Proteolytic single hinge cleavage of pertuzumab impairs its Fc effector function and antitumor activity in vitro and in vivo

Abstract

BackgroundProteolytic impairment of the Fc effector functions of therapeutic monoclonal antibodies (mAbs) can compromise their antitumor efficacy in the tumor microenvironment and may represent an unappreciated mechanism of host immune evasion. Pertuzumab is a human epidermal growth factor receptor 2 (HER2)-targeting antibody and has been widely used in the clinic in combination with trastuzumab for treatment of HER2-overexpressing breast cancer. Pertuzumab susceptibility to proteolytic hinge cleavage and its impact on the drug’s efficacy has not been previously studied.MethodsPertuzumab was incubated with high and low HER2-expressing cancer cells and proteolytic cleavage in the lower hinge region was detected by western blotting. The single hinge cleaved pertuzumab (scIgG-P) was purified and evaluated for its ability to mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro and anti-tumor efficacy in vivo. To assess the cleavage of trastuzumab (IgG-T) and pertuzumab (IgG-P) when simultaneously bound to the same cancer cell surface, F(ab’)2 fragments of IgG-T or IgG-P were combined with the intact IgG-P and IgG-T, respectively, to detect scIgG generation by western blotting.ResultsPertuzumab hinge cleavage occurred when the mAb was incubated with high HER2-expressing cancer cells. The hinge cleavage of pertuzumab caused a substantial loss of ADCC in vitro and reduced antitumor efficacy in vivo. The reduced ADCC function of scIgG-P was restored by an anti-hinge mAb specific for a cleavage site neoepitope. In addition, we constructed a protease-resistant version of the anti-hinge mAb that restored ADCC and the cell-killing functions of pertuzumab when cancer cells exressed a potent IgG hinge-cleaving protease. We also observed increased hinge cleavage of pertuzumab when combined with trastuzumab.ConclusionThe reduced Fc effector function of single hinge-cleaved pertuzumab can be restored by an anti-hinge mAb. The restoration effect indicated that immune function could be readily augmented when the damaged primary antibodies were bound to cancer cell surfaces. The anti-hinge mAb also restored Fc effector function to the mixture of proteolytically disabled trastuzumab and pertuzumab, suggesting a general therapeutic strategy to restore the immune effector function to protease-inactivated anticancer antibodies in the tumor microenvironment. The findings point to a novel tactic for developing breast cancer immunotherapy.

The Proteolytic Cleavage of Therapeutic Monoclonal Antibody Hinge Region: More Than a Matter of Subclass

“…The functional consequences of the proteolytic cleavage have been broadly studied with mAbs including TmAbs such as trastuzumab or pertuzumab, in in vitro and in vivo models (12,13). We explored the ability of the eight TmAbs to bind to the membrane form of FcγRIIIA.…”

“…Therefore, it may be assumed that the reduced FcγRIIIAdependent functions mediated by NK cells reported herein with IgG1 sc-TmAbs, probably extend to FcγRIIIA-and FcγRIIAdependent functions mediated by myeloid cells. This assumption is sustained by the fact that peripheral blood mononuclear cells (instead of NK cells) were used as effector cells, in the previous studies showing the reduced lysis mediated by sc-TmAbs compared with intact TmAbs (12,13). Further studies are warrantied to evaluate specifically the impact of the cleavage on FcγRIIA-mediated function such as ADCP.…”

“…The impact of the cleavage in TmAbs-based therapies is presently unknown. The cleavage of trastuzumab or pertuzumab has been associated with a weakened therapeutic effect in mouse xenograft tumor model (12,13). In humans, increased tumor sc-IgGs were found to be associated with poor breast cancer patient outcomes (35) and high levels of MMP3-/MMP12cleaved IgG were detected in sera from inflammatory bowel diseases patients, who did not respond to anti-TNFα therapy (10).…”

“…The cleavage of the second HC is usually slower and generates F(ab') 2 , which obviously loses the Fc-dependent interactions. The cleavage of trastuzumab or pertuzumab (anti-HER2, used in breast cancer therapy) impairs ADCC and has been associated with a weakened therapeutic effect in a mouse xenograft tumor model (12,13). Furthermore, high levels of cleaved IgG were detected in serum from individuals with inflammatory bowel diseases, who did not respond to anti-TNFα TmAbs (10).…”

“…In this context, we aimed to compare the cleavage of a panel of eight widely used TmAbs (three chimeric, two humanized, and three full-human) regrouping five IgG1s that include the four allotypes, one IgG2 TmAb, and two IgG4 TmAbs. We chose IdeS as a model protease (12)(13)(14)16) and MMP12, secreted by macrophages in the tumor microenvironment or in chronic inflammatory diseases, as a more relevant pathophysiological protease. We used SDS-PAGE to kinetically analyze the proteolytic fragments for each TmAbs.…”