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Immunotherapy in acute myeloid leukemia Treatment of acute myeloid leukemia (AML) with current chemotherapy regimens is still disappointing, with overall survival rates of ≤40% at 5 years. It is now well established that AML cells can evade the immune system through multiple mechanisms, including the expression of the enzyme indoleamine 2,3 dioxygenase. Immunotherapeutic strategies, including both active, such as vaccination with leukemiaassociated antigens, and passive, such as adoptive transfer of allogeneic natural killer cells, may overcome leukemia escape and lead to improved cure. Allogeneic hemopoeitic stem cell transplantation, the most effective treatment of AML, is the best known model of immunotherapy. Following transplant, recipient AML cells are eradicated by donor immune cells through the graft-versus-leukemia (GVL) effect. However, GVL is clinically associated with graft-versus-host disease, the major cause of mortality after transplant. GVL is mediated by donor T cells recognizing either leukemia-associated antigens or minor as well as major histocompatibility antigens. Several innovative strategies have been devised to generate leukemia reactive T cells so as to increase GVL responses with no or little graft-versus-host disease. KEYWORDS: acute myeloid leukemia n allogeneic AML n immunotherapy n NK cells n vaccines

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Mario Arpinati*1 & Antonio Curti1 Department of Hematology & Oncological Sciences ‘Seragnoli’, University of Bologna, Italy *Author for correspondence: Tel.: +39 051 636 3680 Fax: +39 051 636 4037 [email protected] 1

Acute myeloid leukemia Acute myeloid leukemia (AML) is a clonal, hemopoietic stem cell disorder characterized by the accumulation of immature myeloid precursors (blasts) in the bone marrow and blood, along with the suppression of normal hematopoiesis. It has an annual incidence of 3.6 per 100,000, rising to 16.3 per 100,000 in the over 65 years age group. AML may arise de novo or be secondary to preexisting myelodysplasia or previous chemotherapies. The causes of AML include chromosomal translocations resulting in loss of function of genes responsible for myeloid cell differentiation and maturation, along with activation in tyrosine kinases, conferring proliferative advantages to the clone. Treatment for AML is intensive, with multiple cycles of chemotherapy regimens in close succession, with the option of allogeneic stem cell transplantation for eligible patients. Overall, complete remission (CR) rates are good: 70–80% in patients under 60 years of age. However, approximately 60% of patients will subsequently relapse. As a consequence, the total overall 5‑year survival is 40%, which falls to 10% in the over 65 years age group [1]. In patients who are resistant to chemotherapy or who relapse, several different chemotherapy combinations have been administered but the outcome remains poor in those patients unable to receive allogeneic stem cell transplantation as further consolidation therapy.

Interaction between AML & the immune system During tumor progression, tumor cells acquire certain cellular characteristics, such as immortalization, independency of growth signals, and resistance to apoptosis, and profoundly change their interaction with the host microenvironment. The capacity of tumor cells to interact with the immune system is altered in advanced tumors and this may represent a crucial step in the process of malignant transformation. Indeed, several data support the hypothesis that the loss of interaction between tumor cells and the immunity of the host plays a critical role in tumor progression by facilitating tumor escape from immune control [2]. Recent evidence has demonstrated that the immune system can destroy nascent transformed cells, but also can function to promote or select tumor variants with reduced immunogenicity, thus providing developing tumors with a mechanism to escape immunologic detection and elimination. The genetic basis of this process, known as cancer immunoediting, remains poorly understood, as well as its interplay with other aspects of malignant conversion, such as tumor cell proliferation and apoptosis. The leukemogenic process has been widely investigated over the last three decades and major strides have been made in our understanding of the molecular and

