RESEARCH PERSPECTIVE

Clin. lmmunother. 1996 Sap; 6 (3):238-249 1172-7039/96/0009-{)238/S06.00/O

© Adis International Umited. All rights reseNed.

Acute Leukaemia An Overview of New Immunotherapeutic Strategies Robin Faa, Alessandro Cignetti and Anna Guarini Department of Biomedical Science and Human Oncology and CNR Centre for Immunogenetics and Experimental Oncology, University of Turin, Turin, Italy

Contents Summary .. . . . . . . . . . . . . . . . . . . . . . . . . .. .. ... . 1. Why Should Immunotherapy Be Considered for the Management of Acute Leukaemia? . . . . . . . . . . . . . . . . . . . . 2. Interleukin-2 in the Management of Acute Leukaemia . 2.1 Preclinical Studies. 2.2 Clinical Studies . . . . . . . . . . . . . . . . . . . . . 2.3 Further Strategies ... . .. .. . . . . . . . . . . . . 3. Does Interleukin-2 Induce an Activation of the Host Immune System? 4. Donor Leucocyte Infusion . . . . . . 5. Gene Therapy . . . . . . . . .. . . 6. Final Considerations and Prospects

Summary

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After years of major disappointments, the possibility that immunotherapy may be effectively employed in the management of cancer has recently witnessed renewed expectations. This is the case both for patients with solid tumours as well as for those with haematological malignancies, in particular acute leukaemia. This revitalised interest is based on encouraging clinical results obtained in a proportion of patients and on the development of sophisticated technological expertise that allow the design of innovati ve strategies that until very recently would have appeared unrealistic. Clinically, the immunotherapeutic approaches that have yielded the most promising responses in human leukaemia are the administration of high dose interleukin-2 or the infusion of donor leucocytes in patients who have relapsed after allogeneic bone marrow transplant. Biologically, the possibility of transducing a specific gene into the DNA of tumour cells and of normal effector cells has opened the way to the activation of the first cytokine gene therapy protocols in cancer patients. In the present article, we will review some of the most relevant results obtained through the above mentioned immunological strategies in the treatment of patients with acute leukaemia. We will also analyse the openings that are likely to occur in the near future and that should conclusively demonstrate that immunotherapy represents a true therapeutic option in well defined clinical settings.

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As a general concept, one would like to include immunotherapy as a fourth therapeutic strategy in the management of cancer patients. Since surgery, chemotherapy and radiotherapy can effectively debulk consistent tumour masses, it would be theoretically desirable in a condition of minimal residual disease to intervene with a more 'physiological' approach aimed at stimulating the immune system of the tumour-bearing host to control and/or eradicate the neoplastic clone. For many years this long sought goal has been followed by marked disillusionment in both solid tumours and haematological malignancies. This overall scenario appears to have changed quite remarkably over the last few years, and the possibility that immunotherapy may play a role in the management of cancer is being met with renewed enthusiasm. Several clinical and biological observations have contributed to this change of attitude. These include the evidence that clinical responses, even complete, may be obtained following the prolonged infusion of a biological substance such as interferon-a. (IFNa.) both in solid tumours and in haematological malignancies. In haematological malignancies, IFNa. has changed the natural course of hairy cell leukaemia[l] and, possibly, of chronic myeloid leukaemia (CML).[2J In CML, the prolonged administration of IFN a. has allowed complete cytogenetic responses in a proportion of patientsP] Similar results have only exceptionally been achieved with chemotherapy. Although IFNa. is likely to exert its antitumour effect mainly through a direct action on the neoplastic clone rather than through accessory cells, partial and complete responses have been observed in a proportion of solid tumour patients treated only with interleukin-2 (IL-2),[4-6] a cytokine capable of activating immunocompetent cells and of inducing the release of other cytokines in treated patients. It is well known that cytotoxic T lymphocytes (CTL) directed specifically against autologous cancer cells can be found in the blood of solid tumour patients, in particular metastatic melanoma,[7,8] and that these effectors can be amplified following in vitro incubation with IL-2. Cloning of such cells in the pre© Adis International Limited. All rights reserved.

