Conformation-specific antibodies to protein kinases

描述（申请人提供）：为了了解癌症的发病机制，阐明促进致癌转化的细胞信号通路将是至关重要的。蛋白激酶和适配蛋白的复杂网络起着核心作用。当这些分子相互作用并被激活时，它们会经历一系列的构象变化。因此，对单克隆抗体（mab）的需求不断增长，单克隆抗体可以识别依赖于信号蛋白激活状态的构象表位。由于构象变化通常与磷酸化事件相关，因此最广泛使用的方法是培养磷酸化肽特异性抗体。这类抗体是评估靶分子磷酸化状态的有力工具，但它们有一定的局限性：1。由于肽免疫原可能不能反映完整蛋白中相应序列的天然构象，因此产生的单抗通常只在变性条件下与靶蛋白反应，因此许多单抗仅适用于Western blotting系统，无法对完整细胞或组织进行研究。2. 即使肽免疫原确实模仿相应蛋白的构象，磷酸化位点在磷酸化后通常被结合蛋白（例如src同源结构域2）占据，并且在天然条件下无法用于抗体结合。为了弥补靶向免疫（合成肽）和构象比对（肽与相应蛋白）之间的技术差距，我们开发了一系列算法和数据库来优化肽设计，以实现有效的免疫。此外，我们建立了高效的兔杂交瘤系统，并证明兔单克隆抗体培养合成多肽免疫原优于小鼠单克隆抗体和兔多克隆抗体。我们建议将这种兔单抗系统与我们的肽设计方法结合起来，并将这些方法应用于开发与致癌转化相关的信号通路中涉及的蛋白激酶的构象特异性抗体。我们的方法将集中于预测不包括磷酸化位点的免疫原性肽序列。I期研究旨在证明该方法的有效性，并将重点关注Src家族激酶。我们之前已经开发了针对c-Src的构象特异性小鼠单克隆抗体，将与本项目中培养的兔单克隆抗体进行比较。如果该方法被证明是成功的，II期研究将被设计用于开发优化的抗体试剂，以监测Src家族激酶的激活状态，以及参与致癌转化的其他蛋白激酶。建议的商业应用：首先，该项目开发的抗体将作为研究试剂进行商业化。此外，我们将寻求与制药公司合作，将这些单克隆抗体应用于癌症、炎症性疾病和自身免疫性疾病等疾病的诊断试剂。

DESCRIPTION (provided by applicant): In order to understand the pathogenesis of cancer, it will be critical to elucidate the cellular signaling pathways that contribute to oncogenic transformation. Complex networks of protein kinases and adaptor proteins play a central role. These molecules undergo a series of conformational changes, as they interact with each other and become activated. Therefore, there is a growing demand for monoclonal antibodies (mAbs), which recognize conformational epitopes that are dependent on the activation state of the signaling protein. Since conformation changes are often correlated with phosphorylation events, the most widely used approach has been to raise phosphopeptide-specific antibodies. Such antibodies can be powerful tools to assess the phosphorylation status of the target molecule, but they have certain limitations: 1. Since peptide immunogens may not reflect the native conformation of the corresponding sequences in the intact protein, resulting mAbs often react with target proteins under denatured condition exclusively, and thus many mAbs are only applicable to the Western blotting system, precluding studies on intact cells or tissues. 2. Even if the peptide immunogens do mimic the conformation of the corresponding proteins, the phosphorylation sites are often occupied by binding proteins (e.g., Src-homology domain 2) upon phosphorylation, and are unavailable for antibody binding under native conditions. To bridge this technical gap between targeted immunization (synthetic peptide) and conformational alignment (peptide vs. corresponding protein), we have developed a series of algorithms and databases to optimize peptide design for effective immunization. In addition, we have established a highly efficient rabbit hybridoma system, and have shown that rabbit mAbs raised to synthetic peptide immunogens are superior to both mouse mAbs and rabbit polyclonals. We propose to combine this rabbit mAb system with our peptide-design method, and apply these methods to the development of conformation-specific antibodies to protein kinases involved in signaling pathways that are related to oncogenic transformation. Our approach will focus on predicted immunogenic peptide sequences that do not include phosphorylation sites. Phase I studies are designed to demonstrate the validity of the approach, and will focus on Src family kinases. We have previously developed conformation-specific mouse mAbs to c-Src, which will be compared with rabbit mAbs raised in the current project. If the approach proves to be successful, Phase II studies will be designed to develop optimized antibody reagents for monitoring the activation state of Src family kinases, as well as other protein kinases involved in oncogenic transformation. Proposed commercial applications: Initially, antibodies developed in this project will be commercialized as research reagents. In addition, we will seek alliances with pharmaceutical companies to apply these mAbs as diagnostic reagents for diseases such as cancer, inflammatory diseases, and autoimmune diseases.