key: cord-0712644-0r6m9m7x
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: 2021-10-06
journal: Cell Rep
DOI: 10.1016/j.celrep.2021.109869
sha: 228f18dcb1d2fcde2b9caed375b3e616b5fa50ea
doc_id: 712644
cord_uid: 0r6m9m7x

The dramatically expanding COVID-19 needs multiple effective countermeasures. Neutralizing nanobodies (Nbs) are a potential therapeutic strategy for treating COVID-19. Here, we characterize several RBD-specific Nbs isolated from an Nb library derived from an alpaca immunized with SARS-CoV-2 spike glycoprotein (S); among them, three Nbs exhibit picomolar potency against SARS-CoV-2 live virus, pseudotyped viruses, and circulating SARS-CoV-2 variants. To improve the efficacy, various configurations of Nbs are engineered. Nb15-NbH-Nb15, a trimer constituted of three Nbs, is constructed to be bispecific for human serum albumin (HSA) and RBD of SARS-CoV-2. Nb15-NbH-Nb15 exhibits single digit ng/ml neutralization potency against the wild-type and Delta variants of SARS-CoV-2 with a long half-life in vivo. In addition, we show that intranasal administration of Nb15-NbH-Nb15 provides effective 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.

As of Feb. 1 st , 2021, the coronavirus SARS-CoV-2 has caused more than 100 million 56 confirmed cases and over 2.2 million deaths globally. The containment of the 57 expanding COVID-19 pandemic needs multiple countermeasures. Prophylactic 58 vaccines have been recently approved (Dai and Gao, 2020) , and a number of In order to improve efficacy and stability in vivo, we constructed bi-specific Nbs 211 consisting of one Nb specific for HSA (Nb H ) developed by our lab and one or two Nb 15 s 212 specific for RBD with (G4S) 3 as the linker between each Nb ( Figure 3A Figure 4D and 4E ). In addition, the results also showed that Nb 15 -Nb H -Nb 15 could 242 reach lungs, and sustained for more than 168 h (7 d) when administrated i.n.; in contrast 243 the fluorescence could only be detected between 1 and 2 h post i.p. infusion ( Figure 4F  244 and 4G). These results suggest that i.n. administration of Nb 15 -Nb H -Nb 15 will be a 245 favorable route for antibody to reach nasopharynx and lungs where SARS-CoV-2 246

replicates. Therefore, to avoid the potential ADE associated by Fc in the Nb-Fc, we 247 selected Nb 15 -Nb H -Nb 15 for further efficacy evaluation in vivo. prophylactic or therapeutic efficacy ( Figure 6A ). Viral RNA was detected in lungs in 275 the control mice (6.28  10 5 copies/mg on average in SARS-CoV-2 group, n=5) and the 276 isotype treated control mice (7.8  10 4 copies/mg on average in isotype group, n=3,). Figure 6D ). In contrast, the lungs of the mice receiving 294 ( Figure 6D ). Together, Nb 15 -Nb H -Nb 15 at an average of 10 mg/kg via i.n. administrated 296 24 h before or 1 h after challenge provided complete protection against SARS-CoV-2 297 infection, and significantly inhibited SARS-CoV-2 replication when the antibody was 298 administrated 24 h postinfection. Nb 15 -Fc used at an average of 10 mg/kg via i.n. 299 administrated 1 h after challenge significantly reduced viral load but failed to provide 300 complete protection. We noted that those mice receiving Nb 15 s treatment showed less 301 weight loss than the control mice but did not achieve statistical difference (Figure 302 Table S2 ). 318

These evidences demonstrate the neutralizing activity of these Nbs against multiple 319 SARS-CoV-2 variants and suggest that the Nb-Fcs target at a highly conserved epitope 320 on RBD protein. with IC 50 values of 25.1 ng/ml and 8 ng/ml ( Figure 3D and Table 1 ), indicating that Nb 331 configuration has impact on the neutralizing activity. In addition, heterotrimeric 332 bispecific configuration is superior to the bispecific heterodimer. We also noted that the 333 bi-, tri-and tetra-valent configurations exhibited comparable potency with IC 50 values 334 of 2.8, 3.5 and 2.3 ng/ml (11, 9.0, 4.3 pM), respectively. The neutralizing potency did 335 not correspond to the valence increase when there are two or more than two Nb 15 s 336 though monomeric 1xNb 15 had much lower inhibitory activity ( Figure 2C and Mann-Whitney test was performed to compare treatment group with the SARS-CoV-2 511 control group. ns, no significance; *, P < 0.05, **, P < 0.01, ***, P < 0. Further information and requests for resources and reagents should be directed to and 522 will be fulfilled by the Lead Contact, Zhiwei Wu (wzhw@nju.edu.cn). 523

This study did not generate new unique reagents. 525

The data that support the findings of this study are available upon request from the 527 lead contact. 

