ABSTRACT: Tumor heterogeneity and cancer stem cell (CSC) plasticity present a significant challenge to the effective treatment of colorectal cancer (CRC). Several studies have established that Leucine-rich repeat containing, G protein-coupled Receptor 5 (LGR5) marks CSCs and is highly upregulated in CRC. Plasticity of LGR5+ CSCs has been shown to promote therapy resistance, tumor progression, and metastasis. CRC is also dependent on the epidermal growth factor receptor (EGFR) and EGFR-targeted antibodies such as cetuximab (CTX) are routinely used for the treatment of metastatic CRC. However, the clinical efficacy of EGFR-targeted treatment has been limited to a subset of patients that do not harbor KRAS mutations. Antibody-drug conjugates (ADCs) have experienced a recent surge in success within the past few years and several have now been approved for solid tumor indications. The objective of this project is to develop a novel bispecific ADC (BsADC) directed against LGR5 and EGFR and evaluate antitumor and antimetastatic efficacy in patient-derived models of CRC. We propose leveraging the dual-targeting capabilities of an LGR5-EGFR bispecific antibody combined with the ability of ADCs to exert potent drug effects irrespective of tumor mutational status, while minimizing systemic toxicity. In Aim 1, we will generate a lead LGR5-EGFR BsADC with high potency and specificity in vitro. In Aim 2, we will evaluate tolerability and therapeutic efficacy of the lead LGR5-EGFR BsADC in subcutaneous and orthotopic patient-derived tumor models of CRC. Results from this study will lead to the development of a unique dual-targeted ADC and determine if it can overcome therapy resistance due to tumor heterogeneity and CSC plasticity and prevent metastasis. Moreover, an LGR5-EGFR BsADC has the potential to treat other tumor types that express high levels of either tumor antigen.