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 May 15, 2021, Singapore Patent Application No. 10202107013P filed on June 25, 2021, and Singapore Patent Application No. 10202204610V filed on April 28, 2022, the contents of which are incorporated herein by reference.

[0002]

[0002] The present disclosure relates generally to molecules, such as protein antigen-binding molecules, suitable for use in the treatment or prevention of coronavirus infections, particularly SARS-associated betacoronaviruses (sarbecoviruses). [Background technology]

[0003] The following discussion of the background of the present invention is intended only to facilitate an understanding of the present invention. It should be understood that this discussion is not an admission or acknowledgement that any of the material referred to was published, publicly known, or part of the common general knowledge of those skilled in the art as of the priority date of the present invention, to any extent.

[0004] Emerging zoonotic viruses have become a major threat to public health and the global economy in the past two decades, when the world has undergone three major human infectious disease pandemics caused by coronaviruses (CoVs). These include SARS in 2002-2003 (Peiris et al., Nat Med 2004, 10:S88-97), caused by SARS-CoV, 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. With regard to SARS and MERS, we still have no approved treatment or preventive measures against future infections by these viruses. With regard to COVID-19, the unprecedented speed of vaccine development has resulted in many licensed vaccines for human use (Fauci Science 2021, 372:109).

[0005]

[0005] A large number of CoVs are present in wild reservoir animals, especially bats. It is likely that in the future outbreaks (SARS3, SARS4, etc.) will be caused 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 the viruses with the highest risk of infection to humans are all from the genus Betacoronavirus (Nat Microbiol 2020, 5:536-544), specifically the subgenus Sarbecovirus. Sarbecoviruses are the most transmissible coronaviruses in humans, probably because they all use the human angiotensinogen converting enzyme 2 (ACE2) receptor as a portal of entry into human cells. There are hundreds of strains of SARS-related coronavirus (SARSr-CoV) that are known to infect only non-human species, and bats are the primary reservoir for many strains of SARS-related coronaviruses. There are two major clades of sarbecoviruses. Clade 1a contains SARS-CoV-related CoVs (SC2r-CoVs), and clade 1b contains SARS-CoV-2-related CoVs (SC2r-CoVs). Sarbecoviruses 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. Multiple studies have shown limited cross-neutralization between SARS-CoV and SARS-CoV-2 (Yang R et al., EBioMedicine 2020, 58:102890).

[0006] Pandemic preparedness and response require a multi-pronged approach to combat emerging zoonotic viruses, including vaccines, therapeutic monoclonal antibodies, and small molecule drugs. In the case of SARS-CoV-2, the first countermeasure commercial formulation that received FDA emergency use authorization (EUA) was a therapeutic monoclonal antibody (mAb) derived from a COVID-19 patient. Both antibodies and T cell immunity are important to control viral infections, such as SARS-CoV-2 or other sarbecovirus infections. In comparison, neutralizing antibodies (Nabs) are more important to prevent viral entry and thus initial infection, while T cell immunity may later resist and suppress or control disease progression upon infection. Nabs 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 key role in treating severe cases, especially in vulnerable populations (e.g., immunocompromised patients), or in preventing subsequent transmission by isolating targeted high-risk populations. Unfortunately, the first generation of therapeutic mAbs against COVID-19 have proven less effective or ineffective against newly emerging variants of concern (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 variants P.1., delta SARS-CoV-2 B.1.617.2; and the omicron variants SARS-CoV-2 B.1.1.529 BA.1 and BA.2, and the observed decline in immune protection against the new variants obtained from vaccines based on prototype virus strains, 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 reservoirs, e.g., 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, raising the possibility that future outbreaks (SARS3, SARS4, etc.) may be caused by different but related coronaviruses.

[0008]

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

[0009] There is a need to develop molecules for use in the treatment or prevention of human infections caused by sarbecoviruses that alleviate at least one of the above-mentioned problems. Summary of the Invention

[0010]

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

[0011]

[0011] Therefore, one aspect of the present invention is an antigen-binding molecule that binds to sarbecovirus spike proteins derived from two or more different sarbecoviruses, comprising: (i) The following CDR: HC-CDR1 having an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 111 HC-CDR2 having an amino acid sequence with at least 85% sequence identity to SEQ ID NO:2 or SEQ ID NO:112 HC-CDR3 having an amino acid sequence with at least 85% sequence identity to SEQ ID NO:3 or SEQ ID NO:113 and a heavy chain variable (VH) region incorporating (ii) The following CDR: LC-CDR1 having an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 114 LC-CDR2 having an amino acid sequence with at least 85% sequence identity to SEQ ID NO:5 or SEQ ID NO:115 LC-CDR3 having an amino acid sequence with at least 85% sequence identity to SEQ ID NO:6 or SEQ ID NO:116 A light chain variable (VL) region incorporating The present invention refers to an antigen-binding molecule comprising:

[0012] Another aspect of the present invention is an antigen-binding molecule that binds to sarbecovirus spike proteins from two or more different sarbecoviruses, comprising: (i) The following CDR: Formula I: X1-X2-X3-Φ-X4-X n1 -X5-X6 wherein 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 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 sequence Amino acid formula II: X7-X8-X9-X n2 -π-X n3 -X 10 (Wherein, 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 Formula III: Ψ-ζ1-X n4 -X 11 -X n5 -X 12 -X 13 -X 14 -ζ2-X 15 where Ψ is selected from one of A and V; ζ1 is selected from one of R, T, K and LN; X n4 is selected from one of E, HLGGG, GGG, LDIII, DSI, GEAG, RVAIF, LQNG, VTYTS, ADIV, DSLA, DSL, AISQQ, DYYDN, DPL, EGIQG, and DGG; X 11 is selected from one of L, S, Y, T, A, N, V, and W; X n5 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 is selected from one of H, W, G, P, T, S, N, and Y; X 13 is selected from one of Y, P, A, L, S, F, I, V, and G; X 14 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 is selected from one of Y, S, L, N, H, C, V, and F) HC-CDR3 having an amino acid and a heavy chain variable (VH) region incorporating (ii) The following CDR: Formula IV:X 16 -X 17 -X 18 -X n6 -ζ3-X 19 (In the formula, X 16 is selected from one of Q and Y; X 17 is selected from one of G, S, T, N, I, and A; X 18 is selected from one of V, I, T, F and L; Xn6 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 is selected from one of W, Y, S and N LC-CDR1 having an amino acid sequence Formula V:X 20 -X 21 -S (In the formula, X 20 is selected from one of A, W, K, T, G, L, and D; X 21 is selected from one of A, S, G, I, and T) LC-CDR2 having the amino acid Formula VI:X 22 -ζ4-X 23 -X n7 -ζ5-X 24 -X 25 -X n8 -ζ6 (In the formula, X 22 is selected from one of Q, H, and M; ζ4 is selected from one of Q and H; X 23 is selected from one of Y, S, G, A, L, and T; X n7 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 is selected from one of S, Y, T, D, H, F, P, W, and I; X 25 is selected from one of P, I, and R; X n8 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 an amino acid sequence A light chain variable (VL) region incorporating The present invention refers to an antigen-binding molecule comprising:

[0013]

[0013] In another aspect, there is a nucleic acid or a plurality of nucleic acids, optionally isolated, encoding an antigen-binding molecule as discussed herein above.

[0014] In another aspect, there is an expression vector or vectors that comprise the nucleic acid or nucleic acids discussed herein above.

[0014]

[0015] In another aspect, there is a method for producing an antigen binding molecule that binds to a sarbecovirus spike protein from two or more different sarbecoviruses, the method comprising culturing a cell capable of expressing the antigen binding molecule discussed herein above under conditions suitable for expression of the antigen binding molecule by the cell.

[0015]

[0016] In another aspect, there is a composition comprising an antigen binding molecule as discussed herein above, a nucleic acid or nucleic acids as discussed herein above, an expression vector or expression vectors as discussed herein above, and a pharma- ceutically acceptable carrier, diluent, excipient or adjuvant.

[0016]

[0017] In another aspect, there is an antigen binding molecule as discussed herein above, a nucleic acid or nucleic acids as discussed herein above, an expression vector or expression vectors as discussed herein above, or a composition as discussed herein above for use in the treatment or prevention of a disease caused by infection with a sarbecovirus.

[0017]

[0018] In another aspect, there is a use of an antigen binding molecule as discussed herein above, a nucleic acid or nucleic acids as discussed herein above, an expression vector or expression vectors as discussed herein above, or a composition as discussed herein above in the manufacture of a medicament for use in the treatment or prevention of a disease caused by infection with a sarbecovirus.

[0018]

[0019] In another aspect, there is a method of treating or preventing a disease caused by infection with a sarbecovirus, comprising the step of administering to a subject a therapeutically or prophylactically effective amount of an antigen binding molecule as discussed herein above, a nucleic acid or nucleic acids as discussed herein above, an expression vector or expression vectors as discussed herein above, or a composition as discussed herein above.

[0019]

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

[0021] Other aspects and features of the present invention will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying drawings.

