Proteolytic single hinge cleavage of pertuzumab impairs its Fc effector function and antitumor activity in vitro and in vivo
Abstract Background Proteolytic 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...
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| Format: | Article |
| Language: | English |
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BMC
2018-06-01
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| Series: | Breast Cancer Research |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13058-018-0972-4 |
| _version_ | 1818671651713187840 |
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| author | Hao-Ching Hsiao Xuejun Fan Robert E. Jordan Ningyan Zhang Zhiqiang An |
| author_facet | Hao-Ching Hsiao Xuejun Fan Robert E. Jordan Ningyan Zhang Zhiqiang An |
| author_sort | Hao-Ching Hsiao |
| collection | DOAJ |
| description | Abstract Background Proteolytic 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. Methods Pertuzumab 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. Results Pertuzumab 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. Conclusion The 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. |
| first_indexed | 2024-12-17T07:27:24Z |
| format | Article |
| id | doaj.art-26b89b3ab9114b1b8b5399dbfd9a91a6 |
| institution | Directory Open Access Journal |
| issn | 1465-542X |
| language | English |
| last_indexed | 2024-12-17T07:27:24Z |
| publishDate | 2018-06-01 |
| publisher | BMC |
| record_format | Article |
| series | Breast Cancer Research |
| spelling | doaj.art-26b89b3ab9114b1b8b5399dbfd9a91a62022-12-21T21:58:34ZengBMCBreast Cancer Research1465-542X2018-06-0120111210.1186/s13058-018-0972-4Proteolytic single hinge cleavage of pertuzumab impairs its Fc effector function and antitumor activity in vitro and in vivoHao-Ching Hsiao0Xuejun Fan1Robert E. Jordan2Ningyan Zhang3Zhiqiang An4Texas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, the University of Texas Health Science Center at HoustonTexas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, the University of Texas Health Science Center at HoustonTexas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, the University of Texas Health Science Center at HoustonTexas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, the University of Texas Health Science Center at HoustonTexas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, the University of Texas Health Science Center at HoustonAbstract Background Proteolytic 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. Methods Pertuzumab 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. Results Pertuzumab 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. Conclusion The 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.http://link.springer.com/article/10.1186/s13058-018-0972-4PertuzumabHER2Antibody hinge cleavageFc effector functionBreast cancerTumor invasion of humoral immunity |
| spellingShingle | Hao-Ching Hsiao Xuejun Fan Robert E. Jordan Ningyan Zhang Zhiqiang An Proteolytic single hinge cleavage of pertuzumab impairs its Fc effector function and antitumor activity in vitro and in vivo Breast Cancer Research Pertuzumab HER2 Antibody hinge cleavage Fc effector function Breast cancer Tumor invasion of humoral immunity |
| title | Proteolytic single hinge cleavage of pertuzumab impairs its Fc effector function and antitumor activity in vitro and in vivo |
| title_full | Proteolytic single hinge cleavage of pertuzumab impairs its Fc effector function and antitumor activity in vitro and in vivo |
| title_fullStr | Proteolytic single hinge cleavage of pertuzumab impairs its Fc effector function and antitumor activity in vitro and in vivo |
| title_full_unstemmed | Proteolytic single hinge cleavage of pertuzumab impairs its Fc effector function and antitumor activity in vitro and in vivo |
| title_short | Proteolytic single hinge cleavage of pertuzumab impairs its Fc effector function and antitumor activity in vitro and in vivo |
| title_sort | proteolytic single hinge cleavage of pertuzumab impairs its fc effector function and antitumor activity in vitro and in vivo |
| topic | Pertuzumab HER2 Antibody hinge cleavage Fc effector function Breast cancer Tumor invasion of humoral immunity |
| url | http://link.springer.com/article/10.1186/s13058-018-0972-4 |
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