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Expression of Single Chain Variable Fragment (scFv) Molecules in Plants: A Comprehensive Update

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Single chain variable fragments (scFvs) are generated by joining together the variable heavy and light chain of a monoclonal antibody (mAb) via a peptide linker. They offer some advantages over the parental mAb such as low molecular weight, heterologous production, multimeric form, and multivalency. The scFvs were produced against more than 50 antigens till date using 10 different plant species as the expression system. There were considerable improvements in the expression and purification strategies of scFv in the last 24 years. With the growing demand of scFv in therapeutic and diagnostic fields, its biosynthesis needs to be increased. The easiness in development, maintenance, and multiplication of transgenic plants make them an attractive expression platform for scFv production. The review intends to provide comprehensive information about the use of plant expression system to produce scFv. The developments, advantages, pitfalls, and possible prospects of improvement for the exploitation of plants in the industrial level are discussed.

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  1. 1.

    Berger, M., Shankar, V., & Vafai, A. (2002). Therapeutic applications of monoclonal antibodies. American Journal of Medical Sciences,324(1), 14–30.

  2. 2.

    Byrne, H., Conroy, P. J., Whisstock, J. C., & O’Kennedy, R. J. (2013). A tale of two specificities: Bispecific antibodies for therapeutic and diagnostic applications. Trends in Biotechnology,31(11), 621–632. https://doi.org/10.1016/j.tibtech.2013.08.007.

  3. 3.

    Zeng, X., Shen, Z., & Mernaugh, R. (2012). Recombinant antibodies and their use in biosensors. Analytical and Bioanalytical Chemistry,402(10), 3027–3038. https://doi.org/10.1007/s00216-011-5569-z.

  4. 4.

    Bird, R. E., Hardman, K. D., Jacobson, J. W., Johnson, S., Kaufman, B. M., Lee, S. M., et al. (1988). Single-chain antigen-binding proteins. Science,242, 423–426.

  5. 5.

    Huston, J. S., Levinson, D., Mudgett-Hunter, M., Tai, M. S., Novotný, J., Margolies, M. N., et al. (1988). Protein engineering of antibody binding sites: Recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proceedings of the National Academy of Sciences of USA,85(16), 5879–5883.

  6. 6.

    Libyh, M. T., Goossens, D., Oudin, S., Gupta, N., Dervillez, X., Juszczak, G., et al. (1997). A recombinant human scFv anti-Rh(D) antibody with multiple valences using a C-terminal fragment of C4-binding protein. Blood,90, 3978–3983.

  7. 7.

    Liu, M., Wang, X., Yin, C., Zhang, Z., Lin, Q., Zhen, Y., et al. (2007). Targeting TNF-α with a tetravalent mini antibody TNF-TeAb. Biochemical Journal,406, 237–246.

  8. 8.

    Fischer, R., Schumann, D., Zimmermann, S., Drossard, J., Sack, M., & Schillberg, S. (1999). Expression and characterization of bispecific single-chain Fv fragments produced in transgenic plants. European Journal of Biochemistry,262(3), 810–816.

  9. 9.

    Semenyuk, E. G., Stremovskiy, O. A., Edelweiss, E. F., Shirshikova, O. V., Balandin, T. G., Buryanov, Y. I., et al. (2007). Expression of single-chain antibody-barstar fusion in plants. Biochimie,89(1), 31–38.

  10. 10.

    Kelly, M. P., Lee, F. T., Tahtis, K., Power, B. E., Smyth, F. E., Brechbiel, M. W., et al. (2008). Tumor targeting by a multivalent single-chain Fv (scFv) anti-Lewis Y antibody construct. Cancer Biotherapy and Radiopharmaceuticals,23(4), 411–423.

  11. 11.

    Bates, A., & Power, C. A. (2019). David vs. Goliath: The structure, function, and clinical prospects of antibody fragments. Antibodies (Basel). https://doi.org/10.3390/antib8020028.

  12. 12.

    Yusibov, V., Kushnir, N., & Streatfield, S. J. (2016). Antibody production in plants and green algae. Annual Review of Plant Biology,67, 669–701.

  13. 13.

    Streatfield, S. J., Kushnir, N., & Yusibov, V. (2015). Plant-produced candidate countermeasures against emerging and reemerging infections and bioterror agents. Plant Biotechnology Journal,13(8), 1136–1159. https://doi.org/10.1111/pbi.12475.

  14. 14.

    Peters, J., & Stoger, E. (2011). Transgenic crops for the production of recombinant vaccines and anti-microbial antibodies. Human Vaccines,7(3), 367–374.

  15. 15.

    Ma, J. K. C., Drake, P. M. W., & Christou, P. (2003). The production of recombinant pharmaceutical proteins in plants. Nature Reviews (Genetics),4, 794–805.

  16. 16.

    Cox, K. M., Sterling, J. D., Regan, J. T., Gasdaska, J. R., Frantz, K. K., Peele, C. G., et al. (2006). Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor. Nature Biotechnology,24(12), 1591–1597.

  17. 17.

    Rup, B., Alon, S., Amit-Cohen, B.-C., Brill Almon, E., Chertkoff, R., Tekoah, Y., et al. (2017). Immunogenicity of glycans on biotherapeutic drugs produced in plant expression systems—The taliglucerase alfa story. PLoS ONE,12(10), e0186211. https://doi.org/10.1371/journal.pone.0186211.

  18. 18.

    Lonoce, C., Marusic, C., Morrocchi, E., Salzano, A. M., Scaloni, A., Novelli, F., et al. (2019). Enhancing the secretion of a glyco-engineered anti-CD20 scFv-Fc antibody in hairy root cultures. Biotechnology Journal,14(3), e1800081. https://doi.org/10.1002/biot.201800081.

  19. 19.

    Wilson, D. S., Wu, J., Peluso, P., & Nock, S. (2002). Improved method for pepsinolysis of mouse IgG1 molecules to F(ab′)2 fragments. Journal of Immunological Methods,260(1–2), 29–36.

  20. 20.

    Yamaguchi, Y., Kim, H., Kato, K., Masuda, K., Shimada, I., & Arata, Y. (1995). Proteolytic fragmentation with high specificity of mouse immunoglobulin G mapping of proteolytic cleavage sites in the hinge region. Journal of Immunological Methods,181(2), 259–267.

  21. 21.

    Brinkmann, U., Reiter, Y., Jung, S. H., Lee, B., & Pastan, I. (1993). A recombinant immunotoxin containing a disulphide stabilized Fv fragment. Proceedings of the National Academy of Sciences of USA,90, 7538–7542.

