Identification of an anti–SARS–CoV-2 receptor-binding domain–directed human monoclonal antibody from a naïve semisynthetic library

Abstract

There is a desperate need for safe and effective vaccines, therapies, and diagnostics for SARS– coronavirus 2 (CoV-2), the development of which will be aided by the discovery of potent and selective antibodies against relevant viral epitopes. Human phage display technology has revolutionized the process of identifying and optimizing antibodies, providing facile entry points for further applications. Herein, we use this technology to search for antibodies targeting the receptor-binding domain (RBD) of CoV-2. Specifically, we screened a naïve human semisynthetic phage library against RBD, leading to the identification of a high-affinity single-chain fragment variable region (scFv). The scFv was further engineered into two other antibody formats (scFv-Fc and IgG1). All three antibody formats showed high binding specificity to CoV-2 RBD and the spike antigens in different assay systems. Flow cytometry analysis demonstrated specific binding of the IgG1 format to cells expressing membrane-bound CoV-2 spike protein. Docking studies revealed that the scFv recognizes an epitope that partially overlaps with angiotensin-converting enzyme 2 (ACE2)–interacting sites on the CoV-2 RBD. Given its high specificity and affinity, we anticipate that these anti-CoV-2 antibodies will be useful as valuable reagents for accessing the antigenicity of vaccine candidates, as well as developing antibody-based therapeutics and diagnostics for CoV-2. There is a desperate need for safe and effective vaccines, therapies, and diagnostics for SARS– coronavirus 2 (CoV-2), the development of which will be aided by the discovery of potent and selective antibodies against relevant viral epitopes. Human phage display technology has revolutionized the process of identifying and optimizing antibodies, providing facile entry points for further applications. Herein, we use this technology to search for antibodies targeting the receptor-binding domain (RBD) of CoV-2. Specifically, we screened a naïve human semisynthetic phage library against RBD, leading to the identification of a high-affinity single-chain fragment variable region (scFv). The scFv was further engineered into two other antibody formats (scFv-Fc and IgG1). All three antibody formats showed high binding specificity to CoV-2 RBD and the spike antigens in different assay systems. Flow cytometry analysis demonstrated specific binding of the IgG1 format to cells expressing membrane-bound CoV-2 spike protein. Docking studies revealed that the scFv recognizes an epitope that partially overlaps with angiotensin-converting enzyme 2 (ACE2)–interacting sites on the CoV-2 RBD. Given its high specificity and affinity, we anticipate that these anti-CoV-2 antibodies will be useful as valuable reagents for accessing the antigenicity of vaccine candidates, as well as developing antibody-based therapeutics and diagnostics for CoV-2. The recently identified novel human coronavirus, referred to as severe acute respiratory syndrome (SARS)–coronavirus 2 (CoV-2), is the causative agent of the ongoing pandemic of COVID-19. After the report of first case of the CoV-2 infection in December 2019, it has spread all over the globe. To slow down the spread of COVID-19, many countries have introduced lockdown measures. However, these measures will not be enough to eradicate the coronavirus pandemic from the world, and there is an urgent requirement for some medical intervention to control the spread of infection either in the form of a vaccine or other therapeutic options such as small molecule or therapeutic antibodies. So far, no clinically approved therapeutics are available for CoV-2, and only symptomatic treatment is offered (1Felsenstein S. Herbert J.A. McNamara P.S. Hedrich C.M. COVID-19: immunology and treatment options.Clin. Immunol. 2020; 215 (32353634): 10844810.1016/j.clim.2020.108448Crossref PubMed Scopus (407) Google Scholar). One of the approaches is to look for neutralizing antibodies (NAbs) for CoV-2 either from convalescent patient samples or from synthetic antibody library sources. The antibodies can work via two different mechanisms, i.e. by direct neutralization of target viral antigen and also by indirect effector mechanisms such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, wherein antibody binds to infected cells and potentially clears the viral reservoirs (2Hey A. History and practice: antibodies in infectious diseases.Microbiol. Spectr. 2015; 3 (AID-0026-2014, 26104697)Crossref PubMed Scopus (39) Google Scholar, 3Kumar R. Shrivastava T. Samal S. Ahmed S. Parray H.A. Antibody-based therapeutic interventions: possible strategy to counter chikungunya viral infection.Appl. Microbiol. Biotechnol. 