The development of Nanosota-1 as anti-SARS-CoV-2 nanobody drug candidates

Gang Ye; Joseph Gallant; Jian Zheng; Christopher Massey; Ke Shi; Wanbo Tai; Abby Odle; Molly Vickers; Jian Shang; Yushun Wan; Lanying Du; Hideki Aihara; Stanley Perlman; Aaron LeBeau; Fang Li

doi:10.7554/eLife.64815

. 2021 Aug 2;10:e64815. doi: 10.7554/eLife.64815

The development of Nanosota-1 as anti-SARS-CoV-2 nanobody drug candidates

Gang Ye ^1,^2,^†, Joseph Gallant ^3,^†, Jian Zheng ^4,^†, Christopher Massey ⁵, Ke Shi ⁶, Wanbo Tai ⁷, Abby Odle ⁴, Molly Vickers ⁴, Jian Shang ^1,², Yushun Wan ^1,², Lanying Du ⁷, Hideki Aihara ⁶, Stanley Perlman ^4,^✉, Aaron LeBeau ^3,^✉, Fang Li ^1,^2,^✉

Editors: John W Schoggins⁸, Jos W Van der Meer⁹

PMCID: PMC8354634 PMID: 34338634

Abstract

Combating the COVID-19 pandemic requires potent and low-cost therapeutics. We identified a series of single-domain antibodies (i.e., nanobody), Nanosota-1, from a camelid nanobody phage display library. Structural data showed that Nanosota-1 bound to the oft-hidden receptor-binding domain (RBD) of SARS-CoV-2 spike protein, blocking viral receptor angiotensin-converting enzyme 2 (ACE2). The lead drug candidate possessing an Fc tag (Nanosota-1C-Fc) bound to SARS-CoV-2 RBD ~3000 times more tightly than ACE2 did and inhibited SARS-CoV-2 pseudovirus ~160 times more efficiently than ACE2 did. Administered at a single dose, Nanosota-1C-Fc demonstrated preventive and therapeutic efficacy against live SARS-CoV-2 infection in both hamster and mouse models. Unlike conventional antibodies, Nanosota-1C-Fc was produced at high yields in bacteria and had exceptional thermostability. Pharmacokinetic analysis of Nanosota-1C-Fc documented an excellent in vivo stability and a high tissue bioavailability. As effective and inexpensive drug candidates, Nanosota-1 may contribute to the battle against COVID-19.

Research organism: Virus

Introduction

The novel coronavirus SARS-CoV-2 has led to the COVID-19 pandemic, devastating human health and global economy (Li et al., 2020; Huang et al., 2020). Anti-SARS-CoV-2 drugs are urgently needed to treat patients, save lives, and help revive economy. Yet daunting challenges confront the development 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 1—figure-supplement 1; Könning et al., 2017; De Meyer et al., 2014). Because of their small size (2.5 nm by 4 nm; 12–15 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 (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. — A large-sized (diversity 7.5 x 10¹⁰) naive nanobody phage display library was constructed using B cells of over 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 best performing nanobody was subjected to two rounds of affinity maturation.

Figure 1—figure supplement 1. — A large-sized (diversity 7.5 x 10¹⁰) naive nanobody phage display library was constructed using B cells of over 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 best performing nanobody was subjected to two rounds of affinity maturation.

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, the Nanosota-1 series bound potently to the SARS-CoV-2 RBD and were effective at inhibiting SARS-CoV-2 infection in vitro. The best performing drug 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-Fc is easily scalable for mass production. It also demonstrated excellent in vitro thermostability, in vivo stability, and bioavailability. Our data suggest that Nanosota-1c-Fc can potentially contribute to the battle against COVID-19.

Results

Nanosota-1 was identified by phage display

For the rapid identification of virus-targeting nanobodies, we constructed a naive nanobody phage display library using B cells isolated from the spleen, bone marrow, and blood of nearly a dozen non-immunized llamas and alpacas (Figure 1). Recombinant SARS-CoV-2 RBD, expressed and purified from mammalian cells, was screened against the library to identify RBD-targeting nanobodies. Selected nanobody clones were tested in a preliminary screen for their ability to neutralize SARS-CoV-2 pseudovirus entry into target cells (more details about the assay are reported below). The nanobody that demonstrated the highest preliminary neutralization potency was named Nanosota-1A and was subjected to two rounds of affinity maturation. For each round, random mutations were introduced to the whole gene of Nanosota-1A through error-prone polymerase chain reaction (PCR), and mutant phages were selected for enhanced binding to SARS-CoV-2 RBD. Nanobodies contain four framework regions (FRs) as structural scaffolds and three complementarity-determining regions (CDRs) for antigen binding. The nanobody after the first round of affinity maturation, Nanosota-1B, possessed one mutation in CDR3 and two other mutations in FR3 (near CDR3). Affinity maturation of Nanosota-1B resulted in Nanosota-1C, which possessed one mutation in CDR2 and another mutation in FR2. We next made an Fc-tagged version of Nanosota-1C, termed Nanosota-1C-Fc, to create a bivalent construct with increased molecular weight.

Nanosota-1 tightly bound to the SARS-CoV-2 RBD and completely blocked the binding of ACE2

To understand the structural basis for the binding of Nanosota-1 to SARS-CoV-2 RBD, we determined the crystal structure of SARS-CoV-2 RBD complexed with Nanosota-1C. The structure showed that Nanosota-1C binds close to the center of the SARS-CoV-2 RBM (Figure 2A). Among the 14 RBM residues that directly interact with Nanosota-1C, six also directly interact with human ACE2 (Figure 2—figure supplement 1). When the structures of the RBD/Nanosota-1C complex and the RBD/ACE2 complex were superimposed together, significant clashes occurred between ACE2 and Nanosota-1C (Figure 2B), suggesting that Nanosota-1C binding to the RBD blocks ACE2 binding to the RBD. Moreover, trimeric SARS-CoV-2 spike protein is present in two different conformations: the RBD stands up in the open conformation but lies down in the closed conformation (Shang et al., 2020b; Wrapp et al., 2020b; Ke et al., 2020). When the structures of the RBD/Nanosota-1C complex and the closed spike were superimposed, no clash was found between RBD-bound Nanosota-1C and the rest of the spike protein (Figure 2—figure supplement 2A). In contrast, severe clashes were identified between RBD-bound ACE2 and the rest of the spike protein in the closed conformation (Figure 2—figure supplement 2B). Additionally, neither RBD-bound Nanosota-1C nor RBD-bound ACE2 had clashes with the rest of the spike protein in the open conformation (Figure 2—figure supplement 2C; Figure 2—figure supplement 2D). Thus, Nanosota-1C can access the spike protein in both its open and closed conformations, whereas ACE2 can only access the spike protein in its open conformation. Overall, our structural data reveal that Nanosota-1C is an ideal RBD-targeting drug candidate that not only blocks virus binding to its receptor, but also accesses its target in the spike protein in different conformations.

Figure 2. — (A) Structure of SARS-CoV-2 receptor-binding domain (RBD) complexed with *Nanosota-1C*, viewed at two different angles. *Nanosota-1C* is in red, the core structure of RBD is in cyan, and the receptor-binding motif (RBM) of RBD is in magenta. Structure data and refinement statistics are shown in Table 2. (B) Overlay of the structures of the RBD/*Nanosota-1C* complex and RBD/angiotensin-converting enzyme 2 (ACE2) complex (PDB 6M0J). ACE2 is in green. The structures of the two complexes were superimposed based on their common RBD structure. The *Nanosota-1C* loops that have clashes with ACE2 are in blue.

Figure 2—figure supplement 1. — (A) Structure of SARS-CoV-2 receptor-binding domain (RBD) complexed with *Nanosota-1C*, viewed at two different angles. *Nanosota-1C* is in red, the core structure of RBD is in cyan, and the receptor-binding motif (RBM) of RBD is in magenta. Structure data and refinement statistics are shown in Table 2. (B) Overlay of the structures of the RBD/*Nanosota-1C* complex and RBD/angiotensin-converting enzyme 2 (ACE2) complex (PDB 6M0J). ACE2 is in green. The structures of the two complexes were superimposed based on their common RBD structure. The *Nanosota-1C* loops that have clashes with ACE2 are in blue.

To corroborate our structural data on RBD/Nanosota-1 interactions, we performed binding experiments between Nanosota-1 and SARS-CoV-2 RBD using recombinant ACE2 as a comparison. The binding affinity between Nanosota-1 and the RBD was measured using surface plasmon resonance (Table 1; Figure 2—figure supplement 3). Nanosota-1A, -1B, and -1C bound to the RBD with increasing affinity (K_d- 228–14 nM), confirming success of the stepwise affinity maturation. Nanosota-1C-Fc had the highest RBD-binding affinity (K_d - 15.7 pM), which was ~3000 times tighter than the RBD-binding affinity of ACE2. Moreover, compared with ACE2, Nanosota-1C-Fc bound to the RBD with a higher k_on and a lower k_off, demonstrating significantly faster binding and slower dissociation. Next, we investigated the competitive binding among Nanosota-1C, RBD, and ACE2 using protein pull-down assay (Figure 2—figure supplement 4A). Nanosota-1C and ACE2 were mixed together in different ratios in solution, with the concentration of ACE2 kept constant; RBD was added to pull down Nanosota-1C and ACE2 from solution. The result showed that as the concentration of Nanosota-1C increased, less ACE2 was pulled down by the RBD. Thus, Nanosota-1C and ACE2 bound competitively to the RBD. We repeated the above protein pull-down assay, with Nanosota-1C-Fc replacing Nanosota-1C (Figure 2—figure supplement 4B). The result confirmed that Nanosota-1C-Fc and ACE2 bound competitively to the RBD; it further showed that Nanosota-1C-Fc bound to the RBD much more strongly than ACE2 did, consistent with the binding affinity measurement. We then analyzed the competitive binding among Nanosota-1C, RBD, and ACE2 using gel filtration chromatography (Figure 2—figure supplement 4C). Nanosota-1C, RBD, and ACE2 were mixed, with both Nanosota-1C and ACE2 in molar excess over the RBD. Analysis by gel filtration chromatography documented that no ternary complex of Nanosota-1C, RBD, and ACE2 formed; instead, only binary complexes of RBD/ACE2 and RBD/Nanosota-1C were detected. Hence, the bindings of Nanosota-1C and ACE2 to the RBD are mutually exclusive.

Table 1. Binding affinities between Nanosota-1 and SARS-CoV-2 RBD as measured using surface plasmon resonance.

The previously determined binding affinity between human ACE2 and RBD is shown as a comparison (Shang et al., 2020a).

