Protein antigen-binding molecules

Abstract

The present disclosure provides antigen-binding molecules capable of binding to sarbecovirus spike proteins from two or more different sarbecoviruses. Nucleic acids, expression vectors, and cells for producing and using the same. In particular, antigen-binding molecules such as neutralizing antibodies that inhibit the interaction between sarbecovirus spike proteins and ACE2 and thus can act as antagonists of infection of ACE2-expressing cells by sarbecoviruses. The antigen-binding molecules described herein are provided with a combination of advantageous properties over known SARS-CoV-2 antibodies.

Description

[Technical Field]

[0001] Reference to related applications

[0001] This application claims priority to Singapore Patent Application No. 10202105095U filed on 15 May 2021, Singapore Patent Application No. 10202107013P filed on 25 June 2021, and Singapore Patent Application No. 10202204610V filed on 28 April 2022, the contents of which are incorporated herein by reference.

[0002]

[0002] This disclosure relates in general to molecules such as protein antigen-binding molecules suitable for use in the treatment or prevention of coronavirus infections, particularly SARS-related beta-coronavirus (Salvecovirus). [Background technology]

[0003]

[0003] The following discussion relating to the background art of the present invention is intended solely to facilitate understanding of the present invention. It should be understood that this discussion does not acknowledge or admit that any of the materials referred to were publicly disclosed, publicly known, or part of the ordinary general knowledge of those skilled in the art at the priority date of the present invention, in any scope of rights.

[0004]

[0004] Emerging zoonotic viruses have posed a significant threat to public health and the global economy over the past 20 years, as the world has experienced three major human infectious disease pandemics caused by coronaviruses (CoV). The three major human infectious disease pandemics caused by coronaviruses (CoV) are SARS of 2002-2003, caused by SARS-CoV (Peiris et al., Nat Med 2004, 10:S88-97), MERS since 2012 (Zaki et al., N Engl J Med 2012, 367:1814-1820), and the ongoing COVID-19 caused by SARS-CoV-2 (Wang et al., Lancet 2020, 395:470-473). All of these have caused devastating economic and human losses worldwide. Regarding SARS and MERS, we still have no authorized treatment or preventive measures against future infections caused by these viruses. Regarding COVID-19, vaccine development has progressed at an unprecedented pace, resulting in many approved vaccines for human use (Fauci Science 2021, 372:109).

[0005]

[0005] Numerous CoVs exist in wild pathogen-carrying animals, particularly bats. It is highly likely that outbreaks (SARS3, SARS4, etc.) will be caused in the future by different but related CoVs. The current classification of CoVs is shown in Figure 1. Four genera (alpha, beta, delta, and gamma) have been identified, and all viruses with the highest risk of infecting humans belong to the betacoronavirus genus (Nat Microbiol 2020, 5:536~544), especially the subgenus Salvecovirus. Salvecoviruses are the most transmissible coronaviruses in humans, probably because they all use the human angiotensinogen-converting enzyme 2 (ACE2) receptor as an entry point into human cells. There are hundreds of strains of SARS-related coronaviruses (SARSr-CoV) that are known to infect only non-human species, and bats are the main pathogens of many SARS-related coronavirus strains. There are two main clades of Salvecovirus. Clade 1a contains SARS-CoV-related CoVs (SC2r-CoV), and clade 1b contains SARS-CoV-2-related CoVs (SC2r-CoV). Examples of salvecoviruses include SARS-CoV, SC1r-CoV WIV-1, SC1r-CoV RsSHC014, SARS-CoV-2, SARS-CoV-2 B.1.1.7 (alpha), SARS-CoV-2 B.1.351 (beta), SARS-CoV-2 B1.617.2 (delta), SC2r-CoV GD-1, GX-P5L, and SC2r-CoV RaTG13. Several studies have shown that cross-neutralization between SARS-CoV and SARS-CoV-2 is limited (Yang R et al., EBioMedicine 2020, 58:102890).

[0006]

[0006] Pandemic preparedness and response require a multifaceted approach to combating emerging zoonotic viruses, including vaccines, therapeutic monoclonal antibodies, and small molecule drugs. In the case of SARS-CoV-2, the first commercially available countermeasure to receive FDA Emergency Use Authorization (EUA) was therapeutic monoclonal antibody (mAb) derived from COVID-19 patients. Both antibody and T-cell immunity are important for controlling viral infections such as SARS-CoV-2 or other salvecovirus infections. Neutralizing antibodies (Nab) are more important for preventing viral entry, and thus initial infection, while T-cell immunity can later resist infection and suppress or control disease progression. Nab can be induced either through infection or vaccination. Passive immunization using therapeutic mAbs remains an important part of pandemic response and containment, as therapeutic mAbs can play a crucial role, particularly in treating severely ill patients (e.g., immunocompromised patients) in vulnerable populations or in preventing subsequent transmission by isolating targeted high-risk populations. Unfortunately, first-generation mAbs for treating COVID-19 have proven to be less effective or ineffective against newly emerging, potentially harmful variants (VOCs).

[0007]

[0007] The recent emergence of SARS-CoV-2 variants, namely the alpha-COVID-19 variant SARS-CoV-2 B.1.1.7; the beta-COVID-19 variant SARS-CoV-2 B.1.351, also known as the 20H / 501Y.V2 or 501Y.V2 variant; the gamma variant P.1; the delta-SARS-CoV-2 B.1.617.2; and the omicron variant SARS-CoV-2 B.1.1.529 BA.1 and BA.2, along with the observed decline in immune protection against new variants obtained from vaccines based on the original virus strain, has raised new challenges regarding the need for broad-spectrum treatment or prevention against all known and future SARS-CoV-2 variants. Other coronaviruses are known to circulate in wild carriers, such as SC2r-CoV GD-1 and SC2r-CoV GX-P5L in pangolins, and SC2r-CoV RaTG13, SC1r-CoV WIV-1, and SC1r-CoV RsSHC014 in bats, which could potentially trigger outbreaks (SARS3, SARS4, etc.) in the future with different but related coronaviruses.

[0008]

[0008] Most SARS-CoV-2 vaccines currently approved for use in humans were developed against the S protein of the ancestral strain first identified in Wuhan. The emergence and dominance of VOCs pose significant threats and challenges, as some of them, particularly VOC omicrons, have evolved to evade immunity, primarily by nacrofighting NAbs, in individuals who have been infected or vaccinated regardless of vaccine type, and even in individuals who have received booster vaccinations or hybrid immunity derived from infection and vaccination.

[0009]

[0009] It is necessary to develop molecules for use in the treatment or prevention of human infections caused by salvecovirus and to mitigate at least one of the above-mentioned problems. [Overview of the Initiative]

[0010]

[0010] Protein antigen-binding molecules such as monoclonal antibodies, nucleic acids, expression vectors, cells, or compositions suitable for broad-spectrum pan-salvecovirus are envisioned for use in the treatment or prevention of coronavirus infections caused by salvecovirus.

[0011]

[0011] Accordingly, one aspect of the present invention is an antigen-binding molecule that binds to salvecovirus spike proteins derived from two or more different salvecoviruses, (i) The following CDRs: HC-CDR1 having amino acids with at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 111 HC-CDR2 having amino acids with at least 85% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 112 HC-CDR3 having amino acids with at least 85% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 113 A heavy chain variable (VH) region incorporating, and (ii) The following CDRs: LC-CDR1 having amino acids with at least 85% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 114 LC-CDR2 having amino acids with at least 85% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 115 LC-CDR3 having amino acids with at least 85% sequence identity to SEQ ID NO: 6 or SEQ ID NO: 116 Light chain variable (VL) region incorporating This refers to antigen-binding molecules, including [specific example].

[0012]

[0012] Another aspect of the present invention is an antigen-binding molecule that binds to salvecovirus spike proteins derived from two or more different salvecoviruses, (i) The following CDRs: Formula I: X1-X2-X3-Φ-X4-X n1 -X5-X6 (where X1 is selected from one of G and E, X2 is selected from one of F, Y, N, G, D, and V, X3 is selected from one of P, T, S, I and F, Φ is selected from one of F, V, L, and I, X4 is selected from one of S, T, R, N, G, L, and I, X n1 [[ID=I2]]is selected from one of S, SN, N, M, H, T, G, P, G and D, X5 is selected from one of Y, S, N, I and H, X6 is selected from one of Y, G, W, E, A, N, and T) HC-CDR1 having an amino acid having the above, Amino acid formula II: X7-X8-X9-X n2 -π-X n3 -X 10 (where X7 is selected from one of I and T, X8 is selected from one of Y, S, N, G, A and T, X9 is selected from one of S, F, P, N, H, I, Y, G, and T, X n2 is selected from one of G, YN, DD, T, NG, DG, S, SS, D, ST, and NT, π is selected from one of G, S, P, A and E, X n3 is selected from one of S, I, D, G, F, N, RT, L, and RN, X 10 is selected from one of T, R, M, K, S, and P) HC-CDR2 having the above, Formula III: Ψ-ζ1-X n4 -X 11 -X n5 -X 12 -X ;<C 13 -X<C 14 -ζ2-X ; 15 (In the formula, Ψ is selected from one of A and V, ζ1 is selected from one of R, T, K, and LN. X n4 It is selected from one of the following: E, HLGGG, GGG, LDIII, DSI, GEAG, RVAIF, LQNG, VTYTS, ADIV, DSLA, DSL, AISQQ, DYYDN, DPL, EGIQG, and DGG. X 11 It is selected from one of L, S, Y, T, A, N, V, and W. X n5 It is selected from one of R, S, LET, P, SAT, MATIWV, DGY, SY, PLPF, GS, VV, SVT, FDS, GYYY, EGAAS, V, and QLPY. X 12 It is selected from one of H, W, G, P, T, S, N, and Y. X 13 It is selected from one of Y, P, A, L, S, F, I, V, and G. X 14 It is selected from one of F, I, N, Y, L, and M. ζ2 is selected from one of D, E, G, and S. X 15 (This is selected from one of Y, S, L, N, H, C, V, and F.) HC-CDR3 containing amino acids A heavy chain variable (VH) region incorporating, and (ii) The following CDRs: Formula IV:X 16 -X 17 -X 18 -X n6 -ζ3-X 19 (In the formula, X 16 It is selected from one of Q and Y, X 17 It is selected from one of G, S, T, N, I, and A. X 18 It is selected from one of V, I, T, F, and L. Xn6 It is selected from one of S, G, N, V, R, LYSSNNK, LYRSNNK, LQNNGY, VQSNGY, VHSDGN, MQLNGY, and SS. ζ3 is selected from one of S, N, and T. X 19 (This is selected from one of W, Y, S, and N.) LC-CDR1 having amino acids, Formula V:X 20 -X 21 -S (In the formula, X 20 It is selected from one of A, W, K, T, G, L, and D. X 21 (This is selected from one of A, S, G, I, and T.) LC-CDR2 having amino acids, Equation VI:X 22 -ζ4-X 23 -X n7 -ζ5-X 24 -X 25 -X n8 -ζ6 (In the formula, X 22 It is selected from one of Q, H, and M. ζ4 is selected from one of Q and H, X 23 It is selected from one of Y, S, G, A, L, and T. X n7 It is selected from one of F, Y, N, S, L, G, T, YR, and YI. ζ5 is selected from one of S, T, N, Q, and D. X 24 It is selected from one of S, Y, T, D, H, F, P, W, and I. X 25 It is selected from one of P, I, and R, X n8 It is selected from one of F, W, K, G, Y, R, P, L, PY, EY, ED, GY, QY, and QI. ζ6 is selected from one of T and S. LC-CDR3 having amino acids Light chain variable (VL) region incorporating This refers to antigen-binding molecules, including [specific example].

[0013]

[0013] In another embodiment, there exists an optionally isolated nucleic acid or a set of nucleic acids that encodes the antigen-binding molecule discussed above in this specification.

[0014] In another embodiment, there exists an expression vector or a plurality of expression vectors comprising the nucleic acids or plurality of nucleic acids discussed above in this specification.

[0014]

[0015] In another embodiment, there exists a method for producing antigen-binding molecules that bind to salvecovirus spike proteins derived from two or more different salvecoviruses, the method comprising the step of culturing cells capable of expressing the antigen-binding molecules discussed herein under conditions suitable for the expression of the antigen-binding molecules by said cells.

[0015]

[0016] In another embodiment, there are compositions comprising an antigen-binding molecule as discussed above herein, a nucleic acid or multiple nucleic acids as discussed above herein, an expression vector or multiple expression vectors as discussed above herein, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0016]

[0017] In another embodiment, there are antigen-binding molecules, nucleic acids or multiple nucleic acids, expression vectors or multiple expression vectors, or compositions discussed above herein for use in the treatment or prevention of diseases caused by infection with salvecovirus.

[0017]

[0018] In another embodiment, there is the use of the antigen-binding molecules, nucleic acids or multiple nucleic acids, expression vectors or multiple expression vectors, or compositions discussed above herein in the manufacture of a pharmaceutical product for use in the treatment or prevention of diseases caused by infection with salvecovirus.

[0018]

[0019] In another embodiment, there exists a method for treating or preventing a disease caused by infection with salvecovirus, comprising the step of administering a therapeutic or prophylactic amount of an antigen-binding molecule, a nucleic acid or plurality of nucleic acids, an expression vector or plurality of expression vectors, or a composition discussed above herein, to the subject.

[0019]

[0020] In another embodiment, there is the use of the antigen-binding molecules discussed above herein to inhibit infection of ACE2-expressing cells by salvecovirus.

[0021] Other aspects and features of the present invention will become apparent to those skilled in the art by examining the following description of specific embodiments of the present invention together with the accompanying drawings.

[0020]

[0022] The drawings show embodiments of the present invention merely as non-limiting examples. [Brief explanation of the drawing]

[0021] [Figure 1]

[0023] Phylogenetic trees of four known coronavirus species. [Figure 2]

[0024] A) Multiple sVNTs on the Luminex platform. B) As shown, all six RBD proteins can bind to the hACE2 receptor molecule as expected, in the following order (from highest affinity to lowest affinity): SARS-CoV-2 B.1.351 > SARS-CoV-2 B.1.1.7 = SC2r-CoV GX-P5L (pangolin) > SARS-CoV-2 > SARS-CoV > SC2r-CoV RaTG13 (bat). [Figure 3]

[0025] Multiplex sVNT analysis for 10 different salvecoviruses from five panels of human serum (shown above). All serums were used in a 1:20 ratio. A 30% cutoff was set as predetermined. [Figure 4]

[0026] Titration of NAb expressed as NT50 using the same serum panel and viruses as in A. Samples were tested at dilutions of 1:20 to 1:20,480 using a 4-fold serial titration. A cutoff of 1:100 was set as predetermined. [Figure 5]

[0027] Conversion of a non-dominant cross-neutralizing antibody response to an immunodominant cross-neutralizing antibody response through cross-immunization with genetically distant salvecovirus antigens. [Figure 6]

[0028] Inhibition of pan-salvecovirus mAb-RBD interactions by different human serum panels. (A) Inhibition of RBD-ACE2 interactions by rabbit mAb 5D7B7 with pan-salvecovirus RBD binding ability. (B) Inhibition of 5B7D7 binding to different RBDs by the five serum panels shown. Scatter plots show all data points, with the 0th, 25th, 50th, 75th, and 100th percentiles indicated. Statistical significance was determined using the Wilcoxon signed-rank test. "SARS vaccination" was set as the reference group for comparison. [Figure 7]

[0029] Staining of B cells with fluorescently labeled RBD. (A) Representative flow cytometry plots showing the frequency of SARS-CoV-1 and SARS-CoV-2 double-positive cells in the SARS-vaccinated group (n=5), the healthy-vaccinated group (n=6), and the COVID-19-vaccinated group (n=5). (B) Scatter plot of the frequency of SARS-CoV-1 and SARS-CoV-2 double-positive cells relative to all SARS-CoV-1 or SARS-CoV-2 positive cells. The scatter plot shows all data points, with the 0th, 25th, 50th, 75th, and 100th percentiles indicated. Statistical significance was determined using the Wilcoxon signed-rank test. P values ​​are shown at the top of each plot. ns indicates that P > 0.05 is not significant. [Figure 8]

[0030] Neutralization patterns of rabbit superimmune serum targeting different betacoronavirus RBD proteins. A 1:100 cutoff was set as predetermined. [Figure 9]

[0031] A schematic diagram showing the selection of double-positive B cells that produce antibodies that bind to RBD derived from both viruses. [Figure 10]

[0032] Phylogenetic analysis and sequence alignment of ACE2-binding salvecovirus RBDs. (A) Phylogenetic tree based on receptor-binding domain (RBD) sequences of salvecoviruses capable of binding to human ACE2. The phylogenetic tree was constructed using PhyML with a generalized time-reversible (GTR) substitution model and 1,000 bootstrap repeats. The numbers in the branches represent the percentage of the bootstrap value of the relevant node. The scale bar represents the number of substitutions per site. Salvecovirus RBDs involved in this study are shown in bold. (B) Percentage of RBD amino acid sequence identity in descending order (right to left for SARS-CoV-1; left to right for SARS-CoV-2) for different salvecoviruses against SARS-CoV-1 and SARS-CoV-2. (C) Alignment of amino acid sequences of salvecovirus RBDs used in this study. Red indicates mutations / deletions. Amino acids crucial to the SARS-CoV-2 RBD-ACE2 interaction are indicated by blue dots above them. The SARS-CoV-1 clade virus is shown as a gray shadow. [Figure 11]

[0033] CD19+ B cells were selected based on their positive test results for binding to SARS-CoV-1 (SC1+) and SARS-CoV-2 (SC2+) RBD tetramers. [Figure 12]

[0034] Data on the top 6 monoclonal antibodies and 4 control monoclonal antibodies identified in preliminary screening using a multiplex sVNT platform. [Figure 13]

[0035] 50% inhibitory concentration (IC50, ng / ml) of monoclonal antibody blocking RBD-ACE2 binding using a multi-surrogate virus neutralization competition format. [Figure 14]

[0036] The ability of monoclonal antibodies to neutralize different salvecoviruses, including SARS-CoV-2 ancestor and four VOCs (alpha, delta, beta, and gamma), two zoonotic salvecoviruses (GX-P5L and WIV-1), and eight spike pseudotyped reporter viruses, including SARS-CoV-1. [Figure 15]

[0037] Results of monoclonal antibody neutralization tests against ancestral SARS-CoV-2, Omicron BA.1, and Omicron BA.2 viruses using three different platforms: A) multiplex sVNT, B) pseudovirus neutralization test (pVNT), and C) plaque reduction neutralization test (PRNT). [Modes for carrying out the invention]

[0022]

[0038] This disclosure provides antigen-binding molecules capable of binding to salvecovirus spike proteins derived from two or more different salvecoviruses, and in particular, neutralizing antibodies that inhibit the interaction between salvecovirus spike proteins and ACE2, and thus can act as antagonists to infection of ACE2-expressing cells by various salvecoviruses. The antigen-binding molecules described herein are provided with a combination of properties that are more advantageous than known SARS-CoV-2 antibodies.

