Mapping a clinically significant internalizing tumor epitope space

描述(申请人提供)：这项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显微切割(LCM)相结合，以识别新的内化人单链抗体(ScFv)，该单链抗体针对驻留在其自然组织微环境中的原位转移肿瘤细胞。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的方法来识别针对原发性前列腺癌的抗体。这项建议旨在推进我们对前列腺癌骨转移的研究，这是治疗和管理该疾病的主要挑战。这项建议旨在使用我们基于LCM的新型人类抗体库选择策略来识别针对肿瘤细胞原位表达的肿瘤转移相关细胞表面抗原的新型单链抗体，用于成像和治疗开发。此外，基于之前R01研究的新发现，我们发现在转移性前列腺癌细胞中，肿瘤抗原如alcam被积极切割，我们将选择针对切割后的alcam跨膜残留物上的细胞表面新表位的抗体。具体地说，我们的目标是(1)开发针对alcam裂解后跨膜残留物的新型人类抗体。我们将利用这些抗体通过免疫组织化学的方法研究前列腺癌骨转移中Alcam跨膜残留物的分布情况，并利用SPECT/CT成像技术评价抗残留物单链抗体在体内肿瘤靶向中的应用。(2)识别能够在驻留在其组织微环境中的转移性前列腺癌靶细胞中高水平积聚的快速内化抗体。我们建议使用我们先前开发和验证的基于LCM的新型抗体库选择策略来鉴定针对前列腺癌骨转移的新型抗体，其特性如下：(A)与肿瘤细胞原位结合。(B)与活的肿瘤细胞结合，从而识别天然构象中的肿瘤抗原。(C)在肿瘤细胞内迅速内化并积累到高水平。(D)在局部(皮下异种移植)和播散性前列腺癌模型中，显示出体内高水平的肿瘤摄取和靶外组织的最低摄取。(3)鉴定与新抗体结合的肿瘤抗原。我们将筛选实验室开发的一个大型酵母表面展示的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.