As colorectal cancer remains a leading cause of cancer-related death, identifying therapeutic targets and approaches is essential to improve patient outcomes. The EGFR ligand epiregulin (EREG) is highly expressed in RAS wild-type (WT) and mutant colorectal cancer, with minimal expression in normal tissues, making it an attractive target for antibody–drug conjugate (ADC) development. In this study, we produced and purified an EREG mAb, H231, which had high specificity and affinity for human and mouse EREG. H231 also internalized to lysosomes, which is important for ADC payload release. ImmunoPET and ex vivo biodistribution studies showed significant tumor uptake of zirconium-89–labeled H231, with minimal uptake in normal tissues. H231 was conjugated to either cleavable dipeptide or tripeptide chemical linkers attached to the DNA-alkylating payload duocarmycin DM, and the cytotoxicity of EREG ADCs was assessed in a panel of colorectal cancer cell lines. EREG ADCs incorporating tripeptide linkers demonstrated the highest potency in EREG-expressing colorectal cancer cells irrespective of RAS mutations. Preclinical safety and efficacy studies showed that EREG ADCs were well tolerated, neutralized EGFR pathway activity, caused significant tumor growth inhibition or regression, and increased survival in colorectal cancer cell line and patient-derived xenograft models. These data suggest that EREG is a promising target for the development of ADCs for treating colorectal cancer and other cancer types that express high levels of EREG. Although the efficacy of clinically approved anti-EGFR mAbs is largely limited by RAS mutational status, EREG ADCs may show promise for both RAS mutant and WT patients, thus improving existing treatment options. Significance: EREG-targeting antibody-drug conjugates demonstrate acceptable safety and robust therapeutic efficacy in RAS mutant and wild-type colorectal cancer, suggesting their potential as an alternative to EGFR-targeted therapy to benefit a broader patient population.

Triple-negative breast cancer (TNBC) is the most aggressive subtype with high metastasis and mortality rates. Given the lack of actionable targets such as ER and HER2, TNBC still remains an unmet therapeutic challenge. Despite harboring high CDK4/6 expression levels, the efficacy of CDK4/6 inhibition in TNBC has been limited due to the emergence of resistance. The resistance to CDK4/6 inhibition is mainly mediated by RB1 inactivation. Since our aim is to overcome resistance to CDK4/6 inhibition, in this study, we primarily used the cell lines that do not express RB1. Following a screening for activated receptor tyrosine kinases (RTKs) upon CDK4/6 inhibition, we identified the TAM (Tyro3, Axl, and MerTK) RTKs as a crucial therapeutic vulnerability in TNBC. We show that targeting the TAM receptors with a novel inhibitor, sitravatinib, significantly sensitizes TNBC to CDK4/6 inhibitors. Upon prolonged HER2 inhibitor treatment, HER2+ breast cancers suppress HER2 expression, physiologically transforming into TNBC-like cells. We further show that the combined treatment is highly effective against drug-resistant HER2+ breast cancer as well. Following quantitative proteomics and RNA-seq data analysis, we extended our study into the immunophenotyping of TNBC. Given the roles of the TAM receptors in promoting the creation of an immunosuppressive tumor microenvironment (TME), we further demonstrate that the combination of CDK4/6 inhibitor abemaciclib and sitravatinib modifies the immune landscape of TNBC to favor immune checkpoint blockade. Overall, our study offers a novel and highly effective combination therapy against TNBC and potentially treatment-resistant HER2+ breast cancer that can be rapidly moved to the clinic.

Purpose: The high mortality associated with metastatic colorectal cancer (mCRC) illuminates an unmet need for innovative therapeutic modalities. Radiopharmaceutical therapy (RPT) offers a potent, molecular-scale approach for managing and treating cancers with distant micrometastases. However, its clinical use in mCRC remains an unrealized opportunity. We have therefore identified the transmembrane glycoprotein mucin 13 (MUC13) as a promising antigen for developing a targeted RPT and have undertaken preclinical evaluation of its potential by utilizing a monoclonal antibody tool representative of a future class of translatable therapeutics. Experimental Design: The immunoreactivity and transcriptome of patients with colorectal cancer (n = 72 primary, 100 liver metastases) were characterized using annotated clinical datasets. Preclinical assessment of MUC13 as an RPT target for mCRC was then performed in mice using a monoclonal MUC13-targeted antibody C14 labeled with either zirconium-89 for positron emission tomography (PET) measurement of mCRC-associated MUC13 density or terbium-161 for targeted RPT. Results: Strong MUC13 immunoreactivity was observed in ∼70% of mCRC and was inversely correlated with overall survival (P < 0.01). MUC13 levels were visualized by PET and agreed with immunohistochemically determined antigen presence. Furthermore, MUC13-targeted RPT exhibited in vivo proof-of-concept efficacy and enhanced survival in preclinical colorectal cancer models. Resulting imaging, therapeutic, and pathologic analyses elucidated relationships between target density, therapeutic outcome, and a potential genetic signature. Conclusions: MUC13-targeted RPT response was not only associated with radiopharmaceutical accumulation but also seemed to be balanced by DNA damage repair gene expression, suggesting a potential sensitivity signature that could complement a future clinical theranostic approach in MUC13-positive mCRC.

Characterization of DFO-H231 and 89Zr-DFO-H231 conjugates.

Cytotoxicity dose-response curves of EREG-targeting antibody-drug conjugates.

Quantification of surface expressed pro-EREG ligands for a panel of colorectal cancer cell lines.