Sabebe virus spike S2 subunit binding agent

Abstract

The present invention relates to Sabebe virus binding agents, in particular antibodies and antigen binding fragments thereof, which are effective to neutralize Sabebe viruses, in particular to either or both of SARS-CoV-2 (including SARS-CoV-2 variants) and SARS-CoV-1. The binding agents, in particular antibodies and antibody fragments, bind to the heptapeptide repeat 2 (HR2) domain of the Sabebe virus spike protein, more particularly to quaternary epitopes located within two adjacent HR2 domains. The invention also relates to methods of using these binders and uses thereof.

Description

Technical Field

[0001] The present invention relates generally to the field of binding agents, and in particular to the field of antibodies. More specifically, the present invention relates to binding agents (particularly antibodies and antigen-binding fragments thereof) that bind to the spike protein of Sarbecovirus, which are capable of effectively neutralizing Sarbecovirus (e.g., SARS-CoV-2, including SARS-CoV-2 variants and SARS-CoV-1). The present invention also relates to methods of using these binding agents and their uses. Background Art

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen of COVID-19 (Zhu et al., 2020, N Engl J Med 382:727-733). SARS-CoV-2 infection may be asymptomatic, or may present with mild to moderately severe symptoms. However, in approximately 10% of patients, COVID-19 progresses to a more severe stage characterized by dyspnea and hypoxemia, which may further progress to acute respiratory distress, often requiring long-term intensive care, and leading to death in some patients. And, "Long-COVID" refers to the long-term effects of COVID-19 infection, even if the SARS-CoV-2 virus is no longer detectable.

[0003] A specific type of treatment approach may rely on neutralizing antibodies, i.e., passive antibody therapy / immunotherapy. The spike protein of the SARS coronavirus is the primary target of neutralizing antibodies. The spike protein is a class I fusion protein consisting of a membrane-distal S1 subunit and a membrane-proximal S2 subunit. The S1 subunit contains a receptor binding domain (RBD), against which antibodies can have very strong neutralizing activity (Whealey et al., 2021, Cell Rep 37:109822). The S1 subunit, especially the N-terminal domain and RBD, can tolerate mutations that lead to antigenic variation and immune escape. RBD is also immunodominant (Piccoli et al., 2020, Cell 183:1024-1042).

[0004] The S2 subunit is responsible for membrane fusion, during which S2 undergoes major conformational changes (Dodero-Rojas et al., 2021, eLife 10: e70362). The S2 subunit is more conserved and therefore, at least in theory, appears to be an attractive target for the development of neutralizing antibodies with broad anti-Sabemiviral protection potential. Several monoclonal antibodies that recognize conserved epitopes of the SARS coronavirus S2 subunit have been described in the art. However, in general, these monoclonal antibodies exhibit poor virus neutralizing activity. For example, the S2 subunit-specific monoclonal antibody L19 has an IC of 9.9-19.8 μg / ml. 100 Neutralizes the real SARS-CoV-2 virus (Andreano et al., 2021, Cell 184:1821-1835). Wu et al. (2022, JCI Insight 7:ee157597) identified monoclonal antibodies Mab5 and Mab3-2, which target the HR2 domain at an epitope located at the N-terminus of the HR2 domain. Both mAbs have neutralizing ability against SARS-CoV-2, with Mab5's IC 50 The value was 12.3 μg / mL, and the IC 50 The value was 87.4 μg / mL. Single-domain antibodies (also called nanobodies or VHHs) against the SARS-CoV-2 S2 subunit have also been reported in the art (Mast et al., 2021, eLife 110: e73027; Rossotti et al., 2021, DOI: 10.1101 / 2021.12.20.473401). Similarly, the reported VHHs binding to the S2 subunit showed very low SARS-CoV-2 neutralization potency. The S2 subunit-specific VHH S2A3 fused to IgG1-Fc, as described by Rossotti et al. (2021), was able to neutralize SARS-CoV-2 with an IC of 12.2 nM. 50 Neutralizes the Wuhan-Hu-1 strain of SARS-CoV-2, but is not neutralizing outside of this model.

[0005] Therefore, there is still a need in the art for effective neutralizing antibodies targeting the spike protein of Sabeivirus. Summary of the invention

[0006] As demonstrated in the experimental section illustrating certain embodiments of the invention, the inventors have identified Sabeivirus-specific heavy chain antibody variable domains (VHH) that effectively neutralize SARS-CoV-2, including SARS-CoV-2 variants, such as SARS-CoV-2 D614G variant, SARS-CoV-2 Alpha variant, SARS-CoV-2 Omicron BA.1 variant, SARS-CoV-2 Omicron BA.2 variant, SARS-CoV-2 Omicron BA.5 variant, SARS-CoV-2 Omicron BA.2.75.2 variant, SARS-CoV-2 Omicron BA.4.6 variant, SARS-CoV-2 Omicron BF.7 variant, SARS-CoV-2 Omicron BQ.1.1 variant, SARS-CoV-2 Omicron XBB variant and SARS-CoV-2 Omicron XBB.1.5 variant and SARS-CoV-1. Through further analysis, it was found that these VHHs interact with amino acids within the spike protein S2 subunit (especially within the domain of the heptad repeat 2 (HR2) of the S2 subunit, more particularly within the C-terminal region of the HR2 domain close to the viral membrane), which are highly conserved in the spike proteins of Sabei viruses of multiple evolutionary branches. Therefore, this region is expected to be more stable and less susceptible to frequent mutations.

[0007] Therefore, in one aspect, the present invention relates to a binding agent capable of neutralizing Sabeivirus, characterized in that the binding agent specifically binds to the viral membrane-adjacent region of the heptad repeat 2 (HR2) domain of the Sabeivirus spike protein.

[0008] One aspect provides a binding agent capable of neutralizing Sabei virus, characterized in that the binding agent specifically binds to the spike protein of Sabei virus or binds to a region thereof, which corresponds to the region from amino acid E1188 to amino acid Y1206 of the SARS-CoV-2 spike protein, as defined in SEQ ID NO: 86.

[0009] In a particular preferred embodiment, the binding agent specifically binds to the spike protein or to a region thereof, which corresponds to the region from amino acid E1188 to amino acid L1203 of the SARS-CoV-2 spike protein, as defined in SEQ ID NO: 86.

[0010] In a particular preferred embodiment, the binding agent specifically binds to the spike protein or to a region thereof, which corresponds to the region from amino acid E1188 to amino acid L1202 of the SARS-CoV-2 spike protein, as defined in SEQ ID NO: 86.

[0011] Such Sabaevirus neutralizing binders that bind to the more conserved S2 subunit of the spike protein are valuable tools that could be added to the therapeutic options for SARS-CoV-2, which remain limited in number, particularly in light of the multiple emerging SARS-CoV-2 variants, some of which are more contagious and / or cause more severe disease symptoms (including in young people) and / or evade some existing vaccines and / or diagnostic tests.

[0012] In another aspect, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a binding agent according to the present invention, and a vector comprising the nucleic acid molecule; and a cell comprising the nucleic acid molecule or the vector, or a cell expressing a binding agent according to the present invention.

[0013] The invention also relates to pharmaceutical compositions comprising a binding agent according to the invention, or a nucleic acid molecule or vector as described above; and a pharmaceutically acceptable carrier; and to kits, such as diagnostic kits, comprising a binding agent according to the invention.

[0014] Another aspect relates to a binding agent according to the invention, a nucleic acid molecule or a vector as described above, a pharmaceutical composition or a kit as described above for medical use, such as for use in preventing or treating a Sabeivirus infection in a subject or for use in diagnosing a Sabeivirus infection in a subject.

[0015] The present invention also relates to an in vitro or ex vivo method for detecting Sabei virus in a sample, the method comprising:

[0016] - contacting the sample with a binding agent according to the invention, and

[0017] - Determining the binding of said binding agent to Sabeivirus or a part thereof.

[0018] From the detailed description and examples provided below, those skilled in the art will recognize many other effects and advantages of the method, use or product of the present invention, as well as the various possibilities of the end use of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1: VHHs present in E. coli periplasmic extracts (PE) of isolated clones bind to recombinant SARS-CoV-2 spike protein (SC2 S(6P)), SARS-CoV-2 RBD (SC2RBD), SARS-CoV-2 S2 subunit (SC2S2), and SARS-CoV-1 spike protein (SC1 S) in ELISA. (A) ELISA of clones (R3_Cxx) isolated after 3 rounds of biopanning (R1-R3) with SARS-CoV-2 spike protein captured with anti-His antibody coated on ELISA plates. (B) ELISA of clones (R3_DCxx) isolated after 3 rounds of biopanning (R1-2) with SARS-CoV-2 spike protein captured with anti-His antibody followed by biosorting (R3) with directly coated SARS-CoV-2 spike protein. (C) ELISA of clones (R4_Cxx) isolated after 4 rounds of bio-panning (R1-R4) of SARS-CoV-2 spike protein captured with anti-His antibody coated on ELISA plates. (D) ELISA of clones (R4_DCxx) isolated after 4 rounds of bio-panning (R1-2) of SARS-CoV-2 spike protein captured with anti-His antibody followed by bio-sorting (R3-R4) of directly coated SARS-CoV-2 spike protein. The figure shows the ratio of the ELISA OD450 signal of the specified antigen for each PE sample to the ELISA OD450 signal of the control antigen (BSA) for the corresponding PE sample. Periplasmic extracts prepared from E. coli cells expressing VHH, VHH3.83 (3.83) that binds to RBD were used as controls. The buffer used to prepare the periplasmic extracts was used as a negative control (TES).

[0020] Figure 2: Sequence analysis of VHHs capable of binding to the S2 subunit of the SARS-CoV spike protein. (A) Alignment of family 1 VHHs binding to the S2 subunit of the SARS-CoV spike protein. Based on the alignment of the complete VHH sequences, two families of VHHs binding to S2 (family 1 and family 2) could be identified. The sequences of Family 1 VHHs are shown: R3_C4 and R3_DC13 (SEQ ID NO: 1), R3_DC19 (SEQ ID NO: 83), R3_DC21 and R3_DC22 (SEQ ID NO: 84), R3_C22 and R4_DC16 (SEQ ID NO: 2), R3_DC20 (SEQ ID NO: 3), R3_DC1, R3_DC9, R3_DC14 and R3_DC15 (SEQ ID NO: 9), R3_DC2 (SEQ ID NO: 4), R4_DC20 (SEQ ID NO: 5), R3_DC12 and R4_DC13 (SEQ ID NO: 10), R3_DC5 (SEQ ID NO: 85), R4_DC24, R4_DC21, R3_DC11 and R4_DC9 (SEQ ID NO: 6), R3_DC8, R4_DC3 and R4_DC6 (SEQ ID NO: 7). NO: 7), R3_DC23 (SEQ ID NO: 8). Amino acid residue numbering is according to Kabat numbering. CDR1, 2 and 3 according to Kabat annotation are represented by left, middle and right boxes, respectively. (B) Phylogenetic analysis of family 1 VHH based on CDR3 amino acid sequences. VHHs marked in grey were selected for medium-scale production and Ni-NTA purification. VHHs indicated by "*" contain N-glycosylation site motifs.

[0021] Figure 3 :Selected S2-binding VHHs recognize the spike protein of SARS-CoV-1, SARS-CoV-2 Wuhan-Hu-1 variant, SARS-CoV-2 Omicron BA.1 variant and SARS-CoV-2 S2 subunit, but not SARS-CoV-2 RBD. The graph shows the OD450 ELISA signal of the designated VHHs (including GFP-binding VHH control (GBP), S309 control monoclonal antibody) against the spike protein S-6P (A), RBD (E) and S2 subunit (D) of SARS-CoV-2 Wuhan-Hu-1 coated on the matrix, the spike protein of OmicronBA.1 SARS-CoV-2 (B) and the spike protein of SARS-CoV-1 (C), and against the matrix coated with BSA (F).

[0022] Figure 4: VHHs targeting S2 effectively bind to the spike protein expressed on the cell surface. (A) Flow cytometric analysis of the binding of R3_DC23, R4_DC6, and GFP-binding VHH (GBP) controls to cells expressing the SARS-CoV-2 spike protein. The figure shows the mean fluorescence intensity (MFI) of AF647-conjugated anti-mouse IgG for detecting the binding of VHHs to cells expressing GFP, which were transfected with a GFP expression vector in combination with a SARS-CoV-2 spike protein (614G-del18) expression vector (spike protein D614G). (B) Flow cytometric analysis of the binding of R3_DC23, R4_DC6, and GBP control VHHs to cells that do not express the SARS-CoV-2 spike protein. The figure shows the MFI of AF647-conjugated anti-mouse IgG for detecting the binding of VHHs to cells expressing GFP, which were transfected with a GFP expression vector in combination with a control expression vector.

[0023] Figure 5 : VHH targeting S2 neutralizes VSV-GFP reporter virus pseudotyped with SARS-CoV-2 614G spike protein. Vero E6 cells were transduced with VSV-GFP reporter virus pseudotyped with SARS-CoV-2 614G spike protein, which had been pre-incubated with different concentrations of the specified VHH. After 15 hours, GFP fluorescence values ​​were measured using a fluorimeter. (A) The graph shows the average GFP fluorescence intensity of VHH gradient dilutions (N=3±SD), each based on the GFP fluorescence intensity values ​​for uninfected control cells and untreated infected cells, both of which were included in each gradient dilution. (B) For each VHH gradient dilution, the IC50 (log (inhibitor) vs. normalized response, with variable slope) was calculated using a linear regression curve fit. The figure shows the IC50 calculated for each neutralizing VHH tested (N=3±SD).

[0024] Figure 6: VHHs targeting S2 can hinder TMPRSS2-mediated infection of VSV-GFP reporter viruses pseudotyped with SARS-CoV-2 614G spike protein. Vero E6-TMPRSS2 cells were transduced with VSV-GFP reporter viruses pseudotyped with SARS-CoV-2 614G spike protein, which had been pre-incubated with different concentrations of the specified VHHs. After 15 hours, GFP fluorescence values ​​were measured using a fluorimeter. (A) The graph shows the average GFP fluorescence intensity of VHH gradient dilutions (N=3±SD), each of which was normalized based on the GFP fluorescence intensity values ​​of uninfected control cells and untreated infected cells, both of which were included on each plate. (B) For each VHH gradient dilution, the IC50 (log (inhibitor) vs. normalized response, with variable slope) was calculated using linear regression curve fitting. The figure shows the IC50 calculated values ​​for each neutralizing VHH tested (N=3±SD).

[0025] Figure 7 : VHH targeting S2 neutralizes replication-competent VSV-GFP reporter virus, which is pseudotyped with SARS-CoV-2 Wuhan-Hu-1 spike protein. 100 PFU of the replication-competent VSV-GFP reporter virus (VSV-ΔG SC2S EGFP S10a) described by Koenig et al. (2021) was pre-incubated with the specified VHH and used to infect Vero E6 cells. 2 days after infection, GFP fluorescence values ​​were measured with a fluorimeter. (A) The graph shows the average GFP fluorescence intensity of the VHH gradient dilutions (N=2±SD), each of which is normalized based on the GFP fluorescence intensity values ​​of uninfected control cells and untreated infected cells, both of which are included in each gradient dilution. (B) For each VHH gradient dilution, the IC50 (log (inhibitor) vs. normalized response, with variable slope) was calculated using a linear regression curve fit. The figure shows the IC50 calculated values ​​(N=2±SD) for each neutralizing VHH tested.

[0026] Figure 8: VHHs targeting S2 neutralize VSV-GFP reporter viruses pseudotyped with SARS-CoV-2 Omicron BA.1 spike protein. Vero E6 cells were transduced with VSV-GFP reporter viruses pseudotyped with SARS-CoV-2 Omicron BA.1 spike protein, which had been pre-incubated with different concentrations of the indicated VHHs. The S309 monoclonal antibody, which is known to neutralize the SARS-CoV-2 Omicron BA.2 variant, was used as a positive control. After 15 hours, GFP fluorescence values ​​were measured using a fluorimeter. (A) The graph shows the average GFP fluorescence intensity of the VHH gradient dilutions (N=1), each of which was normalized based on the GFP fluorescence intensity values ​​of uninfected control cells and infected cells treated with the lowest VHH concentration, both of which were included in each gradient dilution. (B) For each VHH gradient dilution, the IC50 (log (inhibitor) vs. normalized response, with variable slope) was calculated using a linear regression curve fit. The figure shows the IC50 calculation value (N=1) for each neutralizing VHH tested.

[0027] Fig. 9 : VHH targeting S2 neutralizes VSV-GFP reporter virus pseudotyped with SARS-CoV-1 spike protein. VeroE6 cells (A) or VeroE6-TMPRSS2 cells (B) were transduced with VSV-GFP reporter virus pseudotyped with SARS-CoV-1 spike protein, which had been pre-incubated with different concentrations of the specified VHH. VHH72-S56A nanobody, known to neutralize SARS-CoV-1, was used as a positive control. After 15 hours, GFP fluorescence values ​​were measured using a fluorimeter. The graph shows the average GFP fluorescence intensity of VHH gradient dilutions (N=2±SD), each of which was normalized based on the GFP fluorescence intensity values ​​of uninfected control cells and untreated infected cells, both of which were included in each gradient dilution. (C) For each VHH gradient dilution, the IC50 (log (inhibitor) vs. normalized response, with variable slope) was calculated using linear regression curve fitting. The graph shows the calculated IC50 values ​​for each neutralizing VHH tested in both Vero E6 and Vero E6-TMPRSS2 cells (N=2±SD).

[0028] Fig.10: VHH targeting S2 neutralizes VSV-GFP reporter virus pseudotyped with SARS-CoV-2 Omicron BA.2 spike protein. VeroE6 cells were transduced with VSV-GFP reporter virus pseudotyped with 614G spike protein or Omicron BA.1 or Omicron BA.2 variant spike protein, which had been pre-incubated with different concentrations of the indicated VHH. After 15 hours, GFP fluorescence values ​​were measured using a fluorimeter. (A) The graph shows the average GFP fluorescence intensity of VHH gradient dilutions (N=2±SD), each of which was normalized based on the GFP fluorescence intensity values ​​of uninfected control cells and untreated infected cells, both of which were included in each gradient dilution. (B) For each VHH gradient dilution, the IC50 (log (inhibitor) vs. normalized response, with variable slope) was calculated using linear regression curve fitting. The figure shows the calculated ICS0 values ​​for each tested neutralizing VHH on Vero E6 cells transduced with VSV-GFP reporter virus pseudotyped with SARS-CoV-2 614G spike protein (614G) or Omicron BA.1 (BA.1) or Omicron BA.2 (BA.2) variant spike proteins (N=2±SD).

[0029] Fig.11 : VHHs targeting S2 are able to neutralize authentic 614G and Omicron SARS-CoV-2 viruses. Serial dilutions of R3_DC23 or R4_DC6 were pre-incubated with approximately 40 PFU of 614G variant SARS-CoV-2 or Omicron BA.1 SARS-CoV-2 viruses at 37°C for 1 hour and then used to infect Vero E6-TMPRSS2 cells. Antibody S309, known to neutralize Alpha and Omicron BA.1 SARS-CoV-2 variants, was used as a positive control. Two days after infection, cells were fixed and stained with crystal violet to visualize viral plaques. The figure shows the average number of 614G (A) or Omicron BA.1 (B) viral plaques at the specified VHH or antibody concentration (N=2±SD for R3_DC23 and R4_DC6; N=1 for S309). The curves were fitted using nonlinear regression (log (inhibitor) vs. response (four parameters) with variable slope).

[0030] Fig.12: VHHs targeting S2 do not cause shedding of the S1 subunit. (A) A graph showing an anti-S1 Western blot analysis of growth medium (SN) and cell lysates (LYS) of Raji cells expressing (Raji spike protein) or not expressing (Raji) SARS-CoV-2 spike protein, wherein the Raji cells were incubated with the specified VHH constructs for 30 minutes. CB6 and S309 antibodies, which are known to cause and not cause S1 shedding, respectively, were used as controls. The lower triangle and upper triangle on the right side of the blot represent the S1 spike protein subunit generated after furin-mediated spike protein cleavage and the spike protein that is not cleaved by the cell, respectively. (B) Quantification of S1 shedding. The figure shows the calculated ratio of the S1 Western blot signal detected in the growth medium (shedding) and the S1 Western blot signal detected in the cell lysate (non-shedding + intracellular).

[0031] Fig.13 : VHHs targeting S2 effectively inhibit fusion. VHHR3_DC23 can hinder syncytia formation in confluent monolayers of Vero E6-TMPRSS2 cells infected with replication-competent VSV virus expressing GFP pseudotyped with SARS-CoV-2 spike protein. Vero E6-TMPRSS2 cells were infected with 40 PFU of replication-competent VSV virus pseudotyped with SARS-CoV-2 spike protein, and the indicated mAbs (palivizumab, S309 or CB6) or VHHs (GBP or R3_DC23) were added to a final concentration of 10 μg / ml 2 hours later. Uninfected cells were used as negative controls. Cells were incubated overnight and imaged with a fluorescence microscope. GFP fluorescence values ​​were measured with a fluorimeter. (A) Representative images of GFP expressed by infected cells treated with the indicated VHHs or mAbs. (B) Graphs showing GFP fluorescence intensity (mean ± SEM, N = 4). The GFP fluorescence values ​​measured in the S309-treated samples were significantly lower than those in the samples treated with the palivizumab control antibody (p<0.05, Mann-Whitney test). The GFP fluorescence values ​​measured in the R3_DC23-treated samples were significantly lower than those in the samples treated with the GBP control VHH (p<0.05, Mann-Whitney test). The GFP fluorescence values ​​measured in the R3_DC23-treated samples were significantly lower than those in the samples treated with S309 (p<0.05, Mann-Whitney test).

[0032] Fig.14: VHH targeting S2 effectively inhibits fusion. Vero E6-TMPRSS2 cells were infected with 40 PFU of replication-competent VSV virus pseudotyped with the SARS-CoV-2 spike protein, and gradient dilutions of the specified mAbs (S309 or CB6) or VHHs (GBP, R3_DC23, R3_C4, or R3_DC20) were added 2 hours later. Uninfected cells were used as negative controls. The cells were incubated overnight and imaged with a fluorescence microscope. GFP fluorescence values ​​were measured with a fluorimeter. (A) Representative images of GFP expressed by infected cells treated with 10, 0.4, or 0.0032 μg / ml of the specified VHHs or mAbs. Arrows indicate single GFP-positive infected cells. (B) Graphs showing GFP fluorescence intensity (mean ± SEM, N = 2).

[0033] Fig.15 : VHH targeting S2 effectively inhibits the fusion of Vero E6 cells expressing spike protein. Vero E6 cells were transfected with a GFP expression vector in combination with a control expression vector (no spike protein) or a SARS-CoV-2 spike protein expression vector. 2 hours after transfection, PBS or the specified monoclonal antibody (S309 or palivizumab) or VHH (R3_DC23 or R3_C4) was added to a final concentration of 10 μg / ml. 22 hours after transfection, the cells were fixed and imaged using a fluorescence microscope. The figure shows representative images of the specified samples. No spike protein = PBS-treated cells, which were transfected with a GFP expression vector in combination with a control expression vector; PBS, palivizumab, R3_DC23, R3_C4, and S309 = cells transfected with a GFP expression vector in combination with a SARS-CoV-2 spike protein expression vector and treated with their respective constructs.

[0034] Fig.16:VHH.R3_DC23 binds to the spike protein at a membrane-proximal site in the HR2 region. Viral escape selection was performed on Vero E6-TMPSS2 cells using a replication-competent GFP-expressing VSV virus pseudotyped with the SARS-CoV-2Wuhan-Hu-1 spike protein with an intact furin cleavage site. Single plaques were isolated from wells showing syncytium formation in the presence of 10 μg / ml of VHH.R3_DC23 using limiting dilution. The spike protein coding sequences of the escape variants obtained were sequenced and compared with the sequence of the WT virus. Each selected virus contained an amino acid substitution. (A) Sequence of the R3_DC23 binding region. Viral escape selection of VHH.R3_DC23 was associated with 5 different AA substitutions at 4 positions within the restricted membrane-proximal region within HR2. The sequence shown (SEQ ID NO: 88) corresponds to the spike protein stem region (amino acids 1140-1211) consisting of a stem-helix and a heptad repeat 2 (HR2) domain. The bold and underlined amino acids indicate the positions of mutations in the nine isolated escape variants. The bold and underlined Asn (N) between Asp (N) and Leu (L) is a structural detail of the N-glycosylation site (B) R3_DC23 binding region. The figure on the left represents a model of the full-length SARS-CoV-2 spike protein, with the transmembrane region (TM) and HR2 marked on it (Casalino et al. (2020) ACS Cent Sci. 6: 1722-1734). The middle figure and the figure on the right are enlarged views of the HR2 domain shown in surface and cartoon form, respectively (2FXP, Hakansson-McReynolds et al. (2006) J Biol Chem. 281: 11965-71). In the surface plot images, sticks represent sugar moieties in the model, TMs are shown in gray, and the positions of the selected mutations in the three protomers are shown in black. In the middle figure, arrows indicate the positions of the N1192 and Q1201 mutations in protomer 1, respectively. The other two visible escape mutations (shown in black) are located in protomers 3 and 2, respectively. In one of the protomers shown in the cartoon, the position where the mutation was observed is shown in black sticks.

[0035] Fig.17: VHH.R3_DC23 binds to the spike protein at a site in the HR2 domain that is highly conserved in Sabeivirus. Letko et al. (2020, Nature Microbiology 5:562-569) studied the amino acid sequences of the spike protein stem region of a group of 1, 2 and 3 evolutionary branch saboiviruses, and supplemented them with the amino acid sequence of BtKY72 branch 3 saboivirus, which were aligned to visualize their conservation: Wuhan_SARS-CoV-2, WIV1, Rs4084, SHC014, Rs7327, Rs4231, SARS-CoV_Urbani, Longquan-140, HKU3-8, HKU3-13, Rs4237, Rs4247, As5626, Rp3, 279, Rs4081, Yunnan2011, Hub2013, Shaanxi2011, YN2013 and Rf4098 (SEQ ID NO: 88); LYRa11 (SEQ ID NO: 89); 273-2005, Rf1, HeB2013 and JL2012 (SEQ ID NO: 90); GX2013 (SEQ ID NO: 91); ZC45 (SEQ ID NO: 92); ZXC21 (SEQ ID NO: 93); BM48-31 (SEQ ID NO: 94); and BtKY72 (SEQ ID NO: 95). The linear sequence marked in gray (corresponding to amino acids 1192-1201 of the spike protein of the SARS-CoV2 Wuhan-Hu-1 strain shown in SEQ ID NO: 86) contains the positions that mutated in the isolated R3_DC23 escape variant. The amino acids in bold and underlined are the amino acids that mutated in the isolated R3_DC23 escape variant. The sequences are grouped according to the evolutionary branch of the corresponding Sabei virus (as shown on the right).

[0036] Fig.18 :Fc fusion protein of VHH.R3_DC23 effectively binds to the spike protein expressed on the cell surface. Flow cytometry analysis of the binding of R3_DC23-Fc(YTE) and control antibody palivizumab to cells expressing SARS-CoV-2 spike protein. The figure shows the mean fluorescence intensity (MFI) of anti-human IgG conjugated with AlexaFluor (AF) 633 for detecting the binding of R3_DC23-Fc(YTE) or palivizumab to cells expressing GFP transfected with GFP expression vector in combination with (A) SARS-CoV-2 spike protein (614G-del18) expression vector (spike protein D614G), or in combination with (B) control expression vector combination (empty vector).

[0037] Fig.19 :R3_DC23-Fc(YTE) can effectively neutralize VSV-GFP reporter viruses pseudotyped with SARS-CoV-2 614G spike protein or Omicron BA.1 and BA.2 spike proteins. Vero E6 cells were transduced with VSV-GFP reporter viruses pseudotyped with SARS-CoV-2 spike protein 614G or with spike proteins of SARS-CoV-2 Omicron BA.1 or Omicron BA.2 variants, which were pre-incubated with different concentrations of R3_DC23-Fc(YTE) (N=3), palivizumab (N=1), CB6 (N=1) or monovalent VHH.R3_DC23 (N=1). After 15 hours, GFP fluorescence values ​​were measured with a fluorimeter. (A) The graph shows the average GFP fluorescence intensity of the VHH gradient dilutions (for R3_DC23-Fc (YTE), N = 3 ± SD), each of which is standardized based on the GFP fluorescence intensity values ​​of uninfected control cells and untreated infected cells, both of which are included in each gradient dilution. (B) For each VHH gradient dilution, IC50 (log (inhibitor) vs. standardized response, with variable slope) was calculated using linear regression curve fitting. The figure shows the IC50 calculated values ​​for each antibody construct tested (for R3DC23-Fc (YTE), N = 3 ± SD).

[0038] Fig. 20 : Graphic representation of the VHH R3_DC23 amino acid sequence and the different CDR annotations used herein. The CDR annotations according to AbM, Chothia, Martin, Kabat, IMGT and MacCallum are located in the grey marked box corresponding to the sequence of VHH R3_DC23 (SEQ ID NO: 8).

[0039] Fig.21 : Prophylactic treatment with R3_DC23-Fc protected K18-hACE2 mice from lethal SC2 infection. 2 K18-hACE2 mice were intraperitoneally injected with 100 μg R3_DC23-Fc or isotype control antibody (palivizumab) 20 hours before pFU of SARS-CoV-2 614G variant virus, or left untreated. Animals were monitored daily by measuring body weight changes and humane endpoint scores. (A) The figure shows the mean relative body weight changes of mice treated with R3_DC23-Fc (n=3±SEM), palivizumab (n=2±SEM), or untreated mice (n=6±SEM). Mice treated with R3_DC23-Fc showed significantly less weight loss (using (B) The figure shows the Kaplan-Meier curves of the surviving fraction of the animals in the indicated groups. Mice were euthanized when they lost more than 25% of their body weight (defined by body weight on day 0) or reached a high score at the humane endpoint.

[0040] Fig. 22 : Identification of SARS-CoV-1 and -2 S2 subunit specific VHHs (A) Screening of E. coli periplasmic extracts (PE) of isolated VHH clones after 3 (R3) or 4 (R4) bio-pannings with SARS-CoV-2 spike protein, directly coated (DC) or captured by coated anti-HIS IgG (C) for binding to specified recombinant spike protein or fragments thereof. The heat map shows the ratio of the ELISA OD450 signal of the specified antigen for each PE sample (10-fold dilution) to the ELISA OD450 signal of the control antigen (BSA) of the corresponding PE sample. Periplasmic extracts prepared from E. coli cells expressing VHHs binding to RBD were used as controls. The buffer used to prepare periplasmic extracts (TES) was used as negative controls. (B) Screening of E. coli periplasmic extracts (PE) of VHH clones isolated after 3 (R3) or 4 (R4) biopannings with SARS-CoV-2 spike protein, directly coated (DC) or captured by coated anti-HIS IgG (C), for neutralization of VSV particles pseudotyped with SARS-CoV-2 spike protein. The heat map shows the neutralization level of SARS-CoV-2 spike protein pseudotyped VSV particles for each PE sample (100-fold dilution).

[0041] Fig.23 : VHHs targeting S2 bind to cells expressing SARS-CoV-2 614G, BA.1, BA.2, BA.5, BQ1.1, and MERS spike proteins. VHH72-S56A and S309 binding to SARS-CoV-2 RBD were used as positive controls, and VHHGBP binding to GFP was used as a negative control. Expression of the MERS spike protein was confirmed by binding of the MERS-specific VHH55 (data not shown). The figure shows the ratio of the MFI of transfected (GFP+) cells to the MFI of untransfected (GFP-) cells.

[0042] Fig.24: Neutralization of VSV particles pseudotyped with SARS-CoV-2 614G, BA2, BA.5, XBB, BQ1.1 and the spike protein of SARS-CoV-1 by VHH binding to S2. The figure shows the mean (line) and individual (points) IC50 values ​​calculated from at least 2 independent neutralization assays (E).

[0043] Fig.25 : VHHs targeting S2 effectively neutralize replication-competent SARS-CoV-2 pseudotyped viruses. VeroE6 (A) or Vero E6-TMPRSS2 (B) cells were infected with replication-competent GFP reporter viruses pseudotyped with SARS-CoV-2 VSV-S that had been pre-incubated with serial dilutions of the indicated VHHs. VHH72-S56A served as a positive control and GFP-binding protein (GBP) as a negative control. The average GFP intensity of two technical replicates for each dilution is shown, and the error bars represent standard deviations.

[0044] Fig.26: VHHs targeting S2 do inhibit spike protein-mediated membrane fusion. (A) VHHs targeting S2 do not interfere with the binding of the spike protein to ACE2. The figure shows the OD450 signal of an ELISA that tested the binding of human ACE2-muFc to a coated recombinant spike protein containing an inactive furin cleavage site in the presence of a gradient dilution of R3DC23. A GFP-binding VHH (GBP) was used as a negative control, and VHH72-S56A and CB6, both of which compete with ACE2 for binding to RBD, were used as positive controls. (B) VHHs targeting S2 do not induce shedding of S1. The figure shows anti-S1 Western blot analysis of growth medium (SN) and cell lysates (LYS) of Raji cells expressing (Raji spike protein) or not expressing (Raji) SARS-CoV-2 spike protein, which were incubated with the indicated VHH constructs for 30 minutes. CB6 and S309 antibodies, which are known to cause and not cause S1 shedding, respectively, were used as controls. The lower triangle and upper triangle on the right side of the blot represent the S1 spike protein subunit generated after furin-mediated spike protein cleavage and the cell-uncleaved spike protein, respectively. (C) VHH targeting S2 does not interfere with the binding of human ACE2-muFc to cells expressing spike protein with an intact furin cleavage site. The figure shows the ratio of MFI (detection of cell binding to ACE2-muFc) of GFP+ cells to GFP- cells in the presence of R3DC23. GBP was used as a negative control, and VHH72-S56A, which induces S1 shedding and competes with human ACE2 for binding to RBD, was used as a positive control. The dotted line represents the binding of ACE2-muFc to cells that do not express the spike protein. The dotted line represents the binding of ACE2-muFc to cells expressing the spike protein in the absence of antibodies. (D) VHH targeting S2 effectively prevents infected cells from forming syncytia. The figure shows the mean ± SD of GFP fluorescence in Vero E6 cell wells treated with serial dilutions of R3DC23, GBP or S309 4 hours after infection with replicating VSV pseudotyped with Wuhan spike protein and replicating VSV containing GFP expression cassette (N = 2). The image on the left shows GFP expression of the indicated samples 40 hours after infection. (E) Quantification of syncytium formation of cells expressing spike protein in the presence of R3DC23, GBP or PBS during live cell imaging. The figure shows the mean GFP+ area values ​​± SD (N = 3) of wells treated with the indicated VHHs 4 hours after co-infection with GFP and spike protein expression vectors or co-transfection with GFP expression vector alone (no spike protein).