[0020]

[0022] In the drawings, embodiments of the present invention are illustrated purely by way of non-limiting example. [Brief description of the drawings]

[0021] [Figure 1]

[0023] Phylogenetic tree of the four known coronavirus genera. [Diagram 2]

[0024] A) Multiplexed sVNTs on the Luminex platform. B) As shown, all six RBD proteins are able to bind to the hACE2 receptor molecule as expected, in the following order (from highest 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). [Diagram 3]

[0025] Multiplex sVNT analysis against 10 different sarbecoviruses from five panels of human sera (shown above). All sera were used at 1:20. A 30% cutoff was set as pre-determined. A 30% cutoff was set as pre-determined. [Figure 4]

[0026] Titration of NAbs expressed as NT50 using the same serum panel and viruses as in A. Samples were tested at dilutions from 1:20 to 1:20,480 using a four-fold serial titration. A cutoff of 1:100 was set as previously determined. [Diagram 5]

[0027] Conversion of a nondominant cross-neutralizing antibody response to an immunodominant cross-neutralizing antibody response by cross-immunization with a genetically distant sarbecovirus antigen. [Figure 6]

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

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

[0030] Neutralization patterns of rabbit hyperimmune sera targeting different betacoronavirus RBD proteins. A cutoff of 1:100 was set as previously determined. [Figure 9]

[0031] Schematic diagram of the selection of double-positive B cells that produce antibodies that bind to the RBDs from both viruses. [Figure 10]

[0032] Phylogenetic analysis and sequence alignment of ACE2-binding sarbecovirus RBDs. (A) Phylogenetic tree based on the receptor-binding domain (RBD) sequences of sarbecoviruses capable of binding to human ACE2. The phylogenetic tree was constructed using PhyML with the general time-reversible (GTR) substitution model and 1,000 bootstrap iterations. Numbers in the branches are the percentage of the bootstrap value of the relevant node. The scale bar represents the number of substitutions per site. The sarbecovirus 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) in different sarbecoviruses to SARS-CoV-1 and SARS-CoV-2. (C) Alignment of amino acid sequences of sarbecovirus RBDs used in this study. Red indicates mutations / deletions. Amino acids critical for SARS-CoV-2 RBD-ACE2 interaction are indicated by blue dots above them. Viruses of the SARS-CoV-1 clade are shown in grey shading. [Figure 11]

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

[0034] Data for the top six and four control monoclonal antibodies identified in a preliminary screen using the multiplexed sVNT platform. [Figure 13]

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

[0036] Ability of monoclonal antibodies to neutralize different sarbecoviruses, including the SARS-CoV-2 ancestor and four VOCs (alpha, delta, beta and gamma), two zoonotic sarbecoviruses (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, namely, A. multiplex sVNT, B. pseudovirus neutralization test (pVNT) and C. plaque reduction neutralization test (PRNT). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022]

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

[0023]

[0039] Throughout this document, unless specifically indicated to the contrary, the terms "including," "comprising," "having," and the like, should be construed as open-ended, or in other words, as meaning "including but not limited to."

[0024]

[0040] Furthermore, throughout this document, unless the context requires otherwise, the word "include" or variations such as "includes" or "including" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of 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 commonly understood by one of ordinary skill in the art to which this subject matter belongs.

[0026]

[0042] In various embodiments, an antigen binding molecule that binds to sarbecovirus spike proteins from two or more different sarbecoviruses, (i) The following CDR: HC-CDR1 having an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 111 HC-CDR2 having an amino acid sequence with at least 85% sequence identity to SEQ ID NO:2 or SEQ ID NO:112 HC-CDR3 having an amino acid sequence with at least 85% sequence identity to SEQ ID NO:3 or SEQ ID NO:113 and a heavy chain variable (VH) region incorporating (ii) The following CDR: LC-CDR1 having an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 114 LC-CDR2 having an amino acid sequence with at least 85% sequence identity to SEQ ID NO:5 or SEQ ID NO:115 LC-CDR3 having an amino acid sequence with at least 85% sequence identity to SEQ ID NO:6 or SEQ ID NO:116 A light chain variable (VL) region incorporating There is an antigen-binding molecule comprising:

[0027]

[0043] Throughout this specification, the term "antigen-binding molecule" and its plural forms refer to one or more molecules capable of binding to a target antigen and should be understood to encompass monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), as well as antibody fragments (e.g., Fv, scFv, Fab, scFab, F(ab')2, Fab2, diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g., VhH), etc.), insofar as they exhibit binding to the relevant target molecule and block ACE2-mediated cell entry.

[0028]

[0044] In various embodiments, the two or more different sarbecoviruses can include antigen binding molecules capable of binding to spike proteins of two, or three, or four, or five, or six, or seven, or eight, or nine, or ten or more different sarbecoviruses. In various embodiments, for example, the antigen binding molecule can bind to a SARS-CoV spike protein and can also bind to a SARS-CoV-2 spike protein. In various embodiments, the antigen binding molecule can bind to multiple sarbecovirus spike proteins. For example, the antigen-binding molecule can bind to SARS-CoV spike protein; and / or 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 sarbecovirus spike protein, such as SC2r-CoVRmYN02; RacCS203, or future unknown sarbecoviruses. Broad-spectrum antigen-binding molecules have the advantage of being able to effectively block most sarbecoviruses, facilitating the prevention of infection with both known and unknown sarbecoviruses.

[0029]

[0045] In various embodiments, the term capable of binding can include 30% or greater inhibition or neutralization of binding between the sarbecovirus spike protein and ACE2. In various embodiments, 30% or greater inhibition or neutralization of binding between the sarbecovirus spike protein and ACE2 can be selected from 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 greater inhibition or neutralization. In various embodiments, the antigen binding molecule binds to sarbecovirus spike proteins from two or more different sarbecoviruses and comprises 30% or greater inhibition or neutralization (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 greater inhibition or neutralization) of binding between the sarbecovirus spike protein and ACE2.

[0030]

[0046] In various embodiments, the antigen binding molecule comprises a polyclonal antibody isolated from a patient who had SARS and recovered. In various embodiments, the antigen binding molecule comprises a polyclonal antibody isolated from a patient who had SARS, recovered, and received a 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 sarbecovirus spike proteins. For example, the polyclonal antigen binding molecule can bind to SARS-CoV spike protein; and / or 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 sarbecovirus spike protein, such as SC2r-CoVRmYN02; RacCS203, or future unknown sarbecoviruses. Broad spectrum antigen binding molecules have the advantage of being able to effectively block most sarbecoviruses, facilitating the prevention of infection with both known and unknown sarbecoviruses.

[0031]

[0047] In various embodiments, an antigen binding molecule that binds to sarbecovirus spike proteins from two or more different sarbecoviruses, (i) The following CDR: Formula I: X1-X2-X3-Φ-X4-X n1 -X5-X6 (wherein X1 is selected from one of the amino acids G and E; X2 is selected from one of the amino acids F, Y, N, G, D, and V; X3 is selected from one of the amino acids P, T, S, I, and F; Φ is selected from one of the hydrophobic amino acids F, V, L, or I; X4 is selected from one of the amino acids S, T, R, N, G, L, and I; and X n1 is selected from one of the amino acid sequences S, SN, N, M, H, T, G, P, G and D, X5 is selected from one of the amino acids Y, S, N, I and H, and X6 is selected from one of the amino acids Y, G, W, E, A, N and T). HC-CDR1 having an amino acid sequence Amino acid formula II: X7-X8-X9-X n2 -π-X n3 -X 10 (wherein X7 is selected from one of the amino acids I and T; X8 is selected from one of the amino acids Y, S, N, G, A and T; X9 is selected from one of the amino acids S, F, P, N, H, I, Y, G and T; and X n2 is selected from one of the amino acid sequences G, YN, DD, T, NG, DG, S, SS, D, ST, and NT; π is selected from one of the small amino acids G, S, P, A, and E; X n3 is selected from one of the amino acid sequences S, I, D, G, F, N, RT, L, and RN; 10 is selected from one of the amino acids T, R, M, K, S, and P. HC-CDR2 having Formula III: Ψ-ζ1-X n4 -X 11 -X n5 -X 12 -X 13 -X 14 -ζ2-X 15 (wherein 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 n4is 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 is selected from one of the amino acids L, S, Y, T, A, N, V, and W; n5 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; and X 12 is selected from one of the amino acids H, W, G, P, T, S, N, and Y; and X 13 is selected from one of the amino acids Y, P, A, L, S, F, I, V, and G; and X 14 is selected from one of the amino acids F, I, N, Y, L, and M; ζ2 is selected from one of the hydrophilic amino acids D, E, G, and S; X 16 is selected from one of the amino acids Y, S, L, N, H, C, V, and F. HC-CDR3 having an amino acid and a heavy chain variable (VH) region incorporating (ii) The following CDR: Formula IV:X 16 -X 17 -X 18 -X n6 -ζ3-X 19 (In the formula, X 16 is selected from one of the amino acids Q and Y; X 17 is selected from one of the amino acids G, S, T, N, I, and A; and X 18 is selected from one of the amino acids V, I, T, F and L; n6 is selected from one of the amino acid sequences S, G, N, V, R, LYSSNNK, LYRSNNK, LQNNGY, VQSNGY, VHSDGN, MQLNGY and SS; ζ3 is selected from one of the hydrophilic amino acids S, N and T; X 19 is selected from one of the amino acids W, Y, S and N. LC-CDR1 having an amino acid sequence Formula V:X 20 -X 21 -S (In the formula, 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 the amino acid Formula VI:X 22 -ζ4-X 23 -X n7 -ζ5-X 24 -X 25 -X n8 -ζ6 (In the formula, 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; X 23 is selected from one of the amino acids Y, S, G, A, L, and T; and 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 S, T, N, Q, and D; X 24 is selected from one of the amino acids S, Y, T, D, H, F, P, W, and I; 25 is selected from one of the amino acids P, I, and R; 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 sequence A light chain variable (VL) region incorporating An antigen-binding molecule comprising:

[0032]

[0048] Antigen-binding molecules with CDRs that fall into 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 with CDRs that fall into these formulas. These are some of the best cross-clade neutralizing antibodies reported to date.

