Magenta Therapeutics Research Articles

The Dual PI3Kδγ Inhibitor Duvelisib Potently Inhibits IL-6 Production and Cytokine Release Syndrome (CRS) While Maintaining CAR-T Function in Vitro and In Vivo

Despite remarkable clinical efficacy, CAR-T therapy has been limited by life-threatening toxicities in over 30% of patients (Maude, NEJM 2014 and Davila, SciTransMed 2014). Toxicities primarily manifest as Cytokine Release Syndrome (CRS) characterized by an early phase with fever, hypotension, and elevations of cytokines including IFNγ, GM-CSF, TNF, IL-10, and IL-6. Using a protein kinase inhibitor library containing 644 independent compounds, we aimed to identify compounds that could block CRS-related cytokine production without inhibiting CAR-T function. We identified, duvelisib (kindly provided by Verastem Oncology, Needham, MA), a novel and selective dual PI3K-δ,γ inhibitor as a potent inhibitor of CRS in vitro and in vivo without attenuating CAR-T function. Duvelisib (Copiktra) is approved for the treatment of relapsed/refractory CLL after 2 prior therapies and follicular lymphoma after 2 prior systemic therapies; the latter gained accelerated approval status based on overall response rate and continued approval may be contingent on confirmatory trials. To assess the ability of duvelisib to inhibit CAR-T mediated CRS, we performed an in vitro CRS assay (Singh, Cytotherapy, 2017). CART19 (19-28BBζ, 25,000 cells), Ramos (CD19+, 50,000 cells), and immature dendritic cells (iDC, 2,500 cells) were co-cultured in a 96 well plate for 48hrs in the presence of varying concentrations of duvelisib (0.3nM-1000nM). Secreted IL-6, a surrogate marker of CRS, was determined using a human IL-6 ELISA (R&D Systems). Duvelisib reduced IL-6 levels in a dose-dependent manner with 30nM duvelisib reducing IL-6 secretion more than 10-fold (Fig 1a). To confirm that duvelisib did not inhibit CAR-T function, we performed in vitro killing assays, in which CAR-T efficacy was determined using BLI imaging of luciferase labeled CD19+ Ramos targets. At clinically relevant therapeutic doses (C max of 200 to 500nM) of duvelisib, there was no effect on CAR-T function in vitro. Treatment with 10nM duvelisib resulted in a statistically insignificant ~20% reduction of CART19 efficacy (p>0.05) (Fig 1b). Of interest, although selective inhibitors of either or PI3Kδ (GSK2292767) or PI3Kγ (IPI549) had modest effects on blocking CAR-T induced IL-6 production in this in vitro model, the effect of combining both inhibitors had a more dramatic effect similar to the dual PI3K-δ,γ inhibitor, duvelisib. Next we assessed the ability of duvelisib to block IL-6 secretion in vivo using a fully immunocompetent murine model of CRS. Six-week old BALB/c mice were injected with the mitogenic anti-CD3ε antibody, 145-2C11 (10 µg/mouse). Duvelisib (450µg/mouse) was administered daily intraperitoneally (I.P), with the first dose of duvelisib injected 24 hours prior to injection of 145-2C11. Plasma IL-6 levels were determined using a mouse IL-6 ELISA (R&D Systems). Injection of 145-2C11 acutely elevated plasma IL-6 >32 fold relative to non-treated controls (4hrs; Control 34.4 pg/mL versus vs. 145-2C11 1088±99.6 pg/mL). Duvelisib significantly reduced mean plasma IL-6 >54% at 4hrs (Vehicle 1088±99.6 pg/ml vs duvelisib 492±99.6 pg/ml, p≤ 0.01) and >78% at 24hrs (Vehicle 220±101 pg/ml vs. duvelisib 49.3±16.1, p≤ 0.01) (Fig 1c) consistent with the effect of duvelisib in our in vitro CAR-T-induced CRS model described above. These studies demonstrate that duvelisib can inhibit CAR-T induced IL-6 production from iDC while having no inhibitory effect on CAR-T. Experiments are currently in progress to further characterize PI3K-δ,γ inhibition in humanized mouse models of CRS and CAR-T efficacy. Our preclinical data suggest that dual PI3K-δ,γ inhibition with duvelisib may represent an attractive alternative to IL-6 receptor antagonists, such as tocilizumab, for the treatment of CAR-T-associated cytokine release syndrome in the clinic which warrants further clinical evaluation. Figure 1. Dose dependent effect of duvelisib on IL-6 secretion in co-culture of CART19, Ramosluc, and iDC after 48 hours (a) Dose dependent inhibition of CART19 mediated cytotoxicity using duvelisib (b) Duvelisib effectively lowers IL-6 plasma level in an immunocompetent BALB/cJ mouse model of CRS (c). (** p≤ 0.01 and ns p>0.05). Figure 1 Disclosures Cooper: Wugen: Consultancy, Current equity holder in private company, Patents & Royalties. Pachter:Verastem Therapeutics: Current Employment. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

