RESUMO
BACKGROUND: Animal and clinical studies with plant-produced single-chain variable fragment lymphoma vaccines have demonstrated specific immunogenicity and safety. However, the expression levels of such fragments were highly variable and required complex engineering of the linkers. Moreover, the downstream processing could not be built around standard methods like protein A affinity capture. DESIGN: We report a novel vaccine manufacturing process, magnifection, devoid of the above-mentioned shortcomings and allowing consistent and efficient expression in plants of whole immunoglobulins (Igs). RESULTS: Full idiotype (Id)-containing IgG molecules of 20 lymphoma patients and 2 mouse lymphoma models were expressed at levels between 0.5 and 4.8 g/kg of leaf biomass. Protein A affinity capture purification yielded antigens of pharmaceutical purity. Several patient Igs produced in plants showed specific cross-reactivity with sera derived from the same patients immunized with hybridoma-produced Id vaccine. Mice vaccinated with plant- or hybridoma-produced Igs showed comparable protection levels in tumor challenge studies. CONCLUSIONS: This manufacturing process is reliable and robust, the manufacturing time from biopsy to vaccine is <12 weeks and the expression and purification of antigens require only 2 weeks. The process is also broadly applicable for manufacturing monoclonal antibodies in plants, providing 50- to 1000-fold higher yields than alternative plant expression methods.
Assuntos
Vacinas Anticâncer/biossíntese , Idiótipos de Imunoglobulinas/metabolismo , Linfoma não Hodgkin/imunologia , Linfoma não Hodgkin/terapia , Planticorpos/metabolismo , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/imunologia , Agrobacterium tumefaciens/metabolismo , Animais , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/isolamento & purificação , Clonagem Molecular , Eficiência , Regulação da Expressão Gênica de Plantas , Humanos , Idiótipos de Imunoglobulinas/genética , Idiótipos de Imunoglobulinas/imunologia , Individualidade , Camundongos , Camundongos Endogâmicos C3H , Planticorpos/genética , Planticorpos/isolamento & purificação , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/imunologia , Plantas Geneticamente Modificadas/metabolismo , Fatores de Tempo , Vacinas Sintéticas/biossíntese , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
In elicitor-treated potato cells, 9-lipoxygenase-derived oxylipins accumulate with the divinyl ether colneleic acid as the major metabolite. Here, the identification of a potato cDNA is described, whose predicted amino acid sequence corresponds to divinyl ether synthases, belonging to the recently identified new P450 subfamily CYP74D. The recombinant protein was expressed in Escherichia coli and shown to metabolize 9-hydroperoxy linoleic acid to colneleic acid at pH 6.5. This fatty acid derivative has been implicated in functioning as a plant antimicrobial compound. RNA blot analyses revealed accumulation of divinyl ether synthase transcripts both upon infiltration of potato leaves with Pseudomonas syringae and after infection with Phytophthora infestans.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Plantas , Solanum tuberosum/enzimologia , Solanum tuberosum/microbiologia , Técnicas de Cultura de Células/métodos , Células Cultivadas , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Escherichia coli/genética , Ácidos Graxos Insaturados/metabolismo , Dados de Sequência Molecular , Oxirredutases/efeitos dos fármacos , Phytophthora/patogenicidade , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Pseudomonas/patogenicidade , Solanum tuberosum/citologiaRESUMO
Lanatoside 15'-O-acetylesterase (LAE) from in-vitro-cultivated cells of Digitalis lanata Ehrh. was isolated and partially sequenced. The enzyme was extracted with citrate buffer from acetone dry powder. It was purified in a two-step chromatographical procedure including Phenyl Sepharose hydrophobic interaction chromatography followed by CM Sepharose cation-exchange chromatography to more than 330 mumol.s-1.(g protein)-1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified protein showed a major band at 39 kDa. The protein was identified by correlation of band intensity on SDS-PAGE and enzyme activity of CM Sepharose column fractions. Size-exclusion chromatography on Sephacryl 200 revealed a single activity peak with an apparent molecular mass of about 85 kDa. Electrophoresis under nondenaturating conditions of purified LAE showed only one band with esterase activity. The intensity of this band was correlated with that of the 39-kDa band after SDS-PAGE. About 30% of the protein, including the N-terminus and several fragments obtained by Lys-C protease digestion, was sequenced. A fragment obtained by Lys-C digestion showed partial homology to other hydrolases and apoplasmic proteins. It included the probable location of an active-site histidine. The activity of LAE was high in non-morphogenic D. lanata cell strains selected for high activities in the chemical transformation of cardenolides, but rather low in the proembryogenic masses of the embryogenic cell strain VIII. It increased during the development of somatic embryos. The LAE activity in leaves of D. lanata plants was in the range 4-24 nmol.s-1.(g protein)-1.
