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. The pro-antibody was efficiently activated by native proteases in aorta tissue extracts from ApoE⁻/⁻, but not from normal mice, and accumulated in aortic plaques in vivo with enhanced selectivity when compared to the unmodified antibody. Pro-antibody accumulation in aortic plaques was MMP-dependent, and significantly inhibited by a broad-spectrum MMP inhibitor. These results demonstrate that the activity of disease-associated proteases can be exploited to site-specifically target antibody activity in 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. Isolated antibody specific ligands exhibited varying specificity, revealing critical amino acid determinants for target recognition and nontarget avoidance in complex mixtures. Molecular specificity in the mixture was further enhanced by quantitative directed evolution, yielding a family of epitopes exhibiting improved specificities equivalent, or superior to, the native peptide antigen to which the antibody was raised. Specificity screening simultaneously favored affinity, yielding ligands with three-fold improved affinity relative to the parent epitope. Quantitative specificity screening will be useful to screen, evolve, and characterize the specificity of protein and peptide interactions for molecular recognition applications.

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. Furthermore, nanoparticles functionalized with the identified RBC-binding peptides exhibited nearly 100-fold increased RBC binding relative to nonfunctionalized particles in the presence of physiologically relevant concentrations of human serum albumin, indicating that peptides remained functional in the absence of the protein scaffold used for display. The RBC-binding peptides identified here provide new opportunities for sustained therapeutic delivery applications whereby nanoparticulate drug carriers can be attached to RBCs to achieve long-circulating carrier systems.