ABSTRACT:It is well established that the neonatal Fc receptor (FcRn) plays a critical role in regulating IgG homeostasis in vivo. As such, modification of the interaction of IgG with FcRn has been the focus of protein-engineering strategies designed to generate therapeutic antibodies with improved pharmacokinetic properties. In the current work, we characterized differences in interaction of IgG between mouse and primate receptors using three humanized antitumor necrosis factor ␣ antibodies with variant IgG 1 Fc regions. The wild-type and variant IgG showed a differential combination of improved affinity, modified dissociation kinetics, and altered pHdependent complex dissociation when evaluated on the primate and murine receptors. The observed in vitro binding differences within and between species allowed us to more completely relate these parameters to their influence on the in vivo pharmacokinetics in mice and cynomolgus monkeys. The variant antibodies have different pharmacokinetic behavior in cynomolgus monkeys and mice, which appears to be related to the unique binding characteristics observed with the murine receptor. However, we did not observe a direct relationship between increased binding affinity to the receptor and improved pharmacokinetic properties for these molecules in either species. This work provides further insights into how the FcRn/IgG interaction may be modulated to develop monoclonal antibodies with improved therapeutic properties.The neonatal Fc receptor (FcRn) is a heterodimeric protein consisting of a soluble light chain [ 2 -microglobulin ( 2 m)] and a transmembrane anchored heavy chain (␣-FcRn). FcRn functions as a "salvage receptor" regulating levels of circulating IgG in rodents Junghans and Anderson, 1996). FcRn is expressed ubiquitously in endothelial cells of human tissues (Ghetie and Ward, 2000), and it has been speculated that FcRn also plays a key role in IgG homeostasis in humans Kim et al., 1999).Earlier studies show that the FcRn/IgG interaction is highly pHdependent. IgG binds to FcRn via the Fc region at pH 6.0 (Rodewald, 1976;Raghavan and Bjorkman, 1996) and does not bind to FcRn at neutral pH, and the dissociation of the FcRn/IgG complex is facilitated at pH 7.4 (Rodewald, 1976;Raghavan et al., 1995;Raghavan and Bjorkman, 1996). In endothelium, FcRn appears to have an exclusively intracellular localization within acidified endosomes (Ober et al., 2004a). These observations and cellular studies have suggested of a model of IgG homeostasis, which involves the uptake of IgG into the cell via fluid-phase pinocytosis with subsequent binding to FcRn in endosomes (Ward et al., 2003;Ober et al., 2004b). Unbound IgG is directed down a degradative pathway resulting in proteolysis in lysosomes, whereas FcRn-bound antibody is recycled to the cell surface where the neutral pH facilitates dissociation and release into the circulation (Ward et al., 2003).As monoclonal antibodies continue to show promise as therapeutic agents, there have been a number of studies that have attempte...
A protein engineering strategy based on efficient and focused mutagenesis implemented by codon-based mutagenesis was developed. Vitaxin, a humanized version of the antiangiogenic antibody LM609 directed against a conformational epitope of the ␣ v  3 integrin complex, was used as a model system. Specifically, focused mutagenesis was used in a stepwise fashion to rapidly improve the affinity of the antigen binding fragment by greater than 90-fold. In the complete absence of structural information about the Vitaxin-
The neonatal Fc receptor (FcRn) plays a critical role in regulating IgG homeostasis in vivo. There are mixed reports on whether modification of the interaction with FcRn can be used as an engineering strategy to improve the pharmacokinetic and pharmacodynamic properties of monoclonal antibodies. We tested whether the T250Q/M428L mutations, which improved the pharmacokinetics of humanized IgGs in the rhesus monkey, would translate to a pharmacokinetic benefit in both cynomolgus monkeys and mice when constructed on a different humanized IgG framework (anti-tumor necrosis factor-␣ (TNF␣)). The T250Q/M428L anti-TNF␣ variant displayed an ϳ40-fold increase in binding affinity to cynomolgus monkey FcRn (C-FcRn) at pH 6.0, with maintenance of the pH binding dependence. We also constructed another anti-TNF␣ variant (P257I/Q311I) whose binding kinetics with the C-FcRn was similar to that of the T250Q/M428L variant. The binding affinity of the T250Q/M428L variant for murine FcRn was increased ϳ500-fold, with maintenance of pH dependence. In contrast to the interaction with C-FcRn, this interaction was driven mainly by a decrease in the rate of dissociation. Despite the improved in vitro binding properties of the anti-TNF␣ T250Q/M428L and P257I/Q311I variants to C-FcRn, the pharmacokinetic profiles of these molecules were not differentiated from the wild-type antibody in cynomolgus monkeys after intravenous administration. When administered intravenously to mice, the T250Q/ M428L anti-TNF␣ variant displayed improved pharmacokinetics, characterized by an ϳ2-fold slower clearance than the wildtype antibody. The discrepancy between these data and previously reported benefits in rhesus monkeys and the inability of these mutations to translate to improved kinetics across species may be related to a number of factors. We propose extending consideration to differences in the absolute IgG-FcRn affinity, the kinetics of the IgG/FcRn interaction, and differences in the relative involvement of this pathway in the context of other factors influencing the disposition or elimination of monoclonal antibodies. Monoclonal antibodies (mAbs)2 and Fc fusion proteins have become important therapeutic options in numerous disease indications, including cancer, inflammation, and autoimmune diseases (1). The proven efficacy of these molecular entities in combination with advances in protein engineering and directed evolution strategies has led to efforts to optimize the functional activity of these biologic agents. The goal of many of these approaches is to improve patient convenience and safety by reducing dose and/or dose frequency and potentially to improve efficacy (1). Many reports have suggested that improvement in the pharmacokinetic and pharmacodynamic properties of humanized monoclonal antibodies may be gained through modification of the interaction of the Fc region of IgGs with FcRn (2-6). It has been proposed that optimizing the properties of the receptor interaction may alter the intracellular trafficking of IgGs resulting in reduced cl...
We previously found that injection of a cocaine hydrolase (CocE) engineered from human butyrylcholinesterase will transiently accelerate cocaine metabolism in rats while reducing physiological and behavioral responses. To investigate more extended therapeutic effects, CocE cDNA was incorporated into a replication-incompetent type-5 adenoviral vector with a cytomegalovirus promoter. In rats dosed with this agent (2.2 ϫ 10 9 plaque-forming units), the time course of expression was characterized by reverse transcription polymerase chain reaction for CocE mRNA and by radiometric assay for enzyme activity. Liver and plasma showed comparable expression, beginning 2 days after vector administration and peaking between 5 and 7 days. Plasma CocE content was up to 100 mU/ml, with total cocaine hydrolyzing activity 3000-fold greater than in "empty vector" or untreated controls. This level of expression approximated that found immediately after i.v. injection of purified hydrolase, 3 mg/kg, a dose that shortened cocaine halflife and blunted cardiovascular effects. Sucrose density gradient analysis showed that 96% of the circulating CocE activity was associated with tetrameric enzyme forms, expected to be stable in vivo. Consistent with this expectation, CocE from vector-treated rats showed a plasma t 1/2 of 33 h when reinjected into naive rats. Transduction of another mutant butyrylcholinesterase, Applied Molecular Evolution mutant 359 (AME 359 ), caused plasma cocaine hydrolase activity to rise 50,000-fold. At the point of peak AME 359 expression, cocaine was cleared from the blood too rapidly for accurate measurement, and pressor responses to the injection of drug were greatly impaired.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.