Identifying antigens bound by novel scFvs targeting all subtypes of mesothelioma

描述(由申请人提供)：这项建议的目标是建立一组内化间皮瘤细胞表面抗原的分子身份，这些抗原被一组新型内化人类单链抗体(ScFv)结合，该单链抗体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，我们在活体间皮瘤细胞上选择了一个组合的人抗体库，并鉴定了一组内化单链抗体，它们原位结合所有亚型的间皮瘤细胞，而不与正常间皮细胞结合，并在体外介导小分子有效载荷的细胞内递送到上皮样和肉瘤样间皮瘤细胞。我们假设这些单链抗体定义了新的间皮瘤抗原，这些抗原比目前已知的抗原具有更大的特异性、亚型覆盖率和治疗潜力。鉴定这些新的间皮瘤抗原将有助于(1)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)允许开发针对同一抗原的非重叠表位的额外抗体，这通常是诊断分析开发所必需的，(3)提高我们对涉及细胞膜的肿瘤行为的了解，这可能导致为进一步的治疗开发确定更多新的靶点，以及(4)为临床试验设计提供可靠的理论基础，例如基于靶点表达对患者进行预筛选。我们在实现这一目标方面具有得天独厚的优势，因为我们已经开发并采用了有效的方法来识别单链抗体靶向的肿瘤抗原。这一建议的关键是我们新开发的基于真核细胞表面展示的人类蛋白质组的抗原识别策略。此外，我们还与Burlingame博士和Chalkley博士以及他们在加州大学旧金山分校的国家质谱学设施建立了功能性合作，以识别难以通过表达克隆方法(如翻译后修饰抗原)识别的肿瘤抗原。我们建议使用我们开发和调整的方法和策略来系统地鉴定我们的人单链抗体内化小组靶向的间皮瘤细胞表面抗原。 公共卫生相关性：这项建议的目的是确定一组间皮瘤细胞表面抗原的分子身份，这些抗原被一组新型的内化人类单链抗体结合，这些单链抗体针对上皮样和肉瘤样间皮瘤(一种特别顽固的形式)。鉴于间皮瘤是一种主要的职业性疾病，到目前为止还没有治疗方法，而且对所有疾病亚型所表达的间皮瘤细胞表面抗原知之甚少，因此，我们的内化抗体小组针对间皮瘤抗原的识别满足了一项迫切的需求，并可能对间皮瘤基于抗体的治疗和诊断的发展产生重大影响。

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.