Large Molecule Therapeutics A Novel Therapeutic Strategy to Rescue the Immune Effector Function of Proteolytically Inactivated Cancer Therapeutic Antibodies Xuejun Fan 1 , Randall J. Brezski 2 , Hui Deng 1 , Pooja M. Dhupkar 1 , Yun Shi 1 , Anneliese Gonzalez 3 , Songlin Zhang 4 , Michael Rycyzyn 2 , William R. Strohl 2 , Robert E. Jordan 2 , Ningyan Zhang 1 , and Zhiqiang An 1 Abstract Primary and acquired resistance to anticancer antibody immunotherapies presents significant clinical challenges. Here, we demonstrate that proteolytic inactivation of cancer-targeting antibodies is an unappreciated contributor to cancer immune evasion, and the finding presents novel opportunities for ther- apeutic intervention. A single peptide bond cleavage in the IgG1 hinge impairs cancer cell killing due to structural derange- ment of the Fc region. Hinge-cleaved trastuzumab gradually accumulated on the surfaces of HER2-expressing cancer cell lines in vitro, and was greatly accelerated when the cells were engineered to express the potent bacterial IgG-degrading pro- teinase (IdeS). Similar to cancer-related matrix metalloprotei- nases (MMP), IdeS exposes a hinge neoepitope that we have developed an antibody, mAb2095-2, to specifically target the epitope. In in vitro studies, mAb2095-2 restored the lost anti- body-dependent cell-mediated cytotoxicity functionality of cell-bound single-cleaved trastuzumab (scIgG-T). In vivo, mAb2095-2 rescued the impaired Fc-dependent tumor-sup- pressive activity of scIgG-T in a xenograft tumor model and restored the recruitment of immune effector cells into the tumor microenvironment. More importantly, an Fc-engineered proteinase-resistant version of mAb2095-2 rescued trastuzu- mab antitumor efficacy in a mouse tumor model with human cancer cells secreting IdeS, whereas trastuzumab alone showed significantly reduced antitumor activity in the same model. Consistently, an Fc-engineered proteinase-resistant version of trastuzumab also greatly improved antitumor efficacy in the xenograft tumor model. Taken together, these findings point to a novel cancer therapeutic strategy to rescue proteolytic damage of antibody effector function by an Fc-engineered mAb against the hinge neoepitope and to overcome cancer evasion of antibody immunity. Mol Cancer Ther; 14(3); 1–11. Ó2014 AACR. Introduction Antibodies are becoming a major drug modality for the treat- ment of many human diseases, including cancer (1–3). However, development of resistance to antibody therapies is widespread, and an understanding of the resistance mechanisms is a topic of great interest and clinical urgency (4–7). In the case of breast cancer, it is well established that the humanized IgG1 antibody trastuzumab can inhibit tumor growth by binding via its variable domain to highly expressed HER2 receptors on cancer cells to inhibit cell signaling (4, 8–10). As an IgG1 antibody, trastuzumab can also mediate antibody-dependent cell-mediated cytotoxicity (ADCC) by recruiting immune effector cells to HER2-overexpres- sing tumor cells (11). Resistance to trastuzumab was associated with substantially increased expression of EGFR and HER3 (9). Upregulation of cMET expression (12), loss of PTEN, or other genetic mutations in the network (8) were also proposed as possible resistance mechanisms against trastuzumab. In addition, antibody resistance mechanisms suggestive of an evasion of Fc- mediated ADCC have also been proposed (11, 13–15). Our group has recently reported that a single proteolytic cleavage in the lower hinge region results in a profound loss of Fc immune effector functions and reduced in vivo efficacy of trastuzumab. Moreover, the results indicated that the lower hinge of trastuzumab can be cleaved by matrix metalloproteinases (MMP)—a class of protei- nases associated with the tumor environment (4). In this study, we demonstrated that an anti-hinge antibody against the site of proteinase cleavage can rescue the loss of Fc effect functions of the single-hinge–cleaved trastuzumab (scIgG- T) in vitro and in vivo. By Fc molecular engineering, we showed that trastuzumab itself and the anti-hinge cleavage site antibody mAb2095-2 used in combination with scIgG-T can be rendered resistant to IdeS hinge cleavage by incorporating select mutations in the lower hinge and adjacent CH2 region. The Fc-engineered anti-hinge cleavage site antibody restored immune effector func- tions and the antitumor efficacy of hinge cleaved trastuzumab in vivo. Thus, the recognition of proteolytic inactivation of IgGs 1 Texas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, Houston, Texas. 2 Biologics Research, Janssen R&D, LLC, Spring House, Pennsylvania. 3 Division of Oncology, Department of Internal Medicine, The University of Texas Health Science Center at Houston, Houston, Texas. 4 Department of Pathology and Laboratory Medicine, The University of Texas Health Science Center at Houston, Houston, Texas. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Current address for R.J. Brezski: Antibody Engineering, Genentech, One DNA Way, South San Francisco, CA. Corresponding Authors: Zhiqiang An, Texas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, 1825 Pressler Street, Suite 532, Houston, TX 77030. Phone: 713-500-3011; Fax: 713-500-2447; E-mail: zhiqiang.an@uth.tmc.edu; and Ningyan Zhang, ningyan.zhang@uth.tmc.edu doi: 10.1158/1535-7163.MCT-14-0715 Ó2014 American Association for Cancer Research. Molecular Cancer Therapeutics www.aacrjournals.org OF1 Research. on February 25, 2015. © 2014 American Association for Cancer mct.aacrjournals.org Downloaded from Published OnlineFirst December 31, 2014; DOI: 10.1158/1535-7163.MCT-14-0715