Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jan;106(1):161-171.
doi: 10.1007/s00253-021-11707-y. Epub 2021 Dec 9.

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

Huifang Zong #  1 , Lei Han #  2 , Jie Chen  1 , Zhidi Pan  1 , Lei Wang  1 , Rui Sun  1 , Kai Ding  1 , Yueqing Xie  3 , Hua Jiang  2   3 , Huili Lu  1 , John Gilly  2 , Baohong Zhang  4 , Jianwei Zhu  5   6   7
Affiliations

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

Huifang Zong et al. Appl Microbiol Biotechnol. 2022 Jan.

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.

PubMed Disclaimer

References

    1. Ayoub D, Jabs W, Resemann A, Evers W, Evans C, Main L, Baessmann C, Wagner-Rousset E, Suckau D, Beck A (2013) Correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques. Mabs 5(5):699–710. https://doi.org/10.4161/mabs.25423 - DOI - PubMed - PMC
    1. Camarero JA, Fushman D, Sato S, Giriat I, Cowburn D, Raleigh DP, Muir TW (2001) Rescuing a destabilized protein fold through backbone cyclization. J Mol Biol 308(5):1045–1062. https://doi.org/10.1006/jmbi.2001.4631 - DOI - PubMed
    1. Carter P (2001) Bispecific human IgG by design. J Immunol Methods 248(1):7–15. https://doi.org/10.1016/S0022-1759(00)00339-2 - DOI - PubMed
    1. Chen J, Pan Z, Han L, Zhou Y, Zong H, Wang L, Sun R, Jiang H, Xie Y, Yuan Y, Wu M, Bian Y, Zhang B, Zhu J (2021) A novel bispecific antibody targeting CD3 and Lewis Y with potent therapeutic efficacy against gastric cancer. Biomedicines 9(8):1059. https://doi.org/10.3390/biomedicines9081059 - DOI - PubMed - PMC
    1. Cheriyan M, Pedamallu CS, Tori K, Perler F (2013) Faster protein splicing with the Nostoc punctiforme DnaE intein using non-native extein residues. J Biol Chem 288(9):6202–6211. https://doi.org/10.1074/jbc.M112.433094 - DOI - PubMed - PMC

LinkOut - more resources