Identifying antigens bound by novel scFvs targeting all subtypes of mesothelioma

项目概要/摘要： 本建议的目的是建立一个小组的内化间皮瘤的分子身份 由一组新的内化人单链抗体（scFv）结合的细胞表面抗原 其靶向上皮样和肉瘤样（特别是恶性形式）间皮瘤。 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类 在活的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scFv 原位间皮瘤细胞，不与正常间皮瘤结合，并介导有效的细胞内递送 的小分子有效载荷的上皮样和肉瘤样间皮瘤细胞在体外。我们假设 这些scFv定义了新间皮瘤抗原，其具有显著更高的特异性、亚型 覆盖率和治疗潜力。鉴别这些新的间皮瘤 抗原将（1）允许前导抗体的进一步工程化以提高亲和力，特异性， （2）允许开发靶向非重叠的其他抗体， 相同抗原的表位，这通常是诊断测定开发所需的，（3）增强我们的 了解涉及细胞膜的肿瘤行为，这可能导致识别其他肿瘤细胞。 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依据， 作为基于靶表达的患者的预筛选。 我们处于独特的地位，以实现这一目标，因为我们已经开发和适应有效的 鉴定scFv靶向的肿瘤抗原的方法。这个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的抗原鉴定策略。此外，本发明还提供了一种方法， 我们已经与伯林盖姆博士和乔克利博士以及他们的全国弥撒建立了功能性的合作关系。 UCSF的光谱分析设施用于鉴定难以通过表达克隆鉴定的肿瘤抗原 方法如免疫后修饰的抗原。我们建议使用我们所使用的方法和战略， 已经开发并适应系统地鉴定间皮瘤细胞表面抗原， 内化人scFv的组。

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.