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Harnessing the power of alloreactivity without triggering graft-versus-host disease: how non-engrafting alloreactive cellular therapy might change the landscape of acute myeloid leukemia treatment

Blood Reviews

Abstract

Human leukocyte antigen-mismatched leukocyte infusions outside of the context of transplantation are a promising strategy for acute myeloid leukemia. Recent studies using such non-engrafting alloreactive cellular therapy (NEACT) revealed that survival of elderly patients increased from 10% to 39% when NEACT was given following chemotherapy, and that durable complete remissions were achieved in about a third of patients with relapsed or chemorefractory disease. We review the clinical reports of different NEACT approaches to date and describe how although T-cell and NK alloreactivity could generate immediate anti-leukemic effects, long-term disease control may be achieved by stimulating recipient-derived T-cell responses against tumor-associated antigens. Other variables likely impacting NEACT such as the release of pro-inflammatory cytokines from donor-host bidirectional alloreactivity and the choice of chemotherapeutics as well as future avenues for improving NEACT, such as optimizing the cell dose and potential synergies with adjuvant pharmacologic immune checkpoint blockade, are discussed.

Keywords: Immunotherapy, Tumor-associated antigen, Cytokines, Graft vs host disease, Transplantation, Relapsed acute myeloid leukemia (AML), Alloreactivity, Natural killer cells, T cells.

1. Introduction

For patients with relapsed acute myeloid leukemia (AML), the chance of long-term disease-free survival is below 10% [1] . Therefore, allogeneic hematopoietic cell transplantation (AHCT) is considered for most intermediate- and high-risk fit patients in first complete remission (CR). However, AHCT carries the risk of acute and chronic graft-versus-host disease (GVHD), and many patients are not candidates for AHCT because of older age, comorbidities, or chemorefractory disease. Elderly patients with newly-diagnosed AML and all patients with chemorefractory or relapsed leukemia urgently need better treatment options.

Non-engrafting alloreactive cellular therapy (NEACT) refers to the adoptive transfer of allogeneic immune cells outside the context of transplantation. Others have used the terms “nonengraftment cellular therapy,” “microchimerism” and “microtransplantation”[2], [3], [4], and [5]. Their common features include a focus on HLA- mismatched T or NK cell transfer to mediate strong alloreactivity in order to bring about immediate anti-neoplastic effects in recipients who are not profoundly immunosuppressed as well as the intentional rejection of donor cells such that long-term hematopoiesis overwhelmingly, if not entirely, derives from the recipient's native marrow. Thus, in contradistinction to traditional stem cell transplantation, the risk of GVHD is minimal.

Recent clinical studies indicate that NEACT holds promise as a novel therapeutic strategy for hematologic malignancies, especially for acute myeloid leukemia (AML). To construct this narrative review, we searched PubMed and GoogleScholar using the terms “haploidentical” and “microchimerism” or “microtransplantation” and the MeSH terms “immunotherapy”, “adoptive transfer” and “immunotherapy, adoptive”. Results were restricted to English-language publications and, in PubMed, particular attention was paid to Clinical Trials and Reviews involving human subjects. Relevant articles related to acute myeloid leukemia as well as other cancers were identified and their bibliographies were curated manually for additional references. Focusing on AML, we [1] briefly summarize the clinical literature on NEACT [2] , review possible mechanistic explanations for the anti-leukemic effects [3] , speculate about predictors of response and [4] propose areas where further research is needed in order to optimize this therapeutic approach.

2. Treating AML with alloreactivity

Alloreactivity commonly refers to the immune reaction that occurs following recognition of transplantation antigens (alloantigens) by histo-incompatible T cells. Natural killer (NK) cells can also react against HLA-mismatched cells depending on KIR specificities. The four-decade long experience in AHCT conclusively demonstrated that alloreactivity can eradicate otherwise incurable hematologic cancers. The higher relapse rates observed after autologous or syngeneic stem cell transplantation, and afterex-vivoT cell-depleted compared with non-T cell-depleted AHCT, attest to the necessity of alloreactivity for optimizing cure rates. The fact that donor leukocyte infusions (DLIs) can induce CR in a subset of patients relapsing after AHCT who are not given chemotherapy or who do not respond to chemotherapy further supports the potency of alloreactivity[6], [7], and [8]. The ability of alloreactive T cells to treat AML springs from their vigorous natural response and targeting of non-self antigens. The consequence of such approaches is the unwanted “on-target” toxicity brought by the expression of the alloantigens on normal host tissues and antigen presenting cells (APCs), resulting in GVHD that typically occurs after conversion to full donor chimerism. The impact of GVHD on organ function and immune reconstitution leads to substantial morbidity and mortality, thus restricting the use of AHCT to patients with limited comorbidities.

2.1. The targets of T-cell alloreactivity, “major and minor”

Standard AHCT uses HLA-identical donors in order to limit the risk of GVHD. The molecular basis for alloreactivity in HLA-identical AHCT relies on minor histocompatibility antigen (MiHA) mismatches [9] . The MiHAs are HLA-bound peptides derived from endogenous proteins whose amino acid sequences differ between the donor and the recipient [10] . The frequency of MiHA-specific T cells in the preimmune repertoire is unknown and the number of molecularly defined MiHAs remains scarce at this point. Therefore it is difficult to estimate the degree of mismatching between donors and recipients. Nonetheless, anti-MiHA responses have been shown to provide robust anti-cancer effects in both animal and human systems[8] and [10]. Strategies to select and produce T cell lines with specific reactivity toward MiHAs are being actively explored and represent exciting opportunities to separate GVL from GVH [8] . While such sophisticated approaches are being developed, T cells that recognize foreign HLA molecules are frequent and immediately available. They may constitute up to 3–10% of the naïve T cell repertoire [11] . HLA-incompatible cells have the potential to mobilize extremely brisk and vigorous T-cell as well as NK-cell responses, which may represent an advantage over HLA-matched immune cells[12], [13], and [14]. However, AHCT grafts mismatched at more than one HLA antigen must be T cell-depleted (in vivoorin vitro) in order to avoid fatal GVHD [15] . The subsequent administration of T cells that are devoid of their anti-host component but with preserved anti-leukemic T lymphocytes is, however, an appealing strategy to recover GVL activity removed upon T cell depletion of the graft. Such depletion of anti-host cells can be achieved by eliminating donor cells activated upon exposure to non-malignant recipient cells using immunoconjugates or photodepletion methods[16] and [17]. The other AHCT setting permissible to HLA mismatching at more than one allele is cord blood transplantation, where it is thought that the immaturity of the fetal immune system mitigates the alloresponse [18] .

