Application of peptide barcoding to obtain high-affinity anti-PD-1 nanobodies.

Cancer treatment has been revolutionized by immune checkpoint inhibitors, which regulate immune cell function by blocking the interactions between immune checkpoint molecules and their ligands. The interaction between programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) is a target for immune checkpoint inhibitors. Nanobodies, which are recombinant variable domains of heavy-chain-only antibodies, can replace existing immune checkpoint inhibitors, such as anti-PD-1 or anti-PD-L1 conventional antibodies. However, the screening process for high-affinity nanobodies is laborious and time-consuming. Here, we identified high-affinity anti-PD-1 nanobodies using peptide barcoding, which enabled reliable and efficient screening by distinguishing each nanobody with a peptide barcode that was genetically appended to each nanobody. We prepared a peptide-barcoded nanobody (PBNb) library with thousands of variants. Three high-affinity PBNbs were identified from the PBNb library by quantifying the peptide barcodes derived from high-affinity PBNbs. Furthermore, these three PBNbs neutralized the interaction between PD-1 and PD-L1. Our results demonstrate the utility of peptide barcoding and the resulting nanobodies can be used as experimental tools and antitumor agents.

Peptide barcoding for one-pot evaluation of sequence-function relationships of nanobodies.

Optimisation of protein binders relies on laborious screening processes. Investigation of sequence-function relationships of protein binders is particularly slow, since mutants are purified and evaluated individually. Here we developed peptide barcoding, a high-throughput approach for accurate investigation of sequence-function relationships of hundreds of protein binders at once. Our approach is based on combining the generation of a mutagenised nanobody library fused with unique peptide barcodes, the formation of nanobody-antigen complexes at different ratios, their fine fractionation by size-exclusion chromatography and quantification of peptide barcodes by targeted proteomics. Applying peptide barcoding to an anti-GFP nanobody as a model, we successfully identified residues important for the binding affinity of anti-GFP nanobody at once. Peptide barcoding discriminated subtle changes in KD at the order of nM to sub-nM. Therefore, peptide barcoding is a powerful tool for engineering protein binders, enabling reliable one-pot evaluation of sequence-function relationships.