10.2217/IMT.13.152 © 2014 Future Medicine Ltd

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cellular basis of leukemia. In particular, recent preclinical and clinical reports have clearly demonstrated the role of the immune system in controlling the development of AML, as well as the capacity of AML to shape the antileukemia immune response in a tolerogenic manner. Indeed, it is well known that AML patients harbor an increased number of Tregs, which are induced by AML blasts through a direct and indirect effect [3]. Moreover, a wide range of immunosuppressive cells, such as myeloid-derived suppressive cells and tolerogenic dendritic cells (DCs), have been described as a product of the tolerogenic immuno­logical leukemia-derived microenviron­ment. Such microenvironments are known to shift the balance of the immune response toward leukemia tolerance and immune escape, rather than antileukemia immunity through the suppression of the cytotoxic effector cells, including cytotoxic T cells (CTLs) and NK cells. Among the several mechanisms exerted by AML cells to create its immunosuppressive micro­environment, our group has widely investigated the role of indoleamine 2,3-dyoxigenase (IDO). IDO is a key enzyme in the tryptophan metabolism that catalyzes the initial rate-limiting step of tryptophan degradation along the kynurenine pathway [4]. Tryptophan starvation by IDO consumption inhibits T-cell activation [4,5], while products of tryptophan catabolism, such as kynurenine derivatives and O2 free radicals, regulate T-cell proliferation and survival [4,6]. For these reasons, IDO has immunosuppressive activity. Accordingly, cell populations, including regulatory DCs and bone marrowderived mesenchymal stem cells after exposure to IFN‑g, express the functionally active form of IDO, which has the capacity to suppress T-cell responses to auto- and allo-antigens [7,8]. A wide variety of human tumors, including AML, have been shown to express an active IDO protein. Moreover, transfecting IDO into tumor cells prevents their rejection by preimmunized hosts [9,10]. The antitumor effect of IDO blockade is completely dependent on the presence of a fully competent immune system, thus suggesting that IDO acts by deregulating the host immune response and plays a pivotal role in the inhibition of tumor-specific immunity. The mechanisms that IDO-expressing tumor cells utilize to impair antitumor immunity have been recently investigated. Several studies demonstrated that IDO is expressed and functionally active in placenta, which in turn has been shown to be rich in naturally occurring CD4+CD25+ Treg 96

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cells  [11–13]. More recently, CD4+CD25+ Treg cells have been shown to be increased by Candida albicans infection through IDO activation on host APCs [14]. Moreover, tumor-draining lymph nodes contain IDO-expressing regulatory DCs [15]. We have recently demonstrated that AML cells constitutively express IDO [16], which, in turn, exerts its inhibitory effect on T-cell immunity by inducing the conversion of CD4+CD25 - into CD4+CD25+ Tregs [17]. Our data suggest that IDO expression can be regarded as a novel mechanism of leukemia escape from immune control and its inhibition may represent a novel antileukemia therapeutic strategy.

Vaccination against leukemia Similarly to antimicrobial vaccination, antitumor immunization approaches attempt to activate and expand a population of antigenspecific T cells, which may serve as effectors against tumor cells. Although the best knowledge of the mechanisms that regulate anti­tumor immunity have led to the translation of several immunotherapeutic strategies into the clinical settings (e.g., the use of DC-based vaccines) clinical results have been largely disappointing, with the exception of some clinical studies (see below). Such dismal results may be due to several reasons, including the fact that in most cases immunization protocols have been used as therapeutic strategies in patients with large tumor burden rather than to prevent disease relapse after conventional treatments in patients with nonmeasurable and/or minimal residual disease. Moreover, a large body of evidence seems to demonstrate that within the immune system of cancer patients, the efficacy of vaccineinduced, tumor-specific T cells is significantly hampered by the presence of tumor-induced suppression and regulatory factors [18]. Among these, the capacity of tumor cells, including leukemia cells, to induce and expand a population of Tregs has been clearly elucidated. Indeed, for an effective immuni­z ation against tumors it is necessary to counteract some of these tolerogenic mechanisms. Several strategies have been proposed both at preclinical and clinical level. For example, the induction of hosts’ lymphopenia by cytotoxic drugs, which may act by eliminating Tregs, has been shown to replenish the memory T-cell compartment and allow residual host or adoptively transferred T cells to undergo homeostasis-driven proliferation [19–21]. In such contexts, the use of antigen-specific vaccination may serve to educate the developing T-cell future science group

