Donor Natural Killer Cells Research Articles

Innate Cell Engager AFM13 Combined with Preactivated and Expanded Cord Blood-Derived NK Cells for Patients with Double Refractory CD30+ Lymphoma

Patients with multiple relapsed or refractory (R/R) Hodgkin lymphoma (HL) and other CD30+ lymphoma have few effective therapies available and targeted adoptive natural killer (NK) cell immunotherapy is an active area of investigation. Transfer of non-targeted NK cells has shown limited clinical benefit as target recognition of cancers by NK cells remains a substantial barrier. The innate cell engager (ICE®) construct, AFM13, is a first-in-class CD30/CD16A bispecific antibody construct that induces specific and selective killing of CD30+ tumor cells by engaging and activating NK cells. As a single agent, AFM13 has limited clinical activity against HL, likely due to the reduced effector function of autologous NK cells in these patients, which provides the rationale for the combination of AFM13 with allogeneic NK cells. Our prior preclinical work identified a promising combination of cord blood (CB)-derived NK cells that were first IL-12/IL-15/IL-18-preactivated followed by expansion (P+E) with engineered K562 feeder cells expressing membrane-bound IL-21, 4-1BB ligand and CD48 ex vivo, and subsequently complexed with AFM13 just prior to infusion (Kerbauy et al, Clin Cancer Res 2021; 27:3744-56). These cytokine-induced memory-like CB-derived NK cells precomplexed with AFM13 (AFM13-NK) presented chimeric antigen receptor (CAR)-NK cell-like features and showed an increased in vitro and in vivo antitumor activity compared to either P+E NK or AFM13 alone. Building on this work we wished to clinically study adoptive AFM13-NK cell therapy in pts with R/R CD30+ lymphomas. METHODS: This single-center phase I-II trial (NCT04074746) evaluates the safety and activity of AFM13-NK, followed by AFM13 monotherapy. Patients aged 15-75 years with R/R CD30+ lymphomas refractory to brentuximab vedotin are enrolled. The trial goals are to identify the recommended phase 2 dose (RP2D) of AFM13-NK cells, estimate the overall (ORR) and complete response (CR) rates, event-free (EFS) and overall survival (OS) rates, and studies of NK cell activation and persistence. Each treatment cycle consisted of fludarabine/cyclophosphamide (days −5 to −3), followed by infusion of the AFM13-NK cells, cultured for 14 days as described above (day 0), and three weekly IV infusions of AFM13 (200 mg, days 7, 14 and 21). Two cycles were administered to patients 1-19 and 4 cycles were given starting with patient 20. Response was evaluated on day 28 of each cycle. Patients were enrolled at 3 dose levels: DL1 (106 NK/Kg), DL2 (107NK/Kg) and DL3 (108 NK/Kg). Each patient received the same NK dose in all cycles. RESULTS: As of 7/31/22, 30 pts have been enrolled and treated (Table) at DL1 (N=3), DL2 (N=3) and DL3 (N=24), with 1 (N=1), 2 (N=24), 3 (N=3) or 4 cycles (N=3). All patients had active progressive disease at enrollment and no bridging therapy was given. The cords used for each of the 69 cycles were selected with no consideration for HLA match, which was 0/6 (N=31), 1/6 (N=31), 2/6 (N=5), 3/6 (N=1) and 4/6 (N=1). The product infused consisted of >99% NK cells (expanded >2,700 fold), with only 0.02% T cells. The NK cells were >98% viable and 92% of them were bound to AFM13. The products were infused fresh. There were no infusion-related reactions (IRR) after infusing AFM13-NK and 11 IRR in 182 infusions of AFM13 alone (6%) (1 grade 3, 10 grade 2). We saw no cases of CRS, ICANS or GVHD of any grade. DL3 (108 NK/Kg) was established as the RP2D. The ORR in the entire study is 97% with 63% CRs. All 24 patients treated at the RP2D responded (100% ORR) with 70.8% (17/24) CRs. Five patients had a response consolidated with a SCT. At median follow-up of 8 (range 1-23) months, the EFS and OS rates of all 30 patients are 57% and 83%, respectively. AFM-NK cells were detected in serum from day 1 post infusion and persisted for 3-4 weeks. Donor NK levels followed similar patterns after each cycle, which argues against a sensitization effect in the patient after the first NK infusion. Donor NK cells showed by CyTOF increased binding to AFM13 and expression of activation markers. CONCLUSIONS: This is the first clinical trial to date using an ICE® construct precomplexed with cytokine-induced memory-like CB-NK cells to treat patients with CD30+ R/R HL and NHL. Our preliminary results indicate that this ICE® precomplexed CB-NK cell therapy has an excellent tolerability profile and is highly active in patients with heavily pretreated R/R CD30+ lymphomas and warrants further investigation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Donor natural killer cells trigger production of β-2-microglobulin to enhance post–bone marrow transplant immunity

AbstractAllogeneic hematopoietic transplantation is a powerful treatment for hematologic malignancies. Posttransplant immune incompetence exposes patients to disease relapse and infections. We previously demonstrated that donor alloreactive natural killer (NK) cells ablate recipient hematopoietic targets, including leukemia. Here, in murine models, we show that infusion of donor alloreactive NK cells triggers recipient dendritic cells (DCs) to synthesize β-2-microglobulin (B2M) that elicits the release of c-KIT ligand and interleukin-7 that greatly accelerate posttransplant immune reconstitution. An identical chain of events was reproduced by infusing supernatants of alloreactive NK/DC cocultures. Similarly, human alloreactive NK cells triggered human DCs to synthesize B2M that induced interleukin-7 production by thymic epithelial cells and thereby supported thymocyte cellularity in vitro. Chromatography fractionation of murine and human alloreactive NK/DC coculture supernatants identified a protein with molecular weight and isoelectric point of B2M, and mass spectrometry identified amino acid sequences specific of B2M. Anti-B2M antibody depletion of NK/DC coculture supernatants abrogated their immune-rebuilding effect. B2M knock-out mice were unable to undergo accelerated immune reconstitution, but infusion of (wild-type) NK/DC coculture supernatants restored their ability to undergo accelerated immune reconstitution. Similarly, silencing the B2M gene in human DCs, before coculture with alloreactive NK cells, prevented the increase in thymocyte cellularity in vitro. Finally, human recombinant B2M increased thymocyte cellularity in a thymic epithelial cells/thymocyte culture system. Our studies uncover a novel therapeutic principle for treating posttransplant immune incompetence and suggest that, upon its translation to the clinic, patients may benefit from adoptive transfer of large numbers of cytokine-activated, ex vivo–expanded donor alloreactive NK cells.

