key: cord-0944349-8swqnu46 authors: Wu, Xilin; Cheng, Lin; Fu, Ming; Huang, Bilian; Zhu, Linjing; Xu, Shijie; Shi, Haixia; Zhang, Doudou; Yuan, Huanyun; Nawaz, Waqas; Yang, Ping; Hu, Qinxue; Liu, Yalan; Wu, Zhiwei title: A potent bispecific nanobody protects hACE2 mice against SARS-CoV-2 infection via intranasal administration date: 2021-02-09 journal: bioRxiv DOI: 10.1101/2021.02.08.429275 sha: f0deced014a1e1b1c97769e0a2d1bb0f1a1d7e07 doc_id: 944349 cord_uid: 8swqnu46 The dramatically expanding COVID-19 needs multiple effective countermeasures. Neutralizing antibodies are a potential therapeutic strategy for treating COVID-19. A number of neutralizing nanobodies (Nbs) were reported for their in vitro activities. However, in vivo protection of these nanobodies was not reported in animal models. In the current report, we characterized several RBD-specific Nbs isolated from a screen of an Nb library derived from an alpaca immunized with SARS-CoV-2 spike glycoprotein (S); among them, three Nbs exhibited picomolar potency against SARS-CoV-2 live virus, pseudotyped viruses, and 15 circulating SARS-CoV-2 variants. To improve the efficacy, various configurations of Nbs were engineered. Nb15-NbH-Nb15, a novel trimer constituted of three Nbs, was constructed to be bispecific for human serum albumin (HSA) and RBD of SARS-CoV-2. Nb15-NbH-Nb15 exhibited sub-ng/ml neutralization potency against the wild-type and currently circulating variants of SARS-CoV-2 with a long half-life in vivo. In addition, we showed that intranasal administration of Nb15-NbH-Nb15 provided 100% protection for both prophylactic and therapeutic purposes against SARS-CoV-2 infection in transgenic hACE2 mice. Nb15-NbH-Nb15 is a potential candidate for both prevention and treatment of SARS-CoV-2 through respiratory administration. One sentence summary Nb15-NbH-Nb15, with a novel heterotrimeric bispecific configuration, exhibited potent and broad neutralization potency against SARS-CoV-2 in vitro and provided in vivo protection against SARS-CoV-2 infection in hACE2 transgenic mice via intranasal delivery. Graphical abstract: Highlights We described a novel heterotrimeric configuration of Nb-NbH-Nb (Nb15-NbH-Nb15) that exhibited improved viral inhibition and stability. Nb15-NbH-Nb15 provides ultrahigh neutralization potency against SARS-CoV-2 wild type and 18 mutant variants, including the current circulating variants of D614G and N501Y predominantly in the UK and South Africa. It is the first to demonstrate the Nbs efficacy in preventing and treating SARS-CoV-2 infection in hACE2 transgenic mice via intranasal delivery. As of Feb. 1 st , 2021, the novel coronavirus SARS-CoV-2 has caused more than 100 69 million confirmed cases and over 2. with KD values ranging from 37.6 to 4.25 nM ( Fig. S3C and S3D ). Neutralization 157 analysis showed potent inhibition of pseudotyped SARS-CoV-2 by culture supernatants 158 of RBD-specific Nb15-Fc, Nb22-Fc and Nb31-Fc (Fig. S3E) . 159 160 Epitope analysis of Nb-Fcs 161 The purified Nb15-Fc, Nb22-Fc and Nb31-Fc exhibited dose dependent binding with 162 RBD protein on ELISA (Fig. S4A ). In addition, Nb15-Fc, Nb22-Fc and Nb31-Fc likely 163 reacted with conformational structure as their bindings with reduced RBD protein were 164 almost completely abolished (Fig. S4B) . The kinetic binding of Nb15-Fc, Nb22-Fc and 165 Nb31-Fc with RBD protein ranged from KD of 1.13 to 1.76 nM, indicating tightly 166 clustered binding characteristics ( Fig. 4C-E) , which was substantiated by the 167 superimposed ELISA binding curves (Fig. S4A ). These three Nb-Fcs were next 168 evaluated for epitope specificity in a competition assay by BLI using RBD protein as a 169 capture antigen. The results revealed that the pre-bound Nb-Fcs efficiently blocked the 170 further binding of the other two Nb-Fcs to RBD protein, suggesting that all three Nb-171 Fcs likely recognize an overlapping epitope (Fig. S5) showed an increasing KD ranging from 12 to <0.001 nM as the valence increased ( Table S3 ), suggesting that increasing valence does not confer improved anti-211 viral activity. As such, 3Nb15 was selected for further functional exploration. 