BMT, the patient has dermatological relapse and manifest cytogenetics with 100% of the metaphases observed that reveal the Philadelphia chromosome. The patient received PBL from the same donor at an equivalent dose of 5 x 106 T cells / kg given on December 3, 1991. The donor was three years younger, and therefore, it was not technically possible for complete feresis. The patient received a total dose of PBL from the equivalent donor of 107 T cells / kg on January 15, 1992, with the concomitant administration of rhIL-2 subcutaneously (6 x 106 IU / m2) for 3 consecutive days beginning on the day of the infusion of cells. Subsequently, in February 1992, the patient received ADL at an equivalent dose of 107 T cells / kg. In March 1992, he was given a second dose of 107 ADL / kg with the concomitant administration of rhIL-2 subcutaneously (6 x 106 IU / m2) for 3 consecutive days starting on the day of cell infusion. The ADLs were prepared by treating in vitro donor PBLs with 6,000 IU / ml of rhIL-2 for 4 days. The patient responded hematologically, however, a PCR assay by reverse transcriptase (RT-PCR) indicated the presence of residual cells positive to the Philadelphia chromosome. After treatment with alpha-interferon (Roferon A), all cytogenetic abnormalities disappeared as evidenced by a negative RT / PCR assay for the bcr / abl fusion product.

The patient did not show evidence of GVHD throughout the treatment. The patient is doing very well for 51 months after the Alo-ACT; it is hematologically normal without abnormal karyotypes and is consistently negative by RT-PCR for the fusion product of bcr / abl. He is in excellent clinical condition without signs of chronic GVHD.

Patient No. 4. A 3-year-old girl was diagnosed with adult-type Philadelphia chromosome positive CML in November 1990. The patient was conditioned for allogeneic BMT with busulfan, 16 mg / kg for four consecutive days and with cytoxan, 200 mg / kg for four positive days. The cells for the allogeneic BMT were taken from a younger brother completely matched (one year old). The BMT was performed, without decrease of T cells, on May 2, 1991, with normal anti-GVHD prophylaxis using cyclosporin A. The patient had a quiet result after BMT, without GVHD. At 8 months after BMT, the patient developed relapsed hematologic and cytogenetic manifestations. Cell therapy consisted of donor PBL from the BMT donor at an equivalent dose of 5 x 10 6 T cells / kg administered in February 1992. A similar dose of the donor PBL was administered in March 1992, with the administration concomitant rhIL-2 subcutaneously (6 x 106 IU / m2) for 3 consecutive days starting on the day of cell infusion. In April 1992, the patient received ADL from the BMT donor at an equivalent dose of 5 x 106 T cells / kg. In July 1992, the patient received ADL from the BMT donor at an equivalent dose of 3 x 106 T cells / kg with concomitant administration of rhIL-2 subcutaneously (6 x 106 IU / m2) for 3 days starting at day of the cellular infusion. No signs of GVHD were developed, and perhaps, consequently, the patient showed progressive disease despite cellular immunotherapy. Additional treatment with Roferon A failed to induce cytogenetic remission. The patient underwent a second allogeneic BMT without decreased T cells in September 1994, but died due to progressive disease.

Patient No. 5. A two-year-old girl was diagnosed in August 1992 with refractory anemia of myelodysplastic syndrome (MDS), with excessive concussion exhibiting a clonal translocation t (9: 11), evidence of the transition to leukemia. The allogeneic BMT was carried out on February 10, 1993 from a non-sensitive MLR brother and completely HLA A B DR-DRBl-equaled. Conditioning consisted of busulfan, 16 mg / kg given for four consecutive days, thiotepa, 10 mg / kg data for 2 consecutive days, and cytoxan, 16 mg / kg given for two consecutive days. Allogeneic BMT was not reduced from T cells, and the patient had an unmemorable result with no signs of GVHD after normal anti-GVHD prophylaxis with cyclosporin A. The patient had a complete relapse with the same clonogenic leukemia at five months after the BMT. In August 1993, the patient was treated with donor PBLs from the BMT donor at an equivalent dose of 2.8 x 108 T cells / kg. No evidence of GVHD was developed. In September 1993, the patient received the same PBLs from the donors at an equivalent dose of 4 x 107 T cells / kg, with the concomitant administration of rhIL-2 subcutaneously (6 x 106 IU / m2) for 3 consecutive days beginning in the day of the cell infusion. In November 1993, the patient received the ADL from the BMT donor at an equivalent dose of 1.4 x 108 T cells / kg with the concomitant administration of rhIL-2 subcutaneously (6 x 106 IU / m2) for 3 consecutive days starting in the day in the infusion of cells. No evidence of GVHD was developed. Despite the absence of GVHD, the patient showed a complete hematological and cytogenic response with 20 of the 20 normal male karyotypes, characteristic of metaphase without chromosomal aberrations and with normal morphology of the bone marrow. Unfortunately, the notorious relapse was again reported in January 1994, and the patient died in February 1994 due to the progressive disease. It is noted that in relation to this date, the best method known by the applicant to carry out the present invention, is the conventional one for the manufacture of the objects to which it refers.

