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：
10215525
负责人：
FREDERICK R BLATTNER
金额：
$ 94.45万
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/10215525
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 antibody test 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™技术是一种高效的文化系统，可以产生在ATA中，几个蛋白质的蛋白质仅10升。进一步发展为产生单链抗体疗法的功效系统。抗体碎片，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中和，在大肠杆菌中相对易于生产，尽管非常效率低下在当前的技术中，它们具有巨大的潜在意义在西非对埃博拉病毒的杰出成功。癌症和自身免疫性疾病。制造问题缺乏糖基化，抗体片段在这里短暂高剂量，导致昂贵的药物。低成本需要低成本。链片段抗体，以评估该方法的一般适用性。使用小脚印设备以空前的规模进行水平和持续生产。抗体片段的正确折叠对于功能至关重要。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这些机制将针对所有三个抗体片段进行优化可以通过基因工程来纠正的功率tord缺陷以替代残留物或区域通过测试没有强烈表达的免疫毒素的示例来折叠将探索针对不良生产性的理性抗体设计的软件预测。计算机预测建议的更改将被实施和评估。从爆发到具体目的是：具体的AM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，将后者扩展到至少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结合TOS靶抗原来测试其功能。 3。增强抗体毒素结合物B3FV-PE40（使用大肠杆菌表达的池）的表达生理学和基因工程方法。该蛋白质可用于预测改善的制造。