Abstract 3483: MyTTMplatform-identified PRAME TCRs for T cell therapy demonstrate superior efficacy and best-in-class potential compared to clinical benchmarks

Abstract Introduction: PRAME is a clinically validated target for T cell receptor-modified T cell (TCR-T) therapy with high prevalence in multiple solid tumor indications. T-knife’s MyTTM platform generates best-in-class, high-affinity and high-specificity PRAME-directed TCRs for use in next-generation TCR-T therapy. Methods: We identified PRAME-directed TCRs using the MyT platform, a TCR discovery engine based on transgenic mice expressing the human TCR repertoire with the ability to overcome central tolerance. Reactive TCR candidates were identified by rapid throughput screening using an indicator cell line. Selected TCR candidates were subsequently expressed retrovirally in primary T cells in combination with a CD8αβ coreceptor and characterized for peptide dose-response, cytokine release and cytotoxicity. We performed in-depth TCR safety profiling by rapid alloreactivity screening of single-HLA-transfected cells and by deep mutational cross-reactivity scans (X-scans). Finally, we supercharged TCR-modified T cells by co-expression with an activating CD8 coreceptor with built-in co-stimulation and a FAS-CD40/OX40 switch receptor using CRISPR-Cas9 gene editing combined with knockout of the endogenous TCR. Results: PRAME was highly immunogenic leading to a 100% response rate in mice after immunization. We identified 85 TCRs reactive to the immunodominant HLA-A*02:01 PRAME epitope SLLQHLIGL. Peptide dose-response assays as well as co-cultures with a panel of PRAME-expressing cancer cell lines from a broad range of tumor indications demonstrated high avidity, high cytokine secretion and high cytotoxicity of T cells expressing MyT TCRs. Three lead TCR candidates were selected, which showed no alloreactivity against 149 tested HLA-subtypes covering 98% of class-I HLA alleles. Furthermore, testing over 300 putative off-target peptides predicted in silico or experimentally in X-scans, revealed that MyT TCRs show no off-target recognition. Importantly, benchmarking showed that the selected lead TCR candidate displays higher in-vitro efficacy compared to publicly disclosed clinical-stage TCRs, which was particularly evident at low E:T ratios and retained during multiple rounds of tumor cell restimulation. Finally, as part of the development of a Supercharged PRAME TCR-T product, TCR expression and functionality was further enhanced, by dual T cell armoring and using gene editing combined with endogenous TCR knockout, without impacting safety. Conclusions: Our data demonstrate that the MyT platform delivers best-in-class, high-affinity/high-specificity PRAME TCRs. Administration of Supercharged PRAME-TCR-T cells incorporating these MyT TCRs, a single-chain co-stimulatory CD8 coreceptor and a switch receptor in a non-viral process, provides potential to induce deep and durable responses in hard-to-treat solid tumor indications. Citation Format: Lia Walcher, Lena Immisch, Mira Heichenko, Alexei Leliavski, Robert Hoeltzel, Sharesta Khoenkhoen, Niklas Fellmer, Elisa Kieback, Lorenz Knackstedt, Lucia Poncette. MyTTMplatform-identified PRAME TCRs for T cell therapy demonstrate superior efficacy and best-in-class potential compared to clinical benchmarks [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 3483.