Strategy for global, unbiased mining and discovery of human monoclonal antibodies

全球、公正的人类单克隆抗体挖掘和发现战略

• 批准号: 8570136
• 负责人: Moonsoo M Jin
• 金额: $ 19.56万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-21 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8570136
• 关键词: Antibodies; Antibody Repertoire; Antibody-Producing Cells; Antigen Targeting; Antigens; Autoimmunity; B lymphocyte immortalization; B-Lymphocytes; Bacteriophages; Biological Assay; Blood; Cleaved cell; Clinic; Cloning; Coupled; Detection; Development; Diagnosis; Diagnostic; Disease; Genes; Genetic Engineering; Heavy-Chain Immunoglobulins; Hepatitis B Virus; Human; Human Herpesvirus 4; Hybridomas; Hybrids; Immune; Immunization; Immunoglobulin G; Immunoglobulins; Individual; Infection; Inflammation; Inflammatory; Influenza; Influenza A Virus, H1N1 Subtype; Length; Libraries; Light; Liquid substance; Malignant Neoplasms; Measures; Medicine; Memory B-Lymphocyte; Methodology; Methods; Mining; Monoclonal Antibodies; Mus; Myelogenous; Oligonucleotides; Patients; Peptide Hydrolases; Pertussis; Plasma; Process; Proteins; Proteolysis; Reaction; Recombinant Antibody; Recombinants; Recording of previous events; Robot; Site; Sorting - Cell Movement; Surface; System; TEV protease; Techniques; Technology; Therapeutic; Time; Tumor Antigens; Vaccine Antigen; Validation; Variant; Yeasts; base; cancer therapy; clinical application; combinatorial; design; human monoclonal antibodies; human subject; interest; monoclonal antibody production; novel strategies; pandemic influenza; protein protein interaction; public health relevance; screening; text searching; therapy development; tool; vector

DESCRIPTION (provided by applicant): Human monoclonal antibodies (mAb) are increasingly being used and developed for therapy and diagnosis in treating cancer, infection, and many inflammatory diseases. Despite recent advances in recombinant DNA technology, multiplexed screening tools, and fluid handling robots, it is a long and laborious process to identifying desired mAbs from human subjects. Herein, we propose a strategy for pairing cognate immunoglobulin heavy and light chain genes from the mixture of human B cells for unbiased, global mining of 'natural' antibody repertoires. Subsequently, human antibody libraries will be expressed in a system called, the 'yeast surface 2-hybrid', which we developed for the expression of a pair of proteins for interaction and discovery studies. Full-length antibodies expressed on the surface of yeast can be released by enzymatic proteolysis, which will facilitate downstream assays requiring soluble form of antibodies. Specific aims for the proposed study are 1) Devise a method for the pairing of cognate IgG heavy and light chain genes from a mixture of antibody producing cells; 2) Validate the platform for global, unbiased display and panning of natural human antibody repertoires. We will perform massively parallel sequencing to evaluate the diversity and any bias in our antibody library. Furthermore, we will examine the capacity of the proposed platform to isolate mAbs against selected common vaccine antigens (Pertussis, Hepatitis B virus).

描述（由申请人提供）：人单克隆抗体（mAb）越来越多地用于治疗和诊断癌症、感染和许多炎性疾病。尽管最近在重组DNA技术、多重筛选工具和流体处理机器人方面取得了进展，但从人类受试者中鉴定所需的mAb是一个漫长而费力的过程。在此，我们提出了一种策略，用于配对同源免疫球蛋白重链和轻链基因的混合物的人B细胞的无偏，全球挖掘的'天然'抗体库。随后，人抗体文库将在称为“酵母表面2-杂交”的系统中表达，我们开发该系统用于表达一对蛋白质，用于相互作用和发现研究。在酵母表面上表达的全长抗体可以通过酶促蛋白水解释放，这将促进需要可溶形式抗体的下游测定。所提出的研究的具体目的是1）设计用于从抗体产生细胞的混合物配对同源IgG重链和轻链基因的方法; 2）构建用于天然人抗体库的全局、无偏展示和淘选的平台。我们将进行大规模平行测序，以评估我们的抗体库中的多样性和任何偏倚。此外，我们还将检查拟议平台分离针对选定常见疫苗抗原（百日咳、B型肝炎病毒）的mAb的能力。