RESUMEN
IgG isotypes can differentially bind to Fcγ receptors and complement, making the selection of which isotype to pursue for development of a particular therapeutic antibody important in determining the safety and efficacy of the drug. IgG2 and IgG4 isotypes have significantly lower binding affinity to Fcγ receptors. Recent evidence suggests that the IgG2 isotype is not completely devoid of effector function, whereas the IgG4 isotype can undergo in vivo Fab arm exchange leading to bispecific antibody and off-target effects. Here an attempt was made to engineer an IgG1-based scaffold lacking effector function but with stability equivalent to that of the parent IgG1. Care was taken to ensure that both stability and lack of effector function was achieved with a minimum number of mutations. Among the Asn297 mutants that result in lack of glycosylation and thus loss of effector function, we demonstrate that the N297G variant has better stability and developability compared with the N297Q or N297A variants. To further improve the stability of N297G, we introduced a novel engineered disulfide bond at a solvent inaccessible location in the CH2 domain. The resulting scaffold has stability greater than or equivalent to that of the parental IgG1 scaffold. Extensive biophysical analyses and pharmacokinetic (PK) studies in mouse, rat, and monkey further confirmed the developability of this unique scaffold, and suggest that it could be used for all Fc containing therapeutics (e.g. antibodies, bispecific antibodies, and Fc fusions) requiring lack of effector function or elimination of binding to Fcγ receptors.
Asunto(s)
Sustitución de Aminoácidos , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Mutación Missense , Animales , Humanos , Macaca fascicularis , Ratones , RatasRESUMEN
The stable effector functionLess (SEFL) antibody was designed as an IgG1 antibody with a constant region that lacks the ability to interact with Fcγ receptors. The engineering and stability and pharmacokinetic assessments of the SEFL scaffold is described in the accompanying article (Jacobsen, F. W., Stevenson, R., Li, C., Salimi-Moosavi, H., Liu, L., Wen, J., Luo, Q., Daris, K., Buck, L., Miller, S., Ho, S-Y., Wang, W., Chen, Q., Walker, K., Wypych, J., Narhi, L., and Gunasekaran, K. (2017) J. Biol. Chem 292). The biological properties of these SEFL antibodies were assessed in a variety of human and cynomolgus monkey in vitro assays. Binding of parent molecules and their SEFL variants to human and cynomolgus monkey FcγRs were evaluated using flow cytometry-based binding assays. The SEFL variants tested showed decreased binding affinity to human and cynomolgus FcγRs compared with the wild-type IgG1 antibody. In addition, SEFL variants demonstrated no antibody-dependent cell-mediated cytotoxicity in vitro against Daudi cells with cynomolgus monkey peripheral blood mononuclear cells, and had minimal complement-dependent cytotoxicity activity similar to that of the negative control IgG2 in a CD20+ human Raji lymphoma cell line. SEFL mutations eliminated off-target antibody-dependent monocyte phagocytosis of cynomolgus monkey platelets, and cynomolgus platelet activation in vitro These experiments demonstrate that the SEFL modifications successfully eliminated Fc-associated effector binding and functions.