P727: NK-CELL IMMUNOEDITING BY MESENCHYMAL STROMAL CELLS IN MDS

Background: Disorders of the immune system contribute to the pathophysiology and progression of myelodysplastic syndrome (MDS). Cytotoxic Natural Killer (NK) lymphocytes have been extensively studied for their anti-leukemic activity. However, whether the dysplastic bone marrow (BM) in MDS can directly imprint NK cells and hence alter immunosurveillance is still controversial. Extensive characterizations of MDS patients uncovered deep perturbations in the phenotype and function of cytotoxic lymphocyte subsets and of the stromal compartment, in particular mesenchymal stromal cells (MSC), linked with disease predisposition, initiation and progression. However, few studies have been undertaken to characterize the interactions established within the MDS immune microenvironment between immune and stromal cells. Aims: Recent studies on MDS BM ecosystem suggest a possible symbiotic relationship between cancer, immune and stromal cells. Our project thus aims to underpin the influence of the pathological MSC from MDS BM over NK cells to create a leukemia-permissive microenvironment allowing disease development and progression. Methods: We performed an extensive in vitro characterization of the NK and MSC interactions by co-culture system. Healthy donor (HD) NK cells were co-cultured either with MSC isolated from HD or MDS patient BM samples and were analyzed by high-parametric flow-cytometry, qRT-PCR, secretome and imaging assays. Results: Analyses of supernatants from MSC/NK co-cultures unveiled altered secretion of soluble factors related to inflammation and BM homing such as IL6, CCL2 and CXCL10 in the pathologic condition compared to the healthy one. MDS MSC did not support efficiently NK-cell viability. Moreover, NK cells cultured with MDS MSC were less efficient at eliminating cancer cells compared to NK cells cultured with HD MSC. Finally, metabolic analyses uncovered an altered NK cell energetic profile dictated by the different MSC that could contribute to their poor survival and reduced cytolytic function. Summary/Conclusion: Altogether, our results suggest that MSC from MDS BM edit NK cells to promote an immunosuppressive environment facilitating MDS escape and progression. Further analyses are ongoing to underpin the pivotal role of immune-microenvironment interactions in the treatment response. We expect this study to identify new biomarkers to forecast disease progression and open an avenue for new combination therapies targeting the tumour microenvironment.

Abstract 6169: Identification of molecules mediating natural killer cell-pancreatic stellate cell interactions

Abstract Pancreatic stellate cells (PSCs) are the main sources of cancer-associated fibroblasts in pancreatic ductal adenocarcinoma (PDAC), which account for at least 50% of PDAC’s total tumor volume and its dense desmoplastic stroma. In recent years, immune-stromal cell interactions have been the subject of research in a variety of diseases. Interestingly, natural killer (NK) cells are relatively understudied in PDAC and may be exploited to identify new treatment opportunities. We have explored how NK cell-PSC interactions are mediated by known NK cell ligands. Human PSC (hPSC, CRC-811) or PDAC (MIA-PaCa-2, PANC-1) cell lines were co-cultured with DiO stained NK cells (NK-92, donor NK cells) for 4 hours, then target cells were assessed for apoptosis by flow cytometry using AnnexinV and Sytox stains. We found that although NK cells lyse both PSCs and PDAC cells, there was more NK cell-mediated lysis against PSCs. Using flow cytometry, we found that PSCs express the well-known MICA/B ligand, which binds the activating NKG2D receptor on NK cells to initiate its cytotoxic activity. In co-culture, neutralizing MICA/B in vitro did not completely inhibit NK cell-mediated lysis of PSCs, suggesting other interactions need to be explored. To identify other molecular determinants, NK cell ligand and receptor expression was evaluated by quantitative PCR in human PSC, PDAC, NK, and other leukocytic (K562) cell lines. Cell surface validation of highly expressed NK cell ligands and receptors were confirmed by flow cytometry and/or Western blot. NK cell ligands that were both highly expressed and localized to the cell surface were: NKG2D ligands—MICA and ULBP2 (both activating), KIR2DS4 (CD158I) ligand—HLA-A (activating), and NKp44 ligands—NKp44L and PCNA (activating and inhibitory, respectively). Their corresponding receptors were also expressed and localized to the cell surface by NK cells. These results highlight novel NK cell-PSC interactions that may mediate immune suppression in PDAC. These findings warrant further investigation. Citation Format: Zoe X. Malchiodi, Allison Fitzgerald, Elena Galvano, Louis M. Weiner. Identification of molecules mediating natural killer cell-pancreatic stellate cell interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6169.

Impact of KIR-ligand mismatch on pediatric T-cell acute lymphoblastic leukemia in unrelated cord blood transplantation

Currently, allogeneic hematopoietic stem cell transplantation (allo-HSCT) is considered to be indicated for children and adolescents with high-risk or relapsed T-cell acute lymphoblastic leukemia (T-ALL); however, the outcomes are unsatisfactory. Killer cell immunoglobulin-like receptors (KIRs) are the main receptors on natural killer (NK) cells that play an important role in the graft-versus-leukemia effect after allo-HSCT. In allo-HSCT, when the recipient lacks a donor KIR-ligand (KIR-ligand mismatch in the graft-versus-host [GVH] direction), donor NK cells will be activated against recipient cells. KIR-ligand mismatch in the GVH direction improves outcomes after unrelated cord blood transplantation (UCBT) with acute myeloid leukemia, but the effect in T-ALL is unclear. We evaluated the impact of KIR-ligand mismatch in the GVH direction on the transplantation outcomes of children and adolescents with T-ALL who received UCBT. We conducted a retrospective study using a nationwide registry of the Japanese Society for Transplantation and Cellular Therapy. Patients diagnosed with T-ALL, aged 0 to 19 years, and who underwent first UCBT between 1999 and 2017 were included. A total of 91 patients were included in this study. In all, 23 (25.3%) percent of patients had KIR-ligand mismatch in the GVH direction. The 5-year leukemia-free survival (LFS) and overall survival (OS) rates after UCBT were 65.8% and 69.6%, respectively. In a multivariate analysis, KIR-ligand mismatch in the GVH direction was associated with a significant reduction in the relapse rate (hazard ratio [HR], 0.19; P = .002), resulting in better LFS (HR, 0.18; P =.010) and OS (HR, 0.26; P = .048) without increasing non-relapse mortality (NRM; HR, 1.90; P = .264). The cumulative incidence of GVH disease (GVHD) did not differ between patients with and without KIR-ligand mismatch (grade II-IV acute GVHD, 39.1% versus 36.8%, P = .648, grade III-IV acute GVHD, 13.0% versus 11.8%, P =.857, and chronic GVHD, 26.1% versus 22.9%, P =.736, respectively). Furthermore, acute and chronic GVHD were not associated with good patient outcomes. Notably, no relapse was observed in patients who received KIR-ligand mismatched UCBT in complete remission. KIR-ligand mismatch in the GVH direction improved LFS and decreased relapse rates without increasing NRM in children and adolescents with T-ALL who received UCBT, which was not mediated by GVHD.

