Secretion from bacterial versus mammalian cells yields a recombinant scFv with variable folding properties

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Abstract

Escherichia coli (E. coli) is the most commonly used organism for expressing antibody fragments such as single chain antibody Fvs (scFvs). Previously, we have utilized E. coli to express well-folded scFvs for characterization and engineering purposes with the goal of using these engineered proteins as building blocks for generating IgG-like bispecific antibodies (BsAbs). In the study, described here, we observed a significant difference in the secondary structure of an scFv produced in E. coli and the same scFv expressed and secreted from chinese hamster ovary (CHO) cells as part of a BsAb. We devised a proteolytic procedure to separate the CHO-derived scFv from its antibody-fusion partner and compared its properties with those of the E. coli-derived scFv. In comparison to the CHO-derived scFv, the E. coli-derived scFv was found trapped in a misfolded, but monomeric state that was stable for months at 4 °C. The misfolded state bound antigen in a heterogeneous fashion that included non-specific binding, which made functional characterization challenging. This odd incidence of obtaining a misfolded scFv from bacteria suggests careful characterization of the folded properties of bacterially expressed scFvs is warranted if anomalous issues with antigen-binding or non-specificity occur during an engineering campaign. Additionally, our proteolytic methodology for obtaining significant levels of intact scFvs from highly expressed IgG-like antibody proteins serves as a robust method for producing scFvs in CHO without the use of designed cleavage motifs.

Highlights

► Described is a protocol for producing antibody scFvs using mammalian expression of IgG-like constructs. ► scFv material produced in CHO vs. E. coli is contrasted. ► A soluble and stable misfolded scFv that demonstrates both specific and non-specific binding was identified.

Introduction

Antibody engineering in Escherichia coli (E. coli) has a long and impressive history [1], [2]. Much of the original data regarding the expression of antibody Fv or single-chain Fv (scFv)4 fragments were generated in E. coli [3], [4], [5]. The ability of E. coli to selectively incorporate a single plasmid (i.e., from a library of plasmids) as well as its relatively fast growth phase makes it ideal for the rapid screening or evaluation of protein designs. One caveat is that antibodies and antibody fragments are complex multi-domain proteins with multiple disulfides and multiple cis-proline polypeptide configurations [6], [7], [8], which may not be faithfully reproduced during E. coli expression. For these reasons, large efforts have been made to understand the folding, oxidation, and isomerization (both disulfide and peptide bond) machinery intrinsic to E. coli that can be modified to facilitate the proper secretion of folded and active material [9], [10].

The primary method of E. coli-mediated antibody expression has been to export the nascent and unfolded polypeptide chain(s) across the inner membrane into the highly oxidative E. coli periplasm where secretion and folding is thought to more adequately reflect the natural process in eukaryotic cells [3], [9], [10]. However, the inability to form the correct disulfide pairs or the proper cis-proline configurations [11] can result in proteolysis or misfolded and aggregated material often toxic to the host cell [9], [10]. Thus, both the coexpression of molecular chaperones (with both disulfide- and prolyl-isomerase activities) and the reduction of periplasmic proteases have been employed to enhance functional expression in the periplasm [9], [10]. Secretion of antibody fragments across the bacterial outer membrane and into the media has generally been achieved by non-specific protein release due to a breakdown in the integrity of the outer membrane, which is thought to result from expression of the recombinant protein competing with the production of outer membrane proteins [9].

Antibody fragments such as scFvs continue to be of great interest to the pharmaceutical and biotechnology industries because they can serve as building blocks for targeted-fusion protein (immunotoxin) or bispecific antibody (BsAb) designs [12], [13]. However, nearly all these antibody fragment designs take antibody domains out of the natural context of a full-length immunoglobulin, which can have significant consequences both in terms of protein stability and solubility [14], [15]. Therefore, stability/solubility engineering of antibody fragments to make them suitable as part of a protein therapeutic is often a necessary step – either by requiring stability or refolding during the initial selection or by reverse engineering of stability or solubility into the constructs [14], [15], [16]. In general, the selection and design of antibody fragments are carried out using E. coli as the expression host. We have used the secretion of libraries of scFvs from E. coli to identify variants with improved stability that can be utilized for the generation of BsAbs [17], [18].

We generally have positive experiences expressing natively-folded scFvs in E. coli. Recently, however, we encountered an issue with a particular scFv that we had engineered for stability to be used as part of a potential BsAb therapeutic [19]. The affinity of the scFv as part of the BsAb expressed in chinese hamster overall (CHO) cells was weaker than we had expected based on our experience with the protein expressed in E. coli leading us to investigate the reasons behind this phenomenon. To directly compare the scFv expressed in E. coli with that expressed as part of the BsAb in CHO, we developed a novel proteolytic cleavage method to liberate the scFv from the CHO-produced BsAb. By comparing the two materials, we discovered that while the E. coli derived material was monomeric and stable for months at 4 °C, it differed significantly in structure from the CHO-derived scFv.

Section snippets

Production of the stabilized BIIB4 scFv using both E. coli and CHO

Production of the stabilized BIIB4 scFv in E. coli was performed using a similar procedure (see Methods) to that used for screening the scFv for stabilizing mutations [19]. The stabilized BIIB4 scFv from CHO was expressed as a C-terminal IgG-fusion. Expression levels of IgG-like constructs can be equivalently high as IgGs particularly after cell line selection and engineering (>1 g/L) [19], [20], [21]. We had generated multiple IgG-fusion proteins containing the stabilized BIIB4 scFv including a

Discussion

The misfolded nature of the stabilized BIIB4 scFv produced by E. coli led to non-specific binding and an artificially high apparent affinity for its target IGF-1R. Its misfolded behavior from E. coli was unexpected based on our previous work with scFvs expressed in the periplasmic space and/or secreted into the media – providing the domains of these scFvs were intrinsically stable as was the case for the BIIB4 scFv. Generally, misfolding issues result in aggregation, protein degradation or

Cloning and expression of E. coli derived scFvs

Subcloning and mutagenesis of the BIIB4 scFv plasmid for stability screening in E. Coli was described previously [19]. E. coli strain W3110 (ATCC No. 27325, Manassas, VA) was used for expressing the stability engineered BIIB4 scFv. The E. coli were found to be leaky and the majority of the scFv material was found in the culture supernatant. For these reasons, the supernantant was used directly for screening of (1) the thermal stability of a library of BIIB4 scFvs designed for enhanced stability

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    Current address: Pfizer, 10770 Science Center Drive, San Diego, CA 92121, United States.

    2

    Current address: Centers for Therapeutic Innovation, Pfizer, Inc., 10770 Science Center Drive, San Diego, CA 92121, United States.

    3

    Current address: Centocor R&D, A Division of Johnson & Johnson Pharmaceutical Research & Development, L.L.C., 3210 Merryfield Row, San Diego, CA 92121, United States.

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