Generation and expression in plants of a single-chain variable fragment antibody against the immunodominant membrane protein of Candidatus phytoplasma aurantifolia.

Witches' broom of lime is a disease caused by Candidatus Phytoplasma aurantifolia, which represents the most significant global threat to the production of lime trees (Citrus aurantifolia). Conventional disease management strategies have shown little success, and new approaches based on genetic engineering need to be considered. The expression of recombinant antibodies and fragments thereof in plant cells is a powerful approach that can be used to suppress plant pathogens. We have developed a single-chain variable fragment antibody (scFvIMP6) against the immunodominant membrane protein (IMP) of witches' broom phytoplasma and expressed it in different plant cell compartments. We isolated scFvIMP6 from a naïve scFv phage display library and expressed it in bacteria to demonstrate its binding activity against both recombinant IMP and intact phytoplasma cells. The expression of scFvIMP6 in plants was evaluated by transferring the scFvIMP6 cDNA to plant expression vectors featuring constitutive or phloem specific promoters in cassettes with or without secretion signals, therefore causing the protein to accumulate either in the cytosol or apoplast. All constructs were transiently expressed in Nicotiana benthamiana by agroinfiltration, and antibodies of the anticipated size were detected by immunoblotting. Plant-derived scFvIMP6 was purified by affinity chromatography, and specific binding to recombinant IMP was demonstrated by enzyme-linked immunosorbent assay. Our results indicate that scFvIMP6 binds with high activity and can be used for the detection of Ca. Phytoplasma aurantifolia and is also a suitable candidate for stable expression in lime trees to suppress witches' broom of lime.

Affinity purification of a framework 1 engineered mouse/human chimeric IgA2 antibody from tobacco.

Complex multimeric proteins such as dimeric and secretory immunoglobulin A (IgA) can be difficult to produce in heterologous systems, although this has been achieved using several platforms including plants. As well as topical mucosal applications, dimeric IgA (dIgA), and secretory IgA (sIgA) can be used in tumor and anti-viral therapy, where their more potent cell-killing properties may increase their efficacy compared to current drugs based on IgG. However, the development of therapeutic IgA formats is hampered by the need to co-express four different polypeptides, and the inability to purify such molecules using conventional protein A or protein G affinity chromatography. The light chain (LC)-specific affinity ligand protein L is a potential alternative, but it only recognizes certain kappa light chain (LC(κ)) subtypes. To overcome these limitations, we have adapted a framework-grafting approach to introduce LCs that bind protein L into any IgA. As a model, we used the chimeric anti-human chorionic gonadotropin (hCG) antibody cPIPP, since this contains a murine LC((κ)) subtype that does not bind protein L. Grafting was achieved by replacing selected framework region 1 (FR1) residues in the cPIPP LC(κ) variable domain with corresponding residues from LC(κ) subtypes that can bind protein L. The grafted antibody variants were successfully purified by protein L affinity chromatography. These modifications affected neither their antigen-binding properties nor the yields achieved by transient expression in tobacco plants. Our results therefore show that LC FR1 grafting can be used as generic strategy for the purification of IgA molecules.

Recombinant human tissue transglutaminase produced into tobacco suspension cell cultures is active and recognizes autoantibodies in the serum of coeliac patients.

Human tissue transglutaminase (htTG) is one of the most important member within the transglutaminase family, enzymes that for their capacity of catalyzing post-translational modifications of proteins and peptides, rise an high interest for industrial applications. More recently, for its implication as the major autoantigen in the coeliac disease, availability of human tissue transglutaminase as recombinant form is required for accurate diagnostic tests. The aim of this study was to find an alternative and inexpensive source to produce human tissue transglutaminase. To date, plant systems are proposed as heterologous hosts to produce recombinant proteins for use in disease diagnosis and therapy. Here, we describe the stable expression of human tissue transglutaminase into Nicotiana tabacum cultured cells (cultivar Bright Yellow 2 (BY-2)). The recombinant enzyme was successfully expressed in different plant cell compartments and both apoplast (apo) and chloroplast (chl) purified proteins were shown to be catalytically active and able to bind GTP, a property possessed by the natural counterpart. Importantly, plant produced human tissue transglutaminase recognized autoantibodies in the serum of coeliac patients, suggesting possible applications in the diagnosis of coeliac disease.