[0023]

[0039] Throughout this document, unless otherwise indicated, terms such as “includes,” “consist of,” and “possess” should be interpreted as not being inclusive, or in other words, “includes but not limited to these.”

[0024]

[0040] Furthermore, throughout this document, unless otherwise required by context, the word "include" or its conjugations such as "includes" or "including" will be understood to mean that it includes the integer or group of integers mentioned, but does not exclude any other integer or group of integers.

[0025]

[0041] Unless otherwise defined, all other technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the subject matter herein belongs.

[0026]

[0042] In various embodiments, the antigen-binding molecule binds to salvecovirus spike proteins derived from two or more different salvecoviruses, (i) The following CDRs: HC-CDR1 having amino acids with at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 111 HC-CDR2 having amino acids with at least 85% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 112 HC-CDR3 having amino acids with at least 85% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 113 A heavy chain variable (VH) region incorporating, and (ii) The following CDRs: LC-CDR1 having amino acids with at least 85% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 114 LC-CDR2 having amino acids with at least 85% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 115 LC-CDR3 having amino acids with at least 85% sequence identity to SEQ ID NO: 6 or SEQ ID NO: 116 Light chain variable (VL) region incorporating Antigen-binding molecules, including [specific molecule], exist.

[0027]

[0043] Throughout this specification, the term “antigen-binding molecule” and its plural form should be understood to refer to one or more molecules capable of binding to a target antigen, indicating binding to the relevant target molecule and preventing ACE2-mediated entry into cells, and to that extent, encompass monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fv, scFv, Fab, scFab, F(ab')2, Fab2, diabody, triabody, scFv-Fc, minibody, single-domain antibodies (e.g., VhH), etc.).

[0028]

[0044] In various embodiments, two or more different salvecoviruses may include antigen-binding molecules that can bind to the spike proteins of two, three, four, five, six, seven, eight, nine, or ten or more different salvecoviruses. In various embodiments, for example, the antigen-binding molecule can bind to the SARS-CoV spike protein and can also bind to the SARS-CoV-2 spike protein. In various embodiments, the antigen-binding molecule can bind to multiple salvecovirus spike proteins. For example, antigen-binding molecules can bind to the SARS-CoV spike protein; and / or the SARS-CoV-2 spike protein, and / or SARS-CoV-2 alpha, and / or SARS-CoV-2 beta, and / or SARS-CoV-2 delta, and / or SC2r-CoV RaTG13, and / or SC2r-CoV GX-P5L, and / or SC2r-CoV GD-1, and / or any other salvecovirus spike protein, e.g., SC2r-CoVRmYN02;RacCS203, or any future unknown salvecovirus. Broad-spectrum antigen-binding molecules have the advantage of being able to effectively block most salvecoviruses and promote the prevention of infection with both known and unknown salvecoviruses.

[0029]

[0045] In various embodiments, the term "able to bind" may include inhibition or neutralization of 30% or more of the binding between the Salvecovirus spike protein and ACE2. In various embodiments, inhibition or neutralization of 30% or more of the binding between the Salvecovirus spike protein and ACE2 may be selected from at least 30%, 35%, 40%, 41%, 42%, 43%, 44%, 45%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, or 80% or more of the inhibition or neutralization. In various embodiments, the antigen-binding molecule binds to salvecovirus spike proteins derived from two or more different salvecoviruses, and includes inhibition or neutralization of 30% or more of the binding between the salvecovirus spike protein and ACE2 (e.g., one of at least 30%, 35%, 40%, 41%, 42%, 43%, 44%, 45%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, or 80% or more).

[0030]

[0046] In various embodiments, the antigen-binding molecule comprises polyclonal antibodies isolated from patients who had SARS and recovered. In various embodiments, the antigen-binding molecule comprises polyclonal antibodies isolated from patients who had SARS, recovered, and received COVID-19 vaccination. For example, the polyclonal antigen-binding molecule can bind to the SARS-CoV spike protein and can also bind to the SARS-CoV-2 spike protein. In various embodiments, the polyclonal antigen-binding molecule can bind to multiple SARS-CoV-2 spike proteins. For example, polyclonal antigen-binding molecules can bind to the SARS-CoV spike protein; and / or the SARS-CoV-2 spike protein, and / or SARS-CoV-2 alpha, and / or SARS-CoV-2 beta, and / or SARS-CoV-2 delta, and / or SC2r-CoV RaTG13, and / or SC2r-CoV GX-P5L, and / or SC2r-CoV GD-1, and / or any other salvecovirus spike protein, e.g., SC2r-CoVRmYN02;RacCS203, or any future unknown salvecovirus. Broad-spectrum antigen-binding molecules have the advantage of being able to effectively block most salvecoviruses and promote the prevention of infection with both known and unknown salvecoviruses.

[0031]

[0047] In various embodiments, the antigen-binding molecule binds to salvecovirus spike proteins derived from two or more different salvecoviruses, (i) The following CDRs: Formula I: X1-X2-X3-Φ-X4-X n1 -X5-X6 (In the formula, X1 is selected from one of amino acids G and E, X2 is selected from one of amino acids F, Y, N, G, D, and V, X3 is selected from one of amino acids P, T, S, I, and F, Φ is selected from one of hydrophobic amino acids F, V, L, or I, X4 is selected from one of amino acids S, T, R, N, G, L, and I, X n1 X5 is selected from one of the amino acid sequences S, SN, N, M, H, T, G, P, G, and D; X6 is selected from one of the amino acid sequences Y, S, N, I, and H; and X6 is selected from one of the amino acid sequences Y, G, W, E, A, N, and T. HC-CDR1 having amino acids, Amino acid formula II: X7-X8-X9-X n2 -π-X n3 -X 10 (In the formula, X7 is selected from one of amino acids I and T, X8 is selected from one of amino acids Y, S, N, G, A and T, X9 is selected from one of amino acids S, F, P, N, H, I, Y, G and T, X n2 π is selected from one of the amino acid sequences G, YN, DD, T, NG, DG, S, SS, D, ST, and NT, and π is selected from one of the small amino acids G, S, P, A, and E, and X n3 The amino acid sequence is selected from one of the following: S, I, D, G, F, N, RT, L, and RN, and X 10 (This is selected from one of the amino acids T, R, M, K, S, and P.) HC-CDR2, which has Equation III:Ψ-ζ1-X n4 -X 11 -X n5 -X 12 -X 13 -X 14 -ζ2-X 15 (In the formula, Ψ is selected from one of the aliphatic amino acids A and V, ζ1 is selected from one of the hydrophilic amino acids R, T, K and LN, and X n4X is selected from one of the amino acid sequences E, HLGGG, GGG, LDIII, DSI, GEAG, RVAIF, LQNG, VTYTS, ADIV, DSLA, DSL, AISQQ, DYYDN, DPL, EGIQG, and DGG. 11 X is selected from one of the amino acids L, S, Y, T, A, N, V, and W. n5 X is selected from one of the amino acid sequences R, S, LET, P, SAT, MATIWV, DGY, SY, PLPF, GS, VV, SVT, FDS, GYYY, EGAAS, V, and QLPY. 12 X is selected from one of the amino acids H, W, G, P, T, S, N, and Y. 13 X is selected from one of the amino acids Y, P, A, L, S, F, I, V, and G. 14 is selected from one of the amino acids F, I, N, Y, L, and M, and ζ2 is selected from one of the hydrophilic amino acids D, E, G, and S, and X 16 (This is selected from one of the amino acids Y, S, L, N, H, C, V, and F.) HC-CDR3 containing amino acids A heavy chain variable (VH) region incorporating, and (ii) The following CDRs: Formula IV:X 16 -X 17 -X 18 -X n6 -ζ3-X 19 (In the formula, X 16 X is selected from one of amino acids Q and Y. 17 X is selected from one of the amino acids G, S, T, N, I, and A. 18 is selected from one of the amino acids V, I, T, F, and L, and X n6 ζ3 is selected from one of the amino acid sequences S, G, N, V, R, LYSSNNK, LYRSNNK, LQNNGY, VQSNGY, VHSDGN, MQLNGY, and SS, and ζ3 is selected from one of the hydrophilic amino acids S, N, and T, and X 19 (This is selected from one of the amino acids W, Y, S, and N.) LC-CDR1 having an amino acid having Formula V: X 20 -X 21 -S (wherein X 20 is selected from one of the amino acids A, W, K, T, G, L, and D, and X 21 is selected from one of the amino acids A, S, G, I, and T) LC-CDR2 having an amino acid having Formula VI: X 22 -ζ4-X 23 -X n7 -ζ5-X 24 -X 25 -X n8 -ζ6 (wherein X 22 is selected from one of the amino acids Q, H, and M, ζ4 is selected from one of the hydrophilic amino acids Q and H, and X 23 is selected from one of the amino acids Y, S, G, A, L, and T, X n7 is selected from one of the amino acid sequences F, Y, N, S, L, G, T, YR, and YI, ζ5 is selected from one of the hydrophilic amino acids S, T, N, Q, and D, and X 24 is selected from one of the amino acids S, Y, T, D, H, F, P, W, and I, X 25 is selected from one of the amino acids P, I, and R, X n8 is selected from one of the amino acid sequences F, W, K, G, Y, R, P, L, PY, EY, ED, GY, QY, and QI, and ζ6 is selected from one of the hydrophilic amino acids T and S) LC-CDR3 having an amino acid having A light chain variable (VL) region incorporating An antigen-binding molecule comprising

[0032]

[0048] Antigen-binding molecules having CDRs corresponding to these formulas have been demonstrated to neutralize several sarbecoviruses, including SARS-CoV-2. Antibodies 1 (SS6V1-B5) to 20 (SS6V20-F5) are examples of antigen-binding molecules having CDRs corresponding to these formulas. These are part of the best cross-clade neutralizing antibodies reported to date.

[0033]

[0049] In various embodiments, in the formula, Φ of formula I is selected from one of the hydrophobic amino acids F, L, or I, X5 of formula I is selected from one of Y, S, I, and H, X6 of formula I is selected from one of Y, W, E, A, N, and T, X8 of formula II is I, X n2 of formula II is selected from one of DD, T, NG, DG, S, SS, D, ST, and NT, ζ1 of formula III is selected from one of the hydrophilic amino acids R, T, and K, X n4 of formula III is selected from one of HLGGG, GGG, LDIII, DSI, GEAG, LQNG, VTYTS, ADIV, DSL, AISQQ, DYYDN, DPL, EGIQG, and DGG, X 11 of formula III is selected from one of S, Y, T, A, V, and W, X n5 of formula III is selected from one of S, LET, P, SAT, MATIWV, SY, PLPF, GS, VV, SVT, FDS, GYYY, EGAAS, V, and QLPY, X 12 of formula III is selected from one of W, G, P, T, S, N, and Y, X n6 of formula IV is selected from one of S, G, N, V, R, LYRSNNK, LQNNGY, VQSNGY, VHSDGN, MQLNGY, and SS, X 23 of formula VI is selected from one of Y, S, G, A, and T, X n8However, they are the same as those listed above, except that one of F, W, K, G, Y, R, P, L, EY, ED, GY, QY, and QI is selected. Antigen-binding molecules with CDRs corresponding to these formulas demonstrated pan-salvecovirus neutralization of a wide range of salvecoviruses, including SARS-CoV-1 and SARS-CoV-2. Antigen-binding molecules with CDRs corresponding to these formulas were double-positive for staining with both SARS-CoV-1 and SARS-CoV-2 RBD proteins. Antibodies 1 (SS6V1-B5) and 4-20 (SS6V4-A1, SS6V5-C3, ..., SS6V20-F5) are examples of antigen-binding molecules with CDRs corresponding to these formulas.

[0034]

[0050] In various embodiments, X1 is G, X2 is selected from one of G, F, Y, and V, X3 is selected from one of S, I, T, and F, Φ is selected from one of F, L, and I, X4 is selected from one of R, S, G, L, T, and I, X n1 X is selected from one of P, N, T, D, and G, X5 is selected from one of Y, S, and H, X6 is selected from one of E, N, and Y, X7 is I, X8 is selected from one of G, S, N, and Y, X9 is selected from one of I, N, S, T, and F, X n2 is selected from one of T, S, SS, and NT, and π is selected from one of G, E, S, and A, and X n3 is selected from one of G, S, F, I, and N, and X 10 is selected from one of T, M, and P, Ψ is A, ζ1 is R, and X n4 It is selected from one of VTYTS, GGG;DYYDN, and DGG, and X 11 is selected from one of S, Y, and W, and X n5 It is selected from one of PLPF.LET, GYYY, and QLPY, and X 12 is selected from one of W, Y, and G, and X 13is selected from one of F, P, G, and Y, and X 14 is selected from one of F, M, and L, and ζ2 is selected from one of D and E, and X 15 is selected from one of Y, L, V, F, and S, and X 16 is selected from one of Q and Y, and X 17 is selected from one of G and S, and X 18 is selected from one of I, F, and L, and X n6 is selected from one of G, LQNNGY, R, VQSNGY, S, and MQLNGY, and ζ3 is selected from one of N, S, and T, and X 19 is selected from either Y or S, and X 20 is selected from one of A, L, and G, and X 21 is selected from one of A, S, T, and G, and X 22 is selected from one of Q, M, and L, where ζ4 is Q and X 23 is selected from one of T, S, Y, and G, and X n7 is selected from one of YR, L, and Y, ζ5 is selected from one of T, Q, and S, and X 24 is selected from one of P, I, W, and T, and X 25 P is X n8 X is selected from one of ED, G, QI, and L, and ζ6 is selected from one of S and T. That is, in various embodiments, formula I includes GX2X3ΦX4X5X6X7 (wherein X2 is selected from one of G, F, Y, and V, X3 is selected from one of S, I, T, and F, Φ is selected from one of F, L, and I, X4 is selected from one of R, S, G, L, T, and I, X5 is selected from one of P, N, T, D, and G, X6 is selected from one of Y, S, and H, and X7 is selected from one of E, N, and Y), and formula II is IX9X 10 X n1 πX n2 X 11 (In the formula, X9 is selected from one of G, S, N, and Y, X10 is selected from one of I, N, S, T, and F, and X n1 is selected from one of S, SS, and NT, and π is selected from one of G, E, S, and A, and X n2 is selected from one of G, S, F, I, and N, and X 11 (is selected from one of T, M, and P), and formula III is ARX n3 X 12 X n4 X 13 X 14 X 15 ζ2X 16 (In the formula, X n3 It is selected from one of VTYTS, GGG;DYYDN, and DGG, and X 12 is selected from one of S, Y, and W, and X n4 It is selected from one of PLPF.LET, GYYY, and QLPY, and X 13 is selected from one of W, Y, and G, and X 14 is selected from one of F, P, G, and Y, and X 15 is selected from one of F, M, and L, and ζ2 is selected from one of D and E, and X 16 (is selected from one of Y, L, V, F, and S), and formula IV is X 17 X 18 X 19 X n5 ζ3Ω (in the formula, X 17 is selected from one of the aromatic amino acids Q and Y, and X 18 is selected from one of G and S, and X 19 is selected from one of I, F, and L, and X n5 (where is selected from one of G, LQNNGY, R, VQSNGY, S, and MQLNGY, ζ3 is selected from one of N, S, and T, and Ω is selected from one of Y or S), and formula V is X 20 X 21 S (where X 20 is selected from one of A, L, and G, and X 21(is selected from one of A, S, T, and G), and formula VI is X 22 QX 23 X n6 ζ5X 25 PX n7 ζ6 (where X 22 is selected from one of Q, L, M, and L, and X 23 is selected from one of T, S, Y, and G, and X n6 is selected from one of YR, L, and Y, ζ5 is selected from one of T, Q, and S, and X 25 is selected from one of P, I, W, and T, and X n7 (where is selected from one of ED, G, QI, and L, and ζ6 is selected from one of S and T). Antigen-binding molecules with CDRs corresponding to these formulas demonstrated pan-salbecovirus neutralizing efficacy and pan-salbecovirus neutralizing range over most salbecoviruses, including SARS-CoV-1 and SARS-CoV-2. Antibodies 1 (SS6V1-B5), 11 (SS6V11-E7), 12 (SS6V12-E11), 13 (SS6V13-F1), 19 (SS6V19-F4), and 20 (SS6V20-F5) are examples of antigen-binding molecules with CDRs corresponding to these formulas.

