Therapeutic Antibodies for Bacteria

描述（由申请人提供）：Trellis Bioscience已将其专利CellSpot技术开发成一个强大的平台，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，该平台用于分离一种罕见的高质量呼吸道合胞病毒（RSV）人类抗体，该抗体目前正由Trellis的许可证持有人MedImmune制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，以对抗A组链球菌（GAS）感染。来自克服GAS感染的成人的血液淋巴细胞将被分层到抗体捕获表面上。孵育5小时后，每个细胞分泌的抗体在细胞附近形成一个“足迹”（直径约150 μ m）。然后将这些足迹与缀合至抗原靶标的荧光珠一起孵育，在本项目中，抗原靶标将是GAS的Shr蛋白。Shr是一种铁转运蛋白，在GAS菌株中进化保守，并且是体内生存所必需的。将在自动显微镜检查下检测含有抗Shr抗体的斑点，并分离责任细胞，然后进行抗体的cDNA克隆，以在转染至表达系统后进行进一步表征。为了进一步确保选择的抗体可以检测GAS细胞表面上的Shr，荧光细菌细胞也将包括在CellSpot分析中。将通过体外调理作用测定来筛选这些抗体的生理活性，以测量吞噬血细胞对抗体介导的细菌杀伤，并通过体内动物感染模型来测量对感染的直接保护。在该项目的第一阶段，我们预计分离约15种针对Shr蛋白的人抗体，其中至少一种将在动物模型中显示出保护性，证实了针对Shr产生的兔多克隆血清的保护性所建议的实用性。基于第一阶段目标的成功，我们将提交第二阶段申请，以推进这种抗体的临床试验。该项目的总体目标是一种GAS的治疗性抗体，其耐受性良好，并且在保护免受广泛的GAS菌株方面非常有效。

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.