[0045] Fig. 27: VHH.R3_DC23 binds to the spike protein at the proximal site of the HR2 region. (A and B) Replication of viral escape variants N1192D, L1197P, L1200P, Q1201R and Q1201K in Vero E6-TMPRSS2 and Vero E6 cells in the presence of R3DC23. The figure shows the mean ± SEM (N = 4) of GFP levels normalized by GFP fluorescence values ​​of mock-infected cells and infected cells in the absence of R3DC23. (C) Binding of R3DC23 to cells expressing Wuhan and N1192D, L1197P, L1200P, Q1201R or Q1201K spike protein variants. The figure shows the ratio of the MFI of transfected cells (GFP+) stained with the specified concentration of R3DC23, 10μg / ml VHH55 or 1μg / ml S309 to the MFI of untransfected cells (GFP-). (D) Viral replication kinetics of replication-competent VSV pseudotyped with SARS-CoV-2Wuhan-Hu-1 (parent) or selected escape variants as determined by life cell imaging of infected VeroE6 cells. The figure shows the mean ± SEM (N=5) of the GFP+ area per well of infected cells at the specified time point after infection. (E) Display of the HR2 coiled coil structure (PDB: 2FXP), showing the positions (N1192, L1197, L1200 and Q1201) where substitutions were observed in the escape variants. The polysaccharides coupled at N1194 modeled in 6XVV_1_1_1 are represented by stick figures. The dotted line represents the viral membrane. The alpha helix of the coiled-coil structure in the middle of the left figure corresponds to the sequence underlined in (A). The right figure shows a top view of the HR2 coiled-coil structure with N1192, L1197, L1200 and Q1201. The N1194 glycosylation site is marked, and the first GlcNac of the modeled N-glycan in 6XVV_1_1_1 is indicated as a stick figure (reference is the same as 6vSB_1_1_2). (F) Binding of R3DC23 to cells expressing the specified SARS-CoV-2 spike protein variants. The figure shows the ratio of the MFI of transfected cells (GFP+) stained with the specified concentrations of R3DC23, 10μg / ml GBP or 1μg / ml S309 to the MFI of untransfected cells (GFP-). (G and H) Identification of the R3DC23 epitope on the recombinant spike protein by HDX-MS. The figures show the HDX-MS uptake patterns of two peptides: peptide (1187-1199) (left) and peptide (1200-1205) (right) with a high degree of deuteration protection after binding to R3DC23.The peptide (1187-1199) is glycosylated at residue 1194, and the glycosylated peptide takes up more deuterium than the number of backbone exchangeable sites (11 sites) because glycans can take up and retain deuterium at amide sites similar to the backbone, as pointed out by Guttman, Scian, and Lee (2011, ACS Analytical Chemistry). (H) Woods plot showing the difference in the number of deuterons acquired between the apo spike protein and the R3DC23-bound S-2P spike protein for each designated peptide (indicated by residue number in the x-axis) at the designated time points.

[0046] Fig.28 : X-ray structure of the R3_DC23-HR2 complex. (A) Left: Model of the full-length S protein before fusion (6VSB_1_1_2; Woo et al. (2020 J. Phys. Chem B 124: 7128-7137) superimposed on the R3_DC23-HR2 complex, all shown in the form of molecular surface display, and N-glycans are represented by stick figures. The labeled S protein regions are: cytoplasmic domain (CP), transmembrane domain (TM), heptad repeat 2 (HR2), S2 stem helix (SH), heptad repeat 1 (HR1), central helix and connecting domain (CH-CD). The S1 region containing the N-terminal domain (NTD) and receptor binding domain (RBD) is proteolytically removed before the post-fusion conformational change (transition). Right: Model of the post-fusion S protein treated with proteolysis (7E9T; Tai et al. (2021 PNAS118: e2112703118)). (B) Side and axial views of the R3_DC23-HR2 complex superimposed with the pre-fusion HR2 coiled coil structure (inset). The HR2 binding epitope containing N1192-Y1206 is shown as a stick figure. (C) Close-up of the boxed area of ​​B, which contains one VHH and two HR2 copies (i and ii), forming adjacent binding epitopes. The escape mutation positions (N1192, L1197, Q1201) are marked. (D) Post-fusion S protein Axial view of the HR1-HR2 region with the R3-DC23-HR2 complex superimposed. (E) Structural view of the paratope-epitope contacts in the R3_DC23-HR2 complex. Close-up view of a single copy of R3_DC23 bound to two HR2 helices (i and ii), with key interacting residues in the paratope and epitope shown as stick figures. Candidate hydrogen bonds and salt bridges are shown as dashed lines. Escape mutation positions (N1192, L1197, L1200, and Q1201) are shown.

[0047] Fig.29: R3DC23 binds to a quaternary epitope within the HR2 coiled coil structure. The left and right figures show the binding (OD450nm) of R3DC23 to a trimer full-length spike protein (S-2P) and a monomer SUMO-HR3 coated in different amounts to a half-well 96-well ELISA plate, as shown on the x-axis. VHH (GBP) binding to GFP was used as a negative control.

[0048] Fig.30 :The Fc fusion protein of R3DC23 can effectively neutralize multiple SARS-CoV-2 variants. (A) Neutralization of SARS-CoV-2 614G and BA.5 spike protein VSV pseudotyped viruses by monovalent R3DC23, R3C4 and R4DC20 and their humanized counterparts (huR3DC23-Fc, huR3C4-Fc and huR4DC20-Fc) of human IgG1 Fc fusion proteins containing YTE mutations that extend half-life. These symbols represent the relative infection mean ± SD (N = 3) measured by the GFP fluorescence value of infected cells. (B) Neutralization of SARS-CoV-2 614G spike protein VSV pseudotyped viruses by humanized (huR3DC23-Fc) and non-humanized (R3DC23-Fc) R3DC23 Fc fusion proteins. (C) Graph shows median (line) and individual (diamond) IC50 values ​​calculated from at least 2 independent neutralization assays using VSV pseudotyped with the spike protein of the indicated SARS-CoV-2 variants. (D) Neutralization of authentic SARS-CoV-2 D614G and BA.1 viruses by huR3DC23-Fc and / or R3DC23 VHHs binding to S2. The graph shows the mean ± SEM of plaque counts for each VHH dilution gradient (N=4 for R3DC23 and huR3DC23-Fc, N=2 for S309). (E) Analytical hydrophobic interaction chromatography of R3DC23-Fc, huR3DC23-Fc, huR3C4-Fc, and huR4DC20-Fc compared to the clinically validated VHH-Fc XVR011. Assessment of surface hydrophobicity was performed on a ProPacHIC-10 HPLC by (NH4)2SO4 gradient elution, with shorter retention times indicating lower surface hydrophobicity. The figure shows replicate curves for each designated VHH-Fc construct.

[0049] Fig.31: LS mutants of humanized R3DC23 Fc fusion proteins can effectively neutralize multiple SARS-CoV-2 variants and control viral replication in hamsters. (A) Overlay of analytical hydrophobic interaction chromatograms of LS and YTE variants of huR3DC23-Fc. Surface hydrophobicity was assessed by (NH4)2SO4 gradient elution on ProPac HIC-10 HPLC, and shorter retention times indicated lower surface hydrophobicity. Repeated curves for each VHH-Fc are shown. (B) LS and YTE variants of huR3DC23-Fc were eluted as single (overlapping) peaks in analytical SEC. The molar weight markers of gel filtration standards (Bio-Rad) are shown in gray. Curves and shading represent the average and standard deviation of three replicate runs. (C) FcRn binding of huR3DC23-Fc_LS at pH 6.0 as determined by SPR. After huR3DC23-Fc_LS was fixed to the sensor chip at low density, two-fold gradient dilutions of human FcRn from 250 to 0.97nM were injected into the solution (gray curve). A 1:1 binding model was fitted (black curve). Supporting data in Table 9. (DE) Treatment of SARS-CoV-2Wuhan infection with huR3DC23-Fc_LS in Syrian Golden hamsters. Male Syrian Golden hamsters were infected intranasally with SARS-CoV-2 (Wuhan-Hu-1 strain) on day 0 and received an intraperitoneal injection of 10 or 2 mg / kg of huR3DC23-Fc_LS, 10 mg / kg of bebtelovimab (positive control) or 10 mg / kg of palivizumab (negative control) 4 hours after infection. The animals were euthanized on day 4, and infectious viruses (D) and viral RNA (E) were measured in lung tissue on day 4. Horizontal bars represent median TCID50 / gram (left panel) and RNA copies / gram (right panel) of lung tissue. Dotted horizontal lines represent LLOD. *Two animals in the high dose (10 mg / kg) group were experimentally confirmed to have not received R3DC23hum-Fc_LS treatment. Data were analyzed using one-way ANOVA and Dunn's multiple comparison test (***P<0.0001). (#) Data points corresponding to hamsters in which no or very low levels of huR3DC23-Fc_LS were detected in serum were omitted from the statistical analysis.

[0050] Fig.32: Specificity of R3_DC23-Fc(LS) binding to SARS-CoV-2 spike protein. Binding of 2.5 μg / mL of R3_DC23hum-Fc(LS) to 6101 full-length human plasma membrane proteins and cell surface-bound secretory proteins and 396 human heterodimers expressed on transfected HEK293 cells in a human plasma membrane protein cell array was evaluated. Fixed cell confirmation screening of initial hits in the library screening is shown. (A) Binding of R3_DC23hum-Fc(LS) at 2.5 μg / mL. (B) Binding of Rituximab at 1 μg / mL. (C) Binding of IgG1 isotype control. (D) Binding of secondary antibody (PBS instead of primary antibody). Rep: Replicate.

[0051] Fig.33 : Multispecific constructs comprising VHHs targeting S1 and S2. (A, B, C) Schematic diagram of a bispecific tandem VHHx-VHHy-Fc construct comprising a VHH(C23) targeting S2 (a humanized version of VHHR3_DC23) and a VHH(117) targeting S1 (a humanized version of VHH3.117), separated by a 10 (A), 20 (B) or 30 (C) GS linker and fused to an Fc domain (human Fc (LS)) via a 10 GS linker. (D) Schematic diagram of the VHHx-Fc-VHHy construct, which contains a VHH (C23) targeting S2 (a humanized version of VHH R3_DC23), which is fused to the N-terminus of the Fc domain (human Fc (LS)) via a 10GS linker, and a VHH (117) targeting S1 (a humanized version of VHH3.117), which is fused to the C-terminus via a 15GS linker. (E) Schematic diagram of the tandem VHHx-VHHy-Fc construct, which contains a VHH targeting S1 that binds or competes with the VHH72 epitope (83) (a humanized version of VHH3.83), and a VHH targeting S1 that binds or competes with the VHH3.117 epitope (117) (a humanized version of VHH3.117), which are separated by a 20GS linker and fused to the Fc domain (human Fc (LS)) via a 10GS linker. (F) Schematic diagram of the trispecific tandem VHHx-VHHy-VHHz-Fc construct, which contains a VHH (C23) targeting S2 (a humanized version of VHH R3_DC23), a VHH targeting S1 that binds or competes with the VHH3.117 epitope (117) (a humanized version of VHH3.117), and a VHH targeting S1 that binds or competes with the VHH72 epitope (83) (a humanized version of VHH3.83), which are separated by a 20GS linker and fused to the Fc domain (human Fc (LS)) via a 10GS linker.

[0052] Fig.34 : Composition comprising VHHs targeting S1 and S2. Schematic diagram of a composition comprising a binding agent targeting S1 and S2 (XVR012). The composition comprises a VHH-Fc construct targeting S2 (XVR013) (a humanized version of R3_DC23 fused to a human Fc domain) and a VHHx-Fc-VHHy construct targeting S1 (XVR014), the latter comprising a VHH capable of binding or competing for the VHH3.117 epitope (117) (a humanized version of VHH3.117) and a VHH capable of binding or competing for the VHH72 epitope (83) (a humanized version of VHH3.83), both of which are fused to a human Fc domain.

[0053] Fig.35 : In vivo efficacy of XVR012, XVR013 and XVR014 in the Syrian golden hamster SARS-CoV-2 challenge model. Syrian golden hamsters were infected with SARS-CoV-2 (Wuhan-Hu-1 strain) via the nasal cavity. Four hours after the SARS-CoV2 attack, the molecules XVR012 (4mg / kg and 20mg / kg), XVR013 and XVR014 (2mg / kg and 10mg / kg), palivizumab (10mg / kg, negative control) and bebetocumab (10mg / kg, positive control) were administered by intraperitoneal injection. The graph shows viral replication in the lungs (A) and viral RNA load in lung tissue (B). The median with a 95% confidence interval is reported for each group. The dotted horizontal line represents the lower limit of detection (LLOD).

[0054] Fig.36: ADCC responses mediated by XVR012, XVR013, and XVR014. An FcγRIIIa reporter assay was performed to evaluate the antibody-dependent cellular cytotoxicity (ADCC) of XVR012, XVR013, and XVR014. The CHO-K1 target cell line expressing the SARS CoV-2 spike protein was used as the target cell, and Jurkat FcγRIIIa (CD16) V176-NFAT-RE Luc was used as the reporter cell. Three independent assay runs were performed. The assay used an effector cell to target cell ratio of 40:1, and samples were evaluated in an 8-point serial dilution with a starting concentration of 30 μg / mL for XVR013 and XVR014 and 60 μg / mL for XVR012 in three independent replicates (three assay plates per run). An isotype control was evaluated at a single concentration of 30 μg / ml. The assay plates were incubated overnight (21 hours ± 1 hour) before adding SteadyGlo (luminescence endpoint). Raw luminescence values ​​are expressed as the average of the test sample control wells (reaction) and the negative control wells (wells without test sample, containing the target cell line). The study reports the average values ​​(N = 3 ± SD) for XVR012, XVR013 and XVR014.

[0055] Fig.37 : In vivo preventive efficacy of XVR012, XVR013 and XVR014 in the Syrian golden hamster SARS-CoV-2 challenge model. About 24 hours before SARS-CoV2 challenge, Syrian golden hamsters were administered a mixture of 10mg / kg XVR014 and 1mg / kg XVR013 (XVR012), 1mg / kg XVR013 or 10mg / kg XVR014 by intraperitoneal injection. The graph shows the viral load in the lungs. Each group contained 6 animals, and each group showed the median with a 95% confidence interval. The dashed horizontal line in the figure represents the lower limit of detection (LLOD).

[0056] Fig.38: In vivo therapeutic efficacy of XVR012, XVR013 and XVR014 in the Syrian golden hamster SARS-CoV-2 challenge model. Syrian golden hamsters were infected with SARS-CoV-2 (Wuhan-Hu-1 strain). Four hours after SARS-CoV2 challenge, a mixture of 5mg / kg XVR014 and 0.5mg / kg XVR013, a mixture of 10mg / kg XVR014 and 1mg / kg XVR013, or a mixture of 20mg / kg XVR014 and 2mg / kg XVR013 (XVR012); 0.5mg / kg, 1mg / kg or 2mg / kg XVR013; or 5mg / kg, 10mg / kg or 20mg / kg XVR014 were administered by intraperitoneal injection. Control animals received 10mg / kg palivizumab (negative control), and a group of animals received 10mg / kg bembetocumab (used as a positive control). The graph shows the viral load in the lungs. Each group contained 6 animals and the median value with 95% confidence interval is shown for each group. The dashed horizontal line in the graph represents the lower limit of detection (LLOD).

[0057] Fig.39 .Prophylactic treatment with R3_DC23-Fc protected K18-hACE2 mice from lethal SARS-CoV-2 infection. 2 20 hours before intratracheal infection with PFU SARS-CoV-2 614G variant virus, K18-hACE2 mice were administered 100 μg R3_DC23-Fc, and a second group of K18-hACE2 mice and non-susceptible wild-type (WT) mice were administered 100 μg isotype control antibody (palivizumab). Animals were monitored daily by measuring weight changes and humane endpoint scores. (A) The figure shows the Kaplan-Meier curve of the survival portion of the specified group of animals. When mice lost more than 25% of their weight (defined by weight on day 0) or reached a high score of 25 humane endpoints, they were euthanized. Compared with K18-hACE2 mice treated with palivizumab (n=5), K18-hACE2 mice treated with R3_DC23-Fc (n=5) significantly avoided death (p=0.0016, log rank, Mantel-cox test). (B) The figure shows the average relative body weight of K18-hACE2 mice treated with R3_DC23-Fc (n=5±SEM) or palivizumab (n=5±SEM) and WT mice treated with palivizumab (n=5±SEM). Starting from day 4, mice treated with R3_DC23-Fc showed significantly less weight loss (p<0.001) compared with mice treated with palivizumab (two-way ANOVA, Tukey's multiple comparison test).

[0058] Fig.40 .Prophylactic treatment with R3_DC23-Fc reduced viral replication of SARS-CoV-2 in the lungs of infected K18-hACE2 mice. 2 20 hours before intratracheal infection with PFUSARS-CoV-2 614G variant virus, K18-hACE2 mice were administered 100 μg R3_DC23-Fc, and a second group of K18-hACE2 mice and non-susceptible wild-type (WT) mice were administered 100 ug isotype control antibody (palivizumab). Animals were monitored daily by measuring weight changes and humane endpoint scores. (A) The figure shows the Kaplan-Meier curve of the survival portion of K18 mice treated with R3_DC23-Fc (n=5) compared with K18-hACE2 mice treated with palivizumab (n=4) and WT mice treated with palivizumab (n=5). (B) The figure shows the average relative weight of K18-hACE2 mice treated with R3_DC23-Fc (n=5±SEM) or palivizumab (n=4±SEM) and WT mice treated with palivizumab (n=5±SEM). From the 5th day, K18-hACE2 mice treated with R3_DC23-Fc showed significantly less weight loss (p<0.01) (two-way ANOVA, Tukey multiple comparison test) compared with K18-hACE2 mice treated with palivizumab. (C) The figure shows the median and individual viral titers in the lungs of the specified group of mice killed 5 days after infection. Compared with K18-hACE2 mice treated with palivizumab, the SARS-CoV-2 virus replicated in the lungs of K18-hACE2 mice treated with R3_DC23-Fc and WT mice treated with palivizumab was significantly reduced (p<0.0005, Kruskal-Wallis test, Dunn multiple comparison test). (D) The figure shows the median and individual levels of viral RNA in the lungs of the specified group of mice killed 5 days after infection. K18-hACE2 mice treated with R3_DC23-Fc and WT mice treated with palivizumab had significantly less SARS-CoV-2 virus replicating in the lungs compared with K18-hACE2 mice treated with palivizumab (p<0.005, Kruskal-Wallis test, Dunn's multiple comparison test).

[0059] Fig.41 . Interaction of S-2P with R3_DC23 detected by BLI. S-2P trimer was immobilized on a biolayer interferometry biosensor and then bound to 20 nM R3_DC23 monomer in solution. Raw data from three replicate experiments (no reference subtraction was performed). DETAILED DESCRIPTION

[0060] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0061] As used herein, the terms "comprising, comprises" and "consisting of" are synonymous with "including, includes" or "containing, contains" and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. The terms also encompass "consisting of" and "consisting essentially of", which terms have clear meanings in patent terms.

[0062] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the corresponding ranges, as well as the recited endpoints.

[0063] As used herein, the term "about" or "approximately" when referring to a measurable value (such as a parameter, amount, length of time, etc.) is intended to encompass variations from the specified value, such as ±10% or less, preferably ±5% or less, more preferably ±1% or less, and even more preferably ±0.1% or less, as long as such variations are suitable for implementation in the disclosed invention. It should be understood that the value referred to by the modifier "about" itself is also specifically and preferably disclosed.

[0064] Although the term "one or more" or "at least one" (such as one or more members or at least one member of a group of members) is clear in itself, by way of further example, the term specifically covers any one of the members, or any two or more of the members, such as any ≥3, ≥4, ≥5, ≥6 or ≥7 of the members, etc., as well as up to all of the members. In another example, "one or more" or "at least one" may refer to 1, 2, 3, 4, 5, 6, 7 or more.

[0065] The discussion of the background of the invention herein is for the purpose of explaining the context of the invention. It should not be taken as an admission that any of the material referred to was published, known or became part of the common general knowledge in any country before the priority date of any claim.

[0066] In this specification, various publications, patents and published patent specifications are cited by identifying citations. All documents cited in this specification are incorporated herein by reference in their entirety. In particular, the teachings or chapters of such documents specifically mentioned herein are incorporated by reference.

[0067] Unless otherwise defined, all terms (including technical terms and scientific terms) used in the disclosure of the present invention have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. By way of further guidance, term definitions are included to better understand the teachings of the present invention. When a particular term is defined in conjunction with a particular aspect of the present invention or a particular embodiment of the present invention, unless otherwise defined, such meaning is intended to apply to the entire specification, i.e., also to the context of other aspects or embodiments of the present invention.

[0068] In the following paragraphs, different aspects or embodiments of the present invention will be defined in more detail. Unless otherwise expressly stated, each aspect or embodiment so defined may be combined with any other aspect or embodiment. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0069] References to "one embodiment", "embodiment" throughout the specification mean that a particular feature, structure or characteristic described in conjunction with the embodiment is included in at least one embodiment of the present invention. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing in various places in this specification do not necessarily all refer to the same embodiment, but may refer to the same embodiment. In addition, it will be clear to those skilled in the art from this disclosure that particular features, structures or characteristics may be combined in one or more embodiments in any suitable manner. In addition, as will be appreciated by those skilled in the art, although some embodiments described herein include some features included in other embodiments but not other features, the combination of features of different embodiments is intended to fall within the scope of the present invention and to form different embodiments. For example, in the appended claims, any claimed embodiment may be used in any combination.

[0070] As demonstrated in the experimental section illustrating certain representative embodiments of the invention, the inventors have identified VHHs that specifically bind to the Sabeivirus Spike protein, in particular, VHHs that specifically bind to the Sabeivirus Spike protein S2 subunit (such as the SARS-CoV-2 and SARS-CoV-1 Spike protein S2 subunit). Studies have found that VHH can effectively neutralize SARS-CoV-2 and SARS-CoV-1, including SARS-CoV-2 variants such as SARS-CoV-2D614G variant, SARS-CoV-2Alpha variant, SARS-CoV-2 Omicron BA.1 variant, SARS-CoV-2 Omicron BA.2 variant, SARS-CoV-2Omicron BA.5 variant, SARS-CoV-2 Omicron BA.2.75.2 variant, SARS-CoV-2 Omicron BA.4.6 variant, SARS-CoV-2 Omicron BF.7 variant, SARS-CoV-2 Omicron BQ.1.1 variant, SARS-CoV-2Omicron XBB variant and SARS-CoV-2 Omicron XBB.1.5 variant. The study found that these VHHs interact with the S2 amino acids in the heptad repeat 2 (HR2) domain (more specifically, with the amino acids in the C-terminal region of the HR2 domain close to the viral membrane), which are highly conserved in the spike proteins of Sabeiviruses of multiple evolutionary branches.

[0071] Therefore, on the one hand, it relates to a binding agent (especially an antibody and an antigen-binding fragment thereof) capable of neutralizing Sabeivirus, characterized in that the binding agent (especially an antibody and an antibody fragment) specifically binds to the heptad repeat 2 (HR2) domain of the Sabeivirus spike protein.

[0072] One aspect provides a binding agent capable of neutralizing Sabei virus, characterized in that the binding agent specifically binds to or within a region of the Sabei virus spike protein, which region corresponds to the region from amino acid E1188 to amino acid Y1206 of the SARS-CoV-2 spike protein defined by SEQ ID NO: 86.

[0073] "Binding agent" generally refers to a molecule that is capable of binding to at least one other molecule, wherein the binding is preferably specific binding, such as binding at a defined binding site, pocket or epitope. A binding agent may be of any nature or type and is independent of its source. A binding agent may be chemically synthesized, naturally occurring, recombinantly produced (and optionally purified), and designed and synthetically produced (and optionally purified). Thus, the binding agent may, for example, be a small molecule, a chemical, a peptide, a polypeptide, an antibody, or any derivative of any of them, such as a peptide mimetic, an antibody mimetic, an active fragment, a chemical derivative, etc. A functional fragment of a binding agent or a functional portion of a binding agent refers to a fragment or portion of the binding agent that is functionally equivalent to the binding agent. In particular, such a functional fragment or portion of a binding agent described herein ideally retains one or more of the functional features (1) to (21) of the binding agent as broadly outlined elsewhere herein.

[0074] The term "antibody" refers to an immunoglobulin (Ig) molecule or a molecule comprising an immunoglobulin (Ig) domain that specifically binds to an antigen and its multimers. An "antibody" can be a complete immunoglobulin or an immunoreactive portion of a complete immunoglobulin. The term encompasses antibodies produced naturally, recombinantly, semisynthetically or synthetically. Thus, for example, an antibody can exist in nature or be isolated from nature, such as being naturally or endogenously produced or expressed by a cell or tissue and optionally isolated therefrom; or the antibody can be recombinant, i.e. produced by recombinant DNA technology, and / or can be partially or wholly chemically or biochemically synthesized.

[0075] "Isolated" or "purified" refers to a material that is substantially or essentially free of components that normally accompany it in its native state. For example, an "isolated polypeptide" or "purified polypeptide" refers to a polypeptide that is separated or purified by any suitable method from a mixture of molecules that contains the polypeptide of interest to be separated or purified. The isolated or purified polypeptide of interest can be, for example, an immunoglobulin, an antibody, or a nanobody, and the mixture can be a mixture or molecules present in a cell that produces the immunoglobulin, antibody, or nanobody, and / or a culture medium into which the immunoglobulin, antibody, or nanobody is secreted (possibly mixed with other molecules secreted by the cell).

[0076] The terms "antibody fragment", "antigen binding fragment", "functional antibody fragment" and "active antibody fragment" refer to a portion of any antibody that itself has a high affinity for an antigenic determinant or epitope and contains one or more complementary determining regions (CDRs) that determine such specificity. As used herein, the terms "antibody fragment" and "antigen binding fragment" and "active antibody fragment" and "functional antibody fragment" refer to a protein or peptide comprising an immunoglobulin domain or antigen binding domain that is capable of specifically binding to a Sabeivirus spike protein, such as a SARS-CoV-2 spike protein, in particular the S2 subunit of the Sabeivirus spike protein, and more particularly the HR2 domain of (the S2 subunit of) the Sabeivirus spike protein. Non-limiting examples include immunoglobulin domains, Fab, F(ab)'2, scFv, heavy-light chain dimers, immunoglobulin single variable domains, nanobodies (or VHH antibodies), domain antibodies and single chain structures, such as a complete light chain or a complete heavy chain.

[0077] The term "immunoglobulin (Ig) domain", or more specifically "immunoglobulin variable domain" (abbreviated as "IVD", also referred to herein as "variable domain") refers to an immunoglobulin domain that is essentially composed of four "framework regions", which are referred to in the art and herein below as "framework region 1" or "FR1"; "framework region 2" or "FR2"; "framework region 3" or "FR3"; and "framework region 4" or "FR4", respectively; these framework regions are interrupted by three "complementarity determining regions" or "CDRs", which are referred to in the art and herein below as "complementarity determining region 1" or "CDR1"; "complementarity determining region 2" or "CDR2"; and "complementarity determining region 3" or "CDR3", respectively. Therefore, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulin variable domain (IVD) (particularly the CDR therein, more particularly the CDR3 therein) that confers specificity to the antibody for the antigen by carrying an antigen binding site or epitope binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementary determining regions (CDRs) of VH and VL all contribute (although not necessarily evenly) to the antigen binding site, i.e., a total of 6 CDRs will participate in the formation of the antigen binding site. In view of the above definition, the antigen binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or a Fab fragment, a F(ab′)2 fragment, an Fv fragment such as a disulfide-linked Fv or scFv fragment, or a diabody derived from such a conventional 4-chain antibody (known in the art) will bind to the corresponding epitope of an antigen through a pair of (associated) immunoglobulin domains such as the light chain and heavy chain variable domains (i.e., through a VH-VL pair of immunoglobulin domains), which together bind to the corresponding epitope of the antigen.

[0078] "Immunoglobulin single variable domain" (abbreviated as "ISVD"), equivalent to the term "single variable domain", defines a molecule in which an antigen-binding site is present on and formed by a single immunoglobulin domain. This separates immunoglobulin single variable domains from "conventional" immunoglobulins or fragments thereof, in which two immunoglobulin domains (specifically two variable domains) interact to form an antigen binding site. As used herein, an "immunoglobulin single variable domain" (or "ISVD") refers to a protein or peptide having an amino acid sequence comprising four framework regions (FRs) and three complementary determining regions (CDRs), which is according to the format of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The antigen binding site of an immunoglobulin single variable domain is formed by a single VH / VHH or VL domain. Therefore, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs. Therefore, the single variable domain can be a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit consisting essentially of a single variable domain, such that a single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit). In certain embodiments, the immunoglobulin single variable domain is a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence); more particularly, the immunoglobulin single variable domain can be a heavy chain variable domain sequence derived from a conventional four-chain antibody or a heavy chain variable domain sequence derived from a heavy chain antibody. For example, an immunoglobulin single variable domain may be a (single) domain antibody (or an amino acid sequence suitable for use as a (single) domain antibody), the variable domain of the heavy chain (VH) or light chain (VL) of a conventional antibody (also referred to as a "dAb") (or an amino acid sequence suitable for use as a dAb), or a Nanobody (as defined herein, and including but not limited to VHH); or any suitable fragment of any of them.

[0079] In an embodiment, the immunoglobulin single variable domain may be a Nanobody (as defined herein) or a suitable fragment thereof. Note: and is a registered trademark of Ablynx NV (Sanofi). For a general description of nanobodies, reference is made to the further description below, as well as to the prior art cited herein, such as described in WO2008 / 020079. "VHH domains", also known as VHH, VHH domains, VHH antibody fragments and VHH antibodies, were originally described as antigen-binding immunoglobulin (Ig) (variable) domains of "heavy chain antibodies" (i.e., "antibodies lacking light chains"; Hamers-Casterman et al., 1993, Nature 363: 446-448). The term "VHH domain" is selected to distinguish these variable domains from the heavy chain variable domains present in conventional 4-chain antibodies (referred to herein as "VH domains") and the light chain variable domains present in conventional 4-chain antibodies (referred to herein as "VL domains"). For a further description of VHH and Nanobodies, reference is made to the review article by Muyldermans (2001. Rev Mol Biotechnol 74: 277-302) and to the following patent applications mentioned as general background art: WO 94 / 04678, WO 95 / 04079, WO 96 / 34103; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231, WO 02 / 48193; WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016, WO 03 / 055527; WO 03 / 050531; WO 01 / 90190; WO 03 / 025020 (= EP 1433793); WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825. As described in these references, Nanobodies (particularly VHH sequences and partially humanized Nanobodies) may be characterized inter alia by the presence of one or more "Hallmark residues" in one or more framework sequences.

[0080] According to the present invention, the binders or Sabeivirus binders (which can be used interchangeably) can be described functionally in one aspect by any individual function / embodiment or by any combination of any number of individual functions / embodiments described below, and any number "n" is given between brackets "(n)". The numerical order of these individual functions is random and does not impose any preference on the individual functions; similarly, this random numerical order does not impose any preference on any combination of two or more individual functions. In addition, any such combination should not be considered arbitrary, because each of these individual functions is performed in the binders or Sabeivirus binders herein.

[0081] Thus, the present invention provides binding agents (particularly antibodies or antigen-binding fragments thereof) that (1) specifically bind to Sabeivirus (such as SARS-CoV-2 and SARS-CoV-1) and may also be referred to herein as Sabeivirus binding agents or Sabeivirus antibodies and antibody fragments. In certain embodiments, the binding agent (2) does not bind to Middle East Respiratory Syndrome Coronavirus (MERS-CoV).

[0082] "Binding" refers to any direct or indirect interaction. A direct interaction means a contact (e.g., physical or chemical) between the two binding partners. An indirect interaction refers to any interaction in which the interacting partners interact in a complex of more than two molecules. The interaction can be completely indirect (e.g., two molecules become part of the same complex with the help of one or more bridging molecules, but would not bind without the bridging molecules). An interaction can be partially direct or partially indirect: there is still direct contact between the two interacting partners, but this contact is, for example, unstable and needs to be stabilized by interaction with one or more additional molecules.

[0083] "Specificity of binding" or "binding specificity" or "specific binding" refers to the situation where molecule A binds to a target of interest (e.g., a protein) at a certain concentration (e.g., sufficient to inhibit or neutralize the protein or process of interest) with a higher affinity (e.g., up to at least 2-fold, 5-fold, or at least 10-fold affinity, such as up to at least 20-fold, 50-fold, or 100-fold or more affinity) than it may (if any) bind to other targets (non-targets of interest). Specific binding does not mean exclusive binding. However, specific binding does mean that the binding agent has a certain degree of increased affinity or preference for one or more of its targets. Exclusivity of binding refers to the situation where the binding agent only binds to the target of interest. As used herein, the term "affinity" generally refers to the degree to which one molecule (e.g., a ligand, a chemical substance, a protein or peptide, an antibody or an antibody fragment) binds to another molecule (e.g., a (target) protein or peptide) so as to shift the equilibrium of the unimolecular monomers toward the complex formed by the (specific) (non-covalent) binding of the two molecules. Non-covalent interactions or binding between two or more binding partners may involve interactions such as van der Waals interactions, hydrogen bonds, and salt bridges. The "dissociation constant" or "binding constant" (K D ) is often used to describe the affinity between two molecules and is often expressed by the rate constant for complex formation, called “k on ” value) and the dissociation rate constant (“k off " or "k dis The measurement of the binding affinity of one molecule to another molecule (such as an antibody or antibody fragment to an antigen, or a ligand to a receptor) is known to those skilled in the art and includes, for example, real-time, label-free biolayer interferometry (e.g. RED96 system (ForteBio)), or surface plasmon resonance (SPR) (e.g. BIACORE TM ), or solution affinity ELISA.

[0084] The term "Coronaviridae" and the more common name "coronavirus" refer to a family of viruses that derives its name from the large spike protein molecules present on the surface of the virus, which give the virion a crown-like shape. The Coronaviridae family includes four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. Coronaviruses represent a diverse family of large, enveloped, positive-stranded RNA viruses that infect a variety of animals, multiple vertebrate species, and humans. The spike (S) protein of coronaviruses is essential for host receptor binding and subsequent fusion of the virus with the host cell membrane, effectively leading to the release of the viral nucleocapsid in the host cytoplasm (Letko et al. (2020) Nat Microbiol 5:562-569).

[0085] There are four coronaviruses presumed to be of zoonotic origin that are prevalent in humans: HCoV-NL63 and HCoV-229E (alpha coronaviruses) and HCoV-OC43 and HCoV-HKU1 (beta coronaviruses). In addition, three severe respiratory diseases caused by beta coronaviruses have occurred since 2000: the severe acute respiratory syndrome virus (SARS) caused by SARS-CoV-1 originated from a zoonosis (bats through civets as an intermediate species) and disappeared in 2004 (Drosten et al., 2003, N Engl J Med 348: 1967-1976). More than 8,000 cases of SARS were reported, with a mortality rate of about 10%. In 2012, Middle East Respiratory Syndrome (MERS) appeared in the Arabian Peninsula. MERS is caused by MERS-CoV, with more than 2,500 confirmed cases and a case fatality rate of 34% (deGroot et al., 2013, N Engl J Virol 87: 7790-7792). In recent years, a third zoonotic human coronavirus has emerged, with reports of severe cases of acquired pneumonia caused by a novel betacoronavirus, now called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) because it is genetically related to SARS-CoV-1 (Chen et al. (2020) Lancet 395:507-513). Similar to severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) infections, patients presented with symptoms of viral pneumonia, including fever, dyspnea, and, in the most severe cases, bilateral pulmonary infiltrates (Gralinski et al. (2020) Viruses 12:135).

[0086] As used herein, the term "Sabevirus" refers to a subgenus within the genus Betacoronavirus and includes the severe acute respiratory syndrome-related coronavirus species (SARSr-CoV or SARS-CoV, also referred to as SARS coronavirus, SARS-associated coronavirus, and severe acute respiratory syndrome coronavirus, used synonymously herein). Non-limiting examples of strains belonging to the SARS-CoV species include SARS-CoV-1 and SARS-CoV-2.

[0087] The first available genome sequence places the novel human pathogen SARS-CoV-2 in the subgenus Sabeivirus of the Coronaviridae family, the same subgenus as the SARS virus. Although SARS-CoV-2 belongs to the same genus of betacoronavirus as SARS-CoV (lineage B) and MERS-CoV (lineage C), genomic analysis shows greater similarity between SARS-CoV-2 and SARS-CoV, supporting its classification as a member of lineage B (from the International Committee on Taxonomy of Viruses).

[0088] Among other betacoronaviruses, the virus is characterized by a unique combination of polybasic cleavage sites, a notable feature known to increase pathogenicity and infectivity. Bat CoV RaTG13, collected from horseshoe bats (Rhinolophus affinis), reportedly clusters with SARS-CoV-2 in almost all genomic regions, with a genomic sequence identity of approximately 96% (and more than 93% similarity in the spike protein receptor binding domain (RBD); another mammalian species may have served as an intermediate host. One of the suspected intermediate hosts (Malayan pangolin) carries a coronavirus that is highly similar to SARS-CoV-2 in the receptor binding domain, which contains the following mutations, which are believed to promote binding to the angiotensin-converting enzyme 2 (ACE2) receptor, and shows 97% amino acid sequence similarity.

