ALBERT

Description: Introduction: Conventional CAR-T cells express a single chain antibody variable fragment that restricts recognition to one tumor antigen and a limited set of cancers. This study employs a novel CAR fusing full-length human NKG2D with the CD3z signaling domain. In autologous transduced CM-CS1 T cells, NKG2D CAR receives endogenous costimulation via DAP10 to target multiple NKG2D-ligands that are upregulated in solid and hematologic malignancies but absent or poorly expressed on healthy tissues. Methods: A phase 1 dose-escalation study to establish safety and feasibility of a single infusion of CM-CS1 T cells without lymphodepleting conditioning enrolled subjects with AML/MDS-RAEB or relapsed/refractory progressive multiple myeloma (MM) without standard therapy options (NCT02203825). Eligibility criteria included suitable organ function, no CNS disease, no prior allogeneic SCT or adoptive T-cell therapy, no therapy within 3 weeks prior to infusion, no immune suppression, and no uncontrolled infection. Dose-escalation spanned 4 cohorts [half-log increments from 1x106 to 3x107 CM-CS1 T cells] according to a 3+3 design. DLTs included ≥ Grade 3 non-hematologic toxicity or ≥ Grade 2 autoimmune toxicity related to CAR T cells. Initial assessment was at 28 days. At least 1 AML/MDS and 1 MM subject were mandated in each dose level. Manufacturing included PBMC stimulation with OKT3 and IL-2 followed by 2 rounds of retroviral transduction at DFCI's Cell Manipulation Core Facility. Vector copy number (VCN) and replication-competent retrovirus (RCR) testing were performed on whole blood and PBMCs, respectively, using quantitative PCR. Results: From April 2015 to July 2016, 11 subjects were infused, and 10 completed the DLT period. Eight of 11 were male, 6 had AML/MDS, and median age was 70 (range 44 to 79) (Panel A). Median WBC was 2.3 (range 0.7 to 7.2 K/uL); median ALC was 0.74 (range 0.09-2.37 K/uL). Five had cells manufactured from peripheral blood; 6 underwent apheresis. Median percentage of blasts in bone marrow for AML/MDS patients was 50% (range 4-68%). All myeloma patients had undergone ≥ 5 therapies including ≥1 autologous SCT. Four of the 6 AML/MDS patients had secondary disease, 3 had complex cytogenetics, 3 had p53 mutations, and 1 had a FLT3-ITD mutation. Dose-escalation proceeded from 1x106 to 3x107 CM-CS1 T cells. All 11 products passed release criteria, and there were no infusion reactions. Products consisted of median 97.2% CD3+ cells and 31.0% CD8+ cells, with vector-specific NKG2D expression on median 74.6% of CD3+ and 66.3% of CD8+ cells (Panel B). The first 10 subjects completed their 28 day evaluation period without DLTs. There were no cases of cytokine release syndrome, cell-related neurotoxicity, auto-immunity, or CAR T-related death. SAEs included a Grade 4 intracochlear bleed and an episode each of grade 4 neutropenia and thrombocytopenia deemed related to disease progression. Forty percent of patients experienced some Grade 3 toxicity, all related to underlying disease or a complication thereof (Panel C). At these initial cell doses, no patient to date has had objective tumor response at the 28 day evaluation mark. Nine initiated subsequent therapies; there have been 4 deaths secondary to disease or complications of subsequent therapies. However, cases of unexpected survival without further therapy and responses to subsequent treatments were noted. For example a patient with p53-mutated AML survived 4 months despite 50% blasts at infusion, and another entered PR at 6+months after cells on an IDH-1 inhibitor with

Description: Autologous gene therapy (GT) for beta-hemoglobinopathies has demonstrated encouraging early safety and efficacy using addition of a sickling-resistant globin gene to stem cells. Another therapeutic strategy for sickle cell disease (SCD) is erythroid-specific inactivation of BCL11A, which is a validated repressor of gamma globin expression (Sankaran et al. Science 2011). This approach has the distinct advantage of simultaneously inducing fetal hemoglobin (HbF) while coordinately decreasing sickle hemoglobin (HbS). Since hemoglobin (Hb) polymerization in sickle red cells is highly dependent on the intracellular concentration of HbS and is strongly inhibited by HbF, effective BCL11A repression should prevent the sickling phenotype within red cells. We have shown that erythroid-specific expression of