2020; 104 (32076776): 3209-322810.1007/s00253-020-10437-xCrossref PubMed Scopus (14) Google Scholar). The spike (S) protein on the surface of CoV2 plays the primary role in viral attachment to the host cell receptor followed by fusion and entry. The S protein comprises two components: S1, which contains the distinct RBD (residues 318–510); and S2, which contains the fusion peptide. The virus gains entry into permissive host cells through interactions of the RBD with the cell surface receptor ACE2. Therefore, the RBD of CoV-2 S protein is the most likely target for development of virus attachment inhibitors, NAbs, and vaccines (4Premkumar L. Segovia-Chumbez B. Jadi R. Martinez D.R. Raut R. Markmann A. Cornaby C. Bartelt L. Weiss S. Park Y. Edwards C.E. Weimer E. Scherer E.M. Rouphael N. Edupuganti S. et al.The receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS–CoV-2 patients.Sci. Immunol. 2020; 5 (32527802): eabc841310.1126/sciimmunol.abc8413Crossref PubMed Scopus (0) Google Scholar). An antibody against RBD is expected to block the attachment of the surface spike of virions to the ACE2 receptors on the host-cell surface and is thereby supposed to neutralize virus entry. Recent success with plasma therapy from convalescent patient samples against CoV-2 is a good indicator of possible successful antibody-based therapy to avoid further fatalities (5Casadevall A. Pirofski L.A. The convalescent sera option for containing COVID-19.J. Clin. Invest. 2020; 130 (32167489): 1545-154810.1172/JCI138003Crossref PubMed Scopus (621) Google Scholar, 6Brown B.L. McCullough J. Treatment for emerging viruses: Convalescent plasma and COVID-19.Transfus. Apher. Sci. 2020; 59 (32345485): 10279010.1016/j.transci.2020.102790Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). However, plasma therapy is a crude method of treatment, and a mAb, the main ingredient of plasma therapy, is needed for wide and safe application (7Burnouf T. Seghatchian J. Ebola virus convalescent blood products: where we are now and where we may need to go.Transfus. Apher. Sci. 2014; 51 (25457751): 120-12510.1016/j.transci.2014.10.003Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 8Marano G. Vaglio S. Pupella S. Facco G. Catalano L. Liumbruno G.M. Grazzini G. Convalescent plasma: new evidence for an old therapeutic tool?.Blood Transfus. 2016; 14 (26674811): 152-157PubMed Google Scholar). Thus, novel tools and reagents for therapy and diagnosis are urgently needed. Keeping this need in mind, we here used a scFv phage display library to identify a novel scFv, II62, against the RBD of CoV-2 that targets an epitope immediately adjacent to and slightly overlapping with the ACE2-binding region on RBD. We further characterized the binding properties of II62–scFv and its two other formats, scFv-Fc and IgG1, against both the RBD and S protein of CoV-2. The human synthetic Tomlinson I single fold phage library was used for screening of scFvs against the purified (>95% pure) RBD protein from mammalian expression system. Three rounds of panning were done, and the titers of input and output phage were calculated at the end of each round to monitor the efficiency of enrichment. After three rounds of selection, ∼1100-fold enrichment of antigen-specific clones was observed (Fig. 1A). After the third round of panning, 50 clones were randomly picked and assessed for binding to RBD protein by monoclonal scFv-phage ELISA (Fig. 1B). Twenty clones of the 50 showed five times better binding to RBD in ELISA as compared with the negative controls (BSA and helper phage) and hence were considered as RBD-specific binders. One clone of scFv, II62, was dominantly selected via panning (50% of the selected binders) and was selected for further characterization (Fig. 1C). The complete nucleotide sequence of the heavy and light chain variable region was determined using the immunoglobulin BLAST homology search. The result indicated that the coding sequences are composed of the VH gene (351 bp) and the VL gene (327 bp). The closest germline sequence for the II62 scFv-antibody gene was identified by comparison with the database. The VH gene use the VH3 family-derived germline V3-23, D2-15, and JH4 genes, whereas the VL gene uses the VL1 (Vκ subgroup I, V1–39) family-derived germline and Jk1. Also, a high degree of mutation appeared in VH complementarity-determining region 2. The II62 mAb sequence showed 70.8% (bases) homology to the closest germline match (Fig. 1D and Fig. S1). The His-tagged II62–scFv was overexpressed in a bacterial system and purified as soluble protein using the Ni–NTA affinity column. Overall yield of the II62–scFv was 3 mg/liter with >90% purity. The purified II62–scFv migrated as a single band on SDS-PAGE with an estimated molecular mass of 28 kDa (Fig. S2D) and same was confirmed by Western blotting using HRP-conjugated anti-His antibody (data not shown). The functional activity and specificity of the purified II62–scFv was assessed by its