RESUMEN
Methods to rapidly generate high quality bispecific antibodies (BsAb) having normal half-lives are critical for therapeutic programs. Here, we identify 3 mutations (T307P, L309Q, and Q311R or "TLQ") in the Fc region of human IgG1 which disrupt interaction with protein A while enhancing interaction with FcRn. The mutations are shown to incrementally alter the pH at which a mAb elutes from protein A affinity resin. A BsAb comprised of a TLQ mutant and a wild-type IgG1 can be efficiently separated from contaminating parental mAbs by differential protein A elution starting from either a) purified parental mAbs, b) in-supernatant crossed parental mAbs, or c) co-transfected mAbs. We show that the Q311R mutation confers enhanced FcRn interaction in vitro, and Abs harboring either the Q311R or TLQ mutations have serum half-lives as long as wild-type human IgG1. The mutant Abs have normal thermal stability and Fcγ receptor interactions. Together, the results lead to a method for high-throughput generation of BsAbs suitable for in vivo studies.
Asunto(s)
Anticuerpos Biespecíficos/genética , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Mutación , Receptores de IgG/química , Proteína Estafilocócica A/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Anticuerpos Biespecíficos/biosíntesis , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/aislamiento & purificación , Sitios de Unión , Cromatografía de Afinidad , Expresión Génica , Células HEK293 , Semivida , Humanos , Concentración de Iones de Hidrógeno , Fragmentos Fc de Inmunoglobulinas/biosíntesis , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/aislamiento & purificación , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/química , Inmunoglobulina G/aislamiento & purificación , Cinética , Ratones , Modelos Moleculares , Unión Proteica , Ingeniería de Proteínas/métodos , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Estructura Secundaria de Proteína , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteína Estafilocócica A/inmunología , Proteína Estafilocócica A/metabolismoRESUMEN
Engineering of fragment crystallizable (Fc) domains of therapeutic immunoglobulin (IgG) antibodies to eliminate their immune effector functions while retaining other Fc characteristics has numerous applications, including blocking antigens on Fc gamma (Fcγ) receptor-expressing immune cells. We previously reported on a human IgG2 variant termed IgG2σ with barely detectable activity in antibody-dependent cellular cytotoxicity, phagocytosis, complement activity, and Fcγ receptor binding assays. Here, we extend that work to IgG1 and IgG4 antibodies, alternative subtypes which may offer advantages over IgG2 antibodies. In several in vitro and in vivo assays, the IgG1σ and IgG4σ variants showed equal or even lower Fc-related activities than the corresponding IgG2σ variant. In particular, IgG1σ and IgG4σ variants demonstrate complete lack of effector function as measured by antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, and in vivo T-cell activation. The IgG1σ and IgG4σ variants showed acceptable solubility and stability, and typical human IgG1 pharmacokinetic profiles in human FcRn-transgenic mice and cynomolgus monkeys. In silico T-cell epitope analyses predict a lack of immunogenicity in humans. Finally, crystal structures and simulations of the IgG1σ and IgG4σ Fc domains can explain the lack of Fc-mediated immune functions. These variants show promise for use in those therapeutic antibodies and Fc fusions for which the Fc domain should be immunologically "silent".