microRNA-adapted shRNAs (shRNAmiR) targeting BCL11A effectively induces HbF in human erythroid cells derived from transduced HSCs, largely attenuating the hematologic effects of SCD in a murine model while avoiding negative effects in HSCs and B lymphocytes (Brendel et al. JCI 2016). Here we report the initial results of a pilot clinical study utilizing a shRNAmiR lentiviral vector (LVV) targeting BCL11A for autologous GT in SCD patients. Transduction of hematopoietic cells with GMP lentiviral vector (BCH_BB-LCRshRNA(miR)) expressing the shRNAmiR for BCL11A in an erythroid-specific fashion showed no toxicity in engraftment and genotoxicity assays, efficient transduction rates of 80-95% of CD34+-derived erythroid colony forming cells from healthy donors and SCD patients, and 〉95% of transduced erythroid colonies demonstrating HbF levels of 50-95% of total Hb. Transduction at clinical scale with plerixafor mobilized CD34+ cells from three SCD donors yielded vector copies of 3.7 - 5.2/cell. Patients with severe SCD were screened for eligibility according to an IND enabled, IRB-approved investigator-initiated protocol. The first cohort included patients ≥ 18 years old. After at least 3 months of protocol-required transfusions, autologous CD34+ cells were collected by plerixafor mobilization and apheresis, and then transduced under GMP conditions with the BCH_BB-LCRshRNA(miR) vector. As of July 28, 2018, 3 patients representing the adult cohort had undergone a total of 3 (n=2) or 4 (n=1) days of mobilization. Mean single-day apheresis yields were 3.2 (range 1.5 - 6.8) x 106 CD34+ cells/kg. No Grade 3 or 4 AEs were attributed to mobilization and collection, although one subject developed an incidentally-discovered line-associated atrial clot and pulmonary embolism. Transduced cell products for these 3 patients have cell doses of 3.3 - 6.7 x 106 CD34+ cells/kg, VCN of 3.3 - 5.1 copies per cell and 〉95% vector-positive CD34+-derived colonies. One subject (BCL002), who had been regularly transfused for 17 years, has undergone infusion of gene-modified cells after myeloablative busulfan conditioning and achieved neutrophil engraftment after 22 days. Post-infusion follow-up is 78 days. At the time of the last analysis 76 days after GT and 64 days after last RBC transfusion (Table 1) subject BCL002 had a sustained Hb of 〉10 g/dL and, compared to pre-GT, there was a notable absence of irreversibly sickled cells on peripheral smear and a persistently low absolute reticulocyte count consistent with markedly reduced hemolysis. Hb electrophoresis showed 23.3% HbF, 51.8% HbS and 22.3% HbA (from residual transfused cells) with a HbF/(HbF+HbS) ratio of 29.7%. At day 76, the number of F cells had risen to 59.7% with 12pg HbF/F cell. In flow-sorted immature erythroid cells γ-globin mRNA was 〉80% of total β-like globins in the graft-derived population and BCL11A protein was reduced by ~90%. Adverse events observed from the start of conditioning until latest follow-up were consistent with myeloablative conditioning, and there have been no product-related adverse events and no SCD-related complications. These early results show: (1) feasibility of enrollment, cell procurement, and GMP manufacturing of gene modified CD34+ cells in 3 adult SCD patients; (2) the first proof of principle demonstrating shRNAmiR-based gene knockdown in humans, and (3) successful rapid induction of HbF in the first patient infused, with marked attenuation of hemolysis in the early phase of autologous reconstitution. Based on the trajectory of increasing HbF/(HbF+HbS), near full suppression of the SCD phenotype is expected. Disclosures Esrick: Bluebird Bio: Honoraria. Negre:bluebird bio: Other: Spouse employed by bluebird Bio. Dansereau:Bluebird Bio: Consultancy. Braunewell:Bluebird Bio: Employment, Equity Ownership. Christiansen:Bluebird Bio: Employment, Equity Ownership, Other: Salary. Nikiforow:Kite Pharma: Consultancy. Achebe:Luitpold Pharmaceutical: Consultancy; AMAG Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Syros pharmaceuticals: Consultancy. Negre:Bluebird Bio: Employment, Equity Ownership, Other: Salary. Heeney:Sancilio Pharmaceuticals: Consultancy, Research Funding; Ironwood: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Vertex/Crisper: Other: Data Monitoring Committee; Pfizer: Research Funding; Astra Zeneca: Consultancy, Research Funding. Williams:Bluebird Bio: Research Funding.

