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™ 技术是一种高效的连续培养系统，可以生产几周内以 10 升的规模生产公斤蛋白质该项目将确定 C-Flow 是否可以实现。进一步开发为生产单链抗体疗法的有效系统。抗体片段，尤其是单链变异体，对于诊断和治疗越来越重要用途，例如毒素和病毒中和，在大肠杆菌中相对容易制造，尽管效率非常低在目前的技术中，它们作为疾病爆发和生物威胁的治疗方法具有巨大的潜在意义。其他抗体在西非抗击埃博拉病毒方面取得了显著成功。这些分子具有多种结构和适应症，但都很严重。由于缺乏糖基化，抗体片段在体内的寿命很短，需要多次重复。高剂量，导致药物昂贵得令人望而却步，因此，需要一种快速、高效和的生产方法。该项目将优化三种结构不同的单一产品的 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。水平并利用小型设备以前所未有的规模持续生产。抗体片段的正确折叠对于大肠杆菌的表达和递送机制至关重要。发生蛋白质折叠的周质包括分子伴侣、信号序列和密码子使用这些机制将针对所有三种抗体片段进行优化。揭示折叠缺陷的能力，可以通过基因工程来纠正以替换残基或区域通过测试不强烈表达的免疫毒素的例子，使用的可能性。将探索用于提高可制造性的合理抗体设计的软件预测。计算机预测建议的变化将得到实施和评估。软件和生产技术可以对生物紧急情况（从爆发到爆发）做出快速反应几周内即可进行临床测试的治疗方法具体目标是： 1. 优化结构简单的抗体片段 scFv-SG1 在 1 升和 10 升 C-Flow 中的生产规模，将后者延长至至少 30 天以评估连续抗体生产。 2. 根据目标 1 优化结构更复杂的抗体片段 scFabYMF10 的生产，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. 使用增强抗体-毒素缀合物 B3Fv-PE40（在大肠杆菌中表达不佳）的表达确定是否需要预先进行软件辅助设计。该蛋白质可用于预测可制造性的改善。