Slavin Cv-510 Lancet Oncology

Review

Alloreactive immunotherapy

Immunotherapy of cancer with alloreactive lymphocytes Shimon Slavin

Immunotherapy of cancer with alloreactive lymphocytes is the mainstay of treatment, especially in haematological malignant disease. With donor lymphocyte infusion for immunotherapy, it is essential to induce host-versus-graft tolerance to ensure that the donor lymphocytes are accepted. Engraftment of haemopoietic cells of donor origin can be accomplished with reduced-intensity conditioning. Reducing transplant-related mortality by simplifing the stem-cell transplant procedure with a reduced-intensity regimen, particularly nonmyeloablative conditioning, may have great potential for the treatment of malignant and non-malignant disorders.

Adhesion & homing molecules MHC

mHags

Graft versus malignant disease

Tissue-specific peptides

Viral, oncogenes, mutations, & break-point products

Tumour-specific peptides

Lancet Oncol 2001; 2: 491–98

Standard treatment of patients with haematological malignant diseases and disseminated cancer involves the use of chemotherapy protocols or combinations of chemotherapy with modalities such as surgery, radiation therapy, brachytherapy, and more recently, immunotherapy with monoclonal antibodies to tumours. For patients with tumour cells that are resistant to conventional doses of chemotherapy, or patients who relapse after achieving remission, additional chemotherapy may transiently reduce the tumour bulk, but will not completely eliminate disease. Until recently, attention has focused on maximising the reduction of tumour-cell numbers in patients with resistant or recurrent disease. The aim of this approach is to kill as many tumour cells as possible, disregarding the toxic effects of the treatment on normal marrow stem cells, and repair the damage afterwards by rescuing the patient with stem cells. Extensive tumour debulking should be carried out in vivo before the patient is given autologous stem-cell autografts, which are depleted of tumour cells to reduce the risk of reintroducing disease. Whereas a proportion of patients may respond to newer chemotherapy combinations or myeloablative high-dose chemoradiotherapy supported by autologous bone-marrow or blood-stem-cell transplantation, the large majority of them will retain resistant tumour cells that cause disease recurrence. During the early 1960s, it was shown that tumourbearing mice inoculated with murine leukaemia virus had a lower incidence of leukaemia when reconstituted with bone-marrow allografts or treated with allogeneic lymphocytes, both of which resulted in graft-versus-host disease (GVHD).1,2 Preclinical experiments and clinical observations have confirmed that allogeneic bone-marrow transplant eliminates leukaemia through immuneTHE LANCET Oncology Vol 2 August 2001

Figure 1. Schematic representation of the double barrier in allogeneic bone marrow transplantation.

mediated graft-versus-leukaemia (GVL) effect.3–8 It has also been established that the incidence of GVHD inversely correlates with the incidence of relapse following allogeneic bone-marrow transplant for haematological malignant disease.9–12 Therefore, it was suggested that infusing alloreactive lymphocytes during the course of bone-marrow transplantation might induce GVL effects.9–12 Similarly, several observations pointed to the existence of graftversus-tumour (GVT) effect after allogeneic bone-marrow transplantation in mice13–15 and human beings.16–19

Therapeutic potential of donor-lymphocyte infusion following bone-marrow transplantation Demonstration of the remarkable antitumour effects of allogenic lymphocytes presented the possibility of curing patients that relapse after myeloablative chemoradiotherapy – a condition previously thought to be incurable – and led to a renewed interest in their therapeutic potential. Starting in early 1987, we documented that relapse following bonemarrow transplantation in patients with leukaemia20–24 or multiple myeloma25 could be reversed with donorlymphocyte infusion (DLI). The role of alloreactive lymphocytes in cancer therapy was further substantiated after it was shown that giving DLI after bone-marrow transplantation is beneficial for treatment of overt SS is Professor of Medicine and Chairman of the Department of Bone Marrow Transplantation & Cancer Immunotherapy Correspondence Director, The Danny Cunniff Leukemia Research Laboratory Hadassah University Hospital, Ein Karem Jerusalem 91120, Israel. Tel: +972-2-6776561 Fax: +972-26422731 Email: [email protected] or [email protected]

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Review

Alloreactive immunotherapy

Host versus graft response Rejection T cells NK cells

T cells NK cells?

Graft versus leukaemia/ tumour effects GVL & GVT effects

GVHD Patient

Donor Graft versus host disease

failure, even after patients were given the highest doses of chemoradiotherapy and GVL effects induced by donor lymphocytes.12,38 In hindsight, all commonly practised therapeutic strategies were based on over-estimation of the anticancer potential of available treatments and under-estimation of the efficacy of cancer immunotherapy.

