Peripheral Blood as a Source to Identify Candidate Neoantigens and Reactive TCRs

Abstract Text: Adoptive transfer of neoantigen-specific tumor infiltrating lymphocytes has demonstrated antitumor activity in selected patients. So far, the development of T-cell therapies relies on the obtention of a tumor biopsy to isolate neoantigen-reactive T cells and to extract tumor DNA and RNA to identify neoantigens. Nevertheless, the obtention of a tumor biopsy is often not feasible and it may underrepresent the tumor clonal diversity of the tumor. Last year, we showed that circulating neoantigen-specific CD8 and CD4 T cells could recognize neoantigens derived from non-synonymous mutations detected in circulating tumoral cell free DNA (Garcia-Garijo A et al., FOCIS 23). Moreover, they were preferentially enriched in the PD-1hi T and the PD1hi cells co-expressing CD39.

To further characterize neoantigen-reactive CD8+ with the above phenotypes, we sequenced the TCRs of the corresponding neoantigen reactive lymphocyte from one metastatic breast cancer patient. The most frequent alpha-beta TCR pair were shared by both populations; this TCR and some additional alpha-beta pairs were cloned and transduced into PBMCs. All these evaluated TCRs recognized neoantigen KIAA1524p.S57I but not the wild type peptide. The same procedure was applied to the CD4+ T-cell populations identifying two additional different neoantigens. These results confirmed that the different enriched clonotypes were neoantigen-specific and highlight the polyclonal nature of both PD-1hi and PD-1hiCD39+ KIAA1524p.S57I-reactive populations from PBL. In conclusion, by sorting different T-cell subsets from peripheral blood, we were able to detect eight different TCRs targeting three neoantigens that were originally identified using plasma-derived cell free DNA.