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Chemokine Receptor CCR2b Enhanced Anti-tumor Function of Chimeric Antigen Receptor T Cells Targeting Mesothelin in a Non-small-cell Lung Carcinoma Model.

ABSTRACT: Chimeric antigen receptor (CAR) T cell therapy faces a number of challenges for the treatment of non-small-cell lung carcinoma (NSCLC), and efficient migration of circulating CAR T cells plays an important role in anti-tumor activity. In this study, a CAR specific for tumor antigen mesothelin (Msln-CAR) was co-expressed with cell chemokine receptors CCR2b or CCR4. Findings showed that CCR2b and CCR4 enhanced the migration of Msln-CAR T cell in vitro by transwell assay. When incubated with mesothelin-positive tumor cells, Msln-CCR2b-CAR and Msln-CCR4-CAR T cell specifically exerted potent cytotoxicity and produced high levels of proinflammatory cytokines, including IL-2, IFN-γ, and TNF-α. Furthermore, NSCLC cell line-derived xenograft (CDX) model was constructed by implanting subcutaneously modified A549 into NSG mice. Compared to conventional Msln-CAR T cells, living imaging indicated that Msln-CCR2b-CAR T cells displayed superior anti-tumor function due to enhanced migration and infiltration into tumor tissue shown by immunohistochemistry (IHC) analysis. In addition, histopathological examinations of mice organs showed that no obvious organic damages were observed. This is the first time that CAR T cell therapy combined with chemokine receptor is applied to NSCLC treatment.

SUBMITTER: Wang Y

PROVIDER: S-EPMC7992009 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cancer immunotherapy has made unprecedented breakthrough in the fields of chimeric antigen receptor-redirected T (CAR T) cell therapy and immune modulation. Combination of CAR modification and the disruption of endogenous inhibitory immune checkpoints on T cells represent a promising immunotherapeutic modality for cancer treatment. However, the potential for the treatment of hepatocellular carcinoma (HCC) has not been explored. In this study, the gene expressing the programmed death 1 receptor (PD-1) on the Glypican-3 (GPC3)-targeted second-generation CAR T cells employing CD28 as the co-stimulatory domain was disrupted using the CRISPR/Cas9 gene-editing system. It was found that, in vitro, the CAR T cells with the deficient PD-1 showed the stronger CAR-dependent anti-tumor activity against native programmed death 1 ligand 1-expressing HCC cell PLC/PRF/5 compared with the wild-type CAR T cells, and meanwhile, the CD4 and CD8 subsets, and activation status of CAR T cells were stable with the disruption of endogenous PD-1. Additionally, the disruption of PD-1 could protect the GPC3-CAR T cells from exhaustion when combating with native PD-L1-expressing HCC, as the levels of Akt phosphorylation and anti-apoptotic protein Bcl-xL expression in PD-1 deficient GPC3-CAR T cells were significantly higher than those in wild-type GPC3-CAR T cells after coculturing with PLC/PRF/5. Furthermore, the in vivo anti-tumor activity of the CAR T cells with the deficient PD-1 was investigated using the subcutaneous xenograft tumor model established by the injection of PLC/PRF/5 into NOD-scid-IL-2Rγ-/- (NSG) mice. The results indicated that the disruption of PD-1 enhanced the in vivo anti-tumor activity of CAR T cells against HCC, improved the persistence and infiltration of CAR T cells in the NSG mice bearing the tumor, and strengthened the inhibition of tumor-related genes expression in the xenograft tumors caused by the GPC3-CAR T cells. This study indicates the enhanced anti-tumor efficacy of PD-1-deficient CAR T cells against HCC and suggests the potential of precision gene editing on the immune checkpoints to enhance the CAR T cell therapies against HCC.

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Chemokine Receptor CCR2b Enhanced Anti-tumor Function of Chimeric Antigen Receptor T Cells Targeting Mesothelin in a Non-small-cell Lung Carcinoma Model.

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