It is worth noting that given the recent success of COVID-19 vaccines, future development ofNanosota-1series should focus on its therapeutic efficacy rather than its preventive efficacy

It is worth noting that given the recent success of COVID-19 vaccines, future development ofNanosota-1series should focus on its therapeutic efficacy rather than its preventive efficacy. of such drugs. Though small-molecule drugs could target SARS-CoV-2, it can take years to develop them and their use is often limited by poor specificity and off-target effects. Repurposed drugs, developed against other viruses, also have low specificity against SARS-CoV-2. Therapeutic antibodies have been identified and generally have high specificity; however, their expression in mammalian cells often leads to low yields and high production costs (Salazar et al., 2017;Breedveld, 2000). A realistic therapeutic solution to COVID-19 must be potent and specific, yet easy to produce. Nanobodies (or VHH antibodies) are unique antibodies derived from heavy-chain-only antibodies found in members of the camelidae family (llamas, alpacas, camels, and so on) (Figure 1;Figure 1figure-supplement 1;Knning et al., 2017;De Meyer et al., 2014). Because of their small size (2.5 nm by 4 nm; 1215 kDa) and unique binding domains, nanobodies offer many advantages over conventional antibodies including the ability to bind cryptic epitopes on their antigen, high tissue permeability, ease of production, and thermostability (Muyldermans, 2013;Steeland et al., 2016). Although small, nanobodies bind their targets with high affinity and specificity due to an extended antigen-binding region (Muyldermans, 2013;Steeland et al., 2016). One drawback of nanobodies is their quick clearance by kidneys due to their small size; this can be overcome by adding tags to increase the molecular weight to a desired level that is above the kidney clearance threshold but still much lower than conventional antibodies molecular weight. Underscoring the potency and safety of nanobodies as human therapeutics, a nanobody drug was recently approved for clinical use in treating a blood clotting disorder (Scully et al., 2019). Additionally, due to their superior stability, nanobodies can be inhaled to treat lung diseases CIL56 (Van Heeke et al., 2017) or ingested to treat intestine diseases (Vega et al., 2013). Nanobodies are currently being developed against SARS-CoV-2 to combat COVID-19 (Huo et al., 2020;Hanke et al., 2020;Xiang et al., 2020;Schoof et al., 2020;Wrapp et al., 2020a). However, these reported nanobodies have not been adequately evaluated for therapeutic efficacy in vivo. == Figure 1. Construction of a camelid nanobody phage display library and use of this library for screening of anti-SARS-CoV-2 nanobodies. == A large-sized (diversity 7.5 x 1010) naive nanobody phage display library was constructed using B cells of over CIL56 a dozen llamas and alpacas. Phages were screened for their high binding affinity for SARS-CoV-2 receptor-binding domain (RBD). Nanobodies expressed from the selected phages were further screened for their potency in neutralizing SARS-CoV-2 pseudovirus entry. The Rabbit Polyclonal to RIMS4 best performing nanobody was subjected to two rounds of affinity maturation. == Figure 1figure supplement 1. Schematic drawings of nanobodies and conventional antibodies. == The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a prime target for therapeutic development (Li, 2015). The spike protein guides coronavirus entry into host cells by first binding to a receptor on the host cell surface and then fusing the viral and host membranes (Li, 2016;Perlman and Netland, 2009). The RBDs of SARS-CoV-2 and a closely related SARS-CoV-1 both recognize human angiotensin-converting enzyme 2 (ACE2) as their receptor (Li, 2015;Wan et al., 2020;Li et al., 2003;Zhou et al., 2020). Previously, we have shown that SARS-CoV-1 and SARS-CoV-2 RBDs both contain a core structure and a receptor-binding motif (RBM) and that SARS-CoV-2 RBD has significantly higher ACE2-binding affinity than SARS-CoV-1 RBD due to several structural changes in the RBM (Shang et al., 2020a;Li et al., 2005). We have further shown that SARS-CoV-2 RBD is more hidden than SARS-CoV-1 RBD in the entire spike protein as a possible viral strategy for immune evasion (Shang et al., 2020b). Hence, to block SARS-CoV-2 binding to ACE2, a nanobody drug would need to bind to SARS-CoV-2 RBD more tightly than ACE2. Here we report the development of a series of anti-SARS-CoV-2 nanobody drug candidates,Nanosota-1. Identified by screening a camelid nanobody phage display library against the SARS-CoV-2 RBD, theNanosota-1series bound potently to the SARS-CoV-2 RBD and were effective at inhibiting SARS-CoV-2 infection in vitro. The best performing drug CIL56 candidate,Nanosota-1C-Fc, demonstrated preventative and therapeutic efficacy against SARS-CoV-2 infection in both hamster and mouse models. Produced at high yields,Nanosota-1C-Fcis scalable for mass production easily. It proven superb in vitro thermostability also, in vivo balance, and bioavailability. Our data recommend thatNanosota-1c-Fccan.