[0089] Both SARS-CoV-1 and SARS-CoV-2 use angiotensin-converting enzyme 2 (ACE2) as a receptor on human cells. SARS-CoV-2 binds to ACE2 with higher affinity than SARS-CoV-1 (Wrapp et al. (2020) Science 367:1260-1263). SARS-CoV-2 is distinguished from SARS-CoV-1 and several SARS-related coronaviruses (SARSr-CoV), as outlined by Abdelrahman et al. (2020. Front Immunol 11:552909).

[0090] SARS-CoV-2 refers to the newly emerged Sabei virus, which has been identified as the cause of a severe global outbreak of severe acquired pneumonia. The long-term global spread of SARS-CoV-2, coupled with the selection pressure for immune escape, has led to the virus adapting to the host and producing new SARS-CoV-2 variants. In particular, a variety of mutations have evolved and are evolving in the spike glycoprotein, including mutations located in the spike S1 subunit. For example, a SARS-CoV-2 variant may include a mutation at one or more positions selected from N439, K417, S477, L452, T478, E484, P384, N501 and D614 (relative to the SARS-CoV-2 spike amino acid sequence defined by SEQ ID NO: 86). Further non-limiting examples of SARS-CoV-2 variants include SARS-CoV-2 variants comprising a mutation at position N501, such as the N501Y variant (e.g., SARS-CoV-2 Alpha variants); SARS-CoV-2 variants comprising mutations at positions N501 and E484, such as N501Y and E484K variants (e.g., SARS-CoV-2 Alpha+E484K variants); SARS-CoV-2 variants comprising mutations at positions K417, E484, and N501, such as K417N, E484K, and N501Y variants (e.g., SARS-CoV-2 beta variants); SARS-CoV-2 variants comprising mutations at positions P384, K417, E484, and N501, such as P384L, K417N, E484K, and N501Y variants (e.g., SARS-CoV-2 beta+P384L variants); SARS-CoV-2 variants comprising mutations at positions L452 and E484, Such as L452R and E484Q variants (e.g., SARS-CoV-2 kappa variants); SARS-CoV-2 variants comprising mutations at positions L452 and T478, such as L452R and T478K variants (e.g., SARS-CoV-2 delta variants); SARS-CoV-2 variants comprising mutations at position L452, such as L452R variants (e.g., SARS-CoV-2 epsilon variants); SARS-CoV-2 variants comprising mutations at position K417, such as K417T variants (e.g., SARS-CoV-2 gamma variants); SARS-CoV-2 variants comprising mutations at position D614, such as D614G variants (e.g., SARS-CoV-2 D614G variants, SARS-CoV-2 Omicron BA.1 variants, or SARS-CoV-2 Omicron BA.2 variants); SARS-CoV-2 variants comprising mutations at positions K147, W152R, F157, I210, G257, D339, G446, and N460, such as K147E, W152R, F157L, I210V, G257S, D339H, G446S, and N460K variants (e.g., SARS-CoV-2 Omicron BA.2.75 variant, SARS-CoV-2 Omicron BA.2.75.2 variant); SARS-CoV-2 variants comprising mutations at positions R346, F486, and D1199, such as R346T, F486S, and D1199N variants (e.g., SARS-CoV-2 Omicron BA.2.75.2 variant). 2 variants containing mutations at positions H69, V70, L452, and F486, such as H69-, V70-, L452R, and F486V variants (e.g., SARS-CoV-2 Omicron BA.4 / BA.5 variants); SARS-CoV-2 variants containing mutations at positions R346 and N658, such as R346T and N658S variants (e.g., SARS-CoV-2 Omicron BA.4.6 variants); SARS-CoV-2 variants containing mutations at position R346, such as R346T variants (e.g., SARS-CoV-2 Omicron B F.7 variants); SARS-CoV-2 variants containing mutations at positions R346, such as R346T variants (e.g., SARS-CoV-2 Omicron B F.7 variants); SARS-CoV-2 variants containing mutations at positions R346, K444, and N460, such as R346T, K444T, and N460K variants (e.g., SARS-CoV-2 Omicron BQ.1.1 variants); SARS-CoV-2 variants comprising mutations at positions V83, Y144, H146, Q183, V213, R346, L368, V445, G446, N460, F486, and F490, such as V83A, Y144-, H146Q, Q183E, V213E, R346T, L368I, V445P, G446S, N460K, F486S, and F490S variants (e.g., SARS-CoV-2 Omicron XBB variants) or V83A, Y144-, H146Q, Q183E, V213E, R346T, L368I, V445P, G446S, N460K, F486P, and F490S variants (e.g., SARS-CoV-2 Omicron XBB.1.5 variant). The Alpha variant of SARS-CoV-2 (also known as B.1.1.1.7 lineage) was first discovered in the United Kingdom in late 2020 and was one of the first reported SARS-CoV-2 variants of concern. The SARS-CoV-2 Alpha variant contains several mutations in the spike protein, including the N501Y mutation and the D614G mutation. The Omicron variant of SARS-CoV-2 was first discovered in South Africa and Botswana and was reported to the World Health Organization (WHO) as a new variant on November 24, 2021 (Fan et al., 2022. Signal Transduct Target Ther. 7: 141). The Omicron variant is not a single strain, but has evolved into at least three lineages, including BA.1, BA.2, and BA.3. Up to 60 mutations have been found in the BA.1 lineage, of which up to 38 mutations occur in the spike (S) protein, one mutation occurs in the envelope (E) protein, two mutations occur in the membrane (M) protein, and six mutations occur in the nucleocapsid (N) protein. The BA.2 lineage has 57 mutations, 31 of which occur in the S protein, and the N-terminus of BA.2 is significantly different from that of BA.1. As used herein, the term "SARS-CoV-2" encompasses the original strain and its variants. .

[0091] The binding agent (especially antibodies and antibody fragments) (3) specifically binds or binds to the spike protein of Sabei virus (such as SARS-CoV-2 spike protein or SARS-CoV-1 spike protein); in particular, the binding agent (especially antibodies and antibody fragments) (4) specifically binds or binds to the S2 subunit or a portion of the S2 subunit of the Sabei virus spike protein; more particularly, the binding agent (especially antibodies and antibody fragments) (22) specifically binds or binds to a region of the S2 subunit or binds to the region, which region in the SARS-CoV-2 spike protein defined by SEQ ID NO: 86 is located in the region from amino acid E1188 to amino acid Y1206, preferably in the region from amino acid N1192 to amino acid Y1206 or in the region from amino acid E1188 to amino acid L1203, more preferably in the region from amino acid N1192 to amino acid L1203, even more preferably in the region from amino acid N1194 to amino acid L1203, and most preferably in the region from amino acid N1194 to amino acid Q1201. In certain embodiments, the binding agent (particularly antibodies and antibody fragments) (23) specifically binds to or binds to a region of the Sabeivirus Spike protein or the Sabeivirus Spike protein S2 subunit, which corresponds to the region from amino acid E1188 to amino acid Y1206, preferably amino acid N1192 to amino acid Y1206 or amino acid E1188 to amino acid L1203, more preferably amino acid N1192 to amino acid L1203, even more preferably amino acid N1194 to amino acid L1203, and most preferably amino acid N1194 to amino acid Q1201 of the SARS-CoV-2 Spike protein as defined in SEQ ID NO: 86. More particularly, the binding agent (particularly antibodies and antibody fragments) (5) specifically binds to or binds to the heptad repeat 2 (HR2) domain of the Sabeivirus Spike protein (S2 subunit), or a portion of the HR2 domain.In certain embodiments, the binding agent (particularly antibodies and antibody fragments) (6) specifically binds or binds to a region of the HR2 domain proximal to the viral membrane, which region in the SARS-CoV-2 spike protein defined by SEQ ID NO:86 is preferably located in the region from amino acid A1174 to amino acid E1202, more preferably in the region from amino acid I1179 to amino acid E1202, even more preferably in the region from amino acid D1184 to amino acid E1202, still more preferably in the region from amino acid E1188 to amino acid E1202, or in the region from amino acid V1189 to amino acid E1202, still more preferably in the region from amino acid N1194 to amino acid E1202, and most preferably in the region from amino acid N1194 to amino acid Q1201; or (7) specifically binds or binds to a region of the HR2 domain (or S2 subunit) corresponding to amino acid E1188 to amino acid Y1206 of the SARS-CoV-2 spike protein defined by SEQ ID NO:86, preferably in the region from amino acid D1184 to amino acid E1202, still more preferably in the region from amino acid E1188 to amino acid E1202, and most preferably in the region from amino acid N1194 to amino acid Q1201. The HR2 domain (or S2 subunit) region corresponding to amino acid E1188 to amino acid Y1203 of the SARS-CoV-2 spike protein as defined in SEQ ID NO:86, more preferably the HR2 domain (or S2 subunit) region corresponding to amino acid A1190 to amino acid L1203 of the SARS-CoV-2 spike protein as defined in SEQ ID NO:86 (such as the HR2 domain (or S2 subunit) region corresponding to amino acid K1191 to amino acid E1202 of the SARS-CoV-2 spike protein as defined in SEQ ID NO:86), or the HR2 domain (or S2 subunit) region corresponding to amino acid N1192 to amino acid Q1201 of the SARS-CoV-2 spike protein as defined in SEQ ID NO:86 (such as the region from amino acid N1192 to amino acid Q1201 of the SARS-CoV-2 spike protein as defined in SEQ ID NO:86), even more preferably the HR2 domain (or S2 subunit) region corresponding to amino acid A1190 to amino acid L1203 of the SARS-CoV-2 spike protein as defined in SEQ ID NO:86 The HR2 domain (or S2 subunit) region corresponding to amino acid N1194 to amino acid L1203 of the SARS-CoV-2 spike protein defined in NO:86, most preferably the HR2 domain (or S2 subunit) region corresponding to amino acid N1194 to amino acid Q1201 of the SARS-CoV-2 spike protein defined in SEQ ID NO:86, such as the HR2 domain (or S2 subunit) region corresponding to amino acid S1196 to amino acid Q1201 of the SARS-CoV-2 spike protein defined in SEQ ID NO:86.In a specific embodiment, the binding agent (particularly antibodies and antibody fragments) (8) specifically binds or binds to at least one, at least two, at least three, at least four, at least five, at least six, at least seven or all of the amino acid residues N1192, N1194, S1196, L1197, D1199, L1200, Q1201 and E1202 in the SARS-CoV-2 spike protein as defined in SEQ ID NO: 86; preferably, it binds to at least one, at least two, at least three, at least four or all of the amino acid residues N1194, S1196, D1199, Q1201 and E1202 in the SARS-CoV-2 spike protein as defined in SEQ ID NO: 86; more preferably, it binds to at least one, at least two, at least three or all of the amino acid residues N1194, S1196, D1199 and Q1201 in the SARS-CoV-2 spike protein as defined in SEQ ID NO: 86; most preferably, it binds to At least one or two of amino acid residues S1196 and Q1201 in the SARS-CoV-2 spike protein defined by ID NO: 86. In an embodiment, the binding agent (particularly antibodies and antibody fragments) (24) specifically binds or binds to at least one, at least two, at least three, at least four, at least five, at least six, at least seven or all of the amino acid residues of the Sabei virus spike protein or the Sabei virus spike protein S2 subunit or the HR2 domain corresponding to amino acid residues N1192, N1194, S1196, L1197, D1199, L1200, Q1201 and E1202 in the SARS-CoV-2 spike protein defined in SEQ ID NO: 86; preferably, binds to at least one, at least two, at least three, at least four or all of the amino acid residues corresponding to amino acid residues N1194, S1196, D1199, Q1201 and E1202 in the SARS-CoV-2 spike protein defined in SEQ ID NO: 86; more preferably, binds to at least one, at least two, at least three, at least four or all of the amino acid residues corresponding to amino acid residues N1194, S1196, D1199, Q1201 and E1202 in the SARS-CoV-2 spike protein defined in SEQ ID NO: 86; At least one, at least two, at least three or all of the amino acid residues corresponding to amino acid residues N1194, S1196, D1199 and Q1201 in the SARS-CoV-2 spike protein defined by NO:86; most preferably, it binds to at least one or two amino acid residues corresponding to amino acid residues S1196 and Q1201 in the SARS-CoV-2 spike protein defined by SEQ ID NO:86.In a specific embodiment, the binding agent (particularly antibodies and antibody fragments) (25) specifically binds or binds to amino acid residues S1196 and Q1201 in the SARS-CoV-2 spike protein defined in SEQ ID NO: 86, or the spike protein amino acid residues corresponding to the amino acid residues S1196 and Q1201 in the SARS-CoV-2 spike protein defined in SEQ ID NO: 86; optionally, binds to amino acid residues N1194, S1196, D1199 and Q1201 in the SARS-CoV-2 spike protein defined in SEQ ID NO: 86, or the spike protein amino acid residues corresponding to the amino acid residues N1194, S1196, D1199 and Q1201 in the SARS-CoV-2 spike protein defined in SEQ ID NO: 86.

[0092] In a specific embodiment, the following amino acid residues are essential for the binding of a binding agent (particularly an antibody and antibody fragment) to the spike protein, and the amino acid residues are: (26) at least one, at least two, at least three, at least four, at least five, at least six, at least seven or all of the amino acid residues N1192, N1194, S1196, L1197, D1199, L1200, Q1201 and E1202 in the SARS-CoV-2 spike protein defined in SEQ ID NO: 86; preferably, at least one, at least two, at least three, at least four or all of the amino acid residues N1194, S1196, D1199, Q1201 and E1202 in the SARS-CoV-2 spike protein defined in SEQ ID NO: 86; more preferably, At least one, at least two, at least three or all of the amino acid residues N1194, S1196, D1199 and Q1201 in the SARS-CoV-2 spike protein defined by NO:86; most preferably, at least one or two of the amino acid residues S1196 and Q1201 in the SARS-CoV-2 spike protein defined by SEQ ID NO:86. In an embodiment, the following amino acid residues are essential for binding of a binding agent (particularly an antibody and antibody fragment) to the spike protein, and the amino acid residues are: (27) at least one, at least two, at least three, at least four, at least five, at least six, at least seven or all of the amino acid residues of the Sabeivirus Spike protein or the Sabeivirus Spike protein S2 subunit or the HR2 domain corresponding to amino acid residues N1192, N1194, S1196, L1197, D1199, L1200, Q1201 and E1202 in the SARS-CoV-2 Spike protein defined in SEQ ID NO: 86; preferably, at least one, at least two, at least three, at least four or all of the amino acid residues corresponding to amino acid residues N1194, S1196, D1199, Q1201 and E1202 in the SARS-CoV-2 Spike protein defined in SEQ ID NO: 86; more preferably, At least one, at least two, at least three or all of the amino acid residues corresponding to amino acid residues N1194, S1196, D1199 and Q1201 in the SARS-CoV-2 spike protein as defined in NO:86; most preferably, at least one or two amino acid residues corresponding to amino acid residues S1196 and Q1201 in the SARS-CoV-2 spike protein as defined in SEQ ID NO:86.In a specific embodiment, the following amino acid residues are essential for binding of a binding agent (particularly an antibody and antibody fragment) to the spike protein, and the amino acid residues are: (28) amino acid residues S1196 and Q1201 in the SARS-CoV-2 spike protein defined in SEQ ID NO: 86, or the spike protein amino acid residues corresponding to the amino acid residues S1196 and Q1201 in the SARS-CoV-2 spike protein defined in SEQ ID NO: 86; alternatively, amino acid residues N1194, S1196, D1199 and Q1201 in the SARS-CoV-2 spike protein defined in SEQ ID NO: 86, or the spike protein amino acid residues corresponding to the amino acid residues N1194, S1196, D1199 and Q1201 in the SARS-CoV-2 spike protein defined in SEQ ID NO: 86.

[0093] The assessment of the binding site can be performed in several ways: by determining the crystal structure of a complex of a binding agent (particularly an antibody or antibody fragment) with the spike protein or S2 subunit or a peptide comprising the HR2 domain, for example by applying a crystal structure determination method as shown in the Examples; and / or by selecting and analyzing viral escape variants / mutants, for example by applying a viral escape selection method as shown in the Examples; and / or by analyzing hydrogen-deuterium exchange on a recombinant spike protein (or S2 subunit or HR2-containing peptide) in the presence and absence of a binding agent, for example by applying a hydrogen-deuterium exchange method monitored by mass spectrometry (HDX-MS method) as shown in the Examples.

[0094] Advantageously, these amino acid residues are conserved between different clades of Sabeivirus, in particular between clade 1, clade 2 and clade 3 Sabeivirus. In a preferred embodiment, the binding agent (particularly an antibody or antibody fragment) (9) does not bind to the RBD of the Sabeivirus spike protein.

[0095] The binding agent (particularly antibodies and antibody fragments) (29) specifically binds or binds to a quaternary epitope of the spike protein. In particular, the binding agent (particularly antibodies and antibody fragments) (30) specifically binds or binds to a trimeric HR2 domain (or a trimeric S2 subunit or a trimeric spike protein). In particular, the binding agent (particularly antibodies and antibody fragments) (31) specifically binds or binds to a quaternary epitope within a trimeric HR2 domain (or a trimeric S2 subunit or a trimeric spike protein). More particularly, the binding agent (particularly antibodies and antibody fragments) (32) specifically binds or binds to a quaternary epitope located within two adjacent HR2 domains or helices. In a specific embodiment, the binding agent (particularly antibodies and antibody fragments) (33) specifically binds or binds to a quaternary epitope that comprises or consists of one or more interacting amino acid residues described herein in one HR2 domain or helix and one or more interacting amino acid residues described herein in an adjacent HR2 domain or helix. In specific embodiments, the binding agent (particularly antibodies and antibody fragments) (34) specifically binds to or binds to a quaternary epitope within the trimeric spike protein, wherein amino acid residues from at least two (such as two) monomers of the trimeric spike protein (particularly one or more interacting amino acid residues described herein) contribute to the quaternary epitope.

[0096] As used herein, the term "quaternary epitope" refers to a conformational epitope whose structure depends on or is enhanced by the arrangement of multiple protomers or monomers in a multimeric complex. A quaternary epitope may be located in a single protein (or monomer) in a multimeric complex; or it may span multiple protomers and be re-formed by interactions between multiple protomers.

[0097] Specific binding or binding to a quaternary epitope or a multimeric protein can be assessed by using an enzyme-linked immunosorbent assay (ELISA) assay to assess binding to monomeric and / or (stabilized) multimeric proteins, for example by applying an ELISA assay as shown in the Examples. Stabilization of the trimeric spike protein can be achieved by fusing the spike protein to the foldon domain of the trimeric protein fibritin from bacteriophage T4. Stabilization of the trimeric spike protein is only achieved when the density of the monomeric protein is elevated (such as a density of 1.0 ng / mm 2 or higher, preferably 1.2ng / mm 2 or higher, or 1.5ng / mm 2 or higher), a correlation between binding to monomeric protein and the density of monomeric protein may indicate specific binding or binding to the multimeric conformation of the protein. For monomeric and multimeric proteins of a given density, enhanced binding to multimeric protein compared to monomeric protein may indicate specific binding or binding to the multimeric protein.

[0098] Without being bound by any theory, upon binding to the trimeric spike protein (particularly upon binding to a quaternary epitope within the trimeric spike protein), the binding agents described herein (particularly antibodies and antibody fragments) can stabilize the prefusion conformation of the spike protein. More particularly, the binding agent can stabilize or lock the HR2 coiled coil structure. Thus, the binding agent can prevent the unwinding of the HR2 coiled coil structure, which is considered a critical early step in the process of spike-controlled membrane fusion; or the binding agent can interfere with or prevent the migration of the HR2 α helix to the extended HR1 α helix, which is considered a critical step in the refolding of the spike protein from the pre-hairpin intermediate to the postfusion conformation; and / or the binding agent can prevent the completion of the 6-helix bundle formation, which is considered to be critical for the fusion process. In embodiments, the binding agent (particularly antibodies and antibody fragments) (35) is capable of stabilizing the prefusion conformation of the Sabei virus spike protein. In embodiments, the binding agent (particularly antibodies and antibody fragments) (36) is capable of stabilizing the HR2 coiled coil structure.

[0099] SARS-CoV-2 contains spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins as structural proteins. In addition, sixteen nonstructural proteins (nsp1-16) have been identified that are involved in replication and alter host defense. The Nsp12 protein corresponds to the RNA-dependent RNA polymerase (RdRp).

[0100] Of particular interest to the present invention is the spike protein or S protein, which is a transmembrane glycoprotein that forms a homotrimer that protrudes from the surface of the virus and gives the virus its crown-like appearance. The spike protein has two subunits: S1 and S2.

[0101] The S1 subunit contains the N-terminal domain (NTD), the receptor binding domain (RBD), and subdomains 1 and 2 (SD1, SD2). The S1 subunit is involved in host receptor binding. The spike protein binds to the human host cell receptor angiotensin-converting enzyme 2 (ACE2) through the receptor binding domain (RBD) present in the S1 subunit.

[0102] The S2 subunit is involved in the fusion of virus and host cell and the membrane for virus entry, and contains multiple domains: S2' protease cleavage site (cleavage by host proteases required for fusion), fusion peptide (FP), heptad repeat 1 (HR1) domain, central helix (CH) domain, connector domain (CD), heptad repeat 2 (HR2) domain, transmembrane (TM) domain and cytoplasmic tail (CT) domain (Wang et al., (2020). Front Cell Infect Microbiol 10: 587269).

[0103] The S protein usually exists in a prefusion conformation. In the prefusion conformation, S1 and S2, which are cut at the S1-S2 furin cleavage site during biosynthesis, remain non-covalently bound to each other-this is different from SARS-CoV in which S1 and S2 remain uncut. In the closed state of the S protein (PDB: 6VXX), the three RBD domains in the trimer do not protrude from the trimer, while in the open state (PDB: 6VYB) or "up" conformation, one of the RBDs protrudes from the trimer. The S-trimer extracellular domain with a triangular cross-section has a length of about 160 angstroms, in which the S1 domain adopts a V-type form. Glycosylation occurs in 16 of the 22 N-linked glycosylation sites of each protomer (Walls et al., (2020) Cell 180: 281-292).

[0104] The S1 subunit of the S protein binds to ACE2 through its RBD region, promoting the formation of endosomes, thereby initiating viral fusion activity. After S1-ACE2 binding, S is cleaved by cellular proteases (such as transmembrane protease serine subtype 2 (TMPRSS2) or endosomal cathepsin), exposing the fusion peptide (FP) located in the S2 subunit. FP is inserted into the host cell membrane, thereby shortening the distance between the viral membrane and the host cell membrane, and the HR1 domain of the S protein is close to the host cell membrane, while the HR2 domain is closer to the viral membrane side. Then, HR2 folds back to HR1, so that the two HR domains form a six-helix structure in the reverse parallel form of the fusion core. Therefore, the viral membrane is pulled toward the host cell membrane and tightly bound to it, and the two membranes fuse, resulting in the release of the viral genome into the host cell (Huang et al., (2020) Acta Pharmalogica Sinica 41: 1141-1149).

[0105] The terms "spike protein", "S" or "S protein" used as synonyms herein refer to the spike protein of Sabeivirus, and may refer to specific S proteins, such as SARS-CoV-2 S protein and SARS-CoV-1 S protein. The terms "spike protein" and "SARS-CoV-2 spike protein" include protein variants of Sabeivirus or SARS-CoV-2 spike proteins isolated from different Sabeivirus or SARS-CoV-2 isolates, as well as recombinant Sabeivirus or SARS-CoV-2 spike proteins, or fragments thereof. These terms also encompass Sabeivirus spike proteins or SARS-CoV-2 spike proteins coupled to, for example, histidine tags, mouse or human Fc or signal sequences.

[0106] The SARS-CoV-2 spike protein sequence can be found under / corresponds to the following Genbank accession number: QHQ82464, version QHQ82464.1; and is also defined herein as SEQ ID NO: 86:

[0107]

[0108]

[0109] Herein, the SARS-CoV-2 spike protein HR2 domain corresponds to amino acids 1169-1202 of SEQ ID NO: 86 and is shown as follows (SEQ ID NO: 87): ISGINASVVNIQKEIDRLNEVAKNLNESLIDLQE (SEQ ID NO: 87).

[0110] Herein, the SARS-CoV-2 spike protein TM domain corresponds to amino acids 1214-1237 of SEQ ID NO:86.

[0111] As used herein, the region of the HR2 domain "close to the viral membrane" refers to the region within the HR2 domain that is within 40 amino acids from the viral membrane.

[0112] The SARS-CoV-1 spike protein sequence can be found under / corresponds to the following Genbank accession number: NP_828851.1; and is also defined herein as SEQ ID NO: 111. Herein, the SARS-CoV-1 spike protein HR2 domain corresponds to amino acids 1151-1184 of SEQ ID NO: 111, and is as shown in SEQ ID NO: 87. The amino acids and amino acid numbers referred to herein are relative to / correspond to the SARS-CoV-2 spike protein defined in SEQ ID NO: 86; the corresponding amino acids in spike proteins or spike protein fragments, domains or regions of other Sabeiviruses can be readily determined by aligning multiple amino acid sequences.

[0113] "Angiotensin converting enzyme 2", "ACE2" or "ACE-2" used interchangeably herein refers to a mammalian protein belonging to the dipeptidyl carboxyl dipeptidase family and is sometimes classified as EC: 3.4.17.23. The genomic location of the human ACE2 gene is located at chrX: 15,561,033-15,602,158 (GRCh38 / hg38; negative strand), or alternatively, at chrX: 15,579,156-15,620,271 (GRCh37 / hg19; negative strand). ACE2 serves as a receptor for at least the human coronaviruses SARS-CoV and SARS-CoV-2 and NL63 / HCoV-NL63 (also known as the New Haven coronavirus). UniProtKB identifier for human ACE2 protein: Q9BYF1. Isotype 1 (identifier: Q9BYF1-1) has been selected as the canonical sequence. Reference DNA sequence of human ACE2 gene in GenBank: NC_000023.11. Reference mRNA sequence of human ACE2 in GenBank NM_001371415.1 and NM_021804.3.

[0114] Another functional feature of the binding agents described herein (particularly antibodies and antibody fragments) is that they (10) are able to neutralize Sabeivirus, and in particular (11) are able to neutralize either or both of SARS-CoV-2 and SARS-CoV-1, preferably both.

[0115] As used herein, "neutralizing binders" or "neutralizing antibodies" (or "binding agents or antibodies capable of neutralizing Sabei virus (particularly SARS-CoV-2 and / or SARS-CoV-1)") refers to binding agents or antibodies that bind to Sabei virus (particularly SARS-CoV-2 and / or SARS-CoV-1) to inhibit or suppress the ability of Sabei virus or SARS-CoV-2 or SARS-CoV-1 to initiate and / or persist infection in a host. Neutralizing binders or antibodies can, for example, interfere with the binding of Sabei virus (such as SARS-CoV-2 or SARS-CoV-1) to host receptors (particularly ACE2); and / or interfere with viral entry, for example by inducing S1 shedding and / or interfering with viral fusion. It is not yet entirely clear how the binding agents and antibodies according to the present invention neutralize, inhibit, block or suppress Sabei virus infection. In certain embodiments, the binding agents (particularly antibodies and antibody fragments) described herein (44) do not modulate or interfere with S1 shedding. In certain embodiments, the binding agents (particularly antibodies and antibody fragments) described herein (12) do not induce S1 shedding. In certain embodiments, the binding agents described herein (particularly antibodies and antibody fragments) (45) do not prevent S1 from falling off. In certain embodiments, the binding agents described herein (particularly antibodies and antibody fragments) (13) are capable of inhibiting spike-mediated syncytium formation. Therefore, the binding agents (particularly antibodies and antibody fragments) may (14) be able to inhibit viral fusion, and without being bound by any theory, may thereby prevent the Sabei virus from completing the infection process of the host cell. In certain embodiments, the binding agents described herein (particularly antibodies and antibody fragments) (46) do not prevent HR1 from unfolding. In certain embodiments, the binding agents described herein (particularly antibodies and antibody fragments) (47) do not prevent HR1 from folding onto HR2 (e.g., during the formation of the S2 6-helix bundle). Regardless of their mechanism of action, the binding agents (particularly antibodies and antibody fragments) according to the present invention are able to effectively neutralize Sabei virus infection.

[0116] Neutralization activity can be measured using standard neutralization assays known to those skilled in the art, including but not limited to pseudovirus neutralization assays and plaque reduction assays. Exemplary methods for performing such neutralization assays are described in the Examples herein. Neutralization activity can also be assessed by measuring one or more indicators of infection by Sabeivirus or SARS-CoV-2 or SARS-CoV-1, such as syncytium formation between cells expressing Sabeivirus spike protein and cells expressing Sabeivirus receptor ACE2.

[0117] In certain embodiments, the binding agents (particularly antibodies and antibody fragments) (15) are capable of neutralizing Sabeivirus (particularly SARS-CoV-2 and / or SARS-CoV-1) with a half maximal inhibitory concentration or 50% inhibitory concentration (IC50 ) is 100 ng / ml or less, preferably 50 ng / ml or less or 20 ng / ml or less, more preferably 10 ng / ml or less, even more preferably 1 ng / ml or less, preferably as determined in a Sabeivirus spike protein pseudovirus neutralization assay (such as a vesicular stomatitis virus (VSV)-Sabeivirus spike protein pseudovirus neutralization assay), more preferably as determined in a SARS-CoV-2 spike protein and / or SARS-CoV-1 spike protein pseudovirus neutralization assay (such as a VSV-SARS-CoV-2 spike protein pseudovirus neutralization assay or a VSV-SARS-CoV-1 spike protein pseudovirus neutralization assay). In particular, the pseudovirus neutralization assay may be based on a pseudotyped VSV-delG virus containing a Sabeivirus spike protein (such as a SARS-CoV-2 spike protein, a SARS-CoV-2 variant spike protein, or a SARS-CoV-1 spike protein). As used herein, the "half maximal inhibitory concentration" or "IC" associated with the neutralizing activity of a binding agent or antibody 50 ” refers to an amount, such as the concentration of a binding agent or antibody required to neutralize 50% of Sabei virus.

[0118] In specific embodiments, the binding agents (particularly antibodies and antibody fragments) (16) are capable of neutralizing at least one SARS-CoV-2 variant, such as a SARS-CoV-2 variant comprising a mutation at position D614 (relative to the SARS-CoV-2 spike amino acid sequence defined by SEQ ID NO: 86), such as the D614G variant, particularly at least any one or more, preferably all, of the SARS-CoV-2 Alpha variant, SARS-CoV-2 Omicron BA.1 variant, SARS-CoV-2 Omicron BA.2 variant, SARS-CoV-2 Omicron BA.5 variant, SARS-CoV-2 Omicron BA.2.75.2 variant, SARS-CoV-2 Omicron BA.4.6 variant, SARS-CoV-2 Omicron BF.7 variant, SARS-CoV-2 Omicron BQ.1.1 variant, SARS-CoV-2 Omicron XBB variant, and SARS-CoV-2 Omicron XBB.1.5 variant. In a specific embodiment, the binding agents (particularly the antibodies and antibody fragments described herein) are characterized in that they (17) are capable of neutralizing the SARS-CoV-2 Alpha variant, (18) are capable of neutralizing the SARS-CoV-2 Omicron BA.1 variant, (19) are capable of neutralizing the SARS-CoV-2 Omicron BA.2 variant, (37) are capable of neutralizing the SARS-CoV-2 Omicron BA.5 variant, (38) are capable of neutralizing the SARS-CoV-2 Omicron BA.2.75.2 variant, (39) are capable of neutralizing the SARS-CoV-2 Omicron BA.4.6 variant, (40) are capable of neutralizing the SARS-CoV-2 Omicron BF.7 variant, (41) are capable of neutralizing the SARS-CoV-2 Omicron BQ.1.1 variant, (42) are capable of neutralizing the SARS-CoV-2 Omicron XBB variant, and / or (43) are capable of neutralizing the SARS-CoV-2 Omicron XBB.1.5 variant, wherein the IC 50 100 ng / ml or less, preferably 50 ng / ml or less or 20 ng / ml or less, more preferably 10 ng / ml or less, even more preferably 1 ng / ml or less, preferably as determined in a SARS-CoV-2 variant spike pseudovirus neutralization assay (such as a VSV-SARS-CoV-2 variant spike pseudovirus neutralization assay).

[0119] The binding agents (particularly antibodies and antibody fragments) described herein are further characterized in that they (14) are capable of inhibiting viral fusion. In specific embodiments, the binding agents (particularly antibodies and antibody fragments) described herein (13) are capable of inhibiting spike-mediated syncytium formation, and more particularly, they (20) are capable of inhibiting syncytium formation between cells expressing Sabeivirus spike proteins (such as SARS-CoV-2 and / or SARS-CoV-1 spike proteins) and cells expressing Sabeivirus host receptors (particularly ACE2 receptors).

[0120] As used herein, "viral fusion" refers to the fusion of the viral membrane with the host cell membrane. Viral fusion assays are well known to those skilled in the art, and exemplary methods for performing such methods are described in the Examples herein. It will be clear to those skilled in the art that complete inhibition is not required, and that those skilled in the art will be able to identify binding agents, antibodies, and antibody fragments that significantly inhibit viral fusion or spike-mediated syncytium formation. Preferably, the binding agents (particularly antibodies and antibody fragments) as described herein (21) can induce at least 50% inhibition, preferably at least 60%, at least 70%, at least 80%, or at least 90% inhibition.

[0121] In a specific embodiment, some functional features of the Sabei virus binding agents (particularly Sabei virus antibodies or antibody fragments) described above are combined to characterize such binding agents, antibodies or antibody fragments, for example, binding or specifically binding to the HR2 domain of the Sabei virus spike protein, and being able to neutralize Sabei virus, particularly SARS-CoV-2 (such as SARS-CoV-2 Wuhan-Hu-1 strain, SARS-CoV-2 D6 1 4G variant, SARS-CoV-2 Alpha variant, SARS-CoV-2 Omicron BA.1 variant, SARS-CoV-2 Omicron BA.2 variant, SARS-CoV-2 Omicron BA.5 variant, SARS-CoV-2 Omicron BA.2.75.2 variant, SARS-CoV-2 Omicron BA.4.6 variant, SARS-CoV-2 Omicron BF.7 variant, SARS-CoV-2 Omicron BQ.1.1 variant, SARS-CoV-2 Omicron XBB variant and SARS-CoV-2 Omicron XBB.1.5 variants) and at least one or both of SARS-CoV-1, preferably capable of neutralizing Sabei virus, wherein the 50% inhibitory concentration (IC 50) is 100 ng / ml or less, preferably 10 ng / ml or less, more preferably 1 ng / ml or less, as determined in a vesicular stomatitis virus (VSV)-Sabey virus spike protein pseudovirus neutralization assay. Such binding agents, antibodies or antibody fragments may also have the following characteristics: can inhibit spike-mediated syncytium formation between cells expressing Sabey virus spike protein and cells expressing angiotensin converting enzyme 2 (ACE2) receptors, and / or can inhibit viral fusion; and / or do not bind to Middle East respiratory syndrome coronavirus (MERS-CoV).

[0122] The binding agents described herein may also be structurally defined as polypeptide binding agents (i.e., binding agents that comprise a peptide, polypeptide, or protein portion, or binding agents that comprise a peptide, polypeptide, protein, or protein domain) or polypeptide binding agents (i.e., the binding agent is a peptide, polypeptide, or protein).

