Abstract 3189: CAB-CAR-T: A novel conditionally active biologics approach to minimize on-target off-tumor effects in adoptive immunotherapy (2019)

Adoptive cellular therapy (ACT) using chimeric antigen receptor (CAR) modified T cells has demonstrated promising antitumor effects in hematologic malignancies leading to the recent approval of two CAR-T products targeting the B cell receptor associated protein CD19, however the safe development of CAR-T therapies for solid tumor malignancies remains less advanced in part due to the lack of precision in targeting solid tumor antigens. Identification of clinically viable tumor restricted target antigens while avoiding on-target off-tumor toxicities in normal tissue presents a significant challenge in the solid tumor CAR-T therapeutic scenario. The altered glycolytic pathway of the Warburg effect within the tumor microenvironment (TME) drives lactic acid production leading to an opportunity to develop novel CARs with functional TME dependent switching ability. The resulting acidic extracellular pH is a major feature of the TME and this unique property represents one novel mechanism by which target specificity may be achieved. Here we describe a novel Conditionally Active Biologics approach in adoptive immunotherapy termed CAB-CAR-T. By genetically engineering combinations of amino acid mutations into the antibody variable antigen recognition domains (scFv) of CAR constructs, we demonstrate that TME restricted target antigen engagement can be engineered into chimeric antigen receptors. The described CAB-CARs display minimal antigen dependent activity at the physiological pH of normal tissue. Under TME conditions (low pH) the affinity of CAB-CAR scFv domains against their antigen increases, such that tumor target recognition and CAR-T activity become pH dependent. Thus CAB-CAR-T cells display reversible “AND logic gate” properties, requiring both antigen presence and TME conditions for activity. Comparisons of target cell killing under TME permissive and physiologic pH conditions in a cellular impedance based kinetic killing assay demonstrate that CAB-CAR-T cells efficiently lyse antigen positive target cells at pH 6.7 (TME) with minimal activity at pH 7.4 (normal tissue). pH profiles of CAB-CAR-T were examined through buffering the culture medium down from physiologic pH in 0.1 pH increments, and the data demonstrated that the cytolytic activity of CAB-CAR-T incrementally increased as extracellular pH was decreased compared to control T cells. Similar pH dependence was observed for other measures of CAB-CAR-T activation including the early T cell surface activation marker CD69, the degranulation marker CD107a and cytokine release (IL-2 and IFNγ). Conditionally active chimeric antigen receptor T cells (CAB-CAR-T) harness unique properties of the tumor microenvironment to provide an opportunity to develop safer CAR-T therapeutics for tumor antigen targets also expressed on healthy tissues by minimizing on-target off-tumor activity.Citation Format: Jianfang Hu, Benjamin Lopez, Tiffany Lam, Anirban Kundu, Timothy Mayall, Gregory Schreiber, Farzad Haerizadeh, James Onuffer, Gregory Frost. CAB-CAR-T: A novel conditionally active biologics approach to minimize on-target off-tumor effects in adoptive immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3189.