Reversible blocking of antibodies using bivalent peptide–DNA conjugates allows protease-activatable targeting

Abstract

Antibody-based molecular recognition plays a dominant role in the life sciences ranging from applications in diagnostics and molecular imaging to targeted drug delivery and therapy. Here we report a generic approach to introduce protease sensitivity into antibody-based targeting by taking advantage of the intrinsic ability of antibodies to engage in multivalent interactions. Bivalent peptide ligands with dsDNA as a rigid linker were shown to effectively bridge the relatively large distance between the two antigen binding sites within the same antibody, yielding exclusively the cyclic 1 : 1 antibody–ligand complex. Size exclusion chromatography and small angle X-scattering were used to study the types of complexes formed between a model antibody and peptide–dsDNA conjugates displaying 1 or 2 peptide ligands and different linker lengths. Competitive binding assays using fluorescence anisotropy revealed that the interaction between bivalent peptide–dsDNA conjugate and antibody is 500-fold stronger than that of the monovalent peptide, allowing effective blocking of the antigen binding sites in a non-covalent manner. Cleavage of the linker between the peptide epitope and the DNA by matrix metalloprotease 2 disables this strong bivalent interaction and was shown to effectively restore the binding activity of the antibody in an in vitro binding assay. The approach presented here is broadly applicable, because it takes advantage of the Y-shaped multivalent presentation of antigen binding sites common to all antibodies and could be extended to control antibody activity by other input signals.