key: cord-0976512-tb1p14nf
authors: Stefan, Maxwell A.; Light, Yooli K.; Schwedler, Jennifer L.; McIlroy, Peter R.; Courtney, Colleen M.; Saada, Edwin A.; Thatcher, Christine E.; Phillips, Ashlee M.; Bourguet, Feliza A.; Mageeney, Catherine M.; McCloy, Summer A.; Collette, Nicole M.; Negrete, Oscar A.; Schoeniger, Joseph S.; Weilhammer, Dina R.; Harmon, Brooke
title: Development of Potent and Effective Synthetic SARS-CoV-2 Neutralizing Nanobodies
date: 2021-05-06
journal: bioRxiv
DOI: 10.1101/2021.05.06.442911
sha: 0e1ab871ac735184867d89312fbf93c333423906
doc_id: 976512
cord_uid: tb1p14nf

The respiratory virus responsible for Coronavirus disease 2019 (COVID-19), Severe acute respiratory syndrome coronavirus 2 (SARS-2), has impacted nearly every aspect of life worldwide, claiming the lives of over 2.5 million people globally, at the time of this publication. Neutralizing nanobodies (VHH) represent a promising therapeutic intervention strategy to address the current SARS-2 pandemic and provide a powerful toolkit to address future virus outbreaks. Using a synthetic, high-diversity VHH bacteriophage library, several potent neutralizing VHH antibodies were identified and evaluated for their capacity to tightly bind to the SARS-2 receptor-binding domain (RBD), to prevent binding of SARS-2 spike (S) to the cellular receptor Angiotensin-converting enzyme 2 (ACE2), and to neutralize viral infection. Preliminary preclinical evaluation of multiple nanobody candidates demonstrate that they are prophylactically and therapeutically effective in vivo against wildtype SARS-2. The identified and characterized nanobodies described herein represent viable candidates for further preclinical evaluation and another tool to add to our therapeutic arsenal to address the COVID-19 pandemic. Author Summary To fully address the on-going pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-2), it will be important to have both vaccines and therapeutic strategies to prevent and mitigate the effects of SARS-2. In this study, we describe the identification and characterization of potently neutralizing humanized single domain heavy chain (VHH) antibodies that have binding affinity for both the original Wuhan strain and widely circulating B.1.1.7/UK strain. VHH antibodies have the same therapeutic potential as conventional antibodies in half the size and with greater stability and solubility. Using a synthetic humanized high-diversity VHH phage library we identified several candidates with strong affinity for the SARS-2 spike that block the interaction of SARS-2 spike with the cellular receptor ACE2, and effectively neutralize infection with SARS-2 in vitro. By sequencing viral escape mutants generated in the presence of each VHH we mapped the binding sites of the VHH antibodies and assessed their affinity against newly emerging SARS-2 variants. Finally, we demonstrate that two of these VHH antibodies show prophylactic and therapeutic efficacy in vivo against challenge with SARS-2. This study establishes that screening highly diverse VHH phage libraries against viral threats can yield highly effective therapeutic agents in real time.

