Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Production of a bispecific antibody targeting TNF-α and C5a in Pichia pastoris and its therapeutic potential in rheumatoid arthritis

Abstract

Objective

To provide an alternative therapeutic modality for rheumatoid arthritis (RA), a novel bispecific antibody (BsAb) targeting human tumor necrosis factor α (TNF-α) and human complement component C5a was constructed.

Results

BsAb was expressed in Pichia pastoris and secreted into the culture medium as a functional protein. In vitro functional study demonstrated that BsAb could simultaneously bind to TNF-α and C5a and neutralize their biological actions. Furthermore, BsAb showed significant improvements in both the antigen-binding affinity and the neutralizing ability as compared to its original antibodies produced in E. coli. It was also found that TNF-α and C5a had an additive/synergistic effect on promoting the production of inflammatory cytokines and chemokines and C5a receptor (C5aR) expression in human macrophages. Compared to single inhibition of TNF-α or C5a with respective antibody, BsAb showed a superior efficacy in blocking inflammatory cytokines, chemokines, and C5aR response, as well as in lowering the C5a-mediated chemotaxis of macrophages via C5aR in vitro.

Conclusions

With improved production processing and the ability to simultaneously block TNF-α and C5a action, BsAb has a great potential to be developed into a therapeutic agent and may offer a better therapeutic index for RA.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. Andersson C, Wenander CS, Usher PA, Hebsgaard JB, Sondergaard BC, Rono B, Mackay C, Friedrichsen B, Chang C, Tang R, Hornum L (2014) Rapid-onset clinical and mechanistic effects of anti-C5aR treatment in the mouse collagen-induced arthritis model. Clin Exp Immunol 177(1):219–233. https://doi.org/10.1111/cei.12338

  2. Banda NK, Hyatt S, Antonioli AH, White JT, Glogowska M, Takahashi K, Merkel TJ, Stahl GL, Mueller-Ortiz S, Wetsel R, Arend WP, Holers VM (2012) Role of C3a receptors, C5a receptors, and complement protein C6 deficiency in collagen antibody-induced arthritis in mice. J Immunol 188(3):1469–1478. https://doi.org/10.4049/jimmunol.1102310

  3. Boeggeman EE, Ramakrishnan B, Qasba PK (2003) The N-terminal stem region of bovine and human beta1,4-galactosyltransferase I increases the in vitro folding efficiency of their catalytic domain from inclusion bodies. Protein Expr Purif 30(2):219–229. https://doi.org/10.1016/S1046-5928(03)00093-7

  4. Cavaillon JM, Fitting C, Haeffner-Cavaillon N (1990) Recombinant C5a enhances interleukin 1 and tumor necrosis factor release by lipopolysaccharide-stimulated monocytes and macrophages. Eur J Immunol 20(2):253–257. https://doi.org/10.1002/eji.1830200204

  5. Choy EH, Panayi GS (2001) Cytokine pathways and joint inflammation in rheumatoid arthritis. N Engl J Med 344(12):907–916. https://doi.org/10.1056/NEJM200103223441207

  6. Cregg JM, Cereghino JL, Shi J, Higgins DR (2000) Recombinant protein expression in Pichia pastoris. Mol Biotechnol 16(1):23–52. https://doi.org/10.1385/MB:16:1:23

  7. Firestein GS (1996) Invasive fibroblast-like synoviocytes in rheumatoid arthritis. Passive responders or transformed aggressors? Arthritis Rheum 39(11):1781–1790. https://doi.org/10.1002/art.1780391103

  8. Firestein GS (2004) The T cell cometh: interplay between adaptive immunity and cytokine networks in rheumatoid arthritis. J Clin Invest 114(4):471–474. https://doi.org/10.1172/JCI22651

  9. Firestein GS (2005) Immunologic mechanisms in the pathogenesis of rheumatoid arthritis. J Clin Rheumatol 11(3 Suppl):S39–S44. https://doi.org/10.1097/01.rhu.0000166673.34461.33

