Production of IgG1-based bispecific antibody without extra cysteine residue via intein-mediated protein trans-splicing

Hiroki Akiba,Kouhei Tsumoto,Satoshi Nagata,Haruhiko Kamada,Tomoko Ise,Hiroaki Ohno

doi:10.1038/s41598-021-98855-3

Abstract

A major class of bispecific antibodies (BsAbs) utilizes heterodimeric Fc to produce the native immunoglobulin G (IgG) structure. Because appropriate pairing of heavy and light chains is required, the design of BsAbs produced through recombination or reassembly of two separately-expressed antigen-binding fragments is advantageous. One such method uses intein-mediated protein trans-splicing (IMPTS) to produce an IgG1-based structure. An extra Cys residue is incorporated as a consensus sequence for IMPTS in successful examples, but this may lead to potential destabilization or disturbance of the assay system. In this study, we designed a BsAb linked by IMPTS, without the extra Cys residue. A BsAb binding to both TNFR2 and CD30 was successfully produced. Cleaved side product formation was inevitable, but it was minimized under the optimized conditions. The fine-tuned design is suitable for the production of IgG-like BsAb with high symmetry between the two antigen-binding fragments that is advantageous for screening BsAbs.

Highlights

A major class of bispecific antibodies (BsAbs) utilizes heterodimeric Fc to produce the native immunoglobulin G (IgG) structure
The modified hinge (CH1)...EPKSCFNTHT...(hinge-Fc) without extra amino acids or extra Cys was produced in an intein-mediated protein transsplicing (IMPTS) reaction (Fig. 1c)
Maltose binding protein (MBP) tags were fused at the N-terminus of IntC and at the C-terminus of IntN in addition to the hexahistidine tag [(His)6] for affinity purification

Summary

Introduction

A major class of bispecific antibodies (BsAbs) utilizes heterodimeric Fc to produce the native immunoglobulin G (IgG) structure. Because appropriate pairing of heavy and light chains is required, the design of BsAbs produced through recombination or reassembly of two separately-expressed antigen-binding fragments is advantageous. One such method uses intein-mediated protein transsplicing (IMPTS) to produce an IgG1-based structure. BsAbs are divided into two major classes: low-molecular-weight BsAbs without the Fc part of immunoglobulin G (IgG) proteins, such as diabodies and peptide-linked single-chain variable fragments; and BsAbs bearing the Fc part of IgG so that the fundamental characteristics of the Ig proteins are maintained In the latter class of BsAbs with two different antigen-binding fragments (Fabs), the Fc part is often engineered into heterodimeric structures. Major side reactions to reduce the reaction yield of IMPTS are known as N-cleavage and C-cleavage, which are hydrolytic reactions mediated by reacting nucleophiles at the N-terminus of I ntN and the C-terminus of I ntC, respectively[37,38]

Methods

Results

Conclusion