Identifying antigens bound by novel scFvs targeting all subtypes of mesothelioma

描述（由申请人提供）：本申请的目的是建立一组内化间皮瘤细胞表面抗原的分子特性，这些抗原与一组新型内化人单链抗体（scFvs）结合，该抗体针对上皮样间皮瘤和肉瘤样间皮瘤（一种特别顽固的形式）。这项研究是建立在我们最近的工作基础上的，我们在活的间皮瘤细胞上选择了一个组合人抗体文库，并鉴定了一组内化的scFvs，这些scFvs可以原位结合所有类型的间皮瘤细胞，而不与正常的间皮瘤细胞结合，并且在体外介导小分子有效载荷向上皮样间皮瘤细胞和肉瘤样间皮瘤细胞内的有效递送。我们假设这些scFvs定义了新的间皮瘤抗原，它们比目前已知的抗原具有更大的特异性、亚型覆盖范围和治疗潜力。鉴定这些新的间皮瘤抗原将(1)允许进一步设计导联抗体，以提高亲和力、特异性、药代动力学和生物分布；(2)允许开发针对同一抗原的非重叠表位的额外抗体，这通常是诊断检测开发所必需的；(3)增强我们对涉及细胞膜的肿瘤行为的理解。这可能会导致进一步治疗开发的额外新靶点的识别，并且(4)为临床试验设计提供坚实的基础，例如基于靶点表达的患者预筛选。我们有独特的优势来实现这一目标，因为我们已经开发并适应了有效的方法来识别scFvs靶向的肿瘤抗原。这个建议的关键是我们新开发的基于真核细胞表面显示人类蛋白质组的抗原鉴定策略。此外，我们还与博士建立了功能性合作。Burlingame和Chalkley以及他们在加州大学旧金山分校的国家质谱分析设施来鉴定难以通过表达克隆方法鉴定的肿瘤抗原，如翻译后修饰抗原。我们建议使用我们已经开发和适应的方法和策略来系统地识别间皮瘤细胞表面抗原，这些抗原是由我们的内化人类scfv小组靶向的。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The objective of this proposal is to establish the molecular identities of a panel of mesothelioma cell surface antigens that are bound by a panel of novel internalizing human single chain antibodies that target both epithelioid and sarcomatoid (a particularly recalcitrant form) mesothelioma. Given that mesothelioma is a major occupation-related illness with no treatment to date, and little is known about mesothelioma cell surface antigens expressed by all subtypes of the disease, identification of mesothelioma antigens targeted by our panel of internalizing antibodies addresses a critical need and is likely to have a significant impact on the development of antibody-based therapeutics and diagnostics against mesothelioma.