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Pancreatic cancer is a highly lethal malignancy often presenting with advanced disease and characterized by resistance to standard chemotherapy. Immune-based therapies such checkpoint inhibition have been largely ineffective such that pancreatic cancer is categorized as an immunologically "cold tumor". In the present study, we examine the therapeutic efficacy of a personalized cancer vaccine in which tumor cells are fused with dendritic cells (DC) resulting in the broad induction of antitumor immunity.In the KPC spontaneous pancreatic cancer murine model, we demonstrated that vaccination with DC/KPC fusions led to expansion of pancreatic cancer specific lymphocytes with an activated phenotype. Remarkably, vaccination led to a reduction in tumor bulk and near doubling of median survival in this highly aggressive model. In a second murine pancreatic model (Panc02), vaccination with DC/tumor fusions similarly led to expansion of tumor antigen specific lymphocytes and their infiltration to the tumor site. Having shown efficacy in immunocompetent murine models, we subsequently demonstrated that DC/tumor fusions generated from primary human pancreatic cancer and autologous DCs potently stimulate tumor specific cytotoxic lymphocyte responses.DC/tumor fusions induce the activation and expansion of tumor reactive lymphocytes with the capacity to infiltrate into the pancreatic cancer tumor bed.

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.

Introduction: Our group has pioneered a novel vaccine by fusion of patient-derived tumor with autologous dendritic cells (DCs) that presents an array of tumor antigens generating a polyclonal immune response. DC/AML vaccination led to expansion of leukemia-specific T cells with survival benefit in a phase II clinical trial. We postulated that ex vivo generation of vaccine-educated T cells would provide a powerful platform for adoptive immunotherapy with opportunity to augment T-cell functional potency prior to infusion. We report on an ex vivo system in which vaccine-educated T cells are further enriched for activated antigen-specific effector cells via an agonistic 4-1bb antibody. We report phenotypic and functional characteristics of 4-1bb-enriched vaccine-educated T cells. Methods: DC/AML fusion vaccines were generated from C57BL/6J mice DCs and syngeneic C1498 mCh/luc+ AML cells. Splenic T cells were co-cultured with autologous irradiated DC/AML fusions in presence of IL-2/7/15. Selection with biotinylated agonistic 4-1bb (3H3) was performed on vaccine-educated T cells followed by expansion with anti-CD3/CD28 activation beads. T cells were phenotyped for activation (CD25/CD69), immune checkpoints (PD1/LAG3/TIM3), memory (CD44+CD62L-) and enrichment (anti-rat H&L). Cytotoxicity was evaluated by luminescence. Mice were inoculated with C1498 and injected with T cells 7 days later. BLI imaging was performed and 100-day survival measured. Results: Vaccine-educated T cells demonstrated evidence of immune activation and memory phenotype compared to unstimulated naïve T-cell controls (TN) (7.24-fold, CD4+CD25+CD69+; 1.7-fold, CD3+CD44+CD62L-). Vaccine-educated T cells selected based on 4-1bb expression showed enhanced markers of activation (15.3-fold, CD4+CD25+CD69+) and memory phenotype (5-fold, CD3+CD44+CD62L-) compared to TN. Selection enriched for 4-1bb+ vaccine-educated T cells resulting in enhanced antigen-specific recognition as measured by tumor lysate induction of IFNg expression. Tumor specificity and activation was maintained following CD3/CD28-mediated expansion. The 4-1bb+ vaccine-educated T cells showed enhanced cytotoxicity (1.9-fold increase/TN at 10:1 E/T, P Conclusion: We demonstrate that vaccine-educated T cells subject to selection via an agonist 4-1bb antibody confer enhanced tumor selectivity and potency. Optimal duration for T-cell education was 3-5 days. T-cell stimulation and enrichment by agonistic 4-1bb selection enhanced cytotoxicity and memory phenotype. Thus, 4-1bb selection is a novel approach for antigen-specific T-cell enrichment for superior adoptive immunotherapy in AML. Citation Format: Kathrine S. Rallis*, Jessica Liegel*, Giulia Cheloni, Dina Stroopinsky, Poorva Bindal, Kenel Dufort, Daniela Torres, Isabella Saldarriaga, Samuel Herzlinger, Abigael Morin, Raphael Kesselman, Jeremy Rosenbaum, Georges Chedid, Sophia Adamia, Donald Kufe, Jacalyn Rosenblatt, David Avigan. 4-1bb selection augments DC/AML fusion vaccine-educated T cells for adoptive 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 4076.

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.

We have developed a personalized vaccine in which patientderived leukemia cells are fused to autologous dendritic cells, evoking a polyclonal T-cell response against shared antigens and neoantigens. We postulated that the dendritic cell/acute myeloid leukemia fusion vaccine would demonstrate synergy with checkpoint blockade by expanding tumor antigen-specific lymphocytes that would provide a critical substrate for checkpoint blockade-mediated activation. Using an immunocompetent murine leukemia model, we examined the immunological response to and therapeutic efficacy of vaccination in conjunction with checkpoint blockade with respect to leukemia engraftment, disease burden, survival and the induction of tumor-specific immunity. Mice treated with checkpoint blockade alone had rapid progression of leukemia and only a modest extension of survival. Vaccination with dendritic cell/acute myeloid leukemia fusions resulted in the expansion of tumor-specific lymphocytes and disease eradication in a subset of animals, while the combination of vaccination and checkpoint blockade induced a fully protective tumor-specific immune response in all treated animals. Vaccination followed by checkpoint blockade resulted in upregulation of genes regulating activation and proliferation in memory and effector T cells. Long-term survivors exhibited increased T-cell clonal diversity and were resistant to subsequent tumor challenge. The combination of dendritic cell/acute myeloid leukemia fusion vaccine and checkpoint blockade treatment offers unique synergy, inducing durable activation of leukemia-specific immunity, protection from lethal tumor challenge and the selective expansion of tumor-reactive clones.

8003 Background: Chimeric Antigen Receptor (CAR) T cell therapies targeting B-cell maturation antigen (BCMA) have demonstrated benefit in patients (pts) with relapsed and/or refractory Multiple Myeloma (RRMM). CART-ddBCMA is an autologous anti-BCMA CAR T cell therapy that utilizes a novel, synthetic binding domain, called a D-Domain, instead of a typical scFv binder. The objective of this first-in-human trial is to assess the safety and efficacy of CART-ddBCMA. Methods: This is a Phase 1, multi-center, open label, dose escalation trial for pts with RRMM who have received ≥3 prior regimens or are triple-refractory. After apheresis, bridging therapy is allowed during manufacturing. Pts receive fludarabine and cyclophosphamide (30/300 mg/m2/day) days -5 to -3 and CART-ddBCMA infusion on day 0. Dose escalation was performed at 100 (DL1) and 300 (DL2) x 106 (± 20%) CAR+T cells, followed by expansion of DL1. The primary endpoint is incidence of adverse events (AEs), including dose-limiting toxicities (DLTs). Additional endpoints are depth and duration of response (IMWG Criteria), minimal residual disease (MRD, clonoSEQ), progression-free (PFS) and overall survival (OS). Pts with 1- and 3-months follow-up were eligible for safety and efficacy analysis, respectively. Results: As of January 25, 2022, 25 pts received CART-ddBCMA, with median age 66 (range: 44-76), after a median of 5 prior lines of therapy (3-16), including 10 (40%) with extramedullary disease (EMD). Median follow-up was 9.8 (2-23.7) months. Overall, 25 pts (19 DL1; 6 DL2) were evaluable for safety and 24 (18 DL1; 6 DL2) for efficacy analysis. All pts experienced CRS, but only 1 pt (in DL2) had grade (gr) 3 CRS. All other CRS cases were gr ≤2, with no cases of gr ≥3 CRS in DL1. Four pts experienced ICANS (2, gr ≤2; 2, gr 3), with 1 gr 3 case in each of DL1 (5%) and DL2 (17%). Standard management resulted in resolution of CRS/ICANS within 30 days in all cases without sequelae. The ORR = 100%, sCR/CR rate = 67%, and ≥VGPR rate = 88%. Conversion to CR/sCR has occurred with longer follow-up, as late as month 9 in this trial. At time of data-cut 5 pts in DL1 with PR/VGPR have < 9 months follow-up, with 4 (of 4 evaluable) negative at ≥10-5 for MRD. Overall, 17 of 20 (85%) evaluable pts have achieved best MRD response of ≥10-5. In the dose escalation pts (i.e., those with longest follow-up, n = 12), the ORR and CR/sCR rate was 100% and 75%, respectively, despite 58% (7/12) EMD in this group. Of the first 6 pts dosed in DL1, 4 (67%) continue in ongoing sCR beyond 18 months, including 3 with EMD. Median duration of response, PFS and OS were not evaluable at the time of data-cut because 19 of 24 evaluable pts (79%) remain in ongoing response. Conclusions: CART-ddBCMA administration, to date, has demonstrated clinical activity, including 100% ORR with rates of CR/sCR and ≥VGPR of 67% and 88%, respectively. Durable responses beyond 18 months have been observed, including in pts with EMD. Clinical trial information: NCT04155749.