[0123] The terms "protein", "polypeptide" and "peptide" are used interchangeably herein to refer to polymers of amino acid residues and variants and synthetic analogs thereof; sequences of amino acids are arranged linearly together to produce / form an "amino acid sequence" or "protein sequence". "Peptide" may also refer to a partial amino acid sequence derived from its original protein, for example after enzymatic (e.g., trypsin) digestion. These terms apply to both naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as chemical analogs of the corresponding naturally occurring amino acids. These terms also include proteins that contain one or more post-translational modifications, such as covalent addition of functional groups or proteins, such as glycosylation, phosphorylation, acetylation, ubiquitination, methylation, lipidation, and nitrosylation, or such as proteolytic processing. Based on the amino acid sequence and modifications, the atomic or molecular mass or weight of a polypeptide is expressed in (kilo) Daltons (kDa). Further modifications of the protein include the addition of tags, such as His-tags or sorting tags (sortag). For example, a multi-arm PEG nanobody that neutralizes SARS-CoV2 was constructed by sorting tagging (sortase-mediated transpeptidation; Popp et al., 2007, Nat Chem Biol 3:707-708) (Moliner-Morro et al., 2020, Biomolecules 10:1661).

[0124] A "protein domain" is a unique functional and / or structural unit in a protein, or is a part of a protein. Typically, a protein domain is responsible for a specific function or interaction, thereby contributing to the overall (biological) effect of the protein. Domains can be found in a variety of biological contexts, where similar domains can be found in different proteins with similar or different functions. A protein domain can have a rigid 3D structure if restricted by multiple intramolecular cysteines (e.g., cysteine ​​knot proteins), or can present different 3D conformations depending on, for example, the presence or absence of a binding ligand or, for example, the presence or absence of a post-translational modification, or can have a less defined, more fluid 3D structure.

[0125] Amino acids are referred to herein by the three-letter or one-letter code nomenclature as specified by the IUPAC-IUB Joint Commission on Biochemical Nomenclature (Nomenclature and Symbolism for Amino Acids). and Peptides. Eur. J. Biochem. 138: 9-37 (1984)); as follows: alanine (A or Ala), cysteine ​​(C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or GIn), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp) and tyrosine (Y or Tyr).

[0126] More particularly, the binding agents described herein may be structurally defined as polypeptides or polypeptide binding agents comprising complementarity determining regions (CDRs), such as those contained in any immunoglobulin single variable domain (ISVD) defined herein. In a preferred embodiment, the polypeptide or polypeptide binding agent is an (isolated) antibody or antibody fragment.

[0127] In certain embodiments, the binding agents (particularly antibodies and antibody fragments) according to the invention may be structurally defined as polypeptides or polypeptide binding agents (particularly antibodies and antibody fragments) comprising at least CDR3, such as those contained in an immunoglobulin single variable domain (ISVD) as defined herein. In other embodiments, the binding agents (particularly antibodies and antibody fragments) according to the invention may be structurally defined as polypeptides or polypeptide binding agents (particularly antibodies and antibody fragments) comprising at least two of CDR1, CDR2 and CDR3 (e.g., CDR1 and CDR3, CDR2 and CDR3, CDR1 and CDR2), or all three of CDR1, CDR2 and CDR3, such as those contained in an immunoglobulin single variable domain (ISVD) as defined herein. Such CDRs may be comprised in VHH R3 C4 (defined / listed by SEQ ID NO: 1), VHH R4_DC16 (defined / listed by SEQ ID NO: 2), VHH R3_DC20 (defined / listed by SEQ ID NO: 3), VHHR3_DC2 (defined / listed by SEQ ID NO: 4), VHH R4_DC20 (defined / listed by SEQ ID NO: 5), VHHR4_DC9 (defined / listed by SEQ ID NO: 6), VHH R4_DC6 (defined / listed by SEQ ID NO: 7), VHH R3_DC23 (also referred to herein as VHHR3DC23 or R3DC23; defined / listed by SEQ ID NO: 8), VHHR3_DC9 (defined / listed by SEQ ID NO: 9), or VHHR4_DC13 (defined / listed by SEQ ID NO: 10), as shown below:

[0128] VHH R3_C4:

[0129]

[0130] VHH R4_DC16:

[0131]

[0132] VHH R3_DC20:

[0133]

[0134] VHH R3_DC2:

[0135]

[0136] VHH R4_DC20:

[0137]

[0138] VHH R4_DC9:

[0139]

[0140] VHH R4_DC6:

[0141]

[0142] VHH R3_DC23:

[0143]

[0144] VHH R3_DC9:

[0145]

[0146] VHH R4_DC13:

[0147]

[0148] For the numbering of any IVD or ISVD amino acid residues, different numbering schemes may be applied. For example, all heavy chain variable domains (VH) and light chain variable domains (VL) may be numbered according to the AHo numbering scheme given by Honegger and Plückthun (2001. J Mol Biol 309: 657-70), as applied to the VHH domains of camelids. Alternative methods for numbering the amino acid residues of the VH domains are known in the art, and these methods may also be applied in a similar manner to the VHH domains. For example, the division of FR sequences and CDR sequences may be performed using the Kabat numbering system, as applied to the VHH domains of camelids (Riechmann and Muyldermans (1999. J Immunol Methods 231: 25-38)). It should be noted that - as is well known in the art for VH and VHH domains - the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. The total number of amino acid residues in VH and VHH domains is typically in the range of 110 to 120, often 112 to 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0149] The determination of CDR regions in an antibody / immunoglobulin sequence generally depends on the algorithm / method applied. For example, the CDR regions can be determined according to nomenclature based on contact analysis and binding site topology, as described in MacCallum et al. (J. Mol. Biol. (1996) 262, 732-745), AbM (AbM is an antibody modeling package from Oxford Molecular Ltd, as described at http: / / www.bioinf.org.uk / abs / index.html), Chothia (Chothia and Lesk, 1987; Mol Biol. 196: 901-17), Martin (Abhinandan and Martin. Molecular Immunology 45 (2008) 3832-3839; as shown in http: / / bioinf.org.uk / abs / info.html), Kabat (Kabat et al., 1991; 5th edition, NIH publication 91-3242), or IMGT (LeFranc, 2014; Frontiers in Immunology.5(22):1-22). The annotation also includes the division of CDR and framework regions (FR) in proteins containing immunoglobulin domains, and is a method and system known to those skilled in the art, so they can apply these annotations to any antibody / immunoglobulin sequence without undue burden. As an example, Fig. 20 Different annotation schemes or methods applied to the amino acid sequence of VHH R3_DC23 (SEQ ID NO: 8) are shown.

[0150] Applying different methods to the same antibody / immunoglobulin sequence may result in different CDR amino acid sequences, where the differences may lie in the length and / or division of the CDR sequences within the antibody / immunoglobulin / IVD sequence (e.g. Fig. 20 3_DC23 in ). Thus, the CDRs of the ISVD binders (particularly antibodies and antibody fragments) described herein may be described as CDR sequences present in the ISVD characterized herein. Alternatively, these CDRs may be described as CDR sequences present in the ISVD (as described herein), as determined or divided according to well-known methods, such as according to any of the Kabat-, Martin-, Chothia-, aHo, MacCallum et al. 1996, AbM-, or IMGT numbering schemes or methods, such as preferably the Martin numbering scheme or method.

[0151] VHH or Nb are usually divided into different families or even superfamilies according to the amino acid sequence, so as to cluster clone-related sequences derived from the same progenitor cell during B cell maturation (Deschaght et al., 2017, Front Immunol 8: 420). This classification is usually based on the CDR sequence of VHH or Nb, and each VHH or Nb family is defined as a cluster of (clone) related sequences with a CDR3 region sequence identity threshold. Within a single VHH family defined herein, the CDR3 sequence is therefore identical or very similar in amino acid composition, preferably having at least 80% identity, or at least 85% identity, or at least 90% identity in the CDR3 sequence, so that VHH or Nb of the same family bind to the same binding site and have the same effect such as a functional effect.

[0152] As mentioned above, there are many systems or methods (Kabat, MacCallum, IMGT, AbM, Chothia, Martin) for numbering amino acids in immunoglobulin sequences, including for dividing CDRs and framework regions (FRs) in these protein sequences. These systems or methods are known to those skilled in the art, so they can apply these systems or methods to any immunoglobulin sequence without undue burden (e.g. Fig. 20 ).

[0153] In certain embodiments, the binding agents described herein (particularly antibodies or antibody fragments, more particularly ISVDs) may be characterized in that it comprises a CDR1 defined / listed by any one of SEQ ID NO: 63, SEQ ID NO: 46, SEQ ID NO: 69 or SEQ ID NO: 77. In certain embodiments, the binding agents described herein (particularly antibodies or antibody fragments, more particularly ISVDs) may be characterized in that it comprises a CDR2 defined / listed by any one of SEQ ID NO: 64, SEQ ID NO: 47, SEQ ID NO: 70, SEQ ID NO: 73 or SEQ ID NO: 78. In certain embodiments, the binding agents described herein (particularly antibodies or antibody fragments, more particularly ISVDs) may be characterized in that it comprises a CDR3 defined / listed by any one of SEQ ID NO: 48, SEQ ID NO: 67, SEQ ID NO: 74 or SEQ ID NO: 79. In certain embodiments, the binding agent described herein (particularly an antibody or antibody fragment, more particularly an ISVD) may be characterized in that it comprises a CDR1 defined / listed by any one of SEQ ID NO: 63, SEQ ID NO: 46, SEQ ID NO: 69, or SEQ ID NO: 77; a CDR2 defined / listed by any one of SEQ ID NO: 64, SEQ ID NO: 47, SEQ ID NO: 70, SEQ ID NO: 73, or SEQ ID NO: 78; and a CDR3 defined / listed by any one of SEQ ID NO: 48, SEQ ID NO: 67, SEQ ID NO: 74, or SEQ ID NO: 79.

[0154] In certain embodiments, the binding agents described herein (particularly antibodies or antibody fragments, more particularly ISVDs) may comprise:

[0155] - CDR1 defined / set forth in SEQ ID NO: 63; CDR2 defined / set forth in SEQ ID NO: 64; and CDR3 defined / set forth in SEQ ID NO: 67; or

[0156] - CDR1 defined / set forth in SEQ ID NO: 69, CDR2 defined / set forth in SEQ ID NO: 70; and CDR3 defined / set forth in SEQ ID NO: 67; or

[0157] CDR1 defined / set forth in SEQ ID NO:63, CDR2 defined / set forth in SEQ ID NO:64; and CDR3 defined / set forth in SEQ ID NO:48; or

[0158] CDR1 defined / set forth in SEQ ID NO:46, CDR2 defined / set forth in SEQ ID NO:47; and CDR3 defined / set forth in SEQ ID NO:48; or

[0159] CDR1 defined / set forth in SEQ ID NO:63, CDR2 defined / set forth in SEQ ID NO:73; and CDR3 defined / set forth in SEQ ID NO:74; or

[0160] - CDR1 defined / set forth in SEQ ID NO: 77; CDR2 defined / set forth in SEQ ID NO: 78; and CDR3 defined / set forth in SEQ ID NO: 79.

[0161] Table 1: CDR sequences in VHH according to certain embodiments:

[0162]

[0163]

[0164] In certain embodiments, the binding agent described herein (particularly an antibody or antibody fragment, more particularly an ISVD) may be characterized in that it comprises a CDR1 defined / listed by any one of SEQ ID NO: 65, SEQ ID NO: 71, SEQ ID NO: 49 or SEQ ID NO: 80. In certain embodiments, the binding agent described herein (particularly an antibody or antibody fragment, more particularly an ISVD) may be characterized in that it comprises a CDR2 defined / listed by any one of SEQ ID NO: 66, SEQ ID NO: 72, SEQ ID NO: 50, SEQ ID NO: 75 or SEQ ID NO: 81. In certain embodiments, the binding agent described herein (particularly an antibody or antibody fragment, more particularly an ISVD) may be characterized in that it comprises a CDR3 defined / listed by any one of SEQ ID NO: 51, SEQ ID NO: 68, SEQ ID NO: 76 or SEQ ID NO: 82. In certain embodiments, the binding agent described herein (particularly an antibody or antibody fragment, more particularly an ISVD) may be characterized in that it comprises a CDR1 defined / listed by any one of SEQ ID NO: 65, SEQ ID NO: 71, SEQ ID NO: 49, or SEQ ID NO: 80; a CDR2 defined / listed by any one of SEQ ID NO: 66, SEQ ID NO: 72, SEQ ID NO: 50, SEQ ID NO: 75, or SEQ ID NO: 81; and a CDR3 defined / listed by any one of SEQ ID NO: 51, SEQ ID NO: 68, SEQ ID NO: 76, or SEQ ID NO: 82.

[0165] In certain embodiments, the binding agents described herein (particularly antibodies or antibody fragments, more particularly ISVDs) may comprise:

[0166] - CDR1 defined / set forth in SEQ ID NO: 65; CDR2 defined / set forth in SEQ ID NO: 66; and CDR3 defined / set forth in SEQ ID NO: 68; or

[0167] - CDR1 defined / set forth in SEQ ID NO:71; CDR2 defined / set forth in SEQ ID NO:72; and CDR3 defined / set forth in SEQ ID NO:68; or

[0168] - CDR1 defined / set forth in SEQ ID NO: 65; CDR2 defined / set forth in SEQ ID NO: 66; and CDR3 defined / set forth in SEQ ID NO: 51; or

[0169] - CDR1 defined / set forth in SEQ ID NO:49, CDR2 defined / set forth in SEQ ID NO:50; and CDR3 defined / set forth in SEQ ID NO:51; or

[0170] - CDR1 defined / set forth in SEQ ID NO: 65; CDR2 defined / set forth in SEQ ID NO: 75; and CDR3 defined / set forth in SEQ ID NO: 76; or

[0171] - CDR1 defined / listed by SEQ ID NO: 80; CDR2 defined / listed by SEQ ID NO: 81; and CDR3 defined / listed by SEQ ID NO: 82.

[0172] Table 2: Example definitions / sequences of CDRs in VHHs of certain embodiments described herein using the different annotation methods shown.

[0173]

[0174]

[0175] In certain preferred embodiments, a binding agent or Sabei virus binding agent (particularly an antibody or antibody fragment or Sabei virus antibody or antibody fragment, more particularly an ISVD) as described herein may be characterized in that it comprises a CDR1 as present in any one of SEQ ID NO: 1 to SEQ ID NO: 10, wherein CDR1 is annotated according to any one of Kabat, MacCallum, IMGT, AbM, Martin or Chothia. In certain preferred embodiments, a binding agent or Sabei virus binding agent (particularly an antibody or antibody fragment or Sabei virus antibody or antibody fragment, more particularly an ISVD) as described herein may be characterized in that it comprises a CDR2 as present in any one of SEQ ID NO: 1 to SEQ ID NO: 10, wherein CDR2 is annotated according to any one of Kabat, MacCallum, IMGT, AbM, Martin or Chothia. In certain preferred embodiments, a binding agent or Sabei virus binding agent (particularly an antibody or antibody fragment or Sabei virus antibody or antibody fragment, more particularly an ISVD) as described herein may be characterized in that it comprises a CDR3 as present in any one of SEQ ID NO: 1 to SEQ ID NO: 10, wherein CDR3 is annotated according to any one of Kabat, MacCallum, IMGT, AbM, Martin or Chothia. In certain preferred embodiments, a binding agent or Sabei virus binding agent (particularly an antibody or antibody fragment or Sabei virus antibody or antibody fragment, more particularly an ISVD) as described herein may be characterized in that it comprises CDR1, CDR2 and CDR3, each of which is independently present in any one of SEQ ID NO: 1 to SEQ ID NO: 10, wherein the CDR1, CDR2 and CDR3 are annotated according to any one of Kabat, MacCallum, IMGT, AbM, Martin or Chothia.

[0176] In certain preferred embodiments, the binding agent or Sabei virus binding agent (particularly an antibody or antibody fragment or Sabei virus antibody or antibody fragment, more particularly an ISVD) as described herein may be characterized in that it comprises a combination of CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 are present in a specific sequence among the sequences listed by SEQ ID NO: 1 to SEQ ID NO: 10, wherein the CDR1, CDR2 and CDR3 are annotated according to any one of Kabat, MacCallum, IMGT, AbM, Martin or Chothia.

[0177] In certain embodiments, the binding agent described herein (particularly an antibody or antibody fragment, more particularly an ISVD) may be characterized in that it comprises a CDR1 defined / listed by any one of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 11 or SEQ ID NO: 12. In certain embodiments, the binding agent described herein (particularly an antibody or antibody fragment, more particularly an ISVD) may be characterized in that it comprises a CDR2 defined / listed by any one of SEQ ID NO: 55-62 or SEQ ID NO: 13-20. In certain embodiments, the binding agent described herein (particularly an antibody or antibody fragment, more particularly an ISVD) may be characterized in that it comprises a CDR3 defined / listed by any one of SEQ ID NO: 21-27. In certain embodiments, the binding agent described herein (particularly an antibody or antibody fragment, more particularly an ISVD) may be characterized in that it comprises a CDR1 defined / listed by any one of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 11, or SEQ ID NO: 12; a CDR2 defined / listed by any one of SEQ ID NOs: 55-62 or SEQ ID NOs: 13-20; and a CDR3 defined / listed by any one of SEQ ID NOs: 21-27.

[0178] In certain embodiments, the binding agents described herein (particularly antibodies or antibody fragments, more particularly ISVDs) may comprise:

[0179] - a CDR1 defined / set forth in any one of SEQ ID NOs: 52-54; a CDR2 defined / set forth in any one of SEQ ID NOs: 55-62; and a CDR3 defined / set forth in any one of SEQ ID NOs: 21-27; or

[0180] - a CDR1 defined / set forth in any one of SEQ ID NO: 11 or SEQ ID NO: 12; a CDR2 defined / set forth in any one of SEQ ID NO: 13-20; and a CDR3 defined / set forth in any one of SEQ ID NO: 21-27.

[0181] Table 3: Example definitions / sequences of CDRs in VHHs of certain embodiments described herein, using the different annotation methods shown, in particular CDRs comprised by any of VHH R3_C4, VHH R4_DC16, VHHR3_DC20, VHH R3_DC2, VHH R4_DC20, VHH R4_DC9, VHH R4_DC6, VHH R3_DC23, VHH R3_DC9 and VHH R4_DC13 as determined according to the Kabat or Martin system or method.

[0182]

[0183]

[0184] For example, the polypeptides or polypeptide binding agents described herein (particularly antibodies or antibody fragments, more particularly ISVDs) can be defined as comprising the complementarity determining regions (CDRs) present in any one of SEQ ID NOs: 1-10, wherein the CDRs are defined according to Kabat. In certain embodiments, the binding agent (particularly antibodies or antibody fragments, more particularly ISVDs) comprises one of the following three complementarity determining region (CDR) groups:

[0185] - CDR1 defined / set forth in SEQ ID NO: 11, CDR2 defined / set forth in SEQ ID NO: 13; and CDR3 defined / set forth in SEQ ID NO: 21; or

[0186] - CDR1 defined / set forth in SEQ ID NO: 11, CDR2 defined / set forth in SEQ ID NO: 13; and CDR3 defined / set forth in SEQ ID NO: 22; or

[0187] - CDR1 defined / set forth in SEQ ID NO: 11, CDR2 defined / set forth in SEQ ID NO: 13; and CDR3 defined / set forth in SEQ ID NO: 23; or

[0188] - CDR1 defined / set forth in SEQ ID NO: 11, CDR2 defined / set forth in SEQ ID NO: 14; and CDR3 defined / set forth in SEQ ID NO: 23; or

[0189] - CDR1 defined / set forth in SEQ ID NO: 11, CDR2 defined / set forth in SEQ ID NO: 12; and CDR3 defined / set forth in SEQ ID NO: 23; or

[0190] - CDR1 defined / set forth in SEQ ID NO: 11; CDR2 defined / set forth in SEQ ID NO: 16; and CDR3 defined / set forth in SEQ ID NO: 23;

[0191] - CDR1 defined / set forth in SEQ ID NO: 11; CDR2 defined / set forth in SEQ ID NO: 17; and CDR3 defined / set forth in SEQ ID NO: 24;

[0192] - CDR1 defined / set forth in SEQ ID NO: 11; CDR2 defined / set forth in SEQ ID NO: 18; and CDR3 defined / set forth in SEQ ID NO: 25;

[0193] - CDR1 defined / set forth in SEQ ID NO: 12; CDR2 defined / set forth in SEQ ID NO: 19; and CDR3 defined / set forth in SEQ ID NO: 26;

[0194] - CDR1 defined / listed by SEQ ID NO: 11; CDR2 defined / listed by SEQ ID NO: 20; and CDR3 defined / listed by SEQ ID NO: 27.

[0195] In specific embodiments, the polypeptides or polypeptide binding agents described herein (particularly antibodies or antibody fragments, more particularly ISVDs) may be defined as comprising the complementarity determining regions (CDRs) present in any one of SEQ ID NOs: 1-10, wherein the CDRs are defined according to Martin. In certain embodiments, the binding agent (particularly antibodies or antibody fragments, more particularly ISVDs) comprises one of the following three complementarity determining region (CDR) groups:

[0196] - CDR1 defined / set forth in SEQ ID NO: 52, CDR2 defined / set forth in SEQ ID NO: 55; and CDR3 defined / set forth in SEQ ID NO: 21; or

[0197] - CDR1 defined / set forth in SEQ ID NO: 52, CDR2 defined / set forth in SEQ ID NO: 55; and CDR3 defined / set forth in SEQ ID NO: 22; or

[0198] - CDR1 defined / set forth in SEQ ID NO: 52, CDR2 defined / set forth in SEQ ID NO: 55; and CDR3 defined / set forth in SEQ ID NO: 23; or

[0199] - CDR1 defined / set forth in SEQ ID NO: 52, CDR2 defined / set forth in SEQ ID NO: 57; and CDR3 defined / set forth in SEQ ID NO: 23; or

[0200] - CDR1 defined / set forth in SEQ ID NO:52, CDR2 defined / set forth in SEQ ID NO:58; and CDR3 defined / set forth in SEQ ID NO:23; or

[0201] - CDR1 defined / set forth in SEQ ID NO:53, CDR2 defined / set forth in SEQ ID NO:60; and CDR3 defined / set forth in SEQ ID NO:25; or

[0202] - CDR1 defined / set forth in SEQ ID NO:54; CDR2 defined / set forth in SEQ ID NO:61; and CDR3 defined / set forth in SEQ ID NO:26;

[0203] - CDR1 defined / set forth in SEQ ID NO:53; CDR2 defined / set forth in SEQ ID NO:62; and CDR3 defined / set forth in SEQ ID NO:27;

[0204] - CDR1 defined / set forth in SEQ ID NO:52; CDR2 defined / set forth in SEQ ID NO:56; and CDR3 defined / set forth in SEQ ID NO:23;

[0205] - CDR1 defined / set forth in SEQ ID NO: 52; CDR2 defined / set forth in SEQ ID NO: 59; and CDR3 defined / set forth in SEQ ID NO: 24.

[0206] In a further embodiment, the polypeptide or polypeptide binding agent (particularly antibodies and antibody fragments, more particularly ISVD) according to the present invention may comprise one or more framework regions (FRs) as contained in any one of SEQ ID NOs: 1-10, or a variant of such FRs. More particularly, such binding agents, antibodies or antibody fragments or ISVDs may comprise at least one (such as one, two, three or all) of the FR1, FR2, FR3 and FR4 regions, each of which is independently contained in any one of SEQ ID NOs: 1-10, or a variant of such FRs. For example, such a binding agent, antibody or antibody fragment or ISVD may comprise a FR1 and FR2 region as contained in any one of SEQ ID NOs: 1-10; a FR1 and FR3 region; a FR1 and FR4 region; a FR2 and FR3 region; a FR2 and FR4 region; a FR3 and FR4 region; a FR1, FR2 and FR3 region; a FR1, FR2 and FR4 region; a FR2, FR3 and FR4 region; or a FR1, FR3 and FR4 region, or a variant comprising such FRs. In certain preferred embodiments, such a binding agent, antibody or antibody fragment or ISVD comprises a FR1 region or a FR4 region or a FR2 and FR3 region as contained in any one of SEQ ID NOs: 1-10, or a variant comprising such FRs. To delineate the FRs in these protein sequences, the methods described elsewhere herein, Fig. 20 Any of the following systems or methods shown in for VHH R3_DC23 and known to those skilled in the art for numbering amino acids in immunoglobulin sequences. For example, the FR sequences in certain specific VHHs described herein using Martin or Kabat methodology are shown in Table 4.

[0207] Table 4: Exemplary sequences of FRs in VHHs of certain embodiments described herein using Kabat or Martin methodology.

[0208]

[0209] The polypeptide or polypeptide binding agent described herein (particularly an antibody or antibody fragment, more particularly an ISVD) may be characterized in that it comprises the framework region 1 (FR1) present in any one of SEQ ID NOs: 1-10, wherein FR1 is defined according to any one of AbM, Chothia, Martin, Kabat, IMGT or MacCallum, or it comprises a variant FR1 which is at least 90% or 95% identical to FR1 present in any one of SEQ ID NOs: 1-10, or has up to 3 (such as 1, 2 or 3) amino acid substitutions, deletions or additions, such as preferably conservative and / or humanizing substitutions, wherein FR1 is defined according to any one of AbM, Chothia, Martin, Kabat, IMGT or MacCallum.

[0210] The polypeptide or polypeptide binding agent described herein (particularly an antibody or antibody fragment, more particularly an ISVD) may be characterized in that it comprises a framework region 2 (FR2) as present in any one of SEQ ID NOs: 1-10, wherein FR2 is defined according to any one of AbM, Chothia, Martin, Kabat, IMGT or MacCallum, or it comprises a variant FR2 which is at least 85% or 90% identical to FR2 present in any one of SEQ ID NOs: 1-10, or has up to 2 (such as 1 or 2) amino acid substitutions, deletions or additions, such as preferably conservative and / or humanizing substitutions, wherein FR2 is defined according to any one of AbM, Chothia, Martin, Kabat, IMGT or MacCallum.

[0211] The polypeptide or polypeptide binding agent described herein (particularly an antibody or antibody fragment, more particularly an ISVD) may be characterized in that it comprises a framework region 3 (FR3) as present in any one of SEQ ID NOs: 1-10, wherein FR3 is defined according to any one of AbM, Chothia, Martin, Kabat, IMGT or MacCallum, or it comprises a variant FR3 which is at least 80%, 85%, 90% or 95% identical to FR3 present in any one of SEQ ID NOs: 1-10, or has up to 9 (such as 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions, deletions or additions, such as preferably conservative and / or humanizing substitutions, wherein FR3 is defined according to any one of AbM, Chothia, Martin, Kabat, IMGT or MacCallum.

[0212] The polypeptide or polypeptide binding agent described herein (particularly an antibody or antibody fragment, more particularly an ISVD) may be characterized in that it comprises a framework region 4 (FR4) as present in any one of SEQ ID NOs: 1-10, wherein FR4 is defined according to any one of AbM, Chothia, Martin, Kabat, IMGT or MacCallum, or it comprises a variant FR4 which is at least 90% identical to the FR4 present in any one of SEQ ID NOs: 1-10, or has at most 1 amino acid substitution, deletion or addition, such as preferably conservative and / or humanizing substitutions, wherein FR4 is defined according to any one of AbM, Chothia, Martin, Kabat, IMGT or MacCallum.

[0213] In a further embodiment, the polypeptide or polypeptide binding agent described herein (particularly an antibody or antibody fragment, more particularly an ISVD) may be characterized in that each independently comprises FR1 or variant FR1 as present in any one of SEQ ID Nos: 1-10 as defined above; FR2 or variant FR2 as present in any one of SEQ ID Nos: 1-10 as defined above; FR3 or variant FR3 as present in any one of SEQ ID Nos: 1-10 as defined above; and FR4 or variant FR4 as present in any one of SEQ ID Nos: 1-10 as defined above, wherein FR1, FR2, FR3 and FR4 are defined according to any one of AbM, Chothia, Martin, Kabat, IMGT or MacCallum.

[0214] In a specific embodiment, a polypeptide or polypeptide binding agent (particularly an antibody or antibody fragment, more particularly an ISVD) as described herein may be characterized in that it comprises at least one of the framework regions (FRs) present in any one of SEQ ID NO: 1 to SEQ ID NO: 10, or a specific combination of two, three or all framework regions, or comprises FRs as defined above or any variant of said FRs, wherein FRs are annotated according to any one of Kabat, MacCallum, IMGT, AbM, Martin or Chothia.

[0215] In a specific embodiment, the polypeptide or polypeptide binding agent described herein (particularly an antibody or antibody fragment, more particularly an ISVD) may be characterized in that it comprises at least one framework region present in any one of SEQ ID NO: 1 to SEQ ID NO: 10, or a specific combination of two, three or all framework regions (FRs), wherein the FRs are annotated according to any one of Kabat, MacCallum, IMGT, AbM, Martin or Chothia.

[0216] In further specific embodiments, the polypeptides or polypeptide binding agents described herein (particularly antibodies or antibody fragments, more particularly ISVDs) can be defined as each independently comprising FR1 present in any one of SEQ ID NOs: 1-10; FR2 present in any one of SEQ ID NOs: 1-10; FR3 present in any one of SEQ ID NOs: 1-10; and FR4 present in any one of SEQ ID NOs: 1-10, wherein FR1, FR2, FR3 and FR4 are defined according to any one of AbM, Chothia, Martin, Kabat, IMGT or MacCallum.

[0217] In further specific embodiments, a polypeptide or polypeptide binding agent (particularly an antibody or antibody fragment, more particularly an ISVD) as described herein may be defined as comprising the following FR1, FR2, FR3 and FR4, which FR1, FR2, FR3 and FR4 are present in the same sequence as any of the sequences shown in SEQ ID NOs: 1-10, wherein FR1, FR2, FR3 and FR4 are defined according to any of AbM, Chothia, Martin, Kabat, IMGT or MacCallum.

[0218] In still further specific embodiments, a polypeptide or polypeptide binding agent (particularly an antibody or antibody fragment, more particularly an ISVD) as described herein may be defined as comprising all four framework regions (FRs) present in any one of SEQ ID NOs: 1-10, wherein FRs are defined according to any one of AbM, Chothia, Martin, Kabat, IMGT or MacCallum.

[0219] For example, the polypeptides or polypeptide binding agents described herein (particularly antibodies or antibody fragments, more particularly ISVDs) can be defined as comprising the framework region (FR) present in any one of SEQ ID NOs: 1-10, wherein FR is defined according to Martin. In certain embodiments, the binding agent (particularly antibodies or antibody fragments, more particularly ISVDs) comprises one of the following framework region (FR) groups:

[0220] - FR1 defined / listed by SEQ ID NO:97, FR2 defined / listed by SEQ ID NO:33, FR3 defined / listed by SEQ ID NO:101, and FR4 defined / listed by SEQ ID NO:45; or

[0221] - FR1 defined / listed by SEQ ID NO:97, FR2 defined / listed by SEQ ID NO:33, FR3 defined / listed by SEQ ID NO:102, and FR4 defined / listed by SEQ ID NO:45; or

[0222] - FR1 defined / listed by SEQ ID NO:97, FR2 defined / listed by SEQ ID NO:33, FR3 defined / listed by SEQ ID NO:103, and FR4 defined / listed by SEQ ID NO:45; or

[0223] - FR1 defined / listed by SEQ ID NO:98, FR2 defined / listed by SEQ ID NO:33, FR3 defined / listed by SEQ ID NO:104, and FR4 defined / listed by SEQ ID NO:45; or

[0224] - FR1 defined / listed by SEQ ID NO:98, FR2 defined / listed by SEQ ID NO:33, FR3 defined / listed by SEQ ID NO:105, and FR4 defined / listed by SEQ ID NO:45; or

[0225] - FR1 defined / listed by SEQ ID NO:98, FR2 defined / listed by SEQ ID NO:33, FR3 defined / listed by SEQ ID NO:106, and FR4 defined / listed by SEQ ID NO:45; or

[0226] - FR1 defined / listed by SEQ ID NO:98, FR2 defined / listed by SEQ ID NO:33, FR3 defined / listed by SEQ ID NO:107, and FR4 defined / listed by SEQ ID NO:45; or

[0227] - FR1 defined / listed by SEQ ID NO: 99, FR2 defined / listed by SEQ ID NO: 34, FR3 defined / listed by SEQ ID NO: 108, and FR4 defined / listed by SEQ ID NO: 45; or

[0228] - FR1 defined / listed by SEQ ID NO: 99, FR2 defined / listed by SEQ ID NO: 33, FR3 defined / listed by SEQ ID NO: 109, and FR4 defined / listed by SEQ ID NO: 45; or

[0229] - FR1 defined / listed by SEQ ID NO: 100, FR2 defined / listed by SEQ ID NO: 33, FR3 defined / listed by SEQ ID NO: 110, and FR4 defined / listed by SEQ ID NO: 29.

[0230] For example, the polypeptides or polypeptide binding agents described herein (particularly antibodies or antibody fragments, more particularly ISVDs) can be defined as comprising the framework regions (FRs) present in any one of SEQ ID NOs: 1-10, wherein FRs are defined according to Kabat. In certain embodiments, the binding agent (particularly antibodies or antibody fragments, more particularly ISVDs) comprises one of the following framework region (FR) groups:

[0231] - FR1 defined / listed by SEQ ID NO:28, FR2 defined / listed by SEQ ID NO:33, FR3 defined / listed by SEQ ID NO:35, and FR4 defined / listed by SEQ ID NO:45; or

[0232] - FR1 defined / listed by SEQ ID NO:28, FR2 defined / listed by SEQ ID NO:33, FR3 defined / listed by SEQ ID NO:36, and FR4 defined / listed by SEQ ID NO:45; or

[0233] - FR1 defined / listed by SEQ ID NO:28, FR2 defined / listed by SEQ ID NO:33, FR3 defined / listed by SEQ ID NO:37, and FR4 defined / listed by SEQ ID NO:45; or

[0234] - FR1 defined / listed by SEQ ID NO:29, FR2 defined / listed by SEQ ID NO:33, FR3 defined / listed by SEQ ID NO:38, and FR4 defined / listed by SEQ ID NO:45; or

[0235] - FR1 defined / listed by SEQ ID NO:29, FR2 defined / listed by SEQ ID NO:33, FR3 defined / listed by SEQ ID NO:39, and FR4 defined / listed by SEQ ID NO:45; or

[0236] - FR1 defined / listed by SEQ ID NO:29, FR2 defined / listed by SEQ ID NO:33, FR3 defined / listed by SEQ ID NO:40, and FR4 defined / listed by SEQ ID NO:45; or

[0237] - FR1 defined / listed by SEQ ID NO:29, FR2 defined / listed by SEQ ID NO:33, FR3 defined / listed by SEQ ID NO:41, and FR4 defined / listed by SEQ ID NO:45; or

[0238] - FR1 defined / listed by SEQ ID NO:30, FR2 defined / listed by SEQ ID NO:34, FR3 defined / listed by SEQ ID NO:42, and FR4 defined / listed by SEQ ID NO:45; or

[0239] - FRi defined / listed by SEQ ID NO:31, FR2 defined / listed by SEQ ID NO:33, FR3 defined / listed by SEQ ID NO:43, and FR4 defined / listed by SEQ ID NO:45; or

[0240] - FR1 defined / listed by SEQ ID NO:32, FR2 defined / listed by SEQ ID NO:33, FR3 defined / listed by SEQ ID NO:44, and FR4 defined / listed by SEQ ID NO:29.

[0241] In a specific embodiment, the polypeptide or polypeptide binding agent (particularly an antibody or antibody fragment) comprises one or more ISVDs individually defined or listed in any one of SEQ ID NO: 1 to SEQ ID NO: 10; or comprises one or more ISVDs comprising or consisting of an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 10.

[0242] In a further embodiment, the polypeptide or polypeptide binding agent (particularly an antibody or antibody fragment, more particularly an ISVD) comprises or consists of an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 10; or having at least 95% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 10. Such non-identity or variability is preferably limited to non-identity or variability of FR amino acid residues. In particular, such non-identity or variability may be introduced to obtain a humanized variant of an ISVD defined or listed by any one of SEQ ID NOs: 1-10. In particular, such a humanized variant may be a functional ortholog of the original ISVD, wherein the functional features are one or more of the functional features (1) to (47) broadly outlined above.