Having described the invention as above, the content of the following is claimed as property:

Claims (40)

CLAIMS FOR MEXICO

1. The use of allogeneic stem cells for the preparation of a medicament for the treatment of a human patient with cancer having a solid tumor comprising malignant cells, the patient who has undergone a cancer therapy regimen, comprising the transplantation of allogeneic stem cells, wherein the use comprises: a) administer allogeneic lymphocytes to the patient; and b) inspect the patient for malignant cell levels.

2. The use of claim 1, wherein the solid tumor is a breast carcinoma.

3. The use of claim 1, wherein the allogeneic lymphocytes are activated by exposure to a T cell activator in vitro prior to administration to the patient.

4. The use of claim 3, wherein the T cell activator comprises at least one activator of the T cell signal transduction pathway.

5. The use of claim 4, wherein the T cell activator is selected from the group consisting of IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 , IL-13, IL-15, IFNa, IFN ?, TNFa, anti-CD3, anti-CD28; phytohemagglutinin, concanavalin-A and phorbol esters.

6. The use of claim 5, wherein the T cell activator comprises IL-2.

7. The use of claim 1, wherein the allogeneic lymphocytes are administered to the patient in a series of incrementally increasing amounts, depending on the disease of the graft against the host, controllable or not, between the increments.

8. The use of claim 1, wherein the allogeneic lymphocytes are HLA-compatible with the patient.

9. The use of claim 1 or 3, wherein the administration of the allogeneic lymphocytes is achieved by the in vivo administration of the T cell activator.

10. The use of claim 9, wherein the T cell activator administered in vivo is given to the patient over a period of time of two to four days.

11. The use of claim 9, wherein the T cell activator comprises at least one activator of the T cell signal transduction pathway.

12. The use of claim 11, wherein the T cell activator is selected from the group consisting of IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 , IL-13, IL-15, IFNa, IFN ?, TNFa, anti-CD3, anti-CD28, phytohemagglutinin, concanavalin-A and phorbol esters.

13. The use of claim 12, wherein the T cell activator comprises IL-2.

14. The use of claim 1, wherein the allogeneic lymphocytes have a limited space of life.

15. The use of claim 14, wherein the allogeneic lymphocytes carry a suicide gene which confers to the lymphocytes susceptibility to elimination by a chemotherapeutic agent after the administration of the lymphocytes to the patient.

16. The use of allogeneic stem cells for the manufacture of a medicament for the treatment of a human patient with cancer having a solid tumor comprising malignant cells, the patient who has undergone a regimen of cancer therapy comprising the transplantation of allogeneic stem cells, wherein the use comprises: a) administering allogeneic lymphocytes to the patient, lymphocytes that have been activated by exposure to a T cell activator in vitro prior to administration to the patient; b) inspect the patient for levels of malignant hematopoietic cells.

17. The use of claim 16, wherein step (a) of the use, further comprises in vivo administration of the T cell activator to the patient.

18. The use of claim 16, wherein the patient has been infused with alogeneic lymphocytes at rest prior to performing step (a).

19. The use of claim 16, wherein the patient, before step (a), exhibits malignant cells despite the cancer therapy regimen.

20. The use of claim 19, wherein the patient is in a state of manifest relapse after transplantation of allogeneic stem cells.

21. The use of claim 16, wherein the allogeneic lymphocytes are HLA-compatible with the patient.

22. The use of claim 16, wherein the human patient with cancer has chronic myelogenous leukemia.

23. The use of claim 16, wherein the human patient with cancer has acute lymphocytic leukemia.

24. The use of claim 17, wherein the T cell activator administered in vivo is given to the patient for a period of time of 2 to 4 days.

25. The use of claim 16 or 17, wherein the T cell activator comprises at least one activator of the T cell signal transduction pathway.

