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At least 19 recordsLinked to original sources
PubMed2005

Recent progress in plantibody technology.

Antibodies are an important class of proteins that can be used for the prevention, treatment and diagnosis of many diseases. Consequently, there is an intense and growing demand for recombinant antibodies, placing immense pressure on current production capacity which is based largely on microbial cultures and mammalian cells. Alternative systems for cost effective antibody production would be very welcome, and plants are now gaining widespread acceptance as green bioreactors with advantages in terms of cost, scalability and safety. Several plant-produced antibodies (plantibodies) are undergoing clinical trials and the first commercial approval could be only a few years away. The performance of the first generation of products has been very encouraging so far. In terms of product authenticity, differences in glycosylation between plantibodies and their mammalian counterparts have been defined, and the scientific evaluation of any possible consequences is underway. Ongoing studies are addressing the remaining biochemical constraints, and aim to further improve product yields, homogeneity and authenticity, particularly where the antibody is intended for injection into human patients. A remaining practical challenge is the implementation of large-scale production and processing under good manufacturing practice conditions that are yet to be endorsed by regulatory bodies. The current regulatory uncertainty and the associated costs represent an entry barrier for the pharmaceutical industry. However, the favourable properties of plants are likely to make the plant systems a useful alternative for small, medium and large scale production throughout the development of new antibody-based pharmaceuticals.

Animals↗
PubMed2000

The plantibody approach: expression of antibody genes in plants to modulate plant metabolism or to obtain pathogen resistance.

Immunomodulation is a molecular technique that allows the interference with cellular metabolism or pathogen infectivity by the ectopic expression of genes encoding antibodies or antibody fragments. In recent years, several reports have proven the value of this tool in plant research for modulation of phytohormone activity and for blocking plant-pathogen infection. Efficient application of the plantibody approach requires different levels of investigation. First of all, methods have to be available to clone efficiently the genes coding for antibodies or antibody fragments that bind the target antigen. Secondly, conditions to obtain high accumulation of antigen-binding antibodies and antibody fragments in plants are being investigated and optimized. Thirdly, different strategies are being evaluated to interfere with the function of the target molecule, thus enabling immunomodulation of metabolism or pathogen infectivity. In the near future, optimized antibody gene isolation and expression, especially in reducing subcellular environments, such as the cytosol and nucleus, should turn immunomodulation into a powerful and attractive tool for gene inactivation, complementary to the classical antisense and co-suppression approaches.

Antibodies↗
PubMed2002

Plantibodies: applications, advantages and bottlenecks.

Various strategies have been developed to exploit plants as bioreactors for the production of pharmaceutical antibodies, to engineer antibody-mediated pathogen resistance or to alter the plant phenotype by immunomodulation. Recent research developments focus on the fine-tuning of expression systems and the detailed characterisation of recombinant products, including the implications of plant-specific glycosylation. Meanwhile, the first of these plant-derived antibody products has successfully completed early phase clinical trials.

Antibodies↗
PubMed1999

N-Glycosylation of a mouse IgG expressed in transgenic tobacco plants.

Since plants are emerging as an important system for the expression of recombinant glycoproteins, especially those intended for therapeutic purposes, it is important to scrutinize to what extent glycans harbored by mammalian glycoproteins produced in transgenic plants differ from their natural counterpart. We report here the first detailed analysis of the glycosylation of a functional mammalian glycoprotein expressed in a transgenic plant. The structures of the N-linked glycans attached to the heavy chains of the monoclonal antibody Guy's 13 produced in transgenic tobacco plants (plantibody Guy's 13) were identified and compared to those found in the corresponding IgG1 of murine origin. Both N-glycosylation sites located on the heavy chain of the plantibody Guy's 13 are N-glycosylated as in mouse. However, the number of Guy's 13 glycoforms is higher in the plant than in the mammalian expression system. Despite the high structural diversity of the plantibody N-glycans, glycosylation appears to be sufficient for the production of a soluble and biologically active IgG in the plant system. In addition to high-mannose-type N-glycans, 60% of the oligosaccharides N-linked to the plantibody have beta(1, 2)-xylose and alpha(1, 3)-fucose residues linked to the core Man3GlcNAc2. These plant-specific oligosaccharide structures are not a limitation to the use of plantibody Guy's 13 for topical immunotherapy. However, their immunogenicity may raise concerns for systemic applications of plantibodies in human.