The purified protein or antibody was separated by electrophoresis in a 7.5%-12% 580 polyacrylamide gel. The separated protein or antibody was revealed either using 581

Coomassie blue or transferred to PVDF membrane for WB analysis under reducing or 582 non-reducing conditions with β-mercaptoethanol. The membrane was first blocked and 583 then incubated overnight at 4 o C or 37 o C for one hour with diluted plasma or antibody, 584 followed by incubation with the secondary antibody of either anti-human IgG or 585 anti-rabbit IgG conjugated with an IRDye 800CW (cat.# 926-32232, Rockland). 586

Protein bands were visualized using the Odyssey Image System (Li-COR). 587

Anti-sera titer and antibody characterization or antibody quantification in vivo were 589 examined by ELISA as reported in our previously published method(Wu et al., 2019) 590 with modifications. In brief, the protein was coated to high protein-binding ELISA 591 plates (Corning) at a concentration of 0.5 μg/ml, 100 μl per well at 37 °C for 2 hours (h) 592 or 4 o C overnight. After washing, blocking buffer with 5% non-fat milk in PBS was 593 added and incubated at 37 °C for 1 h. After washing 2-4 times, 100 μl serially diluted 594 NB7242, 1:10000 dilution) was added and incubated at 37 °C for 1 h. Accordingly, 597 3,3′,5,5′-Tetramethylbenzidine (TMB, Sigma) substrate was added at 37 o C for 10 598 minutes (min); and the reaction was stopped by adding 10 μl 0.2 M H 2 SO 4 . The optical 599 densities at 450 nm were measured using the Infinite 200 (Tecan, Ramsey, MN, USA) . 600

Antibody titers were defined as the highest dilution when the diluted serum produced at 601 least 2.1-fold optical density readout as compared to the control serum sample at the 602 same dilution. 

The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice

Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape 797 seen with individual antibodies

Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput

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Nebulised ALX-0171 for respiratory 803 syncytial virus lower respiratory tract infection in hospitalised children: a double-blind, randomised

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Generation and Characterization of ALX-0171, a Potent Novel Therapeutic 808

Nanobody for the Treatment of Respiratory Syncytial Virus Infection

Development 810 of multi-specific humanized llama antibodies blocking SARS-CoV-2/ACE2 interaction with high affinity 811 and avidity

The central role of the 813 nasal microenvironment in the transmission, modulation, and clinical progression of SARS-CoV-2 814 infection

Naturally-Occurring Antibodies Devoid of Light-Chains

An alpaca nanobody neutralizes SARS-CoV-2 by 820 blocking receptor interaction

Preclinical efficacy and safety of mepolizumab 823 (SB-240563), a humanized monoclonal antibody to IL-5, in cynomolgus monkeys

carboxymethylcellulose, CMC) and the plates were then incubated at 37 °C for 24 h. 687Cells were fixed with 4% paraformaldehyde solution for 30 min, and then the overlays 688 were removed. Cells were permeabilized with 0.2% Triton X-100 and incubated with 689 cross-reactive rabbit anti-SARS-CoV-N IgG (Sino Biological, Inc) for 1 h at room 690 temperature before the addition of HRP-conjugated goat anti-rabbit IgG (H+L) 691 antibody (Jackson ImmunoResearch) and further incubated at room temperature. The 692 foci were stained with KPL TrueBlue Peroxidase substrates (SeraCare Life Sciences 693Inc.) and were counted with an EliSpot reader (Cellular Technology Ltd.). 694

Affinity assays were performed on a ForteBio OctetRED 96 biolayer interferometry 696 instrument (Molecular Devices ForteBio LLC, Fremont, CA) at 25 o C with shaking at 697 1,000 rpm. To measure the affinity of Nbs with human Fc tag, anti-human Fc (AHC) 698 biosensors (cat.# 18-5060, Fortebio) were hydrated in water for 30 min prior to 60 699 seconds (sec) incubation in a kinetic buffer (PBS, 0.02% (v/v) Tween-20, pH 7.0). 700Either Nb-Fc in cell supernatant or purified Nb-Fcs were loaded in a kinetic buffer for 701 200 sec prior to baseline equilibration for 200 sec in a kinetic buffer. Association of 702 SARS-CoV-2 RBD in a two-fold dilution series from 20 nM to 2.5 nM was performed 703 prior to dissociation for 180 sec. To measure the affinity of Nbs without Fc tag, RBD 704 protein was coupled to AR2G biosensor (cat.# 18-5092, Fortebio) via BLI instrument 705 according to the instructions of the amino coupling kit. Association of Nbs in a serial 706 dilution was performed prior to dissociation for 180 sec. After each cycle, the 707 biosensors were regenerated via 3 short pulses of 5 sec each of 100 mM pH 2.7 708 glycine-HCL followed by running buffer. The data were baseline subtracted before 709 fitting performed using a 1:1 binding model and the ForteBio data analysis software.