[0035]

[0051] In various embodiments, the heavy chain variable (VH) region is selected from the following CDRs: HC-CDR1 selected from one of the amino acid sequences GFILRNYE, GGFIGPHY, GFTFSTYN, GVSILGSY, GYTFTDYN and GGSIIGYY; HC-CDR2 selected from one of the amino acid sequences IGNTGGT, IYISGST, ISSSSFM, IYFSENT, INTNTGIP and IYFSANT; and amino acid sequences ARVTYTSSPLPFWFLDL, ARGGGYLETGPFEY, ARDYYDNSGYYYYGMDV, ARGGGYLETGPFDS, ARDGGWQLPYWYFDL and AR The light chain variable (VL) region incorporates HC-CDR3 selected from one of GGGYLETGPLDF, LC-CDR1 selected from one of the amino acid sequences QSIGNY, QSLLQNNGYNY, QSIRTY, QGLVQSNGYNY, YSFSSS, and QSLMQLNGYNY; LC-CDR2 selected from one of the amino acid sequences AAS, LSS, GTS, and LGS; and LC-CDR3 selected from one of the amino acid sequences QQTYRTPPEDS, MQSLQIPGT, LQTYSTPQIT, MQGLQTPGT, QQYYSWPLT, and MQGLQIPGT.

[0036]

[0052] In various embodiments, X1 is G, X2 is selected from one of G and V, X3 is selected from one of S and F, Φ is I, X4 is selected from one of G, L and I, X n1 X is selected from P and G, X5 is selected from Y, S and H, X6 is Y, X7 is I, X8 is Y, X9 is selected from I and F, X n2 S is S, and π is selected from one of G, E, and A, and X n3 is selected from one of S and N, and X 10 Ψ is A, ζ1 is R, X n4 It is GGG, X 11 Y is X n5 is LET, X 12 G is X13 P is X 14 is selected from one of F and L, ζ2 is selected from one of D and E, and X 15 is selected from one of Y, F, and S, and X 16 Q is X 17 is selected from one of G and S, and X 18 L is X n6 is selected from one of LQNNGY, VQSNGY, and MQLNGY, where ζ3 is N and X 19 Y is X 20 L is X 21 It is selected from one of S and G, and X 22 is M, ζ4 is Q, and X 23 It is selected from one of S and G, and X n7 is L, ζ5 is Q, and X 24 It is selected from one of I and T, and X 25 P is X n8 ζ₁ is G, and ζ₁ is T. That is, in various embodiments, formula I includes GX₂X₃IX₄X₅X₁₆Y (wherein X₂ is selected from one of G and V, X₃ is selected from one of S and F, X₄ is selected from one of G, L and I, X₅ is selected from one of P and G, and X₁ is selected from one of Y, S and H), and formula II is IYX 10 SπS n2 T (where X 10 is selected from one of I and F, π is selected from one of G, E and A, and X n2 (is selected from one of S and N), and formula III is ARGGGYLETGPX 15 ζ2X 16 (In the formula, X 15 is selected from one of F and L, ζ2 is selected from one of D and E, and X 16 (is selected from one of Y, F, and S), and formula IV is QX 18 LX n5 NY (In the formula, X 18 is selected from one of G and S, and Xn5 (is selected from one of LQNNGY, VQSNGY, and MQLNGY), and formula V is LX 21 S (where X 21 (is selected from one of S and G), and formula VI is MQX 23 LQX 25 PGT(wherein, X 23 It is selected from one of S and G, and X 25 The formulas include (selected from either I or T). Antigen-binding molecules with CDRs corresponding to these formulas demonstrated pan-salvecovirus neutralizing efficacy and pan-salvecovirus neutralizing region against all tested salvecoviruses, including SARS-CoV-1 and SARS-CoV-2. Antibody 11 (SS6V11-E7), antibody 13 (SS6V13-F1), and antibody 20 (SS6V20-F5) are examples of antigen-binding molecules with CDRs corresponding to these formulas. These antibodies demonstrated the highest reported efficacy. The three monoclonal antibodies in this group maintained potent neutralization capability against most SARS-CoV-2 VOCs and VOIs, as well as clade-1a salvecoviruses, across different virus neutralization assay platforms. All three antibodies utilized unique combinations of heavy and light chain gene classes exhibiting over 90% similarity in their heavy and light chain sequences. These sequences have not been previously reported as salvecovirus-specific antibodies.

[0037]

[0053] In various embodiments, the heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 selected from one of the amino acid sequences GGFIGPHY, GVSILGSY, and GGSIIGYY; HC-CDR2 selected from one of the amino acid sequences IYISGST, IYFSENT, and IYFSANT; and HC-CDR3 selected from one of the amino acid sequences ARGGGYLETGPFEY, ARGGGYLETGPFDS, and ARGGGYLETGPLDF. The light chain variable (VL) region incorporates the following CDRs: LC-CDR1 selected from one of the amino acid sequences QSLLQNNGYNY, QGLVQSNGYNY, and QSLMQLNGYNY; LC-CDR2 selected from one of the amino acid sequences LSS and LGS; and LC-CDR3 selected from one of the amino acid sequences MQSLQIPGT, MQGLQTPGT, and MQGLQIPGT.

[0038]

[0054] In various embodiments, X1 is G, X2 is G, X3 is selected from one of S and F, Φ is I, X4 is selected from one of G and I, X n1 X is selected from either P or G, X5 is selected from either Y or H, X6 is Y, X7 is I, X8 is Y, X9 is selected from either I or F, X n2 is S, and π is selected from one of G and A, and X n3 is selected from one of S and N, and X 10 Ψ is A, ζ1 is R, X n4 It is GGG, X 17 Y is X n5 is LET, X 12 G is X 13 P is X 14 is selected from one of F and L, ζ2 is selected from one of D and E, and X 15 is selected from one of Y and F, and X 16 Q is X 17 is S, X 18 L is X n6is selected from one of LQNNGY and MQLNGY, ζ3 is N, and X 19 Y is X 20 L is X 21 It is selected from one of S and G, and X 22 is M, ζ4 is Q, and X 23 It is selected from one of S and G, and X n7 is L, ζ5 is Q, and X 24 is I, X 25 P is X n8 ζ6 is G, and ζ6 is T. That is, in various embodiments, formula I includes GGX3IX4X5X6Y (wherein X3 is selected from one of S and F, X4 is selected from one of G and I, X5 is selected from one of P and G, and X6 is selected from one of Y and H), and formula II is IYX 10 SπS n2 T (where X 10 is selected from one of I and F, π is selected from one of G and A, and X n2 (is selected from one of S and N), and formula III is ARGGGYLETGPX 15 ζ2X 16 (In the formula, X 15 It is selected from one of F and L, and X 16 (where is selected from one of Y and F, and ζ² is selected from one of D and E), and Equation IV is QSLX n5 NY (In the formula, X n5 (is selected from one of LQNNGY and MQLNGY), and formula V is LX 21 S (where X 21 (is selected from one of S and G), and formula VI is MQX 23 LQIPGT(where X 23The formula includes one of S and G). Antigen-binding molecules with CDRs corresponding to these formulas have demonstrated the best pan-salvecovirus neutralizing efficacy and pan-salvecovirus neutralizing region compared to any other antibody reported to date. Epitope mapping studies of antibodies corresponding to these formulas show that these antibodies have a contact footprint specific to RBD. Antibody 11 (SS6V11-E7) and antibody 20 (SS6V20-F5) are examples of antigen-binding molecules with CDRs corresponding to these formulas.

[0039]

[0055] In various embodiments, the heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 selected from one of the amino acid sequences GGFIGPHY and GGSIIGYY; HC-CDR2 selected from one of the amino acid sequences IYISGST and IYFSANT; and HC-CDR3 selected from one of the amino acid sequences ARGGGYLETGPFEY and ARGGGYLETGPLDF. The light chain variable (VL) region incorporates the following CDRs: LC-CDR1 selected from one of the amino acid sequences QSLLQNNGYNY and QSLMQLNGYNY; LC-CDR2 selected from one of the amino acid sequences LSS and LGS; and LC-CDR3 selected from one of the amino acid sequences MQSLQIPGT and MQGLQIPGT.

[0040]

[0056] In various embodiments, the heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 having the amino acid sequence GFILRNYE; HC-CDR2 having the amino acid sequence IGNTGGT; and HC-CDR3 having the amino acid sequence ARVTYTSSPLPFWFLDL. The light chain variable (VL) region incorporates the following CDRs: LC-CDR1 having the amino acid sequence QSIGNY; LC-CDR2 having the amino acid sequence AAS; and LC-CDR3 having the amino acid sequence QQTYRTPPEDS. In various embodiments, these CDRs are formed in the heavy chain sequence number 71 and light chain sequence number 72 of antibody 1 (SS6V1-B5). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, as long as they can bind to salvecovirus spike proteins derived from two or more different salvecoviruses.

[0041]

[0057] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 with amino acid sequence GFTVSSNY; HC-CDR2 with amino acid sequence IYSGGST; and HC-CDR3 with amino acid sequence ARELRHYFDY. The light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with amino acid sequence QGISSY; LC-CDR2 with amino acid sequence AAS; and LC-CDR3 with amino acid sequence QQLNSYPPYS. In various embodiments, these CDRs are formed in heavy chain SEQ ID NO: 73 and light chain SEQ ID NO: 74 of antibody 2 (SS6V2-G1). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, as long as they can bind to salvecovirus spike proteins derived from two or more different salvecoviruses. This antibody was cloned from SC2+ single-positive B cells and demonstrated reactivity to SARS-CoV-2 RBD, while showing minimal reactivity to SARS-CoV-1 RBD.

[0042]

[0058] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 with amino acid sequence GYSFTNSG; HC-CDR2 with amino acid sequence TNFYNGIT; and HC-CDR3 with amino acid sequence ALNRVAIFNDGYNPLGY. The light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with amino acid sequence QSVLYSSNNKNY; LC-CDR2 with amino acid sequence WAS; and LC-CDR3 with amino acid sequence QQYFSSPFS. In various embodiments, these CDRs are formed in heavy chain SEQ ID NO: 75 and light chain SEQ ID NO: 76 of antibody 3 (SS6V3-G2). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, as long as they can bind to salvecovirus spike proteins derived from two or more different salvecoviruses. This antibody was cloned from SC2+ single-positive B cells and demonstrated reactivity to SARS-CoV-2 RBD, while showing minimal reactivity to SARS-CoV-1 RBD.

[0043]

[0059] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 having the amino acid sequence GYTFSMYW; HC-CDR2 having the amino acid sequence IYPDDSDR; and HC-CDR3 having the amino acid sequence ARLQNGYSYGLLEN. The light chain variable (VL) region incorporates the following CDRs: LC-CDR1 having the amino acid sequence QSTLYRSNNKNY; LC-CDR2 having the amino acid sequence WAS; and LC-CDR3 having the amino acid sequence QQYYSYPWT. In various embodiments, these CDRs are formed in the heavy chain sequence number 77 and light chain sequence number 78 of antibody 4 (SS6V4-A1). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, as long as they can bind to salvecovirus spike proteins derived from two or more different salvecoviruses.

[0044]

[0060] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 having the amino acid sequence GYTFTHYW; HC-CDR2 having the amino acid sequence IYPDDSDT; and HC-CDR3 having the amino acid sequence ATADIVVGSNFFDH. The light chain variable (VL) region incorporates the following CDRs: LC-CDR1 having the amino acid sequence QSISTW; LC-CDR2 having the amino acid sequence KAS; and LC-CDR3 having the amino acid sequence QHYNSYIKT. In various embodiments, these CDRs are formed in the heavy chain sequence number 79 and light chain sequence number 80 of antibody 5 (SS6V5-C3). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, as long as they can bind to salvecovirus spike proteins derived from two or more different salvecoviruses.

[0045]

[0061] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 having the amino acid sequence GFTFNTYA; HC-CDR2 having the amino acid sequence ISSNGGIT; and HC-CDR3 having the amino acid sequence VKDSLATVVTLLSY. The light chain variable (VL) region incorporates the following CDRs: LC-CDR1 having the amino acid sequence QTISSY; LC-CDR2 having the amino acid sequence AAS; and LC-CDR3 having the amino acid sequence QQSYSTPGT. In various embodiments, these CDRs are formed in the heavy chain sequence number 81 and light chain sequence number 82 of antibody 6 (SS6V6-C4). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, as long as they can bind to salvecovirus spike proteins derived from two or more different salvecoviruses.

[0046]

[0062] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 having the amino acid sequence ENIFSGYW; HC-CDR2 having the amino acid sequence IYPDDSDT; and HC-CDR3 having the amino acid sequence ARHLGGGSSWPIDY. The light chain variable (VL) region incorporates the following CDRs: LC-CDR1 having the amino acid sequence QGISNY; LC-CDR2 having the amino acid sequence AAS; and LC-CDR3 having the amino acid sequence QQYSSYPFT. In various embodiments, these CDRs are formed in the heavy chain sequence number 83 and light chain sequence number 84 of antibody 7 (SS6V7-C5). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, as long as they can bind to salvecovirus spike proteins derived from two or more different salvecoviruses.

[0047]

[0063] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 having the amino acid sequence GFTFSTYA; HC-CDR2 having the amino acid sequence IASDGGIT; and HC-CDR3 having the amino acid sequence VKDSLTSVTTIFDC. The light chain variable (VL) region incorporates the following CDRs: LC-CDR1 having the amino acid sequence QNINSY; LC-CDR2 having the amino acid sequence TAS; and LC-CDR3 having the amino acid sequence QQSYTDPYT. In various embodiments, these CDRs are formed in the heavy chain sequence number 85 and light chain sequence number 86 of antibody 8 (SS6V8-D3). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, as long as they can bind to salvecovirus spike proteins derived from two or more different salvecoviruses.

[0048]

[0064] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 having the amino acid sequence GGSISSNIW; HC-CDR2 having the amino acid sequence IYHSGST; and HC-CDR3 having the amino acid sequence ARAISQQYFDSSVLGY. The light chain variable (VL) region incorporates the following CDRs: LC-CDR1 having the amino acid sequence QSVVTN; LC-CDR2 having the amino acid sequence GAS; and LC-CDR3 having the amino acid sequence QQYNNWPGYT. In various embodiments, these CDRs are formed in heavy chain sequence number 87 and light chain sequence number 88 of antibody 9 (SS6V9-D11). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, as long as they can bind to salvecovirus spike proteins derived from two or more different salvecoviruses.

