Immunotherapy of hematologic malignancies and metastatic solid tumors in experimental animals and man

Critical Reviews in Oncology/Hematology 46 (2003) 139 /163 www.elsevier.com/locate/critrevonc

Immunotherapy of hematologic malignancies and metastatic solid tumors in experimental animals and man Shimon Slavin *, Shoshana Morecki, Lola Weiss, Reuven Or The Danny Cunniff Leukemia Research Laboratory, Department of Bone Marrow Transplantation & Cancer Immunotherapy, Hadassah University Hospital, Jerusalem 91120, Israel Accepted 20 August 2002

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.

The role of allogeneic lymphocytes in conjunction with BMT in the treatment of hematologic malignancies and solid tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Induction of GVL effects by donor lymphocyte infusion for treatment of relapse post BMT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Induction of GVL effects by immune donor lymphocytes in preclinical animal model . 2.3. Induction of GVL effects by immune donor lymphocytes in clinical practice . . . . . .

141 145 145

3.

Effector cells of GVL effects in mice and man . . . . . . . . . . . . . . . . . . . . . . . . . .

146

4.

Effective graft-versus-leukemia effects independent of graft-versus-host disease in a preclinical animal model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

5.

Allogeneic cell-mediated immunotherapy with donor lymphocyte infusions for prevention of relapse following BMT for hematological malignancies . . . . . . . . . . . . . . . . . . . . . 151

6.

Non-myeloablative stem cell transplantation (NST) for treatment of malignant malignant diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Focusing on lymphoablative therapy pre-transplantation . . . . . . . . . . . . 6.2. Focusing on lymphoablative therapy pre- and post-transplantation . . . . . . 6.3. Focusing on immunosuppressive and cytotoxic chemotherapy . . . . . . . . . 6.4. High-dose chemotherapy supported by autotransplantation followed by NST ication of minimal residual disease . . . . . . . . . . . . . . . . . . . . . . . . .

and non . . . . . . . . . . . . . . . . . . . . for erad. . . . .

7.

Immunotherapy of metastatic solid tumors with alloreactive lymphocytes . . . . . . . . . . .

8.

140 141

152 153 155 156 156 156

Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

158

Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

159

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

159

Biography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

163

Abstract New approaches are needed for maximizing specific responses against tumor cells resistant to chemotherapy. While cytokine therapy may amplify natural resistance against minimal residual disease, more robust anti-leukemia reactivity can be provided by allogeneic bone marrow transplantation (BMT) in conjunction with myeloablative, hence hazardous, conditioning, at the cost of graft-versus-host disease (GVHD). Documentation of the capacity of donor lymphocyte infusion (DLI) given late post BMT, when

* Corresponding author. Tel.:
/972-2-677-7270; fax:
/972-2-642-2731. E-mail address: [email protected] (S. Slavin). 1040-8428/03/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S1040-8428(02)00108-7

140

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

patients were off immunosuppression, in early 1987, with successful reversal of relapse and cure of patients fully resistant to maximally tolerated doses of chemoradiotherapy, with many patients alive and well
/10 /15 years later, indicated two important facts. First, resistant tumors are unlikely to be cured with higher doses of chemoradiotherapy that may harm the patient but not eliminate all his clonogenic tumor cells. Second, that under condition of tolerance to donor alloantigens, DLI may provide a cure to otherwise resistant patients. These observations paved the road for clinical application of non-myeloablative stem cell transplantation (NST), in the early 90s, based on a two-step procedure, first involving induction of transplantation tolerance to donor alloantigens by engraftment of donor stem cells, following safe lymphoablative rather than myeloablative conditioning. Second, use of donor lymphocytes for elimination of residual tumor or otherwise abnormal hematopoietic cells by immunemediated graft-versus-host effects inducible by mobilized blood stem cell allografts containing larger inocula of donor T cells, or supported by post-grafting DLI when patients were off immunosuppressive modalities. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Bone marrow transplantation (BMT); Transplantation tolerance; Stem cell transplantation; Allogeneic cell-mediated immunotherapy; Cytokine-mediated immunotherapy; Recombinant human interleukin 2 (rIL-2); Interferon alpha (IFN); Donor lymphocyte infusion (DLI); Nonmyeloablative stem cell transplantation (NST); Graft-versus-host disease (GVHD); Graft-versus-leukemia (GVL) effects; Graft-versus-tumor (GVT) effects; Graft-versus-malignancy (GVM) effects; Leukemia; Lymphoma; Metastatic solid tumors

1. Introduction Considering lack of specific treatment modalities against cancer, and the well established dose /response effects in treating tumor cells in vitro and in vivo, the dogma over the past years with available anti-cancer modalities has been ‘the more the better’, attempting to eradicate the primary tumor by resection with or without radiation therapy and the secondary metastases with aggressive chemoradiotherapy. These studies lead to the clinical development of myeloablative chemoradiotherapy, followed by rescue with stem cell transplantation as a possible means for eradication of maximal possible number of tumor cells. Over the years it became apparent that none of the available anti-cancer modalities or even combinations thereof could accomplish such a goal, since relapse continued to be the single major obstacle in treatment of hematologic malignancies especially of patients with advanced or resistant disease [1,2]. On the other hand, as the intensity of the regimen used for bone marrow or blood stem cell transplantation (BMT) was escalated, procedure-related toxicity and mortality increased accordingly [3]. In addition the increased incidence and severity of late complications became an important issue too in evaluating the quality of life of long-term survivors. It became apparent that newer modalities must be introduced in order to improve the cure rate of patients with hematologic malignancies as well as to improve the quality of life of successfully treated patients. For patients resistant to available chemotherapy, immunotherapy became an obvious rational alternative. Unfortunately, no tumor-specific antigens were available in patients with hematologic malignancies. Likewise, experiments in animal models of human disease never featured effective humoral control of disease neither were there any positive experiments using tumor cell vaccines [4]. Spontaneous murine leukemias like the human disease were considered ‘non-immunogenic’.

Clearly, the major obstacles in developing effective immunotherapy programs for patients with hematologic malignancies stemmed from either lack of tumor-specific antigens or from failure to recognize them, or alternatively due to failure to recognize the existing tolerance against ubiquitous tumor antigens and failure to induce an ‘autoimmune’ response against tumor cells that were recognized as ‘self’ [5]. Even to date, tumor-associated antibodies, such as anti-CD20 in patients with B cell non-Hodgkin’s lymphoma, that can effectively debulk the number of tumor cells in responding patients, are not sufficient for eradication of the disease [6]. In the present review we will present evidence for tolerance in mice with leukemia that may explain the difficulties in inducing an effective immunotherapy by recipient’s own immune system, as well as the feasibility to eradicate tumor cells and cure otherwise lethal leukemia without chemoradiotherapy using alloreactive lymphocytes following adoptive immunotherapy across minor and major histocompatibility (MHC) barriers. These concepts led to the development of donor lymphocyte infusion (DLI) following induction of transplantation tolerance to donor alloantigens by the BMT procedure, for ensuring durable engraftment of donor lymphocytes, as a means to treat or prevent relapse following maximally tolerated doses of chemoradiotherapy in BMT recipients considered incurable by any of the existing anti-cancer modalities. Finally, we will review the development of the concept of nonmyeloablative conditioning as potential replacement of conventional myeloablative BMT as a safer and more practical means to exploit the use of alloreactive donor lymphocytes as the main tool against malignant cells of hematopoietic origin and possibly non-hematopoietic metastatic solid tumors as well. The main future goal is to maximize the anti-tumor capacity of alloreactive donor lymphocytes while restricting their reactivity against normal tissues, using tumor or tissue specific effector cells. Finally, we will present the scientific basis

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

suggesting the feasibility of such innovative approaches for immunotherapy of malignant and non-malignant diseases.

2. The role of allogeneic lymphocytes in conjunction with BMT in the treatment of hematologic malignancies and solid tumors With the development of antibiotics, anti-fungal and anti-viral agents in parallel with better control of the myeloablated recipient with blood products and isolation, the use of high-dose, myeloablative chemoradiotherapy supported by autologous and allogeneic BMT, explored originally by Thomas and colleagues in the early 1970s became the treatment of choice for patients resistant to conventional doses of chemotherapy, and subsequently for patients at high-risk to relapse following maximally tolerated doses of conventional chemotherapy [7]. Subsequently, BMT was successfully utilized for the treatment of genetic diseases and other life-threatening non-malignant indications using the same therapeutic principles for replacement of abnormal host hematopoietic cells with donor hematopoietic cells. Traditionally, it was considered that high-dose chemoradiotherapy was the main component in the bone marrow transplant procedure and that transplantation of genotypically or phenotypically matched stem cells was mainly indicated for rescue of the lethally treated recipient. Hence, much attention was given to maximize tumor cell kill by maximally tolerated doses of chemotherapy (single agents and combinations of non-cross reactive agents). However, it was recognized for many years that the incidence of relapse was higher among recipients of autologous as well as syngeneic grafts as compared with recipients of allogeneic grafts with graft-versus-host disease (GVHD), suggesting that immune-mediated graft-versus-leukemia (GVL) effects played a major role in elimination of residual tumor cells escaping chemoradiotherapy [8 /11]. The possibility that allogeneic lymphocytes administered in the course of BMT eliminate leukemia through immune-mediated GVL effects has been suggested ever since the earliest days of experimental [12 /19] and clinical BMT [8 /11]. Convincing direct correlation between acute and chronic GVHD and reduced rate of relapse of leukemia in clinical practice was first reported by Weiden et al. [8,9]. Similarly, in analogy to GVL effects graft-versus-tumor (GVT) effects were also described in a murine model of spontaneous sarcoma [20] and more recently in metastatic breast cancer as well [21,22], as well as in preliminary trials in man [23 /25]. The role of immunemediated GVL effects in the course of BMT was further supported by observations suggesting that relapse while patients were on immunosuppressive treatment with

141

cyclosporine A was occasionally reversed by discontinuing immunosuppression [26]. Likewise, it has been documented that the incidence of relapse is lower in patients treated with sub optimal doses of CSA [27]. In support, data in mice inoculated with murine leukemia treated by BMT indicated that GVL effects mediated by mismatched bone marrow cells were totally abrogated by concomitant administration of CSA for 10 days [28]. All of the above suggested that allogeneic BMT provided immunocompetent allogeneic donor T lymphocytes, which could react against residual tumor cells of host origin. Hence, the advantage of BMT over conventional chemotherapy lies in the combined effects of the myeloablative dose of chemoradiotherapy given pretransplantation and the ability of immunocompetent allogeneic donor T lymphocytes to eliminate residual tumor cells of host origin, giving rise to GVL and GVT effects or in fact graft versus any undesirable hematopoietic cells of host origin, including genetically abnormal stem cells or their progeny [29 /32]. Interestingly, similarly to the data first reported in mice [14 /18], GVL effects independently of GVHD were also confirmed in clinical practice either following BMT [11] or following DLI given post transplantation to induce GVL effects to treat or prevent relapse when patients are off any post transplant immunosuppressive agents [33 /40]. Based on the murine data that suggested the feasibility of induction of post-transplant GVL effects induced by T cells present in the allografts, we hypothesized that cell-therapy with donor lymphocytes given post grafting, especially in patients with no spontaneous GVHD following discontinuation of post transplant anti-GVHD prophylaxis, may induce effective antitumor responses [5]. We hypothesized that allogeneic lymphocytes of donor origin can be given post grafting for treatment as well as for prevention of relapse in highrisk cases. The present report documents the first successful case where GVL effects were induced by allogeneic cell-therapy (DLI) in a patient with resistant acute lymphoblastic leukemia (ALL) who relapsed shortly after BMT and the cumulative international experience in a variety of malignant hematologic diseases. 2.1. Induction of GVL effects by donor lymphocyte infusion for treatment of relapse post BMT The first patient successfully treated by DLI for relapse following BMT was a 2-year-old boy that was referred for BMT at the Hadassah University Hospital in Jerusalem in November 1986 [33 /35]. He had been diagnosed as pre-B ALL and relapsed on therapy twice. In December 1986, allogeneic BMT was carried out from a fully matched sister during second resistant relapse. Supra-lethal conditioning included total body irradiation (TBI) 1200 cGy (two daily fractions of 200

142

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

cGy on days -6, -5 and -4) followed by two doses of cyclophosphamide 60 mg/kg (days -3 and -2) and melphalan 60 mg/m2 (day -1) as previously described [24,26]. The patient showed no signs of acute GVHD. At 1 month post-BMT, the patient presented with full hematologic relapse and several bulky masses confirmed as extramedullary disease, including a progressing retro tracheal mass necessitating an emergency tracheotomy. He responded to increments of donor (sister) peripheral blood lymphocytes (PBL) infusions to induce GVL effects. The patient developed grade II GVHD with involvement of the skin and liver. He responded to a short course of corticosteroids and within 2 weeks the palpable masses decreased in size, peripheral blood and bone marrow morphology normalized and cytogenetic analysis confirmed 100% normal female karyotype in all 50 metaphases investigated. To date,
/15 years after his third relapse post BMT, no residual male cells are detected by PCR analysis of either Y-specific or amelogenine markers. On the basis of the success with this first patient, DLI was given to 16 additional patients who relapsed 1 /16 months (median 4) after BMT. In five of 17 patients elimination of all detectable leukemic cells was achieved by DLI. All four patients with minimal cytogenetic relapse responded to DLI given as graded increments of donor PBL. In contrast, out of 13 patients with overt hematologic relapse, four with CML, four with ALL, three with AML, one with Burkitt’s lymphoma and one with myelodysplastic syndrome with excess blasts, only one responded to DLI alone. Based on the preclinical data in mice inoculated with BCL1, it appeared that GVL effects induced with DLI could be amplified with recombinant human interleukin 2 (rIL-2) [5,35]. Hence, it was decided to treat the resistant patients with DLI concomitantly with in vivo activation of donor lymphocytes with rIL-2. Indeed, remission was achieved in five of patients by cell-therapy in combination with rIL-2 resulting in an overall response in ten of the 17 patients (4/6 with ALL, 0/3 with AML, 5/6 with CML and one Table 1 History of allogenic cell-mediated immunotherapy with DLI following BMT for treatment and prevention of relapse First sucessful clinical application of DLI 1987 Slavin et al. [33] Slavin et al. [34] Slavin et al. [35] First successful clinical application of DLI for prevention of relapse following BMT Slavin et al. [33] Naparstek et al. [54] First confirmation of the role of DLI for treatment of relapse in patients with CML Kolb et al. [36]

case of myelodysplastic syndrome with excess blasts) [34,35]. These data, by now confirmed in many centers all over the world, provided a second chance for cure of resistant hematologic malignancies for patients failing all known anti-cancer modalities [33 /40]. The early cumulative international experience with DLI in patients with acute and chronic leukemia is summarized in Tables 1 and 2 and in Fig. 1. As can be seen, complete responses (molecular, cytogenetic or hematologic) can be obtained in about 70 /80% evaluable patients with CML and in 20 /30% of evaluable patients with acute leukemia, with the lesser degree o response in patients with rapidly progressing ALL. Better response rates can be obtained in patients treated at the stage of minimal residual disease [35]. Therefore, it is highly recommended to follow patients at risk very frequently during the first 2 years following transplantation with molecular assessment of host and donor DNA markers. Detection of Y-specific markers can be carried out either by PCR of SRY-specific regions as previously described [41], or by PCR of part of the amelogenine gene located on both the x -chromosome and its shorter copy on the y -chromosome [42] for detection of recurrent host cells in a female to male combinations. Host and donor specific VNTR-PCR can be followed in sex matched pairs. Whenever available, disease specific markers can be also used including cytogenetic or molecular markers such as RT-PCR for detection of bcr/abl in patients with CML or Philadelphia-positive ALL [43]. The cumulative international data document a total incidence of clinically significant GVHD in at least 60% of patients treated with DLI. However, in some reports the frequency of GVHD, including a mild form of the disease (grade II), was much higher among responders and seemed to correlate with induction of remission [33 /40]. Documentation of response to DLI in up to 40% of patients with no clinically significant GVHD, especially those treated at the stage of minimal residual disease, which may require lower doses of donor lymphocytes, supports the existence of GVL effects independently of GVHD [34 /36]. Unfortunately, development of anti-host reactivity following DLI may be unavoidable, unpredictable and has been shown to result in severe, occasionally fatal complications. In addition, similarly to GVHD, DLI is frequently, but not consistently, associated with depression of bone marrow function [34 /37,44 /46]. Overall, some degree of marrow dysfunction can be observed in approximately 30% of evaluable patients; mild marrow aplasia often reverses spontaneously but in some cases, bone marrow aplasia may be severe and occasionally fatal. Marrow dysfunction can be treated with hematopoietic growth factors and occasionally by transfusions with blood products [36,37]. Severe aplasia can be best treated by

