Development of neutralizing nanobodies against SARS-CoV-2

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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.