Abstract
This report describes the stable expression of a medically important antibody in the staple cereal crops rice and wheat. We successfully expressed a single-chain Fv antibody (ScFvT84.66) against carcinoembryonic antigen (CEA), a well characterized tumor-associated marker antigen. scFv constructs were engineered for recombinant antibody targeting to the plant cell apoplast and ER. Up to 30 μg/g of functional recombinant antibody was detected in the leaves and seeds of wheat and rice. We confirmed that transgenic dry seeds could be stored for at least five months at room temperature, without significant loss of the amount or activity of scFvT84.66. Our results represent the first transition from model plant expression systems, such as tobacco and Arabidopsis, to widely cultivated cereal crops, such as rice and wheat, for expression of an antibody molecule that has already shown efficacy in clinical applications. Thus, we have established that molecular pharming in cereals can be a viable production system for such high-value pharmaceutical macromolecules. Our findings provide a strong foundation for exploiting alternative uses of cereal crops both in industrialized and developing countries.
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References
Altpeter, F., Vasil, V., Srivastava, V., Stöger, E. and Vasil, I.K. 1996. Accelerated production of transgenic wheat (Triticum aestivum L.) plants. Plant Cell Rep. 16: 12–17.
Artsaenko, O., Peisker, M., zur Nieden, U., Fiedler, U., Weiler, E.W., Müntz, K. and Conrad, U. 1995. Expression of a singlechain Fv antibody against abscisic acid creates a wilty phenotype in transgenic tobacco. Plant J. 8: 745–750.
Artsaenko, O., Kettig, B., Fiedler, U., Conrad, U. and Düring, K. 1998. Potato tubers as a biofactory for recombinant antibodies. Mol Breed. 4: 313–319.
Christensen, A.H. and Quail, P.H. 1996. Ubiquitin promoter-based vectors for high level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgen. Res 5: 213–218.
Conrad, U. and Fiedler, U. 1998. Compartment-specific accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activity. Plant Mol. Biol. 38: 101–109.
Dellaporta, S.L., Wood, J. and Hicks, J.B. 1984. Maize DNA miniprep. In: R. Malmberg, J. Messing and I. Sussex (Eds.) Molecular Biology of Plants: A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 36–37.
Denecke, J., de Rycke, R. and Botterman, J. 1992. Plant and mammalian sorting signals for protein retention in the endoplasmic reticulum contain a conserved epitope. EMBO J. 11: 2345–2355.
Dorai, H., McCartney, J.E., Hudziak, R.M., Tai, M.-S., Laminet, A.A., Houston, L.L., Houston, J.S. and Oppermann, H. 1994. Mammalian cell expression of single-chain Fv (sFv) antibody proteins and their C-terminal fusions with interleukin-2 and other effector domains. Bio/technology 12: 890–897.
Fiedler, U. and Conrad, U. 1995. High-level production and longterm storage of engineered antibodies in transgenic tobacco seeds. Bio/technology 13: 1090–1093.
Fischer, R., Drossard, J., Liao, Y.C. and Schillberg, S. 1998. Characterization and application of plant-derived recombinant antibodies. In: C. Cunningham and A. Porter (Eds.) Methods in Biotechnology, Vol. 3: Recombinant Proteins from Plants: Production and Isolation of Clinically Useful Compounds, Humana Press, Totowa, NJ, pp. 121–147.
Hiatt, A., Cafferkey, R. and Bowdish, K. 1989. Production of antibodies in transgenic plants. Nature 342: 469–470.
Hughes, J. and Qoronfleh, W. 1991. Perspectives in plant genetic engineering and biopharmacy. BioPharm 4: 18–26.
Klein, T.M., Wolf, E.D., Wu, R. and Sanford, J.C. 1987. High velocity microprojectiles for delivering nucleic acids into living cells. Nature 327: 70–73.
Ma, J.K.-C., Hiatt, A., Hein, M.B., Vine, N., Wang, F., Stabila, P., van Dollewerd, C., Mostov, K. and Lehner, T. 1995. Generation and assembly of secretory antibodies in plants. Science 268: 716–719.
Murray, E.E., Lotzer, J. and Eberle, M. 1989. Codon usage in plant genes. Nucl. Acids Res. 17: 477–498.
Neumaier, M., Shively, L., Chen, F.S., Gaida, F.J., Ilgen, C., Paxton, R.J., Shively, J.E. and Riggs, A.D. 1990. Cloning of the genes for T84.66 an antibody that has high specificity and affinity for carcinoembryonic antigen, and expression of chimeric human mouse T84.66 genes in myeloma and Chinese hamster ovary cells. Cancer Res. 50: 2128–2134.
Owen, M., Gandecha, A., Cockburn, B. and Whitelam, G. 1992. Synthesis of a functional anti-phytochrome single-chain Fv protein in transgenic tobacco. Bio/technology 10: 790–794.
Plückthun, A. 1991. Antibody engineering. Curr. Opin. Biotechnol. 2: 238–246.
Polson, A., Coetzer, T., Kruger, J., von Maltzahn, E. and van der Merwe, K.J. 1985. Improvements in the isolation of IgY from the yolks of eggs laid by immunized hens. Immunol. Invest. 14: 323–756.
Schouten, A., Roosien, J., van Engelen, F.A., de Jong, G.A.M., Borst-Vrenssen, A.W.M., Zilverentant, J.F., Bosch, D., Stiekema, W.J., Gommers, F.J., Schots, A. and Bakker, J. 1996. The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both cytosol and the secretory pathway in transgenic tobacco. PlantMol. Biol. 30: 781–793.
Sudhakar, D., Duc, L.T., Bong, B.B., Tinjuangjun, P., Bano-Maqbool, S., Valdez, M., Jefferson, R. and Christou, P. 1998. An efficient rice transformation system utilizing mature seed-derived explants and a portable, inexpensive particle bombardment device. Transgen. Res. 7: 289–294.
Tavladoraki, P., Benvenuto, E., Trinca, S., de Martinis, D. and Galeffi, P. 1993. Transgenic plants expressing a functional singlechain Fv antibody are specifically protected from virus attack. Nature 366: 469–472.
Vasil, V., Castillo, A.M., Fromm, M.E. and Vasil, I.K. 1992. Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. Bio/technology 10: 667–674.
Voss, A., Niersbach, M., Hain, R., Hirsch, H.J., Liao, Y.C., Kreuzaler, F. and Fischer, R. 1995. Reduced virus infectivity in N. tabacum secreting a TMV-specific full-size antibody. Mol. Breed. 1: 39–50.
Wu, A.M., Chen, W., Raubitschek, A., Williams, L.E., Neumaier, M., Fischer, R., Hu, S., Odom-Maryon, T., Wong, J.Y.C. and Shively, J.E. 1996. Tumor localization of anti-CEA single-chain Fvs: improved targeting by non-covalent dimers. Immunotechnology 2: 21–36.
Zimmermann, S., Schillberg, S., Liao, Y.C. and Fischer, R. 1998. Intracellular expression of TMV-specific single-chain Fv fragments leads to improved virus resistance in Nicotiana tabacum. Mol. Breed. 4: 369–379.
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Stöger, E., Vaquero, C., Torres, E. et al. Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies. Plant Mol Biol 42, 583–590 (2000). https://doi.org/10.1023/A:1006301519427
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DOI: https://doi.org/10.1023/A:1006301519427