Non-myeloablative allogeneic stem cell transplantation focusing on immunotherapy of life-threatening malignant and non-malignant diseases

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1 Critical Reviews in Oncology/Hematology 39 (2001) Non-myeloablative allogeneic stem cell transplantation focusing on immunotherapy of life-threatening malignant and non-malignant diseases Shimon Slavin *, Arnon Nagler, Michael Shapira, Soumya Panigrahi, Simcha Samuel, Arnon Or Department of Bone Marrow Transplantation and Cancer Immunotherapy, The Danny Cunniff Leukemia Research Laboratory, Hadassah Uni ersity Hospital, Jerusalem 91120, Israel Accepted 4 January 2001 Contents 1. Introduction and scientific background Cell therapy by donor lymphocyte infusion Non-myeloablative stem cell transplantation (NST); the mini-transplant transplant program Methods and preliminary results The non-myeloablative stem cell transplantation protocol Clinical application of NST Discussion and conclusions Acknowledgements References Biography Abstract Allogeneic bone marrow transplantation (BMT) represents an important therapeutic tool for treatment of otherwise incurable malignant and non-malignant diseases. Until recently, myeloablative regimens were considered mandatory for eradication of all undesirable host-derived hematopoietic elements. Our preclinical and ongoing clinical studies indicated that much more effective eradication of host immunohematopoietic system cells could be achieved by adoptive allogeneic cell therapy with donor lymphocyte infusion (DLI) following BMT. Thus, eradication of blood cancer cells, especially in patients with CML can be frequently accomplished despite complete resistance of such tumor cells to maximally tolerated doses of chemoradiotherapy. Our cumulative experience suggested that graft versus leukemia (GVL) effects might be a useful tool for eradication of otherwise resistant tumor cells of host origin. The latter working hypothesis suggested that effective BMT procedures may be accomplished without lethal conditioning of the host, using new well tolerated non-myeloablative regimen, thus possibly minimizing immediate and late side effects related to myeloablative procedures considered until recently mandatory for conditioning of BMT recipients. Abbre iations: BMT, bone marrow or blood stem cell transplantation; DLI, donor lymphocyte infusion (DLI); NST, non-myeloablative stem cell transplantation. * Corresponding author. Tel: ; fax: address: slavin@huji.ac.il (S. Slavin) /01/$ - see front matter 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S (01)

2 26 S. Sla in et al. / Critical Re iews in Oncology/Hematology 39 (2001) Recent clinical data that will be presented suggests that safe non-myeloablative stem cell transplantation (NST), with no major toxicity can replace the conventional BMT. Thus, NST may provide an option for cure for a large spectrum of clinical indications in children and elderly individuals without lower or upper age limit, while minimizing procedure-related toxicity and mortality Elsevier Science Ireland Ltd. All rights reserved. Keywords: BMT; Cell therapy; DLI; Fludarabine; Hematologic malignancies; Leukemia; Mini-transplant; NST 1. Introduction and scientific background The list of diseases treatable by allogeneic bone marrow or blood stem cell transplantation (BMT) is growing and includes diseases caused by deficiency of marrow stem cell products (such as aplastic anemia and severe combined immunodeficiency), genetically abnormal stem cells (hemoglobinopathy such as beta thalassemia major; osteopetrosis; chronic granulomatous disease; Wiscott Aldrich syndrome; or enzyme deficiency diseases such as Gaucher s disease or Hurler syndrome, to mention just a few) or malignant hematologic disorders. Acute leukemia, which is one of the commonest malignancies in children, is a highly lethal disease that frequently responds initially to conventional doses of chemotherapy. Unfortunately, leukemia cells frequently become resistant to subsequent chemotherapy and this is the main cause of disease recurrence and unavoidable mortality in children and elderly individuals. An attempt to give more of the same treatment is likely to cause toxicity without improving disease-free survival. A certain proportion of patients with primary resistant leukemia or relapsed leukemia not expected to be cured with additional chemotherapy may still respond to higher than conventional doses of chemotherapy and whole body radiation, delivered in lethal (myeloablative) doses, followed by rescue with bone marrow cells obtained from a genetically matched family member or matched unrelated donor. Myeloablative conditioning with the goal in mind