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Compartment-specific accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activity

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Abstract

Expression and stability of immunoglobulins in transgenic plants have been investigated and optimized by accumulation in different cellular compartments as cytosol, apoplastic space and endoplasmic reticulum (ER) as will be discussed in this review. In several cases described the highest accumulation of complete active antibodies was achieved by targeting into the apoplastic space. High-level expression of active recombinant single-chain Fv antibodies (scFv's) was obtained by retention of these proteins in the lumen of the endoplasmic reticulum. This has been shown for leaves and seeds of transgenic tobacco as well as for potato tubers. Transgenic tobacco seeds, potato tubers and tobacco leaves can facilitate stable storage of scFv's accumulated in the ER over an extended (seeds, tubers) or a short (leaves) period of time. The expression of specific scFv's in different plant species, plant organs and cellular compartments offers the possibility of blocking regulatory factors or pathogens specifically. Examples are scFv's expressed in the cytosol and the apoplastic space of transgenic plant cells modulating the infection process of plant viruses and a cytosolically expressed scFv that influenced the activity of phytochrome A protein. The immunomodulation approach has been shown to be also applicable for investigating the action of the phyto-hormone abscisic acid (ABA). High-level accumulation of specific anti-ABA scFv's in the ER of all leaf cells has been used to block the influence of ABA on the stomatal functions. Seed-specific expression of high amounts of anti-ABA-scFv's at a defined time of seed-development induced a developmental switch from seed ripening to vegetative growth. It has been demonstrated that ER retention is essential for the accumulation of sufficient scFv to bind high concentrations of ABA in the transgenic seeds.

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References

  1. Artsaenko O: Immunomodulation of ABA activity in transgenic tobacco plants. PhD thesis, Martin-Luther-Universität Halle-Wittenberg (1996).

  2. Artsaenko O, Kettig B, Fiedler U, Conrad U, Düring K: Potato tubers as a biofactory for recombinant antibodies. Mol Breed, in press (1998).

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

    Google Scholar 

  4. Bäumlein H, Boerjan W, Nagy I, Bassüner R, van Montagu M, Inze D, Wobus U: A novel seed protein gene from Vicia faba is developmentally regulated in transgenic tobacco and Arabidopsis plants. Mol Gen Genet 225: 459–467 (1991).

    Google Scholar 

  5. Bäumlein H, Boerjan W, Nagy I, Panitz R, Inze D, Wobus U: Upstream sequences regulating legumin gene expression in heterologous transgenic plants. Mol Gen Genet 225: 121–128 (1991).

    Google Scholar 

  6. Biocca S, Pierandreu-Amaldi P, Cattaneo A: Intracellular expression of anti-p21ras single chain Fv fragments inhibits meiotic maturation of Xenopus oocytes. Biochem Biophys Res Comm 197: 422–427 (1993).

    Google Scholar 

  7. Biocca S, Ruberti F, Tafani M, Pierandreu-Amaldi P, Cattaneo A: Redox state of single chain Fv fragments targeted to the endoplasmic reticulum, cytosol and mitochondria. Bio/technology 13: 1110–1115 (1995).

    Google Scholar 

  8. Bird KE, Hardman KD, Jacobsen JW, Johnsen S, Kaufman B, Lee SM, Lee T, Pope SH, Riordan GS, Whitlow M: Singlechain antigen-binding proteins. Science 242: 423–426 (1988).

    Google Scholar 

  9. Brinkmann U, Pastan I: Immunotoxins against cancer. Biochim Biophys Acta 1198: 27–45 (1994).

    Google Scholar 

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

    Google Scholar 

  11. Cheung SC, Dietzschold B, Koprowski H, Notkins AL, Rando RF: A recombinant human Fab expressed in E.coli neutralizes rabies virus. J Virol 11: 6714–6720 (1992).

    Google Scholar 

  12. Clackson T, Hoogenboom HR, Griffith AD, Winter G: Making antibody fragments using phage display libraries. Nature 352: 624–628 (1991).

