Studies of synergistic antibody combination for treatment of patients with HER2-positive cancer

Abstract

Gastric cancer is one of the leading types of cancer worldwide. Although the trend in death rates for gastric cancer is decreasing, prognosis is poor and few therapeutic options are available, particularly in advanced stages. The frequency of HER2-positive gastric cancer is 22.1% based on ToGA trial. Although reports are conflicting, some studies have suggested that HER2-positive status in gastric cancer is associated with poor outcomes and aggressive disease. Trastuzumab, trade name HERCEPTIN, is a HER2-targeting therapeutic antibody and approved for the treatment of HER2-overexpressing breast cancer. Currently, trastuzumab is also approved for the treatment of HER2-overexpressing metastatic gastric cancer based on ToGA trial that proved the clinical benefit of combination treatment of trastuzumab and chemotherapy. Median overall survival was 13.8 months in trastuzumab plus chemotherapy compared with 11.1 months in those assigned to chemotherapy alone. Nonetheless, improving therapeutic efficacy and patient survival is important, particularly in patients with HER2-positive gastric cancer. Recent evidence suggests that particular combinations of non competing antibodies targeting the same receptor increase antitumor activity in vitro and in vivo. One example is the combination of pertuzumab, which binds to sub-domain II of the extracellular domain (ECD) of HER2, with trastuzumab, which binds to sub-domain IV. Pertuzumab, which has limited antitumor activity as a single agent in HER2-overexpressing breast cancer cells, shows increased efficacy in combination with trastuzumab. The increased efficacy of antibody combinations has also been demonstrated with EGFR-targeting or VEGFR3-targeting antibodies. In this thesis, I have investigated on the efficacy and mechanism of a novel HER2-targeting monoclonal antibody 1E11 in HER2-positive gastric and breast cancer. 1E11 shows significant antitumor activity as a single agent in in vitro and in vivo HER2-positive gastric cancer models. Antitumor activity of 1E11 is increased in a highly synergistic manner in combination with trastuzumab. The two antibodies bind to sub-domain IV of the receptor, but have non-overlapping epitopes, allowing them to simultaneously bind to HER2. Treatment with 1E11 alone induced apoptosis in HER2-positive cancer cells, and this effect was enhanced by combination treatment with trastuzumab. Combination treatment with 1E11 and trastuzumab reduced the levels of total HER2 protein and those of aberrant HER2 signaling molecules including phosphorylated HER3 and EGFR. Since 1E11 is murine-derived monoclonal antibodies, murine variable region could be immunogenic in human. So I performed humanization by CDR-grafting of murine CDRs of 1E11 to human antibody germline framework. The humanized antibody by this approach, hz1E11, shows almost identical affinity and biological activity to the parental antibody. To improve the affinity of hz1E11, I performed affinity maturation by site-directed mutagenesis in CDR3 of heavy and light chain. Using antibody phage display technology, I selected 1A12 clone which has 4 amino acid mutations in CDR-L4 and shows improved affinity more than 10-fold. I confirmed that antitumor activity of 1A12 is correlated with HER2 levels in more than 15 gastric and breast cancer cell panels. The antibody-dependent cellular cytotoxicity (ADCC) activity of 1A12 is comparable to that of trastuzumab and pertuzumab. In combination setting with trastuzumab, 1E11 completely inhibits tumor growth in OE-19 xenograft model, while pertuzumab causes partial inhibition of tumor growth. In conclusion, 1E11 showed synergistic antitumor activity in combination with trastuzumab in HER2-positive human gastric cancer in vitro and in vivo. The antitumor activity of 1E11 was mediated by its apoptotic activity and the inhibition of HER2 homo-and hetero-dimerization downstream signaling. This antibody combination could be a novel potent therapeutic strategy for the treatment of patients with HER2-overexpressing gastric and breast cancer.