Abstract 5: Cytosine base editor ameliorates the safety profile of TCR edited T cells for the adoptive cell therapy of gastrointestinal tumors

Abstract Among gastrointestinal tumors, colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) are major unmet medical needs, with a high mortality rate mainly related to the onset of metastases, hindering the therapeutic efficacy of conventional treatments. Adoptive Cell Therapy (ACT) with TCR-edited T cells is a promising therapeutic strategy for patients with late-stage tumors, but its applicability is limited by the paucity of TCRs targeting relevant tumor antigens and the presence of an immunosuppressive tumor microenvironment. Moreover, the safety of the therapeutic cellular products, generated by genome editing tools, is fundamental for an effective and rapid clinical translation. With the aim of improving the safety profile of T-cell based therapeutic products manufactured by genome editing, we evaluated the efficacy and safety of cytosine base editor (CBE). We disrupted the endogenous TCR to redirect T cells specificity toward tumor cells, and TIGIT, a major inhibitory molecule in gastrointestinal tumors to enhancing T cell ability to counteract immunosuppression. Beyond the high efficiency of CBE in simultaneously disrupting the target genes, we observed only marginal sgRNA-dependent and -independent off-target events as measured by targeted sequencing of the top predicted off-target loci and by ultra-deep whole exome sequencing, respectively. In addition, unlike CRISPR/Cas9-treated cells, we could not detect translocations in CBE edited T cells. To select the antigens to target by engineered T cells, we investigated published datasets and identified 19 relevant tumor-associated antigens. By clonal tracking of the TCR repertoire of T cells stimulated with autologous APCs loaded with the selected antigens, we isolated 5 different TCRs specificities, including mesothelin. Importantly, these antigens are expressed in our cohort of primary and metastatic PDAC and CRC patients. TCR-edited T cells displayed anti-tumor activity against PDAC cell lines, CRC and PDAC patient-derived organoids while sparing HLA-unrelated and antigen-negative controls. To boost the efficacy of anti-tumor T cells, we coupled TIGIT disruption with TCR editing. By challenging TCR-edited TIGITKO T cells in vitro and in an orthotopic model of CRC liver metastases, we observed that TIGIT disruption can ameliorate the ability of tumor-specific T cells to control tumor growth. Our findings suggest that base editors can generate cellular products with a better safety profile and a promising therapeutic efficacy in gastrointestinal tumors. Citation Format: Martina Spiga, Alessia Potenza, Stefano Beretta, Alessia Airaghi, Chiara Iozzi, Martina Fiumara, Samuele Ferrari, Neda Mohammadi, Oronza Botrugno, Maximilian Reichert, Maria Pia Protti, Stefano Crippa, Massimo Falconi, Luigi Naldini, Eliana Ruggiero, Chiara Bonini. Cytosine base editor ameliorates the safety profile of TCR edited T cells for the adoptive cell therapy of gastrointestinal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5.