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Development of neutralizing nanobodies against SARS-CoV-2

开发针对 SARS-CoV-2 的中和纳米抗体

基本信息

批准号：
10926408
负责人：
Mitchell Ho
金额：
$ 29.09万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/10926408
关键词：
2019-nCoV ACE2 Affinity Antibodies Antibody Therapy Antigen Targeting Antigens Attention Bacteriophages Binding Binding Proteins Biological Biological Assay CCR COVID-19 COVID-19 diagnosis COVID-19 pandemic Camels Cells Complex Cross Reactions Cryoelectron Microscopy Data Development Dose Dromedaries Etiology Future Human Infection Journals K-18 conjugate Laboratories Libraries Molecular Conformation Nurses Patients Phage Display Protein Engineering Proteins Publishing Receptor Inhibition Research SARS coronavirus SARS-CoV-2 B.1.351 SARS-CoV-2 B.1.617.2 SARS-CoV-2 antibody SARS-CoV-2 spike protein SARS-CoV-2 variant Shark Site Structure Surface Therapeutic Therapeutic antibodies Transgenic Mice Variant Viral Virus Work Writing betacoronavirus clinical efficacy cross reactivity global health improved nanobodies neutralizing antibody novel pandemic disease pathogenic virus receptor receptor binding therapeutic target variants of concern

项目摘要

During the pandemic, Dr Ho has evaluated the challenges for developing antibody therapeutics targeting SARS-Cov-2 and wrote a review/perspective article [Ho Antibody Therapeutics 2020; PMID: 32566896]. SARS-CoV-2 gains entry to human cells through its spike (S) protein binding to angiotensin-converting enzyme 2 (ACE2). Therefore, the S protein is the primary target for neutralizing antibodies. In FY2022, we summarized our work on isolation of dromedary camel nanobodies against SARS-CoV-2 and published the data in PNAS [Hong et al. PNAS. 2022]. We isolated two VHH nanobodies (7A3 and 8A2) from dromedary camels by phage display, which have high affinity for the receptor-binding domain (RBD) and broad neutralization activities against SARS-CoV-2 and its emerging variants. Cryo-EM complex structures reveal that 8A2 binds the RBD in its up mode and 7A3 inhibits receptor binding by uniquely targeting a highly conserved and deeply buried site in the spike regardless of the RBD conformational state. 7A3 at a dose of 5 mg/kg efficiently protects K18-hACE2 transgenic mice from the lethal challenge of B.1.351 or B.1.617.2, suggesting that the nanobody has promising therapeutic potentials to curb the COVID-19 surge with emerging SARS-CoV-2 variants. SARS-CoV-2 is the etiological agent of the COVID-19 pandemic. Antibody-based therapeutics targeting the spike protein, specifically the S1 subunit or the receptor binding domain (RBD) of SARS-CoV-2, have gained attention due to their clinical efficacy in treating patients diagnosed with COVID-19. An alternative to conventional antibody therapeutics is the use of shark new antigen variable receptor domain (VNAR ) antibodies. VNAR s are small (15 kDa) and can reach deep into the pockets or grooves of the target antigen. In FY2023, we isolated 53 VNAR s that bind to the S2 subunit by phage panning from a naive nurse shark VNAR phage display library constructed in our laboratory. Among those binders, S2A9 showed the best neutralization activity against the original pseudotyped SARS-CoV-2 virus. Several binders, including S2A9, showed cross-reactivity against S2 subunits from other beta coronaviruses. Furthermore, S2A9 showed neutralization activity against all variants of concern (VOCs) from alpha to omicron (including BA1, BA2, BA4, and BA5) in both pseudovirus and live virus neutralization assays. Our findings suggest that S2A9 could be a promising lead molecule for the development of broadly neutralizing antibodies against SARS-CoV-2 and emerging variants. The nurse shark VNAR phage library offers a novel platform that can be used to rapidly isolate single-domain antibodies against emerging viral pathogens. We published this work in FASEB Journal in FY2023 [Jesse Buffington, Zhijian Duan, Hyung Joon Kwon, Jessica Hong, Dan Li, Mingqian Feng, Hang Xie, Mitchell Ho. Identification of nurse shark VNAR single-domain antibodies targeting the spike S2 subunit of SARS-CoV-2 FASEB J. 2023 Jun;37(6):e22973. doi: 10.1096/fj.202202099RR.] Ongoing studies aim to improve the potency and coverage of SARS-CoV variants by using protein engineering and combining both S1 and S2 neutralizing nanobodies isolated in the Ho laboratory at the CCR, NCI. This work may also be helpful for future battles against SARS-CoV-like infections.