212 213 Nb15-NbH-Nb15, heterotrimer and bi-specific for RBD and HSA, exhibiting potent 214 neutralization against SARS-CoV-2. In order to improve efficacy and stability in vivo, we constructed bi-specific Nbs 216 consisting of one Nb specific for HSA (NbH) developed by our lab and one or two Nb15s 217 specific for RBD with (G4S)3 as the linker between each Nb ( Fig. 2A ) and analyzed 218 their binding and viral inhibitory activities. In addition to the heterodimeric 219 configuration of Nb-NbH that was previously reported 29 , various new configurations of 220 Nbs were engineered as depicted in Fig. 2A . ELISA analysis showed that all 221 combinations containing Nb15 reacted with RBD protein; among them, heterotrimeric 222 Nb15-Nb15-NbH, NbH-Nb15-Nb15 and Nb15-NbH-Nb15 exhibited better binding with RBD 223 protein than heterodimeric Nb15-NbH and NbH-Nb15 configurations ( Fig. 2A and 2B) . 224 Furthermore, Nb15-NbH, NbH-Nb15-Nb15 and Nb15-NbH-Nb15 were the best HSA binders 225 as compared to other configurations ( Fig. 2A and 2C ). Bi-specific Nbs in various 226 configurations were tested for the inhibition of SARS-CoV-2 infection; among them, 227 Nb15-NbH-Nb15 exhibited the most potent neutralization of the virus with an IC50 of 0.4 228 ng/ml (9.0 pM) ( Fig. 2D and Table S3 ). We next compared Nb15-NbH-Nb15 with 229 homotrimer Nbs (3Nb15) or Nb-Fc for their binding and anti-viral activities and found 230 that 3Nb15, Nb15-NbH-Nb15 and Nb15-Fc exhibited comparable potency with IC50 231 values of 0.4, 0.4, and 0.9 ng/ml (9.0, 9.0 and 11.3 pM), respectively ( Fig. 2E and Table 232 S3). Nb15-NbH-Nb15-YF750 was administered i.n. (6.9 x 10 10 ph/s) than that when administered i.p. or i.v. (1.4 x 10 10 and 4.3 x 10 10 ph/s, respectively) ( Fig. 3D and 3E) . 246 In addition, the results also showed that Nb15-NbH-Nb15 could reach lungs, and 247 sustained for more than 168 h (7 d) when administrated i.n.; in contrast the fluorescence 248 could only be detected between 1 and 2 h post i.p. infusion ( Fig. 3F and 3G ). These 249 results suggest that i.n. administration of Nb15-NbH-Nb15 will be a favorable route for 250 antibody to reach nasopharynx and lungs where SARS-CoV-2 replicates. Therefore, to 251 avoid the potential ADE associated by Fc in the Nb-Fc, we selected Nb15-NbH-Nb15 for 252 further efficacy evaluation in vivo. Table S4 ). Furthermore, Nb15-NbH-268 Nb15 retained 100% activity after aerosolization, indicating the potential application as 269 a nebulized drug (Fig. 4D -E and Table S4 ). 270 271 To evaluate the efficacy of Nb15-NbH-Nb15 in vivo, hACE2 transgenic mice were 273 challenged with SARS-CoV-2, and Nb15-NbH-Nb15 was administrated i.n. either before 274 or after viral challenge for prophylactic or therapeutic efficacy (Fig. 5A ). Viral RNA was detected in lungs in the control mice (6.28  10 5 copies/mg on average in SARS-276 CoV-2 group, n=5) and the isotype treated control mice (7.8  10 4 copies/mg on average 277 in isotype group, n=3,). For the prophylactic group, no viral RNA or infected cells was 278 detected in 100% (5/5) of the mice when 250 μg (average of 10 mg/kg) Nb15-NbH-Nb15 279 was administrated via i.n. 24 hours before SARS-CoV-2 infection (Nb15-NbH-Nb15 -280 24h group, n=5), as evidenced by real-time PCR and immunofluorence staining ( Nb15 24 h group, n=5) than that in the control mice (6.28  10 5 copies/mg on average 287 in SARS-CoV-2 group and 7.8  10 4 copies/mg in isotype control) (Fig. 5B-D) . Nb15-288 Fc inhibited viral replication and reduced the viral copies number (average of 7.59 