Description: Abstract 782 The goal of cellular immunotherapy is to build long-lasting anti-tumor immunologic “memory” in patients and reject tumors for a lifetime. Previously, we and others demonstrated that IL-15 promotes the generation of T cells with a central memory (CM) phenotype which have the capacity to persist and establish effective anti-tumor memory in vivo. Furthermore, it has been shown that CD83 delivers a CD80-dependent T cell stimulatory signal that allows T cells to be long-lived. Based on these findings, we developed a system to generate large numbers of long-lived antigen-specific CD8+ T cells with a memory phenotype. This in vitro culture system utilizes IL-15 and a standardized, renewable artificial antigen presenting cell (aAPC) which was produced by transducing CD80, CD83, and HLA-A*0201 to the human cell line, K562. This aAPC can uniquely support the priming and prolonged expansion of large numbers of antigen-specific CD8+ CTL which display a central/effector memory (CM/EM) phenotype, possess potent effector function, and can be maintained in vitro for 〉1 year without any feeder cells or cloning. We hypothesized that adoptive transfer of these CTL with a CM/EM phenotype should result in anti-tumor memory in humans even without lymphodepletion or high dose IL-2. For our “first-in-human” clinical study, we chose the melanoma antigen MART1 as a target antigen, since MART1-specific HLA-A*0201+-restricted precursor CTL are detectable in some melanoma patients and can be immunophenotyped pre-infusion. Autologous CD8+ T cells were stimulated weekly with peptide-pulsed human cell-based aAPC and expanded with low dose IL-2 and IL-15. After three weeks, polyclonal MART1 CTL were reinfused without additional lymphodepletion, chemotherapy, IL-2, or vaccination. Eight study participants have enrolled and received a total of 15 MART1 CTL infusions (31% MART1 multimer positivity, median). All but one subject received two reinfusions where the 2nd graft was produced from CD8+ T cells harvested two weeks after the 1st reinfusion. To date, ≥2×109 CTL with potent effector function and a CM/EM phenotype were successfully generated for all subjects. No dose limiting toxicities were observed at either Dose Level 1 (2×108/m2) or Dose Level 2 (2×109/m2). Clinical activity was observed with a response by RECIST criteria in 1 subject, which was confirmed by a negative PET/CT 100 days following the last CTL infusion. In addition, 1 patient experienced a mixed response, 1 had stable disease, 3 had progression, and 2 are currently on active therapy. Multimer staining showed that, immediately post infusion, the percentage of CD8+ T cells specific for MART1 temporarily increased in all subjects, with the highest (6.5%) observed in subject #7. In 4 subjects, sustained increases in the frequency of MART1 specific T cells by more than two-fold (range 2.0-10x) for ≥21 days were observed despite the fact that no exogenous cytokines or vaccination was administered. Moreover, an increase of detectable MART1 specific T cells which display a CM phenotype was observed in all evaluable subjects and was observed for ≥35 days in 6 of 8 subjects. In subject #2, the conversion of MART1 CTL immunophenotype from a naïve to a mixture of naïve/memory phenotypes was observed for more than 6 months. We identified 10 individual MART1 T cell clonotypes from peripheral CD45RA- memory T cells on day 21. Clonotypic TCR Vbeta CDR3 analysis revealed that CTL grafts contained 7 out of 10 of these clonotypes. Furthermore, 6 clonotypes persisted in the peripheral CD45RA- memory fraction on days 39, 67 and/or 132. In Subject #3, who showed a mixed clinical response, 5 individual MART1 T cell clonotypes were isolated from lung metastases. 4 out of 5 clones were included in the CTL grafts. This finding supports the possibility that infused CTL can traffic and localize to sites of disease. Intriguingly, in both subjects, we were able to identify MART1 CTL clonotypes that were not detectable in the CTL grafts but possibly emerged after CTL infusion, indicating that adoptive transfer of MART1-specific CTL may provoke a de novo antitumor response. Taken together, these results suggest that CM/EM MART1 CTL generated ex vivo using our cell-based artificial APC in the presence of IL-15 may persist in vivo and induce de novo anti-tumor responses. Further enhancement of anti-tumor activity may be achieved through vaccination, cytokine administration, and/or removal of cytokine sinks and inhibitory factors following appropriate lymphodepletion. Disclosures: No relevant conflicts of interest to declare.