2.2. NK-cell alloreactivity in AML

NK cell reactivity has also been shown to mediate anti-cancer effects. In AHCT settings where donor NK cells lack the KIRs that recognize recipient HLA class I molecules, NK cells are not inhibited and could lyse recipient targets [12] . This is associated with a reduced risk of relapse and a survival advantage in AML[19], [20], [21], and [22]. In T cell-depleted settings, KIR-ligand mismatched AHCT carries a low risk of GVHD, possibly because donor NK cells kill recipient APCs that are necessary for initiating GVHD. However, in T cell-replete, HLA-B or HLA-C mismatched unrelated donor AHCT settings, the benefit of KIR-ligand mismatching in preventing GVHD, in reducing relapse and in prolonging overall survival (OS) is often, but not consistently, observed[23], [24], and [25]. Treatment-related mortality due to GVHD likely stems from the donor T cell compartment. The reasons for treatment failure due to relapse may be more complex. Decreased activating receptor ligand expression [26] and [27]and shedding of those receptors from AML blasts [28] may impair their recognition by KIR-mismatched NK cells. Also, AML blasts can express immunosuppressive ligands[29], [30], and [31]and soluble factors [32] such as transforming growth factor (TGF)-β[33] and [34]capable of directly dampening NK cell cytotoxicity and interferon (IFN)-γ production [35] . Hypothetically, such interactions between host AML and donor NK cells may also impair NK cell-mediated killing of immunosuppressive dendritic cell (DC) subsets [36] , impair T cell stimulation and even induce regulatory T cells, thus facilitating disease relapse [37] . Moreover, assessing NK alloreactivity is not straightforward. Because KIR and KIR ligand genes may be silent, their cell-surface expression in the donor and recipient, respectively, must be confirmed [20] . Also, NK cell responsiveness is affected by their stage of maturation. Additionally, it is “tuned” by soluble signals from monocytes, neutrophils and DCs, by the presence and membrane organization of activating and inhibitory receptors on AML blasts and normal cells, and by co-receptors like NKG2D that are expressed under cell stress modules[38], [39], and [40]. Unless a functional NK cell cytotoxicity assay is performed against recipient target cells and the size of the NK alloreactive repertoire is quantified, donors may be mistakenly thought to be capable of mounting NK-based anti-leukemic responses when in fact they are not [20] . In many clinical studies, such thorough pre-transplantation assessments were not performed. Finally, immunosuppressive drugs administered in the T cell-replete AHCT setting also impair NK cell function.

3. Alloreactivity without engraftment

In order to fully harness the power of anti-leukemic alloreactivity driven by HLA disparities and by KIR-ligand incompatibility, the ideal cell therapy should avoidin vivopharmacologic immunosuppression and preserve T, NK and other cell populations leading to AML cell targeting and elimination. Moreover, it must be used in a context where there is little risk of triggering severe GVHD. Several lines of evidence suggest that it is possible to transfer HLA-mismatched cells safely to patients and induce significant anti-neoplastic effects. Such therapy can be used in patients who are not severely immunosuppressed (as they must be for AHCT), thereby preventing permanent engraftment of the transferred cells. We call this approach NEACT to emphasize that recipient hematopoiesis remains dominant. Moreover, NEACT must not be confused with synonyms that might include autologous T cell, NK cell and DC therapy (e.g., “nonengraftment cellular therapy” [4] ) or natural fetal-maternal microchimerism[2] and [3], or misleadingly suggest a goal of sustainable engraftment (“microtransplantation” [5] ). The early laboratory work leading to this approach has been reviewed by Reagan and colleagues[4] and [41]. To date, NEACT has been tested in human AML subjects in 12 separate studies and was found to be safe and effective at prolonging survival ( Fig. 1 andTable 1 and Table 2).

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Fig. 1 Time-line summary of modern clinical trials of non-engrafting alloreactive cell therapy for acute myeloid leukemia and myelodysplasia, according to year of publication. Unless otherwise stated, patients had relapsed or chemorefractory disease. The numbers treated and the numbers who achieved documented complete remission are displayed. The circles are color-coded according to the type of product received: irradiated peripheral blood mononuclear cells (PBMCs, yellow), steady-state leukapheresis products activated withex vivoand/orin vivointerleukin-2 (blue), PBMCs mobilized with granulocyte-colony stimulating factor (green), and purified NK cells following G-CSF-mobilized leukapheresis (orange). Details of each study are available in the Supplemental Tables 1 and 2.

Table 1 NEACT studies to treat early stage AML.

Author/reference Number of patients Donor Cell dose and number of infusions Outcome Comment
PBMCs as NEACT
Guo, 2011 [2] 30 + 28 well balanced controls (no NEACT infusions) Haploidentical relatives 3 infusions, 0.5 to 2.6 × 108 T cells/kg and 7.5 to 25 × 106 NK cells/kg per infusion. Two year DFS 38.9% versus 10.0%.

Two year OS 39.3% versus 10.3%.
Only fully reported phase III trial of NEACT.

All patients > 60 years of age. G-CSF-mobilized cell product.
Guo, 2012 [3] 101, all in CR1 Haploidentical or higher order HLA-mismatching relatives 3 infusions, 0.4 to 2.4 × 108 T cells/kg and 2.3 to 68.4 × 106 NK cells/kg per infusion. At 6 years, DFS 59.2% and OS 65.2%.

Overall relapse rate 23.8% in intermediate-risk AML patients.
Low or intermediate risk patients.

T cells doses ≥ 1 × 108/kg per course predicted better DFS.

G-CSF-mobilized cell product.
Ai, 2012 [65] 28 Decitabine/ Cytarabine + NEACT 2 control groups (total 44 patients-2 Decitabine regimen) Mismatched (1 to 3 HLA alleles) relatives Unspecified number of infusions of 3.0 to 10.8 × 108 PBMC/kg.

T cell dose not reported.
CR rate 42.9% vs. 14.8%/29.4%. CCR 90.9% vs. 10%/11%. PFS at 12 months 50.1% vs. 9.9%/9.6%. G-CSF-mobilized cell product.

Reported in abstract form only.

Different cytoreductive regimen.
Forès, 2014 [5] 3 CR1, 1 with double induction Haploidentical or higher order HLA-mismatching relatives Two to three infusions, 0.35 to 2.42 × 108 T cells/kg per infusion. 3 CR – MRD negative ongoing at 7, 8 and 11 months.

1 unclear timing of relapse with MRD, alive at 12 months.
G-CSF-mobilized cell product.
Yuan, 2014 [64] 1 Haploidentical relative 1 infusion, dose of T cells not known. No response. G-CSF-mobilized cell product. Skin GVHD on day 20, resolved with corticosteroids.
 
NK cells as NEACT
Rubnitz, 2010 [13] 10, all in CR1 Haploidentical relatives 1 infusion, 5.1 to 80.9 × 106/NK cells/kg. Maintained CR, after a median of 964 days. KIR-mismatching for 9/10 donors.

All pediatric patients.
Curti, 2011 [14] 6 CR, 2 molecular relapse Haploidentical relatives 1 infusion, 1.11 to 5.00 × 106 NK cells/kg. 3/6 ongoing CR at 18, 32 and 34 months. 3/6 early relapses. 2 patients in molecular relapse had CR for 9 and 4 months. KIR mismatched donors.

1 patient in 2nd molecular relapse at 9 months achieved CR with retreatment.

CCR – complete cytogenetic response, CR – complete remission, CR1 – first complete remission, DFS – disease-free survival, MRD – minimal residual disease, PFS – progression-free survival, OS – overall survival.

Table 2 NEACT studies for AML in refractory or late stage disease.

