Conformation-specific antibodies to protein kinases

蛋白激酶构象特异性抗体

• 批准号: 6933722
• 负责人: ROBERT PYTELA
• 金额: $ 9.96万
• 依托单位: EPITOMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-28 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6933722
• 关键词: biological signal transduction; conformation; laboratory rabbit; monoclonal antibody; phosphorylation; protein kinase; protein protein interaction; technology /technique development

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.

描述（由申请人提供）：为了了解癌症的发病机制，阐明有助于致癌转化的细胞信号传导途径至关重要。蛋白激酶和衔接蛋白的复杂网络起着核心作用。这些分子经历一系列构象变化，因为它们彼此相互作用并被激活。因此，对单克隆抗体（mAb）的需求不断增长，所述单克隆抗体识别依赖于信号传导蛋白的活化状态的构象表位。 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产生磷酸肽特异性抗体。这样的抗体可以是评估靶分子磷酸化状态的有力工具，但它们具有一定的局限性：由于肽免疫原可能不反映完整蛋白中相应序列的天然构象，因此所得mAb通常仅在变性条件下与靶蛋白反应，因此许多mAb仅适用于Western印迹系统，排除了对完整细胞或组织的研究。2.即使肽免疫原确实模拟了相应蛋白质的构象，磷酸化位点也常常被结合蛋白（例如，Src-同源结构域2），并且在天然条件下不可用于抗体结合。为了弥合靶向免疫（合成肽）和构象比对（肽与相应蛋白质）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免疫。此外，我们建立了一个高效的兔杂交瘤系统，并已表明，兔单克隆抗体提出的合成肽免疫原是上级优于小鼠单克隆抗体和兔多克隆抗体。我们建议联合收割机，这兔单克隆抗体系统与我们的肽的设计方法，并应用这些方法的构象特异性抗体的蛋白激酶参与信号通路，与致癌转化的发展。我们的方法将集中在预测的免疫原性肽序列，不包括磷酸化位点。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。我们以前已经开发了构象特异性小鼠单克隆抗体的c-Src，这将与兔单克隆抗体在当前项目中提出的。如果该方法被证明是成功的，II期研究将被设计为开发优化的抗体试剂，用于监测Src家族激酶以及参与致癌转化的其他蛋白激酶的活化状态。 拟议的商业应用：最初，本项目中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的联盟，将这些mAb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。