[0033]

[0049] In various embodiments, the formula is: 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; and X of formula II is n2 is selected from one of DD, T, NG, DG, S, SS, D, ST, and NT; ζ1 of formula III is selected from one of hydrophilic amino acids R, T, and K; X of formula III n4 is selected from one of HLGGG, GGG, LDIII, DSI, GEAG, LQNG, VTYTS, ADIV, DSLA, DSL, AISQQ, DYYDN, DPL, EGIQG, and DGG; and X in formula III 11 is selected from one of S, Y, T, A, V, and W; X in formula III n5 is selected from one of S, LET, P, SAT, MATIWV, SY, PLPF, GS, VV, SVT, FDS, GYYY, EGAAS, V, and QLPY; and X in formula III 12 is selected from one of W, G, P, T, S, N, and Y; and X of formula IV n6 is selected from one of S, G, N, V, R, LYRSNNK, LQNNGY, VQSNGY, VHSDGN, MQLNGY, and SS; and X in formula VI 23 is selected from one of Y, S, G, A, and T; and X in formula VI n8The CDRs are the same as those listed above, except that they are selected from one of the following: F, W, K, G, Y, R, P, L, EY, ED, GY, QY, and QI. Antigen-binding molecules with CDRs that fall within these formulas have demonstrated pan-sarbecovirus neutralization, neutralizing a wide range of sarbecoviruses, including SARS-CoV-1 and SARS-CoV-2. Antigen-binding molecules with CDRs that fall within these formulas were double positive for staining with both SARS-CoV-1 RBD protein and SARS-CoV-2 RBD protein. Antibody 1 (SS6V1-B5) and antibodies 4 to 20 (SS6V4-A1, SS6V5-C3, ..., through SS6V20-F5) are examples of antigen-binding molecules with CDRs that fall within 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, and 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, and X n2 is selected from one of T, S, SS and NT, 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, ζ is R, and X n4 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 is selected from one of PLPF.LET, GYYY, and QLPY, and X 12 is selected from one of W, Y, and G; X 13is selected from one of F, P, G, and Y, and X 14 is selected from one of F, M and L, 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; X n6 is selected from one of G, LQNNGY, R, VQSNGY, S and MQLNGY, ζ3 is selected from one of N, S and T, and X 19 is selected from one of Y or S, and X 20 is selected from one of A, L and G; X 21 is selected from one of A, S, T and G, and X 22 is selected from one of Q, M and L, 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 is P and X n8 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 comprises GX2X3ΦX4X5X6X7, where 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 comprises IX9X 10 X n1 πX n2 X 11 (Wherein, 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, 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 includes ARX n3 X 12 X n4 X 13 X 14 X 15 ζ2X 16 (In the formula, X n3 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 is selected from one of PLPF.LET, GYYY, and QLPY, and X 13 is selected from one of W, Y, and G; X 14 is selected from one of F, P, G, and Y, and X 15 is selected from one of F, M and L, 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 includes 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; 18 is selected from one of G and S, and X 19 is selected from one of I, F and L; X n5 is selected from one of G, LQNNGY, R, VQSNGY, S and MQLNGY; ζ3 is selected from one of N, S and T; Ω is selected from one of Y or S); and the formula V includes X 20 X 21 S (where X 20 is selected from one of A, L and G; X 21is selected from one of A, S, T and G, and formula VI includes X 22 QX 23 X n6 Zeta 5X 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 is selected from one of ED, G, QI and L, and ζ6 is selected from one of S and T). Antigen-binding molecules having CDRs falling within these formulas have demonstrated pan-sarbecovirus neutralization potency and pan-sarbecovirus neutralization spectrum that spans the majority of sarbecoviruses, including SARS-CoV-1 and SARS-CoV-2. Antibody 1 (SS6V1-B5), Antibody 11 (SS6V11-E7), Antibody 12 (SS6V12-E11), Antibody 13 (SS6V13-F1), Antibody 19 (SS6V19-F4) and Antibody 20 (SS6V20-F5) are examples of antigen-binding molecules having CDRs falling within these formulas.

[0035]

[0051] In various embodiments, the heavy chain variable (VH) region comprises 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, ISSSSSFM, IYFSENT, INTNTGIP, and IYFSANT; and GGGYLETGPLDF, and the light chain variable (VL) region incorporates the following CDRs: 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; 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, and X n1 is selected from one of P and G, X5 is selected from one of Y, S and H, X6 is Y, X7 is I, X8 is Y, X9 is selected from one of I and F, and X n2 is S, π is selected from one of G, E and A, and X n3 is selected from one of S and N, and X 10 is T, Ψ is A, ζ1 is R, and X n4 is GGG and X 11 is Y and X n5 is LET, and X 12 is G and X13 is P and 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 is Q and X 17 is selected from one of G and S, and X 18 is L and X n6 is selected from one of LQNNGY, VQSNGY, and MQLNGY, ζ3 is N, and X 19 is Y and X 20 is L and X 21 is selected from one of S and G, and X 22 is M, ζ4 is Q, and X 23 is selected from one of S and G, and X n7 is L, ζ5 is Q, and X 24 is selected from one of I and T, and X 25 is P and X n8 is G and ζ6 is T. That is, in various embodiments, formula I comprises GX2X3IX4X5X6Y, where 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 X6 is selected from one of Y, S and H, and formula II comprises IYX 10 SπX 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 comprises 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 includes QX 18 LX n5 NY (wherein 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 includes MQX 23 LQX 25 PGT(wherein, X 23 is selected from one of S and G, and X 25 are selected from one of I and T). Antigen-binding molecules with CDRs falling within these formulas demonstrated pan-sarbecovirus neutralization potency and pan-sarbecovirus neutralization spectrum against all sarbecoviruses tested, 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 falling within these formulas. These antibodies demonstrated the highest reported potency. The three monoclonal antibodies in this group maintained potent neutralization capability across the majority of SARS-CoV-2 VOCs and VOIs as well as clade-1a sarbecoviruses in different virus neutralization assay platforms. All three antibodies utilized unique combinations of heavy and light chain gene classes that exhibited over 90% similarity in their heavy and light chain sequences. These sequences have not previously been reported as sarbecovirus-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; HC-CDR3 selected from one of the amino acid sequences ARGGGYLETGPFEY, ARGGGYLETGPFDS and ARGGGYLETGPLDF, and 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; 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, and X n1 is selected from one of P and G, X5 is selected from one of Y and H, X6 is Y, X7 is I, X8 is Y, X9 is selected from one of I and F, and X n2 is S, π is selected from one of G and A, and X n3 is selected from one of S and N, and X 10 is T, Ψ is A, ζ1 is R, and X n4 is GGG and X 17 is Y and X n5 is LET, and X 12 is G and X 13 is P and 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 is Q and X 17 is S and X 18 is L and X n6is selected from one of LQNNGY and MQLNGY, ζ3 is N, and X 19 is Y and X 20 is L and X 21 is selected from one of S and G, and X 22 is M, ζ4 is Q, and X 23 is selected from one of S and G, and X n7 is L, ζ5 is Q, and X 24 is I and X 25 is P and X n8 is G and ζ is T. That is, in various embodiments, formula I includes GGXIXXYY, where X is selected from one of S and F, X is selected from one of G and I, X is selected from one of P and G, and X is selected from one of Y and H, and formula II includes IYX 10 SπX 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 comprises ARGGGYLETGPX 15 ζ2X 16 (In the formula, X 15 is selected from one of F and L, and X 16 is selected from one of Y and F, and ζ2 is selected from one of D and E, and formula IV comprises QSLX n5 NY (wherein X n5 is selected from one of LX, LQNNGY and MQLNGY, and formula V is 21 S (where X 21 is selected from one of S and G, and formula VI includes MQX 23 LQIPGT(where X 23is selected from one of S and G). Antigen binding molecules with CDRs falling within these formulas have demonstrated the best pan-sarbecovirus neutralization potency and pan-sarbecovirus neutralization spectrum when compared to any other antibodies reported to date. Epitope mapping studies of antibodies falling within these formulas show that these antibodies have unique contact footprints in the RBD. Antibody 11 (SS6V11-E7) and Antibody 20 (SS6V20-F5) are examples of antigen binding molecules with CDRs falling within 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; HC-CDR3 selected from one of the amino acid sequences ARGGGYLETGPFEY and ARGGGYLETGPLDF, and 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; 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 with amino acid sequence GFILRNYE; HC-CDR2 with amino acid sequence IGNTGGT; HC-CDR3 with amino acid sequence ARVTYTSSPLPFWFLDL, and the light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with amino acid sequence QSIGNY; LC-CDR2 with amino acid sequence AAS; LC-CDR3 with amino acid sequence QQTYRTPPEDS. In various embodiments, these CDRs are formed in heavy chain SEQ ID NO: 71 and light chain SEQ ID NO: 72 of antibody 1 (SS6V1-B5). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, so long as they are capable of binding to sarbecovirus spike proteins from two or more different sarbecoviruses.

[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; HC-CDR3 with amino acid sequence ARELRHYFDY, and the light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with amino acid sequence QGISSY; LC-CDR2 with amino acid sequence AAS; 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, so long as they can bind to sarbecovirus spike proteins from two or more different sarbecoviruses. 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 the amino acid sequence GYSFTNSG; HC-CDR2 with the amino acid sequence TNFYNGIT; HC-CDR3 with the amino acid sequence ALNRVAIFNDGYNPLGY, and the light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with the amino acid sequence QSVLYSSNNKNY; LC-CDR2 with the amino acid sequence WAS; LC-CDR3 with the amino acid sequence QQYFSSPFS. In various embodiments, these CDRs are formed in the 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, so long as they are capable of binding to sarbecovirus spike proteins from two or more different sarbecoviruses. 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 with amino acid sequence GYTFSMYW; HC-CDR2 with amino acid sequence IYPDDSDR; HC-CDR3 with amino acid sequence ARLQNGYSYGLLEN, and the light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with amino acid sequence QSTLYRSNNKNY; LC-CDR2 with amino acid sequence WAS; LC-CDR3 with amino acid sequence QQYYSYPWT. In various embodiments, these CDRs are formed in heavy chain SEQ ID NO: 77 and light chain SEQ ID NO: 78 of antibody 4 (SS6V4-A1). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, so long as they are capable of binding to sarbecovirus spike proteins from two or more different sarbecoviruses.

[0044]

[0060] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 with amino acid sequence GYTFTHYW; HC-CDR2 with amino acid sequence IYPDDSDT; HC-CDR3 with amino acid sequence ATADIVVGSNFFDH, and the light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with amino acid sequence QSISTW; LC-CDR2 with amino acid sequence KAS; LC-CDR3 with amino acid sequence QHYNSYIKT. In various embodiments, these CDRs are formed in heavy chain SEQ ID NO: 79 and light chain SEQ ID NO: 80 of antibody 5 (SS6V5-C3). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, so long as they are capable of binding to sarbecovirus spike proteins from two or more different sarbecoviruses.

