Aglycosylated IgG variants expressed in bacteria that selectively bind FcγRI potentiate tumor cell killing by monocyte-dendritic cells

Sang Taek Jung, Sai T. Reddy, Tae Hyun Kang, M. Jack Borrok, Inger Sandlie, Philip W. Tucker, George Georgiou

Research output: Contribution to journalArticle

106 Citations (Scopus)

Abstract

The N-linked glycan of immunoglobulin G (IgG) is indispensable for the interaction of the Fc domain with Fcγ receptors on effector cells and the clearance of target cells via antibody dependent cell-mediated cytotoxicity (ADCC). Escherichia coli expressed, aglycosylated Fc domains bind effector FcγRs poorly and cannot elicit ADCC. Using a novel bacterial display/flow cytometric library screening system we isolated Fc variants that bind to FcγRI (CD64) with nanomolar affinity. Binding was critically dependent on amino acid substitutions (E382V, and to a lesser extent, M428I) distal to the putative FcγRI binding epitope within the CH3 domain. These mutations did not adversely affect its pH-dependent interaction with FcRn in vitro nor its serum persistence in vivo. Remarkably, the anti-Her2 IgG trastuzumab containing the E382V, M428I substitutions and expressed in E. coli exhibited highly selective binding to FcγRI but not to the other activating receptors (FcγRIIa, FcγRIIIa) nor to the inhibitory receptor, FcγRIIb. In contrast, the glycosylated version of trastuzumab (E382V, M428I) purified from HEK293Tcells bound to all Fcγ receptors in a manner similar to that of clinical grade trastuzumab. E. coli-purified trastuzumab (E382V, M428I), but not glycosylated trastuzumab (E382V, M428I) or clinical grade trastuzumab, was capable of potentiating the killing of Her2 overexpressing tumor cells with dendritic cells (DCs) as effectors. These results indicate that aglycosylated IgGs can be engineered to display unique FcγR selectivity profiles that, in turn, mediate ADCC via mechanisms that are not normally displayed by glycosylated monoclonal antibodies.

Original languageEnglish
Pages (from-to)604-609
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume107
Issue number2
DOIs
Publication statusPublished - 2010 Feb 15
Externally publishedYes

Fingerprint

Dendritic Cells
Monocytes
Immunoglobulin G
Bacteria
Antibody-Dependent Cell Cytotoxicity
Neoplasms
Fc Receptors
Escherichia coli
Amino Acid Substitution
Polysaccharides
Trastuzumab
Epitopes
Monoclonal Antibodies
Mutation
Serum

Keywords

  • Antibody engineering
  • Bacterial display
  • Bacterial expression
  • Directed evolution
  • Effector function

ASJC Scopus subject areas

  • General

Cite this

Aglycosylated IgG variants expressed in bacteria that selectively bind FcγRI potentiate tumor cell killing by monocyte-dendritic cells. / Jung, Sang Taek; Reddy, Sai T.; Kang, Tae Hyun; Borrok, M. Jack; Sandlie, Inger; Tucker, Philip W.; Georgiou, George.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 107, No. 2, 15.02.2010, p. 604-609.

Research output: Contribution to journalArticle

Jung, Sang Taek ; Reddy, Sai T. ; Kang, Tae Hyun ; Borrok, M. Jack ; Sandlie, Inger ; Tucker, Philip W. ; Georgiou, George. / Aglycosylated IgG variants expressed in bacteria that selectively bind FcγRI potentiate tumor cell killing by monocyte-dendritic cells. In: Proceedings of the National Academy of Sciences of the United States of America. 2010 ; Vol. 107, No. 2. pp. 604-609.
@article{f0ffbfd443c64e1fae5de16406e00fe5,
title = "Aglycosylated IgG variants expressed in bacteria that selectively bind FcγRI potentiate tumor cell killing by monocyte-dendritic cells",
abstract = "The N-linked glycan of immunoglobulin G (IgG) is indispensable for the interaction of the Fc domain with Fcγ receptors on effector cells and the clearance of target cells via antibody dependent cell-mediated cytotoxicity (ADCC). Escherichia coli expressed, aglycosylated Fc domains bind effector FcγRs poorly and cannot elicit ADCC. Using a novel bacterial display/flow cytometric library screening system we isolated Fc variants that bind to FcγRI (CD64) with nanomolar affinity. Binding was critically dependent on amino acid substitutions (E382V, and to a lesser extent, M428I) distal to the putative FcγRI binding epitope within the CH3 domain. These mutations did not adversely affect its pH-dependent interaction with FcRn in vitro nor its serum persistence in vivo. Remarkably, the anti-Her2 IgG trastuzumab containing the E382V, M428I substitutions and expressed in E. coli exhibited highly selective binding to FcγRI but not to the other activating receptors (FcγRIIa, FcγRIIIa) nor to the inhibitory receptor, FcγRIIb. In contrast, the glycosylated version of trastuzumab (E382V, M428I) purified from HEK293Tcells bound to all Fcγ receptors in a manner similar to that of clinical grade trastuzumab. E. coli-purified trastuzumab (E382V, M428I), but not glycosylated trastuzumab (E382V, M428I) or clinical grade trastuzumab, was capable of potentiating the killing of Her2 overexpressing tumor cells with dendritic cells (DCs) as effectors. These results indicate that aglycosylated IgGs can be engineered to display unique FcγR selectivity profiles that, in turn, mediate ADCC via mechanisms that are not normally displayed by glycosylated monoclonal antibodies.",
keywords = "Antibody engineering, Bacterial display, Bacterial expression, Directed evolution, Effector function",
author = "Jung, {Sang Taek} and Reddy, {Sai T.} and Kang, {Tae Hyun} and Borrok, {M. Jack} and Inger Sandlie and Tucker, {Philip W.} and George Georgiou",
year = "2010",
month = "2",
day = "15",
doi = "10.1073/pnas.0908590107",
language = "English",
volume = "107",
pages = "604--609",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "2",

