Therapeutic Antibodies for Bacteria

描述(由申请人提供)：Trellis Bioscience已将其专利CellSpot技术开发为一个强大的平台，用于表征单个人淋巴细胞分泌的抗体对多种抗原的特异性和亲和力。例如，该平台被用来分离一种罕见的、高质量的呼吸道合胞病毒(RSV)人类抗体，该抗体目前正由Trellis的许可证持有人MedImmune准备用于人类临床测试。在这个项目中，我们建议使用CellSpot来分离治疗性抗体来对抗A组链球菌(GAS)感染。克服GAS感染的成年人的血淋巴细胞将被层叠在抗体捕获表面。孵育5小时后，每个细胞分泌的抗体在细胞附近形成“足迹”(直径约150 5米)。然后，这些足迹将与与抗原靶标相连的荧光珠孵化，在本项目中，该靶标将是GAS的ShR蛋白。SHR是一种铁转运蛋白，在GAS菌株中进化上是保守的，也是体内生存所必需的。含有ShR抗体的斑点将在自动显微镜检查下被检测到，并分离出负责的细胞，然后将抗体导入表达系统，然后克隆抗体以进行进一步鉴定。为了进一步确保选定的抗体可以检测GAS细胞表面的Shr，荧光细菌细胞也将包括在CellSpot分析中。这些抗体将通过体外调理试验和体内动物感染模型进行筛选，以测量抗体对吞噬血细胞的杀菌作用，并测量对感染的直接保护作用。在这个项目的第一阶段，我们预计分离出~15种人类抗Shr蛋白的抗体，其中至少有一种将在动物模型中被证明是保护的，证实了抗Shr的兔多克隆血清的保护性所表明的有效性。在这一第一阶段目标取得成功的基础上，我们将提交第二阶段的申请，将这种抗体的开发提前到临床试验。该项目的总体目标是研制一种对GAS耐受性良好、对多种GAS菌株具有高效保护作用的治疗性抗体。

DESCRIPTION (provided by applicant): Trellis Bioscience has developed its patented CellSpot" technology into a robust platform for characterizing the secreted antibody from single human lymphocytes with regard to specificity and affinity against multiple antigens. For example, the platform was used to isolate a rare, high quality human antibody to Respiratory Syncytial Virus (RSV) that is now being prepared for human clinical testing by Trellis' licensee, MedImmune. In this project, we propose to use CellSpot to isolate therapeutic antibodies to combat Group A Streptococcus (GAS) infections. Blood lymphocytes from adults who have overcome GAS infection will be layered onto an antibody capture surface. After 5 hours of incubation, each cell's secreted antibody forms a "footprint" in the vicinity of the cell (~150 5m diameter). These footprints will then be incubated with fluorescent beads conjugated to the antigen target, which in this project will be the Shr protein of GAS. Shr is an iron transporter protein that is both evolutionarily conserved among GAS strains and required for survival in vivo. Spots that contain antibody to Shr will be detected under automated microscopic examination and the responsible cells will be isolated, followed by cDNA cloning of the antibody for further characterization following transfection into an expression system. To further insure that selected antibodies can detect Shr on the surface of GAS cells, fluorescent bacterial cells will also be included in the CellSpot analysis. These antibodies will be screened for physiological activity both by an in vitro opsonization assay to measure antibody-mediated bacteria killing by phagocytic blood cells and by an in vivo animal infection model to measure direct protection from infection. In Phase I of this project, we anticipate isolating ~15 human antibodies to the Shr protein, of which at least one will be shown to be protective in the animal model, confirming utility suggested by the protective nature of a rabbit polyclonal serum raised against Shr. Based on success in this Phase I objective, we will submit a Phase II application to advance the development of this antibody to clinical trial. The overall objective of this project is a therapeutic antibody to GAS which is well tolerated and highly effective in protecting against a broad range of GAS strains.