BTN1A1 blockade with Nelmastobart potentiates cisplatin-induced tumor suppression in NSCLC

Background: Butyrophilin 1A1 (BTN1A1) is a novel immune-regulatory checkpoint protein expressed in various solid tumors, including non-small cell lung cancer (NSCLC), where it modulates T-cell activation. We previously demonstrated that BTN1A1 inhibition by Nelmastobart enhances immune-mediated tumor suppression. In this study, we evaluated whether combining Nelmastobart with cisplatin could further potentiate anti-tumor immune responses in NSCLC. Methods: A549 NSCLC cells, stably expressing RFP, were cultured as 3D spheroids and co-incubated with human peripheral blood mononuclear cells (PBMCs), Nelmastobart, cisplatin, or their combinations. Anti-tumor immune activity was assessed by monitoring spheroid size reduction and RFP intensity loss. To evaluate translational safety, zebrafish embryos were exposed to Nelmastobart alone, cisplatin alone, or both agents at clinically relevant concentrations. Developmental endpoints, including embryo survival, hatching success, axial and craniofacial morphology, pericardial edema, spinal curvature, cardiac rhythm, and early organogenesis of the eye, heart, and somite structures, were systematically evaluated according to established zebrafish developmental toxicity guidelines. Results: Combination treatment with Nelmastobart and cisplatin induced the strongest PBMC-mediated cytotoxic effect, resulting in substantial reductions in spheroid volume and marked disruption of RFP signals compared to the monotherapies. These findings suggest that BTN1A1 blockade enhances immune recognition, increasing tumor susceptibility to chemotherapy. Zebrafish developmental toxicity testing revealed no significant abnormalities with either Nelmastobart alone or cisplatin alone at clinically relevant exposure ranges. Importantly, no additive or synergistic developmental toxicity was observed in the combination group. Conclusion: BTN1A1 inhibition by Nelmastobart significantly enhances cisplatin-driven immune-mediated cytotoxicity in A549 3D spheroid models while maintaining a strong safety margin in vivo. These data support the continued clinical advancement of Nelmastobart, both as a monotherapy and in combination with standard chemotherapeutic agents.