Abstract: Steroid-refractory chronic graft versus host disease (cGVHD) remains a significant cause of morbidity and mortality following allogeneic transplantation. Abatacept is a selective co-stimulation modulator, used for the treatment of rheumatologic disease, and was recently the first drug to be approved by the FDA for the prophylaxis of acute graft versus host disease. We conducted a Phase II study to evaluate the efficacy of Abatacept in steroid-refractory cGVHD (clinicaltrials.gov #NCT01954979). The overall response rate was 58%, with all responders achieving a partial response. Abatacept was well-tolerated with few serious infectious complications. Immune correlative studies showed a decrease in IL-1-alpha, IL-21, and TNF-alpha as well as decreased PD-1 expression by CD4+ T cells in all patients after treatment with Abatacept, demonstrating the effect of this drug on the immune microenvironment. The results demonstrate that Abatacept is a promising therapeutic strategy for the treatment of cGVHD.

Abstract: Background: Axicabtagene ciloleucel (Axi-cel), a CD19 directed CAR T cell therapy, results in durable response in a subset of patients with relapsed/refractory large B cell lymphoma (LBCL) in the absence of persistent circulating CAR T cells. Aim: We postulated that long-term efficacy of CAR T therapy depends on the downstream triggering of native T cell immunity. We performed single-cell transcriptomics of longitudinal peripheral blood (PB) samples and RNAseq of tumor samples from patients of the ZUMA 1 Axi-cel study. Methods: Single cell immunoprofiling (5’ expression + V(D)J, 10x Genomics) was performed on PB mononuclear cell samples from ZUMA-1 patients (N=32), collected at leukapheresis, 4 weeks, 6 and 12 months post Axi-cel infusion. RNAseq was performed on FFPE lymphoma samples (N=17). Patients were divided into 3 groups: non-responders, relapsed within 1 year from CAR T infusion, and long-term responders. A total of 405,775 cells passed quality check, capturing 73 cellular populations. Results: Long-term responders presented a distinct T cell landscape with increased CD8 T cells and CD8/CD4 ratios prior to CAR T therapy, compared to the other groups, with similar trends observed across time points. They also presented an increased abundance of 3 distinct CD8 T cell populations: (a) cells expressing cytotoxic and NK cell markers, (b) CD8 T effector memory cells characterized by CXCR4, TGFB1, and BCL3, and (c) proinflammatory CD8 T cells. In contrast, patients with early relapse showed increased levels of regulatory T cells pre-/post-CAR T infusion and lower abundance of CD4 cytotoxic T cells.Comparisons of the TCR repertoire pre-/post-CAR T demonstrated a greater clonal expansion of cytotoxic CD4 and CD8 T cell populations in the long-term responders, with high similarity of expanded clones post CAR T. Shared PB T cell clones and tumor antigen sequences derived from the RNAseq samples suggest an antigen-specific response driven by common epitopes. In contrast to the lack of expansion of T reg clones in responders, relapsed patients demonstrated high T reg-clonal expansion 6 months post treatment.Monocyte and NK cells were less prevalent in responders before treatment, driven by differences in phagocytic monocyte and inhibitory NK cell abundance. Modeling the interaction between monocyte and effector cell populations based on ligand receptor expression was suggestive of negative immunoregulatory impact on the T cell populations. Conclusion: The application of single-cell immunoprofiling on longitudinal samples from ZUMA-1 patients demonstrated distinct cellular and clonal profiles among long-term responders. These findings confirm our hypothesis of an important role for the native immune cell repertoire in response to CAR T therapeutics and can be utilized to further our understanding but also to potentially inform patient stratification, management, and treatment. Citation Format: Dimitra Karagkouni, Giulia Cheloni, Yered Pita-Juarez, Daniela Torres, Eleni Kanata, Zachary Avigan, Jessica Liegel, Dina Stroopinsky, Brodie Miles, Gayatri Tiwari, Jenny Kim, Mike Mattie, Jacalyn Rosenblatt, David Avigan, Ioannis Vlachos. Activation and clonotypic expansion of the native T cell repertoire identifies durable response to CD19 CAR T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2257.