	K_d with SARS-CoV-2 RBD (M)	k_off (s⁻¹)	k_on (M⁻¹s⁻¹)
Nanosota-1A (before affinity maturation)	2.28 x 10⁻⁷	9.35 x 10⁻³	4.10 x 10⁴
Nanosota-1B (after first round of affinity maturation)	6.08 x 10⁻⁸	7.19 x 10⁻³	1.18 x 10⁵
Nanosota-1C (after second round of affinity maturation)	1.42 x 10⁻⁸	2.96 x 10⁻³	2.09 x 10⁵
Nanosota-1C-Fc (after second round of affinity maturation; containing a C-terminal human Fc tag)	1.57 x 10⁻¹¹	9.68 x 10⁻⁵	6.15 x 10⁶
ACE2	4.42 x 10⁻⁸	7.75 x 10⁻³	1.75 x 10⁵

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Nanosota-1C-Fc potently neutralized SARS-CoV-2 infection in vitro and in vivo

The ability of Nanosota-1 to neutralize SARS-CoV-2 infection in vitro was investigated next. Both SARS-CoV-2 pseudovirus entry assay and live SARS-CoV-2 infection assay were performed (Figure 3). For the pseudovirus entry assay, retroviruses pseudotyped with SARS-CoV-2 spike protein (i.e., SARS-CoV-2 pseudoviruses) were used to enter human ACE2-expressing HEK293T cells in the presence of an inhibitor. The efficacy of the inhibitor was expressed as the concentration capable of neutralizing either 50 or 90% of the entry efficiency (i.e., ND₅₀ or ND₉₀, respectively). Nanosota-1C-Fc had an ND₅₀ of 0.27 μg/ml and an ND₉₀ of 3.12 μg/ml, both of which were ~10 times more potent than monovalent Nanosota-1C and the first of which was ~160 times more potent than ACE2 (Figure 3A). Additionally, Nanosota-1 potently neutralized SARS-CoV-2 pseudovirus bearing the D614G mutation in the SARS-CoV-2 spike protein (Figure 3—figure supplement 1), which has become prevalent in many strains (Korber et al., 2020). For the live virus infection assay, live SARS-CoV-2 was used to infect Vero cells in the presence of an inhibitor. Efficacy of the inhibitor was described as the concentration capable of reducing the number of virus plaques by 50% (i.e., ND₅₀). Nanosota-1C-Fc had an ND₅₀ of 0.16 μg/ml, which was significantly more potent than monovalent Nanosota-1C and ACE2 (Figure 3B; Figure 3—figure supplement 2). Overall, both Nanosota-1C-Fc and Nanosota-1C are potent inhibitors of SARS-CoV-2 pseudovirus entry and live SARS-CoV-2 infection.

Figure 3. — (A) Neutralization of SARS-CoV-2 pseudovirus entry into target cells by one of three inhibitors: *Nanosota-1C-Fc, Nanosota-1C*, and recombinant human angiotensin-converting enzyme 2 (ACE2). Retroviruses pseudotyped with SARS-CoV-2 spike protein (i.e., SARS-CoV-2 pseudoviruses) were used to enter HEK293T cells expressing human ACE2 in the presence of individual inhibitors at various concentrations. Entry efficiency was characterized via a luciferase signal, indicating successful cell entry. Data are the mean ± SEM (n = 4). Nonlinear regression was performed using a log (inhibitor) versus normalized response curve and a variable slope model (R²>0.95 for all curves). The efficacy of each inhibitor was expressed as the concentration capable of neutralizing pseudovirus entry by 50% (i.e., ND₅₀) or 90% (i.e., ND₉₀). ND₉₀ for ACE2 was not calculated due to insufficient data points. The assay was repeated three times (biological replication: new aliquots of pseudoviruses and cells were used for each repeat). (B) Neutralization of live SARS-CoV-2 infection of target cells by one of two inhibitors: *Nanosota-1C-Fc* and *Nanosota-1C*. The potency of *Nanosota-1* in neutralizing live SARS-CoV-2 infections was evaluated using a SARS-CoV-2 plaque-reduction neutralization test (PRNT) assay. 80 plaque-forming unit (PFU) infectious SARS-CoV-2 particles were used to infect Vero E6 cells in the presence of individual inhibitors at various concentrations. Infection was characterized as the number of virus plaques formed in overlaid cells. Images of virus plaques for each inhibitor at the indicated concentrations are shown. Each image represents data from triplications. The efficacy of each inhibitor was calculated and expressed as the concentration capable of reducing the number of virus plaques by 50% (i.e., ND₅₀). The assay was repeated twice (biological replication: new aliquots of virus particles and cells were used for each repeat).

Figure 3—figure supplement 1. — (A) Neutralization of SARS-CoV-2 pseudovirus entry into target cells by one of three inhibitors: *Nanosota-1C-Fc, Nanosota-1C*, and recombinant human angiotensin-converting enzyme 2 (ACE2). Retroviruses pseudotyped with SARS-CoV-2 spike protein (i.e., SARS-CoV-2 pseudoviruses) were used to enter HEK293T cells expressing human ACE2 in the presence of individual inhibitors at various concentrations. Entry efficiency was characterized via a luciferase signal, indicating successful cell entry. Data are the mean ± SEM (n = 4). Nonlinear regression was performed using a log (inhibitor) versus normalized response curve and a variable slope model (R²>0.95 for all curves). The efficacy of each inhibitor was expressed as the concentration capable of neutralizing pseudovirus entry by 50% (i.e., ND₅₀) or 90% (i.e., ND₉₀). ND₉₀ for ACE2 was not calculated due to insufficient data points. The assay was repeated three times (biological replication: new aliquots of pseudoviruses and cells were used for each repeat). (B) Neutralization of live SARS-CoV-2 infection of target cells by one of two inhibitors: *Nanosota-1C-Fc* and *Nanosota-1C*. The potency of *Nanosota-1* in neutralizing live SARS-CoV-2 infections was evaluated using a SARS-CoV-2 plaque-reduction neutralization test (PRNT) assay. 80 plaque-forming unit (PFU) infectious SARS-CoV-2 particles were used to infect Vero E6 cells in the presence of individual inhibitors at various concentrations. Infection was characterized as the number of virus plaques formed in overlaid cells. Images of virus plaques for each inhibitor at the indicated concentrations are shown. Each image represents data from triplications. The efficacy of each inhibitor was calculated and expressed as the concentration capable of reducing the number of virus plaques by 50% (i.e., ND₅₀). The assay was repeated twice (biological replication: new aliquots of virus particles and cells were used for each repeat).

After the in vitro studies, we next evaluated the therapeutic efficacy of the lead drug candidate Nanosota-1C-Fc in a hamster model challenged with SARS-CoV-2 (at a titer of 1 x 10⁶ Median Tissue Culture Infectious Dose [TCID₅₀]) via intranasal inoculation. Three experimental groups of hamsters (six per group) received a single dose of Nanosota-1C-Fc via intraperitoneal injection: (i) 24-hr pre-challenge at 20 mg/kg body weight, (ii) 4-hr post-challenge at 20 mg/kg, and (iii) 4-hr post-challenge at 10 mg/kg. As previously validated in this model (Sia et al., 2020), body weight and tissue pathology were used as metrics of therapeutic efficacy. In an untreated control group that received phosphate-buffered saline (PBS), weight loss precipitously started on day 1 post-challenge and the lowest weights were recorded on day 6 (Figure 4A). Pathology analysis on tissues collected on day 10 revealed moderate hyperplasia in the bronchial tubes (i.e., bronchioloalveolar hyperplasia) (Figure 4B), with little hyperplasia in the lungs. These data are consistent with previous reports showing that SARS-CoV-2 mainly infects the bronchial epithelial cells of this hamster model (Sia et al., 2020). In contrast, hamsters that received Nanosota-1C-Fc 24 hr pre-challenge were protected from SARS-CoV-2, as evidenced by no weight loss and no bronchioloalveolar hyperplasia (Figure 4A; Figure 4B). When administered 4 hr post-challenge, Nanosota-1C-Fc also effectively protected hamsters from SARS-CoV-2 infections at either dosage (20 or 10 mg/kg), as evidenced by the favorable therapeutic metrics (Figure 4A; Figure 4B). Overall, Nanosota-1C-Fc was effective at curtailing SARS-CoV-2 infections preventively and therapeutically in the hamster model.

(A, B) Hamsters (six per group) were injected with a single dose of *Nanosota-1C-Fc* at the indicated time point and the indicated dosage. At day 0, all groups (experimental and control) were challenged with SARS-CoV-2 (at a titer of 10⁶ Median Tissue Culture Infectious Dose [TCID₅₀]). (A) Body weights of hamsters were monitored on each day and percent change in body weight relative to day 0 was calculated for each hamster. Data are the mean ± SEM (n = 6). Analysis of variance (ANOVA) on group as a between-group factor and day (1-10) as a within-group factor revealed significant differences between the control group and each of the following groups: 24-hr pre-challenge (20 mg/kg) group (F(1, 10) = 17.80, p = 0.002; effect size *η_p*² = 0.64), 4-hr post-challenge (20 mg/kg) group (F(1, 10) = 5.02, p = 0.035; *η_p*² = 0.37), and 4-hr post-challenge (10 mg/kg) group (F(1, 10) = 7.04, p = 0.024; *η_p*² = 0.41). All p-values are two-tailed. (B) Tissues of bronchial tubes from each of the hamsters were collected on day 10 and scored for the severity of bronchioloalveolar hyperplasia: 3 - moderate; 2 - mild; 1 - minimum; 0 - none. Data are the mean ± SEM (n = 6). A comparison between the control group and each of other groups was performed using one-tailed Student’s t-test for directional tests. ***p<0.001; *p<0.05. (C, D) Human ACE2-transgenic mice (seven per group) were injected with a single dose of *Nanosota-1C-Fc* at the indicated time point and the indicated dosage. At day 0, all groups (experimental and control) were challenged with SARS-CoV-2 (at a titer of 5 x 10³ plaque-forming unit [PFU]). (C) Five mice from each group were euthanized on day 2 post-challenge, and the virus titers in their lungs were measured using a plaque assay. A comparison between the control group and each of the other groups was performed using one-tailed Student’s t-test for directional tests. Data are the mean ± SEM (n = 5). **p<0.01; *p<0.05. (D) The remaining two mice from each group were euthanized on day 5 post-challenge. Lung tissues were collected and examined for pathological changes after staining with hematoxylin and eosin. Phosphate-buffered saline (PBS) control group: severe inflammatory cell infiltration and bronchiole infiltration on the top panel; severe alveolar edema filled with liquid (labeled *) and proliferative alveolar epithelium (labeled →) on the bottom panel. 24-hr pre-challenge 20 mg/kg group: close to normal. 24-hr pre-challenge 10 mg/kg group: minor proliferative alveolar epithelium and inflammatory cell infiltration. 4-hr post-challenge 20 mg/kg group: close to normal. 4-hr post-challenge 10 mg/kg group: obvious cell inflammatory cell infiltration and vascular thrombosis (labeled #).

Figure 4—source data 1. Raw images for Figure 4.

