Abstract 895: Coexpression of MHC class I-restricted neoTCRs and ectopic CD8 receptors in precision genome engineered CD4 T cells significantly potentiates antigen-specific effector functions

Neoepitopes (NeoE) from tumor-exclusive mutations represent compelling targets for personalized neoE-specific autologous TCR-T cell therapies for patients with solid tumors. The imPACT Isolation Technology® is an ultra-sensitive and high-throughput process for capturing neoE-specific CD8 T cells from the blood of patients with solid cancers. Leveraging this technology, neoepitope-specific MHC class I-restricted TCRs (MHC-I neoTCRs) are cloned from individually captured CD8 T cells. Using DNA -mediated (non-viral) gene editing, fresh CD8 and CD4 T cells from the same patient with cancer are engineered to express the MHC-I neoTCR (concomitant with elimination of the endogenous TCR). While naturally occurring MHC-I TCRs are presumed to require concurrent CD8 co-receptor help to stabilize peptide-MHC binding, higher affinity TCRs drive CD8-independent target binding and T cell activation. CD4 T cells, when engineered with high affinity neoTCRs, are thus able to recognize peptide-MHC-I targets and trigger effector T cell functions. However, lower affinity TCRs are dependent on CD8 co-receptors to trigger T cell activation. By precision genome engineering CD8 co-receptor genes together with the neoTCR into CD4 T cells, MHC-I neoTCRs are now competent to trigger antigen-specific effector T cell function. In this study, MHC-I neoTCRs were cloned from neoE-specific T cells captured from the blood of a patient with colorectal cancer. Healthy donor CD8 and CD4 T cells were precision genome engineered to express the cloned MHC-I neoTCRs alone or to include engineering of ectopic CD8 co-receptors in the gene-edited T cells. Flow cytometric analysis was used to evaluate surface expression of neoTCRs and ectopic CD8 co-receptors, respectively. Rescue of neoTCR binding among CD4 T cells for lower affinity, CD8-dependent neoTCRs was observed. Importantly, in response to stimulation with cognate antigen, CD107a and intracellular IFNγ staining revealed 10-100-fold increases in the sensitivity of MHC-I neoTCR-induced effector functions by CD4 T cells, with no effect on specificity. No change in functionality or sensitivity was seen on CD8 T cells by the expression of additional CD8 co-receptor. These results demonstrate that simultaneous precision genome engineering of the CD8 co-receptor together with CD8-dependent MHC-I neoTCRs into CD4 T cells significantly increases their sensitivity to neoE-HLA target recognition as well as triggering pro-inflammatory and cytotoxic function, yet without compromising antigen-specificity. Citation Format: Barbara Sennino, Andrew Conroy, Bhamini Purandare, Kyle Jacoby, Olivier Dalmas, Songming Peng, Alex Franzusoff, Stefanie Mandl. Coexpression of MHC class I-restricted neoTCRs and ectopic CD8 receptors in precision genome engineered CD4 T cells significantly potentiates antigen-specific effector functions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 895.