On Target Laboratories (United States)

Abstract Chimeric antigen receptor (CAR) T cell therapies have attracted considerable attention because of their abilities to focus the killing power of T cells specifically onto tumor antigen expressing cancer cells. Despite impressive successes in the clinic, CAR T cell technologies still suffer from an inability to kill cancer cells that mutate to avoid expression of the CAR T cell recognized antigen. While current CAR T cell technologies would require the engineering of a new CAR to recognize a second tumor-specific antigen, we have instead developed a universal CAR that can recognize any tumor antigen. In this modification of standard CAR T cell technology, we fuse an scFv that binds fluorescein to the exoplasmic domain of a T cell receptor that contains the usual 4-1BB and CD3 zeta activation domains within its cytoplasmic domain. We then promote engagement of the resulting CAR T cell with the desired cancer cell by administering a bispecific adapter molecule that contains fluorescein linked to a tumor-specific ligand via a short spacer. Upon addition of this adapter, simultaneous binding of the fluorescein to the CAR and the tumor-specific ligand to the cancer cell mediates multivalent binding of the CAR T cell to the cancer cell. With the simultaneous administration of a cocktail of different tumor-specific adaptors, each comprised of a fluorescein linked to a distinct tumor-specific ligand, multiple orthogonal cancer cells within a heterogeneous solid or liquid tumor can be simultaneously eliminated by a single CAR T cell. To test the ability of our proposed universal CAR T cell to eradicate solid tumors with multiple orthogonal tumor antigens, we first demonstrate the capacity of our anti-fluorescein CAR T cell to kill a variety of cultured human cancer cells in vitro upon addition of the optimal adapter. In this effort, we show that the same anti-fluorescein CAR T cell can kill: i) multiple folate receptor-expressing cancer cells upon addition of folate-fluorescein, ii) PSMA-expressing cancer cells upon addition of a PSMA ligand-fluorescein conjugate, iii) carbonic anhydrase IX-expressing cancer cells upon addition of a CA9 ligand-fluorescein conjugate, and iv) neurokinin-1 receptor expressing cancer cells upon addition of a NK1R ligand-fluorescein conjugate. We then demonstrate that this same universal killing potency is also effective in vivo by transfecting separate MDA-MB-231 breast cancer cell cultures with one of the above tumor antigens and then implanting the MDA-MB-231 cells expressing the orthogonal tumor antigens in NSG mice and showing that our universal CAR T cell can eradicate the resulting tumors upon administration of the appropriate bispecific adapter. Taken together these results establish that a single anti-fluorescein CAR T cell can be exploited to eliminate antigenically heterogeneous tumors upon addition of the correct cocktail of ligand-fluorescein conjugates. Citation Format: Yong Gu Lee, Isaac Marks, Madduri Srinivasarao, Ananda Kumar Kanduluru, Sakkarapalayam M. Mahalingam, Haiyan Chu, Philip S. Low. Use of a universal CAR T cell plus bispecific adapters to eradicate antigenically heterogeneous tumors [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 3571.