Particle generation, functionalization and sortase A-mediated modification with targeting of single-chain antibodies for diagnostic and therapeutic use

Christoph E Hagemeyer; Karen Alt; Angus P R Johnston; Georgina K Such; Hang T Ta; Melissa K M Leung; Sandeep Prabhu; Xiaowei Wang; Frank Caruso; Karlheinz Peter

doi:10.1038/nprot.2014.177

Particle generation, functionalization and sortase A-mediated modification with targeting of single-chain antibodies for diagnostic and therapeutic use

Nat Protoc. 2015 Jan;10(1):90-105. doi: 10.1038/nprot.2014.177. Epub 2014 Dec 11.

Authors

Affiliations

¹ 1] Vascular Biotechnology Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia. [2] Central Clinical School, Monash University, Melbourne, Victoria, Australia.
² 1] Vascular Biotechnology Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia. [2] Atherothrombosis and Vascular Biology Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia.
³ Department of Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Victoria, Australia.
⁴ Department of Chemistry, The University of Melbourne, Melbourne, Victoria, Australia.
⁵ 1] Atherothrombosis and Vascular Biology Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia. [2] Australian Institute of Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland, Australia.
⁶ Department of Chemical and Biomolecular Engineering, The University of Melbourne, Melbourne, Victoria, Australia.
⁷ 1] Central Clinical School, Monash University, Melbourne, Victoria, Australia. [2] Atherothrombosis and Vascular Biology Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia.

PMID: 25502886
DOI: 10.1038/nprot.2014.177

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Aminoacyltransferases / chemistry
Aminoacyltransferases / metabolism*
Bacterial Proteins / chemistry
Bacterial Proteins / metabolism*
Cysteine Endopeptidases / chemistry
Cysteine Endopeptidases / metabolism*
Drug Delivery Systems / methods*
Hydrogen Bonding
Molecular Imaging / methods*
Molecular Structure
Nanoparticles / metabolism*
Polyethylene Glycols
Polymethacrylic Acids
Polyvinyls
Pyrrolidines
Single-Chain Antibodies / metabolism
Single-Chain Antibodies / therapeutic use*

Substances

Bacterial Proteins
Polymethacrylic Acids
Polyvinyls
Pyrrolidines
Single-Chain Antibodies
poly(N-vinylpyrrolidine)
Polyethylene Glycols
Aminoacyltransferases
sortase A
Cysteine Endopeptidases