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Abstract
Objective Colorectal 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.
Design We 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.
Results We 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 (TCRED)) and the CD39 encoding gene (ENTPD1), thus generating TCREDENTPD1KOHER-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 organoids in vitro and in vivo.
Conclusion HER-2-specific CD39 disrupted engineered T cells are promising advanced medicinal products for primary and metastatic CRC.
- colorectal cancer
- liver metastases
- immunotherapy
- cancer immunobiology
- T lymphocytes
Data availability statement
Data are available in a public, open access repository. Qian, J, Olbrecht, S, Boeckx, B et al. (Dataset) from: A Pan-Cancer Blueprint of the Heterogeneous Tumor Microenvironment Revealed by Single-Cell Profiling. Cell Res 2020, 30 (9), 745–762. https://doi.org/10.1038/s41422-020-0355-0. ArrayExpress database available at EMBL-EBI Data generated by Potenza, Balestrieri et al are available in a public, open access repository (GEO Database) under the accession number GSE200133.
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Data availability statement
Data are available in a public, open access repository. Qian, J, Olbrecht, S, Boeckx, B et al. (Dataset) from: A Pan-Cancer Blueprint of the Heterogeneous Tumor Microenvironment Revealed by Single-Cell Profiling. Cell Res 2020, 30 (9), 745–762. https://doi.org/10.1038/s41422-020-0355-0. ArrayExpress database available at EMBL-EBI Data generated by Potenza, Balestrieri et al are available in a public, open access repository (GEO Database) under the accession number GSE200133.
Footnotes
AP and CBa contributed equally.
MS, LA and FP contributed equally.
ER and CBo contributed equally.
Contributors AP designed the study, performed the experiments, analysed and interpreted data and wrote the manuscript. CBa designed the study, performed bioinformatic analyses, interpreted data and wrote the manuscript. MS contributed at organoids preparation, T-cell engineering and functional assays. LA, FP and CD performed pathology analyses on patients’ tissue samples and collected samples for cohorts I and II. FM, LS, ETa and CI participated in experiments and interpreted data. OAB participated in organoids preparation for in vitro and in vivo experiments. SB, DB, FS and DL performed library preparation and sequencing. MR performed NanoString sequencing. GMS and DA performed data analyses. BCC, CDL, GDL, GC, GT, PD and MC participated in data discussion and interpretation. CF performed Tregs evaluation. BC, ETi and CS participated in in vivo experiments. UE, GF, RR and LA collected surgical specimens and participated in data discussion. ER designed and supervised the study, performed TCR sequencing and participated in data discussion and interpretation of results. CBo designed and supervised experiments, discussed data, interpreted results and wrote the manuscript and is the guarantor of this study.
Funding This work was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC 5 per Mille 22737 and AIRC-IG 18458), the Italian Ministry of Education, University and Research (PRIN 2017WC8499) and the Italian Ministry of Health and Alliance Against Cancer (Ricerca Corrente CAR T project: RCR-2019-23669115), EU (T2Evolve) to CBo; by the Italian Ministry of Health (GR-2016-02364847) to ER, by an AIRC Fellowship to BCC.
Competing interests CBo has been member of Advisory Board and Consultant for Intellia, TxCell, Novartis, GSK, Allogene, Kite/Gilead, Kiadis, Evir, Janssen, Genyo, Epsilen and received research support from Intellia Therapeutics. AP, PDB, GC, ER, BCC and CBo are inventors on different patents on cancer immunotherapy and genetic engineering.
Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
Provenance and peer review Not commissioned; externally peer reviewed.
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