key: cord-0919805-ic6fuv6i authors: Xiaojie, Shi; Yu, Li; lei, Yan; Guang, Yang; Min, Qiang title: Neutralizing antibodies targeting SARS-CoV-2 spike protein date: 2020-12-15 journal: Stem Cell Res DOI: 10.1016/j.scr.2020.102125 sha: fb8bde1a15e2b9f063d74a0abee504caa7e54b82 doc_id: 919805 cord_uid: ic6fuv6i SARS-CoV-2 causing the worldwide pandemic has changed people’s life in multiple aspects dramatically since it’s first identified in Wuhan, China at the end of 2019. While the numbers of infected patients and death toll keep vigorous increasing, curbing the progression of the pandemic is an urgent goal. Efforts have been made to search for prophylactic and therapeutic approaches including neutralizing antibodies development. By reviewing dozens of studies on anti-spike antibodies identification, we concluded that (1) promising therapeutic antibodies are being fished out by various approaches, such as screening of single B cells of convalescent patients, recombinant antibody library and B cells of immunized animals; (2) the epitopes are mainly RBD, but also some non-RBD domains, without the requisite of overlapping with ACE2 binding sites; (3) Neutralizing antibodies are convergent to a few germline genes, including IGHV3-30, IGHV3-53, IGHV3-66, with varying levels of somatic mutations. This review summarizes the progress in neutralizing antibodies development and the germline enrichment of effective antibodies, which will shed light on COVID-19 treatment and vaccine design. The pandemic outbreak of COVID-19 starting from the end of 2019, have led to a global 28 disaster with over 58 million people confirmed infected and more than 1 million death around the 29 world (https://covid19.who.int/), which does not seem to end in a short time yet. This unprecedented 30 severe pandemic is caused by a beta coronavirus, highly conserved with the virus causing epidemic 31 SARS in 2003, and now is designated as SARS-CoV-2 by WHO. Same as SARS-CoV, SARS-CoV-2 utilizes the receptor angiotensin-converting enzymes 2 33 (ACE2) on host cells as the entry fusion receptor by its viral spike [1, 2] , while there are some 34 different receptors for other coronavirus, like DPP4 for MERS-CoV [3] . The receptor binding 35 domain (RBD) on viral spike is required for the interaction with ACE2 which was well elaborated 36 by delicate structure resolved [4] [5] [6] . The viral spike (S) is a homo-trimer glycoprotein, with 1 37 monomer in "up" conformation for the binding of ACE2 and the other 2 in "down" conformation. Each S protein monomer consists of cleaved S1 subunit and S2 subunit ( Figure 1A ). Entry of the 39 virus into host cell is a finely regulated process, typically consists of three steps: (i) the RBD in S1 40 subunit make direct contact with the host cell receptor, ACE2 [7] ( Figure 1B ). (ii) S1 will be shed to refold and form a post-fusion conformation and drives membrane fusion of the viral and target cell [8] . Although some reports demonstrated cleavage of S protein prior to binding to ACE2 is 45 essential for the infection of SARS-CoV, it seems not necessary for SARS-CoV-2 but still 46 influential to the cell entry efficiency [1, 9, 10] . (iii) Deliver the viral genetic material inside the cell for replication and reproduction of new virus particles [11] . Different approaches that hold back the above three steps could be effective drug development Although few drug specific to SARS-CoV-2 is approved to the market, amount of 63 pharmaceutical studies for COVID-19 exploded in this area due to the global concern of the 64 coronavirus. Vaccine maybe the most promising reagent to be quickly developed to the market to 65 deal with the situation effectively now, which is mainly for the epidemic prevention and control. To get potential therapeutic antibodies, many efforts were made to isolate monoclonal This rapid process made them discover multiple highly potent neutralizing antibodies (pseudovirus The antigen used for screen is S-RBD and a mechanism-301 based screening strategy that contains a well-designed competition ELISA was adopted [54] . Among the selected neutralizing Fabs, the most attracting one is a monovalent Fab clone 5A6 which 303 could bind both "up" or "down" conformations of S protein via different binding sites in the could bind to two RBDs while a second IgG binds to the remaining RBD as well as an RBD of 307 another spike trimer. This property makes each spike