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.
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Data availability
The data presented in this study are available upon request to the corresponding author. The data may be not publicly available due to their use in further studies.
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Funding
This work was financially supported by the Jecho Laboratories, Inc. This work has been supported by a grant to Jianwei Zhu from Natural Science Foundation # 81773621 and # 82073751.
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Conceptualization: J.Z., B.Z., and L.H.
Experiments and sample preparation: majority performed by H.Z., with contributions by L.H., J.C., Z.P., L.W., and R.S.
Writing – original draft preparation: L.H. and H.Z.
Data analysis: H.Z.
Writing – review and editing: all authors.
All authors had read and agreed to the published version of the manuscript.
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Y.X., H.J., and J.Z. are employees of Jecho Laboratories Inc. L.H., H.J., and J.Z. are employees of Jecho Biopharmaceuticals Inc. J.G. is a paid consultant to Jecho Biopharmaceuticals, Ltd.
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Zong, H., Han, L., Chen, J. et al. Kinetics study of the natural split Npu DnaE intein in the generation of bispecific IgG antibodies. Appl Microbiol Biotechnol 106, 161–171 (2022). https://doi.org/10.1007/s00253-021-11707-y
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DOI: https://doi.org/10.1007/s00253-021-11707-y