Background: Chronic graft versus host disease (cGVHD) remains a significant cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HCT), with a cumulative incidence of 50% (Arora et al, Biol Blood Marrow Transplant 2016). Systemic corticosteroids are considered first-line therapy, and best approach to managing steroid-refractory cGVHD is lacking. Abatacept is a recombinant fusion protein consisting of the extracellular domain of human cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) linked to the modified Fc portion of human immunoglobulin G1 (IgG1) to prevent complement fixation and antibody-dependent cellular cytotoxicity. Abatacept is a selective costimulation modulator that binds specifically to CD80 and CD86 on antigen presenting cells, thus attenuating CD28-mediated T cell activation. We previously reported results from a Phase I clinical trial evaluating the safety and clinical efficacy of abatacept in patients with steroid-refractory cGVHD (Nahas et al, Blood 2018 #NCT01954979). Here we report results of the phase II study evaluating the overall clinical response rate of abatacept in patients with steroid-refractory cGVHD. Methods: Allogeneic bone marrow or stem cell transplantation recipients were eligible if they developed cGVHD as defined by NIH consensus criteria and were treated with prednisone ≥ 0.25 mg/kg/day for at least 4 weeks without complete resolution of signs and symptoms. Peripheral blood was drawn prior to each dose of abatacept and following completion of therapy to assess the effect of treatment on circulating T cells. Abatacept was administered at 10mg/kg for a total of 6 doses. Doses 1-3 were administered at two-week intervals. One month after administration of Dose 3, abatacept was given at four-week intervals for Doses 4-6. Patients who completed 6 doses of abatacept and continued to demonstrate response were eligible to receive extended duration therapy with monthly abatacept at a dose of 10mg/kg for up to a total of 12 additional doses. Primary study endpoint was overall response rate (ORR) of using abatacept in treating cGVHD. Complete response (CR) was defined as resolution of all manifestations in each organ or site, and partial response (PR) was defined as improvement in at least one organ or site without progression in any other organ or site as per the 2014 NIH Chronic GVHD Consensus Response Criteria (Lee et al Biol Blood Marrow Transplant 2015). Results: 39 subjects with median age of 62 years (range 25-77 years) had undergone HCT a median of 43 months (range 6-173 months) prior to study entry. Patients were treated with abatacept and received a median of 8 doses (range 2-18). 17 patients had moderate cGVHD and 22 patients had severe cGVHD at baseline. Baseline cGVHD assessment of the 39 evaluable patients showed involvement of the skin in 85% (n=33), mouth in 44% (n=17), eyes in 69% (n=27), gastrointestinal tract (GI) in 18% (n=7), liver in 23% (n=9), lung in 54% (n=21), joints in 82% (n=32), and genital tract in 8% (n=3). The ORR was 49% (19/39 patients) with CR 0% and PR 49%. The organ sites with greatest improvement included lung (33%), eyes (23%), mouth (21%), and joints (21%). Progression of disease occurred in 26% of patients (n=10), with sites of involvement including skin (8%), mouth (10%), eyes (5%), lung (3%), and joints (5%). Baseline median daily dose of prednisone was 20mg with a 27.5% reduction at 1-month follow-up to 14.5mg (p Conclusions: Abatacept is associated with a 49% ORR in steroid refractory cGVHD and leads to durable reduction in prednisone dosing over time. Severe infections are uncommon and the infusions are well tolerated. Abatacept represents a promising novel therapeutic agent for patients with steroid-refractory cGVHD. Disclosures Stroopinsky: The Blackstone Group: Consultancy. Arnason: Juno/BMS: Honoraria. Cutler: Cimeio: Consultancy; Editas: Consultancy; Kadmon: Consultancy; Pfizer: Consultancy; Mallinckrodt: Consultancy; CareDx: Consultancy; Incyte: Consultancy; Omeros: Consultancy; Syndax: Consultancy; Mesoblast: Consultancy; Deciphera: Consultancy; Jazz: Consultancy. Nikiforow: Kite/Gilead: Other: ad HOC Advisory Boards; Novartis: Other: ad Hoc Advisory Boards; Iovance: Other: ad Hoc Advisory Boards; Glaxo Smith Kline (GSK): Other: ad Hoc Advisory Boards. 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. Soiffer: Rheos Therapeutics, USA: Consultancy; Kiadis, Netherlands: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics, USA: Other: Data Safety Monitoring Board; Precision Biosciences, USA: Consultancy; Jazz Pharmaceuticals, USA: Consultancy; Takeda: Consultancy; Jasper: Consultancy; Gilead, USA: Other: Career Development Award Committee; NMPD - Be the Match, USA: Membership on an entity's Board of Directors or advisory committees. Rosenblatt: Attivare Therapeutics: Consultancy; Parexel: Consultancy; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Imaging Endpoints: Consultancy; Wolters Kluwer Health: Consultancy, Patents & Royalties; Bristol-Myers Squibb: Research Funding.

Introduction: Immunotherapy for AML holds promise in overcoming chemotherapy resistance and in preserving immunologic memory necessary for durable remissions.A bispecific T-cell engaging antibody targeting CD3 and CD123 (CD123-CODV-TCE) has been shown to stimulate T cells to target CD123-expressing leukemic cells in vitroand in mouse models 1. While the short-term immune stimulation mediated by the CD123TCE has the potential to result in clinical response, long-term disease control will require the development of immune memory. We have developed a personalized cancer vaccine in which patient's dendritic cells are fused with autologous leukemia cells resulting in presentation of a wide range of antigens to the immune system. Here, we describe a novel combination of CD123TCE with a DC/AML fusion vaccine ex vivo and in a xenograft murine model. We hypothesized that the CD123TCE will direct the vaccine-educated T cells to not only more effectively eradicate target leukemia cells but also evoke a repertoire of memory T cells and long-term response. Methods/Results: AML cells expressing CD123 were isolated from bone marrow mononuclear cells (BMMCs) from AML patients (n=3). DCs were generated from autologous adherent peripheral blood mononuclear cells (PBMCs) obtained at the time of disease remission as previously described 2,3. Concurrently, CD3+ T cells were isolated from the non-adherent fraction of PBMCs using magnetic bead separation. Fusion cells were generated by co-culturing the DC and tumor cells at a ratio of 3:1 in the presence of polyethylene glycol (PEG). Vaccine educated T cells were then generated by co-culture of T cells with the autologous fusion cells at a ratio of 10:1 for 5-7 days followed by T cell expansion via CD3/CD28 ligation. The capacity of the vaccine-educated T cells to target autologous leukemia cells with the addition of CD123TCE was assessed. The results demonstrated a statistically significant increase in Granzyme B activity in the target AML cells following co-culture with vaccine-educated T cells and the addition of the CD123TCE, compared to T cells + isotype control (n=3). Furthermore, vaccine stimulation in combination with CD123TCE led to a robust increase in induction of tumor specific activated T cells as detected by CD137 expression and intracellular IFN-γ production after co-culture of vaccine-educated T cells with autologous tumor cells in the presence of CD123TCE. The addition of CD123TCE to vaccine-educated T cells resulted in mean 25.4% and 9.6% intracellular IFN-γ expression for CD8 and CD4 T cells, respectively, compared to 8.5 and 3.1% IFN-γ expression following the addition of isotype control (n=3). Next, we examined the efficacy of the combined treatment with vaccine-educated T cells and CD123TCE in-vivo, in two independent xenograft experiments. NSG mice were irradiated with 300rads and challenged with 1x10 6 patient-derived CD123+ tumor cells via retro-orbital injections. After detection of human AML engraftment in the PB on day 76, the mice were inoculated IV with 1X10 6 resting, or ex vivo fusion vaccine educated autologous T cells IV. Subsequently, cohorts of mice were treated with CD123TCE or an appropriate isotype control every 3 days IP. A significant decrease in human leukemia burden was detected in the peripheral blood, spleen and bone marrows of analyzed animals after treatment with vaccine educated T cells and isotype control, or resting T cells and CD123TCE, compared to untreated mice (n=5). Strikingly, no detectable AML was found in peripheral blood, spleens and bone marrows of mice treated with vaccine educated T cells in combination with TCE (n=5). Of note, treatment with vaccine educated T cells led to an expansion of human CD3+ T cells in tissues obtained from the analyzed animals. These human T cells persisted in mice treated with the CD123TCE with a two-fold increase in tumor-specific CD8+ T cells, as assessed by intracellular IFN-γ secretion following ex vivo stimulation with autologous tumor lysate. Conclusions: We demonstrated that the combination of DC/AML fusion vaccine and CD123TCE led to increase in tumor specific T cell immunity, both ex-vivo and in a xenograft murine model when compared to uneducated T cells with CD123TCE or educated T cells with isotype control molecule. Most significantly, the combination treatment was shown to eradicate AML in this model with all animals remaining disease-free several months post inoculation. Disclosures Stroopinsky: The Blackstone Group: Consultancy. Nahas: Kite Pharma: Current Employment. Fraenkel: Sanofi: Current Employment. Yildirim: Sanofi: Current Employment. Bonnevaux: Sanofi: Current Employment. Guerif: Sanofi: Current Employment. Kufe: Genus Oncology: Current equity holder in publicly-traded company; Canbas: Consultancy; REATA: Consultancy, Current equity holder in publicly-traded company; Hillstream BioPharma: Current equity holder in publicly-traded company. Rosenblatt: Parexel: Consultancy; Wolters Kluwer Health: Consultancy, Patents & Royalties; Bristol-Myers Squibb: Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Imaging Endpoints: Consultancy; Attivare Therapeutics: Consultancy. 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.

