Characterization of therapeutic human monoclonal antibodies against SARS

抗SARS治疗性人源单克隆抗体的鉴定 冠状病毒是SARS病原体的发现及其快速基因组测序为开发针对该病毒的有效预防和治疗模式创造了机会。特别感兴趣的是刺突糖蛋白（S蛋白），其表达为表面投影。S蛋白参与病毒的粘附、细胞-细胞融合、诱导保护性免疫反应等 中和该提案将集中于开发可用于预防和/或暴露后治疗SARS-CoV感染的人单克隆抗体。我们已经与Abgenix Inc密切合作，使用其XenoMouse产生了一组能够产生中和人单克隆抗体（HmAb）的杂交瘤。这些杂交瘤已被克隆，并已产生19种HmAb，纯化并在中和试验中重新测试。此外，我们已经产生了几个重组片段的S蛋白，并获得重叠的肽跨越S蛋白，并使用它们来确定HmAb的结合特异性。在目标-1中，我们将进一步表征人单克隆抗体在体外抗SARS-CoV的中和能力，并使用许多不同的方法确定其相对结合亲和力和特异性。在aim-2中，我们将鉴定出能够潜在地中和广泛的 通过在病毒进入试验和细胞融合试验中使用表达突变S蛋白的不同假型病毒对临床分离株进行检测，所述突变S蛋白含有在来自世界各地的90多个临床分离株中鉴定的氨基酸取代。这些研究预期将导致HmAb的表征，其具有作为针对人类SARS-CoV感染的被动免疫治疗剂的潜力。

Characterization of Therapeutic human monoclonal antibodies against SARS The discovery that a corona virus is the causative agent of SARS and its rapid genomic sequencing has created opportunities to develop effective modalities of prevention and treatment against this virus. Of particular interest is the spike glycoprotein (S protein), which is expressed as a surface projection. The S protein is involved in viral attachment, cell-cell fusion, the induction of protective immune response in virus neutralization. This proposal will focus on developing human monoclonal antibodies that can be used for prophylaxis, and/or post-exposure treatment of SARS-CoV infection. We have already generated a panel of hybridomas capable of producing neutralizing human monoclonal antibodies (HmAbs) in close collaboration with Abgenix Inc using their XenoMouse. These hybridomas have been cloned and 19 HmAbs have been produced, purified and re-tested in a neutralization assay. In addition, we have already produced several recombinant fragments of the S protein and obtained overlapping peptides that span the S protein, and used them to determine binding specificities of HmAbs. In aim-1 we will further characterize the neutralizing ability of human monoclonal antibodies against SARS-CoV in vitro and determine their relative binding affinities and specificities using a number of different current approaches. In aim-2, we will identify human monoclonal antibodies that can potentially neutralize a broad range of clinical isolates by testing them in a viral entry and a cell fusion assay using different pseudotyped viruses expressing mutant S proteins that contain amino acid substitutions identified in over 90 clinical isolates from around the world. These studies are expected to result in the characterization of HmAbs with potential as passive immunotherapeutics against SARS-CoV infection in humans.