Specific Human Monoclonal Antibodies for HUS Prophylaxis

本提案是NIH奖#2 R 01-A1-41326延续的第二次修订竞争性更新。我们的目标是开发一种免疫制剂，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STEC的临床分离株主要产生Stxl、Stx 2和/或Stx 2c。儿童特别容易发展Stx介导的HUS。我们的假设是，Stx特异性抗体的管理将防止或修改个人的感染结果在发展HUS的风险。在早期的奖项中，我们已经产生了一组对Stx 1或Stx 2具有特异性的人单克隆抗体（Hu-mAb）。使用gnotobiotic仔猪模型，我们已经表明，Stx特异性Hu-mAb中和Stx，并防止Stx介导的并发症的发展。 我们现在希望确定哪种Hu-mAb应包含在适合临床评价的制剂中。基于上级功效，选择了四种对Stx 2具有特异性的Hu-mAb（3种针对A亚基，1种针对B亚基）和2种对Stx 1具有特异性的Hu-mAb（均针对B亚基）作为候选物。下一步是确定哪种Hu-mAb组合既兼容又高效。 在本提案中，我们计划定义结构和功能特征， Stx-1和Stx 2特异性Hu-mAb的保护功效（特异性目标1）。然后将研究每种HumAb对各自毒素的亲和力和效力（特异性目标2）。然后根据细菌感染后的时间确定给定抗体剂量的保护效力（具体目标3）。最后，将检查对Stx 1的B亚基和Stx 2的A或B亚基具有特异性的Hu-mAb的组合的相对功效和相容性，以确定哪一种是最有效的，从而适合于临床评价（具体目的4）。 在这些实验结束时，我们将确定组分，并优化Hu-mAb的制剂，其将被推荐用于在人类患者中进行测试。Hu-mAb将首先根据其功效、亲和力和彼此相容性进行表征和排序。还将确定制剂中每种Hu-mAb在细菌感染后提供最长保护所需的最佳量。这不是一个假设驱动的建议，而是表征HUS有前途的治疗的重要部分，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.