Production of functional recombinant cyclic citrullinated peptide monoclonal antibody in transgenic rice cell suspension culture

  • Do Van Giap
  • Jae-Wan Jung
  • Nan-Sun KimEmail author
Original Paper


Cyclic citrullinated peptide (CCP) antibody has been shown recently to be a promising marker for early detection and diagnosis of rheumatoid arthritis (RA). In order to exploit newly developed therapies for RA, early intervention is crucial in preventing irreversible joint damage. Here, we describe use of a plant expression system to produce a CCP antibody that could be used in the early diagnosis of RA. Heavy and light chain gene sequences of a CCP monoclonal antibody (CCP mAb) were cloned from the hybridoma cell (12G1) and introduced into two separate plant expression vectors under the control of the rice α-amylase 3D (RAmy3D) promoter system. The vectors were introduced into rice calli (Oryza sativa L. cv. Dongjin) using Agrobacterium tumefaciens mediated transformation. Integration of the CCP mAb genes into rice chromosomes was confirmed by a genomic DNA polymerase chain reaction and expression was verified by northern blot analysis of mRNA. The in vivo assembly and secretion of CCP mAb occurred in transgenic rice cell suspension culture under the RAmy3D expression system; accumulated CCP mAbs in the medium were purified by protein G affinity chromatography. Immunoblot assays and ELISA showed these plant-produced CCP mAbs successfully bound to a synthetic CCP antigen. Taken together, our results suggest that CCP mAb produced in a transgenic rice suspension culture were easily purified and biologically active against their antigen in the RA, and thus may be used a specific serological marker, which is present very early in the RA.


Cyclic citrullinated peptide monoclonal antibody (CCP mAb) Rice α-amylase 3D promoter Transgenic rice cell suspension culture 



This research was supported by the Advanced Production Technology Development Program, Ministry for Food Agriculture, Forestry and Fisheries (312037-05) and Do Van Giap was supported by the BK21 plus program in the Department of Bioactive Material Sciences, Chonbuk National University, Republic of Korea. The authors are appreciative of Dr. JH Ju of Catholic University for providing hybridoma cell line and also grateful to Dr. YS Jang of Chonbuk National University for performing the cloning of heavy and light chain of CCP from hybridoma cell.

Authors’ contribution

DV and JW carried out almost all experiments in this study and prepared the manuscripts. NS provided suggestions for experiments and wrote the manuscript. All the authors have read and approved the final manuscript.

Compliance with ethical standards

Competing interests

The authors declare that they have no competing interests.


