Hepatitis B surface antigen immunopurification using a plant-derived specific antibody produced in large scale

This paper provides an evaluation of a plant-derived HBsAg-specific antibody in the immunopurification of the recombinant HBsAg for vaccine purposes. This plant-derived antibody was obtained from different batches of 100-200kg of tobacco leaves and coupled to Sepharose CL-4B with high efficiency. The plant-derived antibody immunoaffinity matrix purification behavior (elution capacity, antigen purity, purification cycles, and ligand leakage) was comparable to that of its mouse-derived monoclonal antibody homolog. This result supports the feasibility of using this plant-derived antibody for the immunopurification of the Hepatitis B surface antigen for human use, opening a new possibility to overcome the constrain of monoclonal antibody production in mice.     

Fighting cancer with plant-expressed pharmaceuticals

Cancer is one of the most prevalent diseases worldwide, which explains why biological therapies for cancer are forecast to make up 35% of total recombinant pharmaceuticals by 2010. Because of the high demand for cancer drugs, the need to lower production costs and the constraints of present production technologies for recombinant pharmaceuticals (such as the difficulties involved in culturing bacteria, yeast and mammalian cells), attention has recently been focused on recombinant expression of pharmaceutical anti-cancer proteins in plants. This review aims to provide an update on the most recent publications about anti-cancer recombinant pharmaceuticals expressed in plants, as well as on the relevant technical issues, potential and prospects of this emerging production system.     

A new approach for in vitro regeneration of tomato plants devoid of exogenous plant growth hormones

Many available methodologies for in vitro regeneration of commercial tomato varieties promote not only the production of normal shoots but also individual leaves, shoots without apical meristems and vitrified structures. All these abnormal formations influence and diminish the regeneration efficiency. At the basis of this phenomenon lies callus development. We optimized an alternative procedure by which the regeneration occurs without abnormal shoot formation. The portion including the proximal part of hypocotyls and the radicle was cultured on medium consisting of Murashige and Skoog salts, 4 mg/L thiamine, 100 mg/L mio-inositol and 3% sucrose. After two-three weeks, 60% explants showed adventitious shoot formation. No changes in the morphological characteristics of regenerated plants and fruits were observed as compared with parents. Karyotypic analysis of regenerated plants showed no variations in chromosome number. The optimized procedure offers the advantage of tomato plant regeneration avoiding callus formation, which enables normal plant recovery with an efficiency ranging from 1.45 +/- 0.05 to 2.57 +/- 0.06 shoots per explant in Campbell-28, Amalia, Lignon, and Floradel cultivars.     

Proteolytic gut activities in the rice water weevil,Lissorhoptrus brevirostris Suffrian (Coleoptera: Curculionidae)

Digestive endoprotease activities of the rice water weevil, Lissorhoptrus brevirostris Suffrian (Coleoptera: Curculionidae), were characterized based on the ability of gut extracts to hydrolyze specific synthetic substrates, optimal pH, and hydrolysis sensitivity to protease inhibitors. Larvae of this species were found to use a complex proteolytic system that includes cathepsin D-, cathepsin B-, trypsin-, and chymotrypsin-like activities. Trypsin-like activity was evenly distributed among the anterior, middle, and posterior portions of the gut, whereas cathepsin B- and cathepsin D-like activities were mainly located in the anterior and middle sections, and the chymotrypsin-like activity was highest in the middle and posterior sections. Gelatin-containing native-PAGE gels indicated the presence of several aspartyl, cysteine, and serine protease forms and confirmed the spatial organization of the proteolytic digestive process.     

Comparative in vitro and experimental in vivo studies of the anti–epidermal growth factor receptor antibody nimotuzumab and its aglycosylated form produced in transgenic tobacco plants