26. The use of claim 25, wherein the T cell activator is selected from the group consisting of IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 , IL-13, IL-15, IFNa, IFN ?, TNFa, anti-CD3, anti-CD28, phytohemagglutinin, concanavalin-A and phorbol esters. 3

27. The use of claim 26, wherein the T cell activator comprises IL-2.

28. The use of claim 1 or 16, wherein the allogeneic lymphocytes are administered to the patient in the form of a peripheral blood mononuclear cell preparation.

29. The use of allogeneic lymphocytes in the manufacture of a medicament for the treatment of a human patient with cancer having a solid tumor comprising malignant cells, the patient who has undergone a cancer therapy regimen comprising cell transplantation allogeneic stem, wherein the treatment comprises: a) administer allogeneic lymphocytes to the patient; and b) inspecting the patient for malignant cell levels.

30. The use of claim 29, wherein the allogeneic lymphocytes are activated by exposure to a T cell activator in vitro prior to administration to the patient.

31. The use of allogeneic lymphocytes, activated in vitro by exposure to a T-cell activator, in the development of a medicament for the treatment of a human patient with cancer having malignant hematopoietic cells, the patient who has undergone a regimen of cancer therapy comprising the transplantation of allogeneic stem cells, wherein the treatment comprises: a) administering the activated lymphocytes, allogeneic to the patient; and b) inspect the patient for the levels of malignant hematopoietic cells.

32. The use of a T cell activator in the manufacture of a medicament for the treatment of a human patient with cancer having a solid tumor comprising malignant cells, the patient who has undergone a cancer therapy regimen comprising the allogeneic stem cell transplantation, wherein the treatment comprises: a) administering allogeneic lymphocytes and the T cell activator to the patient; and b) inspecting the patient for malignant cell levels.

33. The use of claim 32, wherein the allogeneic lymphocytes are activated by exposure to a T cell activator in vitro prior to administration to the patient.

34. The use of a T cell activator in the manufacture of a medicament for the treatment of a human patient with cancer having malignant hematopoietic cells, the patient who has undergone a regimen of cancer therapy comprising the transplantation of stem cells allogeneic, wherein the treatment comprises: a) administering allogeneic lymphocytes and the T cell activator to the patient, the allogeneic lymphocytes that have been activated by exposure to a T cell activator in vitro prior to administration to the patient; and b) inspect the patient for the levels of malignant hematopoietic cells.

35. A manufacturing article, wherein the packaging material comprises a container within the packaging material, the container containing allogeneic lymphocytes, wherein the packaging material contains a label or instruction piece to the package indicating that the allogeneic lymphocytes can be use for treatment and a human patient with cancer having a solid tumor containing malignant cells, the patient who has undergone a cancer therapy regimen comprises allogeneic stem cell transplantation, wherein the treatment comprises: a) administer allogeneic lymphocytes to the patient; and b) inspecting the patient for malignant cell levels.

36. The article of manufacture according to claim 35, wherein the allogeneic lymphocytes are activated by exposure to a T cell activator in vitro prior to administration to the patient.

37. A manufacturing article, wherein it comprises the packaging material and a container within the packaging material, the container containing lymphocytes activated in vitro, allogeneic, wherein the packaging material contains a label or piece of package instructions indicating that the allogeneic lymphocytes can be used for the treatment of a human patient with cancer, which malignant haematopoietic cells, the patient who has undergone a cancer therapy regimen comprising a allogeneic stem cell transplant, wherein the treatment comprises a) administer the lymphocytes activated in vitro, allogeneic to the patient; and b) inspect the patient for the levels of malignant hematopoietic cells.

38. A manufacturing article, wherein it comprises packaging material and a container within the packaging material, the container containing T-cell activator, wherein the packaging material contains a label or package instruction piece indicating that the activator T cells can be used for the treatment of a human patient with cancer, who has undergone a cancer therapy regimen comprising the transplantation of allogeneic stem cells, wherein the treatment comprises: a) administering the allogeneic lymphocytes and the T cell activator to the patient; and b) inspecting the patient for malignant cell levels.

39. The article of manufacture according to claim 38, wherein the allogeneic lymphocytes are activated by exposure to a T cell activator in vitro prior to administration to the patient.

40. The article of manufacture according to claim 35, 36, 37, 38 or 39, wherein the container is a collapsible container comprising opposite walls and a flexible material and a flexible tube projecting from the container.