Synthesis and self-assembly of a functional monoclonal antibody in transgenic Nicotiana tabacum

Düring, Klaus; Hippe, Sigrun; Kreuzaler, Fritz; Schell, Jeff

doi:10.1007/bf00036914

Cited by 208 publications

(109 citation statements)

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“…Protein secretion, folding, and posttranslational modification are similar in plant and animal cells (4,(40)(41)(42). Antibodies accumulate to high levels in plant cells and are essentially indistinguishable from those produced by hybridomas (7,8). Copurification of bloodborne pathogens and oncogenic sequences is entirely avoided during downstream processing of plant-expressed recombinant proteins.…”

Section: Discussionmentioning

confidence: 99%

“…rAbs are efficiently folded and assembled within the endoplasmic reticulum (ER) of plant cells (4)(5)(6) and retain the antigen binding properties of the antibodies produced by plasma or hybridoma cells (2,5,(7)(8)(9). Since the first report of antibody expression in transgenic plants (7), different engineered antibodies have been produced successfully, including full-size antibodies (8)(9)(10)(11), Fab fragments (12), scFvs (13)(14)(15)(16)(17)(18)(19)(20)(21), and single-domain antibodies (22).…”

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confidence: 99%

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Transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves

Vaquero

Sack

Chandler

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

220

139

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To evaluate the expression of different forms of a tumor-specific antibody in plants, we adapted a recently described Agrobacterium-mediated transient expression system. A recombinant single-chain Fv antibody (scFvT84.66) and a full-size mouse͞human chimeric antibody (cT84.66) derived from the parental murine mAb T84.66 specific for the human carcinoembryonic antigen were engineered into a plant expression vector. Chimeric T84.66 heavy and light chain genes were constructed by exchanging the mouse light and heavy chain constant domain sequences with their human counterparts and cloned into two independent plant expression vectors. In vivo assembly of full-size cT84.66 was achieved by simultaneous expression of the light and heavy chains after vacuum infiltration of tobacco leaves with two populations of recombinant Agrobacterium. Upscaling the transient system permitted purification of functional recombinant antibodies from tobacco leaf extracts within a week. His6-tagged scFvT84.66 was purified by immobilized metal affinity chromatography and cT84.66 by protein A affinity chromatography. Sufficient amounts of recombinant antibodies were recovered for detailed characterization by SDS͞PAGE, Western blotting, and ELISA.Monoclonal antibodies are essential tools in biology, biochemistry, and medicine. Their high affinity and specificity make them invaluable for diagnostic and therapeutic applications. However, the therapeutic use of murine mAbs is limited because they elicit a human anti-mouse antibody response and large amounts of antibody are required for therapy. These limitations may be overcome by engineering humanized antibodies and by producing these proteins in plants.The human anti-mouse antibody response can be reduced by using recombinant antibody (rAb) technology to replace the murine light and heavy chain constant domains with the corresponding human domains and the remaining murine variable domains to maintain the antigen specificity and affinity of the original mAb. A second approach is to use single-chain Fv antibody fragments (scFvs) where the constant domains have been removed and the variable domains are joined by a flexible linker (1). Compared with the full-size antibodies, scFvs display better tumor penetration and faster serum clearance but exhibit no effector functions. A critical step in testing the potential therapeutic use of these molecules is the development of a reproducible and efficient method for large-scale antibody production.Plants are potentially the most economical system for largescale production of rAbs (2, 3). rAbs are efficiently folded and assembled within the endoplasmic reticulum (ER) of plant cells (4-6) and retain the antigen binding properties of the antibodies produced by plasma or hybridoma cells (2,5,(7)(8)(9). Since the first report of antibody expression in transgenic plants (7), different engineered antibodies have been produced successfully, including full-size antibodies (8-11), Fab fragments (12), scFvs (13-21), and single-domain antibodies (22).Regene...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves

Vaquero

Sack

Chandler

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

220

139

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“…Antibody expression in plants [1][2][3][4][5][6] provides a means to 2.1. Construction ofscFv expression plasmids interfere with the biological activity of its antigen.…”

Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

Bacterial and plant‐produced scFv proteins have similar antigen‐binding properties

et al. 1996

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A gene encoding a single-chain variable (scFv)signal peptide sequence was compared, lower or even no acantibody fragment was expressed as a cytoplasmic and cumulation of the cytosol-targeted protein was observed, both endoplasmic reficulum-targeted protein in transgenic tobacco in leaves [11,12] and in seeds [13]. This suggests a lower staplants. In both cases, the scFv accumulated up to 0.01% of total bility of scFv proteins in the cytoplasm. Yet, intracellular soluble protein (TSP). The same scFv fragment was also expression of an anti-artichoke mottled crinkle virus scFv in produced in the periplasm of Escherichia coll. Measurement of Nicotiana benthamiana generated plants with scFv accumulathe affinity by ELISA indicates that the affinity of the tion levels up to 0.1% of TSP, which resulted in reduced inbacterially made scFv is about 80-fold lower than that of the fection incidence and delayed symptom development [9]. parental Fab fragment. The results suggest that the affinity of the However, few data are available on the efficiency of antigenplant-produced scFv fragments is reduced to a similar extent, binding scFv fragment synthesis and on the affinity of the implying that all the plant-produced scFv fragments are antigen produced scFv fragments for the cognate antigen. binding.To investigate the properties of plant-produced scFv mole- The accumulation levels were determined and the antigenbinding properties of the plant-produced scFv fragments were compared to those of a bacterially produced scFv.

