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。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。近年来，我们研究了一类新的肿瘤靶点。Glypican-3 （GPC3）是治疗原发性肝癌中最常见的肝细胞癌（HCC）的新靶点。利用杂交瘤和噬菌体展示技术制备了几种靶向GPC3的抗体。为了分离出针对细胞表面抗原（如GPC3）的高亲和力抗体（如YP7），我们开发了一种新的高通量方法，将流式细胞术的功能细胞结合筛选与传统杂杂瘤技术相结合[Phung et al., MAbs, PMID 22820551, 2012]。此外，我们还开发了一种用于临床开发的人源化非人抗体（包括小鼠和兔子抗体）的新方法[Zhang和Ho， Scientific Reports, 2016；Zhang and Ho, mab, 2017]。此外，我们利用噬菌体展示技术生成了两种人单克隆抗体（HN3和HS20）。HN3是一种人类单域抗体，它识别GPC3核心蛋白的一个新的功能位点，并通过阻断Wnt和Yap癌症信号来抑制HCC细胞的增殖[Feng等，PNAS, PMID: 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。为了进一步开展抗gpc3免疫毒素治疗肝癌的临床开发，我们制作了一种新型的抗gpc3免疫毒素（HN3-mPE24），发现第二代抗gpc3免疫毒素的副作用大大降低，抗肿瘤活性也更好[Wang et al., Oncotarget, 2017]。除了免疫毒素治疗外，我们与合作者一起使用抗gpc3抗体构建了多种肝癌靶向治疗的临床形式，包括嵌合抗原受体（CAR） T细胞免疫治疗和光免疫治疗[Hanaoka等]。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分析glypicans在Wnt信号传导和其他重要生物过程中的作用的研究工具。我们与北卡罗来纳大学刘健博士的实验室合作，使用HS20人抗体鉴定了硫酸肝素中的Wnt结合结构域[Gao等人，Scientific Reports, 2016]。除了GPC3，我们的实验室一直在研究其他glypican成员（例如GPC2）作为儿科癌症的潜在治疗靶点（已于2017年4月1-5日在华盛顿特区举行的2017年美国癌症研究协会年会上提出）。在间皮素项目中，我们使用兔单克隆抗体技术鉴定了一组高亲和力抗体，这些抗体结合了间皮素中的新位点。我们已经将一种最佳候选药物（YP218）人源化，用于治疗间皮瘤和其他间皮瘤阳性癌症[Zhang等，Scientific Reports, 2015；Zhang and Ho, mab, 2017]。

Heparan sulfate proteoglycans (HSPGs) regulate numerous cell surface signaling events. They are extracellular modulators of signal transduction pathways during development and disease. 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 recent years, we have studied glypicans as a new class of cancer targets. Glypican-3 (GPC3) is a new therapeutic target in hepatocellular carcinoma (HCC), the most common form of primary liver cancers. We produced several antibodies targeting GPC3 either by hybridoma and phage display technologies. To isolate high affinity antibodies (e.g. YP7) to the native form of cell surface antigens such as GPC3, we developed a new high-throughput method combining functional cell binding screening by flow cytometry and conventional hybridoma technology [Phung et al., MAbs, PMID 22820551, 2012]. Furthermore, we have developed a new approach to humanize non-human antibodies (including mouse and rabbit antibodies) for clinical development [Zhang and Ho, Scientific Reports, 2016; Zhang and Ho, MAbs, 2017]. In addition, we used phage display technology to generate two human monoclonal antibodies (HN3 and HS20). HN3 is a human single-domain antibody that recognizes a novel functional site in the core protein of GPC3 and inhibits proliferation of HCC cells via blocking Wnt and Yap cancer signaling [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, we generated a new version of the anti-GPC3 immunotoxin (HN3-mPE24) and found that the second generation greatly reduced side effects and had better anti-tumor activity [Wang et al., Oncotarget, 2017]. 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 chimeric antigen receptor (CAR) T cell immunotherapy and photoimmunotherapy [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 glypicans 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 [Gao et al., Scientific Reports, 2016]. In addition to GPC3, our lab has been studying other glypican members (e.g. GPC2) as potential therapeutic targets in pediatric cancers (as presented in 2017 American Association for Cancer Research Annual Meeting in Washington DC, April 1-5, 2017). In the mesothelin project, we have used rabbit monoclonal antibody technology toidentify a panel of high affinity antibodies that bind novel 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, 2015; Zhang and Ho, MAbs, 2017].