Expansion, persistence, and efficacy of donor memory-like NK cells infused for posttransplant relapse.

BackgroundResponses to conventional donor lymphocyte infusion for postallogeneic hematopoietic cell transplantation (HCT) relapse are typically poor. Natural killer (NK) cell–based therapy is a promising modality to treat post-HCT relapse.MethodsWe initiated this ongoing phase I trial of adoptively transferred cytokine-induced memory-like (CIML) NK cells in patients with myeloid malignancies who relapsed after haploidentical HCT. All patients received a donor-derived NK cell dose of 5 to 10 million cells/kg after lymphodepleting chemotherapy, followed by systemic IL-2 for 7 doses. High-resolution profiling with mass cytometry and single-cell RNA sequencing characterized the expanding and persistent NK cell subpopulations in a longitudinal manner after infusion.ResultsIn the first 6 enrolled patients on the trial, infusion of CIML NK cells led to a rapid 10- to 50-fold in vivo expansion that was sustained over months. The infusion was well tolerated, with fever and pancytopenia as the most common adverse events. Expansion of NK cells was distinct from IL-2 effects on endogenous post-HCT NK cells, and not dependent on CMV viremia. Immunophenotypic and transcriptional profiling revealed a dynamic evolution of the activated CIML NK cell phenotype, superimposed on the natural variation in donor NK cell repertoires.ConclusionGiven their rapid expansion and long-term persistence in an immune-compatible environment, CIML NK cells serve as a promising platform for the treatment of posttransplant relapse of myeloid disease. Further characterization of their unique in vivo biology and interaction with both T cells and tumor targets will lead to improvements in cell-based immunotherapies.Trial RegistrationClinicalTrials.gov NCT04024761.FundingDunkin’ Donuts, NIH/National Cancer Institute, and the Leukemia and Lymphoma Society.

Obesity‐induced and LDL‐mediated NK Cell Function Loss is Accompanied by Proteasome‐dependent induction of Lysophagy in an at‐risk population of African American Women

Obesity and related chronic diseases (i.e. cardiovascular disease (CVD) and cancer) disproportionately affect African Americans (AA), shortening life expectancy and impacting quality of life. Life‐long exposure to adverse social determinants of health (SDoH) have been linked to obesity, warranting intensified research to further elucidate the ‘biology of adversity’ or the biologic consequences of adverse social conditions. Evidence suggests that exposure to chronic psychological and environmental stressors as SDoH leads to immune cell dysfunction, particularly for Natural Killer (NK) cells. Therefore, we examined changes in NK cell function in AA women, a group at high risk of Class III obesity (BMI ≥40 kg/m2). We have previously shown that NK cell degranulation is impaired in AA women with Class III obesity accompanied by a decrease in IFNg and TNFa, a loss in function driven by plasma LDL levels (b=‐0.35, p=0.03). A similar phenotype of dysfunctional NK cells could be achieved by overnight LDL treatment (50mg/dl) of healthy donor NK cells. To further determine the underlying signaling pathways, we employed proteomics of control and LDL treated NK cells. Here we identified 18 statistically significant regulated proteins (p<0.05), and 23 proteins approaching significance (p<0.07). Signaling pathway analysis revealed endolysosomal function, actin cytoskeleton regulation, and regulation of innate immune responses as significant pathways. Several proteins associated with autophagy and ubiquitination were also identified. This is of particular importance as LDL treatment of various cell types has been related to damaging lysosomes and a subsequent potential induction of lysophagy. Therefore, we investigated the impact of LDL on NK cell autophagy through western blot analysis of LC3 and p62 and found that enhanced autophagy is present in LDL treated NK cells. By employing the proteasome inhibitor MG‐132, we also found that the LDL‐induced increase in autophagy appears to be dependent on the activation of the 20S proteasome and subsequent ubiquitination of lysosomal proteins which results in depletion of functional lysosomes through lysophagy and is accompanied by NK cell function loss. In summary, we show a novel pathway potentially explaining obesity‐related NK cell function loss as a result of adverse SDoH in a population at highest risk for obesity‐related diseases like CVD and cancer. In the future, more research is needed to further understand the ‘biology of adversity’ and identifying targets for tailored interventions, ultimately reducing health inequities in chronic disease outcomes.

A Phase I Study to Determine the Maximum Tolerated Dose of ex Vivo Expanded Natural Killer Cells Derived from Unrelated, HLA-Disparate Adult Donors