10 3 289 copies/mg in Nb15-Fc 1h group, n=5) but failed to provide complete protection under 290 the same condition as Nb15-NbH-Nb15 (Fig. 5B-D) . Furthermore, histopathological 291 analysis of lung tissues showed that SARS-CoV-2 challenge induced severe lung 292 lesions, as shown by the infiltration of inflammatory cells and thickened alveolar septa 293 ( Fig. 5D ). In contrast, the lungs of the mice receiving Nb15-NbH-Nb15 or Nb15-Fc 294 treatment showed no apparent pathological changes (Fig. 5D ). Together, Nb15-NbH-295 Nb15 at an average of 10 mg/kg via i.n. administrated 24 h before or 1 h after challenge 296 provided complete protection against SARS-CoV-2 infection, and significantly 297 inhibited SARS-CoV-2 replication when the antibody was administrated 24 h 298 postinfection. Nb15-Fc used at an average of 10 mg/kg via i.n. administrated 1 h after 299 challenge significantly reduced viral load but failed to provide complete protection. We 300 noted that those mice receiving Nb15s treatment showed less weight loss than the control 301 mice but did not achieve statistical difference (Fig. 5E-F) . These results indicate that 302 Nb15-NbH-Nb15, when used early during infection, confered higher protection efficacy 303 than used at later time point. In summary, the Nb15-NbH-Nb15 configuration 304 administered via i.n. was superior to Nb15-Fc and exhibited both prophylactic and 305 therapeutic efficacy against SARS-CoV-2 challenge. 306 307 Discussion: 308 In this study, three potent neutralizing Nb-Fcs were isolated from a phage display 309 platform derived from an SARS-CoV-2 S protein immunized alpaca. These three RBD Table S3 ), indicating that Nb configuration has impact on the 329 neutralizing activity. In addition, heterotrimeric bispecific configuration is superior to 330 the bispecific heterodimer. We also noted that the bi-, tri-and tetra-valent 331 configurations exhibited comparable potency with IC50 values of 2.8, 3.5 and 2.3 ng/ml 332 (11, 9.0, 4.3 pM), respectively. The neutralizing potency did not correspond to the 333 valence increase when there are two or more than two Nb15s though monomeric 1xNb15 334 had much lower inhibitory activity ( Fig. S7C and Table S3 ). Noted that Nb15-NbH-Nb15 335 shows higher potency than NbH-Nb15-Nb15, Nb15-Nb15-NbH, and all the homomultimers, 336 suggesting that the position of NbH plays important roles in neutralization activity. We 337 speculate that in Nb15-NbH-Nb15 NbH may space out the two Nb15s to either avoid cross 338 intereference with each other or allow better binding of the trimeric Nb to S proteins on 339 the viral particle. Furthermore, Nb15-NbH-Nb15 displayed comparable neutralizing 340 potency as those of Nb15-Fc and 3xNb15, and higher neutralization potency and longer Viral targets for vaccines against COVID-19 Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape 610 seen with individual antibodies A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2 Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from 614 disease in a small animal model Potent neutralizing antibodies directed to multiple epitopes on SARS-CoV-2 spike Human neutralizing antibodies elicited by SARS-CoV-2 infection Naturally-Occurring Antibodies Devoid of Light-Chains The Therapeutic Potential of Nanobodies Nanobodies as therapeutics: big opportunities 624 for small antibodies Evaluation of Antibody-Dependent Enhancement of SARS-CoV Infection in Rhesus 627 Macaques Immunized with an Inactivated SARS-CoV Vaccine Antibody-dependent enhancement of virus infection and disease Fc receptors in antibody-dependent enhancement of viral infections A single-domain antibody inhibits SFTSV and mitigates virus-induced pathogenesis 634 in vivo Generation and Characterization of ALX-0171, a Potent Novel Therapeutic 636 Nanobody for the Treatment of Respiratory Syncytial Virus Infection