[0243] The term "wild-type" or "native" refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene is a gene that is most commonly observed in a population and is therefore arbitrarily designed to be the "normal" or "wild-type" form of a gene or gene product. In contrast, the term "modified," "mutant," "engineered," or "variant" refers to a gene or gene product that exhibits sequence modifications (such as substitutions, mutations or variations, deletions or additions), post-translational modifications, and / or modifications (i.e., altered characteristics) in a biological or functional property compared to a wild-type gene or gene product. It is noteworthy that naturally occurring mutants or variants can be isolated; these mutants or variants have altered characteristics compared to the wild-type gene or gene product and can therefore be identified. The altered characteristics may exist solely at the sequence level, or may additionally confer altered biological and / or functional properties to the mutant or variant compared to the wild-type gene or gene product. It should be understood that conservative amino acid substitutions may be introduced into a protein or polypeptide without having an essential or substantial effect on the activity of the protein. Preferred conservative substitutions are those that meet the criteria for acceptable point mutations as defined in Dayhofr et al., Atlas of Protein Sequence and Structure, Vol. 5, pp. 345-352 (1978 & Supp.), which is incorporated herein by reference. Examples of conservative substitutions include, but are not limited to, substitutions in the following groups: (a) valine, glycine; (b) glycine, alanine; (c) valine, isoleucine, leucine; (d) aspartic acid, glutamic acid; (e) asparagine, glutamine; (f) serine, threonine; (g) lysine, arginine, methionine; and (h) phenylalanine, tyrosine. A "homolog" or "homologs" of a protein of interest encompasses proteins having amino acid substitutions, deletions, and / or insertions relative to an unmodified (e.g., native, wild-type) protein of interest, and having substantially or substantially similar biological and functional activities to the unmodified protein from which it is derived.

[0244] The "percentage of sequence identity" is calculated by comparing two optimally aligned (amino acid or nucleic acid) sequences within a comparison window, determining the number of positions at which the identical amino acid residue or nucleotide residue occurs in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of (amino acid or nucleic acid) sequence identity.

[0245] Immunoglobulin single variable domains such as domain antibodies and (including VHH domains) can be humanized, i.e., the degree of sequence identity to the closest human germline sequence is increased. In particular, humanized immunoglobulin single variable domains, such as (including VHH domains) may be immunoglobulin single variable domains in which there is at least one amino acid residue (and in particular, at least one framework residue) which is and / or corresponds to a humanizing substitution (as further defined herein). Potentially useful humanizing substitutions may be determined by comparing the sequence of the framework regions of a naturally occurring VHH sequence with the corresponding framework sequences of one or more closely related human VH sequences, after which one or more potentially useful humanizing substitutions (or combinations thereof) so determined may be introduced into the VHH sequence (in any manner known per se, as further described herein), and the resulting humanized VHH sequence may be tested for affinity for the target, stability, ease and level of expression and / or other desired properties. In this way, by a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) may be determined by the skilled person. Furthermore, based on what has been described previously, immunoglobulin single variable domains (such as The (framework regions) of the VHH domain (including the VHH domain)) can be partially humanized or fully humanized.

[0246] Humanized immunoglobulin single variable domain, specifically It may have several advantages, such as reduced immunogenicity compared to the corresponding naturally occurring VHH domain. Humanization refers to mutations, so that the immunogenicity after administration in human patients is small or non-existent. Humanization substitutions should be selected so that the resulting humanized amino acid sequence and / or ISVD or VHH still retains the favorable properties of the parent (non-humanized) VHH, such as antigen binding ability. Based on the description provided herein, the technician will be able to select a suitable combination of humanization substitutions or humanization substitutions, thereby optimizing or achieving the desired or suitable balance between the favorable properties provided by the humanization substitutions on the one hand and the favorable properties of the naturally occurring VHH domains on the other hand. These methods are known to those skilled in the art. The consensus sequence of the people can be used as a target sequence for humanization, but other methods are also known in the art. An alternative includes such a method, in which the technician compares multiple human germline alleles, such as, but not limited to, the comparison of IGHV3 alleles, to use the comparison to identify residues suitable for humanization in the target sequence. In addition, the subset of human germline alleles most homologous to the target sequence can be used as a starting point for comparison to identify suitable humanized residues. Alternatively, VHH is analyzed to identify its closest homologue in human alleles and used for humanized construct design. The humanization technology applied to camelid VHH can also be carried out by including a method for replacing specific amino acids alone or in combination. The replacement can be selected based on the content of the human consensus sequence or the most similar human allele to the VHH sequence compared with the natural VHH sequence known from the literature. From the data on VHH entropy and VHH variability given in Table A-5-A-8 of WO 08 / 020079, it can be seen that some amino acid residues in the framework region are more conservative than other amino acid residues between humans and camelids. Generally, although the present invention is not limited to this in its broadest sense, any substitution, deletion or insertion (or addition) is preferably carried out at a less conservative position. Moreover, amino acid substitution is usually more preferred than amino acid deletion or insertion. For example, human-like camelid single domain antibodies contain hydrophobic FR2 residues that are usually found in conventional antibodies of human origin or from other species, but this loss of hydrophilicity is compensated by an additional substitution at position 103, which replaces the conserved tryptophan residue present in the VH from the diabody. Therefore, peptides belonging to these two categories show a high degree of amino acid sequence homology with human VH framework regions, and the peptides can be directly administered to humans without expecting an unwanted immune response thereby, and without the burden of further humanization. In fact, some camelid VHH sequences show high sequence homology with human VH framework regions, and the VHH can be directly administered to patients without expecting an immune response thereby, and without the burden or need for additional humanization.

[0247] Suitable mutations, in particular substitutions, may be introduced during humanization to generate polypeptides with reduced binding to pre-existing antibodies (see, e.g., WO2012 / 175741 and WO2015 / 173325), for example at at least one of the following positions: 11, 13, 14, 15, 40, 41, 42, 82, 82a, 82b, 83, 84, 85, 87, 88, 89, 103 or 108. The amino acid sequences and / or VHHs of the invention may be suitably humanized at any framework residue, for example at one or more Hallmark residues (as defined below) or at one or more other framework residues (i.e., non-Hallmark residues) or at any suitable combination thereof. Depending on the host organism used to express the amino acid sequences, ISVDs or VHHs or polypeptides described herein, such deletions and / or substitutions may also be designed in a manner to remove one or more post-translational modification sites (such as one or more glycosylation sites), which will be within the capabilities of those skilled in the art. Alternatively, substitutions or insertions may be designed to introduce one or more sites for attachment of functional groups (as described herein), for example to allow site-specific PEGylation.

[0248] In some cases, at least one of the typical camelid hallmark residues with hydrophilic properties at positions 37, 44, 45 and / or 47 is substituted (see Table A-03 of WO2008 / 020079). Another example of humanization includes substitution of residues in the following positions: in FR1, such as positions 1, 5, 11, 14, 16 and / or 28; in FR3, such as positions 73, 74, 75, 76, 78, 79, 82b, 83, 84, 93 and / or 94; and in FR4, such as positions 10 103, 104, 108 and / or 111 (see Table A-05 to Table A08 of WO2008 / 020079; all numbering is according to the Kabat method). In a specific embodiment, the humanized antibody (particularly a humanized ISVD) comprises a substitution of a residue at position 1, 5, 14, 16, 19, 63, 73, 79, 82c, 83 and / or preferably 108 according to the Kabat numbering. In other specific embodiments, the humanized antibody (particularly a humanized ISVD) comprises a substitution of a residue at position 1, 5, 14, 16, 19, 63, 73, 79, 83 and / or preferably 108 according to the Kabat numbering. Humanization generally involves substitutions, deletions or additions only in FRs, and not in CDRs, as this may / will affect the binding affinity and / or potency to the target.

[0249] Specific non-limiting examples of humanized ISVDs described herein include:

[0250] Humanized R3_DC23:

[0251]

[0252] Humanized R3_C4:

[0253]

[0254] Humanized R4_DC20:

[0255]

[0256] In certain embodiments, the antibody comprises one or more ISVDs as described herein (or variants or humanized forms thereof as described herein), wherein the one or more ISVDs (or variants or humanized forms thereof as described herein) are associated with or fused to an Fc domain.

[0257] As used herein, "Fc domain" refers to the fragment crystallizable region (Fc region) of a conventional antibody, which is the following tail region, which is known to interact with some proteins of the cell surface receptors and complement system called Fc receptors. The Fc domain is composed of two identical protein fragments, which are derived from the second and third constant domains of the two heavy chains of the antibody. All conventional antibodies include an Fc domain, and therefore, the Fc domain can be an Fc domain derived from IgG, IgA and IgD antibody Fc regions (even more specifically derived from IgG1, IgG2 or IgG4 antibody Fc regions) or a variant of the Fc region of the antibody. For example, the hinge region of IgG2 can be replaced by the hinge of human IgG1 to produce an ISVD fusion construct, and vice versa. In addition, Fc variants with known half-life extensions can be used, such as M257Y / S259T / T261E

[0258] (also referred to as YTE) or LS variant (M428L combined with N434S). These mutations increase the binding of the Fc domain of conventional antibodies to the neonatal receptor (FcRn). Preferably, a human Fc domain or a humanized Fc domain can be used. Humanized forms include, but are not limited to, IgG humanized variants known in the art, such as C-terminal deletions of lysine, changes or truncations in the hinge region, LALA (L234A and L235A) or LALAPG (L234A, L235A and P329G) mutations and other substitutions in the IgG sequence.

[0259] As used herein, the term "fused to" is used interchangeably with "linked to", "conjugated to", "attached to", and in one aspect refers to "genetic fusion" (e.g., by recombinant DNA technology), as well as "chemical and / or enzymatic conjugation" so that a stable covalent link is formed between two nucleic acid molecules. The same applies to the term "insertion", in which a fragment of one nucleic acid can be inserted into a second nucleic acid molecule by genetically, enzymatically or chemically fusing or linking the two sequences. Peptides or polypeptides can also be fused or linked to each other, such as by peptide bonds or by linking one peptide to an amino acid side chain in a second peptide.

[0260] Linkers can be used to fuse an ISVD, such as an ISVD identified herein (or a variant or humanized form thereof as described herein), to an Fc domain, such as a human IgG1 Fc domain or a LS variant thereof, or a YTE variant thereof, or an IgG2 Fc domain. Non-limiting examples of linkers include Gly-Ser linkers, such as (G4S) n , wherein n=1-6 (SEQ ID NO: 120), preferably 2-3 (SEQ ID NO: 121-122).

[0261] In certain embodiments, antibodies comprising one or more ISVDs described herein (or variants or humanized forms thereof described herein) are in a "multivalent" and / or "multispecific" format, formed by combining two or more identical or variant monovalent ISVDs (or variants or humanized forms thereof described herein) together, for example, by chemical or recombinant DNA techniques.

[0262] Non-limiting examples of multivalent constructs include "bivalent" constructs, "trivalent" constructs, "tetravalent" constructs, etc., comprising two, three or four ISVDs, respectively. The ISVDs contained in the multivalent construct may be the same or different. As used herein, the term "multispecific antibody" specifically refers to a multivalent antibody in which at least one of the two or more ISVDs has a different specificity. Non-limiting examples of multispecific constructs include "bispecific" constructs, "trispecific" constructs, "tetraspecific" constructs, etc. To further illustrate this point, any multivalent and multispecific (as defined herein) antibodies of the present invention may be directed against two or more different antigens, such as against Sabei virus and as an antigen for the half-life extension of serum albumin or Staphylococcal protein A (SpA) and / or against two or more different parts of a specific antigen, such as two or more different parts, regions, subunits or domains of the Sabei virus spike protein.

[0263] In a specific embodiment, the antibody (particularly a multivalent and / or multispecific antibody) may comprise one or more binding agents, such as an ISVD as described herein (or a variant or humanized form thereof as described herein), and one or more binding agents capable of binding to the Sabeivirus spike protein receptor binding domain (RBD), such as an ISVD. Non-limiting examples of ISVDs capable of binding to the Sabeivirus spike protein receptor binding domain (RBD) are described in PCT / EP202I / 052885, PCT / EP2022 / 052919, and PCT / EP2022 / 062980. Advantageously, in a multivalent and / or multispecific antibody, a combination of at least two ISVDs capable of binding to the Sabeivirus spike protein by interacting at two different regions of the spike protein (particularly the S2 subunit, more particularly the HR2 domain and the RBD) can cause cross-reactions and effectively inhibit infection by Sabeivirus, and can further reduce the risk of the emergence of escape mutant viruses.

[0264] In certain further embodiments, one or more ISVDs capable of binding to the Sabeivirus spike protein RBD are capable of binding to or competing for the VHH72 epitope (or an epitope specifically bound by VHH72). The VHH72 epitope has been described in Wrapp et al. (2020, Cell 184: 1004-1015; PCT / EP202I / 052885 and PCT / EP2022 / 062980). The VHH72 epitope defined herein refers to a conformational epitope in the RBD comprising at least one or more of the amino acid residues S371, S375, T376 or C379 as set forth in SEQ ID NO: 86, or even more particularly, comprising at least one or more of L368, Y369, S371, S375, T376, F377, K378, C379 and Y508 as set forth in SEQ ID NO: 86, which is the sequence of the SARS-Cov-2 spike protein. In particular, the ISVD capable of binding to the VHH72 epitope is capable of specifically binding to the SARS-CoV-2 spike protein (SEQ ID NO: 86), binding to at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 or all of the amino acids L368, Y369, S371, S375, T376, F377, K378, C379 and Y508 of the SARS-CoV-2 spike protein shown in SEQ ID NO: 86. An ISVD capable of competing for the VHH72 epitope refers to an ISVD that competes with VHH72 for binding to the spike protein or RBD shown in SEQ ID NO: 86. "Competition" means that in the presence of an ISVD that can compete for the VHH72 epitope, the binding strength of VHH72 to the spike protein or RBD shown in SEQ ID NO: 86 is reduced by at least 30%, or at least 50%, or preferably at least 80%. In particular, the ISVD that can compete for the VHH72 epitope or compete with VHH72 for binding to the RBD epitope can specifically bind to an epitope on the spike protein, which epitope comprises at least three, at least four, at least five, at least six or more of the residues L368, Y369, S371, S375, T376, F377, K378, C379 and Y508 of the SARS-Cov-2 spike protein as shown in SEQ ID NO: 86, thereby providing an overlapping epitope. In an embodiment, an ISVD capable of binding or competing for the VHH72 epitope may be characterized by: (i) competing for binding to a human receptor (ACE-2 in the case of SARS-CoV-1 and SARS-CoV-2) when interacting with the RBD, and / or (ii) not competing with an ISVD capable of binding or competing for the VHH3.117 epitope as defined herein.Non-limiting examples of ISVDs capable of binding or competing with the VHH72 epitope include VHH72 family members (including VHH72 (SEQ ID NO: 124), VHH2.50, VHH3.17, VHH3.77, VHH3.115, VHH3.144 and VHHBE4) and variants thereof, including VHH72 (S56A) and humanized forms thereof; VHH3.83 family members (including VHH3.83 (also referred to herein as VHH83) (SEQ ID NO: 124), VHH2.50, VHH3.17, VHH3.77, VHH3.115, VHH3.144 and VHHBE4) and variants thereof, including VHH72 (S56A) and humanized forms thereof; NO: 125)) and variants and humanized forms thereof; VHH3.38 family members and variants and humanized forms thereof; VHH3.55 family members and variants and humanized forms thereof; VHH3.36 family members and variants and humanized forms thereof; VHH3.149 family members and variants and humanized forms thereof; and VHH3.29 family members and variants and humanized forms thereof, as described in PCT / EP202I / 052885 and PCT / EP2022 / 062980.

[0265] In specific embodiments, antibodies (particularly multivalent and / or multispecific antibodies) may comprise one or more ISVDs as described herein (or variants or humanized forms thereof as described herein), and an ISVD comprising the CDRs present in SEQ ID NO: 125 or SEQ ID NO: 124 (such as an ISVD comprising or consisting of the sequence set forth in SEQ ID NO: 125 (e.g. VHH83) or an ISVD comprising or consisting of the sequence set forth in SEQ ID NO: 124 (e.g. VHH72)), or variants or humanized forms thereof, wherein the CDRs are annotated according to Kabat, Martin, MacCallum, IMGT, AbM or Chothia.

[0266] In certain further embodiments, one or more ISVDs capable of binding to the Sabeivirus spike protein RBD are capable of binding to or competing for the VHH3.117 epitope (or an epitope specifically bound by VHH3.117). The VHH3.117 epitope has been described in PCT / EP2022 / 052919. In particular, the ISVD capable of binding to the VHH3.117 epitope is capable of binding to or specifically binding to SEQ At least one of amino acids Asn394 (or Ser394 in some Sabeiviruses), Tyr396, Phe464, Ser514, Glu516 and Arg355 of the SARS-CoV-2 spike protein as defined by IDNO: 86, or at least two, at least three or at least four of them in increasing order of preference, and optionally capable of further binding or specifically binding to amino acids Arg357 (or Lys357 in some Sabeiviruses) and / or Lys462 (or Arg464 in some Sabeiviruses) 62) and / or Glu465 (or Gly465 in some Sabeiviruses) and / or Arg466 and / or Leu518, such as being able to further bind or specifically bind to at least two of amino acids Arg357 (or Lys357 in some Sabeiviruses) and / or Lys462 (or Arg462 in some Sabeiviruses) and / or Glu465 (or Gly465 in some Sabeiviruses) and / or Arg466 and / or Leu518, or binding to at least three or all four of them in increasing order of preference. An ISVD capable of competing for the VHH3.117 epitope refers to an ISVD that competes with VHH3.117 for binding to the spike protein or RBD shown in SEQ ID NO: 86. "Competition" means that in the presence of an ISVD that can compete for the VHH3.117 epitope, the binding strength of VHH3.117 to the spike protein shown in SEQ ID NO: 86 is reduced by at least 30%, or at least 50%, or preferably at least 80%. In an embodiment, an ISVD that can bind to or compete for the VHH3.117 epitope can be characterized by: (i) it does not inhibit the binding of RBD to a human receptor (ACE-2 in the case of SARS-CoV-1 and SARS-CoV-2), which means that when the ISVD itself binds to the Sabeivirus RBD, it allows the receptor to bind to the Sabeivirus RBD, or the ISVD itself can bind to the Sabeivirus RBD bound by the receptor, and / or (ii) does not compete with an ISVD that can bind to or compete for the VHH72 epitope defined herein.Non-limiting examples of ISVDs capable of binding or competing for the VHH3.117 epitope include VHH3.117 family members (including VHH3.117, 3.42, 3.92, 3.94, 3.180) and variants and humanized forms thereof (as described in PCT / EP2022 / 052919); VHH3.89 family members and variants and humanized forms thereof (as described in PCT / EP2021 / 052885); VHH3_183 family members and variants and humanized forms thereof; and VHH3C_80 family members and variants and humanized forms thereof (as described in PCT / EP2022 / 062980).

[0267] In specific embodiments, antibodies (particularly multivalent and / or multispecific antibodies) may comprise one or more ISVDs as described herein (or variants or humanized forms thereof as described herein), and an ISVD comprising the CDRs present in SEQ ID NO: 127 (such as an ISVD comprising or consisting of the sequence set forth in SEQ ID NO: 127 (e.g., VHH3.117)), or a variant or humanized form thereof, wherein the CDRs are annotated according to Kabat, Martin, MacCallum, IMGT, AbM or Chothia.

[0268] In other further embodiments, the antibody (particularly a multivalent and / or multispecific antibody) comprises more than one ISVD capable of binding to the receptor binding domain (RBD) of the Sabei virus spike protein, wherein at least one ISVD is capable of binding to or competing for the VHH72 epitope as defined herein, and wherein at least one ISVD is capable of binding to or competing for the VHH3.117 epitope as defined herein. Advantageously, the combination of at least two non-competitively targeting RBD ISVDs (capable of binding to the RBD of the spike protein by interacting at two non-competitive different regions of the RBD) and at least one ISVD targeting S2 in the antibody can cause cross-reactions and effectively inhibit Sabei virus infection, which advantageously allows further reduction of the risk of mutational escape.

[0269] In specific embodiments, antibodies (particularly multivalent and / or multispecific antibodies) may comprise one or more ISVDs as described herein (or variants or humanized forms thereof as described herein); and ISVDs comprising the CDRs present in SEQ ID NO: 125 or SEQ ID NO: 124, such as an ISVD comprising or consisting of the sequence set forth in SEQ ID NO: 125 (e.g. VHH83) or an ISVD comprising or consisting of the sequence set forth in SEQ ID NO: 124 (e.g. VHH72), or variants or humanized forms thereof; and ISVDs comprising the CDRs present in SEQ ID NO: 126, such as an ISVD comprising or consisting of the sequence set forth in SEQ ID NO: 126 (e.g. VHH3.117), or variants or humanized forms thereof, wherein the CDRs are annotated according to Kabat, Martin, MacCallum, IMGT, AbM or Chothia.

[0270] The multivalent antibodies described herein can be formed, for example, by directly linking two or more ISVDs or linking them through a linker, such as by chemical means or recombinant DNA technology, and / or by fusing two or more ISVDs (each) to an Fc domain.

[0271] For example, a single ISVD described herein (or a variant or humanized form thereof) can be fused at its C-terminus to an Fc domain (such as an IgG Fc domain) (such as a construct comprising the amino acid sequence defined in SEQ ID NO: 96 or SEQ ID NO: 118), thereby generating a Sabeivirus antibody in a bivalent form, wherein the two ISVDs form a heavy chain-only antibody-type molecule via a disulfide bridge in the hinge region of the Fc portion (such as an IgG Fc portion).

[0272] In specific embodiments, one or more ISVDs described herein (or variants or humanized forms thereof described herein) are linked, fused or conjugated directly or via a linker to one or more ISVDs capable of binding to the Sabei virus spike protein as defined herein. Non-limiting examples of suitable linkers for linking ISVDs include peptide linkers such as (G4S) n , wherein n = 1, 2, 3, 4, 5, or 6. Such multispecific binding agents may also be referred to herein as "head-to-tail fusions."

[0273] In a further embodiment, the C-terminus of the head-to-tail fusion as described herein can be fused to the Fc domain, for example, through a linker, and after the construct is expressed in the host, a multivalent and / or multispecific antibody is formed through a disulfide bridge in the hinge region of the Fc portion. Therefore, in a specific embodiment, one or more ISVDs described herein (or variants or humanized forms thereof as described herein) are directly or through a linker connected, fused or linked to one or more ISVDs capable of binding to the Sabei virus spike protein RBD to form a multispecific binding agent or construct, and the multispecific binding agent or construct is fused to the Fc domain. In a preferred embodiment, the antibody comprises a bispecific binding agent or construct fused to an Fc domain, wherein the bispecific binding agent or construct comprises an ISVD as described herein (or variants or humanized forms thereof as described herein), which is directly or through a linker connected, fused or linked to an ISVD capable of binding to the Sabei virus spike protein RBD, such as an ISVD capable of binding or competing for the VHH3.117 epitope described herein. Fig.33 A to Fig.33 A schematic diagram of such a multispecific antibody (particularly a bispecific antibody (also referred to herein as a "VHH-VHH-Fc fusion")) is shown in C. More specific examples of such multispecific antibodies (particularly bispecific antibodies) (which are capable of binding to the HR2 binding site and the VHH3.117 epitope described herein) are provided herein, such as but not limited to SEQ ID NOs: 112-114, or any functional variants thereof, or variants thereof having at least 90% identity, or humanized forms thereof. The sequences defined by SEQ ID NOs: 112-114 are also shown below.

[0274] SEQ ID NO: 112

[0275]

[0276]

[0277] SEQ ID NO: 113

[0278]

[0279] SEQ ID NO: 114

[0280]

[0281] In a specific embodiment, the antibody comprises a trispecific binding agent or construct fused to an Fc domain, wherein the trispecific binding agent or construct comprises one ISVD described herein (or a variant or humanized form thereof described herein), one ISVD described herein that is capable of binding to or competing for the VHH3.117 epitope described herein, and one ISVD described herein that is capable of binding to or competing for the VHH72 epitope, wherein the ISVDs are connected, fused or associated with each other in any order, directly or through a linker. Fig.33 A schematic diagram of such a multispecific antibody, in particular a trispecific antibody (also referred to herein as a "VHH-VHH-VHH-Fc fusion"), is depicted in F. More specific examples of such multispecific antibodies, in particular trispecific antibodies, which are capable of binding to the HR2 binding site described herein and to the VHH3.117 and VHH72 epitopes, are provided herein, such as but not limited to SEQ ID NO: 117, or any functional variant thereof, or a variant thereof having at least 90% identity, or a humanized form thereof. The sequence defined by SEQ ID NO: 117 is also shown below.

[0282] SEQ ID NO: 117

[0283]

[0284] In other specific embodiments, one or more ISVDs described herein (or variants or humanized forms thereof described herein) are fused to the N-terminus of the Fc domain, and one or more ISVDs capable of binding to the Sabeivirus Spike protein RBD are fused to the C-terminus of the Fc domain, or one or more ISVDs described herein (or variants or humanized forms thereof described herein) are fused to the C-terminus of the Fc domain, and one or more ISVDs capable of binding to the Sabeivirus Spike protein RBD are fused to the N-terminus of the Fc domain. In a preferred embodiment, the antibody comprises an ISVD as described herein (or a variant or humanized form thereof as described herein) fused to the N-terminus of the Fc domain, and an ISVD capable of binding to the RBD of the spike protein of the Sabeivirus, in particular an ISVD capable of binding to or competing for the VHH3.117 epitope as described herein, which is fused to the C-terminus of the Fc domain; or the antibody comprises an ISVD as described herein (or a variant or humanized form thereof as described herein) fused to the C-terminus of the Fc domain, and an ISVD capable of binding to the RBD of the spike protein of the Sabeivirus, in particular an ISVD capable of binding to or competing for the VHH3.117 epitope as described herein, which is fused to the N-terminus of the Fc domain. Fig.33A schematic diagram of such a multispecific antibody (also referred to herein as a "VHH-Fc-VHH fusion" or "moonlander") is depicted in D. More specific examples of such multispecific antibodies (particularly bispecific antibodies) (which are capable of binding to the HR2 binding site described herein as well as the VHH3.117 epitope) are provided herein, such as but not limited to SEQ ID NO: 115, or any functional variant thereof, or a variant thereof having at least 90% identity, or a humanized variant thereof. The sequence defined by SEQ ID NO: 115 is also shown below.

[0285] SEQ ID NO: 115

[0286]

[0287] The multivalent or multispecific antibodies described herein may have (or be engineered and / or selected for) increased affinity and / or improved selectivity for a desired Sabeivirus interaction and / or for any other desired property or combination of desired properties that may be obtained by using such multivalent or multispecific antibodies.

[0288] In a specific embodiment, the binding agent (particularly the multivalent and / or multispecific antibodies described herein, more particularly the multivalent and / or multispecific antibodies comprising the Fc domains described herein) has cell-mediated antibody-dependent cellular toxicity (ADCC) activity. More particularly, the binding agent (particularly the multivalent and / or multispecific antibodies described herein, more particularly the multivalent and / or multispecific antibodies comprising the Fc domains described herein) can induce ADCC of target cells expressing the Sabei virus spike protein. "Cell-mediated antibody-dependent cellular toxicity" or "ADCC" refers to a form of cytotoxicity in which antibodies bind to certain cytotoxic cells (such as NK cells, neutrophils and macrophages). The secretion of Ig on Fcγ receptors enables these cytotoxic effector cells to specifically bind to target cells carrying antigens, and then use, for example, cytotoxins to kill the target cells. In order to evaluate the ADCC activity of the antibody of interest, an in vitro ADCC assay can be performed, such as the method described in the examples of the present application.

[0289] Also disclosed herein are other Sabeivirus binding agents that compete with an ISVD defined by an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 10 for binding to the Sabeivirus Spike protein or a portion thereof (as described above).

[0290] As used herein, the term "competition" or "cross-competition" refers to a compound or binding agent that has the ability to bind to a specific region of an antigen and inhibit or block the binding of another binding agent. In competitive binding assays known to those skilled in the art, "competitive" or "cross-competitive" compounds or binding agents of the present disclosure have the ability to interfere with the binding of an antibody or antigen-binding fragment described herein, particularly the binding of an ISVD defined by an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 10. The term also includes competition between two antibodies or antigen-binding fragments in both directions, i.e., the first antibody binds and blocks the binding of the second antibody, or vice versa. In certain embodiments, the first antigen-binding agent (e.g., antibody or antigen-binding fragment) and the second antigen-binding agent (e.g., antibody or antigen-binding fragment) may bind to the same epitope. Alternatively, the first and second antigen-binding agents (e.g., antibodies or antigen-binding fragments) may bind to different but, for example, overlapping epitopes, wherein binding of one epitope inhibits or blocks binding of the second antibody or antigen-binding fragment, for example, by steric hindrance. Competition between antigen binding agents (e.g., antibodies or antigen binding fragments) can be measured by methods known in the art, such as by ELISA (enzyme-linked immunosorbent assay) or by surface plasmon resonance (SPR). In the presence of a competing binding agent, competition or cross-competition may exist if the strength of the following binding is reduced by at least 30%, or at least 50%, or preferably at least 80%. In the presence of a competing binding agent, competition or cross-competition may exist, the binding of an ISVD defined by an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 10 to a Sabeivirus spike protein (such as a SARS-CoV-2 spike protein consisting of the amino acid sequence set forth in SEQ ID NO: 86 or a SARS-CoV-1 spike protein consisting of the amino acid sequence set forth in SEQ ID NO: 111, or a portion thereof), in particular to a SARS-CoV-2 S2 subunit or a SARS-CoV-1 S2 subunit or a portion thereof, more particularly to a SARS-CoV-1 / SARS-CoV-2 HR2 domain as shown in SEQ ID NO: 87. In particular, such other binding agents desirably retain one or more of the functional features (1) to (47) broadly outlined above.

[0291] Therefore, the present disclosure also relates to the following methods: screening for compounds that bind to the Sabei virus spike protein (particularly the Sabei virus spike protein S2 subunit, more particularly the Sabei virus HR2 domain in the Sabei virus spike protein), and compounds that compete with the ISVD or its functional part (or its variant or humanized form) described herein for binding to the Sabei virus spike protein (particularly the Sabei virus S2 subunit, more particularly the Sabei virus HR2 domain). Such methods generally include one or more of the following steps:

[0292] - providing a compound or a pool of compounds;

[0293] - contacting the compound or compound pool with the Sabeivirus Spike protein or Sabeivirus S2 subunit or Sabeivirus HR2 domain in the absence of an ISVD or a functional part thereof as described herein (or a variant or humanized form thereof);

[0294] - contacting the compound or compound pool with the Sabeivirus Spike protein or Sabeivirus S2 subunit or Sabeivirus HR2 domain in the presence of an ISVD or a functional part thereof as described herein (or a variant or humanized form thereof);

[0295] - measuring, evaluating, determining, analyzing whether the compound or compound pool can reduce the amount of ISVD or its functional part that binds to the Sabei virus spike protein or Sabei virus S2 subunit or Sabei virus HR2 domain; or measuring, evaluating, determining, analyzing whether the ISVD or its functional part can reduce the amount of the compound or compound pool that binds to the Sabei virus spike protein or Sabei virus S2 subunit or Sabei virus HR2 domain;

[0296] - In the presence of a compound, when the amount of ISVD or its functional portion binding to the Sabei virus spike protein or Sabei virus S2 subunit or Sabei virus HR2 domain is reduced, the compound is identified as a competitor of ISVD or its functional portion binding to the Sabei virus spike protein or Sabei virus S2 subunit or Sabei virus HR2 domain; or in the presence of one or more compounds, when the amount of ISVD or its functional portion binding to the Sabei virus spike protein or Sabei virus S2 subunit or Sabei virus HR2 domain is reduced, the compound pool comprising one or more compounds is identified as a competitor of ISVD or its functional portion binding to the Sabei virus spike protein or Sabei virus S2 subunit or Sabei virus HR2 domain or, in the presence of ISVD or a functional portion thereof, when the amount of compound binding to the Sabei virus spike protein, the Sabei virus S2 subunit, or the Sabei virus HR2 domain is reduced, the compound is identified as a competitor of ISVD or its functional portion binding to the Sabei virus spike protein, the Sabei virus S2 subunit, or the Sabei virus HR2 domain; or, in the presence of ISVD or its functional portion, when the amount of compound pool binding to the Sabei virus spike protein, the Sabei virus S2 subunit, or the Sabei virus HR2 domain is reduced, the compound pool comprising one or more compounds is identified as a competitor of ISVD or its functional portion binding to the Sabei virus spike protein, the Sabei virus S2 subunit, or the Sabei virus HR2 domain.

[0297] As used herein, the term "compound" or "test compound" or "candidate compound" or "drug candidate compound" describes any naturally occurring or synthetic molecule that is designed, identified, screened or generated and can be tested in an assay (such as a screening assay or a drug discovery assay), or in particular in a method for identifying a compound that competes with the ISVD described herein (or a variant or humanized form thereof described herein) for binding to the Sabeivirus Spike protein or a portion thereof (as described above). Thus, these compounds include organic and inorganic compounds. To achieve high throughput purposes, test compound libraries, such as combinatorial libraries or random libraries that provide a sufficient range of diversity, can be used. Examples include, but are not limited to, natural compound libraries, allosteric compound libraries, peptide libraries, antibody fragment libraries, synthetic compound libraries, fragment-based libraries, phage display libraries, and the like. Such compounds may also be referred to as binding agents; as noted herein, they may be "small molecules," which refers to organic compounds of low molecular weight (e.g., <900 Da or <500 Da). Compounds or binding agents also include chemicals, polynucleotides, lipids, or hormone analogs that have low molecular weight characteristics. Other biopolymer organic test compounds include small peptides or peptide-like molecules (peptide mimetics) comprising about 2 to about 40 amino acids, and larger polypeptides comprising about 40 to about 500 amino acids, such as antibodies, antibody mimetics, antibody fragments or antibody conjugates.

[0298] As used herein, the terms "determining," "measuring," "evaluating," "identifying," "screening," and "analyzing" are used interchangeably and include both quantitative and qualitative assays.

[0299] In yet another aspect, the present invention provides a nucleic acid molecule such as an isolated nucleic acid, an (isolated) chimeric gene construct, an expression cassette comprising a polynucleotide sequence such as a coding sequence encoding a polypeptide or a polypeptide portion of a polypeptide Sabeivirus binding agent, in particular an antibody or antibody fragment identified herein, more particularly an ISVD as described herein (or a variant or humanized form thereof), or a functional portion thereof.

[0300] As used herein, "nucleic acid" or "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides; the order of the nucleotides is linearly arranged together to produce / form a "nucleotide sequence", "DNA sequence" or "RNA sequence". The term refers only to the primary structure of the molecule. Therefore, the term includes double-stranded and single-stranded DNA, as well as RNA. It also includes known types of modifications, such as methylation, "caps" and substitution of one or more naturally occurring nucleotides with analogs. Modifications to nucleic acids can be introduced at one or more levels: phosphate modifications (e.g., introduction of one or more phosphodiester bonds, phosphoramidate bonds or phosphorothioate bonds), sugar modifications (e.g., introduction of one or more LNA (locked nucleic acid), 2'-O-methyl, 2'-O-methoxyethyl, 2'-fluoro, S-constrained ethyl or tricyclic-DNA) and / or non-ribose modifications (e.g., introduction of one or more phosphorodiamidate morpholinos or peptide nucleic acids).

[0301] "Nucleic acid construct" refers to a nucleic acid molecule that has been constructed to contain one or more functional units that are not found together in nature and therefore has a nucleotide sequence that is not found in nature (non-natural nucleotide sequence). Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes containing non-natural nucleic acid sequences, etc.

[0302] A "coding sequence" is a nucleotide sequence that can be transcribed into mRNA and / or translated into a polypeptide when placed under the control of appropriate (gene) regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5' end and a translation stop codon at the 3' end. A coding sequence may include, but is not limited to, mRNA, cDNA, recombinant nucleotide sequence or genomic DNA, and in some cases introns may also be present.

