Activation of complement by an IgG molecule without a genetic hinge

Abstract

The hinge region links the two Fab arms to the Fc portion of the IgG molecule. It mediates flexibility to the molecule and serves as a connecting structure between the two heavy chains. In addition it provides space between the Fab and Fc parts. All three properties have been proposed to be important for the ability of IgG to initiate complement activation leading to complement-mediated cell lysis (CML). Here we report the construction of a hinge-deleted mouse-human chimaeric IgG3 molecule with specificity for the hapten NIP (3-iodo-4-hydroxy-5-nitrophenacetyl), HM-1. HM-1 lacks the genetic hinge, but has an introduced cysteine between Ala 231 (EU numbering) and Pro 232 in the lower hinge encoded by the CH2 exon. The introduced cysteine forms a disulphide bond between the two heavy chains of the molecule. In CML, HM-1 shows a greater activity than IgG3 wild type. This is the first time an IgG molecule without a genetic hinge has been found to be active in CML. We conclude that the hinge functioning as a spacer is not a prerequisite for complement activation. Rather, its major role seems to be to connect the heavy chains to each other in the amino-terminal part of CH2. Because HM-1 is expected to have low Fab-Fc flexibility, this molecular feature is probably of no importance for complement activation.

Modulation of the Effector Functions of a Human IgG1 through Engineering of Its Hinge Region

“…For instance, conflicting results exist whether at least a portion of the hinge region is needed for complement-dependent cytotoxicity (CDC). 2 Although some studies have suggested that a "spacer" effect between the IgG's Fab and Fc regions is essential for binding of the Fc to the first component of complement activation C1q or to trigger complement mediated cytolysis (1-3), one report describing an IgG whose hinge was partially deleted has disputed this premise (4). Likewise, the exact structural requirements of the hinge region which determine the different aspects of IgG effector functions are still unclear.…”

One disulfide bond in front of the second heavy chain constant region is necessary and sufficient for effector functions of human IgG3 without a genetic hinge.

“…An IgG3 mutant with an IgG4 hinge was also very active in complement-mediated lysis (CML) (14). An IgG3 mutant, mO, without a genetic hinge does not induce CML (12), and IgG1 myeloma proteins without a hinge do not activate complement (15,16), whereas a mutant, mO/ 231C232 (formerly HM-1), without a genetic hinge but with a disulfide bond in front of the second heavy chain constant region (CH2), is active in CML (17). We wanted to do a detailed analysis of the hinge or hinge-related structural requirements for effector functions based on the four mutants mO, mO/C131S, mO/231C232, and mO/C131S/231C232, all without a genetic hinge, and to test these mutants for CML, antibody-dependent cell-mediated cytotoxicity (ADCC), and phagocytosis/respiratory burst.…”

“…mO/231C232 and m0/ C131S/231C232 were positive, whereas mO and mO/C131S were negative for effector functions. (12), and the mutant m0/231C232 originated from mO by inserting a Cys between Ala-231 and Pro-232 (18) as described (17). Schematic structures of the mutants are shown in Fig.…”

A mutant human IgG molecule with only one C1q binding site can activate complement and induce lysis of target cells

“…This underscores a critical role for the hinge region in the positioning of the Fab regions as well as C H 2, and it is suggested that the hinge has a role in limiting C1q recognition. Notably, an IgG3 mutant in which the complete genetic hinge region of 47 amino acids was replaced by a stretch of 3 alanines followed by a cysteine had a disulfide bond between heavy chains and showed no reduction in ADCML activity [28], emphasizing the importance of the disulfide bridges.…”

“…It is conceivable that wild-type human IgG also involve or expose only one C1q binding site for activation of the classical complement pathway, as they do for binding to FccR. The interaction site for C1q on IgG is located to the upper part of the C H 2 domain, near the hinge region where the two heavy chains are covalently linked by disulfide bonds [7,8,25,26], and these disulfide bonds must be intact [27][28][29][30]. Epitope density on the target cell surface, testable in our system [31], is also important for complement activation, underscored by the fact that human IgG2 is unable to activate complement at low epitope density, while it does so at high epitope density [32,33].…”