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repertoire and lead to enhanced T-cell memory against tumor-associated antigens [22,23]. The poor prognosis of patients with high-risk AML (especially in the over 65 years age group who are not eligible for allogeneic stem cell transplantation) points toward the need for novel, not only pharmacological, but therapeutic strategies. Under this viewpoint, antileukemia vaccination may be considered as a potential new tool with high compliance and low toxicity. Indeed, many data suggest that a vaccination approach would be beneficial for preventing disease relapse in AML patients, who achieve a condition of minimal residual disease. In recent years, clinical trials using synthetic peptides derived from tumor-associated antigens, such as proteinase‑1 (PR‑1) and Wilms’ tumor‑1 (WT1), have been conducted in leukemia patients and demonstrated some benefits. In particular, WT1, which is over-expressed in AML, appears as an attractive target for immunotherapy, as outlined by a recent study of the National Cancer Institute that assigned to WT1 the position of best and most suitable target antigen for cancer immunotherapy [24]. Some preliminary and promising results were reported in a first seminal study by Oka et al. [25]. In this paper, a correlation between immunological and clinical responses were observed in a significant number of AML and myelodysplastic syndrome (MDS) patients, who were vaccinated with a WT1 peptide vaccine. Moreover, Keilholz et al. reported that vaccination of a patient with recurrent AML, using HLA‑A0201-restricted WT1 peptide 126 with adjuvant, induced complete remission [26]. This group conducted a Phase II clinical trial in patients with AML and MDS using the same peptide 126 with keyhole lympet hemocyanin and GM‑CSF. In total, 17 AML patients and two refractory anemia with excess blasts patients received a median of 11 vaccinations. Clinical results were encouraging and, importantly, associated with immunological responses as evaluated as the induction/expansion of WT1-specific T cells [27]. A Phase I clinical trial in patients with AML, chronic myeloid leukemia (CML) and MDS, using combined HLA‑A0201-binding peptide vaccines from PR‑1 169–177 and WT1 126–134 was also conducted. Again, the emergence of PR‑1+ or WT1+CD8+ T cells in vaccinated patients was associated with a decrease in WT1 mRNA expression, suggesting a vaccine-driven antileukemia effect [28]. An analog to WT1 peptide 126–134 was generated by substituting R for Y at the position 2 anchor motif (named WT1‑A1) [29]. This analog peptide generated a more potent CD8+ T-cell response, future science group

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which recognized and lysed WT1+ leukemia cells in vitro. Moreover, Asemissen et al. recently identified a highly immunogenic HLA‑A1-binding WT1 peptide (317–327) that was processed and able to induce a CD8+ T-cell response in healthy donors and patients with hematological malignancies [30]. Other approaches to antileukemia vaccination include the use of autologous normal DCs, generated from leukemia patients in CR and loaded with tumor antigens and/or the differentiation of leukemia blasts into leukemic DCs. The latter approach provides a promising tool, which in vitro has been shown to increase the immunogenicity of leukemic cells and to induce CTLs against leukemia. In particular, van Tendeloo and colleagues have recently reported promising clinical and immunological results after vaccination with WT1-loaded autologous DCs in AML patients [31]. However, our group recently demonstrated that during DC differentiation AML blasts upregulate IDO expression, which limits the immunogenicity of AML-derived DCs by expanding a population of Tregs, which are capable of inhibiting antileukemia CD8 and CD4 T-cell responses [32]. Interestingly, vaccination against AML has been tested with very promising clinical results after myeloablative chemotherapy by using a tumorcell based vaccine formulation [33] and may represent an innovative platform to enhance vaccine efficacy. This approach represents the translation into clinics of preclinical results obtained in the murine model, where vaccination performed during the aplastic phase postchemotherapy was associated with the skewing of immune response toward tumor antigen, resulting in increased efficacy of tumor vaccination [34]. Taken together, these preliminary clinical studies, albeit being far too limited, provide the first immunologic, molecular and clinical evidence of a potential efficacy of antileukemia vaccination in AML patients. Future studies are highly warranted to assess the role of active immunotherapy in the clinical management of AML patients, especially in the setting of minimal residual disease.

Adoptive immunotherapy with NK cells in the nontransplant setting Several studies demonstrated that NK cell function has a prominent role for the immune control of tumor development and growth. In recent years, a better knowledge of the mechanisms regulating NK cell effector function, which is MHC-unrestricted, has been provided. In particular, NK cells display a number of activating and inhibitory receptors that interact with a wide www.futuremedicine.com

