Background The recognition of epitopes derived from tumor antigens via T cell receptors (TCR) of T cells is prerequisite for efficient tumor cell destruction. One of the limiting factors in T cell-mediated immunotherapy is the availability of shared targets highly expressed on tumor cells and absent from healthy tissue. Neo-open reading frame peptides (NOPs) arising from insertions or deletions in tumor DNA are a class of antigens providing opportunities for designing T cell receptor therapies.

Methods NOPs were predicted for all patients in The Cancer Genome Atlas and binding of NOP-derived epitopes was predicted for HLA-A*02:01 allele. The top 30 predicted binding epitopes were used to make HLA-A*02:01 fluorescently labeled tetramers. Peripheral blood mononuclear cells of multiple healthy donors were incubated with pooled peptide loaded tetramers. Tetramer positive cells were further expanded and enriched for antigen specific CD8⁺ T cells which were used to identify the alpha and beta chains of TCR. Either in vitro expanded or TCR engineered CD8⁺ T cells were used in co-culture assays with either cells overexpressing selected NOP of interest or with cell lines naturally expressing the same antigen. Additionally, mRNA construct encoding a NOP was used to transfect human monocyte-derived dendritic cells (moDC) and the epitope presentation was detected by co-culture with in vitro expanded antigen specific CD8 T cells. The activation of antigen specific CD8⁺ T cells was detected by increased CD137 and CD107a expression. The engineered T cells were tested in a dose titration experiment to assess the TCR affinity.

Results The in vitro expansion of antigen specific CD8 T cells recognizing HLA-A*02:01 bound peptides derived from NOPs was successful for 15 peptides. Out of all in vitro expanded CD8⁺ T cell populations, only 2 recognized their cognate peptide presented in the context of HLA-A*02:01 allele. One of the TCRs recognized an epitope derived from TP53 NOP. This TCR was cloned into naïve CD8⁺ T cells of a healthy donor which specifically recognized naturally expressed and presented TP53 NOP peptide in all tested models, including mRNA transfected moDC.

Conclusions We identified and validated a TCR recognizing an HLA-A*02:01 epitope from TP53 NOP. TP53 NOP is expressed in 1–3% of patients across multiple cancer types. This study shows feasibility of identifying targets for shared T cell therapies.