Alloreactive NK Cells Research Articles

Comparison of Long-Term Outcomes of Double Unit Cord Blood Versus Haploidentical Donor Transplantation in Adult Patients With Acute Lymphoblastic Leukemia Regarding KIR-Ligand Mismatch

BackgroundAlthough human-leukocyte antigen-matched sibling donor or matched unrelated donor is preferred as a conventional donor, 30%-50% of recipients lack matched sibling donor or matched unrelated donor. In such cases, haploidentical donor or cord blood are commonly considered as alternative donor sources. In addition to donor source, NK cell alloreactivity due to human-leukocyte antigen-mismatch setting is another important factor that may affect outcomes in alternative-donor HCT, possibly through graft-versus-leukemia effect. However, a limited number of studies have evaluated the impacts of these factors, focusing on adult acute lymphoblastic leukemia (ALL). ObjectivesThe objective of this study is to assess the effects of donor source and killer cell immunoglobulin-like receptor ligand mismatch (KIRL-MM) on transplantation outcomes of alternative-donor HCT, with a special focus on adult ALL. Study DesignWe retrospectively compared clinical outcomes between haploidentical donor transplantation ((HIDT), n = 47) and double unit cord blood transplantation ((DCBT), n = 134) in adult patients with ALL. Patients received fludarabine and busulfan-based reduced toxicity conditioning with antithymocyte globulin before HIDT and TBI-based myeloablative conditioning before DCBT. KIR ligands of recipients and donors were determined using a web-based calculator, and KIRL-MM was evaluated based on the KIR ligand missing. For DCBT, donor KIR ligand groups were defined by using the dominant CB unit after engraftment. ResultsCumulative incidences of acute and chronic graft-versus-host disease (GVHD) were similar between HIDT and DCBT, regardless of severity grades. After median follow-up duration of 39.4 months, DCBT showed higher 3-year nonrelapse mortality (NRM) than HIDT (22.8% vs. 9.0%; P = .038), whereas the cumulative incidence of relapse (CIR) was significantly higher in HIDT than DCBT (47.9% vs. 18.9%; P < .001). Estimated 3-year disease-free survival (DFS) was comparable (DCBT 58.5% vs. HIDT 44.3%; P = .106). GVH direction KIRL-MM showed lower incidence of acute GVHD in both HIDT and DCBT. However, GVH direction KIRL-MM was associated with poorer 3-year DFS (37.2% vs. 66.0%; P = .008) only in the DCBT subgroup, mostly due to specifically higher NRM rate (35.0% vs. 18.4%; P = 0.057). ConclusionOur study continues to support the usefulness of DCBT in the HIDT-dominant era and suggests potential ways to improve survival outcomes of DCBT.

Harnessing Immune Response in Acute Myeloid Leukemia.

Despite the results achieved with the evolution of conventional chemotherapy and the inclusion of targeted therapies in the treatment of acute myeloid leukemia (AML), survival is still not satisfying, in particular in the setting of relapsed/refractory (R/R) disease or elderly/unfit patients. Among the most innovative therapeutic options, cellular therapy has shown great results in different hematological malignancies such as acute lymphoblastic leukemia and lymphomas, with several products already approved for clinical use. However, despite the great interest in also expanding the application of these new treatments to R/R AML, no product has been approved yet for clinical application. Furthermore, cellular therapy could indeed represent a powerful tool and an appealing alternative to allogeneic hematopoietic stem cell transplantation for ineligible patients. In this review, we aim to provide an overview of the most recent clinical research exploring the effectiveness of cellular therapy in AML, moving from consolidated approaches such as post- transplant donor's lymphocytes infusion, to modern adoptive immunotherapies such as alloreactive NK cell infusions, engineered T and NK cells (CAR-T, CAR-NK) and novel platforms of T and NK cells engaging (i.e., BiTEs, DARTs and ANKETTM).

Comparison of NK alloreactivity prediction models based on KIR-MHC interactions in haematopoietic stem cell transplantation.

The biological processes underlying NK cell alloreactivity in haematopoietic stem cell transplantation (HSCT) remain unclear. Many different models to predict NK alloreactivity through KIR and MHC genotyping exist, raising ambiguities in its utility and application for clinicians. We assessed 27 predictive models, broadly divided into six categories of alloreactivity prediction: ligand-ligand, receptor-ligand, educational, KIR haplotype-based, KIR matching and KIR allelic polymorphism. The models were applied to 78 NGS-typed donor/recipient pairs undergoing allogeneic HSCT in genoidentical (n=43) or haploidentical (n=35) matchings. Correlations between different predictive models differed widely, suggesting that the choice of the model in predicting NK alloreactivity matters. For example, two broadly used models, educational and receptor-ligand, led to opposing predictions especially in the genoidentical cohort. Correlations also depended on the matching fashion, suggesting that this parameter should also be taken into account in the choice of the scoring strategy. The number of centromeric B-motifs was the only model strongly correlated with the incidence of acute graft-versus-host disease in our set of patients in both the genoidentical and the haploidentical cohorts, suggesting that KIR-based alloreactivity, not MHC mismatches, are responsible for it. To our best knowledge, this paper is the first to experimentally compare NK alloreactivity prediction models within a cohort of genoidentical and haploidentical donor-recipient pairs. This study helps to resolve current discrepancies in KIR-based alloreactivity predictions and highlights the need for deeper consideration of the models used in clinical studies as well as in medical practice.

Association of iKIR-mismatch model and donor aKIRs with better outcome in haploidentical hematopoietic stem cell transplantation for acute myeloid leukemia.