}

TY - JOUR

T1 - Aglycosylated IgG variants expressed in bacteria that selectively bind FcγRI potentiate tumor cell killing by monocyte-dendritic cells

AU - Jung, Sang Taek

AU - Reddy, Sai T.

AU - Kang, Tae Hyun

AU - Borrok, M. Jack

AU - Sandlie, Inger

AU - Tucker, Philip W.

AU - Georgiou, George

PY - 2010/2/15

Y1 - 2010/2/15

N2 - The N-linked glycan of immunoglobulin G (IgG) is indispensable for the interaction of the Fc domain with Fcγ receptors on effector cells and the clearance of target cells via antibody dependent cell-mediated cytotoxicity (ADCC). Escherichia coli expressed, aglycosylated Fc domains bind effector FcγRs poorly and cannot elicit ADCC. Using a novel bacterial display/flow cytometric library screening system we isolated Fc variants that bind to FcγRI (CD64) with nanomolar affinity. Binding was critically dependent on amino acid substitutions (E382V, and to a lesser extent, M428I) distal to the putative FcγRI binding epitope within the CH3 domain. These mutations did not adversely affect its pH-dependent interaction with FcRn in vitro nor its serum persistence in vivo. Remarkably, the anti-Her2 IgG trastuzumab containing the E382V, M428I substitutions and expressed in E. coli exhibited highly selective binding to FcγRI but not to the other activating receptors (FcγRIIa, FcγRIIIa) nor to the inhibitory receptor, FcγRIIb. In contrast, the glycosylated version of trastuzumab (E382V, M428I) purified from HEK293Tcells bound to all Fcγ receptors in a manner similar to that of clinical grade trastuzumab. E. coli-purified trastuzumab (E382V, M428I), but not glycosylated trastuzumab (E382V, M428I) or clinical grade trastuzumab, was capable of potentiating the killing of Her2 overexpressing tumor cells with dendritic cells (DCs) as effectors. These results indicate that aglycosylated IgGs can be engineered to display unique FcγR selectivity profiles that, in turn, mediate ADCC via mechanisms that are not normally displayed by glycosylated monoclonal antibodies.

AB - The N-linked glycan of immunoglobulin G (IgG) is indispensable for the interaction of the Fc domain with Fcγ receptors on effector cells and the clearance of target cells via antibody dependent cell-mediated cytotoxicity (ADCC). Escherichia coli expressed, aglycosylated Fc domains bind effector FcγRs poorly and cannot elicit ADCC. Using a novel bacterial display/flow cytometric library screening system we isolated Fc variants that bind to FcγRI (CD64) with nanomolar affinity. Binding was critically dependent on amino acid substitutions (E382V, and to a lesser extent, M428I) distal to the putative FcγRI binding epitope within the CH3 domain. These mutations did not adversely affect its pH-dependent interaction with FcRn in vitro nor its serum persistence in vivo. Remarkably, the anti-Her2 IgG trastuzumab containing the E382V, M428I substitutions and expressed in E. coli exhibited highly selective binding to FcγRI but not to the other activating receptors (FcγRIIa, FcγRIIIa) nor to the inhibitory receptor, FcγRIIb. In contrast, the glycosylated version of trastuzumab (E382V, M428I) purified from HEK293Tcells bound to all Fcγ receptors in a manner similar to that of clinical grade trastuzumab. E. coli-purified trastuzumab (E382V, M428I), but not glycosylated trastuzumab (E382V, M428I) or clinical grade trastuzumab, was capable of potentiating the killing of Her2 overexpressing tumor cells with dendritic cells (DCs) as effectors. These results indicate that aglycosylated IgGs can be engineered to display unique FcγR selectivity profiles that, in turn, mediate ADCC via mechanisms that are not normally displayed by glycosylated monoclonal antibodies.

KW - Antibody engineering

KW - Bacterial display

KW - Bacterial expression

KW - Directed evolution

KW - Effector function

UR - http://www.scopus.com/inward/record.url?scp=76249100176&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=76249100176&partnerID=8YFLogxK

U2 - 10.1073/pnas.0908590107

DO - 10.1073/pnas.0908590107

M3 - Article

VL - 107

SP - 604

EP - 609

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 2

ER -