Rapid isolation of high-affinity human antibodies from large synthetic libraries

从大型合成文库中快速分离高亲和力人类抗体

• 批准号: 7803512
• 负责人: MATTHEW P DELISA
• 金额: $ 20万
• 依托单位: VYBION, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7803512
• 关键词: Accounting; Affinity; Antibodies; Antibody Affinity; Antibody Therapy; Antigen Targeting; Antigens; Arginine; Binding; Biological; Biotechnology; Cell surface; Cells; Clinical; Clinical Trials; Collection; Complex; Culture Media; Cytoplasm; Development; Diagnostics Research; Disease; Engineering; Ensure; Environment; Enzymes; Evolution; Future; Genes; Goals; Health; Human; Immobilization; Immunization; Immunoglobulin Fragments; Immunotoxins; Inflammatory; Lactamase; Libraries; Light; Malignant Neoplasms; Marketing; Membrane; Methods; Molecular; Monoclonal Antibodies; Nature; Neurodegenerative Disorders; Pathway interactions; Peptide Hydrolases; Peptides; Phage Display; Pharmaceutical Preparations; Production; Property; Protein Export Pathway; Proteins; Quality Control; Reagent; Recombinant Antibody; Recombinant Proteins; Relative (related person); Reporter; Research; Resistance; Screening procedure; Series; Solid; Specificity; Staging; Structure; Surface; System; Techniques; Technology; Twin Multiple Birth; Virus Diseases; Yeasts; antigen antibody binding; antigen binding; base; combinatorial; commercial application; cost; design; directed evolution; drug development; genetic selection; human disease; innovation; interest; new technology; novel; novel therapeutics; periplasm; polypeptide; pre-clinical; protein expression; protein folding; public health relevance; research study; secretory protein; success; tat Protein; therapeutic protein; therapeutic target; tool

DESCRIPTION (provided by applicant): With over 300 therapeutic proteins currently in various stages of clinical trials, the road to a healthier future will require new methods for producing safer and less expensive recombinant proteins. In particular, next generation therapeutics derived from monoclonal antibodies (e.g. Fab, scAb, scFv, immunotoxins, etc) show great clinical promise in treating a range of human disorders including bacterial and viral infections, cancer, inflammatory diseases and neurodegenerative disorders. Currently, the bacterial Sec protein export pathway is the dominant mechanism for protein display (e.g., phage display, cell surface display) and also for the expression and engineering of small, non-glycosylated antibody fragments. However, due to a number of limitations associated with Sec export, bacterial production of stable, high-affinity antibody fragments in high yields for preclinical and clinical trials can be a serious bottleneck in the antibody drug pipeline. Therefore, the overall objective of the proposal is to develop a versatile antibody expression and engineering platform by capitalizing on the remarkable properties of the recently discovered bacterial twin-arginine translocation (Tat) pathway. Towards this objective, the current proposal encompasses the following specific aims: (1) creation of synthetic libraries of single-chain Fv (scFv) antibody fragments; and (2) application of a proprietary Tat-based genetic selection strategy for efficient isolation of super-stable, ultra- high affinity human scFvs against disease-relevant antigens. The proposed studies are expected to result in a rapid, single-step selection platform for the discovery of scFv sequences that fold and function in intra- or extra-cellular environments. Successful completion of the proposed studies is expected to result in a powerful new technology for generating potent antibody-based biopharmaceuticals, diagnostics and research reagents. PUBLIC HEALTH RELEVANCE: By 2008, engineered antibody fragments are predicted to account for >30% of all revenues in the biotechnology market and will be used to treat a wide array of human diseases including bacterial and viral infections, cancer, inflammatory diseases and neurodegenerative disorders. Since antibody therapies are an increasingly large fraction of the drugs in development, with ever escalating increases in the cost of drug development, any improvements to the production or discovery of efficacious antibody fragments will have a significant impact on human health. Thus, this proposal seeks to develop a novel bacterial selection strategy for rapid, low-cost isolation of potent human antibodies against virtually any target antigen of interest.

描述（由申请人提供）：目前有超过300种治疗性蛋白质处于不同的临床试验阶段，通往更健康的未来之路将需要新的方法来生产更安全、更便宜的重组蛋白质。特别是，从单克隆抗体衍生的下一代疗法（如Fab、scAb、scFv、免疫毒素等）在治疗一系列人类疾病（包括细菌和病毒感染、癌症、炎症性疾病和神经退行性疾病）方面显示出巨大的临床前景。目前，细菌Sec蛋白输出途径是蛋白质展示（如噬菌体展示、细胞表面展示）的主要机制，也是小的、非糖基化抗体片段的表达和工程的主要机制。然而，由于与Sec出口相关的一些限制，用于临床前和临床试验的稳定、高亲和力抗体片段的高产量细菌生产可能成为抗体药物管道的严重瓶颈。因此，该提案的总体目标是通过利用最近发现的细菌双精氨酸易位（Tat）途径的显着特性，开发一种多功能抗体表达和工程平台。为了实现这一目标，目前的提案包括以下具体目标：(1)创建单链Fv （scFv）抗体片段的合成文库；(2)应用专利的基于tat的遗传选择策略，高效分离针对疾病相关抗原的超稳定、超高亲和力的人scFvs。所提出的研究有望为发现在细胞内或细胞外环境中折叠和起作用的scFv序列提供一个快速的单步选择平台。拟议研究的成功完成预计将产生一种强大的新技术，用于生产有效的基于抗体的生物制药、诊断和研究试剂。

项目成果

Engineering antibody fitness and function using membrane-anchored display of correctly folded proteins.

• DOI: 10.1016/j.jmb.2011.12.021
• 发表时间: 2012-02-10
• 期刊: JOURNAL OF MOLECULAR BIOLOGY
• 影响因子: 5.6
• 作者: Karlsson, Amy J.;Lim, Hyung-Kwon;Xu, Hansen;Rocco, Mark A.;Bratkowski, Matthew A.;Ke, Ailong;DeLisa, Matthew P.
• 通讯作者: DeLisa, Matthew P.