An engineered human fc variant with exquisite selectivity for FcγRIIIaV158 reveals that ligation of FcγRIIIa mediates potent antibody dependent cellular phagocytosis with GM-CSF-differentiated macrophages

Tae Hyun Kang, Chang Han Lee, George Delidakis, Jiwon Jung, Odile Richard Le Goff, Jiwon Lee, Jin Eyun Kim, Wissam Charab, Pierre Bruhns, George Georgiou

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

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 clearance mechanisms that can be mediated by individual FcγR are not well-understood. Human FcγRIIIa (hFcγRIIIa; CD16a), which exists as two polymorphic variants at position 158, hFcγRIIIaV158 and hFcγRIIIaF158, is widely considered to only trigger ADCC, especially with natural killer (NK) cells as effectors. To evaluate the role of hFcγRIIIa ligation in myeloid-derived effector cells, and in particular on macrophages and monocytes which express multiple FcγRs, we engineered an aglycosylated engineered human Fc (hFc) variant, Fc3aV, which binds exclusively to hFcγRIIIaV158. Antibodies formatted with the Fc3aV variant bind to the hFcγRIIIaV158 allotype with a somewhat lower KD than their wild type IgG1 counterparts, but not to any other hFcγR. The exceptional selectivity for hFcγRIIIaV158 was demonstrated by SPR using increased avidity, dimerized GST-fused versions of the ectodomains of hFcγRs and from the absence of binding of large immune complex (IC) to CHO cells expressing each of the hFcγRs, including notably, the FcγRIIIaF158 variant or the highly homologous FcγRIIIb. We show that even though monocyte-derived GM-CSF differentiated macrophages express hFcγRIIIa at substantially lower levels than the other two major activating receptors, namely hFcγRI or hFcγRIIa, 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 hFcγRIIIa 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 hFcγRs as tools for the detailed molecular understanding of hFcγR biology.

Original languageEnglish (US)
Article number562
JournalFrontiers in immunology
Volume10
Issue numberMAR
DOIs
StatePublished - Jan 1 2019

Fingerprint

IgG Receptors
Granulocyte-Macrophage Colony-Stimulating Factor
Phagocytosis
Ligation
Antibody-Dependent Cell Cytotoxicity
Macrophages
Antibodies
Monocytes
Immunoglobulin G
Antigen-Antibody Complex
CHO Cells
Natural Killer Cells
Neoplasms

Keywords

  • ADCP
  • Fc engineering
  • FcgR
  • Macrophage
  • Monocyte

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Cite this

An engineered human fc variant with exquisite selectivity for FcγRIIIaV158 reveals that ligation of FcγRIIIa mediates potent antibody dependent cellular phagocytosis with GM-CSF-differentiated macrophages. / Kang, Tae Hyun; Lee, Chang Han; Delidakis, George; Jung, Jiwon; Goff, Odile Richard Le; Lee, Jiwon; Kim, Jin Eyun; Charab, Wissam; Bruhns, Pierre; Georgiou, George.

In: Frontiers in immunology, Vol. 10, No. MAR, 562, 01.01.2019.

Research output: Contribution to journalArticle

Kang, Tae Hyun ; Lee, Chang Han ; Delidakis, George ; Jung, Jiwon ; Goff, Odile Richard Le ; Lee, Jiwon ; Kim, Jin Eyun ; Charab, Wissam ; Bruhns, Pierre ; Georgiou, George. / An engineered human fc variant with exquisite selectivity for FcγRIIIaV158 reveals that ligation of FcγRIIIa mediates potent antibody dependent cellular phagocytosis with GM-CSF-differentiated macrophages. In: Frontiers in immunology. 2019 ; Vol. 10, No. MAR.
@article{aa883a0a28b84b10b9c61549f2f4beab,
title = "An engineered human fc variant with exquisite selectivity for FcγRIIIaV158 reveals that ligation of FcγRIIIa 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 (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 clearance mechanisms that can be mediated by individual FcγR are not well-understood. Human FcγRIIIa (hFcγRIIIa; CD16a), which exists as two polymorphic variants at position 158, hFcγRIIIaV158 and hFcγRIIIaF158, is widely considered to only trigger ADCC, especially with natural killer (NK) cells as effectors. To evaluate the role of hFcγRIIIa ligation in myeloid-derived effector cells, and in particular on macrophages and monocytes which express multiple FcγRs, we engineered an aglycosylated engineered human Fc (hFc) variant, Fc3aV, which binds exclusively to hFcγRIIIaV158. Antibodies formatted with the Fc3aV variant bind to the hFcγRIIIaV158 allotype with a somewhat lower KD than their wild type IgG1 counterparts, but not to any other hFcγR. The exceptional selectivity for hFcγRIIIaV158 was demonstrated by SPR using increased avidity, dimerized GST-fused versions of the ectodomains of hFcγRs and from the absence of binding of large immune complex (IC) to CHO cells expressing each of the hFcγRs, including notably, the FcγRIIIaF158 variant or the highly homologous FcγRIIIb. We show that even though monocyte-derived GM-CSF differentiated macrophages express hFcγRIIIa at substantially lower levels than the other two major activating receptors, namely hFcγRI or hFcγRIIa, 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 hFcγRIIIa 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 hFcγRs as tools for the detailed molecular understanding of hFcγR biology.",
keywords = "ADCP, Fc engineering, FcgR, Macrophage, Monocyte",
author = "Kang, {Tae Hyun} and Lee, {Chang Han} and George Delidakis and Jiwon Jung and Goff, {Odile Richard Le} and Jiwon Lee and Kim, {Jin Eyun} and Wissam Charab and Pierre Bruhns and George Georgiou",
year = "2019",
month = "1",
day = "1",
doi = "10.3389/fimmu.2019.00562",
language = "English (US)",
volume = "10",
journal = "Frontiers in Immunology",
issn = "1664-3224",
publisher = "Frontiers Media S. A.",
number = "MAR",

