ObjectiveColorectal tumours are often densely infiltrated by immune cells that have a role in surveillance and modulation of tumour progression but are burdened by immunosuppressive signals, which might vary from primary to metastatic stages. Here, we deployed a multidimensional approach to unravel the T-cell functional landscape in primary colorectal cancers (CRC) and liver metastases, and genome editing tools to develop CRC-specific engineered T cells.DesignWe paired high-dimensional flow cytometry, RNA sequencing and immunohistochemistry to describe the functional phenotype of T cells from healthy and neoplastic tissue of patients with primary and metastatic CRC and we applied lentiviral vectors (LV) and CRISPR/Cas9 genome editing technologies to develop CRC-specific cellular products.ResultsWe found that T cells are mainly localised at the front edge and that tumor-infiltrating T cells co-express multiple inhibitory receptors, which largely differ from primary to metastatic sites. Our data highlighted CD39 as the major driver of exhaustion in both primary and metastatic colorectal tumours. We thus simultaneously redirected T-cell specificity employing a novel T-cell receptor targeting HER-2 and disrupted the endogenous TCR genes (TCR editing (TCR_ED)) and the CD39 encoding gene (ENTPD1), thus generating TCR_EDENTPD1_KOHER-2-redirected lymphocytes. We showed that the absence of CD39 confers to HER-2-specific T cells a functional advantage in eliminating HER-2⁺patient-derived organoidsin vitroandin vivo.ConclusionHER-2-specific CD39 disrupted engineered T cells are promising advanced medicinal products for primary and metastatic CRC.