Abstract 902: Harnessing CD39 for the treatment of colorectal cancer and liver metastases by engineered T cells
Authors: Potenza Alessia , Balestrieri Chiara , Albarello Luca , Pedica Federica , Spiga Martina , Manfredi Francesco , Cianciotti Beatrice C. , De Lalla Claudia , Stasi Lorena , Tassi Elena , Bonfiglio Silvia , Scotti Giulia M. , Redegalli Miriam , Biancolini Donatella , Abbati Danilo , Simeoni Fabio , Lazarevic Dejan , Elmore Ugo , Fiorentini Guido , Lullo Giulia Di , Casorati Giulia , Doglioni Claudio , Tonon Giovanni , Dellabona Paolo , Rosati Riccardo , Aldrighetti Luca , Ruggiero Eliana , Bonini Chiara 
Published: April 2023

Abstract
Colorectal cancer (CRC) is the 2nd cause of cancer-related death. Standard therapies often fail, with more than 50% of patients experiencing relapse, eventually with metastatic disease. Colorectal tumors are densely infiltrated by immune cells that have a role in surveillance and modulation of tumor progression. However, exhaustion mechanisms acting within the tumor microenvironment impede their functional capacity against tumor cells. We paired high-dimensional flow cytometry, RNA sequencing, immunohistochemistry and immunofluorescence to describe the T cell functional landscape in tumor and peritumoral tissues from primary colorectal cancers and liver metastases. Analysis of the healthy, peritumoral and neoplastic tissues of treatment-naïve primary CRCs and of the peritumoral and tumoral tissues of CRC patients undergoing surgery for liver metastasis, revealed extensive transcriptional and spatial remodeling across tumors, being metabolic pathways among the major drivers of this variance. Regarding the immune infiltrate, we found that T cells are mainly localized at the front edge and that tumor-infiltrating T cells co-express multiple inhibitory receptors. Unsupervised analysis of flow cytometry data performed by an advanced pipeline of data handling by dimensionality reduction and clustering algorithms allowed the definition of a peculiar inhibitory receptors signature in TILs enriched both in primary CRCs and liver metastases. Among the highly co-expressed inhibitory receptors, CD39 was found to represent the major driver of exhaustion in both primary and metastatic colorectal tumors. CD39 is a diphosphohydrolase converting ATP into AMP that is emerging as exhaustion marker for tumor-specific T cells, thus highlighting its relevance as molecular target for T cells engineering. We leveraged on these findings to generate a novel cellular product for the adoptive cell therapy of CRC. By CRISPR/Cas9 genome editing tools, we simultaneously redirected T cell specificity by disrupting the alpha and beta genes of the endogenous T cell receptor with 90% efficiency for both genes, and disrupted CD39 with 100% efficiency, generating triple-knockout engineered lymphocytes. By lentiviral transduction, we redirected the specificity of our engineered T cell product employing a novel T-cell receptor targeting the HER-2 antigen. Triple-edited, HER2-redirected T cells were challenged in vitro against HER2+ patient-derived organoids from liver metastases (mPDOs). Measurement of Cas3/7 displayed a functional advantage for CD39-disrupted, HER2-redirected T cells in recognizing and killing mPDOs. We then evaluated the efficacy of our T cell product in two different in vivo models: subcutaneous injection of mPDOs, and intra-hepatic injection of mPDOs. In both models, CD39-disrupted, HER2-redirected T cells displayed a superior capacity of controlling tumor outgrowth long term.
Citation Format: Alessia Potenza, Chiara Balestrieri, Luca Albarello, Federica Pedica, Martina Spiga, Francesco Manfredi, Beatrice C. Cianciotti, Claudia De Lalla, Lorena Stasi, Elena Tassi, Silvia Bonfiglio, Giulia M. Scotti, Miriam Redegalli, Donatella Biancolini, Danilo Abbati, Fabio Simeoni, Dejan Lazarevic, Ugo Elmore, Guido Fiorentini, Giulia Di Lullo, Giulia Casorati, Claudio Doglioni, Giovanni Tonon, Paolo Dellabona, Riccardo Rosati, Luca Aldrighetti, Eliana Ruggiero, Chiara Bonini. Harnessing CD39 for the treatment of colorectal cancer and liver metastases by engineered T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 902.