to eliminate resistant tumor or otherwise abnormal host cells, followed by stem cell transplantation was considered until recently the most effective treatment available for patients with blood cancer not expected to be cured by any alternative therapy. Unfortunately, tumor cells resistant to chemotherapy and radiation, will very unlikely be killed by more of the same, namely, higher doses of chemoradiotherapy. Hence, until recently, no cure was available for patients that have relapsed following BMT Cell therapy by donor lymphocyte infusion In early 1987, Slavin et al. [1 7] introduced the use of a new modality for patients who failed BMT, based on transfer of donor s immune system cells isolated from the blood, known as donor lymphocyte infusion (DLI). In contrast to chemotherapy and radiation therapy, immunotherapy, which operates by different mechanisms, may destroy tumor cells despite their full resistance to all available anti-cancer modalities. Therefore, immunotherapy mediated by DLI may be the most effective modality for treating otherwise resistant blood cancer, especially at the stage of minimal residual disease induced by conventional doses of chemotherapy at an early stage of the disease. For patients at high risk to relapse or at risk for continuous chemotherapy, immunotherapy mediated by donor lymphocytes may thus offer, in principle, the best alternative. Unfortunately, donor lymphocytes, being foreign to the patient, are likely to be rejected if given therapeutically. Therefore, in order to exploit the anti-leukemia potential of donor lymphocytes, which constitute the main therapeutic benefit of a regular BMT procedure as well, one additional step seems mandatory to prevent the rejection of the foreign anti-leukemia effector cells. Post transplant immunotherapy introduced by Slavin et al. for the treatment of hematologic malignancies, was initially developed in animal models of human disease, using the a spontaneous B-cell leukemia and lymphoma in BALB/c mice which resembles the human disease [8]. Data in mice inoculated with BCL1 suggested that amplificaion of graft versus leukemia (GVL) effects cn be accomlished by ril-2 activation of donor lymphocytes in vivo and in vitro [9 11]. Based on recent animal data in a murine model of metastatic breast cancer, various approaches are used in an attempt to prolong the duration of parking of donor lymphocytes in the patient s circulation, to potentiate their possible graft versus tumor (GVT) effect, in analogy to GVL effects [12,13]. 2. Non-myeloablative stem cell transplantation (NST); the mini-transplant transplant program Based on the above, in patients with a matched sibling available, we attempt to induce host versus graft transplantation tolerance in order to enable durable engraftment of donor lymphocytes for more effective GVT effects, in parallel with the well-documented GVL effects in patients with blood cancer. Host vs graft tolerance will be accomplished by engraftment of donor stem cells mobilized from the blood, using a well-tolerated nonmyeloablative ( mini-transplant ) stem cell transplantation procedure. Prevention of rejection of

3 S. Sla in et al. / Critical Re iews in Oncology/Hematology 39 (2001) donor stem cells is based on the use of fludarabine and anti-t lymphocyte immunoglobulin (ATG) thus avoiding the use of radiation. The NST protocol is based on a three-step procedure, the first designed to debulk without necessarily attempting to ablate the last cancer cell, thus avoiding major toxicity, as well as for induction of intensive transient immunosuppression to enable durable acceptance of donor bone marrow cells, isolated from the blood, preferably as an outpatient procedure (step 1). Subsequently, donor lymphocytes are infused together with stem cells collected from the blood (following mobilization with G-CSF) or from the marrow without any mobilization, for induction of host vs graft transplantation tolerance as well as for induction of GVL or GVT effects by donor lymphocytes resent in the graft (step 2). Following the well-tolerated immunosuppressive yet nonmyeloablative procedure, donor T cells, possibly natural killer (NK) cells as well, are expected to eliminate residual leukemia or other tumor cells resistant to chemotherapy by biologic warfare, recognizing tumor cells as being foreign. Patients with more resistant disease may require additional infusions of donor lymphocytes at later stage post transplantation, on an outpatient basis (step 3). Donor lymphocyte infusion (DLI) may be amplified with ril-2 administration or with infusions of donor lymphocytes activated in vitro with ril-2. The success of NST depends on initial engraftment of donor stem cell