Background PRAME is a promising target for immunotherapy due to high prevalence and high expression in multiple tumor indications with high unmet medical need while having limited healthy tissue expression. Therapy with T-cell receptor-modified T-cells (TCR-Ts) targeting PRAME has shown promising early clinical validation. Moreover, accumulating clinical evidence suggests that deep and durable clinical responses in multiple solid tumors require additional T-cell engineering, such as incorporating a CD8 co-receptor (CD8COR) to enable inclusion of CD4 TCR-Ts and engineered receptors to counteract the immunosuppressive tumor microenvironment.

Methods We identified PRAME-reactive TCRs using T-knife’s MyT™ platform – a mouse-based human TCR discovery engine with the ability to overcome central tolerance, the natural immune process eliminating high-affinity TCRs for self-antigens in humans. Selected TCR candidates were assessed for reactivity to PRAME-expressing tumor cell lines in vitro. Several of these candidates were co-expressed with CD8COR or combinations of distinct single-chain CD8CORs and switch receptors. Evaluation was based on phenotype and functional activity against target cell lines expressing distinct levels of PRAME.

Results PRAME was highly immunogenic in the MyT platform leading to a 100% response rate in mice after immunization. Using a rapid-throughput TCR screening assay, we tested the most-expanded HLA-A*02:01-restricted TCR clonotypes isolated from reactive mouse T-cells and identified close to 100 TCRs with reactivity for PRAME. Most TCRs were specific for the epitope SLL425–433 which is highly abundant on PRAME-expressing tumor cells. We selected 22 of these TCRs for in-depth characterization in vitro. Peptide-dose response assays with these selected TCRs demonstrated a large TCR affinity range for the SLL peptide, translating into differential reactivity to a panel of cell lines with low to high PRAME expression as measured by cytokine secretion and cytotoxicity. Importantly, we identified a number of TCRs of similar or higher reactivity when compared to a clinical-stage PRAME TCR and other publicly disclosed PRAME TCRs. TCR affinity and levels of target expression are expected to influence the requirements of TCR-Ts for co-stimulation. We therefore combined selected TCRs with a variety of CD8COR and switch receptor options available in T-knife’s next-generation toolbox and evaluated the constructs for phenotype and functional activity.

Conclusions We demonstrated that the MyT platform can deliver high-affinity, potentially best-in-class PRAME TCR candidates for further clinical evaluation. Combining such high-affinity PRAME TCRs with CD8COR and switch receptor options tailored for TCR, target antigen and targeted indications has the potential to induce deep and durable clinical responses.

Ethics Approval Mouse experiments were conducted according to German law under the license number §9 H 0050_21 approved by the Landesamt für Gesundheit und Soziales Berlin.