Development of a transgenic mouse line engineered to permit selection and cloning

描述（由申请人提供）：申请人提出通过产生转基因小鼠来改进单克隆抗体技术，以促进流式细胞术分离和特异性抗原反应性浆细胞的克隆。单克隆抗体（mab）可以说是生物医学研究、诊断和治疗中最重要的生物试剂，对它们的需求呈指数级增长。单克隆抗体由杂交瘤细胞分泌，杂交瘤细胞是产生抗体的淋巴细胞（B细胞）和不朽的骨髓瘤细胞的融合。abome获得专利的直接选择杂交技术（DiSHTM）代表了对杂交技术的重大改进。通过使用一种新的骨髓瘤亲本，通过基因工程使转基因igg受体蛋白igg - α和igg - β组成性表达，所有衍生的B细胞杂交瘤在细胞表面表达完整的B细胞受体（BCR）复合物。随后，用荧光抗原标记BCR后，通过荧光活化细胞分选（FACS）选择和克隆感兴趣的特异性杂交瘤。然而，杂交瘤技术仍然存在局限性，无法对浆细胞（产生最高亲和力抗体的淋巴细胞）的全部库进行有效采样。在此，我们提出了DiSH技术的扩展，将允许靶向选择浆细胞（TSP）。TSP的概念是建立在基因工程小鼠中ig - α和ig - β受体蛋白的转基因表达上的。TSP技术将导致BCR复合物直接在小鼠的抗体分泌浆细胞表面表达，然后可以使用基于BCR复合物表达及其与荧光标记抗原反应的FACS分离。本一期项目拟建立一个TSP转基因小鼠群体，证明转基因表达，并证明这些小鼠具有免疫功能，从而实现单克隆抗体技术重大商业可行改进的重要里程碑。

DESCRIPTION (provided by applicant): The Applicant proposes an improvement to monoclonal antibody technology through the generation of transgenic mice engineered to facilitate flow cytometric isolation and cloning of specific antigen-reactive plasmacytes. Monoclonal antibodies (mAbs) are arguably the most important biological reagents used in biomedical research, diagnostics, and therapeutics, and the demand for them is increasing exponentially. MAbs are secreted from hybridoma cells that are fusions of antibody-producing lymphocytes (B cells) and immortal myeloma cells. Abeome's patented Direct Selection of Hybridoma (DiSHTM) technology represents a significant improvement to hybridoma technology. Through the use of a novel myeloma parent, genetically engineered for the constitutive expression of transgenic Ig-receptor proteins Ig-alpha and Ig-beta, all of the derived B cell hybridomas express a complete B cell receptor (BCR) complex on the cell surface. Specific hybridomas of interest are subsequently selected and cloned by Fluorescent Activated Cell Sorting (FACS) after labeling the BCR with fluorescent antigen. There remain, however, limitations within hybridoma technology that prevent efficient sampling of the full repertoire of plasmacytes, the lymphocytes producing the highest affinity antibodies. Herein, we are proposing an extension of DiSH technology that will allow the Targeted Selection of Plasmacytes (TSP). The TSP concept is founded on the transgenic expression of Ig-receptor proteins Ig-alpha and Ig-beta in engineered mice. TSP technology will result in surface expression of the BCR complex directly on antibody-secreting plasmacytes in mice, which can then be isolated using FACS based on expression of the BCR complex and its reaction with fluorescently labeled antigen. This Phase I project proposes to establish a colony of TSP transgenic mice, demonstrate expression of the transgene, and show that these mice are immunologically functional and thereby accomplish a major milestone towards a significant commercially feasible improvement in monoclonal antibody technology. PUBLIC HEALTH RELEVANCE: Monoclonal antibodies represent some of the most successful research and diagnostic tools available to scientists and clinicians. More importantly, their high specificity and sensitivity, coupled with favorable pharmacokinetics and safety, make them highly attractive therapeutic agents. The Applicant proposes the generation of transgenic mice that would greatly facilitate the isolation of antigen-specific plasma cells directly from immunized animals, thereby improving the speed and efficiency of obtaining monoclonal antibodies. The proposed project represents a major milestone towards the ultimate goal of generating transgenic mice that eliminate the requirement for the currently required myeloma-B cell fusion step.