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To further examine the in vivo efficacy of Nanosota-1C-Fc, we evaluated its therapeutic efficacy in human ACE2-transgenic mice challenged with SARS-CoV-2 (at a titer of 5 x 10³ plaque-forming unit [PFU]) via intranasal inoculation. Instead of monitoring the body weights of the mice through the viral infection and recovery process, we measured the virus titers in the lungs at the peak of the viral infection. To this end, four experimental groups of mice (seven per group) received a single dose of Nanosota-1C-Fc via intraperitoneal injection: (i) 24-hr pre-challenge at 20 mg/kg body weight, (ii) 24-hr pre-challenge at 10 mg/kg body weight, (iii) 4-hr post-challenge at 20 mg/kg, and (iv) 4-hr post-challenge at 10 mg/kg. Five out of the seven mice from each group were euthanized on day 2 post-challenge, and the virus titers in their lungs were measured using a virus titer plaque assay (Figure 4C). Compared to the untreated control group, the mice that received Nanosota-1C-Fc had much lower virus titers in the lungs (~1000 times lower in the pre-challenge groups and ~100 times lower in the post-challenge groups). In addition to the above virus titer measurements, the remaining two mice in each group were euthanized on day 5 post-challenge for pathologic analysis of lung tissues (Figure 4D). In the untreated control group, histological examination revealed extensive inflammatory cell infiltration, especially in the peribronchial region, alveolar edema, and proliferative alveolar epithelium. These results are consistent with previous reports on the SARS-CoV-2-induced lung pathology of these mice (Zheng et al., 2021). The mice that received Nanosota-1C-Fc showed a near absence of lung pathology for both the pre- and post-challenge groups at 20 mg/kg body weight and minor lung pathological changes for the pre-challenge group at 10 mg/kg body weight. Pathological changes were still present in mice treated post-challenge with 10 mg/kg Nanosota-1C-Fc. Overall, Nanosota-1C-Fc effectively protected the mouse model from SARS-CoV-2 infection of their lungs.

Nanosota-1C-Fc is stable in vitro and in vivo with excellent bioavailability

With the lead drug candidate Nanosota-1C-Fc demonstrating therapeutic efficacy in vivo, we characterized other parameters important for its clinical translation. First, we expressed Nanosota-1C-Fc in bacteria (Figure 5A). After purification on protein A column and gel filtration, the purity of Nanosota-1C-Fc was nearly 100%. With no optimization, the expression yield reached 40 mg/l of bacterial culture. Second, we investigated the in vitro stability of Nanosota-1C-Fc incubated at four temperatures (−80°C, 4°C, 25°C, or 37°C) for 1 week and then measured its remaining SARS-CoV-2 RBD-binding capacity using enzyme-linked immunosorbent assay (ELISA) (Figure 5B). With −80°C as a baseline, Nanosota-1C-Fc retained nearly all of its RBD-binding capacity at the temperatures surveyed. Third, we measured the in vivo stability of Nanosota-1C-Fc (Figure 5C). Nanosota-1C-Fc was injected into mice via tail vein. Sera were obtained at different time points and measured for their SARS-CoV-2 RBD-binding capacity using ELISA. Nanosota-1C-Fc retained significant RBD-binding capability after 10 days in vivo. In contrast, Nanosota-1C was stable for only several hours in vivo (Figure 5—figure supplement 1A; Figure 5—figure supplement 1B). Last, we examined the biodistribution of Nanosota-1C-Fc in mice (Figure 5D). Nanosota-1C-Fc was radiolabeled with zirconium-89 (⁸⁹Zr) and injected systemically into mice. Tissues were collected at various time points and biodistribution of Nanosota-1C-Fc was quantified using a scintillation counter. After 3 days, Nanosota-1C-Fc remained at high levels in the blood, lung, heart, kidney, liver, and spleen, all of which are targets for SARS-CoV-2 (Puelles et al., 2020); moreover, it remained at low levels in the intestine, muscle, and bones. In contrast, Nanosota-1C had poor biodistribution, documenting high renal clearance (Figure 5—figure supplement 1C). Overall, our findings suggest that Nanosota-1C-Fc is a potent anti-SARS-CoV-2 drug candidate with translational values.

(A) Purification of *Nanosota-1C-Fc* from bacteria. The protein was nearly 100% pure after gel filtration chromatography, as demonstrated by its elution profile and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (stained by Coomassie blue). The yield of the protein was 40 mg/l of bacterial culture, without any optimization of the expression. (B) In vitro stability of *Nanosota-1C-Fc*. The protein was stored at indicated temperatures for a week, and then a dilution enzyme-linked immunosorbent assay (ELISA) was performed to evaluate its SARS-CoV-2 receptor-binding domain (RBD)-binding capability. Data are the mean ± SEM (n = 4). (C) In vivo stability of *Nanosota-1C-Fc. Nanosota-1C-Fc* was injected into mice, mouse sera were collected at various time points, and *Nanosota-1C-Fc* remaining in the sera was detected for its SARS-CoV-2 RBD-binding capability as displayed in a dilution ELISA. Data are the mean ± SEM (n = 3). (D) Biodistribution of [⁸⁹Zr]Zr-*Nanosota-1C-Fc. Nanosota-1C-Fc* was radioactively labeled with zirconium-89 (⁸⁹Zr) and injected into mice via the tail vein. Different tissues or organs were collected at various time points (n = 3 mice per time point). The amount of *Nanosota-1C-Fc* present in each tissue or organ was measured through examining the radioactive count of each tissue or organ. Data are the mean ± SEM (n = 3).

Figure 5—source data 1. Raw images for Figure 5.

elife-64815-fig5-data1.zip^{(8.7MB, zip)}

Figure 5—figure supplement 1. — (A) Purification of *Nanosota-1C-Fc* from bacteria. The protein was nearly 100% pure after gel filtration chromatography, as demonstrated by its elution profile and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (stained by Coomassie blue). The yield of the protein was 40 mg/l of bacterial culture, without any optimization of the expression. (B) In vitro stability of *Nanosota-1C-Fc*. The protein was stored at indicated temperatures for a week, and then a dilution enzyme-linked immunosorbent assay (ELISA) was performed to evaluate its SARS-CoV-2 receptor-binding domain (RBD)-binding capability. Data are the mean ± SEM (n = 4). (C) In vivo stability of *Nanosota-1C-Fc. Nanosota-1C-Fc* was injected into mice, mouse sera were collected at various time points, and *Nanosota-1C-Fc* remaining in the sera was detected for its SARS-CoV-2 RBD-binding capability as displayed in a dilution ELISA. Data are the mean ± SEM (n = 3). (D) Biodistribution of [⁸⁹Zr]Zr-*Nanosota-1C-Fc. Nanosota-1C-Fc* was radioactively labeled with zirconium-89 (⁸⁹Zr) and injected into mice via the tail vein. Different tissues or organs were collected at various time points (n = 3 mice per time point). The amount of *Nanosota-1C-Fc* present in each tissue or organ was measured through examining the radioactive count of each tissue or organ. Data are the mean ± SEM (n = 3).

Figure 5—source data 1. Raw images for Figure 5.

elife-64815-fig5-data1.zip^{(8.7MB, zip)}

Discussion

Nanobody therapeutics derived from camelid antibodies has advantages relative to conventional antibodies. Although several studies have reported nanobody drug candidates that specifically target SARS-CoV-2 (Huo et al., 2020; Hanke et al., 2020; Xiang et al., 2020; Schoof et al., 2020; Wrapp et al., 2020a; Pymm et al., 2021; Nambulli et al., 2021), as of this writing, only two studies have evaluated their nanobody drug candidates in an animal model for anti-SARS-CoV-2 therapeutic efficacy (Pymm et al., 2021; Nambulli et al., 2021). None of these studies have evaluated their nanobody drug candidates for in vitro thermostability, in vivo stability, or tissue biodistribution. The current study describes the full development and characterization of a series of nanobody drug candidates that specifically target the SARS-CoV-2 RBD. Our study included screening of nanobody phage display library, two rounds of affinity maturation, structural determination of the RBD/Nanosota-1 complex, and neutralizations of SARS-CoV-2 pseudovirus entry and live SARS-CoV-2 infection in vitro. We also evaluated the protection efficacy in two different animal models (hamsters and mice), characterized the nanobody’s production yields, demonstrated its in vitro thermostability and in vivo stability, and clarified its tissue biodistribution. The extensive scope of the work makes the current study among the most comprehensive on anti-SARS-CoV-2 nanobody drug candidates.

The two best-performing drug candidates from our study are Nanosota-1C and Nanosota-1C-Fc. The latter was constructed from the former through addition of an Fc tag to make a bivalent molecule with increased molecular weight and picomolar RBD-binding affinity. In this study, recombinant viral receptor ACE2 was selected as a comparison for evaluating antiviral potency because Nanosota-1 directly competes with ACE2 for the same binding site on the RBD. Compared with ACE2, Nanosota-1C-Fc bound to the RBD ~3000-fold more strongly and inhibited SARS-CoV-2 pseudovirus entry ~160-fold more effectively. Compared with ACE2, the much higher anti-SARS-CoV-2 potency of Nanosota-1C-Fc was partly due to the small size of its antigen-binding domain and its ideal binding site on the RBD, allowing Nanosota-1C-Fc to access the RBD in both the open spike during viral infection and the closed spike during viral immune evasion. Thus, the Nanosota-1 series are ideal RBD-targeting drug candidates that can inhibit SARS-CoV-2 viral particles regardless of whether the viral spike molecules are in an open or a closed conformation. Importantly, we showed that the effectiveness of Nanosota-1C-Fc was not limited to in vitro experiments, but translated directly to in vivo experiments by demonstrating efficacy in animal models. It is worth noting that the molecular weight of Nanosota-1C-Fc (78 kDa) is above the kidney clearance threshold (60 kDa) (Steeland et al., 2016), but it's still only half of conventional antibodies’ molecular weight (150 kDa). Its ideal size contributed to ease of production, excellent in vitro thermostability, good in vivo stability, and high tissue bioavailability. All of these features are critical for the implementation of Nanosota-1C-Fc as a potential COVID-19 therapeutic.

How can Nanosota-1 drug candidates contribute to the battle against COVID-19? First, as evidenced by both of our animal studies, Nanosota-1C-Fc can be used to prevent SARS-CoV-2 infections. Because of its excellent in vivo stability (significant RBD-binding capacity remained after 10 days in vivo), a single injected dose of Nanosota-1C-Fc can theoretically protect a person from SARS-CoV-2 infection for days or weeks in the outpatient setting, reducing the spread of SARS-CoV-2 in human populations. Second, data from both of our animal studies showed that Nanosota-1C-Fc can be used to treat SARS-CoV-2 infections, thus, potentially saving lives and alleviating symptoms in infected patients in the clinical setting. It is worth noting that given the recent success of COVID-19 vaccines, future development of Nanosota-1 series should focus on its therapeutic efficacy rather than its preventive efficacy. Moreover, to combat emerging SARS-CoV-2 variants with RBM mutations, Nanosota-1 series can be further developed through additional affinity maturation against the RBD of SARS-CoV-2 variants. The series may also be developed to become part of cocktail therapies that target multiple sites on SARS-CoV-2. Third, despite its rapid clearance from the blood, Nanosota-1C could be used as an inhaler to treat infections in the respiratory tracts (Van Heeke et al., 2017; Nambulli et al., 2021) or as an oral drug to treat infections in the intestines (Vega et al., 2013). The worldwide distribution of COVID-19 in the world calls for the large-scale manufacturing of anti-SARS-CoV-2 therapeutics. Molecules such as Nanosota-1, with their high production yields from bacteria and excellent in vitro and in vivo stabilities, are promising drug candidates to meet this need. Therefore, if further validated in clinical trials, Nanosota-1 drug candidates can help minimize the mortality and morbidity of SARS-CoV-2 infections and contribute to the battle against COVID-19.

