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

 描述（由申请人提供）：用于优化抗体工程的抗体膜开关（AMS）技术摘要高生产率细胞系的产生仍然是治疗性抗体开发的主要瓶颈。常规细胞系开发依赖于使用二氢叶酸还原酶（DHFR）或谷氨酰胺合成酶（GS）的基因扩增方法。更高的生产力与增加的基因拷贝数有关。然而，在大规模生产条件下缺乏选择压力导致克隆不稳定。我们开发了一种新的细胞系开发方法，抗体膜开关（AMS）技术，不依赖于基因扩增。这种基于FACS的高通量方法通过细胞表面抗体表达来促进，以快速有效地分离高产细胞。膜表达和分泌之间的转换是通过选择性剪接和特异性DNA重组实现的。感兴趣的抗体最初展示在细胞表面上以促进FACS。然后，在通过DNA重组酶去除膜锚定结构域序列后，将分离的高产细胞无缝转化为生产细胞。AMS技术已应用于许多抗体细胞系开发项目，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，为40-60 pg/ml。细胞/天， 在10天分批培养中生产滴度为2-4 g/L。 在第一阶段的研究中，基本的AMS方法被简化为实践。此外，我们使用两种生物类似抗体优化了AMS过程。在第二阶段，这些优化的方法将被应用于CHO表面展示抗体库的筛选。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。筛选将用高亲和力结合的可溶性抗原或抗原（例如，GPCR）呈递细胞以获得所需的生物活性。分离的阳性细胞将通过DNA重组酶转化为抗体产生细胞，以去除膜锚定。AMS技术的这一成功应用将允许抗体的发现与下游细胞系开发无缝连接，为直接从新型抗体发现平台快速、简便地生产细胞系提供基础。I期：1 R43 AI 109982 - 01 A1

 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