Combining antigen-specific T cells with T-cell engager therapy induces a molecular signature that favors T-cell fitness

Both adoptive cellular therapy with tumor antigen-specific T-cells and redirection of endogenous T-cells with bispecific tumor-specific T-cell engaging antibodies rely on CD8⁺ T-cell effector function. The two approaches employ complementary mechanisms of action (T-cell receptor (TCR)/MHC dependent vs independent tumor cell killing), making them ideal candidates for combinatorial strategies. TCR and T-cell engager (TCE) mediated killing of tumor cells was determined by luciferase assay. T-cell phenotypes were analyzed by multiparameter flow cytometry. NFAT transcriptional activity was measured by Jurkat NFAT luciferase reporter cell assay. Transcriptional changes in antigen-specific T-cells isolated from the bone marrow of THP-1 bearing NSG MHC I/II double knockout mice were analyzed by bulk RNA-seq and pathway analysis was performed by gene set enrichment analysis (GSEA). Compared with the pool of naturally occurring polyclonal (bulk) CD8⁺ T-cells, <i>ex vivo</i> primed and expanded antigen-specific T-cells possess an enhanced capacity to mediate TCE efficacy. Both <i>in vitro</i> and <i>ex vivo</i> analyses confirmed that reduced T-cell activation-induced loss of CD3 correlated with TCE treatment of antigen-specific T-cells, suggesting that CD3 could serve as a surrogate biomarker for TCE activity in this context. TCE treatment of antigen-specific T-cells was associated with reduced NFAT transcriptional activity and decreased PD-1 expression while expression of the transcription factor TCF1 was increased, consistent with a molecular signature favoring CD8⁺ T-cell fate decisions leading to a less exhausted phenotype. In summary, the integration of antigen-specific T-cell and TCE therapies presents an attractive opportunity for combinatorial cancer immunotherapy, and it induces a molecular signature preserving T-cell fitness.