The administration of allogeneic natural killer (NK) cells following a lymphodepleting chemotherapy regimen is emerging as a well-tolerated therapeutic approach in the management of various malignancies. Contrary to the expected complications of allogeneic T cell therapy, there remains no evidence of graft-versus-host disease (GVHD) mediated by NK cells in numerous clinical trials. On the contrary, preclinical and clinical studies suggest that NK cells do not induce GVHD and in fact may prevent its development following allogeneic hematopoietic cell transplantation (HCT). In this study, we sought to determine the maximum tolerated dose of non-HLA-matched donor NK cells derived from peripheral blood and ex vivo expanded using a novel feeder cell platform. In a single-center Phase I clinical trial using a 3×3 design, 9 subjects each received 2 infusions of NK cells 2 weeks apart following a preparative regimen of cyclophosphamide (60 mg/kg i.v.) and fludarabine (25 mg/m2/day i.v for 5 days). No exogenous cytokines were administered. NK cells were administered at 3 dose levels: 1×107/kg, 2.5×107/kg, and 5×107/kg. Three subjects had myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML), and the other 6 subjects had colorectal carcinoma. Recipients were monitored over a 4-week period for GVHD as well as other adverse events and for persistence of donor NK cells in systemic circulation. Disease assessment was started at 28 days following the first NK cell infusion and continued until postinfusion day 100 or disease progression. In all 9 study subjects, there was no occurrence of GVHD and no dose-limiting toxicities that would warrant cohort expansion at any of the 3 planned cell dose levels. Low-level donor NK cell persistence was observed up to 4 weeks after the first NK cell infusion at all dose levels. The best observed response was a complete response with incomplete platelet recovery in a MDS subject who experienced disease relapse after prior allogeneic HCT. Other responses were stable disease in 1 subject with MDS and 2 subjects with colorectal cancer up to postinfusion day 100. This off-the-shelf, third-party NK cell product can be administered safely without inducing GVHD and exhibits in vivo persistence promoted by preparative lymphodepletion alone. The observed clinical responses could be enhanced by administration of exogenous cytokine support, as well as complementary approaches that promote NK cell function in the tumor microenvironment.

Systemic IL-15 promotes allogeneic cell rejection in patients treated with natural killer cell adoptive therapy

Natural killer (NK) cells are a promising alternative to T cells for cancer immunotherapy. Adoptive therapies with allogeneic, cytokine-activated NK cells are being investigated in clinical trials. However, the optimal cytokine support after adoptive transfer to promote NK cell expansion, and persistence remains unclear. Correlative studies from 2 independent clinical trial cohorts treated with major histocompatibility complex-haploidentical NK cell therapy for relapsed/refractory acute myeloid leukemia revealed that cytokine support by systemic interleukin-15 (IL-15; N-803) resulted in reduced clinical activity, compared with IL-2. We hypothesized that the mechanism responsible was IL-15/N-803 promoting recipient CD8 T-cell activation that in turn accelerated donor NK cell rejection. This idea was supported by increased proliferating CD8+ T-cell numbers in patients treated with IL-15/N-803, compared with IL-2. Moreover, mixed lymphocyte reactions showed that IL-15/N-803 enhanced responder CD8 T-cell activation and proliferation, compared with IL-2 alone. Additionally, IL-15/N-803 accelerated the ability of responding T cells to kill stimulator-derived memory-like NK cells, demonstrating that additional IL-15 can hasten donor NK cell elimination. Thus, systemic IL-15 used to support allogeneic cell therapy may paradoxically limit their therapeutic window of opportunity and clinical activity. This study indicates that stimulating patient CD8 T-cell allo-rejection responses may critically limit allogeneic cellular therapy supported with IL-15. This trial was registered at www.clinicaltrials.gov as #NCT03050216 and #NCT01898793.

Recipient HLA-B Leader Genotype, and Its Relationship to Total KIR Missing Ligand, Informs Relapse and Survival Following Haploidentical Transplantation Using Post-Transplant Cyclophosphamide

▪In addition to donor T cells, natural killer (NK) cells are proposed to play a significant role in the graft-versus-leukemia (GVL) effect following haploidentical donor transplantation (HIDT). Following HIDT, donor NK cells express activating (NKG2C) receptors (Ciurea, Leukemia 2021), whose ligand is the non-classical human leukocyte antigen, HLA-E. For surface expression, HLA-E requires binding of leader peptides derived from class I HLA molecules. The rs1050458C/T dimorphism at position -21 of exon 1 of HLA-B gives rise to leader peptides with either methionine (M) or threonine (T) at the second residue of the processed leader peptide. M-containing HLA-B leader peptides promote higher HLA-E expression than T-leaders, potentially favoring more robust natural NK cell recognition of HLA-E-expressing tumor cells. Alternatively, donor NK cells can be activated through inhibitory killer cell immunoglobulin-like receptors (iKIR: 2DL1, 2DL23, 3DL1, 3DL2), if they fail to engage a corresponding class I HLA ligand on recipient leukemia cells (missing ligand (ML) paradigm). We hypothesized that M-containing B-leaders (either MM or MT genotype), and potentially its association with KIR ML, may inform relapse and survival after HIDT using post-transplant cyclophosphamide (PTCy). A total of 322 patients with acute leukemia, MDS, lymphoma, CLL or CML, receiving a HIDT-PTCy from a single institution were evaluated with a median follow-up time of 45 months [range 6, 184]. Baseline characteristics included a median age of 50 years [19, 80], 47% non-white, HCT-CI ≥3 in 50%, PBSC graft in 80%, and myeloablative conditioning in 49%. M-containing B-leader genotype (either MM or MT) was seen in 42% and 44% of recipients and donors, respectively. The B-leader on the unshared donor-recipient haplotypes was matched (either T or M) in 61% of transplants. ML for iKIR 2DL1, 2DL23, 3DL1, and 3DL2 was noted in 29%, 20%, 24% and 72% respectively. Total ML was 0, 1, 2 and 3 in 10%, 43%, 40% and 7% respectively. In univariate analysis, an M-containing recipient B-leader genotype [R(M+)] improved OS and DFS compared to a recipient TT genotype [R(M-)] (75 vs. 53%, p<0.001; 66 vs. 47%, p<0.001), which was primarily due to a lower risk of relapse/progression (24% vs. 38%, p=0.012). In regard to total iKIR ML, the presence of 2-3 ML was associated with better overall (OS) and disease-free (DFS) survival compared with 0-1 (67% vs. 57%, p=0.08; 62% vs. 48%, p=0.019), which was due to lower relapse/progression (25% vs. 38%, p=0.015). There was no association of either recipient B-leader or total iKIR ML with the incidence of NRM, acute or chronic GVHD. When recipient B-leader and ML were combined, the detrimental effect of a R(M-) genotype was seen exclusively in patients with ML 0-1 (see figure). DFS for R(M-)/ML(0-1), R(M+)/ML(0-1), R(M-)/ML(2-3), R(M+)/ML(2-3) was 36%, 68%, 65% and 60%, respectively (p<0.001). Corresponding relapse/progression rates were 49%, 22%, 26% and 25%, respectively (P<0.001). In multivariate analysis, controlling for patient age/sex/race, disease risk index, donor age, HLA-DR mm, HLA-DP npmm and year of transplantation, both recipient B leader and total iKIR ML were independently associated with DFS (HR 0.57, p=0.002 and HR 0.67, p=0.026) and relapse/progression (HR 0.55, p=0.006 and HR 0.55, p=0.007). In summary, a recipient M-containing B-leader genotype (MM or MT) reduces relapse and improves survival following HIDT-PTCy, presumably through HLA-E-mediated effector cell engagement. Furthermore, the negative consequences of low total ML for iKIR, in terms of increased relapse risk and lower DFS, can be mitigated when a R(M+) B-leader is present. In addition to the clinical importance of this novel finding for optimizing HIDT-PTCy, it further supports the role of alloreactive donor NK cells for optimal GVL in this context. [Display omitted] DisclosuresSolh: Partner Therapeutics: Research Funding; Jazz Pharmaceuticals: Consultancy; ADCT Therapeutics: Consultancy, Research Funding; BMS: Consultancy.

