A new technology platform for studying protein function

自1975年单克隆抗体发明以来，以及最近各种体外抗体展示技术的发展，抗体已成为生物研究中最强大的工具之一，也是目前增长最快的一类新药实体。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质。原则上，单克隆抗体在细胞外环境中所能达到的效果，同样可以在细胞内使用体内抗体来达到。由于体内合成可以是组成性的或可诱导的，因此失活水平可以切换，这可能允许比基因缺失、反义或基于rnai的敲低策略观察到的更广泛的表型。此外，由于内体是蛋白质，与RNA相比，它们具有更长的半衰期，并且对目标分子也更有特异性。此外，可以设计或工程体内阻断特定靶蛋白的某些结构域，从而允许单个靶标的多个蛋白活性解耦。这可能对具有不止一种细胞活动的基本靶标特别有用。最后，由于体内可以是多价的，同时功能敲除两个或更多的细胞目标是可能的。基于上述特点，体内体有望在功能基因组学和/或蛋白质组学的靶标鉴定和验证中发挥重要而直接的作用。

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.