}

TY - JOUR

T1 - An engineered human fc variant with exquisite selectivity for FcγRIIIaV158 reveals that ligation of FcγRIIIa mediates potent antibody dependent cellular phagocytosis with GM-CSF-differentiated macrophages

AU - Kang, Tae Hyun

AU - Lee, Chang Han

AU - Delidakis, George

AU - Jung, Jiwon

AU - Goff, Odile Richard Le

AU - Lee, Jiwon

AU - Kim, Jin Eyun

AU - Charab, Wissam

AU - Bruhns, Pierre

AU - Georgiou, George

PY - 2019/1/1

Y1 - 2019/1/1

N2 - 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 clearance mechanisms that can be mediated by individual FcγR are not well-understood. Human FcγRIIIa (hFcγRIIIa; CD16a), which exists as two polymorphic variants at position 158, hFcγRIIIaV158 and hFcγRIIIaF158, is widely considered to only trigger ADCC, especially with natural killer (NK) cells as effectors. To evaluate the role of hFcγRIIIa ligation in myeloid-derived effector cells, and in particular on macrophages and monocytes which express multiple FcγRs, we engineered an aglycosylated engineered human Fc (hFc) variant, Fc3aV, which binds exclusively to hFcγRIIIaV158. Antibodies formatted with the Fc3aV variant bind to the hFcγRIIIaV158 allotype with a somewhat lower KD than their wild type IgG1 counterparts, but not to any other hFcγR. The exceptional selectivity for hFcγRIIIaV158 was demonstrated by SPR using increased avidity, dimerized GST-fused versions of the ectodomains of hFcγRs and from the absence of binding of large immune complex (IC) to CHO cells expressing each of the hFcγRs, including notably, the FcγRIIIaF158 variant or the highly homologous FcγRIIIb. We show that even though monocyte-derived GM-CSF differentiated macrophages express hFcγRIIIa at substantially lower levels than the other two major activating receptors, namely hFcγRI or hFcγRIIa, 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 hFcγRIIIa 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 hFcγRs as tools for the detailed molecular understanding of hFcγR biology.

AB - 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 clearance mechanisms that can be mediated by individual FcγR are not well-understood. Human FcγRIIIa (hFcγRIIIa; CD16a), which exists as two polymorphic variants at position 158, hFcγRIIIaV158 and hFcγRIIIaF158, is widely considered to only trigger ADCC, especially with natural killer (NK) cells as effectors. To evaluate the role of hFcγRIIIa ligation in myeloid-derived effector cells, and in particular on macrophages and monocytes which express multiple FcγRs, we engineered an aglycosylated engineered human Fc (hFc) variant, Fc3aV, which binds exclusively to hFcγRIIIaV158. Antibodies formatted with the Fc3aV variant bind to the hFcγRIIIaV158 allotype with a somewhat lower KD than their wild type IgG1 counterparts, but not to any other hFcγR. The exceptional selectivity for hFcγRIIIaV158 was demonstrated by SPR using increased avidity, dimerized GST-fused versions of the ectodomains of hFcγRs and from the absence of binding of large immune complex (IC) to CHO cells expressing each of the hFcγRs, including notably, the FcγRIIIaF158 variant or the highly homologous FcγRIIIb. We show that even though monocyte-derived GM-CSF differentiated macrophages express hFcγRIIIa at substantially lower levels than the other two major activating receptors, namely hFcγRI or hFcγRIIa, 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 hFcγRIIIa 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 hFcγRs as tools for the detailed molecular understanding of hFcγR biology.

KW - ADCP

KW - Fc engineering

KW - FcgR

KW - Macrophage

KW - Monocyte

UR - http://www.scopus.com/inward/record.url?scp=85064852784&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85064852784&partnerID=8YFLogxK

U2 - 10.3389/fimmu.2019.00562

DO - 10.3389/fimmu.2019.00562

M3 - Article

VL - 10

JO - Frontiers in Immunology

JF - Frontiers in Immunology

SN - 1664-3224

IS - MAR

M1 - 562

ER -