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Development and characterization of small bispecific albumin-binding domains with high affinity for ErbB3

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Abstract

Affinity proteins based on small scaffolds are currently emerging as alternatives to antibodies for therapy. Similarly to antibodies, they can be engineered to have high affinity for specific proteins. A potential problem with small proteins and peptides is their short in vivo circulation time, which might limit the therapeutic efficacy. To circumvent this issue, we have engineered bispecificity into an albumin-binding domain (ABD) derived from streptococcal Protein G. The inherent albumin binding was preserved while the opposite side of the molecule was randomized for selection of high-affinity binders. Here we present novel ABD variants with the ability to bind to the epidermal growth factor receptor 3 (ErbB3). Isolated candidates were shown to have an extraordinary thermal stability and affinity for ErbB3 in the nanomolar range. Importantly, they were also shown to retain their affinity to albumin, hence demonstrating that the intended strategy to engineer bispecific single-domain proteins against a tumor-associated receptor was successful. Moreover, competition assays revealed that the new binders could block the natural ligand Neuregulin-1 from binding to ErbB3, indicating a potential anti-proliferative effect. These new binders thus represent promising candidates for further development into ErbB3-signaling inhibitors, where the albumin interaction could result in prolonged in vivo half-life.

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Acknowledgments

This work was supported by the Swedish Research Council (VR), the Knut and Alice Wallenberg foundation (KAW), and the Royal Swedish Academy of Sciences (KVA). Prof. Per-Åke Nygren is acknowledged for scientific advice and helpful discussion.

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The authors declare that they have no conflicts of interest.

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Authors and Affiliations

  1. Division of Protein Technology, KTH Royal Institute of Technology, AlbaNova University Center, 106 91, Stockholm, Sweden

    Johan Nilvebrant, Mikael Åstrand, John Löfblom & Sophia Hober

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  1. Johan Nilvebrant
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  2. Mikael Åstrand
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  3. John Löfblom
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  4. Sophia Hober
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Correspondence to Sophia Hober.

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Johan Nilvebrant and Mikael Astrand contributed equally to this study.

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Nilvebrant, J., Åstrand, M., Löfblom, J. et al. Development and characterization of small bispecific albumin-binding domains with high affinity for ErbB3. Cell. Mol. Life Sci. 70, 3973–3985 (2013). https://doi.org/10.1007/s00018-013-1370-9

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