Allogeneic transplantation is uniquely curative for a subset of patients with acute myeloid leukemia (AML) however, post-transplant relapse and graft versus host disease remain significant concerns. We have developed a vaccine in which patient-derived AML cells are fused with donor-derived dendritic cells (DCs), thereby presenting a broad array of antigens. Previous work (Rosenblatt Sci Transl Med) has suggested that such vaccines given in the first complete remission setting after chemotherapy consolidation elicit specific immune responses and can possibly lessen the risk of relapse. We hypothesize that donor-derived DC/AML vaccination post-transplant would elicit the durable expansion of leukemia- specific T cells within the donor T cell repertoire to effectively protect against disease relapse. We report the results of a phase 1 clinical trial (NCT03679650) evaluating the use of DC/AML fusion vaccine in patients with acute myeloid leukemia (AML) following allogeneic transplant. DC/AML fusion vaccine is generated with autologous leukemia blasts, cryopreserved at the time of diagnosis with AML, and donor-derived dendritic cells. DCs are collected via leukapheresis in pts in CR 25-70 days after an allogenic matched related or unrelated donor transplant (cohort A) or a haplo-identical donor (Cohort B). In order to proceed with leukapheresis, patients must demonstrate donor hematopoietic recovery and the absence of ongoing grade 2 or higher GVHD. For DC generation, donor-derived adherent mononuclear cells are cultured with GM-CSF, IL-4 and TNFa. Vaccine is administered subcutaneously starting day 70-100 post-transplant. 2 vaccines are given at 3 week intervals, in conjunction with 100 mcg GMCSF daily at the vaccine site for 4 days. A booster vaccine is given 30-60 days following the taper of immune suppression, in the absence of GVHD. Profiling of the immune microenvironment using single cell RNA sequencing from samples obtained before and after vaccination is being carried out. 17 participants have been enrolled. The median age was 59 years (range 23-74). 15 patients were enrolled to cohort A: 8 were transplanted with a matched unrelated donor and 7 were transplanted with a matched sibling donor. 2 participants were enrolled to cohort B. The median yield of leukemia cells was 207x10 6 (range 80-818x10 6)and mean viability was 96.5%. The median yield of DCs was 133x10 6 (range 29-182x10 6) and mean viability 72%. Fusion vaccine was successfully generated in 14/16 patients. One patient had insufficient DC, one patient relapsed prior to leukapheresis and one vaccine is currently in process. Mean fusion efficiency was 52% with viability of 78.6%. Mean fusion vaccine dose was 4.36 x10 6 cells. 3 patients from whom vaccine was generated did not meet eligibility to initiate vaccination due to ongoing toxicity following transplant or GVHD. 11 participants have initiated vaccine administration and are evaluable for toxicity and response. The most common side effects have been vaccine site reactions (n=10 grade 1, n=2 grade 2). 7 patients developed GVHD that was determined to be possibly related to vaccination, at a median time of 19 days after vaccination (range 5-70 days). 2 of these patients developed grade 2 acute GVHD of the skin, and 5 developed chronic GVHD affecting one to up to four organ systems ranging from mild to severe. There were 5 additional GVHD events with a median time of 115 days post vaccination (range 91-126 days), assessed as unlikely related or unrelated to vaccine based on timing of onset. 10 of 11 evaluable patients remain in a CR at a median time of 21 months post-transplant (range 6-33 months). One patient died of relapsed disease 15 months post transplant and another died of GVHD and infection unrelated to vaccine 21 months post transplant. Vaccination using patient-derived autologous leukemia cells and donor derived DC isolated following engraftment of hematopoietic cells appears safe and feasible. Toxicity of vaccination has included injection site reactions as well as graft versus host disease, primarily mild to moderate severity. Outcomes to date demonstrate that vaccination post-transplant may be a promising strategy to reduce relapse as 10 of 11 vaccinated patients remain relapse free. However this approach is limited to patients who have not had early relapse or early GVHD post-transplant. Immune correlates, including single cell RNA sequencing of the immune milieu, are ongoing. Disclosures Stone: Abbvie: Consultancy; Actinium: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Aprea: Consultancy; Arog: Consultancy, Research Funding; Astellas: Membership on an entity's Board of Directors or advisory committees; BerGen Bio: Membership on an entity's Board of Directors or advisory committees; Boston Pharmaceuticals: Consultancy; Bristol Meyers Squibb: Consultancy; Celgene: Consultancy; Elevate Bio: Membership on an entity's Board of Directors or advisory committees; Foghorn Therapeutics: Consultancy; Gemoab: Membership on an entity's Board of Directors or advisory committees; Glaxo Smith Kline: Consultancy; Innate: Consultancy; Janssen: Consultancy; Jazz: Consultancy; Macrogenics: Consultancy; Novartis: Consultancy, Research Funding; OncoNova: Consultancy; Syndax: Membership on an entity's Board of Directors or advisory committees; Syntrix/ACI: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy. Soiffer: Takeda: Consultancy; Jasper: Consultancy; 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. Stroopinsky: The Blackstone Group: Consultancy. Romee: Glycostem: Membership on an entity's Board of Directors or advisory committees; Crispr Therapeutics: Research Funding. Neuberg: Madrigal Pharmaceuticals: Other: Stock ownership; Pharmacyclics: Research Funding. Kufe: Canbas: Consultancy; Hillstream BioPharma: Current equity holder in publicly-traded company; Genus Oncology: Current equity holder in publicly-traded company; REATA: Consultancy, Current equity holder in publicly-traded company. Avigan: Chugai: 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; Parexcel: Consultancy; Janssen: Consultancy; Legend Biotech: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Consultancy, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Juno: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy; Sanofi: Consultancy; Aviv MedTech Ltd: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Rosenblatt: Wolters Kluwer Health: Consultancy, Patents & Royalties; Parexel: Consultancy; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Imaging Endpoints: Consultancy; Bristol-Myers Squibb: Research Funding; Attivare Therapeutics: Consultancy.

Background: In Zuma-1 study, approximately 40% of patients with refractory or relapsed large B cell lymphoma (LBCL) show durable response to Axi-cel. The identification of immunologic factors predictive of therapeutic efficacy and tumor escape is a critical area of investigation. The impact of CAR T cell activation on the native T cell repertoire and lymphoma specific immunity has not been elucidated. Aim: We sought to determine the role of host immune activation in response to tumor-associated antigens and the impact of consequent epitope spreading on CAR-T mediated therapeutic efficacy. To this end, we performed longitudinal single cell immunoprofiling of peripheral blood samples from ZUMA-1 patients to capture immune cell subsets and T cell repertoire during axi-cel treatment. Methods: Single cell immunoprofiling (expression + V(D)J sequencing) was performed on PBMC samples from ZUMA-1 patients (N=32), collected at leukapheresis, 4 weeks, and 6 months post CAR-T cell infusion, to examine potential markers associated with response and resistance. scRNA-seq was performed using 10x Genomics Chromium Next GEM Single Cell 5' Kit v1.1. Full-length paired α/β TCR and BCR libraries were obtained using the Chromium Single Cell V(D)J Enrichment, Human T Cell/B cell kits following manufacturer instructions, while γ/δ TCR libraries were generated using custom primers. Results: Analysis has been completed on the pilot implementation comprising 3 patients. A total of 22,403 cells passed quality-check capturing 31 cellular populations (Figure 1a). In 2 of the 3 patients analyzed, CD8 T cells, after an initial decrease at 4 weeks post CAR T infusion, exhibited an increase at 6 months post CAR T infusion reaching higher levels than those observed prior to CAR T treatment. The third patient presented an increase of the CD8 T cell compartment at 4 weeks compared to pretreatment (Figure 1a). A similar trend was observed for CD4 T cell population, with an increase at 6 months post CAR-T to a level higher than prior to CAR T infusion (Figure 1a). On the contrary, the myeloid cell compartment depicted a gradual decrease from leukapheresis to 6 months post CAR T (Figure 1a). B cells were observed only in 1 of the 3 patients at 6 months (Figure 1a). α/β TCR, γ/δ TCR and BCR clonotypes were identified and projected on the 2-dimensional embedding (Figure 1b). Full-length paired α/β TCR at single cell level showed that some of the most abundant clonotypes at baseline continued to be prominent in post CAR T timepoints (Figure 1c). An extensive expansion of new clonotypes was observed at 6 months after infusion. Moreover, in 2 of the 3 patients, we observed that T cell clonal diversity converged at 4 weeks, and diverged in one patient at 6 months post treatment (Figure 1d). The analysis of the remaining 29 patients (87 samples) is ongoing. Conclusion: The application of single cell immunoprofiling on longitudinal samples from Axi-cel-treated LBCL patients successfully captured the changes in the cellular transcriptional landscape, cell proportions, and TCR/BCR space across the time axis in high resolution. It is anticipated that the full analysis of 32 patients can elucidate the transcriptional program in response to CAR T cell therapy. Figure 1: (A) Two-dimensional uniform manifold approximation and projection (UMAP) of all cells passing QC (n=22,403), separated per patient and timepoint. (B) T and B cell receptor clonality at single cell resolution. T and B cells with one or more clones are colored. (C) α/β clonotype frequency per timepoint for each patient. (D) T Cell receptor Shannon diversity index per timepoint for the 3 profiled patients. Dashed lines connect the different timepoints from the same patient. Figure 1 Figure 1. Disclosures Stroopinsky: The Blackstone Group: Consultancy. Bot: Kite, a Gilead Company: Current Employment; Gilead Sciences: Consultancy, Current equity holder in publicly-traded company, Other: Travel support. Mattie: Kite: Current Employment. Chou: Kite Pharma: Current Employment. Rosenblatt: Karyopharm: Membership on an entity's Board of Directors or advisory committees; Parexel: Consultancy; Wolters Kluwer Health: Consultancy, Patents & Royalties; Bristol-Myers Squibb: Research Funding; Imaging Endpoints: Consultancy; Attivare Therapeutics: Consultancy. 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.