Description: BCL11A regulates the fetal-adult hemoglobin switch by repressing expression at the gamma (γ)-globin locus (Sankaran et al., Science, 2008), and thus it represents an appealing therapeutic target for sickle cell disease (SCD). BCH-BB694 is a lentiviral vector (LVV) encoding a shRNA targeting BCL11A embedded in a microRNA scaffold (shmiR) allowing erythroid-specific knockdown to induce γ-globin expression and concomitantly and coordinately repress β-sickle globin expression (Brendel et al. JCI, 2016). In a pilot and feasibility gene therapy study we are evaluating the safety of infusion of BCH-BB694-transduced autologous CD34+ cells in patients with severe SCD. The study is an IND enabled and IRB approved open label, non-randomized, single center trial (NCT 03282656). We report here data from the full adult cohort which has completed enrollment with 〉 6 months of follow up in all patients. The adult cohort included three patients 〉/= 18 years old. Autologous CD34+ cells were collected by plerixafor mobilization and then transduced ex vivo with the BCH-BB694 shmiR lentiviral vector. Cell doses and vector copy number (VCN) are shown in the Table. After testing and release, gene modified cells were infused into subjects who had received busulfan conditioning. There were no Grade 3 or 4 AEs associated with mobilization, collection or infusion. All three adults (age 21-26 years old) demonstrated neutrophil engraftment on day +22 with adverse events consistent with busulfan conditioning. These patients are now 7, 9, and 17 months post infusion. One subject resumed red cell transfusions at 3 months due to pre-existing moyamoya using a pre-defined conservative trigger value of 40% sickle Hb in whole blood and will be detailed separately. There have been no adverse events related to the gene therapy product. VCN has been stable in bone marrow (BM) and peripheral blood (PB) in all cell lineages during the length of the study, with the latest time point studied at 15 months (BCL002) and ranged from 0.45-2.85 copies per cell in erythroid progenitor cells. BCL11A protein levels evaluated by immunoblot in subject BCL002 at 30 days (PB) and 6 months (BM) post-infusion showed highly effective and selective knockdown of BCL11A in erythroid progenitors with no reduction in BCL11A expression in B lymphoid cells. The number of HbF-containing cells (F cells) was assessed by flow cytometry and the kinetics of F cell production was remarkably similar in all subjects. The two untransfused subjects (BCL002 and BCL004) produced 70% F-cells in PB at 3 and 5 months, which has remained stable until the last point assayed (15 months and 7.5 months, respectively) (table). Calculated average HbF per F cell was 〉10pg in all subjects (table) and quantitative single cell HbF flow analysis showed the majority of F cells had 〉4pg F/cell, a level that is believed to prevent sickling under physiological oxygen saturation (Rakotoson et al., ASH 2017). In both untransfused subjects, total Hb remained stable with evidence of reduced hemolysis by reticulocyte count (slightly elevated) and LDH (normal in one subject, slightly elevated in the other). At the 3-month timepoint before re-starting transfusions, the subject with moyamoya (BCL003) had a pre-transfusion Hb of 11 g/dL with 76% of non-transfused cells containing on average 17pg F/F cell. For all subjects, we estimated the fraction of RBCs containing significant Hb sickle polymers and the amount of polymer in each sickled RBC at physiologic oxygen tension (where 50% of monomeric hemoglobin was oxygen saturated, or the P50) (Di Caprio et al. PNAS 2019, in press). The results for all 3 subjects in this adult cohort showed fewer RBCs with significant Hb polymer than two hydroxyurea-responsive treated comparators and significantly less Hb polymer per sickled RBC than a third highly responsive hydroxyurea-treated comparator. In conclusion, these data demonstrate successful and sustained engraftment in three adult patients treated with LVV-delivered shmiR technology targeting BCL11A. Early results suggest an acceptable safety profile, validation of BCL11A as effective target for HbF induction in humans with high numbers of F cells in circulation containing high levels of HbF per F cell, and mitigation of cellular pathology of SCD. Disclosures