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variety of different ligands on target cells [35]. The engagement of these NK cell receptors results in stimulation or inhibition of NK cell effector function, which ultimately depends on the net effect of activating and inhibitory receptors. Among these, the receptors that recognize antigens at the HLA‑A, -B or -C loci are members of the immunoglobulin super family and are termed killer immunoglobulin receptors (KIRs) [36]. KIRs have been widely investigated and their role has also been recently established in the clinical setting. Indeed, data from a haplo­ identical T-cell-depleted transplantation suggest that KIR mismatch with tumor MHC significantly impacts on tumor cell killing, particularly in AML [37]; and KIR-mismatched NK cells play the main role as antileukemia effector cells [38]. Furthermore, alloreactive KIR mismatched NK cells facilitate hematopoietic engraftment after infusion of haploidentical stem cells, and inhibit the onset of graft-versus-host disease (GVHD) by targeting host APCs [37]. Given these results, haploidentical KIR-mismatch NK cells administered to AML patients as nontransplant cellbased immunotherapy may induce NK cellmediated killing of leukemia cells resulting in the elimination of residual disease in high-risk AML patients. A seminal study demonstrated that NK cells can be safely infused in AML and cancer patients following immunosuppressive chemotherapy and, in some cases, clinical responses without GVHD had been observed [39]. In particular, five out of 19 AML patients with recurrent disease achieved CR. More recently, a study of haploidentical KIR-HLAmismatched NK cell infusion in childhood AML reported that NK cell therapy prolonged diseasefree and overall survival [40]. Our group recently published the results of a Phase I clinical trial of adoptive immunotherapy with haploidentical KIR-mismatched NK cells in elderly patients with AML [41]. A total of 13 AML patients, five with active disease, two in molecular relapse and six in morphological complete remission (CR; median age: 62 years, range: 53–73 years) received highly purified CD56+CD3- NK cells from haploidentical KIR-ligand mismatched donors after f ludarabine/cyclophosphamide immunosuppressive chemotherapy, followed by IL‑2. The median number of infused NK cells was 2.74 × 106/kg; T cells were under 105/kg. No NK cell-related toxicity, including GVHD, was observed. One of the five patients with active disease achieved transient CR, whereas four of the five patients had no clinical benefit. Both patients in molecular relapse achieved CR which 98

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lasted for 9 and 4 months, respectively. Three out of the six patients in CR were disease-free after 34, 32 and 18 months. After infusion, donor NK cells were found in the peripheral blood of all evaluable patients (peak value on day 10). They were also detected in bone marrow in some cases. Donor-versus-recipient alloreactive NK cells were demonstrated in vivo by the detection of donor-derived NK clones that killed recipient’s targets. Adoptively transferred NK cells were alloreactive against recipient’s cells, including leukemia.

Allogeneic hematopoietic stem cell transplantation in acute myeloid leukemia as an in vivo model of immunotherapy Allogeneic hematopoietic stem cell transplantation (HSCT) is the most effective treatment of AML. In registry analyses the event-free survival and overall survival are reported to be as high as 50 and 60%, respectively [42]. However the relapse rate at 5 years has been >30%. The risk of relapse depends on several factors, including the remission status at transplant and the genetic characteristics of the disease. Cure of AML following allogeneic HSCT results from both the use of high doses or radiation or of alkylators before the procedure, and the transfer of donor immunity to the recipient. Pretransplant conditioning prevents the rejection of donor hematopoietic stem cells and contribute to the destruction of leukemic cells. Donor immunity may contribute to the cure of the disease, through the so called graft-versus-leukemia (GVL) effect. However, donor immunity may also lead to the immune-mediated damage of the recipient’s tissues, the GVHD, the major cause of morbidity and mortality following allogeneic HSCT. Increased appreciation of the GVL effect has paved the way in recent years to the use of reduced intensity conditioning regimens, with the aim to exploit the immunotherapeutic potential of allogeneic transplantation in patients unable to tolerate supralethal doses of chemotherapy. However, GVHD is still a major concern. In the end, improvement of the results of allogeneic HSCT in AML will result from the ability to enhance the GVL effect while reducing GVHD rates [43]. Clinical evidence of the GVL effect The initial evidence of a GVL effect was reported by murine experiments performed in 1956, showing that leukemia cells could be eradicated by allogeneic but not syngeneic mononuclear future science group

