Intraperitoneal Co-Delivery of Claudin18.2×41BB and EpCAM×CD3 Bispecific Antibodies via mRNA-LNPs Synergistically Suppresses Gastric Cancer Peritoneal Metastasis Through T Cell Co-Activation

Introductions: Gastric cancer with peritoneal metastasis (GCPM) carries a poor prognosis, constrained by inadequate drug delivery, systemic toxicity, and an immunosuppressive microenvironment. We developed E3C4, a single mRNA-lipid nanoparticle (LNP) co-encoding EpCAM×CD3 and Claudin18.2× 41BB bispecific antibodies (BsAbs), to achieve localized dual-antigen T cell co-activation against GCPM. Methods: E3C4 was prepared by co-encapsulating m 1 Ψ-modified mRNAs using ionizable lipids. Functional characterization included surface plasmon resonance (SPR) for binding affinity, T cell-dependent cytotoxicity (TDCC), and 4– 1BB reporter assays. In vivo efficacy was evaluated in PBMC-humanized mice bearing subcutaneous or orthotopic NCI-N87 tumors. Immune infiltration and cytokine release were analyzed via flow cytometry and ELISA. Safety was assessed in C57BL/6J mice through acute and repeated-dose toxicity studies. Results: Consequently, E3C4 exhibited optimal particle size and encapsulation efficiency. SPR confirmed high affinity for both BsAbs (KD ~10 − 10 M). Synergistic T cell activation achieved 80.9% cytotoxicity in vitro, significantly surpassing EpCAM×CD3 alone (29.1%, P< 0.001). In subcutaneous models, 3 μg E3C4 induced 98.5% tumor growth inhibition with enhanced T cell infiltration and granzyme B expression. Intraperitoneal delivery enabled 96.4% tumor-localized expression, yielding superior efficacy over recombinant BsAbs in orthotopic models. E3C4 showed no mortality or cytokine release syndrome, with only transient elevations in IL-6, AST and ALT, as well as a > 10% body weight loss that resolved within 2 weeks. The maximum tolerated dose exceeded the therapeutic dose by > 200-fold. Conclusion: E3C4 constitutes a localized, synergistic platform that enables dual-antigen T cell co-activation within the peritoneal cavity. This approach maximizes antitumor efficacy while circumventing systemic toxicity, offering a novel immunotherapeutic strategy for GCPM. Keywords: gastric cancer, peritoneal metastasis, mRNA-lipid nanoparticles, bispecific antibodies, T cell co-activation, EpCAM, Claudin18.2, 41BB, CD3