RNA and TCR Sequencing Shed Light on Mechanisms of Treg Suppression in a Murine Model of Acute GvHD

Introduction Regulatory T cell (Treg) based therapies are a promising approach for graft-versus-host disease (GvHD) prevention and treatment. However, mechanisms of Treg suppression of alloreactive conventional T cells (Tcon) in GvHD are incompletely understood. In this study, we performed paired RNA and T cell receptor (TCR) sequencing analysis on Tcon and Treg before and after transplantation to further elucidate Treg suppressive function during in vivo suppression of acute GvHD in an MHC major-mismatch mouse model. Methods CD45.2 Thy1.2 BALB/c mice were lethally irradiated (8.8 Gy) and transplanted with 5x106 T cell- depleted bone marrow cells (TCD-BM) from CD45.1 Thy1.2 C57Bl/6 mice alone or together with CD45.2 Thy1.2 C57Bl/6 FoxP3/GFP+ Treg (1x106) on day 0. On day 2, CD45.1 Thy1.1 C57Bl/6 Tcon (1x106; CD4:CD8 ratio = 2:1) were injected to induce GvHD. Irradiated (11 Gy) syngeneic C57Bl/6 recipients receiving C57Bl/6 TCD-BM and CD45.1 Thy1.1 Tcon alone were used as controls. On day 8, donor Thy1.1+ CD45.1+ CD4 and CD8 Tcon and Thy1.2+CD45.2+ FoxP3/GFP+ Treg were isolated by FACS from spleens and lymph nodes. T cell subsets before injection and recovered at day 8 were analyzed by paired RNA and TCR sequencing. Results The transcriptomic analysis revealed a dominant effect of the allogeneic transplant procedure on the clustering of the different T cell populations. Principal component analysis (PCA) of the top 1000 most differentially expressed genes revealed that 68% of the variance was explained by PC1, which clearly segregated CD4and CD8Tcon recovered at day 8 from allogeneic recipients from cells before injection or recovered from syngeneic recipients (Fig. 1). PC1 was mainly driven by naïve T cell genes (Ccr7, Sell, Il6ra, Il6st, Foxo1) that were progressively downregulated along PC1. Analysis of the TCR repertoire based on sequencing of the TCR alpha chain revealed a progressive clonal restriction along PC1 in CD4 and CD8 T cells (Fig. 1). Accordingly, clonal overlap between cells collected at day 8 and cells analyzed before injection were reduced in allogeneic recipients (Morisita Index [MI], CD4: 0.06±0.01; CD8: 0.02±0.01) compared to syngeneic controls (MI, CD4: 0.23±0.26; CD8: 0.57±0.05). Treg administration did not affect CD4 or CD8 T cell segregation along PC1, suggesting that they minimally interfered with cell activation and differentiation during GvHD. Accordingly, Treg did not inhibit clonal restriction (Fig.1) nor the reduction in clonal overlap (MI, CD4: 0.04±0.01; CD8: 0.01±0.01), indicating that Treg did not inhibit the initial activation of alloreactive T cells clones. Treg impact on CD4 but not CD8 Tcon transcriptome was revealed by PC2 (Fig.1). Treg induced the downregulation of TH1-signature genes (Tbx21, Il12rb1, Il12rb2, Stat4) and proinflammatory genes (Il18rap) while promoting up-regulation of anti-inflammatory genes (Il18bp), TH2 signature genes (Ccr4, Il4) and Il2 in CD4Tcon. Moreover, gene set enrichment analysis (GSEA) revealed that Treg treatment significantly impacted gene sets involved in metabolic processes in CD4and CD8Tcon, leading to a global up-regulation of genes encoding for enzymes involved in oxidative phosphorylation (OXPHOS) and downregulation of genes encoding for enzymes contributing to glycolysis (Slc2a1, Hk1, Pfkl, Pfkp, Pkm). Treg recovered at day 8 preserved a distinct transcriptomic signature observed before injection and further enhanced by the up-regulation of genes involved in Treg activation and suppressive function (Gata3, Tnfrsf18, Tnfrsf4, Icos, Ccr1, Ccr4, Il9r). GSEA in Treg revealed significant up-regulation of genes in the OXPHOS signature. TCR repertoire analysis showed clonal restriction of Treg during GvHD. Direct comparison of clone frequencies in Treg and CD4Tcon showed smaller clonal overlap on day 8 (MI=0.005±0.006) compared to day 0 (MI= 0.11±0.004) suggesting that Treg and CD4Tcon responses during GvHD are engaging different cell clonotypes triggered by different epitopes or antigens. Conclusion Our results indicate that Treg treatment did not interfere with Tcon activation and differentiation of alloreactive Tcon clones in a model of acute GvHD. Treg predominantly affected CD4Tcon and to a lesser extent CD8Tcon transcriptome, modulating the transcription of genes encoding pro- and anti-inflammatory molecules as well as enzymes involved in metabolic processes. Disclosures Turkoz: Adicet Bio: Current Employment, Current equity holder in private company. Negrin:Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company; BioEclipse Therapeutics: Current equity holder in private company; Amgen: Consultancy; KUUR Therapeutics: Consultancy; Biosource: Current equity holder in private company; UpToDate: Honoraria.

