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Tobacco seeds as efficient production platform for a biologically active anti-HBsAg monoclonal antibody

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Abstract

The use of plants as heterologous hosts is one of the most promising technologies for manufacturing valuable recombinant proteins. Plant seeds, in particular, constitute ideal production platforms for long-term applications requiring a steady supply of starting material, as they combine the general advantages of plants as bioreactors with the possibility of biomass storage for long periods in a relatively small volume, thus allowing manufacturers to decouple upstream and downstream processing. In the present work we have used transgenic tobacco seeds to produce large amounts of a functionally active mouse monoclonal antibody against the Hepatitis B Virus surface antigen, fused to a KDEL endoplasmic reticulum retrieval motif, under control of regulatory sequences from common bean (Phaseolus vulgaris) seed storage proteins. The antibody accumulated to levels of 6.5 mg/g of seed in the T3 generation, and was purified by Protein A affinity chromatography combined with SEC-HPLC. N-glycan analysis indicated that, despite the KDEL signal, the seed-derived plantibody bore both high-mannose and complex-type sugars that indicate partial passage through the Golgi compartment, although its performance in the immunoaffinity purification of HBsAg was unaffected. An analysis discussing the industrial feasibility of replacing the currently used tobacco leaf-derived plantibody with this seed-derived variant is also presented.

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References

  • Agraz A, Duarte C, Costa L, Perez L, Paez R, Pujol V, Fontirrochi G (1994) Immunoaffinity purification of recombinant hepatitis B surface antigen from yeast using a monoclonal antibody. J Chromatogr A 672:25–33

    Article  CAS  PubMed  Google Scholar 

  • Andrianov V, Borisjuk N, Pogrebnyak N, Brinker A, Dixon J, Spitsin S, Flynn J, Matyszczuk P, Andryszak K, Laurelli M (2010) Tobacco as a production platform for biofuel: overexpression of Arabidopsis DGAT and LEC2 genes increases accumulation and shifts the composition of lipids in green biomass. Plant Biotechnol J 8:277–287

    Article  CAS  PubMed  Google Scholar 

  • Bigge J, Patel TP, Bruce JA, Goulding PN, Charles SM, Parekh RB (1995) Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. Anal Biochem 230:229–238

    Article  CAS  PubMed  Google Scholar 

  • Cheung S, Sun SSM, Chan JCN, Tong PCY (2009) Expression and subcellular targeting of human insulin-like growth factor binding protein-3 in transgenic tobacco plants. Transgenic Res 18:943–951

    Article  CAS  PubMed  Google Scholar 

  • De Jaeger G, Scheffer S, Jacobs A, Zambre M, Zobell O, Goossens A, Depicker A, Angenon G (2002) Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences. Nat Biotechnol 20:1265–1268

    Article  PubMed  Google Scholar 

  • Fischer R, Schillberg S, Twyman RM (2009) Molecular farming of antibodies in plants. Springer, Recent Advances in Plant Biotechnology, pp 35–63

    Google Scholar 

  • Floss D, Sack M, Arcalis E, Stadlmann J, Quendler H, Rademacher T, Stoger E, Scheller J, Fischer R, Conrad U (2009) Influence of elastin-like peptide fusions on the quantity and quality of a tobacco-derived human immunodeficiency virus-neutralizing antibody. Plant Biotechnol J 7:899–913

    Article  CAS  PubMed  Google Scholar 

  • Giannelos P, Zannikos F, Stournas S, Lois E, Anastopoulos G (2002) Tobacco seed oil as an alternative diesel fuel: physical and chemical properties. Ind Crops Prod 16:1–9

    Article  CAS  Google Scholar 

  • Gorantala J, Grover S, Rahi A, Chaudhary P, Rajwanshi R, Sarin N, Bhatnagar R (2014) Generation of protective immune response against anthrax by oral immunization with protective antigen plant-based vaccine. J Biotechnol 176:1–10

    Article  CAS  PubMed  Google Scholar 

  • Guile G, Rudd PM, Wing DR, Prime SB, Dwek RA (1996) A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Anal Biochem 240:210–226

