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

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 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.

SAMURAI (Solid-phase Assisted Mutagenesis by Uracil Restriction for Accurate Integration) for antibody affinity maturation and paratope mapping

“…The preparation of wild-type template (31) (VH-linker-VL) used a forward biotinylated primer, which annealed at the start of framework 1 (FR1) of VH and a reverse primer, which annealed at FR3 in VL, upstream of CDRL3, omitting CDRL3. Primers used for amplification were ‘2600 TegBio’ and ‘2602 Alexa647’.…”

“…The best binder from each library was re-cloned and expressed as soluble scFvs in Escherichia coli as previously described (31). Chosen clones were amplified with PCR primers containing SfiI and AvrII restriction sites and digested with the corresponding enzymes, cloned into an E. coli expression vector pHP2-15 (33) and transformed into TOP10 E. coli cells for expression of soluble molecules.…”

Applications of flow cytometry sorting in the pharmaceutical industry: A review

“…97,98 However, despite the advantages of cell display in bacteria, phage surface display has been more commonly utilized because of its ability to select more effective sequences for binding peptides and generate larger libraries. 97,98 The display of a yeast surface display was developed as an alternative to that of prokaryotic cells since it solved the problem of expression biases against some eukaryotic proteins and led to more efficiently folded proteins. 99 Cell-display technology has many applications (literature summarized in Table 3), which include vaccine development, 100,102 antibody library screening 101,104 and enzyme library screening.…”

Screening and application of peptamers

“…[33] . 大肠 杆菌遗传背景清晰, 遗传操作技术简单成熟并且兼具 培养简单经济和表达水平高的特点让其成为最常用的 表达系统 [34] , 肉葡萄球菌(Staphylococcus carnosus)和 枯草芽孢杆菌也有用于肽适体筛选的报道 [35,36] .…”