Molecular Imaging of Chimeric Antigen Receptor T Cells By ICOS-Immunopet

Introduction: Immunomonitoring of chimeric antigen receptor (CAR) T cells relies primarily on their quantification in the peripheral blood, which inadequately quantifies their biodistribution and activation status in the tissues. Non-invasive molecular imaging of CAR T cell therapy by positron emission tomography (PET) is a promising approach providing spatial, temporal and functional information. Reported strategies for PET-based monitoring of CAR T cells rely on additional manipulation of the cell product such as the incorporation of reporter transgenes or ex vivo biolabeling, which significantly limits the wider application of CAR T cell molecular imaging. In the present study, we assessed the ability of antibody-based PET (immunoPET) to non-invasively visualize CAR T cells in vivo. Methods: For analysis of human CAR T cell activation, we analyzed publicly available RNA sequencing data (GSE136891) obtained at serial time points during in vitro culture of CD19.CD28z CAR T cells. We analyzed by mass cytometry (CyTOF) the ex vivo ICOS expression on human CD19-28z CAR T cells obtained from 31 patients receiving axicabtagene ciloleucel (Axi-cel) for relapsed/refractory diffuse large B-cell lymphoma (DLBCL). For in vivo murine experiments, CD19-expressing B-cell lymphoma A20 cells (2.5×10e5 cells) were injected by tail vein intravenously (i.v.) into sub-lethally (4.4 Gy) irradiated Thy1.2+ BALB/c mice. Seven days later, murine CD19.CD28z Luc+ Thy1.1+ CAR T cells (1×10e6) were i.v. injected. ICOS expression was analyzed by flow cytometry on CAR T cells recovered from spleen and bone marrow 5 days after injection. For imaging studies, anti-ICOS monoclonal antibody (mAb) specific for murine ICOS (clone:7E.17G9, BioXcell) was modified with the bifunctional chelator deferoxamine (DFO/p-SCN-Bn-Deferoxamine). The DFO-ICOS mAb conjugate was radiolabeled with 37 MBq (~1 mCi) of 89Zr-oxalate (final specific activity 6 µCi/µg/ml and radiochemical purity of 99%). 89Zr-DFO-ICOSmAb (45 μCi ± 3.6, 7.5 μg± 0.6) was injected i.v. 5 days post-CAR T cell administration and PET-CT imaging performed 48 hours later. Following PET-CT, mice were euthanized and radioactivity measured in dissected weighed tissues using a gamma-counter. Results: Analysis of RNA-sequencing data from human CAR T cells identified ICOS as an activation marker whose transcription was up-regulated and sustained during in vitro culture. ICOS was preferentially expressed on CAR+ T cells recovered at day 7 from axi-cel treated patients compared with CAR- cells (p<0.001; Figure 1A). Phenotypic analysis in a murine model of B cell lymphoma infiltrating the spleen and the bone marrow confirmed preferential ICOS expression on murine CAR T cells compared to resident cells in both spleen (p=0.003) and bone marrow (p=0.008). Figure 1B shows representative volume-rendered technique (VRT) PET/CT images of 89Zr-DFO-ICOS mAb-injected tumor-bearing mice either untreated (left panels) or that received mCD19.28z CAR T cells (right panels). 