for induction of host versus graft transplantation tolerance to prevent rejection of donor cells and permit durable engraftment of donor lymphocytes. An optimal and well tolerated procedure for conditioning of recipients of HLA matched sibling or matched unrelated donor (MUD) allografts or even fully haploidentically mismatched family member can be accomplished with a combination of fludarabine (Fludara) and ATG in addition to low dose busulfan or cytoxan which were used until recently in much higher doses against all the aforementioned indications. Following step 1 which is based on well tolerated nonmyeloablative conditioning, tolerated well even by elderly individuals (no upper age limit), donor lymphocytes can be accepted without rejection and thus can mediate graft versus leukemia or graft versus lymphoma effects against residual tumor cells escaping chemotherapy. Following nonmyeloablative stem cell transplantation (NST) donor lymphocytes can eliminate the last tumor cell even in patients who cannot be cured by lethal doses of chemotherapy including whole body irradiation. Basically, patient s own lymphocytes (T cells) ignore tumor cells, which is one of the reasons why the tumor continues to grow indefinitely, whereas donor T cells, being foreign to the tumor, may recognize residual malignant cells and destroy them. This is in essence the whole rationale behind allogeneic cell therapy, which provides an option for cure of patients with otherwise incurable blood cancer or lymphoma. We are currently attempting to use the same rationale for treating patients with metastatic solid tumors for whom no alternative option for cure exists. Slavin s new protocol based on his new concepts involving the use of NST (steps 1 and 2 followed by step 3) has been shown to be effective in all hematologic malignancies, which together with most encouraging animal experiments in a model of metastatic breast cancer, justify a similar approach for consenting patients with otherwise resistant metastatic solid tumors [14 17]. As a rule, since the majority of patients with hematologic malignancies at all age groups are anticipated to relapse even after entering complete remission, with the exception of low risk ALL in children, well tolerated immunotherapy involving NST should be regarded as an additional therapeutic option worthwhile considering at an early stage of the disease for high risk cases. As the experience using NST will grow, provided that it can be proved as being relatively safe, the use of NST for additional indications at an earlier stage of the disease in patients at risk will become acceptable. 3. Methods and preliminary results 3.1. The non-myeloablati e stem cell transplantation protocol The main NST protocol consisted of immunosuppression (step 1) induced by with fludarabine 30 mg/m 2 / day 6; ATG 5 10 mg/kg 4 (Fresenius, AG) and busulfan 4 mg/kg 2 followed by infusion of mobilized blood stem cells from siblings (single locus mismatched allowed) or marrow cells from matched unrelated donors (MUD) to establish host versus graft chimerism and tolerance (step 2) with low dose cyclosporine A (CSA) 3 mg/kg for days [14 17] Clinical application of NST The protocol outlined above was used successfully in 120 patients with a large variety of indications for allogeneic bone marrow or blood stem cell transplantation, including hematologic malignancies and lifethreatening non-malignant diseases. In a recent study, 70 patients with hematologic malignancies, 16 patients with MUD and nine patients receiving an allograft following failure of autobmt. Patients (age range 3 63, median 38; CML (n=19); AML (n=17); ALL (n=10); NHL (n=15); MDS and 2nd leukemia (n= 6); Hodgkin s disease (n=2) and multiple myeloma (n=1). Patients received fully matched (n=66) or single locus mismatched (n=4) stem cells on day 0. The protocol was very well tolerated by patients of all age

4 28 S. Sla in et al. / Critical Re iews in Oncology/Hematology 39 (2001) groups. Day 100 mortality was 4%; 0% in patients with non-malignant diseases and 7% in patients with malignancy. Fast and durable engraftment was observed in all patients with a matched sibling. Persistent evidence of disease or recurrent disease in mixed chimeras was treated by discontinuation of CSA or by graded increments of DLI with 10 of 15 responders. After 3 years, with an observation period of 3 39 months (median 24 months) actuarial probability of survival was 68% and disease-free survival was 48%. Corresponding numbers for MUD at 12 months were 75% and 70%, and at 18 months was 48%, respectively. Additional patients were treated with other modifications of the NST protocol consisting of cytoxan 60 mg/kg for 2 days