The therapeutic potential of sialylated Fc domains of human IgG.
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ABSTRACT:
SUBMITTER: Pleass RJ
PROVIDER: S-EPMC8296966 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:PURPOSE:The Göttingen minipig is a relevant non-rodent species for regulatory toxicological studies. Yet, its use with therapeutic antibodies has been limited by the unknown binding properties of human immunoglobulins (huIgG) to porcine Fc gamma receptors (poFc?R) influencing safety and efficacy readouts. Therefore, knowing IgG-Fc?R interactions in the animal model is a prerequisite for the use of minipigs in preclinical safety and efficacy studies with therapeutic antibodies. METHODS:Here, we describe the cloning and expression of poFc?Rs and their interactions with free and complexed human therapeutic IgG1 by surface plasmon resonance and flow cytometry. RESULTS:We show here that poFc?RIa, poFc?RIIa, and poFc?RIIb bind huIgG1 antibodies with comparable affinities as corresponding huFc?Rs. Importantly, poFc?Rs bind huIgG immune complexes with high avidity, thus probably allowing human-like effector functions. However, poFc?RIIIa binds poIgG1a but not to huIgG1. CONCLUSIONS:The lack of binding of poFc?RIIIa to huIgG1 might cause underestimation of Fc?RIIIa-mediated efficacy or toxicity as mediated by porcine natural killer cells. Therefore, the suitability of minipigs in preclinical studies with human therapeutic antibodies has to be assessed case by case. Our results facilitate the use of Göttingen minipigs for assessment of human therapeutic antibodies in preclinical studies.
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Project description:Non-human primate (NHP) studies are often an essential component of antibody development efforts before human trials. Because the efficacy or toxicity of candidate antibodies may depend on their interactions with Fc? receptors (Fc?R) and their resulting ability to induce Fc?R-mediated effector functions such as antibody-dependent cell-meditated cytotoxicity and phagocytosis (ADCP), the evaluation of human IgG variants with modulated affinity toward human Fc?R is becoming more prevalent in both infectious disease and oncology studies in NHP. Reliable translation of these results necessitates analysis of the cross-reactivity of these human Fc variants with NHP Fc?R. We report evaluation of the binding affinities of a panel of human IgG subclasses, Fc amino acid point mutants and Fc glycosylation variants against the common allotypes of human and rhesus macaque Fc?R by applying a high-throughput array-based surface plasmon resonance platform. The resulting data indicate that amino acid variation present in rhesus Fc?Rs can result in disrupted, matched, or even increased affinity of IgG Fc variants compared with human Fc?R orthologs. These observations emphasize the importance of evaluating species cross-reactivity and developing an understanding of the potential limitations or suitability of representative in vitro and in vivo models before human clinical studies when either efficacy or toxicity may be associated with Fc?R engagement.
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Project description:Protein G is an IgG binding protein that has been widely exploited for biotechnological purposes. Rosetta protein modeling identified a set of favorable polar mutations in Protein G, at its binding interface with the Fc domain of Immunoglobulin G, that were predicted to increase the stability and tighten the binding relative to native Protein G, with only a minor perturbation of the binding mode seen in the crystal structure. This triple mutant was synthesized and evaluated experimentally. Relative to the native protein G, the mutant showed a 3.5-fold enhancement in display level on the surface of yeast and a 5-fold tighter molar affinity for rabbit and human IgG. We attribute the improved affinity to a network of hydrogen bonds exploiting specific polar groups on human and rabbit Fc. The relative specificity increased as well since there was little affinity enhancement for goat and mouse Fc, while the affinity for rat Fc was poorer by half. This designed Protein G will be useful in biotechnological applications as a recombinant protein, where its improved affinity, display and specificity will increase antibody capture sensitivity and capacity. Furthermore, the display of this protein on the surface of yeast introduces the concept of the use of yeast as an affinity matrix.
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Project description:Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fc? receptor (Fc?R). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the Fc?R-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with Fc?R engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.
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Project description:Human IgG is the main antibody class used in antibody therapies because of its efficacy and longer half-life, which are completely or partly due to Fc?R-mediated functions of the molecules. Preclinical testing in mouse models are frequently performed using human IgG, but no detailed information on binding of human IgG to mouse Fc?Rs is available. The orthologous mouse and human Fc?Rs share roughly 60-70% identity, suggesting some incompatibility. Here, we report binding affinities of all mouse and human IgG subclasses to mouse Fc?R. Human IgGs bound to mouse Fc?R with remarkably similar binding strengths as we know from binding to human ortholog receptors, with relative affinities IgG3>IgG1>IgG4>IgG2 and Fc?RI>>Fc?RIV>Fc?RIII>Fc?RIIb. This suggests human IgG subclasses to have similar relative Fc?R-mediated biological activities in mice.
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Project description:The effector activity of antibodies is dependent on engagement with Fc? receptors (Fc?Rs) and activation of the associated intracellular signaling pathways. Preclinical evaluation of therapeutic humanized or chimeric mAbs to study the interactions of their Fc regions with Fc?Rs is hampered by substantial structural and functional Fc?R diversity among species. In this report, we used mice expressing only human Fc?Rs to evaluate the contribution of Fc?R-mediated pathways to the neutralizing activity of an anti-anthrax toxin chimeric mAb. We observed that the protective activity of this mAb was highly dependent upon Fc?R engagement, with minimal protection against anthrax toxin observed in Fc?R-deficient mice following mAb administration. We generated anti-anthrax toxin mAbs with specific Fc domain variants with selectively enhanced affinity for particular human Fc?Rs and assessed their activity in Fc?R-humanized mice. We determined that Fc domain variants that were capable of selectively engaging activating Fc?Rs substantially enhanced the in vitro and in vivo activity of anthrax toxin-neutralizing antibodies. These findings indicate that the application of Fc domain engineering is a feasible strategy to enhance toxin-neutralizing activity and suggest that engineered antitoxin antibodies will have improved therapeutic efficacy.
