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1.
Bioprocess Biosyst Eng ; 42(5): 799-806, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30730009

RESUMO

Stable transfection of mammalian cells using various expression cassettes for exogenous gene expression has been well established. The impact of critical factors in these cassettes, such as promoter and enhancer elements, on recombinant protein production in mammalian cells has been studied extensively to optimize the expression efficiency. However, few studies on the correlation between the strength of selection marker and the expression of gene of interest (GOI) have been reported. Here we investigated the correlation between the strength of a widely used selection marker, glutamine synthetase (GS) gene, and gene of interest in which the expression of GOI is driven by mouse cytomegalovirus (mCMV) major immediate early (MIE) promoter whereas the expression of GS is controlled by SV40E (Simian vacuolating virus 40E) promoter. We used a green fluorescent protein and the adalimumab antibody (heavy and light chain) as two distinct examples for the gene of interest. We then decreased the expression of GS gene by engineering a specific region of its SV40E promoter in these expression cassettes. By comparing the expression of GS and GOI at transcription and translation level before and after the SV40E promoter was weakened, we found that lower GS expression due to weaker SV40E transcription correlated well with the higher expression of recombinant proteins, mainly by increasing the copy number of GS and GOI integration into host cell genome.


Assuntos
Adalimumab , Expressão Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde , Regiões Promotoras Genéticas , Transcrição Genética , Adalimumab/biossíntese , Adalimumab/genética , Animais , Células CHO , Cricetulus , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Camundongos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
2.
Immunol Res ; 66(3): 392-405, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29855993

RESUMO

Tumor necrosis factor alpha (TNFα) is a pro-inflammatory cytokine that mediates the homeostasis of immune responses; its exacerbated production is associated with the pathogenesis of autoimmune and chronic inflammatory diseases. Anti-TNFα drugs have revolutionized the treatment of inflammatory conditions such as rheumatoid arthritis and Crohn's disease. Currently, a worldwide race is on stage for the production of biosimilars moved by patent expiration of monoclonal antibodies (mAbs), such as anti-TNFα adalimumab. Our goal was to develop the first stage of an adalimumab biosimilar candidate with potential for national production, through the generation of a productive and stable cell line and assess its functionality. The robotic system ClonePix was used for screening and isolation of colonies from transfected CHO-S stable pools plated in semisolid medium. Selected clones were expanded based on growth and productivity. Purified mAbs from different clones were tested for binding and functional activity. The binding affinity of the denominated adabut clones to TNFα and FcRγ did not differ statistically when compared to reference adalimumab. One functional activity assay demonstrated the antibody neutralization capacity of the cytotoxicity induced by TNFα in L929 murine fibroblasts. A second assay confirmed adabut as an antagonist of the TNFα activity by the inhibition of the cell adhesion molecule expression in HUVEC cultures. The binding and functional activity analyses performed with selected adabut clones in comparison to reference adalimumab represent an important status of "non-inferiority," part of the process required for a biosimilar development. We generated and selected high-quality adabut clones which mAbs may be further developed as the first in-house made Brazilian biosimilar, demonstrating a success case for our incipient biotechnology industry, or also modified as biobetters, thus representing an innovative strategy for the patients' welfare.


Assuntos
Adalimumab/imunologia , Anticorpos Monoclonais/imunologia , Medicamentos Biossimilares , Proteínas Recombinantes de Fusão/imunologia , Fator de Necrose Tumoral alfa/imunologia , Adalimumab/genética , Adalimumab/metabolismo , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Células CHO , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Células Cultivadas , Cricetinae , Cricetulus , Humanos , Camundongos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
3.
Biochem Biophys Res Commun ; 503(2): 752-756, 2018 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-29909010

