Targeting MET-LGR5 crosstalk using an antibody-drug conjugate combination with diverse payloads to overcome adaptive resistance in colorectal cancer

Off-target side effects and drug resistance often hamper the therapeutic benefits of existing anti-cancer therapies against colorectal cancer (CRC), the second deadliest malignancy worldwide. CRCs contain tumor-initiating cancer stem-like cells (CSCs) that survive toxic drugs and give rise to metastatic disease. Antibody-drug conjugates (ADCs) are a revolutionary class of therapeutics that leverage monoclonal antibody (mAb) specificity to deliver cytotoxic payloads to tumor cells. We generated ADCs targeting leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a bona fide CSC biomarker frequently overexpressed in CRCs. Our LGR5 ADC exhibited high potency in CRC xenograft models with minimal toxicity. However, it failed to prevent tumor relapse after treatment cessation. Interestingly, we found that recurrent tumors evade LGR5 ADC-mediated elimination by transiently converting into an LGR5-negative (LGR5 - ) state, accompanied by concomitant MET upregulation and activation of the downstream pathway. MET is a receptor tyrosine kinase frequently upregulated in CRCs and promotes metastatic progression. We identified that the activation of STAT3, a downstream effector protein in the MET pathway, undermines LGR5 ADC efficacy in CRC cells. To eliminate drug-resistant LGR5 - cells, we generated MET-targeted ADCs by attaching a highly selective MET mAb (ABT700) backbone to the DNA-crosslinking payload pyrrolobenzodiazepine (PBD) via site-specific conjugation. This MET ADC (ABT700-PBD) demonstrated dose-dependent cytotoxicity in CRC cells. Furthermore, ABT700-PBD did not affect CRC cells with genetically induced MET ablation, demonstrating its specificity. Safety studies in immunocompetent mice showed that ABT700-PBD exhibited a favorable safety profile. Moreover, in CRC xenograft models, ABT700-PBD induced marked tumor regression. However, in the residual tumors following dose-dependent ABT700-PBD monotherapy, LGR5 protein expression was upregulated. These observations suggest that the MET-LGR5 crosstalk may be a potential driver of adaptive resistance in CRC. Cytotoxicity assays testing ABT700-PBD and our previously reported camptothecin-derived (CPT2) LGR5-targeted ADC (8E11-CPT2) demonstrated synergistic cell-killing activity in vitro. Combination treatment with ABT700-PBD and 8E11-CPT2 in CRC patient-derived xenograft models extended survival compared to single-agent ADCs. Collectively, our findings support MET and LGR5 ADC combination therapy as a novel treatment strategy to overcome adaptive resistance-driven relapse in CRC.