Abstract: Relapse after allogeneic hematopoietic stem cell transplantation (HSCT) remains an unmet medical need. The ETCTN 9204 study evaluated in 71 study subjects if immune checkpoint blockade with anti-CTLA-4 (Ipilimumab (Ipi), n = 43) or anti-PD-1 (Nivolumab (Nivo), n = 28) antibodies could stimulate graft-versus-leukemia (GvL) responses in this setting. We focused mainly on patients (pts) with relapsed AML/MDS, which constituted the majority of pts (n = 38; 54%). Ipi induced both long-term complete remissions (CR; n = 3) and transient CRs (TR; n = 3), while Nivo did not generate any CRs, but 4 patients demonstrated partial remissions (PR). To define the molecular features associated with response to Ipi, we performed bulk transcriptomic analyses of formalin-fixed paraffin-embedded biopsies of sites of AML/MDS involvement (n = 35) collected before and after Ipi treatment from 13 pts. Our analysis of matched pre- and post-Ipi samples of patients with CRs identified 50 differentially expressed genes. By gene ontology analysis, these were enriched for signatures of 'lymphocyte activation' and 'antigen-receptor signaling'. Principal component analysis using these genes separated post-Ipi CR samples as a distinct cluster, transcriptionally apart from pre-Ipi CR samples and from non-responder (NR, n = 15) pre and post-Ipi samples. Of note, post-Ipi CR samples were similar to both pre- and post-Ipi TR samples as well as samples from GvHD/toxicity biopsies (n = 9). Consistent with these findings, we detected increased CD8+ T cell abundance post-Ipi in CR but not NR samples with CIBERSORTx (pre vs post, median score 0.043 vs. 0.56, p & lt; 0.01). Likewise, reconstruction of T cell receptor (TCR) CDR3 sequences using the tool TRUST showed an increase in TCR clonotypes per million reads post-Ipi in CR samples (pre vs post, median 0.33 vs. 1.65, p & lt; 0.05). In sum, response to CTLA-4 blockade is characterized by transcriptional evidence of T cell infiltration and activation within the tumor microenvironment, with similar gene programs observed in the setting of GvHD. To determine if the changes within tissue sites following Ipi are also observed in peripheral blood (PB), we analyzed the immunophenotype and TCR repertoire of T cells from serially collected PB samples from pts with AML/MDS (n = 14) or non-myeloid malignancy (n = 6). In responders and non-responders, exposure to Ipi was associated with decreased naïve, increased effector memory, and increased expression of HLA-DR on central memory T cells (p & lt; 0.05), consistent with T cell differentiation and activation. From 9 of 14 AML/MDS pts (CR = 4, NR = 5), bulk TCR sequencing before and after 1 cycle of Ipi yielded 572,017 PB TCR sequences. Only 776 clonotypes demonstrated significant change in frequency, and these TCRs were detected in greater proportion among responders (613/776 versus NR 163/776, p & lt; 0.01). Thus, Ipi alters the differentiation states of circulating T cells irrespective of response and leads to higher TCR repertoire turnover in CR patients. Of the AML patients achieving PR following Nivo, we also observed transcriptional evidence of CD8+ T cell infiltration in one patient. This signature, however, was not detected in a second such patient. In a separate instance, we had the opportunity to dissect the molecular features of a partial response in a patient with JAK2V617F+ secondary AML following Nivo through single-cell RNA and ATAC-sequencing (scRNA-seq and scATAC-seq, 10x Genomics) of PB mononuclear cells collected serially across 6 timepoints. In total, we analyzed the transcriptomes and chromatin accessibility data from of 27,777 and 28,713 individual cells, respectively. At time of response, non-exhausted CD4+ T cells expanded while both exhausted CD8+ T cells and a malignant subpopulation with increased expression of PD-L1 and features of megakaryocytic differentiation preferentially contracted. The subsequent expansion of a PD-L1- malignant population at progression suggests that decreased leukemic PD-L1 expression was associated with relapse. Altogether, these data highlight the molecular and cellular features of successful reinvigoration of GvL using CTLA-4 blockade, from increased local T cell infiltration and activation in the leukemic microenvironment to peripheral T cell turnover. In addition, the selection of therapy-resistant subclones after PD-1 blockade underscores the need for further high-resolution studies of GvL responses. Disclosures Zeiser: Novartis: Honoraria; Incyte: Honoraria; Malinckrodt: Honoraria. Ritz:Rheos Medicines: Consultancy; LifeVault Bio: Consultancy; Infinity Pharmaceuticals: Consultancy; Falcon Therapeutics: Consultancy; Avrobio: Consultancy; Kite Pharma: Research Funding; Talaris Therapeutics: Consultancy; Equillium: Research Funding; Amgen: Research Funding; TScan Therapeutics: Consultancy. Neuberg:Madrigak Pharmaceuticals: Current equity holder in publicly-traded company; Celgene: Research Funding; Pharmacyclics: Research Funding. Soiffer:alexion: Consultancy; Gilead: Consultancy; Be the Match/ National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Rheos Therapeutics: Consultancy; Juno: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Mana Therapeutics: Consultancy; Precision Bioscience: Consultancy; VOR Biopharma: Consultancy; Kiadis: Membership on an entity's Board of Directors or advisory committees; Cugene: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees. Liu:GV20 Oncotherapy: Consultancy, Membership on an entity's Board of Directors or advisory committees; 3DMed Care: Consultancy; Sanofi: Research Funding; Takeda: Research Funding. Davids:AbbVie: Consultancy; Gilead Sciences: Consultancy; Sunesis: Consultancy; BeiGene: Consultancy; AstraZeneca: Consultancy, Research Funding; Syros Pharmaceuticals: Consultancy; Research to Practice: Honoraria; Pharmacyclics: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Verastem: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy; Janssen: Consultancy; Surface Oncology: Research Funding; Novartis: Consultancy, Research Funding; MEI Pharma: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Eli Lilly: Consultancy; Zentalis: Consultancy; Celgene: Consultancy; Bristol Myers Squibb: Research Funding; Merck: Consultancy; Ascentage Pharma: Consultancy, Research Funding. Wu:Pharmacyclics: Research Funding; BionTech: Current equity holder in publicly-traded company. OffLabel Disclosure: Ipilimumab and Nivolumab were tested in the setting of relapsed hematological malignancies after allogeneic stem cell transplantation.

