key: cord-0829160-s19pa1z1 authors: Chi, Xiaojing; Liu, Xiuying; Wang, Conghui; Zhang, Xinhui; Ren, Lili; Jin, Qi; Wang, Jianwei; Yang, Wei title: Humanized Single Domain Antibodies Neutralize SARS-CoV-2 by Targeting Spike Receptor Binding Domain date: 2020-04-15 journal: bioRxiv DOI: 10.1101/2020.04.14.042010 sha: 9b7e97e4d064af4903d2bf5f02f5fc85b220c37e doc_id: 829160 cord_uid: s19pa1z1 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread across more than 200 countries and regions, leading to an unprecedented medical burden and live lost. SARS-CoV-2 specific antivirals or prophylactic vaccines are not available. Neutralizing antibodies provide efficient blockade for viral infection and are a promising category of biological therapies. Using SARS-CoV-2 spike RBD as a bait, we have discovered a panel of humanized single domain antibodies (sdAbs). These sdAbs revealed binding kinetics with the equilibrium dissociation constant (KD) of 0.7~33 nM. The monomeric sdAbs showed half maximal inhibitory concentration (IC50) of 0.003~0.3 μg/mL in pseudotyped particle neutralization assay, and 0.23~0.50 μg/mL in authentic SARS-CoV-2 neutralization assay. Competitive ligand-binding data suggested that the sdAbs either completely blocked or significantly inhibited the association between SARS-CoV-2 RBD and viral entry receptor ACE2. Finally, we showed that fusion of the human IgG1 Fc to sdAbs improved their neutralization activity by tens of times. These results reveal the novel SARS-CoV-2 RBD targeting sdAbs and pave a road for antibody drug development. of 0.003∼0.3 µg/mL in pseudotyped particle neutralization assay, and 0.23∼0.50 30 µg/mL in authentic SARS-CoV-2 neutralization assay. Competitive ligand-binding data 31 suggested that the sdAbs either completely blocked or significantly inhibited the 32 association between SARS-CoV-2 RBD and viral entry receptor ACE2. Finally, we 33 showed that fusion of the human IgG1 Fc to sdAbs improved their neutralization 34 activity by tens of times. These results reveal the novel SARS-CoV-2 RBD targeting 35 sdAbs and pave a road for antibody drug development. 36 Coronavirus disease 2019 (COVID-19) is caused by infection of emerging severe 45 acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2) and had been 46 declared by World Health Organization as the first coronavirus pandemic in human 47 6 completely matched (Fig. 2c) . This phenomenon was normally reported in Middle 129 East Respiratory Syndrome coronavirus (MERS-CoV) neutralizing antibodies and 130 may be likely explained by the difference in sdAb recognized RBD spatial epitope or 131 the steric hindrance formed by antigen-antibody complex 9,10 . 132 Within SARS-CoV-2 RBD, the receptor binding motif (RBM) directly contacts ACE2. 133 Recent report demonstrating that SARS-CoV-2 uses ACE2 as its receptor with a 134 much stronger affinity (10-to 20-fold higher) than SARS-CoV 4 . To determine whether 135 sdAbs targeted different antigenic regions on the SARS-CoV-2 RBD surface, we 136 performed a competition-binding assay using a real-time biosensor (Fig. 3) . We tested 137 all five sdAbs in a competition-binding assay in which human ACE2 was attached to a 138 CM5 biosensor. Compared with a non-related isotype control sdAb (Fig. 3a) , addition 139 of 1E2, 3F11 and 4D8 completely prevent binding of SARS-CoV-2 RBD to ACE2 (Fig. 140 3b, 3d, 3e). Whereas, sdAbs 2F2 and 5F8 could partially compete the RBD/receptor 141 association ( Fig. 3c & 3f) . These data suggested that these sdAbs can be divided into 142 RBM targeting or non-RBM targeting groups though it is not directly associated with 143 virus neutralization activity. 144 SdAbs can be readily fused to human Fc-domain to overcome the limitations of the 145 monovalent sdAbs, such as the short blood residential time and lacking 146 antibody-dependent cell-mediated cytotoxicity and complement dependent 147 cytotoxicity 11 . In addition, bivalent sdAbs can be obtained via the disulfide bond 148 formation in Fc hinge area, which was reported to significantly increase sdAb's 149 activity 12 . To further explore the possibility of sdAb-based antiviral therapeutics and 150 enhance neutralization activity, we constructed human heavy chain antibodies by 151 fusing the human IgG1 Fc region to the C-terminus of sdAbs ( Fig. 4a & 4b) . These Fc KDa in the non-reducing condition, but a 40 KDa monomer was observed by prior 157 treatment in reducing condition to break disulfide bonds. This suggests a correct 158 expression and secretion of heavy chain antibodies in consistence with our design. 