Prompting Fab Yeast Surface Display Efficiency by ER Retention and Molecular Chaperon Co-expression.

Meng Mei,Xin Ge,Guimin Zhang,Li Yi,Ki Baek Lee,Brent L Iverson,Shengchen Wang,Junhong Li

doi:10.3389/fbioe.2019.00362

Abstract

For antibody discovery and engineering, yeast surface display (YSD) of antigen-binding fragments (Fabs) and coupled fluorescence activated cell sorting (FACS) provide intact paratopic conformations and quantitative analysis at the monoclonal level, and thus holding great promises for numerous applications. Using anti-TNFα mAbs Infliximab, Adalimumab, and its variants as model Fabs, this study systematically characterized complementary approaches for the optimization of Fab YSD. Results suggested that by using divergent promoter GAL1-GAL10 and endoplasmic reticulum (ER) signal peptides for co-expression of light chain and heavy chain-Aga2 fusion, assembled Fabs were functionally displayed on yeast cell surface with sigmoidal binding responses toward TNFα. Co-expression of a Hsp70 family molecular chaperone Kar2p and/or protein-disulfide isomerase (Pdi1p) significantly improved efficiency of functional display (defined as the ratio of cells displaying functional Fab over cells displaying assembled Fab). Moreover, fusing ER retention sequences (ERSs) with light chain also enhanced Fab display quality at the expense of display quantity, and the degree of improvements was correlated with the strength of ERSs and was more significant for Infliximab than Adalimumab. The feasibility of affinity maturation was further demonstrated by isolating a high affinity Fab clone from 1:103 or 1:105 spiked libraries.

Highlights

Monoclonal antibodies represent the fastest growing class of therapeutics in the last decades
A divergent GAL1-GAL10 promoter derived from previous studies (West et al, 1987; Boder et al, 2005; Jiang and Boder, 2010) is exploited by cloning Fab heavy chain and light chain at downstream of GAL10 and GAL1, respectively (Figure 1A)
Similar to Infliximab Fab, strong ER retention sequences (ERSs) FEHDEL and WEHDEL increased percentages and MFIs of tumor necrosis factor α (TNFα)+ cells and prompted the functional display efficiency of D2E7 Fab for 1.5- and 1.9fold (Figure S5, Table S3), while weak ERSs, HDEL, and KDEL, did not significantly affect display amounts or functional display efficiencies. These results suggested that ERSs with high retention strength improved the quality of yeast surface displayed Fab, presumably due to extended residence time in the endoplasmic reticulum (ER) that facilitated the formation of functional Fabs

Summary

Introduction

Monoclonal antibodies (mAbs) represent the fastest growing class of therapeutics in the last decades. By the end of 2018, at least 116 mAb-based biopharmaceutical products are active on the market (Walsh, 2018; DeFrancesco, 2019). Compared to selection approaches, which rely on overall binding strength such as phage panning, Fab Yeast Display Optimization fluorescence activated cell sorting (FACS) is advantageous by providing quantitative analysis of each library member. During subsequent rounds of sorting, the concentration of a fluorophore-labeled antigen can be fine-tuned in a real-time manner, leading to efficiently distinguish high affinity clones from others. A dual color sorting with one channel for antibody expression and the other for antigen binding has been proven highly effective for enriching affinity improved clones (Feldhaus et al, 2003; van den Beucken et al, 2003)

Methods

Results

Conclusion