Killer cell immunoglobulin like receptor (KIR) can trigger the alloreactivity of NK cells. However, there is no clear consensus as to their function. Here, we investigated the potential influence of KIR mismatch and KIR alleles on the outcome of haploidentical hematopoietic stem cell transplantation (haplo-HSCT) in acute myeloid leukemia (AML) patients. Data from 79 AML patients treated with haplo-HSCT were retrospectively analyzed. HLA-C genotyping was determined by the PCR-rSSO method. KIR, HLA-A and HLA-B genotyping was performed by the PCR-SSP method. Cox proportional hazards model and Kaplan-Meier survival curves were used for analysis. Both KIR ligand mismatch (KLM) group and KIR receptor-ligand mismatch (RLM) group were associated with a decreased risk in aGVHD and relapse rate (RR), and better overall survival (OS) compared to the KIR ligand matching and receptor-ligand matching groups, respectively (aGVHD: KLM: p=0.047, HR:0.235; RLM: p<0.001, HR:0.129; RR: KLM: p=0.049, HR:0.686, RLM: p=0.017, HR:0.200;OS:KLM: p=0.012, HR: 0.298, RLM: p=0.021, HR:0.301). RLM was more accurate at predicting relapse and aGVHD compared with KLM (aGVHD: p=0.009; RR: p=0.039). Patients with greater number of donor activating KIRs (aKIR) had a lower incidence of aGVHD and relapse, and the benefits correlated with the increase in the number of donor aKIRs (aGVHD: p=0.019, HR:0.156; RR: p=0.037, HR:0.211). Patients with RLM and the highest number of donor aKIRs had the lowest RR, lowest incidence of aGVHD and best OS. Both KLM and RLM reduced the risk of aGVHD and relapse after haplo-HSCT in AML patients, and RLM showed superiority in predicting HSCT outcome. The synergistic effects of RLM and donor aKIRs can provide a better donor selection strategy to improve haplo-HSCT outcome in AML patients.

Impact of natural killer cell’s functional reconstruction on the results of allogeneic hematopoietic stem cell transplantation

Introduction.Currently, more and more attention is being paid to possible strategies for preventing the development of graft-versus-host disease (GVHD) and reducing the risk of infections while maintaining the antitumor effect — graft-versus-leukemia effect (GVL). In this context, the study of natural killer cells (NK-cells) seems to be quite promising.Aim– to analyze the biological and functional properties of NK-cells after allo-HSCT, their reconstitution after transplantation and factors affecting this process, as well as the mechanisms of alloreactivity of NK cells in patients after allo-HSCT. Main findings. Various types of activating or inhibiting receptors, which are expressed on NK-cells, regulate the functions of NK-cells. Among them, the main role is played by the killer immunoglobin-like receptor (KIR-receptor), which mediates tolerance to one’s own cells and the immune response, both antitumor and directed against infectious agents. NK-cells can play a decisive role in preventing early relapses and infectious complications, as they are among the first to recover after allo-HSCT. They also have the ability to eliminate the recipient’s T-cells and antigen presenting cells (APCs), thereby preventing the development of graft failure and GVHD. There are several models of NK alloreactivity based on KIR; however, the results of studies in this area are contradictory. This review summarizes the available literature data.

Altered distribution and function of NK-cell subsets lead to impaired tumor surveillance in JAK2V617F myeloproliferative neoplasms.

In cancer, tumor cells and their neoplastic microenvironment can sculpt the immunogenic phenotype of a developing tumor. In this context, natural killer (NK) cells are subtypes of lymphocytes of the innate immune system recognized for their potential to eliminate neoplastic cells, not only through direct cytolytic activity but also by favoring the development of an adaptive antitumor immune response. Even though the protective effect against leukemia due to NK-cell alloreactivity mediated by the absence of the KIR-ligand has already been shown, and some data on the role of NK cells in myeloproliferative neoplasms (MPN) has been explored, their mechanisms of immune escape have not been fully investigated. It is still unclear whether NK cells can affect the biology of BCR-ABL1-negative MPN and which mechanisms are involved in the control of leukemic stem cell expansion. Aiming to investigate the potential contribution of NK cells to the pathogenesis of MPN, we characterized the frequency, receptor expression, maturation profile, and function of NK cells from a conditional Jak2V617F murine transgenic model, which faithfully resembles the main clinical and laboratory characteristics of human polycythemia vera, and MPN patients. Immunophenotypic analysis was performed to characterize NK frequency, their subtypes, and receptor expression in both mutated and wild-type samples. We observed a higher frequency of total NK cells in JAK2V617F mutated MPN and a maturation arrest that resulted in low-numbered mature CD11b+ NK cells and increased immature secretory CD27+ cells in both human and murine mutated samples. In agreement, inhibitory receptors were more expressed in MPN. NK cells from Jak2V617F mice presented a lower potential for proliferation and activation than wild-type NK cells. Colonies generated by murine hematopoietic stem cells (HSC) after mutated or wild-type NK co-culture exposure demonstrated that NK cells from Jak2V617F mice were deficient in regulating differentiation and clonogenic capacity. In conclusion, our findings suggest that NK cells have an immature profile with deficient cytotoxicity that may lead to impaired tumor surveillance in MPN. These data provide a new perspective on the behavior of NK cells in the context of myeloid malignancies and can contribute to the development of new therapeutic strategies, targeting onco-inflammatory pathways that can potentially control transformed HSCs.