Author, year (reference) Number of patients Donor Cell dose and number of infusions Outcome Comment
PBMCs as NEACT
Strair, 2003 [55] 2 Haploidentical relatives 3 infusions, 0.9–3.6 × 108 T cells/kg per infusion. SD for 4 months Irradiated infusion product
Slavin 2005 [66] , 2010 [61] 1 Mismatched (6/8 HLA alleles) relative 3 infusions, 2 × 107 T cells/kg (first dose). CR ongoing at 17 years Cell dose of 2nd and 3rd infusion unknown.
Medina, 2008 [58] 8 Haploidentical relatives 1 infusion, 1.16–2.91 × 108 T-cell/kg. 1 CR for 179 days, 1 CRi with engraftment, GVHD and death in remission at day 108. No other responses. G-CSF- mobilized product.

The patient with GVHD shared an HLA DR with the donor, for which the donor was homozygous.
Colvin, 2009 [60] 13 Haploidentical relatives 1 infusion, dose escalation study (up to 2 × 108 T cells/kg). 3 CR for 8, 11 and 31 months.

7 PR (blast reduction) of 19 days (median).
G-CSF- mobilized product.

One unrelated hemorrhagic death in CR at 8 months.
 
NK Cells as NEACT
Miller, 2005 [67] 19 Haploidentical relatives 1 infusion 2.2–15 × 106 NK cells/kg. 5 CR, duration unspecified for 4. Other responses not reported. 1 patient treated for relapse 100 days post AHCT died of EBV reactivation while in CR at day 126 post NEACT.
Curti, 2011 [14] 5 Haploidentical relatives 1 infusion 1.11–5.00 × 106 NK cells/kg. 1 CR for 6 months. 4 disease persistence. KIR mismatched donors.

CR – complete response, CRi – complete response (incomplete) with failure to normalize blood counts, EBV – Epstein-Barr virus, PR – partial response, SD stable disease

3.1. Historical perspective

In the 1960s–1980s, non-irradiated neutrophils from chronic myeloid leukemia (CML) patients were transfused to unrelated, non-alloimmunized AML patients receiving induction or re-induction chemotherapy as a means of providing transient engraftment for supportive care[42], [43], [44], [45], and [46]. This strategy antedated the use of therapeutic granulocyte transfusions obtained from normal donors (because yields were higher from CML patients [47] ) and antedated granulocyte-colony stimulating factor (G-CSF) therapy. In one report of 24 leukemic patients, 5 of 6 temporary CRs and 3 partial responses were fully attributed to the CML granulocyte transfusions because the recipients were not being treated with chemotherapy due to refractory disease or agranulocytosis. Although perhaps these responses correlated with mild suspected GVHD [43] , the incidence of suspected GVHD was negligible (< 0.5%) in several other large studies[44], [45], and [46]. The fact that CML cells could engraft transiently, eradicate or prevent infection during the neutropenic period, and subsequently be rejected upon endogenous immune recovery of the host illustrates principles that underlie NEACT.

Another historical thread leading up to the development of NEACT the observation that unintentional rejection of related donor (matched and mismatched) grafts during early attempts at non-myeloablative AHCT, without intercurrent clinical GVHD, was associated with objective responses and even cures among patients with myelodysplastic syndrome (MDS) [48] , low-grade and aggressive non-Hodgkin lymphoma (NHL)[49], [50], and [51], Hodgkin lymphoma[50] and [51], and multiple myeloma[51], [52], and [53]. To explain this phenomenon, a role of recipient-derived cytokines, particularly interferon-γ, was demonstrated experimentally [54] . Thus, the idea of deliberately seeking "graft" rejection as a new modality to treat hematologic malignancies as well as metastatic solid tumors was crystallized.

The earliest published deliberate attempt at NEACT employed irradiation in order to virtually eliminate the risk of GVHD [55] . Nevertheless, there remain concerns that radiation would preclude clonal expansionin vivo, which could sharply reduce NEACT anti-leukemic effects. For example, a 1978 study at MD Anderson Cancer Center comparing 38 adult patients with AML or acute lymphoblastic leukemia (ALL) who received non-irradiated granulocyte transfusions as infection prophylaxis to 38 age-matched patients who received no granulocyte transfusions found a not statistically-significant improvement in CR rates (from 69% to 76%) and a significant prolongation of survival (from a median of 50 to a median of 73 weeks) among recipients of non-irradiated products [56] . A 2013 study continuing this line of investigation showed no improvement in remission rates, relapse rates, or survival among 108 AML patients with life-threatening infections randomized to irradiated versus non-irradiated G-CSF-mobilized granulocyte transfusions [57] . While this latest study adds to the proof of safety for non-irradiated white cell products, it lacks a control group that was not treated with granulocyte transfusions and so cannot be used for inferring the anti-leukemic efficacy of granulocyte transfusions (irradiated or not). Moreover, the anti-leukemic effects of granulocyte transfusions are not expected to be comparable to NEACT because the preponderance of anti-leukemic donor T and NK cells is discarded with separation of the mononuclear cell fraction. Only Strair’s group used an irradiated lymphocyte-containing product in NEACT recipients; allo-specific cytotoxicity was retained to some degree as evidenced by partial responses or stable disease in 5 of 11 patients with renal cell carcinoma[55] and [58]

3.2. NEACT is generally well tolerated

Reassuringly, NEACT rarely induces GVHD. Out of 333 patients with hematologic and solid malignancies reported with sufficient detail in modern series (Table 1 and Table 2; Supplementary Table 3) treated with haploidentical or higher-order mismatched NEACT and > 5 × 104non-irradiated T cells/kg/infusion (a threshold previously implicated in GVHD in haploidentical AHCT settings [59] ), the incidence of suspected GVHD was only 2.4%[58], [60], [61], [62], and [63]. Five cases resolved spontaneously[61], [62], [63], and [64], 2 resolved with glucocorticoids (although one of these patients later succumbed to sepsis)[60] and [64], and in 1, GVHD was the primary cause of death [58] . Except for two patients with metastatic cancers whose prior treatments are not described[61] and [63], all patients with suspected GVHD had previously been treated with autologous stem cell transplantation [62] , total body irradiation (TBI) [60] , or radioimmunoconjugates [58] , or had received large doses of donor stem cells leading to engraftment[58], [60], and [64]– and many exhibited more than one of these risk factors.

The same principles that apply to preventing transfusion-associated GVHD should probably apply to NEACT, including the avoidance of donors who are homozygous for allotypes shared with the patient. The reported patient who died of GVHD had a DRB1 allele for which the donor was homozygous [58] . Moreover, the immunocompetence of the recipient should be carefully considered if using a non-irradiated product.

Finally, a “cytokine storm” syndrome that responds quickly to steroids has been described (discussed below). It is also possible that some of the aforementioned cases of GVHD suspected solely on clinical grounds (e.g., rash, transient diarrhea) were in fact manifestations of the cytokine storm. Overall, NEACT is very well tolerated.

3.3. Studies in early-stage AML

The results of studies performed in the setting of early-stage AML are summarized in Table 1 and detailed in Supplemental Table 1. They include 154 patients treated during first induction or consolidation. Both unfractionated PBMC and NK cell preparations were well tolerated. In several of the studies using PBMC, the goal pursued by the investigators was to hasten neutrophil and platelet recovery by providing allogeneic myeloid progenitors after chemotherapy. Hence, the use of G-CSF mobilization was widely used, and the composition and cell doses varied extensively between patients.

