A first cleavage of one heavy chain (HC) generates single-cleaved IgG (sc-IgG), which retains its whole structure via CH3-CH3 and glycan weak interactions ( 9). Previous studies have shown that in addition to the papain and pepsin sensitivity of the hinge region ( 4, 5), the lower hinge region of IgG1 is cleaved by tumor-, inflammatory-, and/or infectious-associated proteases such as matrix metalloproteinases (MMPs) or immunoglobulin-degrading enzyme from Streptococcus pyogenes (IdeS) ( 6– 11). The binding sites of C1q and FcγR on human IgG are relatively close and are partially located in the lower hinge region (defined by the sequence 233PAPELLGGP 241 in IgG1) ( 1– 3). Conversely, the IgG2 or IgG4 format, which weakly binds C1q and FcγR, is usually favored when developing neutralizing/antagonist TmAbs. Therefore, this subclass is used to develop cytolytic mAbs. The IgG1 format efficiently binds C1q and FcγRs and triggers complement-dependent cytotoxicity or antibody-dependent cell-mediated cytotoxicity (ADCC) ( 1, 2). The choice among the three subclasses during the design and development of an mAb is mainly directed by its expected mechanism of action. Therapeutic monoclonal antibodies (TmAbs), which are extensively used for treatment in cancer or chronic inflammatory diseases, are based on a human IgG1, IgG2, or IgG4 structure. Overall, our results indicate that the cleavage of the hinge region should be considered with TmAbs treatment and in the development of new molecules. Finally, with ELISA and flow cytometry, we observed that a single cleavage of IgG1 TmAbs greatly decreased their affinity for FcγRIIIA and C1q and their ability to induce FcγRIIIA-dependent functional responses of NK cells. We propose that the variability in the cleavage sensitivity/resistance balance among TmAbs of IgG1 and IgG4 subclasses results partially, from TmAb characteristics related to and/or located in the Fab region. Importantly, the cleavage kinetic of ipilimumab Fc fragment by IdeS was superimposable to that of trastuzumab, cetuximab and infliximab Fc fragment, showing that the variability observed for intact ipilimumab is unrelated to its Fc portion. In addition the Fc fragment of IgG1 TmAbs were highly resistant to cleavage by MMP12, whereas their cleavage kinetic by IdeS was very similar to that observed with the intact forms (excluding ipilimumab). Ipilimumab was more IdeS-sensitive and MMP12-resistant than the other IgG1 TmAbs, regardless of G1m allotype. Nivolumab and pembrolizumab were cleaved similarly by MMP12, whereas pembrolizumab was more IdeS-resistant. However, we observed intra-subclass variability among IgG4 and IgG1 TmAbs. The latter were usually more protease-sensitive, whereas IgG1 TmAbs were usually cleaved with intermediate kinetics. Panitumumab was more protease-resistant than IgG1 and IgG4 TmAbs. Therefore, we used non-reducing SDS-PAGE to compare the cleavage kinetics of five IgG1 TmAbs (trastuzumab, rituximab, cetuximab, infliximab, ipilimumab), one IgG2 TmAb (panitumumab), and two IgG4 TmAbs (nivolumab and pembrolizumab) by MMP12 and IdeS, which were found to cleave the first and second HCs with different kinetics. The cleavage of therapeutic monoclonal antibodies (TmAbs), which are based on a human IgG1, IgG2 or IgG4 structure, has been poorly investigated, although it may represent an escape mechanism to these treatments. Both heavy chains (HCs) of the hinge region can be cleaved sequentially by several proteases of the tumor/inflammatory/infectious microenvironment, including matrix metalloproteinase 12 (MMP12), or immunoglobulin-degrading enzyme from Streptococcus pyogenes (IdeS), impairing Fc-mediated functions. The hinge region of immunoglobulin G (IgG) is involved in C1q and FcγRIIIA-expressing natural killer (NK) cell recruitment. ![]()
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