Evaluation of the efficacy of cell-penetrating monoclonal antibodies targeting intracellular p-NLRP3S295 in alleviating hepatotoxicant-induced NAFLD

NOD-like receptor protein 3 (NLRP3) is a key driver of hepatotoxicant-induced nonalcoholic fatty liver disease (NAFLD). Phosphorylation of NLRP3 at serine 295 (p-NLRP3^S295) is crucial for pyroptosis. Monoclonal antibodies (mAbs) have been designed to target extracellular molecules or cell membrane surface receptors and have achieved progress in NAFLD treatment. However, research on mAbs targeting intracellular biomarkers for NAFLD treatment remains limited. In this study, aflatoxin B1 (AFB1), lipopolysaccharide (LPS) combined with ATP, or palmitic acid (PA) were used to induce p-NLRP3^S295-dependent pyroptosis and inflammation mediated by lipotoxicity in hepatocytes in vitro. We generated a specific anti-p-NLRP3^S295 mAb (14C7) and internalized it into hepatocytes via an enhanced TAT-based intracellular delivery system (eTAT), which inhibited p-NLRP3^S295-dependent pyroptosis and inflammation in hepatocytes subjected to simulated lipotoxic injury and in the livers of NAFLD mice. The recombinant mAb@p-NLRP3^S295 expression system was constructed with 14C7. The intracellularly expressed recombinant monoclonal antibody (R-mAb) efficiently blocked p-NLRP3^S295-dependent pyroptosis and inflammation in hepatocytes exposed to hepatotoxicant through the proteasome degradation pathway mediated by tripartite motif-containing 40 (TRIM40). In conclusion, this study presents a novel approach for the targeted inhibition of p-NLRP3^S295 through intracellular recombinant mAbs, offering new insights into the treatment of hepatotoxicant-related NAFLD via specific intracellular targeting.