  10. Gonzalez JM, Franzke CW, Yang F, Romero R, Girardi G (2011) Complement activation triggers metalloproteinases release inducing cervical remodeling and preterm birth in mice. Am J Pathol 179(2):838–849. https://doi.org/10.1016/j.ajpath.2011.04.024

  11. Grant EP, Picarella D, Burwell T, Delaney T, Croci A, Avitahl N, Humbles AA, Gutierrez-Ramos JC, Briskin M, Gerard C, Coyle AJ (2002) Essential role for the C5a receptor in regulating the effector phase of synovial infiltration and joint destruction in experimental arthritis. J Exp Med 196(11):1461–1471. https://doi.org/10.1084/jem.20020205

  12. Gross V, Andus T (1992) Human recombinant C5a enhances lipopolysaccharide-induced synthesis of interleukin-6 by human monocytes. Eur J Clin Invest 22(4):271–276. https://doi.org/10.1111/j.1365-2362.1992.tb01462.x

  13. Guo RF, Ward PA (2005) Role of C5a in inflammatory responses. Annu Rev Immunol 23:821–852. https://doi.org/10.1146/annurev.immunol.23.021704.115835

  14. Hornum L, Hansen AJ, Tornehave D, Fjording MS, Colmenero P, Watjen IF, Soe Nielsen NH, Bliddal H, Bartels EM (2017) C5a and C5aR are elevated in joints of rheumatoid and psoriatic arthritis patients, and C5aR blockade attenuates leukocyte migration to synovial fluid. PLoS ONE 12(12):e0189017. https://doi.org/10.1371/journal.pone.0189017

  15. Jose PJ, Moss IK, Maini RN, Williams TJ (1990) Measurement of the chemotactic complement fragment C5a in rheumatoid synovial fluids by radioimmunoassay: role of C5a in the acute inflammatory phase. Ann Rheum Dis 49(10):747–752. https://doi.org/10.1136/ard.49.10.747

  16. Kinne RW, Brauer R, Stuhlmuller B, Palombo-Kinne E, Burmester GR (2000) Macrophages in rheumatoid arthritis. Arthritis Res Ther 2(3):189–202. https://doi.org/10.1186/ar86

  17. Lee H, Zahra D, Vogelzang A, Newton R, Thatcher J, Quan A, So T, Zwirner J, Koentgen F, Padkjaer SB, Mackay F, Whitfeld PL, Mackay CR (2006) Human C5aR knock-in mice facilitate the production and assessment of anti-inflammatory monoclonal antibodies. Nat Biotechnol 24(10):1279–1284. https://doi.org/10.1038/nbt1248

  18. Liu M, Wang X, Yin C, Zhang Z, Lin Q, Zhen Y, Huang H (2006) One-step on-column purification and refolding of a single-chain variable fragment (scFv) antibody against tumour necrosis factor alpha. Biotechnol Appl Biochem 43(Pt 3):137–145. https://doi.org/10.1042/BA20050194

  19. Liu M, Wang X, Yin C, Zhang Z, Lin Q, Zhen Y, Huang H (2007) Targeting TNF-alpha with a tetravalent mini-antibody TNF-TeAb. Biochem J 406(2):237–246. https://doi.org/10.1042/BJ20070149

  20. Liu M, Wang X, Yin C, Zhang Z, Lin Q, Zhen Y, Huang H (2008) A novel bivalent single-chain variable fragment (scFV) inhibits the action of tumour necrosis factor alpha. Biotechnol Appl Biochem 50(Pt 4):173–179. https://doi.org/10.1042/BA20070229

  21. Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22(4):249–270. https://doi.org/10.1002/yea.1208

  22. McInnes IB, Schett G (2011) The pathogenesis of rheumatoid arthritis. N Engl J Med 365(23):2205–2219. https://doi.org/10.1056/NEJMra1004965

