Introduction. Remarkable progress has been made in the treatment of relapsed/refractory Acute Lymphocytic Leukemia and Non-Hodgkin Lymphoma with CAR-CD19 T cells. In contrast, progress against CD19-negative hematological cancers and solid tumors has been limited. Intensive efforts to optimize cellular therapeutics for better efficacy include provision of cytokine support and countering immunosuppression. However, lack of sufficient antigen is a significant additional hurdle that CAR-T therapeutics for solid tumors must overcome. We present a novel strategy to utilize CD19 for sustained antigen presentation in order to promote cellular therapeutic expansion, efficacy and persistence. The strategy, called IMPACT (Integrated Modules oPtimize Adoptive Cell Therapy), employs a methodology that is modular in design and and can be applied to diverse antigens and tumor types, yet retains the well-established advantages of CAR T cells directed to CD19.

Experimental Procedures. A 3rd generation CAR-T was constructed in a lentiviral vector using the FMC63 anti-CD19 scFv for antigen recognition. A second expression cassette, cloned downstream of the CAR-T cassette, was constructed using a secretion signal sequence and exons 1 - 4 of the human CD19 protein linked in frame to a trastuzumab-derived Vh/Vl sequence. This integrated-gene module thus encodes a fusion protein (FP): CD19-anti-HER2-scFv.

Results. ELISA and flow cytometry analyses were used to demonstrate bridging between HER2+ cells and the FMC63 mAb via the CD19 domain of the FP. ELISA analyses further demonstrated wild type binding of the 2 FP domains (i.e. CD19/FMC63 and the scFv/HER2). In vitro cytotoxicity studies showed robust redirected killing of HER2+ (CD19-) tumor cells by CAR19 T cells that is mediated by the secreted FP. The efficiency of cytotoxicity was enhanced by the presence of CD19+ B cells. A tumor xenograft model was used to demonstrate effective killing in vivo when HER2+ BT474 cells were injected in one flank and CD19+ RAMOS cell were injected in the opposite flank, but not when HER2+ cell were injected alone. Thus, in vivo, redirected killing required CD19+ cells for expansion and persistence sufficient to promote anti-HER2 cytotoxicity.

Conclusions. A CD19-anti-HER2-scFv FP was able to redirect the cytotoxicity of CAR19 T cells in order to kill HER2+ tumor cells. In vivo modeling demonstrated that CAR-T cell expansion by CD19 recognition enabled efficient HER2+ cell cytotoxicity. The IMPACT technology is modular, allowing for FPs having diverse protein domains paired with diverse, distinct scFvs to allow CAR T cells to CD19 or other targets to be used to kill any tumor. We have further developed the technology to address antigens of utility for hematopoietic and solid tumors, including BCMA, ROR1, and PD-L1. The technology is broadly applicable and synergistic with existing cellular therapies.

Citation Format: Christine Ambrose, Lihe Su, Lan Wu, Roy R. Lobb, Paul D. Rennert. CAR T cells specific for CD19 can be redirected to kill CD19 negative tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3768. doi:10.1158/1538-7445.AM2017-3768