Disulfide Connectivity of Human Immunoglobulin G2 Structural Isoforms Theresa Martinez, ‡ Amy Guo, ‡ Martin J. Allen, § Mei Han, ‡ Danielle Pace, ‡ Jay Jones, ‡,| Ron Gillespie, ⊥ Randal R. Ketchem, # Yuling Zhang, ‡ and Alain Balland* ,‡ Process and Product DeVelopment, Amgen, Inc., Seattle, Washington 98119 ReceiVed April 2, 2008; ReVised Manuscript ReceiVed May 13, 2008 ABSTRACT: In this communication we present the detailed disulfide structure of IgG2 molecules. The consensus structural model of human IgGs represents the hinge region positioned as a flexible linker connecting structurally isolated Fc and Fab domains. IgG2 molecules are organized differently from that model and exhibit multiple structural isoforms composed of (heavy chain-light chain-hinge) covalent complexes. We describe the precise connection of all the disulfide bridges and show that the IgG2 C H 1 and C-terminal C L cysteine residues are either linked to each other or to the two upper hinge cysteine residues specific to the IgG2 subclass. A defined arrangement of these disulfide bridges is unique to each isoform. Mutation of a single cysteine residue in the hinge region eliminates these natural complexes. These results show that IgG2 structure is significantly different from the conventionally accepted immunoglobulin structural model and may help to explain some of the unique biological activity attributed only to this subclass. The three-dimensional structures of the Fab and Fc domains of IgG molecules have been solved through numer- ous studies by X-ray diffraction studies of crystal fragments (1, 2). The overall structure of the IgG family is organized in 12 immunoglobulin domains each closed by an intrachain disulfide bond (reviewed in refs 3 and 4). Fab and Fc regions are connected by the hinge, an area important for the flexibility of the molecule and its adaptability to various presentations of antigen targets. The hinge is divided into three regions: upper, core, and lower. The core cysteines are involved in inter-heavy chain disulfide bonding responsible for the covalent complex of the form (HC-LC) 2 . 1 Sequence comparison indicates that immunoglobulin subclasses exhibit differences in the genetic hinge region in the length and number of cysteine residues. IgG1 (15 residues) and IgG2 and IgG4 (12 residues each) are relatively comparable in size whereas, due to domain duplication, IgG3 is unique with a long hinge of 62 amino acids including 11 cysteines. IgG1 and IgG4 hinge core sequences are very similar with 2 cysteines on each heavy chain involved in inter-heavy chain connection. Specific residues of the hinge region have been shown to have profound impact on the properties of immunoglobulins. For example, the presence of serine in the canonical core structure, CPXCP, explains the unique property of IgG4 molecules to form HC-LC half-structures (5). A recent report has shown that this property allows a dynamic exchange of the hinge disulfides in ViVo resulting in bispecific IgG4 antibodies (6). IgG2 is unique in presenting 4 cysteine residues in the hinge region, notably two consecu- tive residues, Cys-232 and Cys-233 (Kabat numbering (7)) that have no equivalent in any other immunoglobulin subclass. We show in this report that these residues confer distinctive structural features to the IgG2 subclass. The IgG1 covalent structure is schematically presented in Figure 1A. Resolving intact antibody structures by X-ray diffraction analysis has proved difficult, and only three structures of complete antibodies with hinge are known to date (8–10). In contrast to human IgG1, detailed structural analysis of human IgG2 is very limited, and there are no published human IgG2 structures that include the hinge region. Most information about the hinge of this subclass is derived by homology modeling using human IgG1 and murine IgG1 and IgG2. Based on known X-ray structures of murine antibodies that show the three cysteine residues of the murine hinge region to be involved in inter-heavy chain disulfide bridges (9), a general organization of human IgG2 has been derived as schematically shown in Figure 1B. This consensus model is supported only by limited bio- chemical studies of enzymatic digests of myeloma protein (11). These studies describe the disulfide connection of the cysteine residues in the core region organized in parallel. We recently reported that studies of the covalent structure of IgG2 molecules performed at Amgen indicate that the consensus structural model of human IgG2 is only a minor form, among several disulfide-related structural isoforms (12, 13). In this communication, we investigated the precise cysteine connectivity of each isoform. The experiments were per- formed on a recombinant IgG2 molecule produced at large manufacturing scale and led to a complete description of the * Corresponding author: tel, (206) 265-8603; fax, (206) 217-4692; e-mail, ballanda@amgen.com. ‡ Department of Analytical and Formulation Sciences. § Department of Cell Sciences and Technology. | Present address: Seattle Genetics, 21823 30th Drive SE, Bothell, WA. ⊥ Department of Purification Process Development. # Department of Protein Sciences. 1 Abbreviations: mAb, monoclonal antibody; HC, heavy chain; LC, light chain; RP, reversed phase; LC-MS, liquid chromatography-mass spectrometry; nrCE-SDS, nonreduced capillary electrophoresis sodium dodecyl sulfate; dSEC, denaturing size exclusion chromatography; CEX, cation-exchange chromatography; IAA, iodoacetic acid; NEM, N- ethylmaleimide; Gal, galactose. Biochemistry 2008, 47, 7496–7508 7496 10.1021/bi800576c CCC: $40.75  2008 American Chemical Society Published on Web 06/13/2008