A broad variety of foreign genes can be expressed in transgenic plants, which offer the opportunity for large‐scale production of pharmaceutical proteins, such as therapeutic antibodies. Nimotuzumab is a humanized anti–epidermal growth factor receptor (EGFR) recombinant IgG1 antibody approved in different countries for the treatment of head and neck squamous cell carcinoma, paediatric and adult glioma, and nasopharyngeal and oesophageal cancers. Because the antitumour mechanism of nimotuzumab is mainly attributed to its ability to interrupt the signal transduction cascade triggered by EGF/EGFR interaction, we have hypothesized that an aglycosylated form of this antibody, produced by mutating the N₂₉₇ position in the IgG₁ Fc region gene, would have similar biochemical and biological properties as the mammalian‐cell‐produced glycosylated counterpart. In this paper, we report the production and characterization of an aglycosylated form of nimotuzumab in transgenic tobacco plants. The comparison of the plantibody and nimotuzumab in terms of recognition of human EGFR, effect on tyrosine phosphorylation and proliferation in cells in response to EGF, competition with radiolabelled EGF for EGFR, affinity measurements of Fab fragments, pharmacokinetic and biodistribution behaviours in rats and antitumour effects in nude mice bearing human A431 tumours showed that both antibody forms have very similar in vitro and in vivo properties. Our results support the idea that the production of aglycosylated forms of some therapeutic antibodies in transgenic plants is a feasible approach when facing scaling strategies for anticancer immunoglobulins.     

Tobacco leaf spot and root rot caused by Rhizoctonia solani Kühn

Rhizoctonia solani Kühn is a soil-borne fungal pathogen that causes disease in a wide range of plants worldwide. Strains of the fungus are traditionally grouped into genetically isolated anastomosis groups (AGs) based on hyphal anastomosis reactions. This article summarizes aspects related to the infection process, colonization of the host and molecular mechanisms employed by tobacco plants in resistance against R. solani diseases. TAXONOMY: Teleomorph: Thanatephorus cucumeris (Frank) Donk; anamorph: Rhizoctonia solani Kühn; Kingdom Fungi; Phylum Basidiomycota; Class Agaricomycetes; Order Cantharellales; Family Ceratobasidiaceae; genus Thanatephorus. IDENTIFICATION: Somatic hyphae in culture and hyphae colonizing a substrate or host are first hyaline, then buff to dark brown in colour when aging. Hyphae tend to form at right angles at branching points that are usually constricted. Cells lack clamp connections, but possess a complex dolipore septum with continuous parenthesomes and are multinucleate. Hyphae are variable in size, ranging from 3 to 17 μm in diameter. Although the fungus does not produce any conidial structure, ellipsoid to globose, barrel-shaped cells, named monilioid cells, 10-20 μm wide, can be produced in chains and can give rise to sclerotia. Sclerotia are irregularly shaped, up to 8-10 mm in diameter and light to dark brown in colour. DISEASE SYMPTOMS: Symptoms in tobacco depend on AG as well as on the tissue being colonized. Rhizoctonia solani AG-2-2 and AG-3 infect tobacco seedlings and cause damping off and stem rot. Rhizoctonia solani AG-3 causes 'sore shin' and 'target spot' in mature tobacco plants. In general, water-soaked lesions start on leaves and extend up the stem. Stem lesions vary in colour from brown to black. During late stages, diseased leaves are easily separated from the plant because of severe wilting. In seed beds, disease areas are typically in the form of circular to irregular patches of poorly growing, yellowish and/or stunted seedlings. RESISTANCE: Knowledge is scarce regarding the mechanisms associated with resistance to R. solani in tobacco. However, recent evidence suggests a complex response that involves several constitutive factors, as well as induced barriers controlled by multiple defence pathways. MANAGEMENT: This fungus can survive for many years in soil as mycelium, and also by producing sclerotia, which makes the management of the disease using conventional means very difficult. Integrated pest management has been most successful; it includes timely fungicide applications, crop rotation and attention to soil moisture levels. Recent developments in biocontrol may provide other tools to control R. solani in tobacco.     

A transformation procedure for recalcitrant tomato by addressing transgenic plant-recovery limiting factors

Agrobacterium tumefaciens technology is the battle horse for tomato genetic transformation. However, tomato varieties with low regeneration capacity are very difficult to transform. In the past, tomato transformation through Agrobacterium infection was focused on varieties capable of high regeneration yield, while successful transformation of low regenerable cultivars has not been reported. The genotype response to tissue culture conditions is believed to drive the frequency of regeneration of transgenic plant, whereas the capacity for cell proliferation could determine the transformation efficiency through this technology. The Campbell-28 cultivar is an example of constraints arising from a high morphogenetic potential with low conversion compared to normal plants. In the present work the roles that contribute to improved transgenic plant recovery from this recalcitrant variety were explored for factors like Agrobacterium concentration and antibiotics for bacterial removal and transformant selection. Analysis of the efficiency from independent transformation experiments revealed a more than twofold increase of transformant regeneration after selection on ammonium glufosinate compared to kanamycin selection, showing a transformation efficiency of 21.5%.     

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.