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

143

Table 2 Allogenic cell therapy with DLI for treatment of relapse post allogenic BMT CML

Kolb et al. [36,37] European experience Collins et al. [38] American experience Slavin et al. [34] Jerusalem experience

Fig. 1. Effect of DLI on survival in leukemia & myeloma The EBMT data base 2001 (Provided by H. Kolb)..

Table 3 GVL effect induced with DLI Diagnosis

Number of patients Studied Evaluable* Complete remission (%)

CML Cytogenetic relapse Hematologic relapse Transformed phase Polycythaemia vera/ MPS AML/MDS ALL Multiple myeloma Total

57 124 42 2

50 114 36 1

40(80%) 88(77%) 13(36%) 1

97 55 25 402

58 20 17 296

15(26%) 3(15%) 5(29%) 165(55.7%)

The EBMT data base 2001 (provided by H. Kolb).

up-front infusion with bone marrow cells from the original donor without further conditioning. An update of the European experience based on the European Bone Marrow Transplant Registry (EBMTR) database using DLI for the treatment of relapse post BMT is shown in Table 3, kindly provided by Dr Hans Kolb. Of particular interest were patients with secondary EBV-positive lymphoma. Post-transplant lymphoma induced by EBV is highly malignant and rarely responds

Other

Total

#

CR (%)

#

CR (%)

#

CR (%)

130 57 105

64 60 72

107 78 58

19 19 45

237 135 163

45 36 63

to any of the available anti-lymphoma modalities. As reported by Papadopoulos et al. [47], all patients with post-transplant lymphoma responded to DLI. Interestingly, post transplant lymphoma induced by EBV can now be eradicated by EBV specific cytotoxic cells generated in vitro [48]. Taken together, although relapse following BMT was always considered incurable the cumulative international experience confirms the efficacy and usefulness and even durable cure of resistant acute and chronic leukemia by post-transplant immunotherapy with immunocompetent donor PBL. The only real problem associated with DLI is acute and chronic GVHD, which tend to be more severe if donor lymphocytes are given earlier following BMT. Future studies should focus on generating tumor-specific approaches to maximize GVL and minimize or preferably eliminate GVHD. The response to host tumor cells is thought to be mediated by alloreactive donor T lymphocytes, which might explain the higher incidence of GVHD among responders and vice versa. Nonetheless, GVHD is neither necessary nor sufficient for reversal of relapse post BMT. Interestingly, DLI successfully reversed relapse following both unmanipulated [36 /40] and TCD allogeneic BMT [35]. Since GVHD, which is mediated by immunocompetent donor T lymphocytes, is clearly associated with GVL [8 /10], it is to be expected that recipients of TCD-BMT, in contrast to recipients of non-TCD-BMT, will be able to respond more favorably to DLI. However, post-transplant immunosuppression for prevention, attenuation or treatment of GVHD, which is unavoidable following non-Tcell-depleted BMT, may also abrogate the GVL effect [26,27,49,50]. Since it was previously shown that effective post-BMT immunosuppression by agents such as cyclosporin, with or without methotrexate, may increase the incidence to relapse in experimental animals [28] and man [27,49]. It was also shown that discontinuation of cyclosporin as soon as relapse is diagnosed may lead to reinduction of remission [26,46]. Interestingly, although adult and pediatric patients may respond differently to conventional chemotherapy, both seem equally sensitive to DLI. Anti-leukemic effects induced by DLI are mediated by immunocompetent donor T lymphocytes recognizing minor histocompatibility determinants on the surface of tumor cells of host origin, an effect that

144

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

may most likely depend on the alloreactive capacity of donor PBL rather than the age of the host. Taken together, GVL effects induced by immunocompetent T lymphocytes present in the donor marrow aspirate while administering agents such as cyclosporin, may be insufficient to prevent relapse, justifying additional immunotherapy with donor lymphocytes under no cover of anti-GVHD prophylaxis, especially in patients with no GVHD. Based on earlier animal data suggesting that the incidence of GVHD decreases as the time interval from BMT to DLI increases [51 /53], a simple and cost-effective way to control the incidence, intensity and severity of GVHD, may be by giving graded increments of donor PBL, because different doses of T cells may be required for each patient. Following early evidence of relapse, or in order to prevent relapse in high-risk cases [54,55], DLI could be initiated with a low, sub optimal T cell dose such as 105 T cells per kg, which is escalated at 2 /4 week intervals between each 10-fold increase for patients receiving no anti-GVHD prophylaxis. As shown by our own data all patients with minimal residual disease, with cytogenetic or molecular relapse, responded very effectively, with no marrow aplasia, to small increments with donor PBL, without any need for more aggressive immunotherapy [34]. Our conclusions based on murine data and pilot clinical studies are in agreement with recently published observations in patients with CML, suggesting an advantage for early therapeutic intervention, before the onset of overt hematologic relapse [56]. However, further modifications may be required to improve the benefit-to-risk ratio following DLI. Previous attempts to induce early GVL following non-TCD allogeneic BMT were, however, unsuccessful [57]. Experiments with a murine model of B cell lymphocytic leukemia/lymphoma showed that GVL effects mediated by DLI could be initiated after initial reconstitution of tumor-bearing mice with T-cell-depleted bone marrow allografts [5,52], suggesting that pre-emptive DLI may be initiated under better conditions, late following BMT, in hosts with full hematopoietic reconstitution under stable clinical conditions [54,55]. The only alternative appears to be the use of non-alloreactive or tumor-reactive donor lymphocytes, in analogy to tumor-specific T lymphocytes known as tumor infiltrating lymphocytes (TIL), exerting selective anti-tumor responses in vivo, with no GVHD, as previously described [58]. In principle, tumor-specific GVL effects in the absence of GVHD could be due to donor derived TIL-like cells reacting exclusively or predominantly to tumor cells [59] or tumor-reactive cytotoxic T lymphocytes (CTL) induced in vitro [60]. Furthermore, even adequate non-specific activation of lymphocytes with high dose rIL-2 therapy in mice [61] and man [58,62,63], or activation of lymphocytes following syngeneic BMT, may convert unresponsive

lymphocytes into GVL-like cells without inducing clinically overt GVHD [64]. A similar GVL effect may, therefore, be inducible in principle by MHC-matched donor PBL. Sub optimal or absent GVL responses following allogeneic BMT in patients with non-modified marrow allografts who do not develop overt GVHD may be due to tolerance of donor T cells to host tumor cells, through either clonal reduction, anergy, blockade of T-cell receptors, or active suppression of alloreactivity by immunosuppressive agents used for GVHD prophylaxis. Alternatively, lack of GVHD and GVL may be due to a low number of cytotoxic T-lymphocyte precursors (CTLp) leading to insufficient alloreactivity of donor cells due to a perfect match between donor and host at all minor histocompatibility antigens (mHags). Clearly, mHags do play a major role in GVHD and GVL. The results reported by van Rhee et al. [56] support a direct relationship between the degree of mismatch at mHags, which corresponds to the alloreactive potential of donor PBL, and efficacy of GVL. All recipients of matched unrelated PBL responded while only 5/9 patients receiving fully matched sibling PBL did so. Based on our cumulative experience in preclinical models and ongoing clinical trials, we suggest that optimal treatment of patients at high-risk for relapse should involve a two-step procedure as shown diagrammatically in Fig. 2. First, optimal tumor debulking by non-myeloablative or myeloablative chemoradiotherapy (if indicated, supported by autologous BMT) followed by cell-therapy with DLI, while avoiding, if possible, intensive or prolonged post-BMT immunosuppression, which can negate development of GVL or GVM effects. In the second step, following hematopoietic recovery and stabilization of the patient’s condition, late post transplantation, DLI may be best accomplished by graded increments of donor PBL, for safer control of GVHD, which may required modifications of the dose and schedule of DLI [54,55]. DLI can be given on an outpatient basis, while patient is off any post transplant immunosuppression, starting with an equivalent dose of 105 T cells per kg, which is considered the dose, level at BMT beyond which patients are at risk of developing GVHD. Subsequent doses of DLI can consist of 106, 107, 108 T cells per kg or repeated doses as indicated, until elimination of measurable tumor cell markers or residual host cells, or until GVHD is imminent. Additional activation of donor effector cells, in patients with residual or resistant disease and no GVHD, can be accomplished by cytokines such as rIL-2, alpha IFN or both, which should be considered for patients who do not respond to DLI alone [35].

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

145

Fig. 2. Allogeneic cell therapy following myeloablative ND nonmyeloablative stem cell transplantation.

2.2. Induction of GVL effects by immune donor lymphocytes in preclinical animal model Since treatment of minimal residual resistant disease with DLI, especially when given concomitantly with rIL-2 and interferon which can amplify the anti-tumor effects inducible by DLI [35,61,62], it seemed reasonable to try and amplify the anti-tumor potential as well as the selectivity of anti-tumor reactivity of allogeneic donor lymphocyte by using specifically immune donor lymphocytes rather than polyclonally activated lymphocytes. Hence, we attempted induction of GVL effects with allogeneic T cells obtained from immune donors in recipients of BMT in mice inoculated with BCL1, the equivalent of human B cell leukemia/lymphoma. GVL effects were induced with donor spleen cells from mice immunized across major or minor histocompatibility barriers with BCL1-cells from the spleen of BALB/c mice inoculated with either tumor or normal BALB/c spleen cells [65]. Our data suggest that spleen cells from donor mice immunized against both BCL1 or to lesser extent normal host alloantigens induce better therapeutic GVL effects with less GVHD across both mHag and MHC. Cytokine profiles may help to characterize the nature of GVL effects with respect to GVHD across MHC barriers. A preferential up-regulation of IL-10 secretion and down-regulation of gamma IFN, alpha TNF and IL-2 was detected in donor spleen cells from mice immunized with allogeneic tumor cells compared with normal cells of the same strain [65]. This suggests that immunization of donor mice with host cells, preferentially host tumor cells, prior to isolating spleen cells used for therapy may improve their anti-tumor capacity. Anti-host responses may be down regulated in parallel with a shift of the cytokine profile from Th1 to Th2, thereby reducing GVHD while enhancing GVL. This type of immunotherapy involving the use of specifically immune donor lymphocytes may lead the

way for new approaches to eradicate leukemia, while at the same time reducing procedure-related morbidity and mortality due, primarily, to uncontrolled GVHD. 2.3. Induction of GVL effects by immune donor lymphocytes in clinical practice Based on successful experiments in animal models of human disease, showing that adoptive immunotherapy for leukemia with DLI is much more efficient when donor lymphocytes are obtained from specifically immunized donors, we have recently describe the first successful clinical attempt to treat DLI-resistant relapse with donor lymphocytes pulsed in vitro against host alloantigens [66]. A 7-year-old girl with Philadelphia positive CML underwent BMT from a fully matched 6months-old sibling (male) following conditioning with standard doses of busulfan and cyclophosphamide, relapsed 9 months after BMT (95% of the marrow cells 46XX t(9:22)) and failed to respond to DLI, including to donor lymphocytes activated with rIL-2 both in vivo and in vitro (four attempts). The patient never developed any clinical signs of GVHD. Donor lymphocytes were subsequently pulsed in vitro with a mixture of irradiated PBL obtained from both parents in order to trigger alloactivation of donor lymphocytes against host alloantigens presented by parental cells, using as stimulating cells maternal PBL expressing the shared maternal haplotype and paternal PBL expressing the shared paternal haplotype of the patient. Alpha interferon was administered to the patient in an attempt to increase the immunogenicity of tumor cells by up regulating cell surface expression of MHC. Consequently, hematologic, cytogenetic and molecular remission (negative RTPCR for bcr/abl) were induced for the first time and maintained since 1992 with a normal karyotype consisting of 100% 46XY cells in both blood and marrow. This case report, supported by murine data, suggests that

146

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

superior GVL effects with complete elimination of otherwise resistant tumor cells may be inducible with immune donor PBL, perhaps even partially independently of GVHD [66]. Taken together, using immune rather than naı¨ve donor lymphocytes may open new horizons for cancer immunotherapy by adoptively transferred donor lymphocytes. As indicated above, the use of donor derived CTL sensitized in vitro against tumor antigens was also recently pioneered in man. Successful generation of donor derived anti-leukemic CTL responses for treatment of relapsed leukemia after allogeneic HLA identical BMT was recently reported [60]. In conclusion, the long-term disease-free survival of DLI treated BMT recipients, starting with our first patient with resistant ALL with aggressive and bulky early myeloid and extramyeloid relapse, and many additional cases successfully treated for relapse elsewhere, indicate that allogeneic cell-therapy after inducing chimerism by allogeneic BMT could be the most effective approach to treat chemoradiotherapy-resistant malignant hematologic diseases. Immune-mediated interactions between donor T cells and residual tumor cells may be further increased by rIL-2 or other cytokine combinations for patients who do not respond to DLI alone and probably best by specifically immune donor lymphocytes. Future studies should focus on the development of improved methods to achieve more effective and safer immunotherapy for improving the overall disease-free and relapse-free survival in patients with hematological malignancies and possibly patients with responsive solid tumors as well. Further developments in improved clinical application of DLI with specifically immune donor lymphocytes aiming to intensify GVL and GVT while down-regulating anti-host responses are currently underway. Some potential methods for accomplishing some of these goals include the use of T cells transduced with suicide genes that may make it possible to limit their life-span once untoward GVHD is out of control; use of allogeneic natural killer (NK) cells with potent anti-tumor activity devoid of anti-host reactivity; or tumor or tissue specific T cell clones reactive against tumor-specific or tumor-associated antigens. An innovative agent that may induce apoptosis of alloreactive T cells is another potential way to control GVHD after elimination of tumor cells of host origin.

