RESUMO
The continuous improvement of expression platforms is necessary to respond to the increasing demand for recombinant proteins that are required to carry out structural or functional studies as well as for their characterization as biotherapeutics. While transient gene expression (TGE) in mammalian cells constitutes a rapid and well-established approach, non-clonal stably transfected cells, or "pools," represent another option, which is especially attractive when recurring productions of the same protein are required. From a culture volume of just a few liters, stable pools can provide hundreds of milligrams to gram quantities of high-quality secreted recombinant proteins.In this chapter, we describe a highly efficient and cost-effective procedure for the generation of Chinese Hamster Ovary cell stable pools expressing secreted recombinant proteins using commercially available serum-free media and polyethylenimine (PEI) as the transfection reagent. As a specific example of how this protocol can be applied, the production and downstream purification of recombinant His-tagged trimeric SARS-CoV-2 spike protein ectodomain (SmT1) are described.
Assuntos
Cricetulus , Polietilenoimina , Proteínas Recombinantes , Glicoproteína da Espícula de Coronavírus , Transfecção , Células CHO , Animais , Proteínas Recombinantes/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transfecção/métodos , Polietilenoimina/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/biossíntese , Glicoproteína da Espícula de Coronavírus/isolamento & purificação , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Cricetinae , Meios de Cultura Livres de SoroRESUMO
Protein expression from stably transfected Chinese hamster ovary (CHO) clones is an established but time-consuming method for manufacturing therapeutic recombinant proteins. The use of faster, alternative approaches, such as non-clonal stable pools, has been restricted due to lower productivity and longstanding regulatory guidelines. Recently, the performance of stable pools has improved dramatically, making them a viable option for quickly producing drug substance for GLP-toxicology and early-phase clinical trials in scenarios such as pandemics that demand rapid production timelines. Compared to stable CHO clones which can take several months to generate and characterize, stable pool development can be completed in only a few weeks. Here, we compared the productivity and product quality of trimeric SARS-CoV-2 spike protein ectodomains produced from stable CHO pools or clones. Using a set of biophysical and biochemical assays we show that product quality is very similar and that CHO pools demonstrate sufficient productivity to generate vaccine candidates for early clinical trials. Based on these data, we propose that regulatory guidelines should be updated to permit production of early clinical trial material from CHO pools to enable more rapid and cost-effective clinical evaluation of potentially life-saving vaccines.
Assuntos
COVID-19 , SARS-CoV-2 , Cricetinae , Animais , Humanos , Cricetulus , SARS-CoV-2/metabolismo , Células CHO , Anticorpos Monoclonais , Vacinas contra COVID-19/genética , COVID-19/prevenção & controle , Proteínas Recombinantes/metabolismo , Vacinas de Subunidades Antigênicas/genéticaRESUMO
Some effector functions prompted by immunoglobulin G (IgG) antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC), strongly depend on the N-glycans linked to asparagine 297 of the Fc region of the protein. A single α-(1,6)-fucosyltransferase (FUT8) is responsible for catalyzing the addition of an α-1,6-linked fucose residue to the first GlcNAc residue of the N-linked glycans. Antibodies missing this core fucose show a significantly enhanced ADCC and increased antitumor activity, which could help reduce therapeutic dose requirement, potentially translating into reduced safety concerns and manufacturing costs. Several approaches have been developed to modify glycans and improve the biological functions of antibodies. Here, we demonstrate that expression of a membrane-associated anti-FUT8 intrabody engineered to reside in the endoplasmic reticulum and Golgi apparatus can efficiently reduce FUT8 activity and therefore the core-fucosylation of the Fc N-glycan of an antibody. IgG1-producing CHO cells expressing the intrabody secrete antibodies with reduced core fucosylation as demonstrated by lectin blot analysis and UPLC-HILIC glycan analysis. Cells engineered to inhibit directly and specifically alpha-(1,6)-fucosyltransferase activity allows for the production of g/L levels of IgGs with strongly enhanced ADCC effector function, for which the level of fucosylation can be selected. The quick and efficient method described here should have broad practical applicability for the development of next-generation therapeutic antibodies with enhanced effector functions.
