Specific Human Monoclonal Antibodies for HUS Prophylaxis

这项提案是美国国立卫生研究院奖#2 R01-A1-41326的第二次修订竞争续展。我们的目标是开发一种免疫治疗配方，用于治疗和预防与产生STX的大肠杆菌(STEC)感染相关的并发症。已知的STEC临床分离株主要产生Stx1、Stx2和/或Stx2c。儿童特别容易患上STX介导的HUS。我们的假设是，给予STX特异性抗体将预防或改变有患HUS风险的个人的感染结果。在早期的奖项中，我们已经产生了一组针对STX1或STX2的人类单抗(HU-mAbb)。在仔猪动物模型中，我们已经证明了STX特异性人源单抗可以中和STX并防止STX引起的并发症的发生。 我们现在希望确定哪些HU-mAb应该包括在适合临床评估的配方中。基于较好的疗效，4株针对Stx2的HU-mAb(3株针对A亚基，1株针对B亚基)和2株针对Stx1的HU-mAb(均针对B亚基)被选为候选抗体。下一步是确定HU-mAbs的哪种组合既相容又高效。 在这份提案中，我们计划定义结构和功能特征，以促进 STX-1和STX2特异性人源单抗的保护作用(特指1)。然后将研究每种HumAb与其各自的毒素的亲和力和效力(特定目标2)。然后将根据细菌感染后的时间确定特定抗体剂量的保护效果(具体目标3)。最后，将检查针对STX1的B亚基和STX2的A或B亚基的HU-mAb的组合的相对有效性和配伍，以确定哪一种是最有效的，因此适合于临床评估(特定目标4)。 在这些实验结束时，我们将确定组成成分，并优化HU-mAbs的配方，建议在人类患者身上进行测试。将首先根据它们的效力、亲和力和相互之间的配伍来鉴定和排序Hu-mAbb。还将确定在细菌感染后提供最长保护所需的配方中每种HU-mAb的最佳剂量。这不是一个假说驱动的建议，而是描述HUS有前景的治疗方法的关键部分，目前还没有有效的治疗方法。

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.