3. Effector cells of GVL effects in mice and man Since both GVHD and GVL as well as GVT effects are mediated by T cells, predominantly CD8
/, GVL and GVT effects are also associated with GVHD and thus their clinical potential is somewhat limited, especially in patients with existing spontaneous GVHD, who

may either not respond or aggravate their GVHD. There is no T cell subset that can in duce GVL effects without GVHD, and the T cell phenotype that play the key role in induction of GVL effects may vary, depending on the host, the tumor and the circumstances. It appears that effect cells of GVL effects may vary, depending on the genetic background of the host and the donor as well as expression of MHC class I and class II on the tumor cells in full consideration on the genetic disparity between donor effect cells and the tumor cells serving as target cells. Thus, as a rule, CD4 positive effector cells play the key role against class II positive tumor cells, as much as CD8 positive effector cells play the key role against class I positive tumor cells. In the course of GVHD, alloreactive donor lymphocytes contain a mixture of effector cells, of which some may be tumor-specific, as reported by the Leiden group [59]. Thus, by simple cloning techniques, a variety of donor derived T cell clones could be isolated: CTL that recognize and kill tumor cells but not normal blasts, CTL against normal host blasts but not against tumor cells, CTL that were reactive against both host and tumor cells as well as CTL that were reactive against neither, explaining convincingly the existence of GVL without GVHD, GVL in association with GVHD, and progressive tumors in patients with ongoing GVHD, respectively, [59]. We have investigated the role of CD4
/ and CD8
/ T cells in the development of GVL in mice with B-cell leukemia/lymphoma (BCL1) following allogeneic celltherapy [67,68]. Sublethally irradiated (C57BL/6 / BALB/c)F1 mice were intravenously inoculated with 105 BCL1 cells and given untreated or rIL-2-activated C57BL/6 spleen cells. Effective elimination of clonogenic BCL1 cells was confirmed by adoptive transfer of spleen cells obtained from treated mice into secondary BALB/c recipients. GVL effects were maintained after inactivation of CD4
/ cells with monoclonal anti-CD4 antibodies in the inoculum, while inactivation of CD8
/ cells with monoclonal anti-CD8 antibodies resulted in complete loss of GVL effects induced both by resting and rIL-2 activated allogeneic spleen lymphocytes [68]. Effector cells were also shown to have positive IL-2receptor, sensitive to cyclosporine and deoxyspergualin, resistant to corticosteroids [28,29]. These results indicate that Thy-1 positive, CD8 positive but not CD4 positive T cells play the major role in the induction of GVL effects, mediated by C57BL/6 effector T cells across MHC in this model. However, rIL-2 responsive NK cells may also play an important role, especially double positive CD8
/NK
/ cells, since pretreatment of effector cells, with IL-2-receptor with anti-asialo-GM1 antibody reduced substantially the GVL potential of C57BL/6 cells in BCL1 inoculated BALB/c recipients [70]. In the majority of experimental systems investigated, including the model presented here, CD8
/ -T

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

cells present in the inoculum used for transplantation or for cell-therapy play the key role in the induction of GVL effects, however, no generalization can be made about the nature of the effector cells. Based on all the available experimental data, it appears that it is the tumor in a given host that determines the nature of the GVL effector cells involved, depending on the expression of antigenic determinants, costimulatory and adhesion molecules, major or minor histocompatibility determinants, putative tumor-specific or tumor-associated determinants among many other unknown factors many of which that may be determined by the milieu, such as cytokines belonging to Th1 and Th2, antigen presenting cells and monocytes. The phenotype of GVL effect cells in BALB/c mice inoculated with BCL1 was further characterized recently as ab
/CD8
/, Fas ligand and perforin positive T cells [71]. However, as indicated above, depending on the genetic disparity between the donor, the tumor and the host, both CD4 and CD8 positive effector cells were shown to play a role in mediating GVL effects, although host effector lymphocytes or non-specific inflammatory cells may be also be recruited. In mice, depending on the genetic disparity between the donor and the host, both CD4 and CD8 positive effector cells were shown to play a role in mediating GVL effects. Thus, both CD4
/ and CD8
/ cells were shown to induce equal GVL effects across mHags in C57BL/6 mice inoculated with c-myc transformed myeloid leukemic cells [72]. In addition to T lymphocytes, other cell subsets may also be involved in induction or maintenance of GVL effects following BMT. NK cells [70,73] or even monocytes/macrophages [74] may serve as anti-tumor effector cells acting in a MHC non-restricted fashion. Since many tumor cells, especially metastases, frequently down regulate MHC molecules, NK cells seem to play a key role against tumor cells with low expression of class I, since class I seems to inhibit NK cells [75]. There are several NK inhibitory receptors which recognize class I, some of these receptors recognize specific determinants shared by certain class I alleles, and are clonally-distributed among NK cells. Therefore, in the NK repertoire, some NK cell recognize, and are blocked by, specific class I alleles. Consequently, these NK cells can mount alloreactive reactions against an allogeneic target if the target does not express the class I allele which blocks them. Hence, the target (i.e. the recipient) must fail to express one of the class I groups expressed by the donor. In this way, one of the NK cells in the donor’s repertoire will not find its class I ligand in the recipient and will be activated to kill. In man, one could chose alloreactive NK cells for effective killing of leukemia cells, particularly AML and CML, which also express LFA-1. ALL cells, that are more resistant to cell mediated immu-

147

notherapy did not express LFA-1 and were, therefore, not susceptible to NK cell mediated killing [75]. In chronic myelogenous leukemia (CML) in man, class II positive malignant clonogenic precursor cells (bcr/abl translocated t(9:22) Philadelphia positive cells) may be good targets for CD4
/ effector cells, as shown in CML patients who responded to DLI with CD8
/depleted cells following BMT [76]. Thus, depending on the species, the tumor and the disparity between donor and host cells, the nature of GVL effect cells cannot be predicted, and most likely, several components of the immune system may be involved, with possible combination of specific and non-specific reactivity against a given tumor. In other situations, none of the aforementioned mechanisms may be effective, since relapse or tumor progression may occur despite discontinuation of cyclosporine and development of severe or even lethal GVHD [77]. The existence of GVL effects independent of GVHD suggests that either unresponsive donor T cells may still recognize tumor-specific or associated antigens because different T cell clones may be involved that are highly specific, or alternatively, that under certain conditions, tolerant, anergic donor T cells, may be driven to react against tumor antigens by reversal of the unresponsive state. Indeed, we have already documented that high dose rIL-2 may force GVL-like effects in recipients with tumors [58,61/63] or even after syngeneic BMT [64]. As indicated above, T cell clones, or CTL with potential specific reactivity against tumor cells rather than normal host cells, were documented in different experimental systems supporting a possible clonal basis for GVL independently of GVHD. In addition to the existence of tumor-specific effector cells or targeting tumor cells through MHC non-restricted effector cells, relatively GVL ‘specific’ effector cells may exist against hematopoietic specific antigens present exclusively on hematopoietic cells, such as mHags recently defined as HA-1 [78,79]. Tissue specific antigens may well explain the theoretical basis for existence of GVL independent of GVHD mediated by operationally ‘specific’ or rather harmless effector cells. As such, T cell clones selected for specific reactivity against tumor cells of hematopoietic origin or donor lymphocytes enriched for tumor-specific reactivity may be used for more selective immunotherapy of all hematologic malignancies. As suggested from studies in mice, alloreactive NK cells may also become good candidates for induction of GVL and GVT effects devoid of anti-host reactivity [70,75]. NK cells do not cause GVHD, partly because they are down regulated by KIR receptors that recognize class I epitopes. Tumor cells or metastases with down-regulated class I may serve as good targets for NK cells, especially if mismatched at KIR. Indeed, reduced GVL effects following cell-therapy of tumor bearing BALB/c mice when donor lymphocytes were depleted of

148

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

asialo GM-1, which eliminates NK cells, and the feasibility to induce GVL like responses by using lymphocytes derived from F1 hybrids in tumor bearing parental strains supports in part the possible role of alloreactive NK cells in controlling relapse following BMT [70,73]. After all, resistance to allogeneic stem cell allografts and hematopoietic tumors of parental origin in F1 hybrids, which do not reject donor type skin allografts, is mediated by NK cells [80], so it seems reasonable to assume that the same rules would apply in the direction of graft-versus-host, against hematopoietic tumor cells of host origin, by cells that cannot mediate an immune response against non-hematopoietic tissues which are the main targets of GVHD. In conclusion, although patients with a variety of hematologic malignancies relapsing following BMT, especially CML, may be successfully treated with alloreactive donor lymphocytes, probably mediated primarily by donor T cells with participation of donor NK cells, patients resistant to therapy with donor cells alone might still respond to donor lymphocytes activated non-specifically with rIL-2 in vitro, in vivo, or better both combined. Patients with no GVHD, behaving like identical twins, probably due to low frequency of CTL precursors, may benefit from donor lymphocytes activated against host-type alloantigens, whereas for patients at risk of GVHD, tumor or tissue specific donor lymphocytes, or donor lymphocytes that can be eliminated at will in case of GVHD may be the proper answer. Alloimmune-mediated interactions between immunocompetent donor T cells and residual tumor cells of host origin should best be used for patients receiving no immunosuppressive agents concomitantly in order not to negate the GVL or GVT effects induced by donor lymphocytes. The efficacy of immunotherapy as described here and the lack of a safe alternative modality for treating relapse following BMT, suggest that allogeneic cell-therapy with donor PBL may become an important tool for the treatment of hematologic malignancies and most likely metastatic solid tumors as well, based on alloimmune recognition of host tumor cells, or better tumor-specific, tumor-associated or tissue specific antigens such as hematopoietic restricted mHags. Availability of effective methods to suicide donor T cells, or eliminate alloreactive cells by activation induced cell death by cytotoxicity or apoptosis may make it possible to use mismatched donor lymphocytes for more effective and faster elimination of undesirable target cells of host origin.

4. Effective graft-versus-leukemia effects independent of graft-versus-host disease in a pre-clinical animal model In clinical practice, beneficial GVL effects are usually, although not always, accompanied by GVHD. One of

the most important challenges in clinical BMT is induction of consistent curative GVL effects in the absence of severe GVHD [81]. To date, many investigators at most transplant centers are still reluctant to use T cell depletion as a means of preventing GVHD, due to anticipated increased risks of allograft rejection and relapse, hence, GVHD continues to be the most serious barrier to successful BMT. Unfortunately, as of to date, depletion of T lymphocytes is the only procedure that leads to successful and reproducible prevention of GVHD with no post-transplant anti-GVHD prophylaxis [82,83]. Rejection following T cell depletion can be controlled by using larger inocula of donor stem cells [84,85] but lack of adequate GVL effects continues to be a problem, especially in patients with aggressive lymphoid malignancies [85]. After T cell depletion, regenerating T lymphocytes derived from T cell-depleted, uncommitted donor stem cells are expected to become tolerant to the normal tissues of the host, as well as to residual tumor cells present at the time of reacquisition of immunocompetence, due to central clonal deletion, as normally occurs during the ontogeny of the immune system in establishing self tolerance. Successful induction of GVL after the initial reconstitution of hosts with a T cell-depleted allograft is likely to decrease transplant-related toxicity and improve the quality of life after allogeneic BMT. Several experiments were designed to answer two questions. First, can GVHD-free chimeras, fully reconstituted with T cell-depleted bone marrow allografts, recognize and resist non-immunogenic tumor cells of host origin despite complete tolerance of donor cells to host alloantigens. Second, can GVL effects be amplified in GVHD-free recipients of T cell-depleted marrow allografts later post BMT, by rIL-2, adoptive transfer of immunocompetent naı¨ve donor lymphocytes, in vitro cytokine-activated lymphocytes or specific adoptive immunotherapy with alloimmune or specifically immune donor lymphocytes. The following experiments were conducted to investigate the susceptibility of well-established and fully reconstituted tolerant C57BL/6 0/BALB/c chimeras to murine leukemia (BCL1) of BALB/c origin (Fig. 3) [5]. BALB/c mice were lethally irradiated (TBI 10 Gy) and reconstituted with T cell-depleted C57BL/6 bone marrow cells. Chimerism was confirmed by assaying PBLs shortly after transplantation and again 3 months later, immediately before they were inoculated with BCL1 cells. A total dose of 102 BCL1 cells is sufficient to cause 100% of death from leukemia in normal BALB/c recipients [86,87]. All mice were found to be chimeric. The percentage of donor type cells in their blood ranged between 74 and 100%. None of the chimeras showed any clinical evidence of GVHD and the body weight of chimeras was comparable with the body weight of normal controls. Normal BALB/c mice and C57BL/

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

149

Fig. 3. Induction of GVL effects following T cell depletion in immunocompetent chimeras.

Fig. 4. Development of leukemia in well-established C57BL/60/ BALB/c chimeras.

Fig. 5. Time interval needed for effective GVL effects in tolerant C57BL/60/BALB/c chimeras.

