Antibody Therapy of Cancer

项目一：靶向glypican-3治疗肝细胞癌肝癌是全球第五大常见恶性肿瘤。肝细胞癌（HCC）约占肝癌病例的75%。胆管癌（CCA）是第二常见的原发性肝脏肿瘤。虽然肝癌在美国并不常见，但近年来肝癌的诊断率一直在上升。虽然手术切除是治疗该疾病的标准方法，但只有一小部分患者有资格进行手术。迫切需要能够成功地应用于大量患者的新疗法。我们对glypican-3 （GPC3）的研究是为了研究GPC3在癌症发病机制中的作用，并鉴定用于癌症治疗的人类抗体。GPC3在HCC中高表达，被认为是肝癌治疗的潜在靶点。在FY10，我们制备了不含GPI锚点的重组GPC3蛋白，发现重组GPC3以显性阴性形式直接抑制体外HCC的生长。这一观察结果发表在《国际癌症杂志》上。我们假设，中和GPC3的致癌功能代表了一种治疗HCC的新方法。通过这种方法，我们在2011年通过噬菌体展示成功地分离了人抗体。抗gpc3单克隆抗体（mab）也可用于免疫偶联物、嵌合抗原受体修饰的T细胞治疗、诊断肿瘤成像和其他临床应用。NCI在2011财年基于我们的GPC3研究提交了两项专利申请。我们希望找到一个行业合作伙伴，进一步开发我们的GPC3单克隆抗体，以治疗癌症患者。2011财年，GPC3项目在《国际癌症杂志》、《欧洲癌症杂志》、《生物药物》和《癌症报告》等期刊上发表了四篇论文。2011财年，我们受邀在第七届PEGS蛋白峰会首届癌症治疗抗体研讨会和BIO2011上展示我们的工作。项目2：靶向肝癌、间皮瘤、卵巢癌间皮素在间皮瘤、卵巢癌、胰腺癌、肺癌中均有高水平表达。2010财年，我们与Gregory Gores （Mayo Clinic）、Xin Wei Wang （NCI）合作，分析了间皮素在肝癌中的蛋白表达。我们的研究结果表明，间皮素是胆管癌（CCA）的潜在新靶点。这项研究发表在2011财年的《癌症杂志》上。我们利用我们在噬菌体展示技术方面的专业知识，与Ira Pastan （NCI）合作，鉴定了人类间皮素特异性单抗HN1。HN1单抗与细胞表面相关的间皮素具有高亲和力。HN1 mAb具有出色的抗体依赖细胞介导的细胞毒性（ADCC）杀死癌细胞，有望用于治疗表达间皮素的癌症。2011年，我们在《国际癌症杂志》上发表了这项研究。间皮素是MUC16结合蛋白，间皮素与MUC16的相互作用可能导致肿瘤在腹膜内播散。为了研究这种重要的分子相互作用，我们使用截断和丙氨酸扫描诱变方法鉴定了间皮素中MUC16的独特功能结合域（称为IAB）。我们发现IAB可以阻断间皮素与MUC16的结合并抑制异型癌细胞的粘附。这一发现对我们理解与间皮素有关的癌症转移的新机制具有重要意义。本研究于2009年度发表于《生物化学杂志》。在FY10-11中，我们生成了IAB-human Fc融合蛋白作为一种新的免疫粘附素（HN125）。我们与Manish Patankar（威斯康星大学麦迪逊分校）合作，研究了HN125在前瞻性卵巢癌治疗中的效用，并在2011财年的《癌症杂志》上发表了我们的研究成果。为了评估其在体内的抗肿瘤活性，我们设计了一株人间皮瘤细胞系（命名为LMB-H226-GL）进行体内生物发光肿瘤成像。这是一个非常适合的间皮瘤模型，可能有助于评估其他新的间皮瘤治疗药物在体内的作用。我们于2011年在《癌症杂志》上发表了这项工作，并与Raffit Hassan （NCI）合作使用该模型研究抗胰岛素样生长因子I受体（IGF-IR）抗体在间皮瘤中的疗效。我们的间皮瘤项目得到了间皮瘤应用研究基金会和卵巢癌研究基金个人研究者奖的部分支持。我们在2010 -11财年通过NCI获得了两项与HN1和IAB相关的间皮素工作专利。NCI向科学界提供LMB-H226-GL间皮瘤模型。2011财年，我们受邀在PEGS蛋白峰会、分子医学大会、临床肿瘤药物开发分子医学策略研讨会、胸部肿瘤学多学科研讨会、恶性间皮瘤国际研讨会等多个重要的癌症治疗会议上介绍我们的间皮素工作。项目3：建立体外肿瘤球体，研究抗体渗透。大多数抗癌药物的研究只考虑单细胞水平上的遗传和/或细胞机制。然而，药物渗透是一个非常重要的附加机制，需要更复杂的细胞环境来研究。本研究的目的是建立体外球体，以检测抗体在实体肿瘤中的渗透。与我们的合作者V. Courtney Broaddus（加州大学旧金山分校）和Shuichi Takayama（密歇根大学）一起，我们使用已建立的癌细胞系和从癌症患者中分离的原代细胞系制造了肿瘤球体，并于2011财年在三本期刊（PLoS ONE， Analyst和Tissue Engineering）上发表了该方法和初步观察结果。我们的球体研究在2011财年获得了NCI主任内部创新奖的支持。我们受邀在FY12第四届蛋白质发现与治疗学和蛋白质治疗学论坛上演讲，介绍我们在体外肿瘤球体模型和抗体渗透方面的工作。

Project 1: Targeting glypican-3 in hepatocellular carcinoma Liver cancer is the fifth most common malignant cancer worldwide. Hepatocellular carcinoma (HCC) accounts for approximately 75% of liver cancer cases. Cholangiocarcinoma (CCA) is the second most common primary liver tumor. Although liver cancer is uncommon in the United States, rates of liver cancer diagnosis have been increasing in recent years. While surgical resection offers a standard method for treatment of the disease, only a small portion of patients are eligible for the procedure. There is an urgent need for new treatments that can be successfully applied to a large population of patients. Our work on glypican-3 (GPC3) is to investigate the role of GPC3 in cancer pathogenesis and to identify human antibodies for cancer therapy. GPC3 is highly expressed in HCC and has been suggested as a potential target for liver cancer therapy. In FY10, we produced a recombinant GPC3 protein without the GPI anchor and found that the recombinant GPC3, functioning as a dominant-negative form, directly inhibited the growth of HCC in vitro. This observation was published in International Journal of Cancer. We hypothesize that neutralizing GPC3 oncogenic functions represents a novel therapeutic approach for treating HCC. In this manner, we successfully isolated human antibodies by phage display in FY11. The anti-GPC3 monoclonal antibodies (mAbs) may also be useful for immunoconjugates, chimeric antigen receptor-modified T cell therapy, diagnostic tumor imaging and other clinical applications. The NCI has filed two patent applications based on our GPC3 research in FY11. We hope to find an industrial partner to further develop our GPC3 mAbs in order to treat cancer patients. In FY11, the GPC3 project yielded four publications in the following journals: International Journal of Cancer, European Journal of Cancer, BioDrugs and Cancer Reports. In FY11, we were invited to present our work at 7th PEGS Protein Summit Inaugural Antibodies for Cancer Therapy Symposium and BIO2011. Project 2: Targeting mesothelin