Different mechanisms are involved in the antibody mediated inhibition of ligand binding to the urokinase receptor: a study based on biosensor technology

Certain monoclonal antibodies are capable of inhibiting the biological bind ing reactions of their target proteins. At the molecular level, this type o f effect may be brought about by completely different mechanisms, such as c ompetition for common binding determinants, steric hindrance or interferenc e with conformational properties of the receptor critical for ligand bindin g. This distinction is central when employing the antibodies as tools in th e elucidation of the structure-function relationship of the protein in ques tion. We have studied the effect of monoclonal antibodies against the uroki nase plasminogen activator receptor (uPAR), a protein located on the surfac e of various types of malignant and normal cells which is involved in the d irection of proteolytic degradation reactions in the extracellular matrix. We show that surface plasmon resonance/biomolecular interaction analysis (B IA) can be employed as a highly useful tool to characterize the inhibitory mechanism of specific antagonist antibodies. Two inhibitory antibodies agai nst uPAR, mAb R3 and mAb R5, were shown to exhibit competitive and non-comp etitive inhibition, respectively, of ligand binding to the receptor. The fo rmer antibody efficiently blocked the receptor against subsequent ligand bi nding but was unable to promote the dissociation of a preformed receptor-li gand complex. The latter antibody was capable of binding the preformed comp lex, forming a transient trimolecular assembly, and promoting the dissociat ion of the uPA/uPAR complex. The continuous recording of binding and dissoc iation, obtained in BIA, is central in characterizing these phenomena. The identification of a non-competitive inhibitory mechanism against this recep tor reveals the presence of a determinant which influences the binding prop erties of a remote site in the molecular structure and which could be an im portant target for a putative synthetic antagonist. (C) 1999 Elsevier Scien ce B.V. All rights reserved.