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Combination of phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity binders
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0002-1389-5371
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0003-1096-9061
KTH, School of Biotechnology (BIO), Protein Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Lund University, Sweden.
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2017 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347Article in journal (Refereed) Epub ahead of print
Abstract [en]

Surface display couples genotype with a surface exposed phenotype and thereby allows screening of gene-encoded protein libraries for desired characteristics. Of the various display systems available, phage display is by far the most popular, mainly thanks to its ability to harbour large size libraries. Here, we describe the first use of a Gram-positive bacterial host for display of a library of human antibody genes which, when combined with phage display, provides ease of use for screening, sorting and ranking by flow cytometry. We demonstrate the utility of this method by identifying low nanomolar affinity scFv fragments towards human epidermal growth factor receptor 2 (HER2). The ranking and performance of the scFv isolated by flow sorting in surface-immobilised form was retained when expressed as soluble scFv and analysed by biolayer interferometry, as well as after expression as full-length antibodies in mammalian cells. We also demonstrate the possibility of using Gram-positive bacterial display to directly improve the affinity of the identified binders via an affinity maturation step using random mutagenesis and flow sorting. This combined approach has the potential for a more complete scan of the antibody repertoire and for affinity maturation of human antibody formats.

Place, publisher, year, edition, pages
Elsevier, 2017.
Keywords [en]
Affinity maturation, Antibody, Cell-surface display, Flow cytometry, HER2, Phage display, S. carnosus, Binders, Bins, Cell membranes, Display devices, Genes, Libraries, Mammals, Cell surface displays, Antibodies
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-213019DOI: 10.1016/j.nbt.2017.07.011ISI: 000441913800011Scopus ID: 2-s2.0-85027243712OAI: oai:DiVA.org:kth-213019DiVA, id: diva2:1136462
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20170828

Available from: 2017-08-28 Created: 2017-08-28 Last updated: 2018-09-07Bibliographically approved
In thesis
1. Cell line and protein engineering tools for production and characterization of biologics
Open this publication in new window or tab >>Cell line and protein engineering tools for production and characterization of biologics
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Our increasing understanding of disease mechanisms coupled with technological advances has facilitated the generation of pharmaceutical proteins, which are able to address yet unmet medical needs. Diseases that were fatal in the past can now be treated with novel biological medications improving and prolonging life for many patients. Pharmaceutical protein production is, however, a complex undertaking, which is by no means problem-free. The demand for more complex proteins and the realization of the importance of post-translational modifications have led to an increasing use of mammalian cells for protein expression. Despite improvements in design and production, the costs required for the development of pharmaceutical proteins still are far greater than those for conventional, small molecule drugs. To render such treatments affordable for healthcare suppliers and assist in the implementation of precision medicine, further progress is needed. In five papers this thesis describes strategies and methods that can help to advance the development and manufacturing of pharmaceutical proteins. Two platforms for antibody engineering have been developed and evaluated, one of which allows for efficient screening of antibody libraries whilst the second enables the straightforward generation of bispecific antibodies. Moreover, a method for epitope mapping has been devised and applied to map the therapeutic antibody eculizumab’s epitope on its target protein. In a second step it was shown how this epitope information can be used to stratify patients and, thus, contribute to the realization of precision medicine. The fourth project focuses on the cell line development process during pharmaceutical protein production. A platform is described combining split-GFP and fluorescence-activated droplet sorting, which allows for the efficient selection of highly secreting cells from a heterogeneous cell pool. In an accompanying study, the split-GFP probe was improved to enable shorter assay times and increased sensitivity, desirable characteristics for high-throughput screening of cell pools. In summary, this thesis provides tools to improve design, development and production of future pharmaceutical proteins and as a result, it makes a contribution to the goal of implementing precision medicine through the generation of more cost-effective biopharmaceuticals for well-characterized patient groups.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. p. 91
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:16
Keywords
Pharmaceutical proteins, precision medicine, antibody engineering, epitope mapping, cell line development, split-GFP
National Category
Pharmaceutical Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-212931 (URN)978-91-7729-497-9 (ISBN)
Public defence
2017-09-29, E3, Osquars backe 14, Stockholm, 10:00 (English)
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Note

QC 20170828

Available from: 2017-08-28 Created: 2017-08-24 Last updated: 2017-08-30Bibliographically approved

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Hu, Francis JingxinVolk, Anna-LuisaPersson, HelenaUhlén, MathiasRockberg, Johan

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