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Updated: Apr 19, 2026
Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction
Published on: September 14, 2018
Particle generation, functionalization and sortase A-mediated modification with targeting of single-chain antibodies
Christoph E Hagemeyer1, Karen Alt2, Angus P R Johnston3
11] Vascular Biotechnology Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia. [2] Central Clinical School, Monash University, Melbourne, Victoria, Australia.
Abstract:
Antibody fusion to nonprotein materials such as contrast agents or radio-tracers, nano- or microparticles or small-molecule drugs is attracting major interest for molecular imaging and drug delivery. Nondirected bioconjugation techniques may impair antibody affinity, result in lower amounts of functional antibodies and generate multicomponent mixtures. We present a detailed protocol for the enzymatic bioconjugation of small recombinant antibodies to imaging particles, and we also describe the generation of and conjugation to a low-fouling capsule assembled for drug delivery from PEG and PVPON (poly(N-vinylpyrrolidone) by a layer-by-layer (LbL) technique. The single-chain variable fragment (scFv) is equipped with a short C-terminal LPETG tag and the fusion partners are functionalized with an N-terminal GGG nucleophilic group for sortase A conjugation. The LbL capsules are assembled through hydrogen bonding by depositing alkyne-modified poly(vinylpyrrolidone) and poly(methacrylic acid) layers on silica particles, followed by depositing alkyne-modified PEG. The generation of the antibodies and LbL capsules takes ∼1-2 weeks each. The conjugation and functional testing takes another 3-4 d.