    Article  CAS  PubMed  Google Scholar 

  • He J, Lai H, Engle M, Gorlatov S, Gruber C, Steinkellner H, Diamond MS, Chen Q (2014) Generation and analysis of novel plant-derived antibody-based therapeutic molecules against West Nile virus. PLoS One 9:93541

    Article  Google Scholar 

  • Hernandez A, Lopez A, Ceballo Y, Rosabal L, Rosabal Y, Tiel K, Perez M, Gonzalez EM, Ramos O, Enriquez G (2013) High-level production and aggregation of hepatitis B surface antigen in transgenic tobacco seeds. Biotecnol Apl 30:97–100

    CAS  Google Scholar 

  • Hiatt A, Caffferkey R, Bowdish K (1989) Production of antibodies in transgenic plants. Nature 342:76–78

    Article  CAS  PubMed  Google Scholar 

  • Horsch R, Klee H (1986) Rapid assay of foreign gene expression in leaf discs transformed by Agrobacterium tumefaciens: role of T-DNA borders in the transfer process. PNAS 83:4428–4432

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Huang T, Mcdonald K (2012) Bioreactor systems for in vitro production of foreign proteins using plant cell cultures. Biotechnol Adv 30:398–409

    Article  CAS  PubMed  Google Scholar 

  • Kaiser J (2008) Is the drought over for pharming? Science 320:473–475

    Article  CAS  PubMed  Google Scholar 

  • Köhler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495–497

    Article  PubMed  Google Scholar 

  • Laemmli U (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  • Leyva A, Franco A, González T, Sánchez JC, López I, Geada D, Hernández N, Montañés M, Delgado I, Valdés R (2007) A rapid and sensitive ELISA to quantify an HBsAg specific monoclonal antibody and a plant-derived antibody during their downstream purification process. Biologicals 35:19–25

    Article  CAS  PubMed  Google Scholar 

  • Loos A, Van Droogenbroeck B, Hillmer S, Grass J, Kunert R, Cao J, Robinson DG, Depicker A, Steinkellner H (2010) Production of monoclonal antibodies with a controlled N-glycosylation pattern in seeds of Arabidopsis thaliana. Plant Biotechnol J 9:179–192

    Article  Google Scholar 

  • Loos A, Van Droogenbroeck B, Hillmer S, Grass J, Pabst M, Castilho A, Kunert R, Liang M, Arcalis E, Robinson DG (2011) Expression of antibody fragments with a controlled N-glycosylation pattern and induction of endoplasmic reticulum-derived vesicles in seeds of Arabidopsis. Plant Physiol 155:2036–2048

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Morandini F, Avesani L, Bortesi L, Van Droogenbroeck B, De Wilde K, Arcalis E, Bazzoni F, Santi L, Brozzetti A, Falorni A (2011) Non food/feed seeds as biofactories for the high-yield production of recombinant pharmaceuticals. Plant Biotechnol J 9:911–921

    Article  CAS  PubMed  Google Scholar 

  • Perrin Y, Vaquero C, Gerrard I, Sack M, Drossard J, Stoger E, Christou P, Fischer R (2000) Transgenic pea seeds as bioreactors for the production of a single-chain Fv fragment (scFV) antibody used in cancer diagnosis and therapy. Mol Breed 6:345–352

    Article  CAS  Google Scholar 

  • Petruccelli S, Otegui MS, Lareu F, Tran Dinh O, Fitchette AC, Circosta A, Rumbo M, Bardor M, Carcamo R, Gomord V (2006) A KDEL-tagged monoclonal antibody is efficiently retained in the endoplasmic reticulum in leaves, but is both partially secreted and sorted to protein storage vacuoles in seeds. Plant Biotechnol J 4:511–527

    CAS  PubMed  Google Scholar 

  • Pujol M, Ramírez NI, Ayala M, Gavilondo JV, Valdés R, Rodríguez M, Brito J, Padilla S, Gómez L, Reyes B (2005) An integral approach towards a practical application for a plant-made monoclonal antibody in vaccine purification. Vaccine 23:1833–1837

