key: cord-0717332-6dqyznp4 authors: Tada, Takuya; Dcosta, Belinda M.; Zhou, Hao; Vaill, Ada; Kazmierski, Wes; Landau, Nathaniel R. title: Decreased neutralization of SARS-CoV-2 global variants by therapeutic anti-spike protein monoclonal antibodies date: 2021-02-19 journal: bioRxiv DOI: 10.1101/2021.02.18.431897 sha: c8fc27e4845be0f3c0baa6bdb6fe06f48bc72dfe doc_id: 717332 cord_uid: 6dqyznp4 Monoclonal antibodies against the SARS-CoV-2 spike protein, notably, those developed by Regeneron Pharmaceuticals and Eli Lilly and Company have proven to provide protection against severe COVID-19. The emergence of SARS-CoV-2 variants with heavily mutated spike proteins raises the concern that the therapy could become less effective if any of the mutations disrupt epitopes engaged by the antibodies. In this study, we tested monoclonal antibodies REGN10933 and REGN10987 that are used in combination, for their ability to neutralize SARS-CoV-2 variants B.1.1.7, B.1.351, mink cluster 5 and COH.20G/677H. We report that REGN10987 maintains most of its neutralization activity against viruses with B.1.1.7, B.1.351 and mink cluster 5 spike proteins but that REGN10933 has lost activity against B.1.351 and mink cluster 5. The failure of REGN10933 to neutralize B.1.351 is caused by the K417N and E484K mutations in the receptor binding domain; the failure to neutralize the mink cluster 5 spike protein is caused by the Y453F mutation. The REGN10933 and REGN10987 combination was 9.1-fold less potent on B.1.351 and 16.2-fold less potent on mink cluster 5, raising concerns of reduced efficacy in the treatment of patients infected with variant viruses. The results suggest that there is a need to develop additional monoclonal antibodies that are not affected by the current spike protein mutations. Monoclonal antibody therapies for the treatment of COVID-19 have been found to reduce virus loads and alleviate symptoms when given shortly after diagnosis 1, 2 . The REGN-COV2 therapy developed by Regeneron Pharmaceuticals is a two recombinant monoclonal antibody cocktail consisting of REGN10933 and REGN10987 3, 4 Lilly therapy is based on a single antibody LY-CoV016 5 . The antibodies bind epitopes within the receptor binding domain (RBD) of the Wuhan-Hu-1 spike protein. The rapid evolution of SARS-CoV-2 variants with mutations in the viral S gene that encodes the spike protein raises concerns that monoclonal antibody therapies could lose effectiveness against viruses for which the spike protein has mutations that alter the amino acid sequences of the epitopes bound by the antibodies. Following the isolation of Wuhan-Hu1 SARS-CoV-2 in December 2019, the virus has continued to further evolve as it adapts to the human host. A variant with a D614G mutation 6 the spike protein which was identified in January, 2020 and by May became the predominant strain world-wide with a prevalence of >97%. The amino acid residue, which is located near the S1:S2 processing site, reduces S1 subunit shedding from virions, has increased infectivity and results in higher virus loads 7-9 . Additional variants containing the D614G mutation with increased transmissibility were subsequently identified. The B.1.1.7 lineage (VOC-202012/01) variant identified in patients in the United Kingdom 10-12 encodes a spike protein with 8 mutations in addition to D614G (Δ69-70, Y144Del, N501Y, A570D, P681H, T716I, S982A and D1118H). N501Y is one of six ACE2 contact residues and has been shown to increase affinity for ACE2 13 The increasing prevalence of highly transmissible variants with mutations in the spike protein RBD raises concerns that the therapy could become less effective should any of the mutations lie within the epitopes targeted by the monoclonal antibodies. To address this question, we tested the neutralizing activity of REGN10933 and REGN10987 on Table. 1). The combination of REGN10933 and REGN10987 was highly potent against D614G with an IC50 of 1.69 ng/ml, and appeared to be slightly synergistic as the neutralizing titer was higher than of each antibody alone. Neutralizing titers for the mixture against B.1.351 and mink cluster 5 were reduced 9.14-and 16.2-fold compared to D614G, respectively, a result that reflects the large decrease in neutralizing titer for REGN10933 on both variants combined with a minor decrease in neutralizing titer by REGN10987 on both variants ( Figure. 2 and Table. 1). Analysis of the single point mutations showed that the reduction in neutralizing titer was caused by both E484K and Y453F mutations. We report here that REGN10933, one of the two monoclonal antibodies that constitutes with 10% fetal bovine serum (FBS) and penicillin/streptomycin (P/S) at 37°C in 5% CO2. ACE2.293T cells are clonal cell-line that expresses high levels of human ACE2 and have been previously described 29, 33 . cDNAs encoding REGN10933 and REGN10987 were synthesized using the published sequences of the antibody variable heavy and light chains fused to IgG1 heavy chain and lambda light chain, respectively and cloned into pcDNA3.1 (Invitrogen). The proteins were produced in transfected Freestyle 293 cells and collected from the cell supernatant after four days. The antibodies were purified by on an AKTA prime FPLC with HiTrap Pro A 5cc column. The proteins were tested for purity by SDS-PAGE, quantified by BCA assay and tested for spike protein binding by Bio-layer Interferometry on an Octet Detection System. SARS-CoV-2 spike protein pseudotyped lentiviral stocks were produced by cotransfection of 293T cells with pMDL, pLenti.GFP-NLuc, pcCoV2.S-D19 (or variants thereof) and pRSV.Rev as previously described 33 . Virus stocks were normalized for reverse transcriptase activity 34 . Pseudotyped virus infections were done with 1 X 10 4 cells/well in 96 well tissue culture dishes at an MOI=0.2 33 . Luciferase activity was measured after 2 days using Nano-Glo luciferase substrate (Promega) and plates were read in an Envision 2103 microplate luminometer (PerkinElmer). To measure antibody neutralization, antibodies were serially diluted 5-fold and then incubated for 30 minutes at room temperature with pseudotyped virus (corresponding to approximately 2.5 X 10 7 cps luciferase) in a volume of 50 µl. The mixture was added to 1 X 10 4 ACE2.293T cells (corresponding to an MOI of 0.2) in a volume of 50 µl in a 96 well culture dish. After 2 days, the medium was removed and Nano-Glo luciferase substrate (Nanolight) was added to wells. Luminescence was read in an Envision 2103 microplate luminometer (PerkinElmer). 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(DA046100, AI122390 and AI120898). T.T. was supported by the Vilcek/Goldfarb Fellowship Endowment Fund. The authors declare no competing interests.