sence of IL-2 has recently allowed the identification of the first tumour-associated antigens in humans.[9-ll] Finally, the development of improved technologies has enabled the successful introduction of DNA sequences into the genome of normal and neoplastic cells.[l2,l3] While this has opened potentially revolutionary prospects for the treatment of inherited conditions, it has also allowed the design of new strategies for the management of cancer patients.[14] In cancer, most gene therapy approaches have aimed at boosting, directly or indirectly, the immune compartment of the host)lS] ,These innovative methodological tools have resulted in significant achievements in experimental tumours, which have been followed by a most rapid clinical application through the activation of the first gene therapy protocols in advanced solid tumour patients. 1. Why Should Immunotherapy Be Considered for the Management of Acute Leukaemia? In acute leukaemia, immunotherapeutic approaches have been repeatedly attempted over the last 2 decades. This is largely due to the fact that despite the utilisation of more intensive chemotherapy regimens and of transplantation procedures, for the majority of acute leukaemia patients over the age of 14 years the overall prognosis still remains unfavourable. Several more specific considerations argue in favour of the fact that if suitably educated the host immune system may playa role in acute leukaemia. Biologically, an impairment of the cytotoxic compartment has been reported in patients with acute leukaemia,[16] and the possibility that a close monitoring of natural killer (NK) cell function may have prognostic implications has been suggested)17] Clinically, the great majority of patients achieve a condition of conventionally defined complete remission following induction chemotherapy. However, most of them will sooner or later relapse with the same disease, indicating that the leukaemic clone had not been eradicated, but, rather, that it Clin. Immunother. 1996 Sep; 6 (3)

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had remained 'silent' below the detection limit of conventional monitoring techniques. Some relapses may occur many years after remission, suggesting that the immune system of the host may have contributed to maintain the disease under control. Subsequently, for unknown reasons, the 'silent' leukaemic clone is capable of escaping immune surveillance. Further, though indirect, evidence that the immune compartment may playa role in controlling tumour cell growth is brought about by the high incidence of relapses which occur in leukaemic patients who have received an allogeneic transplant from an identical twin[I8] or with T cell-depleted marrows.[19] In addition, allografted patients who do not develop graft-versus-host disease (GVHD) have a higher risk of relapse.[20] Taken together, these findings clearly point to the existence of a subset of T cells capable of exerting a so-called graft-versus-Ieukaemia effect[21] and, thus, of playing a key role in controlling disease relapse in allotransplanted patients. As discussed in detail in section 4, these indirect observations have been recently matched by convincing and direct clinical confirmations. Several clinical and biological lines of evidence point to the potential in vivo use of immunotherapy in patients with leukaemia, probably to a greater extent compared with solid tumours. Here, we shall analyse the 2 therapeutic approaches, IL-2 and donor leucocyte infusion, which have been accompanied by the most promising clinical responses, as well as the further innovative possibilities made available by gene transfer technologies.

2. Interleukin-2 in the Management of Acute Leukaemia 2.1 Preclinical Studies

The necessary studies aimed at elucidating the preclinical potential of IL-2 in acute leukaemia were performed in the second half of the 1980s. The initial results obtained in experimental models showed that both IL-2 plus ex vivo generated lymphokine activated killer (LAK) cells and IL-2 alone © Adis International Limited. All rights reserved.

could block leukaemic growth in vivo.[22,23] Evidence was then provided that human primary leukaemic blasts, which are resistant to NK cells, can be lysed by IL-2-activated normal peripheral blood lymphocytes.[24,25] Leukaemic blasts are not always killed by normal LAK cells or by the patient's own autologous LAK effectors. In a study conducted in a series of acute leukaemia samples at diagnosis, an overall defect in LAK killing was found in the majority of cases.l 26] At full blown relapse the defect was even more pronounced. However, it may be possible to restore the lytic machinery against autologous blasts of IL-2-activated peripheral blood lymphocytes drawn from acute leukaemia patients in complete remissionP6,27] These observations documented the potential susceptibility of acute leukaemia cells to IL-2-mediated killing, and suggested that should a clinical response to IL-2 be possible this is more likely to occur in patients in apparent remission. These findings were further boosted by the evidence that normal LAK cells, as well as IL-2 alone, can block the in vivo growth of human leukaemic cells in immunosuppressed nude miceP8] Although acute leukaemia cells may express the a and ~ chains of the IL-2 receptor, [29-32] in the large majority of samples recombinant IL-2 fails to trigger a proliferative signal in the leukaemic clone.[28,33] 2.2 Clinical Studies

