Implementation of single B-cell screening to select antibodies in one day based on affinity and multiple binding properties

Purpose: The aim of this study was to establish a rapid and de-risked workflow enabling the selection and functional characterization of antibody-secreting plasma cells based on function, within a single day to streamline therapeutic antibody discovery. Methods: BALB/c and transgenic mice carrying human VH/Vκ repertoires were immunized with the recombinant extracellular domain of a tumor-associated immunomodulatory protein. High-titer animals were screened on the Bruker Beacon microfluidic platform, which isolates single plasma cells in nanowells and enables multiplexed assessment of their secreted antibodies. First, beads coated with the ligand and a soluble target conjugated to a fluorochrome allowed simultaneous evaluation of binding affinity and neutralizing capacity. In a second step, beads coated with a structural analog of the target were used to assess the cross-reactivity. Plasma cells producing antibodies of interest were exported for VH/VL amplification, sequencing, and cloning into a mammalian expression vector. Recombinant antibodies were produced and purified for in-depth characterization. Binding affinity and functional blocking were confirmed by biolayer interferometry, while internalization kinetics were quantified using a lanthanide-based pH-sensitive probe. In-silico sequence analysis and structural modeling were applied to identify potential liabilities and exclude clones at risk of poor developability. Results: The single-day screening workflow enabled high resolution discrimination of plasma cells based on fine differences in affinity and functional binding properties. 215 clones displayed high specificity for the target with no detectable cross-reactivity to analogs and effectively blocked target-ligand interactions. BLI confirmed the accuracy of the phenotypes identified on the microfluidic platform. Internalization assays identified a subset of antibodies with rapid uptake properties compatible with ADC development and computational filtering further refined the panel of antibodies of therapeutic interest by discarding clones with predicted structural liabilities or developability risks. Conclusions: This integrated and accelerated workflow provides high-resolution functional screening of antibody-secreting plasma cells in a single experiment. By combining affinity ranking, specificity and cross-reactivity assessment, neutralization, internalization profiling, and developability evaluation, our antibody discovery process efficiently yields diverse and high-value therapeutic antibody leads with strong potential for development as immunomodulators or ADC candidates.