Animals↗
PubMed2000

Expression of a functional antizearalenone single-chain Fv antibody in transgenic Arabidopsis plants.

The efficacy of cloning a recombinant mycotoxin antibody in plants was tested using Arabidopsis as a model. An antizearalenone single-chain Fv (scFv) DNA fragment was first cloned in the newly constructed phage display vector (pEY.5) and then recloned in the plant transformation vector pKYLX71::35S(2). After transformation, constructs of antizearalenone scFv were introduced into immature Arabidopsis seeds via Agrobacterium tumefaciens mediation by vacuum infiltration. Only plants transformed with the construct containing a PR-1b signal peptide sequence produced transgenic offspring. The antizearalenone scFv "plantibody" from these transgenic plants bound zearalenone with a high affinity (50% inhibitory concentration, 11.2 ng/ml) that was comparable to that of bacterially produced scFv antibody and the parent monoclonal antibody (MAb). By electron microscopic immunogold labeling, the presence of antizearalenone scFv was detected mainly in the cytoplasm and only occasionally outside the cell. Like bacterially produced scFv antibody, antizearalenone scFv plantibody exhibited greater sensitivity to methanol destabilization than did the parent MAb. The sensitivity of antizearalenone scFv plantibody to acidic disassociation was similar to the sensitivities of bacterially produced scFv antibody and MAb. Expression of specific plantibodies in crops might be useful for neutralizing mycotoxins in animal feeds and for reducing mycotoxin-associated plant diseases.

Antibody Formation↗
PubMedDate not supplied

Clinical trial of a plant-derived antibody on recolonization of mutans streptococci.

OBJECTIVE: This double-blinded, placebo-controlled clinical trial tested the safety and efficacy of a topical secretory IgA antibody manufactured in tobacco plants (plantibody) in preventing recolonization of mutans streptococci (MS) in human plaque as measured by whole stimulated saliva samples. METHODS: Following a 9-day antimicrobial treatment with chlorhexidine (CHX), 56 eligible adults (enrollment salivary MS > or = 10(4) CFU/ml; no current caries) were randomized equally to a group receiving 0, 2, 4, or 6 topical applications of plantibody followed by 6, 4, 2, or 0 applications of placebo, respectively, over a 3-week period. RESULTS: Among the 54 subjects who completed the trial, the CHX regimen eliminated salivary MS in 69%. After 6 months, there were no significant differences in MS levels by number of applications, relative to placebo (p > 0.43). No adverse effects were observed. CONCLUSION: Plantibody is safe but not effective at the frequency, concentration, and number of applications used in this study.

Adult↗
PubMed2001

Production of secretory IgA antibodies in plants.

Functional antibodies produced in tobacco plants were first reported over a decade ago (1989). The basic protocol used to generate these 'plantibodies' involved the independent cloning of H and L chain antibody genes in Agrobacterium tumefaciens vectors, the transformation of plant tissue in vitro with the recombinant bacterium, the reconstitution of whole plants expressing individual chains, and their sexual cross. In a 'Mendelian' fashion, a fully assembled and functional antibody was recovered from plant tissue in some double-transgenic plants. In mammalian cells, the antibody H and L chains are produced as precursor proteins that are translocated into the endoplasmic reticulum (ER), under the guidance of signal sequences. Within the ER, the signal peptides are proteolytically cleaved, and several stress proteins act as chaperonins to bind the unassembled antibody chains, and direct subsequent folding and tetramer formation. A similar process occurs in plant cells, and expression can be directed via signal sequences (even of foreign origin) into the aqueous environment of the apoplasm, or to be accumulated in other specific plant tissues, including tubers, fruit, or seed. Plants can facilely assemble secretory IgA, which is comprised of four chains, H and L chains, J chain and secretory component. Plant 'bioreactors' are expected to yield over 10 kg of therapeutic antibody/acre in tobacco, maize, soybean, and alfalfa [(Ann. NY Acad. Sci.)721(1994)235; (Biotechnol. Bioeng.)20(1999)135]. Compared with conventional steel tank bioreactors using mammalian cells, or microorganisms, the costs of GMP plantibodies are expected to perhaps one tenth. The differences in glycosylation patterns of plant and mammalian cell produced antibodies apparently have no effect on antigen-binding or specificity, but there is some concern about potential immunogenicity in humans. N-linked glycans of plants differ from human by having fucose-linked alpha 1,3 and the sugar xylose. No adverse effects or human anti-mouse antibodies (HAMA) have been observed in >40 patients receiving topical oral application of a plant produced secretory IgA specific to Streptococcus mutans, for the control of caries [(Nat. Med.)4(1998)601]. The progressive improvement of expression vectors for plantibodies, and purification strategies, as well as the increase in transformable crop species, is expected to lead to almost limitless availability of inexpensive (even edible forms of) recombinant immunoglobulins free of human pathogens for human and animal therapy, and for novel industrial applications (e.g. catalytic antibodies).

