RESUMO
Critical quality attributes (CQA) are physical, chemical, biological or microbiological properties or characteristics that must be within an appropriate limit, range or distribution to ensure the desired product quality, safety and efficacy. For monoclonal antibody therapeutics that rely on fraction crystalizable (Fc)-mediated effector function for their clinical activity, the terminal sugars of Fc glycans have been shown to be critical for safety or efficacy. Different glycosylation variants have also been shown to influence the pharmacodynamic and pharmacokinetic behavior while other Fc glycan structural elements may be involved in adverse immune reactions. This review focuses on the role of Fc glycans as CQAs. Fc glycan information from the published literature is summarized and evaluated for impact on patient safety, immunogenicity, bioactivity and pharmacodynamics/pharmacokinetics.
Assuntos
Anticorpos Monoclonais/efeitos adversos , Imunização/efeitos adversos , Polissacarídeos/efeitos adversos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Humanos , Dados de Sequência Molecular , Polissacarídeos/imunologia , Polissacarídeos/uso terapêuticoRESUMO
Regulatory bodies worldwide consider N-glycosylation to be a critical quality attribute for immunoglobulin G (IgG) and IgG-like therapeutics. This consideration is due to the importance of posttranslational modifications in determining the efficacy, safety, and pharmacokinetic properties of biologics. Given its critical role in protein therapeutic production, we review N-glycosylation beginning with an overview of the myriad interactions of N-glycans with other biological factors. We examine the mechanism and drivers for N-glycosylation during biotherapeutic production and the several competing factors that impact glycan formation, including the abundance of precursor nucleotide sugars, transporters, glycosidases, glycosyltransferases, and process conditions. We explore the role of these factors with a focus on the analytical approaches used to characterize glycosylation and associated processes, followed by the current state of advanced glycosylation modeling techniques. This combination of disciplines allows for a deeper understanding of N-glycosylation and will lead to more rational glycan control.
Assuntos
Imunoglobulina G/metabolismo , Proteínas Recombinantes/biossíntese , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Linhagem Celular , Retículo Endoplasmático/metabolismo , Glicosilação , Complexo de Golgi/metabolismo , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Activated fibroblasts are thought to play important roles in the progression of many solid tumors, but little is known about the mechanisms responsible for the recruitment of fibroblasts in tumors. Using several methods, we identified platelet-derived growth factor A (PDGFA) as the major fibroblast chemoattractant and mitogen from conditioned medium generated by the Calu-6 lung carcinoma cell line. In addition, we showed that Calu-6 tumors express significant levels of PDGFC, and that the levels of expression of these two PDGFRalpha ligands correlate strongly with the degree of stromal fibroblast infiltration into the tumor mass. The most intense expression of PDGFRalpha was observed in fibroblasts in the tumor outer rim. We subsequently showed that disrupting PDGFRalpha-mediated signaling results in significant inhibition of tumor growth in vivo. Furthermore, analysis of a compendium of microarray data revealed significant expression of PDGFA, PDGFC, and PDGFRalpha in human lung tumors. We propose that therapies targeting this stromal cell type may be effective in treating certain types of solid tumors.
Assuntos
Neoplasias Pulmonares/fisiopatologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Células Estromais/patologia , Células 3T3 , Animais , Anticorpos , Divisão Celular , Linhagem Celular Tumoral , Meios de Cultivo Condicionados , Primers do DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Pulmão/citologia , Pulmão/fisiologia , Neoplasias Pulmonares/patologia , Linfocinas/genética , Camundongos , Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/isolamento & purificação , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
The therapeutic activity of monoclonal antibodies can involve immune cell mediated effector functions including antibody-dependent cellular cytotoxicity (ADCC), an activity that is modulated by the structure of Fc-glycans, and in particular the lack of core fucose. The heterogeneity of these glycostructures and the inherent variability of traditional PBMC-based in vitro ADCC assays, have made it challenging to quantitatively assess the impact of other glycostructures on ADCC activity. We applied a quantitative NK cell based assay to generate a database consisting of Fc-glycostructure and ADCC data from 54 manufacturing batches of a CHO-derived monoclonal antibody. Explorative analysis of the data indicated that, apart from afucosylation, galactosylation levels could influence ADCC activity. We confirmed this hypothesis by demonstrating enhanced ADCC upon enzymatic hypergalactosylation of four different monoclonal antibodies derived using standard CHO manufacturing processes. Furthermore we quantitatively compare the effects of galactosylation and afucosylation in the context of glycan heterogeneity and demonstrate that while galactose can influence ADCC activity, afucosylation remains the primary driver of this activity.
