Design and assessment of an active anti-epidermal growth factor receptor (EGFR) single chain variable fragment (ScFv) with improved solubility

https://doi.org/10.1016/j.bbrc.2018.11.170Get rights and content

Highlights

  • Increased solubility of Anti-EGFR ScFv attached with a SEP tag was predicted by BD.

  • Untagged ScFv expressed in the insoluble fraction of E. Coli.

  • Whereas ∼85% of the SEP-tagged ScFv expressed in the soluble fraction.

  • The SEP tag unexpectedly increased the yield of Ni-NTA purified ScFv by threefold.

  • ScFv's higher solubility was demonstrated by DLS and SLS and EGFR binding by SPR.

Abstract

ScFv is emerging as a therapeutic alternative to the full-length monoclonal antibodies due to its small size and low production cost, but its low solubility remains a limiting factor toward wider use. Here, we increased the solubility of an Anti-epidermal growth factor receptor ScFv (Anti-EGFR ScFv) by attaching, a short 12-residue solubility enhancing peptide (SEP) tag at its C terminus. We first estimated the solubility increase by running 500-ns Brownian dynamics (BD) simulations. We then experimentally evaluated the predictions by producing recombinant Anti-EGFR ScFv with and without a SEP tag (called C9R) in E. coli. At 20 °C, ∼85% of Anti-EGFR ScFv-C9R expressed in the soluble fraction, whereas all of the Anti-EGFR ScFv remained in the insoluble fraction. The total yield of Anti-EGFR ScFv-C9R was 17.15 mg which was ∼3 times higher than that of Anti-EGFR ScFv refolded from the insoluble fraction. Static and dynamic light scattering demonstrated the higher solubility of the purified Anti-EGFR ScFv-C9R, and Circular Dichroism (CD) indicated its high thermal stability, whereas the untagged protein aggregated at 37 °C and pH 6. Finally, the binding activity of Anti-EGFR ScFv-C9R to EGFR was confirmed by surface plasmon resonance (SPR). Altogether, these results illustrate the improved biophysical and biochemical characteristics of Anti-EGFR ScFv-C9R and emphasize the potentials of SEP-tags for enhancing the solubility of aggregation-prone antibody fragments.

Introduction

The epidermal growth factor receptor (EGFR) is a tyrosine kinase family (ErbBs) cell-surface receptor involved in cell proliferation, differentiation, and survival [1]. Mutations in the EGFR gene have been associated with several types of tumors [1], and therapeutic Anti-EGFR monoclonal antibodies (mAbs) such as Panitumumab and Cetuximab have been developed [2,3]. Current mAbs have limitations such as their low tumor penetration, slow blood clearance rate and especially, and their high production cost [4,5]. Recombinant antibody fragments such as ScFv [4] and VHH [6] are much smaller than the full-length mAbs and are expected to address these issues, and additionally to have a rapid tumor penetration rate [7]. ScFv consists of the variable heavy (VH) and light (VL) domains linked by an artificial flexible polypeptide, and its molecular weight is merely ∼28 kDa, which is considerably smaller than the full-length antibodies (∼150 kDa).

Although ScFv has been produced in various expression systems including mammalian [8], yeast [9] and bacterial cells [10], ScFv remains in many instances limited by solubility issues. In particular, ScFv forms inclusion bodies when it is overexpressed in E. coli [10], which is the most popular and common expression system. The solubility of ScFv has been enhanced by fusion to highly soluble proteins such as thioredoxin [11], N-utilization substance (NusA), maltose binding protein (MBP) [12] and small ubiquitin-like modifier (SUMO) [13]. However, due to their large sizes, the fusion proteins often interfere with ScFv's binding to the antigens and need to be removed during purification, reducing its solubility [12].

In this study, we applied our Solubility Enhancing Peptide (SEP) tag strategy [14,15] to increase the solubility of Anti-EGFR ScFv. To this end, we attached a short 12-residue SEP tag (C9R) to the C termini of Anti-EGFR ScFv. The SEP tag increased the solubility of Anti-EGFR ScFv, both during and after purification, without affecting the EGFR binding activity. In addition, the SEP tag unexpectedly increased the yield of Anti-EGFR ScFv by threefold, and we purified 17.15 mg of active Anti-EGFR ScFv-C9R from a 200 mL LB culture.

Section snippets

Computational model

Anti-EGFR ScFv with a SEP tag was constructed with Modeller [16] using ScFv PL-2 as a template (PDB ID 5KOV-chain C). In order to take into account the protein's internal motions in the Brownian dynamics (BD) calculation, where the proteins are treated as a rigid-body particle, we generated 10 Anti-EGFR ScFv structures by all-atom molecular dynamics (MD) simulation. The MD simulation was carried out for 30 ns using Gromacs 4.7 [17] on an HPC5000 server equipped with a NvidiaTesla K40 GPU board

Prediction of the mutant's solubility

Prior to experimental assessments, we predicted the effects of attaching a SEP tag to the C terminus of Anti-EGFR ScFv on its solubility, because we suspected that the solubilization of ScFv, which is a two-domain protein with two disulfide bonds, might be more complex than solubilizing single-domain proteins [14,15]. The solubility of Anti-EGFR ScFv variants was predicted using SDA 7.1, which allows simulating the trajectories of several tens of rigid body proteins and estimate the formation

Funding

This study was supported by a research grant from the TaNeDS global research program and JSPS grant-in-aid for scientific research grants (KAKENHI-21300110, 15H04359, and 18H02385) to Y.K.

Conflicts of interest

None.

Acknowledgments

We thank the members of the Kuroda Laboratory for discussion and suggestions, Dr. Punitha Velmurugan (JSPS) and Dr. Richa Tambi (JSPS) for their kind help with manuscript preparation. We are grateful to Mr. Katsuhiko Hirose and Mr. Satoshi Kosuda for support with MD and BD calculations, Mr. Hiromitsu Sakurai for preliminary experiments, and Ms. Patricia McGahan for English proofreading.

References (30)

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