Anti-EPCAM CAR-T (The First Affiliated Hospital of Chengdu Medical College)

To construct chimeric antigen receptor (CAR)-T cells targeting epithelial cell adhesion molecule (EpCAM) antigen (anti-EpCAM-CAR-T).

A third-generation CAR-T cell construct used a single-chain variable fragment derived from monoclonal antibody against human EpCAM. Peripheral blood mononuclear cells were extracted from volunteers. The proportion of cluster of differentiation 8 positive (CD8+) and CD4 + T cells was measured using flow cytometry. Western blot was used to detect the expression of EpCAM-CAR. The killing efficiency was detected using the MTT assay and transwell assay, and the secretion of killer cytokines tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) was detected using the ELISA. The inhibitory effect of EpCAM-CAR-T on colorectal cancer in vivo was detected using xenografts.

It was found that T cells expanded greatly, and the proportion of CD3+, CD8 + and CD4 + T cells was more than 60%. Furthermore, EpCAM-CAR-T cells had a higher tumour inhibition rate in the EpCAM expression positive group than in the negative group (P < 0.05). The secretion of killer cytokines TNF-α and IFN-γ in the EpCAM expression positive cell group was higher than that in the negative group (P < 0.05). In the experimental group treated with EpCAM-CAR-T cells, the survival rate of nude mice was higher (P < 0.05), and the tumour was smaller than that in the blank and control groups (P < 0.05). The secretion of serum killer cytokines TNF-α and IFN-γ in tumour-bearing nude mice in the experimental group treated with EpCAM-CAR-T cells was higher than that in the blank and control groups (P < 0.05).

This study successfully constructed EpCAM-CAR cells and found that they can target and recognise EpCAM-positive tumour cells, secrete killer cytokines TNF-α and IFN-γ and better inhibit the growth and metastasis of colorectal cancer in vitro and in vivo than unmodified T cells.

CBL-B is an intracellular E3 ubiquitin ligase that acts as a T cell checkpoint by raising activation thresholds and limiting effector function. Here, genetic targeting of CBL-B enhances the performance of adoptively transferred T cells and CAR T cells under tumor microenvironment-like stress. In fully immunocompetent mouse models, Cblb deficiency or transient Cblb silencing improves control of MC-38 colon carcinoma and autochthonous mammary tumors, demonstrating that CBL-B restrains anti-tumor immunity. Cblb-deficient T cells show enhanced expansion and effector/effector-memory differentiation during an in vivo mixed lymphocyte reaction, confirming a cell-intrinsic brake function of CBL-B during sustained antigenic challenge. In a syngeneic Panc02-EpCAM model, Cblb-deficient anti-EpCAM CAR T cells show superior tumor control, enhanced infiltration, prolonged survival, and preserved effector function despite chronic antigen exposure and TGF-β. Mechanistically, Cblb targeting maintains granzyme B and IFN-γ production and is associated in vitro with increased GSDME-linked pyroptotic tumor cell death, consistent with features of immunogenic cell death. These findings extend previous CBL-B CAR T work from lymphocyte-deficient to immunocompetent settings and support CBL-B inhibition as a strategy to engineer CAR T cells that resist suppressive tumor microenvironments while promoting a more inflammatory mode of tumor killing.