Molecular farming of recombinant antibodies in plants.

Antibodies represent a large proportion of therapeutic drugs currently in development. In most cases, they are produced in mammalian cell lines or transgenic animals because these have been shown to fold and assemble the proteins correctly and generate authentic glycosylation patterns. However, such expression systems are expensive, difficult to scale up and there are safety concerns due to potential contamination with pathogenic organisms or oncogenic DNA sequences. Plants represent an inexpensive, efficient and safe alternative for the production of recombinant antibodies. Research over the last 10 years has shown that plants can produce a variety of functional antibodies and there is now intense interest in scaling up production to commercial levels. In this review, we discuss the advantages of plants over traditional expression systems, describe how antibody expression in plants is achieved and optimized and then consider the practical issues concerning large-scale molecular farming in plants. The first plant-produced therapeutic antibodies are already in clinical trials, and, given the economic benefits of this production system, we are likely to see many more recombinant antibodies produced in this manner in the future.

Antibody-based resistance to plant pathogens.

Plant diseases are a major threat to the world food supply, as up to 15% of production is lost to pathogens. In the past, disease control and the generation of resistant plant lines protected against viral, bacterial or fungal pathogens, was achieved using conventional breeding based on crossings, mutant screenings and backcrossing. Many approaches in this field have failed or the resistance obtained has been rapidly broken by the pathogens. Recent advances in molecular biotechnology have made it possible to obtain and to modify genes that are useful for generating disease resistant crops. Several strategies, including expression of pathogen-derived sequences or anti-pathogenic agents, have been developed to engineer improved pathogen resistance in transgenic plants. Antibody-based resistance is a novel strategy for generating transgenic plants resistant to pathogens. Decades ago it was shown that polyclonal and monoclonal antibodies can neutralize viruses, bacteria and selected fungi. This approach has been improved recently by the development of recombinant antibodies (rAbs). Crop resistance can be engineered by the expression of pathogen-specific antibodies, antibody fragments or antibody fusion proteins. The advantages of this approach are that rAbs can be engineered against almost any target molecule, and it has been demonstrated that expression of functional pathogen-specific rAbs in plants confers effective pathogen protection. The efficacy of antibody-based resistance was first shown for plant viruses and its application to other plant pathogens is becoming more established. However, successful use of antibodies to generate plant pathogen resistance relies on appropriate target selection, careful antibody design, efficient antibody expression, stability and targeting to appropriate cellular compartments.

GST fusion proteins cause false positives during selection of viral movement protein specific single chain antibodies.

Glutathione S-transferase (GST) fusion proteins are used frequently for investigating protein-protein and protein-DNA interactions. The present study demonstrates that the use of GST fusion proteins caused false positives during selection of phage-displayed single-chain antibody fragments (scFvs) specific for three domains of the movement protein (NS(M)) of tomato spotted wilt virus (TSWV). To identify and exclude the false positives when using GST as a fusion partner linked to the antigen of interest, indirect phage enzyme-linked immunosorbent assay (ELISA) was compared with capture phage ELISA. Of 210 enriched phage clones, indirect phage ELISA identified 106 clones specific for binding to GST-domain fusions but not to GST. In contrast, using capture phage ELISA, all 106 selected clones were identified as false positives, reacting with the GST fusion proteins and GST. This was confirmed by characterization of soluble scFv antibodies. The data indicate that GST fusion proteins seem unsuitable for screening of phage-displayed antibody fragments and it is essential to use capture phage ELISA, instead of the indirect phage ELISA used commonly to exclude false positives in characterization of selected clones with GST fusion proteins.