A Phase I Clinical Trial Testing the Safety of IL-21-Expanded, Universally Alloreactive Donor-Derived Natural Killer Cells for Relapsed/Refractory Acute Myeloid Leukemia and Myelodysplastic Syndrome

Background: Natural Killer (NK) cells are cytotoxic lymphocytes that are able to exert an anti-tumor effect in an MHC-I independent manner, but are dysfunctional and reduced in number in patients with leukemia. Several studies have shown therapeutic potential for related donor NK cells expanded and/or activated ex vivo when administered to cancer patients, including patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, personalized, donor-derived cell therapies require time for donor identification, manufacturing and product release, resulting in patient attrition. As NK alloreactivity and NKG2C expression play critical roles in mediating anti-tumor effects, we identified ‘ideal’ NK cell donors (in partnership with “Be the Match Biotherapies”) for collection. Human leukocyte antigen (HLA) and Killer immunoglobulin receptor (KIR) genotyping were done to screen and select donors. Following this, a sample was collected from donors to expand NK cells under small-scale conditions. If donor NK cells showed robust expansion, they proceeded with apheresis. To ensure these therapeutic cells would be readily-available, we established a third-party NK cell bank through scalable, affordable mass-production with membrane-bound IL-21 feeder cells (FC21), and cryopreserved large numbers of these NK cells for immediate ‘off-the-shelf’ administration to recipients. Here, we report our initial experience in a Phase I trial of these off-the-shelf (OTS) alloreactive NK cells as immunotherapy for patients with relapsed/refractory AML and MDS.Methods: Patients were treated at Dose level 1, to allow infusion at a dose of 1 x 10e7 NK cells/ kg. Each dose level had two cohorts, stratified by age. Patients were enrolled into Cohort 1 (patients <60 yrs. received Fludarabine (Flu) 30 mg/m2/day & Cytarabine 2 g/m2/day on days -6 to -2) and Cohort 2 (patients ≥ 60 yrs. received Flu 30 mg/m2/day (days -5 to -2) & Decitabine 20 mg/m2/day (days -6 to -2). Patients were evaluated to see if they had any donor-directed antibodies. After chemotherapy, NK cells were infused thrice weekly for 6 doses, to be given over a period of 2 weeks (Clinicaltrials.gov: NCT04220684). Persistence of donor NK cells was determined by flow cytometry using haplotype-discriminating anti-HLA antibodies (Figure 1, 2). NK cells were completely HLA-mismatched between donor and recipients.Results: 6 patients (3/cohort) were treated at the first dose level of 1x10 7 NK cells/kg/dose. All 6 patients had relapsed/refractory AML and had received at least two prior lines of treatment. One patient (005) withdrew from the study prior to infusion of NK cells. 6 patients received the first NK cell dose as an inpatient and were discharged to receive the remaining 5 doses as an outpatient. 5 patients tolerated infusion of all 6 doses of NK cells administered over 2 weeks. One patient (006) developed a Cytokine response (CRS) - like syndrome in the context of streptococcus bacteremia and received only one dose of NK cells. Symptoms suggestive of CRS responded fully with steroids and patient was able to successfully wean off steroids and discharged to the outpatient setting. One patient proceeded to allogeneic hematopoietic cell transplant. No infusion-related reactions, neurotoxicity or graft versus host disease was observed. In vivo persistence/expansion NK cells were superior with FLU/CY lymphodepletion (cohort 1).Conclusions: Cryopreserved, third-party donor-derived NK cells are safe and feasible in relapsed/refractory AML patients, and even at this first dose level, completely HLA-mismatched NK cells can persist and expand to high levels in vivo. [Display omitted] DisclosuresVasu: Kiadis, Inc.: Research Funding; Seattle Genetics: Other: travel support; Boehringer Ingelheim: Other: Travel support; Omeros, Inc.: Membership on an entity's Board of Directors or advisory committees. Walker: Newave: Other: clinical trial support; Geron: Other: clinical trial support; Novartis: Other: clinical trial support. Blachly: INNATE: Consultancy, Honoraria; KITE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. de Lima: Incyte: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotec: Research Funding. Lee: Kiadis Pharma: Divested equity in a private or publicly-traded company in the past 24 months, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Courier Therapeutics: Current holder of individual stocks in a privately-held company.

Improved hematopoietic stem cell transplantation upon inhibition of natural killer cell-derived interferon-gamma.

SummaryHematopoietic stem cell transplantation (HSCT) is a frequent therapeutic approach to restore hematopoiesis in patients with hematologic diseases. Patients receive a hematopoietic stem cell (HSC)-enriched donor cell infusion also containing immune cells, which may have a beneficial effect by eliminating residual neoplastic cells. However, the effect that donor innate immune cells may have on the donor HSCs has not been deeply explored. Here, we evaluate the influence of donor natural killer (NK) cells on HSC fate, concluded that NK cells negatively affect HSC frequency and function, and identified interferon-gamma (IFNγ) as a potential mediator. Interestingly, improved HSC fitness was achieved by NK cell depletion from murine and human donor infusions or by blocking IFNγ activity. Thus, our data suggest that suppression of inflammatory signals generated by donor innate immune cells can enhance engraftment and hematopoietic reconstitution during HSCT, which is particularly critical when limited HSC numbers are available and the risk of engraftment failure is high.

