表皮调节素与双调蛋白作为结直肠癌的双重靶点

Advanced-stage metastatic colorectal cancer is associated with high mortality. Current therapies have shown limited survival benefit and are often constrained by dose-limiting toxicity or drug resistance, including constitutively activating mutations (i.e., KRAS ) limiting epidermal growth factor receptor (EGFR)-targeted therapy efficacy. Thus, novel targeted therapeutic strategies are needed. Antibody-drug conjugates (ADCs) act as guided missiles where a monoclonal antibody (mAb) conjugated to cytotoxic payloads binds its tumor-specific target, internalizes, and traffics to lysosomes for payload release, inducing tumor cell death while sparing normal tissues. We previously developed ADCs targeting the EGFR ligand epiregulin (EREG), which is overexpressed in KRAS wildtype and mutant (MUT) colorectal tumors, that were effective at inhibiting tumor growth in CRC cell line- and patient-derived xenograft models. Yet, tumor regrowth was observed upon treatment termination, and residual tumors showed target downregulation as a potential mechanism of ADC resistance. This indicates EREG ADC monotherapy may not be sufficient to eliminate colorectal tumors. Co-targeting of additional cell-surface antigens may enhance intratumoral payload delivery and address potential resistance mechanisms. Previous studies and our analyses of RNA-seq expression data from The Cancer Genome Atlas (TCGA) colorectal adenocarcinoma cohort show EREG is often co-overexpressed with the related EGFR ligand amphiregulin (AREG) in CRC patients, with their expression predominant of all seven EGFR ligands, suggesting potential functional importance. Therefore, we hypothesize that targeting EREG and AREG with ADCs simultaneously will inhibit tumor growth and enhance ADC efficacy compared to single-target ADC monotherapy. We generated EREG knockout (KO), KRAS -MUT CRC cell lines via lentiviral transduction using CRISPR-Cas9, which show significantly reduced proliferation and tumor growth compared to the parental and vector cells. These results indicate that EREG actively promotes CRC growth, despite oncogenic mutations downstream of EGFR. We are also generating AREG KO and EREG/AREG-double KOs using CRISPR-Cas9 for characterization. Additionally, we have cloned, produced, and purified human AREG-targeted mAbs for the development of novel AREG-targeted ADCs. AREG overexpression cell lines were generated for mAb validation. Our data demonstrate the AREG mAbs bind AREG with high specificity and promote internalization and lysosomal trafficking in CRC cells. These results suggest AREG to be a suitable therapeutic target in CRC, and we are currently in the process of generating AREG-targeted ADCs. Future work will test whether co-targeting EREG and AREG provides additional therapeutic benefit.