key: cord-0271035-5qq6tkey authors: Beaudoin-Bussières, Guillaume; Chen, Yaozong; Ullah, Irfan; Prévost, Jérémie; Tolbert, William D.; Symmes, Kelly; Ding, Shilei; Benlarbi, Mehdi; Gong, Shang Yu; Tauzin, Alexandra; Gasser, Romain; Chatterjee, Debashree; Vézina, Dani; Goyette, Guillaume; Richard, Jonathan; Zhou, Fei; Stamatatos, Leonidas; McGuire, Andrew T.; Charest, Hughes; Roger, Michel; Pozharski, Edwin; Kumar, Priti; Mothes, Walther; Uchil, Pradeep D.; Pazgier, Marzena; Finzi, Andrés title: An anti-SARS-CoV-2 non-neutralizing antibody with Fc-effector function defines a new NTD epitope and delays neuroinvasion and death in K18-hACE2 mice date: 2021-09-08 journal: bioRxiv DOI: 10.1101/2021.09.08.459408 sha: 873f80305cd0716e92bfbd1913d4cf3057d29b5b doc_id: 271035 cord_uid: 5qq6tkey Emerging evidence in animal models indicate that both neutralizing activity and Fc- mediated effector functions of neutralizing antibodies contribute to protection against SARS-CoV-2. It is unclear if antibody effector functions alone could protect against SARS-CoV-2. Here we isolated CV3-13, a non-neutralizing antibody from a convalescent individual with potent Fc-mediated effector functions that targeted the N- terminal domain (NTD) of SARS-CoV-2 Spike. The cryo-EM structure of CV3-13 in complex with SAR-CoV-2 spike revealed that the antibody bound from a distinct angle of approach to a novel NTD epitope that partially overlapped with a frequently mutated NTD supersite in SARS-CoV-2 variants. While CV3-13 did not alter the replication dynamics of SARS-CoV-2 in a K18-hACE2 transgenic mouse model, an Fc-enhanced CV3-13 significantly delayed neuroinvasion and death in prophylactic settings. Thus, we demonstrate that efficient Fc-mediated effector functions can contribute to the in vivo efficacy of anti-SARS-CoV-2 monoclonal antibodies in the absence of neutralization. To test if non-neutralizing antibodies alone can protect from SARS-CoV-2 infection, we 165 characterized a non-neutralizing antibody isolated from the peripheral blood mononuclear 166 cells (PBMCs) of a convalescent individual (CV3) 6 weeks after the onset of symptoms. 167 Using fluorescent SARS-CoV-2 Spike 2P as a probe, we sorted four hundred thirty-two 168 antigen-specific B cells from this donor PBMCs. We successfully generated twenty-169 seven monoclonal antibodies and tested their ability to neutralize pseudoviral particles 170 carrying Spike protein. CV3-13 bound to Spike but did not neutralize SARS-CoV-2 171 pseudovirus (Jennewein et al., 2021) . To determine the epitope recognized by CV3-13, 172 we analyzed its ability to bind different Spike variants on the surface of transfected cells. 173 CV3-13 efficiently bound the WT (Wuhan-Hu-1 reference strain) and the D614G variant 174 but did not recognize the Spike from the B.1.1.7 (alpha) variant ( Figure 1A , B). We took 175 advantage of this differential binding capacity to determine the epitope of CV3-13 by 176 sequentially introducing B.1.1.7 variant mutations into the WT Spike. CV3-13 177 recognized all but the Δ144 mutant that had a single amino acid deletion located in the S1 178 NTD ( Figure 1B ). In agreement with our cell surface binding analyses, surface plasmon 179 resonance (SPR) showed that CV3-13 binds to the SARS-CoV-2 S1 subunit ( Figure 1C ). 180 Monovalent CV3-13 Fab bound to the stabilized Spike trimer ectodomain (Spike-6P) 181 with nanomolar affinity (K D = ~55 nM) ( Figure 1D ). 182 183 184 To confirm that CV3-13 was a non-neutralizing antibody (Jennewein et al., 2021) , we 187 tested its capacity to neutralize pseudoviruses carrying the SARS-CoV-2 Spike. For 188 comparison purposes, we used CV3-1, a potent RBD-targeting NAb, as a positive control 189 (Jennewein et al., 2021) . CV3-1 was recently shown to protect K18-hACE2 mice from a 190 lethal SARS-CoV-2 challenge in a Fc-effector function-dependent manner (Ullah et al., 191 2021) . Our analyses showed that CV3-13 was unable to neutralize pseudoviral particles 192 the spike as a whole) are calculated ( Figure 4B ). The supersite-binding NAbs approach 299 the NTD with a similar