3.3.1. PBMC as NEACT

The evidence for the efficacy of NEACT in early-stage AML derives from one well-designed and well-executed randomized controlled trial [2] , one large prospective cohort [3] , and three smaller case series[5], [13], and [14](see Table 1 and Supplemental Table 1 for detailed information about the trials). A randomized trial in previously untreated MDS has been reported in abstract form [65] . The most impressive results were reported by a group from two hospitals in Beijing, led by Ai [2] . In their first study, 58 AML patients aged 60 to 88 years (43% with ≥ 3 cytogenetic abnormalities) were randomized to receive either standard induction chemotherapy (“3 + 7” with mitoxantrone and cytarabine) or the same chemotherapy followed by G-CSF-mobilized peripheral blood mononuclear cell (PBMC) infusion from HLA-mismatched relatives. Patients achieving CR went on to receive consolidation with 2 cycles of cytarabine with or without subsequent PBMC infusions, at intervals of 3–3.5 months. After 1 or 2 inductions, the CR rate was significantly higher (80%vs.43%, p = 0.006) and 2-year OS was substantially improved in the cell therapy group (39%vs.10%, p = 0.01). Despite the substantial number of CD34+ cells infused (1.2 – 5.2 × 108/kg per course), no donor cell engraftment was detectable by standard techniques (sensitivity threshold ~ 1%) and no GVHD was observed. Another trial by the same group involving induction chemotherapy followed by G-CSF-mobilized PBMCs in younger newly-diagnosed high-risk patients (7 to 60 years old) is anticipated to be completed shortly (ClinicalTrials.gov NCT01484171).

The same group reported a non-randomized study from four hospitals involving 101 patients aged 9 to 65 years with favorable- and intermediate-risk AML in CR1 [3] . Patients received high-dose cytarabine consolidation followed 24 hours later by unmanipulated G-CSF-mobilized PBMCs from an HLA-mismatched relative. The treatment was repeated at 3 month intervals for up to 3 cycles. With a median follow-up of 51 months, standard detection methods identified transient chimerism (lasting < 2 weeks) in only 4 of 101 patients and no occurrences of GVHD were noted. However, in 5 out of 23 female subjects who had male donors, high sensitivity microchimerism based on detection of Y-chromosome markers revealed longstanding microchimerism persisting up to 12–34 months. Six-year leukemia-free survival (LFS) and OS were 84.4% and 89.5% among the 20 favorable-risk patients and 59.2% and 65.2% among the 81 intermediate-risk patients, with a relapse rate of 23.8% overall. A cardinal discovery made retrospectively by the Ai group was that the T-cell dose ≥ 1.1 × 108/kg, rather than microchimerism persistence, NK cell numbers, KIR profiles, or other factors included in the multivariate analysis, was the strongest predictor of LFS [3] . This correlation provides a strong indication that NEACT efficacy might primarily be a consequence of T-cell alloreactivity. The safety profile was excellent, with on average more rapid neutrophil and platelet recovery and fewer infectious complications than expected with chemotherapy alone. Only three patients had delayed neutrophil and platelet recovery, one of whom died of severe infection.

3.3.2. NK cells as NEACT

Taking a different approach to early-stage AML, Rubnitz and colleagues from St. Jude Children’s Research Hospital reported a pilot study in which 10 patients (0.7 to 21 years old) in CR1 received cyclophosphamide and fludarabine, followed by KIR-HLA mismatched NK cells (median 29 × 106/kg) and subcutaneous IL-2. NK cells persisted in all patients, for a median of 10 days. All patients remained in CR at a median follow-up of 964 days (range 569–1162 days), with a 2-year event-free survival of 100% [13] . Ai's findings notwithstanding, the T-cell content of these NK-cell NEACT products was below 103/kg. The laboratory manipulations required to perform the selection/expansion of NK cells pose a challenge for conducting multicenter trials to confirm these results or compare non-manipulated to NK-enriched products. They might likewise preclude many centers from incorporating NK-cell NEACT into routine clinical care. By contrast, although not trivial, the use of G-CSF in healthy donors followed by leukapheresis and use (or cryopreservation) after minimal manipulations could be accessible to a large number of centers.

3.4. Studies in relapsed or refractory AML

Several clinical studies and case reports have described NEACT, using unfractionated PBMC or NK cell-enriched preparations, for the treatment of relapsed or chemorefractory AML. Often these patients had already been exposed to 2–4 lines of therapy (see Fig. 1 , Table 2 and Supplemental Table 2 for detailed information). Durable CRs in such high risk patients have occasionally been reported[58], [60], [61], and [66]. Because of the heterogeneity of patients and prior therapies, the small numbers of patients in each report, variation or vagueness in response criteria, and possible publication bias, the efficacy of NEACT in advanced disease remains anecdotal. Table 2 summarizes 6 published articles that report the use of various NEACT approaches in 48 AML patients. Aggregate CR rates of close to a third are nonetheless comparable to those of other salvage therapies. Large, well-designed studies of NEACT in this difficult-to-treat population would be welcomed. The design of such studies will require consideration of the immunosuppression from previous chemotherapy that could compromise the host’s capacity to ultimately reject the NEACT product.

4. Why would NEACT be most useful in AML?

NEACT has been attempted as a treatment for metastatic and refractory solid tumors[55], [60], [61], [63], and [67], as well as for hematologic malignancies other than AML[58], [60], [61], [62], [63], [66], [67], [68], and [69](often reported along with AML cases in the studies described in Table 2 ). Despite occasional partial responses, the clinical results are muted overall ( Supplemental Fig. 1 and Supplemental Table 3). Several hypotheses can be readily proposed to suggest why NEACT may be most effective in AML. First, HLA expression levels on the surface of hematopoietic cells is much higher than on other tissues, thereby enhancing antigenic density. Second, among all hematologic malignancies, myeloid leukemias are most vulnerable to KIR-mismatch-mediated NK cell lysis for reasons that are still unclear [12] . Lastly, NK cells perturb the protective interaction of AML blasts with the bone marrow stroma, but do not seem to interfere with the interaction between lymphoid blasts and the marrow microenvironment [70] . Furthermore, the myeloid leukemias are those showing the highest response rate to DLI to treat leukemia relapse after AHCT [6] .

In summary, the direct anti-leukemic efficacy of NEACT is probably due to HLA- and NK-mediated alloreactivity. However, indirect effects may be critical for long-term leukemia control ( Fig. 2 ).

gr2

Fig. 2 Possible mechanisms at play during NEACT. Schematic representation of NEACT for AML. Leukemic cells can be directly targeted by partially HLA-matched donor T and NK cells. Following transfer of donor cells, bidirectionnal alloreactivity occurs, resulting in cytokine production and eventual rejection of donor cells. Initial AML blast killing within an inflammatory context may foster the development of autologous T-cell and NK anti-leukemic activity. The use of chemotherapy is likely to alter the course of the immune response by impacting several variables pertaining to the biology of the leukemic blasts, their microenvironment, as well as the lymphoid homeostasis of the host. Abbreviations: HLA - human leukocyte antigen; MiHA - minor histocompatibility antigen; NK – natural killer cells; TAA – tumor-associated antigen; TSA – tumor-specific antigen.