  23. Mehta G, Scheinman RI, Holers VM, Banda NK (2015) A new approach for the treatment of arthritis in mice with a novel conjugate of an anti-C5aR1 antibody and C5 small interfering RNA. J Immunol 194(11):5446–5454. https://doi.org/10.4049/jimmunol.1403012

  24. Nandakumar KS, Jansson A, Xu B, Rydell N, Ahooghalandari P, Hellman L, Blom AM, Holmdahl R (2010) A recombinant vaccine effectively induces C5a-specific neutralizing antibodies and prevents arthritis. PLoS ONE 5(10):e13511. https://doi.org/10.1371/journal.pone.0013511

  25. Rosenfeld RD, Zeni L, Welcher AA, Narhi LO, Hale C, Marasco J, Delaney J, Gleason T, Philo JS, Katta V, Hui J, Baumgartner J, Graham M, Stark KL, Karbon W (1998) Biochemical, biophysical, and pharmacological characterization of bacterially expressed human agouti-related protein. Biochemistry 37(46):16041–16052. https://doi.org/10.1021/bi981027m

  26. Sadik CD, Miyabe Y, Sezin T, Luster AD (2018) The critical role of C5a as an initiator of neutrophil-mediated autoimmune inflammation of the joint and skin. Semin Immunol 37:21–29. https://doi.org/10.1016/j.smim.2018.03.002

  27. Siebert S, Tsoukas A, Robertson J, McInnes I (2015) Cytokines as therapeutic targets in rheumatoid arthritis and other inflammatory diseases. Pharmacol Rev 67(2):280–309. https://doi.org/10.1124/pr.114.009639

  28. Siouti E, Andreakos E (2019) The many facets of macrophages in rheumatoid arthritis. Biochem Pharmacol 165:152–169. https://doi.org/10.1016/j.bcp.2019.03.029

  29. Smolen JS, Maini RN (2006) Interleukin-6: a new therapeutic target. Arthritis Res Ther 8(Suppl 2):S5. https://doi.org/10.1186/ar1969

  30. Smolen JS, Aletaha D, Koeller M, Weisman MH, Emery P (2007) New therapies for treatment of rheumatoid arthritis. Lancet 370(9602):1861–1874. https://doi.org/10.1016/S0140-6736(07)60784-3

  31. Sturfelt G, Truedsson L (2012) Complement in the immunopathogenesis of rheumatic disease. Nat Rev Rheumatol 8(8):458–468. https://doi.org/10.1038/nrrheum.2012.75

  32. Wang Y, Rollins SA, Madri JA, Matis LA (1995) Anti-C5 monoclonal antibody therapy prevents collagen-induced arthritis and ameliorates established disease. Proc Natl Acad Sci USA 92(19):8955–8959. https://doi.org/10.1073/pnas.92.19.8955

  33. Wang L, Han G, Wang R, Chen G, Xu R, Xiao H, Li X, Geng S, Li Y, Li X, Wang J, Feng J, Riedemann NC, Guo R, Shen B, Li Y (2010) Regulation of IL-8 production by complement-activated product, C5a, in vitro and in vivo during sepsis. Clin Immunol 137(1):157–165. https://doi.org/10.1016/j.clim.2010.05.012

Download references

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 30973669) and the Key Science-Technology foundation of Hubei Provincial Department of Education (Grant No. D20141002).

Author information

Correspondence to Mengyuan Liu.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Research involving human and animal rights

This article dose not contain any studies with human participants or animal performed by any of the authors.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Chen, J., He, Z., Fan, Y. et al. Production of a bispecific antibody targeting TNF-α and C5a in Pichia pastoris and its therapeutic potential in rheumatoid arthritis. Biotechnol Lett (2020). https://doi.org/10.1007/s10529-020-02830-3

Download citation

Keywords

  • Bispecific antibody
  • Complement component C5a
  • Rheumatoid arthritis
  • Tumor necrosis factor α