Mapping a clinically significant internalizing tumor epitope space

描述（由申请人提供）：该更新提案的目标是继续我们的方法，该方法将噬菌体抗体展示与激光捕获显微切割（LCM）相结合，以鉴定新型内化人单链抗体（scFv），其靶向原位驻留在其天然组织微环境中的转移性肿瘤细胞。这项工作是建立在我们以前的工作，我们开发和使用这种新的方法来确定针对原发性前列腺肿瘤的抗体。该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。该提案旨在使用我们的新型基于LCM的人抗体文库选择策略来鉴定靶向肿瘤细胞原位表达的肿瘤坏死酶相关细胞表面抗原的新型scFv，用于成像和治疗开发。此外，基于先前R 01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切割后跨膜残留物上的细胞表面新表位的抗体。具体而言，我们的目标是（1）开发靶向ALCAM切割后跨膜残留物的新型人抗体。我们将使用这些抗体通过免疫组织化学研究ALCAM跨膜残留物在前列腺肿瘤骨转移中的患病率，并使用SPECT/CT成像技术评估抗残留物scFv在体内肿瘤靶向中的效用。(2)鉴定能够在组织微环境中的靶向转移性前列腺癌细胞中高水平蓄积的快速内化抗体。我们建议使用我们先前开发和验证的基于LCM的新型抗体文库选择策略来鉴定具有以下性质的靶向前列腺肿瘤骨转移的新型抗体：（a）原位结合肿瘤细胞。(b)与活肿瘤细胞结合，从而识别天然构象的肿瘤抗原。(c)迅速内化并在肿瘤细胞内积累到高水平。(d)在局部（皮下异种移植物）和播散性前列腺肿瘤模型中均显示出体内高水平肿瘤摄取和脱靶组织中的最小摄取。(3)以鉴定与我们的新型抗体结合的肿瘤抗原。我们将筛选在实验室中开发的大的酵母表面展示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.