Development of new antibody-based cancer therapies

硫酸肝素蛋白聚糖（HSPGs）调节许多细胞表面信号转导事件。它们是发育和疾病过程中信号转导通路的细胞外调节剂。然而，人们对它们在癌症发展中的作用知之甚少。HSPGs是细胞表面蛋白，主要由糖基磷脂酰肌醇（GPI）锚定的glypicans和跨膜syndecans组成。由于几种HSPGs在某些肿瘤类型中相对较高的表达，目前正被评估为癌症治疗的潜在靶点。在最近的财政年度，我们专注于评估glypican-3 （GPC3）作为肝细胞癌（HCC）的新靶点，HCC是原发性肝癌最常见的形式。为此，我们制作了识别GPC3中c -末端位点（511-560）的小鼠单克隆抗体（例如YP7） [Phung et al., mab, PMID 22820551, 2012]。此外，我们通过噬菌体展示技术生成了两种人单克隆抗体（HN3和HS20）。HN3是一种人重链抗体，可识别GPC3核心蛋白中的一个新的功能位点，并抑制HCC细胞的增殖。HN3作用的潜在机制涉及肝癌细胞中Wnt和Yap信号的抑制[Feng等，PNAS, pmiid: 23471984, 2013；高等，自然通讯，pmiid: 25758784, 2015]。HS20识别GPC3的硫酸肝素链。人抗体破坏Wnt3a和GPC3的相互作用，抑制Wnt/ β -catenin信号传导[Gao等，肝脏病学，PMID: 24492943, 2014；高等，PLoS One, pmiid: 26332121, 2016；高等，科学报告，pmiid: 27185050, 2016]。我们的抗体在小鼠肝细胞癌异种移植肿瘤生长中表现出显著的抑制作用，并显示出作为治疗候选物的潜力。此外，我们发现GPC3被有效地从细胞表面内化，并且HN3-PE38免疫毒素将毒素带入细胞，从而抑制蛋白质合成。免疫毒素引起小鼠肝癌消退。有趣的是，其新机制涉及抑制癌症信号（Wnt/Yap）和减少蛋白质合成。我们结合抗体和毒素功能的策略可以普遍适用于其他免疫毒素和抗体-毒素/药物偶联物。为了进行抗gpc3免疫毒素治疗肝癌的临床开发，我们在2016财年生产了一种新的抗gpc3免疫毒素（HN3-mPE24），并发现第二代在小鼠高剂量使用时，副作用大大减少，具有良好的抗肿瘤活性。我们总结了抗gpc3免疫毒素治疗的最新研究进展，并在Oncotarget上发表了一篇研究文章[Wang et al., PMID: 27419635, 2016]。基于免疫毒素的肝癌新疗法的效力和缺乏耐药性非常有吸引力。除了免疫毒素治疗外，我们还与合作者一起，利用抗gpc3抗体构建了包括光免疫治疗在内的肝癌靶向治疗的各种临床格式，并在两篇研究论文中发表了我们的合作研究[Hanaoka et al]。Mol Pharm, 2015；Hanaoka等人。纳米医学,2015]。除了靶向治疗外，我们的抗体已被广泛用作研究工具来分析HSPGs在Wnt信号传导和其他重要生物过程中的作用。我们与北卡罗来纳大学刘健博士的实验室合作，利用我们的HS20人抗体鉴定出硫酸肝素中的Wnt结合结构域，并于2016财年发表了一篇研究文章[Gao et al., Scientific Reports, PMID: 27185050, 2016]。在间皮素项目中，我们与Ira Pastan博士的实验室合作，使用兔单克隆抗体技术鉴定了一组有趣的抗体，这些抗体结合间皮素中免疫原性差的位点。我们已经将其中一种最佳候选药物（YP218）人源化，用于治疗间皮瘤和其他间皮素阳性癌症[Zhang等，Scientific Reports, PMID: 25996440, 2015]。

Heparan sulfate proteoglycans (HSPGs) regulate numerous cell surface signaling events. They are extracellular modulators of signal transduction pathways during development and disease. However, their role in cancer development is poorly understood. HSPGs are cell-surface proteins that mainly consist of glycosylphosphatidylinositol (GPI)-anchored glypicans and transmembrane syndecans. Several HSPGs are currently being evaluated as potential targets for cancer therapy because of their relatively high expression in certain tumor types. In the recent fiscal years, we have focused on evaluating glypican-3 (GPC3) as a new target in hepatocellular carcinoma (HCC), the most common form of primary liver cancers. To this end, we produced mouse monoclonal antibodies (e.g. YP7) that recognize a C-terminal site (511-560) in GPC3 [Phung et al., MAbs, PMID 22820551, 2012]. In addition, we generated two human monoclonal antibodies (HN3 and HS20) by phage display technology. HN3 is a human heavy-chain antibody that recognizes a novel functional site in the core protein of GPC3 and inhibits proliferation of HCC cells. The underlying mechanism of HN3 action involves inhibition of Wnt and Yap signaling in liver cancer cells [Feng et al., PNAS, PMID: 