6 0/BALB/c chimeras were injected intravenously with 106 BCL1 cells. All normal BALB/c mice developed leukemia within 21/58 days and died, whereas all ten chimeras tested survived with no evidence of disease for
/6 months (P B/ 0.0001) (Fig. 4). As expected, none of the C57BL/6 0/BALB/c chimeras displayed any clinical evidence of GHVD. To follow the fate of large numbers of clonogenic BCL1 cells given to the C57BL/6 0/BALB/c chimeras, adoptive transfer experiments were conducted. Then, 105 spleen cells (prepared from a pool of three chimeras) were transferred to one secondary naı¨ve BALB/c mice 7, 14 and 21 days after being inoculated with 106 BCL1 cells (Fig. 5). With the exception of a single mouse (1/30), all adoptive recipients of spleen cells, obtained from normal

BALB/c mice 1, 2 and 3 week after inoculation with BCL1 cells, developed leukemia within 37 days and died. Seven of ten secondary recipients of cells obtained from chimeras inoculated 7 days before cell transfer developed leukemia within 44 days (P B/ 0.003). In contrast, none of the adoptive recipients of spleen cells obtained from C57BL/60/BALB/c chimeras at 14 and 21 days after inoculation with BCL1 developed leukemia when monitored for more than 6 months (P B/ 0.001). The data suggest that a period of at least 14 days is required for complete eradication and/or inactivation of 106 BCL1 cells, whereas, at 7 days, eradication of leukemic cells is still incomplete. Additional experiments were done in an attempt to amplify the GVL effects induced by T cell-depleted

150

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

Fig. 6. Amplification of GVL effects in chimeras by rIL-2 & DLI. Adoptive transfer at 1 week after DLI.

allografts in stable GVHD-free chimeras. As shown in Fig. 6, 24 normal BALB/c mice and 24 well-established C57BL/6 0/BALB/c chimeras were injected with 106 BCL1 cells. Injected chimeras were divided into four groups. Group A was comprised of C57BL/6 0/BALB/c chimeras serving as controls with no additional therapy; Group B, C57BL/60/BALB/c chimeras receiving rIL-2 (10 000 Cetus units /3 per day intra-peritoneally for 5 days) starting 1 day after inoculation with leukemic cells; group C, C57BL/6 0/BALB/c chimeras receiving 107 normal immunocompetent C57BL/6 spleen cells; and group D, C57BL/60/BALB/c chimeras receiving both 107 normal C57BL/6 spleen cells and rIL-2. For comparison, several control groups were included: group E, a control group of normal BALB/c mice inoculated with 106 BCL1 cells with no additional therapy; group F, normal BALB/c mice inoculated with 106 BCL1 cells receiving rIL-2; group G, receiving allogeneic C57BL/6 spleen cells; group H, receiving both. Seven days later, all mice were killed and their spleen cells were used for adoptive transfer experiments to assess the presence of clonogenic BCL1 cells. Secondary BALB/c recipients (five in each group) received 105 spleen cells obtained from a pool of three control BALB/c mice or from three C57BL/6 0/BALB/c chimeras of each experimental group. Matching results were obtained when the experiment was duplicated with the remaining three mice of each group. Therefore, the data were pooled and each experimental group shown in Fig. 6 consisted of ten mice. All secondary BALB/c recipients receiving spleen cells obtained from normal BALB/c mice (group E) developed leukemia within 32/ 37 days. As shown in Fig. 6, 70% of secondary recipients receiving spleen cells obtained from C57BL/6 0/BALB/c chimeras (group A) did not develop leukemia for
/6 months, and of the 30% that developed leukemia, the onset of disease was delayed (44 /52 days). C57BL/6 0/

BALB/c chimeras treated with rIL-2 (group B), allogeneic immunocompetent donor-type splenocytes (group C), or both (group D) displayed marked resistance against leukemia, with no evidence of disease being shown for
/6 months in all secondary recipients of spleen cells obtained from groups B and D and with delayed onset of leukemia in only 20% of mice receiving spleen cells from group C. No anti-leukemic effects were detected in normal control BALB/c mice treated with rIL-2, allogeneic splenocytes, or both (groups F, G and H, respectively). The difference between infusion of either spleen cells alone (rIL-2 alone) or spleen cells plus IL-2 was not significant (P /0.47). Results presented herein indicate that full resistance to leukemia can be achieved in stable chimeras reconstituted with complete T cell-depleted bone marrow, despite the complete absence of clinically overt GVHD. Moreover, GVL effects in tolerant chimeras can be amplified further by cell-mediated and/or cytokineactivated immunotherapy in vivo. Our data support the concept that GVL and GVHD are at least partially independent and suggest that GVL effects may be inducible even after initial reconstitution of lethally irradiated recipients with T cell-depleted bone marrow allografts. Furthermore, results show that allogeneic C57BL/6-type immune cells fully tolerant to BALB/c still resist the leukemogenic potential of BCL1 cells in well-established chimeras, despite full tolerance of C57BL/6 immunocompetent cells to normal BALB/c alloantigens after transplantation of T cell-depleted C57BL/6 marrow, with no GVHD. Previously, chimeras were shown to be fully tolerant to host alloantigens and to accept donor-type skin allografts indefinitely [16]. As shown in Fig. 4, 70% of adoptive recipients of spleen cells obtained from C57BL/6 0/BALB/c chimeras 7 days after inoculation with leukemic cells developed leukemia. In contrast, no adoptive recipients of spleen cells obtained from chimeras 14 and 21 days after inoculation with BCL1 cells showed any evidence of disease for more than 6 months. These data suggest that despite the absence of GVHD, tolerant chimeras can recognize tumor-associated or tumor-specific cell surface determinants other than host-type MHC, independently of GVHD. Alternatively, GVL effects could be mediated by MHC-nonrestricted immune cells that may be present in well-established and fully reconstituted chimeras, such as NK cells, rIL-2 activated NK cells, or MHC-nonrestricted gd-positive T cells. Interestingly, enhancement of GVL effects could be achieved in tolerant chimeras without GVHD by posttransplant administration of a short course of relatively low dose rIL-2 at dose levels that are ineffective by themselves, or alternatively, by administration of alloreactive donor type splenocytes. The results shown in Fig. 5 indicate that enhancement of GVL in allogeneic BMT chimeras was achieved by all three treatments,

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

because only 30% of the secondary recipients of the groups that did not receive either rIL-2 or allogeneic cells developed leukemia, whereas all the other secondary recipients were disease-free for more than 6 months. Taken together, our data may provide the evidence for tumor-specific GVL effects after allogeneic BMT induced with cell-therapy with or without rIL-2 at the stage of minimal residual disease, while avoiding early GVHD that could be induced by the BMT procedure.

5. Allogeneic cell-mediated immunotherapy with donor lymphocyte infusions for prevention of relapse following BMT for hematological malignancies Although DLI can reverse overt hematologic relapse following BMT, the chance of cure of patients with bulky or rapidly developing tumor is much smaller than in patients treated at the stage of minimal residual disease [35], hence suggesting that DLI may be most desirable and effective for prevention rather than treatment of relapse following BMT, especially in high-risk patients. Furthermore, even in patients with CML that responds well to DLI, time to achieve a complete molecular response may require several months [88]. In patients with rapidly developing tumors, such as patients with ALL that relapse following BMT, tumor cells may outgrow donor alloreactive T cells and progressive disease may be unavoidable [34,36 /38]. Post-transplant immunosuppression with cyclosporine, or combinations of cyclosporine and other agents used in experimental or clinical BMT as mandatory prophylaxis against GVHD, negate development of optimal GVL effects [27,28,69]. Induction of GVL effects by DLI, albeit its significantly beneficial anti-tumor effects, carries an inevitable risk of GVHD which is the major cause of morbidity and mortality in patients undergoing allogeneic BMT. As indicated earlier, considering the fact that optimal GVL and GVT effects can be mediated against minimal residual disease with the least risk of uncontrolled GVHD, since smaller number of alloreactive donor lymphocytes given the time required for elimination of molecular evidence of disease may be sufficient, especially if the need for rapid and aggressive GVL effects can be avoided. Our results in BALB/c mice inoculated with BCL1 have shown that GVL effects may be effectively accomplished late post BMT in GVHD-free chimeras prepared with T cell-depleted marrow allografts [5,52]. Likewise, resistance to leukemia can be established in recipients of T cell-depleted allografts if recipients were tumor free [5]. Considering the fact that the longer the time interval from BMT to DLI, the stronger the resistance against GVHD [51,52]. Hence, one possible strategy for controlling relapse and GVHD involves the use of T cell depletion for prevention of early GVHD,

151

which is much more aggressive following intensive conditioning due to stress induced cytokine release [89], and induction of GVL or GVT effects late post BMT, by graded increments of donor lymphocytes, as the resistance to GVHD builds up [51,52]. Similar results were demonstrated by Kolb et al. in canine chimeras [90]. Based on these studies, we have attempted to improve the outcome of patients with hematologic malignancy undergoing BMT by T cell depletion while avoiding any post transplant immunosuppression, followed by post transplant immunotherapy with DLI given in graded increments while controlling for GVHD, in order to prevent relapse and improve immunologic reconstitution by adoptive transfer of donor derived memory and immunocompetent cells. Here we summarize the first experience using DLI for prevention rather than treatment of relapse in patients with hematological malignancies transplanted with T cell-depleted marrow allografts. Between 1981 and 1995 we performed 314 allogeneic bone marrow transplants for patients with hematological malignancies who had an HLA identical, MLC nonresponding sibling donors. The overall proportion of patients transplanted in advanced disease was high: 42.1% of ALL and 39.1% of the AML group. Among patients with CML, only 58.6% were in chronic phase at the time of referral for transplantation and the remaining 41.4% were either in accelerated phase or in overt leukemic transformation. Patients were conditioned for transplant with either TBI and cyclophosphamide based protocols, or busulfan and cyclophosphamide regimen, with or without melphalan (60 mg/m2 /1) or cytosar (500 mg/m2 /4), or triple chemotherapeutic combination consisting of single dose of cyclophosphamide 60 mg/kg, etoposide (VP-16) 1500 mg/m2 /2, and melphalan 60 mg/m2. Total lymphoid irradiation (TLI) was administered to 171 patients (600 cGy in four fractions) already scheduled for TBI and to 29 patients scheduled for busulfan, while 15 patients received instead intravenous monoclonal rat anti-human lymphocyte antibody (anti-CD52, Campath-1G) for further immunosuppression for prevention of rejection. CD52 is expressed abundantly on T and B cells and weakly on macrophages and NK cells [91,92]. Eighty-two patients (26.1%), 58 with acute leukemia and 24 with CML, received whole unmanipulated marrow allografts. Of those, 71 patients were in primary non-responding or relapsing acute leukemia or in leukemic phase of CML. T cell depletion for GVHD prevention was initiated in 1983, first by depletion of sheep red blood cell (SRBC) rosetting T cells with or without prior enrichment of stem cells with soybean agglutinin in the first ten patients [93]. Subsequently, as soon as Campath-1 became available (first IgM with fresh donor serum as the source of complement for in vitro killing of lymphocytes and subsequently IgG2b ‘in the bag’ for

152

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

in vivo depletion of T cells through antibody-dependent cell mediated cytotoxicity), bone marrow cells of the next 218 patients were depleted with antibodies. Campath-1M, the IgM isotype resulted in 2/3 logs T cell depletion, which was sufficient for total prevention of GVHD [83]. Campath-1G, IgG2b isotype, is an opsonizing antibody that binds to Fc receptor, thus coated lymphocytes are eliminated in vivo [91,92]. Following BMT, a total of 131 patients, 46 with ALL, 59 with AML and 26 with CML, were scheduled for DLI. Three regimens for graded increments of DLI were applied as previously described [54]: the first cohort of 71 patients received donor T lymphocytes early after marrow transplant, beginning on day
/1 with 103 up to 105 T cell per kg, followed by a general schedule of weekly one log increment for a total of up to four doses. In the second group of 24 patients, T cell administration was started on week 4, pending no signs of acute GVHD developed. Thereafter, the patients received donor’ lymphocytes using gradual increments every 3/4 weeks unless signs or symptoms suggestive of acute GVHD appeared. In the third group of 36 patients, DLI was initiated by week 8. Of the 314 patients with hematologic malignancies, 131 patients (46 with ALL, 59 with AML and 26 with CML) transplanted with T cell-depleted marrow with no signs of acute GVHD, received graded increments of donor lymphocytes for prevention of relapse. Post transplant DLI significantly decreased the incidence of relapse in patients with acute leukemias (ALL and AML) and CML [55]. While the relapse-free survival of patients receiving T depleted marrow allografts without DLI was 56%, patients receiving post transplant DLI featured relapse-free survival of 77% (P /0.05). Patients transplanted with whole marrow had a relapsefree survival of 57%. These data clearly indicate that controlled post transplant DLI can reduce relapse in recipients of T cell-depleted marrow allografts with hematologic malignancies. When analyzed by stage of leukemia at BMT, DLI improved the relapse-free survival in patients transplanted in 1st and 2nd CR (P /0.02 and 0.01, respectively) but no significant effect was observed in patients transplanted in advanced stages of the disease. The relapse-free survival seem to be better in patients with acute leukemia receiving preventive DLI early as compared with late post BMT, suggesting that patients with minimal residual disease benefit much more from pre-emptive immunotherapy. The possible beneficial role of DLI induced with escalating doses of donor lymphocytes for treatment of relapse in CML, a disease which is particularly responsive to DLI, confirms and extends our observations, since it was suggested that a starting dose of 107T cells per kg may be sufficient for elimination of molecular

evidence of disease, while protecting the recipients from severe GVHD [94]. In conclusion, leukemia free survival of patients treated with DLI prophylactically was better when compared with untreated patients, irrespective of the type of T cell depletion or the schedule of T cell repletion. DLI was much more effective in patients with minimal residual disease, especially when initiation of DLI was not delayed beyond week 8 post BMT. Unfortunately, the occurrence of late GVHD following DLI administered in graded increments could not be avoided, however, such GVHD, when patient’s general condition is more stable, with no pancytopenia and susceptibility to infections may be less hazardous. Nevertheless, the overall relapse-free survival, particularly in patients with advanced disease, compared favorably to published reports from centers performing non-T cell-depleted BMT. Taken together it is reasonable to assume that late and well controlled DLI following T cell-depleted BMT may prove effective in patients with acute leukemia as well as with CML. As suggested earlier and as will be featured subsequently, it seems reasonable, though not yet proven, that in the future, tumor [59,60] or hematopoietic specific CTL [78,79]; immune donor lymphocytes [66]; donor-derived alloreactive NK cells [75]; or donor T cells transduced with a suicide gene that will make it possible to limit their life-span in case of uncontrolled GVHD [95], will replace the need for naı¨ve, uncommitted alloreactive donor lymphocytes. Based on conclusive animal data, and considering successful pilot clinical trials reviewed above, either one of these methods is likely to improve the efficacy anti-tumor effects while better controlling GVHD.