Antibody Formation↗
PubMed2003

Large-scale purification of an antibody directed against hepatitis B surface antigen from transgenic tobacco plants.

The application of bioengineering to plants for production of biological products for human and animal use has expanded in recent years. The reasons for this expansion are several and include advances in the technology for novel production systems and the need for very large quantities of therapeutic proteins. The process of growing pharmaceutical proteins in plants, extracting, and purifying is a hard task considering the lack of available information concerning these topics. In this work, a recombinant murine monoclonal antibody specific for the hepatitis B surface antigen, expressed in stably transformed transgenic Nicotiana tabacum plants, was purified by means of a recombinant protein A Streamline chromatography as the main purification step. The antibody expression level varied with the age of the plants and the number of harvests from 40 to 15microg/ml and the maximum process yield was about 25mg of plantibody/kg of biomass. Protein A Streamline chromatography was successfully used in the purification process yielding a recovery of about 60% and a plantibody SDS-PAGE purity of over 90% but unexpectedly, previous clarification steps could not be totally avoided. The amino acid sequence recognized by this affinity purified plantibody was similar to its murine counterpart verifying the potentiality of plants to replace animals or bioreactors for large-scale production of this monoclonal antibody.

Amino Acid Sequence↗
PubMed1999

Functional expression in bacteria and plants of an scFv antibody fragment against tospoviruses.

BACKGROUND: Recombinant antibodies expressed in plants ('plantibodies'), directed against crucial antigens and addressed to the right cell compartment, may be able to protect against viral diseases. Moreover, antibody fragments produced in bacteria or plants may provide low cost reagents for immunodiagnosis. OBJECTIVES: In an attempt to develop genetic immunisation against tomato spotted wilt tospovirus (TSWV), we engineered an scFv fragment starting from a monoclonal antibody (mAb) able to recognise an epitope of the glycoprotein G1 conserved among a large number of tospoviruses. After establishing functional expression in bacteria, we aimed to drive expression of this molecule in the secretory pathway of plants. STUDY DESIGN: An antibody phage display expression system was used to isolate the correct VH and VL binding regions from the hybridoma secreting the original mAb. To assess functional expression in plant, we first used an epichromosomal expression vector derived from potato virus X (PVX). In this vector the scFv gene was cloned to produce a cytosolic or a secretory protein. For secretion, the signal sequence derived from the polygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris was used. Subsequently, the gene encoding the secretory scFv, was used to transform Nicotiana benthamiana plants. RESULTS: High expression levels of fully active molecule were obtained in Escherichia coli. The engineered molecule retained the binding specificity and dissociation rate constant (k(off)) of the cognate monoclonal antibody. Both PVX-infected and transformed plants expressed fully functional scFv molecules in the secretory pathway. CONCLUSION: This engineered scFv may be valuable for inexpensive diagnosis, for studying the role of the glycoproteins in virus transmission and, possibly, for a 'plantibody'-mediated resistance to tospoviruses.

Amino Acid Sequence↗
PubMed2003

Expression and characterization of an anti-(hepatitis B surface antigen) glycosylated mouse antibody in transgenic tobacco (Nicotiana tabacum) plants and its use in the immunopurification of its target antigen.