Background: CAR-T cells are a tremendous breakthrough in the treatment of certain type of blood cancer, demonstrating impressive and durable responses. However, mechanisms of resistance and relapse have been reported. Mechanisms contributing to relapse after CAR-T therapy include the downregulation of the CAR-T target antigen and the rapid extinguishing of CAR-T cells. We have developed a personalized cancer vaccine whereby patient derived tumor cells are fused to autologous dendritic cells (DC). DC/tumor vaccine induces a broad anti-tumor immunity capable of preventing relapse but may not be effective in patients with advanced disease. Aims: We sought to overcome relapse and resistance to CAR-T therapy to improve the current response rate to CAR-T cells. To this end, we combined CAR-T cells treatment with our personalized DC/tumor vaccine. We postulated that the DC/tumor vaccine would demonstrate synergy with CAR-T cells in a setting where CAR-T cells reduce the bulky disease and the fusion vaccine prevents relapse by expanding CAR-T cells and tumor antigen specific lymphocytes. Methods: To investigate the effects of the CAR-T/fusion vaccine combination, we used the A20 murine B-cell lymphoma model. Syngeneic T cells were obtained from BALB/c mice and retrovirally transduced with a second-generation CAR construct composed of an antigen binding domain that recognizes murine CD19, the CD3ζ domain, 4-1BB as costimulatory molecule and GFP (m19BBz-GFP). GFP expression was used to assess gene-transfer efficiency and monitor the CAR-transduced T cells. The DC/tumor vaccine was obtained by PEG-mediated fusions of A20 cells and BALB/c DC. In the in vitro experiments, T cells transduced with the m19BBz-GFP CAR or non-transduced T cells were co-cultured in the presence or the absence of the DC/A20 vaccine for 3 days. Tumor killing was measured by quantifying Firefly luciferase activity (WT A20) or Renilla luciferase activity (CD19- A20). Vaccine was removed before starting the killing assay. In the in vivo experiment, B-cell lymphoma was induced in BALB/c mice by tail vein injection of A20 cells. The mice were lymphodepleted and treated with m19BBz-GFP CAR-T. The mice were then subcutaneous injected with the DC/A20 fusion vaccine or with PBS (control group). CD8+ T-cells specific for the A20 idiotype epitope were quantified by MHC Class I tetramer analysis. Results: In vitro co-culture of CAR-T cells and DC/A20 vaccine induced a memory-like CAR-T phenotype and strongly improved CAR-T persistence (measured as % of GFP+ T cells in culture). These results were confirmed in vivo where increased CAR-T percentage was observed in the bone marrow and spleen of vaccinated mice with respect to the unvaccinated control group. Moreover, in the vaccinated mice we detected CD8+ T-cells specific for the A20 idiotype epitope demonstrating the expansion of tumor-specific lymphocytes in response to the fusion vaccine. To assess whether the vaccine-induced persistence of CAR-T cells was translated in a higher tumor killing capacity, we performed an in vitro killing assay. To mimic the presence of CD19- clones in the tumor bulk, we used a mixture of WT A20 and CD19-A20 as CAR-T target cells. Enhanced killing capacity against CD19+ tumors was induced by education of the CAR-T with the vaccine. No effects on the CAR-T killing capacity against CD19- A20 were elicited by the fusion vaccine. However, when the same killing assay was performed using as effector cells a mixture of CAR-T and naïve T cells or a mixture of CAR-T and vaccine-educated T cells, we observed that the addition of vaccine-educated T cell to the CAR-T, strongly reduced both A20 WT and A20 CD19- in the cultures. Thus, vaccine-educated T cells are able to kill tumor cells independently from the presence or the absence of the CAR-T target antigen on the tumor. Conclusions: The combination of CAR-T and DC/tumor vaccine, increasing the persistence of CAR-T cells and evoking a polyclonal T cell response against tumor antigens in the non-CAR-transduced T cells, may represent a novel therapeutic strategy to overcame therapeutic resistance and improve current response rate to CAR-T therapy. Disclosures Capelletti: Caris Life Sciences: Current Employment. Stroopinsky: The Blackstone Group: Consultancy. Kufe: REATA: Consultancy, Current equity holder in publicly-traded company; Genus Oncology: Current equity holder in publicly-traded company; Hillstream BioPharma: Current equity holder in publicly-traded company; Canbas: Consultancy. Themeli: Fate Therapeutics: Patents & Royalties. Rosenblatt: Attivare Therapeutics: Consultancy; Imaging Endpoints: Consultancy; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Parexel: Consultancy; Wolters Kluwer Health: Consultancy, Patents & Royalties; Bristol-Myers Squibb: Research Funding. Sadelain: Minerva Biotechnologies: Patents & Royalties; Juno Therapeutics: Patents & Royalties; Fate Therapeutics: Other: Provision of Services (uncompensated), Patents & Royalties; Mnemo Therapeutics: Patents & Royalties; Takeda Pharmaceuticals: Other: Provision of Services, Patents & Royalties; Ceramedix: Patents & Royalties; NHLBI Gene Therapy Resource Program: Other: Provision of Services (uncompensated); St. Jude Children's Research Hospital: Other: Provision of Services; Atara Biotherapeutics: Patents & Royalties. 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.

Introduction CAR T cells have demonstrated unique potency for tumor cytoreduction and durable response in hematological malignancies. However, disease relapse remains a concern due to the emergence of antigen negative variants, tolerization of CAR T cell populations and lack of T cell persistence. We postulated that vaccination with a personalized cancer vaccine, that we developed, in which patient derived tumor cells are fused with autologous dendritic cells would enhance CAR T cell efficacy through the expansion of T cell clonal populations and the vaccine mediated enhancement of T cell activation and persistence. In the present study, we examined the potential synergy between CAR T cells targeting CD19 and syngeneic DC/tumor fusions. Methods/Results CD19 CAR T cells and DC/tumor fusions were studied in the context of a murine A20 lymphoma model, and their combination effectively lysed A20 cells in a CTL assay in a higher percentage than CAR T cells alone (20% vs 34%). Moreover, we examined the interaction of vaccine and CAR T cells ex vivo using the IncuCyte S3 Live-Cell Analysis System which allows for live cell visualization of lysis of A20 cells over time. We studied the impact of combining vaccine educated and CAR T cells as well as an individual T cell population that underwent sequential vaccine mediated stimulation followed by transduction with the CD19 CAR. Even in this assay, coculture with either combined vaccine educated and CAR T cells or sequentially vaccine educated, and transduced T cells demonstrated the highest levels of cytotoxicity that was maintained over time. Enhanced lysis by combined vaccine stimulation and CAR T cells was similarly demonstrated in another tumor cell line, 5TGM1, a multiple myeloma cell line transduced to express CD19. On the contrary wild type 5TGM1 cells were recognized by the DC/tumor fusion stimulated cells in contrast to CAR T cells alone (40% vs. 8%). Finally, we examined the capacity of vaccine educated T cells in conjunction with CAR T cells to target A20 cells in an immunocompetent murine model. Mice were challenged with 1 × 106 A20 Mcherry-Luc and after lymphoma engraftment animals were treated with 3 × 106 T cells consisting of CAR T cells, vaccine educated T cells or the combination. Serial bioluminescence imaging demonstrated greatest reduction in tumor burden using combined CAR T and vaccine educated T cells with 4/5 animals still disease free at day 13 after tumor challenge. Conclusions Combined therapy with T cells stimulated by DC/tumor fusions and CAR T cells exhibited potent lysis of murine lymphoma and myeloma cells compared to CAR T cells or vaccine educated T cells alone in in vitro and immunocompetent murine models. These findings suggest potent synergy between these modalities that may overcome recognized pathways of resistance including the broadening of the tumor specific response and vaccine mediated activation of CAR T cell populations.

Introduction In a clinical trial of patients vaccinated after chemotherapy-induced remission with patient-derived AML cells fused with autologous dendritic cells (DC/AML fusions) we demonstrated durable AML-specific immunity. 71% of patients sustained long-term remission from AML at 55-91 months (responders) while those who experienced relapse had recurrence at 2-26 months (non-responders). Objectives We performed genomic analysis of the marrow microenvironment to identify factors associated with durable remission after vaccination. Methods RNA sequencing was done using banked formalin-fixed paraffin embedded (FFPE) marrow at serial time points. Ingenuity Pathways IPA 9.0 was used to define pathways and upstream regulators. To characterize the cellular components, single cell RNA-seq was performed on fresh frozen aspirates with cell cluster annotation performed by Single Cell Wizard software and peripheral blood was used for TCR clonal diversity by NGS. Results Heatmaps depict significant (p-value ≤0.01 and fold change ≥2) differential gene expression both pre- and post-vaccination in responders compared to non-responders. Prior to vaccination there was inhibition of TGF-β in responders. Notable upregulated pathways in responders after vaccination (p value Conclusion In a cohort of AML patients treated with DC/AML fusions, we found distinct gene signatures amongst patients with long term remisison vs those with early relapse. The transcriptomes indicate a microenvironment in which TGF-β-mediated immunosuppression is inhibited after chemotherapy and in which interleukins, B cell and macrophage signaling are activated are associated with vaccine response, and have potential for combinatorial therapeutic strategies. T cell costimulation and activation are key components of response with NK cells and DC activation playing a supporting role. A durable oligoclonal T cell expansion suggests vaccine is capable of enhancing the T cell repertroie which could provide a unique opportunity to identify target antigens by TCR-epitope pairing.

Acute myeloid leukaemia (AML) is a lethal haematological malignancy characterized by an immunosuppressive milieu in the tumour microenvironment (TME) that fosters disease growth and therapeutic resistance. Hypomethylating agents (HMAs) demonstrate clinical efficacy in AML patients and exert immunomodulatory activities. In the present study, we show that guadecitabine augments both antigen processing and presentation, resulting in increased AML susceptibility to T cell-mediated killing. Exposure to HMA results in the activation of the endogenous retroviral pathway with concomitant downstream amplification of critical mediators of inflammation. In an immunocompetent murine leukaemia model, guadecitabine negatively regulates inhibitory accessory cells in the TME by decreasing PD-1 (also termed PDCD1) expressing T cells and reducing AML-mediated expansion of myeloid-derived suppressor cells. Therapy with guadecitabine results in enhanced leukaemia-specific immunity, as manifested by increased CD4 and CD8 cells targeting syngeneic leukaemia cells. We have previously reported that vaccination with AML/dendritic cell fusions elicits the expansion of leukaemia-specific T cells and protects against disease relapse. In the present study, we demonstrate that vaccination in conjunction with HMA therapy results in enhanced anti-leukaemia immunity and survival. The combination of a novel personalized dendritic cell/AML fusion vaccine and an HMA has therapeutic potential, and a clinical trial investigating this combination is planned.