Materials and methods

Cell lines, plasmids, and virus

HEK293T cells (American Type Culture Collection [ATCC]) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies). Vero E6 cells (ATCC) were grown in Eagle's minimal essential medium (EMEM) supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), and 10% FBS. ss320 Escherichia coli (Lucigen), TG1 E. coli (Lucigen), and SHuffle T7 E. coli (New England Biolabs) were grown in TB medium or 2YT medium with 100 mg/l ampicillin. HEK293T cells were authenticated by ATCC using short tandem repeat (STR) profiling. HEK293T cells and Vero E6 cells tested negative for mycoplasma contamination. No commonly misidentified cell lines were used.

SARS-CoV-2 spike (GenBank accession number QHD43416.1) and ACE2 (GenBank accession number NM_021804) were described previously (Shang et al., 2020a). SARS-CoV-2 RBD (residues 319–529) was subcloned into Lenti-CMV vector (Vigene Biosciences) with an N-terminal tissue plasminogen activator (tPA) signal peptide and a C-terminal human IgG4 Fc tag or His tag. The ACE2 ectodomain (residues 1–615) was constructed in the same way except that its own signal peptide was used. Nanosota-1A, -1B, and -1C were each cloned into PADL22c vector (Lucigen) with an N-terminal PelB leader sequence and C-terminal His tag and HA tag. Nanosota-1C-Fc was cloned into pET42b vector (Novagen) with a C-terminal human IgG₁ Fc tag.

SARS-CoV-2 (US_WA-1 isolate) from CDC (Atlanta) was used throughout the study. Experiments involving infectious SARS-CoV-2 were conducted at the University of Texas Medical Branch and the University of Iowa in approved biosafety level 3 laboratories.

Construction of camelid nanobody phage display library

The camelid nanobody phage display library was constructed as previously described (Abbady et al., 2011; Olichon and de Marco, 2012). Briefly, total mRNA was isolated from B cells from the spleen, bone marrow, and blood of over a dozen non-immunized llamas and alpacas. cDNA was prepared from the mRNA. The cDNA was then used in nested PCRs to construct the DNA for the library. The first PCR was to amplify the gene fragments encoding the variable domain of the nanobody. The second PCR (PCR2) was used to add restriction sites (SFI-I), a PelB leader sequence, a His₆ tag, and an HA tag. The PCR2 product was digested with SFI-I (New England Biolabs) and then ligated with SFI-I-digested PADL22c vector. The ligated product was transformed via electroporation into TG1 E. coli (Lucigen). Aliquots of cells were spread onto 2YT agar plates supplemented with ampicillin and glucose, incubated at 30°C overnight, and then scraped into 2YT media. After centrifugation, the cell pellet was suspended into 50% glycerol and stored at −80°C. The library size was 7.5 × 10¹⁰. To display nanobodies on phages, aliquots of the TG1 E. coli bank were inoculated into 2YT media, grown to early logarithmic phase, and infected with M13K07 helper phage.

Camelid nanobody library screening

The above camelid nanobody phage display library was used in the bio-panning as previously described (Hintz et al., 2019). Briefly, four rounds of panning were performed to obtain the SARS-CoV-2 RBD-targeting nanobodies with high RBD-binding affinity. The amounts of the RBD antigen used in coating the immune tubes in each round were 75 μg, 50 μg, 25 μg, and 10 μg, respectively. The retained phages were eluted using 1 ml 100 mM triethylamine and neutralized with 500 µl 1 M Tris-HCl, pH 7.5. The eluted phages were amplified in TG1 E. coli and rescued with M13K07 helper phage. The eluted phages from round 4 were used to infect ss320 E. coli. Single colonies were picked into 2YT media and nanobody expressions were induced with 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). The supernatants were subjected to ELISA for selection of strong binders (described below). The strong binders were then expressed and purified (described below) and subjected to SARS-CoV-2 pseudovirus entry assay for selection of anti-SARS-CoV-2 efficacy (described below). The lead nanobody after initial screening was named Nanosota-1A.

Affinity maturation

Affinity maturation of Nanosota-1A was performed as previously described (Hust and Lim, 2018). Briefly, mutations were introduced into the whole gene of Nanosota-1A using error-prone PCR. Two rounds of error-prone PCR were performed using the GeneMorph II Random Mutagenesis Kit (Agilent Technologies). The PCR product was cloned into the PADL22c vector and transformed via electroporation into the TG1 E. coli. The library size was 6 x 10⁸. Three rounds of bio-panning were performed using 25 ng, 10 ng, and 2 ng RBD-Fc, respectively. The strongest binder after affinity maturation was named Nanosota-1B. A second round of affinity maturation was performed in the same way as the first round, except that three rounds of bio-panning were performed using 10 ng, 2 ng, and 0.5 ng RBD-Fc, respectively. The strongest binder after the second round of affinity maturation was named Nanosota-1C.

Production of Nanosota-1

Nanosota-1A, -1B, and -1C were each purified from the periplasm of ss320 E. coli after the cells were induced by 1 mM IPTG. The cells were collected and re-suspended in 15 ml TES buffer (0.2 M Tris, pH 8, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 0.5 M sucrose), shaken on ice for 1 hr, and then incubated with 40 ml TES buffer (diluted 4 times) followed by shaking on ice for another 1 hr. The protein in the supernatant was sequentially purified using a Ni-NTA column and a Superdex200 gel filtration column (GE Healthcare) as previously described (Shang et al., 2020a). Nanosota-1C-Fc was purified from the cytoplasm of Shuffle T7 E. coli. The induction of protein expression was the same as above. After induction, the cells were collected, re-suspended in PBS, and disrupted using Branson Digital Sonifier (Thermofisher). The protein in the supernatant was sequentially purified on protein A column and Superdex200 gel filtration column as previously described (Shang et al., 2020a).

Production of SARS-CoV-2 RBD and ACE2

HEK293T cells stably expressing SARS-CoV-2 RBD (containing a C-terminal His tag or Fc tag) or human ACE2 ectodomain (containing a C-terminal His tag) were made according to the E and F sections of the pLKO.1 Protocol from Addgene (http://www.addgene.org/protocols/plko/). The proteins were secreted to cell culture media, harvested, and purified on either a Ni-NTA column (for His-tagged proteins) or a protein A column (for Fc-tagged proteins) and then on a Superdex200 gel filtration column as previously described (Shang et al., 2020a).

ELISA

ELISA was performed to detect the binding between SARS-CoV-2 RBD and Nanosota-1 (either as purified recombinant proteins or as proteins in the mouse serum) as previously described (Zhao et al., 2018). Briefly, ELISA plates were coated with recombinant SARS-CoV-2 RBD-His or RBD-Fc and were then incubated sequentially with nanobody proteins, horseradish peroxidase (HRP)-conjugated anti-llama antibody (1:5000) (Sigma), or HRP-conjugated anti-human-Fc antibody (1:5000) (Jackson ImmunoResearch). ELISA substrate (Invitrogen) was added to the plates, and the reactions were stopped with 1N H₂SO₄. The absorbance at 450 nm (A₄₅₀) was measured using a Synergy LX Multi-Mode Reader (BioTek).

Determination of the structure of SARS-CoV-2 RBD complexed with Nanosota-1C

To prepare the RBD/Nanosota-1C complex for crystallization, the two proteins were mixed together in solution and purified using a Superdex200 gel filtration column (GE Healthcare). The complex was concentrated to 10 mg/ml in buffer—20 mM Tris, pH 7.2, and 200 mM NaCl. Crystals were screened at High-Throughput Crystallization Screening Center (Hauptman-Woodward Medical Research Institute) as previously described (Luft et al., 2003) and were grown in sitting drops at room temperature over wells containing 50 mM MnCl₂, 50 mM MES, pH 6.0, and 20% (W/V) polyethylene glycol (PEG) 4000. Crystals were soaked briefly in 50 mM MnCl₂, 50 mM MES, pH 6.0, 25% (W/V) PEG 4000%, and 30% ethylene glycol before being flash-frozen in liquid nitrogen. X-ray diffraction data were collected at the Advanced Photon Source beamline 24-ID-E. The structure was determined by molecular replacement using the structures of SARS-CoV-2 RBD (PDB 6M0J) and another nanobody (PDB 6QX4) as the search templates. Structure data and refinement statistics are shown in Table 2.

Table 2. X-ray data collection and structure refinement statistics (SARS-CoV-2 RBD/Nanosota-1C complex).

Data collection
Wavelength	0.979
Resolution range	45.48–3.19 (3.30–3.19)
Space group	P 43 21 2
Unit cell	60.849 60.849 410.701 90 90 90
Total reflections	64167 (5703)
Unique reflections	13607 (1308)
Multiplicity	4.7 (4.4)
Completeness (%)	96.82 (97.60)
Mean I/sigma(I)	8.41 (1.80)
Wilson B-factor	83.24
R-merge	0.145 (0.928)
R-meas	0.1638 (1.053)
R-pim	0.07385 (0.4858)
CC1/2	0.995 (0.861)
CC*	0.999 (0.962)
Refinement
Reflections used in refinement	13567 (1301)
Reflections used for R-free	674 (62)
R-work	0.2483 (0.3521)
R-free	0.2959 (0.4153)
CC (work)	0.963 (0.819)
CC (free)	0.909 (0.615)
Number of non-hydrogen atoms	4890
Macromolecules	4833
Ligands	57
Protein residues	621
RMS (bonds)	0.002
RMS (angles)	0.45
Ramachandran favored (%)	93.11
Ramachandran allowed (%)	6.89
Ramachandran outliers (%)	0.00
Rotamer outliers (%)	3.23
Clashscore	5.25
Average B-factor	90.29
Macromolecules	89.84
Ligands	127.91

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Statistics for the highest-resolution shell are shown in parentheses.

Surface plasmon resonance assay

Surface plasmon resonance assay using a Biacore S200 system (GE Healthcare) was carried out as previously described (Shang et al., 2020a). Briefly, SARS2-CoV-2 RBD-His was immobilized to a CM5 sensor chip (GE Healthcare). Serial dilutions of purified recombinant Nanosota-1 proteins were injected at different concentrations: 320–10 nM for Nanosota-1A; 80–2.5 nM for Nanosota-1B and Nanosota-1C; 20–1.25 nM for Nanosota-1C-Fc. The resulting data were fit to a 1:1 binding model using Biacore Evaluation Software (GE Healthcare).

Protein pull-down assay

Protein pull-down assay was performed using an immunoprecipitation kit (Invitrogen) as previously described (Shang et al., 2020a). Briefly, to pull down ACE2-His (containing a C-terminal His tag) and Nanosota-1C (containing a C-terminal His tag), 10 µl protein A beads were incubated with 1 µg SARS-CoV-2 RBD-Fc at room temperature for 1 hr. Then different amounts (7.04, 3.52. 1.76, 0.88, 0.44, 0.22, or 0 µg) of Nanosota-1C and 4 µg ACE2-His were added to the RBD-bound beads. After incubation at room temperature for 1 hr, the bound proteins were eluted using elution buffer (0.1 M glycine, pH 2.7). The samples were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed through western blot using an anti-His antibody.

