Abstract 4868: Optimal activation of TCR T cells in solid tumors through addition of best-in-class single chain CD8 coreceptors results in CD4 engagement and improved T cell fitness and persistence

Abstract Background: Clinical response rates in solid tumors have been improved and/or broadened by including either CD8α or wild type CD8αβ co-receptors (CoR) in T cell receptor (TCR) T cell therapy constructs, enabling engagement of CD4 T cells. We designed a small, chimeric, single-chain CD8 CoR maintaining all functional elements of the CD8αβ CoR and used it as a scaffold to add intracellular costimulatory signaling motifs (co-stimCD8 CoR). By providing activating signals simultaneously to TCR engagement, co-stimCD8 CoR prevents TCR-T cell exhaustion caused by the lack of costimulatory molecule expression in tumors. TCR-T cells expressing co-stimCD8 CoR fully engaged CD4 T cells and mediated enhanced and durable TCR-T anti-tumor responses, superior to commonly used wild type CD8 CoRs. Methods: The ActiviT platform consists of different single-chain CD8 CoRs designed by combining building blocks from CD8αβ and CD4 CoRs. In silico 3D modeling using the Rosetta software suite was used to model interactions between TCR-CD8CoR with HLA-I-peptide complexes. HLA-A*02:01-restricted PRAME or MAGE-A1 targeting TCR-T cells with or without different CD8 CoRs were tested for cytokine production (LEGENDplex) and long-term and serial cytotoxicity (Incucyte). Flow cytometry was used to analyze phenotype, activation and exhaustion markers. Results: Structural evaluation by in silico 3D modeling showed that the single-chain CD8 CoR scaffold retains HLA-I binding interface identical to that of the wild type CD8 CoR. The co-stimCD8 CoR was generated by adding tumor necrosis factor receptor associated factor (TRAF) binding motifs. Assessment of cytotoxicity and cytokine secretion showed that co-stimCD8 CoR conferred higher functionality to CD4 T cells incorporating an HLA class-I restricted TCR compared to a wild type CD8 CoR. To evaluate the impact of co-stimCD8 CoR on the anti-tumor activity of TCR T therapies, functional comparison of CD4/CD8 PRAME TCR-T cells co-expressing different CD8 CoRs, including versions currently tested in the clinic, was performed. PRAME TCR-T cells expressing co-stimCD8 CoR improved T cell fitness, resulting in a less exhausted and earlier memory phenotype and enhanced functionality in serial killing experiments at low effector-to-target ratios. Notably, enhanced activity of co-stimCD8 CoR was demonstrated in 3D tumor spheroid models recapitulating hard-to-treat indications. Conclusions: Co-stimCD8 CoR is a chimeric CD8 CoR with built-in co-stimulation. Combination of co-stimCD8 CoR with a PRAME TCR resulted in improved T cell fitness and higher functional activity compared to common PRAME TCR-T approaches, including current clinical development candidates. Inclusion of the co-stimCD8 CoR in TCR-T constructs has the potential to further deepen and broaden responses of hard-to-treat solid tumors. Citation Format: Paul Najm, Alvaro Haroun-Izquierdo, Mikhail Steklov, Panagiota A. Sotiropoulou, Marleen M. van Loenen. Optimal activation of TCR T cells in solid tumors through addition of best-in-class single chain CD8 coreceptors results in CD4 engagement and improved T cell fitness and persistence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4868.