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的抗体疗法的挑战，并撰写了一篇综述/观点文章[Ho抗体疗法2020年；PMID：32566896]。SARS-CoV-2通过其尖峰蛋白(S)与血管紧张素转换酶2(ACE2)结合而进入人类细胞。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抗SARS-CoV-2单峰骆驼纳米抗体方面的工作，并在PNAS上发表了这些数据[Hong等人。美国国家航空航天局。2022年]。我们通过噬菌体展示技术从单峰骆驼中分离到两个VHH纳米体(7A3和8A2)，它们与受体结合域(RBD)具有很高的亲和力，对SARS-CoV-2及其新出现的变异体具有广泛的中和活性。冷冻-EM复合体结构表明，8A2以向上模式与RBD结合，而7A3通过唯一地靶向棘波中高度保守且深埋的位置来抑制受体结合，而无论RBD的构象状态如何。7A3在5 mg/kg的剂量下有效地保护K18-hACE2转基因小鼠免受B.1.351或B.1.617.2的致命攻击，表明该纳米体在抑制出现的SARS-CoV-2变异株的新冠肺炎激增方面具有潜在的治疗潜力。SARS-CoV-2是新冠肺炎大流行的病原体。针对S蛋白，特别是SARS-CoV-2的S1亚单位或受体结合结构域的抗体疗法，因其在治疗确诊为新冠肺炎的患者中的临床疗效而受到关注。传统抗体疗法的替代方法是使用SHARK新抗原可变区(VNAR)抗体。VNAR S很小(15 KDa)，可以深入到目标抗原的口袋或凹槽中。在2023财年，我们通过噬菌体淘汰法从我们实验室构建的护士鲨VNAR噬菌体展示文库中分离到了与S2亚基结合的53个VNAR S。在这些结合剂中，S2A9对原始假型SARS-CoV-2病毒显示出最好的中和活性。包括S2A9在内的几种结合剂显示出与其他贝塔冠状病毒S2亚基的交叉反应。此外，在伪病毒和活病毒中和试验中，S2A9对从α到Omicron的所有变种(VOCs)(包括BA1、BA2、BA4和BA5)都显示出中和活性。我们的发现表明，S2A9可能是一个有希望的先导分子，用于开发针对SARS-CoV-2和新出现的变种的广谱中和抗体。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期刊上发表了这项研究[Jesse Buffington，Zhikin Duan，Hyung Joon Kuan，Jessica Hong，Dan Li，Mingqian Fung，Hang Xie，Mitchell Ho。针对SARS-CoV-2FASEB尖峰S2亚基的护士鲨VNAR单域抗体的鉴定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S1和S2来提高SARS-CoV变异体的效力和覆盖率，中和分离于NCI CCR HO实验室的纳米体。这项工作也可能有助于未来抗击SARS-CoV样感染的斗争。

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.