  22. 22.

    Sundaresan, G., Yazaki, P. J., Shively, J. E., Finn, R. D., Larson, S. M., Raubitschek, A. A., et al. (2003). 124I-labeled engineered anti-CEA minibodies and diabodies allow high-contrast, antigen-specific small-animal PET imaging of xenografts in athymic mice. Journal of Nuclear Medicine,44(12), 1962–1969.

  23. 23.

    Kostelny, S. A., Cole, M. S., & Tso, J. Y. (1992). Formation of a bispecific antibody by the use of leucine zippers. Journal of Immunology,148, 1547–1553.

  24. 24.

    Jorgensen, M. L., Friis, N. A., Just, J., Madsen, P., Petersen, S. V., & Kristensen, P. (2014). Expression of single-chain variable fragments fused with the Fc-region of rabbit IgG in Leishmania tarentolae. Microbial Cell Factories,13, 9. https://doi.org/10.1186/1475-2859-13-9.

  25. 25.

    Jäger, V., Büssow, K., Wagner, A., Weber, S., Hust, M., Frenzel, A., et al. (2013). High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells. BMC Biotechnology,13, 52.

  26. 26.

    Kunert, R., & Reinhart, D. (2016). Advances in recombinant antibody manufacturing. Applied Microbiology and Biotechnology,100(8), 3451–3461. https://doi.org/10.1007/s00253-016-7388-9.

  27. 27.

    Frenzel, A., Hust, M., & Schirrmann, T. (2013). Expression of recombinant antibodies. Frontiers in Immunology,4, 217. https://doi.org/10.3389/fimmu.2013.00217.

  28. 28.

    Balaji, P., Satheeshkumar, P. K., Krishnan, V., & Vijayalakshmi, M. A. (2016). Expression of anti-tumor necrosis factor alpha (TNFα) single chain variable fragment (scFv) in Spirodela punctata plants transformed with Agrobacterium tumefaciens. Biotechnology and Applied Biochemistry,63(3), 354–361. https://doi.org/10.1002/bab.1373.

  29. 29.

    Bruyns, A. M., De Jaeger, G., De Neve, M., De Wilde, C., Van Montagu, M., & Depicker, A. (1996). Bacterial and plant produced scFv proteins have similar antigen-binding properties. FEBS Letters,386, 5–10.

  30. 30.

    Dong, Y., Li, J., Yao, N., Wang, D., Liu, X., Wang, N., et al. (2017). Seed-specific expression and analysis of recombinant anti-HER2 single-chain variable fragment (scFv-Fc) in Arabidopsis thaliana. Protein Expression and Purification,133, 187–192. https://doi.org/10.1016/j.pep.2017.03.009.

  31. 31.

    Sushma, K., Vijayalakshmi, M. A., Krishnan, V., & Satheeshkumar, P. K. (2011). Cloning, expression, purification and characterization of a Single chain variable fragment specific to Tumor Necrosis Factor Alpha in Escherichia coli. Journal of Biotechnology,156, 238–244. https://doi.org/10.1016/j.jbiotec.2011.06.039.

  32. 32.

    Franconi, R., Roggero, P., Pirazzi, P., Arias, F. J., Desiderio, A., Bitti, O., et al. (1999). Functional expression in bacteria and plants of an scFv antibody fragment against tospoviruses. Immunotechnology,4(3–4), 189–201.

  33. 33.

    Weisser, N. E., & Hall, J. C. (2009). Applications of single-chain variable fragment antibodies in therapeutics and diagnostics. Biotechnology Advances,27(4), 502–520.

  34. 34.

    Ahmad, Z. A., Yeap, S. K., Ali, A. M., Ho, W. Y., Alitheen, N. B., & Hamid, M. (2012). scFv antibody: Principles and clinical application. Clinical and Developmental Immunology. https://doi.org/10.1155/2012/980250.

  35. 35.

    Edgue, G., Twyman, R. M., & Beiss, V. (2017). Antibodies from plants for bionanomaterials. WIREs Nanomedicine and Nanobiotechnology,9, e1462. https://doi.org/10.1002/wnan.1462.

  36. 36.

    Geskin, L. J. (2015). Monoclonal antibodies. Dermatologic Clinics,33(4), 777–786. https://doi.org/10.1016/j.det.2015.05.015.

  37. 37.

    Yokota, T., Milenic, D. E., Whitlow, M., & Schlom, J. (1992). Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms. Cancer Research,52(12), 3402–3408.

  38. 38.

    Chowdhury, P. S., Viner, J. L., Beers, R., & Pastan, I. (1998). Isolation of a high-affinity stable single-chain Fv specific for mesothelin from DNA-immunized mice by phage display and construction of a recombinant immunotoxin with antitumor activity. Proceedings of the National Academy of Sciences of USA,95, 669–674.

  39. 39.

    Chames, P., Regenmortel, M. V., Weiss, E., & Baty, D. (2009). Therapeutic antibodies: Successes, limitations and hopes for the future. British Journal of Pharmacology,157(2), 220–233.

  40. 40.

    Monnier, P. P., Vigouroux, R. J., & Tassew, N. G. (2013). In vivo applications of single chain Fv (Variable Domain) (scFv) fragments. Antibodies,2(2), 193–208. https://doi.org/10.3390/antib2020193.

  41. 41.

    Vitaliti, A., Wittmer, M., Steiner, R., Wyder, L., Neri, D., & Klemenz, R. (2000). Inhibition of tumor angiogenesis by a single-chain antibody directed against vascular endothelial growth factor. Cancer Research,60(16), 4311–4314.

  42. 42.

    Ronca, R., Benzoni, P., Leali, D., Urbinati, C., Belleri, M., Corsini, M., et al. (2010). Antiangiogenic activity of a neutralizing human single-chain antibody fragment against fibroblast growth factor receptor 1. Molecular Cancer Therapeutics,9, 3244–3253.

  43. 43.

    Le Gall, F., Kipriyanov, S. M., Moldenhauer, G., & Little, M. (1999). Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: Effect of valency on cell binding. FEBS Letters,453(1–2), 1648.

  44. 44.

    Vaquero, C., Sack, M., Schuster, F., Finnern, R., Drossard, J., Schumann, D., et al. (2002). A carcinoembryonic antigen-specific diabody produced in tobacco. FASEB Journal,16, 408–410.

  45. 45.