The use of stem-cell transplantation as a platform for immunotherapy with donor lymphocytes

As a result of our growing experience of allogeneic-cell therapy in conjunction with bone-marrow transplantation, and recognition of the limited value of more toxic chemoradiotherapy in patients resistant to standard treatment doses, we formulated a new working hypothesis in the early 1980s that bone-marrow transplantaion should be used to induce host-versus-graft transplantation tolerance rather than as a means of giving higher doses of anticancer agents. The rationale behind this hypothesis was that maximum tolerated doses of chemoradiotherapy are unlikely to eliminate all tumour cells in patients with resistant or relapsing disease, whereas donor lymphocytes are effective even in patients who are resistant to all available anticancer modalities.23,24 Thus, myeloablative chemotherapy can be replaced with cell-mediated immunotherapy induced by alloreactive donor lymphocytes, which can be given at the time of transplantation or supplemented as DLI after nonmyeloablative stem-cell transplantation (NST).39–41 In other words, instead of focusing on more aggressive chemoradiotherapy, which causes procedure-related toxic effects and death, we reasoned that it might be both safer and more effective to focus on biological therapy of minimal residual disease with post-transplant immunotherapy.

Figure 2. Possible target molecules for graft-versus-leukaemia or graft-versus-tumour effects.

relapse23,24 or for prevention of relapse in high-risk patients.26 These observations were subsequently confirmed by many transplant centres worldwide.27–29 DLI has become an accepted method for treating overt relapse as well as for eradication of the residual host cells that survive high-dose chemoradiotherapy after bonemarrow transplantation procedures. Interestingly, although GVL effects were originally documented in direct association with GVHD,9–12 experimental3–8 and clinical observations12,23,24 suggest that significant GVL effects may also be associated with bone-marrow transplantation in individuals with no clinically overt GVHD. This implies that GVL effects can be induced independently of clinically significant GVHD.30 Also, allogeneic-cell therapy mediated by DLI is effective in patients who do not develop GVHD,23,24,28,29 suggesting that the two phenomena may be distinct, at least in part.30 As was originally documented in mice, resistance to GVHD is greater with longer intervals between bonemarrow transplantation and DLI.31,32 We therefore suggest that one way to apply allogeneic-cell therapy safely is to use incremental doses of DLI as the time after bone-marrow transplant increases.24,31,32 Indeed, these observations, which have also been confirmed in mice33 and human beings,34 support the use of graded increments of DLI after bonemarrow transplantation, both for treatment23,24 and prevention26 of relapse, while partially controlling GVHD. Although alloreactive lymphocytes are an important part of the transplant procedure, myeloablative chemoradiotherapy – for eradication or debulking tumour cells and preventing rejection of donor bone-marrow cells – was mandatory until recently for conditioning patients before bone-marrow transplants. It was also believed by many that elimination of host stem cells was essential to create a niche for donor stem cells, although experimental work suggested that this was not the case.35–37 So, despite the therapeutic potential of bone-marrow transplantation many patients, especially the elderly, died. A significant proportion of surviviors developed occasionally lethal immediate and late toxic effects as a direct result of myeloablative chemoradiotherapy, in addition to the unavoidable acute and chronic GVHD. Unfortunately, recurrent disease was still the major cause of treatment 492

Scientific rationale for possible replacement of myeloablative with lymphoablative conditioning It became apparent from studies of the mechanisms of induction of transplantation tolerance, both prenatally42 and postneonatally (neonatal tolerance),43 and from our own studies applying the concept of mixed chimerism to induce bilateral transplantation tolerance in animal models, that myeloablative conditioning was not required for engraftment of donor bone-marrow cells. Mixed chimerism is a situation in which hemopoietical cells from both the donor and the host coexist. This results in bilateral transplantation tolerance, which makes it possible for patients to accept tissues from donors without graft-versushost disease. Mixed chimerism probably depends on the capacity of donor cells to neutralise alloreactive cells of host origin, and vice versa. Research published in 1976 had already established that durable engraftment of donor haemopoietical cells and bilateral transplantation tolerance THE LANCET Oncology Vol 2 August 2001

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Alloreactive immunotherapy

(host-versus-graft and graft-versus-host) could be achieved following non-myeloablative conditioning.44,45 These observations were confirmed by many investigations in rodents6,13,31,46–49 and, more recently, in dogs.50,51 Additionally, in the early 1980s, experimental data from murine models suggested that leukaemia could be eradicated by immunotherapy mediated by alloreactive lymphocytes.3,6 Following on from the working hypothesis, NST was developed in the early 1990s. It was based on data supporting the need for immunosuppression rather than myeloablation in ensuring durable engraftment of donor haemopoietical cells, together with documentation of the therapeutic potential of DLI.20–29 NST was put forward as an alternative to conventional bone-marrow transplantation, focusing on induction of a window of immunosuppression without ablative conditioning.

