RESUMO
The current standard of care for antivascular endothelial growth factor (VEGF) treatment requires frequent intravitreal (IVT) injections of protein therapeutics, as a result of limited retention within the eye. A thorough understanding of the determinants of ocular pharmacokinetics (PK) and its translation across species is an essential prerequisite for developing more durable treatments. In this work, we studied the ocular PK in macaques of the protein formats that comprise today's anti-VEGF standard of care. Cynomolgus monkeys received a single IVT injection of a single-chain variable fragment (scFv, brolucizumab), antigen-binding fragment (Fab, ranibizumab), fragment crystallizable-fusion protein (Fc-fusion, aflibercept), or immunoglobulin G monoclonal antibody (IgG, VA2 CrossMAb). Drug concentrations were determined in aqueous humor samples collected up to 42 days postinjection using immunoassay methods. The ocular half-life (t1/2) was 2.28, 2.62, 3.13, and 3.26 days for scFv, Fab, Fc-fusion, and IgG, respectively. A correlation with human t1/2 values from the literature confirmed the translational significance of the cynomolgus monkey as an animal model for ocular research. The relation between ocular t1/2 and molecular size was also investigated. Size was inferred from the molecular weight (MW) or determined experimentally by dynamic light scattering. The MW and hydrodynamic radius were found to be good predictors for the ocular t1/2 of globular proteins. The analysis showed that molecular size is a determinant of ocular disposition and may be used in lieu of dedicated PK studies in animals.
Assuntos
Inibidores da Angiogênese/farmacocinética , Humor Aquoso/metabolismo , Corpo Vítreo/metabolismo , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/química , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/farmacocinética , Meia-Vida , Injeções Intravítreas , Macaca fascicularis , Modelos Animais , Peso Molecular , Ranibizumab/administração & dosagem , Ranibizumab/química , Ranibizumab/farmacocinética , Receptores de Fatores de Crescimento do Endotélio Vascular/administração & dosagem , Receptores de Fatores de Crescimento do Endotélio Vascular/química , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacocinéticaRESUMO
Aim: The work was aimed at developing a bioanalytical approach to identify immunogenic parts of a bispecific F(ab) fragment and to characterize the immune response seen in a preclinical study. Experimental: The bioanalytical method consists of a set of domain detection assays that use germlined variants of the drug. Results: The method demonstrated that anti-drug antibodies (ADAs) were predominantly directed against both antigen-binding sites of the drug. Conclusion: The method was capable to discriminate between ADAs directed against one of the antigen-binding sites, both sites or the constant domain, allowing for an estimation of the relative binding prevalence for these subunits. The developed approach provides a practical and robust solution for exploratory characterization of ADAs against multidomain biotherapeutics.
Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Bioensaio , Preparações Farmacêuticas/química , Animais , Anticorpos Biespecíficos/sangue , Anticorpos Monoclonais/sangue , Reações Antígeno-Anticorpo , Macaca fascicularis , Preparações Farmacêuticas/sangueRESUMO
Despite the availability of antibody libraries for the selection of receptor molecules, the large number of established and well-characterized hybridoma lines still represent a useful source for recombinant antibody genes. This protocol describes the PCR amplification, cloning, and a small-scale expression test for the generation of scFv fragments from hybridoma cell lines. Particular emphasis was placed on frequently observed problems and pitfalls of this method.
