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J. Biol. Chem., Vol. 283, Issue 23, 16206-16215, June 6, 2008

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Structural and Functional Characterization of Disulfide Isoforms of the Human IgG2 Subclass^*

Thomas M. Dillon¹, Margaret Speed Ricci, Chris Vezina, Gregory C. Flynn^¶, Yaoqing Diana Liu^¶, Douglas S. Rehder, Matthew Plant, Brad Henkle, Yu Li, Songpon Deechongkit, Brian Varnum, Jette Wypych^¶, Alain Balland^¶, and Pavel V. Bondarenko²

From the Departments of Pharmaceutics, Inflammation Research, and ^¶Analytical Sciences, Amgen Inc., Thousand Oaks, California 91320

In the accompanying report ( Wypych, J., Li, M., Guo, A., Zhang, Z., Martinez, T., Allen, M. J., Fodor, S., Kelner, D. N., Flynn, G. C., Liu, Y. D., Bondarenko, P. V., Ricci, M. S., Dillon, T. M., and Balland, A. (2008) J. Biol. Chem. 283, 16194-16205[Abstract/Free Full Text] ), we have identified that the human IgG2 subclass exists as an ensemble of distinct isoforms, designated IgG2-A, -B, and -A/B, which differ by the disulfide connectivity at the hinge region. In this report, we studied the structural and functional properties of the IgG2 disulfide isoforms and compared them to IgG1. Human monoclonal IgG1 and IgG2 antibodies were designed with identical antigen binding regions, specific to interleukin-1 cell surface receptor type 1. In vitro biological activity measurements showed an increased activity of the IgG1 relative to the IgG2 in blocking interleukin-1β ligand from binding to the receptor, suggesting that some of the IgG2 isoforms had lower activity. Under reduction-oxidation conditions, the IgG2 disulfide isoforms converted to IgG2-A when 1 M guanidine was used, whereas IgG2-B was enriched in the absence of guanidine. The relative potency of the antibodies in cell-based assays was: IgG1 > IgG2-A > IgG2 >> IgG2-B. This difference correlated with an increased hydrodynamic radius of IgG2-A relative to IgG2-B, as shown by biophysical characterization. The enrichment of disulfide isoforms and activity studies were extended to additional IgG2 monoclonal antibodies with various antigen targets. All IgG2 antibodies displayed the same disulfide conversion, but only a subset showed activity differences between IgG2-A and IgG2-B. Additionally, the distribution of isoforms was influenced by the light chain type, with IgG2 composed mostly of IgG2-A. Based on crystal structure analysis, we propose that IgG2 disulfide exchange is caused by the close proximity of several cysteine residues at the hinge and the reactivity of tandem cysteines within the hinge. Furthermore, the IgG2 isoforms were shown to interconvert in whole blood or a "blood-like" environment, thereby suggesting that the in vivo activity of human IgG2 may be dependent on the distribution of isoforms.

Received for publication, December 7, 2007 , and in revised form, February 22, 2008.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S11, Table S1, and additional text and references.

¹ To whom correspondence may be addressed: Tel.: 805-447-8011; Fax: 805-447-3259; E-mail: Tdillon{at}Amgen.com.

² To whom correspondence may be addressed: Tel.: 805-447-7215; Fax: 805-447-3259; E-mail: Pavel.Bondarenko{at}Amgen.com.

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