RESUMO
Antibody engineering to enhance thermostability may enable further application and ease of use of antibodies across a number of different areas. A modified human IgG framework has been developed through a combination of engineering approaches, which can be used to stabilize antibodies of diverse specificity. This is achieved through a combination of complementarity-determining region (CDR)-grafting onto the stable framework, mammalian cell display and in vitro somatic hypermutation (SHM). This approach allows both stabilization and maturation to affinities beyond those of the original antibody, as shown by the stabilization of an anti-HA33 antibody by approximately 10°C and affinity maturation of approximately 300-fold over the original antibody. Specificities of 10 antibodies of diverse origin were successfully transferred to the stable framework through CDR-grafting, with 8 of these successfully stabilized, including the therapeutic antibodies adalimumab, stabilized by 9.9°C, denosumab, stabilized by 7°C, cetuximab stabilized by 6.9°C and to a lesser extent trastuzumab stabilized by 0.8°C. This data suggests that this approach may be broadly useful for improving the biophysical characteristics of antibodies across a number of applications.
Assuntos
Anticorpos/imunologia , Regiões Determinantes de Complementaridade/imunologia , Imunoglobulina G/imunologia , Engenharia de Proteínas/métodos , Adalimumab , Animais , Anticorpos/química , Anticorpos/genética , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/imunologia , Afinidade de Anticorpos/imunologia , Varredura Diferencial de Calorimetria , Técnicas de Visualização da Superfície Celular , Cetuximab , Regiões Determinantes de Complementaridade/genética , Denosumab , Células HEK293 , Humanos , Imunoglobulina G/genética , Modelos Moleculares , Conformação Proteica , Estabilidade Proteica , Hipermutação Somática de Imunoglobulina , Temperatura , TrastuzumabRESUMO
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.
