Targeting ZP3 with chimeric antigen receptor macrophages: a novel immunotherapeutic strategy for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) remains a major cause of cancer-related mortality worldwide, underscoring the need for effective immunotherapeutic strategies. Here, we generated monoclonal antibodies against zona pellucida glycoprotein 3 (ZP3), developed ZP3-targeting chimeric antigen receptor macrophages (CAR-M), and evaluated their therapeutic potential in HCC. The recombinant extracellular domain of human ZP3 was expressed in HEK293F cells and used to immunize mice. CD138⁺ plasma cells were enriched and subjected to antigen-specific single-cell screening. Paired immunoglobulin variable-region sequences were recovered by nested polymerase chain reaction, followed by antibody cloning, expression, and purification. An anti-ZP3 single-chain variable fragment was incorporated into a lentiviral CAR construct and expressed in THP-1 cells, which were subsequently differentiated into macrophage-like cells. The phagocytic activity, cytotoxicity, polarization phenotype, cytokine secretion, in vivo antitumor efficacy, biodistribution, and preliminary safety of ZP3-targeting CAR-M were evaluated in HCC cell-based assays and Huh7 xenograft models. Highly purified anti-ZP3 monoclonal antibodies were generated and shown to recognize membrane-associated ZP3 on Huh7 cells. Anti-ZP3 antibody treatment significantly suppressed tumor growth in peripheral blood mononuclear cell-reconstituted NOD-Scid mice. ZP3-targeting CAR-M exhibited efficient CAR expression and specific ZP3 binding, together with enhanced phagocytosis and cytotoxicity against HCC cells compared with control macrophages. Antigen engagement promoted an M1-like phenotype, characterized by increased CD80 and CD86 expression, reduced CD163 and CD206 expression, and enhanced secretion of IL-6, IL-1β, TNF-α, and IFN-γ. In Huh7 xenograft-bearing mice, systemic administration of CAR-M significantly delayed tumor progression. After treatment, body weight remained stable, most serum biochemical parameters responded basically normal, and major organs exhibited favorable tolerability. ZP3 is a promising immunotherapeutic target in HCC. ZP3-directed antibodies and CAR-M demonstrated preclinical antitumor activity, supporting further development of ZP3-targeted immunotherapies.