To pull down ACE2-His and Nanosota-1C-Fc (containing a C-terminal Fc tag), 10 µl streptavidin beads were incubated with 1 µg SARS-CoV-2 RBD-His (biotinylated using EZ-LinkTM Sulfo-NHS-LC-Biotinylation Kit; Thermo Scientific) at room temperature for 1 hr. Then different amounts (17.80, 8.90. 4.45, 2.23, 1.11, 0.56, or 0 µg) of Nanosota-1C-Fc and 4 µg ACE2-His were added to the RBD-bound beads. After incubation at room temperature for 1 hr, the bound proteins were eluted using elution buffer (0.1 M glycine, pH 2.7). The samples were then subjected to SDS-PAGE and analyzed through western blot using an anti-His antibody (for detecting ACE2-His) or an anti-Fc antibody (for detecting Nanosota-1C-Fc).

Gel filtration chromatography assay

Gel filtration chromatography assay was performed on a Superdex200 column. 500 µg human ACE2, 109 µg Nanosota-1C, and 121 µg SARS-CoV-2 RBD were incubated together at room temperature for 30 min. The mixture was subjected to gel filtration chromatography. Samples from each peak off the column were then subjected to SDS-PAGE and analyzed through Coomassie blue staining.

SARS-CoV-2 pseudovirus entry assay

The potency of Nanosota-1 in neutralizing SARS-CoV-2 pseudovirus entry was evaluated as previously described (Shang et al., 2020a; Shang et al., 2020b). Briefly, HEK293T cells were co-transfected with a plasmid carrying an Env-defective, luciferase-expressing human immunodeficiency virus-1 (HIV-1) genome (pNL4-3.luc.R-E-) and pcDNA3.1(+) plasmid encoding SARS-CoV-2 spike protein. Pseudoviruses were collected 72 hr after transfection, incubated with individual inhibitors at different concentrations at 37°C for 1 hr, and then used to enter HEK293T cells expressing human ACE2. After pseudoviruses and target cells were incubated together at 37°C for 6 hr, the medium was changed to fresh medium, followed by incubation for another 60 hr. Cells were then washed with PBS buffer and lysed. Aliquots of cell lysates were transferred to plates, followed by the addition of luciferase substrate. Relative light units (RLUs) were measured using an EnSpire plate reader (PerkinElmer). The efficacy of each inhibitor was expressed as the concentration capable of neutralizing 50 or 90% of the entry efficiency (i.e., ND₅₀ or ND₉₀, respectively).

SARS-CoV-2 plaque-reduction neutralization test

The potency of Nanosota-1 in neutralizing live SARS-CoV-2 was evaluated using a SARS-CoV-2 plaque-reduction neutralization test (PRNT) assay. Specifically, the individual drug candidate was serially diluted in DMEM and mixed with SARS-CoV-2 (at a titer of 80 PFU) at 37°C for 1 hr. The mixtures were then added into Vero E6 cells at 37°C for an additional 45 min. After removing the culture medium, cells were overlaid with 0.6% agarose and cultured for 3 days. Plaques were visualized by 0.1% crystal violet staining. The efficacy of each drug candidate was calculated and expressed as the concentration capable of reducing the number of virus plaques by 50% (i.e., ND₅₀) compared to control serum-exposed virus.

SARS-CoV-2 challenge of hamsters

Syrian hamsters (n = 24; equal sex) were obtained from Envigo (IN) and challenged via intranasal inoculation with SARS-CoV-2 (1 x 10⁶ TCID₅₀) in 100 µl DMEM (50 µl per nare). Sample size was constrained by the availability of resources. Four groups of hamsters (n = 6 in each group; randomly assigned) received Nanosota-1C-Fc via intraperitoneal injection at one of the following time points and dosages: (i) 24 hr pre-challenge at 20 mg/kg body weight; (ii) 4 hr post-challenge at 20 mg/kg body weight; (iii) 4 hr post-challenge at 10 mg/kg body weight. Hamsters in the negative control group were administered PBS buffer 24 hr pre-challenge. Body weights were collected daily. Hamsters were humanely euthanized on day 10 post-challenge via overexposure to CO₂. The lungs and bronchial tubes were collected and fixed in formalin for pathological analysis. At a sample size of six animals per group, G*Power analysis indicates that we can detect an effect size of 1.6 with a power of 0.80 (alpha = 0.05, one-tailed).

SARS-CoV-2 challenge of human ACE2-transgenic mice

Human ACE2-transgenic mice (K18-hACE2-transgenic mice) (Zheng et al., 2021; McCray et al., 2007) (n = 35; males and females; 7–8 months old) were obtained from the Jackson Laboratories. Mice were challenged via intranasal inoculation with SARS-CoV-2 (5 x 10³ PFU) in a volume of 50 µl DMEM. Sample size was constrained by the availability of resources. Five groups of mice (n = 7 in each group) were treated with Nanosota-1C-Fc via intraperitoneal injection at one of the following time points and dosages: (i) 24 hr pre-challenge at 20 mg/kg body weight; (ii) 24 hr pre-challenge at 10 mg/kg body weight; (iii) 4 hr post-challenge at 20 mg/kg body weight; (iv) 4 hr post-challenge at 10 mg/kg body weight. Mice in the negative control group were administered PBS buffer 24 hr pre-challenge.

Viral titers in the lungs of mice were measured by a plaque assay. To this end, five mice from each group were euthanized on day 2 post-challenge. Lung homogenate supernatants were collected and then serially diluted in DMEM. 12-well plates of Vero E6 cells were inoculated and then incubated at 37°C in 5% CO₂ for 1 hr and gently rocked every 15 min. After removing the inocula, the plates were overlaid with 0.6% agarose containing 2% FBS. After 3 days, overlays were removed and plaques were visualized via staining with 0.1% crystal violet. Viral titers were quantified as PFU per ml tissue. At a sample size of five animals per group, G*Power analysis indicates that we can detect an effect size of 1.72 with a power of 0.80 (alpha = 0.05, one-tailed).

Histological examination of lungs was performed. For this pupose, the remaining two mice from each group were euthanized on day 5 post-challenge and then perfused transcardially with PBS. Mouse lungs were fixed in formalin. Sections (approximately 4 µm each) were stained with hematoxylin and eosin.

In vivo stability of Nanosota-1

Male C57BL/6 mice (3–4 weeks old) (Envigo) were intravenously injected (tail vein) with Nanosota-1C or Nanosota-1C-Fc (100 μg in 100 μl PBS buffer). At varying time points, mice were euthanized and whole blood was collected. Then sera were prepared through centrifugation of the whole blood at 1500 xg for 10 min. The sera were then subjected to ELISA for evaluation of their SARS-CoV-2 RBD-binding capability.

Biodistribution of Nanosota-1 in mice

To evaluate the in vivo biodistribution of Nanosota-1C-Fc and Nanosota-1C, the nanobodies were labeled with [⁸⁹Zr] and injected into male C57BL/6 mice (5–6 weeks old) (Envigo). Briefly, the nanobodies were first conjugated to the bifunctional chelator p-SCN-Bn-Deferoxamine (DFO, Macrocyclic) as previously described (Zeglis and Lewis, 2015). [⁸⁹Zr] (University of Wisconsin Medical Physics Department) was then conjugated as previously described (Hintz et al., 2020). [⁸⁹Zr]-labeled nanobodies (1.05 MBq, 1–2 μg nanobody, 100 μl PBS) were intravenously injected (tail vein). Mice were euthanized at various time points. Organs were collected and counted on an automatic gamma-counter (Hidex). The total number of counts per minute (cpm) for each organ or tissue was compared with a standard sample of known activity and mass. Count data were corrected to both background and decay. The percent injected dose per gram (%ID/g) was calculated by normalization to the total amount of activity injected into each mouse.

Acknowledgements

The development of Nanosota-1 and animal testing were supported by funding from the University of Minnesota (to FL) and NIH grants R01AI157975 (to FL, AML, LD, SP), R01AI089728 (to FL), and R35GM118047 (to HA). AML is a 2013 Prostate Cancer Foundation Young Investigator and the recipient of a 2018 Prostate Cancer Foundation Challenge Award. Experimental Pathology Laboratories analyzed pathology data on SARS-CoV-2-challenged hamsters. Crystallization screening was performed at Hauptman-Woodward Medical Research Institute and supported by NSF grant 2029943. X-ray diffraction data were collected at Advanced Photon Source beamline 24-ID-E and supported by NIH grants P30 GM124165 and S10OD021527 and DOE contract DE-AC02-06CH11357. We thank Surajit Banerjee for help with X-ray data collection. The University of Minnesota has filed a patent on Nanosota-1 with FL, GY, AML, JPG, JS, and YW as inventors. Coordinates and structure factors have been deposited to the Protein Data Bank with accession number 7KM5.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Stanley Perlman, Email: stanley-perlman@uiowa.edu.

Aaron LeBeau, Email: alebeau@umn.edu.

Fang Li, Email: lifang@umn.edu.

John W Schoggins, University of Texas Southwestern Medical Center, United States.

Jos W Van der Meer, Radboud University Medical Centre, Netherlands.

Funding Information

This paper was supported by the following grants:

National Institutes of Health R01AI157975 to Lanying Du, Stanley Perlman, Aaron LeBeau, Fang Li.
National Institutes of Health R01AI089728 to Fang Li.
National Institutes of Health R35GM118047 to Hideki Aihara.
University of Minnesota to Fang Li.
Prostate Cancer Foundation to Aaron LeBeau.

Additional information

Competing interests

The University of Minnesota has filed a patent on Nanosota-1 drugs with F.L, G.Y., A.M.L., J.P.G., J.S., and Y.W. as inventors.

No competing interests declared.

The University of Minnesota has filed a patent on Nanosota-1 drugs with F.L, G.Y., A.M.L., J.P.G., J.S., and Y.W. as inventors.

Author contributions

Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - review and editing.

Data curation, Validation, Investigation, Visualization, Methodology.

Data curation, Validation, Investigation, Visualization, Methodology, Writing - review and editing.

Data curation, Validation, Investigation, Visualization, Methodology.

Data curation, Investigation.

Conceptualization, Data curation, Validation, Investigation, Visualization, Methodology, Writing - review and editing.

Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - review and editing.

Funding acquisition, Validation, Investigation, Visualization, Methodology.

Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Project administration, Writing - review and editing.

Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols of the University of Texas Medical Branch (protocol number 2007072), of the New York Blood Center (protocol number 194.22), of the University of Iowa (protocol number 9051795), and of the University of Minnesota (protocol number 2009-38426A).

Additional files

Source data 1. Raw data for figures and figure supplements.

elife-64815-data1.xlsx^{(40.5KB, xlsx)}

Transparent reporting form

elife-64815-transrepform.docx^{(70.4KB, docx)}

Data availability

Coordinates and structure factors have been deposited to the Protein Data Bank with accession number 7KM5.