[0049]

[0065] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 having the amino acid sequence EDSFTGYW; HC-CDR2 having the amino acid sequence IYPDDGDT; and HC-CDR3 having the amino acid sequence ARHLGGGSSWPIDS. The light chain variable (VL) region incorporates the following CDRs: LC-CDR1 having the amino acid sequence QGIRNY; LC-CDR2 having the amino acid sequence AAS; and LC-CDR3 having the amino acid sequence QQYNNHPFT. In various embodiments, these CDRs are formed in the heavy chain sequence number 89 and light chain sequence number 90 of antibody 10 (SS6V10-E1). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, as long as they can bind to salvecovirus spike proteins derived from two or more different salvecoviruses.

[0050]

[0066] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 having the amino acid sequence GGFIGPHY; HC-CDR2 having the amino acid sequence IYISGST; and HC-CDR3 having the amino acid sequence ARGGGYLETGPFEY. The light chain variable (VL) region incorporates the following CDRs: LC-CDR1 having the amino acid sequence QSLLQNNGYNY; LC-CDR2 having the amino acid sequence LSS; and LC-CDR3 having the amino acid sequence MQSLQIPGT. In various embodiments, these CDRs are formed in the heavy chain sequence number 91 and light chain sequence number 92 of antibody 11 (SS6V11-E7). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, as long as they can bind to salvecovirus spike proteins derived from two or more different salvecoviruses. These antibodies possessing CDRs demonstrated either the best pan-salvecovirus neutralizing efficacy or pan-salvecovirus neutralizing range compared to any other antibodies reported to date, including being the only antibodies with neutralizing activity against omicron BA.2.

[0051]

[0067] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 having the amino acid sequence GFTFSTYN; HC-CDR2 having the amino acid sequence ISSSSFM; and HC-CDR3 having the amino acid sequence ARDYYDNSGYYYYGMDV. The light chain variable (VL) region incorporates the following CDRs: LC-CDR1 having the amino acid sequence QSIRTY; LC-CDR2 having the amino acid sequence AAS; and LC-CDR3 having the amino acid sequence LQTYSTPQIT. In various embodiments, these CDRs are formed in the heavy chain sequence number 93 and light chain sequence number 94 of antibody 12 (SS6V12-E11). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, as long as they can bind to salvecovirus spike proteins derived from two or more different salvecoviruses.

[0052]

[0068] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 having the amino acid sequence GVSILGSY; HC-CDR2 having the amino acid sequence IYFSENT; and HC-CDR3 having the amino acid sequence ARGGGYLETGPFDS. The light chain variable (VL) region incorporates the following CDRs: LC-CDR1 having the amino acid sequence QGLVQSNGYNY; LC-CDR2 having the amino acid sequence LGS; and LC-CDR3 having the amino acid sequence MQGLQTPGT. In various embodiments, these CDRs are formed in the heavy chain sequence number 95 and light chain sequence number 96 of antibody 13 (SS6V13-F1). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, as long as they can bind to salvecovirus spike proteins derived from two or more different salvecoviruses.

[0053]

[0069] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 having the amino acid sequence GGPISSYY; HC-CDR2 having the amino acid sequence IYYSGST; and HC-CDR3 having the amino acid sequence ARDPLAEGAASSGFDN. The light chain variable (VL) region incorporates the following CDRs: LC-CDR1 having the amino acid sequence QSISSY; LC-CDR2 having the amino acid sequence AAS; and LC-CDR3 having the amino acid sequence QQSYTTPRT. In various embodiments, these CDRs are formed in heavy chain sequence number 97 and light chain sequence number 98 of antibody 14 (SS6V14-F2). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, as long as they can bind to salvecovirus spike proteins derived from two or more different salvecoviruses.

[0054]

[0070] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 having the amino acid sequence GFTFSSYA; HC-CDR2 having the amino acid sequence ISYDGRTK; and HC-CDR3 having the amino acid sequence ARLDIIITPPANDY. The light chain variable (VL) region incorporates the following CDRs: LC-CDR1 having the amino acid sequence QIVSSNY; LC-CDR2 having the amino acid sequence DAS; and LC-CDR3 having the amino acid sequence HQYGDSRRT. In various embodiments, these CDRs are formed in heavy chain sequence number 99 and light chain sequence number 100 of antibody 15 (SS6V15-F6). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, as long as they can bind to salvecovirus spike proteins derived from two or more different salvecoviruses.

[0055]

[0071] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 having the amino acid sequence EFTFSRYT; HC-CDR2 having the amino acid sequence IGGSTPLS; and HC-CDR3 having the amino acid sequence ARDSIASATTLFDL. The light chain variable (VL) region incorporates the following CDRs: LC-CDR1 having the amino acid sequence QAISSY; LC-CDR2 having the amino acid sequence AAS; and LC-CDR3 having the amino acid sequence QQSYITPPEYS. In various embodiments, these CDRs are formed in the heavy chain sequence number 101 and light chain sequence number 102 of antibody 16 (L8N16-C7). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, as long as they can bind to salvecovirus spike proteins derived from two or more different salvecoviruses.

[0056]

[0072] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 having the amino acid sequence GFTFSSYA; HC-CDR2 having the amino acid sequence ISYDGRNK; and HC-CDR3 having the amino acid sequence ARGEAGTMATIWVSSYDY. The light chain variable (VL) region incorporates the following CDRs: LC-CDR1 having the amino acid sequence QSLVHSDGNTY; LC-CDR2 having the amino acid sequence KIS; and LC-CDR3 having the amino acid sequence MQATQFPPT. In various embodiments, these CDRs are formed in the heavy chain sequence number 103 and light chain sequence number 104 of antibody 17 (L8N17-G3). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, as long as they can bind to salvecovirus spike proteins derived from two or more different salvecoviruses.

[0057]

[0073] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 having the amino acid sequence GFTFSSYA; HC-CDR2 having the amino acid sequence ITSNGGGT; and HC-CDR3 having the amino acid sequence AREGIQGWVTYFDY. The light chain variable (VL) region incorporates the following CDRs: LC-CDR1 having the amino acid sequence QSISTN; LC-CDR2 having the amino acid sequence AAS; and LC-CDR3 having the amino acid sequence QQTYTTPQYS. In various embodiments, these CDRs are formed in heavy chain sequence number 105 and light chain sequence number 106 of antibody 18 (SS6V18-E3). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, as long as they can bind to salvecovirus spike proteins derived from two or more different salvecoviruses.

[0058]

[0074] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 having the amino acid sequence GYTFTDYN; HC-CDR2 having the amino acid sequence INTNTGIP; and HC-CDR3 having the amino acid sequence ARDGGWQLPYWYFDL. The light chain variable (VL) region incorporates the following CDRs: LC-CDR1 having the amino acid sequence YSFSSS; LC-CDR2 having the amino acid sequence GTS; and LC-CDR3 having the amino acid sequence QQYYSWPLT. In various embodiments, these CDRs are formed in heavy chain sequence number 107 and light chain sequence number 108 of antibody 19 (SS6V19-F4). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, as long as they can bind to salvecovirus spike proteins derived from two or more different salvecoviruses.

[0059]

[0075] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 with amino acid sequence GGSIIGYY; HC-CDR2 with amino acid sequence IYFSANT; and HC-CDR3 with amino acid sequence ARGGGYLETGPLDF. The light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with amino acid sequence QSLMQLNGYNY; LC-CDR2 with amino acid sequence LGS; and LC-CDR3 with amino acid sequence MQGLQIPGT. In various embodiments, these CDRs are formed in heavy chain SEQ ID NO: 109 and light chain SEQ ID NO: 110 of antibody 20 (SS6V20-F5). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, as long as they can bind to salvecovirus spike proteins derived from two or more different salvecoviruses. Antibodies having these CDRs have demonstrated either the best pan-salvecovirus neutralizing efficacy or the best pan-salvecovirus neutralizing region compared to any other antibodies reported to date.

[0060]

[0076] In various embodiments, the antigen-binding molecule includes a polyclonal antigen-binding molecule. In various embodiments, the antigen-binding molecule includes at least two different antigen-binding domains (i.e., at least two antigen-binding domains including, for example, non-identical VH and VL). The higher neutralizing potency of the antibodies of this disclosure may allow for lower doses of antigen-binding molecules used clinically as individual antigen-binding molecules or mixed in a cocktail of two or more antigen-binding molecules, or antigen-binding molecules having two or more different antigen-binding domains.

[0061]

[0077] In various embodiments, the antigen-binding molecule is at least bispecific because it binds to two different Salvecovirus spike proteins, such as the SARS-CoV spike protein and the SARS-CoV-2 spike protein. The term "bispecific" means that the antigen-binding molecule can specifically bind to at least two distinct antigenic determinants.

[0062]

[0078] In various embodiments, a bispecific antigen-binding molecule may include an antigen-binding molecule that can bind to a target, where the antigen-binding molecule is specific to the target. For example, an antigen-binding molecule that can bind to both the SARS-CoV spike protein and the SARS-CoV-2 spike protein may include a component that can bind to the SARS-CoV spike protein and a second component that can bind to the SARS-CoV-2 spike protein.

[0063]

[0079] In various embodiments, the antigen-binding molecules of the present disclosure include multispecific antigen-binding molecules that may include antigen-binding polypeptides or antigen-binding polypeptide complexes capable of binding to a target, wherein the antigen-binding molecules are specific to the target. In some embodiments, the antigen-binding molecules that are components of a larger antigen-binding molecule may be referred to as the “antigen-binding domain” or “antigen-binding region” of the larger antigen-binding molecule.

[0064]

[0080] In various embodiments, the multispecific antigen-binding molecule can bind to multiple salvecovirus spike proteins. For example, the multispecific antigen-binding molecule can bind to the SARS-CoV spike protein; and / or the SARS-CoV-2 spike protein, and / or SARS-CoV-2 alpha, and / or SARS-CoV-2 beta, and / or SARS-CoV-2 delta (Dealta), and / or SC2r-CoV RaTG13, and / or SC2r-CoV GX-P5L, and / or SC2r-CoV GD-1, and / or any other salvecovirus spike protein, e.g., SC2r-CoVRmYN02;RacCS203, or any future unknown salvecovirus. The broad-spectrum antigen-binding molecule has the advantage of being able to effectively block most salvecoviruses and promote the prevention of infection with both known and unknown salvecoviruses.

[0065]

[0081] Throughout this specification, the term “salvecovirus” and its plural form should be understood to include any betacoronavirus that uses the angiotensinogen-converting enzyme 2 (ACE2) receptor as an entry point into cells. In various embodiments, salvecovirus includes any betacoronavirus that uses the ACE2 receptor as an entry point into cells. In various embodiments, salvecovirus includes any betacoronavirus that uses the human ACE2 receptor as an entry point into human cells. In various embodiments, salvecovirus includes any known or novel salvecovirus. In various embodiments, the sarbecovirus is SARS-CoV-2, SARS-CoV-2, SARS-CoV-2 B.1.1.7, SARS-CoV-2 B.1.351, SARS-CoV-2 B.1.617.2, SARS-CoV-2 C37, SARS-CoV-2 B.1.621, SARS-CoV-2 P.1, SARS-CoV-2 BA.1, SARS-CoV-2 BA.2, SC2r-CoV BANAL-52, SC2r-CoV BANAL-236, SC2r-CoV GX-P5L, SC1r-CoV Rs2018B, SC1r-CoV LYRa11, SC1r-CoV WIV-1, SARS-CoV, SC2r-CoV WIV-1, SC1r-CoV Selected from the group including or consisting of RsSHC014, SARS-CoV-2 alpha, SARS-CoV-2 beta, SARS-CoV-2 delta, SC2r-CoV RaTG13, SC2r-CoV GD-1, and SC2r-CoV GX-P5L.

[0066]

[0082] Throughout this specification, the term “SARS-CoV” should be understood to mean SARSr-CoV having the nucleotide sequence of GenBank:NC_004718.3 ("Severe Acute Respiratory Syndrome Coronavirus Isolate, Complete Genome") and to include its variant strains having nucleotide sequences with at least 85% sequence identity to the nucleotide sequence of GenBank:NC_004718.3 described in Sequence ID No. 7 (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more sequence identity).

[0067]

[0083] Throughout this specification, the term "SARS-CoV-2" refers to SARSr-CoV having the nucleotide sequence of GenBank:NC_045512.2 ("Severe Acute Respiratory Syndrome Coronavirus 2 Isolate Wuhan-Hu1, Complete Genome") reported by Zhou et al., Nature (2020) 579:270-273, and at least 8 of the nucleotide sequence of GenBank:NC_045512.2 described in Sequence ID No. 8. It should be understood that this includes the mutant strain having a nucleotide sequence with 5% sequence identity (for example, one of at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more sequence identity).

[0068]

[0084] Like all coronaviruses, Salvecovirus has a genome that encodes four major structural proteins: the spike (S) protein, the envelope (E) protein, the membrane (M) protein, and the nucleocapsid (N) protein. Generally, the spike protein has a region that incorporates a receptor-binding domain (RBD).

[0069]

[0085] In various embodiments, the salvecovirus spike protein can be characterized by any one of the consensus amino acid sequences described in SEQ ID NOs: 18-25 (SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, or 25).

[0070]

[0086] A fragment of the Salvecovirus spike protein may have a minimum length of one of 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, or 1,200 amino acids, and a maximum length of one of 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, or 1,200 amino acids.

[0071]

[0087] In various embodiments, the SARS-CoV spike protein has the amino acid sequence shown in SEQ ID NO: 9. The SARS-CoV spike protein comprises S1 (SEQ ID NO: 11) and S2 subunits. The S1 subunit includes a receptor-binding domain (RBD), which comprises SEQ ID NO: 12 or SEQ ID NO: 17, through which SARSr-CoV binds to ACE2 expressed by the host cell.

[0072]

[0088] In various embodiments, the SARS-CoV-2 spike protein has the amino acid sequence shown in SEQ ID NO: 10. The SARS-CoV-2 spike protein comprises S1 (SEQ ID NO: 13 or SEQ ID NO: 16) and S2 subunits. The S1 subunit includes a receptor-binding domain (RBD), which comprises SEQ ID NO: 14 or SEQ ID NO: 15, through which SARSr-CoV-2 binds to ACE2 expressed by the host cell.

[0073]

[0089] In various embodiments, the RBD of the salvecovirus spike protein refers to a polypeptide having the amino acid sequence shown in any one of SEQ ID NOs: 12, 14, 15, 17, or 26-30, or a polypeptide having at least 75%, for example, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the amino acid sequence identity to any one of SEQ ID NOs: 12, 14, 15, 17, or 26-30. Such polypeptides may include, for example, isoforms, fragments, variants of the RBD of the spike protein encoded by SARS-CoV-2, and corresponding regions of spike protein homologs derived from other SARSr-CoV or other known salvecoviruses.

[0074]

[0090] In various embodiments, the RBD fragment of the Salvecovirus spike protein may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, or 200 amino acids, or a maximum length of one of 20, 10, 20, 30, 40, 50, 100, 150, or 200 amino acids.

[0075]

[0091] In various embodiments, the isoforms, fragments, variants, or homologs may optionally be functional isoforms, fragments, variants, or homologs that possess the functional properties / activities of a reference protein, which are determined by analysis of a suitable assay for functional properties / activity when they bind to and / or enter host cells, for example, via ACE2. For example, isoforms, fragments, variants, or homologs of the Salvecovirus spike protein may exhibit association with ACE2.

[0076]

[0092] In various embodiments, the salvecovirus spike protein comprises an amino acid sequence having at least 75% amino acid sequence identity with any one of the consensus salvecovirus spike protein SEQ ID NOs. 18-25 (SEQ ID NOs. 18, 19, 20, 21, 22, 23, 24, or 25), for example, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identity. In various embodiments, the Salvecovirus spike protein contains or comprises an amino acid sequence having at least 75% amino acid sequence identity with any one of SEQ ID NOs. 9, 10, or 18-25, for example, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0077]

[0093] In various embodiments, the fragment of the Salvecovirus spike protein contains or comprises an amino acid sequence having at least 75% amino acid sequence identity with SEQ ID NOs. 13-16, for example, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In various embodiments, the fragment of the Salvecovirus spike protein contains or comprises an amino acid sequence having at least 75% amino acid sequence identity with SEQ ID NOs. 12, 14, 15, 17, or 26-30, for example, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In various embodiments, the fragment of the Salvecovirus spike protein contains or comprises an amino acid sequence having at least 75% amino acid sequence identity with respect to SEQ ID NO: 26, for example, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In various embodiments, the fragment of the Salvecovirus spike protein contains or comprises an amino acid sequence having at least 75% amino acid sequence identity with respect to SEQ ID NO: 27, for example, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In various embodiments, the fragment of the Salvecovirus spike protein contains or comprises an amino acid sequence having at least 75% amino acid sequence identity with respect to SEQ ID NO: 28, for example, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In various embodiments, the fragment of the Salvecovirus spike protein contains or comprises an amino acid sequence having at least 75% amino acid sequence identity with respect to SEQ ID NO: 29, for example, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.In various embodiments, the fragment of the Salvecovirus spike protein contains or comprises an amino acid sequence having at least 75% amino acid sequence identity with respect to SEQ ID NO: 30, for example, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0078]

[0094] In some embodiments, the RBD fragment of the SARS-CoV-2 spike protein contains or comprises an amino acid sequence having at least 75% amino acid sequence identity with respect to SEQ ID NOs. 12, 14, 15, 17, or 26-30, for example, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0079]

[0095] In various embodiments, the antigen-binding molecule includes two antigen-binding molecules that bind to the Salvecovirus spike protein.

