リケラボ論文検索は、全国の大学リポジトリにある学位論文・教授論文を一括検索できる論文検索サービスです。

リケラボ 全国の大学リポジトリにある学位論文・教授論文を一括検索するならリケラボ論文検索大学・研究所にある論文を検索できる

リケラボ 全国の大学リポジトリにある学位論文・教授論文を一括検索するならリケラボ論文検索大学・研究所にある論文を検索できる

大学・研究所にある論文を検索できる 「Production of IgG1-based bispecific antibody without extra cysteine residue via intein-mediated protein trans-splicing.」の論文概要。リケラボ論文検索は、全国の大学リポジトリにある学位論文・教授論文を一括検索できる論文検索サービスです。

コピーが完了しました

URLをコピーしました

論文の公開元へ論文の公開元へ
書き出し

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

Akiba, Hiroki Ise, Tomoko Nagata, Satoshi Kamada, Haruhiko Ohno, Hiroaki Tsumoto, Kouhei 京都大学 DOI:10.1038/s41598-021-98855-3

2021

概要

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.

この論文で使われている画像

参考文献

1. Brinkmann, U. & Kontermann, R. E. The making of bispecific antibodies. MAbs 9, 182–212. https://​doi.​org/​10.​1080/​19420​862.​

2016.​12683​07 (2017).

2. Labrijn, A. F., Janmaat, M. L., Reichert, J. M. & Parren, P. W. H. I. Bispecific antibodies: A mechanistic review of the pipeline. Nat.

Rev. Drug Discov. 18, 585–608. https://​doi.​org/​10.​1038/​s41573-​019-​0028-1 (2019).

3. Merchant, A. M. et al. An efficient route to human bispecific IgG. Nat. Biotechnol. 16, 677–681. https://​doi.​org/​10.​1038/​nbt07​

98-​677 (1998).

4. Choi, H. J., Seok, S. H., Kim, Y. J., Seo, M. D. & Kim, Y. S. Crystal structures of immunoglobulin Fc heterodimers reveal the

molecular basis for heterodimer formation. Mol. Immunol. 65, 377–383. https://​doi.​org/​10.​1016/j.​molimm.​2015.​02.​017 (2015).

5. Labrijn, A. F. et al. Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange. Proc. Natl. Acad. Sci. USA 110,

5145. https://​doi.​org/​10.​1073/​pnas.​12201​45110 (2013).

6. Strop, P. et al. Generating bispecific human IgG1 and IgG2 antibodies from any antibody pair. J. Mol. Biol. 420, 204–219. https://​

doi.​org/​10.​1016/j.​jmb.​2012.​04.​020 (2012).

7. Dengl, S. et al. Format chain exchange (FORCE) for high-throughput generation of bispecific antibodies in combinatorial binderformat matrices. Nat. Commun. 11, 4974. https://​doi.​org/​10.​1038/​s41467-​020-​18477-7 (2020).

8. Von Kreudenstein, T. S. et al. Improving biophysical properties of a bispecific antibody scaffold to aid developability. MAbs 5,

646–654. https://​doi.​org/​10.​4161/​mabs.​25632 (2013).

9. Davis, J. H. et al. SEEDbodies: Fusion proteins based on strand-exchange engineered domain (SEED) CH3 heterodimers in an

Fc analogue platform for asymmetric binders or immunofusions and bispecific antibodies†. Protein Eng. Des. Sel. 23, 195–202.

https://​doi.​org/​10.​1093/​prote​in/​gzp094 (2010).

10. Liu, H., Saxena, A., Sidhu, S. S. & Wu, D. Fc engineering for developing therapeutic bispecific antibodies and novel scaffolds. Front.

Immunol. https://​doi.​org/​10.​3389/​fimmu.​2017.​00038 (2017).

11. Sampei, Z. et al. Identification and multidimensional optimization of an asymmetric bispecific IgG antibody mimicking the function of factor VIII cofactor activity. PLoS ONE 8, e57479. https://​doi.​org/​10.​1371/​journ​al.​pone.​00574​79 (2013).

12. Klein, C. et al. Engineering therapeutic bispecific antibodies using CrossMab technology. Methods 154, 21–31. https://​doi.​org/​10.​

1016/j.​ymeth.​2018.​11.​008 (2019).

13. Golay, J. et al. Design and validation of a novel generic platform for the production of tetravalent IgG1-like bispecific antibodies.

J. Immunol. 196, 3199–3211. https://​doi.​org/​10.​4049/​jimmu​nol.​15015​92 (2016).

14. Lewis, S. M. et al. Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nat. Biotechnol.

32, 191–198. https://​doi.​org/​10.​1038/​nbt.​2797 (2014).

15. Mazor, Y. et al. Improving target cell specificity using a novel monovalent bispecific IgG design. MAbs 7, 377–389. https://​doi.​org/​

10.​1080/​19420​862.​2015.​10078​16 (2015).

16. Wagner, K. et al. Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity. Proc. Natl.

Acad. Sci. USA 111, 16820. https://​doi.​org/​10.​1073/​pnas.​14086​05111 (2014).

