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

• Published in: Scientific reports Vol. 11; no. 1; p. 19411
• Main Authors: Akiba, Hiroki, Ise, Tomoko, Nagata, Satoshi, Kamada, Haruhiko, Ohno, Hiroaki, Tsumoto, Kouhei
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 30.09.2021; Nature Publishing Group; Nature Portfolio
• Subjects: 631/154/51/1568; 631/45/612; 639/638/45/612; Antibodies, Bispecific - isolation & purification; Antigens; Bispecific antibodies; CD30 antigen; Conserved sequence; Humanities and Social Sciences; Humans; Immunoglobulin Fab Fragments - immunology; Immunoglobulin G; Immunoglobulin G - immunology; Light chains; multidisciplinary; Protein Engineering - methods; Recombinant Fusion Proteins - isolation & purification; Recombination; Science; Science (multidisciplinary); Tissue engineering
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-021-98855-3

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.