[0045]

[0061] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 with amino acid sequence GFTFNTYA; HC-CDR2 with amino acid sequence ISSNGGIT; HC-CDR3 with amino acid sequence VKDSLATVVTLLSY, and the light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with amino acid sequence QTISSY; LC-CDR2 with amino acid sequence AAS; LC-CDR3 with amino acid sequence QQSYSTPGT. In various embodiments, these CDRs are formed in heavy chain SEQ ID NO: 81 and light chain SEQ ID NO: 82 of antibody 6 (SS6V6-C4). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, so long as they are capable of binding to sarbecovirus spike proteins from two or more different sarbecoviruses.

[0046]

[0062] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 with amino acid sequence ENIFSGYW; HC-CDR2 with amino acid sequence IYPDDSDT; HC-CDR3 with amino acid sequence ARHLGGGSSWPIDY, and the light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with amino acid sequence QGISNY; LC-CDR2 with amino acid sequence AAS; LC-CDR3 with amino acid sequence QQYSSYPFT. In various embodiments, these CDRs are formed in heavy chain SEQ ID NO: 83 and light chain SEQ ID NO: 84 of antibody 7 (SS6V7-C5). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, so long as they are capable of binding to sarbecovirus spike proteins from two or more different sarbecoviruses.

[0047]

[0063] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 with amino acid sequence GFTFSTYA; HC-CDR2 with amino acid sequence IASDGGIT; HC-CDR3 with amino acid sequence VKDSLTSVTTIFDC, and the light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with amino acid sequence QNINSY; LC-CDR2 with amino acid sequence TAS; LC-CDR3 with amino acid sequence QQSYTDPYT. In various embodiments, these CDRs are formed in heavy chain SEQ ID NO: 85 and light chain SEQ ID NO: 86 of antibody 8 (SS6V8-D3). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, so long as they are capable of binding to sarbecovirus spike proteins from two or more different sarbecoviruses.

[0048]

[0064] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 with the amino acid sequence GGSISSNIW; HC-CDR2 with the amino acid sequence IYHSGST; HC-CDR3 with the amino acid sequence ARAISQQYFDSSVLGY, and the light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with the amino acid sequence QSVVTN; LC-CDR2 with the amino acid sequence GAS; LC-CDR3 with the amino acid sequence QQYNNWPGYT. In various embodiments, these CDRs are formed in heavy chain SEQ ID NO: 87 and light chain SEQ ID NO: 88 of antibody 9 (SS6V9-D11). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, so long as they are capable of binding to sarbecovirus spike proteins from two or more different sarbecoviruses.

[0049]

[0065] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 with amino acid sequence EDSFTGYW; HC-CDR2 with amino acid sequence IYPDDGDT; HC-CDR3 with amino acid sequence ARHLGGGSSWPIDS, and the light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with amino acid sequence QGIRNY; LC-CDR2 with amino acid sequence AAS; LC-CDR3 with amino acid sequence QQYNNHPFT. In various embodiments, these CDRs are formed in heavy chain SEQ ID NO: 89 and light chain SEQ ID NO: 90 of antibody 10 (SS6V10-E1). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, so long as they are capable of binding to sarbecovirus spike proteins from two or more different sarbecoviruses.

[0050]

[0066] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 with the amino acid sequence GGFIGPHY; HC-CDR2 with the amino acid sequence IYISGST; HC-CDR3 with the amino acid sequence ARGGGYLETGPFEY, and the light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with the amino acid sequence QSLLQNNGYNY; LC-CDR2 with the amino acid sequence LSS; LC-CDR3 with the amino acid sequence MQSLQIPGT. In various embodiments, these CDRs are formed in heavy chain SEQ ID NO: 91 and light chain SEQ ID NO: 92 of antibody 11 (SS6V11-E7). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, so long as they are capable of binding to sarbecovirus spike proteins from two or more different sarbecoviruses. Antibodies with these CDRs demonstrated one of the best pan-sarbecovirus neutralizing potencies and zones of pan-sarbecovirus neutralization when compared to any other antibodies reported to date, including being the only antibodies with neutralizing ability against Omicron BA.2.

[0051]

[0067] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 with amino acid sequence GFTFSTYN; HC-CDR2 with amino acid sequence ISSSSSFM; HC-CDR3 with amino acid sequence ARDYYDNSGYYYYGMDV, and the light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with amino acid sequence QSIRTY; LC-CDR2 with amino acid sequence AAS; LC-CDR3 with amino acid sequence LQTYSTPQIT. In various embodiments, these CDRs are formed in heavy chain SEQ ID NO: 93 and light chain SEQ ID NO: 94 of antibody 12 (SS6V12-E11). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, so long as they are capable of binding to sarbecovirus spike proteins from two or more different sarbecoviruses.

[0052]

[0068] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 with amino acid sequence GVSILGSY; HC-CDR2 with amino acid sequence IYFSENT; HC-CDR3 with amino acid sequence ARGGGYLETGPFDS, and the light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with amino acid sequence QGLVQSNGYNY; LC-CDR2 with amino acid sequence LGS; LC-CDR3 with amino acid sequence MQGLQTPGT. In various embodiments, these CDRs are formed in heavy chain SEQ ID NO: 95 and light chain SEQ ID NO: 96 of antibody 13 (SS6V13-F1). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, so long as they are capable of binding to sarbecovirus spike proteins from two or more different sarbecoviruses.

[0053]

[0069] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 with amino acid sequence GGPISSYY; HC-CDR2 with amino acid sequence IYYSGST; HC-CDR3 with amino acid sequence ARDPLAEGAASSGFDN, and the light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with amino acid sequence QSISSY; LC-CDR2 with amino acid sequence AAS; LC-CDR3 with amino acid sequence QQSYTTPRT. In various embodiments, these CDRs are formed in heavy chain SEQ ID NO: 97 and light chain SEQ ID NO: 98 of antibody 14 (SS6V14-F2). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, so long as they are capable of binding to sarbecovirus spike proteins from two or more different sarbecoviruses.

[0054]

[0070] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 with amino acid sequence GFTFSSYA; HC-CDR2 with amino acid sequence ISYDGRTK; HC-CDR3 with amino acid sequence ARLDIIITPPANDY, and the light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with amino acid sequence QIVSSNY; LC-CDR2 with amino acid sequence DAS; LC-CDR3 with amino acid sequence HQYGDSRRT. In various embodiments, these CDRs are formed in heavy chain SEQ ID NO: 99 and light chain SEQ ID NO: 100 of antibody 15 (SS6V15-F6). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, so long as they are capable of binding to sarbecovirus spike proteins from two or more different sarbecoviruses.

[0055]

[0071] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 with amino acid sequence EFTFSRYT; HC-CDR2 with amino acid sequence IGGSTPLS; HC-CDR3 with amino acid sequence ARDSIASATTLFDL, and the light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with amino acid sequence QAISSY; LC-CDR2 with amino acid sequence AAS; LC-CDR3 with amino acid sequence QQSYITPPEYS. In various embodiments, these CDRs are formed in heavy chain SEQ ID NO: 101 and light chain SEQ ID NO: 102 of antibody 16 (L8N16-C7). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, so long as they are capable of binding to sarbecovirus spike proteins from two or more different sarbecoviruses.

[0056]

[0072] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 with amino acid sequence GFTFSSYA; HC-CDR2 with amino acid sequence ISYDGRNK; HC-CDR3 with amino acid sequence ARGEAGTMATIWVSSYDY, and the light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with amino acid sequence QSLVHSDGNTY; LC-CDR2 with amino acid sequence KIS; LC-CDR3 with amino acid sequence MQATQFPPT. In various embodiments, these CDRs are formed in heavy chain SEQ ID NO: 103 and light chain SEQ ID NO: 104 of antibody 17 (L8N17-G3). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, so long as they are capable of binding to sarbecovirus spike proteins from two or more different sarbecoviruses.

[0057]

[0073] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 with amino acid sequence GFTFSSYA; HC-CDR2 with amino acid sequence ITSNGGGT; HC-CDR3 with amino acid sequence AREGIQGWVTYFDY, and the light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with amino acid sequence QSISTN; LC-CDR2 with amino acid sequence AAS; LC-CDR3 with amino acid sequence QQTYTTPQYS. In various embodiments, these CDRs are formed in heavy chain SEQ ID NO: 105 and light chain SEQ ID NO: 106 of antibody 18 (SS6V18-E3). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, so long as they are capable of binding to sarbecovirus spike proteins from two or more different sarbecoviruses.

[0058]

[0074] The heavy chain variable (VH) region incorporates the following CDRs: HC-CDR1 with amino acid sequence GYTFTDYN; HC-CDR2 with amino acid sequence INTNTGIP; HC-CDR3 with amino acid sequence ARDGGWQLPYWYFDL, and the light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with amino acid sequence YSFSSS; LC-CDR2 with amino acid sequence GTS; LC-CDR3 with amino acid sequence QQYYSWPLT. In various embodiments, these CDRs are formed in heavy chain SEQ ID NO: 107 and light chain SEQ ID NO: 108 of antibody 19 (SS6V19-F4). In various embodiments, these CDRs are formed in other antigen-binding scaffolds listed below, so long as they are capable of binding to sarbecovirus spike proteins from two or more different sarbecoviruses.

[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; HC-CDR3 with amino acid sequence ARGGGYLETGPLDF, and the light chain variable (VL) region incorporates the following CDRs: LC-CDR1 with amino acid sequence QSLMQLNGYNY; LC-CDR2 with amino acid sequence LGS; 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, so long as they can bind to sarbecovirus spike proteins from two or more different sarbecoviruses. Antibodies with these CDRs have demonstrated one of the best pan-sarbecovirus neutralizing potencies and pan-sarbecovirus neutralizing zones when compared to any other antibodies reported to date.

