Development of an anti-CD171 (L1CAM) antibody-drug conjugate with high avidity and strong activity in resistant tumor models

Background: CD171 (L1CAM) is a cell-adhesion molecule implicated in epithelial-mesenchymal transition (EMT), receptor tyrosine kinase (RTK) activation, and neuroendocrine differentiation across various solid tumors. Spatial transcriptomic profiling of lung and colorectal cancers revealed enrichment of CD171 in EMT- and neuroendocrine-like tumor microenvironments (TMEs), suggesting its involvement in metastasis and therapeutic resistance. Ab612 is a novel anti-CD171 monoclonal antibody developed by APITBIO that demonstrates a favorable developability profile. GLP toxicology studies and GMP manufacturing of Ab612 were completed, providing a robust foundation and enabling accelerated ADC development. Methods: We characterized Ab612 to evaluate its biochemical stability, binding kinetics, internalization, and antitumor efficacy. Comparative analyses were performed against a benchmarking anti-CD171 antibody. Binding affinity and avidity were measured under both physiological and acidic conditions using surface plasmon resonance (SPR) and flow cytometry, while internalization efficiency was assessed via live-cell imaging. Ab612 was conjugated to MMAE (DAR2) and Exatecan (DAR4) using AbTis's proprietary linker, Abclick® Pro, designed to preserve antibody-FcRn interaction, and thereby support extended half-life. These ADCs were evaluated for in vivo efficacy in trastuzumab-resistant breast (JIMT-1) and TKI-resistant lung (Calu-6) xenograft models. Results: Ab612 maintained strong binding under acidic conditions and demonstrated approximately four-fold higher kinetic affinity relative to internal reference values in SPR assays. In CD171-high ovarian (SNU-840) and breast (JIMT-1) cancer cells, flow cytometry analyses showed 22-23% higher avidity, and live-cell imaging confirmed more than 15% increase in internalization efficiency. Ab612-Abclick® Pro-MMAE and Ab612-Abclick® Pro-Exatecan demonstrated high stability in human plasma. In vivo , Ab612-Abclick® Pro-MMAE achieved near-complete tumor regression at ≥3 mpk (minimum effective dose = 3 mpk) and 100% complete responses at 10 mpk in JIMT-1 xenografts without body-weight loss. Ab612-Abclick® Pro-Exatecan induced >100% tumor-growth inhibition in KRAS/TP53-mutant, TKI-resistant Calu-6 models with favorable tolerability. Conclusion: Ab612-ADCs,generated by conjugation of MMAE or Exatecan to the anti-CD171 monoclonal antibody Ab612 with the high-affinity binding within acidic TMEs using the Abclick® Pro linker, demonstrated highly potent preclinical antitumor efficacy at low effective doses. These findings highlight Ab612-ADCs as a promising therapeutic candidate for CD171-expressing refractory solid tumors characterized by EMT and neuroendocrine phenotypes, offering a potential strategy to overcome resistance in aggressive cancer subtypes.