Antibody production by molecular farming in plants.

"Molecular farming" is the production of pharmaceutical proteins in transgenic plants and has great potential for the production of therapeutic anti-cancer antibodies and recombinant therapeutic proteins. Plants make fully functional recombinant human or animal antibodies. Cultivating transgenic plants on an agricultural scale will produce almost unlimited supplies of recombinant proteins for uses in medicine. Combinatorial library technology is a key tool for the generation and optimisation of therapeutic antibodies ahead of their expression in plants. Optimised antibody expression can be rapidly verified using transient expression assays in plants before creation of transgenic suspension cells or plant lines. Subcellular targeting signals that increase expression levels and optimise protein stability can be identified and exploited using transient expression to create high expresser plant lines. When high expresser lines have been selected, the final step is the development of efficient purification methods to retrieve functional antibody. Antibody production on an industrial scale is then possible using plant suspension cell culture in fermenters, or by the propagation of stably transformed plant lines in the field. Recombinant proteins can be produced either in whole plants or in seeds and tubers, which can be used for the long-term storage of both the protein and its production system. The review will discuss these developments and how we are moving toward the molecular farming of therapeutic antibodies becoming an economic and clinical reality.

Transient transformation of the rust fungus Puccinia graminis f. sp. tritici.

The biotrophic rust fungus Puccinia graminis f. sp. tritici (Pgt) was transformed by particle bombardment. The promoter from the Pgt translation elongation factor 1alpha (EF-1alpha) gene was fused to the bacterial marker genes hygromycin B phosphotransferase (hpt) and beta-glucuronidase (GUS). Transformation constructs were introduced into uredospores of Pgt, an obligate pathogen of wheat, by biolistic bombardment. Uredospores transformed with the construct containing the hpt gene germinated and initiated branching on selective medium, indicating that they had acquired resistance to hygromycin B. However, transformants stopped growing 5 days after bombardment. GUS activity in uredospores and germlings was histochemically detected 4-16 h after bombardment. GUS expression was also obtained using the INF24 promoter from the bean rust fungus Uromyces appendiculatus, demonstrating that heterologous genes can be expressed in P. graminis under the control of regulatory sequences from closely related organisms.

Towards molecular farming in the future: moving from diagnostic protein and antibody production in microbes to plants.

Molecular farming of pharmaceuticals in plants has the potential to provide almost unlimited amounts of recombinant proteins for use in disease diagnosis and therapy. Transgenic plants are attracting interest as bioreactors for the inexpensive production of large amounts of safe, functional, recombinant macromolecules, such as blood substitutes, vaccines and antibodies. In some cases, the function of expressed recombinant proteins can be rapidly analysed by expression in microbes or by transient expression in intact or virally infected plants. Protein production can be increased by upscaling production in fermenters, using yeast- or plant-suspension cells or by using transient-expression systems. Stable transgenic plants can be used to produce leaves or seeds rich in the recombinant protein for long-term storage or direct processing. This demonstrates the promise for using plants as bioreactors for the molecular farming of recombinant therapeutics, diagnostics, blood substitutes and antibodies. We anticipate that this technology has the potential to greatly benefit human health by making safe recombinant pharmaceuticals widely available.

Molecular farming of recombinant antibodies in plants.

'Molecular farming' is the production of recombinant proteins in plants. It is intended to harness the power of agriculture to cultivate and harvest transgenic plants producing recombinant therapeutics. Molecular farming has the potential to provide virtually unlimited quantities of recombinant antibodies for use as diagnostic and therapeutic tools in both health care and the life sciences. Importantly, recombinant antibody expression can be used to modify the inherent properties of plants, for example by using expressed antipathogen antibodies to increase disease resistance. Plant transformation is technically straightforward for model plant species and some cereals, and the functional expression of recombinant proteins can be rapidly analyzed using transient expression systems in intact or virally infected plants. Protein production can then be increased using plant suspension cell production in fermenters, or by the propagation of stably transformed plant lines in the field. Transgenic plants can be exploited to produce organs rich in a recombinant protein for its long-term storage. This demonstrates the promise of using transgenic plants as bioreactors for the 'molecular farming' of recombinant therapeutics, blood substitutes and diagnostics, such as recombinant antibodies.