Abstract: Vaccination with dendritic cell (DC)/multiple myeloma (MM) fusions has been shown to induce the expansion of circulating multiple myeloma–reactive lymphocytes and consolidation of clinical response following autologous hematopoietic cell transplant (auto-HCT). Patients and Methods: In this randomized phase II trial (NCT02728102), we assessed the effect of DC/MM fusion vaccination, GM-CSF, and lenalidomide maintenance as compared with control arms of GM-CSF and lenalidomide or lenalidomide maintenance alone on clinical response rates and induction of multiple myeloma–specific immunity at 1-year posttransplant. Results: The study enrolled 203 patients, with 140 randomized posttransplantation. Vaccine production was successful in 63 of 68 patients. At 1 year, rates of CR were 52.9% (vaccine) and 50% (control; P = 0.37, 80% CI 44.5%, 61.3%, and 41.6%, 58.4%, respectively), and rates of VGPR or better were 85.3% (vaccine) and 77.8% (control; P = 0.2). Conversion to CR at 1 year was 34.8% (vaccine) and 27.3% (control; P = 0.4). Vaccination induced a statistically significant expansion of multiple myeloma–reactive T cells at 1 year compared with before vaccination (P = 0.024) and in contrast to the nonvaccine arm (P = 0.026). Single-cell transcriptomics revealed clonotypic expansion of activated CD8 cells and shared dominant clonotypes between patients at 1-year posttransplant. Conclusions: DC/MM fusion vaccination with lenalidomide did not result in a statistically significant increase in CR rates at 1 year posttransplant but was associated with a significant increase in circulating multiple myeloma–reactive lymphocytes indicative of tumor-specific immunity. Site-specific production of a personalized cell therapy with centralized product characterization was effectively accomplished in the context of a multicenter cooperative group study.

Abstract: In a prior phase 2 study, personalized cancer vaccination with autologous dendritic cells (DCs) fused with primary MM tumor cells (DC/MM fusions) induced the expansion of circulating MM-reactive lymphocytes and was associated with conversion to complete response (CR) post-autoHCT in the absence of maintenance therapy. 1 We now present a multicenter randomized phase II study that examined the efficacy of DC/MM fusion vaccination with lenalidomide maintenance therapy after autoHCT, compared with lenalidomide maintenance alone. The study offered a first-of-its-kind academic collaborative effort of personalized cell therapy using an open-source format, site-specific production, and centralized product characterization/release criteria verification. Under the aegis of the BMT CTN (CTN 1401), 203 patients enrolled from 18 participating centers. Sixty-three patients dropped out of the study from enrollment to randomization for an overall dropout rate of 31%, concordant with the expected rate of 30% pre-specified in the protocol. Among the 140 patients, 68 were randomized to the vaccine arm, 37 to the lenalidomide/GM-CSF arm, and 35 to the lenalidomide alone arm. Ninety-one (65.0%) patients had high-risk MM. A collaborative process was established for the standardization of vaccine manufacturing including tumor cell harvest and cryopreservation, DC generation from leukapheresis collection, creation and quantification of the DC/tumor fusion vaccine, and the process of characterizing each cellular product. Of the 140 patients who underwent tumor collection, the median percentage of plasma cells in bone marrow aspirate differential was 45%. Mean CD86 expression and viability of the DC preparations were 80.6% and 79.3%, respectively. The mean fusion efficiency of the DC/MM product, as determined by co-expression of standard DC (CD86) and MM (CD38) markers, was 47.9%. Mean fusion cell viability was 78.6%. Vaccine was successfully generated for 63/68 patients (93%) assigned to the vaccine arm of the study. Thirty-six of the 68 (52.9%) evaluable patients on the vaccine arm (80% confidence interval 44.5%, 61.3%) and 34 of the 68 (50.0%) of the non-vaccine arms (80% confidence interval 41.6%, 58.4%) achieved CR/sCR at 1-year post-transplant (p=0.3). Of the patients not achieving CR at time of randomization post-transplant, conversion to CR at 1-year was 34.8% for the vaccine arm and 27.3% for the non-vaccine arm (p=0.4). sCR/CR/VGPR at 1-year was achieved by 85.3% and 77.8% of patients on the vaccine and non-vaccine arms, respectively (p=0.2). The rates of post-transplant grade 3-4 toxicities were 76.5%, 62.5% for the vaccine, and non-vaccine arms, respectively (p=0.07). There were no grade 5 toxicities in any of the cohorts. The overall grade 2-3 infection rate was 22.9% (23.5% on the vaccine arm, 13.5% on the lenalidomide/GM-CSF arm, and 31.4% on the lenalidomide alone arm). Quantification of circulating MM-reactive T cells was performed