angle (in a range of 6 o -15 o ), substantially different from that of 300 CV3-13 with a calculated angle ~30 o ( Figure 4B ). In contrast, the infectivity-enhancing 301 antibodies use the angle of approach in the range of 45 o -60 o . Also, the fine epitope 302 specificity of CV3-13 is different with contact regions only partially overlapping with 303 neutralization supersite ( Figure 4C ). To summarize, CV3-13 uses the binding angle that 304 positions it somewhere between the binding angles of antibodies recognizing the 305 neutralization supersite, that bind from the top of the spike and antibodies targeting the 306 infectivity-enhancing site, that bind at the bottom of spike, closer to the viral membrane. 307 These features are likely why CV3-13 lacks direct neutralizing activity but has no 308 infectivity-enhancing properties (Figure 2A, B) . The CV3-13 binding mode permits 309 effective engagement of innate immune cells to mediate Fc-effector activity. Indeed, it 310 has been shown that antibodies targeting the NTD supersite have largely overlapping 311 epitope footprints (all engaging the N1, N3 and N5 NTD loops) and a narrow range in 312 their angle of approach that could allow them to sterically disrupt spike-receptor 313 interactions, TMPRSS2-dependent activation and/or viral-host membrane fusion. 314 315 Both CV3-13 and the supersite specific Abs bind to the NTD utilizing highly mobile and 316 conformationally unconstrained NTD loops by an induced-fit mechanism. To assess the 317 impact of antibody recognition on the conformation of NTD, we superimposed the NTD 318 domain from the ligand-free HexaPro Spike (PDB:6XKL) and eleven NTD-directed 319 antibody-spike/NTD complexes and examined mAb-induced structural rearrangements. 320 As described earlier by Cerutti and colleagues (Cerutti et al., 2021) , the highly mobile N1-N5 loops ( Figure 4D ), which are largely disordered in the ligand-free spike, have 322 diverse conformations in response to the bound Fabs at different sites ( Figure 4E ). The 323 degree of local flexibility for the NTD loops is inversely correlated with their 324 contribution to the Fab-NTD interface. These structural changes are a direct consequence 325 of antibody binding, either by a conformational sampling or an induced-fit mechanism. 326 Overall, NTD-directed antibodies induced substantial structural rearrangement of the 327 NTD. The high immunogenicity and high mutational frequency observed in the flexible 328 NTD loops likely results from their increased accessibility on the Spike and their 329 subsequent encounters with both neutralizing and non-neutralizing antibodies. 330 331 CV3-13 does not protect K18-hACE2 mice from a SARS-CoV-2 lethal challenge 332 We next investigated the in vivo efficacy of the non-neutralizing antibody CV3-13 under 333 a prophylactic or therapeutic setting to protect or treat K18-hACE2 transgenic mice from 334 a lethal SARS-CoV-2 infection. CV3-13 was delivered intraperitoneally (ip, 12.5 mg 335 IgG/kg body weight) 24 h before (prophylactic) or one and two days post infection (dpi, 336 therapeutic) in K18-hACE2 mice intranasally challenged with SARS-CoV-2 nLuc, as 337 previously described ( Figure S2 and S3) (Ullah et al., 2021) . Longitudinal non-invasive 338 bioluminescence imaging (BLI), body weight change, survival, viral load estimation in 339 brain, lung and nasal cavity and terminal imaging after necropsy showed no difference 340 between isotype-and CV3-13-treated mice under either prophylactic or therapeutic 341 settings ( Figure S2 and S3, panels A-J). Furthermore, mRNA levels of inflammatory 342 cytokines (IL6, CCL2, CXCL10 and IFNG) in brain and lungs were also similar to 343 isotype-treated cohorts ( Figure S2 and S3, panels K, L). These data suggest that natural Figure 5A ). Non-invasive imaging analyses revealed that 361 CV3-13 GASDALIE-pre-treated mice showed reduced viral spread and delayed 362 neuroinvasion compared to isotype-treated controls ( Figure 5B , D, E). Accordingly, 363 CV3-13 GASDALIE-treated mice displayed a decelerated body-weight loss phenotype 364 and a significant delay in mortality ( Figure 5F , G). Imaging animals at the experimental 365 endpoint after necropsy revealed that systemic virus spread as measured by flux as well 366 as viral loads in the nose, brain and lungs were significantly reduced in CV3-13 GASDALIE-treated compared to isotype-treated mice ( Figure 5B , C, H-J). While the 368 overall inflammatory cytokine mRNA expression profile in the brain and lungs were not 369 different, IFN-gamma mRNA levels were significantly reduced in the brain tissues of 370 CV3-13 GASDALIE-treated mice as compared to controls. 