trimer accommodate 1.5 full-length IgG 308 thus two spike proteins could be bridged by an IgG molecule. This explains the extraordinarily 309 high avidity and neutralizing capacity of 5A6. Moreover, the authors also showed a method to deal 310 with virus escaping by combination of two mutation-insensitive antibodies, 5A6 and 3D11. for immunization to get an alpaca derived single-domain antibody [57] . The structure of this 324 antibody-RBD epitope complex was resolved by cryo-EM at 2.9 Å resolution which revealed that 325 the nanobody directly blocks the binding sterically. Single-domain antibodies could also be screened from constructed VHH libraries besides The VHH format library from animals could also be "human-derived" by methods similar to 337 antibody humanization. Human germline region could be the framework for grafting sequences to promising results in the trials, detailed in Table 1 below. IGHV germline genes revealed that majority of the antibodies binds to RBD of spike protein 392 (Figure 2) , while some of antibodies were found targeting non-RBD regions in S1 subunit, : IGHV 3-30, IGHV3-53, IGHV 1-2, IGHV1-69 and IGHV3-66, while the IGHV3-30 438 is the most enriched germline, consistent with the report of Christoph. More than one paper 439 mentioned that limited somatic mutation occurs in these neutralization antibodies, indicating 440 the change of a few amino acids (3-4) is enough to endow the neutralization ability, which is 441 also reasonable for the antibody maturation in B cell during the acute process of virus infection. As we described in this mini-review, a fruitful antibody development pipeline is under process Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor A 465 pneumonia outbreak associated with a new coronavirus of probable bat origin Coronavirus Spike Protein and Tropism Changes Structural basis for the recognition of SARS-470 CoV-2 by full-length human ACE2 Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Characterization of the 475 receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD 476 protein as a viral attachment inhibitor and vaccine Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor Structures and mechanisms of viral membrane 481 fusion proteins: multiple variations on a common theme TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in 488 human airway cells Physiological and molecular triggers for SARS-CoV membrane 490 fusion and entry into host cells Fruitful Neutralizing Antibody Pipeline Brings Hope To Defeat SARS-Cov-2 Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly 495 potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to 496 mediate membrane fusion A pan-coronavirus fusion inhibitor targeting the HR1 domain of 499 human coronavirus spike REGN-COV2 antibody cocktail prevents and treats SARS-CoV-2 infection in rhesus 505 macaques and hamsters Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase Pharmacologic Treatments for 511 COVID-19): A Review Antibodies may curb pandemic before vaccines The race for coronavirus vaccines: a graphical guide IgG responses in COVID-19 Developing Vaccines and Therapeutic Antibodies For COVID-19 Convalescent plasma as a potential therapy for COVID-19 Treatment of 5 Critically Ill Patients With COVID-19 With 523 Convalescent Plasma Effectiveness of convalescent plasma therapy in severe COVID-19 patients Broad neutralization of SARS-related 535 viruses by human monoclonal antibodies Potent 537 binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal 538 antibody Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody converting enzyme 2 receptor A human neutralizing antibody targets the receptor-binding site of 551 SARS-CoV-2 A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to 555 its receptor ACE2 Analysis of a SARS-CoV-2-Infected 559 Individual Reveals Development of Potent Neutralizing Antibodies with Limited Somatic Mutation Human neutralizing antibodies elicited by SARS-CoV-2 infection Identification of neutralizing human monoclonal antibodies from Italian Covid-19 convalescent 568 patients, bioRxiv Human-IgG-Neutralizing Monoclonal Antibodies Block the SARS-CoV-2 572 Infection Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of 577 Convalescent Patients' B Cells Rapid isolation and profiling of a diverse panel of human monoclonal antibodies 583 targeting the SARS-CoV-2 spike protein of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model