binding to the RBD protein in ELISA. The II62–scFv showed specific binding to the RBD and did not bind to unrelated proteins like BSA and the envelope proteins from HIV and Chikungunya (Fig. 2, B and C). The scFvs are monovalent in nature and lack the Fc-mediated functions. The Fc domain of human IgG1 was introduced to produce a homodimeric II62–scFv-Fc chimeric protein that is capable of bivalent binding and that retains Fc functions (Fig. 2D). The II62–scFv-Fc construct was transiently expressed in Expi293F cells and subsequently purified by using protein G affinity column, with >95% purity and yields of 40–60 mg/liter. The purified II62–scFv-Fc migrated as a single protein band on SDS–PAGE with an estimated molecular mass of 55 kDa (Fig. S2E). The scFv-Fc showed specific binding to RBD in ELISA (Fig. 2, E and F). The full-length version of the II62–scFv (II62–IgG1) was purified from the supernatant of transiently transfected Expi293F cells using protein A/G affinity columns (Fig. 2G). The purified IgG1 migrated as two protein bands on SDS–PAGE with estimated molecular mass of 50 kDa for heavy chain and 25 kDa for light chain (Fig. S2F). Overall yield of the II62–IgG1 was 80–100 mg/liter with >98% purity. The purified II62–IgG1 also showed specific binding to CoV-2 RBD protein (Fig. 2, H and I). To evaluate the affinity of scFv-Fc and IgG1 format of II62 against the RBD, the antibodies were captured on the anti-human Fc biosensor and RBD was used as analyte in concentrations ranging from 10.0 to 0.6 μm with half-fold serial dilution. The scFv-Fc format showed an affinity of ∼700 nm with both slow on (1.5 × 102 (1/Ms)) and off (1.1 × 10−4 (1/s)) rate, suggesting that the scFv-Fc format binds slowly to the RBD, but once bound it also dissociates slowly (Fig. 3A). The full-length format, II62–IgG1, showed an affinity of ∼160 nm, with an on rate of (kon) of 3.3 × 102 (1/Ms) and off rate (koff) of 5.3 × 10−5 (1/s) (Fig. 3B). We investigated the binding of II62–scFv-Fc and IgG1 with the cell surface–expressed CoV-2 S protein in native conformation. For this, HEK293T cells were transiently transfected with plasmid containing the full-length S protein gene to express the same on the cell surface. The cells were then incubated with II62 scFv or IgG1 and analyzed by flow cytometry. The CoV-2 S proteins expressed on the 293T cell surface were readily detected by II62–scFv-Fc and IgG1. The results suggest that both formats of II62 recognizes the CoV-2 S protein on the cell surface (Fig. 3C and Figs. S3 and S4). Specificity of different antibody formats of II62 (scFv, scFv-Fc, and IgG1) were further individually tested for their specific binding to CoV-2 RBD by immunoprecipitation (IP) followed by Western blotting analysis. All the three antibody formats showed specific binding to RBD protein in co-IP (Fig. 3D). Binding specificity of II62 mAb was also tested toward purified soluble spike protein through Western blotting analysis and ELISA. Antibody II62 specifically recognizes the band of ∼180-kDa (uncleaved soluble spike) (Fig. 3E and Fig. S5). Like other coronaviruses, the S protein of CoV-2 contains cleavage sites, and the precursor S protein is cleaved into the S1 and S2 subunits by host-cell proteases (9Shang J. Wan Y. Luo C. Ye G. Geng Q. Auerbach A. Li F. Cell entry mechanisms of SARS–CoV-2.Proc. Natl. Acad. Sci. U.S.A. 2020; 26 (32376634): 11727-1173410.1073/pnas.2003138117Crossref Scopus (1883) Google Scholar), an important step for initializing of infection. To further assess the effect of the binding of II62–IgG1 to cleaved and uncleaved forms of the S protein, 293T cells were transiently transfected with pseudo virus expressing CoV-2 S protein plasmid. Cell lysates of these transfected cells were immunoblotted with II62–IgG1. Immunoblotting of the cell lysate revealed two bands for the CoV-2 S protein at ∼110–130 kDa, which might be the uncleaved S1 protein (Fig. 3F and Fig. S6). The molecular mass of each monomer of trimeric S protein is ∼180 kDa and contains two subunits: S1 (110 kDa) and S2 (70 kDa) (10Qian Z. Dominguez S.R. Holmes K.V. Role of the spike glycoprotein of human Middle East respiratory syndrome coronavirus (MERS-CoV) in virus entry and syncytia formation.PLoS One. 2013; 8 (24098509): e7646910.1371/journal.pone.0076469Crossref PubMed Scopus (170) Google Scholar). The Western blotting showed another band at the position of 90 kDa reflecting the presence of cleaved S1 protein (Fig. 3F and Fig. S6). The band size above 200 kDa likely shows the presence of dimeric or trimeric S proteins as reported previously (11Ou X. Liu Y. Lei X. Li L. L. R. T. J. Z. Z. X. J. et of spike glycoprotein of SARS–CoV-2 on virus entry and its with 2020; PubMed Scopus Google Scholar). The in interactions have identified using we of RBD with these and of RBD were to (Fig. the of the of with were in and The revealed the and i.e. and