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

开发可进行选择和克隆的转基因小鼠品系

• 批准号: 7995915
• 负责人: PAUL W PRICE
• 金额: $ 13.4万
• 依托单位: ABEOME CORPORATION
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-15 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7995915
• 关键词: Address; Animals; Antibodies; Antibody Affinity; Antibody-Producing Cells; Antigens; B-Lymphocytes; Biological; Biological Assay; Biomedical Research; Bypass; Cell Line; Cell Separation; Cell fusion; Cell membrane; Cell surface; Cells; Cloning; Complex; Complex Mixtures; Coupled; DNA; Data; Development; Diagnostic; Diagnostics Research; Drug Kinetics; Engineering; Exhibits; Future; Generations; Goals; Hybridomas; Immune response; Immunoglobulin-Secreting Cells; Inherited; Label; Legal patent; Lymphocyte; Membrane; Monoclonal Antibodies; Multiple Myeloma; Mus; Parents; Phase; Plasma Cells; Price; Production; Reaction; Reagent; Receptors, Antigen, B-Cell; Safety; Sampling; Scientist; Sensitivity and Specificity; Small Business Innovation Research Grant; Speed; Surface; Technology; Testing; Therapeutic; Therapeutic Agents; Time; Transcript; Transgenes; Transgenic Mice; Transgenic Organisms; Work; base; improved; interest; meetings; novel; prevent; public health relevance; receptor; tool; trafficking; transgene expression

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.

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定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，并且对其的需求呈指数级增长。 MAb 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）和永生骨髓瘤细胞的融合体。 Abeome 获得专利的杂交瘤直接选择 (DiSHTM) 技术代表了杂交瘤技术的重大改进。通过使用新型骨髓瘤亲本，经过基因工程改造，可组成性表达转基因 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。在用荧光抗原标记 BCR 后，随后通过荧光激活细胞分选 (FACS) 选择和克隆感兴趣的特定杂交瘤。然而，杂交瘤技术仍然存在局限性，无法对全部浆细胞（产生最高亲和力抗体的淋巴细胞）进行有效采样。在此，我们提议扩展 DiSH 技术，以实现浆细胞 (TSP) 的靶向选择。 TSP 概念基于 Ig 受体蛋白 Ig-α 和 Ig-β 在工程小鼠中的转基因表达。 TSP 技术将导致 BCR 复合物直接在小鼠抗体分泌浆细胞上进行表面表达，然后可以根据 BCR 复合物的表达及其与荧光标记抗原的反应，使用 FACS 进行分离。该第一阶段项目拟建立 TSP 转基因小鼠群体，展示转基因的表达，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性改进的一个重要里程碑。 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，它们的高特异性和敏感性，加上良好的药代动力学和安全性，使它们成为极具吸引力的治疗剂。申请人提出转基因小鼠的产生将极大地促进直接从免疫动物中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。拟议的项目代表了朝着产生转基因小鼠的最终目标迈出的一个重要里程碑，该转基因小鼠消除了目前所需的骨髓瘤-B细胞融合步骤的要求。