[0060]

[0076] In various embodiments, the antigen-binding molecule comprises a polyclonal antigen-binding molecule. In various embodiments, the antigen-binding molecule comprises at least two different antigen-binding domains (i.e., at least two antigen-binding domains comprising non-identical VH and VL, for example). The higher neutralizing potency of the antibody of the present disclosure may allow for smaller doses of antigen-binding molecules to be used clinically as individual antigen-binding molecules or mixed in a cocktail of two or more antigen-binding molecules, or antigen-binding molecules with 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 sarbecovirus spike proteins, e.g., SARS-CoV spike protein and 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 comprise an antigen-binding molecule capable of binding to a target, where the antigen-binding molecule is specific for the target. For example, an antigen-binding molecule capable of binding to SARS-CoV spike protein and SARS-CoV-2 spike protein may comprise a component capable of binding to SARS-CoV spike protein and a second component capable of binding to 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 an antigen-binding polypeptide or an antigen-binding polypeptide complex capable of binding to a target, where the antigen-binding molecule is specific for the target. In some embodiments, an antigen-binding molecule that is a component 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 sarbecovirus spike proteins. For example, the multispecific antigen binding molecule can bind to SARS-CoV spike protein; and / or SARS-CoV-2 spike protein, and / or SARS-CoV-2 alpha, and / or SARS-CoV-2 beta, and / or SARS-CoV-2 delta (Delta), and / or SC2r-CoV RaTG13, and / or SC2r-CoV GX-P5L, and / or SC2r-CoV GD-1, and / or any other sarbecovirus spike protein, such as SC2r-CoVRmYN02; RacCS203, or future unknown sarbecoviruses. Broad spectrum antigen binding molecules have the advantage of being able to effectively block most sarbecoviruses, facilitating the prevention of infection with both known and unknown sarbecoviruses.

[0065]

[0081] Throughout this specification, the term "sarbecovirus" and its plural forms should be understood to include any betacoronavirus that uses the angiotensinogen converting enzyme 2 (ACE2) receptor as a portal of entry into cells. In various embodiments, a sarbecovirus includes any betacoronavirus that uses the ACE2 receptor as a portal of entry into cells. In various embodiments, a sarbecovirus includes any betacoronavirus that uses the human ACE2 receptor as a portal of entry into human cells. In various embodiments, a sarbecovirus includes any known or novel sarbecovirus. 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 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" refers to SARSr-CoV having the nucleotide sequence of GenBank: NC_004718.3 ("Severe Acute Respiratory Syndrome coronavirus isolate, complete genome") and should be understood to encompass variants thereof having a nucleotide sequence having at least 85% sequence identity (e.g., at least one of 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) to the nucleotide sequence of GenBank: NC_004718.3 set forth in SEQ ID NO:7.

[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") as reported in Zhou et al., Nature (2020) 579:270-273, and which has at least 8 nucleotide differences relative to the nucleotide sequence of GenBank:NC_045512.2 as set forth in SEQ ID NO:8. 5% sequence identity (e.g., at least 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] Sarbecoviruses, like all coronaviruses, have genomes that encode 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 portion that incorporates the receptor binding domain (RBD).

[0069]

[0085] In various embodiments, the sarbecovirus spike protein can be characterized by any one of the consensus amino acid sequences set forth in SEQ ID NOs:18-25 (SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25).

[0070]

[0086] A fragment of a sarbecovirus 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 spike protein of SARS-CoV has the amino acid sequence shown in SEQ ID NO: 9. The SARS-CoV spike protein comprises an S1 (SEQ ID NO: 11) and an S2 subunit. The S1 subunit comprises a receptor binding domain (RBD) comprising SEQ ID NO: 12 or SEQ ID NO: 17, through which SARSr-CoV binds to ACE2 expressed by a host cell.

[0072]

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

[0073]

[0089] In various embodiments, the RBD of a sarbecovirus spike protein refers to a polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 12, 14, 15, 17, or 26-30, or a polypeptide having at least 75%, e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more 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 from other SARSr-CoVs or other known sarbecoviruses.

[0074]

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

[0075]

[0091] In various embodiments, the isoforms, fragments, variants, or homologs may optionally be functional isoforms, fragments, variants, or homologs that have a functional property / activity of the reference protein, e.g., when binding to and / or entering a host cell via ACE2, as determined by analysis with a suitable assay for said functional property / activity. For example, an isoform, fragment, variant, or homolog of a sarbecovirus spike protein may exhibit association with ACE2.

[0076]

[0092] In various embodiments, the sarbecovirus spike protein includes a spike protein that comprises or consists of an amino acid sequence having at least 75%, e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of consensus sarbecovirus spike proteins SEQ ID NOs:18-25 (SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25). In various embodiments, the sarbecovirus spike protein comprises or consists of an amino acid sequence having at least 75%, e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: 9, 10, or 18-25.

[0077]

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

[0078]

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

[0079]

[0095] In various embodiments, the antigen binding molecule comprises two antigen binding molecules that bind to the sarbecovirus spike protein.

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

[0080]

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

[0098] In various embodiments, the antigen binding molecule inhibits infection of ACE2-expressing cells by sarbecoviruses.

[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 antibody is selected from the group consisting of WIV-1, and SARS-CoV.

[0082]

[0100] In various embodiments, the sarbecovirus SARS-CoV refers to SARSr-CoV having the nucleotide sequence of GenBank: NC_004718.3, as reported in He et al., Biochem. Biophys. Res. Commun. 316(2), 476-483 (2004), and has at least 85% sequence identity ( For example, variants thereof having a nucleotide sequence with at least 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.

[0083]

[0101] In various embodiments, the sarbecovirus SARS-CoV-2 is as described in Wu et al., Nature 579 (7798), 265-269 (2020), and includes variants thereof having a nucleotide sequence with at least 85% sequence identity (e.g., at least one of 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) to the nucleotide sequence of GenBank: NC_045512.2. In various embodiments, the sarbecovirus SARS-CoV-2 variants 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 the present disclosure may be designed and prepared using the sequence of monoclonal antibodies (mAbs) that can bind to sarbecovirus spike proteins. Antigen-binding regions of antibodies, such as single chain variable fragments (scFv), Fab and F(ab')2 fragments, may also be used / provided. An "antigen-binding region" is any fragment of an antibody that can bind to a target, where a given antibody is specific for the target. mAbs are one of the most efficient and powerful tools for rapid development and deployment in fighting future emerging zoonotic viruses, especially sarbecoviruses.

[0085]

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

[0086]

[0104] The VH and VL regions each contain a framework region (FR) on either side of each CDR, which provides a scaffold for the CDR. From N-terminus to C-terminus, the VH region comprises 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 comprises 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 comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:31, and the VL region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:32. In various embodiments, the VH region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:33, and the VL region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:34. In various embodiments, the VH region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:35, and the VL region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:36. In various embodiments, the VH region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:37, and the VL region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:38. In various embodiments, the VH region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:39, and the VL region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:40. In various embodiments, the VH region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:41, and the VL region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:42. In various embodiments, the VH region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:43, and the VL region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:44. In various embodiments, the VH region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:45, and the VL region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:46.In various embodiments, the VH region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:47, and the VL region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:48. In various embodiments, the VH region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:49, and the VL region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:50. In various embodiments, the VH region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:51, and the VL region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:52. In various embodiments, the VH region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:53, and the VL region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:54. In various embodiments, the VH region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:55, and the VL region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:56. In various embodiments, the VH region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:57, and the VL region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:58. In various embodiments, the VH region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:59, and the VL region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:60. In various embodiments, the VH region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:61, and the VL region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:62. In various embodiments, the VH region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:63, and the VL region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:64. In various embodiments, the VH region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:65, and the VL region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:66. In various embodiments, the VH region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:67, and the VL region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:68.In various embodiments, the VH region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:69, and the VL region comprises amino acids encoded by the nucleic acid sequence set forth in SEQ ID NO:70.

[0087]

[0105] In various embodiments, the antigen binding molecule comprises the CDRs of an antibody capable of binding to the sarbecovirus spike protein described herein, or comprises CDRs derived from an antibody capable of binding to the sarbecovirus spike protein described herein. In some embodiments, the antigen binding molecule comprises the FRs of an antibody capable of binding to the sarbecovirus spike protein described herein, or comprises FRs derived from an antibody capable of binding to the sarbecovirus spike protein described herein. In some embodiments, the antigen binding molecule comprises the CDRs and FRs of an antibody capable of binding to the sarbecovirus spike protein described herein, or comprises CDRs and FRs derived from an antibody capable of binding to the sarbecovirus spike protein described herein. That is, in some embodiments, the antigen binding molecule comprises the VH and VL regions of an antibody capable of binding to the sarbecovirus spike protein described herein, or comprises VH and VL regions derived from an antibody capable of binding to the sarbecovirus spike protein described herein.

[0088]

[0106] In some embodiments, the antigen binding molecule comprises the CDRs, FRs and / or VH and / or VL regions of an antibody capable of binding to a sarbecovirus spike protein selected from any one of the sarbecovirus 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 comprises: (i) The following CDR: 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 and a heavy chain variable (VH) region incorporating (ii) The following CDR: 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 A light chain variable (VL) region incorporating Includes.

[0090]

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

[0091]

[0109] In various embodiments, the antigen binding molecule comprises a VH region comprising an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO:1, e.g., 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 comprises a VH region comprising an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO:2, e.g., 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 comprises a VH region comprising an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO:3, e.g., 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 comprises a VL region comprising an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO:4, e.g., 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 comprises a VL region comprising an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO:5, e.g., 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 comprises a VL region comprising an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO:6, e.g., 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 according to the disclosure in which one or more amino acids are replaced with another amino acid, the substitution may be a conservative substitution, e.g., an aliphatic amino acid is replaced with another aliphatic amino acid, e.g., a non-polar amino acid G, or A, or P, or I, or L, or V is replaced with a different non-polar amino acid; e.g., a polar uncharged amino acid C, or S, or T, or M, or N, or Q is replaced with a different polar uncharged amino acid, e.g., a polar charged amino acid D, or E, or K, or R is replaced with a different polar charged amino acid, or an aromatic amino acid is replaced with another aromatic amino acid, e.g., H, or F, or W, or Y is replaced with a different aromatic amino acid.

