Kinetic Exclusion Analysis (KinExa) of Avidity Enhancement of a Multi-Valent Adnectin Binding to Clustered Receptors on CHO Cells

Multivalency is a strategy used in nature to gain avidity. A variety of cell surface receptors are known to cluster at the cell surface via protein or lipid (raft) interactions. Analytical methods to measure the effect of avidity as it exists at a cell surface are challenging. Kinetic Exclusion Analysis (KinExA) is a sensitive immunodetection analytical technique for measuring solution affinity. AdnectinsTM are a proprietary type of targeted biologic derived from human fibronectin. Adnectin-A was selected with mRNA display (PROfusionTM) to bind specifically to cell surface receptor X, and was formated as a multivalent fusion protein. To determine the affinity and avidity of Adnectin-A for receptor X clustered on cells, both the human and cynomolgus monkey homoloques of receptor X were transfected into CHO cells. The CHO transfectants were characterized by FACS and then scaled up for KinExA binding studies. KinExA has been used to measure binding affinity of Adnectin-A to the cell surface expressed receptor X to measure the effect of avidity of the multivalent adnectin binding to receptor clusters. As controls for the functional activity of the Adnectin-A and the affinity of the monovalent interaction, the same KinExa assay was used, substituting the soluble receptor X extracellular domain for the transfected CHO cells. The binding avidity measured by KinExA for CHO expressed receptor is 14 pM for both species of receptor X. However, the affinity of Adnectin-A for monovalent soluble Receptor X was quite different between the species suggesting that avidity due to receptor clustering equilizes the functional avidity at the cell surface.