Introduction: We are conducting a clinical trial in which patients with acute myeloid leukemia (AML) who are undergoing allogeneic transplant undergo post-transplant vaccination with DC/AML fusion cells. Allogeneic transplantation is uniquely curative for a subset of patients with AML, however, post-transplant relapse and graft versus host disease remain significant concerns. We have developed a promising leukemia vaccine in which patient derived AML cells are fused with donor-derived dendritic cells (DCs), presenting a broad array of antigens that capture the heterogeneity of the leukemia cell population. We hypothesize that DC/AML vaccination post-transplant would elicit the durable expansion of leukemia specific T cells within the donor T cell repertoire to effectively protect against disease relapse. Methods: Patients undergo collection and cryopreservation of leukemia cells at the time of diagnosis with AML. Patients who undergo an allogeneic transplant in complete remission from a matched related or unrelated donor (cohort A) or a haplo-identical donor (Cohort B) are assessed for eligibility to undergo leukapheresis for dendritic cell generation between day 25-45 post-transplant. In order to proceed with leukapheresis, patients must demonstrate donor hematopoietic recovery in the absence of ongoing grade 2 or higher GVHD. Patients initiate vaccination between day 70-100 post-transplant. 2 vaccines are given at 3 week intervals, in conjunction with GMCSF 100 mcg daily at the vaccine site for 4 days. A booster vaccine may be given 30-60 days following the taper of immune suppression, in the absence of GVHD. Results: To date, 12 participants have undergone vaccine generation. The median age is 62 years (range 23-74). 10 participants were enrolled to cohort A: 7 were transplanted with a matched unrelated donor and 3 were transplanted with a matched sibling donor. 2 participants were enrolled to cohort B following transplant from a haplo-identical donor. The mean yield of leukemia cells was 314 x106 (range 95-to 818-x106)and mean viability was 96%. For DC generation, patients underwent leukapharesis and adherent mononuclear cells were cultured with GM-CSF, IL-4 and TNFa. The mean yield of DCs was 131 x106 and viability 77%. Fusion vaccine was successfully generated in 11/12 patients, with mean fusion efficiency of 51% with viability of 76%. One patient had insufficient DC for vaccine generation. Mean Fusion Vaccine Dose was 4.7 x 106 fusion cells. 3 patients did not meet eligibility to initiate vaccination due to ongoing toxicity following transplant (2 patients) and GVHD (1 patient). 8 participants have initiated vaccine administration and are evaluable for toxicity and response. The most common side effects have been grade 1 vaccine site reactions (n=9 grade 1, n=1 grade 2). 4 patients developed GVHD that was determined to be possibly related to vaccination, at a median time of 16.5 days after vaccination (range 5-21 days). 2 of these patients developed grade 2 acute GVHD of the skin, one patient developed grade 2 gastrointestinal GVHD that subsequently evolved into moderate, chronic GVHD affecting the skin, GI tract, eyes and mouth, and one patient developed mild transaminitis attributed to liver GVHD. An additional 3 patients developed GVHD with a median time of 99 days post vaccination (range 91-123 days), assessed as being unlikely related to vaccine. 7 of the 8 patients remain in a CR at a median time of 15.5 months post-transplant (range 4.8-22.4 months). One patient relapsed 14.8 months post haplo-identical transplant. Immunologic response following vaccination is being assessed, with respect to the presence of leukemia-reactive T cells, T cells targeting previously identified leukemia- associated antigens, T cell clonality, T regulatory cells, and PD-1 expressing T cells. In the absence of treatment associated toxicity, a second cohort is planned, in which vaccine will be given in conjunction with decitabine. Conclusions: Vaccine generation using donor derived DC isolated following engraftment is feasible. Mild to moderate graft versus host disease has been observed in a subset of patients, and 7/8 vaccinated patients remain relapse free. Correlative science studies to assess immune response to vaccination, identify neoantigen targets, and characterize the immune milieu, will be reported. Disclosures Stone: Syros: Consultancy; Syntrix: Consultancy; Syndax: Consultancy; Stemline: Consultancy; Hoffman LaRoche: Consultancy; Macrogenics: Consultancy; Janssen: Consultancy; Gemoab: Consultancy; Elevate: Consultancy; Daiichi-Sankyo: Consultancy; Takeda: Consultancy; Trovagene: Consultancy; Pfizer: Consultancy; Otsuka: Consultancy; Novartis: Consultancy, Research Funding; Jazz: Consultancy; Celgene: Consultancy, Other: Data and safety monitoring board; Biolinerx: Consultancy; AstraZeneca: Consultancy; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Arog: Research Funding; Argenx: Consultancy, Other: Data and safety monitoring board; Agios: Consultancy, Research Funding; Actinium: Consultancy; AbbVie: Consultancy, Research Funding. Soiffer:Gilead: Consultancy; Rheos Therapeutics: Consultancy; Cugene: Consultancy; Precision Bioscience: Consultancy; Mana Therapeutics: Consultancy; VOR Biopharma: Consultancy; Novartis: Consultancy; Juno: Membership on an entity's Board of Directors or advisory committees; 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; Kiadis: Membership on an entity's Board of Directors or advisory committees. Neuberg:Pharmacyclics: Research Funding; Celgene: Research Funding; Madrigak Pharmaceuticals: Current equity holder in publicly-traded company.

Our personalized cancer vaccine comprises patient tumor cells fused with autologous dendritic cells (DCs) and limits the risk of antigen escape by presenting a broad array of tumor antigens in the context of DC mediated co-stimulation. In clinical trials of hematologic malignancies patients, vaccination induced expansion of tumor-specific T cells (Tc) and led to prolonged remission in a subset of patients. Currently, we are developing a novel second-generation (2G) vaccine. Fusions are presented in the context of a unique biomatrix expressing high levels of 41BB ligand (41BBL), to further accentuate Tc activation. In this study, we show the efficacy of vaccination in a preclinical lymphoma model and enhanced potency of the 2G vaccine. We first showed the fusions potency in generating anti-tumor immunity in the A20 lymphoma murine model. Bone marrow derived mononuclear cells cultured with GM-CSF and IL-4 were fused to A20 cells. Fusions effectively induced tumor specific immunity as manifested by potent lysis of A20 Tc in vitro as compared to unstimulated Tc in a CTL assay. Consistently, vaccine effectively induced tumor specific immunity in an immunocompetent murine model. Balb/C mice (30) underwent IV inoculation with 750,000, luciferase transduced, A20 cells. 24h post inoculation, 15 mice were vaccinated with 105 fusions. 10 days post inoculation, in the untreated cohort all mice had detectable tumor whereas in the treated group, 5 mice didn't show disease and 5 mice showed minimal disease as detected by BLI imaging. We further showed that patient autologous vaccine stimulated Tc mediated lysis of primary lymphoma cells. DC, generated from patient's peripheral blood mononuclear cells cultured with GM-CSF and IL-4 and matured with TNFa were fused to primary lymphoma cells isolated from resected tumor. In a standard CTL assay, fusion stimulated Tc potently lysed autologous tumor cells as compared to unstimulated Tc (25.7% as compared to 12.66%). To enhance vaccine potency, we developed a biomatrix expressing 41BBL. By carbodiimide chemistry we covalently bonded 41BBL protein to an alginate (Alg)-based scaffold, promoting a supporting microenvironment for Tc and fusions co-culture. We cultured syngeneic Tc with fusions within a scaffold with or without bound 41BBL and examined Tc cytotoxicity by a CTL assay. Tc stimulated by vaccine within the Alg/41BBL scaffold showed higher levels of tumor lysis compared to the percent killed by Tc cultured within an Alg scaffold (22.95% and 13.95 respectively). In this study we succeeded in demonstrating the fusions capacity to generate tumor specific Tc cytotoxicity. We presented patient derived tumor results supporting the applicable nature of the vaccine. In addition, we developed a 2G vaccine comprised of the original vaccine presented to the Tc in an Alg/41BBL scaffold acting as a nurturing microenvironment for Tc tumor specific immune response.

Immunotherapy seeks to restore and augment the unique ability of the immune system to recognize and kill malignant cells. This strategy has previously been incorporated into standard of care in myeloma with the use of immunomodulatory drugs and allogeneic transplant. The following review will discuss the rationale for immunotherapy to reverse critical aspects of the immunosuppressive milieu in myeloma and avenues where cellular therapies are now revolutionizing myeloma treatment. Areas covered: A particular focus is outcomes of clinical trials in myeloma published in PubMed database or abstract form using vaccines or adoptive cell transfer: marrow infiltrating lymphocytes, T-cell receptor and chimeric antigen receptor T cells. Expert commentary: Immunotherapy has extraordinary potential in myeloma. Combinations of cellular therapies with immunomodulatory molecules or checkpoint inhibitors are likely to be synergistic and now underway. Future directions include neoantigen or nanoparticle vaccines and further modifications of engineered T cells such as use of dual-antigens, suicide genes or allogeneic cells.

Graft failure (GF) after hematopoietic cell transplant (HCT) occurs in 5-30% of patients. GF can be accompanied by neutropenia (NGF) or can result with adequate neutrophils, but loss of donor chimerism (non-neutropenic graft failure, NNGF). In this report, we describe the outcomes of 95 patients treated with a second HCT for GF at the University of Minnesota; 62 with NGF and 33 with NNGF. The cumulative incidence of neutrophil recovery at 42 days after second HCT was 45% for NGF and 88% for NNGF. A second GF occurred in 34 NGF (55%) and in 9 NNGF (27%) patients. The incidence of Grade III-IV acute graft versus host disease (GVHD) was 8% (95% confidence interval (CI), 1-16%) and 12% (95% CI, 1-23%) for NGF and NNGF, respectively. From the 2nd HCT, 1-year overall survival (OS) was 44% (95% CI, 34-54%), [NNGF: 76% (95% CI, 57-87%) and NGF: 27% (95% CI, 17-39%)]. The most common cause of death after second HCT was infection (52%). In summary, the outcomes of second HCT after NGF and NNGF are different with much worse outcomes for NGF necessitating new approaches for this complication.

Introduction: Our group has developed a novel vaccine using patient-derived acute myeloid leukemia (AML) cells and autologous dendritic cells (DCs), capable of presenting a broad array of leukemia antigens. In a phase I/II clinical trial DC/AML vaccination led to an expansion of leukemia-specific T cells. We hypothesized that the fusion vaccine offered a unique platform for ex vivo expansion of functionally potent leukemia specific T cells with broad specificity targeting shared and tumor specific neoantigens. We postulated that incorporating 4-1BB (CD137) mediated co-stimulation would further enhance activation of antigen specific T cells and the development of a crucial memory response as well as promote survival and persistence. Here we describe therapeutic exploration of the use of 4-1BB to augment vaccine-educated T cells for adoptive cellular therapy in an immunocompetent murine model. Methods: DC/AML fusion vaccine was generated using DCs obtained from C57BL/6J mice and syngeneic C1498 AML cells as previously described. T cells were obtained from splenocytes after magnetic bead isolation and cultured with irradiated DC/AML fusion vaccine in the presence of IL-15 and IL-7. Following co-culture, 4-1BB positive T cells were ligated using agonistic 4-1BB antibody (3H3 clone, BioXCell) and further selected with RatIgG2a magnetic beads (Easy Sep). Subsequently T cells were expanded with anti-CD3/CD28 activation beads (Dynabeads). In vivo, mice underwent retro-orbital inoculation with C1498 and vaccination with irradiated fusion cells the following day. Agonistic mouse anti-4-1BB antibody was injected intraperitoneally on day 4 and day 7. In addition, C1498 cells were transduced with Mcherry/luciferase and a reproducible model of disease progression was established. Results: DC/fusion stimulated T cells showed increased immune activation as measured by multichannel flow cytometric analysis. Compared to unstimulated T cells, there was 5-fold increase in CD4+CD25+CD69+, and a 10-fold and 7-fold increase in 4-1BB and intracellular IFNƔ expression on CD8+ cells respectively. Following agonistic 4-1BB ligation and bead isolation, the proliferation rate was increased in the 4-1BB positive fraction as compared to both 4-1BB negative cells and unstimulated T cells. In addition, the 4-1BB positive fraction demonstrated increased cytotoxicity, as measured by a CTL assay detecting granzyme B with 1:10 tumor to effector cells. A shift from naïve to memory T cell phenotype was also observed. Following DC/fusion stimulation, CD44+CD62L- cells comprised 67% of CD8+ cells versus 20% without stimulation, the latter reflecting the effect of cytokines alone. Following 4-1BB ligation and anti-CD3/CD28 bead expansion, this phenotype was retained with the CD4+ and CD8+ effector memory and central memory compartments comprising the majority of T cells. Such findings are significant as presence of memory T cell populations are a critical component for successful adoptive cell transfer. The effect of agonistic 4-1BB antibody following vaccination was evaluated in vivo in an aggressive immunocompetent murine AML model. The combination of DC/AML fusion vaccine with 4-1BB antibody was associated with increased long-term survival (>120 days) of 40% versus 20% of mice treated with vaccine alone while all controls required euthanasia by 40 days. Conclusion: In the current study we have demonstrated the ability of DC/AML fusion vaccine to stimulate T cells ex-vivo as demonstrated by both early-activation (CD25,CD69), upregulation of antigen-specific markers (CD137) and cytokine secretion. Further enhancement of the cellular product using agonistic 4-1BB ligation and isolation simultaneously enriches for antigen-activated cells, as demonstrated by more potent cytotoxicity, as well as promoting memory phenotype and survival. Use of 4-1BB ligation for antigen-specific selection while providing an agonistic co-stimulatory signal is a potentially novel approach for development of non-engineered T cells. Ongoing experiments evaluating the efficacy of 4-1BB selected vaccine educated T cells using bioluminescence monitoring will be reported as well as in vitro use of patient-derived T cells. Disclosures Kufe: Canbas: Consultancy, Honoraria; Victa BioTherapeutics: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genus Oncology: Equity Ownership; Hillstream BioPharma: Equity Ownership; Reata Pharmaceuticals: Consultancy, Equity Ownership, Honoraria; Nanogen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Rosenblatt:Dava Oncology: Other: Education; Partner Tx: Other: Advisory Board; Parexel: Consultancy; Celgene: Research Funding; BMS: Research Funding; Amgen: Other: Advisory Board; Merck: Other: Advisory Board; BMS: Other: Advisory Board ; Imaging Endpoint: Consultancy. Avigan:Takeda: Consultancy; Parexel: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partners Tx: 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; Janssen: Consultancy.

