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glypicans作为一个新的癌症靶点家族[Li等人综述]。中国医学进展[j].中国医学进展，2016,33(5):357 - 357。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，我们已经开发了一种新的方法来人源化非人抗体（包括小鼠和兔子抗体），用于2017-2018财年的临床开发[Zhang和Ho， Scientific Reports, 2016；Zhang and Ho, mab, 2017]。2019财年，我们利用人源化YP7 （hYP7）构建了抗体-药物偶联物（ADC），并在多种小鼠模型中显示了单次治疗hYP7 ADC诱导肿瘤消退的效果[Fu等]。中华肝病杂志，2018 [j]。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]。在2019财年，我们利用计算建模和基因编辑技术建立了GPC3的结构模型，该模型在其n端叶含有一个假定的富含半胱氨酸的结构域，并将我们的发现发表在《肝脏学》杂志上[Li等]。中华肝病杂志，2019 [j]。GPC3中F41及其周围残基形成wnt结合槽，与Wnt3a脂质拇指结构域和食指结构域之间的中间区域相互作用。该凹槽中的突变残基显著抑制Wnt3a结合、β -连环蛋白激活和wnt依赖基因的转录激活。具体来说，使用抗体（HN3）阻断该结构域可抑制Wnt的激活。在HCC细胞中，与表达野生型GPC3的细胞相比，GPC3上突变的残基F41在体外抑制β -连环蛋白的激活，并降低小鼠异种移植肿瘤的生长。我们的研究揭示了GPC3与Wnt3a的详细相互作用，揭示了GPC3作为Wnt辅助受体的确切机制，并为Wnt阻断和HCC治疗提供了GPC3上的潜在靶点。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[Gao等]。自然通讯，PMID: 25758784, 2015]。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]。我们在2019财年通过NCI技术转移办公室提交了针对GPC3治疗肝癌的CAR - T细胞的专利申请。除GPC3外，我们还报道了GPC2作为儿科癌症特别是神经母细胞瘤的新治疗靶点[Li等，PNAS, 2017]，并且在2019财年，我们在2019年AACR年会上提出了GPC1作为胰腺癌的潜在靶点。在间皮素项目中，我们之前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]。在2019财年，我们报道了噬菌体展示纳米体文库的创建[Feng等人]。Antibody Therapeutics （PMID: 30627698, 2019）从我们的纳米体文库中分离出一组跨物种纳米体，这些纳米体可以结合人和小鼠间皮素，并通过NCI技术转移办公室申请了用于治疗间皮瘤、肺癌、胰腺癌、卵巢癌和其他癌症的临床应用专利。

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 family of cancer targets [reviewed by Li et al. Trends in Cancer, PMID: 30352677, 2019]. 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 in FY2017-2018 [Zhang and Ho, Scientific Reports, 2016; Zhang and Ho, MAbs, 2017]. In FY2019, we created antibody-drug conjugates (ADCs) using humanized YP7 (hYP7) and showed single treatment of hYP7 ADC induced tumor regression in multiple mouse models [Fu et al. Hepatology, PMID: 30353932, 2018]. 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]. In FY2019, using computational modeling and gene editing technology, we have established a structural model of GPC3 containing a putative cysteine-rich domain at its N-terminal lobe and published our finding in Hepatology [Li et al. Hepatology, PMID: 30963603, 2019]. F41 and its surrounding residues in GPC3 forms a Wnt-binding groove that interacts with the middle region located between the lipid thumb domain and the index finger domain of Wnt3a. Mutating residues in this groove significantly inhibits Wnt3a binding, beta-catenin activation, and the transcriptional activation of Wnt-dependent genes. Specifically, blocking this domain using an antibody (HN3) inhibits Wnt activation. In HCC cells, mutating residue F41 on GPC3 inhibits activation of beta-catenin in vitro and reduced xenograft tumor growth in mice compared with cells expressing wild-type GPC3. Our investigation demonstrates a detailed interaction of GPC3 and Wnt3a, reveals the precise mechanism of GPC3 acting as a Wnt coreceptor, and provides a potential target site on GPC3 for Wnt blocking and HCC therapy. 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. In addition, 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 [Gao et al. Nature Communications, PMID: 25758784, 2015]. 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]. We filed a patent application regarding our CAR T cells targeting GPC3 for treating liver cancer through the NCI Technology Transfer office in FY2019. In addition to GPC3, we reported GPC2 as a new therapeutic target in pediatric cancers in particular neuroblastoma [Li et al., PNAS, 2017] and in FY2019, we presented GPC1 as a potential target in pancreatic cancer in the 2019 AACR annual meeting. In the mesothelin project, we previously used rabbit monoclonal antibody technology to identify 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]. In FY2019, we reported the creation of a phage displayed nanobody library [Feng et al. Antibody Therapeutics, PMID: 30627698, 2019] and isolated a panel of cross-species nanobodies from our nanobody libraries that bind human and mouse mesothelin and filed a patent application for their clinical applications for treating mesothelioma, lung cancer, pancreatic cancer, ovarian cancer and other cancers through the NCI Technology Transfer Office.