[0096] In various embodiments, the antigen-binding molecule binds to the receptor-binding domain (RBD) of the Salvecovirus spike protein.

[0080]

[0097] In various embodiments, the antigen-binding molecule inhibits the interaction between the Salvecovirus spike protein and angiotensinogen-converting enzyme 2 (ACE2).

[0098] In various embodiments, antigen-binding molecules inhibit infection of ACE2-expressing cells by salvecovirus.

[0081]

[0099] In various embodiments, the sarbecovirus is SARS-CoV-2, SARS-CoV-2, SARS-CoV-2 B.1.1.7, SARS-CoV-2 B.1.351, SARS-CoV-2 B.1.617.2, SARS-CoV-2 C37, SARS-CoV-2 B.1.621, SARS-CoV-2 P.1, SARS-CoV-2 BA.1, SARS-CoV-2 BA.2, SC2r-CoV BANAL-52, SC2r-CoV BANAL-236, SC2r-CoV GD-1, SC2r-CoV RaTG13, SC2r-CoV GX-P5L, SC1r-CoV Rs2018B, SC1r-CoV RsSHC014, SC1r-CoV LYRa11, SC1r-CoV The group will be selected from those including WIV-1 and SARS-CoV.

[0082]

[0100] In various embodiments, the salvecovirus SARS-CoV refers to SARSr-CoV having the nucleotide sequence GenBank:NC_004718.3 as reported by He et al., Biochem. Biophys. Res. Commun. 316(2), 476-483 (2004), and having at least 85% sequence identity with respect to the nucleotide sequence of GenBank:NC_004718.3. For example, it includes the mutant strain having a nucleotide sequence with at least one of the following sequence identities: 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more.

[0083]

[0101] In various embodiments, SARS-CoV-2 is described as follows: Wu et al., Nature This refers to SARSr-CoV having the nucleotide sequence of GenBank:NC_045512.2, as reported in 579(7798) and 265-269(2020), and includes mutant strains having a nucleotide sequence with at least 85% sequence identity to the GenBank:NC_045512.2 nucleotide sequence (for example, one of at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more sequence identity). In various embodiments, the SARS-CoV-2 variants of the Salvecovirus include the UK COVID-19 variant SARS-CoV-2 B.1.1.7, the South African COVID-19 variant SARS-CoV-2 B.1.351, also known as the 20H / 501Y.V2 or 501Y.V2 variant, the Indian variant B1.617, and the Brazilian variant P.1.

[0084]

[0102] The antigen-binding molecules of this disclosure may be designed and prepared using sequences of monoclonal antibodies (mAbs) capable of binding to the salvecovirus spike protein. Antigen-binding regions of antibodies, e.g., single-strand variable fragments (scFv), Fab, and F(ab')2 fragments, may also be used / provided. "Antigen-binding region" means any fragment of an antibody capable of binding to a target, where a given antibody is specific to the target. mAbs are one of the most efficient and powerful tools for rapid development and deployment in combating future emerging zoonotic viruses, particularly salvecovirus.

[0085]

[0103] Antibodies generally contain six complementarity-determining regions (CDRs): three located in the heavy chain variable (VH) region: HC-CDR1, HC-CDR2, and HC-CDR3, and three located in the light chain variable (VL) region: LC-CDR1, LC-CDR2, and LC-CDR3. The six CDRs together define the paratope of the antibody, which is the portion of the antibody that binds to the target antigen.

[0086]

[0104] The VH and VL regions include framework regions (FRs) on both sides of each CDR that provide a scaffold for the CDR. From the N-terminus to the C-terminus, the VH region has the following structure: N-terminus-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C-terminus, and the VL region has the following structure: N-terminus-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C-terminus. In various embodiments, the VH region includes amino acids encoded by the nucleic acid sequence described in SEQ ID NO: 31, and the VL region includes amino acids encoded by the nucleic acid sequence described in SEQ ID NO: 32. In various embodiments, the VH region includes amino acids encoded by the nucleic acid sequence described in SEQ ID NO: 33, and the VL region includes amino acids encoded by the nucleic acid sequence described in SEQ ID NO: 34. In various embodiments, the VH region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 35, and the VL region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 36. In various embodiments, the VH region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 37, and the VL region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 38. In various embodiments, the VH region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 39, and the VL region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 40. In various embodiments, the VH region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 41, and the VL region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 42. In various embodiments, the VH region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 43, and the VL region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 44. In various embodiments, the VH region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 45, and the VL region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 46.In various embodiments, the VH region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 47, and the VL region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 48. In various embodiments, the VH region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 49, and the VL region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 50. In various embodiments, the VH region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 51, and the VL region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 52. In various embodiments, the VH region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 53, and the VL region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 54. In various embodiments, the VH region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 55, and the VL region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 56. In various embodiments, the VH region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 57, and the VL region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 58. In various embodiments, the VH region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 59, and the VL region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 60. In various embodiments, the VH region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 61, and the VL region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 62. In various embodiments, the VH region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 63, and the VL region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 64. In various embodiments, the VH region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 65, and the VL region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 66. In various embodiments, the VH region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 67, and the VL region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 68.In various embodiments, the VH region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 69, and the VL region includes an amino acid encoded by the nucleic acid sequence described in SEQ ID NO: 70.

[0087]

[0105] In various embodiments, the antigen-binding molecule includes a CDR of an antibody capable of binding to the Salvecovirus spike protein described herein, or includes a CDR derived from an antibody capable of binding to the Salvecovirus spike protein described herein. In some embodiments, the antigen-binding molecule includes a FR of an antibody capable of binding to the Salvecovirus spike protein described herein, or includes a FR derived from an antibody capable of binding to the Salvecovirus spike protein described herein. In some embodiments, the antigen-binding molecule includes a CDR and FR of an antibody capable of binding to the Salvecovirus spike protein described herein, or includes a CDR and FR derived from an antibody capable of binding to the Salvecovirus spike protein described herein. That is, in some embodiments, the antigen-binding molecule includes a VH region and a VL region of an antibody capable of binding to the Salvecovirus spike protein described herein, or includes a VH and VL region derived from an antibody capable of binding to the Salvecovirus spike protein described herein.

[0088]

[0106] In some embodiments, the antigen-binding molecule includes the CDR, FR, and / or VH and / or VL regions of an antibody that can bind to a salvecovirus spike protein selected from one of the salvecovirus spike proteins of SARS-CoV, SC2r-CoV WIV-1, SC1r-CoV RsSHC014, SARS-CoV-2, SARS-CoV-2 alpha, SARS-CoV-2 beta, SARS-CoV-2 delta, SC2r-CoV RaTG13, SC2r-CoV GD-1, and SC2r-CoV GX-P5L.

[0089]

[0107] In various embodiments, the antigen-binding molecule is (i) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 1 HC-CDR2 having the amino acid sequence of SEQ ID NO: 2 HC-CDR3 having the amino acid sequence of SEQ ID NO: 3 A heavy chain variable (VH) region incorporating, and (ii) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 4 LC-CDR2 having the amino acid sequence of SEQ ID NO: 5 LC-CDR3 having the amino acid sequence of SEQ ID NO: 6 Light chain variable (VL) region incorporating Includes.

[0090]

[0108] In various embodiments, the antigen-binding molecule includes a VH region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 2, 3, or 4, and a VL region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5, 6, or 7.

[0091]

[0109] In various embodiments, the antigen-binding molecule includes a VH region containing an amino acid sequence having at least 75% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, for example, one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0092]

[0110] In various embodiments, the antigen-binding molecule includes a VH region containing an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 2, for example, one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0093]

[0111] In various embodiments, the antigen-binding molecule includes a VH region containing an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 3, for example, one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0094]

[0112] In various embodiments, the antigen-binding molecule includes a VL region containing an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 4, for example, one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0095]

[0113] In various embodiments, the antigen-binding molecule includes a VL region containing an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 5, for example, one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0096]

[0114] In various embodiments, the antigen-binding molecule includes a VL region containing an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 6, for example, one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0097]

[0115] In embodiments of the present disclosure in which one or more amino acids are substituted with other amino acids, the substitutions may be conservative substitutions, for example, an aliphatic amino acid is substituted with another aliphatic amino acid, e.g., a nonpolar amino acid G, or A, or P, or I, or L, or V is substituted with a different nonpolar amino acid; e.g., a polar uncharged amino acid C, or S, or T, or M, or N, or Q is substituted with a different polar uncharged amino acid, e.g., a polar charged amino acid D, or E, or K, or R is substituted with a different polar charged amino acid; or an aromatic amino acid is substituted with another aromatic amino acid, e.g., H, or F, or W, or Y is substituted with a different aromatic amino acid.

[0098]

[0116] In variable embodiments, substitutions may be function-conserving. That is, in some embodiments, substitutions may have no effect (or substantially no effect) on one or more functional properties (e.g., target binding) of the antigen-binding molecule containing the substitution compared to an equivalent unsubstituted molecule.

[0099]

[0117] The VH and VL regions of the antigen-binding domain of the antibody together constitute the Fv region. In some embodiments, the antigen-binding molecule of this disclosure includes or consists of an Fv region that binds to the Salvecovirus spike protein. In various embodiments, the VH and VL regions of Fv may be provided as a single polypeptide linked by a linker region, i.e., a single-stranded Fv (scFv).

[0100]

[0118] The VL and light chain constant (CL) regions, as well as the VH region and heavy chain constant 1 (CH1) region of the antigen-binding region of an antibody, together constitute a Fab region. In some embodiments, the antigen-binding molecule includes a Fab region comprising VH, CH1, VL, and CL (e.g., Cκ or Cλ). In various embodiments, the Fab region comprises a polypeptide comprising VH and CH1 (e.g., a VH-CH1 fusion polypeptide) and a polypeptide comprising VL and CL (e.g., a VL-CL fusion polypeptide). In various embodiments, the Fab region comprises a polypeptide comprising VH and CL (e.g., a VH-CL fusion polypeptide) and a polypeptide comprising VL and CH (e.g., a VL-CH1 fusion polypeptide); that is, in some embodiments, the Fab region is a CrossFab region. In various embodiments, the VH, CH1, VL, and CL regions of the Fab or CrossFab are provided as a single polypeptide linked by a linker region, i.e., as a single-stranded Fab (scFab) or single-stranded CrossFab (scCrossFab).

[0101]

[0119] In various embodiments, the antigen-binding molecule of this disclosure comprises or consists of a Fab region that binds to the Salvecovirus spike protein.

[0120] In various embodiments, the antigen-binding molecules described herein include or consist of a whole antibody that binds to the Salvecovirus spike protein. As used herein, “whole antibody” means an antibody having a structure substantially similar to that of immunoglobulin (Ig).

[0102]

[0121] G-type immunoglobulins (i.e., IgG) are glycoproteins of approximately 150 kDa containing two heavy chains and two light chains. From the N-terminus to the C-terminus, the heavy chains consist of a heavy chain constant region containing VH and three subsequent constant domains (CH1, CH2, and CH3), and similarly, the light chains consist of VL and a subsequent CL. Depending on the heavy chains, immunoglobulins can be classified as IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM. The light chains can be kappa (κ) or lambda (λ).

[0103]

[0122] In some embodiments, the antigen-binding molecules described herein include or consist of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM that bind to the Salvecovirus spike protein.

[0104]

[0123] In some embodiments, the antigen-binding molecule of the Disclosure comprises one or more regions of an immunoglobulin heavy chain constant sequence (e.g., CH1, CH2, CH3, etc.). In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from the heavy chain constant sequences of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM, for example, human IgG (e.g., hIgG1, hIgG2, hIgG3, hIgG4), hIgA (e.g., hIgA1, hIgA2), hIgD, hIgE, or hIgM. In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from the heavy chain constant sequence of a human IgG1 allotype (e.g., G1m1, G1m2, G1m3, or G1m17).

[0105]

[0124] In various embodiments, there are antigen-binding molecules discussed above herein for use in the treatment or prevention of diseases caused by infection with salvecovirus.

[0106]

[0125] In various embodiments, the antigen-binding molecules discussed above herein are suitable for use in individuals who have experienced SARS-CoV-2 infection or vaccination.

[0126] In various embodiments, the antigen-binding molecules discussed above herein are suitable for use in individuals uninfected or unvaccinated against any salvecovirus, including SARS-CoV, SARS-CoV-2, SARS-CoV-2 alpha, or SARS-CoV-2 beta and SARS-CoV-2 delta.

[0107]

[0127] In various embodiments, the antigen-binding molecules discussed above herein are suitable for use in the treatment of individuals diagnosed with salvecovirus infection.

[0128] In various embodiments, the sarbecovirus is SARS-CoV-2, SARS-CoV-2, SARS-CoV-2 B.1.1.7, SARS-CoV-2 B.1.351, SARS-CoV-2 B.1.617.2, SARS-CoV-2 C37, SARS-CoV-2 B.1.621, SARS-CoV-2 P.1, SARS-CoV-2 BA.1, SARS-CoV-2 BA.2, SC2r-CoV BANAL-52, SC2r-CoV BANAL-236, SC2r-CoV GD-1, SC2r-CoV RaTG13, SC2r-CoV GX-P5L, SC1r-CoV Rs2018B, SC1r-CoV RsSHC014, SC1r-CoV LYRa11, SC1r-CoV The group will be selected from those including WIV-1 and SARS-CoV.

[0108]

[0129] In various embodiments, there is use of the antigen-binding molecules discussed above herein in the manufacture of pharmaceuticals for use in the treatment or prevention of diseases caused by infection with salvecovirus.

[0109]

[0130] In various embodiments, the use of antigen-binding molecules discussed above herein in the manufacture of pharmaceuticals is suitable for use in the treatment or prevention of individuals who have experienced SARS-CoV-2 vaccination or infection.

[0110]

[0131] In various embodiments, the use of antigen-binding molecules discussed above herein is suitable for use in the treatment or prevention of individuals uninfected or unvaccinated against any salvecovirus, including SARS-CoV, SARS-CoV-2, SARS-CoV-2 alpha, SARS-CoV-2 beta, or SARs-CoV-2 delta.

[0111]

[0132] In various embodiments, the use of antigen-binding molecules discussed above herein in the manufacture of pharmaceuticals is suitable for the treatment of individuals diagnosed with salvecovirus infection.

[0133] In various embodiments, the sarbecovirus is SARS-CoV-2, SARS-CoV-2, SARS-CoV-2 B.1.1.7, SARS-CoV-2 B.1.351, SARS-CoV-2 B.1.617.2, SARS-CoV-2 C37, SARS-CoV-2 B.1.621, SARS-CoV-2 P.1, SARS-CoV-2 BA.1, SARS-CoV-2 BA.2, SC2r-CoV BANAL-52, SC2r-CoV BANAL-236, SC2r-CoV GD-1, SC2r-CoV RaTG13, SC2r-CoV GX-P5L, SC1r-CoV Rs2018B, SC1r-CoV RsSHC014, SC1r-CoV LYRa11, SC1r-CoV The group will be selected from those including WIV-1 and SARS-CoV.

[0112]

[0134] In various embodiments, there exist methods for treating or preventing diseases caused by infection with salvecovirus, comprising the step of administering a therapeutic or prophylactic dose of the antigen-binding molecules discussed herein above to the target.

[0113]

[0135] In various embodiments, the subjects are individuals who have been vaccinated against or have experienced infection with SARS-CoV-2.

[0136] In various embodiments, the subjects are individuals diagnosed with salvecovirus infection.

[0114]

[0137] In various embodiments, the subjects are individuals who are uninfected or unvaccinated against any salvecovirus, including SARS-CoV, SARS-CoV-2, SARS-CoV-2 alpha, SARS-CoV-2 beta, or SARS-CoV-2 delta.

[0115]

[0138] In various embodiments, the sarbecovirus is SARS-CoV-2, SARS-CoV-2, SARS-CoV-2 B.1.1.7, SARS-CoV-2 B.1.351, SARS-CoV-2 B.1.617.2, SARS-CoV-2 C37, SARS-CoV-2 B.1.621, SARS-CoV-2 P.1, SARS-CoV-2 BA.1, SARS-CoV-2 BA.2, SC2r-CoV BANAL-52, SC2r-CoV BANAL-236, SC2r-CoV GD-1, SC2r-CoV RaTG13, SC2r-CoV GX-P5L, SC1r-CoV Rs2018B, SC1r-CoV RsSHC014, SC1r-CoV LYRa11, SC1r-CoV The group will be selected from those including WIV-1 and SARS-CoV.

