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，所述转基因小鼠经工程改造以促进特异性抗原反应性浆细胞的流式细胞术分离和克隆。单克隆抗体（mAb）可以说是生物医学研究，诊断和治疗中使用的最重要的生物试剂，对它们的需求呈指数级增长。单克隆抗体是从杂交瘤细胞分泌的，杂交瘤细胞是产生抗体的淋巴细胞（B细胞）和永生骨髓瘤细胞的融合体。Abeome的专利杂交瘤直接选择（DiSHTM）技术代表了杂交瘤技术的重大改进。通过使用一种新的骨髓瘤亲本，该亲本经遗传工程改造以组成型表达转基因Ig-受体蛋白Ig-α和Ig-β，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。随后选择感兴趣的特异性杂交瘤，并在用荧光抗原标记BCR后通过荧光激活细胞分选（FACS）克隆。然而，在杂交瘤技术中仍然存在限制，这些限制阻止了对浆细胞的全部库的有效采样，所述浆细胞是产生最高亲和力抗体的淋巴细胞。在此，我们提出了DiSH技术的扩展，其将允许浆细胞（TSP）的靶向选择。TSP概念建立在工程小鼠中Ig受体蛋白Ig-α和Ig-β的转基因表达基础上。TSP技术将导致BCR复合物直接在小鼠中分泌抗体的浆细胞上表面表达，然后可以基于BCR复合物的表达及其与荧光标记抗原的反应使用FACS分离。该I期项目提出建立TSP转基因小鼠的群体，证明转基因的表达，并显示这些小鼠具有免疫功能，从而实现单克隆抗体技术商业上可行的重大改进的重要里程碑。 公共卫生相关性：单克隆抗体代表了科学家和临床医生可用的一些最成功的研究和诊断工具。更重要的是，它们的高特异性和灵敏度，加上有利的药代动力学和安全性，使它们成为极具吸引力的治疗剂。本申请人提出了转基因小鼠的产生，其将极大地促进直接从免疫动物分离抗原特异性浆细胞，从而提高获得单克隆抗体的速度和效率。拟议的项目代表了一个重要的里程碑，朝着产生转基因小鼠的最终目标，消除了目前所需的骨髓瘤-B细胞融合步骤的要求。

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.