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

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 mouse/human 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 SDS/PAGE, Western blotting, and ELISA.

Expression and characterization of bispecific single-chain Fv fragments produced in transgenic plants.

We describe the expression of the bispecific antibody biscFv2429 in transgenic suspension culture cells and tobacco plants. biscFv2429 consists of two single-chain antibodies, scFv24 and scFv29, connected by the Trichoderma reesi cellobiohydrolase I linker. biscFv2429 binds two epitopes of tobacco mosaic virus (TMV): the scFv24 domain recognizes neotopes of intact virions, and the scFv29 domain recognizes a cryptotope of the TMV coat protein monomer. biscFv2429 was functionally expressed either in the cytosol (biscFv2429-cyt) or targeted to the apoplast using a murine leader peptide sequence (biscFv2429-apoplast). A third construct contained the C-terminal KDEL sequence for retention in the ER (biscFv2429-KDEL). Levels of cytoplasmic biscFv2429 expression levels were low. The highest levels of antibody expression were for apoplast-targeted biscFv2429-apoplast and ER-retained biscFv2429-KDEL that reached a maximum expression level of 1.65% total soluble protein in transgenic plants. Plant-expressed biscFv2429 retained both epitope specificities, and bispecificity and bivalency were confirmed by ELISA and surface plasmon resonance analysis. This study establishes plant cells as an expression system for bispecific single-chain antibodies for use in medical and biological applications.

Apoplastic and cytosolic expression of full-size antibodies and antibody fragments in Nicotiana tabacum.

We compared the expression of a functional recombinant TMV-specific full-size antibody (rAb29) in both the apoplast and cytosol of tobacco plants and a single chain antibody fragment (scFv29), derived from rAb29, was expressed in the cytosol. Cloned heavy and light chain cDNAs of full-size rAb29, which binds to TMV coat protein monomers, were integrated into the plant expression vector pSS. The full-size rAb29 was expressed in the cytosol and targeted to the apoplast by including the original murine antibody leader sequences. Levels of functional full-size rAb29 expression were high in the apoplast (up to 8.5 micrograms per gram leaf tissue), whereas cytosolic expression was low or at the ELISA detection limit. Sequences of the variable domains of rAb29 light and heavy chain were used to generate the single chain antibody of scFv29, which was expressed in the periplasmic space of E. coli and showed the same binding specificity as full-size rAb29. In addition, scFv29 was functionally expressed in the cytosol of tobacco plants and plant derived scFv29 maintained same binding specificity to TMV-coat protein monomers as rAb29.

Isolation and characterization of the EF-1 alpha gene of the filamentous fungus Puccinia graminis f. sp. tritici.

A gene of Puccinia graminis f. sp. tritici, coding for the translation elongation factor 1 alpha (EF-1 alpha), was isolated from a P. graminis genomic library using the EF-1 alpha gene sequence of Absidia glauca. The coding region of 1389 nucleotides encodes a polypeptide of 463 amino acids and is interrupted by eight introns. An additional intron is located in the 5' untranslated region. A single transcription start point (tsp) was mapped by primer extension. A cDNA fragment corresponding to P. graminis EF-1 alpha mRNA hybridized with a 1.9-kb-long poly(A+)RNA, sufficient to encode the EF-1 alpha protein. Southern hybridization of digested genomic DNA revealed that two copies of the EF-1 alpha gene exist in the genome of P. graminis.