Asunto(s)
Anticuerpos Monoclonales , Plaquetas/inmunología , Inmunoglobulina G , Monocitos/inmunología , Fagocitosis/efectos de los fármacos , Activación Plaquetaria/efectos de los fármacos , Receptores de IgG , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Línea Celular Tumoral , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Inmunoglobulina G/farmacología , Macaca fascicularis , Ratones , Fagocitosis/inmunología , Activación Plaquetaria/inmunología , Receptores de IgG/genética , Receptores de IgG/inmunologíaRESUMEN
Cynomolgus monkeys dosed with a therapeutic monoclonal antibody (mAbY.1) at ≥ 50 mg/kg had unexpected acute thrombocytopenia (nadir ~3,000 platelets/µl), sometimes with decreases in red cell mass. Increased activated macrophages, mitotic figures, and erythrophagocytosis were observed in the spleen. Binding of mAbY.1 to cynomolgus peripheral blood cells could not be detected in vitro. mAbY.1 induced phagocytosis of platelets by peripheral blood monocytes from cynomolgus monkeys, but not from humans. mAbs sharing the same constant domain (Fc) sequences, but differing from mAbY.1 in their variable domains, bound competitively to and had similar biological activity against the intended target. None of these antibodies had hematologic liabilities in vitro or in vivo. Neither the F(ab')2 portion of mAbY.1 nor the F(ab')2 portion on an aglycosylated Fc (IgG1) framework caused phagocytosis of platelets in vitro. These data suggest that the hematologic effects of mAbY.1 in cynomolgus monkeys likely occurred through an off-target mechanism, shown to be driven by 1 to 3 amino acid differences in the light chain. The hematologic effects made mAbY.1 an unsuitable candidate for further development as a therapeutic agent. This example demonstrates that nonclinical safety studies may be essential for understanding off-target effects of mAbs prior to clinical trials.
Asunto(s)
Anemia/inducido químicamente , Anticuerpos Monoclonales/toxicidad , Trombocitopenia/inducido químicamente , Anemia/sangre , Animales , Anticuerpos Monoclonales/administración & dosificación , Plaquetas/patología , Femenino , Humanos , Macaca fascicularis , Activación de Macrófagos , Masculino , Fagocitosis , Reticulocitos/patología , Bazo/efectos de los fármacos , Bazo/patología , Trombocitopenia/sangreRESUMEN
Human IgG2 antibodies contain three types of disulfide isoforms, classified by the number of Fab arms having disulfide links to the heavy chain hinge region. In the IgG2-B form, both Fab arms have interchain disulfide bonds to the hinge region, and in IgG2-A, neither Fab arm are disulfide linked to the hinge. The IgG2-A/B is a hybrid between these two forms, with only one Fab arm disulfide linked to the hinge. Changes in the relative levels of these forms over time are observed while IgG2 circulates in humans, suggesting IgG2-AâIgG2-A/BâIgG2-B conversion. Using a flow-through dialysis system, we studied the conversion kinetics of these forms in vitro under physiological conditions. For two IgG2κ antibodies, in vivo results closely matched the kinetics observed in vitro, indicating that the changes observed in vivo were solely conversions between isoforms, not differential clearance of specific forms. Moreover, the combined results validate the accuracy of the physiological model for the study of blood redox reactions. Further exploration of the conversion kinetics using material enriched in the IgG2-A forms revealed that the IgG2-AâIgG2-A/B rate was similar between IgG2κ and IgG2λ antibodies. In IgG2κ antibodies, conversion of IgG2-A/BâIgG2-B was slower than the IgG2-AâIgG2-A/B reaction. However, in IgG2λ antibodies, little IgG2-A/BâIgG2-B conversion was detected under physiological conditions. Thus, small differences in the C-terminus of the light chain sequences affect the disulfide conversion kinetics and impact the IgG2 disulfide isoforms produced in vivo.
Asunto(s)
Disulfuros/química , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Animales , Humanos , Inmunoglobulina G/administración & dosificación , Cinética , Ratones , Oxidación-Reducción , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMEN
Protein synthesis and secretion are essential to cellular life. Although secretory activities may vary in different cell types, what determines the maximum secretory capacity is inherently difficult to study. Increasing protein synthesis until reaching the limit of secretory capacity is one strategy to address this key issue. Under highly optimized growth conditions, recombinant CHO cells engineered to produce a model human IgG clone started housing rod-shaped crystals in the endoplasmic reticulum (ER) lumen. The intra-ER crystal growth was accompanied by cell enlargement and multinucleation and continued until crystals outgrew cell size to breach membrane integrity. The intra-ER crystals were composed of correctly folded, endoglycosidase H-sensitive IgG. Crystallizing propensity was due to the intrinsic physicochemical properties of the model IgG, and the crystallization was reproduced in vitro by exposing a high concentration of IgG to a near neutral pH. The striking cellular phenotype implicated the efficiency of IgG protein synthesis and oxidative folding exceeded the capacity of ER export machinery. As a result, export-ready IgG accumulated progressively in the ER lumen until a threshold concentration was reached to nucleate crystals. Using an in vivo system that reports accumulation of correctly folded IgG, we showed that the ER-to-Golgi transport steps became rate-limiting in cells with high secretory activity.