The positive findings gained from the preclinical studies were the starting point for the activation of the clinical trials with recombinant IL-2 in patients with acute leukaemia. A comprehensive overview of the role of IL-2 in the management of acute leukaemia has been published.l34] Based on the clinical data accumulated over the years, several conclusions can be made. First of all, IL-2 can be administered to patients with acute leukaemia. However, the high doses usually employed are associated with a number of adverse effects similar to those recorded in solid tumour patients, which require that IL-2 is administered under closely monitored hospitalisation. Patients with acute leukaemia also experience a C lln. Immunot her. 1996 Sep; 6 (3)

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more or less severe thrombocytopenia, caused by an antimegakaryocytic action exerted by LAK effectors,[35] which may require supportive measures. Systemic toxicity can be better controlled using a continuous intravenous infusion protocol with escalating daily doses. Clinically, IL-2 alone appears oflittle[36] or limited[37] activity in patients with a high marrow blastosis. Remissions have, instead, been reported in a proportion of acute myeloid leukaemias (AML) with advanced disease and a limited number of marrow blasts.[36,38-40] In collaboration with the Department of Haematology in Rome, we have studied a series of patients with AML with less than 30% of residual bone marrow blasts. In 5 of the 8 patients who obtained a complete remission with IL-2 alone, the remission persisted after a median follow-up time of 32 months (range 14 to 68 months»)41] More than 18 months later the patients are in continuous complete remis sion and in all of them the IL2-induced remission (the third or subsequent) is the longest in the natural history of each patient, indicating that IL-2 has had a significant effect on the individual case history. This has also been suggested by a recent report in which bolus IL-2 has been administered as consolidation therapy to AML patients in second complete remission.[42] A clinical situation in which IL-2-based immunotherapy is conceivably likely to prove effective is that of patients who have undergone an autologous transplant. Autografted patients optimally fulfil the condition of minimal residual disease, and an increased proportion of circulating cytotoxic progenitors has been reported.[43-45] The results obtained show that'IL-2 can be given to autografted patients.[46-49] When exactly to start IL-2, the doses to be administered and its effectiveness still need to be defined. In most studies, relatively high doses of IL-2 have been employed and the administration delayed to a time when a satisfactory haemopoietic recovery, particularly the platelet compartment, has been reached. This has meant initiating IL-2 at 60 to 90 days after grafting, a delay which may be too long since most leukaemic relapses are known to occur early after transplant. An alternative strategy © Ad is International Limited. All rights reserved.

utilises early and prolonged daily low dose IL-2, which is capable of inducing a marked amplification of the cytotoxic compartment of the host.[50] Since no randomised studies have to date been conducted, no firm conclusion can be drawn on the clinical impact of IL-2 in autografted patients. Although the results so far obtained are certainly encouraging, in particular for patients with AML, a final word on the role of IL-2 in the treatment of acute leukaemia will come from further studies. The considerable toxicity associated with high doses of IL-2 has been a major limiting factor, which has restricted a broader clinical utilisation of IL-2 and so far has not allowed the completion of the necessary randomised trials. In Italy, a multi centre study for AML patients in second or subsequent remission randomised to receive IL-2 was started over 3 years ago. Due to slow patient accrual, the study has still to be completed. A Southwest Oncology Group/Eastern Cooperative Oncology Group intergroup trial for auto grafted AML patients in first or second complete remission randomised to receive IL-2 or to observation has only recently been activated. It will, thus, be of primary importance to define whether in well defined clinical situations an antileukaemic effect can be obtained also with lower doses of IL-2. Should this be the case, clinical studies could be carried out much more effectively and rapidly on large series of patients and on an outpatient basis. 2.3 Further Strategies

