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Endowing universal CAR T-cell with immune-evasive properties using TALEN-gene editing.

ABSTRACT: Universal CAR T-cell therapies are poised to revolutionize cancer treatment and to improve patient outcomes. However, realizing these advantages in an allogeneic setting requires universal CAR T-cells that can kill target tumor cells, avoid depletion by the host immune system, and proliferate without attacking host tissues. Here, we describe the development of a novel immune-evasive universal CAR T-cells scaffold using precise TALEN-mediated gene editing and DNA matrices vectorized by recombinant adeno-associated virus 6. We simultaneously disrupt and repurpose the endogenous TRAC and B2M loci to generate TCRαβ- and HLA-ABC-deficient T-cells expressing the CAR construct and the NK-inhibitor named HLA-E. This highly efficient gene editing process enables the engineered T-cells to evade NK cell and alloresponsive T-cell attacks and extend their persistence and antitumor activity in the presence of cytotoxic levels of NK cell in vivo and in vitro, respectively. This scaffold could enable the broad use of universal CAR T-cells in allogeneic settings and holds great promise for clinical applications.

SUBMITTER: Jo S

PROVIDER: S-EPMC9247096 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Endowing universal CAR T-cell with immune-evasive properties using TALEN-gene editing.

Jo Sumin S Das Shipra S Williams Alan A Chretien Anne-Sophie AS Pagliardini Thomas T Le Roy Aude A Fernandez Jorge Postigo JP Le Clerre Diane D Jahangiri Billal B Chion-Sotinel Isabelle I Rozlan Sandra S Dessez Emilie E Gouble Agnes A Dusséaux Mathilde M Galetto Roman R Duclert Aymeric A Marcenaro Emanuela E Devillier Raynier R Olive Daniel D Duchateau Philippe P Poirot Laurent L Valton Julien J

Nature communications 20220630 1

Universal CAR T-cell therapies are poised to revolutionize cancer treatment and to improve patient outcomes. However, realizing these advantages in an allogeneic setting requires universal CAR T-cells that can kill target tumor cells, avoid depletion by the host immune system, and proliferate without attacking host tissues. Here, we describe the development of a novel immune-evasive universal CAR T-cells scaffold using precise TALEN-mediated gene editing and DNA matrices vectorized by recombinan ...[more]

PMID: 35773273

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Project description:Adoptive cell therapy based on chimeric antigen receptor (CAR)-engineered T-cells has proven to be lifesaving for many cancer patients. However, its therapeutic efficacy has so far been restricted to only a few malignancies, with solid tumors proving to be especially recalcitrant to efficient therapy. Poor intra-tumor infiltration by T cells and T cell dysfunction due to a desmoplastic, immunosuppressive microenvironment are key barriers for CAR T-cell success against solid tumors. Cancer-associated fibroblasts (CAFs) are critical components of the tumor stroma, evolving specifically within the tumor microenvironment (TME) in response to tumor cell cues. The CAF secretome is a significant contributor to the extracellular matrix and a plethora of cytokines and growth factors that induce immune suppression. Together they form a physical and chemical barrier which induces a T cell-excluding 'cold' TME. CAF depletion in stroma rich solid tumors can thus provide an opportunity to convert immune evasive tumors susceptible to tumor-antigen CAR T-cell cytotoxicity. Using our TALEN-based gene editing platform we engineered non-alloreactive, immune evasive CAR T-cells (termed UCAR T-cells) targeting the unique CAF marker Fibroblast Activation Protein, alpha (FAP). In an orthotopic mouse model of triple-negative breast cancer (TNBC) composed of patient derived-CAFs and tumor cells, we demonstrate the efficacy of our engineered FAP UCAR T-cells in CAF depletion, reduction of desmoplasia and successful tumor infiltration. Furthermore, while previously resistant, pre-treatment with FAP UCAR T-cells now sensitized these tumors to Mesothelin (Meso) UCAR T-cell infiltration and anti-tumor cytotoxicity. Combination therapy of FAP UCAR, Meso UCAR T cells and the checkpoint inhibitor anti-PD-1 significantly reduced tumor burden and prolonged mice survival. Our study thus proposes a novel treatment paradigm for successful CAR T-cell immunotherapy against stroma-rich solid tumors.

Project description:The crosstalk between tumor and immune cells has important implications for cancer treatment, in particular for immunotherapies. Pancreatic ductal adenocarcinoma (PDAC) generally displays a chemoresistant phenotype and is refractory to immune-based therapies. Highly tumorigenic and stem-like cells known as cancer stem cells (CSCs) within PDAC tumors have been demonstrated to contribute to its aggressiveness, metastatic capacity and chemoresistance. Here, we show that CSCs could be also relevant to establish an immunosuppresive environment. Using genetically engineered mouse models (GEMMs) of PDAC we isolated a triple positive (EpCAM+, Sca-1+ and CD133+) population of tumor cells, functionally validated these cells as bona fide CSCs and probed their transcriptional profile by microarray analysis. In addition to stem pathways, signatures related to tumor immune evasion, immune modulation, invasiveness, and innate immune response were enriched in this population. Peptidoglycan recognition protein 1 (Pglyrp1), an innate immune response anti-bacterial protein expressed primarily by neutrophils, was highly expressed in this triple-positive population and in CSC-enriched spheres from human cell lines. Interestingly, modulation of Pglyrp1 expression in murine PDAC primary cultures affected their immune evasive properties, specifically their ability to escape macrophage- and T-cell-mediated killing in vitro and to establish tumors in immunocompetent hosts. Pglyrp1 expression in tumor cells seemed to interfere with TNFα-mediated killing by T cells and to promote activated T-cell cytotoxicity. Importantly, these results could be validated in CSCs from human patient-derived xenografts, and PGLYRP1 protein levels were also significantly higher in PDAC patient samples versus healthy controls. Thus, our study demonstrates a hierarchical organization of cancer cells in a murine model of PDAC, validating the CSC concept, and identifies Pglyrp1 as a putative PDAC biomarker that confers immune privilege properties to CSCs which could have important therapeutic implications.

Dataset Information

Endowing universal CAR T-cell with immune-evasive properties using TALEN-gene editing.

Publications

Endowing universal CAR T-cell with immune-evasive properties using TALEN-gene editing.

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