RESUMO
Protein A affinity chromatography has been widely used for both laboratory scale purification and commercial manufacturing of monoclonal antibodies and Fc-fusion proteins. Protein A purification is specific and efficient. However, there still remain several issues to be addressed, such as incomplete clearance of impurities including host cell proteins, DNA, aggregates, etc. In addition, the effects of wash buffers in protein A purification on the physicochemical characteristics of antibodies have yet to be fully understood. Here we found a new purification protocol for monoclonal antibodies that can improve physicochemical properties of monoclonal antibodies simply by inserting an additional wash step with a basic buffer after the capture step to the conventional protein A purification. The effects of the alkaline wash on monoclonal antibodies were investigated in terms of physicochemical characteristics, yields, and impurity clearance. The simple insertion of an alkaline wash step resulted in protection of antibodies from irreversible aggregation, reduction in free thiols and impurities, an improvement in colloidal and storage stability, and enhanced yields. This new procedure is widely applicable to protein A affinity chromatography of monoclonal antibodies.
Assuntos
Álcalis/química , Anticorpos Monoclonais/química , Proteína Estafilocócica A/isolamento & purificação , Cromatografia de Afinidade , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida/métodos , SoluçõesRESUMO
A phenotypic screening of thienodiazepines derived from a hit compound found through a binding assay targeting co-stimulatory molecules on T cells and antigen presenting cells successfully led to the discovery of a thienotriazolodiazepine compound (7f) possessing potent immunosuppressive activity. A chemical biology approach has succeeded in revealing that 7f is a first inhibitor of epigenetic bromodomain-containing proteins. 7f is expected to become an anti-cancer agent as well as an immunosuppressive agent.
Assuntos
Antineoplásicos/farmacologia , Azepinas/farmacologia , Antígenos CD28/metabolismo , Descoberta de Drogas , Imunossupressores/farmacologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Antineoplásicos/síntese química , Antineoplásicos/química , Azepinas/síntese química , Azepinas/química , Antígenos CD28/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Histona Acetiltransferases , Chaperonas de Histonas , Humanos , Imunossupressores/síntese química , Imunossupressores/química , Estrutura Molecular , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Fenótipo , Relação Estrutura-Atividade , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
The pathogenic role of autoantibodies in rheumatoid arthritis (RA) remains elusive. Anti-glucose-6-phosphate isomerase (GPI) antibodies (Abs) are candidates for arthritogenic Abs because they directly induce arthritis in mice. High titers of anti-GPI Abs are found in some RA patients with severe forms. The aim of this study was to analyze the role of IgG, including anti-GPI Abs, in the joints of RA patients. Synovial tissue was obtained from 6 patients with RA (3 anti-GPI Abs- positive and 3 anti-GPI Abs- negative) and compared histologically and immunohistochemically for IgG and C3 deposition. IgG fractions were separated from the sera of anti-GPI Abs-positive RA patients and healthy subjects, and injected into the metacarpophalangeal joints of 4 cynomolgus monkeys. On day 16, the joints were harvested and examined histologically and immunohistochemically. The expression of the C5a receptor (C5aR) molecule in the synovium was quantified by real-time PCR using cDNA from the monkeys' joints. The synovia of anti-GPI Abs-positive RA patients showed diffuse infiltration of cells, including mast cells, and strong deposition of IgG and C3. In monkeys, IgG from RA patients, including anti-GPI Abs, resulted in recruitment of granulocytes and mononuclear cells, strong deposition of IgG on the articular surface, and overexpression of C5aR, but no joint swelling. No infiltrated cells or IgG deposition were observed in monkeys injected with IgGs from healthy subjects. Our results suggest that IgG fraction from RA patients, including that of anti-GPI Abs, may play a role in the synovitis of RA, although the pathogenesis of human anti-GPI Abs is still uncertain.
Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/toxicidade , Glucose-6-Fosfato Isomerase/imunologia , Imunoglobulina G/toxicidade , Articulações/imunologia , Proteínas de Membrana/metabolismo , Receptores de Complemento/metabolismo , Animais , Artrite Reumatoide/enzimologia , Artrite Reumatoide/genética , Autoanticorpos/sangue , Autoanticorpos/imunologia , Sequência de Bases , Complemento C3/análise , Complemento C3/imunologia , Granulócitos/imunologia , Haplorrinos , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Articulações/efeitos dos fármacos , Articulações/patologia , Leucócitos Mononucleares/imunologia , Mastócitos/imunologia , Proteínas de Membrana/genética , Dados de Sequência Molecular , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptor da Anafilatoxina C5a , Receptores de Complemento/genética , Líquido Sinovial/citologia , Líquido Sinovial/imunologiaRESUMO
Anti-glucose-6-phosphate isomerase (GPI) antibodies (Abs) solely induce arthritis in mice. High titers of anti-GPI Abs are found in some patients with rheumatoid arthritis (RA), but their pathogenic role remains elusive. The aim of this study was to evaluate the pathogenic role of anti-GPI Abs in cynomolgus monkeys. IgG fractions were separated from sera of anti-GPI Abs-positive RA patients and healthy subjects and directly injected into the metacarpophalangeal joints of 4 cynomolgus monkeys. At day 16, the joints were harvested and examined histologically and immunohistochemically. The expression of C5a receptor (C5aR) molecule in the synovium was quantified by real-time PCR using cDNA from monkey joints. In monkey joints, IgG including anti-GPI Abs resulted in recruitment of granulocytes and mononuclear cells, strong deposition of human IgG on the articular surface, and overexpression of C5aR, but no joint swelling. No infiltrated cells or IgG deposition were observed in monkeys injected with IgGs from healthy subjects. Our results suggest that IgG fraction from RA patients including anti-GPI Abs may play a crucial role in the generation of synovitis in monkeys, although the pathogenesis of anti-GPI Abs in RA patients is still uncertain.
Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/administração & dosagem , Glucose-6-Fosfato Isomerase/imunologia , Imunoglobulina G/administração & dosagem , Sinovite/imunologia , Animais , Complexo Antígeno-Anticorpo/metabolismo , Complexo Antígeno-Anticorpo/fisiologia , Artrite Reumatoide/enzimologia , Autoanticorpos/metabolismo , Cartilagem Articular/imunologia , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Humanos , Imunoglobulina G/metabolismo , Macaca fascicularis , Receptor da Anafilatoxina C5a/biossíntese , Receptor da Anafilatoxina C5a/genética , Líquido Sinovial/imunologia , Líquido Sinovial/metabolismo , Sinovite/enzimologia , Sinovite/metabolismoRESUMO
The anaphylatoxin C5a is a classic, potent inflammatory mediator, with biological activities that have been implicated in a variety of diseases. As a result of two decades of study, C5a receptor antagonists that prevent high-affinity binding of C5a to its receptor have been discovered, which show therapeutic effects in various inflammatory disease models. Moreover, the involvement of C5a in disease models has been demonstrated in various experiments using C5/C5a receptor-deficient mice and anti-C5/C5a antibodies. It is therefore anticipated that these antagonists will have potential as novel anti-inflammatory therapeutics.
Assuntos
Anti-Inflamatórios/farmacologia , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Complemento C5a/metabolismo , Modelos Animais de Doenças , Desenho de Fármacos , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Receptor da Anafilatoxina C5a/metabolismo , Relação Estrutura-AtividadeRESUMO
Degenerative lesions were induced in the knee joint of Wistar rats by intraarticular injection of chondrocyte metabolism inhibitor mono-iodoacetate (MIA) at doses of 0, 0.3 or 3 mg/joint. Histopathological examination and the measurement of hind paw weight ratio as an index of joint pain by incapacitance tester were performed. Histological findings that are similar to those observed in human osteoarthritis (OA), such as disorganization of chondrocytes, erosion and fibrillation of cartilage surface, and subchondral bone exposure etc., were observed in a MIA-dose-dependent manner. Saflanin-O fast green staining revealed that marked diffuse reduction of proteoglycan in cartilage tissue of rats treated with MIA. The clinical scores of the joint pain were closely correlated to the grade of histological findings. We conclude that the present experimental model in combination with a novel dual channel weight averager would be very useful for the study of human OA, and could be applied for estimation of therapeutic effect of new anti-OA drugs.
Assuntos
Iodoacetatos/toxicidade , Osteoartrite/patologia , Dor/induzido quimicamente , Animais , Pesos e Medidas Corporais , Modelos Animais de Doenças , Técnicas Histológicas , Osteoartrite/induzido quimicamente , Dor/patologia , Ratos , Ratos Wistar , Tíbia/patologiaRESUMO
The anaphylatoxin C5a is a potent chemotactic factor for neutrophils and other leukocytes, and functions as an important inflammatory mediator. Through a high capacity screening followed by chemical optimization, we identified a novel non-peptide C5a receptor antagonist, N-[(4-dimethylaminophenyl)methyl]-N-(4-isopropylphenyl)-7-methoxy-1,2,3,4-tetrahydronaphthalen-1- carboxamide hydrochloride (W-54011). W-54011 inhibited the binding of (125)I-labeled C5a to human neutrophils with a K(i) value of 2.2 nm. W-54011 also inhibited C5a-induced intracellular Ca(2+) mobilization, chemotaxis, and generation of reactive super oxide species in human neutrophils with IC(50) values of 3.1, 2.7, and 1.6 nm, respectively. In C5a-induced intracellular Ca(2+) mobilization assay with human neutrophils, W-54011 did not show agonistic activity at up to 10 microm and shifted rightward the concentration-response curves to C5a without depressing the maximal responses. Examination on the species specificity of W-54011 revealed that it was able to inhibit C5a-induced intracellular Ca(2+) mobilization in neutrophils of cynomolgus monkeys and gerbils but not mice, rats, guinea pigs, rabbits, and dogs. In gerbils, oral administration of W-54011 (3-30 mg/kg) inhibited C5a-induced neutropenia in a dose-dependent manner. The present report is the first description of an orally active non-peptide C5a receptor antagonist that could contribute to the treatment of inflammatory diseases mediated by C5a.