RESUMEN
BACKGROUND: The biopharmaceutical industry is significantly growing worldwide, and the Chinese hamster ovary (CHO) cells are used as a main expression host for the production of recombinant monoclonal antibodies. Various metabolic engineering approaches have been investigated to generate cell lines with improved metabolic characteristics for increasing longevity and mAb production. A novel cell culture method based on the 2-stage selection makes it possible to develop a stable cell line with high-quality mAb production. RESULTS: We have constructed several design options of mammalian expression vectors for the high production of recombinant human IgG antibodies. Versions for bipromoter and bicistronic expression plasmids different in promoter orientation and cistron arrangements were generated. The aim of the work presented here was to assess a high-throughput mAb production system that integrates the advantages of high-efficiency cloning and stable cell clones to stage strategy selection reducing the time and effort required to express therapeutic monoclonal mAbs. Development of a stable cell line using bicistronic construct with EMCV IRES-long link gave an advantage in high mAb expression and long-term stability. Two-stage selection strategies allowed the elimination of low-producer clones by using metabolic level intensity to estimate the IgG production in the early steps of selection. The practical application of the new method allows to reduce time and costs during stable cell line development.
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The development of and research into new therapies that can selectively and effectively destroy tumor cells that overexpress the ErbB2 receptor is a pressing task. Recently, research into the use of type I interferons in the treatment of cancer has intensified. Cytokine therapy is aimed at activating the cells of the immune system to fight tumors, but it has drawbacks that limit its use because of a number of side effects the severity of which varies depending on the dosage and type of used cytokine. At the moment, a number of studies are being conducted regarding the use of IFNß in oncology. The studies are aimed at mitigating the systemic action of this cytokine. The immunocytokine complex made of a bispecific antibody against the ErbB2 receptor and recombinant IFNß developed in this study underlies the mechanism meant to avoid the systemic action of this cytokine. Part of this study focuses on the development of full-length antibodies that bind to the ErbB2 receptor on the one hand, and bind and neutralize IFNß, on the other hand, which allows us to consider the antibodies as a means of cytokine delivery to tumor cells.
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Currently, there are no approved therapies for targeted prevention and treatment of Ebola hemorrhagic fever. In the present work, we describe the development of a eukaryotic expression system for the production of three full-length chimeric antibodies (IgG1-kappa isotypes) GPE118, GPE325, and GPE534 to the recombinant glycoprotein of the Ebola virus (EBOV GP), which is a key factor in the pathogenicity of the disease. The immunochemical properties of the obtained antibodies were studied by immunoblotting and indirect, direct, and competitive ELISA using the recombinant EBOV proteins rGPdTM, NP, and VP40. The authenticity of the antibodies and the absence of cross-specificity with respect to the structural proteins NP and VP40 of the Ebola virus were proved. The epitope specificity of the resulting recombinant antibodies was studied using commercial neutralizing antibodies against the viral glycoprotein. The recombinant antibodies GPE118, GPE325, and GPE534 were shown to recognize glycoprotein epitopes that coincide or overlap with the epitopes of three well-studied neutralizing anti-Ebola virus antibodies.
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We determined the nucleotide and amino acid sequences of variable domains of three new monoclonal antibodies to the glycoprotein of Ebola virus capsid. The framework and hypervariable regions of immunoglobulin heavy and light chains were identified. The primary structures were confirmed using massspectrometry analysis. Immunoglobulin database search showed the uniqueness of the sequences obtained.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Antivirales/química , Ebolavirus/inmunología , Región Variable de Inmunoglobulina/química , Proteínas del Envoltorio Viral/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Antivirales/genética , Secuencia de Bases , Cartilla de ADN , Electroforesis , Región Variable de Inmunoglobulina/genética , Ratones , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
A panel of ten monoclonal antibodies to aflatoxins B1, B2, and G2 was produced and comprehensively characterized. The affinity and cross reactivity of these antibodies were determined using the methods of direct, indirect, and competitive ELISA. The structures of monoclonal antibody genes were comprehensively studied and the variable and constant domains of the antibody genes were cloned and sequenced. Sequencing analysis confirmed the results of isotyping the light and heavy antibody chains obtained by ELISA. Variable and constant fragments of the antibody genes were cloned into a bicistron expression vector for the recombinant Fab' fragment for one of the antibodies expressed in Escherichia coli and purified. Thus, data were obtained that can be useful for the development of an aflatoxin detection system on the basis of the described monoclonal antibodies and the creation of recombinant antibodies with changed parameters of specificity using protein engineering methods.
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Aflatoxina B1/inmunología , Aflatoxinas/inmunología , Anticuerpos Monoclonales/biosíntesis , Animales , Anticuerpos Monoclonales/genética , Afinidad de Anticuerpos , Reacciones Cruzadas , Escherichia coli/genética , Escherichia coli/metabolismo , Técnicas para Inmunoenzimas , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Fragmentos Fab de Inmunoglobulinas/genética , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Ligeras de Inmunoglobulina/biosíntesis , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
A full-size human antibody to Ebola virus was constructed by joining genes encoding the constant domains of the heavy and light chains of human immunoglobulin with the corresponding DNA fragments encoding variable domains of the single-chain antibody 4D1 specific to Ebola virus, which was chosen from a combinatorial phage display library of single-strand human antibodies. Two expression plasmids. pCH1 and pCL1, containing the artificial genes encoding the light and heavy chains of human immunoglobulin, respectively, were constructed. Their cotransfection into the human embryonic kidney cell line HEK293T provided the production of a full-size recombinant human antibody. The affinity constant for the antibody was estimated by solid-phase enzyme-linked immunoassay to be 7.7 x 10(7) +/- 1.5 x 10(7) M(-1). Like the parent single-chain antibody 4DI, the resulting antibody bound the nucleoprotein of Ebola virus and did not interact with the proteins of Marburg virus.
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Anticuerpos Antivirales/biosíntesis , Ebolavirus/inmunología , Proteínas Recombinantes/biosíntesis , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Línea Celular , Clonación Molecular , Humanos , Nucleoproteínas/inmunología , Plásmidos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Transfección , Proteínas del Núcleo Viral/inmunologíaRESUMEN
The isolation of single-stranded (ss) phagemid DNA using standard protocols often results in impure preparations, which contain undesirable quantities of chromosomal and/or double-stranded (ds) phagemid DNA. Here we report a simple and efficient method for elimination of virtually all dsDNA by incubation of phagemid viral particles with deoxyribonuclease I. In addition to analyzing the ratio of linear-to-circular topological forms of ssDNA after deoxyribonuclease I treatment, we verified that no decrease in transformation efficiency occurred and demonstrated that ssDNA molecules covered by capsid proteins remained intact following such treatment.