Meanwhile, preclinical studies have also suggested the design of further IL-2-based immunotherapeutic strategies. The possibility of activating in experimental tumours bone marrow lymphocytes following culture in vitro with IL-2 to remove contaminating leukaemic cells has been suggested)51] Long term cultures of human marrow cells with IL-2 have enabled the generation of marked antitumour cytotoxicity and, consequently, the induction of a potentially effective purging strategy in different tumour types. [52] Following ex vivo culture of bone marrow samples containing variable proportions of AML blasts in the presence of IL-2, it has been C lin. Immunother. 1996 Sep; 6 (3)

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possible to overgrow the leukaemic population and to document the existence of T cells with cytotoxic activity against the autologous neoplastic clone.[53] Our group has alsofound that after prolonged culture of bone marrow or peripheral blood AML samples with IL-2, a proportion ofT lymphocytes with specific lytic capacity may be progressively 'educated' (Cignetti et aI., unpublished observations). Experiments carried out in animal models have suggested the possibility of utilising IL-2 in the context of allogeneic bone marrow transplantation. In mice with AML, allotransplants with syngeneic marrows activated in vitro for 24 hours with IL-2 and followed by IL-2 given immediately after the transplant induce a graft-versus-Ieukaemia effect.[54] In allogeneic transplants, IL-2 may induce allospecific CTL in a murine minor histoincompatibility model without aggravating GVHD.l55] In another murine leukaemia model, the graft-versus-Ieukaemia effect induced by the administration of allogeneic lymphocytes after a T cell-depleted marrow transplant could be markedly increased by a simultaneous short course of low dose IL-2.[56] In Brown Norway myelocytic rat leukaemia, the graft-versus-leukaemia effect lost by T-cell depletion of the allogeneic graft could be restored by post-transplant administration of IL-2 at 'intermediate' doses. [57] The results of these preclinical experiments have led to the design of pilot studies in humans. Bone marrows 'purged' following in vitro culture for 8 days with IL-2 have been recently utilised in a phase I study in 10 patients with high risk AML auto grafted in first remission.[58] Of the 10 patients, 5 remain in remission between 18 and 32 months after grafting. In addition, it has been shown that the infusion of low doses of IL-2 after a T celldepleted allogeneic bone marrow transplant in an attempt to induce a graft-versus-Ieukaemia effect, and thus to reduce the risk of relapse, is feasible and capable of selectively expanding and activating NK cells.l50] Recently, in a follow-up study it has been suggested that patients who have completed at least 1 month of IL-2 had a lower risk of relapse and a greater disease-free survival compared with a historical group of patients treated with an iden© Adis International Limited. All rights reserved.

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tical ablative regimen and T cell depletion method.[59] In view of the possibility that IL-2 may generate a graft-versus-Ieukaemia effect without inducing GVHD,[60] it is foreseeable that low dose IL-2 will be further employed in leukaemic patients allografted with T cell-depleted marrows.

3. Does Interleukin-2lnduce an Activation of the Host Immune System? IL-2 administered in vivo is capable of inducing marked immunological changes in treated patients,[61 ,62] thus reproducing the biological properties known to occur in vitro. These include: • expression of activation markers (HLA-DR, and the a and ~ chains of the IL-2 receptor) on host lymphocytes • increased NK and LAK activity • more relevantly, the induction of a variable proportion of endogenous LAK effectors. These modifications appear to be greater in autografted patients treated with IL-2.l48] In view of the kinetics of acute leukaemia cells, it is worth noting that these phenotypic and functional changes occur both in the blood and in the bone marrow.[62] A selective increase in phenotypically defined NK cells, associated with an enhanced NK and LAK cytolytic function, has been also observed in patients receiving low dose IL-2 after a T celldepleted allograft. [59] The low doses ofIL-2 did not induce the expression of the low affinity IL-2 receptor chain (p55), but did increase the expression of the intermediate affinity chain (p75). Despite these important phenotypic and functional changes, it should, however, be noted that both in acute leukaemia and in solid tumours no firm correlation has so far been established between immune modulation of the host immune system and response to treatment. [62,63] This issue needs, however, to be further investigated, since no study has addressed in depth the possibility that IL-2 may induce effectors with lytic activity directed against the autologous tumour. Studies are currently in progress to assess in acute leukaemia patients with a documented and prolonged response to IL-2 the effectors of the anti leukaemic activity, and to better Clin. Immunot her. 1996 Sep; 6 (3)