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“…However, the expression of recombinant proteins fused with SPs, leading to dual targeting of the proteins to plastids and cell walls, is being increasingly observed. For example, assembled antibodies were found in chloroplasts, besides being detected in the ER, when mature light and heavy chains of a monoclonal antibody fused to a barley a-amylase SP were expressed in transgenic tobacco (During et al, 1990). Recently, the B-subunit of Escherichia coli heat-labile enterotoxin (LT-B) was found to be associated with starch grains in amyloplasts when this protein, carrying its native SP or fused with a maize (Zea mays) g-zein SP, was overexpressed in transgenic maize endosperm (Chikwamba et al, 2003).…”

Section: Sp Is Sufficient For Directing Cargo Proteins To Plastids Anmentioning

confidence: 99%

Signal Peptide-Dependent Targeting of a Riceα-Amylase and Cargo Proteins to Plastids and Extracellular Compartments of Plant Cells

Chen

Huang

et al. 2004

Plant Physiology

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a-Amylases are important enzymes for starch degradation in plants. However, it has been a long-running debate as to whether a-amylases are localized in plastids where starch is stored. To study the subcellular localization of a-amylases in plant cells, a rice (Oryza sativa) a-amylase, aAmy3, with or without its own signal peptide (SP) was expressed in transgenic tobacco (Nicotiana tabacum) and analyzed. Loss-of-function analyses revealed that SP was required for targeting of aAmy3 to chloroplasts and/or amyloplasts and cell walls and/or extracellular compartments of leaves and suspension cells. SP was also required for in vitro transcribed and/or translated aAmy3 to be cotranslationally imported and processed in canine microsomes. aAmy3, present in chloroplasts of transgenic tobacco leaves, was processed to a product with M r similar to aAmy3 minus its SP. Amino acid sequence analysis revealed that the SP of chloroplast localized aAmy3 was cleaved at a site only one amino acid preceding the predicted cleavage site. Function of the aAmy3 SP was further studied by gain-of-function analyses. b-Glucuronidase (GUS) and green fluorescence protein fused with or without the aAmy3 SP was expressed in transgenic tobacco or rice. The aAmy3 SP directed translocation of GUS and green fluorescence protein to chloroplasts and/or amyloplasts and cell walls in tobacco leaves and rice suspension cells. The SP of another rice a-amylase, aAmy8, similarly directed the dual localizations of GUS in transgenic tobacco leaves. This study is the first evidence of SP-dependent dual translocations of proteins to plastids and extracellular compartments, which provides new insights into the role of SP in protein targeting and the pathways of SP-dependent protein translocation in plants.Two types of starch exist in plants: transitory (assimilatory) starch, which is located in chloroplasts, and reserve starch, which is deposited in amyloplasts. Degradation of starch can be either hydrolytic, mainly catalyzed by a-amylase, b-amylase, and debranching enzymes, or phosphorolytic, catalyzed by starch phosphorylase. Degradation of reserve starch in cereal grains is mainly hydrolytic, whereas degradation of transitory starch in leaves can be hydrolytic and/or phosphorolytic (Beck and Ziegler, 1989). In germinating cereal grains, a-amylases are the most abundant starch-degrading enzymes. The enzymes are secreted by aleurone cells into the starchy endosperm where they degrade the starch grains (Jacobsen et al., 1995). Whether a-amylases also play an essential role in starch degradation in photosynthetic tissues and in tissues other than endosperm is not clear. In most plants, starch can be found in pollens, seeds, leaves, stems, roots, and other tissues. In spinach (Spinacia oleracea), a-amylases are the only enzymes that have been demonstrated to attack starch granules isolated from chloroplasts (Steup et al., 1983).Determination of the subcellular distribution of a-amylases is essential for understanding the physiological function of these enzymes in...

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Synthesis and self-assembly of a functional monoclonal antibody in transgenic Nicotiana tabacum

Cited by 208 publications

References 40 publications

Transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves

Transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves

Bacterial and plant‐produced scFv proteins have similar antigen‐binding properties

Signal Peptide-Dependent Targeting of a Riceα-Amylase and Cargo Proteins to Plastids and Extracellular Compartments of Plant Cells

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