A neutralizing human antibody binds to the N-terminal domain of the Spike protein of 600 SARS-CoV-2 Potent neutralizing 606 antibodies from COVID-19 patients define multiple targets of vulnerability A novel 611 biparatopic hybrid antibody-ACE2 fusion that blocks SARS-CoV-2 infection: implications for therapy Studies in humanized mice and 619 convalescent humans yield a SARS-CoV-2 antibody cocktail Convergent antibody responses to SARS-CoV-2 in convalescent individuals COVID-19: the gendered impacts of the 629 outbreak Near-Germline SARS-CoV-2-Neutralizing Antibodies from COVID-19 Patients SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies Potent SARS-CoV-2 neutralizing 642 antibodies selected from a human antibody library constructed decades ago, bioRxiv A human monoclonal antibody blocking SARS-CoV-2 646 infection Immunoglobulin fragment F(ab')2 against RBD potently neutralizes 649 SARS-CoV-2 in vitro Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with individual 653 antibodies Structural basis for neutralization of SARS-CoV-2 and SARS-CoV by a potent therapeutic 657 antibody Isolation of a human monoclonal antibody specific for the receptor binding domain of SARS-CoV-2 660 using a competitive phage biopanning strategy SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-666 pandemic healthy donors binding at RBD-ACE2 interface, bioRxiv Bivalent binding of a fully human IgG to the SARS-CoV-2 spike 671 proteins reveals mechanisms of potent neutralization, bioRxiv Antibodies Isolated by a site-directed Screening have Potent Protection on SARS-CoV-2 Infection An alpaca nanobody neutralizes SARS-681 CoV-2 by blocking receptor interaction Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block 687 interaction with ACE2 Sybodies targeting the SARS-690 CoV-2 receptor-binding domain, bioRxiv Clinical and 693 immunological assessment of asymptomatic SARS-CoV-2 infections Lack of cross-neutralization by SARS patient sera 696 towards SARS-CoV-2 Inhibition of SARS-CoV-2 Infections in Engineered Human 700 Tissues Using Clinical-Grade Soluble Human ACE2 Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2 Neutralization of SARS-CoV-2 spike 705 pseudotyped virus by recombinant ACE2-Ig A pilot clinical trial of recombinant human angiotensin-converting enzyme 709 2 in acute respiratory distress syndrome Antibody signature induced by SARS-CoV-2 spike protein immunogens in rabbits Molecular Insight into Dengue Virus Pathogenesis and Its 715 Implications for Disease Control CoV-2 vaccines and therapies Rare antibodies from combinatorial libraries suggests an S.O.S. component of the 719 human immunological repertoire Structural basis of a shared 722 antibody response to SARS-CoV-2 Schematic of spike structure of SARS-CoV-2 colored by domain and side 729 views of spike of SARS-CoV-2. SS: signal sequence, S2′: S2′ protease cleavage site, 730 FP: fusion peptide, HR1: heptad repeat 1, CH: central helix, CD: connector domain, 731 HR2: heptad repeat 2, TM, transmembrane domain, CT: cytoplasmic tail. Arrows 732 denote protease cleavage sites. (B) Overall structure of the SARS-CoV-2 RBD bound 733 to ACE2. ACE2 -green Figure 2. Variety of epitopes of antibody CR3022, S309, REGN10987, CC12.1 and 737 S230 on spike RBD Superimposition of RBD with five antibodies belong to different germlines was shown, 739 RBD is displayed in surface representation and antibodies are shown in ribbons. The 740 structure used for superimposition of CR3022 (wheat color 1N343 glycan is shown in sticks). Both REGN10987 (Limon color, PDB 6XDG) and PDB 6NB7) are antibodies of IGHV3-30 germline, and binds to 743 different motif of RBD. Another antibody CC12.1 (Slate color, PDB6XC2) of germline 744 IGHV3-53 binds to similar epitope with that of S230 Figure 3. IGHV germline distribution of SARS-CoV-2 spike-targeting antibodies. 747 (A) Christoph [45] reported the frequencies of VH gene segments of clonal and non-748 clonal sequences from all 12 COVID-19 patients, NGS reference data from 48 healthy 749 individuals Bar and line plots show as mean ± SD. (B) IGHV distribution of 294-RBD targeting 751 antibodies from 12 literatures Writing-original draft preparation. Conceptualization. Li Yu: Information collection Writing-Original draft preparation ☒ The authors declare that they have no known competing financial interests or personal 763 relationships that could have appeared to influence the work