159 Neutralization assay results showed that genetic fusion of human Fc significantly 160 increased the neutralization activity of these sdAbs for 10-to 80-fold in molar 161 concentration of IC 50 using the SARS-CoV-2pp entry assay (Fig. 4d) . Importantly, 162 several Fc-fused sdAbs demonstrated potency with IC 80 at sub-nanomolar level (Fig. 163 4d). Finally, we showed that some of the sdAbs are suitable for immunofluorescence 164 staining ( Supplementary Fig. 1) and Western blot to detect ectopically expressed 165 SARS-CoV-2 S protein ( Supplementary Fig. 2) . PelB peptide leader sequence fused with the sdAbs at N-terminus. Massive 235 electroporation was carried out using E. coli TG1 cells. More than a thousand agar 236 petri dishes (140 mm) were plated to ensure enough size of the library. Quality control 237 was carried out by sequencing more than 1000 clones, and the error rate and diversity 238 were calculated. 239 Screening for SARS-CoV-2 RBD targeting antibodies was performed by panning in 241 both immunotubes and native condition using a proprietary full-synthetic library of 242 humanized sdAbs with high-diversity, according to a standard protocol. Briefly, for the 243 2 nd and 4 th panning rounds, the purified SARS-CoV-2 RBD protein was coated on 244 Nunc MaxiSorp immuno tubes (ThermoFisher) at around 5µg/mL in PBS overnight. For binding affinity assays, the SARS-CoV-2 or SARS-CoV S protein was diluted in 281 10mM sodium acetate buffer, pH5.5, and was immobilized on the chip at about 300 282 response units. Antibodies 1E2, 2F2, 3F11, 4D8 and 5F8 at gradient concentrations (0, 283 1.56nM, 3.125nM, 6.25nM, 12.5nM, 25nM) were flowed over the chip surface. After 284 each cycle, the sensor surface was regenerated with10mM glycine-HCl pH 2.5. The 285 data were fitted to a 1:1 interaction steady-state binding model using the 286 BIAevaluation 1.0 software. 287 For competition-binding assays, the ACE2 protein was diluted in 10mM sodium 288 acetate buffer, pH4.5, and was immobilized on the chip at about 650 response units. 289 For the analyses, RBD protein was diluted in HBS-EP buffer or HBS-EP buffer with 290 100nM antibody (1E2, 2F2, 3F11, 4D8 or 5F8). The RBD in different buffer at gradient 291 concentrations (0, 6.25 25, 100, 400nM) was flowed over the chip surface. After each 292 cycle, the sensor surface was regenerated with 10mM glycine-HCl pH 2.5. The 293 binding kinetics was analyzed with the software of BIAevaluation using a 1:1 binding 294 Production of SARS-CoV-2 spike pseudotyped particle (SARS-CoV-2pp) and 296 virus entry assay. 297 To produce SARS-CoV-2pp, HEK293T cells were seeded 1 day prior to transfection at COVID-19: towards controlling of a pandemic Clinical features of patients infected with 2019 novel 368 coronavirus in Wuhan A pneumonia outbreak associated with a new coronavirus of 371 probable bat origin Cryo-EM structure of the 2019-nCoV spike in the prefusion 374 conformation Naturally occurring antibodies devoid of light 376 chains Nanobodies as therapeutics: 378 big opportunities for small antibodies General strategy to humanize a camelid single-domain 381 antibody and identification of a universal humanized nanobody scaffold Production of Pseudotyped Particles to Study Highly 384 Pathogenic Coronaviruses in a Biosafety Level 2 Setting Prophylactic and postexposure efficacy of a potent human 387 monoclonal antibody against MERS coronavirus Exceptionally potent neutralization of Middle East respiratory 390 syndrome coronavirus by human monoclonal antibodies Fusion of hIgG1-Fc to 111In-anti-amyloid single domain 393 antibody fragment VHH-pa2H prolongs blood residential time in APP/PS1 394 mice but does not increase brain uptake Fusion of 397 the mouse IgG1 Fc domain to the VHH fragment (ARP1) enhances protection 398 in a mouse model of rotavirus Chimeric camel/human heavy-chain antibodies protect 401 against MERS-CoV infection A Novel Nanobody Targeting Middle East Respiratory 404 Syndrome Coronavirus (MERS-CoV) Receptor-Binding Domain Has Potent 405 Cross-Neutralizing Activity and Protective Efficacy against MERS-CoV NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies Construction of Synthetic Antibody Phage-Display 411 Caplacizumab as an emerging treatment option 414 for acquired thrombotic thrombocytopenic purpura Caplacizumab treatment for acquired thrombotic 417 thrombocytopenic purpura (HERCULES trial) Clinical pharmacology of caplacizumab for the 420 treatment of patients with acquired thrombotic thrombocytopenic purpura Molecular Mechanism for Antibody-Dependent Enhancement of 427 SWISS-MODEL: homology modelling of protein 429 structures and complexes Identification of a novel coronavirus causing severe 432 pneumonia in human: a descriptive study The authors declare that they have no competing interests.