    Hornig, N., & Färber-Schwarz, A. (2012). Production of bispecific antibodies: Diabodies and tandem scFv. Methods in Molecular Biology,907, 713–727. https://doi.org/10.1007/978-1-61779-974-7_40.

  46. 46.

    Gallo, E., Snyder, A. C., & Jarvik, J. W. (2015). Engineering tandem single-chain Fv as cell surface reporters with enhanced properties of fluorescence detection. Protein Engineering Design and Selection,28(10), 327–337. https://doi.org/10.1093/protein/gzv016.

  47. 47.

    Helfrich, W., Haisma, H. J., Magdolen, V., Luther, T., Bom, V. J., Westra, J., et al. (2000). A rapid and versatile method for harnessing scFv antibody fragments with various biological effector functions. Journal of Immunological Methods,237(1–2), 131–145.

  48. 48.

    Kato, T., Yui, M., Deo, V. K., & Park, E. Y. (2015). Development of Rous sarcoma virus-like particles displaying hCC49 scFv for specific targeted drug delivery to human colon carcinoma cells. Pharmaceutical Research,32(11), 3699–3707. https://doi.org/10.1007/s11095-015-1730-2.

  49. 49.

    Van Droogenbroeck, B., Cao, J., Stadlmann, J., Altmann, F., Colanesi, S., Hillmer, S., et al. (2007). Aberrant localization and underglycosylation of highly accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds. Proceedings of the National Academy of Sciences of USA,104, 1430–1435.

  50. 50.

    De Jaeger, G., Scheffer, S., Jacobs, A., Zambre, M., Zobell, O., Goossens, A., et al. (2002). Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences. Nature Biotechnology,20, 1265–1268.

  51. 51.

    Stoger, E., Vaquero, C., Torres, E., Sack, M., Nicholson, L., Drossard, J., et al. (2000). Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies. Plant Molecular Biology,42, 583–590.

  52. 52.

    Hiatt, A., Pauly, M., Whaley, K., Qiu, X., Kobinger, G., & Zeitlin, L. (2015). The emergence of antibody therapies for Ebola. Human Antibodies,23(3–4), 49–56. https://doi.org/10.3233/HAB-150284.

  53. 53.

    Rosales-Mendoza, S., Nieto-Gómez, R., & Angulo, C. (2017). A perspective on the development of plant-made vaccines in the fight against Ebola virus. Frontiers in Immunology,8, 252. https://doi.org/10.3389/fimmu.2017.00252.

  54. 54.

    Rival, S., Wisniewski, J. P., Langlais, A., Kaplan, H., Freyssinet, G., Vancanneyt, G., et al. (2008). Spirodela (duckweed) as an alternative production system for pharmaceuticals: A case study, aprotinin. Transgenic Research,17, 503–513.

  55. 55.

    Strasser, R., Stadlmann, J., Schahs, M., Stiegler, G., Quendler, H., Mach, L., et al. (2008). Generation of glycoengineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure. Plant Biotechnology Journal,6, 392–402.

  56. 56.

    Lai, H., He, J., Hurtado, J., Stahnke, J., Fuchs, A., Mehlhop, E., et al. (2014). Structural and functional characterization of an anti-West Nile virus monoclonal antibody and its single-chain variant produced in glycoengineered plants. Plant Biotechnology Journal,12(8), 1098–1107. https://doi.org/10.1111/pbi.12217.

  57. 57.

    Jutras, P. V., Marusic, C., Lonoce, C., Deflers, C., Goulet, M., Benvenuto, E., et al. (2016). An accessory protease inhibitor to increase the yield and quality of a tumour-targeting mAb in Nicotiana benthamiana leaves. PLoS ONE,11(11), e0167086. https://doi.org/10.1371/journal.pone.0167086.

  58. 58.

    Donini, M., & Marusic, C. (2019). Current state-of-the-art in plant-based antibody production systems. Biotechnology Letters,41(3), 335–346. https://doi.org/10.1007/s10529-019-02651-z.

  59. 59.

    Krishna, G., Singh, B. K., Kim, E. K., Morya, V. K., & Ramteke, P. W. (2015). Progress in genetic engineering of peanut (Arachis hypogaea L.)—A review. Plant Biotechnology Journal,13(2), 147–162. https://doi.org/10.1111/pbi.12339.

  60. 60.

    Cardi, T., D’Agostino, N., & Tripodi, P. (2017). Genetic transformation and genomic resources for next-generation precise genome engineering in vegetable crops. Frontiers in Plant Science,8, 241. https://doi.org/10.3389/fpls.2017.00241.

  61. 61.

    Doron, L., Segal, N., & Shapira, M. (2016). Transgene expression in microalgae—From tools to applications. Frontiers in Plant Science,7, 505. https://doi.org/10.3389/fpls.2016.00505.

  62. 62.

    Hiei, Y., Ishida, Y., & Komari, T. (2014). Progress of cereal transformation technology mediated by Agrobacterium tumefaciens. Frontiers in Plant Science,5, 628. https://doi.org/10.3389/fpls.2014.00628.

  63. 63.

    Singh, R. K., & Prasad, M. (2016). Advances in Agrobacterium tumefaciens-mediated genetic transformation of graminaceous crops. Protoplasma,253(3), 691–707. https://doi.org/10.1007/s00709-015-0905-3.

  64. 64.

    Yadava, P., Abhishek, A., Singh, R., Singh, I., Kaul, T., Pattanayak, A., et al. (2017). Advances in maize transformation technologies and development of transgenic maize. Frontiers in Plant Science,7, 1949. https://doi.org/10.3389/fpls.2016.01949.

  65. 65.

    McCormick, A. A., Kumagai, M. H., Hanley, K., Turpen, T. H., Hakim, I., Grill, L. K., et al. (1999). Rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain Fv epitopes in tobacco plants. Proceedings of the National Academy of Sciences of USA,96(2), 703–708.

  66. 66.

    Eto, J., Suzuki, Y., Ohkawa, H., & Yamaguchi, I. (2003). Anti-herbicide single-chain antibody expression confers herbicide tolerance in transgenic plants. FEBS Letters,550, 179–184.

  67. 67.

    Schouten, A., Roosien, J., Bakker, J., & Schots, A. (2002). Formation of disulfide bridges by a single-chain Fv antibody in the reducing ectopic environment of the plant cytosol. Journal of Biological Chemistry,277(22), 19339–19345.

  68. 68.

    Pizzuti, F., & Daroda, L. (2008). Investigating recombinant protein exudation from roots of transgenic tobacco. Environmental Biosafety Research,7, 219–226.