5. Indirect anti-leukemic effects of NEACT

5.1. Tumor-associated and tumor-specific antigens

TAAs include T cell-defined antigens that are overexpressed on neoplastic cells relative to normal cells and oncofetal antigens that are normally expressed only during embryonic and fetal development. Tumor-specific antigens (TSAs) result from mutations that are exclusively expressed on neoplastic cells (i.e., “neo-antigens”) [71] . Notwithstanding the importance of autologous NK cell responses [72] , autologous T cell cytolytic responses against AML blasts also correlate with remission durability in patients treated with chemotherapy alone, and these must be on the basis of TAAs or TSAs[72] and [73]. Although natural TAA-specific CD8+ central memory and effector memory T cells are detectable in most healthy people, they occur at higher frequency, in higher quantity and display stronger reactivity in patients with leukemia[74], [75], and [76].

In AHCT, alloreactivity appears to trigger a specific T-cell response against TAAs. Of particular relevance for NEACT, several lines of evidence suggest that alloreactivity can lead to antigen spreading and foster an anti-TAA response [8] . In ALL [77] , chronic lymphocytic leukemia (CLL) [78], CML [79] , multiple myeloma [80] , and AML [81] , the detection of donor-derived anti-TAA responses after AHCT has been shown to correlate with better disease control. Evidence for the importance of donor-derived T cell-mediated anti-TAA reactivity can also be gleaned from clinical studies that did not directly quantify the occurrence of specific anti-TAA T cells. For example, patients with CML treated with T cell-depleted sibling AHCT had a higher relapse rate compared to those treated with T cell-replete syngeneic AHCT (risk of relapse 5.14 and 2.95 respectively, relative to a reference group of non-T cell-depleted sibling AHCT without GVHD) [82] . Although a few twin transplant patients developed a syndrome consistent with acute GVHD and two progressed to limited chronic GVHD, 90% of these patients never developed GVHD. These data suggest that a GVL effect can be elicited by TAAs. Likewise, TAA-targeted immunotherapeutic strategies may find a role in treating AML outside of AHCT. For example, in several phase I/II studies, chemotherapy followed by vaccination against Wilms’ Tumor 1 (WT1) protein led to temporary partial responses in a substantial proportion of patients [83] . In a promising pilot study, WT1 peptide vaccination was also shown to maintain CR in 4 out of 5 AML patients who were at high risk for relapse post AHCT [84] . It has been argued that the post-chemotherapy and post-AHCT milieu may facilitate effective TAA-targeted immunotherapy because lymphopenic environments promote cytokine-driven T cell proliferation and because of the paucity of regulatory T cells [85] .

In the NEACT literature, both donor- and recipient-derived anti-TAA responses have been proposed to contribute to AML control. Using HLA-A*02:01 pentamers, WT1-specific CD8+ T cells could be detected in 33 out of 39 evaluable cases in Ai’s report [3] . Although not statistically significant in this small subgroup of patients, the presence of a demonstrable anti-WT1 response correlated with better survival. The LFS and OS of patients who showed a substantial increase in WT1+ CD8+ T cells were superior compared with patients who did not mount such a response (66 and 70%vs.33% and 50%, respectively). In 24 cases, these WT1+ CD8+ T cells were potentially of both recipient and donor origin, but in 6 cases they were exclusively of recipient origin (because their donors were HLA-A*02:01 negative). This suggests that NEACT might be capable of stimulating an autologous leukemia-specific response. However, measurements of circulating multimer-positive T cells in patients treated with chemotherapy alone or prior to NEACT were not available, leaving only speculations on the impact of NEACT on the development of a leukemia-specific immune response. Similarly, Strair and colleagues report a patient with relapsed, chemotherapy-resistant AML whose post-NEACT CR was associated with the generation of host-derived cytotoxic T cells that were specific to the TAA proteinase-3 in the absence of detectable donor chimerism [58] .

5.2. Cytokine release and possible bystander effects

It is plausible that “bystander effects” – whereby histo-incompatible cells react against each other – account for the efficacy of NEACT. Clinical evidence for such effects comes from the anecdotal occurrence of antitumor responses despite unintentional rejection of transplanted allogeneic hematopoietic cells[48], [51], [52], [53], and [86]. Similarly, the reduced relapse rates in double umbilical cord compared to single umbilical cord transplantation (UCT) reported in some[87] and [88]but not all studies [89] may be linked to the fact that in the former setting one cord is almost universally rejected by CD8+ T cells from the dominating graft [90] . An ongoing trial at the Fred Hutchinson Cancer Research Center using an “off-the-shelf”ex-vivoexpanded umbilical cord blood product to hasten hematopoietic recovery after chemotherapy in the context of relapsed or refractory AML could be considered as a form of NEACT and therefore shed light on the anti-leukemic role of allo-rejection in double UCT as well as in non-transplantation settings (ClinicalTrials.gov NCT01701323).

Experimental work supports the hypothesis that recipient rejection of donor cells is key to developing specific anti-tumor responses. After non-myeloablative MHC-mismatched AHCT resulting in stable chimerism, host-versus-graft reactions – whether spontaneous or elicited by recipient leukocyte infusions (RLIs) – were associated with strong antitumor effects against hematologic malignancies. These effects were mediated by RLI-derived interferon (IFN)γ-producing CD8+ T cells and non-RLI, recipient-derived CD4+ T cells, and did not involve donor T cells (which were rejected prior to tumor injection)[54] and [91]. This model supports the possibility of mounting an effective recipient-derived anti-leukemic immune response after donor cell rejection. Importantly, this procedure was not associated with GVHD or other detrimental immune-mediated side effects.

On the other hand, cytokines and chemokines released from either donor or recipient cells (or both) during this “bidirectional alloreactivity” might contribute non-specifically to both donor and recipient GVL effects. Slavin and colleagues observed “shaking chills and mild transient fever” in an unspecified number of patients [61] and Colvin and colleagues described a “haploimmunostorm” syndrome affecting 100% of patients at the 1–2 × 108 T cell/kg doses [60] . This syndrome included fever (100%), abnormal liver function tests (93%), morbilliform rash (60%), and diarrhea (73%) with a median onset of 14 hours after DLI. A subsequent report from the same group mentions episodes of hypoxia and hypotension, but it remains unclear whether these occurrences are frequent [41] . None of the skin or colon biopsies were consistent with GVHD, and all cases of pyrexia lasting > 72 hours responded promptly to glucocorticoids. Elevations in serum interleukin (IL)-6, IL-8, IL-10, interferon (IFN)-γ, IL-5, IL-7, IL-13, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-1b post NEACT were also noted [60] . No significant changes were seen for a plethora of other cytokines (including IL-2, IL-4, IL-1β, IL-12, IL-17, IFN-α, granulocyte-macrophage colony stimulating factor, and G-CSF). Although detailed patient-level analysis is needed, this preliminary work suggests that the NEACT cytokine profile may differ from early acute GVHD[92], [93], [94], [95], [96], [97], and [98]and from engraftment syndrome [94] . Intriguingly, it may share the greatest similarities with “autologous GVHD” [99] and with chimeric antigen receptor-transduced autologous T cell immunotherapy[100] and [101]. Other outstanding issues include which fractions of cytokines are released from donor versus recipient cells, how the cytokines in the marrow microenvironment differ from those in circulation and whether a clinical “cytokine storm” syndrome predicts durable CRs.

