Abstract LB029: TIGIT deletion rescues the antitumor activity of low avidity T cells by increasing TCR signal strength
Authors: Martina Spiga, Alessia Potenza, Stefano Beretta, Alessia Airaghi, Barbara Camisa, Neda Mohammadi, Laura Perani, Zulma Magnani, Claudio Doglioni, Chiara Balestrieri, Oronza Botrugno, Giulio Giovannoni, Giovanni Tonon, Danilo Abbati, Chiara Iozzi, Maximilian Reichert, Hana Algül, Arianna Pocaterra, Martina Fiumara, Samuele Ferrari, Giulia Di Lullo, Giovanni Sitia, Maria Pia Protti, Anna Mondino, Michele Reni, Stefano Crippa, Massimo Falconi, Luigi Naldini, Eliana Ruggiero, Chiara Bonini
Journal: Cancer Research (2025)

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a major unmet medical need, with a high mortality rate mainly related to the rapid development of the disease, precipitating the 5-year survival rate to 12%. While conventional treatment often fail, adoptive cell therapy (ACT) is a promising therapeutic option. A key barrier to the clinical success of ACT is the difficulty in isolating high-avidity tumor specific T cells able to resist the signals from the immunosuppressive microenvironment. We exploited the possibility of a broader targetable antigen repertoire of T cell receptors (TCRs) by developing TCR-edited T cells using a cytosine base editor and enhanced their potency by knocking-out TIGIT, a key inhibitory molecule expressed on exhausted T cells infiltrating PDAC. First, we investigated published datasets and identified 19 relevant tumor-associated antigens to target by T cells. By clonal tracking of the TCR repertoire of T cells stimulated with autologous APCs loaded with the selected antigens, we isolated 5 different HLA-A*0201 restricted TCRs specificities. Importantly, all these antigens are expressed in our cohort of primary and metastatic PDAC patients. We confirmed the specificity of each TCR-edited T cell product, which killed PDAC cell lines expressing the antigen and the proper HLA, while sparing negative controls. To boost the efficacy of anti-tumor T cells, we coupled TCR editing with TIGIT knockout (KO). By challenging TIGITKO and TIGITCOMP T cells in orthotopic models of PDAC primary tumors and colorectal cancer liver metastases, established from patient-derived organoids, TIGIT KO induced different effects according to the TCR avidity. We observed that TIGIT KO enhanced the TCR signal strength of low-avidity TCRs only, by increasing the phosphorylation of ZAP70, enforcing cytoskeletal rearrangements, and ultimately stabilizing the immunological synapse with cancer cells. In both low- and high-avidity T cell products TIGIT KO improved T cell costimulation allowing for a faster tumor shrinkage in vivo. We then evaluated the safety profile of our T cell products, employing targeted sequencing of sgRNA-dependent off-target sites and ultra-deep whole exome sequencing to determine sgRNA-independent off-target events. By a direct comparison of CBE and CRISPR/Cas9, we observed that both editing procedures proved highly efficient in simultaneously disrupting TRAC, TRBC1, TRBC2 and TIGIT. While T cell phenotype was unaffected, CRISPR/Cas9 treatment caused translocations between our four target loci and aberrant T cell expansion. We then confirmed that CBE produces high on-target editing with minimal off-target nucleotides conversion and observed that it did not introduce biologically relevant spurious DNA off-target mutations. Our findings suggest that deletion of TIGIT rescues the anti-tumor capacity of low-avidity T cells, highlighting TIGIT KO as a potential strategy to broaden the repertoire of TCRs engineered T cells in the treatment of PDAC and other tumors. Citation Format: Martina Spiga, Alessia Potenza, Stefano Beretta, Alessia Airaghi, Barbara Camisa, Neda Mohammadi, Laura Perani, Zulma Magnani, Claudio Doglioni, Chiara Balestrieri, Oronza Botrugno, Giulio Giovannoni, Giovanni Tonon, Danilo Abbati, Chiara Iozzi, Maximilian Reichert, Hana Algül, Arianna Pocaterra, Martina Fiumara, Samuele Ferrari, Giulia Di Lullo, Giovanni Sitia, Maria Pia Protti, Anna Mondino, Michele Reni, Stefano Crippa, Massimo Falconi, Luigi Naldini, Eliana Ruggiero, Chiara Bonini. TIGIT deletion rescues the antitumor activity of low avidity T cells by increasing TCR signal strength [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB029.