Background T cell receptors (TCRs) have the potential to recognize epitopes derived from both intracellular and cell surface antigens. This facilitates targeting a wide range of tumor antigen classes including overexpressed, differentiation and cancer-testis antigens as well as antigens derived from driver mutations, frameshift mutations, splice variants and human endogenous retroviral elements (HERV). Despite this array of potentially targetable shared antigens, TCR-based anti-tumor therapies have to date only focused on a very limited number of target epitopes. The discovery of clinically relevant TCRs has been challenging due to the limitations in novel tumor antigen discovery and low throughput TCR screening approaches in addition to the inherently low frequency of tumor-reactive T cells in tumor samples and in peripheral blood.
Methods We employ our sensitive and high-throughput Deep Immunomics platform using a mass-cytometry-based multiplexed tetramer staining approach to screen and characterize CD8+ T cells in peripheral blood mononuclear cells (PBMC) for reactivity against >500 potential T cell targets per sample. Downstream single cell sequencing of identified tumor antigen-specific T cells enables a combined analysis of T cell phenotype, transcriptome, TCR specificity and TCR sequence. TCRs identified using this approach are transduced for expression in engineered Jurkat reporter cells and primary T cells and evaluated for activation and effector function, respectively.
Results We have utilized this approach to discover novel TCRs targeting a diverse list of potential tumor epitopes. We identified a TCR recognizing an HLA-A*02-restricted epitope derived from an HERV that is reported to be expressed across many cancer types. In Jurkat reporter cells, this TCR is activated specifically in the presence of the same epitope initially used to identify this TCR, but also through a related epitope variant from the same HERV subfamily. Furthermore, we have identified multiple TCRs that recognize an epitope resulting from alternative splicing of the target antigen in multiple cancers with mutations in a specific splicing factor. Primary T cells expressing these transgenic tumor specific TCR candidates demonstrated killing activity when exposed to target cells presenting the specific cognate peptide.
Conclusions Our Deep Immunomics platform allows us to identify, characterize, and confirm the activity of TCR candidates against novel and therapeutically relevant shared cancer antigens. These findings validate our discovery workflow leading to a portfolio of potential clinical candidates that are currently under preclinical development.
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