Specific Human Monoclonal Antibodies for HUS Prophylaxis

该提案是NIH奖励#2 R01-A1-41326的第二次修订竞争性更新。我们的目标是开发一种免疫治疗制剂，用于治疗和预防与产stx大肠杆菌（STEC）感染相关的并发症。已知产志毒素大肠杆菌的临床分离株主要产生Stxl、Stx2和/或Stx2c。儿童特别容易患上stx介导的溶血性尿毒综合征。我们的假设是，对于有发展成溶血性尿毒综合征风险的个体，给予stx特异性抗体可以预防或改变感染的结果。在早期的奖项中，我们已经生成了一组针对stx1或Stx2的人类单克隆抗体（hu - mab）。通过仔猪模型，我们已经证明了Stx特异性的hu - mab可以中和Stx并预防Stx介导的并发症的发生。

This proposal is the second revised competing renewal for continuation of NIH Award #2 R01-A1-41326. Our objective is to develop an immunotherapeutic formulation for the treatment and prevention of complications associated with Stx-producing Escherichia coli (STEC) infections. Clinical isolates of STEC are known to predominantly produce Stxl, Stx2, and/or Stx2c. Children are particularly susceptible to development of Stx-mediated HUS. Our hypothesis is that administration of Stx-specific antibodies will prevent or modify the outcome of infection for individuals at risk of developing HUS. In the earlier awards, we have generated a panel of human monoclonal antibodies (Hu-mAbs) specific for Stx 1 or Stx2. Using the gnotobiotic piglet model, we have shown that Stx-specific Hu-mAbs neutralize Stx and prevent development of Stx-mediated complications. We now wish to determine which Hu-mAbs should be included in a formulation suitable for clinical evaluation. Based on superior efficacy, four Hu-mAbs specific for Stx2 (3 against the A subunit and 1 against the B subunit), and 2 for Stxl (both against B subunit) have been selected as candidates. The next step is to determine which combination of Hu-mAbs, is both compatible and highly effective. In this proposal we plan to define the structural and functional characteristics, which facilitate protective efficacy of Stx-1 and Stx2-specific Hu-mAbs (Specific Aim 1). Affinity and efficacy of each HumAb will then be studied against their respective toxin (Specific Aim 2). The efficacy of protection of a given antibody dose will then be determined in terms of time after bacterial infection (Specific Aim 3). Finally, combinations of Hu-mAbs specific for B subunit of Stx 1 and A or B subunits of Stx2, will be examined for relative efficacy and compatibility, to determine which is the most effective and thus suitable for clinical evaluation (Specific Aim 4). At the conclusion of these experiments we will have determined the components, and optimized the formulation of Hu-mAbs which will be recommended for testing in human patients. The Hu-mAbs will first be characterized and ranked according to their efficacy, affinity and compatibility with each other. The optimal amount of each Hu-mAb in the formulation required to provide the longest protection after bacterial infection will also be established. This is not a hypothesis-driven proposal, but an essential segment for the characterization of a promising therapy for HUS, against which currently there is no effective treatment.