Site-specific targeting of antibody activity in vivo mediated by disease-associated proteases.

As a general strategy to selectively target antibody activity in vivo, a molecular architecture was designed to render binding activity dependent upon proteases in disease tissues. A protease-activated antibody (pro-antibody) targeting vascular cell adhesion molecule 1 (VCAM-1), a marker of atherosclerotic plaques, was constructed by tethering a binding site-masking peptide to the antibody via a matrix metalloprotease (MMP) susceptible linker. Pro-antibody activation in vitro by MMP-1 yielded a 200-fold increase in binding affinity and restored anti-VCAM-1 binding in tissue sections from ApoE⁻/⁻ mice ex vivo.

Quantitative specificity-based display library screening identifies determinants of antibody-epitope binding specificity.

Despite the critical importance of molecular specificity in bimolecular systems, in vitro display technologies have been applied extensively for affinity maturation of peptides and antibodies without explicitly measuring the specificity of the desired interaction. We devised a general strategy to measure, screen, and evolve specificity of protein ligand interactions analogous to widely used affinity maturation strategies. The specificity of binding to target and nontarget antibodies labeled with spectrally distinct fluorophores was measured simultaneously in protein mixtures via multiparameter flow cytometry, thereby enabling screening for high target antibody specificity.

Identification of peptide ligands facilitating nanoparticle attachment to erythrocytes.

Peptide ligands capable of mediating nanoparticle adhesion to human red blood cells (RBCs) were identified from a large bacterial display peptide library. Peptides were displayed on the surface of fluorescent Escherichia coli, enabling quantitative measurement of RBC binding and high-throughput screening using fluorescence-activated cell sorting. One of the isolated clones remained attached to RBCs under high-shear stresses equivalent to those encountered in vivo.