[0098]

[0116] In variable embodiments, the substitutions may be function-conservative, i.e., in some embodiments, the substitutions may not affect (or may not substantially affect) one or more functional properties (e.g., target binding) of an antigen-binding molecule that contains the substitution, as compared to a comparable unsubstituted molecule.

[0099]

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

[0100]

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

[0101]

[0119] In various embodiments, the antigen binding molecules of the present disclosure comprise or consist of a Fab region that binds to a sarbecovirus spike protein.

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

[0102]

[0121] G-type immunoglobulins (i.e., IgG) are glycoproteins of about 150 kDa that contain two heavy chains and two light chains. From the N-terminus to the C-terminus, the heavy chains contain a VH followed by a heavy chain constant region that contains three constant domains (CH1, CH2, and CH3), and similarly the light chains contain a VL followed by a CL. Depending on the heavy chain, 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 molecule described herein comprises or consists of an IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM that binds to a sarbecovirus spike protein.

[0104]

[0123] In some embodiments, the antigen-binding molecule of the present disclosure comprises one or more regions (e.g., CH1, CH2, CH3, etc.) of an immunoglobulin heavy chain constant sequence. In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from a heavy chain constant sequence of an IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM, such as a 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 a heavy chain constant sequence of a human IgG1 allotype (e.g., G1m1, G1m2, G1m3, or G1m17).

[0105]

[0124] In various embodiments there is an antigen binding molecule as discussed herein above for use in the treatment or prevention of a disease caused by infection with a sarbecovirus.

[0106]

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

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

[0107]

[0127] In various embodiments, the antigen binding molecules discussed herein above are suitable for use in treating an individual diagnosed with a sarbecovirus 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 antibody is selected from the group consisting of WIV-1, and SARS-CoV.

[0108]

[0129] In various embodiments there is the use of an antigen binding molecule as discussed herein above in the manufacture of a medicament for use in the treatment or prevention of a disease caused by infection with a sarbecovirus.

[0109]

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

[0110]

[0131] In various embodiments, the use of the antigen binding molecules discussed herein above is suitable for use in the treatment or prophylaxis of 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.

[0111]

[0132] In various embodiments, the use of the antigen binding molecules discussed herein above in the manufacture of a medicament is suitable for the treatment of an individual diagnosed with a sarbecovirus 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 antibody is selected from the group consisting of WIV-1, and SARS-CoV.

[0112]

[0134] In various embodiments, there are methods of treating or preventing a disease caused by infection with a sarbecovirus, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen binding molecule discussed herein above.

[0113]

[0135] In various embodiments, the subject is an individual who has undergone SARS-CoV-2 vaccination or infection.

[0136] In various embodiments, the subject is an individual diagnosed with a sarbecovirus infection.

[0114]

[0137] 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.

[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 antibody is selected from the group consisting of WIV-1, and SARS-CoV.

[0116]

[0139] In various embodiments, there is a nucleic acid or a plurality of nucleic acids, optionally isolated, encoding an antigen binding molecule discussed herein above.

[0140] In various embodiments, there is an expression vector or vectors that include the nucleic acid or nucleic acids discussed herein above.

[0117]

[0141] In various embodiments, there is a nucleic acid or nucleic acids as discussed herein above, or an expression vector or vectors comprising the nucleic acid or nucleic acids as discussed herein above, capable of expressing an antigen binding molecule as discussed herein above, for use in the treatment or prevention of a disease caused by infection with a sarbecovirus.

[0118]

[0142] In various embodiments, the nucleic acid or nucleic acids discussed herein above, or an expression vector or vectors comprising the nucleic acid or nucleic acids, capable of expressing the antigen-binding molecules discussed herein above, are suitable for use in individuals who have experienced SARS-CoV-2 infection or vaccination.

[0119]

[0143] In various embodiments, the nucleic acid or nucleic acids discussed herein above, or an expression vector or vectors comprising the nucleic acid or nucleic acids capable of expressing the antigen binding molecules discussed herein above, are suitable for use in individuals uninfected or unvaccinated against any sarbecovirus, 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, the nucleic acid or nucleic acids discussed herein above, or an expression vector or vectors comprising the nucleic acid or nucleic acids capable of expressing an antigen binding molecule discussed herein above, are suitable for use in treating an individual diagnosed with a sarbecovirus 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 antibody is selected from the group consisting of WIV-1, and SARS-CoV.

[0122]

[0146] In various embodiments, there is a nucleic acid or nucleic acids as discussed herein above, or an expression vector or vectors comprising a nucleic acid or nucleic acids as discussed herein above, capable of expressing an antigen-binding molecule as discussed herein above, in the manufacture of a medicament for use in the treatment or prevention of a disease caused by infection with a sarbecovirus.

[0123]

[0147] In various embodiments, the use of a nucleic acid or nucleic acids as discussed herein above, an expression vector or expression vectors comprising a nucleic acid or nucleic acids capable of expressing an antigen-binding molecule as discussed herein above in the manufacture of a medicament suitable for use in the treatment or prophylaxis of an individual who has undergone SARS-CoV-2 vaccination or infection.

[0124]

[0148] In various embodiments, the use of the nucleic acid or nucleic acids discussed herein above, an expression vector or expression vectors comprising the nucleic acid or nucleic acids capable of expressing an antigen binding molecule discussed herein above in the manufacture of a medicament suitable for use in the treatment or prophylaxis of an individual 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.

[0125]

[0149] In various embodiments, the use of a nucleic acid or nucleic acids as discussed herein above, an expression vector or expression vectors comprising a nucleic acid or nucleic acids capable of expressing an antigen binding molecule as discussed herein above, in the manufacture of a medicament suitable for use in treating an individual diagnosed with a sarbecovirus 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 antibody is selected from the group including WIV-1, SARS-CoV.

[0127]

[0151] In various embodiments, there are methods of treating or preventing a disease caused by infection with a sarbecovirus, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen binding molecule, wherein the antigen binding molecule is expressed by a nucleic acid or nucleic acids discussed herein above, or is expressed in an expression vector or expression vectors discussed herein above.

[0128]

[0152] In various embodiments, the subject is an individual who has undergone SARS-CoV-2 vaccination or infection.

[0153] 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.

[0129]

[0154] In various embodiments, the subject is an individual diagnosed with a sarbecovirus 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 antibody is selected from the group consisting of WIV-1, and SARS-CoV.

[0130]

[0156] In various embodiments, there is a cell comprising an antigen binding molecule as discussed herein above, a nucleic acid or nucleic acids as discussed herein above, or an expression vector or expression vectors as discussed herein above.

[0131]

[0157] In various embodiments, there is a method for producing an antigen binding molecule that binds to a sarbecovirus spike protein, comprising culturing a cell as discussed herein above under conditions suitable for expression of the antigen binding molecule by the cell.

[0132]

[0158] In various embodiments there is a cell as discussed herein above for use in the treatment or prevention of a disease caused by infection with a sarbecovirus.

[0133]

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

[0160] In various embodiments, the cells discussed herein above are 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.

[0134]

[0161] In various embodiments, the cells discussed herein above are suitable for use in treating an individual diagnosed with a sarbecovirus 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 antibody is selected from the group consisting of WIV-1, and SARS-CoV.

[0135]

[0163] In various embodiments there is the use of a cell as discussed herein above in the manufacture of a medicament for use in the treatment or prevention of a disease caused by infection with a sarbecovirus.

[0136]

[0164] In various embodiments, the use of the cells discussed herein above in the manufacture of a medicament is suitable for use in individuals who have undergone SARS-CoV-2 vaccination or infection.

[0137]

[0165] In various embodiments, the use of the cells discussed herein above 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.

[0138]

[0166] In various embodiments, the use of the cells discussed herein above in the manufacture of a medicament is suitable for use in treating an individual diagnosed with a sarbecovirus 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 antibody is selected from the group consisting of WIV-1, and SARS-CoV.

[0140]

[0168] In various embodiments, there are methods of treating or preventing a disease caused by infection with a sarbecovirus, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen binding molecule discussed herein above, wherein the antigen binding molecule is expressed in a cell discussed herein above.

[0141]

[0169] In various embodiments, the subject is an individual who has undergone a SARS-CoV-2 vaccination.

[0170] 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, 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 antibody is selected from the group consisting of WIV-1, and SARS-CoV.

[0143]

[0172] In various embodiments, there is a composition comprising an antigen binding molecule as discussed herein above, a nucleic acid or nucleic acids as discussed herein above, an expression vector or expression vectors as discussed herein above, or a cell as discussed herein above, and a pharma- ceutically acceptable carrier, diluent, excipient, or adjuvant.

[0144]

[0173] In various embodiments there is a composition as discussed herein above for use in the treatment or prevention of a disease caused by infection with a sarbecovirus.

[0145]

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

[0175] In various embodiments, the compositions discussed herein above are 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.

[0146]

[0176] In various embodiments, the compositions discussed hereinabove are suitable for use in treating an individual diagnosed with a sarbecovirus infection.

[0177] In various embodiments, the sarbecovirus is 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 antibody is selected from the group consisting of WIV-1, and SARS-CoV.

[0147]

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

[0148]

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

[0149]

[0180] In various embodiments, the use of the compositions discussed herein above 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 herein above in the manufacture of a medicament is suitable for use in treating an individual diagnosed with a sarbecovirus infection.

[0151]

[0182] In various embodiments, the sarbecovirus is 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 antibody is selected from the group consisting of WIV-1, and SARS-CoV.

[0152]

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

[0153]

[0184] In various embodiments, the subject is an individual who has undergone 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 sarbecovirus 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 is a use of the antigen binding molecules discussed herein above to inhibit infection of ACE2 expressing cells by sarbecoviruses.

[0189] In various embodiments, the sarbecovirus 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 the above-listed SEQ ID NOs: 31 to 70, the nucleic acids encoding the CDR regions are shown in bold and underlined. In the above-listed SEQ ID NOs: 71 to 110, the amino acids of the CDR regions are shown in bold and underlined.

