Structure‐affinity relationship analysis and affinity maturation of a calprotectin‐binding peptide

Peptide 3 is an 18‐amino acid linear peptide binding with sub‐micromolar affinity to the inflammation marker calprotectin. Its application in point‐of‐care diagnostic assays has shown promising results, yet improving its affinity for calprotectin could facilitate the development of sensitive and robust assays. Herein we report a detailed structure‐activity relationship analysis of Peptide 3 to better understand the importance of each individual amino acid for the binding to calprotectin. Moreover, we have followed two different approaches, one based on prolonging the peptide with random sequences and phage display selection, and one on screening chemically synthesized peptide variants containing non‐canonical amino acids, to increase its binding affinity. Combining several mutations that enhance affinity by small factors yielded Peptide 4 binding human calprotectin with a KD of 39 ± 5 nM measured by surface plasmon resonance spectroscopy, which is a 5‐fold improvement compared to the previously reported Peptide 3.