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identify their NK and/or T cell nature. The possibility that IL-2 may act directly on the neoplastic clone cannot be ruled out. It has, in fact, been reported that IL-2 can reduce the proliferative activity of IL-2 receptor positive human carcinoma cell lines,[64] as well as of K562 cells.[65] A cascade of cytokines is also known to be released in IL-2-treated patients.l66,67] While some, like tumour necrosis factor-a (TNFa) and interferon-,,{, may exert an antiproliferative effect and may also playa role in the subjective complications suffered by the patients, others are most likely to playa primary role in the profound haematological modifications which are known to occur in IL2-treated patients.[36] The measurable levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) probably contribute to the more or less evident leucocytosis, while the increased levels of interleukin-5 help to understand the marked eosinophilia.[68] TNFa is also likely to be involved in the thrombocytopenia observed in acute leukaemia patients treated with IL-2.135]

4. Donor Leucocyte Infusion Animal studies that go back to the 1960s and 1970s demonstrated that donor spleen cells could be infused into recipients of an allogeneic bone marrow transplant without causing GVHD. More recently, it has been shown that the delayed administration of normal immunocompetent donor cells can, in fact, diminish GVHD.l 69-72 ] Over the last few years the potential utilisation of donor leucocyte infusions following a relapsed allograft as a form of immunotherapy for the management of leukaemic patients has been recognised. Using this approach remissions have been induced in patients with CML, and to a lesser extent with AML, in relapse after an allogeneic bone marrow transplant.[73-77] The results from 27 transplant centres within the European Group for Blood and Marrow Transplantation in 135 patients with recurrent leukaemia after bone marrow transplantation treated with transfusions of donor lymphocytes have been recently analysed. [78] Complete remissions were recorded in 73% of patients with CML, as well as in 29% of © Adis International Umited. All rights reserved.

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those with AML. In CML patients treated in cytogenetic and haematological relapse, remissions were durable, with a <20% actuarial probability of relapse at 3 years from the infusion. Results are less encouraging for patients with advanced relapsed CML. Depletion of T cells from the prior graft had little influence on the subsequent clinical response to donor leucocyte administration. Evidence of an antileukaemic effect has also been reported in another study in which donor lymphocytes were administered to acute leukaemia patients who had relapsed after T cell-depleted allotransplants.[79] The results obtained with this new immunotherapeutic strategy are highly encouraging, particularly in view of the overall poor prognosis of leukaemic patients relapsing after an allogeneic transplant and managed with more conventional approaches. In addition, they document directly and unequivocally the existence of a lymphocyte-mediated graftversus-leukaemia effect. The major limitation so far encountered has been the development of significant GVHD and the frequent occurrence of severe myelosuppression, particularly in patients who had been grafted with aT cell-depleted marrow. [78] This is due to the fact that the infused unseparated mononuclear cells contain cells capable of mediating anti leukaemic activity, as well as alloreactive T lymphocytes that induce severe (or fatal) GVHD. Recent data suggest that infusions of donor lymphocytes depleted of CD8+ cells are capable of inducing clinical response, and thus a graft-versus-Ieukaemia response, in the absence of GVHD.[80] These findings indicate that in humans, as well as in experimental tumours,[81] graft-versus-Ieukaemia and GVHD may be mediated by distinct lymphoid subsets. The exact mechanisms through which this impressive immune-mediated antileukaemic effect is exerted are still unknown. Data obtained in 2 CML patients treated with donor buffy coat transfusion suggest that the frequency of CTL, as well as of NK, cells was increased compared with pre-infusion values, pointing to the likely involvement of both MHC-restricted and -unrestricted effectors in the antileukaemic activity.l82] Clin. Immunother. 1996 Sep: 6 (3)