Background: We developed a vaccine consisting of primary AML cells fused with autologous dendritic cells (DC) such that a broad array of antigens are presented in the context of DC-mediated costimulation, thereby expanding the anti-AML T cell repertoire. The reestablishment of tumor tolerance by the microenvironment (TME) can limit vaccine efficacy and reversal of TME-mediated immunosuppression has the potential to enhance vaccine immunogenicity. To this end, using an immunocompetent syngeneic AML murine model, we have shown that hypomethylating agents (HMAs) reverse critical aspects of the immunosuppressive TME, and that HMA + fusion vaccination enhances AML-specific immune responses and survival. The TIGIT-CD155 inhibitory checkpoint pathway is a novel pathway fostering tolerance in the innate and adaptive immune arms. Blockade of this pathway reverses tumor-mediated T cell quiescence and promotes DC and NK cell immune surveillance. We aimed to examine if TIGIT-CD155 pathway inhibition +/- HMA enhances vaccine potency in a murine model. Methods/Results: Using a syngeneic immunocompetent murine AML model, we first investigated the impact of functional TIGIT monoclonal (mAb) blockade +/- decitabine (DAC) +/- vaccine on immune modulation and murine survival. C57BL/6J mice were retro-orbitally inoculated with 5x104 TIB-49 murine AML cells. Animals treated with TIGIT mAb monotherapy had inferior survival as compared to those treated with DAC or DC/AML vaccine monotherapies. Mice treated with vaccine alone, vaccine + TIGIT mAb, and vaccine + DAC + TIGIT mAb had similar survival but each cohort had improved survival compared to untreated animals, suggesting a vaccine-mediated effect not further enhanced by TIGIT mAb. To this end, we sought to investigate if deleting CD155, the native receptor to TIGIT, would impact AML immunogenicity and murine survival. We determined that baseline expression of CD155 in the murine AML cell line, TIB-49, is 72% by Ab staining and flow cytometric analysis. Using CRISPR/cas9 technology, we generated a CD155 knockout TIB-49 cell line. Black mice were retro-orbitally inoculated with 5x104 CD155-deleted or control TIB-49 AML cells. Animals were treated with vaccine, DAC, or both therapies. BLI imaging was performed 1-2/week. Animals inoculated with CD155-deleted AML cells that were treated with both DAC and vaccine show no signs of disease and remain alive 80 days following inoculation as compared to mice treated with vaccine or DAC alone (Fig 1,2). Conclusions: We demonstrate that CD155-specific deletion from a murine AML cell line confers a survival advantage when mice are exposed to HMA + vaccine as compared to either agent alone. Studies including TCR repertoire analyses are underway to better understand the immunologic underpinnings of this findings as targeting this pathway has promising translational potential. Disclosures Rosenblatt: Parexel: Consultancy; Imaging Endpoint: Consultancy; Celgene: Research Funding; BMS: Research Funding; Amgen: Other: Advisory Board; Merck: Other: Advisory Board; BMS: Other: Advisory Board ; Dava Oncology: Other: Education; Partner Tx: Other: Advisory Board. Kufe:Genus Oncology: Equity Ownership; Nanogen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Canbas: Consultancy, Honoraria; Hillstream BioPharma: Equity Ownership; Victa BioTherapeutics: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees; Reata Pharmaceuticals: Consultancy, Equity Ownership, Honoraria. Avigan:Takeda: Consultancy; Parexel: Consultancy; Janssen: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partners Tx: 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.

Introduction: We have pioneered a personalized cancer vaccine in which patient derived tumor cells are fused with autologous dendritic cells (DCs) such that a broad array of shared and neo-tumor antigens is presented in the context of DC mediated co-stimulation, limiting the risk of antigen escape. In clinical trials of patients with hematologic malignancies, vaccination with DC/tumor fusions induced an expansion of tumor-specific T cells, and resulted in prolonged remissions in a subset of patients. In the current study, we have developed a novel second generation vaccine, whereby a DC/lymphoma fusion vaccine is presented in the context of a unique biomatrix that expresses high levels of the 41BB costimulatory molecule, to further accentuate T cell activation and prevent the establishment of tumor tolerance. In this study, we demonstrate efficacy of DC/lymphoma fusion cell vaccination in a preclinical lymphoma model, and show enhanced potency of the second-generation vaccine. Methods/Results: We first demonstrated the potency of the DC/tumor fusion vaccine in generating anti-tumor immunity in the A20 lymphoma model. Murine DC/A20 fusions were generated from bone marrow derived mononuclear cells cultured with GM-CSF and IL-4 then fused to syngeneic A20 lymphoma cells. DC/A20 fusion cells effectively induced tumor specific immunity as manifested by potent lysis of A20 T cells in vitro as compared to unstimulated T cells in a standard CTL assay. Consistent with this observation, vaccination with DC/A20 fusions effectively induced lymphoma specific immunity in an immunocompetent murine model. Balb/C mice (30 animals) underwent IV inoculation with 750,000 syngeneic, luciferase and mCherry transduced, A20 cells. 24 hours after tumor cells challenge, 15 mice were treated subcutaneously with 105 DC/A20 fusions. Tumor burden was detected using BLI imaging. 10 days post inoculation, within the untreated cohort all 15/15 mice had detectable tumor whereas within the treated group, 5 mice did not demonstrate any evidence of disease and 5 mice demonstrated minimal disease. We subsequently demonstrated that patient derived autologous DC/lymphoma fusions stimulated T cell mediated lysis of primary lymphoma cells. DC were generated from patient derived peripheral blood mononuclear cells cultured with GM-CSF and IL-4 and matured with TNFa. Primary lymphoma cells were isolated from resected tumor and fused with DC at a ratio of 10:1. Fusion stimulated T cells potently lysed autologous tumor cells as compared to unstimulated T cells (25.7% as compared to 12.66%) in a standard CTL assay. To further enhance vaccine potency, we developed a biomatrix substrate expressing the costimulatory molecule 41BB. Using carbodiimide chemistry we covalently bonded RGD peptide and 41BBL protein to an alginate (Alg)-based scaffold. The Alg/RGD/41BBL scaffold can serve as a supporting microenvironment for the co-culture of T cells and fusion vaccine. We cultured syngeneic T cells with DC/A20 fusion vaccine within a scaffold with or without bound 41BBL and examined the T cells cytotoxicity by a CTL assay as described above. Vaccine mediated stimulation of T cells in the context of the Alg/RGD/41BBL scaffold demonstrated higher levels of tumor lysis as compared to the percent T cells cultured within an Alg/RGD scaffold (22.95% and 13.95% respectively). Conclusion: In the current study we assessed the efficacy of the DC/Lymphoma fusion vaccine to elicit a tumor specific immune response. We succeeded in demonstrating the capacity of DC/Lymphoma fusion vaccine to generate tumor specific T cell cytotoxicity in vitro as well as in vivo in an immunocompetent murine model. Accordingly, we presented patient derived primary tumor results supporting the applicable nature of the DC/Lymphoma vaccine in lymphoma patients. In addition, we developed a second-generation fusion vaccine comprised of the original DC/Tumor vaccine presented to the T cells in an Alg/RGD/41BBL scaffold acting as a nurturing microenvironment for T cell immune specific response against the tumor cells. Our initial results exhibit promising potential and an in vivo experiment with the second-generation fusion vaccine is ongoing. Disclosures Arnason: Celgene/Juno: Consultancy; Regeneron Pharmaceuticals, Inc.: Consultancy. Kufe:Nanogen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Genus Oncology: Equity Ownership; Reata Pharmaceuticals: Consultancy, Equity Ownership, Honoraria; Hillstream BioPharma: Equity Ownership; Victa BioTherapeutics: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees; Canbas: Consultancy, Honoraria. Rosenblatt:Dava Oncology: Other: Education; BMS: Research Funding; Partner Tx: Other: Advisory Board; Merck: Other: Advisory Board; Parexel: Consultancy; Imaging Endpoint: Consultancy; Celgene: Research Funding; BMS: Other: Advisory Board ; Amgen: Other: Advisory Board. Avigan:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partners Tx: 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; Janssen: Consultancy; Parexel: Consultancy; Takeda: Consultancy.