6. Treating with NEACT, known and unknown variables

6.1. Optimal cell dose and composition

6.1.1. T cells

The optimal dose of each type of accessory and effector cell is not established. Factors influencing the choice of dose may include the type of malignancy, the degree of HLA and KIR incompatibility, disease stage (e.g., minimal residualvs.morphologic disease), the type of chemotherapy to be administered prior to DLI, the degree of lymphopenia in the host, whether the donor will receive G-CSF, whether the recipient will receive immunostimulatory adjuvants, anyex vivoengineering of the cell product, and whether single or repeated infusions are planned. Similar to Ai and colleagues, Colvin and colleagues also found that all responders had received 1 to 2 × 108CD3+/kg [60] . (Two patients developed engraftment followed by GVHD, but what T-cell dose they received is not mentioned.) NEACT T-cell doses above ≈ 2 × 108CD3+/kg have not been carefully studied, and whether the same dose thresholds apply to cellular products collected without G-CSF mobilization is unknown. It may also be interesting to determine whether the absolute number or the proportion of regulatory T cells, dendritic cells or myeloid cells infused affect clinical outcomes.

6.1.2. NK cells

Likewise, the optimal dose of NK cells exerting anti-leukemia activity in this setting is unknown. A seminal NEACT study by Miller and colleagues from the University of Minnesota Cancer Center showed thatin vivoNK cell expansion depended on a more intensive lymphodepleting chemotherapy regimen and on dramatic surges of endogenous IL-15, while KIR ligand mismatching in the graft-versus-host direction predicted CR [67] . It is worth noting that their final NK-cell products bore substantial monocyte (25% ± 1.6%), B-cell (19% ± 2.0%) and T-cell (1.75 ± 0.3 × 105CD3 + cells/kg) fractions. Building on this approach through the use of a purer NK-cell product and treating patients in the consolidation phase rather than during re-induction of florid disease, Rubnitz and colleagues at St. Jude’s Children’s Research Hospital showed that as few as 5.2 × 106CD56+ cells/kg (median 29 × 106CD56+/kg) could effectively consolidate AML CRs, achieving excellent 2-year event-free survival as described above [13] . Similarly, Curti and colleagues from the University of Bologna, Italy treated 13 elderly and poor-prognosis AML patients with much lower doses of NK cells (as expected for adult body mass; median 2.74 × 106CD56+/kg, range 1.11 to 5.00 × 106CD56+/kg) [14] . One of 5 patients with progressive disease obtained a CR lasting 6 months. Three of 6 patients treated in CR were disease free after 18, 32 and 34 months. The two patients in molecular relapse achieved CR lasting 9 and 4 months. The latter achieved a third CR by repeating the entire treatment. Importantly, as in Miller’s study, both these groups administered cells following lymphodepleting chemotherapy and used low-dose IL-2 to achieve maximalin vivodonor NK cell expansion. However, recent data suggest that IL-2 alone is insufficient to induce leukemia control [102] .

6.2. Growth factor mobilization

After Strair's initial attempt to use NEACT for solid malignancies [55] , subsequent studies employing non-irradiated NEACT products often used G-CSF mobilization – including all published NEACT studies focusing on AML, with the exception of one reported patient [66] . The rationale for G-CSF mobilization varied. Sometimes the goal was to mimic the donor products used in traditional AHCT [58] or to polarize donor T cells to a Th2 phenotype in order to minimize the risk of GVHD in the event of engraftment [60] . For others, the main reason for favoring G-CSF mobilization was to hasten hematopoietic recovery [2] . Indeed, G-CSF-mobilized NEACT hastens hematologic recovery after TBI or after chemotherapy in most patients and prolonged aplasia has only rarely been reported in NEACT-treated patients[2], [3], and [60].

Parenthetically, it may be worth noting that G-CSF-primed marrow grafts might have enhanced anti-tumor effects in CML patients compared to unprimed marrow [103] . Even more provocatively, in CML and AML patients who experienced relapse after AHCT, G-CSF administered as a single agent achieved results comparable to those reported for DLI, and superior to the anticipated benefit of simply withdrawing immunosuppression[104] and [105]. Of course, CML is a much more “immunogenic” cancer than AML [106] . Moreover, G-CSF and granulocyte-macrophage colony stimulating factor (GM-CSF) did not prove to be effective adjuvant treatment when priming AML[107] and [108]and no study of their use in supportive care reported decreased relapse rates. Still, the possibility that G-CSF might have a salutary immunomodulatory role in particular subtypes of AML has not been excluded [109] .

Any potential benefit of G-CSF must be balanced against its risks. In clinical AHCT, rates of acute and chronic GVHD are higher in recipients of G-CSF-mobilized grafts compared to steady-state marrow [110] or G-CSF-primed bone marrow[103] and [111]. However, these observations may be explained on the basis of a higher T-cell dose in mobilized PBMC products. Consequently, G-CSF mobilization may not in itself affect the risk of GVHD in NEACT recipients, where the dose of cells is titrated according to T cell content. Nonetheless, G-CSF may expose NEACT donors to discomfort and unnecessary risks [112] . A steady-state, unmanipulated apheresis product is currently being investigated (ClinicalTrials.gov NCT01793025).

6.3. Choice of chemotherapeutics

Most chemotherapeutic regimens used in NEACT trials and cohorts so far reflect standard oncology practice in that the main or sole objective of the treatment is to kill cancer cells. However, chemotherapeutics may differ in their ability to modulate T and NK cell populations (reviewed by Kroemer and colleagues[113] and [114]). Therefore, chemotherapy regimens not traditionally used in AML might hold particular interest for use prior to mismatched cell therapy. For example, low metronomic doses of cyclophosphamide may selectively deplete or inhibit regulatory T cells [115] , thereby freeing recipient anti-leukemic responses. Other drugs, like gemcitabine or 5-flurouracil, may selectively deplete immunosuppressive cells within solid tumor beds [116] . Whether this is also true in the marrow microenvironment remains to be tested.

Chemotherapeutics induce apoptosis of AML blasts. Traditionally, apoptosis was considered non-immunogenic, but recent studies suggest that an immunogenic form of apoptosis also exists. The latter is defined by three molecular events: [1] endoplastic reticulum (ER) stress leads to pre-apoptotic cell-surface exposure of calreticulin and other ER proteins, which in turn trigger DCs bearing a calreticulin receptor to phagocytose the blastic corpse, leading to presentation of TAAs and subsequent CD8 + T cell reactivity: [2] adenosine triphosphate (ATP) release during autophagy serves as a recruitment signal for DCs; and [3] high-mobility group box 1 (HMGB1) release during cell death ultimately improves cross-presentation of dead cell antigens such as foreign HLA molecules, MiHAs and TAAs[113] and [117]. Likewise, hypomethylating agents might synergize with NEACT because they may restore expression of epigenetically-silenced TAAs while potentiating CD8+ T-cell cytotoxicity[118] and [119]. However, they also expand Tregs[120], [121], and [122]. That may partly explain the still-substantial relapse rates seen with azacitidine prophylaxis in AML patients transplanted in CR [123] , and why only ≈ 16–33% of patients with AML and myelodysplasia who relapse post-transplant are successfully salvaged with azacitidine and DLI[122], [124], [125], and [126], a rate comparable to that associated with DLI alone [6] . Results of ongoing attempts to use these agents with NEACT are eagerly awaited[64] and [65].