Mutant SARS-2 RBDs were produced by site-directed mutagenesis. All fragments were 148 synthesized commercially (IDT) and terminal fragments had overlapping sequence with 149 the downstream digested vector. All DNA fragments and pSF-CMV vector restriction 150 digested with EcoRI and BamHI were assembled using NEBuilder HiFi DNA master. 151 Constructs for expression of ACE2-Fc (human crystallizable fragment (Fc) domain) 152 fusion proteins were produced as follows. pAce2-huFc was produced by subcloning the 153 ectodomain on human ACE2 (Sino Biological) into pCR-Fc using the NotI and BamHI 154 restriction sites. To produce Ace2-rbFc (rabbit Fc domain), a gBlock (IDT) of this same 155 region of Ace2 was subcloned into pFUSE rIgG-Fc2. 156 Soluble SARS-2 S and RBD were both produced by transient expression in 157 Expi293F suspension cells. Cells were transfected with 1 μg/mL plasmid at a density of 158 3.0 x 10 6 cells/mL using Expifectamine. Cells were supplemented as instructed by the 159 manufacturer and grown at 37°C with 8% CO 2 . Cell supernatant was harvested on day 4 160 by centrifugation at 4,000 x g for 30 min at 4°C. Clarified supernatant was passed through 161 a 0.22 μm filter and then applied to a 5 mL HisTrap Excel column preequilibrated with 10 162 mM Tris (pH 8.0), 300 mM NaCl for SARS-2 S and 20 mM phosphate (pH 7.4), 300 mM 163 NaCl for SARS-2 RBD. The column was washed with 10 column volumes equilibration 164 buffer followed by 5 column volumes equilibration buffer with 20 mM imidazole. The S 165 and RBD proteins were eluted with a step gradient to 500 mM imidazole. Fractions 166 containing SARS-2 S protein were pooled and dialyzed in 20 mM HEPES (pH 8.0), 200 167 mM NaCl prior to concentration. Protein was then concentrated and filtered prior to 168 application to a Enrich 650 SEC column equilibrated with dialysis buffer for SARS-2 S 169 and PBS for SARS-2 RBD. Purified SARS-2 proteins were stored at -80°C.

ACE2-huFc, ACE2-rbFc, and V H H-huFc antibodies were produced by transient 171 expression in CHO-S cells using the ExpiCHO expression system. In brief, cells were 172 transfected at a density of 6 x 10 6 cells/mL and grown for 18 hours at 37 °C with 8% CO2.

Cells were then supplemented as per the manufacture's guidelines and transferred to 32 174°C with 5% CO2. Cell supernatant containing soluble protein was harvested on day 10 by 175 centrifugation at 4,000 x g for 30 min at 4°C. Clarified supernatant was passed through a 176 0.22 μm filter and the applied to a 1 mL MabSelect PrismA column. The column was 177 washed with 20 mM sodium phosphate (pH 7.4), 150 mM NaCl. ACE2-Fc fusion proteins 178 were eluted with 100 mM sodium citrate (pH 3.0) and immediately neutralized with 1M 179 Tris (pH 9.0). Protein was then concentrated and filtered before application to a Enrich 180 650 SEC column equilibrated with 10 mM sodium phosphate (pH 7.2), 140 mM NaCl. full diversity was used for CDR3. Asparagine was also omitted from CDR1 and CDR2.

The library was also designed such that there would be 3 different lengths of 190 12-, and 15-amino acids Step 2: 98°C, 10 seconds; 68°C, 10 seconds; 72°C, 15 seconds, 5 cycles;

Step 3: 72°C, 198 10 minutes, 1 cycle; Step 4: 12°C, hold). Amplified DNA was pooled and purified by 199 Monarch Nucleic Acid Purification Kit. The library was digested with 5U/μg DNA SfiI at 200 50°C for 16 hours. Digest reactions were again column purified as previously mentioned.

Ligations were set up as follows. pADL20c (205 μg) previously digested with BglI and 202 treated with rSAP was added to a 40 mL reaction containing the linear digested library 203 (42 μg) at a vector to insert ratio of 1:2, 1 mM ATP and 1,100,000 units of T4 Ligase. The 204 reaction was allowed to proceed at 16°C for 16 hours, then at 37°C for 1 hour, and lastly 205 was heat inactivated at 70°C for 20 minutes. DNA was purified and concentrated by a 206 modified ethanol precipitation protocol utilizing tRNA carrier at 15 μg/mL. supplemented with glycerol to 10%, and stored at -80°C in aliquots for further use.

Diversity of the nanobody library was determined by both NGS and colony PCR.

The V H H library was added to 2xYT-GA to a final OD 600 of 0.08 and allowed to 216 grow at 37°C until an OD 600 of 0.5 was reached. Superinfection with CM13 (ADL) was 217 performed with 2.0 x 10 12 helper phage per liter of E. coli culture for 15 minutes without 218 shaking followed by rigorous shaking for 30 minutes. Cell were collected by centrifugation 219 at 5,500 x g for 10 minutes and resuspended in the previous volume used of 2xYT 220 supplemented with 100 μg/mL carbenicillin and 50 μg/mL kanamycin (2xYT-AK). E. coli 221 were grown overnight with shaking at 28°C.

