Simian coronavirus spike S2 subunit binder

Abstract

The present invention relates to a sarbecovirus binder capable of potently neutralizing sarbecoviruses, in particular SARS-CoV-2, including SARS-CoV-2 variants, and / or SARS-CoV-1, and more particularly to antibodies and antigen-binding fragments thereof. The binder, in particular the antibody and antibody fragment, binds to the heptad repeat 2 (HR2) domain of the spike protein of sarbecoviruses, and more specifically to a quaternary epitope located within two adjacent HR2 domains. The present invention also relates to methods of using these binders and their use.

Description

[Technical field]

[0001] The present invention is generally in the field of binding agents, particularly antibodies. More specifically, the present invention relates to binding agents, particularly antibodies and antigen-binding fragments thereof, that bind to the spike protein of Sarbocoviruses, such as SARS-CoV-2, including SARS-CoV-2 variants, and SARS-CoV-1, capable of potently neutralizing the Sarbocoviruses. The present invention also relates to methods of using these binding agents and their uses. [Background technology]

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19 (Zhu et al. 2020, N Engl J Med 382:727-733). SARS-CoV-2 infection can be asymptomatic or result in 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 often leads to acute respiratory distress requiring prolonged intensive care and, in some patients, death. "Long COVID" further refers to the long-term effects of COVID-19 infection that occur even when the SARS-CoV-2 virus is no longer detectable.

[0003] Certain types of therapeutic approaches may rely on neutralizing antibodies, i.e. passive antibody therapy / immunotherapy. The spike of the SARS coronavirus is the primary target of neutralizing antibodies. The spike protein is a class I fusion protein, composed of a membrane-distal S1 subunit and a membrane-proximal S2 subunit. The S1 subunit contains the receptor-binding domain (RBD), and antibodies against this domain can have very strong neutralizing activity (Wheatley et al. 2021. Cell Rep 37:109822). The S1 subunit, especially the N-terminal domain and the RBD, can tolerate mutations that lead to antigenic variation and immune escape. The 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). Because the S2 subunit is more conserved, it seems, at least in theory, an attractive target for the development of neutralizing antibodies with broad anti-sarbecovirus protection. Several monoclonal antibodies that recognize conserved epitopes in the S2 subunit of SARS coronaviruses have been reported. However, in general, these monoclonal antibodies have low virus neutralizing activity. For example, the S2 subunit-specific monoclonal antibody L19 inhibited the authentic SARS-CoV-2 virus with an IC of 9.9–19.8 μg / ml. 100 (Andreano et al. 2021. Cell 184:1821-1835). Wu et al. (2022. JCI Insight 7:ee157597) identified monoclonal antibodies, Mab5 and Mab3-2, that target the HR2 domain at an epitope located at the N-terminus of the HR2 domain. The two mAbs have neutralizing activity against SARS-CoV-2, and the IC of Mab5 was 50 The IC value was 12.3 μg / mL for Mab3-2. 50The neutralization potency was 87.4 μg / mL. Single domain antibodies, also known as nanobodies or VHHs, against the S2 subunit of SARS-CoV-2 have also been reported (Mast et al. 2021. eLife 110:e73027; Rossotti et al. 2021. DOI:10.1101 / 2021.12.20.473401). Again, 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 described in Rossotti et al. (2021) neutralized the Wuhan strain of SARS-CoV-2 with an IC of 12.2 nM. 50 I was able to neutralize it with, but not without the format. Summary of the Invention

[0005] Thus, there remains a need in the art for potent neutralizing antibodies that target the spike protein of sarbecoviruses.

