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Combination of phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity binders

Hu, Francis Jingxin; Volk, Anna Luisa; Persson, Helena LU ; SÄLL, ANNA LU ; Borrebaeck, Carl LU ; Uhlen, Mathias and Rockberg, Johan (2017) In New Biotechnology
Abstract

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... (More)

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.

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author
organization
publishing date
type
Contribution to journal
publication status
epub
subject
keywords
Affinity maturation, Antibody, Cell-surface display, Flow cytometry, HER2, Phage display, S. carnosus
in
New Biotechnology
publisher
Elsevier
external identifiers
  • scopus:85027243712
ISSN
1871-6784
DOI
10.1016/j.nbt.2017.07.011
language
English
LU publication?
yes
id
3c357c36-251f-48ea-8cab-b7d82e8273fa
date added to LUP
2017-08-31 16:47:20
date last changed
2017-09-01 03:00:03
@article{3c357c36-251f-48ea-8cab-b7d82e8273fa,
  abstract     = {<p>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.</p>},
  author       = {Hu, Francis Jingxin and Volk, Anna Luisa and Persson, Helena and SÄLL, ANNA and Borrebaeck, Carl and Uhlen, Mathias and Rockberg, Johan},
  issn         = {1871-6784},
  keyword      = {Affinity maturation,Antibody,Cell-surface display,Flow cytometry,HER2,Phage display,S. carnosus},
  language     = {eng},
  publisher    = {Elsevier},
  series       = {New Biotechnology},
  title        = {Combination of phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity binders},
  url          = {http://dx.doi.org/10.1016/j.nbt.2017.07.011},
  year         = {2017},
}