    Google Scholar 

  13. Denecke J, Goldman MH, Demolder J, Seurinck J, Botterman J: The tobacco luminal binding protein is encoded by a multigene family. Plant Cell 3: 1025–1035 (1991).

    Google Scholar 

  14. De Neve M, De Loose M, Jacobs A, van Houdt H, Kaluza B, Weidle U, Van Montagu M, Depicker A: Assembly of an antibody and its derived antibody fragments in Nicotiana and Arabidopsis. Transgen Res 2: 227–237 (1993).

    Google Scholar 

  15. Duan L, Bagasra O, Laughlin MA, Oakes JW, Pomerantz RJ: Potent inhibition of human immunodeficiency virus type 1 replication by intracellular anti-Rev single-chain antibody. Proc Natl Acad Sci USA 91: 5075–5079 (1994).

    Google Scholar 

  16. Düring K, Hippe S, Kreuzaler F, Schell J: Synthesis and selfassembly of a functional monoclonal antibody in transgenic Nicotiana tabacum. Plant Mol Biol 15: 281–293 (1990).

    Google Scholar 

  17. Fecker L, Kaufmann N, Commandeur U, Commandeur J, Koenig R, Burgemeister W: Expression of single-chain antibody fragments (scFv) specific for beet necrotic yellow vein virus coat protein or 25 kDa protein in Escherichia coli and Nicotiana benthamiana. Plant Mol Biol 32: 979–986 (1996).

    Google Scholar 

  18. Fecker LF, Koenig R, Obermeier C: Nicotiana bentamiana 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. Arch Virol 142: 1857–1863 (1997).

    Google Scholar 

  19. Firek S, Draper J, Owen MRL, Gandecha A, Cockburn B, Whitelam GC: Secretion of a functional single-chain Fv protein in transgenic tobacco plants and cell suspension cultures. Plant Mol Biol 23: 861–870 (1993).

    Google Scholar 

  20. Fiedler U: Hochexpression rekombinanter Antikörperfragmente in transgenen Pflanzen. Ph. D. thesis, Martin-Luther-Universität Halle-Wittenberg (1996).

  21. Fiedler U, Conrad U: High-level production and long-term storage of engineered antibodies in transgenic tobacco seeds. Bio/technology 10: 1090–1094 (1995).

    Google Scholar 

  22. Fiedler U, Phillips J, Artsaenko O, Conrad U: Optimization of scFv antibody production in transgenic plants. Immunotechnology 3: 205–216 (1997).

    Google Scholar 

  23. Fontes EB, Shank BB, Wrobel RL, Moose SP, O'Brian GR, Wurtzel ET, Boston RS: Characterization of an immunoglobulin binding protein homolog in the maize floury-2 endosperm mutant. Plant Cell 3: 483–496 (1991).

    Google Scholar 

  24. Gething MJ, Sambrook J: Protein folding in the cell. Nature 355: 33–45 (1992).

    Google Scholar 

  25. Glaser SM, Yelton DE, Huse WD: Antibody engineering by codon-based mutagenesis in a filamentous phage vector system. J Immunol 149: 3903–3913 (1992).

    Google Scholar 

  26. Griffith AP: Production of human antibodies using bacteriophage. Curr Opin Immunol 52: 263–267 (1993).

    Google Scholar 

  27. Hein MB, Tang Y, McLeod DA, Janda KD, Hiatt A: Evaluation of immunoglobulins from plant cells. Biotechnol Prog 7: 455–461 (1991).

    Google Scholar 

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

    Google Scholar 

  29. Hoogenboom HR, Winter G: Bypassing immunization: human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro. J Mol Biol 227: 381–388 (1992).

    Google Scholar 

  30. Huston JS, Levinson D, Mudgett-Hunter M, Tai MS, Novotny J, Margolies MN, Ridge RJ, Bruccoleri RE, Haber E, Oppermann H: Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in E. coli. Proc Natl Acad Sci USA 88: 5879–5883 (1988).

    Google Scholar 

  31. Kang AS, Barbas CF, Janda KD, Benkovic SJ, Lerner RA: Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces. Proc Natl Acad Sci USA 88: 4353–4366 (1991).

