Background Effective therapy with T-cell receptor-modified T-cells (TCR-Ts) needs to target the right HLA-presented epitope of the tumor antigen. Epitopes must be well processed (leading to high cell surface abundance), as well as immunogenic (allowing effective activation of T-cells). Here we demonstrate that T-knife’s MyT platform can identify the most relevant T-cell epitopes across a range of common HLAs. The MyT platform is a murine model expressing the entire human TCR alpha and beta gene loci along with seven common human class I HLAs. These mice lack central immunological tolerance against human tumor self-antigens. Thus, immunization of the mice with human tumor antigens induces strong T-cell activation and expansion of T-cell clones with fully-human, high-affinity TCRs. Importantly, by immunizing mice with full-length antigens, we can induce epitope-unbiased T-cell responses in vivo.

Methods We aimed to understand potential differences in antigen processing and presentation in the MyT platform versus human tumor cells. To accomplish this, we compared the HLA-A*03:01, B*07:02 and A*02:01-presented T-cell epitopes of MAGE-A1 (the latter currently under clinical development, NCT05430555), that were identified by either i) use of the MyT platform, ii) immunopeptidomics, or iii) in silico prediction.

Results When we analyzed the immunopeptidome of MAGE-A1 expressing human tumor cells via mass spectrometry, we confirmed the existence of several predicted MAGE-A1 epitopes; but also detected novel, unpredicted epitopes showing the limitation of using pure in silico approaches for epitope discovery. Importantly, the most abundant epitopes found via mass spectrometry were also the most immunodominant epitopes after immunization of MyT platform mice with full-length MAGE-A1 inducing strong T-cell activation and highly reactive TCRs.

Conclusions We conclude that the T-cell epitope landscape in the MyT platform and human cells is highly conserved. While mass spectrometry-based epitope discovery informs about epitope abundance, only immunization in the MyT platform can provide additional information on whether the epitope is also immunogenic and can be targeted well by T-cells. Therefore, the MyT platform is a powerful tool for the in vivo discovery and selection of the most relevant T-cell epitopes in the absence of the bias introduced through in silico prediction and central immune tolerance in humans. Targeting only such epitopes of a tumor antigen via TCR-Ts is likely to have an impact on clinical success in cancer therapy. In addition, the MyT platform could also support epitope discovery in autoimmunity and infectious diseases.

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