Abstract: Marrow CD8+ T-cell infiltrates may be a novel predictor of response to donor lymphocyte infusions in patients with relapsed CML. Reversal of T-cell exhaustion is tightly linked to effective antileukemia responses to donor lymphocyte infusions.

Abstract: 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.

Abstract: Vaccination using irradiated, adenovirus transduced autologous myeloblasts to secrete granulocyte–macrophage colony-stimulating factor (GVAX) early after allogeneic hematopoietic stem cell transplantation (HSCT) can induce potent immune responses. We conducted a randomized phase 2 trial of GVAX after HSCT for myelodysplastic syndrome with excess blasts or relapsed/refractory acute myeloid leukemia. Myeloblasts were harvested before HSCT to generate the vaccine. Randomization to GVAX vs placebo (1:1) was stratified according to disease, transplant center, and conditioning. Graft-versus-host disease (GVHD) prophylaxis included tacrolimus and methotrexate. GVAX or placebo vaccination was started between day 30 and 45 if there was engraftment and no GVHD. Vaccines were administered subcutaneously/intradermally weekly × 3, then every 2 weeks × 3. Tacrolimus taper began after vaccine completion. A total of 123 patients were enrolled, 92 proceeded to HSCT, and 57 (GVAX, n = 30; placebo, n = 27) received at least 1 vaccination. No Common Toxicity Criteria grade 3 or worse vaccine-related adverse events were reported, but injection site reactions were more common after GVAX (10 vs 1; P = .006). With a median follow-up of 39 months (range, 9-89 months), 18-month progression-free survival, overall survival, and relapse incidence were 53% vs 55% (P = .79), 63% vs 59% (P = .86), and 30% vs 37% (P = .51) for GVAX and placebo, respectively. Nonrelapse mortality at 18 months was 17% vs 7.7% (P = .18), grade II to IV acute GVHD at 12 months was 34% vs 12% (P = .13), and chronic GVHD at 3 years was 49% vs 57% for GVAX and placebo (P = .26). Reconstitution of T, B, and natural killer cells was not decreased or enhanced by GVAX. There were no differences in serum major histocompatibility chain-related protein A/B or other immune biomarkers between GVAX and placebo. GVAX does not improve survival after HSCT for myelodysplastic syndrome/acute myeloid leukemia. This trial was registered at www.clinicaltrials.gov as #NCT01773395.

Abstract: Cytokine release syndrome (CRS) following haploidentical hematopoietic cell transplantation (HCT) resembles CRS after chimeric antigen receptor-T therapy. We conducted this single-center retrospective study to evaluate the association of posthaploidentical HCT CRS with clinical outcomes and immune reconstitution. One hundred sixty-nine patients who underwent haploidentical HCT between 2011 and 2020 were identified. Of these, 98 patients (58%) developed CRS after HCT. CRS was diagnosed based on the presence of fever within the first 5 days after HCT without evidence of infection or infusion reaction and was graded according to established criteria. The development of posthaploidentical HCT CRS was associated with a lower incidence of disease relapse (P = .024) but with an increased risk of chronic graft-versus-host disease GVHD (P = .01). The association of CRS with a lower incidence of relapse was not confounded by graft source or disease diagnosis. Neither CD34 nor total nucleated cell dose was associated with CRS independently of graft type. In patients developing CRS, CD4+ Treg (P  & lt; .0005), CD4+ Tcon (P  & lt; .005), and CD8+ T cells (P  & lt; .005) increased 1 month after HCT compared with those who did not develop CRS, but not at later time points. The increase in CD4+ regulatory T cells 1 month after HCT was most notable among patients with CRS who received a bone marrow graft (P  & lt; .005). The development of posthaploidentical HCT CRS is associated with a reduced incidence of disease relapse and a transient effect on post-HCT immune reconstitution of T cells and their subsets. Therefore, the validation of these observations in a multicenter cohort is required.