17. Han, L. et al. Efficient generation of bispecific IgG antibodies by split intein mediated protein trans-splicing system. Sci. Rep. 7,

8360. https://​doi.​org/​10.​1038/​s41598-​017-​08641-3 (2017).

18. Hofmann, T. et al. Intein mediated high throughput screening for bispecific antibodies. MAbs 12, 1731938. https://​doi.​org/​10.​

1080/​19420​862.​2020.​17319​38 (2020).

19. Yumura, K. et al. Use of SpyTag/SpyCatcher to construct bispecific antibodies that target two epitopes of a single antigen. J. Biochem.

162, 203–210. https://​doi.​org/​10.​1093/​jb/​mvx023 (2017).

20. Shibuya, Y. et al. Generation of camelid VHH bispecific constructs via in-cell intein-mediated protein trans-splicing. Protein Eng.

Des. Sel. 30, 15–21. https://​doi.​org/​10.​1093/​prote​in/​gzw057 (2016).

21. Perler, F. B. Protein splicing mechanisms and applications. IUBMB Life 57, 469–476. https://​doi.​org/​10.​1080/​15216​54050​01633​43

(2005).

22. Alam, M. K. et al. Synthetic modular antibody construction by using the SpyTag/SpyCatcher protein-ligase system. ChemBioChem

18, 2217–2221. https://​doi.​org/​10.​1002/​cbic.​20170​0411 (2017).

23. Sutherland, A. R., Alam, M. K. & Geyer, C. R. Post-translational assembly of protein parts into complex devices by using SpyTag/

SpyCatcher protein ligase. ChemBioChem 20, 319–328. https://​doi.​org/​10.​1002/​cbic.​20180​0538 (2019).

24. Wood, D. W. & Camarero, J. A. Intein applications: From protein purification and labeling to metabolic control methods. J. Biol.

Chem. 289, 14512–14519. https://​doi.​org/​10.​1074/​jbc.​R114.​552653 (2014).

25. Liu, C. et al. Convenient method of producing cyclic single-chain Fv antibodies by split-intein-mediated protein ligation and

chaperone co-expression. J. Biochem. 168, 257–263. https://​doi.​org/​10.​1093/​jb/​mvaa0​42 (2020).

26. Hemmi, S. et al. Construction of a circularly connected VHH bispecific antibody (cyclobody) for the desirable positioning of

antigen-binding sites. Biochem. Biophys. Res. Commun. 523, 72–77. https://​doi.​org/​10.​1016/j.​bbrc.​2019.​12.​018 (2020).

27. Sarmiento, C. & Camarero, J. A. Biotechnological applications of protein splicing. Curr. Protein Pept. Sci. 20, 408–424. https://​doi.​

org/​10.​2174/​13892​03720​66619​02081​10416 (2019).

28. Tornabene, P. et al. Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina. Sci.

Transl. Med. 11, eaav4523. https://​doi.​org/​10.​1126/​scitr​anslm​ed.​aav45​23 (2019).

29. Khoo, K. K. et al. Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing. Nat.

Commun. 11, 2284. https://​doi.​org/​10.​1038/​s41467-​020-​16208-6 (2020).

Scientific Reports |

Vol:.(1234567890)

(2021) 11:19411 |

https://doi.org/10.1038/s41598-021-98855-3

www.nature.com/scientificreports/

A Self-archived copy in

Kyoto University Research Information Repository

https://repository.kulib.kyoto-u.ac.jp

30. Nanda, A., Nasker, S. S., Mehra, A., Panda, S. & Nayak, S. Inteins in science: Evolution to application. Microorganisms 8, 2004.

https://​doi.​org/​10.​3390/​micro​organ​isms8​122004 (2020).

31. Han, L. et al. Naturally split intein Npu DnaE mediated rapid generation of bispecific IgG antibodies. Methods 154, 32–37. https://​

doi.​org/​10.​1016/j.​ymeth.​2018.​10.​001 (2019).

32. Zettler, J., Schütz, V. & Mootz, H. D. The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein transsplicing reaction. FEBS Lett. 583, 909–914. https://​doi.​org/​10.​1016/j.​febsl​et.​2009.​02.​003 (2009).

33. Iwai, H., Züger, S., Jin, J. & Tam, P.-H. Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme. FEBS Lett. 580, 1853–1858. https://​doi.​org/​10.​1016/j.​febsl​et.​2006.​02.​045 (2006).

34. Shah, N. H., Eryilmaz, E., Cowburn, D. & Muir, T. W. Extein residues play an intimate role in the rate-limiting step of protein

trans-splicing. J. Am. Chem. Soc. 135, 5839–5847. https://​doi.​org/​10.​1021/​ja401​015p (2013).

35. Stevens, A. J. et al. A promiscuous split intein with expanded protein engineering applications. Proc. Natl. Acad. Sci. USA 114,

8538. https://​doi.​org/​10.​1073/​pnas.​17010​83114 (2017).