371 To confirm the ability of CV3-13 GASDALIE to reduce virus spread and delay 373 neuroinvasion, we terminated the experiment at 4 dpi ( Figure 6A ). Our BLI analyses 374 confirmed a significant decrease in nLuc signals in the brain both non-invasively and 375 after necropsy ( Figure 6B Figure S6A ). This is in line with the interpretation that NTD 421 mutations from the emerging variants may be a result of NTD-directed antibody 422 selection, suggesting that non-neutralizing NTD antibodies like CV3-13 influence virus 423 evolution through the Fc-mediated effector functions. 424 425 CV3-13 did not neutralize pseudoviral particles or authentic SARS-CoV-2 viruses but did 426 mediate Fc effector functions against Spike-expressing cells. We suggest that differences 427 in fine epitope specificity and the angle of approach used by CV3-13 as compared to 428 neutralizing NTD-specific mAbs limit its ability to sterically hinder Spike-co-429 receptor/auxiliary receptor interactions, the prefusion-to-postfusion transition of Spike 430 and/or membrane fusion as has been suggested as a neutralization mechanism for other 431 NTD binding antibodies. 432 Our data demonstrates that an antibody devoid of neutralizing activity is able to reduce 433 virus dissemination and delay death in mice from lethal SARS-CoV-2 challenge via its 434 Fc-mediated effector functions. While wild-type CV3-13 IgG1 did not provide any 435 protection, CV3-13 GASDALIE mutant delayed death in prophylactically treated mice. 436 These data suggest that a threshold of Fc-mediated effector function activity was required 437 to impede virus progression. For all in vivo experiments, the 6 to 8 weeks male and female hACE2-K18 mice were 828 intranasally challenged with 1 x 10 5 PFU in 25-30 µl volume under anesthesia (0.5 -5 % 829 isoflurane delivered using precision Dräger vaporizer with oxygen flow rate of 1 L/min). 830 For mAb treatment using prophylaxis regimen, mice were treated with 250 µg (12.5 µg/g 831 body weight) of indicated antibodies (CV3-13 WT or CV3-13 GASDALIE) via 832 intraperitoneal injection (i.p.) 24 h prior to infection. For mAb treatment under 833 therapeutic regimen, mice were treated at 1 and 2 dpi intraperitoneally with CV3-13 (12.5 834 µg/g body weight). Body weight was measured and recorded daily. The starting body 835 weight was set to 100 %. For survival experiments, mice were monitored every 6-12 h 836 starting six days after virus administration. Lethargic and moribund mice or mice that had 837 lost more than 20 % of their body weight were sacrificed and considered to have 838 succumbed to infection for Kaplan-Meier survival plots. 839 COVID-19 Severity Is Cao Potent Neutralizing Antibodies against SARS-CoV-2 Identified 1179 by High-Throughput Single-Cell Sequencing of Convalescent Patients' B Cells Potent SARS-CoV-2 neutralizing antibodies directed 1183 against spike N-terminal domain target a single supersite MolProbity: all-atom 1187 structure validation for macromolecular crystallography Human monoclonal antibodies block the binding of SARS-CoV-2 spike 1191 protein to angiotensin converting enzyme 2 receptor A neutralizing human antibody binds to the N-terminal domain of 1194 the Spike protein of SARS-CoV-2 Differential Fc-Receptor Engagement Drives an 1196 Anti-tumor Vaccinal Effect Structure, Function, and Antigenicity: Implications 1199 for the Design of Spike-Based Vaccine Immunogens SAbPred: a structure-based antibody prediction 1202 server High-level and high-throughput 1204 recombinant