Supernatant containing packaged phagemid was clarified by centrifugation at 223 6,000 x g for 15 minutes and supplemented with one-fourth volume 20% PEG-8000 and 224 2.5 M NaCl. Phage were precipitated overnight at 4°C and were collected by 225 centrifugation at 5,500 x g for 60 minutes at 4°C. The pellet containing phage was 226 resuspended in PBS and centrifugated for 15 minutes at 5,500 x g to remove E. coli 227 particulates. Clarified phage was again precipitated as mentioned above and incubated 228 on ice for 60 minutes. The phage was finally centrifuged at 17,900 x g for 10 minutes at 229 4°C, the supernatant was removed, and the pellet was again centrifuged at 17,900 x g for were added to plates for 2 hours after washing. Secondary antibody, goat anti-human 299 H+L IgG HRP (Thermo) was added for an additional hour before washing. Plates were 300 developed with TMB Ultra (Thermo) and the reaction stopped by addition of equal volume 301 2 M H 2 SO 4 after washing. Absorbance was read at 450 nm.

Competition of soluble ACE2-huFc by V H H-huFc candidates identified from display 303 screening was performed as follows. SARS-2 antigen was immobilized and blocked as 304 described above. After blocking, serial dilutions of V H H-huFc antibodies were prepared in 305 blocking solution and allowed to incubate for at least 2 hours, after which ACE2-rbFc was 306 added to a final concentration of 0.1 μg/mL (SARS-CoV-1 S) or 0.06 μg/mL (SARS-2 S) 307 and allowed to incubate for 1 hour with shaking at 25°C. Plates were washed and 308 1:10,000 HRP-conjugated goat anti-rabbit IgG (Thermo) was added. Plates were 309 developed as described above. In order to obtain sufficient diversity coverage for the library (i.e. transformants), 441 150 electroporations were performed yielding approximately 3.38 x 10 10 transformants.

To determine the level of success for the ligation of the library into the vector backbone, 443 colony PCR was performed. Of the 408 colonies selected, 395 contained the correct size 444 amplified DNA fragment (95.9%). This value was used to adjust the calculated value for 445 library diversity to 3.24 x 10 10 . Finally, library diversity, quality, and the distribution of 446 CDR3 lengths were assessed by NGS from a total of 39,870,360 reads (S1 and S2 447   Table) . The 9-amino acid CDR3 was the most prevalent at 40%, followed by 12-amino 448 acid CDR3 at 34%, and lastly the 15-amino acid CDR3 at 25% of the observed diversity.

Overall, there was good coverage of all represented CDR3s. Approximately 1% of 450 sequences contained a stop codon and 99% of reads were unique sequences 451 (38,592,027 reads). Roughly 1% of reads were duplicates, and 0.01% (1,095 sequences) 452 were present in triplicate. With these corrections the adjusted diversity for this nanobody 453 library is 3.18 x 10 10 .

Panning against SARS-2 S and RBD 455 Four rounds of biopanning were used to identify clones that bind to the SARS-2 S 456 protein RBD. For the first three rounds, full-length soluble purified SARS-2 S protein was 457 used, ensuring that conformational integrity of the RBD was maintained for initial 458 selection. A 15-minute heat denaturing step at 70°C was used to remove unstable 459 sequences and a final round of biopanning against SARS-2 RBD was conducted to 460 identify therapeutically relevant V H H antibodies (Fig. 1B) . Enrichment of phage against 461 SARS-2 S was observed over the initial 3 rounds of biopanning ( Fig. 1C and S3 Table) 462 and there was a significant loss in phage recovered when the antigen was shifted to RBD 463 (0.0005% compared to 0.004%).