36. Cheriyan, M., Pedamallu, C. S., Tori, K. & Perler, F. Faster protein splicing with the Nostoc punctiforme DnaE intein using nonnative extein residues. J. Biol. Chem. 288, 6202–6211. https://​doi.​org/​10.​1074/​jbc.​M112.​433094 (2013).

37. Martin, D. D., Xu, M.-Q. & Evans, T. C. Characterization of a naturally occurring trans-splicing intein from Synechocystis sp.

PCC6803. Biochemistry 40, 1393–1402. https://​doi.​org/​10.​1021/​bi001​786g (2001).

38. Aranko, A. S., Züger, S., Buchinger, E. & Iwaï, H. In vivo and in vitro protein ligation by naturally occurring and engineered split

DnaE inteins. PLoS ONE 4, e5185. https://​doi.​org/​10.​1371/​journ​al.​pone.​00051​85 (2009).

39. Stevens, A. J. et al. Design of a split intein with exceptional protein splicing activity. J. Am. Chem. Soc. 138, 2162–2165. https://​doi.​

org/​10.​1021/​jacs.​5b135​28 (2016).

40. Akiba, H., Satoh, R., Nagata, S. & Tsumoto, K. Effect of allotypic variation of human IgG1 on the thermal stability of disulfidelinked knobs-into-holes mutants of the Fc for stable bispecific antibody design. Antibody Ther. 2, 65–69. https://​doi.​org/​10.​1093/​

abt/​tbz008 (2019).

41. Nagata, S., Salvatore, G. & Pastan, I. DNA immunization followed by a single boost with cells: A protein-free immunization protocol

for production of monoclonal antibodies against the native form of membrane proteins. J. Immunol. Methods 280, 59–72. https://​

doi.​org/​10.​1016/​S0022-​1759(03)​00192-3 (2003).

42. Nagata, S. et al. Rapid grouping of monoclonal antibodies based on their topographical epitopes by a label-free competitive

immunoassay. J. Immunol. Methods 292, 141–155. https://​doi.​org/​10.​1016/j.​jim.​2004.​06.​009 (2004).

43. Oeemig, J. S., Beyer, H. M., Aranko, A. S., Mutanen, J. & Iwaï, H. Substrate specificities of inteins investigated by QuickDrop-cassette

mutagenesis. FEBS Lett. 594, 3338–3355. https://​doi.​org/​10.​1002/​1873-​3468.​13909 (2020).

44. Cerutti, A. et al. Engagement of CD153 (CD30 ligand) by C

­ D30+ T cells inhibits class switch DNA recombination and antibody

production in human I­ gD+ ­IgM+ B cells. J. Immunol. 165, 786. https://​doi.​org/​10.​4049/​jimmu​nol.​165.2.​786 (2000).

45. Hargreaves, P. G. & Al-Shamkhani, A. Soluble CD30 binds to CD153 with high affinity and blocks transmembrane signaling by

CD30. Eur. J. Immunol. 32, 163–173. https://​doi.​org/​10.​1002/​1521-​4141(200201)​32:1%​3c163::​AID-​IMMU1​63%​3e3.0.​CO;2-T

(2002).

46. Ise, T. et al. Immunoglobulin superfamily receptor translocation associated 2 protein on lymphoma cell lines and hairy cell leukemia

cells detected by novel monoclonal antibodies. Clin. Cancer Res. 11, 87–96 (2005).

47. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins

and proteomes. Nat. Protoc. 1, 2856–2860. https://​doi.​org/​10.​1038/​nprot.​2006.​468 (2006).

48. Adachi, J. et al. Proteome-wide discovery of unknown ATP-binding proteins and kinase inhibitor target proteins using an ATP

probe. J. Proteome Res. 13, 5461–5470. https://​doi.​org/​10.​1021/​pr500​845u (2014).

Acknowledgements

We thank Reiko Satoh and Yuko Yamanaka for their technical assistance. This study was supported partly by

the Japan Agency for Medical Research and Development (grant number JP20ak0101099; H.A. and H.K.), the

Kyoto University Foundation (H.A.), and the Takeda Science Foundation (H.A.).

Author contributions

H.A. and K.T. designed the research; H.A. conducted the experiments; T.I. and S.N. provided biological materials;

all authors analyzed the results; H.A. wrote the manuscript; all authors reviewed the manuscript.

Competing interests The authors declare no competing interests.

Additional information

Supplementary Information The online version contains supplementary material available at https://​doi.​org/​

10.​1038/​s41598-​021-​98855-3.

Correspondence and requests for materials should be addressed to H.A. or K.T.

Reprints and permissions information is available at www.nature.com/reprints.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and

institutional affiliations.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International

License, which permits use, sharing, adaptation, distribution and reproduction in any medium or

format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the

Creative Commons licence, and indicate if changes were made. The images or other third party material in this

article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the

material. If material is not included in the article’s Creative Commons licence and your intended use is not

permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/.

© The Author(s) 2021

Scientific Reports |

(2021) 11:19411 |

https://doi.org/10.1038/s41598-021-98855-3

Vol.:(0123456789)

...

参考文献をもっと見る