A novel CLDN6 nanobody-based bispecific T-cell engager delivered by self-amplifying RNA platform exhibits potent antitumor efficacy

Ovarian cancer remains a highly lethal gynecologic malignancy with limited early detection and frequent relapse. Claudin-6 (CLDN6), minimally expressed in normal tissues but frequently upregulated in ovarian cancer, represents a promising tumor-specific target. Bispecific T-cell engagers (BiTEs) effectively redirect cytotoxic T cells toward tumor cells. However, previous studies have reported that protein-based BiTEs, particularly scFv-based constructs, may encounter challenges related to protein folding, aggregation, and in vivo stability. To address these limitations, CLDN6-nanobodies were developed through our screening platform, which display cross-species reactivity to human, murine and non-human primate CLDN6. Using this binder, we constructed CLDN6 nanobody-based BiTE (CLDN6-Nb-BiTE) encoded by self-amplifying mRNA (sa-mRNA) and delivered by lipid nanoparticles (LNPs), providing a functional alternative to protein administration. The LNP-sa-mRNA formulation was stable and uniform, enabling robust CLDN6-Nb-BiTE expression, antigen-dependent cytotoxicity, and cytokine release in vitro, while sustained CLDN6-Nb-BiTE production in vivo promoted T-cell recruitment and durable tumor control without systemic toxicity. Overall, our work introduces the first CLDN6-Nb-BiTE delivered by sa-mRNA, establishing a foundation for next-generation ovarian cancer immunotherapy.