Recombinant Fc fusions for treatment of uropathogenic E. coli

尿路感染是最常见的细菌感染之一，影响1.5亿人 世界各地的人们每年都会。在美国，社区获得性尿路感染每年约占1100万例 这每年花费美国公共卫生预算50亿美元。尿路致病性大肠杆菌(UPEC)占 高达80%的尿路感染。虽然尿路感染目前使用抗生素治疗，但多重耐药的频率为 越来越多的尿路感染预示着未来无法治愈的尿路感染。抗生素的一个有希望的替代方案是直接靶向 细菌毒力因子对细菌与尿路上皮细胞的黏附和侵袭至关重要。这些 毒力因子包括细菌粘附素FimH，在大多数致尿路病原性大肠杆菌菌株中都有表达。 FimH与一种特殊的糖形式--寡糖-3结合，由一种丰富的膜糖蛋白携带。 尿路上皮细胞。 我们已经生产了一种针对FimH的重组蛋白。它是人类蛋白质的分子融合， 二肽基肽酶4(DPP4)与IgG1Fc结合(DPP4-Fc)，携带8个含甘露糖的N-糖链并结合 以甘露糖特有的方式作用于FimH。我们的研究表明，DPP4-Fc能促进致尿性E。 以补体的形式在大肠杆菌中表达。本研究的目的是为了证明DPP4-Fc能够促进 激活耐药尿路致病性大肠杆菌表面补体从而导致增加 补体和多形核细胞(PMN)杀死细菌。 我们将生产三种新的DPP4-FC变体，每个单体含有不同数量的N-糖链和 不同的糖型。我们将修改DPP4-Fc的Fc以改善C1q结合，从而提高C1q的活性 经典的补体途径。修改将包括点突变和将IgG1Fc替换为 IgG3Fc。我们还将产生阴性对照DPP4-Fc，其中Fc包含两个点突变 取消C1q结合，因此没有调理活性。我们将测试所有这些新的DPP4-FC变种 能力：i)与致尿性大肠杆菌、肠杆菌和肺炎克雷伯氏菌菌株结合，ii)抑制细菌 附着于尿路上皮细胞，iii)促进血清介导的细菌杀灭和iv)促进吞噬细胞功能 细菌的数量。 我们期待DPP4-Fc能被开发成一种新的治疗泌尿系致病性大肠杆菌肠杆菌的方法。 和克雷伯氏菌感染。未来的研究项目将在动物模型和动物模型中测试这些分子的活性 后来在人体临床试验中。

Urinary tract infections (UTIs) are among the most common bacterial infections, affecting 150 million people worldwide each year. In the USA, community-acquired UTIs account for about 11 million cases each year that cost the U.S. public health budget $5 billion annually. Uropathogenic Escherichia coli (UPEC) accounts for up to 80% of UTIs. While UTIs are currently treated with antibiotics, the frequency of multi-drug resistance is increasing, portending a future of untreatable UTIs. A promising alternative to antibiotics is to directly target bacterial virulence factors that are critical for bacterial adhesion to and invasion of uroepithelial cells. These virulence factors include the bacterial adhesin FimH, expressed by the majority of uropathogenic E. coli strains. FimH binds to a specific glycoform, oligomannose-3, carried by an abundant membrane glycoprotein on uroepithelial cells. We have produced a recombinant protein targeting FimH. It is a molecular fusion of human protein, dipeptidyl peptidase 4 (DPP4) with IgG1 Fc (DPP4-Fc), carries eight mannose-containing N-glycans and binds in a mannose-specific manner to FimH. We have shown that that DPP4-Fc promotes killing of uropathogenic E. coli by complement. The purpose of this research project is to demonstrate the ability of DPP4-Fc to promote activation of complement on the surface of antibiotic-resistant uropathogenic E. coli thereby leading to increased bacterial killing by complement and polymorphonuclear cells (PMNs). We will produce three new variants of DPP4-Fc carrying different numbers of N-glycans per monomer and different glycoforms. We will modify the Fc of DPP4-Fc to improve C1q binding and thus increase activation of the classical complement pathway. Modifications will include point mutations and replacement of IgG1 Fc with IgG3 Fc. We will also produce a negative control DPP4-Fc in which the Fc contains two point mutations that abrogate C1q binding and thus has no opsonizing activity. We will test all these new DPP4-Fc variants for their ability to: i) bind to uropathogenic E.coli, Enterobacter and Klebsiella pneumonia strains, ii) inhibit bacterial attachment to urothelial cells, iii) promote serum mediated killing of bacteria and iv) promote opsonophagocytosis of bacteria. We expect that DPP4-Fc can be developed into a novel treatment for uropathogenic E. coli, Enterobacter and Klebsiella infections. Future research projects will test the activity of these molecules in animal models and later in human clinical trials.