Clinical application of NST in bone-marrow transplantation

Review given two doses of cyclophosphamide. Engraftment was confirmed in all patients receiving ⭓ 3 doses of cyclophosphamide (50 mg/kg each). Three of 17 patients were alive and disease-free more than 5 years later. Following this first NST study, we focused on the use of a fludarabine-based regimen, primarily for the induction of host-versus-graft transplantation tolerance, but also to enable GVL and GVT effects by preventing rejection of alloreactive donor lymphocytes.40,41 Our aim was to prevent rejection of donor-blood stem cells with the minimum toxic effects and achieve acute immunosuppression. Once the principle was confirmed, fludarabine was used at 30 mg/m2/day x 6 in combination with busulfan 4 mg/kg/day x 2 (initially given orally, but recently we have also given 3.3 mg/kg/day intravenously), in conjunction with Fresenius ATG 5–10 mg/kg/day x 4.40,41 Equally effective protocols involved the use of similar doses of fludarabine with lowdose cyclophosphamide 60 mg/kg/day x 2 in individuals with a normal immune system, or 5 mg/kg/day x 2 in patients with Fanconi’s anaemia (who are known to be sensitive to alkylating agents instead of busulfan), or equal doses of fludarabine with a single dose of well-tolerated low-dose total body irradiation (TBI) 200 cGy without ATG.52 Following lymphoablation, transplantation tolerance was induced against donor stem cells and lymphocytes by infusion of bone-marrow or G-CSF mobilised peripheral-blood stem cells obtained from a fully matched donor (sibling or matched unrelated donor) and treated with Neupogen 5 ␮g twice daily for 5 days. This procedure eliminates residual malignant or normal stem cells of host origin. Although the first protocol we practised on a large scale was a fludarabine and busulfan-based regimen, we reasoned that the same approach could be applied to all indications of bone-marrow transplantation, including patients with non-malignant diseases. This goal was accomplished successfully.41 In the future, we anticipate that more disease-oriented regimens will be used, eg a busulfan-based regimen for myeloid malignant disorders, a

The concept that the donor’s immunocompetent T cells may have the key role in elimination of malignant cells of host origin, especially at the stage of minimal residual disease, led to the development of new protocols, which used escalating, yet non-myeloablative, doses of immunosuppressive (rather than myeloablative) agents, specifically aiming at engraftment of donor stem cells. Subsequently, fludarabine – a potent purine analogue used mainly against malignant lymphocytes in patients with lymphoid malignant disorders – was found to react against normal as well as diseased lymphocytes. In parallel with the transplant team at MD Anderson Cancer Center, TX, USA, we introduced the use of fludarabine to intensify pretransplant immunosuppression enabling stem-cell engraftment and avoiding conventional myeloablative conditioning.39–41 The first NST protocol consisted of increments of intravenous cyclophosphamide, starting at 50 mg/kg/day x1, x2, x3, and finally x4 after immunosuppression with rabbit antibodies against T lymphocytes (ATG, Fresenius) which Days before transplantation was given at 10 mg/kg/day for 4 days. This treatment is directed at both host and donor immunocompetent cells, GVHD prophylaxis: because ATG remains in the patient’s Cyclosporine Cyclosporine ⫾ MMF ⫾ MTX circulation after grafting (Slavin et al., unpublished). In proof-of-principle studies, ⫾ Campath-1H ⫾ ATG NST was offered to patients with advanced malignant disease (haematological malignant disorders Fludarabine G-CSF mobilised and metastatic solid tumours) who blood stem cells were not eligible for conventional bone-marrow transplantation. The G-CSF Alkylating agent 5 ␮g/kg twice studies included ten patients with (Busulfan, cytoxan, daily for 5 days resistant haematological malignant melphalan, or low-dose TBI) disease and seven with metastatic solid tumours that were not responding to chemotherapy. With all patients receiving four doses of ATG, durable Figure 3. The principles of currently available non-myeloablative stem-cell transplantation focusing engraftment was seen after they were on reduced-intensity conditioning before transplantation of mobilised blood stem cells. THE LANCET Oncology Vol 2 August 2001

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Review cyclophosphamide-based protocol for low-grade lymphoid malignant disease, a melphalan-based protocol for multiple myeloma and lymphoma, etc.

