Background Human cancers can express both private and shared mutation-associated neoantigens, the latter being derived from recurrent cancer driver gene mutations, with the R175H mutation in the human TP53 tumor-suppressor gene being the most frequently shared between patients across cancer types. This makes the generation and subsequent adoptive transfer of TCR-engineered T cells reactive to an HLA-A*02:01-restricted TP53R175H epitope a highly attractive cancer immunotherapy approach. However, the combined immunosuppressive tumor-microenvironment and the low levels of neoantigen presentation by solid cancers compromise the activation and reactivity of TCR-engineered T cells, posing a major hurdle for these cells to eradicate tumor lesions. A substantial body of work has highlighted that inactivation of negative regulators of TCR signaling can augment T cell functionality and anti-tumor reactivity.
Methods Aiming to develop a highly potent anti-cancer T cell product, we applied CRISPR genome engineering to specifically introduce a TP53R175H-specific TCR into the TRAC locus of primary human T cells, while simultaneously inactivating negative regulators of TCR signaling. After validating successful introduction of the TP53R175H TCR and inactivation of endogenous TCR chains and the TCR inhibiting genes of interest, the ability of edited T cells to proliferate, produce cytokines and control tumor growth were assessed in vitro.
Results We found that inactivation of TCR signaling inhibitory genes significantly enhanced the ability of TCR-edited T cells to proliferate and produce the cytokines IFN-γ, TNF and IL-2, both in response to anti-CD3/anti-CD28 stimulation and HLA-A*02:01+ tumor cell lines that express the TP53R175H mutation. Notably, T cells that had lost expression of negative regulators of TCR signaling were also superior in serial killing of TP53R175H-positive tumor cells in vitro, while these T cells did not inhibit tumor cells that overexpressed a TP53 R175 WT epitope.
Conclusions Taken together, our findings show that inactivation of negative regulators of the TCR signaling pathway can enhance the reactivity of TCR-edited T cells against shared neoantigen-expressing tumor cells, thereby providing a promising new approach to increase the efficacy of TCR therapy directed against solid human cancers.
Ethics Approval Samples were obtained through a supply agreement with Sanquin Bloedvoorziening Amsterdam. These samples were collected only from voluntary, non-remunerated, adult donors who provided written informed consent as part of routine donor selection and blood collection procedures, that were approved by the Ethics Advisory Council of Sanquin Blood Supply Foundation.
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