Antibody Therapy of Cancer

特异性目标1：制备人单克隆抗体。抗体工程通常是通过在微生物（如噬菌体、细菌和酵母）表面展示人抗体或抗体片段来进行的。利用人HEK-293T细胞的高效蛋白表达和流式细胞术的高通量筛选特点，我们开发了一种新的哺乳动物细胞展示方法。使用这种方法，功能性单链抗体可变片段（scFvs）在人HEK-293细胞中表达，高亲和力抗原结合物随后通过流式细胞术从组合文库中鉴定和分离。HEK 293细胞比微生物更容易正确表达抗体。我们和其他人已经证明，哺乳动物细胞展示也可以通过靶向内在热点或共同表达激活诱导胞苷脱氨酶（AID）来模拟抗体亲和成熟的体细胞超突变，这是B细胞体细胞超突变的关键酶。在09-10财年，我们在哺乳动物细胞展示方面的工作导致在《分子生物学方法》上发表了两篇方法论文，并在国内和国际治疗性抗体会议上发表了几篇报告。由于其在患者体内的免疫原性较低，完全人源性单克隆抗体（mAb）是临床应用中最理想的抗体格式。间皮素在包括间皮瘤和卵巢癌在内的多种癌症中高表达，是一种很有前景的新的治疗靶点。Ira Pastan及其同事之前的研究导致了抗间皮素免疫毒素（SS1P）的发展。它含有与细菌毒素融合的小鼠ss1fv。一种含有相同小鼠Fv的嵌合抗体（MORAb-009）也被开发出来，目前正在进行间皮瘤和胰腺癌的II期临床试验。在08-09年度，我们(1)发现HN1是一种高亲和力的间皮素特异性Fv，(2)基于HN1 Fv产生了一个完全的人IgG分子，(3)发现HN1能够在间皮瘤和卵巢癌细胞上以高亲和力特异性结合细胞表面相关的间皮素。这项工作产生了一项专利申请。在2010财年，我们继续对HN1进行表征，并证明它可以通过强大的抗体依赖细胞介导的细胞毒性（ADCC）特异性杀死癌细胞。在2010财年，我们关于HN1的工作发表在《国际癌症杂志》上，并在几次国际癌症和抗体会议上发表。这项工作得到了间皮瘤应用研究基金会和卵巢癌研究基金会的部分支持。在2009财年，我们对glypican-3 （GPC3）作为一种新的肝癌特异性抗原产生了兴趣。由于GPC3在正常组织中不表达，而在肝细胞癌（HCC）中高表达，因此它是一个很有希望的肝癌治疗候选者。GPC3的功能缺失突变导致辛普森-戈拉比-贝梅尔综合征，这是一种罕见的x连锁过度生长疾病。GPC3通过其n端与IGF-2和IGF-1R结合，并通过其c端与Wnt和Hedgehog蛋白结合。GPC3也通过硫酸肝素聚糖链结合FGF-2。2010财年，我们在HEK-293细胞中制备了重组可溶性GPC3 （sGPC3）蛋白，发现sGPC3蛋白能够抑制HCC细胞的增殖。这项工作发表在《国际癌症杂志》上，并于2010财年申请了专利。我们正在进行的研究重点是开发针对GPC3的新型单克隆抗体来抑制HCC的生长。特异性目标2：设计新的免疫偶联物。卵巢癌和间皮瘤经常表达间皮素和粘蛋白CA125/MUC16。间皮素与CA125/MUC16的相互作用可能通过细胞粘附促进肿瘤的植入和腹膜扩散。然而，人们对这种分子相互作用的详细性质知之甚少。在2009财年，我们在间皮素中发现了CA125/MUC16的一个新的独特功能结合域（称为IAB）。我们进一步发现IAB可以有效阻断间皮素与CA125的结合，并通过一种新机制抑制癌细胞粘附。IAB是一种很好的候选拮抗剂，用于治疗表达ca125的肿瘤。我们针对CA125的工作在2009财年发表了一篇论文并申请了一项国际专利。人类单抗的分离并不总是可能的，特别是对那些在人类中具有低免疫原性的抗原。其中一种抗原是CA125。自1981年产生OC125以来，已经开发了许多针对CA125的小鼠单克隆抗体。一种名为B43.13（也称为Oregovomab）的小鼠单抗首次被开发为肿瘤显像剂，用于检测复发性卵巢癌，并已在卵巢癌治疗的II期临床研究中进行了评估。然而，尚未描述完全针对CA125的人单抗，可能是由于CA125在免疫系统下调中的作用。2010年，我们制作了名为免疫粘附素HN125的IAB-human Fc杂交分子，发现它可以高亲和力地结合表达ca125的癌细胞，并能功能阻断ca125 -间皮素的相互作用。我们目前正在研究它的抗肿瘤活性。

Specific Aim 1: Creating human monoclonal antibodies. Antibody engineering is typically carried out by displaying human antibodies or antibody fragments on the surface of microorganisms (e.g. phage, bacteria and yeast). By taking advantage of highly efficient protein expression in human HEK-293T cells and high throughput screening features of flow cytometry, we have developed a new method called mammalian cell display. Using this approach, functional single-chain antibody variable fragments (scFvs) are expressed in human HEK-293 cells and high affinity antigen binders are subsequently identified and isolated from a combinatory library via flow cytometry. HEK 293 cells are more likely than microorganisms to correctly express antibodies. We and others have shown that mammalian cell display can also be used to mimic somatic hypermutation for antibody affinity maturation by targeting intrinsic hotspots or co-expressing activation-induced cytidine deaminase (AID), the key enzyme for somatic hypermutation in B cells. In FY09-10, our work on mammalian cell display resulted in two method papers published in Methods in Molecular Biology and in several presentations at national and international therapeutic antibody conferences. Due to their lower immunogenicity in patients, a fully human monoclonal antibody (mAb) is the most desirable antibody format for clinical application. Mesothelin is a promising new therapeutic target given its high expression in a variety of cancers including mesothelioma and ovarian cancer. Previous research by Ira Pastan and colleagues led to the development of an anti-mesothelin immunotoxin (SS1P). It contains a murine SS1 Fv fused to a bacterial toxin. A chimeric