Kinetics study of the natural split Npu DnaE intein in the generation of bispecific IgG antibodies

Huifang Zong; Lei Han; Jie Chen; Zhidi Pan; Lei Wang; Rui Sun; Kai Ding; Yueqing Xie; Hua Jiang; Huili Lu; John Gilly; Baohong Zhang; Jianwei Zhu

doi:10.1007/s00253-021-11707-y

Kinetics study of the natural split Npu DnaE intein in the generation of bispecific IgG antibodies

Appl Microbiol Biotechnol. 2022 Jan;106(1):161-171. doi: 10.1007/s00253-021-11707-y. Epub 2021 Dec 9.

Authors

Huifang Zong^#¹, Lei Han^#², Jie Chen¹, Zhidi Pan¹, Lei Wang¹, Rui Sun¹, Kai Ding¹, Yueqing Xie³, Hua Jiang^{2

3}, Huili Lu¹, John Gilly², Baohong Zhang⁴, Jianwei Zhu^{5

6

7}

Affiliations

¹ Engineering Research Center of Cell & Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
² Jecho Biopharmaceuticals Co., Ltd., Tianjin, China.
³ Jecho Laboratories, Inc., Frederick, MD, USA.
⁴ Engineering Research Center of Cell & Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China. bhzhang@sjtu.edu.cn.
⁵ Engineering Research Center of Cell & Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China. jianweiz@sjtu.edu.cn.
⁶ Jecho Biopharmaceuticals Co., Ltd., Tianjin, China. jianweiz@sjtu.edu.cn.
⁷ Jecho Laboratories, Inc., Frederick, MD, USA. jianweiz@sjtu.edu.cn.

^# Contributed equally.

PMID: 34882254
DOI: 10.1007/s00253-021-11707-y

Abstract

Rapid and efficient bispecific antibody (BsAb) production for industrial applications is still facing many challenges. We reported a technology platform for generating bispecific IgG antibodies, "Bispecific Antibody by Protein Trans-splicing (BAPTS)." While the "BAPTS" method has shown potential in high-throughput screening of BsAbs, further understanding and optimizing the methodology is desirable. A large number of BsAbs were selected to illustrate the conversion efficiency and kinetics parameters. The temperature of reaction makes no significant influence in conversion efficiency, which can reach more than 70% within 2 h, and CD3 × HER2 BsAb can reach 90%. By fitting trans-splicing reaction to single-component exponential decay curves, the apparent first-order rate constants at a series of temperatures were determined. The rate constant ranges from 0.02 to 0.11 min^-1 at 37 °C, which is a high rate reported for the protein trans-splicing reaction (PTS). The reaction process is activated rapidly with activation energy of 8.9 kcal·mol^-1 (CD3 × HER2) and 5.2 kcal·mol^-1 (CD3 × EGFR). The BsAbs generated by "BAPTS" technology not only had the similar post-translation modifications to the parental antibodies, but also demonstrated excellent in vitro and in vivo bioactivity. The kinetics parameters and activation energy of the reaction illustrate feasible for high-throughput screening and industrial applications using the "BAPTS" approach. KEY POINTS: • The trans-splicing reaction of Npu DnaE intein in "BAPTS" platform is a rapid process with low reaction activation and high rate. • The BsAb generated by "BAPTS" remained effective in tumor cell killing. • The kinetics parameters and activation energy of the reaction illustrate feasible for high-throughput screening and industrial applications using the "BAPTS" approach.

Keywords: BAPTS; Bispecific antibody; Glycosylation; In vitro and in vivo activity; Kinetics; Split intein.

MeSH terms

Antibodies, Bispecific*
Immunoglobulin G
Inteins*
Kinetics
Protein Splicing

Substances

Antibodies, Bispecific
Immunoglobulin G