Transgenic plants expressing recombinant immunoglobulins have arisen as an alternative technology for the large-scale production of antibodies useful in therapeutics and in industrial processes. In the present paper we report the expression in transgenic tobacco ( Nicotiana tabacum ) of an anti-HBsAg [anti-(hepatitis B virus surface antigen)] mouse IgG1 mAb (monoclonal antibody), currently used for the industrial purification of the recombinant vaccine antigen. Using the sweet potato sporamin signal peptide, a KDEL (Lys-Asp-Glu-Leu) ER (endoplasmic reticulum) anchorage domain, and a heavy- and light-chain gene tandem construction, we generated F1 plants in which the expression of the antibody accounted for 0.5% of the total soluble proteins. The 'plantibody' (functional IgG antibody produced in plants) was easily purified by Protein A-Sepharose chromatography with a yield of approximately 35 microg/g of fresh leaf material, and its glycosylation indicated that, irrespective of the KDEL signal, the molecule is modified in both the ER and Golgi. Finally, a successful comparison of the plantibody with the ascites-derived mAb in the immunoaffinity purification of the vaccine recombinant HBsAg was performed. Taken as a whole, our results show that the large-scale production of this antibody of industrial relevance in transgenic tobacco is feasible.

Animals↗
PubMed2003

Monoclonal C5-1 antibody produced in transgenic alfalfa plants exhibits a N-glycosylation that is homogenous and suitable for glyco-engineering into human-compatible structures.

Structural analysis of the N-glycosylation of alfalfa proteins was investigated in order to evaluate the capacity of this plant to perform this biologically important post-translational modification. We show that, in alfalfa, N-linked glycans are processed into a large variety of mature oligosaccharides having core-xylose and core alpha(1,3)-fucose, as well as terminal Lewis(a) epitopes. In contrast, expression of the C5-1 monoclonal antibody in alfalfa plants results in the production of plant-derived IgG1 which is N-glycosylated by a predominant glycan having a alpha(1,3)-fucose and a beta(1,2)-xylose attached to a GlcNAc2Man3GlcNAc2 core. Since this core is common to plant and mammal N-linked glycans, it therefore appears that alfalfa plants have the ability to produce recombinant IgG1 having a N-glycosylation that is suitable for in vitro or in vivo glycan remodelling into a human-compatible plantibody. For instance, as proof of concept, in vitro galactosylation of the alfalfa-derived C5-1 mAb resulted in a homogenous plantibody harbouring terminal beta(1,4)-galactose residues as observed in the mammalian IgG.

Journal Article↗
PubMed2005

Transient expression in tobacco leaves of an aglycosylated recombinant antibody against the epidermal growth factor receptor.

When generating stably transformed transgenic plants, transient gene expression experiments are especially useful to rapidly confirm that the foreign molecule of interest is correctly assembled and retains its biological activity. TheraCIM(R) (CIMAB S.A., Havana) is a recombinant humanized antibody against the Epidermal Growth Factor receptor (EGF-R), now in clinical trials for cancer therapy in Cuba and other countries. An aglycosylated version (Asn 297 was mutated for Gln 297) of this antibody was transiently expressed in tobacco leaves after vacuum-mediated infiltration of recombinant Agrobacterium tumefaciens that carried a binary plasmid bearing the antibody heavy and light chain genes and plant regulation signals. Protein extracts from "agroinfiltrated" leaves were tested by ELISA and Western blot, showing that the fully assembled antibody was accumulated in plant tissues. The absence of plant specific glycans did not interfere in the assembling or in the activity of the plantibody, as demonstrated in this work. Indirect immunofluorescence demonstrated that the aglycosylated antibody expressed in plants recognizes the EGF-R expressed on the surface of A431 human tumor culture cells.

Antibodies, Monoclonal↗
PubMed1996

Antibody production in plants.

Production of heterologous proteins in plants has become increasingly efficient due to recent advances in plant biotechnology. Heterologous proteins that have specifically attracted a great deal of attention are plant-produced monoclonal antibodies. A variety of applications for these so-called plantibodies have been explored since they were first expressed in tobacco seven years ago. Both full length antibodies and antibody fragments produced in transgenic plants offer many intriguing possibilities to plant molecular biologists and plant breeders. However, questions such as how cellular targeting influences the expression and accumulation of these proteins in plants still need to be answered before the technology can be used commercially, on a large-scale.

Journal Article↗
PubMed2004

Standardization and validation of an alkaline phosphatase-linked immunoassay to quantify a plant-derived antibody directed against the hepatitis B virus surface antigen.