6. Non-myeloablative stem cell transplantation (NST) for treatment of malignant and non malignant diseases Although the use of myeloablative doses of chemotherapy with or without TBI followed by rescue with allogeneic BMT was considered mandatory for durable engraftment of marrow allografts, the experience over the years suggested that regardless of the intensity of chemoradiotherapy, the ‘last tumor cell’ is unlikely to be eradicated by cytoreduction alone. Indeed, the BMT procedure offers a most important advantage in the form of alloreactivity against host leukemia cells, the GVL and GVT effects, which unfortunately may be also associated with undesirable acute and chronic GVHD. Hence, it seems unlikely that a substantial improvement in the treatment of high-risk hematologic malignancies, which may require eradication of all tumor cells, may be accomplished merely by increasing the intensity of the conditioning based on the well known ‘log-dose’ relationship between the dose of

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

cytoreductive agents and the degree of tumor cell kill. Moreover, by comparing numerous protocols comprising a wide range for intensities for each of the cytoreductive components used for over 20 000 transplants reported to the International Bone Marrow Transplant Registry, no difference or clear advantage could be documented for different regimens administered as preparation for autologous BMT or allogeneic BMT, including or excluding TBI [96]. As indicated earlier, the importance of immunemediated reactions between donor-derived immunocompetent, alloreactive T lymphocytes and host-type tumor cells has been recognized to be of major therapeutic importance, accounting for the significantly better antitumor effects induced by allogeneic BMT compared with autologous BMT and transplants from an identical twin [11,81], and mostly, by the remarkable therapeutic potential of GVL effects induced by DLI [33 /40]. All this indicated that the main therapeutic component of allogeneic BMT could be ascribed to T cell mediated GVL effects rather than to physical elimination of all tumor cells by high doses of cytoreductive agents given as part of the conditioning prior to transplantation. The possibility to completely eradicate tumor cells (hematologic malignancies and solid tumors) by adoptive allogeneic cell-therapy in preclinical animal models [5,15,20,97] and in patients relapsing following maximally tolerated doses of chemoradiotherapy by DLI [33 /40], suggested that alloreactive T lymphocytes of donor origin may be the strongest tool available against tumor cells of hematopoietic origin. Hence, we have developed a working hypothesis suggesting that the main role of the transplant procedure may be in the induction of a state of host versus graft tolerance for accomplishing durable engraftment of donor-derived T lymphocytes, thus providing alloreactive T cells the opportunity to recognize and eradicate over time, late post BMT, host-derived tumor cells or abnormal stem cells, optimally when the recipient is off immunosuppressive treatment. This working hypothesis prompted us and other centers to develop a new approach for the treatment of both malignant and non-malignant hematologic diseases, avoiding the use of myeloablative conditioning, in order to improve the immediate and long-term outcome of the patients by preventing or minimizing procedure-related toxicity and mortality. Thus, several protocols were designed based on minimizing the intensity of the conditioning regimen to the range of non-myeloablative levels, followed by infusion of donor stem cells, preferably G-CSF-mobilized blood stem cells enriched with circulating T lymphocytes collected by apheresis, or bone marrow cells. The main new component of most of the new regimen was based on the use of intensive immunosuppression with fludarabine pre-grafting.

153

As recently reported [97,98], the nonmyeloablative approaches can be roughly divided into three categories: [1] focusing on lymphoablative conditioning, pre-and/or post-transplantation; [2] Focusing on immunosuppressive therapy and tumor-specific cytotoxic agents; [3] high-intensity chemotherapy for tumor debulking followed by non-myeloablative stem cell transplantation (NST) for eradication of minimal residual disease. In all these settings, NST served as a platform for subsequent adoptive cell-mediated immunotherapy in case of residual or recurrent malignancies, using donor lymphocytes. Reduced-intensity allotransplantation regimens can be divided as follows. 6.1. Focusing on lymphoablative therapy pretransplantation At the Hadassah University Hospital in Jerusalem we focused on induction of a window of immunosuppression (step 1) followed by induction of host versus graft tolerance accompanied by GVL effects mediated by donor lymphocytes infused with the mobilized blood stem cells (step 2) or DLI given later as an outpatient procedure (step 3), reasoning that the same approach may offer the prospect of safer treatment of malignant and nonmalignant diseases at all age groups with minimal and controllable early and late procedurerelated toxicity and minimal mortality [99]. Intensive pre-transplant immunosuppressive therapy was accomplished with a combination of fludarabine 30 mg/m2 per day for 6 days, busulfan 4 mg/kg per day for 2 days or cyclophosphamide for 2 days (60 mg/kg for aplastic anemia and other indications; cyclophosphamide 5-10 mg/kg for patients with Fanconi’s anemia) without or with anti-T lymphocyte globulin (ATG, Fresenius) 5 or 10 mg/kg per day for 4 days to control residual host T cells and/or partially control donor T cells causing GVHD [99 /103]. Following the conditioning, each patient received one or two infusions of G-CSF mobilized blood stem cell collections. Low dose cyclosporine (CSA) (3 mg/kg per day) was used as the sole GVHD prophylaxis for B/100 days. Our preliminary data in patients with standard indications for allogeneic BMT, including acute leukemia; chronic leukemia; non-Hodgkin’s lymphoma; myelodysplastic syndrome; multiple myeloma and a variety of non-malignant indications for BMT [99,100] including genetic diseases [101,102]; severe aplastic anemia [99]; Fanconi’s anemia [103,104] and other diseases suggest that non-myeloablative conditioning based on the use of fludarabine, was extremely well tolerated. Engraftment of allografts obtained from matched siblings and matched unrelated donors (MUD) following infusion of G-CSF mobilized blood stem cells was consistent and durable [105]. Analysis of host and donor markers using VNTR-PCR or amelogenine gene based

154

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

PCR suggested a transient stage of mixed chimerism and full replacement of host with donor hematopoietic cells. The only problem encountered using low dose cyclosporin as the sole anti-GVHD prophylaxis was acute and chronic GVHD, which was occasionally severe. Absolute neutrophil count (ANC) did not decrease below 0.1 /109/l whereas several patients never experienced ANC B/0.5 /109/l. Platelet counts did not decrease below 20/109/l, thus requiring no transfusions in 10/ 20% of the patients. The incidence of relapse did not appear to be higher than following conventional myeloablative BMT, and relapse could be reversed by DLI. To date with an observation period extending over 4 years, it can be confirmed that successful eradication of malignant and genetically abnormal host hematopoietic cells can be accomplished by alloreactive donor PBL, under conditions of host-versus-graft transplantation tolerance. Based on our preliminary experience, that needs to be confirmed in a larger series of patients observed for a longer time period, major advantages are to be expected if it can be confirmed that allogeneic NST can safely replace allogeneic BMT. Due to the patients excellent general feeling throughout the procedure, independence of hyperalimentation and the low incidence of common immediate complications (mucositis; fever due to intercurrent infections with no or shorter period of agranulocytosis; shorter period of platelet dependence; smaller risk of severe veno-occlusive disease of the liver, interstitial pneumonitis and multi-organ failure resulting from combination of some or all of the above) we anticipate that allogeneic NST may eventually become an outpatient procedure. Perhaps even more important, the state of transient or stable mixed chimerism that results from allogeneic NST may help design newer strategies for better control of GVHD. The use of allogeneic NST may also help bypass frequent late complications, which result from the combined effects of high-dose chemo-radiotherapy in addition to prior conventional treatments, especially in the low and high age groups. In the low age group, in contrast to myeloablative allogeneic BMT, allogeneic NST may reduce the incidence of growth retardation and infertility due to the unique sensitivity to chemoradiotherapy of the growth centers in the bones, the gonads and testicles. Indeed, early recovery of menstrual bleeding observed in a 19-years-old female (data not shown) was indeed encouraging in these regards. In elderly individuals, who normally may not be eligible for a standard BMT, allogeneic NST may permit a relatively safe clinical application of a potentially curative procedure based primarily on adoptive immunotherapy rather than high-dose chemo-radiotherapy. In the long run, induction of a state of mixed chimerism may help reduce the incidence and severity of GVHD. Based on animal data, mixed chimerism seems to be a reliable

recipe for engraftment of allogeneic hematopoietic cells while avoiding GVHD, as was previously shown by our earlier work [51,106] and confirmed by others [107,108]. Apparently, as suggested by experimental data in mice, host hematopoietic cells can veto donor anti-host alloreactivity while donor hematopoietic cells can veto residual alloreactive host cells, hence explaining why mixed chimeras can result in bilateral transplantation tolerance [109,110]. Availability of a relatively safe protocol for adoptive cell-therapy using matched allogeneic stem cells and T cells may offer treating physicians another therapeutic tool that may be considered with fewer hesitations for a larger number of patients in need at an optimal stage of their disease. Many clinicians would agree that as far as utilizing chemotherapy and other available cytoreductive anti-cancer agents, whatever cannot be achieved at an early stage of treatment is unlikely to be accomplished later. In addition to preventing the development of resistant tumor cell clones by continuous courses of conventional doses of chemotherapy, clinical application of a final curative modality at an earlier stage of disease may avoid the need for repeated courses of chemotherapy with cumulative multi-organ toxicity, while preventing development of platelet resistance induced by repeated sensitization with blood products and development of resistant strains of various infective agents that frequently develops in the course of antimicrobial protocols given for treatment of infections that are unavoidable during repeated courses of conventional anti-cancer modalities. In summary, we propose that immunotherapy mediated by allogeneic lymphocytes in tolerant hosts at an early stage of the disease, for every patient with a fully matched sibling, may result in a significant improvement of disease-free survival, quality of life and cost-effectiveness for candidates of allogeneic BMT. Once confirmed, these observations may open new avenues for the treatment of hematologic malignancies and genetic diseases at an earlier stage of the disease, avoiding the need for repeated courses of chemotherapy or alternative replacement therapy, respectively. Tumor cells or genetically abnormal stem cells may be effectively eliminated by an optimal combination of intense immunosuppression with relatively low dose chemotherapy, followed by infusion of donor stem cells enriched with immunocompetent T cells, aiming for induction of bilateral transplantation tolerance, thus enabling gradual elimination of all hosttype cells by donor T cells over time, while controlling for GVHD. It remains to be seen whether a similar therapeutic approach can be developed for patients with matched unrelated donor available and whether a similar modality may be extrapolated for a large number of malignancies other than those originating from hematopoietic stem cells.

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

6.2. Focusing on lymphoablative therapy pre- and posttransplantation In Seattle, conditioning focused on the use of low dose TBI. Canine studies have been used in Seattle for several decades to study immunosuppressive regimens for achieving engraftment across minor and major histocompatibility barriers and for the prevention of GVHD [36]. An advantage of this model is the ready availability of dog lymphocyte antigen (DLA) identical littermates as a model for HLA identical transplants in humans. The feasibility of using TBI as the main component of the conditioning was supported by the following studies: TBI delivered in a single fraction at a dose rate of 7 cGy per min in dogs given intensive supportive care but no stem cell grafts invariably caused lethal marrow failure at 5/400 cGy whereas at doses B/ 200 cGy dogs spontaneously recovered after a period of myelosuppression [111,112]. Single dose TBI at 920 cGy was sufficiently immunosuppressive to allow stable engraftment of marrow from 95% of DLA-identical littermate donors in the absence of post grafting immunosuppression. At 450 cGy, only 41% of dogs had stable engraftment. Additional experiments evaluated whether immunosuppressive agents given post transplant could facilitate engraftment. CSA alone was effective at a dose of 450 cGy with stable engraftment in seven of seven dogs, and the combination of CSA and MMF allowed for stable engraftment in ten of 11 dogs after a non-myeloablative dose of only 200 cGy TBI [112]. Engraftment was achieved as stable mixed chimerism with the donor component comprising 45/80% of hematopoietic cells. Dogs were followed for up to several years and continued to show stable donor engraftment. At 100 cGy TBI, MMF and CSA did not allow for stable engraftment. To evaluate mechanism by which low dose TBI was contributing to engraftment six dogs were given marrow grafts after 450 cGy lymph node irradiation at 200 cGy/min and then post grafting MMF and CSA [113,114]. The radiation was given to target cervical, thoracic and upper abdominal lymph nodes while shielding the remainder of the dogs including most marrow spaces with lead blocks. Each dog showed evidence of initial mixed chimerism. Two dogs rejected their grafts, one died with full engraftment from GVHD, and three remained stable mixed chimeras with follow up of 1 /2 years. Marrow samples from unirradiated bones showed chimerism from as early as 4 weeks post transplant that persisted for the period of observation. These results supported the hypothesis that marrow grafts could create their own space and that myelosuppressive therapy was not necessary to establish allogeneic engraftment. These observations together with results of additional preclinical studies involving second T cell activation signal blockade [115], suggest that in the future TBI may be replaced with immuno-

155

suppressive agents that lack the undesirable side effects of ionizing radiation. The safety and efficacy of the canine pre-clinical studies helped develop a conceptual scheme of studies in patients with hematologic malignancy. Immunosuppression is divided into two components, one directed at host cells before the transplant, and the other at both donor and host cells after the transplant to provide simultaneous control of both GVH and HVG reactions. The goal is to establish bi-directional graft-host tolerance as manifested by stable mixed donor-host hematopoietic chimerism. The goal of these studies was to evaluate whether allogeneic hematopoietic cell engraftment could be established in patients with malignancies using peripheral blood stem cell grafts from HLA identical donors using an immunosuppressive conditioning regimen of 200cGy TBI pre-transplant and shortterm post-grafting immunosuppression with MMF and CSA [116]. CSA was given at 6.25 mg/kg orally twice daily from day -1 through day
/35 or day
/56 and targeted to blood levels at the upper end of the therapeutic range, with supratherapeutic levels tolerated in the absence of CSA toxicities. MMF was given at a dose of 15 mg orally twice daily from day 0 to
/27 and discontinued without tapering. Eligibility for the study required a contraindication to the use of conventional allografting because of age, prior high-dose therapy, or organ dysfunction. A growing number of patients from several American and European centers over the age of 50 years (range of 31/72) or with other high-risk features were already treated. Diagnoses were acute and chronic leukemias, multiple myeloma, Hodgkin’s disease, non-Hodgkin’s lymphoma, myelodysplastic syndromes, breast cancer, and amyloidosis. Many patients were transplanted in an outpatient setting; the median number of days of subsequent hospitalization during the initial 60 days post transplant was 0 (range 0 /26). Transplants were very well tolerated with mild myelosuppression, no development of mucositis and no additional alopecia. The only significant regimen induced toxicities were reversible hepatotoxicity in three patients. In two of these, predisposing factors were liver cirrhosis in one patient and concomitant amphotericin therapy in another. Non-fatal graft rejection occurred in 16% of patients. Spontaneous acute GVHD requiring treatment occurred in 36% of patients. Transplantrelated deaths occurred in 6.5%. Significant disease responses have been already observed in the majority of patients with sustained engraftment after transplant. This has included molecular remissions in CML patients and in CLL patients and disappearance of paraprotein in myeloma patients. Many of the patients are still being monitored for disease responses. Responses have frequently been gradual in onset occurring over a periods of 4 /12 months. This is an important difference from conventional allografting in which detection of disease

