Facile Method for the Site‐Specific, Covalent Attachment of Full‐Length IgG onto Nanoparticles

Abstract

Antibodies, most commonly IgGs, have been widely used as targeting ligands in research and therapeutic applications due to their wide array of targets, high specificity and proven efficacy. Many of these applications require antibodies to be conjugated onto surfaces (e.g. nanoparticles and microplates); however, most conventional bioconjugation techniques exhibit low crosslinking efficiencies, reduced functionality due to non-site-specific labeling and random surface orientation, and/or require protein engineering (e.g. cysteine handles), which can be technically challenging. To overcome these limitations, we have recombinantly expressed Protein Z, which binds the Fc region of IgG, with an UV active non-natural amino acid benzoylphenyalanine (BPA) within its binding domain. Upon exposure to long wavelength UV light, the BPA is activated and forms a covalent link between the Protein Z and the bound Fc region of IgG. This technology was combined with expressed protein ligation (EPL), which allowed for the introduction of a fluorophore and click chemistry-compatible azide group onto the C-terminus of Protein Z during the recombinant protein purification step. This enabled crosslinked-Protein Z-IgG complexes to be efficiently and site-specifically attached to aza-dibenzycyclooctyne-modified nanoparticles, via copper-free click chemistry.

Optimization of Photoactive Protein Z for Fast and Efficient Site-Specific Conjugation of Native IgG

“…25−28 Derived from the IgG-binding B domain of Protein A, Protein Z exhibits nanomolar affinity for a binding site in the CH2-CH3 region of most IgG isotypes. Upon binding to IgG and exposure to long wavelength UV (365 nm), the engineered Protein Z constructs can be site-specifically and covalently conjugated to the Fc region of the IgGs.…”

Bioconjugation of therapeutic proteins and enzymes using the expanded set of genetically encoded amino acids

“…Since it does not require a Cu catalyst, SPAAC is quite biocompatible allowing both in vivo and in vitro bioconjugation without deleterious effects (Beatty et al, 2010;Hui et al, 2014;. To overcome a relatively slow reaction rate of a simple cyclooctyne (0.0024 M À1 s À1 ), a benzene ring or a cyclopropane was fused to yield dibenzoazacyclooctyne (DIBAC) or bicyclononyne (BCN), each of which exhibits a rate constant of 0.31 and 0.14 M À1 s À1 , respectively (Dommerholt et al, 2010;Jewett et al, 2010), and is most commonly employed in SPAAC.…”

Nanogel carrier design for targeted drug delivery

“…In fact, a variety of enzymes have already been employed for the site-specific modification of proteins for the preparation of protein-drug conjugates 162 . To enable the attachment of full-length antibodies to a nanoparticle surface, EPL-click chemistry was recently combined with non-natural amino acid incorporation to produce an antibody-binding protein, Protein Z, with a photocrosslinker in the Fc-biinding domain and an azide at the c-terminus (Figure 2B) 163 . This allowed Protein Z to be covalently linked to IgG upon photoactivation and for the subsequent site-specific attachment of IgG to alkyne-modified nanoparticles.…”