Improving NST protocols As anticipated, NST caused the elimination of all malignant or genetically abnormal stem cells, as well as the entire immune system of the patient, going through a transient stage of mixed chimerism. Low-dose cyclosporine (3 mg/kg/day) treatment started 1 day before transplantation and was discontinued within 1–3 months in the absence of GVHD. Thus, the alloreactive donor lymphocytes were capable of inducing optimum expression of GVL, graftversus-lymphoma (GVLy), graft-versus-myeloma (GVM), or in a much broader sense GVT effects at an early stage after NST. Alloreactive responses may also occur against genetically abnormal host cells in genetic diseases,53,54 as well as against self-reactive lymphocytes in autoimmune diseases.55 Our cumulative experience from over 200 cases along with results from several hundred cases from other centres following the original protocol or using minor variations of NST strategy, suggest that NST may be most effective for standard-risk patients. As NST is well tolerated, it could be administered on an outpatient basis and, for the first time, bone-marrow transplantation could be provided safely in elderly individuals and other patients with bad performance status who would not qualify for a standard myeloablative bone-marrow transplantation procedure. As NST is rapidly becoming popular, several modifications are being incorporated into the protocols originally described by the MD Anderson and the Hadassah teams. This is making it more difficult to define the differences between myeloablative and non-myeloablative regimens, as the boundaries are becoming blurred.56 Depending on the protocol used, patients may develop aplasia or minimal pancytopenia of short duration, especially using fludarabine with low-dose cytoxan or TBI 200 cGy.57–60 So, even using the mildest NST regimen patients may be converted to 100% donor-type chimerism, probably going through a transient stage of mixed chimerism, converting spontaneously and rapidly into full donor type immunohaemopoiesis, with no severe or durable aplasia. The benign nature of the NST regimen is emphasised by its avoidance of high-dose chemoradiotherapy and the lack of aplasia. Mucositis is avoided and normal oral intake maintained throughout the post-transplant period. Furthermore, in some cases, no blood-product transfusions are required, and there are no fever episodes, so no antibiotic treatment is necessary after transplantation. One of our female patients became pregnant and gave birth (with no gynaecological intervention) to a normal baby 18 months after NST, suggesting that fertility may be preserved after NST, unlike the large majority of patients who go through bone-marrow transplantation. Although NST protocols seem effective and well tolerated for donor peripheral blood stem-cell engraftment, GVHD is still the major barrier. Up to one third of patients develop significant GVHD; it is the most frequently cited cause of death in reported series, despite the overall 494

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reductions in procedure-related toxic effects and mortality. GVHD cannot be completely prevented by adding methotrexate (MTX)9 or mycophenolate mofetil (MMF), in addition to treatment with the standard dose of cyclosporine A (CSA).58,59 At the Hadassah Hospital in Jerusalem, among the first 100 patients treated with NST using the fludarabine/busulfan regimen, transplant-related mortality at day 100 was 2.6% for all patients and 4% among patients with haematological malignant diseases. Nonetheless, the majority of patients (56% in Jerusalem) had mild (grade 1) or no GVHD while on CSA. In some patients, GVHD is activated and aggravated when CSA or other GVHD treatments are discontinued, or when DLI is attempted. It is therefore recommended to use posttransplant immunosuppression until chimerism is well established and to avoid the necessity of using DLI when there has been no response. In case DLI is indicated due to persisting or reappearance of host markers, it should be given as graded increments while monitoring for GVHD. Larger doses of DLI should be reserved for patients with bulky or rapidly developing relapse, in which case GVHD may be unavoidable. Indolent, low-grade, and to a lesser extent, high-grade and resistant lymphoma (including related lymphoid malignant disorders) also appear to respond to NST. Good responses have been seen with fludarabine and cyclophosphamide regimens and with fludarabine, busulfan, and ATG regimens, as reported from MD Anderson57 and our own institution.41 Although engraftment of matched sibling stem-cell allografts was observed in most patients with low-grade lymphoid malignant disorders who were conditioned with lower doses of both agents, including fludarabine 30 mg/m2/day for 3 days and cyclophosphamide 300 mg/m2/day for 3 days, consistent and stable engraftment was only observed when the dose of cyclophosphamide was raised to 750 mg/m2/day x 3.57 Interestingly, despite reduced conditioning, GVLy effects were still observable against malignant lymphocytes, especially in patients with lowgrade non-Hodgkin lymphoma. This is in agreement with our earlier observations in patients treated with DLI after conventional bone-marrow transplantation,24 confirming that malignant lymphocytes can be good targets for donor alloreactive T cells, despite contrary claims.28 There are several instances where GVL effects may not be adequate – when donor lymphocytes are disadvantaged, when they are challenged against a large tumour mass, or when the rate of tumour growth is excessive – which explains the difference between the intensity of GVL effects against low-grade (low grade non-Hodgkin lymphoma and chronic lymphocytic leukaemia) and high-grade (high grade non-Hodgkin lymphoma and acute lymphoblastic leukaemia) lymphoid malignant disorders.

Clinical application of NST for treatment of metastatic solid tumours All the aforementioned data support the view that rejection, prevention, and engraftment of donor stem cells, as well as eradication of malignant or otherwise abnormal haemopoietic cells of host origin, can be accomplished THE LANCET Oncology Vol 2 August 2001

