Development of neutralizing nanobodies against SARS-CoV-2

在大流行期间，何博士评估了开发针对SARS-Cov-2的抗体疗法所面临的挑战，并撰写了一篇综述/观点文章[Ho antibody therapeutics 2020；PMID: 32566896)。SARS-CoV-2通过其刺突(S)蛋白与血管紧张素转换酶2 （ACE2）结合进入人体细胞。因此，S蛋白是中和抗体的主要靶点。在2022财年，我们总结了我们在分离单峰骆驼纳米体对抗SARS-CoV-2方面的工作，并在PNAS上发表了相关数据[Hong等]。《美国国家科学院学报》上。2022]。我们通过噬菌体展示从单峰骆驼中分离出两个VHH纳米体（7A3和8A2），它们对受体结合域（RBD）具有高亲和力，对SARS-CoV-2及其新变体具有广泛的中和活性。Cryo-EM复合物结构显示，8A2以其上向模式结合RBD，而7A3通过独特地靶向RBD中高度保守和深埋的位点来抑制受体结合，而不管RBD的构象状态如何。5mg /kg剂量的7A3可有效保护K18-hACE2转基因小鼠免受B.1.351或B.1.617.2的致命攻击，这表明该纳米体在抑制新出现的SARS-CoV-2变体的COVID-19激增方面具有良好的治疗潜力。SARS-CoV-2是COVID-19大流行的病原体。针对刺突蛋白（特别是SARS-CoV-2的S1亚基或受体结合域（RBD））的抗体治疗方法因其治疗COVID-19患者的临床疗效而受到关注。一种替代常规抗体治疗的方法是使用鲨鱼新抗原可变受体结构域（VNAR）抗体。VNAR很小（15 kDa），可以深入到目标抗原的口袋或凹槽中。20123财年，我们通过噬菌体筛选，从实验室构建的初代护士鲨VNAR噬菌体展示文库中分离出53个与S2亚基结合的VNAR。在这些结合物中，S2A9对原始伪型SARS-CoV-2病毒表现出最好的中和活性。包括S2A9在内的几种结合剂对来自其他β冠状病毒的S2亚基表现出交叉反应性。此外，在假病毒和活病毒中和实验中，S2A9对从α到组粒的所有VOCs变体（包括BA1、BA2、BA4和BA5）都有中和活性。我们的研究结果表明，S2A9可能是一种有希望的先导分子，用于开发针对SARS-CoV-2和新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出现的病毒病原体的单域抗体。我们在2023财年的FASEB Journal上发表了这项研究[Jesse Buffington， Zhijian Duan, Hyung Joon Kwon, Jessica Hong, Dan Li, Mingqian Feng, Hang Xie, Mitchell Ho]。SARS-CoV-2亚型特异性VNAR单结构域抗体的鉴定[j] .浙江大学学报（自然科学版）；2009;37(6):591 - 591。doi: 10.1096 / fj.202202099RR。正在进行的研究旨在通过蛋白质工程和结合从NCI CCR Ho实验室分离的S1和S2中和纳米体来提高SARS-CoV变体的效力和覆盖范围。这项工作也可能有助于未来对抗sars冠状病毒感染。

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.