Proteolytic single hinge cleavage of pertuzumab impairs its Fc effector function and antitumor activity in vitro and in vivo.
Proteolytic single hinge cleavage of pertuzumab impairs its Fc effector function and antitumor activity in vitro and in vivo.
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DOI：
10.1186/s13058-018-0972-4
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发表时间：
2018-06-01
期刊：
影响因子：
--
通讯作者：
An Z
中科院分区：
文献类型：
--
作者：
Hsiao HC;Fan X;Jordan RE;Zhang N;An Z
治疗性单克隆抗体（mAb）的Fc效应子功能的蛋白水解损伤可能会损害其在肿瘤微环境中的抗肿瘤疗效，并可能代表宿主免疫逃避的一种未被认识到的机制。帕妥珠单抗是一种人表皮生长因子受体2（HER 2）靶向抗体，临床上已广泛与曲妥珠单抗联合用于治疗HER 2过表达乳腺癌。帕妥珠单抗对蛋白水解铰链切割的敏感性及其对药物疗效的影响先前尚未研究。将帕妥珠单抗与高和低HER 2表达癌细胞一起孵育，并通过蛋白质印迹法检测下铰链区中的蛋白水解裂解。纯化单铰链切割的帕妥珠单抗（scIgG-P），并评价其体外介导抗体依赖性细胞毒性（ADCC）的能力和体内抗肿瘤功效。为了评估曲妥珠单抗（IgG-T）和帕妥珠单抗（IgG-P）在同时结合至相同癌细胞表面时的切割，将IgG-T或IgG-P的F（ab '）2片段分别与完整IgG-P和IgG-T组合，以通过蛋白质印迹检测scIgG产生。当mAb与高HER 2表达癌细胞一起孵育时，发生帕妥珠单抗铰链切割。帕妥珠单抗的铰链切割导致体外ADCC的实质性损失和体内抗肿瘤功效的降低。scIgG-P降低的ADCC功能通过对切割位点新表位具有特异性的抗铰链mAb得以恢复。此外，我们构建了抗铰链mAb的蛋白酶抗性版本，当癌细胞表达有效的IgG铰链切割蛋白酶时，其恢复了帕妥珠单抗的ADCC和细胞杀伤功能。我们还观察到当与曲妥珠单抗组合时帕妥珠单抗的铰链切割增加。单铰链切割的帕妥珠单抗的降低的Fc效应子功能可通过抗铰链mAb恢复。修复效果表明，当受损的一抗与癌细胞表面结合时，免疫功能可以容易地增强。抗铰链mAb还恢复了蛋白水解失活的曲妥珠单抗和帕妥珠单抗混合物的Fc效应子功能，这表明在肿瘤微环境中恢复蛋白酶失活的抗癌抗体的免疫效应子功能的一般治疗策略。这些发现为开发乳腺癌免疫疗法提供了一种新的策略。
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. 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. 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. 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.
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曲妥珠单抗较低铰链内的单个蛋白水解裂解可降低免疫效应子功能和体内功效。
DOI：
10.1186/bcr3240
发表时间：
2012-08-08
期刊：
Breast cancer research : BCR
影响因子：
--
作者：
Fan X;Brezski RJ;Fa M;Deng H;Oberholtzer A;Gonzalez A;Dubinsky WP;Strohl WR;Jordan RE;Zhang N;An Z
通讯作者：
An Z
影响因子：
5.7
作者：
Fan, Xuejun;Brezski, Randall J.;An, Zhiqiang
通讯作者：
An, Zhiqiang
影响因子：
4.4
作者：
Falkenburg, Willem J. J.;van Schaardenburg, Dirkjan;Rispens, Theo
通讯作者：
Rispens, Theo
影响因子：
11.5
作者：
Yamashita-Kashima, Yoriko;Iijima, Shigeyuki;Fujimoto-Ouchi, Kaori
通讯作者：
Fujimoto-Ouchi, Kaori
影响因子：
64.8
作者：
Cho, HS;Mason, K;Leahy, DJ
通讯作者：
Leahy, DJ