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Alloreactive immunotherapy

without myeloablative conditioning. These observations paved the way for the use of NST in patients with metastatic solid tumours to induce GVT effects with the minimum procedure-related risks, as documented in experimental and pilot clinical trials.13–19 Based on experience using NST for haematological malignant disease, the clinical application of NST for metastatic solid tumours was a natural development. Childs and colleagues at the NCI, MD, USA,60,61 pioneered the use of NST in patients with metastatic renal-cell cancer who were resistant to all other treatments. Data reported recently by the same group suggest that allogeneic cell-mediated immunotherapy in conjunction with NST may be an effective method of treatment for chemotherapy-resistant metastatic renal-cell cancer. Responses were documented in over half of the patients and complete durable remission was seen in three of 19 patients.61 The process of elimination of tumour cells with immunocompetent donor lymphocytes is time consuming, so durable engraftment of donor lymphocytes is a mandatory first step. Durable engraftment of donor cells can be accomplished following induction of host-versus-graft unresponsiveness which is mediated by engraftment of donor stem cells. These observations may provide new hope for patients with metastatic renal-cell cancer and provide new options for the treatment of patients with other ‘incurable’ metastatic solid tumours. Unfortunately, the most serious drawback in treating renal-cell cancer, as with haematological malignant disorders, is that adoptive immunotherapy with donor lymphocytes causes GVHD, which is occasionally severe or even fatal. In future protocols, the most desirable goal should be to induce GVL and GVT effects while avoiding, minimising, or at least controlling GVHD. Several studies in animal models and many clinical observations have indicated that both GVL and GVT effects can operate independently of GVHD, and once the mechanism of antitumour immunity and the target molecules for GVL and GVT are better understood, this is a likely goal for future treatments.

Prevention or better control of GVHD following NST There seems little doubt that the progress of NST and wider clinical application of reduced-intensity conditioning for more indications will depend on better control or prevention of GVHD. Induction of graft-versus-host tolerance can be accomplished by T-cell depletion25,26 or by using non-alloreactive donor stem cells.62 However, elimination of alloreactive donor T cells may have a negative impact on engraftment as well as early GVL and GVT, which could be counteracted by DLI given in graded increments, while controlling GVHD.24–26 Alternatively, donor T cells engineered in vitro with transduction of herpes simplex thymidine kinase suicide gene would be a safer option. With this approach, GVT effects could be maximised and followed by elimination of GVHD in patients with uncontrolled responses to host cells.63 The antitumour effects mediated by DLI could be amplified by using donor lymphocytes activated with recombinant interleukin 2 in vivo or in vitro.24,32 The use of allogeneic THE LANCET Oncology Vol 2 August 2001

Review natural-killer cells activated with recombinant interleukin2 may thus also provide a practical approach for induction of GVL and GVT effects with no GVHD.

Application of innovative immunotherapy to improve the efficacy of NST Following induction of host-versus-graft and graft-versushost transplantation tolerance, supplemental immunotherapy may be applied – preferably at the stage of minimal residual disease accomplished by the transplant procedure. This treatment may involve antibodies against tumour cells presenting cancer-specific or cancerassociated antigens (eg monoclonal antibodies against tumour cell-surface antigens such as CD20, CD19, CEA, HER2/neu, etc.) or antibodies against tumour cells presenting tissue-specific antigens (eg minor histocompatibility antigens present on haemopoietic but not somatic cells such as HA-1).64,65 Since the effectiveness of cell-mediated antitumour responses has already been established, immunotherapy mediated by alloreactive lymphocytes may become an important tool for fighting cancer, particularly for patients with metastatic renal-cell cancer. Furthermore, the outstanding results reported by the NCI team may encourage application of similar therapeutic strategies for the treatment of other incurable metastatic tumours, further exploiting the therapeutic potential of allogeneic lymphocytes. Recommendations for large-scale clinical application of NST and DLI for additional solid tumours await confirmation of the results and proof that there are no safer methods of controlling GVHD. The results reported by Childs and colleagues60,61 seem most promising, providing for the first time a realistic hope for patients with metastatic renal-cell cancer with a matched donor available, but they are far from fully satisfactory. Under half of the patients responded and others still suffer from GVHD and/or progressive disease, with lethal consequences in two individuals. This suggests that the technique needs further development to minimise procedure-related complications and increase anticancer activity. The association between GVT and GVHD implies that the antitumour effects of donor lymphocytes are, at least in part, mediated by MHC determinants such as minor histocompatibility antigens in the case of HLA-identical siblings. Future studies should focus on maximising the anticancer effects of DLI under conditions of host-versusgraft transplantation tolerance, while minimising antihost responses – possibly by using immune donor lymphocytes. Indeed, ongoing preclinical and clinical studies show that GVL and GVT effects may be accomplished in the absence of GVHD, although the occurrence of GVHD is always associated with a better disease response. Generation of a graft-versus-tumour effect with no GVHD at the time of disease-regression was reported in two patients from the trial of patients with renal-cell cancer. Likewise, diseaseregressions were often delayed by months from the onset of GVHD, suggesting that the relevant effector cells may be distinct from GVHD effector cells. It remains to be seen whether clinically effective GVT effects can be mediated against tumour-specific antigens.61 495