Introduction Our group has pioneered a personalized vaccine in which patient-derived acute myeloid leukemia (AML) cells are fused with autologous dendritic cells (DC/AML fusion), presenting a broad array of leukemia associated antigens with DC mediated costimulation. In a clinical trial of AML patients who were vaccinated after chemotherapy-induced remission, 71% remained free of disease at median follow up of 57 months. We sought to identify factors associated with durable remission after vaccination using genomic analysis of the bone marrow microenvironment including single cell RNA-seq and TCR clonal diversity analysis. Methods Banked bone marrow samples both prior to and 1 month post-vaccination were selected from patients who maintained long disease remission for greater than 5 years and those who had early relapse. FFPE marrow core biopsy samples (N=10) were the source for gene expression analysis. NEBNext ultra II directional library prep kit and Illumina NextSeq 500/550 system were used to generate reliable high quality RNA sequencing data. Differentially expressed genes were identified by p-value (≤0.01) and fold change (≥2) using Linear Models for Microarray (Limma) approach. Ingenuity Pathways IPA 9.0 was then used to define pathways and upstream regulators. Flash frozen samples (N=4) were analyzed by RNAseq at the single cell level using a standard 10X genomics approach with cell cluster annotation performed with Single Cell Wizard software. Banked peripheral blood was used to evaluate TCR diversity with Takara SMART-Seq next-generation sequencing to amplify variable regions of TCR- α/β subunits. Results Heatmaps depict significant differential gene expression in bone marrow biopsies both pre- and post-vaccination in patients who remained in long-term remission (responders) compared to those who relapsed (non-responders). Prior to vaccination there was modest upregulation of immune activation pathways including IL-7, IL-17A as well as inhibition of TGF-b in responders, suggesting a role of the micro-environment in modulating response. Significantly upregulated pathways in responders after vaccination (p value To characterize the cellular components of the immune micro-environment, single cell analysis was assessed in bone marrow aspirates both pre- and post-vaccination (N=2). Increased cellular heterogeneity pre-vaccination, and increases in T and NK populations post-vaccination, were noted in responding patients who had durable remissions. Furthermore, the temporal changes in expression of TCR clonotypes showed an increase in TCR diversity post-vaccination (N=2). Of note, a patient who achieved a durable remission had (i) loss of specific clonal populations present at the time of diagnosis and (ii) the emergence of newly expanded TCR signatures that were further expanded with subsequent vaccinations and remained present during the follow up period. In contrast, the TCR diversity in a non-responder was low and static with no difference in the TCR clonotypes after vaccination. Conclusions In a cohort of AML patients vaccinated after chemotherapy-induced remission, we found distinct gene signatures amongst patients with long term response as compared to those with early relapse. These signatures have potential to serve as predictive and early biomarkers of vaccine response, and will be investigated in a larger cohort from an ongoing trial. The transcriptomes indicate that vaccine response is dependent on a robust immune microenvironment, as characterized by upregulation of cytokines, activation of T cells, B cells and macrophages, and reduction of TGF-b-mediated negative immunoregulation. We also found that vaccine response is associated with durable oligoclonal expansion within the T cell repertoire, which purportedly represent functionally potent anti-AML shared- and neo-antigen specific T cell populations. This provides an especially unique opportunity to identify target antigens by TCR-epitope pairing. Indeed, information regarding AML antigens targeted by the immune system in the induction of durable remissions could further advance the field of AML treatment by integration in combinatorial therapeutic strategies. Figure Disclosures Stone: Roche: Consultancy; Arog: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Stemline: Consultancy; Takeda: Other: DSMB; Agios: Consultancy, Research Funding; Astra-Zeneca: Consultancy; Macrogenics: Consultancy; Argenix: Other: DSMB; Arog: Consultancy, Research Funding; Biolinerx: Consultancy; Astellas: Consultancy; Daiichi-Sankyo: Consultancy; Celgene: Consultancy, Other: DSMB; Jazz: Consultancy; Abbvie: Consultancy, Research Funding; Trovagene: Consultancy; Biosight: Consultancy; Pfizer: Consultancy; Otsuka: Consultancy; Pfizer: Consultancy; Trovagene: Consultancy; Stemline: Consultancy; Jazz: Consultancy; Actinium: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Biolinerx: Consultancy; Biosight: Consultancy; Novartis: Consultancy, Research Funding; Astra-Zeneca: Consultancy; Abbvie: Consultancy, Research Funding; Biolinerx: Consultancy; Agios: Consultancy, Research Funding; Roche: Consultancy; Macrogenics: Consultancy; Trovagene: Consultancy; Argenix: Other: DSMB; Argenix: Other: DSMB; Otsuka: Consultancy; Takeda: Other: DSMB. Kufe:Genus Oncology: Equity Ownership; Reata Pharmaceuticals: Consultancy, Equity Ownership, Honoraria; Nanogen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Victa BioTherapeutics: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees; Canbas: Consultancy, Honoraria; Hillstream BioPharma: Equity Ownership. Avigan:Takeda: Consultancy; Parexel: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partners Tx: 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; Janssen: Consultancy. Rosenblatt:Partner Tx: Other: Advisory Board; Dava Oncology: Other: Education; Parexel: Consultancy; BMS: Other: Advisory Board ; Merck: Other: Advisory Board; Amgen: Other: Advisory Board; BMS: Research Funding; Celgene: Research Funding; Imaging Endpoint: Consultancy.

Introduction: CAR T cells have demonstrated unique potency for tumor cytoreduction and the potential for durable response in patients with advanced hematological malignancies. However, disease relapse remains a significant concern due to the emergence of antigen negative variants, tolerization of CAR T cell populations and lack of T cell persistence. We have developed a personalized cancer vaccine in which patient derived tumor cells are fused with autologous dendritic cells such that a broad array of tumor antigens is expressed in the context of DC mediated co-stimulation. Vaccination of patients with acute leukemia and multiple myeloma has been associated with the durable expansion of tumor specific lymphocytes in the bone marrow and peripheral blood, targeting of residual disease, and durable remission. We postulated that vaccination with DC/tumor fusions would enhance CAR T cell efficacy through the expansion of T cell clonal populations targeting tumor cells via the native TCR and the vaccine mediated enhancement of T cell activation and persistence. In addition, ex vivo engineered CAR T cells provide a substrate of functionally competent T cells with cytoreductive capacity in the setting of advanced disease. In the present study, we examined the potential synergy between CAR T cells targeting CD19 and syngeneic DC/tumor fusions. Methods/Results: CAR T cells and DC/tumor fusions were studied in the context of a murine A20 lymphoma model. CD19 CAR T cells were established through retroviral transduction of a CD19 CAR construct expressing CD28 and 41BBL syngeneic DC/A20 fusions were generated as previously described. Vaccine stimulated T cells were generated by coculturing splenocyte derived T cells with syngeneic DC/A20 fusion cells over a period of three days in a 10:1 ratio in the presence of low dose IL2. While CD19 CAR T cells effectively lysed a subset of A20 cells in a CTL, the addition of vaccine educated T cells increased the percentage of tumor cells undergoing CTL mediated lysis (20% vs 34%). We subsequently examined the interaction of vaccine and CAR T cells ex vivo using the IncuCyte S3 Live-Cell Analysis System which allows for live cell visualization of lysis of A20 cells over time. We studied the impact of combining vaccine educated and CAR T cells as well as an individual T cell population that underwent sequential vaccine mediated stimulation followed by transduction with the CD19 CAR. While vaccine educated and CAR T cells demonstrated potent lysis of A20 cells over time, coculture with either combined vaccine educated and CAR T cells or sequentially vaccine educated and transduced T cells demonstrated the highest levels of cytotoxicity that was maintained over time (1786 and 2338 signal overlap count per image at 23 hours compared to 123 of the control). Enhanced lysis by combined vaccine stimulation and CAR T cells was similarly demonstrated in another tumor cell line, 5TGM1, a multiple myeloma cell line transduced to express CD19. Cytotoxic killing of the 5TGM1-CD19 cells was most pronounced when combining vaccine educated and CAR T cells as compared to CAR T cells alone (33% vs 14%). Consistent with the broad targeting of vaccine educated as compared to the CAR T cell population, wild type 5TGM1 cells were recognized by the DC/tumor fusion stimulated cells in contrast to CAR T cells alone (40% vs. 8%). We subsequently examined the capacity of vaccine educated T cells in conjunction with CAR T cells to target A20 cells in an immunocompetent murine model. Mice were challenged with 1 x 10(6) A20 Mcherry-Luc and lymphoma engraftment was demonstrated at Day 7. Animals were then treated with 3 x 10(6) T cells consisting of CAR T cells, vaccine educated T cells or the combination. Serial bioluminescence imaging demonstrated greatest reduction in tumor burden using combined CAR T and vaccine educated T cells with 4/5 animals without BLI evidence of disease at day 13 after tumor challenge. Conclusions: In in vitro and immunocompetent murine models, we have demonstrated that combined therapy with T cells stimulated by DC/tumor fusions and CAR T cells exhibited potent lysis of murine lymphoma and myeloma cells as compared to the efficacy of CAR T cells or vaccine educated T cells alone. These findings suggest potent synergy between these modalities that may overcome recognized pathways of resistance including the broadening of the tumor specific response and vaccine mediated activation of CAR T cell populations. Disclosures Themeli: Covagen: Consultancy. Mutis:Janssen Research and Development: Research Funding; Celgene: Research Funding; Onkimmune: Research Funding; Genmab: Research Funding. Munshi:Adaptive: Consultancy; Amgen: Consultancy; Oncopep: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Abbvie: Consultancy. Kufe:Genus Oncology: Equity Ownership; Reata Pharmaceuticals: Consultancy, Equity Ownership, Honoraria; Nanogen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Hillstream BioPharma: Equity Ownership; Victa BioTherapeutics: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees; Canbas: Consultancy, Honoraria. Rosenblatt:BMS: Research Funding; Amgen: Other: Advisory Board; Merck: Other: Advisory Board; BMS: Other: Advisory Board ; Parexel: Consultancy; Imaging Endpoint: Consultancy; Partner Tx: Other: Advisory Board; Dava Oncology: Other: Education; Celgene: Research Funding. Sadelain:Fate Therapeutics: Consultancy, Patents & Royalties; Memorial Sloan Kettering Cancer Center: Employment; Juno Therapeutics: Consultancy, Patents & Royalties, Research Funding. Avigan:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partners Tx: 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; Janssen: Consultancy; Parexel: Consultancy; Takeda: Consultancy.