156

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

post transplant usually represents failure of the treatment approach, and is potentially important in the development of optimal DLI strategies for use after nonmyeloablative transplants. 6.3. Focusing on immunosuppressive and cytotoxic chemotherapy Giralt et al. reported a study combining melphalan (180 mg/m2) and either fludarabine (125 mg/m2) or cladribine (60 mg/m2) for treatment of advanced acute leukemia; patients with refractory relapse usually recurred rapidly, but 56% of patients with chemotherapy sensitive disease remained in continuous remission beyond 1 year [117,118]. Indolent lymphoid malignancies also appear to be amenable to this strategy. Khouri et al. treated 15 heavily pretreated patients with CLL or lymphoma using a nonmyeloablative regimen of fludarabine/cyclophosphamide or fludarabine/cytarabine/cisplatin [119]. All patients had failed to respond or relapsed after primary chemotherapy. Nine patients had CLL in relapse after prior fludarabine treatment and six had lymphoma. Eleven of the 15 patients had durable engraftment, with 50 /100% donor cells at 1 month post-transplant, typically converting to 100% over the next 2 months spontaneously or after infusion of additional donor lymphocytes. Hematopoietic recovery was prompt and, with the exception of a patient with hepatitis C infection, no patient had non-hematologic toxicity of greater than grade II. The patients failing to engraft recovered endogenous hematopoiesis promptly and had no serious adverse effects. All 11 patients with engraftment have responded and eight have achieved complete remission. Maximal responses were slow to develop and gradually occur over a period of several months to 1 year. The strategy of a nonablative preparative regimen was applied also to multiple myeloma still harnessing a graft-versus-myeloma effect while reducing regimen related toxicities. The same team in Houston explored a regimen of melphalan (140 mg/m2) and fludarabine (30 mg/m2 for 4 days). This appears to be a promising strategy since seven of 13 patients with far advanced myeloma have achieved complete remissions. Use of less toxic, nonmyeloablative preparative regimens produced engraftment and generated graftversus-malignancy effects. This approach allowed the use of stem cell transplantation for older patients and those with co morbidities, which precluded high dose chemoradiotherapy. A regimen comparable to the one reported from Houston and consisting of cyclophosphamide/fludarabine was used with success by investigators in Bethesda [120,121]. Investigators in Boston used cyclophosphamide 150/ 200 mg/kg along with ATG and thymic irradiation before to HLA-identical sibling BMT in 21 patients with advanced, refractory hematologic malignancies. Grade

II /IV GVHD was seen in only one of 21 patients not receiving DLI. Prophylactic DLI was given to patients in whom no GVHD was present by day 35. Seven patients were alive and free of disease progression 105/ 548 (median 445) days after transplantation. Durable chimerism has been seen in about 90% of patients receiving HLA-identical and mismatched transplants with this protocol, and lasting mixed chimerism has been demonstrated
/1.5 years in an extensively mismatched transplant recipient [122]. The sustained remissions obtained in this group of patients with advanced and refractory disease suggest that this is a promising approach to achieving disease eradication, possibly with less GVHD than is seen with conventional transplants. 6.4. High-dose chemotherapy supported by autotransplantation followed by NST for eradication of minimal residual disease Investigators in Genoa designed a combined protocol consisting of autografting followed by mini-allografting for patients with advanced hematologic neoplasia and metastatic breast cancer [97,98,123,124]. Patients with high-risk Hodgkin’s disease, non-Hodgkin’s lymphoma, blastic or accelerated phase CML; liver/bone metastatic breast cancer already entered various modifications of this combined approach. After engraftment of autologous stem cells, all patients were conditioned for allografting with fludarabine 30 mg/m2 per day /3 days with cyclophosphamide 300 mg/m2 per day/3 days, designated the fludarabine
/cyclophosphamide protocol. Progenitor cells from HLA-matched donors were mobilized with G-CSF and infused to the patients. GVHD prophylaxis comprised CSA and methotrexate. After autografting, the results in the different diseases were encouraging, with many patients achieving CR after mini-allografting and others only after DLI. Most patients did not develop severe neutropenia. In summary, these observations confirmed the efficacy of immunosuppressive therapy with fludarabine and low dose cyclophosphamide for engraftment of hematopoietic progenitor cells from HLA-matched sibling donors with minimal toxicity and low mortality rate.

7. Immunotherapy of metastatic solid tumors with alloreactive lymphocytes Tumor cells are uniformly rejected when planted in allogeneic recipients, it seems reasonable to assume that the mirror image is also true, that is, that donor lymphocytes can also react against tumor cells of host origin, as long as rejection of such potentially antitumor effect cells can be prevented. Indeed, we have already documented in the early 80’s that following induction of host-versus-graft transplantation tolerance

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

in female (NZB /NZW)F1 recipients (mice susceptible to develop clinical and laboratory manifestations of systemic lupus erythematosus as well as solid sarcoma) with BMT from BALB/c mice, donor bone marrow cells containing alloreactive lymphocytes were also responsible for complete prevention of development of spontaneous sarcoma, which developed in 24% of untreated controls and in none of the chimeras [20]. The data suggested induction of GVT effects following nonmyeloablative conditioning prior to allogeneic stem cell transplantation independently of clinically overt GVHD. Recently, we have been able to demonstrate the efficacy of allogeneic cell-therapy in a murine mammary carcinoma model (4T1) of BALB/c mice, using naive MHC-mismatched splenocytes [21,22]. The encouraging clinical results with cell-therapy in hematologic malignancies using MHC identical donor cells that differ from the phenotype of the tumor by mHags only, led us to test the feasibility of inducing GVT effects across mHag barriers in a murine model of mammary carcinoma, in order to apply this modality to patients who have received immunocompetent cells from related mHagmismatched donors. In the following study, naive or immune donor cells sensitized with either tumor or normal minor mismatched splenocytes, were tested for their ability to exert more effective anti-tumor activity in mice inoculated with a minimal dose of mammary carcinoma cells. Cell-therapy with allogeneic donor cells mismatched for mHag was tested in a murine mammary carcinoma (4T1) model for metastatic epithelial tumors [125]. BALB/c mice bearing the 4T1 tumor of BALB/c origin were given MHag-mismatched DBA/2-derived splenocytes. GVT effects were assayed in secondary recipients of adoptively transferred lung cells derived from primary hosts previously inoculated intravenously with 4T1 cells and injected with either: [1] naive BALB/c splenocytes [2] naive DBA/2 splenocytes, [3] 4T1immune DBA/2 splenocytes, or [4] DBA/2 splenocytes immunized with host-derived BABL/c spleen cells. Naive DBA/2 splenocytes inhibited tumor growth only slightly and hardly prolonged the survival of secondary recipients, in comparison with fully matched tumor/host BALB/c spleen cells. An efficient GVT reaction was demonstrated in vitro and in vivo with MHag-mismatched DBA/2 splenocytes from mice pre-sensitized by multiple injections of irradiated tumor or BALB/cderived spleen cells. All 30 mice adoptively inoculated with lung cells from primary hosts that had previously been treated with these pre-sensitized effector cells were tumor-free for
/250 days. Secondary recipients inoculated with lung cells from mice given naive BALB/c or DBA/2 spleen cells died of metastatic tumors within 33/ 46 days. Pre-immunization with MHag-mismatched tumor or spleen cells activated effector cells to induce an anti-tumor response. We suggest that cell-therapy

157

with pre-immunized effector cells may be clinically applicable in cases where naive allogeneic cells have not been able to eradicate residual tumor cells in mammary carcinomas or other metastatic solid tumors. Taken together, our data provide the scientific background to consider the possibility of using donor lymphocytes activated against tumor antigens or hosttype alloantigens as a new clinical tool, both for overcoming residual disease in metastatic breast cancer and possibly other solid tumors as well as well as for the treatment of recurrent disease, especially for patients failing all available anti-cancer modalities. Measurable response against liver metastases which disappeared transiently following cell-therapy, were also documented in a patient with liver metastases that developed following myeloablative combination chemotherapy supported by autologous blood stem cell transplantation, following administration of fully matched donor lymphocytes without any additional conditioning [25]. The data suggests that in analogy to GVL effects, GVT effects may also occur against solid tumors, as long as donor lymphocytes are accepted by the host. GVT effects following standard myeloablative conditioning was also reported in patients with metastatic breast cancer, however, the procedure proved toxic, hence, widespread clinical application could not be recommended [23,24]. Considering the toxicity and mortality resulting from conventional myeloablative preparatory regimen, introducing non-myeloablative conditioning in preparation for allogeneic stem cell transplantation may provide new therapeutic options for patients with otherwise incurable malignant disorders. Indeed, it was recently documented by Childs et al. that NST may be used also for elimination of metastatic renal cell cancer, including in patients fully resistant to all available anti-cancer modalities [120,121]. These observations, also supported by earlier reports suggesting that renal cell cancer may be even responsive to rIL-2 activated autologous lymphocytes [58,126], may open new potential therapeutic options for other solid tumors that are resistant to conventional chemotherapy and radiotherapy. Although the rationale for this treatment is well based on documentation of GVT effects induced by alloreactive donor lymphocytes, provided that rejection of donor lymphocytes can be prevented, such an approach seemed unacceptable in cancer patients treated with conventional myeloablative BMT, due to severe toxicity and high mortality rates [23,24]. As the clinical experience with NST for the treatment of hematologic malignancies grew, it became evident that this method may be applicable for immunotherapy of metastatic solid tumors. This prospect was reinforced by existing data in animals suggesting that GVT effects could be induced following nonmyeloablative conditioning [20]. For this reason, the partial success of NST in treating

158

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

metastatic renal cell cancer may provide hope for adoptive immunotherapy of cancer by allogeneic lymphocytes. A measurable response was observed in approximately 50% of the patients who enrolled in the study, and durable complete responses were accomplished in several patients (10 /15%). Although these results are promising, and should encourage similar strategies for other metastatic tumors, the procedure involving NST followed by mandatory GVHD as a result of non-specific anti-host responses, should not be regarded as the final protocol, since it was not fully satisfactory both in terms of the efficacy of anti-tumor effects accomplished, as well as the GVHD related toxicity and mortality. Indeed, despite tumor regression in some patients, there was severe, occasionally fatal, GVHD as well as progressive tumor growth or both in others. Two patients died after receiving this treatment. Clearly, although the principle was well supported by the regression of metastatic lesions, the procedure needs further improvement to minimize complications such as uncontrolled GVHD, while in parallel, improving anticancer efficacy. The association between GVT and the occurrence of GVHD in most of the patients with renal cell cancer studied suggests that the anti-tumor effect of donor lymphocytes is mediated at least in part by an immune reaction against tumor cells that display the recipient’s histocompatibility antigens. Nevertheless, pre-clinical [5,115] and early clinical studies [11] indicate that both GVL and GVT effects may occur in the absence of clinically significant GVHD. Indeed, in several patients with renal cell cancer too, GVT effect was observed in the absence of GVHD at the time of disease regression [121]. Likewise, regression of metastases was often delayed by months from the onset of GVHD [121], again suggesting that the anti-tumor effector mechanisms may be distinct from those that cause anti-host responses. The clinical responses observed in patients with renal cell cancer were not yet documented in any other type of metastatic solid tumor. Several patients with metastatic malignant melanoma failed to respond, but they were all in advanced disease state and since the time to response may last several months, up to 1 year, it is possible that failure to respond was due to the short life expectancy. It remains to be seen if the response observed thus far in patients with renal cell cancer are reproducible and whether they represent the rule rather than the exception. Clearly, once the principle can be confirmed in clinical practice, several newer approaches may enhance the efficacy and reduce the risks of immunotherapy with donor lymphocytes. Recent experiments in our laboratory suggest that anti-cancer effects can be significantly improved while eliminating or reducing the severity of GVHD by using specifically immune rather than naı¨ve donor T lymphocytes. Such immune T cells can be

generated in vitro by culture of lymphocytes with tumor-specific peptides or the patient’s tumor cells as long as tumor antigens are presented by antigen presenting cells. Donor cytotoxic and helper T cells can be generated in vitro by a method that disables reactivity of the T cells against the prospective host’s histocompatibility antigens, leaving a population of tumor-specific T cells. Another approach, which could apply to patients without a histocompatible sibling, uses T cell-depleted hematopoietic stem cells to render the recipient incapable of rejecting the graft, followed by administration of graded increments of donor T cells (or tumor-specific T cells), with careful titration until elimination of all tumor cells of host origin or until the first evidence of GVHD. It is also possible to manipulate the donor lymphocytes in vitro by insertion of a suicide gene, such as the herpes simplex virus thymidine kinase gene, which provides an option for limiting the life-span of the infused lymphocytes once elimination of all tumor cells was accomplished, thus reducing the risk of uncontrolled GVHD. Further future progress in treating metastatic solid tumors with adoptive allogeneic cell-mediated immunotherapy will depend on improving the methods for induction of antitumor immunotherapy on the one hand, and better control of transplant-related complications, particularly GVHD, on the other. Clearly, targeting donor anticancer effector cells to the tumor by using immune specific donor lymphocytes, or by targeting killer cells to cell surface tumor markers not shared by normal somatic cells may be the next obvious step to induce GVT independently of GVHD.

8. Conclusions and future directions Allogeneic cell-therapy, occasionally cytokine therapy and certainly both combined, provide a most effective therapeutic tool for patients resistant to chemoradiotherapy. Whereas patient’s naı¨ve immune system may ignore tumor cells and considering the fact that activation of immune responses against autologous tumor cells may be ineffective or difficult to accomplish, adoptive allogeneic cell-therapy with alloreactive, or even much better, tumor-reactive lymphocytes, may provide a safe and most effective therapeutic option. Engraftment of donor lymphocytes seems essential for durable anti-tumor effects. Whereas myeloablative conditioning was considered until recently mandatory for engraftment of marrow or blood stem cell allografts and for eradication of the malignant disease, recent investigations suggest that effective treatment can be accomplished by reduced intensity conditioning, since engraftment of donor stem cells which results in induction of host-versus-graft transplantation tolerance permits engraftment of donor derive immunocompetent

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

lymphocytes, thus potentially mediating GVL and GVT effects, through a transient stage of mixed chimerism followed by rapid replacement of host with donor hematopoietic cells. Depending on the components used for cytoreduction, NST appears to be well tolerated, with minimal procedure-related toxicity and mortality. Although larger number of patients and longer observation period is required to fully assess the efficacy of NST and to compare the overall results of NST to conventional BMT, grossly speaking, the incidence of relapse did not appear to be higher than following conventional myeloablative BMT. Likewise, based on nearly 15 years of experience with DLI and nearly 8 years of experience with NST, it can be concluded that such modalities may cure patients considered incurable by any of the alternative anti-cancer modalities. The existing experience, which needs to be substantiated, suggests that the overall disease-free survival of patients treated with NST is superior or equal to BMT, depending on the disease category and tumor bulk. On some occasions, reversal of partial to complete chimerism, elimination of residual host hematopoietic cells or reversal of early relapse could be accomplished by discontinuation of CSA or by adding graded increments of DLI post grafting, but frequently at the cost of GVHD. To date, with an observation period extending over 15 years since the first successful clinical application of DLI, it can be confirmed that successful and durable eradication of malignant or otherwise abnormal host hematopoietic cells can be accomplished by alloreactive donor lymphocytes following induction of hostversus-graft transplantation tolerance. Thus, it appears that myeloablative conditioning is neither mandatory for durable engraftment of stem cell allografts obtained from fully matched sibling and unrelated donors, nor is it required for replacement of malignant or otherwise abnormal host hematopoietic cells with normal donor cells. Following successful NST, engraftment of donor stem cells is rapid and consistent and the GVL, GVT or GVM effects induced by donor immunocompetent lymphocytes may successfully replace the need for myeloablative conditioning. Reduced incidence of procedure-related toxicity and mortality may serve as an incentive for cell-therapy at an earlier stage of the of disease, when the chance of uneventful recovery is optimal, for all patients with a matched donor available in need of BMT, regardless of age. Furthermore, using a well-tolerated reduced intensity conditioning, even elderly individuals or patients with poor performance status may be eligible for BMT, thus providing an option for cure for a larger number of patients in need. However, optimal clinical application of NST will depend on prospective randomized trials to document advantage over conventional BMT on the one hand, and much better control of acute and especially chronic GVHD on the other. Several innovative approaches are

159

currently in under investigation in an attempt to accomplish the final goal in adoptive allogeneic celltherapy: maximizing anti-tumor effects while minimizing anti-host responses.

