Therapeutic antibody fragments from glycoengineered Escherichia coli

描述(申请人提供)：奥马利单抗(XolairTM)是一种重组的抗IgE单抗，用于治疗严重过敏性哮喘，每年产生5亿美元。然而，每1000名接受奥马珠单抗治疗的患者中，有2人出现过敏反应，这是一种对治疗药物的严重过敏反应。此外，奥马珠单抗免疫疗法的费用高得令人望而却步，每年高达近3万美元。因此，存在对改进的奥马珠单抗组合物的巨大未得到满足的需求，该组合物是医疗保健消费者负担得起的并且在流通中耐受。基因泰克目前在哺乳动物细胞培养中生产奥马珠单抗，这种药物价格昂贵，而且容易受到病毒污染。或者，大肠杆菌培养成本低，特性好，生长快，不容易受到病毒污染。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抗体，如抗原结合片段(Fabs)。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是有限的。尽管有这些缺点，但治疗性抗体片段经常在大肠杆菌中产生，包括Genentech的ranibizumab(LucentisTM)，这是一种设计用于眼内使用的Fab。一种新出现的延长流通中纤维半衰期的解决方案是人型低聚糖的共价连接。与人类ABO血型O型抗原相同的脂联寡糖是在O86血清群的大肠杆菌菌株中自然合成的。这项提议的假设是，非致病性大肠杆菌菌株可以通过工程产生O型抗原，并将其转移到重组FEB中的特定位置。为了验证这一假设，本方案的目的是通过以下方式产生具有改善血清耐受性的抗IgE重组抗体：(1)克隆并在E.coliK12中表达人血型O型寡糖的生物合成机制；(2)克隆并在大肠杆菌中表达重组抗IgE Fab；(3)在糖工程大肠杆菌中将O型血型抗原与抗IgE Fab偶联。预计这些研究将产生一种高效的大肠杆菌表达平台，用于以受控、快速和经济有效的方式生产与人血型O型寡糖相连的抗IgE Fabs。这些研究意义重大，因为它们探索了一种范式转换技术，用于生产用于治疗哮喘和其他免疫疾病的治疗性纤维蛋白。 与公共卫生相关：在对抗包括严重过敏性哮喘在内的许多免疫性疾病方面，克隆抗体是重要的疗法。不幸的是，单抗的生产过程非常昂贵，导致医疗保健消费者负担不起的治疗费用。建议的研究集中于在大肠杆菌发酵中生产耐受性良好的治疗性抗体结合物，而不需要昂贵的哺乳动物细胞培养或体外化学修饰。

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.