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cells [44]. The first hint that such an effect could occur in human allogeneic HSCT came in 1979, when it was observed that the development of GVHD (both chronic and/or acute) correlated with reduced relapse rates [45,46]. Subsequent studies showed that relapse rates are even higher in recipients of T-cell-depleted transplants [47,48]. Several anecdotal reports of remission of relapsed AML following withdrawal of immuno­ suppressive treatment or GVHD flares have been published over the years [49]. However, the most convincing evidence of the GVL effect is demonstrated by the curative potential of donor lymphocyte infusions (DLIs) in patients relapsing after transplantation [50]. Although DLI has been effective in the treatment of relapsed CML, with response rates as high as 60–80% [51], its efficacy in AML is still a matter of debate [52]. Responses vary from 10 to 50%, but they are usually of short duration [53], although some reports have shown long-term leukemia-free survival of up to 40% [54]. The reasons of the reduced sensitivity of AML cells, as compared with CML cells, to DLI, are still poorly known, and possibly depend on the higher proliferation rate of AML cells and on differences in the size of the stem/proliferating pool among the two diseases [52]. Furthermore, DLI is regularly associated with high rates of GVHD, often with bone marrow involvement [55]. However, responses may be higher in patients developing GVHD. Chemotherapy may improve the response of AML to DLI [56,57]. The timing of DLI administration is also important; when administered early after allogeneic HSCT it induces a high incidence of severe GVHD. Instead, delaying DLI until more than 6 months after transplant portends a lower incidence of GVHD. The chimerism status is also relevant to the administration of DLI as preemptive DLI in patients with mixed chimerism has been associated with prevention of relapse in patients with high-risk AML [58] receiving allogeneic transplantation with reduced intensity conditioning. In conclusion, although DLI may be effective in patients with AML [59], it has the potential to induce severe life-threatening GVHD and has not been associated with a significant survival benefit in patients with relapsed AML following allogeneic HSCT [60]. It is therefore necessary to devise strategies to separate its GVL from its graft-versus-host (GVH) potential.

Biologic mechanisms of GVL effect The clinical data described above strongly suggest that GVL and GVH reactions are widely future science group

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overlapping and that most responses against leukemia cells are probably directed against normal recipient cells as well. However, pathophysiological data both in humans and animal models suggest that some mechanism may be restricted to GVL responses, and could therefore be exploited to selectively promote GVL responses without causing GVHD. The immunologic mechanisms of GVHD, as well as the effectors contributing to its pathophysiology, has been thoroughly described elsewhere [61]. Several animal models have been set up and employed to study the mechanisms of GVL as opposed to GVH effects. In this manuscript we will focus on the studies performed in patients. GVL is mostly mediated by donor T cells recognizing antigens on the surface of recipient leukemic cells. Mature donor T cells are transfused with the graft at transplantation. Moreover, donor T cells reconstitute from donor hematopoietic stem cells. Based on speculation and on clinical data as well, mature donor T cells in the graft may mediate a more potent alloresponse against recipient minor histocompatibility antigens (mHAs) and leukemia-associated antigens (LAAs). Although leukemia cells are killed by antigen-specific effector CTL, CD4+ helper T cells have been shown to be necessary in the differentiation of adequate numbers of CTL [62]. Th1 and Th17 effector CD4+ T cells may be more relevant in the induction of GVL responses [63]. Although B cells have not been commonly considered effectors of GVL reactions, there is recent accumulating evidence linking humoral immune responses to GVL effects [64]. Most T-cell responses mediate both GVH and GVL effects. MHC class I (HLA‑A, -B and -C) and class II (HLA‑DR, -DQ and -DP) antigens are highly polymorphic antigens involved in antigen presentation to T cells. Disparities in HLA antigens can evoke powerful T cell responses and are associated with a high risk of GVHD and rejection following allogeneic HSCT. However, MHC antigens are involved in GVL responses as well, as recently shown by Vago et al., who demonstrated that relapse following HLA‑mismatched allogeneic HSCT may be caused by loss of recipient specific HLA antigens on leukemic cells, leading to immune escape [65]. As most transplants involve HLA‑matched donors and recipients, T-cell responses mediating GVL and GVH effects are directed against mHA. mHA are peptides derived from polymorphic proteins that are differentially expressed in the donor and the recipient. Proteins encoded www.futuremedicine.com