Lineage-Specific Relapse Prediction After Haploidentical Transplantation With Post-Transplant Cyclophosphamide Based on Recipient HLA-B-Leader Genotype and HLA-C-Group KIR Ligand

The role of NK cell alloreactivity on outcomes after T cell-replete haploidentical donor transplantation (HIDT) remains uncertain. After transplantation, newly formed NK cells are licensed through interactions of donor inhibitory KIR (iKIR) and NKG2A receptors with their cognate ligands on recipient cells. Donor NKG2A recognizes HLA-E bound by recipient HLA class I leader peptides, a process requiring methionine (M) at position -21 of the leader sequence. An rs1050458C/T dimorphism results in approximately 40% of individuals expressing at least one copy of -21M HLA-B (M/M or M/T [M+]), allowing ligand expression. We assessed the impact of recipient HLA-B-leader genotype (M+ versus M- [T/T]) and HLA-C-group iKIR missing ligand (ML, C1C1/C2C2 versus C1C2) on relapse and disease-free survival (DFS) in recipients of post-transplantation cyclophosphamide (PTCy)-based HIDT. Based on preclinical data, we hypothesized that the relative impact of each variable may depend on disease lineage (lymphoid versus myeloid). To this end, we analyzed outcomes of 322 consecutive PTCy-based HIDT recipients with hematologic malignancy who underwent transplantation at a single institution using standardized supportive care measures with mature follow-up (median 45 months). Primary endpoints were relapse and DFS of patients based on HLA-B-leader genotype and HLA-C-group iKIR ML. Planned subgroup analysis included patient with lymphoid versus myeloid malignancy. M+ HLA-B-leader genotype and HLA-C-group iKIR ML were seen in 42% and 49% of recipients, respectively. The presence of a recipient M+ B-leader (versus M-) improved overall survival (OS) and DFS and lowered cumulative incidence of relapse (CIR), an effect primarily seen in lymphoid malignancies (80% versus 51%, 72% versus 41%, 16% versus 42%, respectively). In contrast, myeloid malignancy patients benefited most from HLA-C-group iKIR ML with better OS and DFS and lower CIR (67% versus 51%, 64% versus 44%, 25% versus 45%, respectively). Multivariate analysis confirmed the disease-specific associations of improved relapse/DFS with M+ HLA-B-leader in lymphoid malignancy (hazard ratio [HR] 0.20, P < .001/HR 0.34, P <.001) and HLA-C-group iKIR ML in myeloid malignancy (HR 0.44, P=.004/HR 0.54, P=.009). Neither HLA-B-leader nor iKIR ML was associated with the incidence of non-relapse mortality or acute or chronic graft-versus-host disease. Two distinct NK cell education pathways predict relapse and DFS after HIDT-PTCy in a disease-specific manner: the presence of recipient M+ HLA-B-leader genotype improves outcome in patients with lymphoid malignancies, whereas HLA-C-group iKIR ML improves outcome in patients with myeloid malignancies. These findings strengthen the essential role of NK cells for optimal GVL in the context of HIDT-PTCy and may suggest different approaches to improving transplant outcome depending on disease type.

24 - Disease-Specific Pathways for NK Cell Alloreactivity Informs Relapse Risk after Haploidentical Donor Transplantation with Post-Transplant Cyclophosphamide: Relative Effect of Recipient HLA-C-Group KIR Ligand and HLA-B-Leader Allotype

24 - Disease-Specific Pathways for NK Cell Alloreactivity Informs Relapse Risk after Haploidentical Donor Transplantation with Post-Transplant Cyclophosphamide: Relative Effect of Recipient HLA-C-Group KIR Ligand and HLA-B-Leader Allotype

Exploiting Natural Killer Cell Engagers to Control Pediatric B-cell Precursor Acute Lymphoblastic Leukemia.

Natural killer (NK) cells represent a promising cell type in antitumor immunotherapy for efficacy and safety, particularly in the treatment of hematologic malignancies. NK cells have been shown to exert antileukemia activity in the context of haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Products have been developed to boost the activation of NK cells only when cross-linked by tumor cells, avoiding any off-target effect. Here, we tested the in vitro effect of different NK-cell engagers (NKCE), which trigger either NKp46 or NKp30 together with CD16A, and target either CD19 or CD20 to induce killing of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Target cells were NALM-16 and MHH-CALL-4 cell lines and four primary leukemias, while effector cells were resting NK cells derived from healthy donors and pediatric patients with leukemia after αβT/B-depleted haplo-HSCT. The NK cell-resistant MHH-CALL-4 was efficiently killed using all NKCEs. Boosting of NK activity against MHH-CALL-4 was also evident by degranulation and IFNγ production. Because of the lack of CD20 and high expression of CD19 on primary BCP-ALL, we focused on NKCEs targeting CD19. NKp46- and NKp30-based NKCEs displayed similar potency at inducing NK-cell activity, even when challenged with primary BCP-ALL blasts. Their efficacy was shown also using NK cells derived from transplanted patients. NKCE-induced activation against BCP-ALL can override HLA-specific inhibitory interactions, although the strongest response was observed by the alloreactive NK-cell subset. These data support the therapeutic use of NKp46/CD16A/CD19-NKCE to fight refractory/relapsed leukemia in pretransplantation or posttransplantation settings.

KIR in Allogeneic Hematopoietic Stem Cell Transplantation: Need for a Unified Paradigm for Donor Selection.