antibody (MORAb-009) containing the same murine Fv was also developed and is currently being examined in a Phase II clinical trial for mesothelioma and pancreatic cancer. In FY08-09, we (1) identified HN1 as a high affinity Fv specific for mesothelin, (2) produced a fully human IgG molecule based on the HN1 Fv, and (3) showed that HN1 was able to bind specifically to cell surface-associated mesothelin on mesothelioma and ovarian cancer cells with high affinity. This work yielded a patent application. In FY10, we continued to characterize HN1 and demonstrated it could specifically kill cancer cells with strong antibody-dependent cell-mediated cytotoxicity (ADCC). In FY10, our work on HN1 was published in the International Journal of Cancer and presented at several international cancer and antibody meetings. This work was in part supported by the Mesothelioma Applied Research Foundation and the Ovarian Cancer Research Fund. In FY09, we became interested in glypican-3 (GPC3) as a new liver cancer-specific antigen. GPC3 is a promising candidate for liver cancer therapy given that it is not expressed in normal tissues and shows high expression in hepatocellular carcinoma (HCC). Loss-of-function mutations of GPC3 cause Simpson-Golabi-Behmel syndrome, a rare X-linked overgrowth disorder. GPC3 binds, through its N-terminus, to IGF-2 and IGF-1R, and through its C-terminus to Wnt and Hedgehog proteins. GPC3 also binds FGF-2 through its heparan sulfate glycan chains. In FY10, we made recombinant soluble GPC3 (sGPC3) protein in HEK-293 cells and found that sGPC3 protein could inhibit the proliferation of HCC cells. This work yielded a publication in the International Journal of Cancer and a patent application in FY10. Our ongoing studies are focused on the development of novel mAbs targeting GPC3 to inhibit HCC growth. Specific Aim 2: Designing novel immunoconjugates. Ovarian cancer and mesothelioma frequently express both mesothelin and mucin CA125/MUC16. The interaction between mesothelin and CA125/MUC16 may facilitate the implantation and peritoneal spread of tumors by cell adhesion. However, the detailed nature of this molecular interaction is poorly understood. In FY09, we identified a novel distinct functional binding domain (called IAB) in mesothelin for CA125/MUC16. We further showed that IAB could effectively block the binding of mesothelin to CA125 and inhibit cancer cell adhesion by a novel mechanism. IAB is a good candidate for use as an antagonist to treat CA125-expressing tumors. Our work targeting CA125 yielded one publication and an international patent application in FY09. Isolation of a human mAb is not always possible, especially for those antigens with low immunogenicity in humans. One such antigen is CA125. Since the generation of OC125 in 1981, numerous mouse mAbs directed against CA125 have been developed. A mouse mAb called B43.13 (also called Oregovomab) was first developed as a tumor-imaging agent to detect recurrent ovarian cancer and has been evaluated in Phase II clinical studies for ovarian cancer treatment. However, a fully human mAb against CA125 has not been described, possibly due to the role of CA125 in down-regulation of the immune system. In FY10, we made the IAB-human Fc hybrid molecule called immunoadhesin HN125 and showed it could bind CA125-expressing cancer cells with high affinity and functionally block the CA125-mesothelin interaction. We are currently examining its anti-tumor activity.