  69. 69.

    Urakami, E., Yamaguchi, I., Asami, T., Conrad, U., & Suzuki, Y. (2008). Immunomodulation of gibberellin biosynthesis using an anti-precursor gibberellin antibody confers gibberellin-deficient phenotypes. Planta,228(5), 863–873. https://doi.org/10.1007/s00425-008-0788-z.

  70. 70.

    Yuan, Q., Hu, W., Pestka, J. J., He, S. Y., & Hart, P. (2000). Expression of a functional antizearalenone single-chain Fv antibody in transgenic Arabidopsis plants. Applied and Environmental Microbiology,66, 3499–3505.

  71. 71.

    Kathuria, S., Sriraman, R., Sack, M., Pal, R., Artsaenko, O., Talwar, G. P., et al. (2002). Efficacy of plant-produced recombinant antibodies against HCG. Human Reproduction,17, 2054–2061.

  72. 72.

    Dobhal, S., Chaudhary, V. K., Singh, A., Pandey, D., Kumar, A., & Agrawal, S. (2013). Expression of recombinant antibody (single chain antibody fragment) in transgenic plant Nicotiana tabacum cv. Xanthi. Molecular Biology Reports,40(12), 7027–7037. https://doi.org/10.1007/s11033-013-2822-x.

  73. 73.

    Zhang, M.-Y., Zimmermann, S., Fischer, R., & Schillberg, S. (2008). Generation and evaluation of movement protein-specific single-chain antibodies for delaying symptoms of Tomato spotted wilt virus infection in tobacco. Plant Pathology,57, 854–860. https://doi.org/10.1111/j.1365-3059.2008.01863.x.

  74. 74.

    Yang, J. G., Hwang, K. H., Kil, E. J., Park, J., Cho, S., Lee, Y. G., et al. (2017). PVX-tolerant potato development using a nucleic acid-hydrolyzing recombinant antibody. Acta Virologica,61(1), 105–115. https://doi.org/10.4149/av_2017_01_105.

  75. 75.

    Marusic, C., Pioli, C., Stelter, S., Novelli, F., Lonoce, C., Morrocchi, E., et al. (2018). N-glycan engineering of a plant-produced anti-CD20-hIL-2 immunocytokine significantly enhances its effector functions. Biotechnology and Bioengineering,115, 565–576. https://doi.org/10.1002/bit.26503.

  76. 76.

    De Jaeger, G., Buys, E., Eeckhout, D., De Wilde, C., Jacobs, A., Kapila, J., et al. (1999). High level accumulation of single-chain variable fragments in the cytosol of transgenic Petunia hybrida. European Journal of Biochemistry,259, 426–434.

  77. 77.

    Scheller, J., Leps, M., & Conrad, U. (2006). Forcing single-chain variable fragment production in tobacco seeds by fusion to elastin-like polypeptides. Plant Biotechnology Journal,4, 243–249. https://doi.org/10.1111/j.1467-7652.2005.00176.x.

  78. 78.

    Zimmermann, J., Saalbach, I., Jahn, D., Giersberg, M., Haehnel, S., Wedel, J., et al. (2009). Antibody expressing pea seeds as fodder for prevention of gastrointestinal parasitic infections in chickens. BMC Biotechnology,9, 79. https://doi.org/10.1186/1472-6750-9-79.

  79. 79.

    Yao, N., Ai, L., Dong, Y. Y., Liu, X. M., Wang, D. Z., Wang, N., et al. (2016). Expression of recombinant human anti-TNF-α scFv-Fc in Arabidopsis thaliana seeds. Genetics and Molecular Research. https://doi.org/10.4238/gmr.15027726.

  80. 80.

    Ritala, A., Leelavathi, S., Oksman-Caldentey, K. M., Reddy, V. S., & Laukkanen, M. L. (2014). Recombinant barley-produced antibody for detection and immunoprecipitation of the major bovine milk allergen, β-lactoglobulin. Transgenic Research,23(3), 477–487. https://doi.org/10.1007/s11248-014-9783-2.

  81. 81.

    Wang, D., Ma, J., Sun, D., Li, H., Jiang, C., & Li, X. (2015). Expression of bioactive anti-CD20 antibody fragments and induction of ER stress response in Arabidopsis seeds. Applied Microbiology and Biotechnology,99(16), 6753–6764.

  82. 82.

    Lonoce, C., Salem, R., Marusic, C., Jutras, P. V., Scaloni, A., Salzano, A. M., et al. (2016). Production of a tumor-targeting antibody with a human compatible glycosylation profile in N. benthamiana hairy root cultures. Biotechnology Journal,11, 1209–1220. https://doi.org/10.1002/biot.201500628.

  83. 83.

    David, K. M., Couch, D., Braun, N., Brown, S., Grosclaude, J., & Perrot-Rechenmann, C. (2007). The auxin-binding protein 1 is essential for the control of cell cycle. Plant Journal,50, 197–206.

  84. 84.

    Gomes, M., Alvarez, M. A., Quellis, L. R., Becher, M. L., Castro, J. M. A., Gameiro, J., et al. (2019). Expression of an scFv antibody fragment in Nicotiana benthamiana and in vitro assessment of its neutralizing potential against the snake venom metalloproteinase BaP1 from Bothrops asper. Toxicon,160, 38–46. https://doi.org/10.1016/j.toxicon.2019.02.011.

  85. 85.

    Schillberg, S., Raven, N., Spiegel, H., Rasche, S., & Buntru, M. (2019). Critical analysis of the commercial potential of plants for the production of recombinant proteins. Frontiers in Plant Science,10, 720. https://doi.org/10.3389/fpls.2019.00720.

  86. 86.

    Canto, T. (2016). Transient expression systems in plants: Potentialities and constraints. Advances in Experimental Medicine and Biology,896, 287–301. https://doi.org/10.1007/978-3-319-27216-0_18.

  87. 87.

    Mortimer, C. L., Dugdale, B., & Dale, J. L. (2015). Updates in inducible transgene expression using viral vectors: From transient to stable expression. Current Opinion in Biotechnology,32, 85–92. https://doi.org/10.1016/j.copbio.2014.11.009.

  88. 88.

    Marsian, J., & Lomonossoff, G. P. (2016). Molecular pharming—VLPs made in plants. Current Opinion in Biotechnology,37, 201–206.

  89. 89.