Even “non-immunogenic” apoptotic modules might help specific adjuvant chemotherapeutics to recruit immune effectors. These include (1) exposure of heat shock proteins (e.g., seen with a variety of chemotherapeutics, valproic acid and bortezomib)[127] and [128]; (2) p53 activation within neoplastic cells that promotes NKG2D ligand (MHC class I chain-related genes MICA and MICB) expression in tumors and hence facilitates NK-cell recognition (seen with DNA-damaging agents, proteasome inhibitors and histone deacetylase inhibitors) [129] ; and (3) upregulation of classical HLA molecules that likewise makes neoplastic cells more “visible” to T cells (e.g., seen with cyclophosphamide, gemcitabine, and oxaliplatin) [114] . Intriguingly, alloreactivity has been shown to restore cell surface expression of HLA, CD40 (a costimulatory protein required for antigen presentation), and CD54 (a T cell recruiter) on the surface of AML blasts [130] . The aspects pertaining to immunological death and plausible synergy with NEACT approaches remain to be explored. These observations lead to the unanswered question of whether NEACT should precede, follow, or “sandwich” chemotherapy.

Another key factor to consider is the degree of lymphodepletion achieved prior to NEACT treatment. A large body of evidence supports the use of lymphodepletion prior to adoptive transfer in order to increase the concentration of endogenous homeostatic cytokines (IL-7 and IL-15) and facilitate donor cell expansion (reviewed by Rezvani and Barrett [131] ). Moreover, lymphodepletion may be conducive to the development of an anti-TAA response [132] . As a note of caution however, intense host lymphodepletion and immunosuppression can also lead to donor cell engraftment and GVHD.

7. Future directions

According to the largest studies so far, NEACT may show its greatest promise in early stage disease. In fact, this approach has several theoretical and practical advantages over established therapies and presents several opportunities for research ( Table 3 ). In order to further improve the efficacy of NEACT and maximize its potential, especially for late stage AML, several avenues besides what has been described in the previous sections may be particularly attractive to fully harness immune mechanisms in AML. Table 4 summarizes the how NEACT contrasts with other emerging immunotherapies and how it could synergize with some of them.

Table 3 Main advantages of NEACT and niches for future trials.

Potential advantage of NEACT Opportunities/open questions Limitations/uncertainties
Strong alloreactivity based on HLA-mismatching with no pharmacologic immunosuppression -Role of T-cell and NK subsets -Temporary effect: allogeneic cells are rejected.
-Dosing of cell subtypes  
-Enrichment or priming of alloreactive T cells  
-Optimal donor selection  
-Biological assessment of bi-directional alloreactivity  
NEACT conducive to host or donor-derived TAA-specific T cells generation -Vaccination with TAA post-NEACT -Finding requires confirmation.
  -Bystander effect or genuine anti-leukemic effect?
Good safety profile/Tolerability -Use in majority of AML patients unfit for transplant (early or late disease) -Minimum safe level of host immunocompetence unknown.
-Use as bridge to transplantation -Alloimmunization and impact on future transplant?
Few cellular manipulations (unfractionated PBMC products) -Amenable to multi-institutional trials and broadly applicable -Heterogeneity of cellular product
  -Effect of cryopreservation?
Accelerated hematologic recovery post chemotherapy (G-CSF primed products) -Use of intensified chemotherapy in vulnerable patients - Finding requires confirmation.
  -G-CSF related immunomodulation of strength of allo-response.
  -Risk of engraftment.
  -Donor discomfort.

Table 4 NEACT and other novel immunotherapy strategies.

Alternative immunotherapy Advantage of alternative immunotherapy Potential advantage of NEACT Potential synergy with NEACT
Immune checkpoint inhibition -Precedent success with other malignancies -Tolerability -Enhancement of the donor-versus-leukemia alloresponse, Risk of engraftment/GVHD?
  -Targeting high avidity antigens (HLA) -Activation/expansion of TAA-specific T cells observed after NEACT
  -Cost -Activation/expansion of TAA-specific T cells observed after NEACT
    - Decreased effect of PD-L1 expressed by leukemic blast might sensitize blasts to donor and host immune-mediated killing
TAA-directed cellular or vaccine therapy -Persistence of anti-TAA immunity -Targeting high avidity alloantigen antigens -Bidirectional alloreactivity, “haploimmunostorm” may provide right inflammatory milieu to enhance anti-TAA T-cell effect
-Response easier to characterize and follow -No risk of target epitope loss/editing -Ex-vivo expansion of TAA-specific T cells detected after NEACT for adoptive immunotherapy
-No risk of GVHD -No requirement for extensive ex-vivo manipulation  
Chimeric antigen-receptor T cell therapy -Proven track record of efficacy in other diseases -To this date, lack of suitable surface antigen for CAR-based therapy in AML -Combined targeting of alloantigen and surface antigen?
-Persistence of CAR-modified T cells -No requirement for extensive ex-vivo manipulation -Combined toxicity (cytokine release)?
  -Tolerability  

7.1. Adjuvant pharmacologic and cellular therapies

On the patient side, some the most attractive adjuvant therapies that could enhance the potency of NEACT entail blockade of inhibitory pathways or agonism of co-stimulatory pathways in immune effector cells[133] and [134]. Follow-up infusions of chimeric antigen-receptor directed T cells (CART) or autologous TAA-specific T cells generated by other methods might also prove to be effective maintenance therapy, gradually leading to a cure in patients unsuitable for immediate leukemia eradication through AHCT due to anticipated toxicities or patient preference. Some examples of these potential future directions are discussed below.

7.1.1. CTLA-4 blockade

Upon T-cell activation, cytotoxic T-lymphocyte antigen 4 (CTLA-4) is expressed on effector CD4+ and CD8+ T cells and engages with the B7 receptor on APCs to inhibit T-cell activation through a negative feedback loop. CTLA-4 also enables regulatory T cells to induce peripheral tolerance. CTLA-4 blockade is intended to promote both helper and cytotoxic T-lymphocyte activation while diminishing regulatory T-cell activity  [134] . The utility of ipilimumab in advanced melanoma [135] and other tumor types [136] raises the possibility that CTLA-4 blockade itself may prevent AML relapse. AML relapse is more frequent with a particular genotype associated with increased production of soluble CTLA-4, which is thought to have an anti-proliferative effect on T cells [137] . Blocking or deletion of CTLA-4 promoted T cell-mediated rejection of AML cell lines that express one of its cognate ligands, CD80, in a pre-clinical model [138] . Also, at least 25% of AML express CTLA-4 and inhibiting CTLA-4 promotes apoptosis of such AML blasts [139] . Moving to the clinical arena, a phase 1 trial found that CTLA-4 blockade in patients with relapsed malignancies post AHCT was safe [140] . The patients were off immunosuppression at the time of treatment and did not develop GVHD or graft rejection. A study of ipilimumab in advanced AML (in non-transplanted patients) is ongoing (NCT01757639) and another has been terminated but not yet reported (NCT00039091).

