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Proc. Natl. Acad. Sci. USA, January 12, 2010 vol. 107 no. 2 604-609. Published online before print December 18, 2009, doi: 10.1073/pnas.0908590107
Aglycosylated IgG variants expressed in bacteria that selectively bind FcγRI potentiate tumor cell killing by monocyte-dendritic cells
Abstract
Sang Taek Junga, Sai T. Reddya, Tae Hyun Kangb, M. Jack Borroka, Inger Sandliec, Philip W. Tuckerb,d and George Georgioua,b,d,e,1
aDepartment of Chemical Engineering,
bInstitute for Cellular and Molecular Biology,
dSection of Molecular Genetics and Microbiology, and
eDepartment of Biomedical Engineering, University of Texas, Austin, TX 78712; and
cDepartment of Molecular Biosciences and Center for Immune Regulation, University of Oslo, 0316 Oslo, Norway
Edited by Frances H Arnold, California Institute of Technology, Pasadena, CA, and approved November 12, 2009 (received for review July 31, 2009)
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 HEK293T cells 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.
antibody engineering, bacterial display, bacterial expression, directed evolution, effector function
Footnotes
1To whom correspondence should be addressed.
Author contributions: S.T.J. and G.G. designed research; S.T.J., S.T.R., T.H.K., and M.J.B. performed research; I.S. contributed new reagents and analytic tools; S.T.J., S.T.R., T.H.K., M.J.B., P.W.T., and G.G. analyzed data; and S.T.J. and G.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at
www.pnas.org/cgi/content/full/0908590107/DCSupplemental.
aDepartment of Chemical Engineering,
bInstitute for Cellular and Molecular Biology,
dSection of Molecular Genetics and Microbiology, and
eDepartment of Biomedical Engineering, University of Texas, Austin, TX 78712; and
cDepartment of Molecular Biosciences and Center for Immune Regulation, University of Oslo, 0316 Oslo, Norway
Edited by Frances H Arnold, California Institute of Technology, Pasadena, CA, and approved November 12, 2009 (received for review July 31, 2009)
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 HEK293T cells 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.
antibody engineering, bacterial display, bacterial expression, directed evolution, effector function
Footnotes
1To whom correspondence should be addressed.
Author contributions: S.T.J. and G.G. designed research; S.T.J., S.T.R., T.H.K., and M.J.B. performed research; I.S. contributed new reagents and analytic tools; S.T.J., S.T.R., T.H.K., M.J.B., P.W.T., and G.G. analyzed data; and S.T.J. and G.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at
www.pnas.org/cgi/content/full/0908590107/DCSupplemental.
논문정보
- Research article
- 2009년 12월 (BRIC 등록일 )
- 국외 연구진
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