Antibody Therapy of Cancer

靶向癌症治疗的最大挑战是缺乏好的治疗靶点。除了开发新的治疗性抗体外，我们目前和未来的研究都集中在研究肿瘤特异性靶点及其在肿瘤发生和转移中的作用。我们正在研究三种肿瘤抗原：(1)间皮蛋白，(2)CA125(也称为MUC16)和(3)一种新的肝癌抗原。间皮蛋白是一种细胞表面生物标记物，在间皮瘤、卵巢癌等恶性肿瘤中高表达，有望成为肿瘤免疫治疗的新靶点。Ira Pastans实验室鉴定了一种针对间皮蛋白的小鼠单抗(MAb)可变区(Fv)，称为SS1。它已经被开发为一种免疫毒素或嵌合抗体，目前正在进行癌症治疗的临床试验。由于其在患者体内的免疫原性较低，全人单抗是临床应用的最理想的抗体形式。我们已经筛选了噬菌体展示文库，并分离了几个前导Fv分子。在2009财年，我们(1)确定HN1是一种针对间皮蛋白的高亲和力抗体，(2)开发了基于HN1 FV的全人免疫球蛋白分子，(3)表明HN1与间皮瘤和卵巢癌细胞上的细胞表面相关间硫蛋白特异结合，以及(4)证明了其对癌细胞的特异性细胞毒活性。我们目前正在评估HN1作为治疗间皮蛋白表达肿瘤的候选治疗剂。我们在HN1方面的工作在几次国际会议上提出，并在09财年获得了一项国际专利申请。CA125是在大多数卵巢癌和间皮瘤细胞上发现的另一个重要的生物标志物。卵巢癌和间皮瘤细胞通常通过种植到有间皮衬里的结构而在腹膜内扩散。CA125和间充质蛋白的相互作用被认为促进了肿瘤的种植和转移。我们假设，阻断间充质蛋白-CA125的相互作用可能会阻止或逆转转移，并导致癌症患者总体生存率的提高。更好地了解这两种蛋白质是如何相互作用的，最终可能有助于开发这种疗法。为此，我的实验室大力研究了间皮蛋白和CA125的分子相互作用。在2008财年，我们(1)确定了间皮蛋白与CA125的结合部位，(2)揭示了与CA125相互作用的间皮蛋白结合部位内的特定氨基酸。在2009财年，我们基于对CA125的研究开发了新的抗癌疗法。在一种方法中，我们已经(1)开发了一种新的全人分子，具有高亲和力和良好的特异性，针对表达CA125的癌细胞，以及(2)发现该分子可以有效地阻断CA125与间硫蛋白的结合，从而表明它可以作为一种拮抗剂用于抑制肿瘤的进展和在腹膜腔内的扩散。我们现在正在研究它对癌细胞的潜在细胞毒活性。在另一种方法中，我们开发了一种新型的杂化分子，含有促凋亡多肽，可以诱导表达CA125的癌细胞死亡。我们针对CA125的工作在09财年获得了一份出版物和一项国际专利申请。在2009财年，我们开始对最近发现的一种肝癌特异性抗原感兴趣。由于在肝肿瘤中高表达，而在正常肝组织中不表达，我们能够以重组Fc融合蛋白的形式表达和纯化肝癌抗原。我们目前正在研究它与一个可能的伴侣蛋白的分子相互作用。这项工作可能会对肝癌的起源以及肝癌与肝炎病毒感染的关系提供机械性的见解。我们计划利用杂交瘤和噬菌体展示技术分离高亲和力的单抗。为了评估抗体疗法在临床相关肿瘤模型中的疗效，我的实验室最近开发了一种新的小鼠恶性间皮瘤移植瘤。该模型可能对研究抗体和其他与恶性间皮瘤治疗有关的治疗方法非常有用。此前，我们的工作导致了哺乳动物细胞展示的发展，这是一种新的抗体工程方法。基于这种方法，可以在人细胞上表达功能性单链抗体(ScFv)，并从组合Fv库中快速鉴定和分离高亲和力的scFv结合子。我们正在改进这一方法，以建立一个大的、永久的Fv文库，目的是分离出高亲和力的抗体用于癌症治疗。在2009财年，我们在抗体工程方面的工作产生了三份出版物，并与人合著了两份出版物。

The greatest challenge in targeted cancer therapy is the lack of good therapeutic targets. Our current and future research is focused on investigating tumor-specific targets and their roles in tumorigenesis and metastases in addition to developing new therapeutic antibodies. We are studying three tumor antigens: (1) mesothelin, (2) CA125 (also known as MUC16) and (3) a new liver cancer antigen. As a promising new target for cancer immunotherapy, mesothelin is a cell surface biomarker that is highly expressed in mesothelioma, ovarian cancer and other malignant tumors. A mouse monoclonal antibody (mAb) variable fragment (Fv) specific for mesothelin, called SS1, was identified by Dr. Ira Pastans lab. It has been developed as an immunotoxin or a chimeric antibody and is currently in clinical trials for cancer therapy. Due to their low immunogenicity in patients, fully human monoclonal antibodies are the most desirable antibody format for clinical applications. We have screened phage display libraries and isolated several lead Fv molecules. In FY09, we (1) identified HN1 as a high affinity Fv specific for mesothelin, (2) developed a fully human IgG molecule based on the HN1 Fv, (3) showed that HN1 binds specifically to cell surface-associated mesothelin on mesothelioma and ovarian cancer cells, and (4) demonstrated its specific cytotoxic activity on cancer cells. We are currently evaluating HN1 as a candidate therapeutic agent to treat mesothelin-expressing tumors. Our work on HN1 was presented in several international meetings and yielded an international patent application in FY09. CA125 represents