Abstract:The neonatal Fc receptor (FcRn) binds maternal immunoglobulin G (IgG) from ingested milk in the gut (pH 6.0-6.5) and delivers it to the bloodstream of the newborn (pH 7.0-7.5). A soluble version of FcRn reproduces the physiological pH-dependent interaction with IgG, showing high-affinity binding at pH 6.0-6.5 but weak or no binding at pH 7.0-7.5. We have studied the pH dependence of the FcRn/IgG interaction using a surface plasmon resonance assay to measure kinetic and equilibrium constants. We show that the a… Show more
“…The increased affinity of P257I/Q311I variant was attributable to effects on both the rate of association and dissociation ( Table 2). The improved affinity of these variant IgG complexes is consistent with previous reports indicating the involvement of the C H 2 and C H 3 Fc regions in the interaction of IgG with FcRn (6,12,17,18,21,22,24,(31)(32)(33)(34)(35)(36). At the highest IgG concentration tested for pH 6.0 binding (4 M), neither the WT nor variant anti-TNF␣ mAbs bound at measurable levels to any species of FcRn at pH 7.4 (data not shown), indicting that these mutations did not influence the characteristic pH binding dependence of the FcRn/IgG interaction (22).…”
Section: Interaction Characteristics Of the Wt And Fc Variant Anti-tnsupporting
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...
“…The increased affinity of P257I/Q311I variant was attributable to effects on both the rate of association and dissociation ( Table 2). The improved affinity of these variant IgG complexes is consistent with previous reports indicating the involvement of the C H 2 and C H 3 Fc regions in the interaction of IgG with FcRn (6,12,17,18,21,22,24,(31)(32)(33)(34)(35)(36). At the highest IgG concentration tested for pH 6.0 binding (4 M), neither the WT nor variant anti-TNF␣ mAbs bound at measurable levels to any species of FcRn at pH 7.4 (data not shown), indicting that these mutations did not influence the characteristic pH binding dependence of the FcRn/IgG interaction (22).…”
Section: Interaction Characteristics Of the Wt And Fc Variant Anti-tnsupporting
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...
“…Mutation of either of the two histidine residues significantly reduces the binding affinity of Fc with FcRn. 20,21 In addition to His310 and His435, several other Fc residues are involved in making molecular contacts with FcRn (Figure 1). 10.1080/19420862.2018.1490119-F0001Figure 1.( top ) Structural view of human IgG CH2-CH3 domains ( yellow ) in complex with human FcRn α ( green ) and β2M ( cyan ) domains.…”
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.
“…In many instances, however, the responses to these variables are the inverse of those seen with the CCR3/eotaxin interactions. For example, neonatal Fc receptors bind maternal IgG from ingested milk in the gut (pH 6.0 -6.5) with a high affinity and deliver them to the bloodstream (pH 7.0 -7.5) where the binding affinity drops 2 orders of magnitude (19). There are several examples also where the difference in binding affinity at acidic pH, such as would be present in subcellular compartments, might have a biological significance.…”
Section: Ph and Ionic Strength Modification Of Ccr3 Binding 28208mentioning
The CC chemokine receptor 3 (CCR3) plays an important role in the regulation of the migration of eosinophils, a leukocyte population involved in many inflammatory pathologies including asthma. CCR3 binds to the CC chemokine eotaxin, a promigratory cytokine originally isolated as the key component in a model of eosinophil-induced airway inflammation. We show here that eotaxin/CCR3 binding interactions exhibit a marked sensitivity to relatively small changes in the extracellular environment. In particular, modest variations in the pH and the level of sodium chloride over a range of physiologic and near physiologic conditions had dramatic effects on eotaxin binding and CCR3-mediated cytoplasmic Ca 2؉ mobilization. These biochemical indices were reflected at the functional level as well; small changes in pH and salt also resulted in striking changes in the migration of primary human eosinophils in vitro. These results reveal that relatively small perturbations in extracellular buffer conditions can yield widely disparate interpretations of CCR3 ligand binding and affinities and suggest that modulation of the tissue microenvironment might be utilized to control the affinity and efficacy of chemokine-mediated cell migration.Eosinophils are involved in many inflammatory pathologies including asthma, urticaria, and hypereosinophilic syndrome (1-3). The CC chemokine receptor 3 (CCR3) 1 and its ligands, most notably eotaxin, have been shown to play a central role in controlling eosinophil migration (4 -12). Eotaxin was originally isolated as a protein responsible for eosinophil chemoattraction in the bronchoalveolar fluid of allergen-sensitized guinea pigs (9). Subsequently, the human and mouse homologs were identified and also shown to be chemoattractants for eosinophils (10,12). Eotaxin has been shown to be a primary ligand for the seven-transmembrane G protein-coupled receptor CCR3. The genes for CCR3 and the related receptors CCR1-CCR5 are encoded within a 130-kilobase region of human chromosome 3 (13) indicating recent gene duplication and divergence and perhaps explaining the partial overlap of chemokine ligand repertoires. CCR3 is highly expressed on primary human eosinophils (50,000 -400,000 sites/cell) (5-7), consistent with the potent ability of eotaxin to selectively influence the migration of these cells.The mechanisms by which specific cells are attracted by specific chemokines have been a topic of intense interest and clinical relevance. The question is complicated by the fact that a number of closely related chemokine receptors with overlapping specificities are expressed on many different classes of leukocytes. In addition, the affinity of a specific receptor/ligand interaction can vary depending upon the specific cell type expressing the chemokine receptor, perhaps due to posttranslational modification events or G protein coupling. Thus it appears that cells can modulate their responses to chemokines, but how this is done is not yet clear. While most investigations have focused on factors intrinsic to the...
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