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by genes located in the Y chromosome are the best known mHAs, as they are expressed only in the male recipient of a female donor and have been shown to mediate both GVH and GVL responses [66]. However, there is clinical evidence of GVL responses occurring in the absence of GVHD. Targeting of mHA with hematopoieticrestricted expression may allow to reduce GVH while preserving GVL activity [67]. Hematopoiesis-restricted mHAs include all polymorphic proteins that are selectively expressed by cells belonging to the hematopoietic lineage. Warren et al. have discovered at least 38 previously undescribed mHA by in vitro isolating specific CD8+ cells from transplant patients [68]. The expression of mHA on leukemic cells needs to be tested by evaluating the ability of mHA-specific T cell clones to target leukemic cells both in vitro and in vivo in xenogeneic models of GVL [69]. More recently, LRH‑1 has been characterized as a potential mHA, restricted to the hematopoietic tissues [70]. LRH‑1-specific CD8+ T cells have been isolated in vivo in patients receiving DLI and have been shown to target CD34+ leukemic cells in vitro. It should be noted that mHA are good targets for immunotherapy of leukemia only in the context of allogeneic HSCT and only in the context of certain HLA alleles. Therefore, the best targets of adoptive immunotherapy are LAA. LAAs include leukemiaspecific fusion proteins, such as Bcr/Abl or PML/RARa [71] , or mutated proteins, germ cell antigens, and also antigens that are expressed more on AML cells than on normal cells, such as CD45, WT1 and PR3. These proteins have been convincingly shown to elicit CD8+ T-cell responses [72 ,73]. Moreover, CD8+ CTL targeting PR3 expressing leukemia cells have been detected in patients with CML following transplantation [74]. The immunogenicity of each antigen depend on their expression on the leukemic cells, as well as on the sequence of the peptide(s) derived from each antigen, resulting in differential binding to the patient’s HLA molecules. Both GVHD and GVL are triggered by recipient APCs surviving the conditioning regimen [75]. GVL responses may be elicited also by cross presentation of leukemic antigens by reconstituting donor APC following allogeneic HSCT [76]. Also, GVL responses may be induced by the leukemic cells themselves, thus potentially leading to GVL responses in the absence of GVHD. This is most pronounced when leukemic cells have APC potential, such as in CML and in certain types of AML [77]. Animal models suggest a prominent role of recipient derived APC 100

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in the induction of GVL responses following transplantation [78]. The clinical observation that responses are more common in mixed rather than full chimeric patients suggest that recipient derived hematopoietic cells are required for optimal GVL responses [79]. However, unlike in animal models, cross presentation of LAAs by donor derived APC may be increased by chemotherapy, leading to the immunologic death of leukemic cells and their uptake by APC, as described previously.

NK cell-mediated GVL effects Seminal observations made by Ruggeri et al. have convincingly shown that donor NK cells may contribute to GVL activity in HLA‑mismatched allogeneic HSCT [37]. This issue has been extensively reviewed by Velardi (in Perugia) [80] and Cooley and Weisdorf [81]. It is known that NK cell activation is negatively regulated by inhibitory receptors, the KIRs, that recognize certain groups of HLA class I molecules. Donor NK cells expressing KIRs that are not inhibited by the HLA molecules of the recipient can be predicted to be alloreactive [36]. Donor NK cell alloreactivity toward the recipient has been associated with decreased relapse following T cell-depleted HLA‑mismatched, and even HLA‑matched [82], HSCT in patients with AML. The role of NK cell alloreactivity in T-cell replete HLA‑mismatched transplantation is still a matter of debate, although some reports suggest a correlation with a reduced relapse rate [83]. Furthermore, the donor KIR haplotype may influence the outcome of both T-cell replete HLA‑mismatched and HLA‑matched HSCT. Patients receiving transplants from KIR B haplotype unrelated donors had improved overall survival in a retrospective ana­lysis [84]. More recently, the same authors reported that only patients with AML, but not acute lymphoblastic leukemia, seem to benefit of receiving the transplant from KIR B haplotype donors [85]. Higher B gene dosage (i.e., B/B vs B/x) appeared to further decrease the risk of relapse. Also, the presence of certain KIR activating genes, such as 2DL5A, 2DS1 and 3DS1, in the donor genome has been correlated with reduced relapse in AML patients receiving HLA‑matched T cell-depleted HSCT [86]. Finally, higher NK cell counts following transplant (>150/µl at 30 days) have also been correlated with reduced relapse after allogeneic T celldepleted HSCT in patients with AML [87]. Thus the available data convincingly demonstrate that donor NK cells mediate antileukemic activity in AML patients receiving allogeneic T cell depleted future science group

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HSCT. Their role in T-cell replete transplantation appears more limited, possibly due to the higher potency of concurrent alloreactive T-cell responses.