Reviewers Professor A.M. Carella, Chief, Department of Hematology & BMT unit, IRCSS, Casa Sollieno della Sofferenza, S. Giovanni Rotondo, I-71030 Foggia, Italy. Sergio Giralt, M.D. Associate Professor of Medicine, M.D. Anderson Cancer Center, The University of Texas, 1515 Holcombe Blvd., Box 423, Houston, TX 77030, USA.

References [1] Thomas ED. Marrow transplantation for malignant diseases. J Clin Oncol 1983;1:517 /31. [2] Chronic Leukemia Working Party of the EBMT, Gratwohl A, Hermans J, Goldman JM, et al. Chronic leukemia. Lancet 1998;352:1087. [3] Passweg JR, Rowlings PA, Armitage JO, et al. In: Cecka JM, Teraskaki PI, editors. Report from the international bone marrow transplant registry and autologous blood and marrow transplant registry */North America. Clinical transplants, 1995. Los Angeles, California: UCLA Tissue Typing Laboratory, 1996:117 /27. [4] Bast CR. Principles of cancer biology: tumour immunology. In: DeVitta VT, Hellman S, Rosenberg SA, editors. Cancer; principles & practice of oncology. Philadelphia: J.B. Lippincott Company, 1985:125 /41. [5] 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):2562 /7. [6] Grillo-Lopez AJ, White CA, Dallaire BK, et al. Rituximab: the first monoclonal antibody approved for the treatment of lymphoma. Curr Pharm Biotechnol 2000;1(1):1 /9 (Review). [7] Thomas ED. The role of marrow transplantation in the eradication of malignant disease. Cancer 1963;49:1963 /8. [8] Weiden PL, Sullivan KM, Fluornoy N, Strob R, Thomas ED. Antileukemic effect of chronic graft-versus-host disease: contribution to improved survival after allogeneic marrow transplantation. New Engl J Med 1981;304:1529 /33. [9] Weiden PL, Fluornoy N, Sanders JE, Sullivan KM, Thomas ED. Antileukemic effect of graft-versus-host disease contributes to improved survival after allogeneic marrow transplantation. Transplantation 1981;13:248 /51. [10] Sullivan KM, Weiden PL, Storb R, et al. Influence of acute and chronic graft-versus-host disease on relapse and survival after bone marrow transplantation from HLA-identical siblings as treatment of acute and chronic leukemia. Blood 1989;73:1720 /6. [11] Horowitz M, Gale RP, Sondel PM, et al. Graft-versus-leukemia reactions after bone marrow transplantation. Blood 1990;75:555 /62. [12] Sinkovics JG, Shullenberger CC, Howe CD. Prolongation and prevention of Rauscher virus mouse leukemia by spleen cells of naturally resistant or actively immunized mice. Clin Res 1965;13:36 /9.

160

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

[13] Boranic M, Tonkovic I. Time pattern of the antileukemia effect of graft-versus-host reaction in mice, I. Cellular events. Cancer Res 1971;31:1140 /7. [14] Bortin MM, Truitt RL, Rimm AA, Bach FH. Graft-versusleukaemia reactivity induced by alloimmunization without augmentation of graft-versus-host reactivity. Nature 1979;281:490 /1. [15] Slavin S, Weiss L, Morecki S, Weingensberg M. Eradication of murine leukemia with histoincompatible marrow grafts in mice conditioned with total lymphoid irradiation (TLI). Cancer Immunol Immunother 1981;11:155 /61. [16] Truitt RL, Shih F-H, LeFever AV, Tempelis LD, Andreani M, Bortin MM. Characterization of alloimmunization-induced T lymphocytes reactivated against AKR leukemia in vitro and correlation with graft-vs-leukemia activity in vivo. J Immunol 1983;131:2050 /8. [17] Meredith RF, O’Kunewick JP. Possibility of graft-vs-leukemia determinants independent of the major histocompatibility complex in allogeneic marrow transplantation. Transplantation 1983;35:378 /85. [18] Weiss L, Weigensberg M, Morecki S, et al. Characterization of effector cells of graft vs leukemia (GVL) following allogeneic bone marrow transplantation in mice inoculated with murine Bcell leukemia (BCL1). Cancer Immunol Immunother 1990;31:236 /42. [19] Truitt RL, Atasoylu AA. Impact of pretransplant conditioning and donor T cells on chimerism, graft-versus-host disease, graftvs-leukemia reactivity, and tolerance after bone marrow transplantation. Blood 1991;77:2515 /23. [20] Moscovitch M, Slavin S. Anti-tumor effects of allogeneic bone marrow transplantation in (NZB/NZW)F1 hybrids with spontaneous lymphosarcoma. J Immunol 1984;132:997 /1000. [21] Morecki S, Moshel Y, Gelfend Y, et al. Induction of graft vs tumor effect in a murine model of mammary adenocarcinoma. Int J Cancer 1997;71:59 /63. [22] Morecki S, Yacovlev E, Diab A, Slavin S. Allogeneic cell therapy for a murine mammary carcinoma. Cancer Res 1998;58:3891 /5. [23] Eibl B, Schwaighofer H, Nachbaur D, et al. Evidence for a graftversus-tumor effect in a patient treated with marrow ablative chemotherapy and allogeneic bone marrow transplantation for breast cancer. Blood 1996;88:1501 /8. [24] Ueno NT, Rondo´n G, Mirza NQ, et al. Allogeneic peripheralblood progenitor-cell transplantation for poor-risk patients with metastatic breast cancer. J Clin Oncol 1998;16:986 /93. [25] Or R, Ackerstein A, Nagler A, et al. Allogeneic cell mediated immunotherapy for breast cancer after autologous stem cell transplantation: a clinical pilot study, Cytokines, Cellular & Molecular Therapy 1998;4:1 /6. [26] Higano CS, Brixey M, Bryant EM. Durable complete remission of acute nonlymphocytic leukemia associated with discontinuation of immunosuppression following relapse after allogeneic bone marrow tranplantation. A case report of a probable graftversus-leukemia effect. Transfusion 1990;50:175 /8. [27] Bacigalupo A, Van Lint MT, Occhini D, et al. Increased risk of leukemia relapse with high dose cyclosporine A after allogeneic marrow transplantation for acute leukemia. Blood 1991;77:1423. [28] Weiss L, Reich S, Slavin S. Effect of cyclosporine A and methylprednisolone on the GVL effect across major histocompatibility barriers in mice following allogeneic bone marrow transplantation. Bone Marrow Transplant 1990;6:229 /33. [29] Kapelushnik J, Or R, Aker M, et al. Allogeneic cell therapy of severe beta thalassemia major by displacement of host stem cells in mixed chimera by donor blood lymphocytes. Bone Marrow Transplant 1996;19:96 /8. [30] Kapelushnik J, Aker M, Or R, et al. Allogeneic cell therapy as a new modality for displacement of genetically abnormal stem cells as part of the conditioning for allogeneic bone marrow

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

transplantation.Correction of genetic diseases by transplantation (Chapter 4). Cogent Press, 1997:111 /9. Aker M, Kapelushnik J, Pugatsch T, et al. Donor lymphocyte infusions to displace residual host hematopoietic cells after allogeneic BMT for beta thalassemia major. J Pediatr Hematol/Oncol 1998;20(2):145 /8. Slavin S, Nagler A, Naparstek E, et al. Minitransplants and cellbased therapies for malignant and non-malignant disorders. Curr Opin Organ Transplant 1999;4(3):184 /8. 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. 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;23:1553 /62. 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;87:2195 /204. Kolb HJ, Mittermuller J, Clemm C, et al. Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood 1990;76:2462 /5. Working Party Chronic Leukemia, Kolb HJ, Schattenberg A, Goldman JM, et al. Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients: European Group for blood and marrow transplantation. Blood 1995;86:2041 /50. Collins RH, Shpilberg O, Drobyski WR, et al. Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. J Clin Oncol 1997;15:433 / 44. Porter DL, Roth MS, McGarigle C, et al. Induction of graftversus-host disease as immunotherapy for relapsed chronic myeloid leukemia. New Engl J Med 1994;330:100 /6. Mackinnon S, Papadopoulos EB, Carabassi MH, et al. Adoptive immunotherapy evaluating escalating doses of donor leukocytes for relapse of chronic myeloid leukemia after bone marrow transplantation: separation of graft-versus-leukemia responses from graft-versus-host disease. Blood 1995;86:1261 /8. Gubbay I, Collignon J, Koopman P, et al. A gene mapping to the sex-determining region of the mouse y chromosome is a member of a novel family of embryologically expressed genes. Nature 1990;346:245 /50. Pugatsch T, Oppenheim A, Slavin S. Improved single-step PCR assay for sex identification post-allogeneic sex-mismatched BMT. Bone Marrow Transplant 1996;17:273 /5. Kawasaki ES, Clark SS, Coyne MY, et al. Diagnosis of chronic myeloid and acute lymphocytic leukemias by detection of leukemia-specific mRNA sequences amplified in vitro. PNAS 1988;85:5698 /702. De Witte T, Schattenberg A, Preijers F, Mensink E. Treatment of relapse in recipients of lymphocyte depleted grafts with infusion of lymphocytes of the original bone marrow donor. Exp Hematol 1992;20:723. Bar BM, Schattenberg A, Mensink EJ, et al. Donor leukocyte infusions for chronic myeloid leukemia relapsed after allogeneic bone marrow transplantation. J Clin Oncol 1993;11:513 /9. Leber B, Walker IR, Rodrigues A, McBride JA, Carter R, Brian MC. Reinduction of remission of chronic myeloid leukemia by donor leukocyte transfusion following relapse after bone marrow transplantation: recovery complicated by initial pancytopenia and late dermatomyositis. Bone Marrow Transplant 1993;12:405 /7. Papadopoulos EB, Ladanyi M, Emanual D, et al. Infusions of donor leukocytes to treat Epstein /Barr virus-associated lympho-

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58] [59]

[60]

[61]

[62]

[63]

proliferative disorders after allogeneic bone marrow transplantation. New Engl J Med 1994;330:1185 /91. Kuzushima K, Yamamoto M, Kimura H, et al. Establishment of anti-Epstein /Barr virus (EBV) cellular immunity by adoptive transfer of virus specific cytotoxic T lymphocytes from an HLA matched sibling to a patient with severe chronic active EBV infection. Clin Exp Immunol 1996;103:192 /8. Stockschlaeder M, Storb R, Pepe M, et al. A pilot study of lowdose cyclosporin for graft-versus-host prophylaxis in marrow transplantation. Br J Haematol 1992;80:49 /54. Odom LF, August CS, Githens JH, et al. Remmission of relapsed leukaemia during a graft-versus-host reaction. A ‘graft-versus-leukaemia reaction’ in man. Lancet 1978;2:537 /8. Slavin S, Fuks Z, Kaplan HS, Strober S. Transplantation of allogeneic bone marrow without graft vs host disease using total lymphoid irradiation. J Exp Med 1978;147:963 /72. 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;10:19 /26. Johnson BD, Drobyski WR, Truitt RL. Delayed infusion of normal donor cells after MHC-matched bone marrow transplantation provides an antileukemia reaction without graftversus-host disease. Bone Marrow Transplant 1993;11:329 /36. 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;89:506 /15. Naparstek E, Nagler A, Or R, Slavin S. In: Cecka JM, Teraskaki PI, editors. Allogeneic cell mediated immunotherapy using donor lymphocytes for prevention of relapse in patients treated with allogeneic BMT for hematological malignancies. North America. Clinical transplants, 1995. Los Angeles, California: UCLA Tissue Typing Laboratory, 1996:281 /90. van Rhee F, Lin F, Cullis JO, et al. Relapse of chronic myeloid leukemia after allogeneic bone marrow transplant: the case for giving donor leukocyte transfusions before the onset of hematological relapse. Blood 1994;83(11):3377 /83. Sullivan KM, Storb R, Buckner CD, et al. Graft-versus-host disease as adoptive immunotherapy in patients with advanced hematologic neoplasms. New Engl J Med 1989;320:828 /34. Rosenberg SA. The immunotherapy and gene therapy of cancer. J Clin Oncol 1992;10:180 /92. Van Lochem E, De Gast B, Goulmy E. In vitro separation of host specific graft-versus-host and graft-versus-leukemia cytotoxic-T cell activities. Bone Marrow Transplant 1992;10:181 /3. Falkenburg JHF, Faber LM, van den Elshout M, et al. Generation of donor derived antileukemic cytotoxic T lymphocyte responses for treatment of relapsed leukemia after allogeneic HLA identical bone marrow transplantation. J Immunother 1993;14:305. Slavin S, Ackerstein A, Weiss L. Adoptive immunotherapy in conjunction with bone marrow transplantation-amplification of natural host defence mechanisms against cancer by recombinant IL2. Natural Immunity & Cell Growth Regul 1988;7:180 /4. Nagler A, Ackerstein A, Or R, Naparstek E, Slavin S. Immunotherapy with recombinant human interleukin-2 (rIL-2) and recombinant a-interferon in lymphoma patients post autologous marrow or stem cell transplantation. Blood 1997;89(11):3951 /9. Nagler A, Ackerstein A, Barak V, Slavin S. Treatment of chronic myelogenous leukemia with recombinant human interleukin-2 and interferon-2a. J Hematother 1994;3:75 /82.

