Generation of Clinical Diagnostic Antibodies Against Common Lymphatic Endothelial and Vascular Endothelial Receptor 1

Abstract

Background: Common lymphatic endothelial and vascular endothelial receptor 1 (CLEVER-1) is a scavenger glycoprotein receptor expressed on the surface of a subset of immunosuppressive macrophages. CLEVER-1 promotes tumor growth and spread in cancer. Immunotherapeutic blockade of CLEVER-1 delays tumor growth by activating cytotoxic CD8+ T-cells. Bexmarilimab, a novel anti-CLEVER-1 antibody, has shown promising efficacy and safety in phase II clinical trials. This study aimed to understand the impact of bexmarilimab dosing on blood CLEVER-1 levels by developing a companion diagnostic quantitative time-resolved fluorescence immunoassay (TRFIA) using novel non-competitive antibody fragments. Methods: The University of Turku Fab phage display libraries were panned against the in-house generated recombinant CLEVER-1 protein. The non-competitive binding of the newly discovered Fabs to recombinant human CLEVER-1 protein was explored using TRFIA, to confirm their non-competitive binding to bexmarilimab epitope. The best antibody fragments were sequenced and affinity-measured with biolayer interferometry. They were then used for validation in a TRFIA to measure the concentration and target engagement of CLEVER-1 protein in human plasma. Results: After three rounds of Fab phage library panning, considerable enrichment of the libraries was observed with a specific CLEVER-1 binding to background ratio of 86. Followed by screening for the best Fabs, six clones demonstrated over 150 signal-to-background ratio in specific binding to CLEVER-1. After sequencing, the fragments were identified as having different CDR regions. Bexmarilimab epitope blocking did not significantly affect binding of the Fabs to CLEVER-1, with an average Fab binding decrease of 9%. The dissociation constant of the best Fabs ranged from 2.1 nM to 16.5 nM. The results from the final validation TRFIA on healthy serum samples were in line with the CLEVER-1 normal range in serum. Conclusion: Six unique Fab antibody fragments were selected against CLEVER-1, which bound to non-competitive epitopes. Using the novel fragments, a TRFIA was developed to measure the CLEVER-1 concentration in cancer patients without interference from bexmarilimab treatment.