[0185]

[0192] Working Example

[0193] In the following, we present, merely by way of example, antigen-binding molecules that bind to a wide range of sarbecovirus spike proteins. 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 sera collected from SARS survivors in Singapore at different time points (2003, 2012 and 2020) before the vaccination program was launched in February 2021; (2) COVID-19 patients (n=40): this group of sera was collected during 2020 as part of a national longitudinal study (Chia et al: Lancet Microbe 2021); (3) Healthy individuals vaccinated with a COVID-19 vaccine (in this case, the Pfizer mRNA vaccine) (n=20): these were sera collected 14 days after the second dose of the Pfizer-BioNTech BNT162b2 mRNA vaccine (or 35 days after the first dose). (4) SARS survivors who received a COVID-19 vaccine (in this case, the Pfizer mRNA vaccine) (n=9): Serum obtained from SARS survivors 21 to 62 days after the first dose of the Pfizer-BioNTech BNT162b2 mRNA vaccine.

[0187]

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

[0188] [Table 1]

[0189]

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

[0190]

[0198] A multiplex surrogate virus neutralization test (sVNT) based on the RBD from six different sarbecoviruses

[0199] Viral RBD was immobilized on a solid phase (magnetic beads) and used together with a fluorescent dye conjugated to ACE2, in this case phycoerythrin (PE), to measure virus-receptor binding, allowing for multiplex detection of NAbs against different sarbecoviruses (Figure 2). A total of six RBD proteins from six different sarbecoviruses were used, namely 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 AviTag biotinylated RBDs from different sarbecoviruses were coated onto MagPlex Avidin microspheres (Luminex) at 5 μg per million beads. In multiplex sVNTs, RBD-coated microspheres (600 beads / antigen) were pre-incubated with serum at a final concentration of 1:20 or greater for 1 h at 37°C with agitation at 800 rpm. After 1 h incubation, 50 μl of PE-conjugated hACE2 (GenScript, 1000 ng / ml) was added to the wells and incubated for 30 min at 37°C with agitation, followed by two washes with PBS-1% BSA. Data were acquired using the MAGPIX system.

[0192]

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

[0193]

[0202] It has been demonstrated that mRNA vaccines have an extraordinary ability to induce very high levels of neutralizing antibodies (NAbs) against SARS-CoV-2. However, from the data in the reported breakthrough infections (Hacisuleyman et al.: N Engl J Med 2021) and [Figure 3C], it is clear that NAbs have a relatively narrow spectrum, i.e., NAbs are highly specific for the viral sequence used in the mRNA vaccine, since some individuals had lower NAbs 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. The same was observed in COVID-19 patient sera ([Figure 3B]).

[0194]

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

[0195]

[0204] Pan-sarbecovirus mAb inhibition assay

[0205] Using the multiplex sVNT developed above, we expanded our studies to examine cross-NAbs against SARS-CoV-2 variants and, more importantly, against other sarbecoviruses that have been detected in bats and pangolins that are considered to be at potential risk for human infection (Lam et al.: Nature 2020, 583:282-285).

[0196]

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

[0197]

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

[0198]

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

[0199]

[0209] mRNA vaccines have been demonstrated to have an extraordinary ability to induce very high levels of NAbs against SARS-CoV-2. However, from the reported breakthrough infections (Hacisuleyman et al., N Engl J Med 2021) and data in [Figure 4], it is clear that NAbs have a relatively narrow spectrum, as some individuals had lower NAbs 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, and even lower NAbs against unemerged bat and pangolin viruses, with the lowest NAs against SARS-CoV-1.

[0200]

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

[0201]

[0211] SARS-CoV-1 survivors who received the BNT162b2 mRNA vaccine generated broadly neutralizing antibodies against 10 sarbecoviruses in clades 1a and 1b, including multiple VOCs of SARS-CoV-2 and zoonotic sarbecoviruses. EXAMPLES

[0202]

[0212] Example 2. Mouse studies

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

[0203]

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

[0204]

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

[0216] For flow cytometry analysis, cryopreserved PBMCs were thawed and surface stained for SARS-CoV-1 and SARS-CoV-2 specific B cells using bait tetramers (custom made by GenScript) prepared with biotinylated SARS-CoV-1 RBD or SARS-CoV-2 RBD tetramerized with streptavidin conjugated to BV421 (Biolegend, Cat#405225) or streptavidin conjugated to PE (BD Pharmigen, Cat#554061). Briefly, thawed PBMCs were incubated with SARS-CoV-1-RBD tetramers and SARS-CoV-2-RBD tetramers in FACS staining buffer (PBS supplemented with 2mM EDTA and 2% FBS) at 10% FBS for 40 minutes at room temperature before proceeding to stain with surface panel fluorochrome-conjugated antibodies. Surface staining was performed with a viability dye (Invitrogen, LIVE / DEAD® Fixable AQUA Dead Cell Stain), anti-human CD3 conjugated with FITC, anti-human CD14 conjugated with FITC, anti-human CD56 conjugated with FITC, anti-human CD19 conjugated with PE-Cyanine5, anti-human CD27 conjugated with APC-H7, and anti-human CD38 conjugated with BV786 for 30 minutes in FACS staining buffer at 4° C. Stained cells were washed twice with FACS staining buffer and acquired on the same day. Samples were acquired on a BD LSR Fortessa™ analyzer or a BD FACS Aria III equipped with 355, 405, 488, 561, and 640 nm lasers. SARS-CoV-1- and SARS-CoV-2-specific B cells were quantified by gating on CD19+ B cells after exclusion of AQUA-positive dead cells and CD3+, CD14+, and CD56+ cells. Cross-NAb boosting in the SARS-vaccinated group was further confirmed by direct staining of B cells with virus-specific RBD proteins.As shown in [Figure 7], dual stained B cells, i.e., B cells that bind RBDs derived from both SARS-CoV-1 and SARS-CoV-2, were significantly enriched by more than 10-fold in the SARS-vaccinated group compared to the healthy-vaccinated group. EXAMPLES

[0205]

[0217] Example 3 Rabbit Study

[0218] Virus / strain-specific immunodominant antibody responses were further confirmed using rabbit hyperimmune sera targeting specific viruses / strains. In addition to the four important 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 sarbecovirus with a very close genetic relationship to SARS-CoV-2, but its RBD was unable to bind hACE2 (Wacharapluesadee et al.: Nat Commun 2021, 12:972). RmYN02 is also very closely related to another bat sarbecovirus, RacCS203, found in bats in Thailand (Wacharapluesadee et al.: Nat Commun 2021, 12:972). HKU1 is a human betacoronavirus, not a sarbecovirus, and is included here as a negative control.

[0206]

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

[0220] Rabbit anti-RBD serum was generated by commercial agreement with GenScript Biotech using the RBD of each virus as an antigen. The test was essentially performed as described above. Rabbit serum was used in 4-fold serial dilutions starting from 1:20.

[0207]

[0221] The data shown in [Figure 8] demonstrate that cross-neutralization is restricted to the strain / lineage level only in the five sarbecoviruses SC2r-CoV. There was no cross-neutralization between SC2r-CoV and SARS-CoV, and the negative control HKU1 did not neutralize any virus / strain as shown. It is noteworthy that RmYN02 has been shown not to bind hACE2 (Wacharapluesadee et al.: Nat Commun 2021, 12:972), but is nevertheless capable of generating neutralizing antibodies against the bat sarbecovirus RaTG13.

[0208]

[0222] Based on the various embodiments described above from SARS-vaccinated donors, human pan-sarbecovirus NAbs are formed as follows.

[0223] Select SARS1-SARS2 double positive B cells: As shown in [Figure 9], use the RBDs from SARS-CoV-1 and SARS-CoV-2 to select B cells that produce antibodies that can recognize both RBDs (type C in the figure) and test them for neutralization of different sarbecoviruses.

[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 of mAb into the supernatant for initial screening and subsequent B cell receptor cloning of the best clones. Second, sorted B cells are directly lysed and the B cell receptor sequence is detected from the RNA, followed by subcloning the sequence into a mAb expression plasmid.

[0210]

[0225] Testing pan-sarbecovirus neutralizing activity: Supernatants containing individual mAbs are used to test cross-NAb activity using the multiplex sVNT platform described herein above.

[0211]

[0226] Large-scale production and further characterization: The top candidates are obtained for further characterization, including structural analysis for epitope mapping, determination of neutralizing activity against live virus, and confirmation of in vivo protection in antigen-challenged animal models. EXAMPLES

[0212]

[0227] Example 4. Isolation of broadly sarbecovirus-neutralizing mAbs from a SARS1 survivor donor vaccinated with BNT162b2 vaccine.

[0228] Blood was obtained from SARS-CoV-1 survivor SS6V, and the neutralizing capacity 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 were isolated from EDTA whole blood 23 days after the first vaccination and frozen 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 cell sorted into 96-well PCR plates (Axygen) pre-filled with 10 μl / well of RT-PCR catch buffer containing 10 mM TRIS pH 8.0 and 10 U of RNasin ribonuclease inhibitor (Promega) using a BD FACSAria III equipped with 355 nm, 405 nm, 488 nm, 561 nm and 640 nm lasers (BD Biosciences). Plates were then flash frozen on dry ice and kept at −80° C. until use.

[0213]

[0229] Reverse transcription was then performed on each plate of sorted 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, specific primers were then used to amplify individual gene families, and the PCR products were cloned into pTRIOZ expression vector (Invivogen).

