Introduction. CAR-CD19 T cell therapeutics are approved to treat B cell leukemias and lymphomas. Previously we showed that providing CAR19 T cells with a retargeting fusion protein (FP), consisting of soluble CD19 protein linked to an scFv, redirects CAR19 T cell cytotoxic activity to other tumor antigens. Further, exposure of CAR19 T cells to normal or malignant B cells expressing CD19 improves effector function and phenotype, due to the provision of costimulatory signals. Such signals are missing when CAR-T cells engage antigens on solid tumor cells. In addition, lack of sufficient antigen for expansion and persistence is a critical issue for CAR T therapeutics targeting solid tumors in the clinical setting. We have redirected CAR19 T cells to other tumor antigens via novel FP and bispecific FP (biFP) expression cassettes. CAR19 T cells that express FP or biFP solve critical issues in the cell therapy field by 1) promoting CAR19 T cell expansion, efficacy and persistence by engaging CD19 on B cells; 2) addressing antigen heterogeneity/escape in hematologic malignancies and solid tumors. FP- and biFP-mediated cytotoxicity is extremely potent at pM concentrations. Here we provide examples of in vitro and in vivo modeling to characterize the activity of the CAR19 T cells that are retargeted to kill other tumor types.
Experimental Procedures. A CAR-CD19 construct was created as the recipient for FP and biFP expression cassettes, in a lentiviral vector with an MCSV promoter. The CAR consists of the FMC63 scFv, a stalk, TM and CD28 and/or 4-1BB cytoplasmic signaling domains, and the CD3ε cytoplasmic signaling domain. FP and biFP cassettes were designed to encode the extracellular domain of the CD19 protein, followed by an scFv or VHH to one (FP) or two (biFP) antigens, separated from the CAR sequence by a P2A cleavage site.
Results. Flow cytometry and binding analyses demonstrated FP and bi-FP-mediated bridging between target tumor cells and CAR19 T cells. In vitro cytotoxicity studies showed robust redirected killing of antigen+ (CD19-) tumor cells by CAR19 T cells. This cytotoxicity is specifically mediated by the secreted FP or biFP and correlated with antigen affinity. The efficiency of cytotoxicity was enhanced by the presence of CD19+ B cells. Tumor xenograft models using Her2+ and CD38+ tumor lines demonstrated effective tumor killing in vivo, with or without the presence of CD19+ cells.
Conclusions. Redirected CAR19 T cells potently kill diverse tumor cells in vitro and in vivo. The strategy of redirecting CAR19 T cells to other tumor cell types by encoding novel FP and biFP expression cassettes solves critical issues in cell therapy by supporting CAR19 T cell activation and expansion and by providing a simple and modular multi-antigen targeting system. Further technology refinements include the creation of inducible promoters for FP expression, adding anti-PD-L1 activity, and providing cytokine support.
Citation Format: Paul Rennert, Fay Dufort, Lihe Su, Lan Wu, Alyssa Birt, Roy Lobb, Christine Ambrose. CAR19 T cells secreting antigen-retargeting fusion proteins have remarkable potency against diverse tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2569.