89Zr-DFO-ICOS mAb similarly accumulated in highly vascularized organs (heart, liver and spleen) of both untreated and CAR T cell treated mice, consistent with the biodistribution and clearance of intact antibodies. We detected pronounced 89Zr-DFO-ICOS mAb-PET signals in the bones of CAR T cell treated mice, particularly prominent in the lumbar spine, iliac bones, femur, tibia and humeral heads (Figure 1B). Region of interest analysis confirmed markedly increased radiotracer uptake in bones rich in bone marrow from CAR T treated mice compared with those of untreated mice (lumbar spine vertebrae p<0.001; iliac bones p=0.001; femur p=0.002; tibia p=0.002). Moreover we observed a slight, but statistically significant increase in radiotracer accumulation in the heart of CAR T cell-treated mice (p=0.004) while no significant differences were detected in spleen and liver. As expected, there was no significant signal difference in the muscle, considered background. Biodistribution analysis using gamma counting of tissues confirmed the PET results. Conclusions: We describe for the first time an immunoPET approach to monitor the in vivo dynamics of CAR T cell migration, expansion, and persistence that does not require the addition of reporter genes or ex vivo labeling, being therefore applicable to the clinical setting for the study of any commercially available and investigational CAR T cell products. Disclosures Miklos: Novartis: Consultancy, Other: Travel support, Research Funding; Allogene Therapeutics Inc.: Research Funding; Pharmacyclics: Consultancy, Other: Travel support, Patents & Royalties, Research Funding; Juno-Celgene-Bristol-Myers Squibb: Consultancy, Other: Travel support, Research Funding; Janssen: Consultancy, Other: Travel support; Miltenyi Biotec: Research Funding; Kite-Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Adaptive Biotech: Consultancy, Other: Travel support, Research Funding. Mackall:BMS: Consultancy; Allogene: Current equity holder in publicly-traded company; Lyell Immunopharma: Consultancy, Current equity holder in private company; NeoImmune Tech: Consultancy; Nektar Therapeutics: Consultancy; Apricity Health: Consultancy, Current equity holder in private company. Gambhir:CellSight Inc: Current equity holder in private company. Negrin:Amgen: Consultancy; BioEclipse Therapeutics: Current equity holder in private company; Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company; Biosource: Current equity holder in private company; KUUR Therapeutics: Consultancy; UpToDate: Honoraria.

Efficacy and Safety of Ponatinib (PON) in Patients with Chronic-Phase Chronic Myeloid Leukemia (CP-CML) Who Failed One or More Second-Generation (2G) Tyrosine Kinase Inhibitors (TKIs): Analyses Based on PACE and Optic

Efficacy and Safety of Ponatinib (PON) in Patients with Chronic-Phase Chronic Myeloid Leukemia (CP-CML) Who Failed One or More Second-Generation (2G) Tyrosine Kinase Inhibitors (TKIs): Analyses Based on PACE and Optic

Clinical Outcomes of Patients with Secondary Acute Myeloid Leukemia (sAML) Treated with Hypomethylating Agent Plus Venetoclax (HMA-Ven) or Liposomal Daunorubicin Cytarabine (CPX-351)

Clinical Outcomes of Patients with Secondary Acute Myeloid Leukemia (sAML) Treated with Hypomethylating Agent Plus Venetoclax (HMA-Ven) or Liposomal Daunorubicin Cytarabine (CPX-351)

Upfront Alternative Donor Transplant Versus Immunosuppressive Therapy in Patients with Severe Aplastic Anemia Who Lack Fully HLA Matched Related Donor: Systematic Review and Meta-Analysis of Retrospective Studies. on Behalf of the Severe Aplastic Anemia Working Party of European Group for Blood and Marrow Transplantation (SAAWP of EBMT)

Upfront Alternative Donor Transplant Versus Immunosuppressive Therapy in Patients with Severe Aplastic Anemia Who Lack Fully HLA Matched Related Donor: Systematic Review and Meta-Analysis of Retrospective Studies. on Behalf of the Severe Aplastic Anemia Working Party of European Group for Blood and Marrow Transplantation (SAAWP of EBMT)