Polyclonal ELISA showed that over each round of biopanning there was 465 enrichment for SARS-2 S binders. Interestingly, there was relatively little SARS-2 RBD 466 binding until panning was conducted against RBD specifically, indicating preferred 467 epitopes are outside the RBD (Fig. 1C) . Additionally, neutralization of SARS-2 468 pseudotyped vesicular stomatitis virus encoding eGFP (VSV-SARS2-GFP)(19) was only 469 observed after RBD biopanning (R4) showing enrichment for RBD binders is required to 470 remove clones binding to epitopes outside the RBD and to identify virus neutralizing 471 clones (Fig. 1D) . Monoclonal characterization was performed using ELISA, 222 of the 472 384 clones were designated as hits. Clones that had OD 450 ≥ 2.5 in the phage coat 473 monoclonal ELISA against both SARS-2 S and RBD and that did not bind BSA were 474 designated as hits (S2 Fig.) . Of those designated as hits, 54 sequences were identified 475 as unique. Those which showed positive binding by ELISA were then tested for their ability to 485 compete with ACE2-huFc for binding to SARS-2 S by a competition ELISA (S4 Table) . 486 Finally, 46 candidates were evaluated for their ability to neutralize VSV-SARS-2-GFP 487 infection in vitro, with 34 V H H-huFc antibodies demonstrating full or partial neutralization 488 of infection (S4 Table) . in viral neutralization with EC 50 s of 0.33, 0.45, and 0.14 nM respectively, against VSV-496 SARS-2-GFP and EC 50 s of 3.14, 1.12, and 0.70 nM respectively, against WT SARS-2 by 497 a plaque neutralization assay (Fig. 2E-F) . The neutralization values were obtained with 498 three independent virus-based assays (S5 Table) , suggesting that SP1B4, SP1D9, and 499 SP3H4 are potent neutralizers of SARS-2, and this observed potency correlates with their 500 strong competition with ACE2 for binding to SARS-2 S protein (Fig. 2D) . The thermal 501 stability of V H H-huFc antibodies was evaluated by differential scanning fluorimetry (DSF).

Melting temperatures for SP1B4, SP1D9, and SP3H4 were 60.4°C, 64.0°C, and 61.0°C 503 respectively and are comparable to previously reported SARS-2 neutralizing V H H-huFc 504 antibodies ( Fig. 2J; S5 Fig.) .(25) 505 Dissociation constants were determined for the V H H-huFc antibodies using 506 biolayer interferometry (BLI) revealing high affinity for the SARS-2 RBD of 39.5 nM and 507 8.9 nM for SP1B4 and SP1D9 respectively (Fig. 2G-H) . SP3H4 exhibited biphasic 508 association and dissociation kinetics which could not be reconciled with a 1:1 global fit 509 analysis and it was therefore not given kinetic parameters (Fig. 2I) . 510 

