RFdiffusion-designed CLDN18.2 T-cell engager coupled with dendritic-cell supplementation enhances antitumor immunity in gastric cancer

CLDN18.2 is a validated therapeutic target in gastric cancer, but T-cell redirection in solid tumors can be limited by poor immune infiltration. We designed a CLDN18.2-targeting T-cell engager using an RFdiffusion-assisted workflow and investigated why its activity waned in immune-cold gastric cancer models. Candidate CLDN18.2 binders were screened computationally and incorporated into human and murine CD3-engaging TCE formats. Binding, CLDN18.2 selectivity, T cell-dependent cytotoxicity, and CD8 + T-cell activation were tested in engineered gastric cancer cells, patient-derived tumor cells, and patient-derived organoids. Antitumor activity was then evaluated in syngeneic and hPBMC-reconstituted PDX models. Dendritic-cell supplementation, MHC-II blockade, and IL-12 blockade were used to probe the immune-support signals that shaped TCE response. The selected TCE bound CLDN18.2-positive tumor cells and CD3 + T cells, with limited detectable binding to CLDN18.1-expressing cells in cell-surface assays. It induced CLDN18.2-dependent tumor-cell killing and CD8 + T-cell activation in cell-line, patient-derived tumor-cell, and organoid co-cultures. In vivo, TCE treatment produced early tumor inhibition, but tumor control weakened over time. This loss of durability was accompanied by limited intratumoral T-cell accumulation, increased PD-1 expression, sparse CD11c+ dendritic cells, and weak CXCL9/CXCL10-associated chemokine signals. Adding dendritic cells improved TCE-mediated killing in vitro and tumor control in vivo. MHC-II blockade had modest effects, whereas IL-12 blockade more consistently reduced the DC-associated enhancement, indicating that added DCs acted mainly through cytokine-associated support rather than antigen presentation alone. RFdiffusion-assisted binder design generated a CLDN18.2-targeting TCE with activity across gastric cancer models. Under the tested conditions, tumor-T-cell bridging alone did not achieve durable control in cold tumor models, whereas DC-associated IL-12/CXCL9 signals strengthened TCE activity. These findings support further testing of CLDN18.2-directed TCEs together with strategies that rebuild local DC-T-cell immune circuits.