for an initial cohort of 20 patients who completed 1-year post-transplant assessments. Patients in the vaccine arm demonstrated a significant expansion of MM-reactive CD8 cells, representing 2.9%, 3.5%, and 15.9% of the lymphocyte population prior to post-transplant maintenance, following 1 cycle of lenalidomide and following 3 vaccinations at 1-year post-transplant, respectively (Figure). In contrast, there was no increase in MM-specific CD8 T cells in the control arm with levels of 1.1, 2.0. and 0.6, respectively. Single-cell transcriptomic analysis performed on the peripheral T-cell repertoire of 14 patients from the vaccine arm at serial time points demonstrated progressive expansion of dominant CD8, CD4, and NKT cell clones with an activated phenotype. Vaccination was associated with the recovery of T-cell clonal diversity. This multicenter trial demonstrated successful site-specific production. DC/MM fusion vaccination with lenalidomide maintenance after autoHCT did not result in a significant increase in CR rates at 1-year but was associated with measurable anti-MM immune reactivity for which the impact on response duration will require longer term follow-up. 1. Rosenblatt, J. et al. Vaccination with dendritic cell/tumor fusions following autologous stem cell transplant induces immunologic and clinical responses in multiple myeloma patients. Clin. Cancer Res. 19, 3640-3648 (2013) Figure 1 Figure 1. Disclosures Shah: CareDx: Consultancy; GSK: Consultancy; Teneobio: Research Funding; Kite: Consultancy; Nektar: Research Funding; Poseida: Research Funding; Oncopeptides: Consultancy; Bluebird Bio: Research Funding; CSL Behring: Consultancy; Indapta Therapeutics: Consultancy; Janssen: Research Funding; Sanofi: Consultancy; Sutro Biopharma: Research Funding; Precision Biosciences: Research Funding; BMS/Celgene: Research Funding; Karyopharm: Consultancy; Amgen: Consultancy. Stroopinsky: The Blackstone Group: Consultancy. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Devine: Magenta Therapeutics: Current Employment, Research Funding; Tmunity: Current Employment, Research Funding; Johnsonand Johnson: Consultancy, Research Funding; Orca Bio: Consultancy, Research Funding; Be the Match: Current Employment; Sanofi: Consultancy, Research Funding; Vor Bio: Research Funding; Kiadis: Consultancy, Research Funding. Holmberg: Sanofi: Research Funding; Millennium-Takeda: Research Funding; Seattle Genetics: Research Funding; Bristol Myers Squibb: Research Funding; Merck: Research Funding; Janssen: Research Funding; Up-To-Date: Patents & Royalties. Johnson: Miltenyi Biotec: Research Funding. Lazarus: Bristol Myer Squibb: Membership on an entity's Board of Directors or advisory committees. Malek: Janssen: Other: Advisory board ; Medpacto Inc.: Research Funding; Takeda: Honoraria; BMS: Honoraria, Research Funding; Amgen: Honoraria; Cumberland Inc.: Research Funding; Bluespark Inc.: Research Funding; Sanofi: Other: Advisory Board. McCarthy: Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bluebird: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Juno: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. McKenna: Qihan Bio: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Other: all manufacturing of cell therapy products for clinical trials; Intima: Other: all manufacturing of cell therapy products for clinical trials; Gamida: Other: all manufacturing of cell therapy products for clinical trials; Magenta: Other: all manufacturing of cell therapy products for clinical trials. Munshi: Takeda: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Amgen: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy; Abbvie: Consultancy; Adaptive Biotechnology: Consultancy; Legend: Consultancy; Pfizer: Consultancy; Bristol-Myers Squibb: Consultancy. Nooka: Janssen Oncology: Consultancy, Research Funding; Adaptive technologies: Consultancy; Sanofi: Consultancy; GlaxoSmithKline: Consultancy, Other: Travel expenses; Takeda: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy; Amgen: Consultancy, Research Funding; Oncopeptides: Consultancy; Karyopharm Therapeutics: Consultancy. Soiffer: Jazz Pharmaceuticals, USA: Consultancy; Precision Biosciences, USA: Consultancy; Juno Therapeutics, USA: Other: Data Safety Monitoring Board; Kiadis, Netherlands: Membership on an entity's Board of Directors or advisory committees; Rheos Therapeutics, USA: Consultancy; Gilead, USA: Other: Career Development Award Committee; NMPD - Be the Match, USA: Membership on an entity's Board of Directors or advisory committees; Jasper: Consultancy; Takeda: Consultancy. Uhl: Grifols: Consultancy, Speakers Bureau; Abbott: Consultancy, Speakers Bureau; UpToDate: Patents & Royalties. Young: Amgen: Current equity holder in publicly-traded company; Pfizer: Current equity holder in publicly-traded company; Merck: Current