Introduction: Acute myeloid leukemia (AML) is a lethal hematologic malignancy in which standard therapy is often inadequate in sustaining durable remissions. In part due to an immunosuppressive milieu characterized by the upregulation of checkpoint inhibitory pathways leading to T cell anergy, ineffective antigen (Ag) presentation, and increased numbers of myeloid derived suppressor cells (MDSCs). We have pioneered a novel vaccine that targets an array of leukemic antigens by fusing whole patient-derived AML cells to autologous dendritic cells (DCs) in the presence of polyethylene glycol (PEG). In a phase I/II clinical trial in AML, vaccination induced an expansion of tumor-specific T cells and resulted in prolonged remissions in a subset of patients. Checkpoint blockade is being investigated in combination with cancer vaccines to overcome the suppressive effects of the tumor microenvironment on cytotoxic tumor-specific T cells. Here we describe the addition of a novel combination of checkpoint inhibitors with DC/AML fusion vaccine in an immunocompetent murine model. Methods/Results: C57BL/6J mice underwent retro-orbital inoculation with 50,000 syngeneic luciferase and mCherry labeled TIB-49 AML cells. 24 hours later a cohort of mice was treated with 100,000 DC/AML irradiated fusion cells. An additional cohort was treated with 6 doses of a combination of anti-PD1/anti-TIM3/anti-RGMb mAbs administered IP every 3 days with and without vaccine. Control mice were treated with appropriate isotype control. AML burden was evaluated in peripheral blood (PB) 14 days post inoculation using flow cytometric analysis for mCherry expression. AML cells were present in PB of control mice and mice treated with check point inhibitors alone (mean 1.2% and 1.5% respectively). Lower disease burden was detected in mice treated with the DC/AML fusion vaccine (0.5%) or combination of fusion vaccine with check point inhibitors (0.5%). Furthermore, to measure tumor-specific T cell response, PB cells were cultured in the presence of autologous TIB-49 tumor lysate for 3 days and analyzed for intracellular IFN-γ expression using multicolor flow cytometric analysis. Mice treated with combination of DC/AML fusion vaccine and checkpoint inhibition showed a significant increase in IFN-γ expression by CD8+ T cells with mean values of 6.4%. Mice treated with vaccine, checkpoint inhibitors alone or IgG control demonstrated mean levels of 3.4%, 1.9% and 1.9% respectively (n=5; p=0.01). Additional immunologic assessment was performed from PB of surviving animals 37 days after tumor challenge. Combination treatment with DC/AML fusion vaccine and PD1/anti-TIM3/anti-RGMb mAbs led to a statistically significant increase in CD4/CD44+/CD62L- memory T cells with concurrent decrease in naïve CD8/ CD44-/CD62L+ T cells as compared to single agent treatments. Furthermore, a statistically significant decrease in CD4+CD25+FOXP3+ Tregs was detected after combination treatment compared to single treatment groups. The mice were followed for survival and disease progression using BLI imaging. All control mice developed visible AML by luminescence following luciferin injection as well as symptomatic disease requiring euthanasia by day 31 after initial challenge with tumor cells (Figure 1). Mice treated with the anti-PD1/anti-TIM3/anti-RGMb mAbs alone demonstrated a survival benefit but all required euthanasia by day 44. 3 of 5 mice treated with vaccine alone remain disease free at more than 65 days. And strikingly, the entire cohort of mice treated with the combination of DC/AML fusion vaccine and PD1/anti-TIM3/anti-RGMb mAbs remain alive and disease free in this aggressive AML model. Conclusion: In the current study we have demonstrated the capacity of a combination of PD-1, TIM-3 and RGMb checkpoint inhibition to create an enhanced environment for immune response to the DC/AML fusion vaccine in an immunocompetent murine AML model. Treatment with this combination led to an increase in tumor specific T cell immunity, decrease in circulating Tregs and shift toward a memory phenotype. Most significantly, mice who received the combination treatment remain disease-free several months post inoculation. This synergistic approach has promising translational potential and a phase 1 clinical trial is planned. Figure. Figure. Disclosures Rosenblatt: Bristol-Myers Squibb: Research Funding; Merck: 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; Celgene: Research Funding. Stone:Astellas: Consultancy; Otsuka: Consultancy; Arog: Consultancy, Research Funding; Argenx: Other: Data and Safety Monitoring Board; Pfizer: Consultancy; Fujifilm: Consultancy; Celgene: Consultancy, Other: Data and Safety Monitoring Board, Steering Committee; Orsenix: Consultancy; Amgen: Consultancy; Merck: Consultancy; Agios: Consultancy, Research Funding; AbbVie: Consultancy; Novartis: Consultancy, Research Funding; Jazz: Consultancy; Cornerstone: Consultancy; Ono: Consultancy; Sumitomo: Consultancy. Freeman:Roche: Patents & Royalties; Merck: Patents & Royalties; Origimed: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers-Squibb: Patents & Royalties; Xios: 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; Novartis: Patents & Royalties; Dako: Patents & Royalties; Boehringer-Ingelheim: Patents & Royalties; Roche: Membership on an entity's Board of Directors or advisory committees; EMD-Serono: Patents & Royalties; AstraZeneca: Patents & Royalties.

Introduction: Adoptive cellular transfer using engineered cells in hematologic malignancies often rely on single antigens as targets but antigen downregulation has been described as a mechanism of tumor resistance. Furthermore, identification of optimal tumor-specific antigens that are immunogenic and have tolerable off-target effects remain an area of active investigation. Our group has developed a novel vaccine using whole patient-derived AML cells and autologous dendritic cells (DCs), capable of presenting a broad array of leukemia antigens. Here we describe the ability of DC/AML fusion vaccine to stimulate T cells ex-vivo and provide a framework for therapeutic exploration of vaccine stimulated T cells as a cellular therapy. Methods/Results: Vaccine was generated with C57BL/6J bone marrow matured DCs and syngeneic TIB-49 AML cells as previously described. T cells were obtained from C57BL/6J splenocytes after magnetic bead isolation and cultured with vaccine plus IL-15 and IL-7. Vaccine stimulated T cells showed increased immune activation as measured by multicolor flow cytometric analysis. Compared to unstimulated T cells, there was 8-fold increase in CD3+CD69+ expression, which is detected upon TCR ligation. Likewise, there was a 12-fold and 3-fold increase in CD137 expression on CD4+ and CD8+ cells, which is up-regulated upon antigen recognition. In addition, a shift from naïve to memory T cell phenotype occurred, which has important implications for response to adoptive cell transfer. Amongst CD4+ cells, the CD44+CD62L- subset comprised 71% after vaccine stimulation. Amongst CD8+ cells, there was increase in both effector and memory subsets. Furthermore, there was enhanced Th1 polarization in ex-vivo culture with vaccine, as IFN-γ expression was increased 2-fold and 5-fold in CD4+ and CD8+ subsets respectively. A corresponding 1.5-fold increase in cytotoxicity was detected using a standard granzyme B CTL assay. The efficacy of adoptive therapy with vaccine stimulated T cells was demonstrated in a xenograft model in which NSG mice were engrafted with patient derived AML cells following sublethal total body irradiation. T cells autologous to the primary tumor were obtained at the time of disease remission and DC/AML fusions were generated. T cells were cultured with or without fusion vaccine at 1:10 ratio. Intracellular IFN-γ expression increased 9-fold and 11-fold on CD8+ and CD4+ cells respectively with ex-vivo vaccine stimulation compared to unstimulated T cells. On day 14 after tumor inoculation, mice were injected with 1.5 x 106 T cells that had been cultured with or without fusion vaccine. Untreated mice were used as a control. The mice were sacrificed two weeks later at which time bone marrow and spleen cells were harvested. AML engraftment in bone marrow was significantly reduced in mice treated with ex -vivo vaccine stimulated T cells compared to control as measured by presence of hCD45+hCD33+ leukemia cells with mean levels of 18% and 38% respectively (n=4). Increased marrow infiltration of human CD8+ T cells was detected in animals treated with ex vivo stimulated T cells and corresponded with lower AML burden. In contrast, both control mice and those given unstimulated T cells demonstrated low hCD8+ marrow T cell infiltration and higher disease burden. We propose this was due to tumor-specific T cell activation of vaccine educated T cells as evidenced by their increased levels of hCD8+IFN-γ expression following exposure to autologous tumor lysate at the time of splenocyte harvesting. Conclusion: In the current study we have shown the ability of DC/AML fusion vaccine to stimulate T cells ex-vivo, as demonstrated by increased CD69, CD137 and IFN-γ expression, and enhance memory and effector subsets when co-cultured in the presence of cytokines. Results of treatment with vaccine stimulated T cells to date have shown reduced engraftment of primary AML in NSG mice. This provides a framework to evaluate the therapeutic use of adoptive transfer of ex-vivo fusion vaccine stimulated T cells in AML. Further in vitro work incorporates marrow-infiltrating T cells and circulating tumor-specific T cells to assess efficacy in active disease. In vivo models are also ongoing including both xenograft and an immunocompetent murine model and subsequent survival analyses will be reported. Disclosures Rosenblatt: Celgene: Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding.

Graft failure (GF) after hematopoietic cell transplant (HCT) occurs in 5-30% of patients. GF can be accompanied by neutropenia (NGF) or can result with adequate neutrophils, but loss of donor chimerism (non-neutropenic graft failure, NNGF). In this report, we describe the outcomes of 95 patients treated with a second HCT for GF at the University of Minnesota; 62 with NGF and 33 with NNGF. The cumulative incidence of neutrophil recovery at 42 days after second HCT was 45% for NGF and 88% for NNGF. A second GF occurred in 34 NGF (55%) and in 9 NNGF (27%) patients. The incidence of Grade III-IV acute graft versus host disease (GVHD) was 8% (95% confidence interval (CI), 1-16%) and 12% (95% CI, 1-23%) for NGF and NNGF, respectively. From the 2nd HCT, 1-year overall survival (OS) was 44% (95% CI, 34-54%), [NNGF: 76% (95% CI, 57-87%) and NGF: 27% (95% CI, 17-39%)]. The most common cause of death after second HCT was infection (52%). In summary, the outcomes of second HCT after NGF and NNGF are different with much worse outcomes for NGF necessitating new approaches for this complication.

Internal tandem duplication (ITD) mutation of the FLT3 receptor tyrosine kinase ( FLT3/ITD ) is one of the most common gene mutations in patients with acute myeloid leukemia (AML) and is a notoriously poor prognostic factor. The ligand-independent constitutive activation of the FLT3 kinase and its