Development and Utilization of Splice-specific Antibodies

摘要 在人类基因组中，选择性剪接事件有助于产生具有 比在蛋白质编码基因库中观察到的更大的多样性。换句话说，广大的 大多数人类基因都可以产生许多不同的转录本，随后会产生多个 蛋白质异构体。然而，异常剪接可以产生新的亚型或正常情况下的增加 低水平的亚型导致潜在的有害影响，包括蛋白质功能改变，蛋白质- 蛋白质相互作用以及蛋白质复合体和途径的重塑。事实上，这一增长 在这些选择性剪接的异构体中，现在被认为是致癌的主要贡献者 表型，如肿瘤的发展和支持细胞侵袭的新血管 和扩散。实现生物影响、预后和治疗的中心挑战 跨多种癌症类型的选择性剪接异构体的潜力一直是缺乏技术 可用于明确区分天然形式中高度相似的蛋白质变体。当前 在蛋白质水平上检测和研究交替剪接异构体的方法仅依赖于 间接方法，如基于标签的检测和非特异性亲和试剂，无法 区分多种蛋白质异构体。在此，我们提出了一种新的方法来开发高 使用靶向方法针对选择性剪接蛋白异构体 具有近氨基酸特异性的剪接位点连接。利用这项技术，可以产生抗体 专门检测所需的、选择性剪接的蛋白质异构体，而不与天然的 全长表格。这一技术进步将加速肿瘤学研究，并使科学家 研究高价值和新的蛋白质异构体及其在特定肿瘤中的直接作用 表型、转移潜能、肿瘤分级特异性和生存率，并提供验证 评估抗癌选择性剪接抑制剂治疗潜力的方法。

ABSTRACT In the human genome, alternative splicing events facilitate the generation of a proteome with a greater diversity than is observed in the protein-coding gene repertoire. In other words, the vast majority of human genes can each produce numerous different transcripts and subsequently multiple protein isoforms. However, aberrant splicing can produce an increase in novel isoforms or normally low-level isoforms leading to potentially detrimental effects including altered protein function, protein- protein interactions as well as remodeling of protein complexes and pathways. In fact, the increase in these alternatively-spliced isoforms is now recognized as a major contributor to oncogenic phenotypes, such as the development of tumors and new blood vessels by supporting cell invasion and proliferation. A central challenge to realizing the biological impact, prognostic and therapeutic potential of alternatively-spliced isoforms across multiple cancer types has been a lack of technology available for unambiguously differentiating highly similar protein variants in their native forms. Current methods of detecting and studying alternatively-spliced isoforms at the protein level rely solely on indirect methods such as tag-based detection and nonspecific affinity reagents that are unable to discriminate among multiple protein isoforms. Herein, we propose a novel approach to develop highly specific antibodies against alternatively-spliced protein isoforms using a targeting method at the splice site junction with near amino acid specificity. With this technology, antibodies can be generated to specifically detect a desired, alternatively-spliced protein isoform without cross-reactivity to the native full-length form. This technological advance will accelerate oncology research and enable scientists to study both high-value and novel protein isoforms and their direct role in specific cancer phenotypes, metastatic potential, tumor grade specificity and survival rates, and provide a validation means for assessing the therapeutic potential of anticancer alternative-splicing inhibitors.