Peptide barcoding for high‐throughput functional evaluation of antibodies without immobilization

Background Display technologies such as phage display and yeast display can evaluate a large number of antibodies in a high‐throughput manner, and have been used to isolate various antibody medicines. In display technologies, the immobilization of antibodies on the cell surface is essential to establish genotype–phenotype linkages, but this immobilization can affect functions of antibodies. Here, we developed a novel methodology, “peptide barcoding”, for high‐throughput quantitative measurement of binding properties of antibodies without immobilization. In the scheme of “peptide barcoding”, unique peptide barcodes are fused with each antibody, and they represent genotype–phenotype linkages. A mixture of different peptide‐barcoded antibodies is reacted with antigens, and peptide barcodes of functional antibodies are identified by mass spectrometry after cleavage by specific proteases. This methodology will enable high‐throughput development of antibodies for experimental reagents, diagnostics, and pharmaceuticals. Methods Unique peptide barcodes were designed to have high detectability for mass spectrometry. Peptide‐barcoded nanobodies (anti‐CD4 or anti‐GFP) were produced by Pichia pastoris . A mixture of peptide‐barcoded nanobodies were reacted with antigen‐coated magnetic beads. Peptide barcodes of functional nanobodies were cleaved by a specific protease, enterokinase, and released peptides were identified by mass spectrometry. Precise dissociation constants of nanobodies were measured by BIACORE T200. Results & Discussion We designed peptide‐barcoded anti‐CD4 nanobody or anti‐GFP nanobody, and successfully produced them using P. pastoris . We measured the values of dissociation constants of the peptide‐barcoded nanobodies using surface plasmon resonance, and confirmed that addition of peptide barcodes didn’t affect affinities of these nanobodies. To demonstrate high‐throughput evaluation of free nanobodies, we produced peptide‐barcoded nanobody mutants, and mixed them with antigen‐coated magnetic beads. Released peptide barcodes from functional nanoboides were quantified by selected reaction monitoring, and we successfully ranked nanobody mutants in order of affinity. This methodology has potential scalability and could evaluate affinities of hundreds of antibodies in one shot [ 1, 2]. Support or Funding Information This research was supported by JST, CREST (grant number JPMJCR16G2), Japan.[1] Miyamoto et al, PLOS ONE 14 ( 4 ): e0215993 ( 2019 ) [2] Matsuzaki et al, submitted