Nanobodies (R)dagger as inhaled biotherapeutics for lung diseases Nebulised ALX-0171 for respiratory syncytial virus lower respiratory tract 641 infection in hospitalised children: a double-blind, randomised, placebo-controlled, phase 2b 642 trial A pneumonia outbreak associated with a new coronavirus of probable bat origin Detection of SARS-CoV-2 in Different Types of Clinical Specimens Preclinical efficacy and safety of mepolizumab (SB-240563), a humanized 648 monoclonal antibody to IL-5, in cynomolgus monkeys Topically Administered Neutralizing Antibodies in Experimental Immunotherapy of Respiratory 652 Syncytial Virus-Infection in Cotton Rats Versatile and multivalent nanobodies efficiently neutralize SARS-CoV-2 An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing 657 inactive Spike Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with 659 An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction Identification of Human Single-Domain Antibodies against SARS-CoV-2 Development of multi-specific humanized llama antibodies blocking SARS-CoV-666 /ACE2 interaction with high affinity and avidity. 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(E) The 755 cell supernatants of 21 various Nb-Fcs were tested for neutralization against CoV-2 infection, the cell supernatant displaying outstanding neutralizing curve was 757 labeled as the color-coded curve. Data of B represent as mean ± SEM Purified Nb-Fcs 761 binding with RBD identified by ELISA. Data represent as mean ± SEM. (B) RBD 762 protein under reducing condition (R) or non-reducing condition (NR) was detected by 763 WB with Nb15-Fc, Nb22-Fc and Nb31-Fc. Kinetic binding curve of RBD with Nb15-Fc 764 (C), Nb22-Fc (D) and Nb31-Fc (E), respectively. Binding curves are colored black Epitope analysis of Nb-Fcs by BLI. RBD protein was coated 768 on the sensor, Nb15-Fc (A), Nb22-Fc (B) or Nb31-Fc(C) as the first antibody was added 769 to bind for 300 s, followed by the addition of Nb15-FC, Nb22-FC and Nb31-FC as the 770 second antibody for another 300 s Nb31-Fc(C), SNB02 (isotype control antibody) (D) was detected 774 based on authentic SARS-CoV-2 plaque reduction neutralization test Characterization of Nb15s with multivalent or various 779 formats. (A) The binding curve of multivalent Nb15s with RBD protein detected by 780 BLI. (B) The table summary of the binding of Nb15s with RBD protein tested by BLI Multivalent Nb15s and various formats were evaluated for neutralization potency 782 against pseudotyped SARS-CoV-2 infection Supplemental Figure 8. Kinetic binding curve of Nb15-NbH-Nb15 with MSA Kinetic binding curve of Nb15-NbH-Nb15 at the concentration of 300 nM, 100nM, 33.3 786 nM,11.1nM, 3.7nM and 1.2 nM with MSA by BLI Supplemental Table 1. Summary of CDR sequences of positive Nb clones Supplemental Table 2. Summary of Nbs inhibiting SARS-CoV-2 variants Supplemental Table 3. Summary of various Nbs inhibiting pseudotyped SARS-CoV-792 2 The experimental schedule for immunization. The titer of anti-sera specific for SARS-728CoV-2 S protein (B) and RBD protein (C) was evaluated one week after the 729 immunization in alpaca receiving SARS-CoV-2 spike protein, respectively. The titer of 730 the third anti-serum was indicated as blue line. The blue # indicates the anti-serum titer 731after the third immunization. 3 rd anti-serum and 2 nd anti-serum represent the anti-sera 732 collected from alpaca one week after the 3 rd and 2 nd immunization. Blank serum 733represents the alpaca serum collected before immunization, which was taken as a in (E) was plotted. Data represent mean ± SEM; One-way or two-way ANOVA were 879 performed to compare treatment group with the isotype control group. ns, no 880 significance; **, P < 0.01, ***, P < 0.001. Data of B, C, E and F represent as mean ± 881 SEM. All experiments of B and C were repeated twice. 882 883 884