in liver cancer, mesothelioma and ovarian cancer Mesothelin is expressed at high levels in mesothelioma, as well as ovarian, pancreatic, and lung cancers. In FY10, in collaboration with Gregory Gores (Mayo Clinic) and Xin Wei Wang (NCI), we analyzed protein expression of mesothelin in liver cancer. Our results show that mesothelin is a potential novel target in cholangiocarcinoma (CCA). This work was published in the Journal of Cancer in FY11. We applied our expertise in phage display technology to identify HN1, a human mAb specific for mesothelin, by collaborating with Ira Pastan (NCI). The HN1 mAb binds cell surface-associated mesothelin with high affinity. The HN1 mAb kills cancer cells with excellent antibody-dependent cell mediated cytotoxicity (ADCC) and is promising for the treatment of mesothelin-expressing cancers. In FY11, we published this work in the International Journal of Cancer. Mesothelin is a MUC16 binding protein and the mesothelin-MUC16 interaction may lead to intra-peritoneal dissemination of tumors. To investigate this important molecular interaction, we used a truncation and alanine scanning mutagenesis approach to identify a distinct functional binding domain (named IAB) in mesothelin for MUC16 We show that IAB can block the binding of mesothelin to MUC16 and inhibit heterotypic cancer cell adhesion. This finding is important for us to understand the novel mechanisms of cancer metastasis involving mesothelin. In FY09, this work was published in the Journal of Biological Chemistry. In FY10-11, we generated the IAB-human Fc fusion protein as a novel immunoadhesin (HN125). We collaborated with Manish Patankar (University of Wisconsin-Madison) to investigate the utility of HN125 for prospective ovarian cancer therapy and publish our work in the Journal of Cancer in FY11. To evaluate the anti-tumor activity in vivo, we engineered a human mesothelioma cell line (named LMB-H226-GL) for in vivo bioluminescence tumor imaging. This is a well-suited model for mesothelioma, and may be useful for evaluating other novel agents for mesothelioma treatment in vivo. We published this work in the Journal of Cancer in FY11 and collaborated with Raffit Hassan (NCI) to use this model to study efficacy of an anti-insulin like growth factor I receptor (IGF-IR) antibody in mesothelioma. Our mesothelin project is supported in part by a Mesothelioma Applied Research Foundation grant and the Ovarian Cancer Research Fund Individual Investigator Award. We obtained two patents through the NCI on our mesothelin work related to HN1 and IAB in FY10-11. NCI provides the LMB-H226-GL mesothelioma model to the scientific community. In FY11, we were invited to present our mesothelin work in several important cancer therapy meetings such as PEGS Protein Summit, Congress of Molecular Medicine, Molecular Medicine Strategies for Clinical Oncology Drug Development symposium, Multidisciplinary Symposium in Thoracic Oncology and International Symposium on Malignant Mesothelioma. Project 3: Establish in vitro tumor spheroids to investigate antibody penetration. Most studies of anticancer drugs consider only genetic and/or cellular mechanisms at the level of the single cell. However, drug penetration is a highly important additional mechanism and requires a more complex cellular environment to study. The goal of this research is to establish in vitro spheroids in order to examine antibody penetration in solid tumors. Along with our collaborators V. Courtney Broaddus (University of California San Francisco) and Shuichi Takayama (University of Michigan), we made tumor spheroids using established cancer cell lines and primary lines isolated from cancer patients and published the method and initial observations in three journals (PLoS ONE, Analyst and Tissue Engineering) in FY11. Our spheroid research is supported by a NCI Directors Intramural Innovation Award for Principal Investigators in FY11. We were invited to give lectures at 4th Protein Discovery & Therapeutics and Protein Therapeutics Forum in FY12 to present our work on in vitro tumor spheroid model and antibody penetration.