[0214]

[0230] pTRIOZ constructs were transfected into HEK293 cells using Fugene6 (Promega), and supernatants were harvested to confirm small-scale efficacy screening using SARS-CoV-1 and SARS-CoV-2 RBD binding ELISAs and surrogate virus neutralization tests (sVNT). For binding ELISA assays, 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 per well and incubated for 1 h at 37°C. Goat anti-human IgG-HRP (Bethyl) was then added at 1:5000 diluted in OptEIA, which can convert the TMB substrate into a colorimetric readout for quantification using a Cytation5 reader (BioTek). 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 antibodies against SARS-CoV-1 was measured by substituting 6 ng / well of SARS-CoV-1 RBD-HRP (Genscript) instead of 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 the initial binding and neutralization screen 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) 23 days after the first dose of BNT162b2 vaccine were obtained for B cell enrichment and isolation. CD19+ B cells that were positive for binding to SARS-CoV-1 (SC1+) and SARS-CoV-2 (SC2+) RBD tetramers [Figure 11] 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 from double-positive (SC1+SC2+) B cells, but two were from SC2+, and none were recovered from SC1+ single-positive B cells.

[0218]

[0233] For control and comparison studies, four published broad-spectrum mAbs, namely, S309 (Sotrovimab, 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 against SARS-CoV-1 RBD [Table 3]. All 17 mAbs recovered from SC1+SC2+ B cells showed binding to both SARS-CoV-1 RBD and SARS-CoV-2 RBD, but their neutralizing capacity varied among the 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 IGHV4-59 heavy chains and IGKV2-28 / IGKJ5 light chains, suggesting that the B cells most likely originated from the same clonal family and that the subtle differences in the heavy and light chains arose by hypersomatic mutation.

[0219] [Table 3-1]

[0220] [Table 3-2] EXAMPLES

[0221]

[0234] Example 5 Potency, Spectrum and Mutant Escape Ability of Pan-Sarbecovirus mAbs

[0235] An 18-plex sVNT was performed based on the RBDs derived from the SARS-CoV-2 ancestral virus and its variants (alpha, beta, delta, delta plus, gamma, lambda, mu); the zoonotic sarbecoviruses BANAL-52, BANAL-236, GD-1, RaTG13, GX-P5L, Rs2018B, LYRa11, RsSHC014, WIV-1; and SARS-CoV-1. Data for the top six mAbs identified in the preliminary screening and three control mAbs are shown in [Figure 12]. SS6V11-E7, SS6V13-F1, and SS6V20-F5 mAbs exhibited highly potent ability to neutralize all 18 sarbecoviruses, 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 were less active / inactive against some of the viruses tested, i.e., SS6V12-E11 against lambda, SS6V19-F4 against lambda, GX-P5L, and RsSHC014. These are listed above as antibodies 1, 12, and 19. Their NT50 could be in the range of higher than 1,000 ng / ml (6.67 nM) for some clade-1b sarbecoviruses and higher than 200 ng / ml (1.33 nM) for clade-1a sarbecoviruses. For the four control mAbs, some degree of immune escape was also observed, i.e., by lambda and WIV-1 for S309, and by some clade-1b zoonotic sarbecoviruses (RaTG13 and GX-P5L) and all clade-1a sarbecoviruses (WIV-1, RsSHC014, Rs2018B, and SARS-CoV-1) for LyCoV-1404 [Figure 13]. It was therefore concluded that LyCoV-1404 retains potent activity against multiple variants, i.e., it is variant-proof, but not a pan-sarbecovirus mAb.As the primary objective was to develop a broadly pan-sarbecovirus neutralizing mAb, LyCoV-1404 was not included as a comparison in the remaining assays. S2X259 was the only control mAb tested that retained pan-sarbecovirus neutralizing activity against all 18 strains tested, with NT50s ranging between 47.39 and 370.50 ng / ml (0.316 to 2.47 nM). EXAMPLES

[0222]

[0236] Example 6 Functionality of Pan-Sarbecovirus Neutralizing mAbs

[0237] Based on the data from multiple assays presented above, SS6V11-E7, SS6V13-F1 and SS6V20-F5 were selected for further characterization along with three control mAbs, S309, CR3022 and S2X257, all of which were supplied by commercial suppliers either directly purchased or contract manufactured.

[0223]

[0238] The functionality of these mAbs in their ability to neutralize different sarbecoviruses was further evaluated against eight spike-pseudotyped reporter viruses, including the SARS-CoV-2 ancestral and four VOCs (alpha, delta, beta and gamma), two zoonotic sarbecoviruses (GX-P5L and WIV-1), and SARS-CoV-1. It was observed that all three tested mAbs retained highly potent neutralizing activity against all eight pseudoviruses, with relative half-maximal neutralization titers (NT50) below 10 ng / ml (0.067 nM) (Figure 14). S309 has an NT50 of 100-1000 ng / ml (0.667-6.67 nM) against SARS-CoV-2 VOCs and unemerged sarbecoviruses. Overall, S2X259 performed better than S309. However, it was approximately 10-fold less potent than the three mAbs identified in this study.CR3022 was only able to neutralize some clade-1a sarbecoviruses (WIV-1 and SARS-CoV-1) and failed to neutralize any clade-1b sarbecoviruses. EXAMPLES

[0224]

[0239] Example 7. Ability to neutralize different substrains of Omicron virus

[0240] During the final phase of this study, a new sublineage, Omicron BA.2, emerged that is becoming equally dominant to the prototype Omicron BA.1 virus. To determine the neutralizing capacity of the newly identified mAbs against these two virus variants, they were tested using three different platforms: multiplex sVNT, pseudovirus neutralization test (pVNT), and plaque reduction neutralization test (PRNT).

[0225]

[0241] Serum samples were tested in a newly developed multiplex sVNT assay.Briefly, AviTag biotinylated RBD proteins from ancestral SARS-CoV-2 and SARS-CoV-1, nine VOCs / VOIs (alpha, delta, beta, gamma, delta plus, lambda, mu, omicron BA.1, omicron BA.2), and ten zoonotic sarbecoviruses (BANAL-52, BANAL-236, GD-1, RaTG13, GX-P5L, Rs2018B, LYRa11, RsSHC014, and WIV-1) were coated onto MagPlex Avidin microspheres (Luminex) at 5 μg per million beads. RBD-coated microspheres (600 beads / antigen) were preincubated with 4-fold serially diluted mAbs at a starting concentration of 10,000 ng / ml for 15 min at 37°C with agitation at 250 rpm. After 15 min incubation, 50 μL of 2 μg / mL phycoerythrin (PE)-conjugated hACE2 (GenScript) was added to the wells and incubated at 37°C for 15 min with agitation, followed by two washes with PBS-1% BSA. Final readings were taken using a MAGPIX system (Luminex Corporation). Data shown in [Figure 15] indicate that SS6V11-E7 and SS6V20-F5 retained potent activity against both BA.1 omicron and BA.2 omicron, with average NT50s ranging between 177-315 ng / ml (1.18-2.10 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 SARS-CoV-1 full-length spike pseudotyped viruses were generated and packaged. Briefly, 5 million HEK293T cells were transfected with 20 μg of pCAGGS spike plasmid using FuGENE6 (Promega). 24 h after transfection, cells were incubated with VSVΔG luc seed virus (MOI of 5) for 2 h. 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. Pseudoviruses were harvested 24 h after infection by centrifugation at 2,000 × g for 5 min. For pVNT assays, 3 × 10 6 RLU of pseudovirus was preincubated with 4-fold serially diluted mAbs in a final volume of 50μl at a starting concentration of 20ug / ml for 1 hour at 37℃ and then infected into ACE2 stably expressing A549 cells. 20-24 hours after infection, an equal volume of ONE-Glo luciferase substrate (Promega) was added and luminescence signal was measured using a Cytation5 microplate reader (BioTek) with Gen5 software, version 3.10.

[0227]

[0243] The mAbs were serially diluted 4-fold from a starting concentration of 20ug / ml using DMEM containing 2% FBS. SARS-CoV-2 virus (ancestral or Omicron BA.1 and BA.2 strains) was then diluted to 500PFU / ml, mixed with the diluted mAbs and incubated at 37°C for 1 hour to allow binding of the mAbs to the virus. After 1 hour, the mAb-virus mixture was added to the A549-ACE2 monolayer and incubated at 37°C for an additional 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% carboxylmethycellulose) 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 virus neutralization studies, only SS6V11-E7, listed above as antibody 11, continued to show the ability to neutralize BA.2 at NT50 of 1400ng / ml (9.3nM) or 500ng / ml (3.33nM) using pVNT or PRNT assays, respectively (see [Figures 15B and 15C]). The approximately 2-4 fold difference in neutralization potency against BA.2 mutants measured by live virus assays (pVNT and PRNT) compared to the biochemical multiplex sVNT assay suggests that there may be additional antibody escape effects facilitated by mutations present in the complete BA.2 spike, which became evident in the live virus assay. Nevertheless, we observed that only SS6V11-E7 maintained neutralization capacity against Omicron BA.2, while all other mAbs tested, including three control mAbs, completely lost potency.

[0229]

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

[0230]

[0247] As can be understood by one of ordinary skill in the art, each embodiment can be used in combination with other embodiments or with several embodiments.

Claims

1. An antigen-binding molecule that binds to sarbecovirus spike proteins derived from two or more different sarbecoviruses, (i) the following CDRs: HC-CDR1 having the amino acid sequence GGFIGPHY HC-CDR2 having the amino acid sequence IYISGST HC-CDR3 having the amino acid sequence ARGGGYLETGPFEY and a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence QSLLQNNGYNY LC-CDR2 having the amino acid sequence LSS LC-CDR3 having the amino acid sequence MQSLQIPGT A light chain variable (VL) region incorporating An antigen-binding molecule comprising:

2. The antigen-binding molecule described in 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. The antigen-binding molecule of claim 1 or 2, which binds to the receptor binding domain (RBD) of a sarbecovirus spike protein.

4. The antigen-binding molecule of claim 1 or 2, which inhibits the interaction between sarbecovirus spike protein and angiotensinogen converting enzyme 2 (ACE2).

5. The antigen-binding molecule of claim 1 or 2, which inhibits infection of ACE2-expressing cells by a sarbecovirus.

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

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

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

12. 13. A pharmaceutical for treating or preventing a disease caused by infection with a sarbecovirus, comprising, as an active ingredient, an antigen-binding molecule according to any one of claims 1 to 6, a nucleic acid or multiple nucleic acids according to claim 7, an expression vector or multiple 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, 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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