Allogeneic hematopoietic stem cell transplantation (aHSCT) is a lifesaving therapy for hematological malignancies. For years, a fully matched HLA donor was a requisite for the procedure. However, new immunosuppressive strategies have enabled the recruitment of viable alternative donors, particularly haploidentical donors. Over 95% of patients have at least two potential haploidentical donors available to them. To identify the best haploidentical donor, the assessment of new immunogenetic criteria could help. To this end, the clinical benefit of KIR genotyping in aHSCT has been widely studied but remains contentious. This review aims to evaluate the importance of KIR-driven NK cell alloreactivity in the context of aHSCT and explain potential reasons for the discrepancies in the literature. Here, through a non-systematic review, we highlight how the studies in this field and their respective predictive models or scoring strategies could be conceptually opposed, explaining why the role of NK cells remains unclear in aHCST outcomes. We evaluate the limitations of each published prediction model and describe how every scoring strategy to date only partly delivers the requirements for optimally effective NK cells in aHSCT. Finally, we propose approaches toward finding the optimal use of KIR genotyping in aHSCT for a unified criterion for donor selection.

A Randomized Phase II Trial Comparing a Calcineurin Inhibitor-Free Graft-Versus-Host Disease Prophylaxis Regimen with Post-Transplantation Cyclophosphamide and Abatacept to Standard of Care

Background: Graft versus host disease (GVHD) is one the major causes of mortality and morbidity after an allogeneic stem cell transplantation. We hypothesized that we can induce post-transplant tolerance by using the combination of post-transplant cyclophosphamide (PTCy) and abatacept (CTLA4Ig) for GVHD prophylaxis. PTCy when given on Days +3 and +4 can eliminate host-reactive donor T cells. CTLA4Ig blocks the costimulatory signals given through CD28 to naïve donor T cells thus favoring an anergic phenotype that promotes tolerance towards recipient derived antigens. CTLA4Ig gives an activating signal to NK cells and therefore has the ability to preserve the graft-versus-tumor effect.Methods: We have initiated a 50 patient randomized clinical trial. Patients with hematologic malignancies in need of a transplant and with an 8/8 matched donor are randomized 1:1 to tacrolimus and methotrexate for GVHD prophylaxis (standard of care arm) or PTCy on days +3 and +4 followed by CTLA4Ig on days +5, +14,+28, +56, +84, +112, +140 and +168. Patients are stratified by conditioning regimen (myeloablative vs reduced intensity) and by donor type (matched sibling vs matched unrelated donor). The primary endpoint is chronic GVHD at 1 year as a marker of tolerance induction. Secondary endpoints include acute GVHD rate, relapse rate, overall survival, GVHD-relapse-free-survival, transplant related mortality and infection rate. Post-transplantation immune reconstitution studies include measuring T cell and NK cell phenotype, PD-1 expression, and alloreactivity to recipient and third party at predetermined time points.Results: 25 patients have been treated on this study, 10 of which are on the experimental arm. Patients on the experimental arm have been followed for up to 516 days post-transplant. So far no cases of chronic GVHD or grade 3-4 acute GVHD have been observed in the experimental arm. All the patients have engrafted and there have been no treatment related deaths.Conclusions: The combination of PTCy and CTLA4Ig for GVHD prophylaxis is feasible. This ongoing study will examine its ability to induce post-transplant tolerance. DisclosuresTzachanis: Kite, a Gilead Company: Consultancy, Research Funding, Speakers Bureau; Partner: Consultancy; Fate Therapeutics: Research Funding; Genentech: Research Funding; Bristol Myers Squibb: Research Funding; Incyte: Research Funding; EUSA: Consultancy; Takeda: Consultancy, Speakers Bureau; Magenta: Consultancy; Kyowa Kirin: Consultancy. Goodman: Seattle Genetics: Consultancy, Speakers Bureau; EUSA Pharma: Consultancy, Honoraria. Mangan: Elevate Bio: Other: ad board. OffLabel Disclosure:abatacept: using as part of graft versus host disease prophylaxis

ADCC-Inducing Antibody Trastuzumab and Selection of KIR-HLA Ligand Mismatched Donors Enhance the NK Cell Anti-Breast Cancer Response.

Simple SummaryNatural killer (NK) cells are potent killers of tumor cells. Many tumors, including breast cancers, develop mechanisms to suppress anti-tumor immune responses, requiring the development of strategies to overcome suppression. Here, we tested a combination therapy that aims to (1) enhance NK cell activation and (2) reduce NK cell inhibition mediated by suppressive factors in tumors or in the tumor microenvironment. We cultured cell lines under hypoxia to mimic the tumor microenvironment or used patient-derived breast cancer cells that were primed by the patient’s tumor environment. Our results demonstrated that cytokine-activated NK cells remained active under hypoxia and that tumor-targeting antibodies enhanced the NK cell anti-breast cancer response. Moreover, we observed that NK cell suppression by inhibitory ligands on the tumor cells can be reduced by the selection of NK cell donors with NK receptors that are incompatible with these ligands. Collectively, we present two powerful strategies to enhance the NK cell responses against breast cancer.Natural killer (NK)-cell-based immunotherapies are an attractive treatment option for cancer. We previously showed that alloreactive mouse NK cells cured mice of 4T1 breast cancer. However, the tumor microenvironment can inhibit immune responses, and these suppressive factors must be overcome to unfold the NK cells’ full anti-tumor potential. Here, we investigated the combination of antibody-dependent cellular cytotoxicity (ADDC) and the selection of KIR-HLA-ligand mismatched NK cells to enhance NK cell anti-breast cancer responses in clinically relevant settings. Donor-derived and IL-2-activated NK cells were co-cultured with patient-derived breast cancer cells or cell lines MCF7 or SKBR3 together with the anti-HER2 antibody trastuzumab. NK cells mediated anti-breast cancer cytotoxicity under normoxic and hypoxic conditions. Under both conditions, trastuzumab vigorously enhanced NK cell degranulation (CD107a) against HER2-overexpressing SKBR3 cells, but we observed a discrepancy between highly degranulating NK cells and a rather modest increase in cytotoxicity of SKBR3. Against patient-derived breast cancer cells, the anti-tumor efficacy was rather limited, and HLA class I expression seemed to contribute to inhibited NK cell functionality. KIR-ligand-mismatched NK cells degranulated stronger compared to the matched NK cells, further highlighting the role of HLA. In summary, trastuzumab and KIR-ligand-mismatched NK cells could be two strategies to potently enhance NK cell responses to breast cancer.