Addressing Recruitment Challenges in the Engage-HU Trial in Young Children with Sickle Cell Disease

Addressing Recruitment Challenges in the Engage-HU Trial in Young Children with Sickle Cell Disease

Safety Analysis of Patients Who Received Ruxolitinib for the Treatment of Steroid-Refractory Chronic Graft-Versus-Host Disease in an Expanded Access Program

Safety Analysis of Patients Who Received Ruxolitinib for the Treatment of Steroid-Refractory Chronic Graft-Versus-Host Disease in an Expanded Access Program

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

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

Predictors of Cytopenia after Treatment with Axicabtagene Ciloleucel in Patients with Large Cell Lymphoma

Predictors of Cytopenia after Treatment with Axicabtagene Ciloleucel in Patients with Large Cell Lymphoma

Long-Term Outcomes of Patients with Peripheral T-Cell Lymphoma after Autologous Hematopoietic Cell Transplantation

Introduction: Patients with T-cell lymphoma have variable clinical manifestations and outcomes depending on the histology and their response to therapies. However, the overall outcomes are not as good as their B-cell lymphoma counterpart with induction chemotherapy alone. Therefore, autologous transplant is often used as consolidation in first remission or at relapse because of the poor outcomes with conventional therapy. We have reported previously on Stanford experience of these patients who underwent autologous transplant before 2007 (BBMT 2008, 14:741). Here, we reported a retrospective review of patients in the modern era (2008-2018) with emphasis on the impact of pre-transplant disease status on outcomes and post-transplant relapse management. Method: Between July 1, 2008 and July 31, 2018, 102 consecutive patients with T-cell lymphoma received high dose chemotherapy/autologous hematopoietic cell rescue at Stanford and constitute the study cohort (Figure 1). This study cohort was selected for adequate follow-up (>2 years) after transplant. Progression free survival (PFS) and overall survival (OS) was estimated from the date of transplant using the Kaplan-Meier method. PFS and OS were compared between groups with different pre-transplant disease status based on response to the last pre-transplant therapies (CR1 vs. PR1 vs. CR2). Result: This study cohort included patients with peripheral T-cell lymphoma, non-specified (n=21), angioimmunoblastic T-cell lymphoma (n=50), ALK-negative anaplastic large-cell lymphoma (n=14), ALK-positive anaplastic large-cell lymphoma (n=5), extranodal NK/T cell lymphoma (n=9), enteropathy-type T-cell lymphoma (n=1), adult T-cell leukemia/lymphoma (n=1) and hepatosplenic T-cell lymphoma (n=1). It had a male/female ratio of 61/41, and a median age of 58 years (range 23-71). At diagnosis the majority of the patients had stage III/IV disease (70%) and B symptoms (56%). The median time from diagnosis to transplant was 8.1 months (range 4-176). The majority of patients were in first complete remission (CR1, n=79) at the time of transplant, while others were in PR1 (n=11) or in CR2 (n=12) from last pre-transplant therapies. Ninety-one (89%) patients received high dose cyclophosphamide/carmustine/etoposide(CBV) and 