Impact of Molecular Processing in the Hinge Region of Therapeutic IgG4 Antibodies on Disposition Profiles in Cynomolgus Monkeys

Abstract

ABSTRACT:The IgG4 isotype antibody is a potential candidate for immunotherapy when reduced effector functions are desirable. However, antigen binding fragment (Fab) arm exchange leads to functional monovalency with potentially reduced therapeutic efficacy. Mutagenesis studies suggested that the CH3 domain and not the core hinge is dominantly involved in in vivo molecular processing. This work investigated whether stabilization of the core hinge of a therapeutic IgG4 antibody by mutation of Ser228 to Pro ( Since the first approval of a therapeutic monoclonal antibody in 1986, more than 20 therapeutic antibodies were approved in the United States, and over 200 therapeutic monoclonal antibodies (mAb) were undergoing clinical evaluation in (Reichert, 2008. Therapeutic mAbs have become an increasingly important class of therapeutic compounds in a variety of diseases. A number of technologies have been successfully used to engineer mAbs (Lonberg, 2008). Of the 21 approved therapeutic mAbs, the majority (15) are of the IgG1 isotype, whereas three mAbs are IgG2 antibodies and three are IgG4 or hybrid IgG2/4 antibodies (Reichert, 2008).

Species-Specific Determinants in the IgG CH3 Domain Enable Fab-Arm Exchange by Affecting the Noncovalent CH3–CH3 Interaction Strength

“…Therefore, mutations that completely prevent Fab-arm exchange in vivo should be considered when designing therapeutic IgG4 Abs. We and others have previously shown that a S228P mutation in the IgG4 corehinge represents a tool for preventing Fab-arm exchange to less than detection limits (8,9,15). The mutations in the CH3 domain identified in this article, R409K, R409L, R409M, K370E, and K370Q, may be used for additional stabilization or as an alternative approach to hinge stabilization.…”

“…3). Previous studies suggested that IgG from rhesus monkeys (Macaca mulatta) (6), cynomolgus monkeys (Macaca fascicularis) (8,9), and IgG3 from mice (Mus musculus) (8) could engage in Fab-arm exchange with human IgG4. In accordance, we now demonstrate that a variety of other species share this ability.…”

“…In accordance, mutating the core-hinge sequence (S228P) inhibited Fab-arm exchange to undetectable levels in vivo (8,9,15), whereas the molecular determinants in the CH3 enabling Fab-arm exchange remain to be elucidated. In this article, we identify R409 as the crucial CH3 residue governing Fab-arm exchange in human IgG4.…”

“…Furthermore, through Fab-arm exchange, natural IgG4 Abs acquire the ability to cross-link two different Ags (heterologous cross-linking), making them bispecific (5). More recent studies suggest that IgG subclasses from other mammalian species share this ability (6,(8)(9)(10), making it a mechanism that could impact humoral immunity more generally.…”

Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies

“…A large variety of formats for bispecific antibodies has been described (1-3). The most prominent examples are tetravalent IgG-single-chain variable fragment (scFv) fusions (4-6), catumaxomab, a trifunctional rat/mouse hybrid bispecific epithelial cell adhesion molecule-CD3 antibody (7), the bispecific CD19-CD3 scFv antibody blinatumomab (8), "dualacting Fab" (DAF) antibodies (9), tetravalent bispecific formats such as the IgG-like dual-variable-domain antibodies (DVD-Ig) (10), covalently linked pharmacophore peptides to catalytic antibodies (11), or use of the dynamic exchange between half IgG4 molecules to generate bispecific antibodies (12,13). Each of these approaches has advantages but also has limitations such as immunogenicity, poor pharmacokinetic properties, or loss of effector functions caused by the lack of a fragment crystallizable (Fc) region; also, they may tend to aggregate because of the presence of linkers or may contain potentially immunogenic nonhuman domains.…”