Generation of Neutralizing Monoclonal Antibodies Against SARS-CoV2 for Immunotherapy of Patients with COVID-19

生成针对 SARS-CoV2 的中和单克隆抗体，用于 COVID-19 患者的免疫治疗

• 批准号: 10272298
• 负责人: Patrizia Farci
• 金额: $ 7.13万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10272298
• 关键词: 2019-nCoV; Affinity; Animal Model; Antibodies; Antibody Repertoire; Antibody-Producing Cells; Area; B-Lymphocytes; Bacteriophages; Binding; Biological Assay; Blood Banks; COVID-19; Cell Culture Techniques; Cessation of life; Clone Cells; Complementary DNA; Enzyme-Linked Immunosorbent Assay; Generations; Genes; Human; Immune response; Immunocompromised Host; Immunoglobulin G; Immunoglobulin Genes; Immunotherapy; Length; Libraries; Measurement; Memory B-Lymphocyte; Messenger RNA; Methods; Monoclonal Antibodies; Nucleocapsid Proteins; Patients; Peripheral Blood Mononuclear Cell; Phage Display; Plasma; Plasma Cells; Plasmablast; Process; Proteins; RNA; Recombinants; Research; Structural Protein; Surface Plasmon Resonance; System; Technology; Testing; Transgenic Mice; United States; United States National Institutes of Health; Vaccines; Virus; Whole Blood; base; combinatorial; effective therapy; neutralizing antibody; neutralizing monoclonal antibodies; recruit; screening

Screening of more than 120 convalescent patients from COVID-19, recruited at the NIH Blood Bank, identified 14 convalescent patients with high titers of neutralizing antibodies against SARS-CoV2. These donors were recalled and whole blood was processed (isolation of PBMC, B-cell purification, extraction of mRNA, cDNA synthesis, and PCR amplification of VH, VK and VL). Four phage antibody-display libraries were constructed from isolated B-cell RNA. All 4 libraries were panned against the recombinant trimeric S protein of SARS-CoV-2. So far, this has led to the identification of 384 positive antibody clones that specifically bind to the S protein. Sequencing analysis is in progress. The selected monoclonal antibodies will be functionally characterized by ELISA and affinity measurement by using surface plasmon resonance (SPR), followed by expression of Fabs as full-length IgG and determination of the neutralizing activity against SARS-CoV2, using initially a pseudovirus-based assay system and then a neutralization assay with the entire virus. In addition, the SARS-CoV2 virus has several other structural proteins including M (matrix), E (envelope) and N (nucleocapsid) proteins. Once these proteins are available or prove to be valid targets, our phage libraries can be used again to screen for mAbs against these targets. The last step will include testing of these antibodies in small animal models.

在NIH血库招募了来自Covid-19的120多名COVID患者的筛查，确定了14名康复性患者，具有对SARS-COV2的中和抗体的高滴度。召回这些供体并处理全血（分离PBMC，B细胞纯化，MRNA的提取，cDNA合成和VH，VK和VL的PCR扩增）。由分离的B细胞RNA构建了四个噬菌体抗体 - 播放库。所有4个文库都针对SARS-COV-2的重组三聚体S蛋白。到目前为止，这导致了特异性结合S蛋白的384个阳性抗体克隆的鉴定。测序分析正在进行中。所选的单克隆抗体将在功能上以ELISA和亲和力测量，通过使用表面等离子体共振（SPR），然后表达Fabs作为全长IgG，并确定针对SARS-COV2的中和活性，最初使用基于伪病毒的分析系统，然后使用基于Pseudovirus的分析系统进行中性分析，然后对整个病毒性进行中性分析。此外，SARS-COV2病毒具有其他几种结构蛋白，包括M（矩阵），E（Invelope）和N（Nucleocapsid）蛋白。一旦这些蛋白质可用或被证明是有效的靶标，我们的噬菌体库可以再次使用以筛选对这些目标的mAb。最后一步将包括对小动物模型中这些抗体的测试。