protein production by transient transfection of suspension-growing human 1205 293-EBNA1 cells Efficacy of ChAdOx1 nCoV-19 1208 (AZD1222) vaccine against SARS-CoV-2 variant of concern 202012/01 (B.1.1.7): an 1209 exploratory analysis of a randomised controlled trial Coot: model-building tools for molecular graphics Features and development 1213 of Coot Major role of IgM in the neutralizing 1216 activity of convalescent plasma against SARS-CoV-2 Visualizing density maps with 1218 UCSF Chimera UCSF ChimeraX: Meeting modern challenges in visualization and 1221 analysis SARS-CoV-2 1224 variants, spike mutations and immune escape SARS-CoV-2 Cell 1227 Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease 1228 Inhibitor Structure-based design of 1231 prefusion-stabilized SARS-CoV-2 spikes Clinical features of patients infected with 2019 novel coronavirus in 1234 Wuhan Structural basis for potent 1237 neutralization of SARS-CoV-2 and role of antibody affinity maturation A Study of Ad26.COV2.S for the Prevention of 1240 SARS-CoV-2-Mediated COVID-19 in Adult Participants Isolation and 1243 characterization of cross-neutralizing coronavirus antibodies from COVID-19+ subjects Human neutralizing antibodies elicited by SARS-CoV-2 infection Inference of macromolecular assemblies from 1249 crystalline state A novel coronavirus associated with 1252 severe acute respiratory syndrome Structure of the SARS-CoV-2 spike receptor-binding domain bound to 1255 the ACE2 receptor Engineered antibody Fc variants with enhanced 1258 effector function CoV-2 infection and influence the neutralizing activity of different classes of antibodies vitro and in vivo functions of SARS-1265 CoV-2 infection-enhancing and neutralizing antibodies Structural Basis and Mode of Action for Two 1268 Broadly Neutralizing Antibodies Against SARS-CoV-2 Emerging Variants of Concern Macromolecular structure 1272 determination using X-rays, neutrons and electrons: recent developments in Phenix Potent neutralizing antibodies against multiple epitopes on 1276 SARS-CoV-2 spike An infectivity-enhancing site on the 1279 SARS-CoV-2 spike protein targeted by antibodies Clinical and immunological assessment of asymptomatic 1282 SARS-CoV-2 infections Real-Time Conformational Dynamics of SARS-CoV-2 1285 Spikes on Virus Particles Serological Assays 1288 Estimate Highly Variable SARS-CoV-2 Neutralizing Antibody Activity in Recovered 1289 COVID-19 Patients N-terminal domain antigenic 1292 mapping reveals a site of vulnerability for SARS-CoV-2 Recurrent deletions in the SARS-CoV-2 spike 1295 glycoprotein drive antibody escape A Study to Evaluate Efficacy, Safety, and Immunogenicity of 1298 mRNA-1273 Vaccine in Adults Aged 18 Years and Older to Prevent Longitudinal Serological 1301 Analysis and Neutralizing Antibody Levels in Coronavirus Disease 2019 Convalescent 1302 Patients The 1305 Advisory Committee on Immunization Practices' Interim Recommendation for Moderna COVID-19 Vaccine -United States The Advisory 1310 Committee on Immunization Practices' Interim Recommendation for Use of Janssen 1311 COVID-19 Vaccine -United States SARS-CoV-2 1315 Infections and Serologic UCSF ChimeraX: Structure visualization for 1319 researchers, educators, and developers Pfizer and BioNTech Confirm High Efficacy and No Serious 1321 Safety Concerns Through Up to Six Months Following Second Dose in Updated Topline 1322 Analysis of Landmark COVID-19 Vaccine Study Mapping 1325 Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding 1326 Domain by Structure-Guided High-Resolution Serology Safety and Efficacy of the 1329 BNT162b2 mRNA Covid-19 Vaccine Cross-Sectional 1332 Evaluation of Humoral Responses against SARS-CoV-2 Spike cryoSPARC: 1337 algorithms for rapid unsupervised cryo-EM structure determination Broad and potent 1341 activity against SARS-like viruses by an engineered human monoclonal antibody Optimization of antibody binding to FcgammaRIIa enhances macrophage phagocytosis 1345 of tumor cells Convergent antibody responses 1348 to SARS-CoV-2 in convalescent individuals Alignment of cryo-EM movies of 1350 individual particles by