在大流行期间，何博士评估了开发针对SARS-Cov-2的抗体疗法的挑战，并撰写了一篇综述/展望文章[Ho Antibody Therapeutics 2020; PMID：32566896]。SARS-CoV-2通过与血管紧张素转换酶2（ACE 2）结合的刺突蛋白（S）进入人体细胞。因此，S蛋白是中和抗体的主要靶标。在2022财年，我们总结了我们关于分离单峰骆驼纳米抗体以对抗SARS-CoV-2的工作，并在PNAS上发表了数据[Hong et al. PNAS. 2022年]。我们通过噬菌体展示从单峰骆驼中分离出两种VHH纳米抗体（7A 3和8A 2），它们对受体结合结构域（RBD）具有高亲和力，并且对SARS-CoV-2及其新出现的变体具有广泛的中和活性。Cryo-EM复合物结构揭示，8A 2以其向上模式结合RBD，7A 3通过独特地靶向刺突中高度保守且深埋的位点而抑制受体结合，而不管RBD构象状态如何。5 mg/kg剂量的7A 3有效地保护K18-hACE 2转基因小鼠免受B.1.351或B.1.617.2的致死性攻击，这表明纳米抗体具有抑制COVID-19激增和新出现的SARS-CoV-2变体的有希望的治疗潜力。SARS-CoV-2是COVID-19大流行的病原体。靶向刺突蛋白（特别是SARS-CoV-2的S1亚基或受体结合结构域（RBD））的基于抗体的治疗方法因其在治疗诊断为COVID-19的患者中的临床疗效而受到关注。常规抗体疗法的替代方案是使用鲨鱼新抗原可变受体结构域（VNAR）抗体。VNAR很小（15 kDa），可以深入靶抗原的口袋或凹槽。在FY 2023中，我们通过噬菌体淘选从我们实验室构建的幼稚护士鲨VNAR噬菌体展示文库中分离出53个与S2亚基结合的VNAR。在这些结合剂中，S2 A9显示出对原始假型SARS-CoV-2病毒的最佳中和活性。包括S2 A9在内的几种结合剂显示出与其他β冠状病毒S2亚基的交叉反应性。此外，在假病毒和活病毒中和试验中，S2 A9显示出对从α到Omicron的所有相关变体（VOC）（包括BA 1、BA 2、BA 4和BA 5）的中和活性。我们的研究结果表明，S2 A9可能是一个有前途的领导分子，用于开发针对SARS-CoV-2和新出现的变体的广泛中和抗体。护士鲨VNAR噬菌体文库提供了一个新的平台，可用于快速分离针对新兴病毒病原体的单域抗体。我们于2023财年在FASEB Journal上发表了这项工作[Jesse Buffington，Zhijian Duan，Hyung Joon Kwon，Jessica Hong，Dan Li，Mingqian Feng，Hang Xie，Mitchell Ho。鉴定护士鲨VNAR单域抗体靶向SARS-CoV-2的刺突S2亚基FASEB J. 2023 Jun;37（6）：e22973. doi：10.1096/fj.202202099RR.]正在进行的研究旨在通过使用蛋白质工程并结合在CCR，NCI的Ho实验室中分离的S1和S2中和纳米抗体来提高SARS-CoV变体的效力和覆盖率。这项工作也可能有助于未来对抗SARS-CoV样感染的斗争。