The following dataset was generated:

Ye G, Shi K, Aihara H, Li F. 2021. Crystal structure of SARS-CoV-2 RBD complexed with Nanosota-1. RCSB Protein Data Bank. 7KM5

References

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eLife. doi: 10.7554/eLife.64815.sa1

Decision letter

Editor: John W Schoggins¹

Reviewed by: Zhiwei Wu²

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Camelid-derived nanobodies share some unique properties such as high affinity, thermal stability, ease of production etc. and can be applied as both therapeutic and diagnostic agents. This study describes a nanobody that inhibits SARS-CoV-2 and has the potential to be a drug candidate for COVID-19.

Decision letter after peer review:

Thank you for submitting your article "The Development of a Novel Nanobody Therapeutic for SARS-CoV-2" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen Jos van der Meer as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Zhiwei Wu (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Please note that the major concern for all reviewers was lack of in vivo efficacy data with respect to viral titers. This will be the most critical data needed for competitive resubmission. Each reviewer also had additional comments and suggestions to improve the text, each of which should be addressed upon revision.

Title: As noted by reviewer 2, the phrase in the title "novel nanobody therapeutic" has the potential to be misleading as several other similar nanobodies have been reported. Accordingly, we would request that the authors revise the title of any potential revision.

Reviewer #1:

Summary: The authors aimed to develop camelid nanobodies for treatment of SARS-CoV-2.

Strengths: The camelid nanobody developed has promising antiviral efficacy in vitro and reduced viral disease symptoms in vivo. This appears to be one of the first demonstrations of in vivo efficacy for a SARS-CoV-2 nanobody.

Weaknesses: The reduction in viral titers is not as robust as would be predicted for a highly effective antiviral antibody. It is not clear whether this nanobody would be more effective than other monoclonal antibody therapies already in use.

Appraisal: The authors have largely achieved their aim of developing a camelid nanobody that shows anti-SARS-CoV-2 efficacy.

Impact: This work may provide impetus for further development of nanobodies in human clinical settings.

1. Figure S7. The impact of the nanobody on viral titers seems quite model relative to the impact on weight loss. It would seem that a highly effective nanobody would more robustly reduce viral burden, by more than the half-log drop shown here. By comparison maybe therapy in a hamster model results in 2-3 log drop in SARS-CoV-2 viral RNA. Can the authors examine viral RNA from these samples? What else might explain why the viral load is not diminished more than it is? Also, the statistical analyses of these data are not clear. Only one p value for each group is mentioned in legend. It would seem that a multiple comparison test should be able to provide a p-value for every dose, at each time point.

2. Figure 4. It would seem appropriate to include some histology images corresponding to the clinical scoring parameter.

3. Figure 5C. Is there a negative control that can be included for comparison?

Reviewer #2:

In this manuscript by Ye et al., the authors employed a phage display library based on naive camelids to identify nanobodies that target the receptor-binding domain (RBD) of SARS-CoV-2. A clone named Nanosota-1A was identified from the initial screen, which was further optimized for RBD binding by in vitro affinity maturation. The resulting nanobody Nanosota-1C binds moderately the RBD at 14 nM. The authors determined the crystal structure of Nanosota-1C in complex with the RBD and found that Nanosota-1C partially overlap with the ACE2 binding sites.

To potentially enable bivalent binding and increase the pharmacological properties, the authors fused the lead construct with an Fc domain (Nanosota-1C-Fc) and positively evaluated its neutralization potential (with an NC50 of sub-microgram/ml) using a clinical isolate of the virus. Moreover, Ye and colleagues performed experiments to evaluate the in vivo efficacy of Nanosota-1C-Fc (for prophylaxis and potential treatment) in a COVID model using golden hamsters. For treatment, they evaluated two doses of a lead construct (10 mg/kg and 20 mg/kg) by intraperitoneal (IP) injection 6 hours post-infection (IN/intranasal-based inoculation). The therapeutic efficacy was evaluated based on body weight, lung pathology, and virus tilter from nasal swabs. Both prophylaxis (pre-IN) and treatment (post-IN) groups post-infection showed potential protections on body weights compared to the control group. The lead construct also appears to mitigate lung infections with decreased BAL hyperplasia, which is especially true for pre-IN animals, and less so for the post-IN(treatment) group, consistent with in vivo studies using monoclonal antibodies. Moreover, no significant decrease in nasal viral titers was detected.

The authors further expanded the study by evaluating the solubility, stability, and biodistribution of the lead construct. They provided data to indicate that Nanosota-1C-Fc can be expressed in E. coli with high yield, and is potentially stable both in vitro and in vivo. Using a wild type mouse model, they found that following i.v., Nanosota-1C-Fc is widely distributed in the major organs.

While the lead construct is not among the most potent nanobodies developed to date, this study is highly comprehensive including in vivo evaluations in the hamster model. Clearly, there were lots of efforts and I appreciate the amount of data presented here, from screening/ in vitro affinity maturation, structural characterization, bioengineering, biophysics to in vivo efficacy studies. However, I do have the following concerns that need to be addressed.

1. The overall flow is fine, however, the manuscript will need to be improved for clarity and conciseness. There is grammar throughout the text. Finally, the Discussion section seems to be a bit awkward and lengthy.

2. The major advantages of nanobodies over monoclonal IgG antibodies include the small size, biophysical properties for COVID, potential inhalation delivery, as well as high bioengineering potentials. In the present work, the lead construct (Nanosota-1C-Fc) was back-engineered into a full-length antibody comparable with the monoclonal antibodies directly isolated from COVID patients. The in vitro neutralization potency of the Fc fusion construct (Nanosota-1C-Fc) appears to be considerably inferior to other monoclonal COVID antibodies under clinical evaluations(e.g., single-digit nanogram/ml). Would the authors elaborate on the key conceptual advances of this work?

3. The affinity of Nanosota-1C-Fc: the authors claimed that there was a three-log improvement on the RB binding affinity, after fusing monomeric Nanosota-1C to an Fc (from 14 nM to ~ 16 pM). This raises the possibility of avidity binding. However, since the RBD does not dimerize, how could bivalent binding occur? Along the same line, I wonder if the ACE2 competition assay could be performed on Nanosota-1C-Fc to substantiate the KD measurement.

4. Line 187: the NC50 curves were plotted without sufficient data points and dilutions ( Figure 3B and S6), the quantitative differences (20 fold and 6,000 fold) therefore can not be accurately derived.

5. Literature: please expand the published literature on COVID nanobodies:

Hanke, L., Vidakovics Perez, L., Sheward, D.J., Das, H., Schulte, T., Moliner-Morro, A., Corcoran, M., Achour, A., Karlsson Hedestam, G.B., Hallberg, B.M., et al. (2020). An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction. Nat Commun 11, 4420.

Xiang, Y., Nambulli, S., Xiao, Z., Liu, H., Sang, Z., Duprex, W.P., Schneidman-Duhovny, D., Zhang, C., and Shi, Y. (2020). Versatile and multivalent nanobodies efficiently neutralize SARS-CoV-2. Science 370, 1479-1484.

Schoof, M., Faust, B., Saunders, R.A., Sangwan, S., Rezelj, V., Hoppe, N., Boone, M., Billesbolle, C.B., Puchades, C., Azumaya, C.M., et al. (2020). An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike. Science 370, 1473-1479.

Wrapp, D., De Vlieger, D., Corbett, K.S., Torres, G.M., Wang, N., Van Breedam, W., Roose, K., van Schie, L., Team, V.-C.C.-R., Hoffmann, M., et al. (2020). Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies. Cell 181, 1436-1441.

6. Regarding the weight increase of hamsters: there was evidence to indicate that weight recovery/increase in the infected hamsters is likely non-specifically related to the IP administration. What is the placebo/control used for Figure 4A? Is that PBS injection? It is not clear what "control (no drug)" indicates here. Did the author perform any post-mortem lung viral titer analysis, especially 3-4 days post-infection? This key data may provide direct evidence about how efficiently the construct can inhibit the virus in vivo.

7. Related to question 6. Since there were marginal differences (generally less than one log) of viral titers in the nasal swabs, I'd appreciate it if the author can provide more direct evidence to demonstrate the virus reductions. Minimally, examining the virus RNA levels in the lung would help substantiate the study.

8. Fc and vector construct information is missing. Is it a glycosylation variant that enables E. coli expression?

9. Pharmacokinetics and in vivo half-life: the authors claim that the T_1/2 of Nanosota-1C-Fc in mice (by tail vein injection) is over 10 days. However, it is hard to conclude simply based on Figure 5C. Would it the possibility that the authors show the standard PK curve to indicate the distribution phase and to carefully derive the clearance rate?

Reviewer #3:

Geng Ye et al. in "The development of a Novel Nanobody Therapeutic for SARS-CoV-2" reported an isolation and characterization of nanobodies from a camelid antibody library and identified and generated Nanosota-I nanobodies for their SARS-CoV-2 inhibitory activities. The authors found that Nanosota-1C bound to both the closed and open conformations of the RBD with high affinities. The nanobody also exhibited inhibitory activity against pseudotyped SARS-CoV-2 infection of cells in vitro and inhibited live virus replication and lessened pathology in a hamster model. In addition, the authors also demonstrated that Nanosota-1C-Fc exhibited excellent stability in vivo and bioavailability, and suggest that Nanosota-1C-Fc is a potential therapeutic and preventative candidate for COVID-19. The study is well designed and executed with evidence that supports some of the conclusions.

The study demonstrated biochemically that Nanosota-1C-Fc binds both the close and open conformation of the RBD of SARS-CoV-2 and that the nanobody neutralized pseudotyped SARS-CoV-2 in in vitro assay. However, the in vivo viral inhibitory activity of the nanobody was not supported by the data since no convincing evidence was presented.

The study also suffers from incomplete investigation of the antibody role in controlling the virus since the authors did not investigate the nanobody in the nasal compartment where virus was easily detected.

Due to the unique immunological and biochemical charactertistics of the nanobodies, this study will have some impact on the therapeutic antibody development against COVID-19.

However, there remain a number of serious concerns that need to be addressed before the manuscript can be accepted for publication:

1. For the in vitro neutralization study, the authors only calculated ND50 values. The ND90 values are more meaningful biologically with respect to the efficacy of inhibiting viral infection and reflect the potency of an antibody in suppressing the virus. The authors should calculate ND90 values and presented in the text.

2. For the in vivo study, the viral load in various settings showed essentially no differences across all time points as shown in Figure S7, suggesting that the Nanosota-1C-Fc either did not inhibit the viral infection or the intravenous-administrated nanobody did not reach functional concentrations in the nasal compartment. Did the authors measure Nanosota-1C-Fc concentrations in the nasal cavity ? Figure 5D clearly indicated that there is little antibody in the lungs.

3. In view of the Figure S7 data, did the authors measure viral titers in other compartments, particularly lungs, to see if virus was inhibited by Nanosota-1C-Fc. It is critical that the mitigation of the pathology is indeed caused by the reduced viral infection/replication rather than other mechanisms.

