Intrachain disulfide bond in the core hinge region of human IgG4

Abstract

IgG is a tetrameric protein composed of two copies each of the light and heavy chains. The four-chain structure is maintained by strong noncovalent interactions between the amino-terminal half of pairs of heavy-light chains and between the carboxyl-terminal regions of the two heavy chains. In addition, interchain disulfide bonds link each heavy-light chain and also link the paired heavy chains. An engineered human IgG4 specific for human tumor necrosis factor-cy (CDP571) is similar to human myeloma IgG4 in that it is secreted as both disulfide bonded tetramers (approximately 75% of the total amount of IgG) and as tetramers composed of nondisulfide bonded half-IgG4 (heavy chain disulfide bonded to light chain) molecules. However, when CDP571 was genetically engineered with a proline at residue 229 of the core hinge region rather than serine, CDP57 I(S229P), or with an IgGl rather than IgG4 hinge region, CDP571(yl), only trace amounts of nondisulfide bonded half-lgG tetramers were observed. Trypsin digest reversephase HPLC peptide mapping studies of CDP57 1 and CDP57 1 (y I ) with on-line electrospray ionization mass spectroscopy supplemented with Edman sequencing identified the chemical factor preventing inter-heavy chain disulfide bond formation between half-IgG molecules: the two cysteines in the IgG4 and IgGl core hinge region KPSCP and GPPCP, respectively) are capable of forming an intrachain disulfide bond. Conformational modeling studies on cyclic disulfide bonded CPSCP and CPPCP peptides yielded energy ranges for the low-energy conformations of 31-33 kcallmol and 40-42 kcal/mol, respectively. In addition, higher torsion and angle bending energies were observed for the CPPCP peptide due to backbone constraints caused by the extra proline. These modeling results suggest a reason why a larger fraction of intrachain bonds are observed in IgG4 rather than IgGl molecules: the serine in the core hinge region of IgG4 allows more hinge region flexibility than the proline of IgGl and thus may permit formation of a stable intrachain disulfide bond more readily.

Engineered anti-CD70 antibody-drug conjugate with increased therapeutic index

“…Under nonreducing conditions, all IgG variants electrophoresed as a single major band, as anticipated. The IgG4 variant gave rise to an additional minor band, consistent with the presence of ''half-IgG,'' as reported for other recombinant IgG4 and attributed to inefficient interheavy chain disulfide bond formation (23,24). Mutation of the IgG4 hinge sequence, CPSC, to mimic the IgG1 hinge, CPPC, creates a strong preference for interheavy over intraheavy chain disulfide bonding (23 -25).…”

Antibody C Region Influences TGN1412-like Functional Activity In Vitro

“…Interactions in both the IgG4 core hinge sequence and the IgG4 CH3 domain are critical for this process to occur (31,32). Substitution of serine with proline at position 228, as found in IgG1 and IgG2 core hinge sequences (i.e., CPSCP to CPPCP) stabilized the inter-H chain disulfide linkage in NIB(28SA)-G4-S228P with the resulting loss of half-molecules after SDS-PAGE, as shown for other IgG4 mAbs with "wild-type" hinges (10,16,33,34). Exchange of the IgG4 for the IgG1 core hinge was previously shown to abrogate Fab arm-exchange in vitro and in vivo (10,32).…”

“…The greater superagonistic activity of NIB(28SA)-G4-S228P in cocultures compared with the IgG4 molecules with wild-type hinges may thus be explained, at least in part, by its inability to engage in Fab arm-exchange. Conformational modeling has indicated that proline in the core hinge region allows less torsional flexibility than serine, eliminating the potential for intrachain disulfide bridge formation between the two hinge cysteines at positions 226 and 229 (33). As immobilized IgG4 mAb could not have engaged in Fab arm-exchange, the effects of restricted hinge flexibility may also have enhanced the in vitro functional activity of NIB(28SA)-G4-S228P in both functional assay formats, potentially through optimizing and stabilizing CD28 ligation and subsequent superagonistic signaling.…”

Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange

“…Thus, whereas replacing either of these residues in IgG4 by their IgG1 counterpart (i.e., P228 or K409) blocked FAE both in vitro and in vivo, introducing both residues enabled IgG1 molecules to engage in FAE (21). Because breaking the covalent linkage between half-molecules is a prerequisite for FAE, residue S228 (opposed to P228) is thought to act by making the interheavy chain disulfide bonds more susceptible to reducing conditions (22). Dissociation of the noncovalently associated CH3 domains has been shown to be the rate-limiting step in the FAE reaction (23) and heavily dependent on the composition of the CH3-CH3 interface residues (24).…”