[0006] As demonstrated in the experimental section illustrating certain embodiments of the present invention, the inventors have identified 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 ... We identified sarbecovirus-specific variable domains of heavy chain antibodies (VHHs) that potently neutralized SARS-CoV-2, including SARS-CoV-2 variants such as the 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, as well as SARS-CoV-1. Further analysis revealed that these VHHs interact with amino acids within the S2 subunit of the spike protein, particularly within the heptad repeat 2 (HR2) domain of the S2 subunit, and more specifically within the C-terminal region of the HR2 domain proximal to the viral membrane, and that these amino acids are highly conserved in the spike proteins of multiple clades of sarbecoviruses. Therefore, this region is expected to be more stable and less susceptible to frequent mutations.

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

[0008] One embodiment provides a binding agent capable of neutralizing a sarbecovirus, characterized in that the binding agent specifically binds to a region or within a region of a sarbecovirus spike protein corresponding 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 certain preferred embodiments, the binder specifically binds to a region or within a region of the spike protein corresponding to the region of the SARS-CoV-2 spike protein from amino acid E1188 to amino acid L1203 as defined in SEQ ID NO: 86.

[0010] In certain preferred embodiments, the binder specifically binds to a region or within a region of the spike protein corresponding to the region of the SARS-CoV-2 spike protein from amino acid E1188 to amino acid L1202 as defined in SEQ ID NO: 86.

[0011] Given that the overall number of currently available SARS-CoV-2 treatment options remains limited, and in particular, that multiple SARS-CoV-2 variants have emerged, some of which are more infectious and / or cause more severe disease symptoms (including in younger individuals), and / or evade some of the existing vaccines and / or diagnostic tests, such a sarbecovirus neutralizing binder that binds to the more highly conserved S2 subunit of the spike protein is a valuable tool that should be added.

[0012] In a further aspect, the invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a binder according to the invention, as well as a vector comprising such a nucleic acid molecule, and a cell comprising such a nucleic acid molecule or such a vector, or a cell expressing a binder according to the invention.

[0013] The invention further relates to a pharmaceutical composition comprising a binder according to the invention, or a nucleic acid molecule or vector as described above, and a pharmaceutically acceptable carrier, as well as a kit such as a diagnostic kit comprising a binder according to the invention.

[0014] A further aspect is directed to the binder according to the invention, a nucleic acid molecule or vector as described above, a pharmaceutical composition, or a kit as described above, for use in the prevention or treatment of sarbecovirus infection in a subject, or for use in the diagnosis of sarbecovirus infection in a subject, etc. in medicine.

[0015] The present invention further relates to an in vitro or ex vivo method for detecting sarbecovirus in a sample, the method comprising: - contacting the sample with a binder according to the invention; and - determining the binding of the binder to sarbecovirus or a part thereof.

[0016] Those skilled in the art will recognize many other effects and advantages of the methods, uses or products of the present invention, as well as numerous possibilities for the end uses of the present invention, from the detailed description and examples provided below. BRIEF DESCRIPTION OF THE DRAWINGS

[0017]

Figure 1-1

Figure 1-2

Figure 2-1

Figure 2-2

Figure 3-1

Figure 3-2

Figure 3-3

Figure 3-4

Figure 3-5

Figure 3-6

Figure 4

Figure 5-1

Figure 5-2

Figure 6-1

Figure 6-2

Figure 7-1

Figure 7-2

Figure 8-1

Figure 8-2

Figure 9-1

Figure 9-2

Figure 9-3

Figure 10-1

Figure 10-2

Figure 11

Figure 12

Figure 13

Figure 14-1

Figure 14-2

Figure 15

Figure 16-1

Figure 16-2

Figure 17

Figure 18

Figure 19-1

Figure 19-2

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26-1

Figure 26-2

Figure 26-3

Figure 27-1

Figure 27-2

Figure 27-3

Figure 27-4

Figure 28-1

Figure 28-2

Figure 28-3

Figure 28-4

Figure 29

Figure 30-1

Figure 30-2

Figure 31-1

Figure 31-2

Figure 31-3

Figure 32-1

Figure 32-2

Figure 33

Figure 34

Figure 35

Figure 36

Figure 37

Figure 38

Figure 39-1

Figure 39-2

Figure 40-1

Figure 40-2

Figure 40-3

Figure 41

DETAILED DESCRIPTION OF THE INVENTION

[0018] As used herein, the singular forms "a", "an", and "the" include references to both the singular and plural forms unless the context clearly dictates otherwise.