    Henquet, M., Eigenhuijsen, J., Hesselink, T., Spiegel, H., Schreuder, M., van Duijn, E., et al. (2011). Characterization of the single-chain Fv-Fc antibody MBP10 produced in Arabidopsis alg3 mutant seeds. Transgenic Research,20(5), 1033–1042. https://doi.org/10.1007/s11248-010-9475-5.

  90. 90.

    Eeckhout, D., Fiers, E., Sienaert, R., Snoeck, V., Depicker, A., & De Jaeger, G. (2000). Isolation and characterization of recombinant antibody fragments against CDC2a from Arabidopsis thaliana. European Journal of Biochemistry,267, 6775–6783.

  91. 91.

    McCormick, A. A., Reinl, S. J., Cameron, T. I., Vojdani, F., Fronefield, M., Levy, R., et al. (2003). Individualized human scFv vaccines produced in plants: Humoral anti-idiotype responses in vaccinated mice confirm relevance to the tumor Ig. Journal of Immunological Methods,278(1), 95–104.

  92. 92.

    Capodicasa, C., Chiani, P., Bromuro, C., De Bernardis, F., Catellani, M., Palma, A. S., et al. (2011). Plant production of anti-β-glucan antibodies for immunotherapy of fungal infections in humans. Plant Biotechnology Journal,9, 776–787. https://doi.org/10.1111/j.1467-7652.2010.00586.x.

  93. 93.

    Kopertekh, L., Meyer, T., Freyer, C., & Hust, M. (2019). Transient plant production of Salmonella typhimurium diagnostic antibodies. Biotechnology Reports (Amsterdam),21, e00314. https://doi.org/10.1016/j.btre.2019.e00314.

  94. 94.

    Landry, N., Ward, B. J., Trépanier, S., Montomoli, E., Dargis, M., Lapini, G., et al. (2010). Preclinical and clinical development of plant-made virus-like particle vaccine against avian H5N1 influenza. PLoS ONE,5(12), e15559. https://doi.org/10.1371/journal.pone.0015559.

  95. 95.

    De Wilde, K., De Buck, S., Vanneste, K., & Depicker, A. (2013). Recombinant antibody production in Arabidopsis seeds triggers an unfolded protein response. Plant Physiology,161(2), 1021–1033. https://doi.org/10.1104/pp.112.209718.

  96. 96.

    De Muynck, B., Navarre, C., & Boutry, M. (2010). Production of antibodies in plants: Status after twenty years. Plant Biotechnology Journal,8, 529–563. https://doi.org/10.1111/j.1467-7652.2009.00494.x.

  97. 97.

    Marschall, L., Sagmeister, P., & Herwig, C. (2017). Tunable recombinant protein expression in E. coli: Promoter systems and genetic constraints. Applied Microbiology and Biotechnology,101(2), 501–512.

  98. 98.

    Leavitt, J. M., & Alper, H. S. (2015). Advances and current limitations in transcript-level control of gene expression. Current Opinion in Biotechnology,34, 98–104.

  99. 99.

    Park, S. H., Ong, R. G., & Sticklen, M. (2016). Strategies for the production of cell wall-deconstructing enzymes in lignocellulosic biomass and their utilization for biofuel production. Plant Biotechnology Journal,14(6), 1329–1344.

  100. 100.

    Peeters, K., Wilde, C. D., & Depicker, A. (2001). Highly efficient targeting and accumulation of a Fab fragment within the secretory pathway and apoplast of Arabidopsis thaliana. European Journal of Biochemistry,268, 4251–4260.

  101. 101.

    Morandini, F., Avesani, L., Bortesi, L., Van Droogenbroeck, B., De Wilde, K., Arcalis, E., et al. (2011). Non-food/feed seeds as biofactories for the high-yield production of recombinant pharmaceuticals. Plant Biotechnology Journal,9, 911–921. https://doi.org/10.1111/j.1467-7652.2011.00605.x.

  102. 102.

    Vaquero, C., Sack, M., Chandler, J., Drossard, J., Schuster, F., Monecke, M., et al. (1999). Transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves. Proceedings of the National Academy of Sciences of USA,96, 11128–11133.

  103. 103.

    Zimmermann, S., Schillberg, S., Liao, Y. C., & Fischer, R. (1998). Intracellular expression of TMV-specific single-chain Fv fragments leads to improved virus resistance in Nicotiana tabacum. Molecular Breeding,4, 369–379.

  104. 104.

    Heim, U., Wang, Q., Kurz, T., Borisjuk, L., Golombek, S., Neubohn, B., et al. (2001). Expression patterns and subcellular localization of a 52 kDa sucrose-binding protein homologue of Vicia faba (VfSBPL) suggest different functions during development. Plant Molecular Biology,7, 461–474.

  105. 105.

    Fiedler, U., Filistein, R., Wobus, U., & Bäumlein, H. (1993). A complex ensemble of cis-regulatory elements controls the expression of a Vicia faba non-storage protein gene. Plant Molecular Biology,22, 669–679.

  106. 106.

    Saalbach, I., Giersberg, M., & Conrad, U. (2001). High-level expression of a single chain Fv fragment (scFv) antibody in transgenic pea seeds. Journal of Plant Physiology,158, 529–533.

  107. 107.

    Lee, W. S., Tzen, J. T., Kridl, J. C., Radke, S. E., & Huang, A. H. (1991). Maize oleosin is correctly targeted to seed oil bodies in Brassica napus transformed with the maize oleosin gene. Proceedings of the National Academy of Sciences of USA,88, 6181–6185.

  108. 108.

    Winichayakul, S., Pernthaner, A., Livingston, S., Cookson, R., Scott, R., & Roberts, N. (2012). Production of active single-chain antibodies in seeds using trimeric polyoleosin fusion. Journal of Biotechnology,161(4), 407–413. https://doi.org/10.1016/j.jbiotec.2012.07.195.

  109. 109.

    Yamamoto, T., Hoshikawa, K., Ezura, K., Okazawa, R., Fujita, S., Takaoka, M., et al. (2018). Improvement of the transient expression system for production of recombinant proteins in plants. Scientific Reports,8, 4755. https://doi.org/10.1038/s41598-018-23024-y.

  110. 110.

    Von Schaewen, A., Sturm, A., Oneill, J., & Chrispeels, M. J. (1993). Isolation of a mutant Arabidopsis plant that lacks N-acetyl glucosaminyl transferase-I and is unable to synthesize Golgi-modified complex N-linked glycans. Plant Physiology,102, 1109–1118.

  111. 111.

