Therapeutic antibody fragments from glycoengineered Escherichia coli

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1000名接受omalizumab治疗的患者中有2名患有过敏反应，这是对治疗的严重过敏反应。此外，omalizumab免疫疗法非常昂贵，每年将近3万美元。因此，对于医疗保健消费者负担得起且在流通中耐受的改进的omalizumab组合物，存在很大的未满足需求。基因泰克目前在哺乳动物细胞培养中生产omalizumab，这种方法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，生长迅速，不易受病毒污染。然而，大肠杆菌通常不用于生产全长抗体，而是用于表达较小的工程化抗体，如抗原结合片段（fab）。fab与目标抗原的结合同样或更好，但它们在人体中的持久性是有限的。尽管存在这些缺点，但治疗性抗体片段在大肠杆菌中定期产生，包括基因泰克的雷尼单抗（LucentisTM），一种设计用于眼内使用的Fab。一种新兴的延长循环中fab半衰期的解决方案是人型低聚糖的共价附着。与人类血型ABO o型抗原相同的脂联寡糖在O86血清群大肠杆菌菌株中自然合成。这一提议的假设是，非致病性大肠杆菌菌株可以被改造产生o型抗原并将其转移到重组fab的特定位点。为了验证这一假设，本研究的目的是通过以下方法产生具有改善血清耐受性的抗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）在糖工程大肠杆菌中将血型o型抗原与抗ige Fab结合。预计这些研究将产生一个高效的大肠杆菌表达平台，以一种可控、快速和经济的方式生产与人类血型o型低聚糖相关的抗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，用于生产治疗性fab，以治疗哮喘和其他免疫疾病。

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.