23471984, 2013; Gao et al., Nature Communications, PMID: 25758784, 2015]. HS20 recognizes the heparan sulfate chains of GPC3. The human antibody disrupts the interaction of Wnt3a and GPC3 and inhibits Wnt/beta-catenin signaling [Gao et al., Hepatology, PMID: 24492943, 2014; Gao et al., PLoS One, PMID: 26332121, 2016; Gao et al., Scientific Reports, PMID: 27185050, 2016 ]. Our antibodies exhibit significant inhibition of HCC xenograft tumor growth in mice and show potential for use as therapeutic candidates. Furthermore, we found that GPC3 was efficiently internalized from the cell surface and that the HN3-PE38 immunotoxin brought the toxin into the cell, resulting in inhibition of protein synthesis. The immunotoxin caused regression of liver cancer in mice. Interestingly, Its novel mechanism involved both inhibition of cancer signaling (Wnt/Yap) and reduction in protein synthesis. Our strategy combining both antibody and toxin functions could be applicable generally to other immunotoxins and antibody-toxin/drug conjugates. To pursue clinical development of our anti-GPC3 immunotoxin for the treatment of liver cancer, in FY16 we generated a new version of the anti-GPC3 immunotoxin (HN3-mPE24) and found that the second generation greatly reduced side effects and had good anti-tumor activity when used at high doses in mice. We summarized the most recent research in the development of anti-GPC3 immunotoxin therapy and published a research article in Oncotarget [Wang et al., PMID: 27419635, 2016]. The potency and lack of drug resistance makes the new immunotoxin-based liver cancer therapy very attractive. In addition to the immunotoxin therapy, along with our collaborators, we used our anti-GPC3 antibodies to construct various clinical formats for targeted therapy of liver cancer including photoimmunotherapy and published our collaborative research in two research articles [Hanaoka et al. Mol Pharm, 2015; Hanaoka et al. Nanomedicine, 2015]. In addition to targeted therapies, our antibodies have been widely used as a research tool to analyze the role of HSPGs in Wnt signaling and other important biological processes. We have identified the Wnt binding domain in heparan sulfate using our HS20 human antibody in collaboration with Dr. Jian Liu's lab in the University of North Carolina and published a research article in FY16 [Gao et al., Scientific Reports, PMID: 27185050, 2016]. In the mesothelin project, we have collaborated with Dr. Ira Pastan's lab and used rabbit monoclonal antibody technology to identify a panel of interesting antibodies that bind poorly immunogenic sites in mesothelin. We have humanized one of the best candidates (YP218) for the treatment of mesothelioma and other mesothelin-positive cancers [Zhang et al., Scientific Reports, PMID: 25996440, 2015].