Identifying antigens bound by novel scFvs targeting all subtypes of mesothelioma

项目摘要/摘要： 这项建议的目的是确定一组内化性间皮瘤的分子身份。 一组新型内化人单链抗体(ScFv)结合的细胞表面抗原 它同时针对上皮样和肉瘤样间皮瘤(一种特别顽固的形式)。 这项拟议的研究建立在我们最近的工作基础上，我们选择了一个组合人 活体间皮瘤细胞上的抗体库，并鉴定了一组与所有亚型结合的内化单链抗体 不与正常间皮细胞结合的原位间皮瘤细胞，并介导有效的细胞内递送 体外对上皮样和肉瘤样间皮瘤细胞的小分子有效载荷。我们假设 这些单链抗体定义了新的间皮瘤抗原，这些抗原具有明显更强的特异性、亚型 比目前已知的抗原更具覆盖率和治疗潜力。这些新型间皮瘤的鉴定 抗原将(1)允许进一步改造先导抗体以提高亲和力、特异性、 药代动力学和生物分布，(2)允许开发针对非重叠的额外抗体 相同抗原的表位，这通常是诊断分析发展所必需的，(3)增强我们的 对涉及细胞膜的肿瘤行为的理解，这可能导致识别其他 为进一步的治疗开发提供新的靶点，以及(4)为临床试验设计提供合理的理论基础 作为基于目标表达的患者预筛选。 我们处于独特的地位，能够实现这一目标，因为我们已经开发和调整了有效的 方法鉴定单链抗体靶向的肿瘤抗原。这一提议的关键是我们新开发的小说 基于真核细胞表面展示的人类蛋白质组抗原识别策略。此外, 我们已经与Burlingame博士和Chalkley博士以及他们的全国弥撒建立了职能合作 加州大学旧金山分校的光谱设备用于鉴定通过表达克隆难以识别的肿瘤抗原 翻译后修饰抗原等方法。我们建议使用我们所采用的方法和策略 已经开发和改造成系统地鉴定我们的靶向的间皮瘤细胞表面抗原 内化人单链抗体的小组。

Project Summary/Abstract: The objective of this proposal is to establish the molecular identities of a panel of internalizing mesothelioma cell surface antigens that are bound by a panel of novel internalizing human single chain antibodies (scFvs) that target both epithelioid and sarcomatoid (a particularly recalcitrant form) mesothelioma. The proposed study is built upon our recent work where we have selected a combinatorial human antibody library on live mesothelioma cells and identified a panel of internalizing scFvs that bind to all subtypes of mesothelioma cells in situ with no binding to normal mesothelium, and mediate efficient intracellular delivery of small molecule payloads to both epithelioid and sarcomatoid mesothelioma cells in vitro. We hypothesize that these scFvs define novel mesothelioma antigens that have significantly greater specificity, subtype coverage and therapeutic potential than currently known antigens. Identification of these novel mesothelioma antigens would (1) allow further engineering of the lead antibodies to improve affinity, specificity, pharmacokinetics and biodistribution, (2) allow development of additional antibodies targeting non-overlapping epitopes of the same antigen, which is often required for diagnostic assay development, (3) enhance our understanding of tumor behaviors that involve the cell membrane, which may lead to identification of additional novel targets for further therapeutic development, and (4) provide a sound rationale for clinical trial design such as pre-screening of patients based on target expression. We are uniquely positioned to accomplish this goal as we have developed and adapted effective methods to identify tumor antigens targeted by scFvs. Key to this proposal is our newly developed novel antigen identification strategies based on eukaryotic cell surface display of the human proteome. In addition, we have established functional collaborations with Drs. Burlingame and Chalkley and their National Mass Spectrometry Facility at UCSF to identify tumor antigens that are difficult to identify by expression cloning methods such as post-translationally modified antigens. We propose to use the methods and strategies that we have developed and adapted to systematically identify mesothelioma cell surface antigens targeted by our panel of internalizing human scFvs.