Objective

Affimer® biotherapeutics are a new protein scaffold with great potential for the generation of new medicines. Based on the human protease inhibitor Stefin A, the scaffold is small (12 kDa), and lacks any post translational modifications such as disulphide bonds and glycosylation. Affimer proteins display two 9 amino acid peptide loops that can be randomized to bind to desired target proteins with high affinity and specificity in a similar manner to antibody fragments. We performed phage display selections against human PD-L1 using a synthetic naive library of a diversity of 10^11 Affimer clones. After two rounds of solution selections using biotinylated antigen approximately 20 % of clones that were picked were positive for binding to PD-L1, resulting in 38 unique sequences with affinities ranging from 31 nM down to 2.6 nM (Biacore analysis). Additional screening from third round output (70 % hit rate) increased the panel of Affimer proteins to 64 unique sequences in total. Clones were shown to compete with both human PD-1 and CD80 for binding to human PD-L1 in a competition ELISA. The top 8 Affimer antagonists also blocked PD-1 binding to PD-L1 in a Promega PD-1/PD-L1 blockade cell assay, measuring the TCR signaling through NFAT-mediated luciferase activity. This data demonstrates that high affinity Affimer therapeutics can be directly isolated from a diverse naïve library using phage display technology.