RESUMO

The production of therapeutic monoclonal antibodies is costly; therefore, antigen-binding fragments (Fabs) can be used instead. However, their tendency toward aggregation can reduce the half-life in the plasma and the therapeutic effectiveness. To examine the effect of glycosylation on the properties of the Fab of a therapeutic antibody, an N-glycosylation site was introduced at position 178 of the H-chain constant region of adalimumab Fab through site-directed mutagenesis of L178 N (H:L178 N Fab), and then H:L178 N Fab was expressed in Pichia pastoris. SDS-PAGE analysis with treatment of N-glycosidase F or periodic acid-Schiff reagent showed that H:L178 N Fab contained a relatively low glycan level. Moreover, the H:L178 N mutation did not decrease the binding activity and thermal stability of Fab, and H:L178 N Fab was more resistant to protease digestion than wild-type Fab. The aggregation of Fab induced by pH-shift stress was measured by monitoring the optical density at 350 nm. Although the wild-type Fab showed a large increase in optical density with an increase of protein concentration, no such increase of turbidity during aggregation was found in H:L178 N Fab. These results demonstrated that glycosylation at position 178 of the H-chain constant region of adalimumab Fab can prevent protein aggregation, and therefore serve as a potentially effective platform for drug development.


Assuntos
Adalimumab/química , Anti-Inflamatórios/química , Fragmentos Fab das Imunoglobulinas/química , Agregados Proteicos , Adalimumab/genética , Anti-Inflamatórios/metabolismo , Expressão Gênica , Glicoproteínas/química , Glicoproteínas/genética , Glicosilação , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Mutação , Pichia/genética
4.
Mol Biotechnol ; 60(6): 387-395, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29616400

RESUMO

Production of monoclonal antibodies and pharmaceutical proteins in transgenic plants has been the focus of many research efforts for close to 30 years. Use of plants as bioreactors reduces large-scale production costs and minimizes risk for human pathogens contamination. Stable nuclear transformation of the plant genome offers a clear advantage in agricultural protein production platforms, limited only by the number of hectares that can be cultivated. We report here, for the first time, successful and stable expression of adalimumab in transgenic Nicotiana tabacum plants. The plant-derived adalimumab proved fully active and was shown to rescue L929 cells from the in vitro lethal effect of rhTNFα just as effectively as commercially available CHO-derived adalimumab (Humira). These results indicate that agricultural biopharming is an efficient alternative to mammalian cell-based expression platforms for the large-scale production of recombinant antibodies.


Assuntos
Adalimumab/genética , Tabaco/genética , Adalimumab/biossíntese , Adalimumab/isolamento & purificação , Adalimumab/metabolismo , Reatores Biológicos , Engenharia Genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Tabaco/metabolismo
5.
Biochem Biophys Res Commun ; 495(1): 7-11, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29097200

RESUMO

We constructed a system for expressing the Fab of the therapeutic human monoclonal antibody adalimumab at a yield of 20 mg/L in the methylotrophic yeast Pichia pastoris. To examine the contribution of interchain disulfide bonds to conformational stability, we prepared adalimumab Fab from which the interchain disulfide bond at the C-terminal region at both the CH1 and CL domains was deleted by substitution of Cys with Ala (FabΔSS). DSC measurements showed that the Tm values of FabΔSS were approximately 5 °C lower than those of wild-type Fab, suggesting that the interchain disulfide bond contributes to conformational thermostability. Using computer simulations, we designed a novel interchain disulfide bond outside the C-terminal region to increase the stability of FabΔSS. The resulting Fab (mutSS FabΔSS) had the mutations H:V177C and L:Q160C in FabΔSS, confirming the formation of the disulfide bond between CH1 and CL. The thermostability of mutSS FabΔSS was approximately 5 °C higher than that of FabΔSS. Therefore, the introduction of the designed interchain disulfide bond enhanced the thermostability of FabΔSS and mitigated the destabilization caused by partial reduction of the interchain disulfide bond at the C-terminal region, which occurs in site-specific modification such as PEGylation.


