RESUMO
Antibody dependent cellular cytotoxicity (ADCC) is an Fc-dependent effector function of IgG important for anti-viral immunity and anti-tumor therapies. NK-cell mediated ADCC is mainly triggered by IgG-subclasses IgG1 and IgG3 through the IgG-Fc-receptor (FcγR) IIIa. Polymorphisms in the immunoglobulin gamma heavy chain gene likely form a layer of variation in the strength of the ADCC-response, but this has never been studied in detail. We produced all 27 known IgG allotypes and assessed FcγRIIIa binding and ADCC activity. While all IgG1, IgG2, and IgG4 allotypes behaved similarly within subclass, large allotype-specific variation was found for IgG3. ADCC capacity was affected by residues 291, 292, and 296 in the CH2 domain through altered affinity or avidity for FcγRIIIa. Furthermore, allotypic variation in hinge length affected ADCC, likely through altered proximity at the immunological synapse. Thus, these functional differences between IgG allotypes have important implications for therapeutic applications and susceptibility to infectious-, allo- or auto-immune diseases.
Assuntos
Alótipos de Imunoglobulina/metabolismo , Imunoglobulina G/metabolismo , Sinapses Imunológicas/metabolismo , Células Matadoras Naturais/imunologia , Receptores de IgG/metabolismo , Citotoxicidade Celular Dependente de Anticorpos , Células Cultivadas , Glicosilação , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Polimorfismo Genético , Ligação Proteica , Receptores de IgG/genéticaRESUMO
Therapeutic concepts exploiting tumor-specific antibodies are often established in pre-clinical xenograft models using immuno-deficient mice. More complex therapeutic paradigms, however, warrant the use of immuno-competent mice, that more accurately capture the relevant biology that is being exploited. These models require the use of (surrogate) mouse or rat antibodies to enable optimal interactions with murine effector molecules. Immunogenicity is furthermore decreased, allowing longer-term treatment. We recently described controlled Fab-arm exchange (cFAE) as an easy-to-use method for the generation of therapeutic human IgG1 bispecific antibodies (bsAb). To facilitate the investigation of dual-targeting concepts in immuno-competent mice, we now applied and optimized our method for the generation of murine bsAbs. We show that the optimized combinations of matched point-mutations enabled efficient generation of murine bsAbs for all subclasses studied (mouse IgG1, IgG2a and IgG2b; rat IgG1, IgG2a, IgG2b, and IgG2c). The mutations did not adversely affect the inherent effector functions or pharmacokinetic properties of the corresponding subclasses. Thus, cFAE can be used to efficiently generate (surrogate) mouse or rat bsAbs for pre-clinical evaluation in immuno-competent rodents.
Assuntos
Anticorpos Biespecíficos/biossíntese , Imunoglobulina G/imunologia , Neoplasias/terapia , Animais , Anticorpos Biespecíficos/imunologia , Humanos , Imunoglobulina G/genética , Imunoglobulina G/uso terapêutico , Camundongos , Modelos Animais , Neoplasias/genética , Neoplasias/imunologia , Mutação Puntual/genética , Mutação Puntual/imunologia , Ratos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The generation of bispecific antibodies (bsAbs) with natural IgG architecture in a practical and efficient manner has been a longstanding challenge. Here we describe controlled Fab-arm exchange (cFAE), which is an easy-to-use method to generate bispecific IgG1 (bsIgG1). The protocol involves the following: (i) separate expression of two parental IgG1s containing single matching point mutations in the CH3 domain; (ii) mixing of parental IgG1s under permissive redox conditions in vitro to enable recombination of half-molecules; (iii) removal of the reductant to allow reoxidation of interchain disulfide bonds; and (iv) analysis of exchange efficiency and final product using chromatography-based or mass spectrometry (MS)-based methods. The protocol generates bsAbs with regular IgG architecture, characteristics and quality attributes both at bench scale (micrograms to milligrams) and at a mini-bioreactor scale (milligrams to grams) that is designed to model large-scale manufacturing (kilograms). Starting from good-quality purified proteins, exchange efficiencies of ≥95% can routinely be obtained within 2-3 d (including quality control).
Assuntos
Anticorpos Biespecíficos/metabolismo , Imunoglobulina G/metabolismo , Engenharia de Proteínas/métodos , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/genética , Reatores Biológicos , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulina G/genética , Espectrometria de Massas/métodos , Oxirredução , Mutação Puntual , Engenharia de Proteínas/instrumentação , Controle de QualidadeRESUMO
The promise of bispecific antibodies (bsAbs) to yield more effective therapeutics is well recognized; however, the generation of bsAbs in a practical and cost-effective manner has been a formidable challenge. Here we present a technology for the efficient generation of bsAbs with normal IgG structures that is amenable to both antibody drug discovery and development. The process involves separate expression of two parental antibodies, each containing single matched point mutations in the CH3 domains. The parental antibodies are mixed and subjected to controlled reducing conditions in vitro that separate the antibodies into HL half-molecules and allow reassembly and reoxidation to form highly pure bsAbs. The technology is compatible with standard large-scale antibody manufacturing and ensures bsAbs with Fc-mediated effector functions and in vivo stability typical of IgG1 antibodies. Proof-of-concept studies with HER2×CD3 (T-cell recruitment) and HER2×HER2 (dual epitope targeting) bsAbs demonstrate superior in vivo activity compared with parental antibody pairs.