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Another clinical setting in which the infusion of donor leucocytes has proven beneficial is in the context of Epstein-Barr virus (EBV)-associated B lymphoproliferative disorders originating in patients who have undergone T cell-depleted allotransplants. [83] It is likely that such clinical responses have been mediated by anti-EBV-specific cytotoxic T cells present in the blood of the donor and capable of lysing EBV-infected B cells. This suggestion is confirmed by the evidence that clinical responses have been obtained following infusion of EBV-specific CTL lines obtained from donor leucocytes and expanded ex vivoJ84] It is worth noting that by using specific CTL lines the treated patients did not develop GVHD, again pointing to the existence of different effectors of the graftversus-leukaemia and GVHD responses. 5. Gene Therapy Over the last few years great interest has been raised by the possibility of introducing genomic sequences into the DNA of normal and neoplastic cells.[l3-15] As mentioned in the introduction, the large majority of results obtained by gene transfer in experimental tumours, as well as the early clinical applications in man, represent innovative immunotherapeutic strategies. Considerable attention has been placed on cytokine genes and on the effects induced in cytokine gene-transduced animal tumours. Particular attention has been focused on transducing the IL-2 gene,[85] because of the encouraging results obtained in a proportion of cancer patients and of the limitations associated with the infusion of high dose IL-2, such as: • toxicity • heterogeneous and unpredictable clinical response • lack of evidence that IL-2 can induce the in vivo generation of specific antitumour cytotoxic effectors. Experiments carried out in different animal models have enabled the realisation of 2 of the most desired goals of an ideal immunotherapeutic protocol, i.e. the generation of specific antitumour CTL and of an immunological memory against sub© Adis International Limited. All rights reserved.

sequent challengesJ86.87] More recently, human cancer lines of different cell origin have also been successfully transduced with various cytokine genes, including IL_2J15,88-91] Our group has succeeded in the retroviral vector-mediated insertion of the IL-2 gene into the DNA of human leukaemic cell lines of both myeloid and lymphoid origin.[92] The constitutive release of variable amounts ofIL-2 did not modify the phenotypic profile of the leukaemic cells, promote the expression of the IL-2 receptor a and Pchains or induce a proliferative signal. On the other hand, growth in immunosuppressed nude mice was decreased or abolished compared with that of parental cells, indicating that the tumorigenic potential of acute leukaemia cells can be reversed following transfer of the IL-2 gene. Transduction of specific genes into the DNA of neoplastic cells has witnessed a most rapid clinical application. The design of pilot studies has been largely aimed at activating, through vaccination with cytokine gene-transduced allogeneic or, more rarely, autologous cell lines, the immune system of the host. Most cytokine gene therapy trials have utilised the IL-2 gene for the treatment of patients with advanced metastatic melanoma and renal cell carcinoma. The results so far obtained have documented the feasibility of these vaccination protocols, which are devoid of systemic toxicity and can be carried out in an outpatient setting. Based on the in vitro evidence that human melanoma cell lines engineered to release IL-2 may induce the generation of autologous CTL more effectively than administration of exogenous IL-2,[93] it will be of primary importance to document whether in the cytokine gene treated patients the in vivo release of low, but prolonged, doses ofIL-2 is capable of generating or amplifying anti tumour CTL. Should this occur, cytokine gene therapy protocols are likely to be extended to patients with less advanced disease, in whom the immune compartment is less compromised, and to different tumours. The above reported clinical results with exogenous IL-2, together with the in vitro evidence that lymphocytes with antileukaemic activity can be generated in the presence of IL-2, suggest that cytokine Clin. Immunother. 1996 Sep; 6 (3)