or a single dose total body irradiation (TBI) 200 cgy instead of busulfan. All patients transplanted with a fully matched sibling showed early engraftment, some with no aplasia at all, with rapid conversion of host to donor cells within days. Patients with Fanconi s anemia were treated with much lower doses of cytoxan consisting of two daily doses of 5 mg/kg which was sufficient for engraftment with 100% donor cells in all three patients treated with this protocol. However, GVHD remains the single major problem. Based on our preliminary data, NST appears promising, but larger cohorts of patients and longer observation periods will be required to confirm the overall advantage of NST compared with conventional BMT [14 17]. 4. Discussion and conclusions In conclusion, for patients with high risk diseases (resistant or relapsing hematologic malignancies and possibly other drug-resistant metastatic solid tumors) failing front-line chemotherapy or even high-dose chemoradiotherapy there is very little benefit that can be accomplished by more of the same conventional modalities. Innovative immunotherapy offers a rationale chance for eradication of tumor cells or otherwise abnormal host cells by introduction of foreign immune system cells that can eliminate undesirable host hematopoietic cells much more effectively, at the risk of graft versus host disease GVHD. The use of NST as replacement of conventional BMT needs to be further investigated and formally proven. However, it is already clear that using a combination of fludarabine and ATG with low dose cytoxan or busulfan stable engraftment and full donor chimerism can be established, thus avoiding the need for myeloablative doses of chemoradiotherapy that in children are associated with retardation of growth and development, cataract formation and sterility, to mention just a few. Conventional conditioning, should still be considered for patients with bulky disease at high risk to accomplish better tumor debulking prior to immunotherapy with donor lymphocytes or post transplant DLI. The use of well tolerated NST, while minimizing procedure related toxicity and mortality may open the door for safer early application of an ultimate therapeutic modality that may involve allogeneic stem cell therapy in conjunction with additional innovative post-transplant immunotherapy based on the use of allogeneic donor lymphocytes, may thus provide a new platform for new modalities based on biologic tools rather than relying solely on chemoradiotherapy for the treatment of life threatening malignant, genetic and autoimmune diseases at all age groups. Acknowledgements We would like to thank the following for their ongoing support: Baxter International Corporation; Max & Adi Moss Research laboratory; Ryna & Melvin Cohen; The Szydlowsky Foundation; The Cancer Treatment Foundation; the German Israel Foundation; Donald & Ronne Hess and Joanne & David Morrison; Pep & Jerry Silverstein; The Gabrielle Rich Leukemia Research Foundation; The Himmelfarb Foundation. The work was done at the Danny Cunniff Leukemia Research Laboratory. References [1] Slavin S, Or R, Naparstek E, Ackerstein A, Weiss L. Cellularmediated immunotherapy of leukemia in conjunction with autologous and allogeneic bone marrow transplantation in experimental animals and man. Blood 1988;72(suppl 1):407a. [2] Slavin S, Ackerstein A, Nagler A, Naparstek E, Weiss L. Cellmediated cytokine-activated immunotherapy (CCI) of malignant hematological disorders for eradication of minimal residual disease (MRD) in conjunction with conventional chemotherapy or bone marrow transplantation (BMT). Blood 1990;76(suppl 1):566a. [3] Slavin S, Nagler A. New developments in bone marrow transplantation. Curr Opin Oncol 1991;3: [4] Slavin S, Or R, Kapelushnik Y, et al. Immunotherapy of minimal residual disease in conjunction with autologous and allogeneic bone marrow transplantation (BMT). Leukemia 1992;6: [5] Naparstek E, Or R, Nagler A, et al. T-cell-depleted allogeneic bone marrow transplantation for acute leukaemia using Campath-1 antibodies and post-transplant administration of donor s peripheral blood lymphocytes for prevention of relapse. Br J Haematol 1995;89: [6] Slavin S, Naparstek E, Nagler A, Ackerstein A, Kapelushnik Y, Or R. Allogeneic cell therapy for relapsed leukemia following bone marrow transplantation with donor peripheral blood lymphocytes. Exp Hematol 1995;23: [7] Slavin S, Naparstek E, Nagler A, et al. Allogeneic cell therapy with donor peripheral blood cells and recombinant human interleukin-2 to treat leukemia relapse post allogeneic bone marrow transplantation. Blood 1996;87: [8] Slavin S, Strober S. Spontaneous murine B-cell leukemia. Nature 1978;272:624 6.