| S-EPMC3904629 | biostudies-literature
Project description:Fc receptor-like (FCRL) 5 regulates B cell Ag receptor signaling and has been reported to bind aggregated IgG. Using surface plasmon resonance, we analyzed the interaction of native IgG samples with FCRL5, revealing a complex binding mechanism, where isotype is just one factor. FCRL5 bound IgG1 and IgG4 with ~1 ?M KD, whereas the interaction with IgG3 was a magnitude weaker. However, IgG2 samples displayed a wide range of affinities, indicating that additional factors affect binding. We used a panel of 19 anti-FCRL5 mAbs with defined reactivity to identify domains involved in ligand binding. Six mAbs blocked IgG binding, indicating critical roles of FCRL5 domains 1 and 3, as well as epitopes at the domain 1/2 and domain 2/3 boundaries. We found that only glycosylated IgG containing both Fab arms and the Fc region bound with high affinity. Furthermore, the presence of sialic acid in the IgG carbohydrate altered FCRL5 binding. The interaction of IgG and FCRL5 consisted of two kinetic components, suggesting a complex binding mechanism. We established that the IgG-Fc and IgG-F(ab')2 fragments bind FCRL5 independently but with low affinity, revealing the mechanism behind the two-step binding of whole IgG. This complex binding mechanism is distinct from that of Fc receptors, which bind through the Fc. We propose that FCRL5 is a new type of receptor that recognizes intact IgG, possibly enabling B cells to sense Ig quality. Recognition of undamaged IgG molecules by FCRL5 could allow B cells to engage recently produced Abs.
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Project description:Today, monoclonal immunoglobulin gamma (IgG) antibodies have become a major option in cancer therapy especially for the patients with advanced or metastatic cancers. Efficacy of monoclonal antibodies (mAbs) is achieved through both its antigen-binding fragment (Fab) and crystallizable fragment (Fc). Fab can specifically recognize tumor-associated antigen (TAA) and thus modulate TAA-linked downstream signaling pathways that may lead to the inhibition of tumor growth, induction of tumor apoptosis, and differentiation. The Fc region can further improve mAbs' efficacy by mediating effector functions such as antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent cell-mediated phagocytosis. Moreover, Fc is the region interacting with the neonatal Fc receptor in a pH-dependent manner that can slow down IgG's degradation and extend its serum half-life. Loss of the antibody Fc region dramatically shortens its serum half-life and weakens its anticancer effects. Given the essential roles that the Fc region plays in the modulation of the efficacy of mAb in cancer treatment, Fc engineering has been extensively studied in the past years. This review focuses on the recent advances in therapeutic Fc engineering that modulates its related effector functions and serum half-life. We also discuss the progress made in aglycosylated mAb development that may substantially reduce the cost of manufacture but maintain similar efficacies as conventional glycosylated mAb. Finally, we highlight several Fc engineering-based mAbs under clinical trials.
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Project description:Binding of the Fc domain of Immunoglobulin G (IgG) to Fc? receptors on leukocytes can initiate a series of signaling events resulting in antibody-dependent cell-mediated cytotoxicity (ADCC) and other important immune responses. Fc domains lacking glycosylation at N297 have greatly diminished Fc? receptor binding and lack the ability to initiate a robust ADCC response. Earlier structural studies of Fc domains with either full length or truncated N297 glycans led to the proposal that these glycans can stabilize an "open" Fc conformation recognized by Fc? receptors. We determined the structure of an E. coli expressed, aglycosylated human Fc domain at 3.1 Å resolution and observed significant disorder in the C'E loop, a region critical for Fc? receptor binding, as well as a decrease in distance between the C(H)2 domains relative to glycosylated Fc structures. However, comparison of the aglycosylated human Fc structure with enzymatically deglycosylated Fc structures revealed large differences in the relative orientations and distances between C(H)2 domains. To provide a better appreciation of the physiologically relevant conformation of the Fc domain in solution, we determined Radii of Gyration (R(g)) by small-angle X-ray scattering (SAXS) and found that the aglycosylated Fc displays a larger R(g) than glycosylated Fc, suggesting a more open C(H)2 orientation under these conditions. Moreover, the R(g) of aglycosylated Fc was reduced by mutations at the C(H)2-C(H)3 interface (E382V/M428I), which confer highly selective binding to Fc?RI and novel biological activities.
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Project description:A cDNA clone encoding a human receptor for the Fc portion of IgG (Fc gamma R), Fc gamma RIII or CD16, was isolated from a human leukocyte library by a transient expression-immunoselection procedure. This cDNA (pGP5) encodes a 46-kDa phosphatidylinositol-linked cell surface protein with CD16 determinants and affinity for human IgG. The deduced protein sequence is most homologous to the murine receptor Fc gamma RII alpha, with slightly less homology to the human receptors Fc gamma RII and Fc epsilon RI. The cDNA hybridizes to a 2.2 kilobase mRNA in human leukocytes and a cloned human natural killer cell line. Fc gamma RIII is mapped to chromosome 1 by spot-blot analysis of sorted human chromosomes. Hybridization of Fc gamma RII and Fc gamma RIII probes to restriction digests of human genomic DNA separated by pulsed-field gel electrophoresis demonstrates physical linkage of the two genes within a maximum distance of 200 kilobases. The results identify a locus for at least two Fc gamma R genes on human chromosome 1.
| S-EPMC286611 | biostudies-other