Asunto(s)
Retículo Endoplásmico/metabolismo , Expresión Génica , Inmunoglobulina G/biosíntesis , Pliegue de Proteína , Proteínas Recombinantes/biosíntesis , Animales , Células CHO , Cricetinae , Cricetulus , Retículo Endoplásmico/genética , Humanos , Concentración de Iones de Hidrógeno , Inmunoglobulina G/genética , Proteínas Recombinantes/genéticaRESUMEN
In this study, we characterized the chemical modifications in the monoclonal antibody (IgG(2)) aggregates generated under various conditions, including mechanical, chemical, and thermal stress treatment, to provide insight into the mechanism of protein aggregation and the types of aggregate produced by the different stresses. In a separate study, additional biophysical characterization was performed to arrange these aggregates into a classification system (Joubert, M. K., Luo, Q., Nashed-Samuel, Y., Wypych, J., and Narhi, L. O. (2011) J. Biol. Chem. 286, 25118-25133). Here, we report that different aggregates possessed different types and levels of chemical modification. For chemically treated samples, metal-catalyzed oxidation using copper showed site-specific oxidation of Met(246), His(304), and His(427) in the Fc portion of the antibody, which might be attributed to a putative copper-binding site. For the hydrogen peroxide-treated sample, in contrast, four solvent-exposed Met residues in the Fc portion were completely oxidized. Met and/or Trp oxidation was observed in the mechanically stressed samples, which is in agreement with the proposed model of protein interaction at the air-liquid interface. Heat treatment resulted in significant deamidation but almost no oxidation, which is consistent with thermally induced aggregates being generated by a different pathway, primarily by perturbing conformational stability. These results demonstrate that chemical modifications are present in protein aggregates; furthermore, the type, locations, and severity of the modifications depend on the specific conditions that generated the aggregates.
Asunto(s)
Cobre/química , Peróxido de Hidrógeno/química , Fragmentos Fc de Inmunoglobulinas/química , Animales , Calor , Humanos , Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Ratones , Oxidación-Reducción , Conformación ProteicaRESUMEN
Proteins are susceptible to degradation upon exposure to a variety of stresses during product manufacturing, transportation and storage. In this study, we investigated the aggregation properties of a monoclonal antibody during agitation stress. Agitation exclusively led to insoluble aggregates, or particle formation. Removal or modification of the air-liquid interface with a surfactant (e.g., polysorbate) abrogated particle formation. The supernatant postagitation was analyzed using SE-HPLC, FTIR, and AUC analyses and revealed no changes in conformation and aggregation profile when compared to the nonagitated antibody sample. The antibody particles were comprised of a combination of nonnative intermolecular disulfide-linked covalent as well as noncovalent interactions. Analysis of the antibody's unpaired cysteines revealed that the nonnative intermolecular disulfide bonds were formed through buried cysteines, which suggested at least partial unfolding of the antibody domains. FTIR analysis indicated that the particulated antibody maintained significant native-like secondary structure suggesting that particle formation led to minimal structure changes, but capable of exposing free cysteines to solvent to form the nonnative intermolecular disulfide bonds. The results presented in this study indicate the importance of the interactions between the antibody and the air-liquid interface during agitation in the formation of particles and suggests that reduced disulfide bonds may play a significant role in the particulation reaction. This phenomenon can be applicable to other proteins with similar free cysteine and structural characteristics.