[0019] The terms "comprising", "comprise", and "comprised of", as used herein, are synonymous with "including", "include", or "containing", "contain", and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps. This term also encompasses "consisting of" and "consisting essentially of", which enjoy well-established meanings in patent terminology.

[0020] The recitation of numerical ranges by endpoints includes not only the recited endpoints but also all numbers and fractions subsumed within each range.

[0021] The terms "about" or "approximately", as used herein when referring to measurable values such as parameters, amounts, durations of time, etc., mean that variations of the specified value, and variations from the specified value, such as variations of up to ±10%, preferably up to ±5%, more preferably up to ±1%, and even more preferably up to ±0.1% of the specified value, are included as long as they are appropriate for carrying out the disclosed invention. It should be understood that the value itself to which the modifier "about" refers is also specifically, preferably, disclosed.

[0022] The terms "one or more" or "at least one" are clear in themselves, such as at least one member among one or more members or a group of members. By way of further illustration, this term encompasses, inter alia, any one of said members, or any two or more of said members, for example, any three or more, four or more, five or more, six or more, or seven or more of said members, and references to all of the said members up to the maximum. In another example, "one or more" or "at least one" may refer to 1, 2, 3, 4, 5, 6, 7 or more.

[0023] The discussion of the background of the invention in this specification is included to explain the context of the invention. This should not be taken as an admission that any of the documents referred to were published, known, or part of common general knowledge in any country at the priority date of any of the claims.

[0024] Throughout this disclosure, various publications, patents, and published patent specifications are referenced by distinguishable citation. All documents cited in this specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents specifically referred to in this specification are incorporated by reference.

[0025] Unless otherwise defined, all terms used in disclosing the present invention, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As a further guide, definitions of terms are included to better understand the teachings of the present invention. Where a particular term is defined in relation to a particular aspect or a particular embodiment of the present invention, such meaning is meant to apply throughout this specification, i.e., in the context of other aspects or embodiments of the present invention, unless otherwise specifically defined.

[0026] In the following text, different aspects or embodiments of the present invention are defined in more detail. Each aspect or embodiment thus defined may be combined with other aspects or embodiments, unless the contrary is clearly indicated. In particular, any requirement shown to be preferred or advantageous may be combined with other requirements (s) shown to be preferred or advantageous.

[0027] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular requirement, structure, or feature described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the phrases "in one embodiment" or "in an embodiment" appear in various places throughout this specification, and although not necessarily all refer to the same embodiment, they may. Further, the particular requirements, structures, or features may be combined in any suitable manner in one or more embodiments, as will be apparent to those skilled in the art from the present disclosure. Further, some embodiments described herein include some requirements included in other embodiments but not others, and combinations of requirements of different embodiments are within the scope of the present invention and form different embodiments, as will be understood by those skilled in the art. For example, in the appended claims, any of the claimed embodiments may be used in any combination.

[0028] As demonstrated by an experimental section exemplifying certain representative embodiments of the present invention, the inventors have identified VHHs that specifically bind to the S2 subunit of the sarbecovirus spike protein, particularly the S2 subunit of the SARS-CoV-2 and SARS-CoV-1 spike proteins. The VHHs were found to potently neutralize SARS-CoV-2 and SARS-CoV-1, including SARS-CoV-2 variants such as the 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. These VHHs interact with the S2 amino acids of the heptad repeat 2 (HR2) domain, more specifically, the amino acids within the C-terminal region of the HR2 domain proximal to the viral membrane, and these amino acids were found to be highly conserved within the spike proteins of sarbecoviruses of multiple clades.