[0303] "Chimeric gene" or "chimeric construct" or "chimeric gene construct" interchangeably refers to a recombinant nucleic acid sequence in which a (gene) promoter or regulatory nucleic acid sequence is operably or effectively linked or associated with a nucleic acid sequence of interest encoding an RNA (e.g., a coding sequence, shRNA, etc.) such that the regulatory nucleic acid sequence is capable of regulating the transcription or expression of the nucleic acid of interest.

[0304] An operable or effective linkage between a regulatory nucleic acid sequence and a nucleic acid sequence of interest in a chimeric gene.

[0305] An "expression cassette" comprises any nucleic acid construct capable of directing the expression of a gene / coding sequence of interest, which is operably linked to a (gene) promoter. An expression cassette is typically a DNA construct, preferably comprising (transcription direction 5' to 3'): a (gene) promoter region, a polynucleotide sequence of interest having a transcription initiation region, and a termination sequence comprising an RNA polymerase termination signal and a polyadenylation signal; all of these elements are operably or effectively linked, which means that when transformed into a cell such as a prokaryote (e.g., bacteria) or a eukaryote (e.g., mammals, yeast, insects, fungi, plants, algae), all of these regions should be able to operate (express) in the cell. The promoter region comprising a transcription initiation region (preferably comprising an RNA polymerase binding site) and a polyadenylation signal may be natural to the cell to be transformed, may be derived from an alternative source, or may be synthetic, as long as it is functional in the cell. Such an expression cassette may be constructed, for example, in a "vector" or "expression vector" (linear or circular nucleic acid, plasmid, cosmid, viral vector, phagemid, etc.).

[0306] The present invention also provides a vector comprising the above-mentioned nucleic acid molecule inserted therein.

[0307] As used herein, the terms "vector," "vector construct," "expression vector," "recombinant vector," or "gene transfer vector" are intended to refer to a nucleic acid molecule capable of carrying another nucleic acid molecule to which it has been linked.

[0308] The vector may include a clone or expression vector, and a delivery vector such as a virus, a slow virus or an adenovirus vector. The expression vector may include a plasmid and a viral vector, and is generally included in a specific host organism (e.g., bacteria, yeast, plant, insect or mammal) or in an in vitro expression system to express an operably connected coding sequence and a suitable DNA sequence. In particular, an expression vector as described herein may include a nucleic acid molecule as described herein, which includes a nucleic acid sequence encoding an antibody or antigen binding fragment as described herein, which is operably connected to at least one regulatory sequence. The regulatory sequence is selected to guide the expression of the protein of interest (particularly an antibody or antigen binding fragment) in a suitable host cell, and includes a promoter, an enhancer and other expression control elements known to those skilled in the art. Therefore, in an embodiment, the vector includes a promoter for driving the expression of the nucleic acid of interest, a nucleic acid sequence encoding a signal peptide of the secretory antibody or antigen binding fragment, and a nucleic acid sequence encoding a terminator. When the expression vector is operated in a production strain or cell line, the vector may or may not be integrated into the genome of the host cell when introduced into the host cell. Cloning vectors are generally used for engineering and amplification of a certain required DNA fragment. Therefore, the cloning vector may include a replication origin that matches the cell type specified by the cloning vector, and may lack the functional sequence required for expressing the desired DNA fragment. Preferably, the vector includes one or more selective markers. The selection of selective markers may depend on the selected host cell, although this is not important for the present invention, as is well known to those skilled in the art. The construction of the expression vector for transfected cells is also well known in the art, and therefore can be achieved by standard techniques (see, for example, Sambrook, Fritsch and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, EJ Murray edited, The Humana Press Inc., Clifton, NJ) and Ambion 1998 catalogue (Ambion, Austin, Tex.).

[0309] More specifically, the vector may comprise any vector known to those skilled in the art, including any suitable type, but not limited to, for example, a plasmid vector, a cosmid vector, a phage vector (such as lambda phage), a viral vector (even more specifically a lentiviral, adenoviral, AAV or baculoviral vector), or an artificial chromosome vector (such as a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC) or a P1 artificial chromosome (PAC). The choice of vector may depend, among other things, on the properties of the selected host cell.

[0310] Another aspect of the present invention provides a host cell comprising an antibody or antigen-binding fragment thereof, such as an ISVD (or a variant or humanized form thereof) or a portion thereof of an antibody or antigen-binding fragment as described herein. Thus, the host cell may comprise a nucleic acid molecule encoding the antibody or antigen-binding fragment. The host cell may be prokaryotic or eukaryotic. The host cell may also be a recombinant host cell comprising a cell that has been genetically modified to contain an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment of the present invention. Representative host cells that can be used to produce the antibody or antigen-binding fragment, such as an ISVD, include, but are not limited to, bacterial cells, yeast cells, plant cells, and animal cells. Bacterial host cells suitable for producing the antibodies or antigen-binding fragments of the invention include Escherichia spp. cells, Bacillus spp. cells, Streptomyces spp. cells, Erwinia spp. cells, Klebsiella spp. cells, Serratia spp. cells, Pseudomonas spp. cells, and Salmonella spp. cells. Yeast host cells suitable for use in the present invention include species within the genera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia (e.g., Pichia pastoris), Hansenula (e.g., Hansenula polymorpha), Yarowia, Schwaniomyces, Schizosaccharomyces, Zygosaccharomyces, and the like. Saccharomyces cerevisiae, S. carlsbergensis, and Kluyveromyces lactis are the most commonly used yeast hosts and are convenient fungal hosts. Animal host cells suitable for use in the present invention include insect cells and mammalian cells (e.g., derived from Chinese hamsters (e.g., CHO) and human cell lines (such as HeLa)). Exemplary insect cell lines include, but are not limited to, Sf9 cells, baculovirus-insect cell systems (e.g., review by Jarvis, Virology, Vol. 310 No. 1, May 25, 2003, pp. 1-7). Alternatively, the host cell may also be a transgenic animal or plant.

[0311] Introduction of the vector into the host cell can be achieved by, for example, calcium phosphate transfection, viral infection, DEAE-dextran-mediated transfection, liposome transfection or electroporation, and any person skilled in the art can select and use an introduction method suitable for the expression vector and host cell used.

[0312] Another aspect of the invention relates to a composition comprising a binding agent, such as an antibody or antigen-binding fragment thereof, comprising one or more ISVDs (or variants or humanized forms thereof) as described herein, or a portion thereof. As used herein, a "composition" refers to a combination of one or more molecules present in a formulation that retains binding agent activity, particularly in this case HR2 (or S2) binding and Sabeivirus neutralization activity, and is therefore a functional composition. Thus, the composition comprises one or more molecules constituting one or more binding agents as described herein that specifically bind to Sabeivirus Spike protein by interacting with its HR2 domain (a binding agent targeting S2 or a binding agent targeting an HR2 domain). In a specific embodiment, the composition may comprise a bivalent antibody comprising an ISVD as described herein (or a variant or humanized form thereof), such as a binding agent, particularly an antibody comprising the amino acid sequence defined by SEQ ID NO: 118, fused to an Fc domain. The composition may be a soluble composition or a solid composition.

[0313] In addition to the binding agent molecule targeting S2, particularly targeting HR2 domain, the composition may further comprise, for example but not limited to, a buffer component, an adjuvant or an additional molecule, which may be a functional molecule.

[0314] In a specific embodiment, the composition may further comprise one or more binding agents capable of binding to the Sabeivirus spike protein receptor binding domain (RBD) as described elsewhere herein. In a specific embodiment, the composition may further comprise one or more binding agents such as antibodies or antigen-binding fragments thereof, the one or more binding agents comprising one or more (such as two, three, four or more) ISVDs (or variants or humanized forms thereof) capable of binding to the Sabeivirus spike protein receptor binding domain (RBD) as described elsewhere herein. Thus, the composition may comprise at least two binding agents, characterized in that one binding agent specifically binds to the HR2 domain and the second binding agent specifically binds to the RBD region, thereby producing a composition having at least two binding agents that bind to the spike protein in a non-competitive manner, possibly simultaneously.

[0315] In a preferred embodiment, the binding agent capable of binding to the Sabeivirus spike protein RBD is capable of binding to two non-competitive binding sites of the RBD, preferably through two different ISVDs present in the binding agent, wherein the binding agent can be a bispecific binding agent or a multispecific binding agent. More particularly, the binding agent may comprise one or more ISVDs capable of binding or competing with the VHH72 epitope as defined herein, and one or more ISVDs capable of binding or competing with the VHH3.117 epitope as defined herein. Non-limiting examples of binding agents comprising one or more ISVDs capable of binding or competing with the VHH72 epitope, and one or more ISVDs capable of binding or competing with the VHH3.117 epitope are described in PCT / EP2022 / 062980.

[0316] In a specific embodiment, the composition may comprise (i) a binding agent (particularly an antibody or an antigen-binding fragment thereof) comprising one or more ISVDs comprising the CDRs present in SEQ ID NO: 8, such as one or more ISVDs comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 8 (e.g., VHHR3-DC23) or a variant or humanized form thereof; and (ii) a binding agent (particularly an antibody or an antigen-binding fragment thereof) comprising one or more ISVDs comprising the CDRs present in SEQ ID NO: 125 or SEQ ID NO: 124, such as an ISVD comprising or consisting of the sequence set forth in SEQ ID NO: 125 (e.g., VHH83) or an ISVD comprising or consisting of the sequence set forth in SEQ ID NO: 124 (e.g., VHH72) or a variant or humanized form thereof, and an ISVD comprising the CDRs present in SEQ ID NO: 126, such as an ISVD comprising SEQ ID NO: 127. An ISVD consisting of or having a sequence as set forth in NO: 126 (eg, VHH3.117) or a variant or humanized form thereof, wherein the CDRs are annotated according to Kabat, Martin, MacCallum, IMGT, AbM or Chothia.In a further specific embodiment, the composition may comprise (i) a binding agent, in particular a (bivalent) antibody, comprising: an ISVD comprising the CDRs present in SEQ ID NO: 8, such as an ISVD comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 8 (e.g., VHHR3-DC23) or a variant or humanized form thereof, such as an antibody comprising the amino acid sequence set forth in SEQ ID NO: 118, fused to an Fc domain; and (ii) a binding agent, in particular a bispecific antibody, comprising: an ISVD comprising the CDRs present in SEQ ID NO: 126, such as an ISVD comprising or consisting of the sequence set forth in SEQ ID NO: 126 (e.g., VHH3.117) or a variant or humanized form thereof, fused to the N-terminus of an Fc domain as defined herein, and an ISVD comprising the CDRs present in SEQ ID NO: 125 or SEQ ID NO: 124, such as an ISVD comprising SEQ ID NO: 125 fused to the N-terminus of an Fc domain. An ISVD comprising a sequence set forth in SEQ ID NO: 125 or consisting thereof (e.g., VHH83) or an ISVD comprising a sequence set forth in SEQ ID NO: 124 or consisting thereof (e.g., VHH72) or a variant or humanized form thereof; or an ISVD comprising the CDRs presented in SEQ ID NO: 126, such as an ISVD comprising a sequence set forth in SEQ ID NO: 126 or consisting thereof (e.g., VHH3.117) or a variant or humanized form thereof that can be fused to the N-terminus of the Fc domain, and an ISVD comprising the CDRs presented in SEQ ID NO: 125 or SEQ ID NO: 124, such as an ISVD comprising a sequence set forth in SEQ ID NO: 125 or consisting thereof (e.g., VHH83) or an ISVD comprising a sequence set forth in SEQ ID NO: 124 or consisting thereof (e.g., VHH72) or a variant or humanized form thereof that can be fused to the C-terminus of the Fc domain. NO: 124 or an ISVD (e.g., VHH72) or a variant or humanized form thereof, wherein the CDRs are annotated according to Kabat, Martin, MacCallum, IMGT, AbM or Chothia. In a further specific embodiment, the bispecific antibody (ii) comprises or consists of the amino acid sequence set forth in SEQ ID NO: 119, or any functional variant thereof, or a variant having at least 90% identity thereto, or a humanized variant thereof.

[0317] In an embodiment, the molecular ratio of the (targeted S2) binding agent as described herein (such as an antibody or antigen-binding fragment thereof comprising one or more ISVDs (or variants or humanized forms thereof) or a portion thereof) to the (targeted S1) binding agent capable of binding to the Sabeivirus Spike protein receptor binding domain (RBD) in the composition (such as an antibody or antigen-binding fragment thereof comprising one or more ISVDs (or variants or humanized forms thereof)) can be in the range of 3:1 to 1:3, preferably 2:1 to 1:2, and more preferably a molecular ratio of about 1:1.

[0318] In addition, the composition may also comprise additional binding agents or molecules, which optionally bind to additional binding regions on the same or different epitopes of the spike protein or other viral proteins, or may even target completely unrelated target proteins.

[0319] Another aspect of the invention relates to a medicament or pharmaceutical composition comprising a binding agent (particularly an antibody or antigen-binding fragment), a nucleic acid encoding the binding agent and / or a (recombinant) vector comprising the nucleic acid as described herein, and / or a composition comprising a binding agent (particularly an antibody or antigen-binding fragment). In particular, the pharmaceutical composition is a pharmaceutically acceptable composition; such a composition preferably further comprises a (pharmaceutically) suitable or acceptable carrier, diluent, adjuvant, excipient, stabilizer, etc.

[0320] "Pharmaceutically acceptable" refers to a substance that is not biologically or otherwise undesirable, i.e., the substance can be administered to an individual together with a compound (particularly a Sabeivirus binder, more particularly a Sabeivirus antibody or antigen-binding fragment) without causing any undesirable biological effect or interacting in a harmful manner with any other component of a pharmaceutical composition containing the substance. A pharmaceutically acceptable carrier is preferably a carrier that is relatively nontoxic and harmless to the patient at a concentration consistent with the effective activity of the active ingredient, so that any side effects caused by the carrier do not impair the beneficial effects of the active ingredient. Suitable carriers or adjuvants typically contain one or more compounds included in the following non-exhaustive list: large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive viral particles. As used herein, the term "excipient" is intended to include all substances that may be present in a pharmaceutical composition and are not active ingredients but may contribute to, for example, the long-term stability or therapeutic enhancement of the active ingredient (e.g., by promoting drug absorption, reducing viscosity, or enhancing solubility). Excipients may include, for example, salts, binders (e.g., lactose, glucose, sucrose, trehalose, sorbitol, mannitol), lubricants, thickeners, surfactants, preservatives, emulsifiers, buffer substances, stabilizers, flavoring agents or coloring agents. "Diluents", such as particularly "pharmaceutically acceptable diluents", include vehicles such as water, saline, physiological saline solutions, glycerol, ethanol, etc. Auxiliary substances such as wetting agents or emulsifiers, pH buffer substances, preservatives may be included in such vehicles.

[0321] A pharmaceutically effective amount of a binding agent (particularly an antibody or antigen-binding fragment) of the invention is preferably an amount that produces a result or exerts an effect on the particular condition being treated.

[0322] The pharmaceutical composition of the present invention can be freeze-dried for storage and reconstituted in a suitable carrier before use. When prepared as a lyophilisate or liquid, it is necessary to add a physiologically acceptable carrier, excipient, stabilizer (Remington's Pharmaceutical Sciences 22nd edition, Allen edited, Loyd V, Jr. (2012) to the pharmaceutical composition of the present invention. The preparation containing the pharmaceutical composition of the present invention should be sterilized before injection. The procedure can be performed using a sterile filtration membrane before or after freeze-drying and reconstitution. The pharmaceutical composition can be packaged in a container or vial with a sterile access port, such as an intravenous solution bottle-pharmaceutical composition with a rubber stopper can exist in liquid form, or a container or vial is filled with a liquid pharmaceutical composition, which is then freeze-dried or dried; or it can be packaged in a pre-filled syringe.

[0323] Another aspect of the invention relates to a binding agent (particularly an antibody or antigen-binding fragment) as described herein, a nucleic acid encoding the binding agent, a vector comprising such a nucleic acid, a composition comprising a binding agent (particularly an antibody or antigen-binding fragment) as described herein, or a pharmaceutical composition comprising a binding agent (particularly an antibody or antigen-binding fragment), a nucleic acid encoding the binding agent, a (recombinant) vector comprising such a nucleic acid and / or a composition comprising a binding agent (particularly an antibody or antigen-binding fragment) as described herein for use as a medicament or pharmaceutical agent. Alternatively, the use of a binding agent (particularly an antibody or antigen-binding fragment) as described herein, a nucleic acid encoding the binding agent, a vector comprising such a nucleic acid, or a composition comprising a binding agent (particularly an antibody or antigen-binding fragment) as described herein in the manufacture of a medicament or pharmaceutical agent is contemplated, or a pharmaceutical composition comprising a binding agent (particularly an antibody or antigen-binding fragment), a nucleic acid encoding the binding agent, a vector comprising such a nucleic acid and / or a composition comprising a binding agent (particularly an antibody or antigen-binding fragment) as described herein in the manufacture of a medicament or pharmaceutical agent is contemplated.

[0324] In particular, a binding agent (particularly an antibody or antigen-binding fragment) as described herein, a nucleic acid encoding the binding agent, a vector comprising such a nucleic acid, or a composition comprising the binding agent (particularly an antibody or antigen-binding fragment) as described herein, or a medicament or pharmaceutical composition comprising a binding agent (particularly an antibody or antigen-binding fragment), a nucleic acid encoding the binding agent, a (recombinant) vector comprising such a nucleic acid, and / or a composition comprising a binding agent (particularly an antibody or antigen-binding fragment) as described herein is used for passive immunization, for treating a subject suffering from a Sabeivirus infection, for preventing a subject from being infected with a Sabeivirus, or for protecting a subject from an infection with a Sabeivirus.

[0325] When used for passive immunization, the subject may be infected with Sabei virus (therapeutic passive immunization) or may not be infected with Sabei virus (prophylactic passive immunization).

[0326] A related aspect relates to a method for treating a subject suffering from / infected with / infected with Sabei virus, the method comprising administering to the subject a binding agent (particularly an antibody or antigen-binding fragment) as described herein, a nucleic acid encoding the binding agent, a (recombinant) vector comprising such a nucleic acid, or a composition comprising the binding agent (particularly an antibody or antigen-binding fragment), or comprising administering to the subject a medicament or pharmaceutical composition comprising a binding agent (particularly an antibody or antigen-binding fragment), a nucleic acid encoding the binding agent, a (recombinant) vector comprising such a nucleic acid and / or a composition comprising the binding agent (particularly an antibody or antigen-binding fragment) as described herein.

[0327] Another aspect of the present invention relates to a method for protecting a subject from infection with Sabeivirus or preventing a subject from infection with Sabeivirus, the method comprising administering to the subject a binding agent (particularly an antibody or antigen-binding fragment) as described herein, a nucleic acid encoding the binding agent, a (recombinant) vector comprising such a nucleic acid, or a composition comprising a binding agent (particularly an antibody or antigen-binding fragment) as described herein before infection, or comprising administering to the subject a medicament or pharmaceutical composition comprising a binding agent (particularly an antibody or antigen-binding fragment), a nucleic acid encoding the binding agent, a (recombinant) vector comprising such a nucleic acid and / or a composition comprising a binding agent (particularly an antibody or antigen-binding fragment) as described herein before infection.

[0328] In the above-mentioned medical aspects, nucleic acids encoding binding agents (particularly antibodies or antigen-binding fragments) as described herein or (recombinant) vectors comprising such nucleic acids can be used, for example, in a gene therapy setting. As used herein, "gene therapy" refers to treatment by administering an expressed or expressible nucleic acid to a subject. For such applications, nucleic acid molecules or vectors as described herein allow the production of binding agents, antibodies or antibody fragments in cells. There are a large number of gene therapy methods in the art, including, for example, (adeno-associated) virus-mediated gene silencing or virus-mediated gene therapy (e.g., US 20040023390; Mendell et al., 2017, N Eng J Med 377: 1713-1722). A variety of delivery methods are well known to those skilled in the art, including but not limited to viral delivery systems, microinjection of DNA plasmids, gene guns of naked nucleic acids, the use of liposomes or artificial exosomes, and the administration of nucleic acids or vectors formulated in nanoparticles or lipids or lipid-containing particles. In vivo delivery to an individual patient typically employs systemic administration (eg, intravenous, intraperitoneal infusion, or brain injection; e.g., Mendell et al., 2017, N Eng J Med 377: 1713-1722).

[0329] "Therapeutic agent" generally refers to any molecule that has or can have a therapeutic effect (i.e., a therapeutic or preventive effect) in the case of disease treatment. Preferably, the therapeutic agent is a disease modifier, which can be a cytotoxic agent such as a toxin, or a cytotoxic drug, or an enzyme that can convert a prodrug into a cytotoxic drug, or a radionuclide, or a cytotoxic cell, or it can be a non-cytotoxic agent. Even more preferably, the therapeutic agent has a therapeutic effect on the disease. The binding agent (particularly an antibody or antibody fragment) or pharmaceutical composition of the present invention can serve as a therapeutic agent when it is beneficial to treat patients infected with Sabei virus (e.g., SARS-CoV-2 or SARS-CoV-1) or patients suffering from COVID-19. The binding agent (particularly an antibody or antibody fragment) may include a variant of the ISVD combined with Sabei virus as described herein, preferably an improved variant combined with the same binding region of the HR2 domain, and more preferably a humanized variant thereof, and may include or be coupled to additional functional groups, which is advantageous when applied to a subject. Examples of such functional groups and examples of techniques for incorporating them will be clear to those skilled in the art and may generally include all functional groups and techniques mentioned in the art as well as those known per se for modifying pharmaceutical proteins, in particular antibodies or antibody fragments, for which reference may be made, for example, to Remington's Pharmaceutical Sciences, 16th edition, Mack Publishing Co., Easton, PA (1980). Such functional groups may, for example, be directly (e.g., covalently) attached to an ISVD or active antibody fragment, or alternatively attached via a suitable linker or spacer, which will also be apparent to those skilled in the art. One of the most widely used techniques for increasing the half-life of a pharmaceutical protein and / or reducing its immunogenicity involves attachment of a suitable pharmacologically acceptable polymer, such as poly(ethylene glycol) (PEG) or a derivative thereof (such as methoxypoly(ethylene glycol) or mPEG). For example, for this purpose, PEG may be attached to a naturally occurring cysteine ​​residue in an immunoglobulin single variable domain as described herein (or a variant or humanized form thereof as described herein), an immunoglobulin single variable domain as described herein (or a variant or humanized form thereof as described herein) may be modified so as to appropriately introduce one or more cysteine ​​residues for attachment of PEG, or an amino acid sequence comprising one or more cysteine ​​residues for attachment of PEG may be fused to the N-terminus and / or C-terminus of an ISVD or active antibody fragment as described herein (or a variant or humanized form thereof as described herein), all using protein engineering techniques known per se to the skilled person. Another generally less preferred modification includes N-linked or O-linked glycosylation, typically as part of a co-translational and / or post-translational modification, depending on the host cell used to express the antibody or active antibody fragment.Another technique for increasing the half-life of a binding domain (particularly an antibody or antibody fragment) may include engineering into a bifunctional or bispecific domain (e.g., an ISVD or active antibody fragment for the target Sabeivirus HR2 domain and an ISVD for a serum protein such as albumin or Staphylococcus protein A (SpA)-which is a surface protein present in large quantities in the lungs, helping to extend the half-life) or engineering into a fusion of an antibody fragment (particularly an immunoglobulin single variable domain) with a peptide (e.g., a peptide for a serum protein such as albumin). In yet another example, an ISVD as described herein (or a variant or humanized form thereof as described herein) may be fused to an immunoglobulin Fc domain as described elsewhere herein. Examples are further shown in the experimental section and are also described in the sequence table. In embodiments, in the above-mentioned medical aspects, the Sabeivirus is SARS-CoV-2 such as a SARS-CoV-2 variant, or SARS-CoV-1. SARS-CoV-2 variants may be variants at positions N439, K417, S477, L452, T478, E484, P384, N501 and / or D614 (relative to SEQ ID NO: 86 as defined in the SARS-CoV-2 spike protein amino acid sequence), more particularly variants at position N501 such as N501Y variants (e.g., SARS-CoV-2 Alpha variants), variants at positions N501 and E484 such as N501Y and E484K variants (e.g., SARS-CoV-2 Alpha+E484K variants), variants at positions K417, E484 and N501 such as K417N, E484K and N501Y variants (e.g., SARS-CoV-2 beta variants), variants at positions P384, K417, E484 and N501 such as P384L, K417N, E484K and N501Y variants (e.g., SARS-CoV-2 b eta+P384L variant), variants at positions L452 and E484 such as L452R and E484Q variants (e.g., SARS-CoV-2 kappa variants), variants at positions L452 and T478 such as L452R and T478K variants (e.g., SARS-CoV-2 delta variants), variants at positions L452 such as L452R variants (e.g., SARS-CoV-2 epsilon variants), variants at positions K417 such as K417T variants (e.g., SARS-CoV-2 gamma variants), or variants at positions D614 such as D614G variants (e.g., SARS-CoV-2 Omicron variants or SARS-CoV-2 BA.1 variants). In specific embodiments, the Sabei virus is either or both of SARS-CoV-2 and SARS-CoV-2.In a further specific embodiment, SARS-CoV-2 is the SARS-CoV-2 Wuhan-Hu-1 strain or a SARS-CoV-2 variant, in particular a SARS-CoV-2 variant selected from the group consisting of the SARS-CoV-2 Alpha variant, the SARS-CoV-2 Omicron BA.1 variant and the SARS-CoV-2 Omicron BA.2 variant.

[0330] As used herein, the term "therapy" or "treatment" refers to relieving or measurably alleviating one or more symptoms or measurable markers of a pathological condition (such as a disease or illness, particularly a Sabei virus infection). Measurably alleviating includes any statistically significant decline in measurable symptoms or markers. In general, these terms encompass curative treatments and treatments intended to alleviate symptoms and / or slow down disease progression. These terms encompass therapeutic treatments for already developed pathological conditions (particularly Sabei virus infections), as well as preventive or defensive measures, wherein the goal is to prevent or reduce the incidence of pathological conditions (particularly Sabei virus infections). Beneficial or desired clinical results include, but are not limited to, preventing disease, reducing disease incidence, alleviating symptoms associated with the disease, alleviating disease extent, stabilizing disease, delaying or slowing disease progression, improving or alleviating disease, or a combination thereof. In certain embodiments, these terms may relate to therapeutic treatments. In certain other embodiments, these terms may relate to preventive treatments.

[0331] For example, treatment may refer to passive immunization (therapeutic treatment) of a subject infected with Sabei virus. Prevention of infection with Sabei virus may be useful, for example, in the context of an epidemic or pandemic, during which subjects known to be most susceptible to developing severe disease symptoms may be treated prophylactically (defensive or prophylactic immunization) with a binding agent (particularly an antibody or antigen-binding fragment) as described herein, or a nucleic acid encoding the binding agent, or a vector comprising such a nucleic acid, or a composition comprising a binding agent (particularly an antibody or antigen-binding fragment), in order to prevent infection in general, or to prevent the development or occurrence of severe disease symptoms.

[0332] In an embodiment, a therapeutically effective amount of a binding agent (particularly an antibody or antigen binding fragment), a nucleic acid, a vector or a pharmaceutical composition is administered to a subject in need. In other embodiments, a prophylactic effective amount of a binding agent (particularly an antibody or antigen binding fragment), a nucleic acid, a vector or a pharmaceutical composition is administered to a subject in need. "Therapeutically effective amount" or "therapeutically effective dose" means that when a binding agent (particularly an antibody or antigen binding fragment), a nucleic acid, a vector or a pharmaceutical composition is administered to a subject, the amount that produces a clinically positive response, such as curing a Sabeivirus infection, for therapeutic treatment of a subject with Sabeivirus infection. Similarly, "prophylactic effective amount" or "prophylactic effective dose" refers to the amount of a binding agent (particularly an antibody or antigen binding fragment), a nucleic acid, a vector or a pharmaceutical composition that prevents, inhibits or delays the occurrence of Sabeivirus infection in a subject and / or prevents or reduces the risk of clinical manifestations of Sabeivirus infection and / or reduces the severity, symptoms and / or duration of Sabeivirus infection. In order to achieve a therapeutic effect or a defensive or preventive effect, the binding agent (particularly an antibody or antigen-binding fragment) or a nucleic acid encoding the binding agent or a vector comprising such a nucleic acid or a composition comprising the binding agent (particularly an antibody or antigen-binding fragment) may need to be administered to the subject multiple times, for example at intervals of 1 or 2 weeks; the intervals are determined by the pharmacokinetic behavior or characteristics of the binding agent (particularly an antibody or antigen-binding fragment), nucleic acid or vector (for example, half-residence time or half-life in the blood circulation of the subject). Alternatively, therapeutic treatment and preventive treatment are envisioned, in which a single dose of a binding agent (particularly an antibody or antigen-binding fragment) as described herein is administered to the subject. A single dose may be in the range of 0.5 mg / kg to 25 mg / kg.

[0333] The terms "subject", "individual" or "patient" are used interchangeably herein and relate to any organism such as a vertebrate, specifically any mammal, including humans and other mammals, for which diagnosis, treatment or prevention is required, for example, animals such as rodents, rabbits, cattle, sheep, horses, dogs, cats, alpacas, pigs or non-human primates (e.g., monkeys). Rodents can be mice, rats, hamsters, guinea pigs or chinchillas. In one embodiment, the subject is a human, rat or non-human primate. Preferably, the subject is a human. In a specific embodiment, the subject is a subject infected or suspected of being infected with a Sabei virus, such as a human subject, also referred to herein as a "patient" or "subject". However, it should be understood that the above terms do not mean that there are symptoms. In a specific embodiment, the subject is a mammal susceptible to infection with a Sabei virus, such as a human subject susceptible to infection with SARS-CoV-2 such as a SARS-CoV-2 variant or SARS-CoV-1.

[0334] Pharmaceutical composition of the present invention can be applied to any patient according to standard techniques. Can be used by any appropriate mode, including oral, parenteral, topical, intranasal, intraocular, intrathecal, intraventricular, sublingual, rectal, vaginal etc. Other formulation techniques such as nanotechnology and aerosol and inhalant are also within the scope of the present invention. The dosage and frequency of use will depend on the patient's age, sex and condition, other drugs used simultaneously, contraindications and other parameters that clinicians will consider.

[0335] In specific embodiments of the medical aspects described herein, the binding agent (particularly an antibody or antigen-binding fragment), nucleic acid, vector or pharmaceutical composition can be administered to a subject by intravenous, subcutaneous or intranasal injection, or by inhalation or pulmonary delivery.

[0336] Another aspect of the invention relates to a binding agent (particularly an antibody or antigen-binding fragment) as described herein for use in diagnosing a Sabeivirus infection as a diagnostic agent. Nucleic acids encoding a Sabeivirus binding agent (particularly a Sabeivirus antibody or antigen-binding fragment) as described herein, (recombinant) vectors comprising such nucleic acids, or compositions comprising a Sabeivirus binding agent (particularly a Sabeivirus antibody or antigen-binding fragment) as described herein can also be used.

[0337] It is also contemplated that the use of a binding agent (particularly an antibody or antigen binding fragment) as described herein in the preparation of an (in vitro) diagnostic agent or diagnostic kit is used. In particular, a binding agent (particularly an antibody or antigen binding fragment) as described herein can be used to detect the presence (or absence) of Sabeivirus or a portion thereof (such as Sabeivirus spike protein or a portion thereof) in a sample, such as a sample obtained from a subject (such as a subject suspected of being infected with Sabeivirus). Nucleic acids encoding a binding agent (particularly an antibody or antigen binding fragment) as described herein, a (recombinant) vector comprising such a nucleic acid, or a composition comprising a binding agent (particularly an antibody or antigen binding fragment) as described herein can also be used to prepare a diagnostic agent or diagnostic kit, such as an in vitro diagnostic agent or kit.

[0338] Another aspect relates to a method for detecting Sabeivirus in a sample, such as a sample obtained from a subject, such as a sample obtained from a subject suspected of being infected with Sabeivirus infection. Such methods generally include the steps of obtaining a sample; contacting the sample with a binding agent (particularly an antibody or antigen-binding fragment) as described herein; and detecting, determining, evaluating, determining, identifying or measuring the binding of the binding agent (particularly an antibody or antigen-binding fragment) to Sabeivirus or a portion thereof (such as Sabeivirus spike protein or a portion thereof).

[0339] In specific embodiments of the diagnostic aspects described herein, the Sabeivirus is selected from the group consisting of clade 1a, 1b, 2 and / or clade 3 Sabeiviruses, such as SARS-Cov-2, GD-Pangolin, RaTG13, WIV1, LYRa11, RsSHC014, Rs7327, SARS-CoV-1, Rs4231, Rs4084, Rp3, HKU3-1 or BM48-31 viruses, preferably SARS-CoV-2 such as a SARS-CoV-2 variant or SARS-CoV-1.

[0340] In the embodiments of the diagnostic aspects described herein, the binding agents (particularly antibodies or antibody fragments) as described herein comprise a detectable portion fused, bound, coupled, connected, compounded or chelated thereto. "Detectable portion" generally refers to a portion that emits a signal or is capable of emitting a signal when appropriately stimulated, or refers to a portion that can be detected by binding or interacting with another molecule (e.g., a label, such as an affinity label, which is specifically recognized by a labeled antibody) or by any means (preferably by a non-invasive means if the detection is in vivo / in vivo). In addition, the detectable portion can allow computerized synthetic images, so the detectable portion can be referred to as an imaging agent. The detectable portion includes, but is not limited to, fluorescent emitters, phosphorescent emitters, positron emitters, radioactive emitters, etc., enzymes (capable of measurably converting substrates) and molecular tags. Examples of radioactive emitters / radiolabels include 68Ga, 110mIn, 18F, 45Ti, 44Sc, 47Sc, 61Cu, 60Cu, 62Cu, 66Ga, 64Cu, 55Ca, 72As, 86Y, 90Y, 89Zr, 125I, 74Br, 75Br, 76Br, 77Br, 78Br, 111In, 114m1n, 114In, 99mTc, 11C, 32Cl, 33Cl, 34Cl, 123I, 124I, 131I, 186Re, 188Re, 177Lu, 99Tc, 212Bi, 213Bi, 212Pb, 225Ac, 153Sm, and 67Ga. Fluorescent emitters include, but are not limited to, cyanine dyes (e.g., Cy5, Cy5.5, Cy7, Cy7.5), FITC, TRITC, coumarins, indolenine-based dyes, benzoindoline-based dyes, phenoxazines, BODIPY dyes, rhodamine, Si-rhodamine, Alexa dyes, and any derivatives thereof. Non-limiting examples of molecular tags include: affinity tags, such as chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), poly (His) (e.g., 6x His or His6); biotin or streptavidin, such as Strep-tag and Solubilizing tags, such as thioredoxin (TRX), poly (NANP) and SUMO; chromatographic tags, such as FLAG-tags; epitope tags, such as V5-tags, myc-tags and HA-tags; fluorescent markers or tags (i.e. fluorescent dyes / groups), such as fluorescent proteins (e.g. GFP, YFP, RFP, etc.); luminescent markers or tags, such as luciferase, bioluminescent or chemiluminescent compounds (such as luminol, isoluminol, theromatic acridinium esters, imidazoles, acridinium salts, oxalates, dioxetanes or GFP and their analogs); phosphorescent labels; metal chelators; and (other) enzyme labels (e.g. peroxidase, alkaline phosphatase, β-galactosidase, urease or glucose oxidase).

[0341] Binding agents (particularly antibodies and antibody fragments) as described herein and comprising a detectable moiety can be used, for example, in in vitro, in vivo or in situ assays (including immunoassays known per se, such as ELISA, RIA, EIA and other "sandwich assays", etc.), as well as for in vivo imaging purposes, depending on the choice of the particular label.

