Development of single-cell sequencing based method for mAb identification and validation

该项目的总体目标是开发更具成本效益的方法来产生针对癌症生物标志物的单克隆抗体（mAb）。虽然MédiMabs等公司使用的mAb生成的传统方案已经建立了几十年，但最近的技术发展为新的和潜在的更有效的方法打开了大门。在这个项目中，我们将研究使用单细胞DNA测序作为一种方法来快速鉴定mAb生产细胞，而无需进行传统的杂交瘤融合。为了做到这一点，我们将对来自用感兴趣的抗原免疫的小鼠的B细胞进行单细胞RNA测序。当制备这些细胞用于测序时，添加相同的抗原，然而这种形式的抗原也具有附着的DNA条形码。当能够识别抗原的B细胞结合它时，附着的DNA条形码也被添加到单细胞测序反应中。这允许同时测序条形码（如果存在）和编码可识别抗原的抗体的区域。有了这个序列信息，任何感兴趣的抗体都可以克隆到新的稳定细胞系中进行有效生产。与此同时，我们将评估各种方法来优化这一部分的过程，包括快速表达和翻译测序克隆，以允许其功能验证。这些实验将在Wilhelm实验室鉴定的儿科急性髓性白血病（AML）亚组中鉴定的几种生物标志物的背景下进行。由于这种方法在mAb生产的时间和成本方面具有许多潜在优势，因此支持拟议工作的科学和经济价值都很强。

The overall goal of this project is to develop more cost-effective methods for generating monoclonal antibodies (mAb) against cancer biomarkers. While the traditional protocols for mAb generation used by companies such as MédiMabs have been established decades ago, recent technological developments have opened the door to new and potentially far more efficient methodologies. In this project, we will research the use of single cell DNA sequencing as a method to rapidly identify mAb producing cells without performing traditional hybridoma fusions. In order to do this, we will perform single cell RNA sequencing on B-cells from mice that have been immunized with an antigen of interest. When these cells are prepared for sequencing, the same antigen is added, however this version of the antigen also has a DNA barcode attached. When B cells that can recognize the antigen bind it, the attached DNA barcode is also added to the single cell sequencing reaction. This allows the simultaneous sequencing of both the barcode (if present) and the region encoding the antibody that can recognize the antigen. With this sequence information available, any antibodies of interest can be cloned into a new stable cell line for efficient production. At the same time, we will assess various approaches to optimize this part of the process, including rapid expression and translation of sequenced clones to allow their functional validation. These experiments will be done in the context of several biomarkers that have been identified in a subgroup of pediatric acute myeloid leukemia (AML) identified by the Wilhelm lab. Because of the numerous potential advantages of this approach with respect to time and cost of mAb production, there is both a strong scientific as well as economic value underpinning the proposed work.