Adoptive immunotherapy following allogeneic transplantation As described above, it is possible to identify antigens as well as functional or phenotypic subsets of immune cells mediating GVL but not GVH effects. Attempts to enhance GVL activity while sparing GVH effects have been the object of intense investigation for several decades. Studies of GVHD prevention, either with drugs or adoptive infusion of suppressor cells (e.g., with Tregs), are beyond the scope of this review and will not be detailed. Strategies to enhance GVL have mostly focused on manipulation of DLI products. Selective depletion of either CD4+ or CD8+ T cells have been associated with reduced GVHD with preserved GVL effects [88]. In vitro activation of T lymphocytes in the DLI product with beadconjugated anti-CD3 and anti-CD28 antibodies may induce polyclonal expansion of alloreactive T cells, leading to possible GVL responses [89]. The straightest way to enhance GVL activity is to identify and expand T cells specifically recognizing LAAs [90]. The technology of in vitro generation of antigen-specific T cell clones or lines has been set up starting in the 1990s, by employing T cells directed against viruses such as cytomegalovirus and Epstein–Barr virus [91]. T cell clones specific for hematopoieticrestricted mHA have been employed for adoptive immunotherapy in patients with AML, resulting in the achievement of complete remission [92]. More easily, antigen-specific T cells may be selected based on the expression of activation molecules such as CD137 and CD154 following in vitro stimulation [93]. Generation of T cells specific for WT1 [71,94], CD45 [95] and PR‑1 [96], all resulting in targeting of leukemic cells in vitro and in animal models, have been described. LAA-specific T cells can be expanded in vitro using patient APC loaded with LAA, or AML blasts stimulated so as to enhance their immunostimulatory potential [97], for example, through transfection with costimulatory molecules, such as CD80 [98]. Moreover, AML cells have been shown to differentiate to DCs in vitro [77]. However, in vitro generation of high avidity LAA-specific T cells is difficult [99], and infusion of enhanced T cells to patients does not lead to the persistence of sufficient numbers of functioning CTL cells in vivo. future science group

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Many groups have attempted to circumvent these difficulties by directly translating T cells with the gene encoding for a T-cell receptor (TCR) recognizing either mHA or LAA. T cell gene transfer via retrovirus has been pioneered by Bonini et al. starting in the 1990s [100]. The authors have transfected donor T cells with a ‘suicide’ TK gene, making them sensitive to killing by ganciclovir, resulting in the effective treatment of GVHD following infusion of transfected T cells [101]. An elegant method of T-cell gene therapy is to transfer the gene encoding for a LAA-specific TCR into T cells in vitro [102]. Transfer of a TCR-recognizing mHAs, such as HA-2 [103], or LAA, such as WT‑1 [104,105] has led to the generation of leukemia-reactive T cells. The major limitation of all the approaches mentioned so far is that the antigen-specific T-cell lines are restricted to a specific HLA allele, thereby making it necessary to perform costly patient-tailored T-cell generation procedures for each patient in need. Moreover, technical concerns, including the possible pairing of the transduced with the native a- and b-chains, leading to the generation of potentially alloreactive TCRs [106]. To overcome this technical difficulty, a sophisticated approach to T-cell gene therapy has been devised at the San Raffaele Institute (Milan, Italy). To avoid potentially dangerous pairings between the transfected and the endogenous TCR-a and -b chains, the authors have specifically deleted endogenous TCR coding sequences by using zinc finger protein, a procedure called ‘TCR editing’ [107]. However, although very attractive, it is unlikely that such difficult and time-consuming procedures may become standard practice, even in resourceful tertiary centers involved in allogeneic HSCT. Regulatory issues may also be a concern, considering the potential difficulty in standardization. The technology of chimeric antigen receptors (CARs) may represent a more easily generalizable approach to T-cell gene therapy [108]. CARs are genetically engineered molecules, including an antigen specific extracellular binding domain, fused to a transmembrane domain and to one or more intracellular T cell-specific signaling domains. Tumor targeting with CARmodified T cells is HLA‑independent and may therefore be used in a broad range of patients. Also, CARs can be directed at any cell surface antigens, including carbohydrate and lipid moieties. Finally, CAR-modified T cells could be generated in advance and stored for future use in experimental trials. Several clinical trials of www.futuremedicine.com

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T8

T4

NK

BC

APC

Figure 1. Immunologic mechanisms of graft-versus-leukemia reactions. Leukemiaassociated antigens as well as minor histocompatibility antigens are presented by recipient- or donor-derived (cross-priming) APCs (A) to donor CD4 + Th1 and Th17 cells (B), which in turn promote the generation of mature cytotoxic T lymphocytes and NK cells (C), finally targeting leukemia cells and preventing disease relapse (D). BC: Blast cells.

