Mapping a clinically significant internalizing tumor epitope space

描述（由申请人提供）：本更新提案的目标是继续我们的方法，将噬菌体抗体展示与激光捕获微解剖（LCM）相结合，以鉴定新的内在化人单链抗体（scFvs），该抗体靶向转移性肿瘤细胞原位居住在其自然组织微环境中。这项工作是建立在我们之前的工作基础上的，我们开发并使用了这种新方法来识别针对原发性前列腺肿瘤的抗体。本提案旨在推进我们对前列腺肿瘤骨转移的研究，这是该疾病治疗和管理的主要挑战。本研究旨在利用我们基于lcm的人类抗体文库选择策略，识别新的靶向肿瘤细胞原位表达的肿瘤转移相关细胞表面抗原的scFvs，用于成像和治疗开发。此外，基于先前R01研究的新发现，我们发现肿瘤抗原如ALCAM在转移性前列腺癌细胞中被主动切割，我们将选择靶向ALCAM切割后跨膜残余的细胞表面新表位的抗体。具体来说，我们的目标是(1)开发针对ALCAM切割后跨膜残留物的新型人抗体。我们将利用这些抗体通过免疫组化研究ALCAM跨膜残余在前列腺肿瘤骨转移中的流行，并利用SPECT/CT成像技术评估抗残余scFvs在体内肿瘤靶向中的应用。(2)鉴定能够在转移性前列腺癌细胞组织微环境中高水平积累的快速内化抗体。我们建议使用我们之前开发和验证的基于lcm的新型抗体库选择策略来鉴定针对前列腺肿瘤骨转移的新型抗体，这些抗体具有以下特性：(a)与肿瘤细胞原位结合。(b)与活肿瘤细胞结合，从而识别肿瘤抗原的天然构象。(c)迅速内化并在肿瘤细胞内高水平积累。(d)在局部（皮下异种移植）和播散性前列腺肿瘤模型中，均表现出高水平的体内肿瘤摄取和低水平的非靶组织摄取。(3)鉴定结合新抗体的肿瘤抗原。我们将筛选一个在实验室开发的大型酵母表面显示的cDNA文库，以鉴定与我们的新型噬菌体抗体结合的肿瘤抗原。同时，我们将通过免疫沉淀和质谱分析来鉴定肿瘤抗原，从而鉴定翻译后修饰的抗原。

DESCRIPTION (provided by applicant): The goal of this renewal proposal is to continue our approach that combines phage antibody display with laser capture micro dissection (LCM) to identify novel internalizing human single chain antibodies (scFvs) that target metastatic tumor cells in situ residing in their natural tissue microenvironment. The work is built on our previous work where we developed and used this novel approach to identify antibodies targeting primary prostate tumor. This proposal aims to advance our study to prostate tumor bone metastases, a major challenge for treatment and management of the disease. This proposal aims to use our novel LCM-based human antibody library selection strategy to identify novel scFvs that target tumor metastases-associated cell surface antigens expressed by tumor cells in situ for imaging and therapy development. In addition, based on novel findings of the previous R01 study where we found that tumor antigens such as ALCAM are actively cleaved in metastatic prostate cancer cells, we will select antibodies that target cell surface neoepitopes on the post-cleavage transmembrane remnants of ALCAM. Specifically, we aim (1) to develop novel human antibodies that target ALCAM post-cleavage transmembrane remnants. We will use these antibodies to study the prevalence of ALCAM transmembrane remnants on prostate tumor bone metastases by immunohistochemistry, and evaluate utility of anti-remnant scFvs in tumor targeting in vivo using SPECT/CT imaging techniques. (2) To identify rapidly internalizing antibodies that are capable of high-level accumulation in target metastatic prostate cancer cells residing in their tissue microenvironment. We propose to use our previously developed and validated novel LCM-based antibody library selection strategies to identify novel antibodies targeting prostate tumor bone metastases with the following properties: (a) binding to tumor cells in situ. (b) Binding to live tumor cells and thus recognizing tumor antigens in their native conformations. (c) rapidly internalizing and accumulating to high levels inside tumor cells. (d) Exhibiting high-level tumor uptake in vivo and minimal uptake in off target tissues in both localized (subcutaneous xenograft) and disseminated prostate tumor models. (3) To identify tumor antigens bound by our novel antibodies. We will screen a large yeast surface displayed cDNA library developed in the lab to identify tumor antigens bound by our novel phage antibodies. In parallel, we will identify tumor antigens by immunoprecipitation and mass spectrometry analysis that allow identification of post- translational modified antigens.