Abstract
Chimeric antigen receptor (CAR)-T cell therapy for solid tumors faces challenges of insufficient efficacy and a high recurrence rate. Mesothelin (MSLN) is a membrane glycoprotein highly expressed in various solid tumors that has restricted low expression in normal tissues such as the pleura, peritoneum, and pericardium. We previously performed affinity maturation based on the parental antibody M912, and constructed the phage display library. In this study we identified four novel human anti-MSLN antibodies (LP12, HP4-11, HP4-41/LP6, and HP4-44/LP2) with varying degrees of enhanced affinity. These third-generation CARs targeting MSLN were packaged into lentiviral vectors to generate stable CAR-T cells. The CAR-T variants induced robust cytolytic activity, significant cytokine production, and activation-induced clonal proliferation against various MSLN-positive tumors in vitro, and effectively cleared disseminated tumors in mice. A single administration of the CAR-T variant LP12 potently eradicated various types of MSLN-positive solid tumors, achieved long-term persistence in vivo, effectively prevented tumor recurrence, and exhibited no non-specific toxicity. Therefore, optimizing the affinity of antigen-binding domain in CAR represents a promising strategy for advancing the development of safe and effective CAR-T cell therapies. The LP12 CAR-T cells developed in this study have potential applications in patients with MSLN-positive solid tumors. Schematic illustration of the generation and antitumor mechanism of affinity-tuned MSLN-targeted CAR-T cells. The expression plasmids carrying different anti-MSLN CAR genes were packaged into lentiviral vectors. Lentiviral transduction of human CD3+ T cells was performed to generate CAR-T cells, which were then expanded. After injection of moderately affinity-tuned MSLN-targeted CAR-T cells into mice, they enter the bloodstream, recognize, and infiltrate the solid tumors. They specifically recognize and bind to MSLN on the surface of tumor cells, and upon activation, release IFN-γ, IL-2, and TNF-α to exert cytolytic activity. Subsequently, they undergo clonal proliferation and primarily differentiate into effector memory CAR-T cells, maintaining long-term antitumor immunity and effectively preventing recurrence.