Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation

E. T. J. van den Bremer, F. J. Beurskens, M. Voorhorst, P. J. Engelberts, R. N. de Jong, B. G. van der Boom, E. M. Cook, M. A. Lindorfer, R. P. Taylor, P. H. C. van Berkel, Paul W.H.I. Parren

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

Human IgG is produced with C-terminal lysines that are cleaved off in circulation. The function of this modification was unknown and generally thought not to affect antibody function. We recently reported that efficient C1q binding and complement-dependent cytotoxicity (CDC) requires IgG hexamerization at the cell surface. Here we demonstrate that C-terminal lysines may interfere with this process, leading to suboptimal C1q binding and CDC of cells opsonized with C-terminal lysine-containing IgG. After we removed these lysines with a carboxypeptidase, maximal complement activation was observed. Interestingly, IgG1 mutants containing either a negative C-terminal charge or multiple positive charges lost CDC almost completely; however, CDC was fully restored by mixing C-terminal mutants of opposite charge. Our data indicate a novel post-translational control mechanism of human IgG: human IgG molecules are produced in a pro-form in which charged C-termini interfere with IgG hexamer formation, C1q binding and CDC. To allow maximal complement activation, C-terminal lysine processing is required to release the antibody's full cytotoxic potential.
OriginalsprogEngelsk
TidsskriftmAbs
Vol/bind7
Udgave nummer4
Sider (fra-til)672-680
ISSN1942-0862
DOI
StatusUdgivet - 2015

Emneord

  • complement activation herapeutic antibody post-translational control ADCC antibody-dependent cell-mediated cytotoxicity CDC complement-dependent cytotoxicity CEX cation-exchange cIEF capillary isoelectric focusing CPB carboxy peptidase B ESI-MS electrospray ionization mass spectrometry IEF isoelectric focusing SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis HUMAN MONOCLONAL-ANTIBODY MAMMALIAN-CELL CULTURE MASS-SPECTROMETRY IN-VIVO THERAPEUTIC ANTIBODIES CHARGE VARIANTS BINDING-SITE RECOMBINANT PROTEINS MOLECULE

Citer dette

van den Bremer, E. T. J., Beurskens, F. J., Voorhorst, M., Engelberts, P. J., de Jong, R. N., van der Boom, B. G., ... Parren, P. W. H. I. (2015). Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation. mAbs, 7(4), 672-680. https://doi.org/10.1080/19420862.2015.1046665
van den Bremer, E. T. J. ; Beurskens, F. J. ; Voorhorst, M. ; Engelberts, P. J. ; de Jong, R. N. ; van der Boom, B. G. ; Cook, E. M. ; Lindorfer, M. A. ; Taylor, R. P. ; van Berkel, P. H. C. ; Parren, Paul W.H.I. / Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation. I: mAbs. 2015 ; Bind 7, Nr. 4. s. 672-680.
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title = "Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation",
abstract = "Human IgG is produced with C-terminal lysines that are cleaved off in circulation. The function of this modification was unknown and generally thought not to affect antibody function. We recently reported that efficient C1q binding and complement-dependent cytotoxicity (CDC) requires IgG hexamerization at the cell surface. Here we demonstrate that C-terminal lysines may interfere with this process, leading to suboptimal C1q binding and CDC of cells opsonized with C-terminal lysine-containing IgG. After we removed these lysines with a carboxypeptidase, maximal complement activation was observed. Interestingly, IgG1 mutants containing either a negative C-terminal charge or multiple positive charges lost CDC almost completely; however, CDC was fully restored by mixing C-terminal mutants of opposite charge. Our data indicate a novel post-translational control mechanism of human IgG: human IgG molecules are produced in a pro-form in which charged C-termini interfere with IgG hexamer formation, C1q binding and CDC. To allow maximal complement activation, C-terminal lysine processing is required to release the antibody's full cytotoxic potential.",
keywords = "complement activation herapeutic antibody post-translational control ADCC antibody-dependent cell-mediated cytotoxicity CDC complement-dependent cytotoxicity CEX cation-exchange cIEF capillary isoelectric focusing CPB carboxy peptidase B ESI-MS electrospray ionization mass spectrometry IEF isoelectric focusing SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis HUMAN MONOCLONAL-ANTIBODY MAMMALIAN-CELL CULTURE MASS-SPECTROMETRY IN-VIVO THERAPEUTIC ANTIBODIES CHARGE VARIANTS BINDING-SITE RECOMBINANT PROTEINS MOLECULE",
author = "{van den Bremer}, {E. T. J.} and Beurskens, {F. J.} and M. Voorhorst and Engelberts, {P. J.} and {de Jong}, {R. N.} and {van der Boom}, {B. G.} and Cook, {E. M.} and Lindorfer, {M. A.} and Taylor, {R. P.} and {van Berkel}, {P. H. C.} and Parren, {Paul W.H.I.}",
note = "Genmab 1 26037225",
year = "2015",
doi = "10.1080/19420862.2015.1046665",
language = "English",
volume = "7",
pages = "672--680",
journal = "mAbs",
issn = "1942-0862",
publisher = "Taylor & Francis",
number = "4",

