RESUMEN
While the concept of Quality-by-Design is addressed at the upstream and downstream process development stages, we questioned whether there are advantages to addressing the issues of biologics quality early in the design of the molecule based on fundamental biophysical characterization, and thereby reduce complexities in the product development stages. Although limited number of bispecific therapeutics are in clinic, these developments have been plagued with difficulty in producing materials of sufficient quality and quantity for both preclinical and clinical studies. The engineered heterodimeric Fc is an industry-wide favorite scaffold for the design of bispecific protein therapeutics because of its structural, and potentially pharmacokinetic, similarity to the natural antibody. Development of molecules based on this concept, however, is challenged by the presence of potential homodimer contamination and stability loss relative to the natural Fc. We engineered a heterodimeric Fc with high heterodimeric specificity that also retains natural Fc-like biophysical properties, and demonstrate here that use of engineered Fc domains that mirror the natural system translates into an efficient and robust upstream stable cell line selection process as a first step toward a more developable therapeutic.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Animales , Anticuerpos/química , Anticuerpos/genética , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/genética , Células CHO , Cromatografía Liquida , Cricetinae , Cricetulus , Diseño de Fármacos , Humanos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/genética , Espectrometría de Masas , Modelos Moleculares , Mutación , Ingeniería de Proteínas/métodos , Multimerización de Proteína , Estructura Terciaria de Proteína , Electricidad Estática , TemperaturaRESUMEN
The E1 helicase from BPV and HPV16 interacts with Ubc9 to facilitate viral genome replication. We report that HPV11 E1 also interacts with Ubc9 in vitro and in the yeast two-hybrid system. Residues in E1 involved in oligomerization (353-435) were sufficient for binding to Ubc9 in vitro, but the origin-binding and ATPase domains were additionally required in yeast. Nuclear accumulation of BPV E1 was shown previously to depend on its interaction with Ubc9 and sumoylation on lysine 514. In contrast, HPV11 and HPV16 E1 mutants defective for Ubc9 binding remained nuclear even when the SUMO pathway was inhibited. Furthermore, we found that K514 in BPV E1 and the analogous K559 in HPV11 E1 are not essential for nuclear accumulation of E1. These results suggest that the interaction of E1 with Ubc9 is not essential for its nuclear accumulation but, rather, depends on its oligomerization and binding to DNA and ATP.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Papillomavirus Humano 11/enzimología , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/fisiología , Papillomavirus Humano 11/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Organismos Modificados Genéticamente , Conformación Proteica , Transporte de Proteínas/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Proteínas Virales/genéticaRESUMEN
Human papillomavirus (HPV) DNA replication is initiated by recruitment of the E1 helicase by the E2 protein to the viral origin. Screening of our corporate compound collection with an assay measuring the cooperative binding of E1 and E2 to the origin identified a class of small molecule inhibitors of the protein interaction between E1 and E2. Isothermal titration calorimetry and changes in protein fluorescence showed that the inhibitors bind to the transactivation domain of E2, the region that interacts with E1. These compounds inhibit E2 of the low risk HPV types 6 and 11 but not those of high risk HPV types or of cottontail rabbit papillomavirus. Functional evidence that the transactivation domain is the target of inhibition was obtained by swapping this domain between a sensitive (HPV11) and a resistant (cottontail rabbit papillomavirus) E2 type and by identifying an amino acid substitution, E100A, that increases inhibition by approximately 10-fold. This class of inhibitors was found to antagonize specifically the E1-E2 interaction in vivo and to inhibit HPV DNA replication in transiently transfected cells. These results highlight the potential of the E1-E2 interaction as a small molecule antiviral target.