Production of antibody therapeutic fragments by reduced genome E. coli in continuous culture

在连续培养中通过减少基因组大肠杆菌生产抗体治疗片段

基本信息

批准号：
10081714
负责人：
FREDERICK R BLATTNER
金额：
$ 100万
依托单位：
SCARAB GENOMICS, LLC
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-13 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10081714
关键词：
Affinity Affinity Chromatography Africa Antibodies Antibody Formation Antibody Therapy Antigen Targeting Antigens Autoimmune Diseases Bacillus anthracis Behavior Binding Biological Biological Assay Characteristics Clinic Codon Nucleotides Complex Computer software Computers Defect Development Diagnostic Disease Outbreaks Dose Ebola Emergency Situation Engineering Enzyme-Linked Immunosorbent Assay Equipment Escherichia coli Event Fermentation Foundations Future Genes Genetic Genetic Engineering Genome Genomics Goals Growth High Pressure Liquid Chromatography Immunoglobulin Fragments Immunotoxins Inclusion Bodies Industry Kilogram Mass Spectrum Analysis Medical Methodology Methods Modification Molecular Chaperones Molecular Weight Nickel Peptide Hydrolases Peptide Sequence Determination Peptide Signal Sequences Pharmaceutical Preparations Physiological Plant Resins Plasmids Process Production Protein Engineering Proteins Readiness Regimen Rest Running Solubility Structure Surface System Techniques Technology Temperature Testing Therapeutic Therapeutic Uses Therapeutic antibodies Toxin Variant Virus biothreat cancer therapy cost cost effective design experimental study flasks glycosylation improved in vivo interest manufacturability periplasm personalized medicine protein folding research clinical testing response scale up screening software systems success

项目摘要

Scarab Genomics’ C-Flow™ technology is a highly efficient continuous culture system that can produce kilograms of protein in a few weeks at a mere 10-liter scale. This project will determine whether C-Flow can be further developed as an efficient system for production of single-chain antibody therapeutics. Antibody fragments, especially single-chain variants, are increasingly important for diagnostic and therapeutic use such as toxin and virus neutralization, being relatively easy to manufacture in E. coli, albeit very inefficiently in current techniques. They are of great potential significance as therapies for outbreaks and biothreats, with notable success against Ebola in West Africa. Other antibodies are remarkably successful for e.g. treatment of cancer and autoimmune diseases. These molecules have a variety of structures and indications but severe problems in manufacturing. Lacking glycosylation, antibody fragments are short-lived in vivo, requiring multiple high doses, leading to prohibitively expensive drugs. Therefore, a method of production that is fast, efficient, and low in cost is critically needed. This project will optimize C-Flow production of three structurally distinct single chain fragment antibodies, to evaluate the general applicability of the approach. The goal is high expression levels and sustained production on an unprecedented scale with small-footprint equipment. Correct folding of antibody fragments is critical for function. E. coli mechanisms for expression and delivery into the periplasm, where protein folding occurs, include chaperones, signal sequences, and codon usage and distribution. These mechanisms will be optimized for all three antibody fragments. Computer predictions have the power to reveal folding defects that could be corrected by genetic engineering to replace residues or regions that do not fold well. By testing an example immunotoxin that does not express strongly, the possibility of using software predictions for rational antibody design for increased manufacturability will be explored. Engineered changes suggested by computer predictions will be implemented and evaluated. Such an integrated system of software and production technology could provide a rapid response to a bio-emergency, going from outbreak to therapeutic ready for clinical testing in weeks. The Specific Aims are: 1. Optimize production of a structurally simple antibody fragment, scFv-SG1, at 1-liter then 10-liter C-Flow scales, extending the latter to at least 30 days to evaluate continuous antibody production. 2. Optimize production of a more structurally complex antibody fragment, scFabYMF10 as per Aim 1, purify the antibody and test its function by evaluating scFabYMF10 binding to its target antigen. 3. Enhance expression of the antibody-toxin conjugate B3Fv-PE40 (poorly expressed in E. coli) using physiological and genetic engineering approaches. Determine whether up-front software-assisted design of the protein can be used to predict improved manufacturability.

Scarab Genomics的C-Flow™技术是一种高效的连续文化系统，可以产生几周内仅10升尺度上的蛋白质公斤蛋白质。该项目将确定C-Flow是否可以进一步发展为产生单链抗体治疗的有效系统。抗体片段，尤其是单链变体，对于诊断和治疗越来越重要使用例如毒素和病毒神经化，在大肠杆菌中相对易于生产，尽管效率很低在当前技术中。作为爆发和生物治疗的疗法，它们具有很大的潜在意义，在西非对埃博拉病毒的杰出成功。其他抗体对于例如处理癌症和自身免疫性疾病。这些分子具有多种结构和适应症，但严重制造业问题。缺乏糖基化，抗体片段在体内短暂，需要多个高剂量，导致禁止的昂贵药物。因此，一种快速，高效的生产方法低成本是至关重要的。该项目将优化三个结构上独特单一的C-Flow生产链片段抗体，以评估该方法的一般适用性。目标是高表达使用小脚印设备以空前的规模进行水平和持续生产。正确折叠抗体片段对于功能至关重要。大肠杆菌的表达和传递到发生蛋白质折叠的周期包括伴侣，信号序列和密码子的使用以及分配。这些机制将针对所有三种抗体片段进行优化。计算机预测有揭示折叠缺陷的功能，可以通过基因工程来纠正替换保留或区域折叠不好。通过测试一个没有强烈表达的示例免疫毒素，使用的可能性将探索用于增加制造的理性抗体设计的软件预测。设计将实施和评估计算机预测建议的更改。这样的集成系统从爆发到治疗准备在数周内进行临床测试。具体目的是： 1。优化结构简单的抗体片段SCFV-SG1的产生，1升然后10升C流量鳞片，将较晚的时间延长至至少30天，以评估连续抗体的产生。 2。优化一个更结构复杂的抗体片段的生产，scfabymf10符合AIM 1，纯化抗体并通过评估SCFABYMF10与其靶抗原的结合来测试其功能。 3。增强抗体毒素结合物B3FV-PE40（在大肠杆菌中表达不佳）的表达生理和基因工程方法。确定是否具有前期软件辅助设计该蛋白质可用于预测改进的制造。