Achebe: Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pharmacosmos: Membership on an entity's Board of Directors or advisory committees; Fulcrum Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees. Bartolucci:Novartis: Membership on an entity's Board of Directors or advisory committees; AddMedica: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; HEMANEXT: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees. Heeney:AstraZeneca: Research Funding; Micelle Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Novartis: Consultancy, Research Funding; Ironwood / Cyclerion: Research Funding; Vertex / Crisper Therapeutics: Other: Data Safety Monitoring Board. Higgins:Sanofi: Consultancy, Research Funding. Nikiforow:Kite/Gilead: Honoraria; Novartis: Honoraria; NKarta: Honoraria. Wood:Sanofi: Consultancy, Research Funding. Williams:Alerion Biosciences: Other: Co-founder; Novartis: Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder, Patents & Royalties: Potential for future royalty/milestone income, X-SCID., Research Funding; bluebird bio: Patents & Royalties: Licensed certain IP relevant to hemoglobinopathies to bluebird bio. Received payment in the past bluebird bio through a BCH institutional licensing agreement and there is a potential for future royalty/milestone income from this agreement., Research Funding.

Description: Introduction: Chimeric Antigen Receptor (CAR) T cell therapies directed against B-cell maturation antigen (BCMA) have demonstrated compelling clinical activity and manageable safety in subjects with relapsed and refractory Multiple Myeloma (RRMM). These CAR T cells encode humanized or murine scFvs, or camelid heavy chain antibody fragments with CD3-zeta in combination with 41BB or CD28 co-stimulatory domains. In contrast, CART-ddBCMA is an anti-BCMA investigational CAR T cell therapy encoding a non-scFv, synthetic binding domain targeting BCMA with a 4-1BB costimulatory motif and CD3-zeta T cell activation domain. The binding domain is a small stable protein comprising 73 amino acids engineered to reduce the risk of immunogenicity. CART-ddBCMA is being studied as part of a Master Phase 1 Cell Therapy protocol for RRMM and is a first-in-human clinical study to assess the safety, pharmacokinetics, immunogenicity, efficacy, and duration of effect. Methods: ARC-101 (NCT04155749), ARM 1 (CART-ddBCMA) is a Phase 1, multi-center, open label, dose escalation trial enrolling approximately 12 subjects with RRMM who have received ≥ 3 prior regimens, including a proteasome inhibitor, an immuno-modulatory agent, and a CD38 antibody or are triple-refractory. There is no prescreening or requirement for BCMA expression on tumor cells. Peripheral blood mononuclear cells are collected via leukapheresis and sent to a central facility for selection, transduction, and expansion on the CliniMACS Prodigy® system. The drug product is cryopreserved and undergoes release testing prior to being returned to the site for infusion. Subjects undergo lymphodepletion with fludarabine (30 mg/m2) and cyclophosphamide (300 mg/m2) daily for 3 days, then receive CART-ddBCMA as a single infusion. Planned dose levels are 100, 300, and 900 x 106 CAR+ T cells. The primary outcome measure is incidence of adverse events (AEs), including dose-limiting toxicities (DLTs). Additional outcome measures are quality and duration of clinical response assessed according to the IMWG Uniform Response Criteria for MM, evaluation of minimal residual disease (MRD), progression-free and overall survival, and quantification of CAR+ cells in blood. Results: As of July 1, 2020, 4 subjects (median age 73.5 [min;max 73 to 75]) were enrolled and 4 received CART-ddBCMA. Median follow-up after CART-ddBCMA infusion was 100 days (min:max 9 to 142 days), 3 subjects were evaluable for initial safety and clinical response and 1 subject was pending assessment. All subjects received a dose of 100 x 106 CAR+ T cells±20%, median drug product CAR+ expression was 76% (min:max 72-78%) of total CD3+ T cells. Subjects had a median of 5 (min;max 5 to 7) prior lines of therapy and one had prior autologous stem cell transplant; one had high-risk cytogenetics. All 4 subjects had previously received Bort/Len/Car/Pom/Dara and 2 were penta-refractory. Three subjects had high tumor burden, with 95, 95, and 70% bone marrow plasma cells pre-infusion, respectively. Three subjects developed