RESUMEN
Various types of structural variants have been observed in recombinant DNA - derived products. These isoforms include variations in post translational carbohydrate modifications where variations in site occupancy or unoccupied sites may occur. In addition, varying degrees of C-terminal processing and N-terminal substitutions have been observed. Isoforms may also be generated during processing and can include aggregated and/or chemically modified forms of the protein. Sophisticated analytical techniques exist for the identification and characterization of these structural variants. Several strategies have been used to isolate or enrich the isoform before molecular characterization. However, the effect these structural variations have on the biological activity of the product is less well understood. This may, in part, be due to the specificity and variability of the bioassay employed. This presentation describes the isolation and characterization of specific molecular isoforms for a monoclonal antibody product as well as an assessment of effects on biological activity.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Proteínas Recombinantes/química , Animales , Anticuerpos Monoclonales/metabolismo , Industria Farmacéutica/métodos , Electroforesis , Humanos , Espectrometría de Masas , Isoformas de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismoRESUMEN
A Primatized anti-CD4 monoclonal antibody (MAb), CE9.1, with V-domain from cynomolgus macaque (showing 92% homology with human consensus sequence V-domains), and a human IgG1 constant region, was characterized in vitro and in vivo in chimpanzees. This MAb binds human CD4 with Kd of 1.0 nM and was also able to bind to human IgG Fc receptors (Fc gamma R). However, despite being of the IgG1 subclass, CE9.1 did not bind to complement component C1q, nor did it mediate complement-dependent cytotoxicity. Examination of T cells from a number of species showed restricted reactivity for CE9.1, recognizing only human and chimpanzee CD4. In both human and chimpanzee MLRs, it had an IC50 of about 10.0 ng/mL. Therefore, a chimpanzee in vivo model was used to characterize CE9.1, CE9.1 caused transient decrease in the number of lymphocytes bearing the CD4 receptor starting at doses of 0.3 mg/kg in an in vivo dose ranging study in one chimpanzee. This effect was reversed within approximately 7 days. In a multiple high-dose study in which 10.0 mg/kg of CE9.1 was administered at intervals of 1-3 months, there was a dramatic loss of CD4 marker with a reciprocal increase in the number of CD3+ CD8- CD4- cells. The CD4 receptor was totally undetectable on these lymphocytes for 1-2 weeks, with a gradual, but complete, reversal within 4 weeks. We interpret these observations as receptor modulation because, although there was apparent loss of CD4+ lymphocytes, an equivalent number of CD3+CD8- T lymphocytes were present in circulation in all four chimpanzees treated with 10.0 mg/kg CE9.1. Even at this high dose, only limited reduction of CD4+ T lymphocytes was observed in these animals. These observations are in sharp contrast to what has been reported in rodents or in human clinical studies using other IgG1 mAbs to human CD4. CD8 counts, although variable, remained unaffected by CE9.1 treatment. No adverse events were observed following administration of CE9.1 to chimpanzees, and there was no detectable host immune responses to the Primatized MAb.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacología , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/inmunología , Proteínas Recombinantes de Fusión/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Sitios de Unión de Anticuerpos , Linfocitos T CD4-Positivos/metabolismo , Supresión Clonal , Pruebas de Fijación del Complemento , Reacciones Cruzadas , Humanos , Inyecciones Intravenosas , Prueba de Cultivo Mixto de Linfocitos , Pan troglodytes , Receptores Fc/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacología , Especificidad de la EspecieRESUMEN
Rationally designed synthetic inhibitors of retroviral proteases inhibit the processing of viral polypeptides in cultures of human T lymphocytes infected with human immunodeficiency virus type 1 (HIV-1) and therefore suppress the infectivity of HIV-1 in vitro. We have previously reported the antiviral activity in vitro of HIV-1 protease inhibitors against the C-type retrovirus Rauscher murine leukemia virus (RMuLV) and the lentivirus simian immunodeficiency virus (SIV). The same compounds which blocked the infectivity of HIV-1 also inhibited the infectivity of RMuLV and SIV in vitro. This report extends these findings by testing the antiviral activity of HIV-1 protease inhibitors in vivo in the RMuLV model. RMuLV-infected mice were treated twice a day (bid) with either an active (SKF 108922) or inactive (SKF 109273) compound for fourteen days by the intraperitoneal (i.p.) route. Compared with excipient control, SKF 108922, formulated with hydroxypropyl-beta-cyclodextrin (HPB), reduced virus-induced splenomegaly, viremia, and serum reverse transcriptase (RT) levels, while SKF 109273 was inactive. The HPB vehicle by itself enhanced replication of RMuLV. The effects of changing the formulation and the route of administration were examined. SKF 108922, formulated in HPB, had similar antiviral activity when administered by the i.p. or subcutaneous (SC) routes. However, SKF 108922 administered as a colloidal suspension in cholesterol sulfate (CS) had no detectable antiviral effect. Measurements of the circulating levels of the protease inhibitor in plasma explained this result. Plasma concentrations of SKF 108922 exceeded 1000 nM within 10 min after SC administration of the compound solubilized in HPB, but SKF 108922 was not detected in plasma after SC administration of the same dose formulated with CS. Information on optimal conditions for administering these agents should prove useful in guiding their clinical application Therefore, RMuLV should provide a good model for the preclinical evaluation and development of this class of agents for the treatment of HIV.