11 patients received high dose carmustine/etoposide/cytarabine/melphalan (BEAM) prior to autograft infusion. Median follow-up post-transplant was 36.8 months (range 0.7-130) for the entire cohort. The estimated 3-year PFS and OS were 60% (95% CI 49-68%) and 75% (95% CI 65-82%), respectively (Figure 2A). Patients who were in CR1 had significantly better median PFS compared to patients in PR1 or CR2 (7.04 vs 1.19 years, p=0.039; 7.04 vs 0.48 years p=0.004, Figure 2B). The estimated 3-year PFS were 67% (95% CI 55-76%), 36% (95% CI 11-63%), and 29% (95% CI 8-56%) for the CR1, PR1 and CR2 groups respectively. Patients who were in CR1 also had significantly better median OS compared to patients in PR1 or CR2 (not reached vs 2.30 years, p=0.018; not reached vs 3.76 years p=0.045, Figure 2C).The estimated 3-year OS were 82% (95% CI 71-89%), 44% (95% CI 14-70%), and 53% (95% CI 21-78%) for the CR1, PR1 and CR2 groups respectively. In this cohort, there were no significant differences in either PFS or OS between different histology. Forty patients experienced disease relapse after transplant. The majority (n=28, 70%) of these patients received additional therapies including chemotherapy (n=13), brentuximab vedotin (n=12), HDAC inhibitor (n=7), and radiation (n=3) with a median systemic therapy of 2 (range 1-5). Thirteen patients eventually underwent allogeneic hematopoietic cell transplantation. The median OS after post-transplant relapse was 21.3 months (Figure 3). Both brentuximab vedotin and allogeneic transplant seemed to provide prolonged survival for these relapsed patients, with estimated 2-year post-relapse OS were 75% (95% CI 13-96%) and 63% (95% CI 28-84%) for the two groups respectively. Conclusion: Autologous transplant remains to be a good option as consolidation for patients with T-cell lymphoma, mostly in patients with first complete remission. While close to 40% of the patients experienced relapse after autologous transplant, additional therapies such as brentuximab vedotin or/and allogeneic transplant can provide long-term benefit for these patients. Disclosures Shizuru: Jasper Therapeutics, Inc: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Shiraz:Kite, a Gilead Company: Research Funding; ORCA BioSystems: Research Funding. Muffly:Servier: Research Funding; Amgen: Consultancy; Adaptive: Research Funding. Sidana:Janssen: Consultancy. Meyer:Orca Bio: Research Funding. Rezvani:Pharmacyclics: Research Funding. Miklos:Novartis: Consultancy, Other: Travel support, Research Funding; Pharmacyclics: Consultancy, Other: Travel support, Patents & Royalties, Research Funding; Allogene Therapeutics Inc.: Research Funding; Kite-Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Juno-Celgene-Bristol-Myers Squibb: Consultancy, Other: Travel support, Research Funding; Janssen: Consultancy, Other: Travel support; Miltenyi Biotec: Research Funding; Adaptive Biotech: Consultancy, Other: Travel support, Research Funding. Negrin:Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company; Biosource: Current equity holder in private company; Amgen: Consultancy; BioEclipse Therapeutics: Current equity holder in private company; UpToDate: Honoraria; KUUR Therapeutics: Consultancy.

