Development of new antibody-based cancer therapies

硫酸乙酰肝素蛋白多糖(HSPGs)调节多种细胞表面信号事件。它们是发育和疾病过程中信号转导通路的细胞外调节器。HSPGs是一种细胞表面蛋白，主要由糖基磷脂酰肌醇(GPI)锚定的葡聚糖和跨膜联糖组成。几种HSPG目前正被评估为癌症治疗的潜在靶点，因为它们在某些肿瘤类型中相对高表达。近年来，我们将石首作为一类新的癌症靶点进行了研究。GPC3是治疗肝细胞癌的新靶点，肝细胞癌是最常见的原发性肝癌。我们通过杂交瘤和噬菌体展示技术制备了几种针对GPC3的抗体。为了分离针对GPC3等天然形式的细胞表面抗原的高亲和力抗体(如YP7)，我们开发了一种新的高通量方法，结合功能细胞结合流式细胞仪筛选和传统杂交瘤技术[Phung等人，单抗，PMID 22820551,2012年]。此外，我们还开发了一种新的方法来人源化非人类抗体(包括小鼠和兔抗体)用于临床开发[Zhang和Ho，科学报告，2016；Zhang和Ho，MAbs，2017]。此外，我们利用噬菌体展示技术制备了两株人源单抗(HN3和HS20)。HN3是一种人源单结构域抗体，它识别GPC3核心蛋白中的一个新的功能位点，并通过阻断WNT和YAP癌症信号来抑制肝癌细胞的增殖[冯等，PNAS，PMID：23471984,2013年；Gao等，自然通讯，PMID：25758784,2015年]。HS20识别GPC3的硫酸乙酰肝素链。人类抗体破坏Wnt3a和GPC3的相互作用，并抑制Wnt/β-catenin信号转导[高等人，肝病学，PMID：24492943,2014年；高等人，PLoS one，PMID：26332121,2016年；高等，科学报告，PMID：27185050,2016年]。我们的抗体对小鼠肝细胞癌移植瘤生长有显著的抑制作用，并显示出作为候选治疗药物的潜力。此外，我们还发现GPC3能有效地从细胞表面内化，HN3-PE38免疫毒素将毒素带入细胞内，导致蛋白质合成受到抑制。免疫毒素可使小鼠肝癌消退。有趣的是，它的新机制涉及抑制癌症信号(WNT/YAP)和减少蛋白质合成。我们的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/药物结合物。为了追求我们的抗GPC3免疫毒素用于治疗肝癌的临床开发，我们产生了一种新版本的抗GPC3免疫毒素(HN3-mPE24)，并发现第二代大大减少了副作用，具有更好的抗肿瘤活性[Wang等，OncoTarget，2017]。除了免疫毒素疗法，我们与我们的合作者一起，使用我们的抗GPC3抗体构建了多种临床形式的肝癌靶向治疗，包括嵌合抗原受体(CAR)T细胞免疫疗法和光免疫疗法[Hanaoka等人。莫尔·帕姆，2015；Hanaoka等人。纳米医学，2015年]。除了靶向治疗外，我们的抗体还被广泛用作研究工具，分析Glypicans在Wnt信号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，使用我们的HS20人抗体确定了硫酸肝素中的Wnt结合域[高等人，2016年科学报告]。除了GPC3，我们的实验室一直在研究其他英国成员(例如GPC2)作为儿科癌症的潜在治疗靶点(如2017年4月1日至5日在华盛顿举行的2017年美国癌症研究协会年会上提出的)。在间皮蛋白项目中，我们使用兔单抗技术来鉴定一组高亲和力抗体，这些抗体结合间皮蛋白中的新位点。我们已经人性化了治疗间皮瘤和其他间皮蛋白阳性癌症的最佳候选药物之一(YP218)[Zhang等人，科学报告，2015；Zhang and Ho，MAbs，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].