A single proteolytic cleavage within the lower hinge of trastuzumab reduces immune effector function and in vivo efficacy

Abstract

IntroductionRecent studies reported that human IgG antibodies are susceptible to specific proteolytic cleavage in their lower hinge region, and the hinge cleavage results in a loss of Fc-mediated effector functions. Trastuzumab is a humanized IgG1 therapeutic monoclonal antibody for the treatment of HER2-overexpressing breast cancers, and its mechanisms of action consist of inhibition of HER2 signaling and Fc-mediated antibody-dependent cellular cytotoxicity (ADCC). The objective of this study is to investigate the potential effect of proteinase hinge cleavage on the efficacy of trastuzumab using both a breast cancer cell culture method and an in vivo mouse xenograft tumor model.MethodsTrastuzumab antibody was incubated with a panel of human matrix metalloproteinases, and proteolytic cleavage in the lower hinge region was detected using both western blotting and mass spectrometry. Single hinge cleaved trastuzumab (scIgG-T) was purified and evaluated for its ability to mediate ADCC and inhibition of breast cancer cell proliferation in vitro as well as anti-tumor efficacy in the mouse xenograft tumor model. Infiltrated immune cells were detected in tumor tissues by immunohistochemistry.ResultsscIgG-T retains HER2 antigen binding activity and inhibits HER2-mediated downstream signaling and cell proliferation in vitro when compared with the intact trastuzumab. However, scIgG-T lost Fc-mediated ADCC activity in vitro, and had significantly reduced anti-tumor efficacy in a mouse xenograft tumor model. Immunohistochemistry showed reduced immune cell infiltration in tumor tissues treated with scIgG-T when compared with those treated with the intact trastuzumab, which is consistent with the decreased ADCC mediated by scIgG-T in vitro.ConclusionTrastuzumab can be cleaved by matrix metalloproteinases within the lower hinge. scIgG-T exhibited a significantly reduced anti-tumor efficacy in vivo due to the weakened immune effector function such as ADCC. The results suggest that the lower hinge cleavage of trastuzumab can occur in the tumor microenvironment where matrix metalloproteinases often have high levels of expression and scIgG-T might compromise its anti-tumor efficacy in the clinic. However, further studies are needed to validate these hypotheses in the clinical setting.

Proteinase-nicked IgGs: an unanticipated target for tumor immunotherapy

“…38 However, mice treated with single-cleaved trastuzumab had markedly inhibited macrophage recruitment to HER2 overexpressing xenografts as compared to mice treated with intact trastuzumab. 37 Similarly, decreased recruitment of macrophages was observed in human tumors when host antibodies exhibited evidence of proteinolytic cleavage. 38 Thus, not only does proteinolytic hinge cleavage of an anti-tumor mAb prevent effector functions such as ADCC and CDC while simultaneously blocking competent mAb binding (below), it can also further inhibit the arrival of effective immune clearance cells.…”

“…31 The anti-HER2 mAb, trastuzumab, was found to be substantially impaired compared to the intact mAb in blocking the growth of a human xenograft tumor in mice. 36,37 The proteinolytic impairment of Fc-mediated cell clearance was thus replicated using multiple human mAbs in the immune effector systems in animal models. 36,37 …”

“…36,37 The proteinolytic impairment of Fc-mediated cell clearance was thus replicated using multiple human mAbs in the immune effector systems in animal models. 36,37 …”

“…Low expressing cells did not yield trastuzumab cleavage and none was detected in the media. 37 Confirmation of a preferential accumulation of cell surface scIgG (rather than F(ab′) 2 ) was made when cell-bound mAbs were exposed to endogenous and exogenous proteinases or when the cells were engineered to express the IgG cleaving enzyme, IdeS. 31,36–38,41 These findings indicated that an initially more rapid first and then slower second chain cleavage of the IgG hinge, previously noted in solution at low enzyme to IgG ratios, was even more exaggerated with cell surface-bound IgGs.…”

“…31,36–38,41 Important in vivo correlations were provided by demonstrations that clinically administered trastuzumab was detectable as scIgG-T in tumor extracts from patients with breast cancer or by IHC in biopsy samples. 36,37 …”