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van den Bremer, ETJ, Beurskens, FJ, Voorhorst, M, Engelberts, PJ, de Jong, RN, van der Boom, BG, Cook, EM, Lindorfer, MA, Taylor, RP, van Berkel, PHC & Parren, PWHI 2015, 'Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation', mAbs, bind 7, nr. 4, s. 672-680. https://doi.org/10.1080/19420862.2015.1046665

Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation. / van den Bremer, E. T. J.; Beurskens, F. J.; Voorhorst, M.; Engelberts, P. J.; de Jong, R. N.; van der Boom, B. G.; Cook, E. M.; Lindorfer, M. A.; Taylor, R. P.; van Berkel, P. H. C.; Parren, Paul W.H.I.

I: mAbs, Bind 7, Nr. 4, 2015, s. 672-680.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation

AU - van den Bremer, E. T. J.

AU - Beurskens, F. J.

AU - Voorhorst, M.

AU - Engelberts, P. J.

AU - de Jong, R. N.

AU - van der Boom, B. G.

AU - Cook, E. M.

AU - Lindorfer, M. A.

AU - Taylor, R. P.

AU - van Berkel, P. H. C.

AU - Parren, Paul W.H.I.

N1 - Genmab 1 26037225

PY - 2015

Y1 - 2015

N2 - Human IgG is produced with C-terminal lysines that are cleaved off in circulation. The function of this modification was unknown and generally thought not to affect antibody function. We recently reported that efficient C1q binding and complement-dependent cytotoxicity (CDC) requires IgG hexamerization at the cell surface. Here we demonstrate that C-terminal lysines may interfere with this process, leading to suboptimal C1q binding and CDC of cells opsonized with C-terminal lysine-containing IgG. After we removed these lysines with a carboxypeptidase, maximal complement activation was observed. Interestingly, IgG1 mutants containing either a negative C-terminal charge or multiple positive charges lost CDC almost completely; however, CDC was fully restored by mixing C-terminal mutants of opposite charge. Our data indicate a novel post-translational control mechanism of human IgG: human IgG molecules are produced in a pro-form in which charged C-termini interfere with IgG hexamer formation, C1q binding and CDC. To allow maximal complement activation, C-terminal lysine processing is required to release the antibody's full cytotoxic potential.

AB - Human IgG is produced with C-terminal lysines that are cleaved off in circulation. The function of this modification was unknown and generally thought not to affect antibody function. We recently reported that efficient C1q binding and complement-dependent cytotoxicity (CDC) requires IgG hexamerization at the cell surface. Here we demonstrate that C-terminal lysines may interfere with this process, leading to suboptimal C1q binding and CDC of cells opsonized with C-terminal lysine-containing IgG. After we removed these lysines with a carboxypeptidase, maximal complement activation was observed. Interestingly, IgG1 mutants containing either a negative C-terminal charge or multiple positive charges lost CDC almost completely; however, CDC was fully restored by mixing C-terminal mutants of opposite charge. Our data indicate a novel post-translational control mechanism of human IgG: human IgG molecules are produced in a pro-form in which charged C-termini interfere with IgG hexamer formation, C1q binding and CDC. To allow maximal complement activation, C-terminal lysine processing is required to release the antibody's full cytotoxic potential.

KW - complement activation herapeutic antibody post-translational control ADCC antibody-dependent cell-mediated cytotoxicity CDC complement-dependent cytotoxicity CEX cation-exchange cIEF capillary isoelectric focusing CPB carboxy peptidase B ESI-MS electrospr

U2 - 10.1080/19420862.2015.1046665

DO - 10.1080/19420862.2015.1046665

M3 - Journal article

VL - 7

SP - 672

EP - 680

JO - mAbs

JF - mAbs

SN - 1942-0862

IS - 4

ER -

van den Bremer ETJ, Beurskens FJ, Voorhorst M, Engelberts PJ, de Jong RN, van der Boom BG et al. Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation. mAbs. 2015;7(4):672-680. https://doi.org/10.1080/19420862.2015.1046665