NK-Cell Alloreactivity Is Associated with Acute Gvhd and Decreased Relapse Incidence after T-Replete Haplo-Identical Allotransplant with High-Dose Post-Transplant Cyclophosphamide

Background: NK cells express activating (act) and inhibitory (inh) receptors, which recognize MHC class-I alleles, termed “Killer cell Immunoglobulin-like Receptors” (KIRs). Previous clinical data from haplo-identical T-cell-depleted allotransplant have demonstrated that donor vs recipient NK cell alloreactivity (or KIR/HLA incompatibility, KIR/HLA inc) plays a major role as effectors against acute myeloid leukemia (AML). Recently, T-replete haplo-transplant using high-dose post-transplant cyclophosphamide (PTCY) has been a revolution in the field as it is associated with good rate of engraftment and very low incidence of severe acute and chronic GVHD.Methods: The main objective of this prospective study was to study the impact of NK alloreactivity after T-replete haplo-transplant using PTCY. The Baltimore conditioning regimen (Luznik, 2008) was used for lymphoid malignancies while fludarabine as part of this regimen was replaced by clofarabine (Clo-Baltimore) for patients with myeloid malignancies.KIR/HLA inc were studied between donor and recipient considering various act and inh KIR receptors. We then studied the correlation between the presence of KIR/HLA inc and inh KIR/HLA inc and the occurrence of GVHD and relapse post-transplant. Finally, we compared reconstitution of various NK cell subsets (KIR2DL3, KIR2DL1, NKp46+2B4-, NKp46-2B4+, NKG2A+) between cases with or without acute GVHD, KIR/HLA inc, and between Baltimore vs Clo-Baltimore patients. For this purpose, peripheral blood samples were collected at days+10, +20, +30, +60 and +90/100 post-transplant. All patients gave informed consent.Results: Between August 2014 and March 2017, 34 patients (males n=23, median age: 62.5 years) were included in the study. Twelve cases received a Baltimore regimen vs 22 a Clo-Baltimore regimen. All patients except one received peripheral blood stem cell as source of graft. 56% and 14.7% of patients developed grade 2-4 and 3-4 acute GVHD, respectively, at a median of 35 days post-transplant. In July 2017, 9 patients had relapsed and 10 are dead. No differences were observed in terms of acute GVHD incidence, relapse or deaths between Baltimore and Clo-Baltimore sub-groups.KIR/HLA inc was documented in 17 cases of whom 13 presented with acute grade 2-4 while it was only 6/17 cases among patients with no KIR/HLA inc (p=0.001). The same was observed when considering only inhKIR/HLA inc (n=11/13 vs n=8/21, p=0.006). Conversely, relapse was less frequent in patients with KIR/HLA inc (n=2/17 vs n=7/17, p=0.05). However, incidence of relapse was similar between patients developing GvHD (n 5/19) vs no (n=4/15), suggesting that decreased incidence of relapse was not due to GVHD but was related to KIR/HLA inc.The proportions of NK cells at each kinetic point considered after transplant were similar regardless of GVHD, KIR/HLA inc, or conditioning. Phenotypic analyses of NK cell subsets showed that patients developing GVHD had higher mature/activated NKp46+2B4- and NKG2A+NK cells at day +20 post-transplant. The proportion of KIR2DL3+ NK cells was also significantly higher in absence of KIR/HLA inc at day+30, in contrast to KIR2DL1+ NK cells that were similar between each sub-groups at any time considered.Conclusion: KIR/HLA inc are associated with acute GVHD and decreased relapse incidence after T-repleted haplo-identical allotransplantation using PTCY. NKp46+2B4- and NKG2A+NK cells were significantly higher in cases developing GVHD at day+20, suggesting a role of these activated NK cells in the occurrence of GVHD. Also, KIR/HLA inc impact the phenotypic NK cell reconstitution and may favor NK cell expansion with better leukemic effect. These results may be of crucial importance regarding the selection of haplo-donor. DisclosuresNo relevant conflicts of interest to declare.

Natural killer cell alloreactivity in HLA-haploidentical hematopoietic transplantation: a study on behalf of the CTIWP of the EBMT.