4. Based on the data presented, there is no evidence that the nanobody inhibited virus in the in vivo model. How did the administration of Nanosota-1C-Fc resulted in the reduction of disease severity ? The authors need to provide an explanation.

5. SARS-CoV-2 infection induces marked inflammatory cytokine secretion which causes extensive organ damage and is the major pathogenic mechanisms of the viral infection. Did the authors look into the inflammatory cytokine production before and after Nanosota-1C-Fc administration ? Using bronchioloalveolar hyperplasia alone is not sufficient to indicate the efficacy of the antibody.

6. The authors overstated the roles of Nanosota-1C-Fc as a protective agent against SARS-CoV-2 infection. With 1-2 weeks of in vivo life, using antibody for prevention is both costly and ineffective. In particular, if Nanosota-1C-Fc is not present in the nasal compartment or oral/upper respiratory mucosa in functional concentrations, intravenously administrated Nanosota-1C-Fc will not prevent the viral transmission. I suggest that the authors remove these statements.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "The Development of Nanosota-1as anti-SARS-CoV-2 nanobody drug candidates" for further consideration by eLife. Your revised article has been evaluated by Jos van der Meer as the Senior Editor, and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

1. At least two published studies have evaluated the preclinical efficacy in rodents, including innovative use of nanobody aerosolization for inhalation therapy of SARS-CoV-2 infection.

i) Nanobody cocktails potently neutralize SARS-CoV-2 D614G N501Y variant and protect mice

ii) Inhalable Nanobody (PiN-21) prevents and treats SARS-CoV-2 infections in Syrian hamsters at ultra-low doses.

These studies should be discussed in the context of your work.

2. Since Nanosota targets highly variable ACE2 binding sites, it may not resist many circulating VOCs (especially E484K/Q that shares with beta and the prevalent delta variants). It would be useful to discuss this vis-a-vis the evolving virus.

eLife. 2021 Aug 2;10:e64815. doi: 10.7554/eLife.64815.sa2

Author response

Please note that the major concern for all reviewers was lack of in vivo efficacy data with respect to viral titers. This will be the most critical data needed for competitive resubmission. Each reviewer also had additional comments and suggestions to improve the text, each of which should be addressed upon revision.

Summary of new data collections:

1) To further examine the in vivo efficacy of Nanosota-1C-Fc, we evaluated its therapeutic efficacy in a human ACE2-transgenic mouse model challenged with SARS-CoV-2. We measured the virus titer in the mouse lungs and also took photos of the lung histology (Figure 4C; Figure 4D). The results showed that compared to the untreated control group, the mice that received Nanosota-1C-Fc had much lower virus titers in the lungs (2~3 logs lowers) and showed improvement in the lung pathology. Combined with the data from the hamster study, the current manuscript showed that Nanosota-1C-Fc demonstrated preventive and therapeutic efficacy against live SARS-CoV-2 infection in hamster and mouse models.

2) We repeated the protein pull-down assay, with Nanosota-1C-Fc replacing Nanosota-1C (Figure 3—figure supplement 3B). The result confirmed that Nanosota-1C-Fc and ACE2 bound competitively to the RBD; it further showed that Nanosota-1C-Fc bound to the RBD much more strongly than ACE2 did, consistent with the binding affinity measurement.

Reviewer #1:

[…] 1. Figure S7. The impact of the nanobody on viral titers seems quite model relative to the impact on weight loss. It would seem that a highly effective nanobody would more robustly reduce viral burden, by more than the half-log drop shown here. By comparison maybe therapy in a hamster model results in 2-3 log drop in SARS-CoV-2 viral RNA. Can the authors examine viral RNA from these samples? What else might explain why the viral load is not diminished more than it is? Also, the statistical analyses of these data are not clear. Only one p value for each group is mentioned in legend. It would seem that a multiple comparison test should be able to provide a p-value for every dose, at each time point.

We recently learned that nasal wash, which we used to assess the hamsters, is a poor source for detecting viral titers in hamsters using RT-PCR. This is based on Zhou et al. 2021 (Cell Host and Microbe 29, 551–563). The RT-PCR results of that study showed that in hamsters receiving neutralizing antibodies and challenged by SARS-CoV-2, “viral RNA copy numbers were reduced in the lungs by an average of 3 logs (range, 0.7–4.5) (Figure 3C). In contrast, there was no significant viral load reduction in nasal turbinates and trachea in both dose groups”. Because nasal wash is not a good source, the data are uninformative and not included in the revised manuscript.

A superior index of viral titers in treated animals is to measure virus titers in the animal lungs during the peak of viral infections. Although limited resources prevented us from repeating the hamster study, we were able to conduct this critical test in a mouse model. Specifically, to further examine the in vivo efficacy of Nanosota-1C-Fc, we evaluated its therapeutic efficacy in a human ACE2-transgenic mouse model challenged with SARS-CoV-2. Instead of monitoring the body weights of the mice through the viral infection and recovery process, we measured the virus titers in the lungs at the peak of the viral infection. To this end, four experimental groups of mice (seven per group) were injected with a single dose of Nanosota-1C-Fc. Five out of the seven mice from each group were euthanized on day 2 post-challenge, and the virus titers in their lungs were measured using a virus titer plaque assay (Figure 4C). The remaining two mice in each group were euthanized on day 5 post-challenge for pathology analysis of their lung tissues (Figure 4D). Compared to the untreated control group, the mice that received Nanosota-1C-Fc had much lower virus titers in the lungs (2-3 logs lower) and showed improvement in their lung pathology. Combined with the weight and pathology data from the hamster study, these findings showed that Nanosota-1C-Fc demonstrated preventive and therapeutic efficacy against live SARS-CoV-2 infection in both hamster and mouse models.

2. Figure 4. It would seem appropriate to include some histology images corresponding to the clinical scoring parameter.

The initial histology experiment on the hamsters was done by a company that didn’t provide the histology images despite our repeated requests. Instead, we collected histology images on the mouse lungs. These histology images have been added as Figure 4D in the revised manuscript.

3. Figure 5C. Is there a negative control that can be included for comparison?

PBS buffer was used as a negative control for the in vivo stability experiment. The data have been added as Figure 5—figure supplement 1B in the revised manuscript.

Reviewer #2:

[…] 1. The overall flow is fine, however, the manuscript will need to be improved for clarity and conciseness. There is grammar throughout the text. Finally, the Discussion section seems to be a bit awkward and lengthy.

We shortened the discussion and carefully edited the manuscript to increase its clarity and to minimize grammatical errors.

2. The major advantages of nanobodies over monoclonal IgG antibodies include the small size, biophysical properties for COVID, potential inhalation delivery, as well as high bioengineering potentials. In the present work, the lead construct (Nanosota-1C-Fc) was back-engineered into a full-length antibody comparable with the monoclonal antibodies directly isolated from COVID patients. The in vitro neutralization potency of the Fc fusion construct (Nanosota-1C-Fc) appears to be considerably inferior to other monoclonal COVID antibodies under clinical evaluations(e.g., single-digit nanogram/ml). Would the authors elaborate on the key conceptual advances of this work?

Regarding the size of the Nanosota-1C-Fc, we added the following discussion:

“It is worth noting that the molecular weight of Nanosota-1C-Fc (78 kDa) is above the kidney clearance threshold (60 kDa) (8), but still only half of conventional antibodies’ molecular weight (150 kDa). […] All of these features are critical for the implementation of Nanosota-1C-Fc as a potential COVID-19 therapeutic.”

Regarding the comparison between nanobodies and conventional antibodies, we included the following discussions:

“Unlike conventional antibodies, Nanosota-1C-Fc was produced at high yields in bacteria and had exceptional thermostability.”

“The widespread of COVID-19 in the world calls for large-scale manufacturing of anti-SARSCoV-2 therapeutics. Molecules such as Nanosota-1, with its high production yields from bacteria and excellent in vitro and in vivo stabilities, are promising drug candidates to meet this need.”

Regarding the key conceptual advances of this work, we added the following discussions:

“Nanobody therapeutics derived from camelid antibodies have advantages relative to conventional antibodies. […] The extensive scope of the work makes the current study among the most comprehensive on anti-SARS-CoV-2 nanobody drug candidates.”

3. The affinity of Nanosota-1C-Fc: the authors claimed that there was a three-log improvement on the RB binding affinity, after fusing monomeric Nanosota-1C to an Fc (from 14 nM to ~ 16 pM). This raises the possibility of avidity binding. However, since the RBD does not dimerize, how could bivalent binding occur? Along the same line, I wonder if the ACE2 competition assay could be performed on Nanosota-1C-Fc to substantiate the KD measurement.

The binding affinities were measured using surface plasmon resonance. All of the measurement curves were shown in Figure 2—figure supplement 3.

To further characterize the binding interactions between Nanosota-1C-Fc and RBD, we repeated the protein pull-down assay, with Nanosota-1C-Fc replacing Nanosota-1C (Figure 3—figure supplement 3B). The result confirmed that Nanosota-1C-Fc and ACE2 bound competitively to the RBD; it further showed that Nanosota-1C-Fc bound to the RBD much more strongly than ACE2 did, consistent with the binding affinity measurement.

4. Line 187: the NC50 curves were plotted without sufficient data points and dilutions ( Figure 3B and S6), the quantitative differences (20 fold and 6,000 fold) therefore cannot be accurately derived.

In the revised manuscript, we no longer compare the Nanosota-1C-Fc and ACE2 in their capability to neutralize live SARS-CoV-2 infections. Instead, we focus on their differences in neutralizing pseudotyped SARS-CoV-2 entry, where sufficient data points were acquired.

5. Literature: please expand the published literature on COVID nanobodies:

Hanke, L., Vidakovics Perez, L., Sheward, D.J., Das, H., Schulte, T., Moliner-Morro, A., Corcoran, M., Achour, A., Karlsson Hedestam, G.B., Hallberg, B.M., et al. (2020). An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction. Nat Commun 11, 4420.

Xiang, Y., Nambulli, S., Xiao, Z., Liu, H., Sang, Z., Duprex, W.P., Schneidman-Duhovny, D., Zhang, C., and Shi, Y. (2020). Versatile and multivalent nanobodies efficiently neutralize SARS-CoV-2. Science 370, 1479-1484.

Schoof, M., Faust, B., Saunders, R.A., Sangwan, S., Rezelj, V., Hoppe, N., Boone, M., Billesbolle, C.B., Puchades, C., Azumaya, C.M., et al. (2020). An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike. Science 370, 1473-1479.

Wrapp, D., De Vlieger, D., Corbett, K.S., Torres, G.M., Wang, N., Van Breedam, W., Roose, K., van Schie, L., Team, V.-C.C.-R., Hoffmann, M., et al. (2020). Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies. Cell 181, 1436-1441.

We have included these references in the revised manuscript.

6. Regarding the weight increase of hamsters: there was evidence to indicate that weight recovery/increase in the infected hamsters is likely non-specifically related to the IP administration. What is the placebo/control used for Figure 4A? Is that PBS injection? It is not clear what "control (no drug)" indicates here. Did the author perform any post-mortem lung viral titer analysis, especially 3-4 days post-infection? This key data may provide direct evidence about how efficiently the construct can inhibit the virus in vivo.