Third Generation CD20 Targeted CAR T-Cell Therapy (MB-106) for Treatment of Patients with Relapsed/Refractory B-Cell Non-Hodgkin Lymphoma

Third Generation CD20 Targeted CAR T-Cell Therapy (MB-106) for Treatment of Patients with Relapsed/Refractory B-Cell Non-Hodgkin Lymphoma

High Incidence of Venous Thromboembolism in Patients with Chronic Graft-Versus-Host Disease

High Incidence of Venous Thromboembolism in Patients with Chronic Graft-Versus-Host Disease

Myeloma Cell Associated Therapeutic Protein Discovery Using Single Cell RNA-Seq Data

Multiple myeloma (MM) is a hematological cancer of the antibody-secreting plasma cells. Despite therapeutic advancements, MM remains incurable due to high incidence of drug-resistant relapse. In recent years, targeted immunotherapies, which take advantage of the immune system's cytotoxic defenses to specifically eliminate tumor cells expressing certain cell surface and intracellular proteins have shown promise in combating this and other B cell hematologic malignancies. A major limitation in the development of these therapies lies in the discovery of optimal candidate targets, which require both high expression in tumor cells as well as stringent tissue specificity. In an effort to identify potential myeloma-specific target antigens, we performed an unbiased search for genes with specific expression in plasma and/or B cells using single-cell RNA-sequencing (scRNAseq) of 53 bone marrow samples taken from 42 patients. By comparing >40K plasma cells to >97K immune cells across our cohort, we were able to identify a total of 181 plasma cell-associated genes, including 65 that encode cell-surface proteins and 116 encoding intracellular proteins. Of particular interest is that the plasma cells from each patient were shown to be transcriptionally distinct with unique sets of genes expressed defining each patient's malignant plasma cells. Using pathway enrichment analysis, we found significant overrepresentation of cellular processes related to B-Cell receptor (BCR) signaling, protein transport, and endoplasmic reticulum (ER) stress, involving genes such as DERL3, HERPUD1, PDIA4, PDIA6, RRBP1, SSR3, SSR4, TXNDC5, and UBE2J1. To note, our strategy successfully captured several of the most promising MM therapeutic targets currently under pre-clinical and clinical trials, including TNFRSF17(BCMA), SLAMF7, and SDC1 (CD138). Among these, TNFRSF17 showed very high plasma cell expression, with concomitant sharp exclusion of other immune cell types. To ascertain tissue specificity of candidate genes outside of the bone marrow, we analyzed gene and protein expression data from the Genotype-Tissue Expression (GTEx) portal and Human Protein Atlas (HPA). We found further support for several candidates (incl. TNFRSF17,SLAMF7, TNFRSF13B (TACI), and TNFRSF13C) as being both exclusively and highly expressed in lymphoid tissues. While several surface candidates were not found to be lymphocyte-restricted at the protein level, they remain relevant considerations as secondary targets for bi-specific immunotherapy approaches currently under development. To further investigate potential combinatorial targeting, we examine sample-level patterns of candidate co-expression and mutually-exclusive expression using correlation analysis. As the majority of our detected plasma cell-specific genes encode intracellular proteins, we investigated the potential utility of these epitopes as therapeutic targets via MHC presentation. Highly expressed candidates include MZB1, SEC11C, HLA-DOB, POU2AF1, and EAF2. We analyzed protein sequences using NetMHC and NETMHCII to predict high-affinity peptides for common class-I and class-II HLA alleles. To correlate MHC allelic preference with candidate expression in our cohort, we performed HLA-typing for 29 samples using Optitype. To support our scRNAseq-driven findings, we cross-referenced gene expression data with 907 bulk RNA-sequencing samples, including 15 from internal studies and 892 from the Multiple Myeloma Research Foundation (MMRF), as well as bulk global proteomics data from 4 MM cell lines (TIB.U266, RPMI8226, OPM2, MM1ST) and 4 patients. We see consistent trends across both cohorts, with high positive correlation (Pearson R ranging between 0.60 and 0.99) for a majority of genes when comparing scRNA and bulk RNA expression in the same samples. Our experimental design and analysis strategies enabled the efficient discovery of myeloma-associated therapeutic target candidates. In conclusion, this study identified a set of promising myeloma CAR-T targets, providing novel treatment options for myeloma patients. Disclosures Goldsmith: Wugen Inc.: Consultancy. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Vaccination of Hematopoietic Stem Cell (HCT) Donors with Triplex, a Novel Cytomegalovirus Vaccine: Safety, Feasibility and Adoptive Transfer of Protective CMV-Specific T Cells in HCT Recipients

