RESUMO
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.
Assuntos
Anticorpos Monoclonais/genética , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Antígenos/imunologia , Sequência de Bases , Separação Celular , Primers do DNA/genética , Evolução Molecular Direcionada , Descoberta de Drogas , Citometria de Fluxo , Biblioteca Gênica , Células HEK293 , Humanos , Dados de Sequência Molecular , Ligação Proteica , Engenharia de ProteínasRESUMO
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.
Assuntos
Processamento Alternativo , Anticorpos Monoclonais/biossíntese , Afinidade de Anticorpos/genética , Evolução Molecular Direcionada , Expressão Gênica , Imunoglobulina G/biossíntese , Anticorpos Monoclonais/genética , Células HEK293 , Humanos , Imunoglobulina G/genética , Precursores de RNA/biossíntese , Precursores de RNA/genéticaRESUMO
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.
Assuntos
Anticorpos de Cadeia Única/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Afinidade de Anticorpos , Especificidade de Anticorpos , Proteínas do Capsídeo/imunologia , Técnicas de Visualização da Superfície Celular , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Simulação por Computador , Cistina/química , Cistina/genética , Evolução Molecular Direcionada , Células HEK293 , Temperatura Alta , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Cinética , Levivirus/imunologia , Camundongos , Modelos Moleculares , Método de Monte Carlo , Mutagênese Sítio-Dirigida , Ligação Proteica , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Desdobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Anticorpos de Cadeia Única/genética , Temperatura de TransiçãoRESUMO
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.