equity holder in publicly-traded company. Rosenblatt: Attivare Therapeutics: Consultancy; Bristol-Myers Squibb: Research Funding; Parexel: Consultancy; Wolters Kluwer Health: Consultancy, Patents & Royalties; Imaging Endpoints: Consultancy; Karyopharm: Membership on an entity's Board of Directors or advisory committees. Waller: Verastem Oncology: Consultancy, Research Funding; Cambium Oncology: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Pasquini: GlaxoSmithKline: Research Funding; Kite Pharma: Research Funding; Novartis: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding. Avigan: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Kite Pharma: Consultancy, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Aviv MedTech Ltd: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Legend Biotech: Membership on an entity's Board of Directors or advisory committees; Chugai: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Parexcel: Consultancy; Takeda: Consultancy; Sanofi: Consultancy. OffLabel Disclosure: Dendritic Cell/Tumor Fusion Vaccine and GM-CSF

Abstract: We have developed a promising leukemia vaccine in which patient derived AML cells are fused with autologous dendritic cells (DCs), presenting a broad array of antigens. We are conducting a clinical trial in which AML patients who are not candidates for allogeneic transplantation undergo vaccination with DC/AML fusion cells following chemotherapy induced remission. Twenty-six patients (14 males, 12 females) underwent collection of AML cells at disease presentation for vaccine generation and immune monitoring studies. Median age of the patients is 66 years. Tumor was collected from either a bone marrow aspirate (N=16), 20 cc of peripheral blood (N=7), or leukapheresis product (N=3) at the time of presentation with newly diagnosed AML (N=25) or first relapsed AML (N=1). The mean yield of AML cells was 109 x 106 cells with a mean viability of 91%. Eligible patients achieving CR following chemotherapy (N=16) underwent leukapheresis for DC generation and vaccine preparation. Adherent peripheral blood mononuclear cells were isolated, cultured in the presence of GM-CSF and IL-4 for 5-7 days, and exposed to TNFα for 48-72 hours to generate mature DCs. The mean yield of DCs was 177 x106 cells with a mean viability of 89%. Fusion cells were generated by co-culture of DCs with AML cells in the presence of 50% polyethylene glycol and identified as cells co-expressing antigens that were unique to the DC and tumor population. Mean fusion efficiency and viability was 38% and 85%, respectively. As a measure of their activity as antigen presenting cells, the capacity of fusion cells to stimulate allogeneic T cell proliferation ex vivo was quantified. In contrast to the leukemia preparation (mean stimulation index (SI) 3.81), the DC and fusion cell preparation were potent stimulators (mean SI 19.61 and 13.48, respectively). Vaccination with DC/leukemia fusion cells was initiated within 12 weeks from count recovery following the final cycle of chemotherapy. 13 patients received at least two monthly vaccinations at a dose of 5x106 fusion cells. 8 patients had intermediate risk cytogenetics, 3 patients had good risk cytogenetics, and 2 patients had a complex karyotype. Vaccination was well tolerated, and importantly, was not associated with clinically significant auto-immunity. Possibly related adverse events were transient and of grade 1-2 intensity, including vaccine site reactions, pruritis, arthalgias, myalgias, eosinophilia, leukopenia, thrombocytopenia. Biopsy of vaccine site reactions demonstrated a dense infiltrate of CD4 and CD8 T cells consistent with recruitment of reactive T cell populations to the vaccine bed. To date, 9 patients remain in remission (69%), with a mean follow up of 23 months. Peripheral blood samples were collected prior to each vaccination and at 1, 3, and 6 months following completion of vaccination. Vaccination resulted in the potent induction of leukemia specific immunity as measured by an increase in CD8 T cells expressing IFNγ in response to ex vivo exposure to autologous leukemia cell lysates (mean fold increase 8, n=6). Bone marrow derived T cells were isolated prior to and following vaccination in patients who are HLA2.1+. Vaccination resulted in the expansion of bone marrow infiltrating T cells recognizing MUC1 (9 fold increase), WT1 (5 fold increase), PRAME (12 fold increase) tumor antigens by tetramer analysis (n=2). In conclusion, DC/AML fusion cell vaccination results in the potent expansion of leukemia reactive T cells and durable remissions following chemotherapy. Enrollment to a second cohort is being initiated, in which patients with be treated with DC/AML fusion cell vaccination in conjunction with PD1 blockade. Disclosures: No relevant conflicts of interest to declare.

