key: cord-0728417-nzf83xwx authors: Wang, Yingdan; Zhang, Xiang; Liu, Jiangyan; Wang, Yanqun; Zhan, Wuqiang; Liu, Mei; Zhang, Meng; Wang, Qimin; Liu, Qianying; Zhu, Tongyu; Wen, Yumei; Chen, Zhenguo; Zhao, Jincun; Wu, Fan; Sun, Lei; Huang, Jinghe title: Combating the SARS-CoV-2 Omicron variant with non-Omicron neutralizing antibodies date: 2022-01-31 journal: bioRxiv DOI: 10.1101/2022.01.30.478305 sha: d49393d9f95ff525974d48af80e99ab5dcb41aed doc_id: 728417 cord_uid: nzf83xwx The highly mutated and transmissible Omicron variant has provoked serious concerns over its decreased sensitivity to the current coronavirus disease 2019 (COVID-19) vaccines and evasion from most anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies (NAbs). In this study, we explored the possibility of combatting the Omicron variant by constructing bispecific antibodies based on non-Omicron NAbs. We engineered ten IgG-like bispecific antibodies with non-Omicron NAbs named GW01, 16L9, 4L12, and REGN10987 by fusing the single-chain variable fragments (scFvs) of two antibodies through a linker and then connecting them to the Fc region of IgG1. Surprisingly, eight out of ten bispecific antibodies showed high binding affinity to the Omicron receptor-binding domain (RBD) and exhibited extreme breadth and potency against pseudotyped SARS-CoV-2 variants of concern (VOCs) including Omicron, as well as authentic Omicron(+R346K) variants. Six bispecific antibodies containing the cross-NAb GW01 neutralized Omicron variant and retained their abilities to neutralize other sarbecoviruses. Bispecific antibodies inhibited Omicron infection by binding to the ACE2 binding site. A cryo-electron microscopy (cryo-EM) structure study of the representative bispecific antibody FD01 in complex with the Omicron spike (S) revealed 5 distinct trimers and one unique bi-trimer conformation. The structure and mapping analyses of 34 Omicron S variant single mutants elucidated that two scFvs of the bispecific antibody synergistically induced the RBD-down conformation into 3-RBD-up conformation, enlarged the interface area, accommodated the S371L mutation, improved the affinity between a single IgG and the Omicron RBD, and hindered ACE2 binding by forming bi-trimer conformation. Our study offers an important foundation for anti-Omicron NAb design. Engineering bispecific antibodies based on non-Omicron NAbs may provide an efficient solution to combat the Omicron variant. We sorted and cultured SARS-CoV-2 S-specific memory B cells from two 82 recovered coronavirus disease 2019 (COVID-19) patients and discovered three 83 anti-SARS-CoV-2 NAbs, designated GW01, 4L12, and 16L9. The germlines 84 and CDR3 of these antibodies are listed in Table S1 . All three antibodies 85 showed strong binding to the RBD of SARS-CoV-2 (Fig. 1A) . However, they (180 kDa, Fig. 2B ). 113 We constructed ten bispecific antibodies and tested their binding abilities to the the tested VOCs and sarbecoviruses (Fig. 2E ). FD01 and GW01-REGN10987 plaque reduction neutralization assays with an authentic Omicron variant containing the R346K mutation, which escapes more SARS-CoV-2 NAbs than 140 the Omicron variant 3 . All five representative bispecific antibodies efficiently 141 neutralized the live Omicron variant (Fig. 2F) To further investigate the neutralization mechanism of the bispecific antibodies, 157 we chose FD01 (GW01-16L9) as a representative antibody for structural study. Local refinement focused on the RBD and ScFvs improved the interface region 159 to 3.51 Å resolution and allowed us to unambiguously build the RBD and scFvs 160 ( structure (bi-trimer, all wide_up RBDs, 12 scFvs, 6.11 Å) (Fig. 3, Fig. S2 protomers of a spike trimer are clockwisely defined as 1, 2, and 3 (Fig. 3, Fig. 183 4 hydrophobic interactions (Fig. 5C ). In addition, the hydrogen bond between S96 215 of CDRL3 and R403 from the RBD and the salt bridge between E52 of CDRL2 216 and R493 from the RBD further enhance the interaction (Fig. 5C ). Coincidentally, residues Y453, A475, Y489, R493, T500, Y501, and H505 of the between V503 and N370, respectively (Fig. 5D) . 