RESUMEN
Fc engineering to increase the binding affinity of IgG antibodies to FcRn has been reported to reduce the elimination of IgG antibodies. Herein, we present a novel non-FcRn-dependent approach to reduce the elimination of IgG antibodies. Pharmacokinetic studies conducted in normal mice of various humanized IgG4 antibodies, which had identical constant regions but different variable region sequences, revealed that an antibody with a lower isoelectric point (pI) has a longer half-life. These antibodies exhibited comparable binding affinity to FcRn, and with the antibodies with lower pIs, a longer half-life was also observed in beta2-microglobulin knockout mice, suggesting that differences in the pharmacokinetics were due to a non-FcRn-dependent mechanism. On the basis of our findings, we attempted to engineer the pharmacokinetic properties of a humanized anti-IL6 receptor IgG1 antibody. Selected substitutions in the variable region, without substitution in the Fc region, lowered the pI but did not reduce the biological activity and showed a significant reduction in the clearance of the antibody in cynomolgus monkey. These results suggest that lowering the pI by engineering the variable region could reduce the elimination of IgG antibodies and could provide an alternative to Fc engineering of IgG antibodies.
Asunto(s)
Ingeniería Genética/métodos , Inmunoglobulina G/genética , Región Variable de Inmunoglobulina/genética , Ingeniería de Proteínas/métodos , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacocinética , Células CHO , Cricetinae , Cricetulus , Semivida , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inmunoglobulina G/metabolismo , Punto Isoeléctrico , Ratones , Receptores Fc/metabolismoRESUMEN
The immunoreactivity (specific binding) of three Gly(A1)-sulfobenzoxadiazole-labeled insulins which have different spacer groups, Gly(A1)-[3-(4-sulfobenzoxadiazol-7-ylthio)propanoyl]-insulin , Gly(A1)-[2-(4-sulfobenzoxadiazol-7-ylthio)acetyl]-insulin, Gly(A1)-[3-carboxy-2(or 3)-(4-sulfobenzoxadiazol-7-ylthio)propanoyl]-insulin, toward an antiporcine insulin monoclonal antibody was investigated, where solid-phase fluoroimmunoassay technique was utilized. The immunoreactivities of three labeled insulins were 2.1, 1.8 and 1 in that order.
Asunto(s)
Anticuerpos Insulínicos/inmunología , Insulina/análogos & derivados , Insulina/química , Animales , Anticuerpos Monoclonales/inmunología , Cromatografía Líquida de Alta Presión , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Indicadores y Reactivos , Insulina/síntesis química , Insulina/inmunología , PorcinosRESUMEN
The preparation of insulins labeled with a fluorescent moiety at a definite position (Gly(A1) or Lys(B29)) on the molecule is described. Gly(A1)- and Lys(B29)-S-acetylthioglycoloyl-insulins and Gly(A1)-2(or 3)-acetylmercapto-3-carboxypropranoyl-insulin were deacetylated and then reacted with 7-chloro-4-sulfobenzoxadiazole, a fluorogenic reagent, to afford the corresponding fluorescence labeled insulins. The preparative separation of the fluorescence labeled insulins from the insulin derivatives that remained unreacted was carried out by anion-exchange high-performance liquid chromatography on a TSKgel DEAE-2SW column.
Asunto(s)
Insulina/química , Cromatografía Líquida de Alta Presión , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Indicadores y Reactivos , Insulina/inmunología , Oxadiazoles , Espectrometría de FluorescenciaRESUMEN
A method for the preparation of insulin derivatives which have protected sulfhydryl group(s) at definite site(s) on the molecule is described. Porcine insulin reacts with S-acetylmercaptosuccinic anhydride to afford four species of insulin derivatives that have 2 (or 3)-acetylmercapto-3-carboxypropanoyl group(s) at i) Gly(A1), ii) Gly(A1) and Phe(B1), iii) Gly(A1) and Lys(B29), and iv) Gly(A1), Phe(B1) and Lys(B29) positions. The derivatives are efficiently separated in a preparative scale by anion-exchange high-performance liquid chromatography on a TSKgel DEAE-2SW column. The four derivatives are all readily deacetylated with hydroxylamine to give the corresponding sulfhydryl insulin derivatives.
Asunto(s)
Ácido 3-Mercaptopropiónico/análogos & derivados , Insulina/análogos & derivados , Ácido 3-Mercaptopropiónico/síntesis química , Animales , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Insulina/síntesis química , PorcinosRESUMEN
A method for the preparation of insulin derivatives having protected sulfhydryl group(s) on definite site(s) on the molecule which uses anion-exchange high performance liquid chromatography on a TSKgel DEAE-2SW column for separation is described. Porcine insulin reacts with N-succinimidyl S-acetylthioacetate to afford four species of insulin derivatives that have S-acetylthioglycoloyl group(s) at: i) Gly(A1), ii) Gly(A1) and Phe(B1), iii) Gly(A1) and Lys(B29), and iv) Gly(A1), Phe(B1) and Lys(B29) positions. An insulin derivative which has a group at the Lys(B29)-position is prepared by the S-acetylthioglycoloylation of Gly(A1), Phe(B1)-dicitraconyl insulin followed by decitraconylation. The five derivatives are readily deacetylated with hydroxylamine to yield the corresponding sulfhydryl insulin derivatives.
Asunto(s)
Insulina/análogos & derivados , Tioglicolatos/síntesis química , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Insulina/síntesis química , Insulina/aislamiento & purificación , Tioglicolatos/aislamiento & purificaciónRESUMEN
Three horseradish peroxidase (HRP)-labeled porcine insulins which have definite labeling site(s) were compared regarding sensitivity in a solid-phase enzyme-immunoassay (EIA) of anti-insulin antibodies. The standard curves obtained with LysB29-HRP-insulin and GlyA1-HRP-insulin were steeper than that with GlyA1,LysB29-diHRP-insulin for both polyclonal and monoclonal antibodies. Thus, the mono-HRP-labeled insulins can afford higher sensitivities in the EIA. The importance of the HRP-labeling site in insulin and of the number of labeled HRP was first demonstrated by using HRP-labeled insulins having definite labeling site(s).