    Ramirez, N., Ayala, M., Lorenzo, D., Palenzuela, D., Herrera, L., Doreste, V., et al. (2002). Expression of a single-chain Fv antibody fragment specific for the hepatitis B surface antigen in transgenic tobacco plants. Transgenic Research,11, 61–64.

  112. 112.

    Loose, A., Van Droogenbroeck, B., Hillmer, S., Grass, J., Pabst, M., Castilho, A., et al. (2011). Expression of antibody fragments with a controlled N-glycosylation pattern and induction of endoplasmic reticulum-derived vesicles in seeds of Arabidopsis. Plant Physiology,155(4), 2036–2048. https://doi.org/10.1104/pp.110.171330.

  113. 113.

    Petruccelli, S., Otegui, M. S., Lareu, F., Tran Dinh, O., Fitchette, A. C., Circosta, A., et al. (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 Biotechnology Journal,4, 511–527.

  114. 114.

    De Wilde, C., De Neve, M., De Rycke, R., Bruyns, A.-M., De Jaeger, G., Van Montagu, M., et al. (1996). Intact antigen-binding MAK33 antibody and Fab fragment accumulate in intercellular spaces of Arabidopsis thaliana. Plant Science,114, 233–241. https://doi.org/10.1016/0168-9452(96)04331-2.

  115. 115.

    Le Gall, F., Bové, J. M., & Garnier, M. (1998). Engineering of a single-chain variable-fragment (scFv) antibody specific for the stolbur phytoplasma (mollicute) and its expression in Escherichia coli and tobacco plants. Applied Environmental Microbiology,64, 4566–4572.

  116. 116.

    Hensel, G., Floss, D. M., Arcalis, E., Sack, M., Melnik, S., Altmann, F., et al. (2015). Transgenic production of an anti HIV antibody in the Barley endosperm. PLoS ONE,10(10), e0140476. https://doi.org/10.1371/journal.pone.0140476.

  117. 117.

    Zakharov, A., Giersberg, M., Hosein, F., Melzer, M., Muntz, K., & Saalbach, I. (2004). Seed-specific promoters direct gene expression in non-seed tissue. Journal of Experimental Botany,55(402), 1463–1471.

  118. 118.

    Boothe, J., Nykiforuk, C., Shen, Y., Zaplachinski, S., Szarka, S., Kuhlman, P., et al. (2010). Seed-based expression systems for plant molecular farming. Plant Biotechnology Journal,8, 588–606.

  119. 119.

    Geertsma, E. R., Groeneveld, M., Slotboom, D. J., & Poolman, B. (2008). Quality control of overexpressed membrane proteins. Proceedings of the National Academy of Sciences of USA,105, 5722–5727.

  120. 120.

    Schlegel, S., Klepsch, M., Gialama, D., Wickström, D., Slotboom, D. J., & de Gier, J. (2010). Revolutionizing membrane protein overexpression in bacteria. Microbial Biotechnology,3(4), 403–411. https://doi.org/10.1111/j.1751-7915.2009.00148.

  121. 121.

    Dvorak, P., Chrast, L., Nikel, P. I., Fedr, R., Soucek, K., Sedlackova, M., et al. (2015). Exacerbation of substrate toxicity by IPTG in Escherichia coli BL21 (DE3) carrying a synthetic metabolic pathway. Microbial Cell Factories,14, 201. https://doi.org/10.1186/s12934-015-0393-3.

  122. 122.

    Hattab, G., Moncoq, K., Warschawski, D. E., & Miroux, B. (2014). Escherichia coli as host for membrane protein structure determination: A global analysis. Biophysical Journal,106(2 Suppl 1), 46a.

  123. 123.

    Abdeen, A., Schnell, J., & Miki, B. (2010). Transcriptome analysis reveals absence of unintended effects in drought-tolerant transgenic plants overexpressing the transcription factor ABF3. BMC Genomics,11, 69. https://doi.org/10.1186/1471-2164-11-69.

  124. 124.

    Pons, E., Peris, J. E., & Pena, L. (2012). Field performance of transgenic citrus trees: Assessment of the long-term expression of uidA and nptII transgenes and its impact on relevant agronomic and phenotypic characteristics. BMC Biotechnology,12, 41. https://doi.org/10.1186/1472-6750-12-41.

  125. 125.

    Gullì, M., Salvatori, E., Fusaro, L., Pellacani, C., Manes, F., & Marmiroli, N. (2015). Comparison of drought stress response and gene expression between a GM Maize variety and a near-isogenic Non-GM variety. PLoS ONE,10(2), e0117073. https://doi.org/10.1371/journal.pone.0117073.

  126. 126.

    Marusic, C., Novelli, F., Salzano, A. M., Scaloni, A., Benvenuto, E., Pioli, C., et al. (2016). Production of an active anti-CD20-hIL-2 immunocytokine in Nicotiana benthamiana. Plant Biotechnology Journal,14, 240–251.

  127. 127.

    Villani, M. E., Morgun, B., Brunetti, P., Marusic, C., Lombardi, R., Pisoni, I., et al. (2009). Plant pharming of a full-sized, tumour-targeting antibody using different expression strategies. Plant Biotechnology Journal,7, 59–72. https://doi.org/10.1111/j.1467-7652.2008.00371.x.

  128. 128.

    de Muynck, B., deNavarre, C., Nizet, Y., Stadlmann, J., & Boutry, M. (2009). Different subcellular localization and glycosylation for a functional antibody expressed in Nicotiana tabacum plants and suspension cells. Transgenic Research,18, 467–482. https://doi.org/10.1007/s11248-008-9240.

  129. 129.

    Mandal, M. K., Ahvari, H., Schillberg, S., & Schiermeyer, A. (2016). Tackling unwanted proteolysis in plant production hosts used for molecular farming. Frontiers in Plant Science,7, 267. https://doi.org/10.3389/fpls.2016.00267.

  130. 130.

    Li, X.-G., Chen S.-B., Lu, Z.-X., Chang, T.-J., Zeng, Q.-C., & Zu, Z. (2002). Impact of copy number on transgene expression in tobacco. Acta Botanica Sinica,44, 120–123.

  131. 131.

    Kohli, A., Gonzales-Melendi, P., Abranches, R., Capell, T., Stoger, E., & Christou, P. (2006). The quest to understand the basis and mechanisms that control expression of introduced transgenes in crop plants. Plant Signaling and Behavior,1(4), 185–195.

  132. 132.