Abstract: Abstract 948 Patients with acute myeloid leukemia (AML) achieve remission following chemotherapy; however, curative outcomes remain elusive due to relapse with chemotherapy-resistant disease. Allogeneic transplantation remains a potentially curative therapy for AML patients, but is associated with significant morbidity and mortality due to the lack of specificity of the alloreactive response. A promising area of investigation is the development of cancer vaccines that educate host immunity to more selectively target leukemia cells, including the stem cell compartment. Our group has developed a cancer vaccine model in which dendritic cells (DCs) are fused to autologous tumor cells, resulting in the presentation of multiple tumor antigens with the capacity to elicit a broad anti-tumor response. A fundamental challenge to developing a more effective tumor vaccine is overcoming the immunosuppressive milieu by which tumor cells evade host immunity. Key elements contributing to tumor-mediated immune suppression are the increased presence of regulatory T cells in patients with malignancy, and upregulation of the PD-1/PDL1 pathway. Tumor expression of PD-L1 promotes T cell tolerance by binding PD-1 on activated T cells and suppressing their capacity to secrete stimulatory cytokines. In addition, the PD-1/PDL-1 pathway has been shown to inhibit T cell-mediated lysis of tumor cells, potentially preventing a clinically meaningful immunologic response to vaccination. We are conducting a clinical trial in which AML patients who are in a first or second complete remission following chemotherapy receive three monthly doses of DC/AML fusion cells alone (Cohort 1) or in conjunction with anti-PD1 antibody, CT-011 (cohort 2). To date, 16 patients (9 males, 7 females; mean age 55 years) have been enrolled to the first cohort. All patients underwent successful tumor collection from either a bone marrow aspirate (N=12), collection of 20 cc of peripheral blood (N=3), or leukapheresis product (N=1) at the time of presentation with newly diagnosed AML (N=15) or first relapsed AML (N=1). The mean yield was 1.45×108 cells, and the mean viability was 90%. Tumor cells were subjected to immunohistochemical analysis to identify antigens unique to the leukemia fusion partner. Those patients achieving complete remission following 1–2 cycles of induction chemotherapy underwent leukapheresis for dendritic cell generation. Adherent peripheral blood mononuclear cells were isolated, cultured in the presence of GM-CSF and IL-4 for 5–7 days, and then exposed to TNFα for 48–72 hours to generate mature DCs. Mean viability of the DC preparation was 92%. DCs strongly expressed the co-stimulatory molecule CD86 (mean 75% expression). One patient died during remission induction chemotherapy and 3 patients were removed from study after induction chemotherapy to undergo allogeneic transplantation. Vaccine was successfully generated in 9 patients at a dose of 5×106 fusions cells, mean fusion efficiency of 30%, and viability of 87%. As a measure of their activity as antigen presenting cells, the capacity of the fusion cell preparation to stimulate allogeneic T cell proliferation ex vivo was quantified. In contrast to the leukemia preparation (mean stimulation index (SI) 3.7), the DC and fusion cell preparation were potent stimulators (mean SI 20.8 and 13.1, respectively). Vaccination with the DC/leukemia fusion vaccine was initiated within 12 weeks from count recovery following their final cycle of chemotherapy. 4 patients have completed vaccinations and are 2, 4, 5 and 6 months following the final vaccine. One patient was taken off study for disease progression one week after receiving his first vaccine. 4 patients experienced grade 1 vaccine site reactions. Biopsy of a vaccine site reaction demonstrated a dense T cell infiltrate. Additional vaccine related adverse events have included grade 1 ankle pain and edema. The remaining patients are undergoing chemotherapy, and when complete, will initiate vaccination. Peripheral blood samples are being collected prior to each vaccination and at 1, 3, and 6 months following completion of vaccination. Immune response targeting leukemia cells, leukemic stem cells, and leukemia associated antigens will be assessed. Levels of circulating regulatory T cells and T cell expression of PD1 will be measured. Time to disease progression will also be determined. Disclosures: Avigan: Curetech: Research Funding.