Abstract A110: Enhancing TCR Potency While Maintaining Specificity Through CD8 Co-receptor Engineering

Abstract TCR-based therapies are emerging for solid tumors, but there are limitations due to potency and off target reactivities. The CD8 co-receptor stabilizes the TCR/peptide-MHC complex, enhancing TCR signaling, especially for lower-affinity TCRs. While affinity-enhanced TCRs, such as the clinically relevant 1G4 TCR (95:LY) targeting NY-ESO-1, improve T cell responses, their CD8-independent behavior increases the risk of off-target reactivity and potential autoimmunity due to bypassed thymic negative selection. We hypothesized we could enhance the efficacy of native TCRs by engineering the CD8 co-receptor to reduce off-target effects while maintaining or improving potency. We initially demonstrated that CD8αα homodimers paired with the affinity-enhanced 1G4 TCR were safer than CD8αβ combinations, reducing off-target effects and minimizing inappropriate T cell activation. This research led to the hypothesis that CD8β modulates TCR sensitivity and specificity potentially compromising antigen discrimination. Building on this finding, we sought to determine whether native TCRs could be enhanced by modifying CD8α itself, rather than the TCR sequence, to achieve a more controlled enhancement in T cell response. To explore this, we generated a saturation mutagenesis library targeting the complementarity-determining regions (CDRs) of CD8α involved in MHC binding. This strategy aims to identify mutations to improve native TCR potency, enhancing anti-tumor activity without triggering undesired immune responses. Through multiple rounds of enrichment in a Jurkat NFAT reporter system transduced with the CD8α mutant library and native 1G4 TCR, we identified several mutations that significantly enhanced TCR potency. The mutations are being validated through live-cell imaging T cell cytotoxicity assays, which allow real-time monitoring of T cell killing activity, and advanced flow cytometry to assess functional avidity (including TNFα, IFNγ, IL-2, and CD107a expression) and intracellular signaling (Nur77). By studying Nur77 expression, we aim to investigate the relationship between TCR binding affinity and downstream signaling pathways, which remains an understudied area in the field of T cell immunology. Additionally, we are performing IFN-γ ELISPOT assays to confirm that enhanced potency maintains the natural safety mechanisms of native TCRs, such as the appropriate regulation of cytokine responses. Furthermore, we are testing other CD8-TCR combinations in the context of HLA-A02 and with other HLA types beyond HLA-A02. Since CD8α interacts with highly conserved regions of MHC class I molecules, we hypothesize that these engineered combinations could have broad applicability across different patient populations and HLA types, potentially expanding the therapeutic utility of TCR-based immunotherapies. Our findings suggest that engineering the CD8 co-receptor represents a safer and more effective alternative to current TCR engineering strategies, offering enhanced tumor targeting while minimizing the risks of off-target reactivity and autoimmune complications. Citation Format: Zita EH Aretz, Neel Panchwagh, David A Scheinberg. Enhancing TCR Potency While Maintaining Specificity Through CD8 Co-receptor Engineering [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A110.