    Article  CAS  PubMed  Google Scholar 

  • Rademacher T, Sack M, Arcalis E, Stadlmann J, Balzer S, Altmann F, Quendler H, Stiegler G, Kunert R, Fischer R (2008) Recombinant antibody 2G12 produced in maize endosperm efficiently neutralizes HIV-1 and contains predominantly single GlcNAc glycans. Plant Biotechnol J 6:189–201

    Article  CAS  PubMed  Google Scholar 

  • Ramessar K, Sabalza M, Capell T, Christou P (2008) Maize plants: an ideal production platform for effective and safe molecular pharming. Plant Sci 174:409–419

    Article  CAS  Google Scholar 

  • Ramírez N, Rodríguez M, Ayala M, Cremata J, Pérez M, Martínez A, Linares M, Hevia Y, Páez R, Valdés R, Gavilondo JV, Selman-Housein G (2003) Expression and characterization of an anti-(hepatitis B surface antigen) glycosylated mouse antibody in transgenic tobacco (Nicotiana tabacum) plants and its use in the immunopurification of its target antigen. Biotechnol Appl Bioc 38:223–230

    Article  Google Scholar 

  • Rhonda F, Ling-Ling G, Lena S, Danica G, Craig A, Karam S (2011) Identification and characterization of seed storage protein transcripts from Lupinus angustifolius. BMC Plant Biol 11:59

    Article  Google Scholar 

  • Rodriguez M, Pérez L, Gavilondo JV, Garrido G, Bequet-Romero M, Hernández I, Huerta V, Cabrera G, Pérez M, Ramos O (2013) Comparative in vitro and experimental in vivo studies of the anti-epidermal growth factor receptor antibody nimotuzumab and its aglycosylated form produced in transgenic tobacco plants. Plant Biotechnol J 11:53–65

    Article  CAS  PubMed  Google Scholar 

  • Rossi L, Fusi E, Baldi G, Fogher C, Cheli F, Baldi A, Dell´Orto V (2013) Tobacco seeds by-product as protein source for piglets. Open J Vet Med 3:73

    Article  Google Scholar 

  • Schillberg S, Fischer R, Emans N (2003) Molecular farming of recombinant antibodies in plants. Cell Mol Life Sci 60:433–445

    Article  CAS  PubMed  Google Scholar 

  • Sengupta-Gopalan C, Reichert NA, Barker RF, Hall TC, Kemp JD (1985) Developmentally regulated expression of the ß-phaseolin gene in tobacco seed. PNAS 82:3320–3324

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Shenoy V, Kwon K, Rathinasabapathy A, Lin S, Jin G, Song C, Shil P, Nair A, Qi Y, Li Q (2014) Oral delivery of angiotensin-converting enzyme 2 and angiotensin-(1-7) bioencapsulated in plant cells attenuates pulmonary hypertension. Hypertension 64:1248–1259

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Shukla A, Thommes J (2010) Recent advances in large-scale production of monoclonal antibodies and related proteins. Trends Biotechnol 28:253–261

    Article  CAS  PubMed  Google Scholar 

  • Stoger E, Fischer R, Moloney M, Ma J (2014) Plant molecular pharming for the treatment of chronic and infectious diseases. Annu Rev Plant Biol 65:743–768

    Article  CAS  PubMed  Google Scholar 

  • Stöger E, Vaquero C, Torres E, Sack M, Nicholson L, Drossard J, Williams S, Keen D, Perrin Y, Christou P (2000) Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies. Plant Mol Biol 42:583–590

    Article  PubMed  Google Scholar 

  • Stöger E, Sack M, Perrin Y, Vaquero C, Torres E, Twyman RM, Christou P, Fischer R (2002) Practical considerations for pharmaceutical antibody production in different crop systems. Mol Breed 9:149–158

    Article  Google Scholar 

  • Stöger E, Ma JKC, Fischer R, Christou P (2005) Sowing the seeds of success: pharmaceutical proteins from plants. Curr Opin Biotech 16:167–173

    Article  PubMed  Google Scholar 

  • Taheripour F, Hertel TW, Tyner WE, Beckman JF, Birur DK (2010) Biofuels and their by-products: global economic and environmental implications. Biomass Bioenerg 34:278–289