Grade 2 cytokine release syndrome (CRS) and 1 subject developed Grade 2 ICANS. These adverse effects resolved quickly after intervention; 3 subjects received tocilizumab and 2 received steroids (dexamethasone). All 3 evaluable subjects have demonstrated clinical response per IMWG criteria: currently 1 sCR (MRD-10-4), 1 sCR, 1 sCR (MRD-10-6). MRD negative results were obtained by next-generation sequencing (Adaptive clonoSEQ), 1 subject did not have baseline bone marrow involvement. Extramedullary disease resolved in three subjects. CAR+ T cell expansion during the first 30 days was observed in evaluable subjects by ddPCR. No post treatment ADA were detected in the first 3 subjects, through M1. Conclusions: In the initial cohort receiving 100 x 106 CAR+ T cells of CART-ddBCMA, no subjects experienced severe CRS and/or ICANs. Early efficacy results are encouraging, with all 3 evaluable subjects demonstrating deep clinical responses of sCR, with 2 MRD negative bone marrow responses at 1 month. No evidence of ADA has been detected to date. These data are encouraging in a small group of elderly subjects who did not initially receive autologous transplant following induction therapy. Subjects continue to be enrolled and treated. Additional subjects, and longer follow-up will establish whether treatment with CART-ddBCMA results in durable CAR+ T responses. Disclosures Frigault: Celgene: Consultancy; Novartis: Consultancy, Research Funding; Arcellx: Consultancy; Gilead/Kite: Consultancy, Research Funding. Bishop:Kite: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Honoraria, Speakers Bureau; Autolus: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; CRSPPR Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Speakers Bureau. O'Donnell:Celgene: Consultancy. Raje:Celgene: Consultancy. LeFleur:Arcellx: Current Employment, Current equity holder in private company. Buonato:arcellx: Current Employment, Current equity holder in private company. Edwards:arcellx: Current Employment, Current equity holder in private company. Richman:arcellx: Current equity holder in private company. Polianova:arcellx: Current Employment, Current equity holder in private company. Sabatino:Arcellx: Current equity holder in private company. Currence:Arcellx: Current Employment, Current equity holder in private company. Shen:Arcellx: Current Employment, Current equity holder in private company. Quigley:Arcellx: Current Employment, Current equity holder in private company. Maus:arcellx: Consultancy, Research Funding; kite: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

Description: Background Relapse of acute myeloid leukemia (AML) after allogeneic stem cell transplant has a poor prognosis with limited treatment options. Cytokine-induced memory like natural killer (CIML NK) cells are a novel therapy with enhanced cytotoxicity independent of KIR ligand interactions able to induce remission of relapsed/refractory AML (Romee et al, Science TM 2016). We are evaluating the safety and potential efficacy of donor-derived CIML NK cells in patients with relapsed myeloid malignancies after haploidentical donor transplant (haploSCT) in a phase 1/1b study (clinicaltrials.gov: NCT040247761). Here we report on manufacturing, safety, and in vivo correlative biology of the adoptively transferred CIML NK cells in the first 3 enrolled patients. Methods The primary endpoint is to identify the maximum-tolerated dose (MTD) of CIML NK cells in patients with MDS, MPN, or AML who relapsed after haplo-SCT. NK cells were enriched from donor-derived non-mobilized leukapheresis product via two-step CD3 depletion followed by selection of CD56+ cells using the CliniMACS reagent system (Miltenyi Biotec). The enriched NK cell product was cultured for 12-16 hours in X-VIVO 15 media containing rhIL-12, rhIL-15, and rhIL-18 to generate CIML NK cells (Figure 1A). Patients in the current cohort were lymphodepleted with fludarabine 25mg/m2 daily for 3-5 days and cyclophosphamide 60mg/kg daily for 2 days followed by CIML NK cells at a dose of 5-10 x 106 cells/kg and IL-2 106 IU/m2 QOD for 7 doses. Dose-limiting toxicities were evaluated for 6 weeks following NK cell infusion. Response to therapy was assessed at day+28 following CIML NK cell infusion. Results Patient #001 has FLT3-ITD AML that relapsed 5 months after a reduced intensity (RIC) haploSCT. She