Potent Synergy between Combination of Chimeric Antigen Receptor (CAR) Therapy Targeting CD19 in Conjunction with Dendritic Cell (DC)/Tumor Fusion Vaccine in Hematological Malignancies
Authors: Capelletti Marzia , Liegel Jessica , Themeli Maria , Mutis Tuna , Stroopinsky Dina , Orr Shira , Torres Daniela , Morin Adam , Gunset Gertrude , Ghiasuddin Haider , Rahimian Maryam , Nahas Myrna , Bisharat Lina , Meng Shufen , Munshi Nikhil C. , Kufe Donald , Rosenblatt Jacalyn , Sadelain Michel , Avigan David E. 
Published: November 2019

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.