In both the figure legend and methods, we have clarified that PBS buffer was the negative control in animal experiments.

7. Related to question 6. Since there were marginal differences (generally less than one log) of viral titers in the nasal swabs, I'd appreciate it if the author can provide more direct evidence to demonstrate the virus reductions. Minimally, examining the virus RNA levels in the lung would help substantiate the study.

We undertook an important step by measuring virus titers in the lungs in a mouse model. The data did provide more direct evidence for virus reductions after treatment. Please see our response to point #6.

8. Fc and vector construct information is missing. Is it a glycosylation variant that enables E. coli expression?

We included Fc and vector construct information in the Methods:

“Nanosota-1C-Fc was cloned into pET42b vector (Novagen) with a C-terminal human IgG₁ Fc tag.”

We didn’t mutate the glycosylation site in the Fc tag.

9. Pharmacokinetics and in vivo half-life: the authors claim that the T_1/2 of Nanosota-1C-Fc in mice (by tail vein injection) is over 10 days. However, it is hard to conclude simply based on Figure 5C. Would it the possibility that the authors show the standard PK curve to indicate the distribution phase and to carefully derive the clearance rate?

In the revised manuscript, we have removed discussions on the half-life of Nanosota1C-Fc. Instead, we only discuss “its excellent in vivo stability (significant RBD-binding capacity remained after 10 days in vivo).”

Reviewer #3:

[…] There remain a number of serious concerns that need to be addressed before the manuscript can be accepted for publication:

1. For the in vitro neutralization study, the authors only calculated ND50 values. The ND90 values are more meaningful biologically with respect to the efficacy of inhibiting viral infection and reflect the potency of an antibody in suppressing the virus. The authors should calculate ND90 values and presented in the text.

In the revision, we calculated ND90 for Nanosota-1C-Fc and Nanosota-1C in neutralizing SARS-CoV-2 pseudovirus entry and presented them in both the text and Figure 3A/ Figure 3—figure supplement 1.

We didn’t calculate ND90 for the proteins in neutralizing live SARS-CoV-2 infection because, as Reviewer 2 pointed out, the data points in these experiments were insufficient for reliable calculations.

2. For the in vivo study, the viral load in various settings showed essentially no differences across all time points as shown in Figure S7, suggesting that the Nanosota-1C-Fc either did not inhibit the viral infection or the intravenous-administrated nanobody did not reach functional concentrations in the nasal compartment. Did the authors measure Nanosota-1C-Fc concentrations in the nasal cavity ? Figure 5D clearly indicated that there is little antibody in the lungs.

3. In view of the Figure S7 data, did the authors measure viral titers in other compartments, particularly lungs, to see if virus was inhibited by Nanosota-1C-Fc. It is critical that the mitigation of the pathology is indeed caused by the reduced viral infection/replication rather than other mechanisms.

4. Based on the data presented, there is no evidence that the nanobody inhibited virus in the in vivo model. How did the administration of Nanosota-1C-Fc resulted in the reduction of disease severity ? The authors need to provide an explanation.

5. SARS-CoV-2 infection induces marked inflammatory cytokine secretion which causes extensive organ damage and is the major pathogenic mechanisms of the viral infection. Did the authors look into the inflammatory cytokine production before and after Nanosota-1C-Fc administration ? Using bronchioloalveolar hyperplasia alone is not sufficient to indicate the efficacy of the antibody.

In our human ACE2 transgenic model challenged by SARS-CoV-2, most cytokine/chemokine increase occurred at 4 and 6 dpi. (J. Zheng et al., COVID-19 treatments and pathogenesis including anosmia in K18-hACE2 mice. Nature 589, 603-607 (2021).). In the current study, lungs of five mice from each group were collected at 2 dpi for the purpose of measuring virus titers in lungs. Consistent with our previously published results, for samples collected at this time point, we didn’t find a significant increase in cytokine secretion even in the PBS control group. Nevertheless, in the revised manuscript, we showed that compared to the untreated control group, the mice that received Nanosota-1C-Fc had much lower virus titers in the lungs (2-3 logs lower) and showed improvement in their lung pathology. Combined with the data from the hamster study, the current manuscript showed that Nanosota-1C-Fc demonstrated preventive and therapeutic efficacy against live SARS-CoV-2 infection in both hamster and mouse models.

6. The authors overstated the roles of Nanosota-1C-Fc as a protective agent against SARS-CoV-2 infection. With 1-2 weeks of in vivo life, using antibody for prevention is both costly and ineffective. In particular, if Nanosota-1C-Fc is not present in the nasal compartment or oral/upper respiratory mucosa in functional concentrations, intravenously administrated Nanosota-1C-Fc will not prevent the viral transmission. I suggest that the authors remove these statements.

We removed these statements and added the following discussion:

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

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

1. At least two published studies have evaluated the preclinical efficacy in rodents, including innovative use of nanobody aerosolization for inhalation therapy of SARS-CoV-2 infection.

i) Nanobody cocktails potently neutralize SARS-CoV-2 D614G N501Y variant and protect mice

ii) Inhalable Nanobody (PiN-21) prevents and treats SARS-CoV-2 infections in Syrian hamsters at ultra-low doses.

These studies should be discussed in the context of your work.

In the revised manuscript, we have revised the discussion as follows:

“Although several studies have reported nanobody drug candidates that specifically target SARS-CoV-2 (12-16, 32, 33), as of this writing, only two studies have evaluated their nanobody drug candidates in an animal model for anti-SARS-CoV-2 therapeutic efficacy (32, 33). None of these studies have evaluated their nanobody drug candidates for in vitro thermostability, in vivo stability, or tissue biodistribution.”

We also added one new reference (in red) to the following discussion:

“Third, despite its rapid clearance from the blood, Nanosota-1C could be used as an inhaler to treat infections in the respiratory tracts (10, 33) or as an oral drug to treat infections in the intestines (11).”

2. Since Nanosota targets highly variable ACE2 binding sites, it may not resist many circulating VOCs (especially E484K/Q that shares with beta and the prevalent delta variants). It would be useful to discuss this vis-a-vis the evolving virus.

In the revised manuscript, we have added the following discussion:

“Moreover, to combat emerging SARS-CoV-2 variants with RBM mutations, Nanosota-1 series can be further developed through additional affinity maturation against the RBD of SARS-CoV2 variants. The series may also be developed to become part of cocktail therapies that target multiple sites on SARS-CoV-2.”

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Citations

Ye G, Shi K, Aihara H, Li F. 2021. Crystal structure of SARS-CoV-2 RBD complexed with Nanosota-1. RCSB Protein Data Bank. 7KM5

Supplementary Materials

Figure 2—figure supplement 4—source data 1. Raw images for Figure 2—figure supplement 4.

elife-64815-fig2-figsupp4-data1.zip^{(10.3MB, zip)}

Figure 4—source data 1. Raw images for Figure 4.

elife-64815-fig4-data1.zip^{(60.2MB, zip)}

Figure 5—source data 1. Raw images for Figure 5.

elife-64815-fig5-data1.zip^{(8.7MB, zip)}

Source data 1. Raw data for figures and figure supplements.

elife-64815-data1.xlsx^{(40.5KB, xlsx)}

Transparent reporting form

elife-64815-transrepform.docx^{(70.4KB, docx)}

Data Availability Statement

Coordinates and structure factors have been deposited to the Protein Data Bank with accession number 7KM5.

The following dataset was generated:

Ye G, Shi K, Aihara H, Li F. 2021. Crystal structure of SARS-CoV-2 RBD complexed with Nanosota-1. RCSB Protein Data Bank. 7KM5

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PERMALINK

The development of Nanosota-1 as anti-SARS-CoV-2 nanobody drug candidates

Gang Ye

Joseph Gallant

Jian Zheng

Christopher Massey

Ke Shi

Wanbo Tai

Abby Odle

Molly Vickers

Jian Shang

Yushun Wan

Lanying Du

Hideki Aihara

Stanley Perlman

Aaron LeBeau

Fang Li

Roles

Abstract

Introduction

Figure 1. Construction of a camelid nanobody phage display library and use of this library for screening of anti-SARS-CoV-2 nanobodies.

Figure 1—figure supplement 1. Schematic drawings of nanobodies and conventional antibodies.

Results

Nanosota-1 was identified by phage display

Nanosota-1 tightly bound to the SARS-CoV-2 RBD and completely blocked the binding of ACE2

Figure 2. Crystal structure of SARS-CoV-2 RBD complexed with Nanosota-1C.

Figure 2—figure supplement 1. Footprint of Nanosota-1C on SARS-CoV-2 RBD.

Figure 2—figure supplement 2. The binding of Nanosota-1C to SARS-CoV-2 spike protein in different conformations.

Figure 2—figure supplement 3. Measurement of the binding affinities between Nanosota-1 and SARS-CoV-2 RBD by surface plasmon resonance assay using Biacore.

Figure 2—figure supplement 4. Binding interactions between Nanosota-1 and SARS-CoV-2 RBD.

Table 1. Binding affinities between Nanosota-1 and SARS-CoV-2 RBD as measured using surface plasmon resonance.

Nanosota-1C-Fc potently neutralized SARS-CoV-2 infection in vitro and in vivo

Figure 3. Efficacy of Nanosota-1 in neutralizing SARS-CoV-2 infections in vitro.

Figure 3—figure supplement 1. Neutralization of SARS-CoV-2 pseudovirus, which contains the D614G mutation in the spike protein, by Nanosota-1.

Figure 3—figure supplement 2. Detailed data on the neutralization of live SARS-CoV-2 infection of target cells by Nanosota-1.

Figure 4. Efficacy of Nanosota-1 in protecting both hamsters and mice from SARS-CoV-2 infections.

Nanosota-1C-Fc is stable in vitro and in vivo with excellent bioavailability

Figure 5. Analysis of expression, purification, and pharmacokinetics of Nanosota-1C-Fc.

Figure 5—figure supplement 1. Pharmacokinetics of Nanosota-1C.

Discussion

Materials and methods

Cell lines, plasmids, and virus

Construction of camelid nanobody phage display library

Camelid nanobody library screening

Affinity maturation

Production of Nanosota-1

Production of SARS-CoV-2 RBD and ACE2

ELISA

Determination of the structure of SARS-CoV-2 RBD complexed with Nanosota-1C

Table 2. X-ray data collection and structure refinement statistics (SARS-CoV-2 RBD/Nanosota-1C complex).

Surface plasmon resonance assay

Protein pull-down assay

Gel filtration chromatography assay

SARS-CoV-2 pseudovirus entry assay

SARS-CoV-2 plaque-reduction neutralization test

SARS-CoV-2 challenge of hamsters

SARS-CoV-2 challenge of human ACE2-transgenic mice

In vivo stability of Nanosota-1

Biodistribution of Nanosota-1 in mice

Acknowledgements

Funding Statement

Contributor Information

Funding Information

Additional information

Competing interests

Author contributions

Ethics

Additional files

Data availability

References

Decision letter

Roles

Author response

Associated Data

Data Citations

Supplementary Materials

Data Availability Statement

ACTIONS

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