Mapping a clinically significant internalizing tumor epitope space.

描述（由申请人提供）：本提案的目标是开发基于抗体文库的方法，以促进临床相关肿瘤特异性细胞表面抗原的有效鉴定，这些方法一般适用于细胞类型特异性谱系标记的鉴定。肿瘤细胞的异常生理部分反映在其细胞表面化学和分子组成的改变上。表面分子表达的进行性变化使肿瘤细胞能够有效地响应外部信号以生长和生存，与宿主组织相互作用，实现转移，并避免免疫监视。然而，肿瘤特异性细胞表面抗原的鉴定和靶向受到肿瘤细胞表面表位空间复杂性的阻碍。除蛋白质外，相关抗原还包括碳水化合物和其他翻译后修饰产物，这些产物无法通过基因组拷贝数或mRNA表达水平的研究来预测。本提案旨在开发基于组合抗体库的策略，允许有效识别肿瘤特异性细胞表面抗原，用于诊断，预后和治疗应用。具体而言，我们的目标是(1)开发一种基于流式细胞术分选的高通量减法选择策略，以显着提高选择效率并获得更多数量的肿瘤靶向噬菌体抗体。(2)在合适的基质环境下，通过原位选择针对前列腺癌细胞的噬菌体抗体来鉴定临床相关的肿瘤抗原。我们建议将抗体库技术与激光捕获显微解剖（LCM）相结合，以鉴定组织载玻片上针对单个肿瘤细胞的抗体。由此产生的抗体将具有非常高的识别临床相关肿瘤抗原的可能性。(3)建立肿瘤特异性抗体识别的肿瘤细胞表面抗原的分子鉴定。我们开发了一种新的组合酵母cDNA展示文库。特异性抗原抗体对将从该文库中通过流程分选进行鉴定。同时，我们将采用能够分析翻译后修饰的质谱方法对肿瘤抗原进行分析。这项研究将增加我们对肿瘤生理学的认识，并将有助于设计有效的治疗方法；这也将有助于确定翻译后修饰对肿瘤表位空间最终构成的确切贡献。

DESCRIPTION (provided by applicant): The goal of this proposal is to develop antibody library-based methods which promote efficient identification of clinically relevant tumor specific cell surface antigens, methods which are applicable to the identification of cell type-specific lineage markers in general. The abnormal physiology of tumor cells is reflected in part in the altered chemical and molecular composition of their cell surface. Progressive changes in surface molecule expression allow tumor cells to respond efficiently to external signals for growth and survival, to interact with host tissues, to achieve metastasis, and to avoid immune surveillance. The identification and targeting of tumor-specific cell surface antigens, however, is hampered by the complexity of the epitope space at the tumor cell surface. In addition to proteins, relevant antigens include carbohydrates and other post-translational modification products that cannot be predicted from studies of genomic copy number or mRNA expression levels. This proposal aims to develop combinatorial antibody library-based strategies that allow efficient identification of tumor specific cell surface antigens for diagnostic, prognostic and therapeutic applications. Specifically, we aim (1) To develop a high throughput subtractive selection strategy based on flow cytometric sorting to significantly improve selection efficiency and to obtain greater numbers of tumor- targeting phage antibodies. (2) To identify clinically relevant tumor antigens by selecting phage antibodies against prostate cancer cells in situ, within their proper stromal contexts. We propose to combine antibody library technology with laser capture microdissection (LCM) to identify antibodies against individual tumor cells on tissue slides. The resulting antibodies will have a very high likelihood of recognizing clinically relevant tumor antigens. (3) To establish the molecular identify of tumor cell surface antigens recognized by tumor-specific antibodies. We have developed a novel combinatorial yeast cDNA display library. Specific antigen-antibody pairs will be identified from this library following flow sorting. In parallel, we will pursue the analysis of tumor antigens by mass spectrometry methods which are capable of analyzing post-translational modifications. This study will increase our knowledge of tumor physiology and will facilitate the design of effective therapy; it will also help determine the exact contribution of post-translational modifications to the final makeup of the tumor epitope space.