[0116]

[0139] In various embodiments, there are optionally isolated nucleic acids or a set of nucleic acids encoding the antigen-binding molecules discussed above herein.

[0140] In various embodiments, there are expression vectors or multiple expression vectors containing the nucleic acids or multiple nucleic acids discussed above in this specification.

[0117]

[0141] In various embodiments, there are nucleic acids or multiple nucleic acids, or expression vectors or multiple expression vectors containing nucleic acids or multiple nucleic acids, that can express the antigen-binding molecules discussed herein for use in the treatment or prevention of diseases caused by infection with salvecovirus.

[0118]

[0142] In various embodiments, the nucleic acids or multiple nucleic acids, or expression vectors or multiple expression vectors containing the nucleic acids or multiple nucleic acids, which can express the antigen-binding molecules discussed above herein, are suitable for use in individuals that have experienced SARS-CoV-2 infection or vaccination.

[0119]

[0143] In various embodiments, the nucleic acids or multiple nucleic acids, or expression vectors or multiple expression vectors containing the nucleic acids or multiple nucleic acids, which can express the antigen-binding molecules discussed herein, are suitable for use in individuals uninfected or unvaccinated against any salvecovirus, including SARS-CoV, SARS-CoV-2, SARS-CoV-2 B.1.1.7, or SARS-CoV-2 B.1.351.

[0120]

[0144] In various embodiments, nucleic acids or multiple nucleic acids, or expression vectors or multiple expression vectors containing nucleic acids or multiple nucleic acids, which can express the antigen-binding molecules discussed above herein, are suitable for use in the treatment of individuals diagnosed with salvecovirus infection.

[0121]

[0145] In various embodiments, the sarbecovirus is SARS-CoV-2, SARS-CoV-2, SARS-CoV-2 B.1.1.7, SARS-CoV-2 B.1.351, SARS-CoV-2 B.1.617.2, SARS-CoV-2 C37, SARS-CoV-2 B.1.621, SARS-CoV-2 P.1, SARS-CoV-2 BA.1, SARS-CoV-2 BA.2, SC2r-CoV BANAL-52, SC2r-CoV BANAL-236, SC2r-CoV GD-1, SC2r-CoV RaTG13, SC2r-CoV GX-P5L, SC1r-CoV Rs2018B, SC1r-CoV RsSHC014, SC1r-CoV LYRa11, SC1r-CoV The group will be selected from those including WIV-1 and SARS-CoV.

[0122]

[0146] In various embodiments, there are nucleic acids or multiple nucleic acids, or expression vectors or multiple expression vectors containing nucleic acids or multiple nucleic acids, that can express the antigen-binding molecules discussed herein, for use in the manufacture of pharmaceuticals for use in the treatment or prevention of diseases caused by infection with salvecovirus.

[0123]

[0147] In various embodiments, the use of a nucleic acid or multiple nucleic acids, an expression vector containing a nucleic acid or multiple nucleic acids, or multiple expression vectors capable of expressing the antigen-binding molecule discussed herein in the manufacture of pharmaceuticals is suitable for use in the treatment or prevention of individuals who have experienced SARS-CoV-2 vaccination or infection.

[0124]

[0148] In various embodiments, the use of nucleic acids or multiple nucleic acids, expression vectors comprising nucleic acids or multiple nucleic acids, or multiple expression vectors, which can express the antigen-binding molecules discussed herein, in the manufacture of pharmaceuticals, is suitable for use in the treatment or prevention of individuals uninfected or unvaccinated against any salvecovirus, including SARS-CoV, SARS-CoV-2, SARS-CoV-2 alpha, SARS-CoV-2 beta, or SARS-CoV-2 delta.

[0125]

[0149] In various embodiments, the use of a nucleic acid or multiple nucleic acids, an expression vector containing a nucleic acid or multiple nucleic acids, or multiple expression vectors, capable of expressing the antigen-binding molecule discussed herein, in the manufacture of pharmaceuticals, is suitable for use in the treatment of individuals diagnosed with salvecovirus infection.

[0126]

[0150] In various embodiments, the sarbecovirus is SARS-CoV-2, SARS-CoV-2, SARS-CoV-2 B.1.1.7, SARS-CoV-2 B.1.351, SARS-CoV-2 B.1.617.2, SARS-CoV-2 C37, SARS-CoV-2 B.1.621, SARS-CoV-2 P.1, SARS-CoV-2 BA.1, SARS-CoV-2 BA.2, SC2r-CoV BANAL-52, SC2r-CoV BANAL-236, SC2r-CoV GD-1, SC2r-CoV RaTG13, SC2r-CoV GX-P5L, SC1r-CoV Rs2018B, SC1r-CoV RsSHC014, SC1r-CoV LYRa11, SC1r-CoV The group will be selected from those containing WIV-1 and SARS-CoV.

[0127]

[0151] In various embodiments, there exist methods for treating or preventing diseases caused by infection with salvecovirus, comprising the step of administering a therapeutic or prophylactic effective amount of an antigen-binding molecule to a target, wherein the antigen-binding molecule is expressed by the nucleic acids or multiple nucleic acids discussed herein, or by the expression vectors or multiple expression vectors discussed herein.

[0128]

[0152] In various embodiments, the subjects are individuals who have been vaccinated against or have experienced infection with SARS-CoV-2.

[0153] In various embodiments, the subjects are individuals who are uninfected or unvaccinated against any salvecovirus, including SARS-CoV, SARS-CoV-2, SARS-CoV-2 alpha, SARS-CoV-2 beta, or SARS-CoV-2 delta.

[0129]

[0154] In various embodiments, the subjects are individuals diagnosed with salvecovirus infection.

[0155] In various embodiments, the sarbecovirus is SARS-CoV-2, SARS-CoV-2, SARS-CoV-2 B.1.1.7, SARS-CoV-2 B.1.351, SARS-CoV-2 B.1.617.2, SARS-CoV-2 C37, SARS-CoV-2 B.1.621, SARS-CoV-2 P.1, SARS-CoV-2 BA.1, SARS-CoV-2 BA.2, SC2r-CoV BANAL-52, SC2r-CoV BANAL-236, SC2r-CoV GD-1, SC2r-CoV RaTG13, SC2r-CoV GX-P5L, SC1r-CoV Rs2018B, SC1r-CoV RsSHC014, SC1r-CoV LYRa11, SC1r-CoV The group will be selected from those including WIV-1 and SARS-CoV.

[0130]

[0156] In various embodiments, there are cells containing the antigen-binding molecules discussed above herein, the nucleic acids or multiple nucleic acids discussed above herein, or the expression vector or multiple expression vectors discussed above herein.

[0131]

[0157] In various embodiments, there exist methods for producing antigen-binding molecules that bind to the Salvecovirus spike protein, the methods comprising the step of culturing the cells discussed above herein under conditions suitable for the expression of the antigen-binding molecules by said cells.

[0132]

[0158] In various embodiments, there are cells discussed above herein for use in the treatment or prevention of diseases caused by infection with salvecovirus.

[0133]

[0159] In various embodiments, the cells discussed above herein are suitable for use in individuals that have experienced SARS-CoV-2 infection or vaccination.

[0160] In various embodiments, the cells discussed above herein are suitable for use in individuals that are uninfected or unvaccinated against any salvecovirus, including SARS-CoV, SARS-CoV-2, SARS-CoV-2 alpha, SARS-CoV-2 beta, or SARS-CoV-2 delta.

[0134]

[0161] In various embodiments, the cells discussed above herein are suitable for use in the treatment of individuals diagnosed with salvecovirus infection.

[0162] In various embodiments, the sarbecovirus is SARS-CoV-2, SARS-CoV-2, SARS-CoV-2 B.1.1.7, SARS-CoV-2 B.1.351, SARS-CoV-2 B.1.617.2, SARS-CoV-2 C37, SARS-CoV-2 B.1.621, SARS-CoV-2 P.1, SARS-CoV-2 BA.1, SARS-CoV-2 BA.2, SC2r-CoV BANAL-52, SC2r-CoV BANAL-236, SC2r-CoV GD-1, SC2r-CoV RaTG13, SC2r-CoV GX-P5L, SC1r-CoV Rs2018B, SC1r-CoV RsSHC014, SC1r-CoV LYRa11, SC1r-CoV The group will be selected from those including WIV-1 and SARS-CoV.

[0135]

[0163] In various embodiments, there is the use of the cells discussed above herein in the manufacture of pharmaceuticals for use in the treatment or prevention of diseases caused by infection with salvecovirus.

[0136]

[0164] In various embodiments, the use of cells discussed above herein in the manufacture of pharmaceuticals is suitable for use in individuals that have been vaccinated with or infected with SARS-CoV-2.

[0137]

[0165] In various embodiments, the use of cells discussed above herein in the manufacture of pharmaceuticals is suitable for use in individuals that are uninfected or unvaccinated against any salvecovirus, including SARS-CoV, SARS-CoV-2, SARS-CoV-2 alpha, SARS-CoV-2 beta, or SARS-CoV-2 delta.

[0138]

[0166] In various embodiments, the use of cells discussed above herein in the manufacture of pharmaceuticals is suitable for use in the treatment of individuals diagnosed with salvecovirus infection.

[0139]

[0167] In various embodiments, the sarbecovirus is SARS-CoV-2, SARS-CoV-2, SARS-CoV-2 B.1.1.7, SARS-CoV-2 B.1.351, SARS-CoV-2 B.1.617.2, SARS-CoV-2 C37, SARS-CoV-2 B.1.621, SARS-CoV-2 P.1, SARS-CoV-2 BA.1, SARS-CoV-2 BA.2, SC2r-CoV BANAL-52, SC2r-CoV BANAL-236, SC2r-CoV GD-1, SC2r-CoV RaTG13, SC2r-CoV GX-P5L, SC1r-CoV Rs2018B, SC1r-CoV RsSHC014, SC1r-CoV LYRa11, SC1r-CoV The group will be selected from those including WIV-1 and SARS-CoV.

[0140]

[0168] In various embodiments, there exist methods for treating or preventing diseases caused by infection with salvecovirus, comprising the step of administering a therapeutic or prophylactic dose of an antigen-binding molecule discussed herein above to a target, wherein the antigen-binding molecule is expressed in the cells discussed herein above.

[0141]

[0169] In various embodiments, the subjects are individuals who have received SARS-CoV-2 vaccination.

[0170] In various embodiments, the subjects are individuals who are uninfected or unvaccinated against any salvecovirus, including SARS-CoV, SARS-CoV-2, SARS-CoV-2 alpha, SARS-CoV-2 beta, SARS-CoV-2 B.1.1.7, SARS-CoV-2 B.1.351, SARS-CoV-2 B.1.617.2, SARS-CoV-2 C37, SARS-CoV-2 B.1.621, SARS-CoV-2 P.1, SARS-CoV-2 BA.1, SARS-CoV-2 BA.2, or SARS-CoV-2 delta.

[0142]

[0171] In various embodiments, the sarbecovirus is SARS-CoV-2, SARS-CoV-2, SARS-CoV-2 B.1.1.7, SARS-CoV-2 B.1.351, SARS-CoV-2 B.1.617.2, SARS-CoV-2 C37, SARS-CoV-2 B.1.621, SARS-CoV-2 P.1, SARS-CoV-2 BA.1, SARS-CoV-2 BA.2, SC2r-CoV BANAL-52, SC2r-CoV BANAL-236, SC2r-CoV GD-1, SC2r-CoV RaTG13, SC2r-CoV GX-P5L, SC1r-CoV Rs2018B, SC1r-CoV RsSHC014, SC1r-CoV LYRa11, SC1r-CoV The group will be selected from those including WIV-1 and SARS-CoV.

[0143]

[0172] In various embodiments, compositions exist comprising an antigen-binding molecule as discussed above herein, a nucleic acid or multiple nucleic acids as discussed above herein, an expression vector or multiple expression vectors as discussed above herein, or a cell as discussed above herein, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0144]

[0173] In various embodiments, there are compositions discussed above herein for use in the treatment or prevention of diseases caused by infection with salvecovirus.

[0145]

[0174] In various embodiments, the compositions discussed above herein are suitable for use in individuals who have experienced SARS-CoV-2 infection or vaccination.

[0175] In various embodiments, the compositions discussed above herein are suitable for use in individuals that are uninfected or unvaccinated against any salvecovirus, including SARS-CoV, SARS-CoV-2, SARS-CoV-2 alpha, SARS-CoV-2 beta, or SARS-CoV-2 delta.

[0146]

[0176] In various embodiments, the compositions discussed above herein are suitable for use in the treatment of individuals diagnosed with a sarbecovirus infection.

[0177] In various embodiments, the sarbecovirus is selected from the group consisting of SARS-CoV-2, SARS-CoV-2 B.1.1.7, SARS-CoV-2 B.1.351, SARS-CoV-2 B.1.617.2, SARS-CoV-2 C37, SARS-CoV-2 B.1.621, SARS-CoV-2 P.1, SARS-CoV-2 BA.1, SARS-CoV-2 BA.2, SC2r-CoV BANAL-52, SC2r-CoV BANAL-236, SC2r-CoV GD-1, SC2r-CoV RaTG13, SC2r-CoV GX-P5L, SC1r-CoV Rs2018B, SC1r-CoV RsSHC014, SC1r-CoV LYRa11, SC1r-CoV WIV-1, and SARS-CoV.

[0147]

[0178] In various embodiments, there is use of the compositions discussed above herein in the manufacture of a medicament for use in the treatment or prevention of a disease caused by a sarbecovirus infection.

[0148]

[0179] In various embodiments, the use of the compositions discussed above herein in the manufacture of a medicament is suitable for use in individuals who have experienced SARS-CoV-2 vaccination.

[0149]

[0180] In various embodiments, the use of the compositions discussed above herein in the manufacture of a medicament is suitable for use in individuals who are uninfected or unvaccinated against any sarbecovirus, including SARS-CoV, SARS-CoV-2, SARS-CoV-2 alpha, SARS-CoV-2 beta, or SARS-CoV-2 delta.

[0150]

[0181] In various embodiments, the use of the compositions discussed above herein in the manufacture of a medicament is suitable for use in the treatment of an individual diagnosed with a sarbecovirus infection.

[0151]

[0182] In various embodiments, the sarbecovirus is selected from the group consisting of SARS-CoV-2, SARS-CoV-^{2} B.1.1.7, SARS-CoV-^{2} B.1.351, SARS-CoV-^{2} B.1.617.2, SARS-CoV-^{2} C37, SARS-CoV-^{2} B.1.621, SARS-CoV-^{2} P.1, SARS-CoV-^{2} BA.1, SARS-CoV-^{2} BA.2, SC2r-CoV BANAL-52, SC2r-CoV BANAL-236, SC2r-CoV GD-1, SC2r-CoV RaTG13, SC2r-CoV GX-P5L, SC1r-CoV Rs2018B, SC1r-CoV RsSHC014, SC1r-CoV LYRa11, SC1r-CoV WIV-1, and SARS-CoV.

[0152]

[0183] In various embodiments, there is a method of treating or preventing a disease caused by a sarbecovirus infection, the method comprising administering to a subject a therapeutically or prophylactically effective amount of the composition discussed above herein.

[0153]

[0184] In various embodiments, the subject is an individual who has experienced SARS-CoV-2 vaccination or infection.

[0185] In various embodiments, the subject is an individual who is uninfected or unvaccinated against any sarbecovirus, including SARS-CoV, SARS-CoV-2, SARS-CoV-2 alpha, SARS-CoV-2 beta, or SARS-CoV-2 delta.

[0154]

[0186] In various embodiments, the subject is an individual diagnosed with a sarbecovirus infection.

[0187] In various embodiments, the salvecovirus is selected from the group including SARS-CoV, SC2r-CoV WIV-1, SC1r-CoV RsSHC014, SARS-CoV-2, SARS-CoV-2 alpha, SARS-CoV-2 beta, SARS-CoV-2 delta, SC2r-CoV RaTG13, SC2r-CoV GD-1, and SC2r-CoV GX-P5L.

[0155]

[0188] In various embodiments, there are uses of the antigen-binding molecules discussed above herein to inhibit infection of ACE2-expressing cells by salvecovirus.

[0189] In various embodiments, the salvecovirus is selected from the group including SARS-CoV, SC2r-CoV WIV-1, SC1r-CoV RsSHC014, SARS-CoV-2, SARS-CoV-2 alpha, SARS-CoV-2 beta, SARS-CoV-2 delta, SC2r-CoV RaTG13, SC2r-CoV GD-1, and SC2r-CoV GX-P5L.