[0342] Another aspect relates to a kit comprising a binding agent (particularly an antibody or antigen-binding fragment) as described herein, a nucleic acid encoding the binding agent, a vector comprising such a nucleic acid, or a composition comprising a binding agent (particularly an antibody or antigen-binding fragment) as described herein, or a pharmaceutical composition comprising a binding agent (particularly an antibody or antigen-binding fragment), a nucleic acid encoding the binding agent, a vector comprising such a nucleic acid and / or a composition comprising a binding agent (particularly an antibody or antigen-binding fragment) as described herein.

[0343] Such a kit may be a pharmaceutical kit or a medicament kit comprising a container or vial (any suitable container or vial, such as a pharmaceutically acceptable container or vial) containing an amount of a binding agent (particularly an antibody or antigen-binding fragment) as described herein, or a nucleic acid encoding the binding agent, or a vector comprising such a nucleic acid, or a composition comprising a binding agent (particularly an antibody or antigen-binding fragment) as described herein, and further comprising, for example, a kit insert such as a medical leaflet or packaging leaflet containing information about, for example, the intended indication (prophylactic or therapeutic treatment of Sabeivirus infection) and potential side effects. The pharmaceutical kit or medicament kit may further comprise, for example, a syringe for administering a binding agent (particularly an antibody or antigen-binding fragment) as described herein, a nucleic acid encoding the binding agent, a vector comprising such a nucleic acid, or a composition comprising a binding agent (particularly an antibody or antigen-binding fragment) as described herein to a subject.

[0344] Such kits may also be diagnostic kits, which include a container or vial (any suitable container or vial, such as a pharmaceutically acceptable container or vial) containing an amount of a binding agent (particularly an antibody or antigen-binding fragment) as described herein, such as a binding agent (particularly an antibody or antigen-binding fragment) comprising a detectable portion. Such diagnostic kits may further include, for example, one or more reagents to detect the detectable portion and / or instructions, for example, on how to use the binding agent (particularly an antibody or antigen-binding fragment) to detect Sabei virus in a sample.

[0345] Although the present invention has been described in conjunction with its specific embodiments, it is apparent that many substitutions, modifications and variations will be apparent to those skilled in the art based on the foregoing description. Therefore, it is intended to encompass all such substitutions, modifications and variations as follows within the spirit and broad scope of the appended claims.

[0346] Certain aspects and embodiments of the invention are set forth in the following numbered statements:

[0347] (1) A binding agent capable of neutralizing Sabei virus, characterized in that the binding agent specifically binds to the region of the heptad repeat 2 (HR2) domain of the Sabei virus spike protein close to the viral membrane.

[0348] (2) The binding agent according to (1), wherein the binding agent specifically binds to or to a region of the HR2 domain, which region is located from amino acid 11179 to amino acid E1202, preferably from amino acid D1184 to amino acid E1202, and more preferably from amino acid V1189 to amino acid E1202 of the SARS-CoV-2 spike protein defined by SEQ ID NO: 86.

[0349] (3) The binding agent according to (1) or (2), wherein the binding agent specifically binds to a region of the HR2 domain corresponding to the region from amino acid N1192 to amino acid Q1201 of the SARS-CoV-2 spike protein defined by SEQ ID NO: 86.

[0350] (4) The binding agent according to any one of (1) to (3), wherein:

[0351] - The binding agent is capable of neutralizing Sabei virus, wherein the 50% inhibitory concentration (IC 50 ) is 100 ng / ml or less, preferably 10 ng / ml or less, more preferably 1 ng / ml or less, as determined in a Sabeivirus Spike protein pseudovirus neutralization assay (such as a vesicular stomatitis virus (VSV)-Sabeivirus Spike protein pseudovirus neutralization assay);

[0352] - The binding agent is capable of neutralizing any or both of SARS-CoV-2, such as one or more of the SARS-CoV-2 Wuhan-Hu-1 strain, the SARS-CoV-2 Alpha variant, the SARS-CoV-2 Omicron BA.1 variant, and the SARS-CoV-2 Omicron BA.2 variant; and SARS-CoV-1;

[0353] - the binding agent is capable of inhibiting spike protein-mediated syncytium formation between cells expressing the Sabei virus spike protein and cells expressing angiotensin converting enzyme 2 (ACE2) receptor; and / or

[0354] - The binding agent does not bind to Middle East Respiratory Syndrome Coronavirus (MERS-CoV).

[0355] (5) The binding agent according to any one of (1) to (4), which comprises or consists of an antibody or an antibody fragment.

[0356] (6) The binding agent according to any one of (1) to (5), comprising an immunoglobulin single variable domain (ISVD), preferably VHH.

[0357] (7) The binding agent according to (6), wherein the ISVD comprises a complementarity determining region 1 (CDR1) defined by any one of SEQ ID NO: 63, SEQ ID NO: 46, SEQ ID NO: 69 or SEQ ID NO: 77, a complementarity determining region 2 (CDR2) defined by any one of SEQ ID NO: 64, SEQ ID NO: 47, SEQ ID NO: 70, SEQ ID NO: 73 or SEQ ID NO: 78, and a complementarity determining region 3 (CDR3) defined by any one of SEQ ID NO: 48, SEQ ID NO: 67, SEQ ID NO: 74 or SEQ ID NO: 79; preferably a CDR1 defined by any one of SEQ ID NO: 65, SEQ ID NO: 71, SEQ ID NO: 49 or SEQ ID NO: 80, a CDR2 defined by any one of SEQ ID NO: 66, SEQ ID NO: 72, SEQ ID NO: 50, SEQ ID NO: 75 or SEQ ID NO: 81, and a CDR3 defined by any one of SEQ ID NO: 51, SEQ ID NO: 52. The CDR3 is defined by any one of SEQ ID NO:68, SEQ ID NO:76 or SEQ ID NO:82.

[0358] (8) The binding agent according to (6) or (7), wherein the ISVD comprises CDR1, CDR2 and CDR3, each independently present in any one of SEQ ID NO: 1 to SEQ ID NO: 10, wherein the CDR1, CDR2 and CDR3 are annotated according to any one of Kabat, MacCallum, IMGT, AbM, Martin or Chothia; preferably, wherein the ISVD comprises a combination of CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 are present in a specific sequence of the sequences listed by SEQ ID NO: 1 to SEQ ID NO: 10, wherein the CDR1, CDR2 and CDR3 are annotated according to any one of Kabat, MacCallum, IMGT, AbM, Martin or Chothia.

[0359] (9) The binding agent according to any one of (6) to (8), wherein the ISVD comprises a CDR1 defined by SEQ ID NO: 63, a CDR2 defined by SEQ ID NO: 64, and a CDR3 defined by SEQ ID NO: 48; preferably a CDR1 defined by SEQ ID NO: 65, a CDR2 defined by SEQ ID NO: 66, and a CDR3 defined by SEQ ID NO: 51; more preferably a CDR1 defined by any one of SEQ ID NOs: 52-54, a CDR2 defined by any one of SEQ ID NOs: 55-62, and a CDR3 defined by any one of SEQ ID NOs: 21-27.

[0360] (10) The binding agent according to any one of (7) to (9), wherein the ISVD comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 10.

[0361] (11) The binding agent according to any one of (1) to (10), comprising an ISVD comprising an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 10.

[0362] (12) The binding agent according to any one of (1) to (11), which is in a multivalent form, preferably wherein the binding agent comprises an ISVD fused to an Fc domain.

[0363] (13) A nucleic acid molecule comprising a polynucleotide sequence encoding the binding agent according to any one of (1) to (12); a vector comprising the nucleic acid molecule; or a cell expressing the binding agent according to any one of (1) to (12) or comprising the nucleic acid molecule or the vector.

[0364] (14) A pharmaceutical composition comprising the binding agent according to any one of (1) to (12), the nucleic acid molecule according to (13) or the vector according to (13), and a pharmaceutically acceptable carrier; or a kit, such as a diagnostic kit, comprising the binding agent according to any one of (1) to (12).

[0365] (15) The binding agent according to any one of (1) to (12), the nucleic acid molecule according to (13), the vector according to (13), the pharmaceutical composition according to (14) or the kit according to (14) for pharmaceutical use; preferably for preventing or treating Sabeivirus infection in a subject, or for diagnosing Sabeivirus infection in a subject.

[0366] (16) An in vitro or ex vivo method for detecting Sabei virus in a sample, the method comprising:

[0367] - contacting the sample with a binding agent according to any one of (1) to (12), and

[0368] - Determining the binding of said binding agent to Sabeivirus or a part thereof.

[0369]

[00136] The aspects and embodiments of the invention disclosed herein are further supported by the following non-limiting examples.

[0370] Example

[0371] Materials and methods

[0372] Isolation of SARS-CoV-2 VHH phage

[0373] To obtain SARS-CoV-1 and SARS-CoV-2 cross-reactive VHHs, llamas previously immunized with recombinant prefusion stabilized SARS-CoV-1 and MERS-CoV spike proteins were additionally immunized three times with their prefusion stabilized recombinant SARS-CoV-2 spike proteins (S-2P) (Wrapp, D. et al., (2020) "Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies." Cell 181: 1004-1015.e15; Wrapp et al., (2020) "Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation." Science 367: 1260-1263). After immunization, peripheral blood lymphocytes were isolated from llamas, and immune VHH display phagemid libraries were constructed. Phages displaying SARS-CoV-2 specific VHHs were enriched from the phage library by two rounds of biopanning against 100 ng of His-tagged SARS-CoV-2 spike 6P protein (Hsieh et al., (2020) "Structure-based design of prefusion-stabilized SARS-CoV-2 spikes." Science 369: 1 501-1 505), which was immobilized on the wells of a microtiter plate (type II, F96 Maxisorp, Nunc) via coated anti-His antibodies in the presence of 1 0 μg / ml RBD-SD 1-mouse IgG (Sionobiological). In addition, two additional rounds of biopanning were performed using anti-His captured spike proteins (R3_C and R4_C series) or directly coated spike proteins (R3_DC and R4_DC series). In addition, in additional rounds of both series, biopanning was performed in the presence of 10 μg / ml RBD-SD1-mouse IgG (Sionobiological). For each round of panning, uncoated wells were used as negative controls.The wells were then washed 5 times with phosphate buffered saline (PBS) + 0.05% Tween 20 in the first round of panning and blocked with 4% milk powder in PBS SEA BLOCK blocking buffer (Thermo Scientific), blocked with Pierce protein-free blocking buffer (Thermo Scientific) in the second round of panning, blocked with SEABLOCK blocking buffer (Thermo Scientific) in the third round of panning, and blocked with 1% BSA in the fourth round of panning. Phages that were non-specifically bound were removed by washing thoroughly with PBS + 0.05% Tween 20. The retained phages were eluted with TEA solution (14% trimethylamine (Sigma), pH 10) and then neutralized with 1M Tris-HCl (pH 8). The collected phages were amplified in exponentially growing E. coli TG1 cells, infected with VCS M13 helper phages, and then purified using PEG 8,000 / NaCl precipitation for the next round of selection. Enrichment after each round of panning was determined by infecting TG1 cells with 10-fold serial dilutions of collected phages, and then plating the bacteria on LB agar plates containing 100 μg / mL ampicillin and 1% glucose.

[0374] Preparation of periplasmic extract (PE)

[0375] After 3 or 4 rounds of panning, a single colony of phage-infected bacteria was randomly selected for further analysis. A single colony was inoculated in a 24-well deep-well plate in a 2mL super broth (TB) medium containing 100 μg / mL ampicillin. After culturing a single colony at 37°C for 5 hours, isopropyl β-D-1-thiogalactoside (IPTG) (1mM) was added to induce VHH expression during overnight incubation at 37°C. In order to prepare periplasmic extracts, bacterial cells were precipitated and resuspended in 250 μL TES buffer (0.2M Tris-HCl pH 8, 0.5mM EDTA, 0.5M sucrose), and incubated at 4°C for 30 minutes. 350 μL of water was subsequently added to induce osmotic shock. After incubation at 4°C for 1 hour, centrifugation was subsequently performed to collect periplasmic extracts.

[0376] Periplasmic extract-ELISA

[0377] The wells of a half-well microtiter plate were coated with 50 ng of recombinant SARS-CoV-2 S-2P protein, SARS-CoV-2S2 subunit (Biopsy, S2N-C52H5), SARS-CoV-2RBD (Sino-Biologics), SARS-CoV S, MERS-CoV S, HKU1S, and BSA at 4°C overnight. The plate was blocked with a 5% milk powder in PBS solution. The periplasmic extract was diluted 1 / 10 in PBS and then added to the blocked wells. The binding of VHH was detected with mouse anti-HA antibody (BioLegend901501, 1 / 2000), followed by anti-mouse IgG-HRP (GE Healthcare, NA931V, 1 / 2000). After washing, 50 μl of TMB substrate (tetramethylbenzidine, BD OptEIA) was added to the plate, and the reaction was stopped by adding 50 μl of 1M H2SO4. The absorbance at 450 nm was measured using an iMark microplate absorbance spectrophotometer (Bio-Rad). Curve fitting was performed using nonlinear regression (Graphpad 8.0).

[0378] Periplasmic extract-pseudovirus neutralization assay

[0379] The pseudovirus expressing the SARS-CoV-2 spike protein (D614G) was incubated at 37°C for 30 minutes with a 1 / 100 dilution of the periplasmic extract in Fluorobrite DMEM medium (Invitrogen), and supplemented with 5% heat-inactivated FBS, 1% penicillin, 1% streptomycin, 2mM L-glutamine, non-essential amino acids (Invitrogen) and 1mM sodium pyruvate. The incubated pseudovirus was then added to the sub-confluent monolayer of Vero E6, and the initial growth medium was removed therefrom. Sixteen hours later, the cells were lysed using passive lysis buffer (Promega). The transduction efficiency was quantified by measuring the GFP fluorescence value in the prepared cell lysate using a Tecan infinite 200 pro microplate reader. The GFP fluorescence value was standardized using the GFP fluorescence value of uninfected cells and infected cells treated with PBS.

[0380] Cell lines

[0381] FreeStyle293F cells (Thermo Fisher Scientific) and HEK293-S cells (Thermo Fisher Scientific) were cultured in FreeStyle 293 Expression Medium (Life Technologies) at 37°C, 8% CO2, and 130 rpm. HEK293-T cells (ATCC) and Vero E6 cells (ATCC) were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin, 1% streptomycin, 2 mM l-glutamine, non-essential amino acids (Invitrogen), and 1 mM sodium pyruvate at 37°C and 5% CO2. ExpiCHO-S cells (GIBCO) were cultured in ExpiCHO Expression Medium (GIBCO) at 37°C, 8% CO2, and 130 rpm. Vero E6-TMPRSS2 cells (NIBIOHN, JCRB1819) stably expressing human TMPRSS2 (Matsuyama et al., PNAS, 2020) were cultured in DMEM containing 10% FBS, penicillin (100 units / mL), streptomycin (100ug / mL), and geneticin (G418) (1mg / ml). When Vero E6-TMPRSS2 cells were inoculated for assay, medium without geneticin was used.

[0382] Raji cells and Raji cells stably expressing the SARS-CoV-2 spike protein were cultured in RPMI-1640 medium supplemented with 10% FCS, 0.1 μg / ml puromycin, 1% penicillin and 1% streptomycin at 37°C, 5% CO2.

[0383] Sotovir, cegavir, bemecticomab, and palivizumab

[0384] Biosimilar bebectinomab (PX-TA1750), biosimilar celgavir (PX-TA1033), and biosimilar sotovir (PX-TA1637) were all commercially available from Proteogenix. Clinical grade palivizumab was obtained from Ghent University hospital.

[0385] Generation of R3_DC23-Fc(YTE) (also referred to herein as huR3DC23-Fc) (SEQ ID NO: 96)

[0386] The humanized (Q1D, Q5V, A14P, D16G, T19R, M63V, S73N, D79Y, T82cL, K83R and Q108L, according to Kabat numbering; the T82cL modification is particularly useful for inactivating glycosylation at position N82a and can be used in both humanized and non-humanized versions for expression in mammalian cells) version of R3_DC23 was fused to human IgG1 Fc (EPKSCdel_YTE_K447del) ordered as gBlock at IDT Synthesis via a (G4S)2 linker. Once obtained, gBlock was dissolved in ultrapure water at a concentration of 20 ng / μL. gBlock was A-tailed using the NEBNext dA-tailing module (NEB), purified using CleanPCR magnetic beads (CleanNA), and inserted into the pcDNA3.4-TOPO vector (Thermo Fisher Scientific). The ORFs of positive clones were completely sequenced, and pDNA of selected clones was prepared using the NucleoBond Xtra Midi Kit (Machery-Nagel).

[0387] huR3DC23-Fc_LS (also referred to herein as R3_DC23hum-Fc(LS) or XVR013) (SEQ ID NO: 118) Instantaneous generation

[0388] The gene encoding huR3DC23-Fc_LS was codon optimized, synthesized and cloned into the pXLG6 backbone vector in the ATUM laboratory. After gene and codon optimization, the R3DC23 DNA sequence was inserted into the pXLG6 expression vector and transfected into the CHOExpress at a cell density of 4.00E+6 cells / ml. TM The TGE supernatant was harvested by centrifugation and clarified by filtration (0.2 μm) after 10 days when the cell viability dropped below 10%. The protein was further purified by Protein A.

[0389] Produces VHH73S56A, GBP, CB6 and S309

[0390] Production of VHH73_S56A, GBP, CB6 and S309 was performed as described by Schepens et al. (Schepens et al., (2021) Sci. Transl Med. 13:eabi7826).

[0391] HEK S transfection and protein purification protocol; production of the YTE variant of VHH-Fc in mammalian cells

[0392] HEK293-S cells were transfected with VHH-Fc(S) encoding plasmid using polyethyleneimine (PEI). Briefly, suspension-adapted, serum-free HEK293-S cells were cultured at 3 × 10 6Cells / mL were inoculated in FreeStyle 293 medium (Thermo Fisher Scientific). Next, 4.5 μg of pcDNA3.3-VHH-Fc plasmid DNA was added to the cells and incubated for 5 minutes on a shaking platform at 37°C and 8% CO2. Next, 9 μg of PEI was added to the culture and the cells were further incubated for 5 hours, after which an equal culture volume of Ex-Cell-293 (Sigma) was added. The transfectants were incubated for 4 days, after which the cells were pelleted (10′, 300 g) and the supernatant was filtered before further use. To purify the VHH-Fc protein, the supernatant was loaded onto a 5 mL MAbSelectSuRe column (GE Healthcare). Unbound proteins were washed away using McIlvaine buffer (pH 7.2), and bound proteins were eluted using McIlvaine buffer (pH 3). Immediately after elution, the protein-containing fraction was neutralized with 30% (v / v) saturated Na3PO4 buffer. Next, the fractions were pooled and loaded onto a HiPrep desalting column for buffer exchange into PBS (pH 7.4).

[0393] In addition, huR3DC23-Fc_YTE was cultured in ExpiCHO-S according to the manufacturer's protocol. TM cells (Thermo Fisher Scientific). Briefly, ExpiFectamine TM CHO reagent was used to transfect 6×10 6 One day after transfection, add 300 μL of ExpiCHO TM Enhancer and 8mL ExpiCHO TM Feed and further incubate at 32°C and 5% CO2. Feed the cells a second time on day 5 post-transfection. Once cell viability drops below 75%, the product is harvested.

[0394] To purify the VHH-Fc protein, the supernatant was loaded onto a 5 mL MAbSelect SuRe column (GE Healthcare). Unbound proteins were washed off using McIlvaine buffer (pH 7.2), and bound proteins were eluted using McIlvaine buffer (pH 3). Immediately after elution, the protein-containing fractions were neutralized with 30% (v / v) saturated Na3PO4 buffer. Next, these fractions were combined and loaded onto a HiPrep desalting column for buffer exchange to PBS (pH 7.4).

[0395] huR3DC23-Fc_LS was produced in 1L scale from steady state to fixed pool feed batches

[0396] The gene encoding huR3DC23-Fc LS was codon-optimized, synthesized, and cloned into the pXLG6 backbone vector in the ATUM laboratory. 6 When the expression vector and pXLG5 auxiliary vector were co-transfected into the parent CHOExpress TM Cells. Stable pools were generated and further expanded under 50 mg / L puromycin selection pressure (applied daily). Stable pool research cell banks were stored on day 14 when cell viability reached 95%.

[0397] The RCB pool was then expanded for 1L scale protein production and cultured until day 12 (cell density 3.5 × 10 7 Cells / mL, cell viability 96%). The supernatant was harvested by centrifugation and clarified by filtration (0.2 μm). The protein was further purified by ProteinA using MabSelectSuRe LX resin. Continuous washing was performed with 20 mM sodium phosphate and 110 mM NaCl (pH 7.2), 100 mM sodium acetate and 500 mM NaCl (pH 5.5), and 20 mM sodium phosphate (pH 7.2). The eluate in 100 mM sodium acetate (pH 3.5) was neutralized to pH 7.0 by adding 1 M Tris (pH 11, 10% v / v). After filtration sterilization (0.22 μm), the protein was aliquoted at 2 mg / ml.

[0398] Protein preparation for biophysical analysis

[0399] Prior to biophysical characterization, MAbSelect SuRe purified protein samples were further purified by size exclusion chromatography (SEC) on a Superdex 200 column (GE Healthcare) cooled at 12° C., equilibrated with Dulbecco's phosphate buffered saline (PBS, Sigma-Aldrich) supplemented with 0.02% sodium azide to prevent microbial growth, or equilibrated with sample buffer containing 50 mM L-histidine and 150 mM L-arginine (Sigma-Aldrich), 0.02% polysorbate-20, and 0.02% sodium azide, set to pH 7.0 at 25° C. After filter sterilization (0.22 μm), 1 mg / ml aliquots were snap-frozen in liquid nitrogen in polypropylene tubes and stored at 80° C.

[0400] Sample composition

[0401] Purified VHH-Fc samples were characterized by analytical SEC to determine the molecular composition of each sample. After rapid thawing in a warm water bath at 25°C, centrifugation was performed at 16,000 x g for 10 min, and the supernatant was transferred to a new tube. 5 μg was injected into a 4.6 × 300 mm AdvanceBio SEC column (Agilent) with a porous particle size of 2.7 μm and a pore size of Calibration was performed with PBS. Separation was monitored by absorbance at 280 nm in a 1 6 nm bandwidth without reference subtraction. For additional quality control, proteins were separated on reducing 15% SDS-PAGE and stained with Coomassie Brilliant Blue.

[0402] Generate spike protein expression vectors for production of spike proteins expressing RBD mutations containing SARS-CoV-2 variants White VSVdelG pseudovirion

[0403] The pCG1 expression vector of the SARS-CoV-2 spike protein containing the D614G mutation was generated from the pCG1-SARS-2-Sdel18 vector by introducing specific RBD mutations via QuickChange mutagenesis using appropriate primers according to the manufacturer's instructions (Aligent). For the pCG1-SARS-2-Sdel18 expression vector of the Omicron BA.1 variant, the pCG1-SARS-2-Sdel18 vector containing the BA.1 mutations (A67V, Δ69-70, T95I, G142D, Δ143-145, N211I, Δ212, ins215EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496 The spike protein nucleotide sequence was optimized with codons flanking BamHI and SalI restriction sites and cloned into the pCG1 vector as a BamHI / SalI fragment. For the pCG1-SARS-2-BA.2Sdel18 expression vector, the nucleotide sequence was purified by Geneart (Thermo Fischer-Weiss, Germany). The codon-optimized spike protein nucleotide sequence containing BA.2 mutations (T19I, ΔL14-P26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K) and flanking BamHI and SalI restriction sites was ordered from Genentech Scientific and cloned into the pCG1 vector as a BamHI / SalI fragment. For the pCG1-SARS-2-BA.2.75Sdel18 expression vector, the codon-optimized spike protein nucleotide sequence containing the BA.2.75 mutations (K147E, W152R, F157L, 1210V, G257S, D339H, G446S, N460K, R493Q) and flanking BamHI and SalI restriction sites was ordered from Geneart (Thermo Fischer Scientific) and cloned into the pCG1 vector as a BamHI / SalI fragment.The pCG1 expression vectors of the OmicronBA.2.75.2 variants were generated by introducing the R346T, F486S, D1199N mutations from pCG1-SARS-2-BA.2.75Sdel18 via Gibson assembly cloning using appropriate gBlocks (ordered from IDT) according to the manufacturer's instructions (New England BioLabs).

[0404] The pCG1 expression vectors of the Omicron BA.4 / BA.5 variants were generated by introducing the H69 deletion, V70 deletion and L452R, F486V, R493Q mutations from the pCG1-SARS-2-BA.2 Sdel18 vector via QuickChange mutagenesis using appropriate primers (ordered at IDT) according to the manufacturer's instructions (Agilent).

[0405] The pCG1 expression vector of the Omicron BA.4.6 variant was generated by introducing the R346T and N658S mutations from pCG1-SARS-2-BA.4Sdel18 via QuickChange mutagenesis using appropriate primers (ordered at IDT) according to the manufacturer's instructions (Agilent).

[0406] The pCG1 expression vector for the Omicron BF.7 variant was generated by introducing the R346T mutation from pCG1-SARS-2-BA.4Sdel18 via QuickChange mutagenesis using appropriate primers (ordered at IDT) according to the manufacturer's instructions (Agilent).

[0407] The pCG1 expression vectors for the Omicron BQ.1.1 variants were generated by introducing R346T, K444T and N460K mutations from pCG1-SARS-2-BA.5Sdel18 via QuickChange mutagenesis using appropriate primers (ordered at IDT) according to the manufacturer's instructions (Agilent).

[0408] The pCG1 expression vectors for the Omicron XBB variants were generated from pCG1-SARS-2-BA.2 Sdel18 by introducing V83A, Y144, H146Q, Q183E, V213E, D339H, R346T, L368I, V445P, G446S, N460K, F486S, F490S, R493Q mutations by Gibson assembly cloning using appropriate gBlocks (ordered from IDT) according to the manufacturer's instructions (New England BioLabs). The pCG1 expression vectors for the Omicron XBB.1.5 variants were generated from pCG1-SARS-2-XBB Sdel18 by introducing the F486P mutation by QuickChange mutagenesis using appropriate primers (ordered from IDT) according to the manufacturer's instructions (Agilent).

[0409] After sequencing, clones containing the correct spike protein coding sequence were prepared using the Qiagen plasmid kit. Before use, the spike protein coding sequence of the prepared pCG1 vector was confirmed by Sanger sequencing.

[0410] Hydrophobic interaction chromatography (HIC) assay

[0411] Apparent hydrophobicity was assessed using a hydrophobic interaction chromatography (HIC) assay using a 100 mm × 4.6 mm Dionex ProPac HIC-10 column (Thermo Fisher 063655) containing a stationary phase consisting of mixed ethyl and amide functional groups bound to silica. All separations were performed on an Agilent 1100 / 1260 HPLC equipped with a UV / VIS detector. The column temperature was maintained at 25°C and the flow rate was 0.8 ml / min throughout the run. The mobile phases used for HIC were (A) 1.6 M ammonium sulfate and 50 mM phosphate (pH 7.0), and (B) 50 mM phosphate (pH 7.0). Protein and calibrant sample A were diluted 1:1 with buffer and injected into the column. After holding at 50% B for 5 minutes, bound proteins were eluted using a linear gradient from 50% B to 100% B over 50 minutes, followed by a 5-minute hold at 100% B. The column was washed with 100% B, then 50 mM ammonium acetate pH 5, and re-equilibrated in 50% B for 10 min before the next sample was added. The separation was monitored by absorbance at 280 nm in a 16 nm bandwidth without reference subtraction.

[0412] Mass spectrometry analysis of proteins.

[0413] The intact VHH protein (10 μg) was first reduced with tris(2-carboxyethyl)phosphine (TCEP; 10 mM) at 37°C for 30 min, and the reduced protein was then separated on an Ultimate 3000 HPLC system (Thermo Fisher Scientific, Bremen, Germany) connected online to a LTQ Orbitrap XL mass spectrometer (Thermo Fischer Scientific). Briefly, approximately 8 μg of protein was injected onto a Zorbax 300SB-C18 column (5 μm, 1×250mmIDxL; Agilent) and separated using a 30-min gradient from 5% to 80% solvent B at a flow rate of 100 μl / min (solvent A: 0.1% formic acid and 0.05% trifluoroacetic acid in water; solvent B: 0.1% formic acid and 0.05% trifluoroacetic acid in acetonitrile). The column temperature was maintained at 60°C. The eluted proteins were directly sprayed into a mass spectrometer with an ESI source using the following parameters: spray voltage of 4.2 kV, surface induced dissociation of 30 V, capillary temperature of 325°C, capillary voltage of 35 V, and sheath gas flow rate of 7 (arbitrary units). The mass spectrometer was operated in MS1 ​​mode using an orbitrap analyzer with a resolution of 100,000 (m / z 400), a mass range of 600-4000 m / z, and in profile mode. The obtained MS spectra were deconvoluted using BioPharma Finder™ 3.0 software (Thermo Fischer Scientific) with the Xtract deconvolution algorithm (isotope resolved spectroscopy). The deconvoluted spectra were annotated manually.

[0414] Production of VHH by E. coli

[0415] In order to produce VHH in E. coli, the pMECS vector containing the VHH of interest was transformed into WK6 cells (non-inhibitory E. coli strain) and plated on LB plates containing ampicillin. The next day, clones were picked and grown overnight at 37°C in 2mL LB containing 100 μg / mL ampicillin and 1% glucose, while shaking at 200rpm. 25ml of TB (super broth) supplemented with 100 μg / ml ampicillin, 2mMMgCl and 0.1% glucose was inoculated with 1ml of this pre-culture, and incubated at 37°C with shaking (200rpm-250rpm) until an OD600 of 0.6-0.9 was reached. VHH was produced by adding IPTG to a final concentration of 1mM. These induced cultures were incubated overnight at 28°C, while shaking at 200rpm. The VHH produced was extracted from the periplasm and purified as described by Wrapp et al. (2020. Cell 181: 1004-1015.e15). Briefly, VHHs were purified from solution using Ni agarose beads (GE Healthcare). After elution with 500 mM imidazole, the flow-through fraction containing VHHs was buffer exchanged with PBS using a Vivaspin column (5 kDa cutoff, GE Healthcare). The purified VHHs were analyzed by SDS-PAGE and Coomassie staining as well as by intact mass spectrometry.

[0416] ELISA

[0417] Wells of microtiter plates (Type II, F96 Maxisorp, Nunc) were coated with 100 ng of recombinant SARS-CoV S-6P protein (Hsieh et al., 2020), SARS-CoV-1 S-2P protein (with foldon), His-tagged SARS-CoV-2 RBD (Sino-Bio), SARS-CoV-2 spike protein S2 subunit (Biopsy), recombinant SARS-Cov-2 S-2P, SARS-CoV-2 S-6P protein, recombinant SARS-CoV-1 spike protein, recombinant MERS-CoV spike protein, recombinant HKU1 spike protein, SARS-CoV-2 Omicron BA.1 S protein (Biopsy), mouse Fc-tagged SARS-CoV-2 RBD (Sino-Bio), or BSA overnight at 4°C. The coated plates were blocked with 5% milk powder in PBS. Serial dilutions of VHH or VHH-Fc or antibodies were added to the wells and incubated for 90 minutes. After washing, the plates were incubated with HRP-conjugated rabbit anti-camelid VHH antibody (Genscript) or mouse anti-HA antibody (BioLegend901501, 1 / 2000) and then anti-mouse IgG-HRP (GE Healthcare, NA931V, 1 / 2000) to detect binding. Binding of VHH-Fc or conventional human monoclonal antibodies was detected by rabbit anti-human IgG (Sigma) and then by HRP-conjugated anti-rabbit IgG (Southern Biotech). After washing, 50 μL of TMB substrate (tetramethylbenzidine, BD OptETA) was added to the plate and the reaction was stopped by adding 50 μL of 1M H2SO4. The absorbance at 450 nM was measured with an iMark microplate absorbance spectrophotometer (Bio-Rad). Curve fitting was performed using nonlinear regression (Graphpad 8.0).

[0418] Flow cytometric analysis of binding to HEK293 cells expressing SARS-CoV spike protein

[0419] To study the binding of VHHs to the spike protein on the surface of mammalian cells by flow cytometry, we used the pCG1 expression plasmid containing the SARS-CoV-2 spike protein coding sequence, in which the C-terminal 18 amino acids were deleted and the D614G substitution (614G) was introduced by QuickChange site-directed mutagenesis (Agilent) according to the manufacturer's instructions. Two days after HEK293-T cells were transfected with a combination of GFP expression plasmid and spike protein expression plasmid or control expression plasmid, the cells were collected. All further procedures were performed on ice. Cells were washed once with PBS and blocked with 1% BSA. Cells were stained with antibodies or VHH serial dilutions for 90 minutes and subsequently washed three times with PBS containing 1% BSA. The binding of VHHs was detected using mouse anti-His tag antibody (Bio-Rad) and AF647 conjugated donkey anti-mouse IgG antibody (Invitrogen). Donkey anti-human IgG antibody (Bio-Rad) was used to detect the binding of VHH-Fc or antibodies, and Live / Dead stain (Bio-Rad) was used to stain dead cells. After washing three times with PBS containing 0.5% BSA, cells were analyzed by flow cytometry using a BD LSRII flow cytometer (BD Biosciences). Binding curves were fitted using nonlinear regression (Graphpad 8.0).

[0420] FcRn binding affinity

[0421] Surface plasmon resonance (SPR) analysis of antibody binding to purified recombinant human FcRn / FCGRT-B2M protein was performed by FairJourney Biologics (Porto, Portugal) on a Biacore 8K+ instrument. Recombinant human FcRn / FCGRT-B2M heterodimer protein with a His tag was purchased from Biopsys. Biosimilar bebetelomonab and human IgG1 isotype control antibody were used as controls in the assay. Briefly, R3_DC23hum-Fc (LS) or control antibody was immobilized on a CM5 sensor chip (Cytiva) at low density by amine coupling, with an immobilization level of 130RU to 283RU. At pH 6.0, human FcRn / FCGRT-B2M heterodimer protein was injected into the solution at 1.5 μM (anchor point), and eight steps of 2-fold gradient dilution were performed in the range of 1000nM-7.8nM for the control antibody immobilized channel, and nine steps of 2-fold gradient dilution were performed in the range of 250nM-0.97nM for the R3 DC23hum-Fc (LS) immobilized channel. At pH 7.4, human FcRn / FCGRT-B2M heterodimer protein was injected into the solution at 1.5 μM (anchor point), and eight steps of 2-fold gradient dilution were performed in the range of 1000nM-7.8nM for all immobilized channels. Analyte injection was performed at 30 μl / min in running buffer for 1 minute; the measurement run at 0nM concentration was included as a blank reference. The SPR running buffer contained 1xPBS and 0.05Tween20 at pH 6.0 / pH 7.4. A multi-cycle kinetic protocol was applied (90 s off-rate measurement); data obtained from antibody or VHH-Fc concentrations above 250 nM were omitted due to volume effects. After double reference subtraction, data were analyzed using the steady-state affinity predefined evaluation method of the Biacore Insight evaluation software or by fitting a 1:1 binding model in the same software.

[0422] Human plasma membrane protein cell array

[0423] Retrogenix cell microarray technology (Charles River Laboratories, UK) was used to screen the specific off-target binding interactions of the test antibodies. First, a pre-screening was performed to determine the background binding level of each test antibody to fixed untransfected HEK293 cells and cells covered with SARS-CoV-2FL spike 6-HIS protein. These data are used to assess the suitability and optimal concentration for further screening. Secondly, in the library screening, the test antibody pool was screened for binding to fixed HEK293 cells, which overexpressed 6101 individual full-length human plasma membrane proteins, secreted and cell surface bound human secretory proteins, and additional 396 human heterodimers. This determined the library interactions. Finally, in the confirmation screening, all library interactions were re-expressed and probed with test antibodies alone or in control treatment to determine which interactions, if any, were repeatable and specific for each test antibody. This was performed on both fixed cells and living cells.

