Work

Unique monoclonal antibody allowing dual recognition of cell surface antigens via binding short 2’-O-methyl RNA oligonucleotide duplexes 4238

Year: 2025

Type: article

Abstract: Description We have produced a unique antibody binding short 2’-O-methyl RNA duplexes, which enables novel proximity-based strategies with living cells. An scFv phage library was initially screened for binding to click reaction duplex targets comprised of specific 2’-O-methyl RNA oligonucleotides. A unique scFv was discovered, specifically directed towards short 2’-O-methyl RNA duplexes without binding of single strands. This scFv was subsequently converted to a human IgG1 antibody (‘DS5’), whose novel properties suggested applications based on single-strand molecular proximity. Initially, we enabled cell surface placement of biotins via azide metabolic labeling. Subsequent treatment with tetravalent neutravidin [NAV]) carrying sub-saturating levels of biotinylated 2’-O-methyl RNA target duplexes allowed strong DS5 flow signals, demonstrating cell-surface recognition. We then used biotinylated anti-EGFR antibody linked with biotinylated 2’-O-methyl RNA single strands, via NAV protein as an adaptor. Flow cytometry showed that DS5 binding was only obtained when combinations of distinct antibodies carrying top and bottom target strands were applied sequentially to EGFR-positive cells. These results show that proximity-based surface annealing of DS5 target single strands can define cell populations via recognition of surface antigens on living cells. If DS5 is derivatized with either a fluorescent moiety or a cytotoxic drug, DS5 could be used in diagnostic or therapeutic tumor targeting. Funding Sources Supported in part by contract to HaplomeRx LLC and Royalties from Takeda Pharmaceuticals paid via license to Massachusetts General Hospital Topic Categories Technological Innovations in Immunology (TECH)

Source: The Journal of Immunology

DOI: 10.1093/jimmun/vkaf283.1944