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gene therapy strategies may also have a rationale for acute leukaemia patients. This is further underlined by the evidence that, using as a model a leukaemic T cell line obtained from a living patient and transduced with the IL-2 gene, it has been possible to generate both specific and nonspecific effectors capable of blocking the growth of the cell line (Cignetti et aI., submitted). These results were obtained more effectively following transduction of the IL-2 gene than in the presence of corresponding amounts of exogenous IL-2. Should IL-2 gene transfer be considered for the management of acute leukaemia, different methodological problems will be encountered since the likelihood of generating long term cell lines is extremely low. The possibility of transducing primary acute leukaemia blasts will have to be approached. In this respect, it is likely that the presently available retroviral vectors will prove ineffective. New generation retroviral vectors with a more efficient transduction capacity or different means of infection, e.g. adenoviruses, will need to be studied. Effective tumour cell recognition appears to be a multifactorial process. Failure to recognise the tumour can, thus, be a consequence of multiple events which encompass: • defective expression of MHC molecules • lack of suitable tumour-specific or associated antigen(s) • deficient antigen processing and presentation • release of inhibitors of the host immune compartment by neoplastic cells • lack of costimimulatory molecules on tumour cells. Absence of costimulatory molecules is believed to result in T cell anergy or apoptosis. Conceptually, tumour immunity could be induced following transfer into the neoplastic cells of genes encoding for instance for MHC molecules or for costimulatory molecules. Recently, it has been reported that the B7-1 molecule plays an important role in mediating an immune response to murine myeloid leukaemia cellsJ94) B7-1 gene modified leukaemic cells induced protective immunity against subsequent challenges, suggesting that myeloid leu© Adis International Limited. All rights reserved,

kaemic cells may be candidates for B7 -1 gene transfer approaches. As discussed above, a key issue remains the establishment of long term cultures of human blasts or the transduction of primary leukaemic populations.

6. Final Considerations and Prospects Following years of disillusionment, the immunotherapeutic prospects for patients with leukaemia are improving. In the present article we have reviewed some of the strategies which, in our opinion, have already offered tangible clinical results, such as IL-2 and donor leucocyte infusion, as well as new approaches, for example gene transfer, which are likely to open new therapeutic prospects. What conclusions can be drawn and what are the likely future extensions? The strategies hereby discussed point to the potential immunogenicity of (some) human leukaemic cells. The knowledge that a proportion of AML patients with limited disease have benefited from IL-2 treatment suggests that in these patients IL-2 has induced a host immune activation capable of controlling or eradicating the disease. It will be important to understand why only some patients respond and to identify the effectors of such responses. The evidence that prolonged culture of AML samples with IL-2 may lead to the generation of a proportion of T cells directed against autologous blasts (Jahn et aI.,[53] and our unpublished observations) further addresses the potential immunogenicity of AML blasts.[95] These findings, together with the evidence that IL-2 gene transduced leukaemic cells can induce the recognition of the engineered cells (Cignetti et aI., submitted), point to the existence of putative leukaemia-associated antigens and suggest that attempts aimed at their identification and cloning need to be readdressed. The results obtained with the infusion of donor leucocytes in allograft relapses also open relevant biological and clinical implications. Biologically, it will be of primary importance to more finely characterise the effectors of the graft-versusleukaemia activity and to try to unravel why such Clin, Immunother. 1996 Sep; 6 (3)

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cells are tolerated and highly effective following an allogeneic marrow relapse. Similar to what occurs in EBV-related disorders, in which the transfer of virus-specific CTL induced disease regression, it is conceivable that the graft-versus-Ieukaemia effect observed in CML and acute leukaemia is mediated by CTL directed against leukaemiaassociated or specific antigens. Although in CML the bcr/abl fusion protein is the putative immunogenic candidate, the complete responses observed in a proportion of acute leukaemia patients further indicate the likely existence of so far unrecognised leukaemia-related antigenic sequences. As mentioned in section 4, a key issue is that of increasing tolerance without losing the graft-versusleukaemia effect. Since the effectors of graft-versus-leukaemia and GVHD are likely to be different,[81] efforts aimed at separating the 2 populations and to treat leukaemic patients only with graft-versus-leukaemia effectors[80] will need to be further pursued. Gene marking experiments have recently documented that EBV-specific donor lymphocytes persist for several weeks in recipients of allogeneic marrow transplantsJ84] Since such effectors are the likely mediators of the clinical response, a further potential strategy is that of transducing a suicide gene, e.g. thymidine kinase, in an attempt to control the degree of GVHD when this becomes too severe. Should the development of graft-host tolerance be broken, it is not farfetched to imagine prophylactic treatment of allografted acute leukaemia patients with low doses of donor leucocytes in an attempt to prevent relapse. In view of the recent clinical acquisitions and of the always more sophisticated technologies becoming available, it is easy to foresee that the next few years will witness an increasing exploitation of immunotherapeutic approaches, such as cytokines, adoptive immunotherapy and gene transfer, in the management of acute (and chronic) leukaemia. It is also likely that the possibility of inducing a specific recognition of the leukaemic cell by autologous effectors williead to the identification of further leukaemia-associated or -specific peptides. Should shared leukaemia antigens be identified, © Adis International Limited. All rights reserved.