allogeneic CAR-modified T cells are currently being performed at various centers, including the Memorial Sloan-Kettering Cancer Center (NY, USA) and the MD Anderson Cancer Center (TX, USA) [108]. Recently, a case report of the treatment of a child with acute lympho­blastic leukemia relapsing following allogeneic HSCT has been published [109]. CAR-modified T cells targeting several myeloid antigens, including CD33 [110]. However, CAR T cell-based therapy may result in major potentially life-threatening side effects. Moreover, the use of CAR T cells directed against other antigens besides CD19 has not been adequately studied as yet. Based on the antileukemic effects of donor NK alloreactivity, infusion of donor NK cells may be considered a possible strategy to prevent or treat

relapse following allogeneic HLA‑mismatched HSCT. As previously described, most trials of adoptive immunotherapy with NK cells have been restricted to AML patients outside allogeneic transplantation. More recently, the group at the MD Anderson Cancer Center has reported the preliminary results of a Phase I trial of adoptive immunotherapy with third party purified NK cells as part of the conditioning regimen of patients undergoing allogeneic HLA‑matched HSCT for high risk AML. At doses as high as 3 × 107/kg, the infusion of NK cells did not appear to increase the short term morbidity, including acute GVHD, of the patients [111]. An interesting approach to adoptive immunotherapy with killer cells has been recently developed by Introna et al. [112]. The authors generated cytokine-induced killer cells in vitro by culturing unfractionated donor T cells with appropriate amounts of cytokines for 21 days, and showed that they could selectively kill leukemia cells. In vivo infusion of cytokine-induced killer cells was shown to induce remission of relapsed AML with very little or no GVHD [113]. Stimulation of antileukemic T-cell responses following transplant could be achieved by vaccination. These strategies include administration of synthetic peptides, LAAs, or with APCs loaded with LAAs or with apoptotic malignant cells. Administration of adjuvants, such as GM‑CSF, following transplant has been shown to enhance immune responses in murine models [114]. Recently, WT1 peptide vaccination of patients with high-risk leukemia following allogeneic HSCT has been shown to be associated with prolonged remission and a low incidence of relapse [115,116].

Executive summary ƒƒ Several mechanisms, including the expression of indoleamine 2,3-dyoxigenase, have been described to demonstrate the capacity of leukemia cells of evading leukemia-rejecting immunity. ƒƒ Antileukemia vaccination has been proved as feasible and safe. For acute myeloid leukemia (AML), future clinical studies should consider simple strategies and target the objective of clinical efficacy in the management of minimal residual disease after conventional chemotherapy. ƒƒ The infusion of purified NK cells is feasible in young as well as elderly patients with AML. NK cell-based adoptive immunotherapy represents a novel and promising strategy to be used as a consolidation program for the eradication of minimal residual disease. ƒƒ Cure of AML with allogeneic hematopoetic stem cell transfer depends on the eradication of AML cells by donor immunity (graft-versus-leukemia [GVL] effect). GVL is demonstrated by the induction of clinical responses following the infusion of donor lymphocytes. ƒƒ Graft-versus-host disease is mediated by donor T cells recognizing either histocompatibility antigens or leukemia-associated antigens. Cross priming of the antigens by recipient APCs is essential to the generation of GVL responses. NK cells may contribute to GVL in HLA‑mismatched allogeneic hematopoetic stem cell transfer. ƒƒ T-cell lines specific for minor histocompatibility or leukemia-associated antigens may kill leukemic cells in vitro and in vivo but their clinical use has been hampered by technical difficulties. ƒƒ T-cell gene therapy through transfer of leukemia-associated antigen-specific T-cell receptor allows higher specificity and has led to clinical responses in clinical trials.

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A very simple and attractive option would be to define a post-transplant drug-mediated maintenance treatment to enhance GVL responses and reduce relapse. IL‑2 is an obvious choice and has been associated with reduced relapse following transplant [117]. IFN‑a has also been shown GVL responses following DLI [118]. Azacytidine has been shown to prevent relapse after allogeneic HSCT [119] possibly due in part to immunoregulatory effects [120]. In conclusion, based on the mechanisms of leukemia-directed immune responses following allogeneic HSCT, as shown in Figure 1, several potential strategies might be developed for the immunotherapy of AML patients, including stimulation of LAA-carrying APCs, infusion of antigen-specific T-helper cells, adoptive treatment with CTL or NK cells, or development of monoclonal antibodies.

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