Antibody-Membrane Switch (AMS) technology for optimized antibody engineering

用于优化抗体工程的抗体膜开关 (AMS) 技术

基本信息

批准号：
8978257
负责人：
JAMES W LARRICK
金额：
$ 49.98万
依托单位：
PANORAMA RESEARCH, INC.
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-06-01 至 2017-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8978257
关键词：
Affinity Agonist Alternative Splicing Antibodies Antibody Formation Antigens Bacteria Bacteriophages Binding Cell Line Cell surface Cells Characteristics Chinese Hamster Ovary Cell Clinical DNA Development Dihydrofolate Reductase Exhibits Exons Expression Library Failure Fc domain Fluorescence-Activated Cell Sorting G Protein-Coupled Receptor Genes GNAI2 gene Gene Amplification Gene Dosage Generations Genetic Recombination Glutamate-Ammonia Ligase Health Human IgG1 Immunoglobulin Constant Region Introns Libraries Link Mammalian Cell Mediating Membrane Methodology Methods Molecular Phage Display Phase Post-Translational Protein Processing Process Production Productivity Rodent Site Sorting - Cell Movement Specificity Surface System Technology Therapeutic antibodies Time Transmembrane Domain Yeasts abstracting antibody engineering antigen binding base belimumab constant region gene cost infliximab interest member method development novel phase 1 study pre-clinical pressure recombinase research clinical testing screening vector

项目摘要

DESCRIPTION (provided by applicant): Antibody-Membrane Switch (AMS) technology for optimized antibody engineering Abstract Generation of high-productivity cell lines remains a major bottleneck in therapeutic antibody development. Conventional cell line development depends on gene amplification methodologies using dihydrofolate reductase (DHFR) or glutamine synthetase (GS). Higher productivity is associated with an increased gene copy number. However, lack of selection pressure under the conditions of large scale manufacturing leads to clonal instability. We have developed a novel method for cell line development, Antibody Membrane Switch (AMS) technology that does not rely on gene amplification. This FACS-based, high-throughput method is facilitated by cell surface antibody expression to rapidly and efficiently isolate high producing cells. The switch between membrane expression and secretion is achieved by alternative splicing and specific DNA recombination. The antibody of interest is initially displayed on the cell surface to facilitate FACS. Isolated high producing cells are then seamlessly transformed into production cells after removing the membrane-anchoring domain sequence via a DNA recombinase. AMS technology has been applied in a number of antibody cell line development projects which typically last 2-3 months. The top manufacturing cell lines exhibit very high specific productivity of 40-60 pg./cell/day resulting in production titers of 2-4 g/L in 10-day batch culture. In Phase I studies the underlying AMS methods were reduce to practice. Furthermore, we have optimized the AMS process using two biosimilar antibodies. In Phase II, these optimized methods will be applied to screening of CHO surface displayed antibody libraries. An affinity maturation library and a naïve human antibody library will be constructed and utilized to screen by FACS for therapeutic antibody candidates. The screening will be carried out with soluble antigen for high affinity binding or antigen (e.g., GPCR) presenting cells for desired bio-activities. The isolated positive cells will be transformed into antibody production cells by DNA recombinase to remove the membrane anchorage. This successful application of AMS technology will permit the discovery of antibodies to be seamlessly linked to downstream cell line development providing the basis for rapid, facile cell line production directly from a novel antibody-discovery platform. Phase I: 1R43AI109982-01A1

描述（由申请人提供）：用于优化抗体工程的抗体膜转换（AMS）技术摘要高生产力细胞系的产生仍然是治疗性抗体开发的主要瓶颈，传统的细胞系开发依赖于使用二氢叶酸还原酶（DHFR）的基因扩增方法。）或谷氨酰胺合成酶（GS）。较高的生产率与基因拷贝数的增加有关。然而，在大规模生产的条件下缺乏选择压力会导致克隆不稳定。抗体膜开关（AMS）技术是一种细胞系开发的新方法，这种基于 FACS 的高通量方法通过细胞表面抗体表达来快速有效地分离高产细胞。膜表达和分泌之间的平衡是通过选择性剪接和特异性 DNA 重组实现的。目标抗体最初展示在细胞表面，以促进 FACS，然后在去除膜锚定结构域后将分离的高产细胞无缝转化为生产细胞。通过 DNA 重组酶进行的序列已应用于许多抗体细胞系开发项目，这些项目通常持续 2-3 个月，顶级生产细胞系表现出非常高的比生产率，可达 40-60 pg./cell/day。在 10 天的批量培养中，生产滴度为 2-4 g/L。应用于筛选 CHO 表面展示的抗体文库。将构建亲和力成熟文库和天然人抗体文库，并用于通过 FACS 筛选治疗性抗体候选物。筛选将使用高亲和力结合的可溶性抗原或抗原进行。（例如，GPCR）呈递具有所需生物活性的细胞，通过 DNA 重组酶将其转化为抗体生产细胞，以去除膜锚定，AMS 技术的成功应用将允许发现与抗体无缝连接的细胞。下游细胞系开发为直接从新型抗体发现平台快速、简便地生产细胞系提供了基础：1R43AI109982-01A1。