Human leukocyte antigen (HLA) class-I mismatches that trigger donor-versus-recipient natural killer (NK)-cell alloreactivity reduce the incidence of leukemia relapse and improve survival of acute myeloid leukemia patients after T-cell-depleted HLA-haplotype mismatched ("haploidentical") hematopoietic transplantation. In murine graft-versus-host disease (GvHD) models, alloreactive NK-cells also prevent GvHD. Here we report the results of a non-interventional, prospective study performed on behalf of the Cellular Therapy and Immunobiology Working Party of the European Society for Blood and Marrow Transplantation. The study was aimed at re-assessing the role of NK-cell alloreactivity in a cohort of haploidentical transplants performed in Europe between 2012 and 2015 and composed of unmanipulated, as well as T-cell-depleted transplants. One hundred thirty-eight patients with acute myeloid or lymphoid leukemias were analyzed. Eighty-six patients received ex-vivo T-cell-depleted transplants, 52 patients received unmanipulated transplants. Fifty patients were transplanted from NK alloreactive donors, 88 from non-NK alloreactive donors. NK cell alloreactivity did not impact on GvHD/relapse-free survival (GRFS) in unmanipulated transplants (HR: 1.66 (0.9-3.1), p = 0.1). In contrast, it did impact beneficially on GRFS in T-cell-depleted transplants (HR: 0.6, (0.3-1.2), p = 0.14, interaction p < 0.001). This effect was the consequence of reduced incidences of acute and chronic GvHD and non-relapse mortality.

531 - BMT CTN 1803: Haploidentical Natural Killer Cells (K-NK002) to Prevent Post-Transplant Relapse in AML and MDS (NK-REALM)

Background and Rationale: Relapse remains the leading cause of treatment failure for patients with high-risk acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) undergoing allogeneic bone marrow transplantation (BMT). Although the risk depends on parameters such as age and conditioning intensity, relapse is experienced by 30-50% of patients after conventional BMT in high-risk AML/MDS. Initial safety and post-BMT relapse risk reduction results were reported from a phase I study of haploidentical (haplo) donor-derived natural killer (NK) cells expanded ex vivo on feeder cells expressing IL-21 and 4-1BBL (FC21-NK) in conjunction with haploBMT. Of 13 patients with high-risk myeloid malignancies treated with FC21-NK cells, no infusion reactions or dose-limiting toxicities occurred and only 1 patient, treated at the lowest dose of 1×105 cells/kg, relapsed (Ciurea, Blood, 2017). This experience supports investigation of K-NK002, a product derived from haploidentical donor NK cells and expanded ex vivo in a feeder cell-free system using plasma membrane (PM21) particles bearing membrane-bound IL-21 and 4-1BBL. We used contemporary data from the Center for International Blood and Marrow Transplant Research registry to determine baseline relapse rates that informed the statistical design. K-NK002 will be given in the peri-transplant period to test the hypothesis that haploidentical NK cells can mediate an effective anti-leukemia response. Trial Design and Methods: BMT CTN 1803 is a phase II, single-arm, open-label, multicenter trial designed to investigate the safety and efficacy of K-NK002 for the treatment of patients with high-risk AML or MDS undergoing haploBMT (NCT04395092). An initial 6 patient safety run-in phase will precede enrollment into the full study of approximately 60 patients. Major inclusion criteria of patients and donors are listed in Table 1. Production of the haploidentical donor NK-cells is completed prior to the planned haploBMT. BMT conditioning will consist of 140 mg/m2 (100 mg/m2 for patients ≥60 years old) melphalan on Day -7; 40 mg/m2 fludarabine on Days -7, -6, -5, and -4; and 2 Gy of total body irradiation on Day -3. Donor bone marrow will be harvested and given on Day 0. Three doses of K-NK002 (1×108 NK cells/kg) will be administered IV on Days -2, +7, and +28, relative to the haploBMT. Graft-versus-host disease (GVHD) prophylaxis consists of post-transplantation cyclophosphamide with tacrolimus and mycophenolate mofetil. The primary endpoint is cumulative incidence of relapse at 1 year post haploBMT in patients receiving at least 1 infusion of K-NK002. Secondary endpoints are safety and tolerability of K-NK002; overall survival; non-relapse mortality; relapse-free survival; GVHD-free survival; cumulative incidence of acute GVHD and chronic GVHD; hematologic recovery; donor-cell engraftment; primary and secondary graft failure; overall incidence of toxicity; and cumulative incidence of infections including cytomegalovirus re-activation and symptomatic BK virus hemorrhagic cystitis. Exploratory endpoints are systemic immunosuppression-free survival; immune reconstitution at Days 28, 100, and 365 post haploBMT; proportion of patients with detectable minimal residual disease at Days 28 and 100 post haploBMT; feasibility of administering the planned K-NK002 doses; and impact of NK-cell alloreactivity on relapse and survival. Download : Download high-res image (319KB) Download : Download full-size image Disclosures Bejanyan: Kiadis Pharma: Membership on an entity’s Board of Directors or advisory committees. Devine: Magenta Therapeutics: Consultancy. Luznik: WindMil Therapeutics: Patents & Royalties: Patent holder; Genentech: Research Funding; Merck: Research Funding, Speakers Bureau; AbbVie: Consultancy. Sandler: Kiadis Pharma: Current Employment. Krakow: HighPass Bio: Research Funding. Fitzgerald: Kiadis Pharma: Current Employment. Tracey: Kiadis Pharma: Current Employment. Champlin: DKMS America: Membership on an entity’s Board of Directors or advisory committees; Takeda: Patents & Royalties; Actinium: Consultancy; Johnson and Johnson: Consultancy; Cytonus: Consultancy; Omeros: Consultancy; Genzyme: Speakers Bureau.