    Article  CAS  Google Scholar 

  • Triguero A, Cabrera G, Cremata J, Yuen C-T, Wheeler J, Ramírez NI (2005) Plant-derived mouse IgG monoclonal antibody fused to KDEL endoplasmic reticulum-retention signal is N-glycosylated homogeneously throughout the plant with mostly high-mannose-type N-glycans. Plant Biotechnol J 3:449–457

    Article  CAS  PubMed  Google Scholar 

  • Triguero A, Cabrera G, Rodriguez M, Soto J, Zamora Y, Perez M, Wormald MR, Cremata J (2011) Differential N-glycosylation of a monoclonal antibody expressed in tobacco leaves with and without endoplasmic reticulum retention signal apparently induces similar in vivo stability in mice. Plant Biotechnol J 9:1120–1130

    Article  CAS  PubMed  Google Scholar 

  • Valdés R, Gómez L, Padilla S, Brito J, Reyes B, Álvarez T, Mendoza O, Herrera O, Ferro W, Pujol M (2003a) Large-scale purification of an antibody directed against hepatitis B surface antigen from transgenic tobacco plants. BBRC 308:94–100

    PubMed  Google Scholar 

  • Valdés R, Reyes B, Alvarez T, García J, Montero JA, Figueroa A, Gómez L, Padilla S, Geada D, Abrahantes MC (2003b) Hepatitis B surface antigen immunopurification using a plant-derived specific antibody produced in large scale. BBRC 310:742–747

    PubMed  Google Scholar 

  • Van Droogenbroeck B, Cao J, Stadlmann J, Altmann F, Colanesi S, Hillmer S, Robinson DG, Van Lerberge E, Terryn N, Van Montagu M (2007) Aberrant localization and underglycosylation of highly accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds. PNAS 104:1430–1435

    Article  PubMed Central  PubMed  Google Scholar 

  • Wilken L, Nikolov Z (2012) Recovery and purification of plant-made recombinant proteins. Biotechnol Adv 30:419–433

    Article  CAS  PubMed  Google Scholar 

  • Wright A, Morrison S (1997) Effect of glycosylation on antibody function: implications for genetic engineering. Trends Biotechnol 15:26–32

    Article  CAS  PubMed  Google Scholar 

  • Zhang Y, Li D, Jin X, Huang Z (2014) Fighting Ebola with ZMapp: spotlight on plant-made antibody. Sci China Life Sci 57:987–988

    Article  PubMed  Google Scholar 

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Acknowledgments

We thank the Department of Plant Systems Biology of Ghent University Belgium for supplying the signals for expression in seeds. In addition, we would like to extend our appreciation to the staff of the experimental area at CIGB for their help in cultivating the tobacco plants used in this study and to the Monoclonal Antibody Production Department at CIGB for their help with the immunoaffinity purification of HBsAg. No potential conflicts of interest are declared in this work.

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Authors and Affiliations

  1. Plant Biotechnology Department, Center for Genetic Engineering and Biotechnology (CIGB), PO Box 6162, 10600, Havana, Havana, Cuba

    Abel Hernández-Velázquez, Alina López-Quesada, Yanaysi Ceballo-Cámara, Kenia Tiel-González, Liliana Mirabal-Ortega, Marlene Pérez-Martínez, Rosabel Pérez-Castillo, Yamilka Rosabal-Ayán, Osmani Ramos-González, Gil Enríquez-Obregón & Merardo Pujol-Ferrer

  2. Department of Carbohydrate Chemistry, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba

    Gleysin Cabrera-Herrera

  3. Department of Plant Systems Biology, VIB, Plant-made Antibodies and Immunogens, Ghent, Belgium

    Ann Depicker

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  1. Abel Hernández-Velázquez
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  2. Alina López-Quesada
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Correspondence to Abel Hernández-Velázquez.

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Hernández-Velázquez, A., López-Quesada, A., Ceballo-Cámara, Y. et al. Tobacco seeds as efficient production platform for a biologically active anti-HBsAg monoclonal antibody. Transgenic Res 24, 897–909 (2015). https://doi.org/10.1007/s11248-015-9890-8

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