Non-Invasive Theranostics for Endometriosis 2259283

Abstract Introduction Endometriosis (EM) presents significant clinical challenges due to associated pain, infertility, presentation and pathophysiology. There is an urgent unmet need for improved diagnostics and therapeutic approaches to EM. Here, we report the identification of EvA1C as a potential biomarker displayed on the luminal surface of endothelial cells (ECs) in EM lesions. Targeting of EVA1C by engineered nanobodies (NAbs) could have therapeutic and/or diagnostic utility. Methods Public single-cell RNAseq data of human EM were analyzed bioinformatically and compared to a comprehensive atlas of transcriptional profiles of EC from 20 normal tissues. EvA1C mRNA was robustly expressed in ECs in EM, but not in other tissues, including uterus. We screened a yeast display library of camelid NAbs to isolate clones against the EvA1C ectodomain. By alternating between human and murine EVA1C as a bait followed by a single cell sorting, we screened for NAbs recognizing EVA1C in both species. Candidate subclones were expressed in E. coli and assessed for their antigen binding properties by ELISA, flow cytometry and confocal microscopy. Results NAb clones were identified that recognize the ectodomain of human and mouse EVA1C with similar binding affinity. Immunohistochemistry confirmed at the protein level preferential endothelial EvA1C expression in ectopic EM lesions but not in eutopic endometrium. Further experiments will assess the utility of anti-EvA1C NAbs for non-invasive diagnosis and therapy of EM. Conclusion We developed NAbs that recognize EVA1C, a candidate microvascular target in EM. In vivo imaging of EvA1C expression after IV injection of NAbs carrying a radiotracer or contrast agent may be useful as a biomarker to enable the diagnosis, localization and monitoring of EM by routine medical imaging. Ultimately, NAb mediated targeting of endothelial EVA1C may also allow delivery of a therapeutic payload to reduce or ablate EM lesions. Funding Source Q-FASTR Pilot grant Topic Categories Immune Mechanisms of Human Disease (HUM)