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An Engineered Human Fc variant With Exquisite Selectivity for Fc?RIIIa_V158 Reveals That Ligation of Fc?RIIIa Mediates Potent Antibody Dependent Cellular Phagocytosis With GM-CSF-Differentiated Macrophages.

ABSTRACT: IgG antibodies mediate the clearance of target cells via the engagement of Fc gamma receptors (Fc?Rs) on effector cells by eliciting antibody-dependent cellular cytotoxicity and phagocytosis (ADCC and ADCP, respectively). Because (i) the IgG Fc domain binds to multiple Fc?Rs with varying affinities; (ii) even low Fc:Fc?Rs affinity interactions can play a significant role when antibodies are engaged in high avidity immune complexes and (iii) most effector cells express multiple Fc?Rs, the clearance mechanisms that can be mediated by individual Fc?R are not well-understood. Human Fc?RIIIa (hFc?RIIIa; CD16a), which exists as two polymorphic variants at position 158, hFc?RIIIa_V158 and hFc?RIIIa_F158, is widely considered to only trigger ADCC, especially with natural killer (NK) cells as effectors. To evaluate the role of hFc?RIIIa ligation in myeloid-derived effector cells, and in particular on macrophages and monocytes which express multiple Fc?Rs, we engineered an aglycosylated engineered human Fc (hFc) variant, Fc3aV, which binds exclusively to hFc?RIIIa_V158. Antibodies formatted with the Fc3aV variant bind to the hFc?RIIIa_V158 allotype with a somewhat lower K_D than their wild type IgG1 counterparts, but not to any other hFc?R. The exceptional selectivity for hFc?RIIIa_V158 was demonstrated by SPR using increased avidity, dimerized GST-fused versions of the ectodomains of hFc?Rs and from the absence of binding of large immune complex (IC) to CHO cells expressing each of the hFc?Rs, including notably, the Fc?RIIIa_F158 variant or the highly homologous Fc?RIIIb. We show that even though monocyte-derived GM-CSF differentiated macrophages express hFc?RIIIa at substantially lower levels than the other two major activating receptors, namely hFc?RI or hFc?RIIa, Fc3aV-formatted Rituximab and Herceptin perform ADCP toward CD20- and Her2-expressing cancer cells, respectively, at a level comparable to that of the respective wild-type antibodies. We further show that hFc?RIIIa activation plays a significant role on ADCC by human peripheral monocytes. Our data highlight the utility of Fc3aV and other similarly engineered exquisitely selective, aglycosylated Fc variants toward other hFc?Rs as tools for the detailed molecular understanding of hFc?R biology.

SUBMITTER: Kang TH

PROVIDER: S-EPMC6448688 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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An Engineered Human Fc variant With Exquisite Selectivity for FcγRIIIa<sub>V158</sub> Reveals That Ligation of FcγRIIIa Mediates Potent Antibody Dependent Cellular Phagocytosis With GM-CSF-Differentiated Macrophages.

Kang Tae Hyun TH Lee Chang-Han CH Delidakis George G Jung Jiwon J Richard-Le Goff Odile O Lee Jiwon J Kim Jin Eyun JE Charab Wissam W Bruhns Pierre P Georgiou George G

Frontiers in immunology 20190327

IgG antibodies mediate the clearance of target cells via the engagement of Fc gamma receptors (FcγRs) on effector cells by eliciting antibody-dependent cellular cytotoxicity and phagocytosis (ADCC and ADCP, respectively). Because (i) the IgG Fc domain binds to multiple FcγRs with varying affinities; (ii) even low Fc:FcγRs affinity interactions can play a significant role when antibodies are engaged in high avidity immune complexes and (iii) most effector cells express multiple FcγRs, the clearan ...[more]

PMID: 30984171

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Project description:Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm) infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ) stained positive for CD206 and M-CSF-derived macrophages (M-Mφ) for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages.

Project description:IntroductionEndogenous granulocyte-macrophage colony-stimulating factor (GM-CSF), identified by its ability to support differentiation of hematopoietic cells into several types of myeloid cells, is now known to support maturation and maintain the metabolic capacity of mononuclear phagocytes including monocytes, macrophages, and dendritic cells. These cells sense and attack potential pathogens, present antigens to adaptive immune cells, and recruit other immune cells. Recombinant human (rhu) GM-CSF (e.g., sargramostim [glycosylated, yeast-derived rhu GM-CSF]) has immune modulating properties and can restore the normal function of mononuclear phagocytes rendered dysfunctional by deficient or insufficient endogenous GM-CSF.MethodsWe reviewed the emerging biologic and cellular effects of GM-CSF. Experts in clinical disease areas caused by deficient or insufficient endogenous GM-CSF examined the role of GM-CSF in mononuclear phagocyte disorders including autoimmune pulmonary alveolar proteinosis (aPAP), diverse infections (including COVID-19), wound healing, and anti-cancer immune checkpoint inhibitor therapy.ResultsWe discuss emerging data for GM-CSF biology including the positive effects on mitochondrial function and cell metabolism, augmentation of phagocytosis and efferocytosis, and immune cell modulation. We further address how giving exogenous rhu GM-CSF may control or treat mononuclear phagocyte dysfunction disorders caused or exacerbated by GM-CSF deficiency or insufficiency. We discuss how rhu GM-CSF may augment the anti-cancer effects of immune checkpoint inhibitor immunotherapy as well as ameliorate immune-related adverse events.DiscussionWe identify research gaps, opportunities, and the concept that rhu GM-CSF, by supporting and restoring the metabolic capacity and function of mononuclear phagocytes, can have significant therapeutic effects. rhu GM-CSF (e.g., sargramostim) might ameliorate multiple diseases of GM-CSF deficiency or insufficiency and address a high unmet medical need.

Dataset Information

An Engineered Human Fc variant With Exquisite Selectivity for Fc?RIIIa_V158 Reveals That Ligation of Fc?RIIIa Mediates Potent Antibody Dependent Cellular Phagocytosis With GM-CSF-Differentiated Macrophages.

Publications

An Engineered Human Fc variant With Exquisite Selectivity for FcγRIIIa<sub>V158</sub> Reveals That Ligation of FcγRIIIa Mediates Potent Antibody Dependent Cellular Phagocytosis With GM-CSF-Differentiated Macrophages.

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Dataset Information

An Engineered Human Fc variant With Exquisite Selectivity for Fc?RIIIaV158 Reveals That Ligation of Fc?RIIIa Mediates Potent Antibody Dependent Cellular Phagocytosis With GM-CSF-Differentiated Macrophages.

Publications

An Engineered Human Fc variant With Exquisite Selectivity for FcγRIIIa<sub>V158</sub> Reveals That Ligation of FcγRIIIa Mediates Potent Antibody Dependent Cellular Phagocytosis With GM-CSF-Differentiated Macrophages.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

An Engineered Human Fc variant With Exquisite Selectivity for Fc?RIIIa_V158 Reveals That Ligation of Fc?RIIIa Mediates Potent Antibody Dependent Cellular Phagocytosis With GM-CSF-Differentiated Macrophages.