A new technology platform for studying protein function

描述(申请人提供)：自从1975年单抗发明以来，以及最近各种体外抗体展示技术的发展，抗体已经成为生物学研究中最强大的工具之一，目前是增长最快的新药实体类别。一种显示出巨大前景的分子形式是细胞内抗体或体内抗体，它利用免疫球蛋白的特异性和多样性，通过在体内表达抗体来靶向广泛的细胞内蛋白。原则上，在细胞外环境中通过单抗可以实现的任何东西，都可以类似地使用体内在细胞内实现。由于体内合成可以是结构性的或可诱导的，可以切换失活水平，这可能允许比基因缺失、反义或基于RNAi的敲除策略观察到的更广泛的表型。此外，由于体内是蛋白质，与RNA相比，它们的半衰期要长得多，而且对其目标分子也更具特异性。此外，有可能设计或设计体内以阻断特定靶蛋白的某些区域，从而允许单个靶蛋白的多个蛋白质活动的解偶联。这可能被证明对具有不止一种细胞活动的基本目标特别有用。最后，由于体内可以是多价的，两个或多个细胞靶点的同时功能敲除是可能的。基于上述特征，体内有望在功能基因组学和/或蛋白质组学的靶标识别和验证方面发挥重要和直接的作用。 这项研究的长期目标是开发一种蛋白质组范围的体内蛋白质组，用于探测和调节活细胞内的蛋白质活动。这一特殊应用的目标是创建一种基于细菌双精氨酸易位(TAT)途径的新型平台技术，使单链体内针对几乎任何细胞内靶蛋白的快速、一步遗传选择成为可能。为了实现这一应用的总体目标，提出了以下具体目标：(1)基于细菌TAT系统的独特机制特征开发基因选择，以分离体内-抗原配对；以及(2)设计特异性抑制生物过程的体内。胞内抗体是一类在细胞内发挥功能(例如，结合同源抗原)的抗体分子，由于其特异性和多样性，有可能阻止、抑制、改变甚至增强大量的生物过程。因此，这些研究的重点是开发一个快速、大规模合成体内的技术平台，可以用作(I)功能基因组学试剂，使新的基因产品能够表征并确认这些基因产品是潜在的药物靶点，以及(Ii)用于治疗癌症、艾滋病或神经退行性疾病等人类疾病的药物实体。

DESCRIPTION (provided by applicant): Ever since the invention of monoclonal antibodies in 1975 and, more recently, the development of various in vitro antibody display technologies, antibodies have become one of the most powerful tools in biological research and are presently the fastest growing category of new drug entities. One molecular format that shows great promise is the intracellular antibody or intrabody that exploits the specificity and diversity of immunoglobulins to target a wide range of intracellular proteins by expressing the antibody in vivo. In principle, whatever can be achieved by a monoclonal antibody in the extracellular environment can be similarly achieved inside of a cell using an intrabody. Since intrabody synthesis can be constitutive or inducible, the level of inactivation can be toggled which might allow for a wider range of phenotypes than can be observed with gene deletion, antisense or RNAi-based knockdown strategies. Further, since intrabodies are proteins, they possess a much longer half-life compared to RNA and are also more specific to their target molecules. Also, it is possible to design or engineer intrabodies to block certain domains of a particular target protein, thus allowing for the decoupling of multiple protein activities of a single target. This might prove particularly useful for essential targets that have more than one cellular activity. Finally, since intrabodies can be multivalent, simultaneous functional knockout of two or more cellular targets is possible. Based on the above features, intrabodies are expected to play an important and immediate role for target identification and validation in functional genomics and/or proteomics. The long-term objective of this research effort is to develop a proteome-wide repertoire of intrabodies for probing and modulating protein activities inside living cells. The objective of this particular application, which is the first step towards our long-term goal, is to create a novel platform technology based on the bacterial twin-arginine translocation (Tat) pathway that enables rapid, one-step genetic selection of single-chain intrabodies against virtually any intracellular target protein. To accomplish the overall objective of this application, the following specific aims are proposed: (1) develop a genetic selection based on unique mechanistic features of the bacterial Tat system for isolating intrabody-antigen pairings; and (2) engineer intrabodies that specifically inhibit biological processes. Intrabodies are an emerging class of antibody molecules that function (e.g., bind their cognate antigen) intracellularly and, owing to their specificity and diversity, have the potential to block, suppress, alter or even enhance a vast array of biological processes. Therefore, the focus of these studies is to develop a technology platform for rapid, large-scale synthesis of intrabodies that could be used as (i) functional genomics reagents that enable characterization of novel gene products and validation of these gene products as potential drug targets and (ii) drug entities that be used in the treatment of human disorders such as cancer, AIDS or neuro-degenerative disorders.