Assuntos
Adalimumab/química , Adalimumab/genética , Adalimumab/metabolismo , Dissulfetos/química , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Modelos Moleculares , Pichia/genética , Conformação Proteica , Engenharia de Proteínas , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinâmica , Fator de Necrose Tumoral alfa/antagonistas & inibidores
6.
Cytokine ; 101: 56-63, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-27567553

RESUMO

Tumor necrosis factor (TNF)-α is a potent pro-inflammatory and pathological cytokines in inflammatory diseases such as rheumatoid arthritis and inflammatory bowel diseases. Anti-TNF-α therapy has been established as an efficacious therapeutic strategy in these diseases. In clinical settings, three monoclonal anti-TNF-α full IgG1 antibodies infliximab, adalimumab, and golimumab, PEGylated Fab' fragment of anti-TNF-α antibody certolizumab pegol, extracellular domain of TNF receptor 2/IgG1-Fc fusion protein etanercept, are almost equally effective for rheumatoid arthritis. Although monoclonal full IgG1 antibodies are able to induce clinical and endoscopic remission in inflammatory bowel diseases, certolizumab pegol without Fc portion has been shown to be less effective for inflammatory bowel diseases compared to full IgG1 antibodies. In addition, there are no evidences that etanercept leads clinical remission in inflammatory bowel diseases. Besides the common effect of anti-TNF-α agents on neutralization of soluble TNF-α, each anti-TNF-α agent has its own distinctive pharmacological properties which cause the difference in clinical efficacies. Here we focus on the distinctions of action of anti-TNF-α agents especially in following points; (1) blocking ability against ligands, transmembrane TNF-α and lymphotoxin, (2) effects toward transmembrane TNF-α-expressing cells, (3) effects toward Fcγ receptor-expressing cells, (4) degradation and distribution in inflamed tissue. Accumulating evidence will give us the idea how to modify anti-TNF-α agents to enhance the clinical efficacy in inflammatory diseases.


Assuntos
Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/genética , Artrite Reumatoide/tratamento farmacológico , Imunoglobulina G/uso terapêutico , Doenças Inflamatórias Intestinais/tratamento farmacológico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adalimumab/efeitos adversos , Adalimumab/genética , Adalimumab/uso terapêutico , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/efeitos adversos , Anti-Inflamatórios/uso terapêutico , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/uso terapêutico , Antirreumáticos/administração & dosagem , Antirreumáticos/efeitos adversos , Antirreumáticos/uso terapêutico , Artrite Reumatoide/imunologia , Certolizumab Pegol/efeitos adversos , Certolizumab Pegol/genética , Certolizumab Pegol/uso terapêutico , Modelos Animais de Doenças , Etanercepte/efeitos adversos , Etanercepte/uso terapêutico , Humanos , Fragmentos Fab das Imunoglobulinas/efeitos adversos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Imunoglobulina G/efeitos adversos , Imunoglobulina G/genética , Fatores Imunológicos/efeitos adversos , Fatores Imunológicos/genética , Fatores Imunológicos/uso terapêutico , Imunossupressores/administração & dosagem , Imunossupressores/efeitos adversos , Imunossupressores/uso terapêutico , Doenças Inflamatórias Intestinais/imunologia , Infliximab/efeitos adversos , Infliximab/genética , Infliximab/uso terapêutico , Camundongos , Polietilenoglicóis/uso terapêutico , Fator de Necrose Tumoral alfa/imunologia
7.
Biotechnol J ; 12(1)2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27440252

RESUMO

As a possible viable and non-invasive method to identify high producing cells, Confocal Raman Microscopy was shown to be able to differentiate CHO host cell lines and derivative production clones. Cluster analysis of spectra and their derivatives was able to differentiate between different producer cell lines and a host, and also distinguished between an intracellular region of high lipid and protein content that in structure resembles the Endoplasmic Reticulum. This ability to identify the ER may be a major contributor to the identification of high producers. PCA enabled the discrimination even of host cell lines and their subclones with inherently higher production capacity. The method is thus a promising option that may contribute to early, non-invasive identification of high potential candidates during cell line development and possibly could also be used for proof of identity of established production clones.


