Background Generation of dual CD4/CD8 TCR-T products by redirecting CD4+ T-cells to peptide-MHC class I complexes through incorporation of a CD8 co-receptor (CD8COR) has established early clinical and extensive preclinical validation. Key approaches employed include utilizing the wild-type (WT) CD8COR, comprised of a CD8-alpha (CD8a) and a CD8-beta (CD8b) chain, or a single CD8a chain. The WT CD8COR displays superior preclinical activity, endowing CD4+ T-cells with cytotoxic activity while maintaining immune modulating functions, but construct size and incorporation of multiple elements remain challenging. We therefore designed chimeric CD8CORs that combine building blocks from the CD8a and CD8b chains, and the CD4 co-receptor. The smaller constructs allow for the addition of signaling motifs from the tumor necrosis factor receptors CD30 and CD40, combining engagement of CD4+ T-cells with enhancement of both CD4+ and CD8+ T-cells. We combined the different CD8CORs with a MAGE-A1-targeting TCR currently under clinical development (NCT05430555) and determined the design enabling the highest anti-tumor response and T-cell fitness.

Methods CD4+ and CD8+ T-cells were transduced with a MAGE-A1 TCR alone, or together with each CD8COR. Constructs were analyzed for transgene expression levels and phenotype by flow cytometry, and cellular metabolism by Seahorse. Real-time cytotoxicity by Incucyte and cytokine secretion profile by LEGENDplex were assessed upon long-term or repeated stimulation.

Results We identified a chimeric CD8COR endowing similar functional activity to the WT CD8COR as demonstrated by cytotoxicity and cytokine secretion profile using SAOS2, and HLA-A*02:01-overexpressing NCI-H2030 and CorL23 cells at 0.5:1 effector-to-target ratio. This comprised the CD8a and CD8b MHC-binding domains connected through a linker and the CD4 co-receptor intracellular domain, maintaining all functional elements of the WT CD8COR with smaller transgene size. The selected chimeric CD8COR design was further optimized and used as a scaffold to add CD30 and CD40 cytoplasmic signaling motifs, creating an enhanced chimeric CD8COR. T-cells expressing the MAGE-A1 TCR and the WT or enhanced chimeric CD8COR were assessed for strength and durability of anti-tumor response. Both TCR-T candidates exhibited robust cytotoxicity upon repeated stimulation at low effector-to-target ratios. Moreover, the enhanced chimeric CD8COR triggered elevated secretion of proinflammatory cytokines and cytotoxic effector molecules.

Conclusions Our results show that the incorporation of a CD8COR to MAGE-A1 TCR-T therapy enhances in vitro anti-tumor activity and T-cell fitness. Detailed comparison between the WT and enhanced chimeric CD8COR, including their impact on polyfunctionality, memory phenotype, metabolic fitness and modulation of other immune cells will be presented.