    Google Scholar 

  32. Ma JKC, Hein MB: Immunotherapeutic potential of antibodies produced in plants. Trends Biotechnol 13: 522–527 (1995).

    Google Scholar 

  33. Ma JKC, Hiatt A, Hein M, Vine ND, Wang F, Stabila P, van Dolleweerd C, Mostov K, Lehner T: Generation and assembly of secretory antibodies in plants. Science 268: 716–719 (1995).

    Google Scholar 

  34. Ma JKC, Lehner T, Stabila P, Fux CI, Hiatt A: Assembly of monoclonal antibodies with IgG1 and IgA heavy chain domains in transgenic tobacco plants. Eur J Immunol 24: 131–138 (1994).

    Google Scholar 

  35. Mallender, WD, Voss EW: Construction, expression, and activity of a bivalent bispecific single-chain antibody. J Biol Chem 269: 199–206 (1994).

    Google Scholar 

  36. Marks JD, Griffith AP, Malmquist M, Clackson TP, Bye JM, Winter G: By-passing immunization: building high affinity human antibodies by chain shuffling. Bio/technology 10: 779–783 (1992).

    Google Scholar 

  37. Melnick J, Aviel S, Argon YJ: The endoplasmic reticulum stress protein GRP94, in addition to BiP, associates with unassembled immunoglobulin chains. J Biol Chem 267: 21303–21306 (1992).

    Google Scholar 

  38. Muench DG, Wu Y, Zhang Y, Li X, Boston RS, Okita TW: Molecular cloning, expression and subcellular localization of a BiP homolog from rice endosperm tissue. Plant Cell Physiol 38: 404–412 (1997).

    Google Scholar 

  39. Nissim A, Hoogenboom H, Tomlinson I, Flynn G, Midgley C, Lane D, Winter G: Antibody fragments from a 'single pot’ phage display library as immunochemical reagents. EMBO J 13: 692–698 (1994).

    Google Scholar 

  40. Owen MRL, Cockburn B, Whitelam G: The expression of recombinant antibody fragments in plants. In: Owen MR, Pen J (eds) Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins, pp. 245–260. John Wiley, Chichester, UK (1996).

    Google Scholar 

  41. Owen MRL, Gandecha A, Cockburn B, Whitelam G: Synthesis of a functional anti-phytochrome single-chain Fv protein in transgenic tobacco. Bio/technology 10: 790–794 (1992).

    Google Scholar 

  42. Pack P, Plückthun A: Miniantibodies: use of amphipathic helices to produce functional, flexibly linked dimeric Fv fragments with high avidity in Escherichia coli. Biochemistry 31: 1579–1584 (1992).

    Google Scholar 

  43. Pen J, Molendyk L, Quax WJ, Symons PC, van Qoijen AJJ, van den Elzen PJM, Rietveld K, Hoekema A: Production of active Bacillus licheniformis alpha-amylase in tobacco and its application in starch liquefaction. Bio/technolygy 10: 292–296 (1992).

    Google Scholar 

  44. Pen J, Vewoerd TC, van Paridon PA, Beudeker RF, van den Elzen PJM, Geerse K, van den Klirs JD, Versteegh H, van Qoijen AJJ, Hoekema A: Phytase-containing transgenic seeds as a novel feed additive for improved phosphorus utilization. Bio/technology 11: 811–814 (1993).

    Google Scholar 

  45. Phillips J, Artsaenko O, Fiedler U, Horstmann C, Mock HP, Müntz K, Conrad U: Seed-specific immunomodulation of abscisic acid activity induces a developmental switch. EMBO J 16: 4489–4496 (1997).

    Google Scholar 

  46. Richardson JH, Sodroski JG, Waldmann TA, Marasco WA: Phenotypic knockout of the high-affinity human interleukin 2 receptor by intracellular single-chain antibodies against the α subunit of the receptor. Proc Natl Acad Sci USA 92: 3137–3141 (1995).

