RESUMEN
Recent advances are described for the isolation and affinity maturation of antibodies that couple in vitro somatic hypermutation (SHM) with mammalian cell display, replicating key aspects of the adaptive immune system. SHM is dependent on the action of the B cell specific enzyme, activation-induced cytidine deaminase (AID). AID-directed SHM in vitro in non-B cells, combined with mammalian display of a library of human antibodies, initially naïve to SHM, can be used to isolate and affinity mature antibodies via iterative cycles of fluorescence-activated cell sorting (FACS) under increasingly stringent sort conditions. SHM observed in vitro closely resembles SHM observed in human antibodies in vivo in both mutation type and positioning in the antibody variable region. In addition, existing antibodies originating from mouse immunization, in vivo based libraries, or alternative display technologies such as phage can also be affinity matured in a similar manner. The display system has been developed to enable simultaneous high-level cell surface expression and secretion of the same protein through alternate splicing, where the displayed protein phenotype remains linked to genotype, allowing soluble secreted antibody to be simultaneously characterized in biophysical and cell-based functional assays. This approach overcomes many of the previous limitations of mammalian cell display, enabling direct selection and maturation of antibodies as full-length, glycosylated IgGs.
Asunto(s)
Anticuerpos Monoclonales/genética , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/metabolismo , Afinidad de Anticuerpos , Antígenos/inmunología , Secuencia de Bases , Separación Celular , Cartilla de ADN/genética , Evolución Molecular Dirigida , Descubrimiento de Drogas , Citometría de Flujo , Biblioteca de Genes , Células HEK293 , Humanos , Datos de Secuencia Molecular , Unión Proteica , Ingeniería de ProteínasRESUMEN
A mammalian expression system has been developed that permits simultaneous cell surface display and secretion of the same protein through alternate splicing of pre-mRNA. This enables a flexible system for in vitro protein evolution in mammalian cells where the displayed protein phenotype remains linked to genotype, but with the advantage of soluble protein also being produced without the requirement for any further recloning to allow a wide range of assays, including biophysical and cell-based functional assays, to be used during the selection process. This system has been used for the simultaneous surface presentation and secretion of IgG during antibody discovery and maturation. Presentation and secretion of monomeric Fab can also be achieved to minimize avidity effects. Manipulation of the splice donor site sequence enables control of the relative amounts of cell surface and secreted antibody. Multi-domain proteins may be presented and secreted in different formats to enable flexibility in experimental design, and secreted proteins may be produced with epitope tags to facilitate high-throughput testing. This system is particularly useful in the context of in situ mutagenesis, as in the case of in vitro somatic hypermutation.
Asunto(s)
Empalme Alternativo , Anticuerpos Monoclonales/biosíntesis , Afinidad de Anticuerpos/genética , Evolución Molecular Dirigida , Expresión Génica , Inmunoglobulina G/biosíntesis , Anticuerpos Monoclonales/genética , Células HEK293 , Humanos , Inmunoglobulina G/genética , Precursores del ARN/biosíntesis , Precursores del ARN/genéticaRESUMEN
Antibodies are important tools for a broad range of applications due to their high specificity and ability to recognize virtually any target molecule. However, in order to be practically useful, antibodies must be highly stable and bind their target antigens with high affinity. We present a combinatorial approach to generate high-affinity, highly stable antibodies through the design of stable frameworks, specificity grafting and maturation via somatic hypermutation in vitro. By collectively employing these methods, we have engineered a highly stable, high-affinity, full-length antibody with a T(m) over 90°C that retains significant activity after heating to 90°C for 1 h, and has ~95-fold improved antigen-binding affinity. The stabilized IgG framework is compatible with affinity maturation, and should provide a broadly useful scaffold for grafting a variety of complementarity-determining region loops for the development of stable antibodies with desired specificities.
Asunto(s)
Anticuerpos de Cadena Única/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Proteínas de la Cápside/inmunología , Técnicas de Visualización de Superficie Celular , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Simulación por Computador , Cistina/química , Cistina/genética , Evolución Molecular Dirigida , Células HEK293 , Calor , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/genética , Cinética , Levivirus/inmunología , Ratones , Modelos Moleculares , Método de Montecarlo , Mutagénesis Sitio-Dirigida , Unión Proteica , Ingeniería de Proteínas , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Desplegamiento Proteico , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Anticuerpos de Cadena Única/genética , Temperatura de TransiciónRESUMEN
A novel approach has been developed for the isolation and maturation of human antibodies that replicates key features of the adaptive immune system by coupling in vitro somatic hypermutation (SHM) with mammalian cell display. SHM is dependent on the action of the B cell specific enzyme, activation-induced cytidine deaminase (AID), and can be replicated in non-B cells through expression of recombinant AID. A library of human antibodies, based on germline V-gene segments with recombined human regions was used to isolate low-affinity antibodies to human ß nerve growth factor (hßNGF). These antibodies, initially naïve to SHM, were subjected to AID-directed SHM in vitro and selected using the same mammalian cell display system, as illustrated by the maturation of one of the antibodies to low pM K(D). This approach overcomes many of the previous limitations of mammalian cell display, enabling direct selection and maturation of antibodies as full-length, glycosylated IgGs.