    Phoolcharoen, W., Prehaud, C., van Dolleweerd, C. J., Both, L., da Costa, A., Lafon, M., et al. (2017). Enhanced transport of plant-produced rabies single-chain antibody-RVG peptide fusion protein across an in cellulo blood–brain barrier device. Plant Biotechnology Journal,15(10), 1331–1339. https://doi.org/10.1111/pbi.12719.

  133. 133.

    Phoolcharoen, W., Banyard, A. C., Prehaud, C., Selden, D., Wu, G., Birch, C. P. D., et al. (2019). In vitro and in vivo evaluation of a single chain antibody fragment generated in planta with potent rabies neutralization activity. Vaccine,37, 4673–4680. https://doi.org/10.1016/j.vaccine.2018.02.057.

  134. 134.

    Foetisch, K., Westphal, S., Lauer, I., Retzek, M., Altmann, F., Kolarich, D., et al. (2003). Biological activity of IgE specific for cross-reactive carbohydrate determinants. Journal of Allergy and Clinical Immunology,111, 889–896.

  135. 135.

    Mari, A. (2002). IgE to cross-reactive carbohydrate determinants: Analysis of the distribution and appraisal of the in vivo and in vitro reactivity. International Archives of Allergy and Immunology,129, 286–295.

  136. 136.

    Wong-Arce, A., González-Ortega, O., & Rosales-Mendoza, S. (2017). Plant-made vaccines in the fight against cancer. Trends in Biotechnology,35(3), 241–256. https://doi.org/10.1016/j.tibtech.2016.12.002.

  137. 137.

    Hiatt, A., Bohorova, N., Bohorov, O., Goodman, C., Kim, D., Pauly, M. H., et al. (2014). Glycan variants of a respiratory syncytial virus antibody with enhanced effector function and in vivo efficacy. Proceedings of the National Academy of Sciences of USA,111(16), 5992–5997. https://doi.org/10.1073/pnas.1402458111.

  138. 138.

    Bosch, D., Castilho, A., Loos, A., Schots, A., & Steinkellner, H. (2013). N-glycosylation of plant-produced recombinant proteins. Current Pharmaceutical Design,19, 5503–5512.

  139. 139.

    Webster, D. E., & Thomas, M. C. (2012). Post-translational modification of plant-made foreign proteins; glycosylation and beyond. Biotechnology Advances,30, 410–418.

  140. 140.

    Gomord, V., Fitchette, A. C., Menu-Bouaouiche, L., Saint-Jore-Dupas, C., Plasson, C., Michaud, D., et al. (2010). Plant-specific glycosylation patterns in the context of therapeutic protein production. Plant Biotechnology Journal,8, 564–587.

  141. 141.

    Budzianowski, J. (2015). Tobacco against Ebola virus disease. Przegl Lek, 72(10), 567–571 (only abstract available).

  142. 142.

    Na, W., Park, N., Yeom, M., & Song, D. (2015). Ebola outbreak in Western Africa 2014: What is going on with Ebola virus? Clinical and Experimental Vaccine Research,4, 17–22.

  143. 143.

    McCormick, A. A., Reddy, S., Reinl, S. J., Cameron, T. I., Czerwinkski, D. K., Vojdani, F., et al. (2008). Plant-produced idiotype 667 vaccines for the treatment of non-Hodgkin’s lymphoma: Safety and immunogenicity in a phase I clinical study. Proceedings of the National Academy of Sciences of USA,105, 10131–10136.

  144. 144.

    Tusé, D., Ku, N., Bendandi, M., Becerra, C., Collins, R., Jr., Langford, N., et al. (2015). Clinical safety and immunogenicity of tumor-targeted, plant-made Id-KLH conjugate vaccines for follicular lymphoma. Biomed Research International,2015, 648143. https://doi.org/10.1155/2015/648143.

  145. 145.

    Monger, W., Alamillo, J. M., Sola, I., Perrin, Y., Bestagno, M., Burrone, O. R., et al. (2006). An antibody derivative expressed from viral vectors passively immunizes pigs against transmissible gastroenteritis virus infection when supplied orally in crude plant extracts. Plant Biotechnology Journal,4, 623–631. https://doi.org/10.1111/j.1467-7652.2006.00206.x.

  146. 146.

    Tusé, D., Tu, T., & McDonald, K. A. (2014). Manufacturing economics of plant-made biologics: Case studies in therapeutic and industrial enzymes. Biomedical Research International,2014, 256135. https://doi.org/10.1155/2014/256135.

  147. 147.

    Buyel, J. F., Twyman, R. M., & Fischer, R. (2017). Very-large-scale production of antibodies in plants: The biologization of manufacturing. Biotechnology Advances,35(4), 458–465. https://doi.org/10.1016/j.biotechadv.2017.03.011.

  148. 148.

    Lim, J. A. C., Patkar, A., McDonagh, G., Sinclair, A., & Lucy, P. (2010). Modeling bioprocess cost. BioProcess. International,8, 62–70.

  149. 149.

    Buyel, J. F. (2019). Plant molecular farming—Integration and exploitation of side streams to achieve sustainable biomanufacturing. Frontiers in Plant Science,9, 1893. https://doi.org/10.3389/fpls.2018.01893.

  150. 150.

    Kelley, B. (2007). Very large scale monoclonal antibody purification: The case for conventional unit operations. Biotechnology Progress,23, 995–1008. https://doi.org/10.1021/bp070117s.

  151. 151.

    Madeira, L. M., Szeto, T. H., Henquet, M., Raven, N., Runions, J., Huddleston, J., et al. (2016). High-yield production of a human monoclonal IgG by rhizosecretion in hydroponic tobacco cultures. Plant Biotechnology Journal,14, 615–624. https://doi.org/10.1111/pbi.12407.

  152. 152.

    Tran, D. T., Cho, S., Hoang, P. M., Kim, J., Kil, E., Lee, T., et al. (2016). A codon-optimized nucleic acid hydrolyzing single-chain antibody confers resistance to Chrysanthemums against Chrysanthemum stunt viroid infection. Plant Molecular Biology Reporter,34, 221. https://doi.org/10.1007/s11105-015-0915-5.

  153. 153.

    Jung, Y., Rhee, Y., Auh, C. K., Shim, H., Choi, J. J., Kwon, S. T., et al. (2009). Production of recombinant single chain antibodies (scFv) in vegetatively reproductive Kalanchoe pinnata by in planta transformation. Plant Cell Reports,28(10), 1593–1602. https://doi.org/10.1007/s00299-009-0758-3.

