Engineering intrabodies for knockdown of target proteins in cancer cells

工程化体内抗体以敲低癌细胞中的靶蛋白

• 批准号: 8125436
• 负责人: Amy J Karlsson
• 金额: $ 4.86万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2012-05-22
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8125436
• 关键词: Antibodies; Arginine; Biological Process; Cell Differentiation process; Cell Nucleus; Cell model; Cells; Cytoplasm; Development; Engineering; Environment; Escherichia coli; Gene Deletion; Generations; Genes; Goals; HL60; Half-Life; Human; Immunoglobulins; Intracellular Membranes; Knock-out; Life; MAP Kinase Gene; Mediating; Membrane; Molecular; Monoclonal Antibodies; Pathway interactions; Phenotype; Proteins; Proteome; Quality Control; RNA; RNA Interference; Research; Research Training; Role; Screening procedure; Signal Transduction; Specificity; Therapeutic; Therapeutic Intervention; Time; Training Programs; Twin Multiple Birth; Work; base; biological research; cancer cell; cellular targeting; design; disulfide bond; in vivo; leukemia; loss of function; novel; prevent; protein function; proto-oncogene protein c-cbl; tool

DESCRIPTION (provided by applicant): Intracellular antibodies or "intrabodies" exploit the specificity and diversity of immunoglobulins to knockdown the function of intracellular proteins through antibody expression inside cells. In addition to their specificity, intrabodies offer several advantages over the gene deletion and RNAi-based strategies that are commonly used to eliminate protein function. For example, intrabodies can be engineered to block only certain domains of a protein, allowing the ability to target specific functions of a protein for knockdown. The ability to decouple functions of a protein target could be especially useful when the protein targeted for knockdown has an essential function. In addition, intrabodies can be multivalent, so a single intrabody could be engineered to simultaneously eliminate the function of two or more intracellular targets. Although intrabodies show great promise, generating antibodies for specific intracellular proteins is generally more time-consuming and labor- intensive than gene-based knockout approaches. The reducing environment of the cell cytoplasm and nucleus also presents a technical challenge, since it prevents the formation of intrachain disulfide bonds that are essential for the folding of nearly all antibodies. The ultimate objective for this work is to overcome these limitations and develop a proteome-wide set of intrabodies that can be used to study and modulate the activity of intracellular proteins. This research training program works toward the long-term objective by engineering functional intrabodies for defined protein targets in human cancer cells. The specific aims of this research training program are to (1) isolate novel intrabodies against targets involved in MAPK signaling and differentiation and (2) validate the engineered intrabodies against their targets in HL-60 cells. Intrabodies will be engineered in Escherichia coli using an inner membrane antibody display platform that incorporates the intracellular folding quality control mechanism of the bacterial twin-arginine translocation (Tat) pathway. The intrabodies will target CD38 and c-Cbl, proteins that have critical roles in MAPK signaling. Following screening of intrabodies displayed on the E. coli inner membrane, the intrabodies will be expressed in HL-60 cells to validate their functionality and determine their effect on HL-60 differentiation. This research will result in an intrabody-mediated protein knockdown strategy that will provide valuable information about the molecular mechanisms involved in HL-60 differentiation, and the information gained could be used to determine whether specific intracellular proteins are viable targets for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Because of their diversity and specificity, intracellular antibodies (intrabodies) have the potential to alter a vast array of biological processes by functioning inside of living cells. Intrabodies that target signaling and differentiation targets in human leukemia cells will be engineered and validated. The project will develop strategies that can be applied to other cellular models to determine whether specific intracellular proteins are viable targets for therapeutic intervention.

描述（由申请人提供）：细胞内抗体或“胞内抗体”利用免疫球蛋白的特异性和多样性，通过细胞内的抗体表达来敲低细胞内蛋白质的功能。除了它们的特异性之外，胞内抗体还提供了几个优于基因缺失和基于RNAi的策略的优点，这些策略通常用于消除蛋白质功能。例如，胞内抗体可以被工程化以仅阻断蛋白质的某些结构域，从而允许靶向蛋白质的特定功能以进行敲低的能力。当靶向敲低的蛋白质具有基本功能时，使蛋白质靶标的功能解耦的能力可能特别有用。此外，胞内抗体可以是多价的，因此可以工程化单个胞内抗体以同时消除两个或更多个胞内靶标的功能。尽管胞内抗体显示出巨大的前景，但是产生针对特定胞内蛋白的抗体通常比基于基因的敲除方法更耗时且劳动密集。细胞质和细胞核的还原环境也提出了一个技术挑战，因为它阻止了链内二硫键的形成，而链内二硫键是几乎所有抗体折叠所必需的。这项工作的最终目标是克服这些局限性，并开发一组蛋白质组范围内的胞内抗体，可用于研究和调节细胞内蛋白质的活性。该研究培训计划通过为人类癌细胞中定义的蛋白质靶点设计功能性胞内抗体来实现长期目标。本研究培训计划的具体目的是（1）分离针对MAPK信号传导和分化相关靶点的新型胞内抗体，以及（2）在HL-60细胞中验证针对其靶点的工程化胞内抗体。胞内抗体将在大肠杆菌中使用内膜抗体展示平台进行工程化，该平台结合了细菌双精氨酸易位（达特）途径的胞内折叠质量控制机制。胞内抗体将靶向CD 38和c-Cbl，这些蛋白在MAPK信号传导中具有关键作用。在筛选在E. coli内膜，胞内抗体将在HL-60细胞中表达，以验证其功能并确定其对HL-60分化的影响。这项研究将导致胞内抗体介导的蛋白质敲低策略，这将提供有关HL-60分化的分子机制的有价值的信息，所获得的信息可用于确定特定的细胞内蛋白质是否是治疗干预的可行靶点。 公共卫生相关性：由于它们的多样性和特异性，细胞内抗体（胞内抗体）有可能通过在活细胞内发挥作用来改变大量的生物过程。靶向人白血病细胞中的信号传导和分化靶点的胞内抗体将被工程化和验证。该项目将开发可应用于其他细胞模型的策略，以确定特定的细胞内蛋白质是否是治疗干预的可行靶点。