received 7.4x106 NK cells/kg followed by IL-2 106 IU/m2 QOD for 7 doses. Her day+28 bone marrow had no leukemia blasts although FLT3-ITD mutation remained detectable. Two months post-CIML NK the leukocyte and granulocyte chimerism were 88% and 89%, respectively. Patient #002 has AML with multiple pathogenic variants, including a potentially pathogenic variant in TP53. His disease relapsed 15 months post haplo-SCT with repeat marrow showing all the original mutations, including the TP53 variant (VAF 50.5%). He received 9.5x106 NK cells/kg followed by IL-2 106 IU/m2 QOD for 7 doses. His day+28 marrow had trilineage hematopoiesis without any mutations, including no TP53 mutation (Figure 1B). Two months post-CIML NK the leukocyte and granulocyte chimerism were both 99%. Patient #003 has MDS whose disease relapsed 8 months post RIC haploSCT with persistence of all her diagnostic pathogenic mutations. She received approximately 9.2x106 NK cells/kg and has only just completed IL-2 106 IU/m2 QOD for 7 doses. Among the 3 patients, the main toxicity was prolonged cytopenia requiring stem cell boost in one case. Donor NK cells demonstrated a dramatic shift from a predominantly CD56dimCD16hi (88% of NK cells) to a CD56dimCD16lo phenotype (49% of NK cells) as CIML NK cells. Infused CIML NK cells expanded massively, with approximately 50-fold, 10-fold, and 15-fold maximum in vivo expansion in the first three patients, respectively (Figure 1C). CIML NK cells were the major population in the day+28 marrows in both patients #001 and #002, with CD56+CD7+ cells constituting 89% of the cellularity in the former and 48% in the latter (Figure 1C). CIML NK cells persisted for 3 and 6 months post-infusion in the first two patients. The NK cells were predominantly mature, with most expressing CD16 and low levels of the inhibitory receptor NKG2A. PD-1 expression was much lower on the expanded NK cells vs the pre-infusion donor-derived NK cells. There was minimal concurrent expansion of CD4+CD25+ T-regulatory cells (Figure 1D). Conclusion We show with the first 3 patients in this trial that CIML NK cells can be generated and infused safely, can expand massively in the peripheral blood and bone marrow within the first 30 days post-infusion, and can persist for several months. In addition, CIML NK cell infusion can reduce the burden of pathogenic variant alleles to below the limit of detection, including the burden of high-risk mutations such as in TP53. Though our results are preliminary, the massive in vivo expansion and long-term persistence of adoptively transferred CIML NK cells underscores the unique biology of these cells that makes them an attractive option for cellular therapy protocols. Disclosures Nikiforow: Kite: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Nkarta: Membership on an entity's Board of Directors or advisory committees. Rambaldi:Equillium: Research Funding. Cutler:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kadmon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Medsenic: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Mesoblast: Consultancy, Membership on an entity's Board of Directors or advisory committees. Koreth:Cugene: Membership on an entity's Board of Directors or advisory committees; Regeneron: Other: Research Support; Clinigen: Other; Miltenyi: Other: Research Support; BMS: Other: Research Support; Therakos: Membership on an entity's Board of Directors or advisory committees; Equillium: Consultancy; EMD Serono: Consultancy; Biolojic Design Inc: Consultancy; Amgen: Consultancy; Moderna Therapeutics: Consultancy. Wu:Pharmacyclics: Research Funding; BionTech: Current equity holder in publicly-traded company. Soiffer:Juno: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; VOR Biopharma: Consultancy; Mana Therapeutics: Consultancy; Precision Bioscience: Consultancy; Cugene: Consultancy; Rheos Therapeutics: Consultancy; Kiadis: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; alexion: Consultancy; Be the Match/ National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees. Ritz:TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Rheos Medicines: Consultancy; LifeVault Bio: Consultancy; Avrobio: Consultancy; Kite Pharma: Research Funding; Equillium: Research Funding; Amgen: Research Funding; Falcon Therapeutics: Consultancy; Infinity Pharmaceuticals: Consultancy.