Therapeutic antibody fragments from glycoengineered Escherichia coli

来自糖工程大肠杆菌的治疗性抗体片段

• 批准号: 8002633
• 负责人: Adam Charles Fisher
• 金额: $ 15.35万
• 依托单位: GLYCOBIA, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-15 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8002633
• 关键词: ABO blood group system; Adrenal Cortex Hormones; Affect; Affinity; Aleuria aurantia lectin; Allergic Reaction; Anabolism; Anaphylaxis; Antibodies; Antibody Formation; Antigen Targeting; Antigens; Asparagine; Asthma; Bacteria; Binding; Blood Circulation; Blood Group Antigens; Blood Transfusion; Blood typing procedure; Cell Culture Techniques; Cessation of life; Chemicals; Chinese Hamster Ovary Cell; Cloning; Engineering; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Escherichia coli K12; Extrinsic asthma; Fab Immunoglobulins; Fermentation; Genes; Genetic; Glycoproteins; Half-Life; Healthcare; Human; Human body; IgE; Immune; Immune System Diseases; Immunoglobulin Fragments; Immunoglobulin G; Immunoglobulins; Immunotherapy; In Vitro; Individual; Laboratories; Length; Link; Location; Mammalian Cell; Marketing; Modification; Monoclonal Antibodies; O Antigens; Oligosaccharides; Patients; Plasmids; Population; Process; Production; Proteins; Recombinant Antibody; Recombinants; Sales; Serotyping; Serum; Site; Solutions; Surface Plasmon Resonance; Technology; Testing; Therapeutic; Therapeutic antibodies; Time; United States; Viral; Western Blotting; allergic response; antibody conjugate; antibody engineering; base; biomaterial compatibility; blood group; cost; design; fight against; glycosylation; glycosyltransferase; immunoregulation; improved; lipooligosaccharide; manufacturing process; omalizumab; periplasm; public health relevance; ranibizumab; response; stoichiometry; success

DESCRIPTION (provided by applicant): Omalizumab (XolairTM) is a recombinant monoclonal anti-IgE antibody used in the fight against severe allergic asthma that generates $500 million per year. However, 2 out of every 1,000 patients treated with omalizumab suffer from anaphylaxis, a severe allergic reaction to the therapeutic. Moreover, omalizumab immunotherapy is prohibitively expensive, reaching nearly $30,000 per year. Thus, there is a great unmet need for an improved omalizumab composition that is affordable to health care consumers and tolerated in circulation. Genentech currently produces omalizumab in mammalian cell culture, which is expensive and susceptible to viral contamination. Alternatively, Escherichia coli culture is inexpensive, well-characterized, fast-growing, and not susceptible to viral contamination. However, E. coli is not typically used for full-length antibody production and instead is relegated to expression of smaller, engineered antibodies such as antigen binding fragments (Fabs). Fabs bind equally or better to target antigens, but their persistence in the human body can be limited. In spite of these shortcomings, therapeutic antibody fragments are regularly produced in E. coli including Genentech's ranibizumab (LucentisTM), a Fab designed for intraocular use. An emerging solution to prolong the half-life of Fabs in circulation is the covalent attachment of human-type oligosaccharides. Lipid-linked oligosaccharides identical to human blood group ABO O-type antigens are naturally synthesized in E. coli strains of serogroup O86. The hypothesis of this proposal is that non-pathogenic strains of E. coli can be engineered to produce and transfer O-type antigens to specific sites in recombinant Fabs. To test this hypothesis, the objective of this proposal is to generate anti-IgE recombinant antibodies with improved serum tolerance in by: (i) cloning and expressing the biosynthetic machinery for the human blood group O-type oligosaccharide in E. coli K12, (ii) cloning and expressing a recombinant anti-IgE Fab in E. coli, and (iii) conjugating blood group type O-type antigens to anti-IgE Fabs in glycoengineered E. coli. It is anticipated that these studies will result in an efficient E. coli expression platform for the production of anti-IgE Fabs linked to human blood group O-type oligosaccharides in a controlled, rapid, and cost-effective manner. These studies are significant because they explore a paradigm-shifting technology for the production of therapeutic Fabs for the treatment of asthma and other immunological diseases. PUBLIC HEALTH RELEVANCE: Monoclonal antibodies are prominent therapeutics in the fight against many immunological diseases including severe allergic asthma. Unfortunately, the production process for monoclonal antibodies is expensive, resulting in a cost of therapy that is unaffordable for the healthcare consumer. The proposed studies focus on producing well-tolerated therapeutic antibody conjugates in Escherichia coli fermentation without the need for costly mammalian cell culture or in vitro chemical modification.

描述（由申请人提供）：奥马珠单抗（XolairTM）是一种重组单克隆抗IgE抗体，用于治疗严重过敏性哮喘，每年产生5亿美元。然而，每1,000名接受奥马珠单抗治疗的患者中就有2名患有过敏反应，这是一种对治疗剂的严重过敏反应。此外，奥马珠单抗免疫疗法非常昂贵，每年接近30，000美元。因此，对于保健消费者负担得起并且在流通中耐受的改进奥马珠单抗组合物存在巨大的未满足的需求。Genentech目前在哺乳动物细胞培养中生产奥马珠单抗，这是昂贵的，易受病毒污染。或者，大肠杆菌培养物是廉价的、良好表征的、快速生长的，并且不易受病毒污染。然而，E.大肠杆菌通常不用于全长抗体生产，而是用于表达较小的工程抗体，如抗原结合片段（Fab）。Fab与靶抗原的结合相等或更好，但它们在人体中的持久性可能有限。尽管存在这些缺点，但治疗性抗体片段通常在E.包括基因泰克公司的雷珠单抗（LucentisTM），一种设计用于眼内使用的Fab。延长Fab在循环中的半衰期的新兴解决方案是人型寡糖的共价连接。与人血型ABO O型抗原相同的脂联寡糖在E.大肠杆菌O 86血清群。这一建议的假设是，非致病性E。大肠杆菌可以被工程化以产生O型抗原并将其转移到重组Fab中的特定位点。为了验证这一假设，本发明的目的是通过以下方法产生具有改善的血清耐受性的抗IgE重组抗体：（i）在大肠杆菌中克隆和表达人血型O型寡糖的生物合成机制; coli K12;（ii）在E.大肠杆菌中，和（iii）在糖工程改造的大肠杆菌中将血型类型O型抗原缀合至抗IgE Fab。杆菌预计这些研究将导致一个有效的E。大肠杆菌表达平台，用于以受控、快速和具有成本效益的方式生产与人血型O型寡糖连接的抗IgE Fab。这些研究意义重大，因为它们探索了一种范式转变技术，用于生产治疗哮喘和其他免疫性疾病的治疗性Fab。 公共卫生相关性：单克隆抗体在对抗许多免疫性疾病包括严重过敏性哮喘中是突出的治疗剂。不幸的是，单克隆抗体的生产过程是昂贵的，导致医疗保健消费者负担不起的治疗成本。所提出的研究集中于在大肠杆菌发酵中产生耐受性良好的治疗性抗体缀合物，而不需要昂贵的哺乳动物细胞培养或体外化学修饰。