optimization of image translations Conceptual Approaches to Modulating Antibody Effector 1352 Functions and Circulation Half-Life Antibody 1355 potency, effector function, and combinations in protection and therapy for SARS-CoV-2 1356 infection in vivo Prevention of overfitting in cryo-EM structure 1358 determination Analysis of a SARS-CoV-2-1361 Infected Individual Reveals Development of Potent Neutralizing Antibodies with Limited 1362 Somatic Mutation Cell entry 1364 mechanisms of SARS-CoV-2 Safety and Efficacy of the BNT162b2 1366 mRNA Covid-19 Vaccine Mouse model 1368 recapitulating human Fcgamma receptor structural and functional diversity The N-terminal domain of spike glycoprotein mediates 1372 SARS-CoV-2 infection by associating with L-SIGN and DC-SIGN Humoral 1376 and cellular immune responses against SARS-CoV-2 variants and human coronaviruses 1377 after single BNT162b2 vaccination Structure-based development of three-and four-antibody 1380 cocktails against SARS-CoV-2 via multiple mechanisms Neutralizing and 1383 protective human monoclonal antibodies recognizing the N-terminal domain of the 1384 SARS-CoV-2 spike protein A single dose of the 1387 SARS-CoV-2 vaccine BNT162b2 elicits Fc-mediated antibody effector functions and T 1388 cell responses Human 1391 monoclonal antibody combination against SARS coronavirus: synergy and coverage of 1392 escape mutants Potent binding of 2019 novel coronavirus spike protein by a SARS 1395 coronavirus-specific human monoclonal antibody Ultrapotent human 1398 antibodies protect against SARS-CoV-2 challenge via multiple mechanisms Live Imaging CoV-2 Infection in Mice Reveals Neutralizing Antibodies Require Fc Function for 1403 Optimal Efficacy Prevalent, protective, and 1406 convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes Safety and efficacy 1410 of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis 1411 of four randomised controlled trials in Brazil, South Africa, and the UK Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein A human 1418 monoclonal antibody blocking SARS-CoV-2 infection CoV-2 require intact Fc effector functions for optimal therapeutic protection COVID-19 CORONAVIRUS PANDEMIC Structural 1426 Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Cryo-EM structure of the 2019-nCoV spike in the 1430 prefusion conformation Evaluating the Association of Clinical Characteristics With Neutralizing Antibody Levels in Patients Who Have Recovered From Mild COVID-19 in Shanghai, 1434 China A new coronavirus associated with human respiratory 1437 disease in China A noncompeting pair of human neutralizing antibodies block COVID-19 1440 virus binding to its receptor ACE2 An Infectious cDNA Clone of 1443 SARS-CoV-2 CoV-2 for rapid neutralization testing and screening of anti-infective drugs for COVID-1447 19 A highly conserved cryptic epitope in the receptor binding domains of 1450 SARS-CoV-2 and SARS-CoV Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia A Novel Coronavirus from Patients with Pneumonia in China New tools for automated high-resolution cryo-EM structure 1459 Compromised Humoral Functional Evolution 1462 Tracks with SARS-CoV-2 Mortality The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger All standard operating procedures and protocols for IVIS imaging of SARS-CoV-2 842infected animals under ABSL-3 conditions were approved by IACUC, IBSCYU and 843 YARC. All the imaging was carried out using IVIS Spectrum® (PerkinElmer) in XIC-3 844 animal isolation chamber (PerkinElmer) that provided biological isolation of anesthetized 845 mice or individual organs during the imaging procedure. All mice were anesthetized via 846 isoflurane inhalation (3 -5 % isoflurane, oxygen flow rate of 1.5 L/min) prior and during 847 BLI using the XGI-8 Gas Anesthesia System. Prior to imaging, 100 µL of nanoluciferase 848 substrate, furimazine (NanoGlo TM , Promega, Madison, WI) diluted 1:40 in endotoxin-849 free PBS was retro-orbitally administered to mice under anesthesia. The mice were then 850 placed into XIC-3 animal isolation chamber (PerkinElmer) pre-saturated with isothesia 851 