Immunopurification is one of the most effective chromatography steps to purify the hepatitis B surface antigen, which have successfully been used as an active pharmaceutical ingredient of hepatitis B vaccines. Plant-derived antibodies could be an appropriated ligand for such purposes because plants are the most cost-effective production systems and have the additional advantage that plant viruses cannot infect humans. In this work, a polyclonal antibody alkaline phosphatase-linked immunoassay was standardized and validated to quantify a plant-derived antibody directed against the HBsAg. The validation of an immunoassay to quantify plantibodies is a relatively complex task due to the complexity of the plant extract, the low level of expression of this molecule, and the potential interferences of endogenous peroxidases contributed by plants. These results allow estimating the plant-derived antibody concentration up to 3.81 ng/mL with high specificity, precision, and repeatability. The working range of the standard curve was between 3.81 and 60 ng/mL, and the intra- and inter-variation coefficients were between 10% and 20% in a production process's sample dependent way. This enzyme-linked immunosorbent assay is considered valuable to improve the design of the purification process and also to obtain a better estimation of the antibody expression level and process's recovery.

Alkaline Phosphatase↗
PubMed2004

Identification of a 49-bp fragment of the HvLTP2 promoter directing aleurone cell specific expression.

Identification of regulatory elements directing definite and specific spatiotemporal expression patterns is a prerequisite to the next generation of transgenic plants with commercial and ethical feasibility for producing plantibodies or other pharmaceutically important compounds. Here we describe the functional dissection of the barley nonspecific lipid transfer protein gene promoter, HvLTP2. The gene is specifically expressed in aleurone cells of cereals and used as an aleurone marker in maize and rice. The transcript is uniformly localised in the barley aleurone cells from around 10 DAP. Patchy expression in aleurone cells of transgenic rice has been reported and explained by silencing of transgenes. We have performed deletion analyses of the 801-bp HvLTP2 promoter to gain insight into the molecular basis of its regulation and the presence of putative regulatory elements. From the deletion studies, a 49-bp promoter region directing aleurone-specific expression was identified. Simultaneously, in vivo footprinting was carried out to identify promoter elements bound by putative regulatory proteins. Within the 49-bp fragment, the most promising candidate for a minimal cis-acting regulatory region directing aleurone specificity is the ds-sequence. Based on our results, we hypothesise that the ds-sequence directs aleurone specificity, possibly through a concerted action with elements directing general expression in the seed. Moreover, we present an overview of LTP2 elements putatively involved in directing seed, endosperm, and aleurone expression. Additionally, we report HvLTP2 expression in the embryo, not previously detected. The regulatory element(s) directing expression in embryo is located downstream of the 49-bp fragment directing aleurone specificity, thus demonstrating independent control of aleurone and embryo-localised expression. Finally, we discuss the existence of several endosperm-specific boxes and whether alternative promoter elements and combinations of them may direct aleurone expression, explaining why comparing different genes expressed in aleurone fail to identify only one required, common promoter element.

Base Sequence↗
PubMed2003

Resistance mechanisms to plant viruses: an overview.

To obtain virus-resistant host plants, a range of operational strategies can be followed nowadays. While for decades plant breeders have been able to introduce natural resistance genes in susceptible genotypes without knowing precisely what these resistance traits were, currently a growing number of (mostly) dominant resistance genes have been cloned and analyzed. This has led not only to a better understanding of the plant's natural defence systems, but also opened the way to use these genes beyond species borders. Besides using natural resistance traits, also several novel, "engineered" forms of virus resistance have been developed over the past 15 years. The first successes were obtained embarking from the principle of pathogen-derived resistance (PDR) by transforming host plants with viral genes or sequences with the purpose to block a specific step during virus multiplication in the plant. As an unforeseen spin-off of these investments, the phenomenon of post-translational gene silencing (PTGS) was discovered, which to date is by far the most successful way to engineer resistance. It is generally believed that PTGS reflects a natural defence system of the plant, and part of the hypothesized components required for PTGS have been identified. As counteracting strategy, and confirming PTGS to be a natural phenomenon, a considerable number of viruses have acquired gene functions by which they can suppress PTGS. In addition to PDR and PTGS, further strategies for engineered virus resistance have been explored, including the use of pokeweed antiviral protein (PAP), 2',5'-oligoadenylate synthetase and "plantibodies". This paper will give a brief overview of the major strategies that have become operational during the past 10 years.

Gene Silencing↗