Haploidentical Hematopoietic Cell Transplantation Followed By Donor Natural Killer Cell Infusion and Sirolimus Maintenance Can Successfully Treat Patients with High-Risk Ewing Sarcoma

Background: Approximately 30-40% of patients with newly diagnosed localized Ewing Sarcoma (EWS), and 60-80% with disseminated EWS, will eventually relapse. Patients with late relapse have a 5 year overall survival (OS) of 30%, while patients with early relapse (<2 years from initial diagnosis) have a more dismal 5 year OS of <10% (Stahl Ped Blood Cancer 2011). Unfortunately, conventional treatments including chemotherapy, autologous transplantation, surgery, and radiation therapy often lead to additional morbidities without substantial gains in disease remissions or survival. Allogeneic hematopoietic cell transplantation (HCT) using human leukocyte antigen (HLA)-matched related donors has shown some success for patients with high-risk EWS (Baird BBMT 2012). Additionally, EWS cells are exquisitely sensitive to natural killer (NK) cell cytotoxic activity (Cho CCR 2010), and many EWS tumors have upregulated the mammalian target of rapamycin (mTOR) pathway making them susceptible to anti-mTOR agents (Wan et al The Oncologist 2007). With growing literature supporting the safety and efficacy of HLA-haploidentical HCT with post-transplant cyclophosphamide (CY) for hematological malignancies, we developed a novel treatment regimen adding a single infusion of donor NK cells on day +7 after HLA-haploidentical HCT, followed by sirolimus as part of a GVHD prophylaxis strategy. We hypothesized that this multi-faceted immunotherapy approach, in combination with an mTOR-inhibitor post-transplant, would be well-tolerated and efficacious in patients with relapsed EWS.Methods: Patients received reduced-intensity conditioning using fludarabine (150 mg/m2), CY (29 mg/kg) and 3 Gy total body irradiation, followed by infusion of HLA-haploidentical related marrow (n=4) or peripheral blood stem cells (n=2). Post-transplant immune suppression consisted of CY (50 mg/kg, days +3 and +4), tacrolimus (beginning day +5, and transitioned to sirolimus from day +28 to 6 months), and mycophenolate mofetil (MMF) (day +9 to +84). MMF initiation was delayed until day +9 to decrease concerns of inhibiting NK activity. For NK cell therapy, non-mobilized peripheral blood mononuclear cells were collected from the same haplo donors on day +6 using apheresis. NK cells were selected on day +7 using the Miltenyi CliniMACS system (CD3 depletion followed by CD56 selection) and were administered as a single, fresh infusion that same day without prior culturing, expansion, or exogenous cytokine exposure.Results: Four pediatric and 2 adult patients with median age of 18.5 (10.5-34.0) years having relapsed EWS were enrolled on this Phase II study. All patients had stable or undetectable gross disease at HCT. Patients had 1 (n=3), 2 (n=2), or 5 (n=1) relapses prior to HCT. Median time from EWS diagnosis to first relapse was 22.5 (15-56) months. Two patients had prior autologous HCTs. Median HCT-CI score at time of transplant was 4 (2-5), demonstrating increased risk for transplant-related mortality (TRM). Patients received a median CD34 dose of 5.24 (1.3-8.3) x 106/kg. NK cell products had a median log T cell depletion of 6.25 (5.18-6.42), median NK recovery of 54% (47-71%), and median NK purity of 91% (78-96%). Excellent viability (>99%) was seen in all NK products. Donor NK infusions were well-tolerated without fevers or other adverse reactions. Sustained full donor chimerism (>95% CD3) was achieved in all 6 patients. One patient developed stage 1 (grade II) gut graft-versus-host disease (GVHD) at day +52 which was treated with budesonide. To date, no patients have developed chronic GVHD nor have died from TRM. With a median follow-up of 1.3 years (range, 180 days - 2.7 years), 1 year overall survival is estimated at 75% (see figure). Three patients have relapsed or progressed at 187, 284, and 455 days after HCT, of which 1 patient (who had 5 prior relapses), died of progressive disease at day +350. The other 2 patients have received salvage treatments. They remain alive with full-donor chimerism and low-level/absent disease at 1.7 and 2.7 years post-HCT.Summary: This dual immunotherapy approach, using HLA-haploidentical HCT with donor NK cell infusion, followed by sirolimus maintenance, was well-tolerated by all patients. While longer follow-up is needed, preliminary results of this ongoing novel approach demonstrate feasibility in EWS patients, with better than expected 1 year OS and promising disease control. [Display omitted] DisclosuresThakar:Miltenyi Biotec: Other: funding of partial costs - disposable agents. Hari:Janssen: Honoraria; Sanofi: Honoraria, Research Funding; Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy; Takeda Pharmaceuticals: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Spectrum: Consultancy, Research Funding; Amgen: Consultancy, Honoraria, Research Funding.

Neurological Consequences of Cytokine Release Syndrome Following Subcutaneous Recombinant IL-15 and Haploidentical Donor Natural Killer Cell Therapy for Advanced Acute Myeloid Leukemia