and oxygen mix. The mice were imaged in both dorsal and ventral position at indicated 852 days post infection. The animals were then imaged again after euthanasia and necropsy 853 by spreading additional 200 µL of substrate on to exposed intact organs. Infected areas of 854 interest identified by carrying out whole-body imaging after necropsy were isolated, 855 washed in PBS to remove residual blood and placed onto a clear plastic plate. Additional 856 droplets of furimazine in PBS (1:40) were added to organs and soaked in substrate for 1-2 857 min before BLI. 858Images were acquired and analyzed with the manufacturer's Living Image v4.7.3 859 in vivo software package. Image acquisition exposures were set to auto, with imaging 860 parameter preferences set in order of exposure time, binning, and f/stop, respectively. 861Images were acquired with luminescent f/stop of 2, photographic f/stop of 8. Binning was 862 set to medium. Comparative images were compiled and batch-processed using the image 863 browser with collective luminescent scales. Photon flux was measured as luminescent 864 radiance (p/sec/cm 2 /sr). During luminescent threshold selection for image display, 865 luminescent signals were regarded as background when minimum threshold levels 866 resulted in displayed radiance above non-tissue-containing or known uninfected regions. 867To determine the pattern of virus spread, the image sequences were acquired every day 868 following administration of SARS-CoV-2 (i.n). Image sequences were assembled and 869 converted to videos using Image J. 870 SARS-CoV RBD-SD1(residue 306-577) using polyethylenimine (PEI). One-week post-894 transfection, supernatants were clarified and filtered using a 0.22 µm filter (Thermo 895Fisher Scientific). The crude S-6P was purified on strep-tactin resin (IBA) followed by 896 size-exclusion chromatography on Superose 6 10/300 column (GE Healthcare) in 10 mM 897Tris pH 8.0 and 200 mM NaCl (SEC buffer). RBD was purified on a Ni-NTA column 898 (Invitrogen) followed by size-exclusion chromatography on a Hiload 16/600 Superdex 899 200pg column using the same SEC buffer. For CryoEM protein sample preparation, the 900 expression plasmid encoding S-6P was transfected into 293F GnT1-cells using PEI. The 901 protein was harvested and purified with the same protocol as above. The C-terminal twin-902Strep-Tag and 8xHis tag on S-6P was removed by HRV3C (Sigma) digestion as 903 described in (Wrapp et al., 2020b) and the cleaved protein was further purified on a Ni-904 NTA column followed by gel filtration on a Superose 6 10/300 in SEC buffer. Purified 905 proteins were snap-frozen in liquid nitrogen and stored in aliquots at -80°C until further 906 use. Protein purity was confirmed by SDS-PAGE. 907The expression plasmids encoding the heavy and light chains of CV3-13 IgG were 908 transiently transfected into Expi293F cells (Thermo Fisher) using ExpiFectamine 293 909 transfection reagent as per the manufacturer's protocol (Thermo Fisher). After 6-days 910 post-transfection, the antibody was purified on Protein A resin from cell supernatant 911 (Thermo Fisher) before the overnight papain digestion at 37°C using immobilized papain 912 agarose (Thermo Fisher). The resulting Fab was separated from Fc and undigested IgG 913 by passage over protein A resin. Fab was further purified by gel filtration using a 914 Superose 6 10/300 column before use in SPR binding or cryo-EM sample preparation. and CV3-13 GASDALIE monoclonal antibodies (0.0025 µg/mL, 0.01 µg/mL, 0.05 1063 µg/mL, 0.25 µg/mL, 1 µg/mL and 5 µg/mL) were added to the appropriate wells. The 1064plates were subsequently centrifuged for 1 min at 300 x g, and incubated at 37°C, 5% 1065 CO 2 for 5 hours before being fixed in a 2% PBS-formaldehyde solution. Since 1066 CEM.NKr-Spike cells express GFP, ADCC activity was calculated using the formula: 1067 Targets processing workflow is shown in Figure S4 and statistics of data collection, 1120 reconstruction and refinement is described in Table S1 . The epitope interface analysis 1121 was performed in PISA (Krissinel and Henrick, 2007) .