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）：Omalizumab (XolairTM) 是一种重组单克隆抗 IgE 抗体，用于对抗严重过敏性哮喘，每年产生 5 亿美元的收入。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，即对治疗的严重过敏反应。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，对于卫生保健消费者负担得起并且在循环中耐受的改进的奥马珠单抗组合物存在巨大的未满足的需求。基因泰克目前在哺乳动物细胞培养物中生产奥马珠单抗，这种方法价格昂贵且容易受到病毒污染。另外，大肠杆菌培养物价格低廉、特性良好、生长快速且不易受到病毒污染。然而，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，例如抗原结合片段 (Fab)。 Fab 与目标抗原的结合相同或更好，但它们在人体内的持久性可能有限。尽管存在这些缺点，治疗性抗体片段还是经常在大肠杆菌中产生，包括基因泰克的雷珠单抗 (LucentisTM)，这是一种专为眼内使用而设计的 Fab。一种延长循环中 Fab 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是在血清群 O86 的大肠杆菌菌株中自然合成的。该提议的假设是，可以对大肠杆菌的非致病菌株进行改造，以产生 O 型抗原并将其转移到重组 Fab 中的特定位点。为了检验这一假设，本提案的目的是通过以下方式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 K12 中克隆和表达人血型 O 型寡糖的生物合成机制，(ii) 在大肠杆菌中克隆和表达重组抗 IgE Fab，以及 (iii) 结合血型 糖工程大肠杆菌中抗 IgE Fab 的 O 型抗原。预计这些研究将产生一个有效的大肠杆菌表达平台，用于以受控、快速且经济高效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换技术，用于生产治疗哮喘和其他免疫疾病的治疗性 Fab。 公共卫生相关性：单克隆抗体是对抗许多免疫疾病（包括严重过敏性哮喘）的重要疗法。不幸的是，单克隆抗体的生产过程非常昂贵，导致医疗保健消费者无法承受治疗费用。拟议的研究重点是在大肠杆菌发酵中生产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。