Abstract 5560: KIR-based CAR design showed durable antitumor efficacy by altered internalization kinetics following persistent antigenic stimulation
Authors: Zhang Qian , Nigel Eric , Yu Alvin , Hresko Morgan , Minutolo Nicholas , Powell Daniel , Milone Michael C. 
Published: June 2022

Poor persistence of functional chimeric antigen receptor (CAR) T cells in the tumor microenvironment can limit the effectiveness of the antitumor immune response. We previously showed that T cells expressing a multichain CAR design that uses an activating killer immunoglobulin-like receptor (KIR) transmembrane (TM) and cytoplasmic domain co-expressed with the ITAM-containing adapter, DAP12 (KIR-CAR) are able to control large, pre-established tumors that cannot be controlled by 2nd generation CD3ζ-based CAR T cells. This improved function is associated with maintenance of KIR-CAR expression on the tumor infiltrating lymphocytes compared with CD3ζ-based CARs. In the present study, we explored the mechanism for the improved KIR-CAR expression, which we hypothesized was related to reduced receptor internalization following receptor activation compared with standard CD3ζ-based CARs. To monitor receptor dynamics at the T cell surface, we took advantage of a surface protein tagging method that employs the SpyCatcher-SpyTag system to construct a KIR-based or CD3ζ-based CAR with the ability to pulse label the CAR at the cell surface with a fluorescent molecule. In this approach, a CAR ectodomain comprised of a SpyCatcher domain is linked to either the KIR TM and cytoplasmic domain (SpyKIR-CAR) or CD28 TM and cytoplasmic domain with CD3ζ (SpyCAR). The antigenic specificity of the CAR is conferred by addition of a fluorescently-labeled binder comprised of an antibody-like Designed Ankyrin Repeat Protein (DARPin) fused to a SpyTag sequence (SpyDARPin) that specifically binds the spyCatcher domain covalently via its spyTag and recognizes the EGFR antigen. The SpyKIR-CAR or Spy-CAR expression is confirmed by flow cytometry using an antibody to a myc-tag within the DARPin sequence. Uniform fluorescence labeling at the plasma membrane is observed following addition of the SpyDARPin as assessed by image cytometry and confocal microscopy. The kinetics of SpyCAR or SpyKIR-CAR fluorescence loss were similar in the absence of EGFR ligand with a half-life of 2 days consistent with normal membrane turnover by pinocytosis. Upon ligand-induced CAR activation, the SpyCAR fluorescence rapidly reduced beginning at 30 min post-activation to background fluorescence levels of unlabeled SpyCAR cells within 8 hours of activation. In contrast, the mean SpyKIR-CAR fluorescence showed no change in fluorescence at 30 min, while exhibiting a reduction to 80% of the mean baseline fluorescence at 8 hours and 4-fold above the background fluorescence. Confocal imaging confirmed the retention of labeled KIR-CAR at the plasma membrane over the time frame in which the labeled SpyCAR is lost. Our data support differences in CAR internalization upon activation as an important mechanism underlying the enhanced function of KIR-CAR T cells in solid tumors compared with traditional CD3ζ-based CAR designs.

Citation Format: Qian Zhang, Eric Nigel, Alvin Yu, Morgan Hresko, Nicholas Minutolo, Daniel Powell, Michael C. Milone. KIR-based CAR design showed durable antitumor efficacy by altered internalization kinetics following persistent antigenic stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5560.