Abstract LB069: Repurposing anti-mesothelin CAR-NK immunotherapy against colorectal cancer

Abstract Colorectal cancer (CRC) is the third most common cancer worldwide, with highly variable prognosis and response to treatment. A large subset of patients does not respond to standard treatments or develops resistance. As an alternative, adoptive immunotherapy based on chimeric antigen receptor (CAR)-transduced immune cells has been proposed, however with significant adverse events. We therefore evaluated alternative CAR targets already tested in other tumour types, and employed the natural killer cell line NK-92 for CAR transduction because of its more favourable toxicity profile. As an alternative antigen, we considered mesothelin (MSLN), the most represented in CAR-based clinical studies for solid tumours. MSLN RNA expression was analysed in large series of CRC patient-derived xenografts (PDXs, n=500), cell lines (n=150) and tumours (n=640), to evaluate its distribution and to identify MSLN-overexpressing models. NK-92 cells were transduced with anti-MSLN CAR, and subsequently sorted and cloned. Activity of CAR-NK-92 cells against target-expressing ovarian and CRC cells was assessed in vitro and in vivo. Statistical significance of efficacy was evaluated by t-test. The large-scale expression analysis highlighted that about 10% of CRCs overexpress MSLN at levels comparable to those of ovarian cancer, a typical target of MSLN-CAR-based therapy. Intriguingly, MSLN overexpression is more frequent in the CRIS-B poor prognosis subtype and KRAS/BRAF-mutant CRC. Lentiviral transduction of NK-92 cells with the MSLN-CAR, followed by sorting and cloning, led to the identification of one clone, NK-92cl45, stably expressing the CAR and retaining the NK phenotype. As expected, the clone demonstrated significant in vitro and in vivo activity against ovarian cancer cells. When repurposed against models of CRC expressing high MSLN levels, it displayed comparable efficacy, both in vitro and in vivo. Specificity of NK-92cl45 was confirmed by the absence of activity on control models with low or absent MSLN. Our results provide preclinical evidence that a subset of colorectal cancers expressing high mesothelin levels can be effectively targeted by MSLN-CAR-based immunotherapy. The potential therapeutic impact of these findings is enhanced by the fact that frequently MSLN-overexpressing CRCs display worse prognosis and resistance to standard care. Citation Format: Erica Torchiaro, Marco Cortese, Alice D'Andrea, Consalvo Petti, Enzo Medico. Repurposing anti-mesothelin CAR-NK immunotherapy against colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB069.