161

[64] Ackerstein A, Kedar E, Slavin S. Use of recombinant human interleukin-2 in conjunction with syngeneic bone marrow transplantation as a model for control of minimal residual disease in malignant hematological disorders. Blood 1991;78:1212 /5. [65] Weiss L, Weigensberg M, Morecki S, et al. Characterization of effector cells of graft vs leukemia (GVL) following allogeneic bone marrow transplantation in mice inoculated with murine Bcell leukemia (BCL1). Cancer Immunol Immunother 1990;31:236 /42. [66] Slavin S, Ackerstein A, Morecki S, Gelfand Y, Cividalli G. Immunotherapy of relapsed resistant chronic myelogenous leukemia post allogeneic bone marrow transplantation with alloantigen pulsed donor lymphocytes, Bone Marrow Transplant. 2001;28:795 /8. [67] Weiss L, Reich S, Slavin S. The role of non-activated and rIL-2 activated CD4
/ and CD8
/ T cells in immunotherapy for leukemia. Cytokines, Cell Mol Therapy 1999;5:153 /8. [68] Weiss L, Reich S, Slavin S. Effect of deoxyspergualin on graft vs host disease and graft vs leukemia in mice. Bone Marrow Transplant 1996;17:789 /92. [69] Weiss L, Reich S, Slavin S. The role of antibodies to IL-2 receptor and anti-Asialo GMI antibodies on GVL effects induced by BMT in murine B cell leukemia. Bone Marrow Transplant 1995;16(3):457 /61. [70] Palathumpat V, Dejbakhsh-Jones S, Strober S. The role of purified CD8
/ T cells in graft-versus-leukemia activity and engraftment after allogeneic bone marrow transplantation. Transplantation 1995;60(4):355 /61. [71] Korngold R, Leighton C, Manser T. Graft-vs-myeloid leukemia responses following syngeneic and allogeneic bone marrow transplantation. Transplantation 1994;58(3):278 /87. [72] Zeis M, Uharek L, Glass B, et al. Allogeneic MHC-mismatched activated natural killer cells administered after bone marrow transplantation provide a strong graft-versus-leukemia effect in mice. Br J Haematol 1997;96:757. [73] Greenberg PD. Adoptive T cell therapy of tumors: Mechanisms operative in the recognition and elimination of tumor cells. Adv Immunol 1991;49:281. [74] Ruggeri L, Capanni M, Casucci M, et al. Role of natural killer cell alloreactivity in HLA-mismatched hematopoietic stem cell transplantation. Blood 1999;94(1):333 /9. [75] Giralt S, Hester J, Huh Y, et al. CD8-depleted donor lymphocyte infusions as treatment for relapsed chronic myelogenous leukemia after allogeneic bone marrow transplantation. Blood 1995;86:4337 /43. [76] Collins RH, Rogers ZR, Bennett M, Kurser V, Nikein A, Fay JW. Hematologic relapse of chronic myelogenous leukemia following abrupt discontinuation of immunosuppression. Bone Marrow Transplant 1992;10:391 /5. [77] Goulmy E. Human minor histocompatibility antigens: new concepts for marrow transplantation and adoptive immunotherapy. Immunol Rev 1997;157:125 /40. [78] Mutis T, Verdijk R, Schrama E, Esendam B, Brand A, Goulmy E. Feasibility of immunotherapy of relapsed leukemia with ex vivo-generated cytotoxic T lymphocytes specific for hematopoietic system-restricted minor histocompatibility antigens. Blood 1999;93:2336 /41. [79] Cudkowicz G, Rossi GB. Hybrid resistance to parental DBA-2 grafts: independence from the H-2 locus. I. Studies with normal hematopoietic cells. J Natl Cancer Inst 1972;48(1):131 /9. [80] Ringden O, Horowitz MM. Graft-vs-leukemia reactions in humans. Transplant Proc 1989;21:2989. [81] Reisner Y, Kapoor N, et al. Transplantation for severe combined immunodeficiency with HLA-A,B,D,DR incompatible parental marrow cells fractionated by soybean agglutinin and sheep red blood cells. Blood 1983;61(2):341 /8.

162

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163

[82] Waldmann H, Polliack A, Hale G, et al. Elimination of graftversus-host disease by in-vitro depletion of alloreactive lymphocytes with a monoclonal rat anti-human lymphocyte antibody (CAMPATH-1). Lancet 1984;2:483 /5. [83] Reisner Y, Martelli MF. Bone marrow transplantation across HLA barriers by increasing the number of transplanted cells. Immunol Today 1995;16:437. [84] Aversa F, Tabilio A, Velardi A, et al. Treatment of high-risk acute leukemia with T-cell-depleted stem cells from related donors with one fully mismatched HLA haplotype. New Engl J Med 1998;339:1186 /93. [85] Slavin S, Strober S. Spontaneous murine B-cell leukemia. Nature 1978;272:624. [86] Slavin S, Weiss L, Morecki S, et al. Ultrastructural, cell membrane and cytogenetic characteristics of B-cell leukemia (BCL1), a murine model of chronic lymphocytic leukemia. Cancer Res 1981;41:4162 /6. [87] Raanani P, Dazzi F, Sohal J, et al. The rate and kinetics of molecular response to donor leucocyte transfusions in chronic myeloid leukaemia patients treated for relapse after allogeneic bone marrow transplantation. Br J Haematol 1997;99(4):945 / 50. [88] Hill GR, Crawford JM, Cooke KR, Brinson YS, Pan L, Ferrara JL. Total body irradiation and acute graft-vs-host disease; the role of gastrointestinal damage and inflammatory cytokines. Blood 1997;90:3204 /13. [89] Kolb HJ, Beisser K, Holler E, et al. Donor buffy coat transfusion for adoptive immunotherapy in human and canine chimeras. Periodicum Biologorum 1991;93:81. [90] Slavin S, Waldmann H, Or R, et al. Prevention of graft vs host disease in allogeneic bone marrow transplantation for leukemia by T cell depletion in vitro prior to transplantation. Transplant Proc 1985;17:465 /7. [91] Cobold S, Martin G, Waldmann H. Monoclonal antibodies for the prevention of graft versus host disease and marrow graft rejection. Transplantation 1986;42:239. [92] Hale G, Xia MQ, Tighe HP, Dyer MJS, Waldmann H. The CAMPATH-1 antigen (CDw52). Tissue Antigens 1990;35:118. [93] Naparstek E, Or R, Nagler A, et al. Allogeneic BMT for leukemia using Campath-1 monoclonal antibodies & post transplant alloimmunization with donor lymphocytes. Exp Hematol 1993;21:1061 (Abs. 189). [94] Mackinnon S, Papadopoulos EB, Carabasi MH, et al. Adoptive immunotherapy evaluating escalating doses of donor leukocyte for relapse of chronic myeloid leukemia after bone marrow transplantation: separation of graft versus leukemia response from graft versus disease. Blood 1995;86:1261. [95] Bonini C, Ferrari G, Verzeletti S, et al. HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft versus leukemia. Science 1997;276:1719 /24. [96] Passweg JR, Rowlings PA, Armitage JO, et al. In: Cecka JM, Teraskaki PI, editors. Report from the international bone marrow transplant registry and autologous blood and marrow transplant registry */North America. Clinical transplants, 1995. Los Angeles, California: UCLA Tissue Typing Laboratory, 1996:117 /27. [97] Carella AM, Giralt S, Slavin S. Low intensity regimens with allogeneic hematopoietic stem cell transplantation as treatment of hematologic neoplasia. Haematologica 2000;85(3):304 /13. [98] Carella AM, Champlin R, Slavin S, McSweeney P, Strob R. ‘Mini-allografts’: ongoing trials in humans. Bone Marrow Transplant 2000;25(4):345 /50. [99] Slavin S, Nagler A, Naparstek E, et al. Non-myeloablative 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):756 /63.

[100] Nagler A, Slavin S, Varadi G, Naparstek E, Samuel S, Or R. Allogeneic peripheral blood stem cell transplantation using a fludarabine-based low intensity conditioning regimen for a malignant lymphoma. Bone Marrow Transplant 2000;25(10):1021 /8. [101] Kapelushnik J, Aker M, Pugatsch T, Samuel S, Slavin S. Bone marrow transplatation from a cadaveric donor. Bone Marrow Transplant 1998;21(8):857 /8. [102] Nagler A, Ackerstein A, Kapelushnik J, Or R, Naparstek E, Slavin S. Donor lymphocyte infusion post non-myeloablative allogeneic peripheral blood stem cell transplantation for chronic granulomatous disease. Bone Marrow Transplant 1999;24:339 / 42. [103] Kapelushnik J, Or R, Slavin S, Nagler A. Fludarabine based protocol for BMT in Fanconi anemia. Bone Marrow Transplant 1997;29(12):1109 /10. [104] Aker A, Varadi G, Slavin S, Nagler A. A fludarabine based nonmyeloablative protocol for human umbilical cord blood transplantation in Fanconi’s anemia. Fludarabine-based protocol for human umbilical cord blood transplantation in children with Fanconi’s anemia. J Pediatr Hematol Oncol 1999;21(3):237 /9. [105] Nagler A, Aker M, Or R, et al. Low-intensity conditioning is sufficient to ensure engraftment in matched unrelated bone marrow transplantation. Exp Hematol 2001;29:362 /70. [106] Slavin S. Total lymphoid irradiation (TLI). Immunol Today 1987;8:88 /92. [107] Ildstad ST, Sachs DH. Reconstitution with syngeneic plus allogeneic or xenogeneic bone marrow leads to specific acceptance of allografts and xenografts. Nature 1984;307:168. [108] Sykes M, Sachs DH. Mixed allogeneic chimerism as an approach to transplantation tolerance. Immunol Today 1988;9:23. [109] Weiss L, Slavin S. Prevention and treatment of graft vs host disease by down-regulation of anti-host reactivity with veto cells of host origin. Bone Marrow Transplant 1999;23:1139 /43. [110] Prigozhina T, Gurevitch O, Slavin S. Non-myeloblative conditioning to induce tolerance after allogeneic bone marrow transplantation in mice. Exp Hematol 1999;27:1503 /10. [111] Storb R, Raff RF, Appelbaum FR, et al. Comparison of fractionated to single-dose total body irradiation in conditioning canine littermates for DLA-identical marrow grafts. Blood 1989;74:1139. [112] Storb R, Yu C, Wagner JL, et al. Stable mixed hematopoietic chimerism in DLA-identical littermate dogs given sublethal total body irradiation before and pharmacological immunosuppression after marrow transplantation. Blood 1997;89:3048. [113] Yu C, Storb R, Mathey B, et al. DLA-identical bone marrow grafts after low-dose total body irradiation: effects of high-dose corticosteroids and cyclosporine on engraftment. Blood 1995;86:4376 /81. [114] Storb R, Yu C, Barnett T, et al. Stable mixed hematopoietic chimerism in DLA-identical littermate dogs given lymph node irradiation before and pharmacological immunosuppression after marrow transplantation. Blood 1999;90:1131 /6. [115] Storb R, Yu C, Zaucha JM, et al. Stable mixed hematopoietic chimerism in dogs given donor antigen, CTLA4Ig, and 100 cGy TBI before and pharmacological immunosuppression after marrow transplant, Blood 1994;94(7):2523 /9. [116] McSweeney PA, Wagner JL, Maloney DG, et al. Outpatient PBSC allografts using immunosuppression with low-dose TBI before, and cyclosporine (CSP) and mycophenolate mofetil (MMF) after transplant. Blood 1998;92(Suppl 1):519a (abstr. 2133). [117] Giralt S, Gajewski J, Khouri I, et al. Induction of graft-versusleukemia as primary treatment of chronic myelogenous leukemia. Blood 1997;90(suppl.1):1857a.

S. Slavin et al. / Critical Reviews in Oncology/Hematology 46 (2003) 139 /163 [118] Giralt S, Cohen A, Mehra R, et al. Preliminary results of fludarabine/melphalan or 2CDA/melphalan as preparative regimens for allogeneic progenito cell transplantation in poor candidates for conventional myeloablative conditioning. Blood 1997;90(suppl.1):1853a. [119] Khouri IF, Keating M, Korbling M, et al. Transplant-lite: induction of graft-versus-malignancy using fludarabine-based nonablative chemotherapy and allogeneic blood progenitor-cell transplantation as treatment for lymphoid malignancies. J Clin Oncol 1998;16:2817 /24. [120] Childs RW, Clave E, Tisdale J, et al. Successful treatment of metastatic renal cell carcinoma with a non-myeloablative allogeneic peripheral-blood progenitor-cell transplant: evidence for a graft-versus-tumor effect. J Clin Oncol 1999;17:2044 /50. [121] Childs R, Chernoff A, Contentin N, et al. Regression of metastatic renal-cell carcinoma after nonmyeloablative allogeneic peripheral-blood stem-cell transplantation. New Engl J Med 2000;14, 343(11):750 /8. [122] Sykes M, Preffer F, McAfee S, et al. Mixed lymphohematopoietic chimerism and graft versus lymphoma effect are achievable in adult recipients following non-myeloablative therapy and HLAmismatched donor bone marrow transplantation. The Lancet 1999;353:1755 /9. [123] Carella AM, Lerma E, Dejana A, et al. Engraftment of HLAmatched sibling hematopoietic stem cells after immunosuppressive conditioning regimen in patients with hematologic neoplasias. Haematologica 1998;83:904 /9. [124] Carella AM, Lerma E, Corsetti MT, et al. Evidence of cytogenetic and molecular remission by allogeneic cells after immunosuppressive therapy alone. Br J Haematol 1998;103:565 /7. [125] Morecki S, Yacovlev E, Gelfand Y, Uzi I, Slavin S. Cell therapy with pre-immunized effector cells mismatched for minor histocompatible antigens, in the treatment of a murine mammary carcinoma. J Immunother 2001;24(2):114 /21. [126] Rosenberg SA, Lotze MT. Cancer immunotherapy using interleukin-2 and interleukin-2-activated lymphocytes. Ann Rev Immunol 1986;4:681 /709.

Biography Shimon Slavin MD graduated from the Hadassah Hebrew University School of Medicine in Jerusalem, Israel, in 1967. He specialized in internal medicine and subsequently in clinical immunology at Stanford University, California, and the Bone Marrow Transplant Center at the Fred Hutchinson Cancer Research Center,

163

Seattle, and USA. Upon returning to Israel in 1979, he opened 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 Cancer Immunotherapy & Immunobiology Research Center at the Hadassah University Hospital in Jerusalem. Dr Slavin introduced successfully the use of donor lymphocyte infusion (DLI) in early 1987 and subsequently pioneered adoptive allogeneic cell-mediated immunotherapy with immunocompetent and cytokine-activated donor lymphocytes, for both treatment and prevention of relapse following allogeneic and autologous bone marrow transplantation for leukemia and other hematologic malignancies. Additional preclinical research and clinical activities carried out at Dr Slavin’s research center include new approaches for immunotherapy of hematologic malignancies and metastatic solid tumors based on adoptive allogeneic cell-mediated immunotherapy and more recently also using tumor cell vaccines. More recently, Dr Slavin introduced new approaches for safer clinical application of nonmyeloablative stem cell transplantation based on induction of specific unresponsiveness of host-vs-graft and graft-vs-host, aiming at developing safer and more effective methods for allogeneic stem cell transplantation for malignant and non-malignant indications. In parallel, new modalities based on upregulation of immune responses in conjunction with adoptive allogeneic cell-therapy are being considered for treatment of AIDS, while newer approaches designed for downregulation of the immune system are being introduced for immunotherapy of autoimmunity and induction of permanent and specific transplantation tolerance to cellular and perfused organ allografts. Dr Slavin serves on many editorial boards and national and international advisory boards. He is a member of the Executive Committee of the IBMTR and a member of the Immunotherapy Committees of the IBMTR and EBMT.