An innovative approach to protect hematopoietic cell transplant (HCT) recipients from cytomegalovirus (CMV) viremia early after transplant is vaccinating the donor prior to stem cell harvest, before the recipient can achieve adequate immune responses. Triplex vaccine, a recombinant modified vaccinia Ankara expressing immunodominant CMV antigens (pp65, IE1 and IE2), is being evaluated in a Phase 1 trial (NCT03560752) to immunize donors of CMV seropositive (CMV+) recipients. Triplex, developed to elicit and enhance protective CMV-specific T cells, is a promising vaccine that demonstrated excellent tolerability and immunogenicity in healthy adults (La Rosa et al., Blood 2017), and in HCT CMV seropositive recipients, who had a 50% reduction in CMV reactivation requiring preemptive antiviral therapy, and significantly enhanced CMV-specific immune responses (Aldoss et al. AIM, 2020). Aim of the current Phase I trial is to assess a proof of concept and preliminary results of safety, feasibility and transfer of CMV-specific immunity, in recipients receiving an HCT from a matched related donor (MRD), vaccinated with Triplex. After obtaining informed consent, 18-75 year old HCT recipients (CMV seropositive) with matched related donors underwent T cell replete HCT. Donors received one Triplex injection 10-60 days prior to start of stem cell mobilization, and recipients underwent HCT within 9 weeks of donor vaccination. As per standard of care, Triplex vaccinated donors were mobilized with granulocyte colony-stimulating factor, prior to apheresis. The study has a target of 18 HCT donor/recipient pairs. All HCT recipients are followed until day 365 post-HCT for safety, virologic and immunologic assessment. All donors are monitored for vaccine induced adverse events. Antiviral treatment for viremia is considered a failure of donor vaccination to provide protection to the recipient. As of July 2020, we have safely vaccinated 9 MRD with Triplex, and infused mobilized peripheral blood stem cells, with T cells by volume approximately 10-25% to their recipients. No adverse events, possibly or likely related to the vaccine were reported. No preemptive antiviral treatment was administered to any of the HCT recipients. Six donor/recipient pairs have reached study end, and immune monitoring has been completed. Two donor/recipient pairs have been evaluated immunologically for up to 6 months and one to day 100 post-HCT. The flow cytometry panel of cellular immune assays included measuring concentrations of CMV-specific T cells expressing the 4-1BB (CD137) activation marker, and assessing the memory phenotype profiles (performed as detailed in La Rosa et al., Blood 2017). Peripheral blood mononuclear cells (PBMC) were stimulated for 24 hours with either pp65, IE1 and IE2 overlapping 15mer peptide libraries and then stained with fluorescently tagged antibodies against CD137, CD3, CD8, CD4, combined with CD28 and CD45RA memory markers by using a Beckman-Coulter Gallios cytometer with Kaluza software. Early post-HCT development of robust and long-lasting frequency of pp65-, IE1- and IE2-specific CD4 and CD8 T cells was observed in all recipients (Figure 1A). Memory profiles of CMV specific T cells had marked prevalence of effector memory phenotype early post-HCT, which persisted over time. Interestingly, on day 28 post-HCT, pp65-specific CD8 T cell levels were significantly higher in the 9 recipients who received an HCT from a Triplex vaccinated MRD compared to a control cohort of consecutively enrolled MRD HCT recipients (Figure 1B). In summary, this is the first clinical trial to assess the possible benefit of vaccinating MRD, to elicit early CMV immune recovery in CMV seropositive HCT recipients, at high risk for developing CMV serious complication after HCT. The approach is feasible and highly tolerable, with promising evidence of efficacy, indicating that Triplex vaccination of the MRD confers early protective anti-CMV immunity to the HCT recipient. Clinical and immunological outcomes from the ongoing study indicate that this novel immune therapeutic strategy provides a safer alternative to antivirals, including letermovir prophylaxis and may find application in higher CMV risk patients, such as haploidentical HCT recipients. Figure 1 Disclosures Nakamura: Viracor: Consultancy; Celgene: Other: Support on seminar; Kyowa-Kirin: Other: Support on a meeting presentation; Alexion: Other: Support on a meeting presentation; Merck: Other: advisory board meeting; Magenta Therapeutics: Other: Advisory board meeting; NapaJen Pharma: Consultancy; Kadmon Corporation: Other: Advisory board meeting. Diamond:Helocyte: Consultancy.

Total Marrow and Lymphoid Irradiation (TMLI) at a Dose of 2000cGy in Combination with Post-Transplant Cyclophosphamide (PTCy)-Based Graft Versus Host Disease (GvHD) Prophylaxis Is Safe and Associated with Favorable GvHD-Free/Relapse-Free Survival at 1 Year in Patients with Acute Myeloid Leukemia (AML)

Total Marrow and Lymphoid Irradiation (TMLI) at a Dose of 2000cGy in Combination with Post-Transplant Cyclophosphamide (PTCy)-Based Graft Versus Host Disease (GvHD) Prophylaxis Is Safe and Associated with Favorable GvHD-Free/Relapse-Free Survival at 1 Year in Patients with Acute Myeloid Leukemia (AML)

Outcomes after Second Allogeneic Transplantation and Donor Lymphocyte Infusion for Relapse after a First Allogeneic Transplant

Outcomes after Second Allogeneic Transplantation and Donor Lymphocyte Infusion for Relapse after a First Allogeneic Transplant

Flotetuzumab As Salvage Therapy for Primary Induction Failure and Early Relapse Acute Myeloid Leukemia

Flotetuzumab As Salvage Therapy for Primary Induction Failure and Early Relapse Acute Myeloid Leukemia

Bleeding and Thrombosis Are Associated with Endothelial Dysfunction in CAR-T Cell Therapy and Are Increased in Patients Experiencing Neurologic Toxicity

Bleeding and Thrombosis Are Associated with Endothelial Dysfunction in CAR-T Cell Therapy and Are Increased in Patients Experiencing Neurologic Toxicity

Comparison of Bone Marrow Versus Peripheral Blood in Haploidentical Transplantation Using Post-Transplant Cyclophosphamide- a Retrospective Analysis

Comparison of Bone Marrow Versus Peripheral Blood in Haploidentical Transplantation Using Post-Transplant Cyclophosphamide- a Retrospective Analysis