Llama mRNA-LNP immunization enables rapid isolation of target specific nanobodies using cell-based panning

Abstract Background As a clinical vaccine platform, lipid nanoparticle (LNP)-formulated mRNA has demonstrated potent and broad antibody responses, prompting speculation about its potential for antibody discovery. Membrane proteins remain among the most challenging targets for antibody development, highlighting the urgent need for technologies that preserve their native conformation during immunization and screening. Nanobodies (VHHs), the single-domain fragments of heavy-chain-only antibodies (HCAbs) naturally found in camelids, offer unique advantages over conventional antibodies, including high solubility, stability, and the ability to access cryptic or membrane-proximal epitopes. Methods Here, we report the first demonstration of target specific VHH discovery in a llama using an mRNA–LNP immunization platform. A llama was immunized with LNPs encapsulating mRNA encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, a well-characterized model antigen. The resulting immune VHH library was screened using a cell-based panning strategy that maintains antigen conformation and enables recovery of binders recognizing native epitopes. Results This approach yielded multiple high-affinity spike-specific VHHs, including two clones that effectively blocked the spike–ACE2 interaction in a surrogate cell-based assay. Conclusions These findings demonstrate the feasibility of mRNA-LNP immunization as a rapid and efficient method for isolating target specific, functionally promising VHHs from camelids. When combined with conformationally relevant screening, this platform may provide a versatile and accelerated route for generating high-quality VHHs against complex or recalcitrant targets, particularly membrane proteins, thereby expanding the potential of VHH-based therapeutics and diagnostics.