Engineered α9-nAChR-targeting antibodies attenuate nicotine-enhanced triple-negative breast cancer progression and exhibit favorable short-term tolerability in cynomolgus macaques.

Cigarette smoking and nicotine exposure promote breast cancer progression and poor outcomes by aberrantly activating nicotinic acetylcholine receptors (nAChRs), with α9-nAChR serving as a key oncogenic driver, especially in triple-negative breast cancer (TNBC). However, effective therapeutic strategies that directly target α9-nAChR remain limited. In this study, we engineered an antigen comprising the extracellular loops of α9-nAChR (α9CLP) and used mouse immunization followed by phage display to isolate an α9CLP-specific single-chain variable fragment (scFv) antibody, mCLPS1. The dominant clone mCLPS1 showed specific binding to α9-nAChR and was further engineered into an Fc-fusion antibody (mCLPS1-Fc) and an anti-mPEG bi-specific antibody (mCLPS1/anti-mPEG BsAb) to improve its affinity and therapeutic potential. The mCLPS1/anti-mPEG BsAb significantly enhanced the therapeutic efficacy of mPEGylated liposomal doxorubicin (LD) in targeting α9-nAChR-positive breast cancer. In TNBC patient-derived xenograft (PDX) models, mCLPS1-Fc markedly inhibited tumor growth, mitigated nicotine-induced tumor acceleration, and enhanced docetaxel's antitumor efficacy. Preliminary tolerability assessments of mCLPS1/anti-mPEG BsAb and mCLPS1 Fc antibody administration in non-human primates, cynomolgus macaque (Macaca fascicularis). did not reveal neuromuscular toxicity, myasthenia gravis-like symptoms, hematologic abnormalities, or overt organ injury within the observation period, supporting a favorable short-term tolerability profile under the tested conditions. In summary, this study identifies α9-nAChR as a key molecular target in nicotine-induced tumor progression and highlights α9-nAChR-specific antibodies as promising targeted therapeutic candidates for TNBC, particularly in patients with a history of nicotine exposure.