7.1.2. PD-1/PD-L1 blockade

Programmed-death receptor-1 (PD-1) is also a member of the CTLA-4 family but it is expressed on a wider variety of activated immune cells (including B cells, NK cells, monocytes and myeloid cells) and is primarily involved in limiting effector T cell function at peripheral sites of inflammation rather than inhibiting central activation of T cells[133] and [134]. Experimental evidence suggests that PD-1 or its L1 ligand would be potential targets in AML. PD-L1 (also called B7-H1) is expressed by 24-54% of AML depending on histologic subtype [141] , is induced by the leukemic "microenvironment" (hence expression is higherin vivothanin vitro), and has been linked to immune escape of persistent residual disease[141] and [142]. PD-1 or PD-L1 blockade also reversed exhaustion of MiHA-specific T cells from relapsed AML patients post AHCT [143] . Despite promising pre-clinical data[143], [144], and [145], the PD-1 inhibitor CT-011 used as a single agent led to a brief partial response in only 1 of 8 patients with advanced AML in a phase 1 study [146] . A trial using PD-1 blockade to potentiate vaccine immunotherapy in patients with newly-diagnosed or relapsed AML is ongoing (NCT01096602). Combined CTLA-4 and PD-1/PD-L1 blockade could be envisioned to increase efficacy and enable lower doses of each agent, perhaps reducing adverse effects, but the same caveats regarding the promulgation of GVHD apply.

7.1.3. Chimeric antigen-receptor T cells

T cells can be engineered to express transmembrane chimeric antigen receptors (CAR) to direct them to cells expressing a specific surface antigen. This approach proved successful in B-cell malignancies, where CD19 is commonly targeted. The paucity of AML-specific surface markers with broad population coverage has impeded the introduction of CART therapy for myeloid leukemia. A team led by Kalos used CD123-directed CART cells in a humanized mouse model, but their construct unexpectedly [147] eradicated normal hematopoiesis alongside primary AML [148] . While CART123 might find a role as a novel conditioning regimen for AHCT, the extreme potency makes it unsuitable for less intensive AML therapy. By contrast, using a different construct, Mardiros and colleagues found that CD123-specific CART cells led to transient anti-leukemic responses that prolonged survival in their animal model only incrementally [149] . These two groups used different viral vectors incorporating different co-stimulatory domains (4-1BB versus CD28), as well as different expansion techniques (anti-CD3/CD28 beads versus OKT3); any of these differences could potentially have impacted the potency of the resulting CART cells.

Other target antigens have been evaluated. A CD33-directed CART construct was found to ablate normal stem cellsin vitroso further development was not pursued [150] . Because of demonstrated induction of monocytopenia and concern for xenogeneic GVHD due to expression of the target antigen on keratinocytes, a CD44v6-directed CART cell line was transduced with a suicide gene and showed promise in a pre-clinical study [151] . A pilot study of a CART cells directed against the Lewis Y antigen (expressed by 46% of AML samples [152] ) showed limited results. The 3 patients with cytogenetic minimal residual disease (MRD) attained a transient cytogenetic remission, disease stability at 23 months of follow-up, and stability followed by relapse 7 weeks after infusion respectively, while the patient with chemorefractory disease attained only a fleeting reduction in blasts [153] . Because the potency of deliberately non-myeloablative CART cell therapy might be limited by the lack of suitable AML target antigens, CART cells might find a role as adjuvant therapy. The cytokine milieu created by recipient rejection of a donor NEACT product might be particularly conducive to CART cell expansion and effector functionin vivo.

7.1.4. TAA-specific adoptive immunotherapy

As already discussed, the generation of T-cell lines or clones targeting TAA (either through the natural TCR repertoire or through a transgenic TCR) and the peptide vaccines in AML have been developed mostly for use in the context of allogeneic transplantation because the lymphopenic milieu might be permissive toin vivoexpansion of adoptively transferred or native vaccine-induced TAA-specific T cells [85] , yet these strategies have met with limited success [83] . However, the finding that TAA-specific T cells emerge after NEACT could indicate that NEACT fosters the priming and expansion of leukemia-reactive T cells. Whether this could be improved by vaccination after NEACT is an open question [154] . An interesting possibility would be to expand TAA-reactive cells after NEACT for subsequent adoptive immunotherapy of TAA-reactive T-cell lines/clones[155] and [156].

7.2. Donor optimization

On the donor side,ex vivomanipulation of the cell product could be attempted using checkpoint inhibitors as discussed above or co-stimulatory pathway agonists. The desirability of sex and ABO mismatching, as well as the choice of mother or father, sibling, offspring, or unrelated volunteer, should be investigated [157] . For instance, parous donor immunity to paternal antigens been hypothesized to reduce the risk of relapse if recipients share those antigens [158] . Genetically-programmed thresholds for triggering cytotoxicity and amplifying cytokine release may also be incorporated into future donor selection algorithms (“strong alloresponders”) [159] .

8. Conclusion

Alloreactivity is a basis for effective AML treatment. NEACT using haploidentical and higher-order mismatched donors has the potential to harness the potency of alloreactivity without entraining GVHD. This approach is still in the early stages of development. Basic questions such as donor selection, the choice of pre-treatment chemotherapy, optimal composition of the cell product, the utility of repeated infusions, and what mechanisms underlie durable responses ought to be addressed in order to generate consistent results. Whether NEACT will become a “blockbuster” treatment or more of a “personalized” therapy, it is a promising platform worthy of careful and creative attention.

 

gr3

Supplemental Fig. 1 Time-line summary of clinical trials of non-engrafting alloreactive cell therapy for cancers other than acute myeloid leukemia, according to year of publication. Patients had metastatic, relapsed, or chemorefractory disease. The numbers treated and the best responses in each trial are displayed. Tumors included renal cell carcinoma (RC), melanoma (mel), multiple myeloma (MM), Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL) and others. The time points are color-coded according to the type of product received: irradiated peripheral blood mononuclear cells (PBMCs, yellow), steady-state leukapheresis product activated withex vivoand/orin vivointerleukin-2 (blue), PBMCs mobilized with granulocyte-colony stimulating factor (green), and purified and enriched NK cell products (orange and mixed orange/blue respectively). Details of each study are available in the Supplemental Table 3.

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NEACT for cancers other than AML.

Conflict of interest statement

The authors declare no potential conflict of interest.

Acknowledgements

The authors are grateful to the members of the Hematology-Oncology service and research coordinators at Hôpital Maisonneuve-Rosemont for helpful discussions. EFK is supported by a post-doctoral training award from the Cole Foundation and JSD by a Junior 1 salary award from the Fonds de recherche du Québec-Santé (FRQS). This work is supported by an early career Transition award from the Cole Foundation and funds from the Fondation de l’Hôpital Maisonneuve-Rosemont held by JSD. The sponsors did not contribute to the manuscript or to the data discussed in this review.

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Footnotes

Department of Medicine, Division of Hematology and Oncology, Hôpital Maisonneuve-Rosemont Research Center, Université de Montréal, 5415 de l’Assomption, Montreal, Quebec, H1T 2M4, Canada

lowast Corresponding author. Tel.: + 1 514 252 3404; fax: + 1 514 252 5904.