  154. 154.

    Villani, M. E., Roggero, P., Bitti, O., Benvenuto, E., & Franconi, R. (2005). Immunomodulation of cucumber mosaic virus infection by intrabodies selected in vitro from a stable single-framework phage display library. Plant Molecular Biology,58(3), 305–316.

  155. 155.

    Galeffi, P., Lombardi, A., Donato, M. D., Latini, A., Sperandei, M., Cantale, C., et al. (2005). Expression of single-chain antibodies in transgenic plants. Vaccine,23(15), 1823–1827.

  156. 156.

    Safarnejad, M. R., Fischer, R., & Commandeur, U. (2009). Recombinant-antibody-mediated resistance against Tomato yellow leaf curl virus in Nicotiana benthamiana. Archives of Virology,154(3), 457–467. https://doi.org/10.1007/s00705-009-0330-z.

  157. 157.

    Fecker, L. F., Koenig, R., & Obermeier, C. (1997). Nicotiana benthamiana plants expressing beet necrotic yellow vein virus (BNYVV) coat protein-specific scFv are partially protected against the establishment of the virus in the early stages of infection and its pathogenic effects in the late stages of infection. Archives of Virology,142(9), 1857–1863.

  158. 158.

    Boonrod, K., Galetzka, D., Nagy, P. D., Conrad, U., & Krczal, G. (2004). Single-chain antibodies against a plant viral RNA-dependent RNA polymerase confer virus resistance. Nature Biotechnology,22(7), 856–862.

  159. 159.

    He, J., Lai, H., Engle, M., Gorlatov, S., Gruber, C., Steinkellner, H., et al. (2014). Generation and analysis of novel plant-derived antibody-based therapeutic molecules against West Nile virus. PLoS ONE,9(3), e93541. https://doi.org/10.1371/journal.pone.0093541.

  160. 160.

    Artsaenko, O., Peisker, M., zur Nieden, U., Fiedler, U., Weiler, E. W., Müntz, K., & Conrad, U. (1995). Expression of a single chain Fv, antibody against abscisic acid creates a wilty phenotype in transgenic tobacco. Plant Journal,8, 745–750.

  161. 161.

    Longstaff, M., Newell, C. A., Boonstra, B., Strachan, G., Learmonth, D., Harris, W. J., et al. (1998). Expression and characterization of single-chain antibody fragments produced in transgenic plants against the organic herbicides atrazine and paraquat. Biochimica Biophysica Acta,1381, 147–160.

  162. 162.

    Schouten, A., Rossien, J., Van Engelen, F. A., De Jong, G. A., Borst-Vrenssen, A. W., Zilverentant, J. F., et al. (1996). The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobacco. Plant Molecular Biology,30, 781–793.

  163. 163.

    Fiedler, U., & Conrad, U. (1995). High-level production and long-term storage of engineered antibodies in transgenic tobacco seeds. Biotechnology (NY),13(10), 1090–1093.

  164. 164.

    Owen, M., Gandecha, A., Cockburn, B., & Whitelam, G. (1992). Synthesis of a functional anti-phytochrome single-chain Fv protein in transgenic tobacco. Biotechnology (NY),10(7), 790–794.

  165. 165.

    Schillberg, S., Zimmermann, S., Voss, A., & Fischer, R. (1999). Apoplastic and cytosolic expression of full-size antibodies and antibody fragments in Nicotiana tabacum. Transgenic Research,8, 255–263.

  166. 166.

    Olea-Popelka, F., McLean, M. D., Horsman, J., Almquist, K., Brandle, J. E., & Hall, J. C. (2005). Increasing expression of an anti-picloram single-chain variable fragment (ScFv) antibody and resistance to picloram in transgenic tobacco (Nicotiana tabacum). Journal of Agricultural and Food Chemistry,53(17), 6683–6690.

  167. 167.

    Makvandi-Nejad, S., McLean, M. D., Hirama, T., Almquist, K. C., Mackenzie, C. R., & Hall, J. C. (2005). Transgenic tobacco plants expressing a dimeric single-chain variable fragment (scfv) antibody against Salmonella enterica serotype Paratyphi B. Transgenic Research,14(5), 785–792.

  168. 168.

    Almquist, K. C., McLean, M. D., Niu, Y., Byrne, G., Olea-Popelka, F. C., Murrant, C., et al. (2006). Expression of an anti-botulinum toxin A neutralizing single-chain Fv recombinant antibody in transgenic tobacco. Vaccine,24(12), 2079–2086.

  169. 169.

    Morgun, B. V., Deshmuk, S., Stepaniuk, V., Pasternak, T., & ukach, N. (2005). Site-specific expression of single-stranded antibody fragments in Nicotiana tabacum. Tsitologica Genetica, 39(3), 43–49 (article in Russian).

  170. 170.

    Lee, G., Shim, H.-K., Kwon, M.-H., Son, S.-H., Kim, K.-Y., Park, E.-Y., et al. (2013). RNA virus accumulation is inhibited by ribonuclease activity of 3D8 scFv in transgenic Nicotiana tabacum. Plant Cell Tissue and Organ Culture,115, 189–197.

  171. 171.

    Xu MQ, Li HP, Wang M, Wu ZC, Borth WB, Hsu HT, Hu JS (2006) Transgenic plants expressing a single-chain fv antibody to tomato spotted wilt virus (tswv) are resistant to tswv systemic infection. Acta Horticulturae 722:337–348. https://doi.org/10.17660/actahortic.2006.722.43

  172. 172.

    Sunilkumar, G., Waghela, S. D., Campbell, L. M., & Rathore, K. S. (2009). Expression of anti-K99 scFv in transgenic rice tissues and its functional characterization. Transgenic Research,18(3), 347–360. https://doi.org/10.1007/s11248-008-9223-2.

  173. 173.

    Schouten, A., Roosien, J., de Boer, J. M., Wilmink, A., Rosso, M. N., Bosch, D., et al. (1997). Improving scFv antibody expression levels in the plant cytosol. FEBS Letters,415, 235–241.

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The author acknowledges the support from the Centre for Advanced Study, Department of Botany, Banaras Hindu University. Varanasi, India.

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Satheeshkumar, P.K. Expression of Single Chain Variable Fragment (scFv) Molecules in Plants: A Comprehensive Update. Mol Biotechnol 62, 151–167 (2020). https://doi.org/10.1007/s12033-020-00241-3

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  • Recombinant protein
  • Targeted expression
  • Glycoengineering
  • scFv