5 S. Sla in et al. / Critical Re iews in Oncology/Hematology 39 (2001) [9] Slavin S, Weiss L, Morecki S, Weigensberg M. Eradication of murine leukemia with histoincompatible marrow grafts in mice conditioned with total lymphoid irradiation (TLI). Cancer Immunol Immunother 1981;11: [10] Weiss L, Reich S, Slavin S. Use of recombinant human interleukin-2 in conjunction with bone marrow transplantation as a model for control of minimal residual disease in malignant hematological disorders. I. Treatment of murine leukemia in conjunction with allogeneic bone marrow transplantation and IL2-activated cell-mediated immunotherapy. Cancer Invest 1992;10: [11] Weiss L, Lubin I, Factorowich Y, et al. Effective graft vs leukemia effects independent of graft vs host disease after T-cell depleted allogeneic bone marrow transplantation in a murine model of B cell leukemia/lymphoma. Role of cell therapy and ril-2. J Immunol 1994;153(6): [12] Morecki S, Moshel Y, Gelfend Y, Pugatsch T, Slavin S. Induction of graft vs tumor effect in a murine model of mammary adenocarcinoma. Int J Cancer 1997;71: [13] Morecki S, Yacovlev E, Diab A, Slavin S. Allogeneic cell therapy for a murine mammary carcinoma. Cancer Research 1998;58: [14] Slavin S, Nagler A, Naparstek E, et al. Immunotherapy of leukemia in conjunction with non-myeloablative conditioning: engraftment of blood stem cells and eradication of host leukemia with nonmyeloablative conditioning based on Fludarabine and anti-thymocyte globulin (ATG). Blood 1996;88(10):614a. [15] Slavin S, Nagler A, Naparstek E, et al. Non-myeloablative conditioning in preparation for allogeneic stem cell transplantation: the future treatment of choice of hematologic malignancies and genetic diseases. Exp Hematol 1997;25(8):787. [16] Slavin S, Nagler A, Naparstek E, et al. Successful replacement of conventional bone marrow transplantation with high-dose chemoradiotherapy with well tolerated nonmyeloablative conditioning in preparation for allogeneic blood stem cell transplantation for the treatment of malignant and non-malignant diseases. Blood 1997;90(10):534a. [17] Slavin S, Nagler A, Naparstek E, et al. Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and non-malignant hematologic diseases. Blood 1998;91(3): Biography Shimon Sla in MD graduated from the Hadassah Hebrew University School of Medicine in Jerusalem, Israel, in 1967, specialized in internal medicine and subsequently trained in clinical immunology and rheumatology and basic immunology research at Stanford University, California. Before arrival to Israel in 1979 he trained at the Fred Hutchinson Bone Marrow Transplantation Center at Seattle, Washington, and subsequently established Israel s first bone marrow transplantation unit, later officially recognized as the National Bone Marrow Transplantation Center. Dr Slavin is currently Professor and Chairman of the Department of Bone Marrow Transplantation and the Research Center he established at the Hadassah University Hospital in Jerusalem. Dr Slavin pioneered the use of mixed chimerism following non-myeloablative conditioning as a means for induction of bilateral transplantation tolerance of host-vs-graft and graft-vs-host for bone marrow and organ transplantation in the 70 s. In early 1987, he introduced the use of donor lymphocyte infusion (DLI), pioneering the clinical use of adoptive allogeneic cell-mediated immunotherapy and cytokine-activated immunotherapy, for both treatment and prevention of relapse following allogeneic and autologous stem cell transplantation, which are now being practiced for hematologic malignancies and metastatic solid tumors. He showed that lymphocytes activated nonspecifically by ril-2, and more recently, specifically immune donor lymphocytes, could exert much more potent yet selective anti-tumor responses. Dr Slavin has also pioneered the use of new approaches for clinical application of non-myeloablative stem cell transplantation (NST), based on adoptive immunotherapy with DLI following induction of specific unresponsiveness of host-vs-graft responses, focusing on safer yet more effective immunotherapy of malignant and nonmalignant diseases, rather than using more aggressive chemoradiotherapy. New approaches based on NST are now being introduced for down-regulation of the immune system, towards induction of specific transplantation tolerance to cellular and organ allografts, as well as self-tolerance for treating autoimmune disorders. Dr Slavin serves on many editorial boards and national and international advisory boards..