another important biomarker found on a majority of ovarian cancer and mesothelioma cells. Ovarian cancer and mesothelioma cells commonly spread within the peritoneal cavity via seeding to mesothelium-lined structures. The interaction between CA125 and mesothelin has been suggested to facilitate implantation and metastasis of tumors. We hypothesize that blocking the mesothelin-CA125 interaction may prevent or reverse metastasis and lead to an overall improved survival in cancer patients. A better understanding of how these two proteins interact may eventually aid in developing such therapy. To this end, my laboratory has vigorously examined the molecular interaction of mesothelin and CA125. In FY08, we (1) identified the binding site on mesothelin for CA125 and (2) revealed specific amino acids within the CA125-binding site of mesothelin that are involved in the interaction with CA125. In FY09, we developed novel anti-cancer therapeutics based on our work on CA125. In one approach, we have (1) developed a new fully human molecule specific for CA125-expressing cancer cells with high affinity and excellent specificity, and (2) found that this molecule can effectively block the binding of CA125 to mesothelin, thereby suggesting it could be used as an antagonist to inhibit tumor progression and spread in the peritoneal cavity. We are now studying its potential cytotoxic activity on cancer cells. In another approach, we have developed a novel hybrid molecule containing a pro-apoptotic peptide to induce cell death in CA125-expressing cancer cells. Our work targeting CA125 yielded one publication and an international patent application in FY09. In FY09, we became interested in a recently-identified liver cancer-specific antigen. Highly expressed in liver tumors but not in normal liver tissues, we were able to express and purify the liver cancer antigen as a recombinant Fc fusion protein. We are currently investigating its molecular interaction with a possible partner protein. This work may provide mechanistic insights on the origin of liver cancer and how liver cancer is associated with hepatitis viral infection. We plan to isolate high affinity monoclonal antibodies using hybridoma and phage display technology. In order to evaluate the efficacy of antibody therapeutics in a clinically relevant tumor model, my laboratory recently developed a novel malignant mesothelioma xenograft in mice. This model may be very useful for investigating antibodies and other therapeutics pertaining to the treatment of malignant mesothelioma. Previously, our work led to the development of mammalian cell display, a new method for antibody engineering. Based on this approach, functional single chain Fv antibodies (scFv) may then be expressed on human cells and high affinity scFv binders are rapidly identified and isolated from a combinatory Fv library. We are improving this method in order to make a large and permanent Fv library with the purpose of isolating high affinity antibodies for cancer therapy. In FY09, our work on antibody engineering yielded three publications and co-authored two publications.