    Google Scholar 

  47. Schouten A, Roosien J, van Engelen FA, de Jong GAM, Borst-Vrenssen AWM, Zilverentant JF, Bosch D, Stiekema WJ, Gommer FJ, Schots A, Bakker J: 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 Mol Biol 30: 781–793 (1996).

    Google Scholar 

  48. Schouten A, Roosien J, de Boer JM, Wilmink A, Rosso MN, Bosch D, Stiekema WJ, Gommers FJ, Bakker J, Schots A: Improving scFv antibody expression levels in the plant cytosol. FEBS Lett 415: 235–241 (1997).

    Google Scholar 

  49. Senter PD, Sautmer MG, Schreiber GJ, Hirschberg DL, Brown JP, Hellström I, Hellström KE: Anti-Tumor effects of antibody-alkaline phosphatase conjugates in combination with etoposide phosphate. Proc Natl Acad Sci USA 85: 4842–4846 (1988).

    Google Scholar 

  50. Skerra A: Bacterial expression of immunoglobulin fragments. Curr Opin Immunol 2: 250–262 (1993).

    Google Scholar 

  51. Somerville CR: Production of industrial materials in transgenic plants. In Bevan MW, Harrison BD, Leaver CJ (eds) The production and uses of genetically transformed plants, Cambridge University Press, Cambridge, UK (1994).

    Google Scholar 

  52. Stempfer G, Höll-Neugebauer B, Rudolph R: Improved refolding of an immobilized fusion protein. Nature Biotechnol 14: 329–334 (1996).

    Google Scholar 

  53. Tavladoraki P, Benvenuto E, Trinca S, Martinis DD, Cattaneo A, Galeffi P: Transgenic plants expressing a functional singlechain Fv antibody are specifically protected from virus attack. Nature 366: 469–472 (1993).

    Google Scholar 

  54. Vandekerckhove J, van Damme J, van Lijsebettens M, Botterman J, de Block M, Vandewiele M, de Clercq A, Leemans J, van Montagu M, Krebbers E: Encephalins produced in transgenic plants using modified 2S seed storage proteins. Bio/technology 7: 929–932 (1989).

    Google Scholar 

  55. van Engelen FA, Schouten A, Molthoff JW, Roosien J, Salinas J, Dirkse WG, Schots A, Bakker J, Gommers FJ, Jongsma MA et al.: Coordinate expression of antibody subunit genes yields high levels of functional antibodies in roots of transgenic tobacco. Plant Mol Biol 26: 1701–1710 (1994).

    Google Scholar 

  56. Vaughan TJ, Williams AJ, Pritchard K, Osbourn JK, Pope AR, Earnshaw JC, McCafferty J, Hodits RA, Wilton J, Johnson KS: Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nature Biotechnol 14: 309–314 (1996).

    Google Scholar 

  57. von Schaewen A, Stitt M, Schmidt R, Sonnewald U, Willmitzer L: Expression of a yeast-derived invertase in the cell wall of tobacco and Arabidopsis plants leads to accumulation of carbohydrate and inhibition of photosynthesis and strongly influences growth and phenotype of transgenic tobacco plants. EMBO J 9: 3033–3044 (1990).

    Google Scholar 

  58. Voss A, Niersbach M, Hain R, Hirsch HJ, Liao Y, Kreuzaler F, Fischer R: Reduced virus infectivity in N. tabacum secreting a TMV-specific full-size antibody. Mol Breed 1: 15–26 (1995).

    Google Scholar 

  59. Walther-Larsen H, Brandt J, Collinge DB, Thordal-Christensen H: A pathogen-induced gene of barley encodes a HSP90 homologue showing striking similarity to vertebrate forms resident in the endoplasmic reticulum. Plant Mol Biol 21: 1097–1108 (1993).

    Google Scholar 

  60. Willmitzer L, Töpfer R: Manipulation of oil, starch and protein composition. Curr Opin Biotechnol 3: 176–180 (1992).

    Google Scholar 

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Conrad, U., Fiedler, U. 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 (1998). https://doi.org/10.1023/A:1006029617949

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