Assuntos
Células CHO/citologia , Células CHO/ultraestrutura , Microscopia Confocal/métodos , Engenharia de Proteínas/métodos , Adalimumab/genética , Adalimumab/metabolismo , Amina Oxidase (contendo Cobre)/genética , Amina Oxidase (contendo Cobre)/metabolismo , Animais , Análise por Conglomerados , Cricetulus , Retículo Endoplasmático/ultraestrutura , Humanos , Lipídeos/química , Metais/química , Imagem Molecular/métodos , Análise de Componente Principal , Proteínas/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise Espectral Raman/métodos
8.
Arthritis Res Ther ; 17: 63, 2015 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-25885039

RESUMO

INTRODUCTION: We have hypothesized that incompatibility between the G1m genotype of the patient and the G1m1 and G1m17 allotypes carried by infliximab (INX) and adalimumab (ADM) could decrease the efficacy of these anti-tumor necrosis factor (anti-TNF) antibodies in the treatment of rheumatoid arthritis (RA). METHODS: The G1m genotypes were analyzed in three collections of patients with RA totaling 1037 subjects. The first, used for discovery, comprised 215 Spanish patients. The second and third were successively used for replication. They included 429 British and Greek patients and 393 Spanish and British patients, respectively. Two outcomes were considered: change in the Disease Activity Score in 28 joint (ΔDAS28) and the European League Against Rheumatism (EULAR) response criteria. RESULTS: An association between less response to INX and incompatibility of the G1m1,17 allotype was found in the discovery collection at 6 months of treatment (P = 0.03). This association was confirmed in the replications (P = 0.02 and 0.08, respectively) leading to a global association (P = 0.001) that involved a mean difference in ΔDAS28 of 0.4 units between compatible and incompatible patients (2.3 ± 1.5 in compatible patients vs. 1.9 ± 1.5 in incompatible patients) and an increase in responders and decrease in non-responders according to the EULAR criteria (P = 0.03). A similar association was suggested for patients treated with ADM in the discovery collection, but it was not supported by replication. CONCLUSIONS: Our results suggest that G1m1,17 allotypes are associated with response to INX and could aid improved therapeutic targeting in RA.


Assuntos
Adalimumab/genética , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Imunoglobulina G/genética , Infliximab/genética , Adalimumab/uso terapêutico , Adulto , Antirreumáticos/uso terapêutico , Artrite Reumatoide/imunologia , Sequência de Bases , Feminino , Genótipo , Humanos , Alótipos de Imunoglobulina , Infliximab/uso terapêutico , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Resultado do Tratamento , Fator de Necrose Tumoral alfa/antagonistas & inibidores
9.
Plant Cell Rep ; 34(6): 959-68, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25689888

RESUMO

KEY MESSAGE: We successfully developed a method for metabolic isotope labeling of recombinant proteins produced in transgenic tobacco. This enabled assessment of structural integrity of plant-derived therapeutic antibodies by NMR analysis. A variety of expression vehicles have been developed for the production of promising biologics, including plants, fungi, bacteria, insects, and mammals. Glycoprotein biologics often experience altered folding and post-translational modifications that are typified by variant glycosylation patterns. These differences can dramatically affect their efficacy, as exemplified by therapeutic antibodies. However, it is generally difficult to validate the structural integrity of biologics produced using different expression vehicles. To address this issue, we have developed and applied a stable-isotope-assisted nuclear magnetic resonance (NMR) spectroscopy method for the conformational characterization of recombinant antibodies produced in plants. Nicotiana benthamiana used as a vehicle for the production of recombinant immunoglobulin G (IgG) was grown in a (15)N-enriched plant growth medium. The Fc fragment derived from the (15)N-labeled antibody thus prepared was subjected to heteronuclear two-dimensional (2D) NMR measurements. This approach enabled assessment of the structural integrity of the plant-derived therapeutic antibodies by comparing their NMR spectral properties with those of an authentic IgG-Fc derived from mammalian cells.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Tabaco/genética , Adalimumab/genética , Sequência de Aminoácidos , Sequência de Carboidratos , Glicosilação , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Dados de Sequência Molecular , Isótopos de Nitrogênio , Plantas Geneticamente Modificadas/genética , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Tabaco/metabolismo
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