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Review Immunotherapy with tumour-specific cytotoxic lymphocytes Several approaches may be applied in the future to improve the efficacy of immunotherapy with donor lymphocytes. Recent experiments in our laboratory suggest that anticancer activity can be significantly improved in parallel with reducing or eliminating the severity of GVHD, using lymphocytes obtained from immune, rather than naïve, donors. Using immune-donor lymphocytes or cytotoxic T lymphocytes (CTLs) generated against common diseasespecific peptides and presented by antigen-presenting cells or the patient’s own tumour cells, may be the best approach for future studies. Allogeneic CTL66,67 and minor antigenspecific CTL68 based on the presence of minor histocompatibility antigens on haemopoietic cells and not on other somatic cells, have recently been shown to eliminate residual disease in vivo.69 Also, there seems to be hope that CTL-specific CTLs for antigens only present on tumour cells such as chronic myeloid leukaemia may also induce tumour-specific immune responses.70 Most effective GVL effects, predominantly against tumour cells can also be accomplished by using donor lymphocytes stimulated in vitro against tumour cells or normal host alloantigens represented by parental stimulating cells using one-way mixed lymphocyte cultures.71 Immune-donor cells may thus be obtained either by in vitro generation of cytotoxic or helper T cells or using tumour-cell vaccines. Based on animal studies, another option is to optimise the conditioning and induce bilateral transplantation tolerance of host-versus-graft and graftversus-host. By balancing residual host with donor immunocompetent T cells, or using large T cell depleted stem cell inocula to overcome resistance by residual immunocompetent cells of host origin, stable mixed chimerism can be established, facilitating this approach. The former method is based on down-regulation of GVHD and graft rejection by host and donor veto cells, respectively.45,72 Another approach, which could be applied to patients with no matched siblings, uses T-cell-depleted stem-cell allografts to induce host-versus-graft transplantation tolerance by central clonal deletion, followed by graded increments of tumour-specific or minor histocompatibility-specific donor CTLs, for minimising the risk of GVHD. Immunotherapy with tumour-specific CTLs is likely to maximise antitumour effects, while potentially minimising antihost responses, as recently confirmed in a preclinical animal model (Ji, Slavin, submitted for publication 2001). Successful clinical application of innovative cell therapy may be required before patients with cancer can be safely and effectively treated with alloreactive lymphocytes, in conjunction with other biological therapies, to maximise the therapeutic potential of donor lymphocytes.

Conclusions Although much work remains ahead, the principle has been proven. First, consistent engraftment of donor stem cells and donor lymphocytes can be accomplished following well-tolerated non-myeloablative conditioning. Second, alloreactive T cells can eradicate malignant haemopoietical cells of host origin, genetically abnormal stem cells and 496

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Search and strategy Published data for this review were identified by searching MEDLINE. The search terms included: donor lymphocyte infusion, non-myeloablative stem cell transplantation, and bone marrow transplantation. Papers and book chapters from private collections are also referenced. Only papers published in English were used.

their progeny, self-reactive T and B cells in autoimmune diseases, and somatic tumour cells such as renal-cell cancer cells, eventually replacing the host immunohaemopoietical system with that of the donor. The feasibility of inducing host-versus-graft and graft-versus-host transplantation tolerance following conditioning with NST raises the possibility that similar techniques could be used to induce transplantation tolerance to allogeneic tissues and organ allografts. In the future, the progress of cell therapy for all clinical indications will depend on improving the strategies for safer and better regulation of antihost responses to avoid uncontrolled GVHD. Development of effective antitumour immunotherapy independently of anti-host responses will remain a challenge for future research. Results from our ongoing studies in animal models and pilot clinical trial, suggest that these goals are feasible and may become applicable at the patient’s bedside in the near future. References

1 Sinkovics JG, Shullenberger CC. Effect of hematopoietic chimerism on the course of Rauscher’s viral mouse leukemia. Proc Am Assoc Cancer Res 1963; 4: 62–72. 2 Boranic M, Tonkovic I. Time pattern of the antileukemia effect of graft-versus-host reaction in mice: I. Cellular events. Cancer Res 1971; 31: 1140–47. 3 Slavin S, Weiss L, Morecki S, Weigensberg M. Eradication of murine leukemia with histoincompatible marrow grafts in mice conditioned with total lymphoid irradiation (TLI). Cancer Immunol Immunother 1981; 11: 155–61. 4 Truitt RL, Shih F-H, LeFever AV, et al. 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: 2050–58. 5 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. 6 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 B-cell leukemia (BCL1). Cancer Immunol Immunother 1990; 31: 236–242. 7 Truitt RL, Atasoylu AA. Impact of pretransplant conditioning and donor T cells on chimerism, graft-versus-host disease, graft-vsleukemia reactivity, and tolerance after bone marrow transplantation. Blood 1991; 77: 2515–23. 8 Weiss L, Reich S, Slavin S. Use of recombinant human interleukin2 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 cellmediated immunotherapy. Cancer Invest 1992; 10: 19–26. 9 Weiden PL, Sullivan KM, Fluornoy N, et al. Antileukemic effect of chronic graft-versus-host disease: Contribution to improved survival after allogeneic marrow transplantation. N Engl J Med 1981; 304: 1529–33. 10 Weiden PL, Fluornoy N, Sanders JE, et al. Antileukemic effect of graft-versus-host disease contributes to improved survival after allogeneic marrow transplantation. Transplantation 1981; 13: 248–51. 11 Sullivan KM, Weiden PL, Storb R, et al. Influence of acute and chronic graft-versus-host disease on relapse and survival after bone THE LANCET Oncology Vol 2 August 2001

