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

描述(申请人提供)：人类单抗(MAb)越来越多地被用于治疗和诊断癌症、感染和许多炎症性疾病。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mAbs是一个漫长而费力的过程。在这里，我们提出了一种策略，从人类B细胞的混合物中配对同源免疫球蛋白重链和轻链基因，以无偏见地、全局地挖掘“天然”抗体谱系。随后，人类抗体库将在一种名为“酵母表面2-杂交体”的系统中表达，这是我们为表达一对蛋白质而开发的，用于相互作用和发现研究。酵母表面表达的全长抗体可以通过酶蛋白分解释放，这将有助于下游需要可溶性抗体的检测。这项研究的具体目标是：1)设计一种从抗体产生细胞的混合物中配对同源的免疫球蛋白G重链和轻链基因的方法；2)验证用于全球、无偏见地展示和平移天然人类抗体库的平台。我们将进行大规模的并行测序，以评估我们抗体库中的多样性和任何偏见。此外，我们将检查拟议平台的能力，以分离针对选定的常见疫苗抗原(百日咳、乙肝病毒)的单抗。

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).