Assuntos
Fucose , Fucosiltransferases , Animais , Anticorpos Monoclonais/química , Células CHO , Cricetinae , Cricetulus , Fucose/metabolismo , Fucosiltransferases/genética , Imunoglobulina G/química , PolissacarídeosRESUMO
Antibody-based drugs are an increasingly important part of the therapeutic arsenal against a wide variety of medical conditions. As the number of commercial products and pipeline candidates grows, a crucial issue facing the industry is the current and future state of biomanufacturing. The productivity of the protein expression platforms, along with the performance of the technologies impacting upstream and downstream bioprocessing, are critical factors affecting the cost and time of therapeutic antibody development and commercialization. Cell engineering strategies are being used to improve the production of antibodies and to better control their quality in terms of posttranslational modifications, in particular with regards to their glycosylation state, as this can influence their therapeutic activity. Additionally, the advance of "omics" technologies have recently given rise to new possibilities in improving these expression platforms. We review here the various advances in biomanufacturing essential to the continued growth of the therapeutic antibody market.
TITLE: La bioproduction des anticorps monoclonaux. ABSTRACT: Les anticorps monoclonaux font désormais partie intégrante de l'arsenal thérapeutique pour une multitude de maladies. Étant donné le nombre croissant de produits commerciaux et de candidats en développement, l'état actuel des systèmes de bioproduction est une préoccupation majeure de l'industrie. La productivité des plateformes d'expression, ainsi que la performance des technologies utilisées dans les procédés en amont et en aval, sont des facteurs critiques qui ont un impact sur le coût et la durée du développement des anticorps thérapeutiques. De multiples stratégies de génie cellulaire peuvent être utilisées pour l'amélioration de la production des anticorps et pour un contrôle accru des modifications post-traductionnelles, comme la glycosylation, particulièrement importante, car elle peut avoir un effet prononcé sur l'activité thérapeutique des anticorps. Les avancées des techniques « omiques ¼ rendent maintenant possibles de nouvelles approches pour l'amélioration de ces plateformes. Nous passons ici en revue les progrès en bioproduction essentiels à la croissance continue du marché des anticorps thérapeutiques.
Assuntos
Anticorpos Monoclonais , Composição de Medicamentos , Engenharia de Proteínas/métodos , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Composição de Medicamentos/métodos , Desenvolvimento de Medicamentos/métodos , Indústria Farmacêutica/métodos , Humanos , Manufaturas/provisão & distribuiçãoRESUMO
Individuals with neurofibromatosis type-1 (NF1) can manifest focal skeletal dysplasias that remain extremely difficult to treat. NF1 is caused by mutations in the NF1 gene, which encodes the RAS GTPase-activating protein neurofibromin. We report here that ablation of Nf1 in bone-forming cells leads to supraphysiologic accumulation of pyrophosphate (PPi), a strong inhibitor of hydroxyapatite formation, and that a chronic extracellular signal-regulated kinase (ERK)-dependent increase in expression of genes promoting PPi synthesis and extracellular transport, namely Enpp1 and Ank, causes this phenotype. Nf1 ablation also prevents bone morphogenic protein-2-induced osteoprogenitor differentiation and, consequently, expression of alkaline phosphatase and PPi breakdown, further contributing to PPi accumulation. The short stature and impaired bone mineralization and strength in mice lacking Nf1 in osteochondroprogenitors or osteoblasts can be corrected by asfotase-α enzyme therapy aimed at reducing PPi concentration. These results establish neurofibromin as an essential regulator of bone mineralization. They also suggest that altered PPi homeostasis contributes to the skeletal dysplasias associated with NF1 and that some of the NF1 skeletal conditions could be prevented pharmacologically.