Asunto(s)
Antivirales/uso terapéutico , Inhibidores de la Proteasa del VIH/uso terapéutico , Leucemia Experimental/tratamiento farmacológico , Oligopéptidos/uso terapéutico , Virus Rauscher/efectos de los fármacos , Infecciones por Retroviridae/tratamiento farmacológico , Infecciones Tumorales por Virus/tratamiento farmacológico , beta-Ciclodextrinas , 2-Hidroxipropil-beta-Ciclodextrina , Animales , Antivirales/sangre , Antivirales/farmacocinética , Línea Celular , Ciclodextrinas/farmacología , Femenino , Inhibidores de la Proteasa del VIH/sangre , Inhibidores de la Proteasa del VIH/farmacocinética , Humanos , Inyecciones Intraperitoneales , Inyecciones Subcutáneas , Leucemia Experimental/virología , Ratones , Ratones Endogámicos BALB C , Oligopéptidos/sangre , Oligopéptidos/farmacocinética , Vehículos Farmacéuticos/farmacología , ADN Polimerasa Dirigida por ARN/sangre , Infecciones por Retroviridae/virología , Infecciones Tumorales por Virus/virologíaRESUMEN
Dopamine beta-hydroxylase (EC 1.14.17.1) is inactivated by p-hydroxybenzylcyanide (PHBC) in a manner characteristic of a suicide substrate. The inactivation 1) is first order in inhibitor (Kd = 1.9 mM, k2 = 0.05 min-1, pH 5.0), 2) exhibits saturation kinetics, and 3) is dependent on O2 and ascorbate. Restoration of activity could not be achieved by dialysis. The substrate, p-tyramine, protects the enzyme from inactivation, while in initial velocity kinetic experiments, PHBC is a linear competitive inhibitor (Kis = 2.6 mM) versus p-tyramine. The Kis value for PHBC is in good agreement with the Kd value obtained from analysis of the inactivation reaction. PHBC (in the presence of O2 and ascorbate) is also a substrate for the enzyme, being converted to p-hydroxymandelonitrile (PHMN) at a relative Vmax 13% that of p-tyramine. Under these conditions, the ratio of product formation to inactivation is 8000:1, suggesting that PHMN (or a tautomer of PHMN) is the species responsible for inactivation. Indeed, incubation of the enzyme with PHMN in the absence of ascorbate leads to irreversible inactivation. Since PHMN breaks down to p-hydroxybenzaldehyde and cyanide, experiments were conducted to determine whether these compounds may have been responsible for the inactivation. Incubation of the enzyme with p-hydroxybenzaldehyde did not lead to inactivation, whereas the inactivation produced with cyanide could be reversed by dialysis. Thus, the data point to PHMN as the molecule responsible for time-dependent loss of activity of the enzyme. Together, these experiments demonstrate that the newly discovered inhibitor, PHBC, meets the critieria for a suicide substrate for dopamine beta-hydroxylase.