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, a detailed structure-activity relationship analysis of Peptide 3 is reported to better understand the importance of each individual amino acid for the binding to calprotectin. Moreover, two different approaches have been followed here, 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 K_D of 39 ± 5 nM measured by surface plasmon resonance spectroscopy, which is a five-fold improvement compared to the previously reported Peptide 3.