Identifying antigens bound by novel scFvs targeting all subtypes of mesothelioma

描述（由申请人提供）：本提案的目的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单链抗体（scFv）结合，这些抗体靶向上皮样间皮瘤和肉瘤样间皮瘤（一种特别是恶性间皮瘤）。 所提出的研究建立在我们最近的工作基础上，其中我们选择了活间皮瘤细胞上的组合人抗体文库，并鉴定了一组内化scFv，其原位结合间皮瘤细胞的所有亚型，而不结合正常间皮瘤，并在体外介导小分子有效载荷向上皮样和肉瘤样间皮瘤细胞的有效细胞内递送。我们假设这些scFv定义了新的间皮瘤抗原，其具有比目前已知的抗原显著更高的特异性、亚型覆盖率和治疗潜力。这些新的间皮瘤抗原的鉴定将（1）允许进一步工程化先导抗体以改善亲和力、特异性、药代动力学和生物分布，（2）允许开发靶向相同抗原的非重叠表位的另外的抗体，这通常是诊断测定开发所需的，（3）增强我们对涉及细胞膜的肿瘤行为的理解，这可能导致鉴定用于进一步治疗开发的额外的新靶点，和（4）为临床试验设计提供合理的理论基础，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处于独特的地位，因为我们已经开发和调整了有效的方法来鉴定scFv靶向的肿瘤抗原。这个建议的关键是我们新开发的新的抗原鉴定策略的基础上真核细胞表面展示的人类蛋白质组。此外，我们还与Burlingame和Chalkley博士及其在UCSF的国家质谱设施建立了功能合作，以鉴定难以通过表达克隆方法鉴定的肿瘤抗原，例如后修饰抗原。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鉴定由我们的内化人scFv组靶向的间皮瘤细胞表面抗原。 公共卫生相关性：该提案的目的是建立一组间皮瘤细胞表面抗原的分子身份，所述间皮瘤细胞表面抗原被一组新的内化人单链抗体结合，所述抗体靶向上皮样间皮瘤和肉瘤样间皮瘤（特别是肉瘤形式）。鉴于间皮瘤是一种主要的职业相关疾病，迄今为止没有治疗方法，并且对该疾病的所有亚型所表达的间皮瘤细胞表面抗原知之甚少，我们的内化抗体组靶向的间皮瘤抗原的鉴定解决了一个关键需求，并且可能对基于抗体的间皮瘤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.