gene-modified vaccines may well find a place in the treatment of patients with acute leukaemia. Acknowledgements Work supported by Consiglio Nazionale delle Ricerche (CNR), Progetto Finalizzato 'Applicazioni Cliniche della Ricerca Oncologica' , Roma, Associazione Italiana per la Ricerca sui Cancro (AIRC), Special Project on 'Gene Therapy ' , Milano, by Ministero dell'Universita e della Ricerca Scientifica, Roma, and by Istituto Superiore di Sanita, Roma. A.C. is a doctoral student in Oncology at the University of Torino, Italy.

References 1. Golomb HM, Ratain MJ, Fefer A, el a\. Randomized study of the duration of treatment with_interferon alpha-2b in patients with hairy cell leukemia. J Nat! Cancer Inst 1988; 80: 369-72 2. The Italian Cooperative Study Group on Chronic Myeloid Leukemia. Interferon alfa-2a as compared with conventional chemotherapy for the treatment of chronic myeloid leukemia. N Engl J Med 1994; 330: 820-5 3. Talpaz M, Kantarjian HM, McCredie K, et a\. Hematologic remission and cytogenetic improvement induced by recombinant human interferon alphaA in chronic myelogenous leukentia. N Engl J Med 1986; 314: 1065-9 4. Rosenberg SA, Lotze MT, Muul LM, et a\. A progress report on the treatment of 157 patients with advanced cancer using Iymphokine-activated killer cells and interleukin-2 or highdose interleukin-2 alone. N Engl J Med 1987; 316: 889-97 5. West WH, Tauer KW, Yannelli JR, et a\. Constant-infusion recombinant interleukin-2 in adoptive immunotherapy of advanced cancer. N Engl J Med 1987; 316: 898-905 6. Rosenberg SA, Lotze MT, Yang JC, et a\. Experience with the use of high-dose interleukin-2 in the treatment of 652 cancer patients. Ann Surg 1989; 210: 474-84 7. De Vries JE, Spitz H. Cloned human cytotoxic T lymphocyte (CTL) lines reactive with autologous melanoma cells. J Immuno11984; 132: 510-9 8. Coulie PG, Somville M, Lehmann F, et al. Precursor frequency analysis of human cytolytic T lymphocytes directed against autologous melanoma cells. Int J Cancer 1992; 50: 289-97 9. Van Der Bruggen P, Traversari C, Chomez P, et a\. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 1991; 254: 1643-7 10. Brichard V, Van Pel A, Wolfel T, et a\. The tyrosinase gene codes for an antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J Exp Med 1993; 178: 489-95 II. Cox AL, Skipper J, Chen Y, et a\. Identification of a peptide recognized by five melanoma-specific human cytotoxic T cell lines. Science 1994; 264: 716-9 12. Miller AD. Human gene therapy comes of age. Nature 1992; 357:455-60 13. Pardoll D. New strategies for active immunotherapy with genetically engineered tumor cells. CUff Opin Immunol 1992; 4: 619-23 14. Forni G, Parntiani G, Guarini A, et a\. Gene transfer in tumor therapy. Ann Onco11994; 5: 789-94

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Correspondence and reprints: Dr Robin Faa, Dipartimento di Scienze Biomediche ed Oncologia Umana, Sezione Clinica, Via Genova 3, 10126 Torino, Italy.

Clin. Immunother. 1996 Sep; 6 (3)