Epitopes were mapped by sequencing VSV-SARS-2-GFP virus containing escape 512 mutants generated in the presence of SP1B4, SP1D9, or SP3H4, followed by 513 confirmation with BLI binding studies of escape mutant SARS-2 RBDs (Fig. 3) . Within 48-514 60 hours of the first passage, viral escape was apparent from all V H H-huFc antibodies at 515 the highest concentration tested, with all cells expressing GFP. The viral supernatant 516 used for the second passage was completely resistant to neutralization from a 15-fold 517 excess of the corresponding V H H-huFc (Fig. 3C) . Escape mutations were characterized 518 by RNAseq for each of the VSV-SARS-2-GFP supernatants (Fig. 3D) . Interestingly, for 519 SP1B4 and SP1D9, E484K (the mutation found in the variant B.1.351 first isolated in 520 South Africa) seems to be selected against in the second passage and Q493R and S494P 521 seem to stabilize as the predominant mutations observed (Fig. 3D) . This may indicate and WT RBD combined with the 1:1 binding kinetics of SP3H4 for RBD with the L452R 538 mutation may indicate that SP3H4 has multiple binding sites. SP1D9 maintained the 539 ability to bind many of the point mutations generated, though binding was abrogated by 540 Q493R and S494P (Fig. 3D) as expected based on the escape mutant analysis. All 541 mutations generated in RBD abrogated binding by SP1B4. prophylactically dosed mice. Although SP1B4 was a potent neutralizer in vitro it did not 560 demonstrate efficacy against challenge with SARS-2 in vivo ( Fig. 4A and 4B) and was showed 94% survival (n=16), and the SP3H4 group showed 87.5% survival (n=16) at ten 564 days post infection, indicating effective neutralizing activity in vivo (Fig. 4A) . While 81.3% 565 (n=16) of control treated mice lost more than 20% of their starting weight by 6 days post 566 infection (dpi), requiring euthanasia, those pretreated with SP1D9 lost an average of only 567 3.9% (+/-4.8%) of their starting weight ( Fig. 4B and S10 Fig.) . Similarly, the SP3H4 dosed 568 group exhibited a 5.4% average (+/-9.3%) weight loss. Animals dosed prophylactically control. The SP1D9 group showed 58% survival (n=8), and the SP3H4 group showed 580 75% survival (n=8) at ten dpi (Fig. 4C) . Of note, several surviving individuals in each of 581 the post-exposure V H H-huFc-treated groups exhibited 10-15% weight loss up to 5 dpi, 582 but then rebounded from 6-10 dpi, gaining back 90-95% of their starting weight (Fig. 4D   583 and S10 Fig.) . Taken together, these data demonstrate protection from lethal infection 584 after a single 10 mg/kg dose, and further suggest that neutralization of SARS-2 by V H H-585 huFc antibodies in vivo can promote recovery from an ongoing infection. Fig. 4D and S10 Fig.) . In this study, viral escape was readily apparent in the presence of our top 645 candidates, with several mutations in the RBD. Interestingly, many of the initial mutations 646 were selected against in the second passage (Fig. 3) and none of these individual 647 mutations in the RBD significantly impacted binding to ACE2 ( Fig. 3D and S6 Fig.) . This 648 suggests that these positions of the S gene are highly susceptible to single-nucleotide 649 polymorphisms and should be considered when identifying future preclinical antibody 650 candidates as they may be present in future variant strains. Escape mutations observed 651 for our two most promising candidates are all found in proximity to each other likely 652 indicating overlapping epitopes, though they may engage this epitope with different 653 geometries (Fig. 3) 

Medical 701 Branch for generously providing the infectious clone of SARS-CoV-2 expressing a 702 NeonGreen reporter gene. We would also like to thank Robert Meager for critically 703 reviewing the paper. This work was supported by the Laboratory Directed Research

706 consortium of DOE national laboratories focused on response to COVID-19, with 707 funding provided by the Coronavirus CARES Act. Sandia National Laboratories is a 708 multi-mission laboratory managed and operated by National Technology & Engineering 709 Solutions of Sandia, LLC, a wholly owned subsidiary of Honeywell International Inc., for 710 the U.S. Department of Energy's National Nuclear Security Administration under 711 contract DE-NA0003525

Any subjective views or opinions that might be expressed in the paper do not 713 necessarily represent the views of the U.S. Department of Energy or the United States 714 Government. All work performed at Lawrence Livermore National Laboratory is 715 performed under the auspices of the

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Triage of VHH-huFc candidates: A) ELISA with 54 VHH-huFc with SARS-2 S 912 (left), SARS-2 RBD (center), and BSA (right). The data are from experimental conditions 913

Reconfirmation of Purified VHH-huFc candidates: A) ELISA with 16 VHH-huFc 915 antibodies with SARS-2 S. B) Binding of 16 VHH-huFc candidates to SARS-2 RBD. C)

Competition ELISA with the VHH-huFc antibodies. D) Neutralization of VSV-SARS-2-917 GFP infection of Vero cells. MM57 is a control anti-SARS-2 neutralizing monoclonal 918 antibody from Sino Biological. The anti-VSV monoclonal antibody was produced from 919 hybridoma line CRL-2700 (ATCC). E) VHH-huFc antibodies do not

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 697 We would like to thank Prof. Sean Whelan from Washington University School of 698 Medicine St. Louis for graciously providing access to the pseudotyped VSV-SARS-GFP 699 virus used in this study. We would also like to thank Dr. Pei Yong Shi from the World