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

Nature Protocols volume 10, pages 90–105 (2015)Cite this article

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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.

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Figure 1: Illustration of the conjugation process between scFv-LPETG and the GGG-coupling partner (probe).
Figure 2: Generation and specific binding evaluation of scFv-coupled iron oxide particles to platelet aggregates in a flow chamber system.
Figure 3: Generation of sortase A–mediated scFv-functionalized LbL capsules.
Figure 4: Targeting of sortase A–mediated scFv-functionalized LbL capsules to platelet-rich thrombi.

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Acknowledgements

This work was funded by the National Health and Medical Research Council (grant nos. 472665, 472667 and 487922), the National Heart Foundation (grant no. G09M4345) and an Australian Research Council (ARC) Discovery Project (DP0877360). The work was supported in part by the Victorian Government's Operational Infrastructure Support Program. C.E.H. is supported by a National Heart Foundation Career Development Fellowship (CR 11M 6066), K.A. is supported by the German Research Foundation (Al 1521/1-1), A.P.R.J. is supported by an ARC Future Fellowship (FT110100265), H.T.T. is supported by a National Heart Foundation Postdoctoral Fellowship (PF09M4688), M.K.M.L. is supported by an ARC Australian Postgraduate Award, F.C. is supported by an ARC Federation Fellowship (FF0776078) and K.P. is supported by an ARC Future Fellowship (FT0992210). We thank G. Krippner for his help with Figure 1.

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Author notes

  1. Christoph E Hagemeyer, Karen Alt, Angus P R Johnston, Frank Caruso and Karlheinz Peter: These authors contributed equally to this work.

Authors and Affiliations

  1. Vascular Biotechnology Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia

    Christoph E Hagemeyer & Karen Alt

  2. Central Clinical School, Monash University, Melbourne, Victoria, Australia

    Christoph E Hagemeyer, Sandeep Prabhu, Xiaowei Wang & Karlheinz Peter

  3. Atherothrombosis and Vascular Biology Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia

    Karen Alt, Hang T Ta, Sandeep Prabhu, Xiaowei Wang & Karlheinz Peter

  4. Department of Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Victoria, Australia

    Angus P R Johnston

  5. Department of Chemistry, The University of Melbourne, Melbourne, Victoria, Australia

    Georgina K Such

  6. Australian Institute of Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland, Australia

    Hang T Ta

  7. Department of Chemical and Biomolecular Engineering, The University of Melbourne, Melbourne, Victoria, Australia

    Melissa K M Leung & Frank Caruso

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Contributions

C.E.H. designed and produced the antibodies, supervised the work and oversaw preparation of the manuscript; K.A. prepared the manuscript and made the figures; A.P.R.J. generated the nanocapsules and supervised the LbL capsule work; G.K.S. made the polymers for the nanocapsules; H.T.T. performed the MPIO; M.K.M.L. performed the nanocapsule and thrombus-targeting work; S.P. established sortase A–mediated scFv conjugation; X.W. cloned and produced scFv antibodies; F.C. supervised the nanocapsule work; K.P. generated the scFvs and supervised the MPIO work. All authors contributed to writing of the manuscript and approved the final version.

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Correspondence to Christoph E Hagemeyer.

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Hagemeyer, C., Alt, K., Johnston, A. et al. Particle generation, functionalization and sortase A–mediated modification with targeting of single-chain antibodies for diagnostic and therapeutic use. Nat Protoc 10, 90–105 (2015). https://doi.org/10.1038/nprot.2014.177

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