231 Interestingly, the simultaneous binding of 16L9 and GW01 with the RBD 232 introduces additional interactions. Hydrogen bonds are formed between N178 233 and N196 of GW01 and S26, Y93, S96 and N98 of 16L9 (Fig. 5B) , which further 234 enhances the interaction between FD01 and S. Structural alignment of the 16L9-GW01-RBD complex with the ACE2-RBD 236 complex indicated that both 16L9 and GW01 were able to compete with ACE2 237 when binding to the RBD (Fig. 5E) , which is consistent to the competition assay. 409 The human primary embryonic kidney cell lines (HEK293T) and 293T-hACE2 All the data processing was carried out using either modules on, or through, To get clear interfaces of RBD with Fabs, we did local-refinement focused on 598 that region. We first selected all good 3D-classes with relatively complete RBD 599 and FD01 density within all states. We auto-refined these particles with a C3-600 aligned reference, but the auto-refinement procedure was not applied any Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron 632 antigenic shift Omicron extensively but incompletely escapes Pfizer BNT162b2 634 neutralization SARS-CoV-2 Omicron Variant Neutralization in Serum from Vaccinated 640 and Convalescent Persons Activity of convalescent and vaccine serum against SARS-CoV-2 mRNA-based COVID-19 vaccine boosters induce neutralizing 644 immunity against SARS-CoV-2 Omicron variant Reduced neutralisation of SARS-CoV-2 omicron B.1.1.529 variant 647 by post-immunisation serum Resistance of SARS-CoV-2 Omicron Variant to Convalescent and 650 CoronaVac Vaccine Plasma The significant immune escape of pseudotyped SARS-CoV-2 variant Omicron-B.1.1.529 leads to widespread escape from neutralizing 655 antibody responses. bioRxiv Structural basis for potent antibody neutralization of SARS-CoV-2 variants 657 including B.1.1.529 An infectious SARS-CoV-2 B.1.1.529 Omicron virus escapes 660 neutralization by therapeutic monoclonal antibodies Structural analysis of receptor binding domain mutations in SARS-CoV-663 2 variants of concern that modulate ACE2 and antibody binding Omicron escapes the majority of existing SARS-CoV-2 neutralizing 666 antibodies Human neutralizing antibodies elicited by SARS-CoV-2 infection Evaluating the Association of Clinical Characteristics With Neutralizing 670 Antibody Levels in Patients Who Have Recovered From Mild COVID-19 in Shanghai Isolation of human monoclonal antibodies from peripheral blood B cells Structure-based design of prefusion-stabilized SARS-CoV-2 spikes Amorphous nickel titanium alloy film: A new choice for cryo electron of specimen movements New tools for automated high-resolution cryo-EM structure 685 determination in RELION-3 A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure 687 determination MotionCor2: anisotropic correction of beam-induced motion for 689 improved cryo-electron microscopy Real-time CTF determination and correction UCSF Chimera--a visualization system for exploratory research and 697 analysis Structure visualization for researchers, educators, 699 and developers DeepEMhancer: a deep learning solution for cryo-EM volume 701 post-processing SWISS-MODEL: homology modelling of protein structures and 703 complexes Real-space refinement in PHENIX for cryo-EM and crystallography Cryo-EM data collection and processing of FD01 bound SARS-719 A) Representative electron micrograph and 2D 720 classification results of FD01 bound SARS-CoV-2 S. (B) The reconstruction 721 map of the complex structures at six states. (C) The local-refined map of the 722 NRF region. (D) Gold-standard Fourier shell correlation curves generated in 723 RELION for structures of six states. The 0.143 cut-off is Data processing flowchart of local refinement of RBD-FD01