Abstract 5448: Single domain antibodies, EGFR and cancer: a small solution to a big problem

Abstract The primary purpose of our work is to identify high-affinity, efficacious single-domain antibodies (sdAbs, also known as nanobodies) targeting significant molecular drivers of cancer. The epidermal growth factor receptor (EGFR) family play a prolific role in the development and progression of numerous cancers. The clinical importance of these receptors is exemplified by the fact that 14 small molecule tyrosine kinase inhibitors as well as several monoclonal antibodies (mAbs) have received approval for therapeutic intervention. While these medications have undoubtedly had significant, positive impacts on patient outcomes, loss of efficacy due to mutagenic changes is a persistent issue. Unlike mAbs, sdAbs bind to a substantially smaller epitope, reducing the chances of mutation-induced loss of activity. Additionally, the distinct structure of sdAbs imparts them with highly desirable drug characteristics, such as exceptional tissue penetration, solubility, stability, and binding affinity. Considering these favorable traits, it is no surprise that the pharmaceutical industry is now allocating significant resources towards the development of sdAb-based drugs, with over a dozen in clinical trials, and 2 FDA approvals since 2019. While several anti-EGFR sdAbs have been described, at the time of this abstract submission, we were only aware of one sdAb-based drug targeting an EGFR family member under clinical investigation (I-GMIB-Anti-HER2-VHH1, Precirix). To address this currently unmet need, we first generated a novel sdAb library from 24 naïve crias (immature alpacas). Utilizing an iterative process of magnetic bead panning, mammalian cell display in a high EGFR-expressing cell line, and flow cytometric sorting, we were able to identify several candidates of interest via sequence analysis. Individual evaluation in our reporter cells showed that our candidates were capable of suppressing EGFR signaling, as determined by our fluorometric reporter. Following this validation, we assessed the impact of sdAb-mediated signaling regulation on classical cancer hallmarks. Data from in vitro migration and proliferation assays demonstrated that sdAb-antagonism caused significant inhibition of both hallmark properties. These findings were further supported by differential gene expression data, which highlighted significant changes in the expression of well-established regulators of these processes. Taken together, these data demonstrate our sdAb development pipeline can accurately identify multiple candidates capable of inhibiting crucial cancer hallmarks via EGFR antagonism. Ultimately, our goal is to utilize our process across a spectrum of cancers in order to provide patients with a new generation of high-efficacy, low-side effect therapeutics that more precisely and effectively target the primary molecular driver of their malignancy. Citation Format: Adam T. Nelczyk, Kimberly J. Anderson, Nicholas Wu. Single domain antibodies, EGFR and cancer: a small solution to a big problem [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5448.