ABSTRACT: Colorectal cancer stem cells (CSCs) are believed to mediate therapeutic resistance and relapse of residual disease due to their increased capacity for survival and ability to self-renew and differentiate into heterogeneous lineages of tumor cells. Recent therapeutic efforts have been focused on the targeting of CSCs to improve treatment efficacy. However, due to the plastic nature of CSCs and tumor heterogeneity, it is becoming more apparent that in order to successfully eliminate colorectal cancer (CRC), treatment may require a dual- or multi- targeted approach. Several reports have established that LGR5 (Leucine-rich repeat containing, G protein- coupled Receptor 5) is highly upregulated in CRC and marks highly plastic CSCs. The dynamic interconversion of LGR5-positive CSCs to LGR5-negative cancer cells has been shown to be essential during tumor progression and metastasis. We and others generated LGR5-targeted antibody-drug conjugates (ADCs) that were highly effective in eliminating LGR5-positive colorectal tumors without major adverse effects, however a fraction of tumors eventually relapsed. These findings suggest that targeting LGR5-positive CSCs alone may not be sufficient to eliminate colorectal tumors due to their plasticity. Epiregulin (EREG) is a cell surface expressed, EGFR ligand that is upregulated in CRC and highly expressed on both LGR5-postive CSCs and LGR5-negative cancer cells. EREG was also shown to drive colitis-associated CRC and be predictive of CRC-associated liver metastasis. Interesting, EGFR-targeted therapies have been shown to increase LGR5 expression in CRC models and LGR5 knockdown in CRC cells increased EREG levels. Thus, therapeutic targeting of both LGR5 and EREG/EGFR may be a more effective strategy to eliminate LGR5-positive CSCs and overcome plasticity. In Aim 1, we will generate anti-EREG ADCs and evaluate safety and therapeutic efficacy against multiple CRC models. In Aim 2, we will determine if ADC combination treatment can improve therapeutic response and overcome cancer cell plasticity. We will characterize different EREG monoclonal antibodies and screen different linker-payloads to develop an optimal anti-EREG ADC with high specificity, affinity, and potency. Anti-EREG ADCs will be tested as a monotherapy and in combination with anti-LGR5 ADCs linked to the same or different payloads against patient-derived xenograft models of CRC. ADCs will also be evaluated in combination with other EGFR-targeted therapies. This project will lead to the generation of unique ADCs and an innovative ADC combination therapy to potentially overcome CSC plasticity and resistance for the improved treatment of CRC.

PROJECT NARRATIVE: Metastasis and relapse of residual disease continue to be leading causes of colorectal cancer-related deaths. Treatment failure has been attributed to the presence and plasticity of cancer stem cells, or their ability to reversibly interchange between distinct cell types to avoid and survive therapy. This proposed project aims to generate unique antibody-drug conjugates and evaluate a novel combination therapy to target plasticity for the improved treatment of colorectal cancer.