  1. Alexiou I, Germenis A, Zioqas A, Thedoridou K, Sakkas LI (2007) Diagnostic value of anti-cyclic citrullinated peptide antibodies in Greek patients with rheumatoid arthritis. BMC Musculoskelet Disord 8:37CrossRefGoogle Scholar
  2. Aljanabi SM, Martinez I (1997) Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques. Nucl Acids Res 25:4692–4693CrossRefGoogle Scholar
  3. Brar HK, Bhattacharyya MK (2012) Expression of a single-chain variable-fragment antibody against a Fusarium virguliforme toxin peptide enhances tolerance to sudden death syndrome in transgenic soybean plants. Mol Plant Microbe Interact 25:817–824CrossRefGoogle Scholar
  4. Bukhari MA, Wiles NJ, Lunt M, Harrison BJ, Scott DG, Symmons DP, Silman AJ (2003) Influence of disease modifying therapy on radiographic outcome in inflammatory polyarthritis at five years: results from a large observational inception study. Arthritis Rheum 48:46–53CrossRefGoogle Scholar
  5. Buyel JF, Twyman RM, Fischer R (2017) Very-large-scale production of antibodies in plants: the biologization of manufacturing. Biotechnol Adv 35:458–465CrossRefGoogle Scholar
  6. Casadevall A (2002) Passive antibody administration (immediate immunity) as a specific defense against biological weapons. Emerg Infect Dis 8:833–841CrossRefGoogle Scholar
  7. Chen MH, Liu LF, Chen YR, Wu HK, Yu SM (1994) Expression of α-amylases, carbohydrate metabolism, and autophagy in cultured rice cells is coordinately regulated by sugar nutrient. Plant J 6:625–636CrossRefGoogle Scholar
  8. Gelvin SB (2017) Integration of Agrobacterium T-DNA into the plant genome. Annu Rev Genet 51:195–217CrossRefGoogle Scholar
  9. Giritch A, Marillonnet S, Engler C, van Eldik G, Botterman J, Klimyuk V, Gleba Y (2006) Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors. Proc Natl Acad Sci USA 103:14701–14706CrossRefGoogle Scholar
  10. Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–282CrossRefGoogle Scholar
  11. Higel F, Seidl A, Sorgel F, Friess W (2016) N-glycosylation heterogeneity and the influence on structure, function and pharmacokinetics of monoclonal antibodies and Fc fusion proteins. Eur J Pharm Biopharm 100:94–100CrossRefGoogle Scholar
  12. Hong SY, Lee TS, Kim J, Jung JH, Cho CW, Kim TG, Kwon TH, Jang YS, Yang MS (2008) Tumor targeting of humanized fragment antibody secreted from transgenic rice cell suspension culture. Plant Mol Biol 68:413–422CrossRefGoogle Scholar
  13. Huang N, Chandler J, Thomas BR, Koizumi N, Rodriguez R (1993) Metabolic regulation of α-amylase gene expression in transgenic cell cultures of rice (Oryza sativa L.). Plant Mol Biol 23:737–747CrossRefGoogle Scholar
  14. Huang Z, Phoolcharoen W, Lai H, Piensook K, Cardineau G, Zeitlin L, Whaley KJ, Arntzen CJ, Mason HS, Chen Q (2010) High-level rapid production of full-size monoclonal antibodies in plants by a single-vector DNA replicon system. Biotechnol Bioeng 106:9–17Google Scholar
  15. Itty S, Pulido JS, Bakri S, Baratz KH, Matteson EL, Hodge DO (2008) Anti-cyclic citrullinated peptide, rheumatoid factor, and ocular symptoms typical of rheumatoid arthritis. Trans Am Ophthalmol Soc 106:75–81Google Scholar
  16. Ju JH, Kim YK (2015) Rheumatoid arthritis diagnosis kit. United States Patent Application Publication. US2015/0080245 A1Google Scholar
  17. Jung JW, Kim NS, Jang SH, Shin YJ, Yang MS (2016) Production and characterization of recombinant human acid α-glucosidase in transgenic rice cell suspension culture. J Biotechnol 226:44–53CrossRefGoogle Scholar
  18. Kim NS, Yu HY, Chung ND, Shin YJ, Kwon TH, Yang MS (2011) Production of functional recombinant bovine trypsin in transgenic rice cell suspension cultures. Protein Expr Purif 76:121–126CrossRefGoogle Scholar
  19. Kim NS, Jang SH, Yu HY, Chung ND, Kwon TH, Yang MS, Kim TG (2013) Amylase and cysteine proteinase gene knockdown in rice cells using RNA interference for enhancing production of recombinant proteins. Plant Cell Tiss Organ Cult 114:97–107CrossRefGoogle Scholar
  20. Kim BG, Kim SH, Kim NS, Huy NX, Choi YS, Lee JY, Jang YS, Yang MS, Kim TG (2014) Production of monoclonal antibody against FimA protein from Porphyromonas gingivalis in rice cell suspension culture. Plant Cell Tiss Organ Cult 118:293–304CrossRefGoogle Scholar
  21. Kim YK, Lee J, Jung H, Hyoju Yi, Rim YA, Jung SM, Ju JH (2015) Development of synthetic anti-cyclic citrullinated peptide antibody and its arthritogenic role. Clin Transl Immunol 4:e51CrossRefGoogle Scholar