Assuntos
Fosfatase Alcalina/uso terapêutico , Desenvolvimento Ósseo/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Imunoglobulina G/uso terapêutico , Neurofibromatose 1/tratamento farmacológico , Neurofibromina 1/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Adolescente , Fosfatase Alcalina/biossíntese , Animais , Doenças do Desenvolvimento Ósseo/genética , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Células Cultivadas , Criança , Pré-Escolar , Colágeno Tipo I/biossíntese , Cadeia alfa 1 do Colágeno Tipo I , Colágeno Tipo II/genética , Difosfatos/metabolismo , Modelos Animais de Doenças , Durapatita/metabolismo , Humanos , Lactente , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Osteoblastos/enzimologia , Osteogênese/genética , Proteínas de Transporte de Fosfato/biossíntese , Proteínas de Transporte de Fosfato/genética , Diester Fosfórico Hidrolases/biossíntese , Diester Fosfórico Hidrolases/genética , Pirofosfatases/biossíntese , Pirofosfatases/genética , Fator de Transcrição Sp7 , Fatores de Transcrição/genéticaRESUMO
The E2 protein of human papillomavirus (HPV) binds to specific sites in the viral genome to regulate its transcription, replication, and maintenance in infected cells. Like most regulatory proteins, E2 is rapidly turned over. A high-throughput assay was developed to quantify the expression and stability of E2 in vivo, based on its fusion to Renilla luciferase (RLuc). The steady-state levels of Rluc-E2 were quantified by measuring the amounts of associated luciferase activity, and its degradation was measured by monitoring the decrease in enzymatic activity occurring after a block of translation with cycloheximide. Using this assay, the E2 proteins from a low-risk (HPV11) and a high-risk (HPV31) human papillomavirus (HPV) type were found to have short half-lives of 60 min in C33A cervical carcinoma cells and to be ubiquitinated and degraded by the proteasome. Analysis of mutant proteins showed that the instability of E2 is independent of its DNA-binding and transcriptional activities but is encoded within its transactivation domain, the region that binds to the cellular chromatin factor bromodomain-containing protein 4 (Brd4) to regulate viral gene transcription. Overexpression of Brd4, or of its C-terminal E2-interaction domain, was found to increase the steady-state levels and stability of wild-type E2 but not of E2 mutants defective for binding Brd4. These results indicate that the stability of E2 is increased upon complex formation with Brd4 and highlight the value of the luciferase assay for the study of E2 degradation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Papillomaviridae/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Genes Reporter/genética , Humanos , Proteínas Nucleares/genética , Papillomaviridae/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional/genética , Ubiquitinação , Proteínas Virais/genéticaRESUMO
Due to the limited coding capacity of their small genomes, human papillomaviruses (HPV) rely extensively on host factors for the completion of their life cycles. Accordingly, most HPV proteins, including the replicative helicase E1, engage in multiple protein interactions. The fact that conserved regions of E1 have not yet been ascribed a function prompted us to use tandem affinity protein purification (TAP) coupled to mass spectrometry to identify novel targets of this helicase. This method led to the discovery of a novel interaction between the N-terminal 40 amino acids of HPV type 11 (HPV11) E1 and the cellular WD repeat protein p80 (WDR48). We found that interaction with p80 is conserved among E1 proteins from anogenital HPV but not among cutaneous or animal types. Colocalization studies showed that E1 can redistribute p80 from the cytoplasm to the nucleus in a manner that is dependent on the E1 nuclear localization signal. Three amino acid substitutions in E1 proteins from HPV11 and -31 were identified that abrogate binding to p80 and its relocalization to the nucleus. In HPV31 E1, these substitutions reduced but did not completely abolish transient viral DNA replication. HPV31 genomes encoding two of the mutant E1 proteins were not maintained as episomes in immortalized primary keratinocytes, whereas one encoding the third mutant protein was maintained at a very low copy number. These findings suggest that the interaction of E1 with p80 is required for efficient maintenance of the viral episome in undifferentiated keratinocytes.
Assuntos
DNA Helicases/metabolismo , DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Queratinócitos/virologia , Papillomaviridae/fisiologia , Proteínas/metabolismo , Proteínas Virais/metabolismo , Substituição de Aminoácidos/genética , Animais , Linhagem Celular , Núcleo Celular/química , Cromatografia de Afinidade , Proteínas de Ligação a DNA/genética , Haplorrinos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Espectrometria de Massas , Mutagênese Sítio-Dirigida , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Virais/genéticaRESUMO
SV40 large T antigen (T-ag) is a multifunctional protein that successively binds to 5'-GAGGC-3' sequences in the viral origin of replication, melts the origin, unwinds DNA ahead of the replication fork, and interacts with host DNA replication factors to promote replication of the simian virus 40 genome. The transition of T-ag from a sequence-specific binding protein to a nonspecific helicase involves its assembly into a double hexamer whose formation is likely dictated by the propensity of T-ag to oligomerize and its relative affinities for the origin as well as for nonspecific double- and single-stranded DNA. In this study, we used a sensitive assay based on fluorescence anisotropy to measure the affinities of wild-type and mutant forms of the T-ag origin-binding domain (OBD), and of a larger fragment containing the N-terminal domain (N260), for different DNA substrates. We report that the N-terminal domain does not contribute to binding affinity but reduces the propensity of the OBD to self-associate. We found that the OBD binds with different affinities to its four sites in the origin and determined a consensus binding site by systematic mutagenesis of the 5'-GAGGC-3' sequence and of the residue downstream of it, which also contributes to affinity. Interestingly, the OBD also binds to single-stranded DNA with an approximately 10-fold higher affinity than to nonspecific duplex DNA and in a mutually exclusive manner. Finally, we provide evidence that the sequence specificity of full-length T-ag is lower than that of the OBD. These results provide a quantitative basis onto which to anchor our understanding of the interaction of T-ag with the origin and its assembly into a double hexamer.
Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Vírus 40 dos Símios/metabolismo , Proteínas Virais/metabolismo , Substituição de Aminoácidos , Antígenos Transformantes de Poliomavirus/química , Sítios de Ligação/genética , Sequência Consenso , DNA de Cadeia Simples/metabolismo , Polarização de Fluorescência , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Guanylyl cyclases catalyze the formation of cGMP from GTP. This family of enzymes includes soluble (sGC) and particulate guanylyl cyclases (pGC). The sGC are heterodimers containing one active catalytic site and one inactive pseudo-site. They are activated by nitric oxide. The pGC are homodimers whose activity is notably regulated by peptide binding to the extracellular domain and by ATP binding to the intracellular kinase homology domain (KHD). The catalytic mechanism of the pGC is still not well understood. Homology modeling of the structure of the homodimeric guanylyl cyclase domain, based on the crystal structure of adenylyl cyclase, suggests the existence of two functional sites for the substrate GTP. We used a purified and fully active recombinant catalytic domain from mammalian pGC, to document its enzyme kinetics properties in the absence of the KHD. The enzyme presents positive cooperativity with the substrate Mg-GTP. However, a heterodimeric catalytic domain mutant (GC-HET) containing only one active catalytic site is non-cooperative and is more similar to sGC. Structure-activity studies of purine nucleoside analogs indicate that 2'd3'GMP is the most potent inhibitor of pGC tested. It displays mixed non-competitive inhibition properties that are potentiated by the second catalytic product inorganic pyrophosphate (PPi). It appears to be equivalent to purinergic site (P-site) inhibitors characterized on particulate adenylyl cyclase. Inhibition of pGC by 2'd3'GMP in the presence of PPi is accompanied by a loss of cooperative enzyme kinetics. These results are best explained by an allosteric dimer model with positive cooperativity for both the substrate and inhibitors.
Assuntos
Motivos de Aminoácidos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Receptores de Peptídeos/metabolismo , Animais , Fator Natriurético Atrial/metabolismo , Sítios de Ligação , Dimerização , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Insetos/citologia , Conformação Proteica , Receptores de Peptídeos/química , Receptores de Peptídeos/genéticaRESUMO
The natriuretic peptide receptor-A (NPR-A) is composed of an extracellular ligand-binding domain, a transmembrane-spanning domain, a kinase homology domain (KHD) and a guanylyl cyclase domain. Because the presence of ATP or adenylylimidodiphosphate reduces atrial natriuretic peptide (ANP) binding and is required for maximal guanylyl cyclase activity, a direct interaction of ATP with the receptor KHD domain is plausible. Therefore, we investigated whether ATP interacts directly with a binding site on the receptor by analyzing the binding of a photoaffinity analog of ATP to membranes from human embryonic kidney 293 cells expressing the NPR-A receptor lacking the guanylyl cyclase moiety (DeltaGC). We demonstrate that this receptor (NPR-A-DeltaGC) can be directly labeled by 8-azido-3'-biotinyl-ATP and that labeling is highly increased following ANP treatment. The mutant receptor DeltaKC, which does not contain the KHD, is not labeled. Photoaffinity labeling of the NPR-A-DeltaGC is reduced by 50% in the presence of 550 microm ATP, and competition curve fitting studies indicate a Hill slope of 2.2, suggestive of cooperative binding. This approach demonstrates directly that the interaction of ANP with its receptor modulates the binding of ATP to the KHD, probably through a conformational change in the KHD. In turn, this conformational change is essential for maximal activity. In addition, the ATP analog, 8-azido-adenylylimidodiphosphate, inhibits guanylyl cyclase activity but increases ANP binding to the extracellular domain. These results suggest that the KHD regulates ANP binding and guanylyl cyclase activity independently.