[0156]

[0190]

[0157] [Table A-1]

[0158] [Table A-2]

[0159] [Table A-3]

[0160] [Table A-4]

[0161] [Table A-5]

[0162] Table A-6

[0163] Table A-7

[0164] Table A-8

[0165] Table A-9

[0166] Table A-10

[0167] Table A-11

[0168] Table A-12

[0169] Table A-13

[0170] Table A-14

[0171] Table A-15

[0172] Table A-16

[0173] Table A-17

[0174] Table A-18

[0175] Table A-19

[0176] Table A-20

[0177] Table A-21

[0178] Table A-22

[0179] Table A-23

[0180] Table A-24

[0181] Table A-25

[0182] Table A-26

[0183] Table A-27

[0184]

[0191] In sequence numbers 31-70 listed above, the nucleic acids encoding the CDR region are shown in bold and underlined. In sequence numbers 71-110 listed above, the amino acids in the CDR region are shown in bold and underlined.

[0185]

[0192] Examples

[0193] In the following, the present inventors present antigen-binding molecules that bind to a wide range of Salvecovirus spike proteins simply as examples. [Examples]

[0186]

[0194] Example 1: Human serum panel

[0195] The four serum panels included in this study were described as follows: (1) SARS patients (n=11): These were serums collected from SARS survivors in Singapore at different points in time (2003, 2012, and 2020) prior to the start of the vaccination program in February 2021; (2) COVID-19 patients (n=40): Serums from this group were collected during 2020 as part of a nationwide longitudinal study (Chia et al.: Lancet Microbe 2021); (3) Healthy individuals vaccinated with the COVID-19 vaccine (in this case, Pfizer mRNA vaccine) (n=20): These were serums collected 14 days after the second dose of the Pfizer-BioNTech BNT162b2 mRNA vaccine (or 35 days after the first dose). (4) SARS survivors (n=9) who received the COVID-19 vaccine (in this case, Pfizer mRNA vaccine): Serum obtained from SARS survivors 21 to 62 days after the first dose of Pfizer-BioNTech BNT162b2 mRNA vaccine.

[0187]

[0196] Surprisingly, when SARS survivors were immunized with a COVID-19 vaccine (in this case, a Pfizer mRNA vaccine), the inventors found an unexpectedly high boost in anti-SARS-CoV NAb (see Table 1).

[0188] [Table 1]

[0189]

[0197] Given that SARS-CoV and SARS-CoV-2 share approximately 80% genomic identity, some level of boosting was expected (Zhou et al.: Nature 2020, 579:270~273), but the multiplier increase in SARS-CoV-specific NAb (5-6 times) was unexpected. Importantly, this observation was not limited to the first two SARS survivors tested, and this trend persisted when more individuals (N=9) were tested (see below).

[0190]

[0198] Multiple surrogate virus neutralization (sVNT) test based on RBD derived from six different salvecoviruses.

[0199] Viral RBDs were immobilized on a solid phase (magnetic beads) and used with a fluorescent dye conjugated to ACE2, in this case phycoerythrin (PE), to measure virus-receptor binding, enabling multiple detection of NAbs against different salvecoviruses ([Figure 2]). A total of six RBD proteins were used, derived from six different salvecoviruses: SARS-CoV, SARS-CoV-2, SARS-CoV-2 B.1.1.7, SARS-CoV-2 B1.351, SC2r-CoV GX-P5L, and SC2r-CoV RaTG13.

[0191]

[0200] Six different AviTag biotinylated RBDs derived from different salvecoviruses were coated onto MagPlex Avidin microspheres (Luminex) at a rate of 5 μg per 1 million beads. In multiplex sVNTs, RBD-coated microspheres (600 beads / antigen) were pre-incubated at 37°C for 1 hour with serum at a final concentration of 1:20 or higher, while agitated at 800 rpm. After 1 hour of incubation, 50 μl of PE conjugate hACE2 (GenScript, 1000 ng / ml) was added to the wells, incubated at 37°C for 30 minutes with agitation, and then washed twice with PBS-1% BSA. Data were acquired using the MAGPIX system.

[0192]

[0201] Cross-NAb data demonstrated two very important features: 1) Vaccination of SARS survivors resulted in very high NAb levels against all viruses studied, even bat and pangolin viruses ([Figure 3D]); 2) they neutralized SARS-CoV-2 variants better than naive individuals who received the usual two doses ([Figure 3C]); 3) SARS patients had minimal cross-NAb levels against any of the other five viruses before vaccination ([Figure 3A]), while COVID-19 patients had cross-NAb levels against the other viruses (all of which are SARS-CoV-2 related viruses) and very little cross-NAb level against SARS-CoV ([Figure 3B]).

[0193]

[0202] mRNA vaccines have been demonstrated to have an exceptional ability to induce very high levels of neutralizing antibodies (Nab) against SARS-CoV-2. However, data from a reported breakthrough infection (Hacisuleyman et al.: N Engl J Med 2021) and [Figure 3C] show that some individuals had lower NAb levels against the alpha COVID-19 variant SARS-CoV-2 B.1.1.7 and / or the beta COVID-19 variant SARS-CoV-2 B.1.351, also known as the 20H / 501Y.V2 or 501Y.V2 variant, clearly indicating that NAb has a relatively narrow spectrum, i.e., NAb is highly specific to the viral sequence used in mRNA vaccines. The same was observed in the serum of COVID-19 patients ([Figure 3B]).

[0194]

[0203] SARS-CoV and SARS-CoV-2 share 80% genomic identity, and cross-NAb epitopes have been found in the past (in both humans and animals). However, most of the key immunodominant neutralizing epitopes in the RBD region of their spike protein (S) are highly virus or mutant-specific. Unexpectedly, however, if cross-immunization occurs (either through infection or vaccination), it is possible to make cross-NAb epitopes more immunodominant.

[0195]

[0204] Pan-salvecovirus mAb Inhibition Assay

[0205] Using the multiple sVNTs developed above, we expanded our research to investigate cross-NAbs against SARS-CoV-2 variants and, more importantly, against other salvecoviruses detected in bats and pangolins that are thought to pose a potential risk of human infection (Lam et al.: Nature 2020, 583:282~285).

[0196]

[0206] RBD-coated microspheres (600 beads / antigen) were pre-incubated at 37°C for 1 hour with serum diluted 1:100, while agitated. Unbound antibodies were removed by two washes with PBS-1% BSA. Pan-salvecovirus mAb (1000 ng / ml) was then added, followed by incubation at 37°C for 1 hour with agitation, and then washing. Binding of pan-salvecovirus mAb to RBD was detected by PE-conjugated anti-mouse IgG antibody. Data were acquired using the MAGPIX system.

[0197]

[0207] Using serial dilutions, the best performance of pan-salvecovirus cross-neutralization by the SARS-vaccinated group was further demonstrated ([Figure 4]). This is the first real-world human study / data demonstrating the feasibility of a pan-salvecovirus antigen-binding molecule.

[0198]

[0208] Previous studies have shown that cross-neutralization between SARS-CoV and SARS-CoV-2 is limited (Yang R et al., EBioMedicine 2020, 58:102890). However, when SARS survivors were immunized with a COVID-19 vaccine (in this case, a Pfizer mRNA vaccine), the inventors found that high levels of cross-NAbs capable of neutralizing the six different salvecoviruses used in this study were generated ([Figure 4]). This is the first human study / data to demonstrate the feasibility of producing pan-salvecovirus NAbs with high efficacy and broad spectrum.

[0199]

[0209] mRNA vaccines have demonstrated an exceptional ability to induce very high levels of NAb against SARS-CoV-2. However, data from reported breakthrough infections (Hacisuleyman et al., N Engl J Med 2021) and [Figure 4] clearly indicate that NAb has a relatively narrow spectrum, as some individuals had lower NAb against the alpha COVID-19 variant SARS-CoV-2 B.1.1.7 and / or the beta COVID-19 variant SARS-CoV-2 B.1.351, also known as the 20H / 501Y.V2 or 501Y.V2 variant, as well as even lower NAb against unemerged bat and pangolin viruses, with the lowest NAb against SARS-CoV-1.

[0200]

[0210] As shown in [Figure 5], the most likely mechanism of this cross-neutralizing boosting is hypothesized to be through exposure to two separate epitopes derived from two different salvecoviruses. SARS-CoV-1 and SARS-CoV-2 share 80% genomic identity, and cross-NAb epitopes have been found in the past (in both humans and animals), but most of the key immunodominant neutralizing epitopes in the RBD region of their spike protein (S) are highly virus or mutant-specific. However, if cross-immunization occurs (either through infection or vaccination), it is possible to make cross-NAb epitopes more immunodominant.

[0201]

[0211] SARS-CoV-1 survivors who received the BNT162b2 mRNA vaccine produced broad-spectrum neutralizing antibodies against 10 salvecoviruses in clades 1a and 1b, including multiple VOCs of SARS-CoV-2 and zoonotic salvecoviruses. [Examples]

[0202]

[0212] Example 2. Mouse study

[0213] In this study, we used the mouse mAb 5B7D7(Genscript). As shown in [Figure 6A], this mAb can neutralize all six viruses, although its efficacy against GX-P5L is relatively low. We used a blocking assay on the same principle as sVNT, replacing PE-hACE2 with the mAb, to determine the ability of the mAb to block neutralization in four different serum panels ([Figure 6B]). Several important findings were obtained from this analysis. First, it is clear that the cross-neutralizing ability of the SARS-vaccinated group was the best among the four groups. Second, during natural infection (either SARS or COVID-19), the activation of cross-neutralizing antibodies across the two lineages between SARS-CoV-2 (and related viruses) and SARS-CoV-1 was minimal. Third, mRNA vaccination enhanced the overall neutralizing ability against SARS-CoV-2 related viruses, but had minimal effect on cross-neutralization against SARS-CoV-1.

[0203]

[0214] Cross-neutralization between SARS-CoV-1 and SARS-CoV-2 is not common, but this monoclonal antibody binds to RBD and cross-neutralizes both SARS-CoV-1 and SARS-CoV-2, as well as other salvecoviruses.

[0204]

[0215] B-cell profiling by staining with RBD derived from SARS-CoV-1 and SARS-CoV-2

[0216] For flow cytometry analysis, cryopreserved PBMCs were thawed, and SARS-CoV-1 and SARS-CoV-2 specific B cells were surface-stained using bait tetramers (custom-made by GenScript) prepared with biotinylated SARS-CoV-1 RBD or SARS-CoV-2 RBD, which were tetramerized using streptavidin conjugated with BV421 (Biolegend, Cat#405225) or streptavidin conjugated with PE (BD Pharmigen, Cat#554061). Briefly, thawed PBMCs were incubated with SARS-CoV-1-RBD tetramers and SARS-CoV-2-RBD tetramers in 10% FBS in FACS staining buffer (PBS supplemented with 2 mM EDTA and 2% FBS) at room temperature for 40 minutes, followed by staining with surface panel fluorescent dye-conjugated antibodies. Surface staining was performed for 30 minutes in FACS staining buffer at 4°C using a survival dye (Invitrogen, LIVE / DEAD® Fixable AQUA Dead Cell Stain), anti-human CD3 antibody conjugated with FITC, anti-human CD14 antibody conjugated with FITC, anti-human CD56 antibody conjugated with FITC, anti-human CD19 antibody conjugated with PE-cyanine 5, anti-human CD27 antibody conjugated with APC-H7, and anti-human CD38 antibody conjugated with BV786. Stained cells were washed twice with FACS staining buffer and acquired on the same day. Samples were acquired using a BD LSR Fortessa® analyzer equipped with 355, 405, 488, 561, and 640 nm lasers or a BD FACS Aria III. SARS-CoV-1 and SARS-CoV-2 specific B cells were quantified by excluding AQUA-positive dead cells and CD3+, CD14+, and CD56+ cells, followed by gating for CD19+ B cells. Cross-NAb boosting in the SARS-vaccinated group was further confirmed by direct staining of B cells using virus-specific RBD proteins.As shown in [Figure 7], double-stained B cells, i.e., B cells that bind to RBD derived from both SARS-CoV-1 and SARS-CoV-2, were significantly enriched more than 10-fold in the SARS-vaccinated group compared to the healthy-vaccinated group. [Examples]

[0205]

[0217] Example 3: Rabbit Study

[0218] We further confirmed virus / strain-specific immunodominant antibody responses using rabbit superimmune sera targeting specific viruses / strains. In addition to the four key viruses used in this study (i.e., SARS-CoV, RaTG13, GX-P5L, and SARS-CoV-2), we also included RmYN02 and HKU1. RmYN02 is a bat salvecovirus with a very close genetic relationship to SARS-CoV-2, but its RBD could not bind to hACE2 (Wacharapluesadee et al.: Nat Commun 2021, 12:972). RmYN02 is also very closely related to RacCS203, another bat salvecovirus found in bats in Thailand (Wacharapluesadee et al.: Nat Commun 2021, 12:972). HKU1 is a human betacoronavirus, not a salvecovirus, and is included here as a negative control.

[0206]

[0219] Multiplex sVNT analysis using rabbit superimmune sera targeting RBD of six different betacoronaviruses.

[0220] Rabbit anti-RBD serum was prepared using RBD of each virus as the antigen, under a commercial agreement with GenScript Biotech. The tests were performed essentially the same as those described above. Rabbit serum was used in 4-fold serial dilutions starting at 1:20.

[0207]

[0221] The data shown in [Figure 8] demonstrate that cross-neutralization is limited to the strain / lineage level only among the five salvecovirus SC2r-CoV strains. No cross-neutralization was observed between SC2r-CoV and SARS-CoV, and the negative control HKU1 did not neutralize any of the viruses / strains, as shown. It is noteworthy that RmYN02, although shown not to bind to hACE2 (Wacharapluesadee et al.: Nat Commun 2021, 12:972), can nevertheless produce neutralizing antibodies against bat salvecovirus RaTG13.

[0208]

[0222] Based on the various embodiments described above from SARS-vaccinated donors, the human pan-salvecovirus NAb is formed as follows:

[0223] Select SARS1-SARS2 bipositive B cells: As shown in [Figure 9], B cells (Type C in the figure) that produce antibodies capable of recognizing both RBDs (Reactive B cells) derived from SARS-CoV-1 and SARS-CoV-2 are selected and tested for neutralization of different salvecoviruses.

[0209]

[0224] Cloning and culturing B cells for small-scale monoclonal antibody production: Two methods are used to detect the B cell receptor. First, single B cells are grown in a 3T3 feeder system that allows continuous secretion into the supernatant of mAbs for initial screening and subsequent cloning of the best clones of the B cell receptor. Second, the selected B cells are directly lysed, the B cell receptor sequence is detected from the RNA, and the sequence is subsequently subcloned into an mAb expression plasmid.

[0210]

[0225] Testing pan-salvecovirus neutralizing activity: Use the supernatant containing individual mAbs to test cross-NAb activity using the multiplex sVNT platform described above in this specification.

[0211]

[0226] Large-scale production and further characterization: The top candidate substances are obtained for further characterization, including structural analysis for epitope mapping, determination of neutralizing activity against live viruses, and confirmation of in vivo defense in antigen-exposed animal models. [Examples]

[0212]

[0227] Example 4. Isolation of broad-spectrum salvecovirus neutralizing mAbs from SARS-1 survivor donors vaccinated with BNT162b2 vaccine.

[0228] Blood was obtained from SARS-CoV-1 survivor SS6V, and the neutralizing ability of the donor's serum against SARS-CoV-1 and SARS-CoV-2 was confirmed before and after BNT162b2 vaccination using a surrogate virus neutralization test. PBMCs and plasma 23 days after the first vaccination were isolated from EDTA whole blood and cryopreserved for long-term storage. PBMCs were thawed and first stained for SARS-CoV-1 RBD and SARS-CoV-2 RBD tetramers, then surface-stained with LIVE / DEAD Fixable aqua dead cell strain (Invitrogen), anti-human CD3-FITC, anti-human CD14-FITC, anti-human CD56-FITC, anti-human CD19-PE-Cy5, anti-human CD27-APC-H7, and anti-human CD38-BV786. These were single cells sorted into 96-well PCR plates (Axygen) pre-filled with 10 μl / well of RT-PCR capture buffer containing 10 mM TRIS pH 8.0 and 10 U of the RNasin ribonuclease inhibitor (Promega), using a BD FACSAria III (BD Biosciences) equipped with 355 nm, 405 nm, 488 nm, 561 nm, and 640 nm lasers. The plates were then rapidly frozen on dry ice and kept at -80°C until use.

