“…Previously, two models of IgG engagement with mFcRn have been proposed: the “standing up” and “lying down” positioning of engaged Fc, relative to mFcRn ( Supplementary Figure 4 ). 50,51 Since albumin and IgG molecules can bind simultaneously to membrane-positioned FcRn, we propose that the IgG molecule is likely to bind in an inverted orientation, the “standing up” orientation, with its Fab domains close to the membrane bilayer surface and possibly, in certain circumstances, interacting with the membrane (Figure 6). We note that this close membrane proximity of Fab domains to the cell membrane offers a possible structure-based explanation for the reduced affinity to membrane-bound, native mFcRn of most IgG molecules and Fc-fusion proteins, as well as antibodies bound to large antigens.10.1080/19420862.2018.1490119-F0006Figure 6. Current best-fit model of IgG-FcRn complex formation .…”
Engineering of antibodies for improved pharmacokinetics through enhanced binding to the neonatal Fc receptor (FcRn) has been demonstrated in transgenic mice, non-human primates and humans. Traditionally, such approaches have largely relied on random mutagenesis and display formats, which fail to address related critical attributes of the antibody, such as effector functions or biophysical stability. We have developed a structure- and network-based framework to interrogate the engagement of IgG with multiple Fc receptors (FcRn, C1q, TRIM21, FcγRI, FcγRIIa/b, FcγRIIIa) simultaneously. Using this framework, we identified features that govern Fc-FcRn interactions and identified multiple distinct pathways for enhancing FcRn binding in a pH-specific manner. Network analysis provided a novel lens to study the allosteric impact of half-life-enhancing Fc mutations on FcγR engagement, which occurs distal to the FcRn binding site. Applying these principles, we engineered a panel of unique Fc variants that enhance FcRn binding while maintaining robust biophysical properties and wild type-like binding to activating receptors. An antibody harboring representative Fc designs demonstrates a half-life improvement of > 9 fold in transgenic mice and > 3.5 fold in cynomolgus monkeys, and maintains robust effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity.
“…Previously, two models of IgG engagement with mFcRn have been proposed: the “standing up” and “lying down” positioning of engaged Fc, relative to mFcRn ( Supplementary Figure 4 ). 50,51 Since albumin and IgG molecules can bind simultaneously to membrane-positioned FcRn, we propose that the IgG molecule is likely to bind in an inverted orientation, the “standing up” orientation, with its Fab domains close to the membrane bilayer surface and possibly, in certain circumstances, interacting with the membrane (Figure 6). We note that this close membrane proximity of Fab domains to the cell membrane offers a possible structure-based explanation for the reduced affinity to membrane-bound, native mFcRn of most IgG molecules and Fc-fusion proteins, as well as antibodies bound to large antigens.10.1080/19420862.2018.1490119-F0006Figure 6. Current best-fit model of IgG-FcRn complex formation .…”
Engineering of antibodies for improved pharmacokinetics through enhanced binding to the neonatal Fc receptor (FcRn) has been demonstrated in transgenic mice, non-human primates and humans. Traditionally, such approaches have largely relied on random mutagenesis and display formats, which fail to address related critical attributes of the antibody, such as effector functions or biophysical stability. We have developed a structure- and network-based framework to interrogate the engagement of IgG with multiple Fc receptors (FcRn, C1q, TRIM21, FcγRI, FcγRIIa/b, FcγRIIIa) simultaneously. Using this framework, we identified features that govern Fc-FcRn interactions and identified multiple distinct pathways for enhancing FcRn binding in a pH-specific manner. Network analysis provided a novel lens to study the allosteric impact of half-life-enhancing Fc mutations on FcγR engagement, which occurs distal to the FcRn binding site. Applying these principles, we engineered a panel of unique Fc variants that enhance FcRn binding while maintaining robust biophysical properties and wild type-like binding to activating receptors. An antibody harboring representative Fc designs demonstrates a half-life improvement of > 9 fold in transgenic mice and > 3.5 fold in cynomolgus monkeys, and maintains robust effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity.
“…29 To avoid avidity effects during SPR analyses, all affinities were determined with IgG immobilized and FcRn in solution. In earlier studies 25 where, in general, the FcRn-IgG interactions were of lower affinity than here, the analyte (FcRn) concentrations used were such that the major contribution to the interactions came from the higher-affinity binding sites on IgGs.…”
“…Molecular Modeling-Molecular models of the human Fc⅐FcRn complex were generated based on the crystal structures (18,19) and a model (20) of the rat Fc⅐FcRn complex. First, the rat 2m and FcRn ␣ chains of the complex were replaced, respectively, with the human 2m (21) and FcRn ␣ chains (22).…”
Antibody therapy is coming of age, with 15 monoclonal antibodies approved for therapeutic use in the United States and many others currently undergoing clinical trials (1). The advent of antibody engineering over the past two decades has contributed to the recent clinical success of therapeutic antibodies. The development of chimeric (2) and humanized (3) antibodies not only reduced the potent immunogenicity of rodent antibodies in humans but also improved the serum halflives and efficacy of such therapeutics compared with rodent antibodies. Phage display (4) and other display technologies have led to the ability to increase the affinity of antibodies for their target antigens. More recently, antibody engineering has been used to modify the effector functions of antibodies by altering their binding to C1q (5) and various Fc␥ receptors (6).The neonatal Fc receptor (FcRn) 1 is a heterodimer that comprises a transmembrane ␣ chain with structural homology to the extracellular domains of the ␣ chain of major histocompatibility complex class I molecules, and a soluble light chain consisting of 2-microglubulin (2m) (7). FcRn mediates both transcytosis of maternal IgG to the fetus or neonate and IgG homeostasis in adults (8). Evidence for the latter role initially came from studies indicating an unusually short serum halflife for IgG antibodies in 2m-deficient mice (9 -11). This observation led to the generation of mutant mouse hinge-Fc fragments with enhanced binding to FcRn and increased serum persistence in mice (12). Recently, several studies have identified human IgG 1 mutants with enhanced FcRn binding (6, 13), although no improvement in the serum half-lives of these mutants was observed in mice (13) or reported in primates.The binding of IgG to FcRn is sharply pH-dependent; IgG binds to FcRn under mildly acidic conditions and is released under slightly basic conditions (14). It has been hypothesized that pinocytosed IgG antibodies are captured by FcRn in acidified endosomes, rescued from degradation in lysosomes, recycled back to the cell surface, and returned to the circulation (8). Mutagenesis studies have identified both the mouse (15, 16) and human (17) Fc residues believed to be important in mediating pH-dependent binding. The results of the mutagenesis studies are consistent with the interpretation of a crystallographic study of the Fc⅐FcRn interaction (18). In the current study, molecular modeling was used to identify residues in the human IgG Fc near the FcRn binding site that, when mutated, might alter binding to FcRn without affecting the pH dependence of this interaction. Following exhaustive mutagenesis at these positions, several IgG 2 mutants were identified with improved binding to FcRn at pH 6.0 that retained the property of pH-dependent release. A pharmacokinetics study in rhesus monkeys showed that two mutant IgG 2 antibodies with increased FcRn binding affinity had considerably longer serum half-lives than the wild-type antibody.
EXPERIMENTAL PROCEDURESMolecular Modeling-Molecular models o...
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