that this mAb is the (Fig. and The RBD of and CoV-2 S protein uses ACE2 on the host cell surface as The can block this and virus entry into the host cell R. C. X. Z. Liu J. T. X. C. L. G. X. Y. Z. et human neutralizing antibody targets the receptor binding of 2020; Scopus Google Scholar). display is a that has used for for the discovery of therapeutic against infectious R. R. A. N. A. L. S. novel strategy for of human scFvs against Biotechnol. PubMed Scopus Google Scholar). The of of such is that it the direct use of samples in a pandemic and and R. Parray H.A. Shrivastava T. S. display antibody for of human monoclonal J. PubMed Scopus Google Scholar). of Tomlinson I library against CoV-2 RBD captured II62 as clone that showed specific binding to CoV-2 RBD protein. We further engineered and characterized three different formats of the selected i.e. scFv, scFv-Fc, and IgG1, We the binding of different formats to CoV-2 RBD protein by ELISA and by co-IP followed by Western blotting analysis. The results that all three formats and specifically bind to the CoV-2 RBD protein. we tested these formats bind with cell surface–expressed S protein by flow cytometry analysis of transiently transfected HEK293T cells expressing membrane-bound CoV-2 S protein. suggest that the II62 antibody formats (scFv-Fc and IgG1) bind to a epitope on the full-length S protein as on the cell surface. we determined the binding affinity of these mAb formats using scFv-Fc and IgG1 bind with affinity to CoV-2 RBD. The RBD in the S1 domain of CoV-2 S protein with the ACE2 receptors expressed on the host cell that a of the S2 in of and of fusion into the host is reported that most of the CoV-2 S proteins in are and results suggest that II62 mAb binds to forms of S protein. mAb be potentially used to on CoV-2 S protein and expression and will to identify to of vaccine have previously reported for (10Qian Z. Dominguez S.R. Holmes K.V. Role of the spike glycoprotein of human Middle East respiratory syndrome coronavirus (MERS-CoV) in virus entry and syncytia formation.PLoS One. 2013; 8 (24098509): e7646910.1371/journal.pone.0076469Crossref PubMed Scopus (170) Google Scholar). of sequence and CoV-2, most of the and against and with CoV-2 B. T. C. of B third variable region for of neutralizing antibodies against primary PubMed Scopus Google Scholar). it will be important to this mAb can also with and S the gene sequence of II62 only compared with the closest germline The II62 mAb did not in neutralization in and assay tested to against CoV-2 virus and Docking that the II62–scFv epitope on RBD partially overlaps with the ACE2-binding was further by the assay (Fig. The II62–scFv-Fc antibody showed a of RBD binding to ACE2 from 5 to The other possible is that the epitope is in the form of S protein and only in conformation. et Park S. S. F. A. A. B. R. E. et of SARS–CoV-2 by a human monoclonal 2020; PubMed Scopus Google also reported with and that of CoV-2 S glycoprotein and were to be have recently reported for of to CoV-2 X. So highly epitope in the receptor binding of SARS–CoV-2 and 2020; PubMed Scopus Google Scholar). Human and studies for viral infection and vaccine studies that in may an important role to block cell surface infection in virus entry J. functional a new for HIV Immunol. 2014; PubMed Scopus Google Scholar). The high affinity and specificity of II62 mAb it an target in to the virus with infection. the it has that application of mAb was times effective of neutralizing antibodies in of respiratory virus infection in PubMed Google Scholar). also a to different engineered of a mAb and their for different applications. The of scFv format is that it is of Fc-mediated functions that the infection through S. to and Immunol. PubMed Scopus Google Scholar). Wan et Y. J. S. J. Geng Q. L. Y. J. Z. Y. L. Li F. for antibody-dependent of coronavirus 2020; PubMed Scopus Google that also with anti-CoV-2 To the is to either scFv or antibodies that are of functions S. C. 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Z. of single chain antibody binding to the domain of receptor by phage display Scopus Google Scholar). for this has in the After the third round of were picked and tested for specificity by phage ELISA. phage ELISA was as by et R. R. A. N. A. L. S. novel strategy for of human scFvs against Biotechnol. PubMed Scopus Google and et A. C. T. T. A. N. A. S. of by an of human antibodies from 2016; PubMed Scopus Google and in the from third round of were in containing and and infected with helper phage in The was then incubated at with 50 of the supernatant from each well was used for phage ELISA. His-tagged for mammalian expression were used for of CoV-2 RBD and S protein in Expi293F cells to produce the proteins and purified by Ni–NTA affinity A. J. F. B. of His-tagged 2015; PubMed Scopus Google Scholar). 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