[0213]

[0229] Next, reverse transcription was performed on each plate of selected B cells using the Qiagen OneStep RT-PCR kit. Nested PCR was performed using Q5 polymerase (NEB), and wells containing the corresponding heavy and light chains were purified for sequencing. After sequencing, individual gene families were amplified using specific primers, and the PCR products were cloned into pTRIOZ expression vectors (Invivogen).

[0214]

[0230] The pTRIOZ construct was transfected into HEK293 cells using Fugene6 (Promega), and the supernatant was collected to verify small-scale efficacy screening using SARS-CoV-1 and SARS-CoV-2 RBD-binding ELISA and surrogate virus neutralization assay (sVNT). For the binding ELISA assay, 100 ng of protein was coated onto Maxisorp plates (Nunc) overnight at 4°C. After blocking with OptEIA blocking buffer (BD), 50 μl of each supernatant was added directly to each well and incubated at 37°C for 1 hour. Then, 1:5000 goat anti-human IgG-HRP (Bethyl) diluted in OptEIA was added, which can be converted to a colorimetric readout for quantification using a Cytation5 reader (BioTek) as the TMB substrate. For sVNT, a commercially available kit for SARS-CoV-2 (cPass, Genscript) was used, following the manufacturer's protocol. Using the same kit, the amount of neutralizing antibody against SARS-CoV-1 was measured by using 6 ng / well of SARS-CoV-1 RBD-HRP (Genscript) instead of the SARS-CoV-2 RBD-HRP reagent. The best mAbs (SS6V1-B5, SS6V11-E7, SS6V12-E11, SS6V13-F1, SS6V19-F4, and SS6V20-F5) and control mAbs (S309, CR3022, S2X259, and LyCoV-1404) from initial binding and neutralization screening were then batch-produced by transfection into EXPI293 cells for large-scale expression and purified using protein G agarose beads (Millipore) for downstream characterization.

[0215]

[0231] The results are summarized in [Table 2].

[0216] [Table 2]

[0217]

[0232] Peripheral blood mononuclear cells (PBMCs) were obtained 23 days after the first dose of the BNT162b2 vaccine for B cell enrichment and isolation. CD19+ B cells [Figure 11] that were positive for binding to SARS-CoV-1 (SC1+) and SARS-CoV-2 (SC2+) RBD tetramers were selected, and their antibody genes were amplified and cloned. A total of 19 pairs of heavy and light chain kappa fragments were recovered from single B cells, reconstituted into pTRIOZ expression vectors, and monoclonal antibodies (mAbs) were expressed in vitro. The majority (17 of 19) were derived from double-positive (SC1+SC2+) B cells, two were derived from SC2+ cells, and none were recovered from SC1+ sole-positive B cells.

[0218]

[0233] For control and comparative studies, four publicly available broad-spectrum mAbs, namely S309 (sotorovimab, GSK), CR3022, S2X259 (VirBiotech), and LyCoV-1404 (Eli Lily), were also included in this study. Two mAbs cloned from SC2+ single-positive B cells showed minimal reactivity to SARS-CoV-1 RBD [Table 3]. All 17 mAbs recovered from SC1+SC2+ B cells showed binding to both SARS-CoV-1 and SARS-CoV-2 RBD, but their neutralizing ability varied among different mAbs. Six of the most potent neutralizers (SS6V1-B5, SS6V11-E7, SS6V12-E11, SS6V13-F1, SS6V19-F4, and SS6V20-F5) were selected for large-scale production and further characterization. These are listed above as antibodies 1, 11-13, 19, and 20. The most potent mAbs (SS6V11-E7, SS6V13-F1, and SS6V20-F5) utilize unique heavy and light chain gene family combinations not reported to date. These are listed above as antibodies 11, 13, and 20. All three mAbs use the IGHV4-59 heavy chain and IGKV2-28 / IGKJ5 light chain, suggesting a very high probability that B cells originate from the same clonal family, and that the slight differences in the heavy and light chains arose from hypersomatic mutations.

[0219] [Table 3-1]

[0220] [Table 3-2] [Examples]

[0221]

[0234] Example 5: Efficacy, range, and mutant evasion ability of pansalvecovirus mAb

[0235] 18-fold sVNT was performed based on SARS-CoV-2 ancestral virus and its variants (alpha, beta, delta, delta plus, gamma, lambda, mu); zoonotic salvecoviruses BANAL-52, BANAL-236, GD-1, RaTG13, GX-P5L, Rs2018B, LYRa11, RsSHC014, WIV-1; and RBD derived from SARS-CoV-1. Data for the top six mAbs and three control mAbs identified in preliminary screening are shown in [Figure 12]. SS6V11-E7, SS6V13-F1, and SS6V20-F5 mAbs exhibited very potent ability to neutralize all 18 salvecoviruses, with 50% neutralization titers (NT50) ranging from 10.44 to 120.30 ng / ml (0.070 to 0.802 nM). These are listed above as antibodies 11, 13, and 20. The remaining three mAbs, SS6V1-B5, SS6V12-E11, and SS6V19-F4, also showed broad-spectrum neutralizing activity, but with lower activity / inactivity against some of the viruses tested; namely, SS6V12-E11 was less active against lambda, and SS6V19-F4 was less active against lambda, GX-P5L, and RsSHC014. These are listed above as antibodies 1, 12, and 19. Their NT50s were in the range higher than 1,000 ng / ml (6.67 nM) for some clade-1b sarbcoviruses and higher than 200 ng / ml (1.33 nM) for clade-1a sarbcoviruses. Some degree of immune evasion was observed for the four control mAbs; specifically, immune evasion was observed for S309 by lambda and WIV-1, and for LyCoV-1404 by some clade-1b zoonotic salvecoviruses (RaTG13 and GX-P5L) and all clade-1a salvecoviruses (WIV-1, RsSHC014, Rs2018B, and SARS-CoV-1) [Figure 13]. Therefore, it was concluded that LyCoV-1404 is variant-proof, meaning it retains potent activity against multiple variants, but is not a pan-salvecovirus mAb.Since the primary objective was to develop a pan-salvecovirus broad-spectrum neutralizing mAb, LyCoV-1404 was not included as a control in the remaining assays. S2X259 was the only test control mAb that retained pan-salvecovirus neutralizing activity against all 18 strains tested, with an NT50 ranging from 47.39 to 370.50 ng / ml (0.316 to 2.47 nM). [Examples]

[0222]

[0236] Example 6 Functionality of Pan-Salvecovirus Neutralizing mAb

[0237] Based on data from the multiple assays shown above, SS6V11-E7, SS6V13-F1, and SS6V20-F5 were selected for further characterization along with three control mAbs, S309, CR3022, and S2X257. All control mAbs were supplied by commercial suppliers either through direct purchase or contract manufacturing.

[0223]

[0238] The functionality of these mAbs in neutralizing different salvecoviruses was further evaluated against SARS-CoV-2 ancestral and four VOCs (alpha, delta, beta, and gamma), two zoonotic salvecoviruses (GX-P5L and WIV-1), and eight spike pseudotyping reporter viruses, including SARS-CoV-1. All three test mAbs were observed to retain highly potent neutralizing activity against all eight pseudoviruses, with relative maximum half-volume neutralization titers (NT50) less than 10 ng / ml (0.067 nM) (Figure 14). S309 had an NT50 of 100–1000 ng / ml (0.667–6.67 nM) against SARS-CoV-2 VOCs and unemerged salvecoviruses. Overall, S2X259 performed better than S309. However, its potency was about one-tenth that of the three mAbs identified in this study. CR3022 was only able to neutralize some clade-1a salvecoviruses (WIV-1 and SARS-CoV-1), and was unable to neutralize any clade-1b salvecoviruses. [Examples]

[0224]

[0239] Example 7: Ability to neutralize different sub-strains of omicron virus

[0240] During the final stages of this study, a new sub-strain, Omicron BA.2, emerged, which is becoming equally dominant over the original Omicron BA.1 virus. To determine the neutralizing ability of the newly identified mAb against these two viral variants, we performed tests using three different platforms: multiplex sVNT, pseudovirus neutralization test (pVNT), and plaque reduction neutralization test (PRNT).

[0225]

[0241] Serum samples were tested using a newly developed multiplex sVNT assay. Briefly, AviTag biotinylated RBD proteins derived from ancestral SARS-CoV-2 and SARS-CoV-1, nine VOC / VOI (alpha, delta, beta, gamma, delta plus, lambda, mu, omicron BA.1, omicron BA.2), and ten zoonotic salvecoviruses (BANAL-52, BANAL-236, GD-1, RaTG13, GX-P5L, Rs2018B, LYRa11, RsSHC014, and WIV-1) were coated onto MagPlex Avidin microspheres (Luminex) at a rate of 5 μg per 1 million beads. RBD-coated microspheres (600 beads / antigen) were pre-incubated at 37°C for 15 minutes with 4-fold serial dilutions of mAb at an initial concentration of 10,000 ng / ml, while stirring at 250 rpm. After 15 minutes of incubation, 50 μL of 2 μg / mL phycoerythrin (PE) conjugate hACE2 (GenScript) was added to the wells and incubated at 37°C for 15 minutes with stirring, followed by two washes with PBS-1% BSA. Final readings were obtained using a MAGPIX system (Luminex Corporation). The data shown in [Figure 15] indicate that SS6V11-E7 and SS6V20-F5 retained potent activity against both BA.1 and BA.2 omicrons, with mean NT50 ranging from 177 to 315 ng / ml (1.18 to 2.10 nM) in multiple sVNTs.

[0226]

[0242] SARS-CoV-2 Wuhan-hu-1 (ancestor), alpha, delta, beta, gamma, omicron BA.1, omicron BA.2, GX-P5L, WIV-1, and full-length spike pseudotyped SARS-CoV-1 viruses were constructed and packaged. Briefly, 5 million HEK293T cells were transfected with 20 μg of pCAGGS spike plasmid using FuGENE6 (Promega). 24 hours after transfection, cells were incubated with VSVΔG luc seed virus (MOI of 5) for 2 hours. After washing twice with PBS, infected cells were supplemented with complete growth medium supplemented with anti-VSV-G mAb (clone 8GF11, Kerafast) diluted 1:5000. 24 hours after infection, pseudoviruses were recovered by centrifugation at 2000 × g for 5 minutes. For the pVNT assay, 3 × 10⁶ 6 RLU pseudoviruses were pre-incubated at 37°C for 1 hour with mAbs serially diluted fourfold to a final volume of 50 μl at an initial concentration of 20 ug / ml, and then used to infect ACE2-stable-expressing A549 cells. 20–24 hours after infection, an equal volume of ONE-Glo luciferase substrate (Promega) was added, and the luminescence signal was measured using a Cytation5 microplate reader (BioTek) with Gen5 software, version 3.10.

[0227]

[0243] The mAbs were serially diluted fourfold from an initial concentration of 20 ug / ml using DMEM containing 2% FBS. Next, SARS-CoV-2 virus (ancestral or Omicron BA.1 and BA.2 strains) was diluted to 500 PFU / ml, mixed with the diluted mAbs, and incubated at 37°C for 1 hour to conjugate the mAbs to the virus. After 1 hour, the mAb-virus mixture was added to an A549-ACE2 monolayer and incubated at 37°C for a further 1 hour. The inoculum was then decontaminated. The cells were supplemented with plaque medium (DMEM supplemented with 2% FBS, 0.8% Avicel, and 0.2% carboxymethylcellulose) and incubated at 37°C for 3 days. The plaques were fixed and stained with 10% buffered formalin and 0.2% crystal violet, respectively.

[0228]

[0244] In reliable viral neutralization tests, only SS6V11-E7, listed above as antibody 11, consistently demonstrated the ability to neutralize BA.2 at NT50 concentrations of 1400 ng / ml (9.3 nM) or 500 ng / ml (3.33 nM), respectively, using pVNT or PRNT assays (see Figures 15B and 15C). The approximately 2-4-fold difference in neutralizing efficacy against BA.2 mutants measured by live virus assays (pVNT and PRNT) compared to biochemical multiplex sVNT assays suggests the possibility of additional antibody evasion facilitated by mutations present in the complete BA.2 spike, which was evident in the live virus assays. Nevertheless, we observed that only SS6V11-E7 maintained neutralizing ability against omicron BA.2, while all other mAbs tested, including three control mAbs, lost their efficacy completely.

[0229]

[0246] It should be further understood by those skilled in the art that variations and combinations of the features described above, which are neither substitutes nor replacements, can be combined to form further embodiments that fall within the intended scope of the present invention.

[0230]

[0247] As can be understood by those skilled in the art, each embodiment can be used in combination with other embodiments or several embodiments.

Claims

1. An antigen-binding molecule that binds to salvecovirus spike proteins derived from two or more different salvecoviruses, (i) The following CD-Rs: HC-CDR1 having the amino acid sequence GGFIGPHY HC-CDR2 having the amino acid sequence IYISGST HC-CDR3 having the amino acid sequence ARGGGYLETGPFEY A heavy chain variable (VH) region incorporating, and (ii) The following CD-Rs: LC-CDR1 having the amino acid sequence QSLLQNNNGYNY LC-CDR2 having amino acid sequence LSS LC-CDR3 having the amino acid sequence MQSLQIPGT Light chain variable (VL) region incorporating Antigen-binding molecules, including those mentioned above.

2. The antigen-binding molecule according to claim 1, comprising a VH region having the amino acid sequence of SEQ ID NO: 91 and a VL region having the amino acid sequence of SEQ ID NO:

92.

3. An antigen-binding molecule according to claim 1 or 2, which binds to the receptor-binding domain (RBD) of the Salvecovirus spike protein.

4. An antigen-binding molecule according to claim 1 or 2, which inhibits the interaction between the salvecovirus spike protein and angiotensinogen-converting enzyme 2 (ACE2).

5. An antigen-binding molecule according to claim 1 or 2, which inhibits infection of ACE2-expressing cells by salvecovirus.

6. The two or more different sarbecoviruses include SARS-CoV-2, SARS-CoV-2 B. 1.1.7, SARS-CoV-2 B. 1.351, SARS-CoV-2 B. 1.617.2, SARS-CoV-2 C37, SARS-CoV-2 B. 1.621, SARS-CoV-2 P.

1. SARS-CoV-2 BA.

1. SARS-CoV-2 BA.

2. An antigen-binding molecule according to claim 1 or 2, selected from the group comprising SC2r-CoV BANAL-52, SC2r-CoV BANAL-236, SC2r-CoV GD-1, SC2r-CoV RaTG13, SC2r-CoV GX-P5L, SC1r-CoV Rs2018B, SC1r-CoV RsSHC014, SC1r-CoV LYRa11, SC1r-CoV WIV-1, and SARS-CoV.

7. An optionally isolated nucleic acid or a plurality of nucleic acids encoding an antigen-binding molecule according to any one of claims 1 to 6.

8. An expression vector or a plurality of expression vectors comprising the nucleic acid or plurality of nucleic acids described in claim 7.

9. A cell comprising an antigen-binding molecule according to any one of claims 1 to 6, a nucleic acid or a plurality of nucleic acids according to claim 7, or an expression vector or a plurality of expression vectors according to claim 8.

10. A method for producing an antigen-binding molecule that binds to the Salvecovirus spike protein, comprising the step of culturing cells containing the nucleic acid or a plurality of nucleic acids described in claim 7, or the expression vector or a plurality of expression vectors described in claim 8, under conditions suitable for the expression of the antigen-binding molecule by the cells.

11. A composition comprising an antigen-binding molecule according to any one of claims 1 to 6, a nucleic acid or a plurality of nucleic acids according to claim 7, an expression vector or a plurality of expression vectors according to claim 8, or a cell according to claim 9, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

12. A pharmaceutical for the treatment or prevention of a disease caused by infection with Salvecovirus, comprising as an active ingredient: an antigen-binding molecule according to any one of claims 1 to 6, a nucleic acid or plurality of nucleic acids according to claim 7, an expression vector or plurality of expression vectors according to claim 8, a cell according to claim 9, or a composition according to claim 11.

13. The sarbecoviruses include SARS-CoV-2, SARS-CoV-2 B. 1.1.7, SARS-CoV-2 B. 1.351, SARS-CoV-2 B. 1.617.2, SARS-CoV-2 C37, SARS-CoV-2 B. 1.621, SARS-CoV-2 P.

1. SARS-CoV-2 BA.

1. SARS-CoV-2 BA.

2. The pharmaceutical product according to claim 12, selected from the group comprising SC2r-CoV BANAL-52, SC2r-CoV BANAL-236, SC2r-CoV GD-1, SC2r-CoV RaTG13, SC2r-CoV GX-P5L, SC1r-CoV Rs2018B, SC1r-CoV RsSHC014, SC1r-CoV LYRa11, SC1r-CoV WIV-1, and SARS-CoV.

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