Assuntos
Anticorpos Monoclonais/metabolismo , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Galactose/metabolismo , Imunoglobulina G/metabolismo , Receptores de IgG/imunologia , Anticorpos Monoclonais/química , Fucose/metabolismo , Glicosilação , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/química , Células Matadoras Naturais/imunologia , Receptores de IgG/química , Receptores de IgG/metabolismoRESUMO
Antibody-dependent cell-mediated cytotoxicity (ADCC) is a relevant characteristic to measure for a number of therapeutic monoclonal antibodies (mAbs) under development. ADCC is a mechanism by which antibody-opsonized, infected, or cancerous cells are destroyed by FcγRIII (CD16)-expressing effector cells. Here we describe three methods that can be used to quantify the ADCC activity of mAbs by measuring distinct aspects of the ADCC mechanism.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos/imunologia , Testes Imunológicos de Citotoxicidade/métodos , Citocinas/biossíntese , Humanos , Células Matadoras Naturais/imunologiaRESUMO
Antibody dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action (MoA) for many monoclonal antibody (mAb) therapeutics. As such, quantitative measurement of ADCC activity is key to drug development. Traditional cell lysis based ADCC assays using PBMCs or NK cell lines can be challenging to develop and implement for routine testing. To provide an alternative to the cell lysis based ADCC assay, a non-cell based measure of ADCC activity was developed to determine the ADCC activity of an anti-CD20 mAb, which measures the ability of the mAb to bind to CD20 antigen and FcγRIIIa, simultaneously. The bridging of CD20 and FcγRIIIa is an essential interaction for the initiation of ADCC activity. This ADCC bridging method is simple, offers the ease of use of a standard ELISA, and shows reproducible dose-response curves in the concentration range of 50-1000 ng/mL. With interassay variability of 7-10% and recovery of 89-115%, the assay demonstrates acceptable precision and accuracy. The assay is able to detect degradative changes in anti-CD20 mAb samples subjected to light and acid exposure, suggesting that it is suitable for use as a stability-indicating method. The assay is also sensitive to mAb fucose levels. A linear relationship between the ADCC bridging assay and the cell lysis ADCC assay was demonstrated, strongly suggesting that the ADCC bridging assay can be used as a surrogate measure of ADCC.
Assuntos
Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos CD20/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Receptores de IgG/imunologia , Anticorpos Monoclonais/metabolismo , Antígenos CD20/metabolismo , Fucose/imunologia , Fucose/metabolismo , Glicosilação , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Cinética , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ligação Proteica , Receptores de IgG/metabolismo , Reprodutibilidade dos TestesRESUMO
In fission yeast, nutrient starvation induces physiological, biochemical, and morphological changes that enable survival. Collectively these changes are referred to as stationary phase. We have used a green fluorescent protein random insertional mutagenesis system to isolate two novel stress-response proteins required in stationary phase. Ish1 is a nuclear envelope protein that is present throughout the cell cycle and whose expression is increased in response to stresses such as glucose and nitrogen starvation, as well as osmotic stress. Expression of Ish1 is regulated by the Spc1 MAPK pathway through the Atf1 transcription factor. Although overexpression of Ish1 is lethal, cells lacking ish1 exhibit reduced viability in stationary phase. Bis1 is a novel interacting partner of Ish1. Bis1 is the Schizosaccharomyces pombe member of the ES2 nuclear protein family found in Mus musculus, Drosophila melanogaster, Homo sapiens, and Arabidopsis thaliana. Overexpression of Bis1 results in a cell elongation phenotype, whereas bis1(-) cells exhibit a reduced viability in stationary phase similar to that seen in ish1(-) cells.