  22. Kolotilin I, Topp E, Cox E, Devriendt B, Conarad U, Joensuu J, Stöger E, Warzecha H, McAllister T, Potter A, McLean MD, Hall JC, Menassa R (2014) Plant-based solutions for veterinary immunotherapeutics and prophylactics. Vet Res 45:117CrossRefGoogle Scholar
  23. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  24. Lallemand J, Bouche F, Desiron C, Stautemas J, De Lemos Esteves F, Perilleux C, Tocquin P (2015) Extracellular peptidase hunting for improvement of protein production in plant cells and roots. Front Plant Sci 6:1–10CrossRefGoogle Scholar
  25. Landewe RB (2003) The benefits of early treatment in rheumatoid arthritis: confounding by indication, and the issue of timing. Arthritis Rheum 48:1–5CrossRefGoogle Scholar
  26. Lard LR, Visser H, Speyer I, vander Horst-Bruinsma IE, Zwinderman AH, Breedveld FC, Hazes JM (2001) Early versus delayed treatment in patients with recent-onset rheumatoid arthritis: comparison of two cohorts who received different treatment strategies. Am J Med 111:446–451CrossRefGoogle Scholar
  27. Larrick JW, Yu L, Naftzger C, Wycoff K (2001) Production of secretory IgA antibodies in plants. Biomol Eng 18:87–94CrossRefGoogle Scholar
  28. Magy B, Tollet J, Laterre R, Boutry M, Navarre C (2014) Accumulation of secreted antibodies in plant cell cultures varies according to the isotype, host species and culture conditions. Plant Biotechnol J 12:457–467CrossRefGoogle Scholar
  29. Martinez EO, Ramirez DFH, Nunez-Alvarez CA, Cabiedes J (2011) Citrullinated proteins in rheumatoid arthritis. Reumatol Clin 7:68–71CrossRefGoogle Scholar
  30. McDonald KA, Hong LM, Trombly DM, Xie Q, Jackman AP (2005) Production of alpha-1-antitrypsin from transgenic rice cell culture in a membrane bioreactor. Biotechnol Prog 21:728–734CrossRefGoogle Scholar
  31. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, vol 1, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  32. Santos RB, Abranches R, Fischer R, Sack M, Holland T (2016) Putting the spotlight back on plant suspension cultures. Front Plant Sci 7:297CrossRefGoogle Scholar
  33. Schellekens GA, de Jong BA, van den Hoogen FH, van de Putte LB, van Venrooij WJ (1998) Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies. J Clin Invest 101:273–281CrossRefGoogle Scholar
  34. Schellekens GA, Visser H, de Jong BA, van den Hoogen FH, Hazes JM, Breedveld FC, van Venrooij WJ (2000) The diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide. Arthritis Rheum 43:155–163CrossRefGoogle Scholar
  35. Schillberg S, Raven N, Fischer R, Twyman RM, Schiermeyer A (2013) Molecular farming of pharmaceutical proteins using plant suspension cell and tissue cultures. Curr Pharm Des 19:5531–5542CrossRefGoogle Scholar
  36. Serdaroglu M, Cakirbay H, Deger O, Cengiz S, Kul S (2008) The association of anti-CCP antibodies with disease activity in rheumatoid arthritis. Rheumatol Int 28:965–970CrossRefGoogle Scholar
  37. Shin YJ, Hong SY, Kwon TH, Jang YS, Yang MS (2003) High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture. Biotechnol Bioeng 82:778–783CrossRefGoogle Scholar
  38. Simmons CR, Huang N, Cao Y, Rodriguez RI (1991) Synthesis and secretion of α-amylase by rice callus: evidence for differential gene expression. Biotechnol Bioeng 38:545–551CrossRefGoogle Scholar
  39. Van Haute E, Joos H, Maes M, Warren G, Van Montagu M, Schell J (1983) Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. EMBO J 2:411–417CrossRefGoogle Scholar
  40. Vossenaar ER, Van Venrooij WJ (2004) Anti-CCP antibodies, a highly specific marker for (early rheumatoid arthritis. Clin Applied Immunol Rev 4:239–262CrossRefGoogle Scholar
  41. Weiner GJ (2015) Building better monoclonal antibody-based therapeutics. Nat Rev Cancer 15:361–370CrossRefGoogle Scholar
  42. Wycoff KL (2005) Secretory IgA antibodies from plants. Curr Pharm Des 11:2429–2437CrossRefGoogle Scholar
  43. Xu J, Zhang N (2014) On the way to commercializing plant cell culture platform for biopharmaceuticals: present status and prospect. Pharm Bioprocess 2:499–518CrossRefGoogle Scholar
  44. Zeitli L, Olmsted SS, Moench TR, Co MS, Martinel BJ, Paradkar VM, Russell DR, Queen C, Cone RA, Whaley KJ (1998) A humanized monoclonal antibody produced in transgenic plants for immunoprotection of the vagina against genital herpes. Nat Biotechnol 16:1361–1364CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Department of Molecular BiologyChonbuk National UniversityJeonju-siRepublic of Korea
  2. 2.National Institute of Horticultural & Herbal Science (NIHHS)Rural Development Administration (RDA)WanjuRepublic of Korea

Personalised recommendations