[0424] For prescreening, slides were spotted with expression vectors encoding ZsGreen1 and human CD20 or EGFR and used to reverse transfect HEK293 cells. The slides were fixed and subsequently spotted with gelatin + / - SARS-CoV-2 spike protein (SARS-CoV-2FL spike 6-HIS protein provided by PeakProteins, spotted at 0.2mg / mL). After fixation, 1μg / mL, 2.5μg / mL, or 10μg / mL R3_DC23hum-Fc (LS) and 1μg / mL rituximab biosimilar or PBS alone were added to the above cells / slides. Antibodies were detected using AlexaFluor 647-labeled anti-human IgG Fc (AF647 anti-hIgGFc), followed by fluorescence imaging to assess binding to target-expressing and untransfected cells. For library screening, 6101 expression vectors (encoding ZsGreen1 and full-length human plasma membrane proteins, secreted or cell surface-bound human secretory proteins) and an additional 396 human heterodimers were individually arrayed in duplicate on a cell microarray slide. Subsequently, gelatin + / - SARS-CoV-2 spike protein (0.2 mg / mL) was spotted on another slide. Expression vectors (pIRES-hEGFR-IRES-ZsGreen1) were spotted in quadruplicate on each slide to ensure that the minimum threshold of transfection efficiency was reached or exceeded on each slide. Reverse transfection / expression was performed using human HEK293 cells. After cell fixation, the test antibody pool was added to each slide. Binding detection was performed using the same fluorescent secondary antibody (AF647 anti-hIgG Fc) used in the pre-screening. The test antibody pool was screened for 2 replicate slide groups. Fluorescent images (for transfection) were analyzed and quantified using ImageQuant software (GE Healthcare, version 8.2). Protein interactions were defined as replicate spots showing an increase in signal compared to background levels. This was achieved by visual inspection. Depending on the intensity of the replicate spots, interactions were classified as “strong, moderate, weak, or very weak”. Significant interactions were defined as signals of weak or greater intensity. For confirmation screening, vectors encoding all interactions identified in the library screen, as well as control vectors encoding CD20 (positive control) and EGFR (transfection and negative control), were arrayed and expressed in HEK293 cells on new slides. Confirmation screening slides and analysis of library screening were performed after cell fixation (n=2) or without fixation (n=1). Gelatin + / - SARS-CoV-2 spike protein (0.2mg / mL) was also spotted on fixed slides. Slides were treated with 2.5μg / mL R3_DC23hum-Fc (LS) or 20μg / mL IgGkappa antibody (negative control), 1μg / mL rituximab biosimilar (array positive control), or no test molecule (secondary antibody only; negative control).Binding to target-expressing and untransfected cells was again assessed by fluorescence imaging.

[0425] SARS-CoV pseudovirus neutralization assay

[0426] To generate replication-defective VSV pseudotyped viruses, HEK293-T cells transfected with SARS-CoV-1 S or SARS-CoV-2 S were inoculated with replication-defective VSV vectors containing eGFP and firefly luciferase expression cassettes (Berger and Zimmer 2011, PloS One 6: e25858 and Hoffmann et al., (2020) Cell 181: 271-280.e8). After incubation at 37°C for 1 hour, the inoculum was removed, the cells were washed with PBS, and incubated in medium supplemented with anti-VSV G mAb (ATCC) for 16 hours. The pseudotyped particles were then harvested and clarified by centrifugation.

[0427] For VSV pseudotype neutralization experiments, pseudoviruses were incubated with different dilutions of purified VHH or VHH-Fc fusions or with GFP binding protein (GBP: VHH specific for GFP) at 37°C for 30 minutes. The incubated pseudoviruses were then added to subconfluent monolayers of Vero E6 or Vero E6-TMPRSS2 cells. Sixteen hours later, the cells were lysed using passive lysis buffer (Promega). The transduction efficiency was quantified by measuring the GFP fluorescence values ​​in the prepared cell lysates using a Tecan infinite 200pro microplate reader. GFP fluorescence was normalized with the GFP fluorescence of uninfected cells and PBS-treated infected cells or the lowest and highest GFP fluorescence values ​​of each serial dilution. IC 50 Calculated by nonlinear regression curve fitting (log(inhibitor) vs. response) (four parameters).

[0428] Alternatively, a gradient dilution of VHH or antibody was mixed with 100 PFU of GFP-expressing, replication-competent VSV virus particles pseudotyped with the SARS-CoV-2 spike protein derived from an early isolate. It is noteworthy that during the propagation of this viral clone (S1-10a) on Vero E6 cells, the furin protease cleavage site mutated and was therefore inactivated (Koenig et al., (2021) Science 371:eabe6230). After incubation at 37°C for 30 minutes, the virus-antibody mixture was added to the Vero E6 cell monolayer and allowed to infect and replicate for three days.

[0429] In all neutralization assays using pseudotyped VSV viral particles, VHH or antibody-virus mixtures were prepared using FluoroBrite DMEM medium (Invitrogen) supplemented with 5% heat-inactivated FBS, 1% penicillin, 1% streptomycin, 2 mM l-glutamine, non-essential amino acids (Invitrogen) and 1 mM sodium pyruvate. The mixtures were added to the cells from which the original growth medium had been removed.

[0430] SARS-CoV-2 Plaque Reduction Neutralization Test (PRNT)

[0431] Plaque reduction assays using authentic viruses were performed using the SARS-CoV-2 strain SARS-CoV-2 / human / FRA / 702 / 2020 (obtained from the European Virus Archive (EVAG)) and the SARS-CoV-2 BA.1 virus (Planas et al., (2022) Nature 602: 671-675) and grown on Vero E6 cells. Further propagation of the virus was performed on Vero E6-TMPRSS2 cells.

[0432] Both viruses were titrated using a plaque assay, where Vero E6-TMPRSS2 cell monolayers were infected with serial dilutions prepared in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 2% fetal bovine serum (FBS), 2 vials per virus, in duplicate. Two hours after infection, Avicel was added to a final concentration of 0.3% (w / v).

[0433] The dose-dependent neutralization of different constructs was evaluated by mixing different concentrations (5-fold serial dilutions) of the constructs with 40 PFU of SARS-CoV-2 and incubating the mixture at 37°C for 1 hour. The VHH virus mixture was then added to a Vero E6-TMPRSS2 cell monolayer in a 12-well plate and incubated at 37°C for 1 hour. Subsequently, Avicel was added to a final concentration of 0.3% (w / v). After incubation at 37°C for 2 days, the overlay was removed, the cells were fixed with 3.7% paraformaldehyde (PFA), and stained with 0.5% crystal violet. The half-maximal neutralization titer (PRNT 50 ) was defined as the VHH-Fc concentration that resulted in a 50% reduction in plaques on two independent plates.

[0434] Live virus assay

[0435] Live virus assays were performed on SARS-CoV-2 viruses belonging to different lineages (614G, Delta, Omicron BA.1, Omicron BA.2, and Omicron BA.5) isolated from nasopharyngeal swabs obtained from patients / travelers between January 2020 and July 2022.

[0436] name Separation date WT SARS-CoV-2 isolate BavPat1 / 2020; Germany; February 9, 2020 January 1, 2020 Delta SARS-related coronavirus 2, isolate hCoV-19 / USA / MD-HP05647 / 2021 April 27, 2021 BA.1 SARS-CoV-2hCoV-19 / Netherlands / NH-RIVM-72291 / 2021 November 29, 2021 BA.2 Clinical isolate hCoV-19 / Netherlands / VCB-20220303-1 / 2022 March 3, 2022 BA.5 Clinical isolate hCov19 / NL / VCB-20220714-2 / 2022 July 14, 2022

[0437] Dose-dependent neutralization of test items, positive controls (bebetocumab biosimilar, segavir monoclonal antibody biosimilar, sotovir monoclonal antibody biosimilar), and negative controls (isotype controls) were evaluated in live virus neutralization assays. For all assays testing live D614G, Delta, BA.1, and BA.2 SARS-CoV-2, huR3DC23-Fc_LS produced by transiently transfected cells was used. For all assays testing SARS-CoV-2BA.5, huR3DC23_Fc_LS produced by stable cell pools was used. For each variant, three independent runs were performed. Different system controls were included in the assay: cells only (medium only), virus only, and internal positive control (human serum). Briefly, 5-fold or 7-fold dilutions of the test items and controls were incubated with a fixed amount of virus at room temperature for 1 hour.

[0438]

[0439]

[0440] Vero-E6 cell monolayers were inoculated with the virus-antibody cocktail for 1 hour at 37°C. Next, the inoculum was removed and the cells were incubated with infection medium (minimum essential medium (MEM) supplemented with 2mM L-glutamine, 1x non-essential amino acids, 25mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid), 1% heat-inactivated fetal bovine serum (FBS), and 1x antibiotic-antimycotic (Gibco)) at 37°C (up to 18-24 hours after infection). SARS-CoV2-infected cells were then fixed and immunostained with a SARS-CoV nucleocapsid antibody (Sino-Biological, catalog number: 40143-MM05), followed by immunostaining with an HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (Invitrogen, catalog number A16072). Use Plaques (infected cells) were counted on an S6 Ultimate Analyzer (CTL). For each antibody / construct, the concentration that reduced infection by 50% (IC 50 ) was calculated based on the Zielinska method (Zielinska et al., (2005) Virology Journal 2: 84). The geometric mean was calculated based on three independent runs. S1 shedding assay

[0441] The antibody or VHH was added to 1 million Raji cells that did not express the spike protein or expressed the SARS-CoV-2 spike protein at a final concentration of 10 μg / ml. The antibody / VHH cell mixture was incubated at 37°C and 5% CO2 for 30 minutes or 1 hour. After incubation, the cells were precipitated by centrifugation, the supernatant was transferred to a fresh tube, and the cell pellet was lysed with RIPA lysis buffer (50mMTris-HCl pH 8.0, 100mM NaCl, 1mM EDTA, 1mM EGTA, 0.1% SDS, 1% NP-40). 20 μμl supernatant and lysate samples were separated on 8% SDS-PAGE gels and electroblotted onto nitrocellulose membranes. The membrane was blocked with 4% milk, stained with rabbit anti-SARS-S1 antibody (1 / 1000, Sino Biological, 40591-T62), followed by anti-rabbit IgG-HRP (1 / 2000, GE Healthcare, NA934V), and stained using Pierce TM ECL Western blotting substrate (Thermo Fisher Scientific) was developed.

[0442] Use of SARS-CoV-2 Wuhan-Hu-1 spike protein (del-18) pseudotyped, replication-competent Fusion inhibition assay with VSV-GFP reporter virus

[0443] Vero E6-TMPRSS2 cells were infected with 40 PFU of GFP-expressing, replication-competent VSV-GFP virus pseudotyped with the SARS-CoV-2 spike protein (Koenig et al., 2021). Two hours later, the indicated monoclonal antibodies or VHHs were added. Uninfected cells were used as negative controls. The cells were infected overnight and imaged with a fluorescence microscope. GFP fluorescence values ​​were measured with a fluorimeter. It is noteworthy that, unlike the clones of replication-competent pseudotyped VSV particles used in the neutralization assay, the furin cleavage site of the virus used in the fusion assay had an intact furin cleavage site, which was confirmed by Sanger sequencing.

[0444] Using Vero expressing the spike protein Fusion inhibition assay with E6 cells

[0445] Using Fugene, Vero E6 cells were transfected with a GFP expression vector in combination with a control expression vector (no spike protein) or a SARS-CoV-2 spike protein expression vector. Two hours after transfection, PBS, monoclonal antibodies, or VHHs were added to a final concentration of 10 μg / ml. Twenty-two hours after transfection, cells were fixed with 3.7% paraformaldehyde and imaged using a fluorescence microscope after washing with PBS.

[0446] Virus escape selection

[0447] Vero E6-TMPRSS2 cell monolayers seeded in 96-well plates were infected with 400 PFU of GFP-expressing, replication-competent VSV virus particles pseudotyped with the SARS-CoV-2 spike protein containing an intact furin cleavage site (Koenig et al., 2021). Two hours after infection, 10 μg / ml VHH.R3_DC23 was added. No VHH was added to a control well.

[0448] The growth medium of wells showing syncytia formation or viral replication in the presence of VHH.R3_DC23 was collected and isolated by limiting dilution in the presence of 2 μg / ml VHH.R3_DC23 for single plaques of escaped viruses. The growth medium was used to propagate the virus on a monolayer of Vero E6-TMPRSS2 cells inoculated in a 6-well plate in the presence of VHH.R3_DC23. RNA was prepared from these infected cells using a nucleospin RNA virus kit (Macherey Nagel Bioanalysis), and cDNA was generated using random hexamer primers. The cDNA was used to amplify the spike protein S2 coding sequence by PCR. These PCR fragments were purified and sequenced using Sanger sequencing. The obtained nucleotide sequences were analyzed and compared with the spike proteins of WT SARS-CoV-2 and clades 1, 2, and 3 Sabei viruses using CLCMainWorkbench20.0.4 (Letko et al., (2020) NatureMicrobiology 5: 562-569). Mutations were observed on the full-length glycosylated spike protein model obtained from Charmm-gui.org (PDB: 6VXX_1_1_1 model) or the SARS-CoV-2 HR2 coiled-coil structure as determined by NMR using pymol (PDB: 3FXP).

[0449] Growth dynamics of viral escape variants

[0450] Vero E6 cells seeded in 96-well plates were infected with 50 PFU of GFP-expressing, replication-competent VSV viral particles pseudotyped with the SARS-CoV-2 spike protein obtained during escape selection. GFP expression was monitored hourly using the Incucyte Zoom instrument and analyzed using the included software.

[0451] HDX-MS epitope mapping

[0452] 3.33 μM SARS-CoV-2 S-2P trimer was incubated at 37 ° C overnight. The protein was then diluted to 1.66 μM trimer in 1x PBS (pH 7.4, Sigma Aldrich P4417) in the presence or absence of 6.25 μM R3DC23. To start the exchange, the protein was diluted tenfold into a temperature-equilibrated deuterated buffer made by lyophilizing 1x PBS and resuspending in D2O (Sigma Aldrich 151882). At each time point (15 seconds, 3 minutes, 30 minutes, 3 hours), 60 ul of the exchange reaction was mixed with 60 ul of ice-cold 2x quenching buffer (3.6 M guanidine chloride, 500 mM TCEP, 200 mM glycine pH 2.4) to quench the sample. The quenched samples were incubated on ice for 1 min and then snap frozen in liquid nitrogen and stored at -80°C until LC-MS. LC-MS and data analysis were performed as previously described (Costello, Shoemaker et al., 2022).

[0453] HR2 expression and purification

[0454] For structural biology purposes, HR2 protein is expressed in a bacterial expression system. Therefore, a synthetic gene encoding HR2 protein residues H1159-K1211 was cloned into the pFloat-SUMO vector to generate a His-tagged SUMO-HR2 fusion protein. The construct also contains a 3C protease cleavage site to remove the His-SUMO tag. The pFloat-SUMO-HR2 plasmid was transformed into BL21 (DE3) cells and plated on LB agar plates containing kanamycin (100 μg / ml). A single colony of BL21 (DE3) (pFloat-SUMO-HR2) was inoculated into a small amount of LB culture supplemented with 100 μg / ml kanamycin and grown at 37°C. Subsequently, 20 ml of this preculture was inoculated into 1 L LB culture and grown at 37°C until OD 600The expression of protein was induced by 0.5mM isopropyl β-D-1-thiogalactoside (IPTG). The cells were further incubated overnight at 20°C and harvested by centrifugation (Beckman rotor 8.1000, 5000rpm, 15 minutes, 4°C). The precipitation was resuspended in PBS, 500mM NaCl, 10mM imidazoles, 5mM β-mercaptoethanol, 0.1mg / mL 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), 1μg / mL leupeptin, 50μg / mL DNaseI and 20mM MgCl2. The cells were lysed at 20kpsi using a French press (Constant Systems), and cell debris was removed by centrifugation. The cell lysate was loaded onto a Ni-sepharose FF HiLoad column (GE Healthcare) equilibrated in 20 mM Tris-HCl pH 7.5, 500 mM NaCl, 10 mM imidazole, 5 mM β-mercaptoethanol. The bound protein was eluted using a linear gradient to 500 mM imidazole. The fractions containing His-SUMO-HR2 protein were combined, dialyzed to 20 mM Tris-HCl pH 7.5, 150 mM NaCl overnight at 4 °C, and then incubated with 3C protease for 2 hours at room temperature. The cleaved sample was loaded onto a Ni-sepharose FF HiLoad column again and equilibrated in the same buffer. The flow-through containing HR2 protein was concentrated and applied to a BioRad Enrich70 10 / 30 size exclusion column (SEC) equilibrated in 20 mM Tris-HCl pH 7.5, 150 mM NaCl. The SEC fractions containing HR2 were combined.

[0455] R3DC23-HR2 crystallization, X-ray data collection, processing and structure determination

[0456] For crystallization, R3DC23 was added to HR2 at a 1.2-fold molar excess and concentrated to 19 mg / ml using an Amicon Ultra 3 kDa cutoff centrifugal filter device. Using the sitting drop vapor diffusion technique, 0.1 ul of R3DC23-HR2 and 0.2 μl of the bottom solution were mixed to set up a crystallization screen. Crystals were grown by Molecular Dimensions Proplex crystallization screen in 0.1 M magnesium chloride hexahydrate, 0.1 M sodium citrate (pH 5.0), 15% PEG4000. For X-ray data collection, crystals were flash frozen in liquid nitrogen. X-ray data were collected at the i24 beamline at the Diamond Light Source synchrotron facility (Didcot, UK). X-ray data were processed using autoPROC+Staraniso (Vonrhin et al., 2011 ActaCrystallogrD Biol Crystallogr 67:293-302; Vonrhein et al., 2023 ActaCrystallographica Section A:Foundations and Advances). The structure of the R3DC23-HR2 complex was solved using the automated molecular replacement workflow in CCP4 Cloud (Krissinel et al., 2022 Acta Cryst D 78: 1079-1089). The initial model was further manually built in Coot (Emsley and Cowtan 2004 Acta Crystallogr D Biol Crystallogr 60: 2126-2132) and refined using phenix.refine (Afonine et al., 2012 Acta Crystallogr D Biol Crystallogr 68: 352-367), which is from the Phenix crystallographic software suite (Adams et al., 2010 Acta Crystallogr D Biol Crystallogr 66: 213-221). Data collection parameters and processing and refinement statistics are shown in Table 6.

[0457] Table 6. Data collection statistics and refinement parameters for R3_DC23-HR2. [a] R cryst =S||F obs |-|F calc || / S|F obs |, F obs and F calc are the observed and calculated structure factor amplitudes. [b] R freeWith R cryst Again, use a random subset of the data that is excluded from refinement. (c) The data in brackets are those of the highest resolution shell

[0458]

[0459]

[0460] In vivo viral challenge in K18-hACE2 mice

[0461] K18-hACE2 mice: B6.Cg-Tg(K18-ACE2)2Prlmn / J (7-9 weeks old) were purchased from The Jackson Laboratory and housed in-house under specific pathogen-free (SPF) conditions. SARS-CoV-2 infection was performed under biosafety level 3 (BSL3) conditions. Antibody treatment was performed by intraperitoneal injection using a 100 μl volume. Animals were anesthetized by inhalation of isoflurane and 3*10 2 pFU of SARS-CoV-2 614G variant virus (SARS-CoV-2 / human / FRA / 702 / 2020, from the European Virus Archive). Animals were monitored daily by a blinded observer, and weight changes were measured and scored for humane endpoints: hunchback (1 point), piloerection (1 point), reduced movement when opening the cage (1 point), immobility when touched (2 points), neurological signs (tremors, balance, 3 points), heavy breathing (3 points). Mice that lost more than 25% of their initial body weight or reached the humane endpoint (score of 5 points) were euthanized.

[0462] Titration of SARS-Co V-2 Virus Titers in Mouse Lung Homogenates

[0463] After dissection, the right lung lobe was stored at -80 ° C. To quantify lung virus titers, lung samples were homogenized using a Precellys Evolution tissue homogenizer (Bertin-technologies). The lung homogenate was centrifuged (1,000 × g) at 4 ° C for 15 minutes to clarify it, and then a 12-well plate was used to perform a plaque assay on VeroE6-TMPRSS2 cells in duplicate to determine the viral titer. After adding the gradient dilution of the lung homogenate to the cells, the plate was incubated at 37 ° C for 2 hours. The cells were then washed twice and incubated in medium containing Avicel at a concentration of 0.3% (w / v). After incubation at 37 ° C for 2 days, the cover was removed, the cells were fixed with 3.7% paraformaldehyde (PFA), and stained with 0.5% crystal violet dye to observe viral plaques.

[0464] To quantify SARS-CoV-2 viral RNA levels, RNA was prepared from lung homogenates and analyzed by qRT-PCR. TM cDNA was prepared using a cDNA synthesis kit and random hexamer primers. qPCR was performed using the SARS-CoV-2 Research Specific (RUO) qPCR Primers and Probe Kit (IDT) according to the manufacturer’s instructions.

[0465] Syrian golden hamster attack model

[0466] Male Syrian golden hamsters (Mesocricetus auratus) weighing 89.8 g to 132.3 g were purchased from Janvier (France). Six hamsters per group were infected intranasally with 10^2.050% tissue culture infection dose (TCID50) / dose SARS-CoV-2 (Wuhan-Hu-1 strain), with a total dose of 100 μl, equally distributed in both nostrils. The specified doses of XVR012 (a mixture of XVR014 and XVR013), XVR013 or XVR014, palivizumab (10 mg / kg) or bebeteloizumab (10 mg / kg) were administered by intraperitoneal injection 4 hours after SARS-CoV2 challenge (treatment setting) or about 24 hours before infection (prevention setting). An unrelated antibody palivizumab (Synagis, anti-RSV antibody) was used as a negative control, and bebeteloizumab was used as a positive control. The huR3DC23-Fc LS used in the hamster studies was produced from a stable cell pool. The behavior, appearance and weight of the hamsters were monitored daily. On the fourth day after infection, the animals were euthanized. At autopsy, gross pathological examinations were performed and abnormalities were recorded. Samples from the right lung lobe were collected and frozen for virological analysis. To determine the viral titer, quadruplicate 10-fold serial dilutions were used in confluent layers of Vero E6 cells. To this end, serial dilutions of the samples (lung tissue homogenates) were incubated on Vero E6 monolayers at 37°C for 1 hour. The Vero E6 monolayers were then washed and incubated at 37°C for 5 or 6 days, after which the plates were stained and scored based on the cytopathic effect (CPE) by using the viability marker WST8 (colorimetric readout). Viral titers (Log10TCID50 / g) were calculated using the Spearman-Karber method ( 1931 Archiv F. Experiment. Pathol. u. Pharmakol. 162: 480-483). For viral titers, the lower limit of detection (LLOD) ranged from 1.1 to 1.3 log10 TCID / g. For the detection of viral RNA, lung tissue homogenates were used. RNA was isolated and Taqman PCR was performed. The number of copies (Log10 CP / g) in the different samples was calculated based on the standards included in each run. For viral RNA in lung tissue, the LLOD was 3.5 Log10 CP / g. Blood samples were collected at -2 days before the start of the study (approximately 200 μl of blood was collected under isoflurane anesthesia for serum) and at autopsy on day 4 post-infection (pi) for pharmacokinetic analysis. Blood samples for serum were immediately transferred to appropriate test tubes containing a coagulation activator. Serum was collected and stored frozen. In order to inactivate any potentially infectious material and allow the testing of serum samples in a BSL-2 environment, the serum on day 4 post-infection was heat treated at 56°C for 30 minutes.

[0467] ADCC assay / FcγRIIIa reporter gene assay

[0468] ADCC was evaluated by AntibodyAnalytics using the CHO-K1 target cell line expressing the SARS CoV-2 spike protein as the target cell and Jurkat FcγRIIIa (CD16) V176-NFAT-RE Luc as the reporter cell.

[0469] Biolayer Interferometry (BLI)

[0470] Biolayer interferometry was performed on an Octet RED96 system (FortéBio). Streptavidin (SA) biosensors (Sartorius) were soaked in 1x kinetic buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.1 mg / ml bovine serum albumin, 0.02% Tween-20 and 0.02% sodium azide) for 20 min before use. Trimeric S-2P-foldon-His-Strep was thawed from -20°C and incubated at 37°C for 24 h, then at 30°C for 30 min, and then immobilized on the SA biosensor at 36.5 μg / ml (80 nM) for 450 s, giving a signal from 1.5 nm to 1.7 nm. Binding (120 s) and dissociation (480 s) of two-fold serial dilutions starting from 20 nM monomeric R3DC23 VHH in 1x kinetic buffer were measured at 30°C. Between analyses, the biosensor was regenerated by three 5-s exposures to regeneration buffer (10 mM glycine pH 3). The stoichiometry of the nanoscale responses for the analyses was determined from the raw data curves. The data from the kinetic analyses were double reference subtracted and compared to each other in Octet Data Analysis Software v9.0 (FortéBio). Both association and dissociation of the full gradient dilutions were fitted with a global 1:1 model.

[0471] Example 1: Isolation of VHHs that bind to the SARS-CoV-2 and SARS-CoV-1 spike proteins at their S2 subunits.

[0472] A llama that had been previously vaccinated with MERS-CoV and SARS-CoV-1 spike proteins (Wrapp, D. et al., (2020) "Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies." Cell 181:1004-1015.e15) was boosted with three consecutive immunizations with SARS-CoV-2 spike protein S-2P, specifically recombinant prefusion stabilized SARS-CoV-2-2P spike protein.

[0473] After vaccination, phagemid libraries containing VHH coding sequences were constructed and used for phage display biopanning. To obtain VHHs targeting spike proteins at sites other than RBD, phages were pre-incubated with 10 μg / ml RBD-SD1 in each round of biopanning. After two rounds...

Claims

1. A binding agent capable of neutralizing Sarbecovirus, characterized in that The binding agent specifically binds to or within a region of the Sabeivirus spike protein, which corresponds to the region from amino acid E1188 to amino acid Y1206 in the SARS-CoV-2 spike protein defined by SEQ ID NO:

86.

2. The binding agent according to claim 1, which specifically binds to or within a region of the spike protein, which corresponds to the region from amino acid E1188 to amino acid L1203 in the SARS-CoV-2 spike protein defined by SEQ ID NO:86, or preferably the region from amino acid E1188 to amino acid L1202.

3. A binding agent according to claim 1 or 2, which specifically binds to or within a region of the spike protein, which corresponds to the region from amino acid N1194 to amino acid L1203, or preferably the region from amino acid E1194 to amino acid L1202 in the SARS-CoV-2 spike protein defined by SEQ ID NO:

86.

4. The binding agent according to any one of claims 1 to 3, wherein the binding agent specifically binds to the region of the heptad repeat 2 (HR2) domain of the Sabei virus spike protein close to the viral membrane.

5. The binding agent of any one of claims 1 to 4, wherein the binding agent specifically binds to a quaternary epitope in the spike protein trimer.

6. A binding agent according to claim 5, wherein amino acid residues from at least two monomers of the spike protein trimer contribute to the quaternary epitope.

7. The binding agent of any one of claims 1 to 6, wherein the binding agent specifically binds to at least one amino acid residue of the spike protein corresponding to amino acid residue N1194, S1196, D1199 or Q1201 in the SARS-CoV-2 spike protein as defined in SEQ ID NO: 86; or wherein at least one amino acid residue of the spike protein corresponding to the amino acid residue N1194, S1196, D1199 or Q1201 in the SARS-CoV-2 spike protein as defined in SEQ ID NO: 86 is essential for the binding of the binding agent to the spike protein.

8. The binding agent of any one of claims 1 to 6, wherein the binding agent specifically binds to at least one amino acid residue of the spike protein corresponding to amino acid residue S1196 or Q1201 in the SARS-CoV-2 spike protein defined by SEQ ID NO: 86; or wherein at least one amino acid residue of the spike protein corresponding to the amino acid residue S1196 or Q1201 in the SARS-CoV-2 spike protein defined by SEQ ID NO: 86 is essential for the binding of the binding agent to the spike protein.

9. The binding agent of any one of claims 1 to 8, wherein the binding agent specifically binds to the spike protein amino acid residues corresponding to amino acid residues S1196 and Q1201 in the SARS-CoV-2 spike protein as defined in SEQ ID NO:86, optionally specifically binds to the spike protein amino acid residues corresponding to amino acid residues N1194, S1196, D1199 and Q1201 in the SARS-CoV-2 spike protein as defined in SEQ ID NO:86; or wherein the spike protein amino acid residues S1196 and Q1201 in the SARS-CoV-2 spike protein as defined in SEQ ID NO:86, optionally the spike protein amino acid residues corresponding to amino acid residues N1194, S1196, D1199 and Q1201 in the SARS-CoV-2 spike protein are essential for the binding of the binding agent to the spike protein.

10. The binding agent of any one of claims 1 to 9, wherein the binding agent stabilizes the pre-fusion conformation of the spike protein, for example, wherein the binding agent stabilizes the HR2 coiled-coil structure in the spike protein.

11. The binding agent according to any one of claims 1 to 10, wherein: - The binding agent is capable of neutralizing Sabei virus, wherein the 50% inhibitory concentration (IC 50 ) is 100 ng / ml or less, preferably 10 ng / ml or less, more preferably 1 ng / ml or less, as determined in a Sabeivirus Spike protein pseudovirus neutralization assay (such as a vesicular stomatitis virus (VSV)-Sabeivirus Spike protein pseudovirus neutralization assay); - the binding agent is capable of neutralizing either or both of SARS-CoV-2 and SARS-CoV-1, such as one or more of the SARS-CoV-2 Wuhan-Hu-1 strain, the SARS-CoV-2 Alpha variant, the SARS-CoV-2 Omicron BA.1 variant, the SARS-CoV-2 Omicron BA.2 variant, the SARS-CoV-2 Omicron BA.5 variant, the SARS-CoV-2 Omicron BA.2.75.2 variant, the SARS-CoV-2 Omicron BA.4.6 variant, the SARS-CoV-2 Omicron BF.7 variant, the SARS-CoV-2 Omicron BQ.1.1 variant, the SARS-CoV-2 Omicron XBB variant, and the SARS-CoV-2 Omicron XBB.1.5 variant; - the binding agent is capable of inhibiting spike protein-mediated syncytium formation between cells expressing the Sabei virus spike protein and cells expressing angiotensin converting enzyme 2 (ACE2) receptor; and / or - The binding agent does not bind to Middle East Respiratory Syndrome Coronavirus (MERS-CoV).

12. The binding agent according to any one of claims 1 to 11, comprising or consisting of an antibody or an antibody fragment.

13. The binding agent according to any one of claims 1 to 12, comprising an immunoglobulin single variable domain (ISVD), preferably a VHH.

14. The binding agent of claim 13, wherein the ISVD comprises a complementarity determining region 1 (CDR1) defined by any one of SEQ ID NOs: 63, 46, 69 or 77, a complementarity determining region 2 (CDR2) defined by any one of SEQ ID NOs: 64, 47, 70, 73 or 78, and a complementarity determining region 3 (CDR3) defined by any one of SEQ ID NOs: 48, 67, 74 or 79; preferably a CDR1 defined by any one of SEQ ID NOs: 65, 71, 49 or 80, a CDR2 defined by any one of SEQ ID NOs: 66, 72, 50, 75 or 81, and a CDR3 defined by any one of SEQ ID NOs: 51, 68, 76 or 82.

15. The binding agent of claim 13 or 14, wherein the ISVD comprises CDR1, CDR2 and CDR3, each independently present in any one of SEQ ID NOs: 1-10, wherein the CDR1, CDR2 and CDR3 are annotated according to any one of Kabat, MacCallum, IMGT, AbM, Martin or Chothia; preferably, wherein the ISVD comprises a combination of CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 are present in a specific sequence of the sequences listed by SEQ ID NOs: 1-10, wherein the CDR1, CDR2 and CDR3 are annotated according to any one of Kabat, MacCallum, IMGT, AbM, Martin or Chothia.

16. The binding agent of any one of claims 13 to 15, wherein the ISVD comprises a CDR1 defined by SEQ ID NO: 63, a CDR2 defined by SEQ ID NO: 64, and a CDR3 defined by SEQ ID NO: 48; preferably a CDR1 defined by SEQ ID NO: 65, a CDR2 defined by SEQ ID NO: 66, and a CDR3 defined by SEQ ID NO: 51; more preferably a CDR1 defined by any one of SEQ ID NOs: 52-54, a CDR2 defined by any one of SEQ ID NOs: 55-62, and a CDR3 defined by any one of SEQ ID NOs: 21-27.

17. The binding agent of any one of claims 14 to 16, wherein the ISVD comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 1-10 and 127-129.

18. The binding agent of any one of claims 1 to 17, comprising an ISVD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10 and 127-129.

19. The binding agent according to any one of claims 1 to 18, which is in a multivalent form, preferably wherein the binding agent comprises an ISVD fused to an Fc domain.

20. The binding agent of any one of claims 1 to 19, comprising an amino acid sequence defined by any one of SEQ ID NOs: 96, 118, 130 or 131.

21. A multispecific binding agent comprising a binding agent as defined in any one of claims 1 to 20 and further comprising a binding agent that specifically binds to a Sabeivirus spike protein receptor binding domain (RBD).

22. The multispecific binding agent of claim 21, wherein the binding agent that specifically binds to the RBD comprises at least one ISVD capable of binding to or competing for an epitope specifically bound by VHH72 defined by SEQ ID NO: 124, such as an ISVD comprising a sequence set forth by SEQ ID NO: 125 or SEQ ID NO: 124, or a humanized form thereof.

23. The multispecific binding agent of claim 21 or 22, wherein the binding agent that specifically binds to the RBD comprises at least one ISVD capable of binding to or competing for an epitope specifically bound by VHH3.117 defined by SEQ ID NO: 126, such as an ISVD comprising the sequence set forth by SEQ ID NO: 126 or a humanized form thereof.

24. The multispecific binding agent of any one of claims 21 to 23, comprising an amino acid sequence defined by any one of SEQ ID NOs: 112-117.

25. A nucleic acid molecule comprising a polynucleotide sequence encoding a binding agent according to any one of claims 1 to 24; a vector comprising the nucleic acid molecule; or a cell expressing the binding agent according to any one of claims 1 to 24 or comprising the nucleic acid molecule or the vector.

26. A composition comprising a binding agent as defined in any one of claims 1 to 20 and further comprising a binding agent that specifically binds to the receptor binding domain (RBD) of the Sabei virus spike protein.

27. A composition according to claim 26, wherein the binding agent that specifically binds to the RBD comprises at least one ISVD capable of binding to or competing for the epitope specifically bound by VHH72 defined by SEQ ID NO: 124 and at least one ISVD capable of binding to or competing for the epitope specifically bound by VHH3.117 defined by SEQ ID NO: 126, such as a binding agent defined by SEQ ID NO:

119.

28. A composition according to claim 26 or 27, comprising a binding agent defined by SEQ ID NO: 118 and a binding agent defined by SEQ ID NO:

119.

29. A pharmaceutical composition comprising a binding agent according to any one of claims 1 to 20, a multispecific binding agent according to any one of claims 21 to 24, a nucleic acid molecule according to claim 25, a vector according to claim 25, or a composition according to any one of claims 26 to 28, and a pharmaceutically acceptable carrier; or a kit, such as a diagnostic kit, comprising a binding agent according to any one of claims 1 to 20, a multispecific binding agent according to any one of claims 21 to 24, or a composition according to any one of claims 26 to 28.

30. The binding agent of any one of claims 1 to 20, the multispecific binding agent of any one of claims 21 to 24, the nucleic acid molecule of claim 25, the vector of claim 25, the composition of any one of claims 26 to 28, the pharmaceutical composition of claim 29, or the kit of claim 29 for pharmaceutical use; preferably for preventing or treating Sabeivirus infection in a subject, or for diagnosing Sabeivirus infection in a subject.

31. An in vitro or ex vivo method for detecting Sabei virus in a sample, the method comprising: - contacting the sample with a binding agent according to any one of claims 1 to 20 or a multispecific binding agent according to any one of claims 21 to 24, and - Determining the binding of said binding agent to Sabeivirus or a part thereof.

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