Increased Inhibitory KIR-Ligand Interactions Confer Relapse Protection Following HLA Matched Unrelated Donor HCT for AML

Killer immunoglobulin-like receptor (KIR) and KIR-ligand (KIRL) interactions play an important role in natural killer cell-mediated graft versus leukemia effect (GVL) after hematopoietic stem cell transplant (HCT) for AML. There is considerable heterogeneity in the KIR gene and KIRL content of individuals, making it difficult to estimate the full clinical impact of NK cell alloreactivity following HCT. Herein, we validate a mathematical model accounting for KIR-KIRL interactions identifying donors with optimal NK cell-mediated alloreactivity and GVL. This retrospective study was performed on de-identified donor and recipient demographic and clinical outcomes data provided by the Center for International Blood and Marrow Transplant Research (CIBMTR). Donor recipient pairs (DRP) who underwent unrelated donor (URD) HCT for early and intermediate AML were included. KIR-KIRL interaction values were assigned as follows; if an inhibitory KIR (iKIR) on the NK cell encounters a ligand on its target, this will give the NK cell an inhibitory signal and this is scored as a single interaction(Figure 1b), as is the case, if there is no ligand for an inhibitory KIR, i.e., missing KIRL (mKIRL) (Figure 1c). Finally, activating KIR (aKIR) interacting with its ligands is similarly scored(Figure 1a). The absolute values of the iKIR and mKIR scores were summed to calculate the composite inhibitory-missing ligand (IM)-KIR score (Figure 1d). The study cohort was comprised of 2365 donor-recipient pairs (DRP) who underwent URD HCT for early or intermediate AML. Mean age was 53 years; 85% of DRPs were high-resolution 8/8 HLA-matched for HLA-A, -B, -C, and -DRB1. All patients received T cell replete grafts; 42% (n=996) received in vivo T cell depletion, 937 (94%) with anti-thymocyte globulin (ATG); 86% received a graft of mobilized peripheral blood stem cells (PBSC), 59% received myeloablative conditioning. This cohort was primarily of Caucasian descent (89%). When adjusted for recipient age, donor age, CMV status, KPS, GVHD prophylaxis, cytogenetics, disease status, conditioning regimen, in vivo T cell depletion, graft source, and sex match, relapse risk was significantly reduced in donor-recipient pairs (DRP) with higher inhibitory KIR-KIRL interaction and missing KIRL (mKIR) scores, with HR=0.86 (P=0.01) &amp; HR=0.84 (P=0.02) respectively. This effect was not observed with activating KIR-KIRL interactions. Chronic GVHD and TRM were not significantly affected by iKIR, mKIR or aKIR. Given the significant individual impact of iKIR and mKIR, the summed inhibitory-missing ligand (IM-KIR) score was next assessed, and when this score was 5 (as opposed to &amp;lt;5), the IM-KIR score was also associated with lower relapse risk, HR 0.8 (P=0.004) (Figure 2a). Acute and chronic graft vs. host disease (GVHD), survival, GRFS, and relapse-free survival were not significantly different, likely due to increased TRM among these patients, HR 1.32 (P=0.01). Interaction analysis indicated that amongst the HLA matched DRP, ATG recipients with IM-KIR=5, had a lower relapse rate compared to those with an IM-KIR&amp;lt;5, HR 0.61 (p=0.001) (Figure 2b), among the cohort who did not receive ATG there was no significant difference in relapse among IM-KIR=5 and IM-KIR&amp;lt;5; thus, the use of ATG significantly modified the effect of IM KIR score in an interaction analysis (p=0.049), suggesting higher NK cell magnitude of KIR-KIRL interaction may compensate for the general increase of relapse in those who receive in vivo T cell depletion. Nevertheless, TRM was also increased in these patients, HR 1.49 (p=0.034), likely abrogating survival advantage from a lower relapse risk. This large international study confirms that unrelated DRPs with greater magnitude of inhibitory KIR-KIRL interactions confer significant relapse protection after MUD HCT in standard-risk AML. This challenges the notion that KIR are irrelevant to donor selection. Future clinical trials evaluating donor selection for URD HCT should include this measure to evaluate its value prospectively in uniformly treated patient cohorts, with adequate GVHD and antiviral prophylaxis to mitigate TRM. Disclosures No relevant conflicts of interest to declare.

BMT CTN 1803: Haploidentical Natural Killer Cells (K-NK002) to Prevent Post-Transplant Relapse in AML and MDS (NK-REALM)