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marrow transplantation from HLA-identical siblings as treatment of acute and chronic leukemia. Blood 1989; 73: 1720–26. Horowitz M, Gale RP, Sondel PM, et al. Graft-versus-leukemia reactions after bone marrow transplantation. Blood 1990; 75: 555–62. Moscovitch M, Slavin S. Anti-tumor effects of allogeneic bone marrow transplantation in (NZB x NZW)F1 hybrids with spontaneous lymphosarcoma. J Immunol 1984; 132: 997–1000. 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. Morecki S, Yacovlev E, Diab A, Slavin S. Allogeneic cell therapy for a murine mammary carcinoma. Cancer Res 1998; 58: 3891–95. 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: 1501–08. Ben-Yosef R, Or R, Nagler A, Slavin S. Graft vs tumor and graft vs leukemia in patient with concurrent breast cancer and acute myelocytic leukemia. Lancet 1996; 348: 1242–43. Ueno NT, Rondòn G, Mirza NQ, et al. Allogeneic peripheral-blood progenitor-cell transplantation for poor-risk patients with metastatic breast cancer. J Clin Oncol 1998; 16: 986–93. 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 Cell Mol Ther 1998; 4: 1–6. Slavin S, Or R, Naparstek E, et al. Cellular-mediated 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, Nagler A. New developments in bone marrow transplantation. Curr Opin Oncol 1991; 3: 54–271. Slavin S, Or R, Kapelushnik Y, et al. Immunotherapy of minimal residual disease in conjunction with autologous and allogeneic bone marrow transplantation (BMT). Leukemia 1992; 6: 164–66. Slavin S, Naparstek E, Nagler A, et al. 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 interleukin2 to treat leukemia relapse post allogeneic bone marrow transplantation. Blood 1996; 87: 2195–204. Or R, Mehta J, Naparstek E, et al. Successful T cell-depleted allogeneic bone marrow transplantation in a child with recurrent multiple extramedullary plasmacytomas. Bone Marrow Transplant 1992; 10: 381–82. 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. 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: 2462–65. Kolb HJ, Schattenberg A, Goldman JM, et al. Graft-versusleukemia effect of donor lymphocyte transfusions in marrow grafted patients. Blood 1995; 86: 2041–50. Collins RH, Shpilber 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. Slavin S, Ackerstein A, Naparstek E, et al. Phenomenon: the graftversus-leukemia (GVL) phenomenon: is GVL separable from GVHD? Bone Marrow Transplant 1990; 6: 155–61. 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 interleukin2 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 cellmediated 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 anti-leukemia reaction without graft-versus-host disease. Bone Marrow Transplant 1993; 11: 329–36. Mackinnon S, Papadopoulos EB, Carabasi MH, et al. Adoptive