Abstract Cytokine Release Syndrome (CRS) is associated with elevated cytokine levels, macrophage activation, and IL-6 production after antibody or cell-based immunotherapy. We have observed over 200 patients with no CRS following adoptively transferred haploidentical donor natural killer (haplo-NK) cell products after lymphodepleting chemotherapy plus IL-2; and achieved complete remission in 30-50% of patients with refractory AML. To promote NK cell expansion without stimulation of Treg that may compromise NK function, we tested the substitution of IL-2 with the NCI manufactured rIL-15. Using the same lymphodepleting preparative regimen (Fludarabine 25 mg/m2IV for 5 days and Cyclophosphamide 60 mg/kg IV for 2 days) we infused CD3- and CD19-depleted haplo-NK incubated overnight with rIL-15 (10 ng/mL) prior to infusion. Two cohorts received rIL-15; either intravenously x 12 daily doses (at the Phase I MTD of 0.75 mcg/kg) or subcutaneously x 10 doses (5 days on, 2 days off, 5 days on at 2 mcg/kg SQ, the MTD when used as monotherapy in solid tumor patients). CRS was defined by modified CRS grading criteria (Lee et al. Blood, 2014). Results: Forty patients with relapsed/refractory AML were treated with haplo-NK plus IV rIL-15 (n=24) or SQ rIL-15 (n=16). Patients received an average of 11.6 IV doses [9-12, 96% of planned doses] or 7.5 SQ doses [1-10, 75% of planned doses]. Dosing was stopped for toxicity. No CRS events were observed after IV dosing. However, after SQ dosing, 9 of 16 patients [56%] had grade 1-4 CRS; [4 (25%) with grade 3-4 CRS) (Table 1). The median time from NK infusion to onset of CRS was 8 days [2-44]. Six of the 9 CRS patients (66%) had neurological events. Neurological toxicities included grade 1-2 encephalopathy (n=2), intracranial hemorrhage (ICH) (n=2, grade 1 and grade 5), and grade 4 seizures (n=2). One additional event (a patient with a fall resulting in ICH due to pre-existing gait abnormality) without evidence of CRS was not considered as IL-15 associated neurotoxicity CRS. Symptoms resolved in 4 of 5 patients who received treatment for neurologic CRS (3 received both steroids and Tocilizumab, 1 received steroids only, 1 patient did not require treatment as symptoms resolved with stopping IL-15). Non-neurologic toxicity included hypoxia (n=2) and hypotension (n=3). No significant correlations were found between development of CRS and absolute lymphocyte or monocyte count, presence or absence of haplo-NK expansion at day 14, number of rIL-15 doses received, or disease burden at baseline as measured by blast percentage in bone marrow. In the IV rIL-15 cohort, only 2 of 14 patients at the MTD (14%) cleared leukemia and underwent transplantation. In contrast, 8 of 16 patients (50%, P=0.04) in the SQ rIL-15 cohort achieved CR (n=7) or CRp (n=1), with 6 patients proceeding to alloHCT consolidation. CRS did not limit the clinical benefit in the SQ cohort as 4 of 9 [44%] patients with CRS achieved CR/PR compared to 4 of 7 [57%] patients without CRS. Pharmacokinetic data showed slower clearance of rIL-15 in SQ compared to IV dosing, as shown by higher trough levels prior to dose 2 (501 vs. 233 pg/mL, p Download : Download high-res image (163KB) Download : Download full-size image Disclosures Bachanova: Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Seattle-Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Zymogen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oxis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Miller: Celegene: Consultancy; Oxis Biotech: Consultancy; Fate Therapeutics: Consultancy, Research Funding.

Complete Remission and Signs of Immunoediting Following Haploidentical NK Cell Therapy in Refractory High-Risk MDS and AML Ell Therapy in Refractory High-Risk MDS and AML

Haploidentical IL-2-activated natural killer (NK) cells can induce remission in patients with acute myeloid leukemia (AML). Here, we report results of a phase I/II clinical trial in which short-term IL-2-activated haploidentical NK cells were given to patients with primary chemotherapy-refractory or relapsed high-risk myelodysplastic syndrome (MDS), secondary AML (MDS/AML), and AML. The patients received conditioning with cyclophosphamide/fludarabine combined with total lymphoid irradiation. To avoid expansion of regulatory T cells (Treg), no post-NK cell infusion IL-2 was given. Using this protocol, nine of sixteen patients responded. Of the nine patients, six achieved a major response with complete remission (CR), marrow CR or partial response in combination with improved hematological parameters. Five of these patients had high-risk MDS or MDS/AML, and one had relapsed de novo AML. These patients all became eligible for, and proceeded to, allogeneic hematopoetic stem cell transplantation (HSCT). Four of the patients are still free from disease >4 years after treatment. The patients with a major responses had detectable donor NK cells at days 7 to 14 following infusion, displayed reduction of allelic burden, and partial or complete eradication of MDS/AML subclones. Residual lin-CD34+CD123+CD45RA+ blast cells showed an increase in total HLA class I- and HLA-E-expression suggesting immunoediting by the NK cells. Non/minor responders displayed an activation of CD4+ and CD8+ T cells as well as Treg cells accompanied by a marked increase in inflammatory cytokines. Together, this study supports the use of haploidentical NK cell infusions as a bridge to HSCT, especially for patients with high-risk MDS and MDS/AML. DisclosuresMiller:Celegene: Consultancy; Fate Therapeutics: Consultancy, Research Funding; Oxis Biotech: Consultancy. Hansson:Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janssen-Cilag: Honoraria, Research Funding.

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.

DONOR KIR3DL1/RECEPTOR HLA-BW4-80I COMBINATION REDUCES ACUTE LEUKEMIA RELAPSE AFTER UMBILICAL CORD BLOOD TRANSPLANTATION WITHOUT IN VITRO T-CELL DEPLETION

BackgroundDonor natural killer (NK) cell alloreactivity in umbilical cord bone marrow transplantation (UCBT) can lead to leukemic relapse. However, NK cell function is calibrated by interaction with human leukocyte antigens (HLAs). This study aimed to investigate graft-resistant leukemia after transplantation and compared specific genotypes of killer immunoglobulin-like receptors (KIRs) in donors and human leukocyte antigen ligands in patients.MethodsWe retrospectively analyzed 232 patients with acute leukemia from a single center. Patients had undergone UCBT with myeloablative conditioning and without anti-thymocyte globulin. We identified the KIR genotypes of cord blood donors using polymerase chain reaction with sequence-specific primers. All of the donors contained KIR3DL1.ResultsThe patients were divided into three groups according to the HLA-B locus. The donor KIR3DL1 and recipient HLA-Bw4-80I combination was predictive of being highly educated and was associated with a lower relapse (P=0.006) and better overall survival (probability of relapse=0.13, P < 0.001) than the uneducated group. We found no significant increase in the incidence of acute or chronic graft-versus-host disease.ConclusionsOur data suggest that the donor KIR3DL1/receptor and HLA-Bw4-80I combination in UCBT results in stronger graft-versus-leukemia effects and improved outcomes in patients with acute leukemia.

