Abstract: 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.

Abstract: 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 〈 0.01) were related to immune activation including NO and Reactive Oxygen Species in Macrophages, IL-2, IL-15, IL-6, IL-7,IL17A, and B cell activation. TGF-b was also downregulated in responders post-vaccination. 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.

Abstract: 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.

Abstract: 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.

Abstract: We have developed a personalized vaccine whereby patient derived leukemia cells are fused to autologous dendritic cells, evoking a polyclonal T cell response against shared and neo-antigens. We postulated that the dendritic cell (DC)/AML 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 immunologic response 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 leukemia progression and demonstrated only a modest extension of survival. Vaccination with DC/AML 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 combined DC/AML fusion vaccine and checkpoint blockade treatment offers unique synergy inducing the durable activation of leukemia specific immunity, protection from lethal tumor challenge and the selective expansion of tumor reactive clones.

Abstract: 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.