Background and Rationale: Relapse remains the leading cause of treatment failure for patients with high-risk acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) undergoing allogeneic bone marrow transplantation (BMT). Although the risk depends on parameters such as age and conditioning intensity, relapse is experienced by 30-50% of patients after conventional BMT in high-risk AML/MDS. Initial safety and post-BMT relapse risk reduction results were reported from a phase I study of haploidentical (haplo) donor-derived natural killer (NK) cells expanded ex vivo on feeder cells expressing IL-21 and 4-1BBL (FC21-NK) in conjunction with haploBMT. Of 13 patients with high-risk myeloid malignancies treated with FC21-NK cells, no infusion reactions or dose-limiting toxicities occurred and only 1 patient, treated at the lowest dose of 1×105 cells/kg, relapsed (Ciurea, Blood, 2017). This experience supports investigation of K-NK002, a product derived from haploidentical donor NK cells and expanded ex vivo in a feeder cell-free system using plasma membrane (PM21) particles bearing membrane-bound IL-21 and 4-1BBL. We used contemporary data from the Center for International Blood and Marrow Transplant Research registry to determine baseline relapse rates that informed the statistical design. K-NK002 will be given in the peri-transplant period to test the hypothesis that haploidentical NK cells can mediate an effective anti-leukemia response. Trial Design and Methods: BMT CTN 1803 is a phase II, single-arm, open-label, multicenter trial designed to investigate the safety and efficacy of K-NK002 for the treatment of patients with high-risk AML or MDS undergoing haploBMT (NCT04395092). An initial 6 patient safety run-in phase will precede enrollment into the full study of approximately 60 patients. Major inclusion criteria of patients and donors are listed in Table 1. Production of the haploidentical donor NK-cells is completed prior to the planned haploBMT. BMT conditioning will consist of 140 mg/m2 (100 mg/m2 for patients ≥60 years old) melphalan on Day -7; 40 mg/m2 fludarabine on Days -7, -6, -5, and -4; and 2 Gy of total body irradiation on Day -3. Donor bone marrow will be harvested and given on Day 0. Three doses of K-NK002 (1×108 NK cells/kg) will be administered IV on Days -2, +7, and +28, relative to the haploBMT. Graft-versus-host disease (GVHD) prophylaxis consists of post-transplantation cyclophosphamide with tacrolimus and mycophenolate mofetil. The primary endpoint is cumulative incidence of relapse at 1 year post haploBMT in patients receiving at least 1 infusion of K-NK002. Secondary endpoints are safety and tolerability of K-NK002; overall survival; non-relapse mortality; relapse-free survival; GVHD-free survival; cumulative incidence of acute GVHD and chronic GVHD; hematologic recovery; donor-cell engraftment; primary and secondary graft failure; overall incidence of toxicity; and cumulative incidence of infections including cytomegalovirus re-activation and symptomatic BK virus hemorrhagic cystitis. Exploratory endpoints are systemic immunosuppression-free survival; immune reconstitution at Days 28, 100, and 365 post haploBMT; proportion of patients with detectable minimal residual disease at Days 28 and 100 post haploBMT; feasibility of administering the planned K-NK002 doses; and impact of NK-cell alloreactivity on relapse and survival. Disclosures Bejanyan: Kiadis Pharma: Membership on an entity's Board of Directors or advisory committees. Devine:Magenta Therapeutics: Consultancy. Luznik:WindMil Therapeutics: Patents &amp; Royalties: Patent holder; Genentech: Research Funding; Merck: Research Funding, Speakers Bureau; AbbVie: Consultancy. Sandler:Kiadis Pharma: Current Employment. Krakow:HighPass Bio: Research Funding. Fitzgerald:Kiadis Pharma: Current Employment. Tracey:Kiadis Pharma: Current Employment. Champlin:DKMS America: Membership on an entity's Board of Directors or advisory committees; Takeda: Patents &amp; Royalties; Actinium: Consultancy; Johnson and Johnson: Consultancy; Cytonus: Consultancy; Omeros: Consultancy; Genzyme: Speakers Bureau.

Haploidentical Mbil-21 Ex Vivo Expanded NK Cells (FC21-NK) for Patients with Multiple Relapsed and Refractory Acute Myeloid Leukemia

Haploidentical Mbil-21 Ex Vivo Expanded NK Cells (FC21-NK) for Patients with Multiple Relapsed and Refractory Acute Myeloid Leukemia

Phenotypic and Functional Characterization of NK Cells in αβT-Cell and B-Cell Depleted Haplo-HSCT to Cure Pediatric Patients with Acute Leukemia.

NK cells can exert remarkable graft-versus-leukemia (GvL) effect in HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Here, we dissected the NK-cell repertoire of 80 pediatric acute leukemia patients previously reported to have an excellent clinical outcome after αβT/B-depleted haplo-HSCT. This graft manipulation strategy allows the co-infusion of mature immune cells, mainly NK and γδT cells, and hematopoietic stem cells (HSCs). To promote NK-cell based antileukemia activity, 36/80 patients were transplanted with an NK alloreactive donor, defined according to the KIR/KIR-Ligand mismatch in the graft-versus-host direction. The analysis of the reconstituted NK-cell repertoire in these patients showed relatively high proportions of mature and functional KIR+NKG2A−CD57+ NK cells, including the alloreactive NK cell subset, one month after HSCT. Thus, the NK cells adoptively transfused with the graft persist as a mature source of effector cells while new NK cells differentiate from the donor HSCs. Notably, the alloreactive NK cell subset was endowed with the highest anti-leukemia activity and its size in the reconstituted repertoire could be influenced by human cytomegalovirus (HCMV) reactivation. While the phenotypic pattern of donor NK cells did not impact on post-transplant HCMV reactivation, in the recipients, HCMV infection/reactivation fostered a more differentiated NK-cell phenotype. In this cohort, no significant correlation between differentiated NK cells and relapse-free survival was observed.

The Role of Natural Killer Cells in the Immune Response in Kidney Transplantation.

Natural killer cells (NK) represent a population of lymphocytes involved in innate immune response. In addition to their role in anti-viral and anti-tumor defense, they also regulate several aspects of the allo-immune response in kidney transplant recipients. Growing evidence suggests a key role of NK cells in the pathogenesis of immune-mediated graft damage in kidney transplantation. Specific NK cell subsets are associated with operational tolerance in kidney transplant patients. On the other side, allo-reactive NK cells are associated with chronic antibody-mediated rejection and graft loss. Moreover, NK cells can prime the adaptive immune system and promote the migration of other immune cells, such as dendritic cells, into the graft leading to an increased allo-immune response and, eventually, to chronic graft rejection. Finally, activated NK cells can infiltrate the transplanted kidney and cause a direct graft damage. Interestingly, immunosuppression can influence NK cell numbers and function, thus causing an increased risk of post-transplant neoplasia or infection. In this review, we will describe how these cells can influence the innate and the adaptive immune response in kidney transplantation and how immunosuppression can modulate NK behavior.