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40

41

42 43 44 45 46 47 48 49

50

51

52

53

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56

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: 1261–68. Takada A, Takada Y, Ambrus JL. Proliferation of donor spleen and marrow cells in the spleens and bone marrows of unirradiated and irradiated adult mice. Proc Soc Exp Biol Med 1970; 136: 222–26. Brecher, G, Ansell JD, Micklem HS, et al. Special proliferative sites are not needed for seeding and proliferation of transfused bone marrow cells in normal syngeneic mice. PNAS 1982; 79: 5085–87. Ramshaw HS, Rao SS, Crittenden RB, et al. Engraftment of bone marrow cells into normal unprepared hosts: effects of 5fluorouracil and cell cycle status. Blood 1995; 86: 924–29. Ringden O, Horowitz MM, for the Advisory Committee of the International BMT Registry. Graft-versus-leukemia reactions in humans. Transplant Proc 1989; 21: 2989–92. Giralt S, Estey E, Albitar M, et al. Engraftment of allogeneic hematopoietic progenitor cells with purine analog-containing chemotherapy: Harnessing graft-versus-leukemia without myeloablative therapy. Blood 1997; 89: 4531–36. Slavin S, Nagler A, Naparstek E, et al. Successful replacement of conventional bone marrow transplantation with high-dose chemoradiotherapy with well tolerated nonmyeloablative conditioning in preparation for allogeneic blood stem cell transplantation for the treatment of malignant and non-malignant diseases. Blood 1997; 90: 534a. Slavin S, Nagler A, Naparstek E, et al. Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and non-malignant hematologic diseases. Blood 1998; 91: 756–63. Owen RD. Immunogenetic consequences of vascular anastomoses between bovine twins. Science 1945; 102: 400. Billingham RE, Brent L, Medavar PB. Actively acquired tolerance to foreign cells. Nature 1953; 172: 606. Slavin S, Strober S, Fuks Z, Kaplan HS. Long-term survival of skin allogafts in mice treated with fractionated total lymphoid irradiation. Science 1976; 193: 1252–54. Slavin S. Total lymphoid irradiation (TLI). Immunol Today 1987; 8: 88–92. Ildstad ST, Sachs DH. Reconstitution with syngeneic plus allogeneic or xenogeneic bone marrow leads to specific acceptance of allografts or xenografts. Nature 1984; 307: 168. Sykes M, Sachs DH. Bone marrow transplantation as a means of inducing tolerance. Semin Immunol 1990; 2: 401–17. Prigozhina T, Gurevitch O, Slavin S. Non-myeloablative conditioning to induce bilateral tolerance after allogeneic bone marrow transplantation in mice. Exp Hematol 1999; 27: 1503–10. Wekerle T, Kurtz J, Ito H, et al. Allogeneic bone marrow transplantation with co-stimulatory blockade induces macrochimerism and tolerance without cytoreductive host treatment. Nat Med 2000; 6: 464–69. 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: 1131–36. 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. Slavin S, Nagler A, Naparstek E, et al. A new non-myeloablative protocol using fludarabine and low-dose TBI in preparation for allogeneic blood stem cell transplantation for high risk patients with malignant and non-malignant disorders. Blood 1999; 94: 388b (abstr 4965). 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: 96–98. Kapelushnik J, Or R, Slavin S, Nagler A. Fludarabine based protocol for BMT in Fanconi anemia. Bone Marrow Transplant 1997; 29:1109–10. Slavin S, Nagler A, Varadi G, Or R. Graft vs autoimmune lymphocytes (GVA) following allogeneic bone marrow transplantation in a patient with chronic myelogenous leukemia and severe systemic psoriasis and psoriatic polyarthritis. Exp Hematol. 2000; 28: 853–57. Slavin S, Nagler A, Or R. New developments in Therapy AML. Treatment of myeloid leukemias with non-myeloablative stem cell

497

For personal use. Only reproduce with permission from The Lancet Publishing Group.

Review

57

58

59 60

61 62 63 64

transplantation (NST): accomplishments and future goals. Hematol 2000: 85–90. 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. McSweeney P, Niederwieser D, Shizuru J, et al. Outpatient allografting with minimally myelosuppressive, immunosuppressive conditioning of low-dose TBI and postgrafting cyclosporine (CSP) and mycophenolate mofetil (MMF). Blood 1999; 94 (suppl 1): 393a. Carella AM, Champlin R, Slavin S, et al. “Mini-allografts”: ongoing trials in humans. Bone Marrow Transplant 2000; 25: 345–50. Childs RW, Clave E, Tisdale J, et al. Successful treatment of metastatic renal cell carcinoma with a nonmyeloablative allogeneic peripheral-blood progenitor-cell transplant: evidence for a graftversus-tumor effect. J Clin Oncol 1999; 17: 2044–44. Childs R, Chernoff A, Contentin N, et al. Regression of metastatic renal cell cancer following nonmyeloablative allogeneic peripheral blood stem cell transplantation. N Engl J Med 2000; 343: 750–58. Guinan EC, Boussiotis VA, Neuberg D, et al. Transplantation of anergic histoincompatible bone marrow allografts. N Engl J Med 1999; 340: 1704–14. 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. de Bueger M, Bakker A, van Rood JJ, et al. Tissue distribution of human minor histocompatibility antigen. Ubiquitous versus

498

Alloreactive immunotherapy

65

66 67 68

69

70 71 72

restricted tissue distribution indicates heterogeneity among human CTLs defined non-MHC antigens. J Immunol 1992; 149: 1788–94. Mutis T, Gillespie G, Schrama E, et al. Tetrameric HLA class Iminor histocompatibility antigen peptide complexes demonstrate minor histocompatibility antigen-specific cytotoxic T lymphocytes in patients with graft-versus-host disease. Nat Med 1999; 5: 839–42. Strauss HJ. Immunotherapy with CTLs restricted by nonself MHC. Immunol Today 1999; 20: 180–83. Gao L, Bellantuono I, Elsasser A, et al. Selective elimination of leukemic CD34+ progenitor cells by cytotoxic T lymphocytes specific for WT1. Blood 2000; 95: 2198–203. Mutis T, Verdijk R, Schrama E, et al. 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. 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: 305–09. Moldrem JJ, Lee PP, Wang C, et al. Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukemia. Nat Med 2000; 6: 1018–23. Slavin S, Ackerstein A, Gelfand Y, et al. Immunotherapy with immune donor lymphocytes for relapsed leukemia post allogeneic bone marrow transplantation. Submitted for publication. 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.

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