Study on the in vitro and in vivo killing effects of PD-1 antibody-secreting c-Met-targeted CAR-T cells on esophageal cancer cell line ECA109.

Background: This study was designed to investigate the in vitro and in vivo killing effects of PD-1 antibody-secreting c-Met-targeted CAR-T cells on esophageal cancer cell line ECA109.

Methods: We employed the TCGA and GTEx databases to analyze the expression levels of c-Met and PD-L1 in esophageal cancer tissues. Immunohistochemistry was employed to detect the expression of c-Met and PD-L1 in clinical esophageal cancer tissues and adjacent normal tissues. Flow cytometry was employed to verify the expression of c-Met and PD-L1 in ECA109 cells. T cells from healthy volunteers were extracted and activated, and c-Met/PD-1 CAR-T cells were prepared utilizing lentivirus. Flow cytometry was employed to detect the positive rate, subtype, antibody secretion, and anti-apoptotic effects of c-Met/PD-1 CAR-T cells. The experimental group consisted of c-Met/PD-1 CAR-T cells, with c-Met CAR-T and CD19 CAR-T as control groups, and Active T cells as the blank control group. The proliferation, cytokine secretion, and killing ability of CAR-T cells co-cultured with ECA109 cells were assessed utilizing cell viability assays (CCK-8), enzyme-linked immunosorbent assays (ELISA), and lactate dehydrogenase release assays (LDH). In vivo experiments were conducted by subcutaneously injecting ECA109 cells into nude mice to establish tumor-bearing models. CAR-T cells were administered via in situ injection, and changes in body weight and tumor volume were measured. Hematoxylin and eosin (HE) staining was performed on heart, liver, spleen, lung, and kidney tissues from sacrificed mice.

Results: c-Met and PD-L1 were highly expressed in esophageal cancer tissues but were low or not expressed in adjacent normal tissues. ECA109 cells expressed c-Met and PD-L1 on their surface. Successfully prepared c-Met/PD-1 CAR-T cells secreted PD-1 antibodies that blocked PD-1 on the surface of CAR-T cells. Under stimulation by c-Met antigens, c-Met/PD-1 CAR-T cells exhibited stronger proliferation, cytokine release, and killing ability. In vivo experiments demonstrated significant tumor inhibition in nude mice treated with c-Met/PD-1 CAR-T cells without evident off-target effects.

Conclusion: The c-Met/PD-1 CAR-T cells were successfully constructed and demonstrated significant in vitro and in vivo killing effects on ECA109 esophageal cancer cells.