Cellular immunotherapy with multiple infusions of in vitro-expanded haploidentical natural killer cells after autologous transplantation for patients with plasma cell myeloma

Background aimsTo investigate the feasibility and safety of haploidentical natural killer (NK) cell infusions as consolidation immunotherapy after autologous stem cell transplant (ASCT) in patients with plasma cell myeloma. MethodsTen patients (median age, 59 years) received induction treatment followed by high-dose melphalan (200 mg/m2) at day –1, ASCT at day 0 and increasing NK cell doses (1.5 × 106, 1.5 × 107 and multiple doses of 1.0 × 108 cells/kg body weight) from day +1 to day +30 after ASCT. NK cells were harvested and purified from peripheral blood of haploidentical donors and expanded for 19 days with interleukin (IL)-2 and IL-15 under Good Manufacturing Practice conditions. ResultsNK cell numbers increased 56.0-fold (37.4- to 75.5-fold). Patients received a median of 3.8 × 108 (0.9–5.7 × 108) NK cells/kg body weight in six (three to eight) infusions. Multiparametric mass cytometry analysis demonstrated an altered surface receptor repertoire of expanded NK cells with increased degranulation and cytokine production activities but diminished expression of perforin. Donor NK cells were detectable in the peripheral blood, peaking 1 h after each dose (up to 90% donor NK cells). The treatment was safe and well tolerated, without evidence of graft-versus-host disease. Comparison with a control patient population receiving ASCT without NK cell infusions showed no significant difference in relapse, progression-free survival and overall survival. ConclusionsThis study demonstrates reliable manufacturing of high numbers of activated NK cells for multiple-dose infusions and safe administration of these cellular products. The trial was registered at ClinicalTrials.gov (identifier no. NCT01040026).

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

Results of a Phase 1 Trial of Gda-201, Nicotinamide-Expanded Allogeneic Natural Killer (NK) Cells in Patients with Refractory Non-Hodgkin Lymphoma (NHL) and Multiple Myeloma

Background: The innate capacity of natural killer (NK) cells to kill tumor targets has been translated into cancer immunotherapy. GDA-201 is a novel allogeneic NK cell product derived from NK cells from healthy donors, expanded ex-vivo with nicotinamide (NAM) and IL-15. We previously reported improved killing function, in vivo proliferation, organ trafficking, and augmented resistance against exhaustion in pre-clinical models. We conducted a phase 1 study of GDA-201 in combination with monoclonal antibodies to enhance NK cell targeting through antibody-dependent cellular cytotoxicity (ADCC). We now report safety data in patients (pts) with relapsed or refractory (R/R) non-Hodgkin lymphoma (NHL) and multiple myeloma (MM), and report efficacy outcomes in pts with NHL. Methods: Following donor apheresis, CD3-depleted mononuclear cells were cultured for 14-16 days with NAM (5mM) and IL-15 (20ng/ml), resulting in a 40-fold increase in NK cells and increased expression of CD62L from 2.9% to 21%. GDA-201 contained ~98% NK cells, and CD3 content was maintained at &amp;lt;0.5% (&amp;lt;5x105/kg/dose). Pts with R/R B-cell NHL or MM received lymphodepleting (LD) therapy with cyclophosphamide (400mg/m2 IV x 3d) and fludarabine (30 mg/m2 /d IV x 3d), followed by GDA-201 (days 0 and 2) and low-dose IL-2 (6 million units sc x 3 doses). Pts with NHL or MM received rituximab (375 mg/m2) or elotuzumab (10 mg/kg), respectively, x 3 weekly infusions. Results: 30 pts were enrolled:15 with NHL and 15 with MM, in 3 cohorts of escalating GDA-201 dose; 15 pts received the maximum target dose (median dose 12.4 [range 2.0-26.0] x 107 cells/kg). There were no dose limiting toxicities. The most common grade 3/4 adverse events were thrombocytopenia (n=9), hypertension (n=5), neutropenia (n=4), febrile neutropenia (n=4), and anemia (n=3). There were no neurotoxic events, confirmed cytokine release syndrome, graft versus host disease, or marrow aplasia. One patient died of E-coli sepsis. In pts with NHL, histologies included diffuse large B cell lymphoma (DLBCL) (de novo n=5, transformed n=3), follicular lymphoma (FL) (n=6), and mantle cell lymphoma (n=1). Median age was 64 (range 48-83 years). Pts had a median of 3 lines of prior therapy (range 1-8); most were multiply relapsed or refractory (n=2), and 87% had advanced stage. Median follow-up was 10.8 months (range 4.3-27.5 months). Ten pts had complete response (CR): 6/6 pts with FL and 4/8 with DLBCL; 1 pt had partial response (PR), and overall response rate in pts with NHL was 73.3%. Median duration of response was 8.7 months (range 4.3-25 months). Flow cytometry confirmed the persistence of GDA-201 in peripheral blood for 7-10 days (range 2-92% donor NK cells on day 7), as well as enhanced in vivo proliferation (median Ki 67 99%). Flow cytometry of biopsied tissues at day 4 demonstrated trafficking to bone marrow and lymph nodes. Four pts underwent re-treatment with GDA-201 without LD chemotherapy; GDA-201 cells were detectable in blood after the re-treatment and likely contributed to deepening of response in 2 patients. Post-GDA-201 therapy included allogeneic (n=2) and autologous (n=1) hematopoietic stem cell transplantation. One-year estimates of progression-free survival and overall survival were 66% (95% CI 36-84%) and 82% (95% CI 42-95%), respectively. Conclusions: Cellular therapy using GDA-201 with monoclonal antibodies to enhance ADCC was well-tolerated, and demonstrated significant clinical activity in heavily pretreated pts with advanced NHL. Data support the future testing of multiple infusions to potentially enhance anti-tumor effect. The omission of lymphodepleting chemotherapy is feasible and contributes to safety of this approach. Phase II studies in aggressive and indolent NHL cohorts are planned. Disclosures Bachanova: Incyte: Research Funding; FATE: Research Funding; Kite: Membership on an entity's Board of Directors or advisory committees; Karyopharma: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Gamida Cell: Membership on an entity's Board of Directors or advisory committees, Research Funding. McKenna:Gamida: Other: Cell Manufacturing; Fate Therapeutics: Other: Cell Manufacturing; Intima: Other: Cell Manufacturing; Magenta: Other: Cell Manufacturing. Janakiram:Takeda, Fate, Nektar: Research Funding. Simantov:Gamida Cell: Current Employment. Lodie:Gamida Cell: Current Employment. Miller:Vycellix: Consultancy; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; GT Biopharma: Consultancy, Patents &amp; Royalties, Research Funding; Fate Therapeutics, Inc: Consultancy, Patents &amp; Royalties, Research Funding.