[0029] Accordingly, one aspect relates to a binding agent capable of neutralizing a sarbecovirus, particularly an antibody and antigen-binding fragment thereof, which specifically binds to the heptad repeat 2 (HR2) domain of the spike protein of a sarbecovirus, particularly an antibody and antibody fragment.

[0030] One aspect provides a binding agent capable of neutralizing a sarbecovirus, which specifically binds to a region or within a region of the spike protein of a sarbecovirus corresponding 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.

[0031] The term "binding agent" generally refers to a molecule capable of binding to at least one other molecule, and the binding is preferably a specific binding, such as on a defined binding site, pocket or epitope. The binding agent can be of any nature or type and is independent of its origin. The binding agent may not only be chemically synthesized, naturally occurring, recombinantly produced (and optionally purified), but may also be designed or generated by synthesis (and optionally purified). Thus, the binding agent may be, for example, a small molecule, chemical substance, peptide, polypeptide, antibody, or derivative thereof, especially peptidomimetics, antibody mimetics, active fragments, chemical derivatives, etc. A functional fragment or a functional part of a binding agent refers to a fragment or part of a binding agent that is functionally equivalent to that binding agent. In particular, the functional fragments or parts of the binding agents described herein preferably retain one or more of the functional requirements (1)-(21) of that binding agent outlined extensively elsewhere herein.

[0032] The term "antibody" refers to an immunoglobulin (Ig) molecule or a molecule containing an immunoglobulin (Ig) domain that specifically binds to an antigen, and multimers thereof. An "antibody" can be an intact immunoglobulin or an immunoreactive part of an intact immunoglobulin. This term encompasses antibodies produced naturally, recombinantly, semi-synthetically or synthetically. Thus, for example, an antibody may exist in nature or be isolated from nature, for example, produced or expressed naturally or endogenously by a cell or tissue and optionally isolated therefrom; or an antibody may be a recombinant, i.e., produced by recombinant DNA technology, and / or may be chemically or biochemically synthesized, in whole or in part.

[0033] "Isolated" or "purified" means a substance that is substantially or essentially free of the components that are normally associated with it in its native state. For example, an "isolated polypeptide" or "purified polypeptide" refers to a polypeptide that has been isolated or purified by any suitable means from a mixture of molecules containing the polypeptide to be isolated or purified. The isolated or purified polypeptide of interest may be, for example, an immunoglobulin, an antibody or a nanobody, and the mixture may be a mixture or molecule present in cells that produce immunoglobulins, antibodies or nanobodies, and / or in a culture medium in which immunoglobulins, antibodies or nanobodies are secreted (which is likely to be present together with other molecules secreted by the cells).

[0034] The terms "antibody fragment", "antigen-binding fragment", "functional antibody fragment" and "active antibody fragment" refer to a part of any antibody that itself has a high affinity for an antigenic determinant, or epitope, and contains one or more complementarity-determining regions (CDRs) that account for such specificity. The terms "antibody fragment" and "antigen-binding fragment" and "active antibody fragment" and "functional antibody fragment", as used herein, refer to a protein or peptide that includes an immunoglobulin domain or antigen-binding domain that is capable of specifically binding to a sarbecovirus spike protein, such as the SARS-CoV-2 spike protein, particularly the S2 subunit of the sarbecovirus spike protein, and more particularly the HR2 domain of the sarbecovirus spike protein (of the S2 subunit). 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 complete heavy chain.

[0035] The term "immunoglobulin (Ig) domain", or more specifically "immunoglobulin variable domain" (abbreviated as "IVD" and also referred to herein as "variable domain") means, in the art and hereinafter in this specification, an immunoglobulin domain consisting essentially of four "framework regions" referred to respectively as "framework region 1" or "FR1"; "framework region 2" or "FR2"; "framework region 3" or "FR3"; and "framework region 4" or "FR4", and these framework regions are divided by three "complementary determining regions" or "CDRs" referred to respectively as "complementary determining region 1" or "CDR1"; "complementary determining region 2" or "CDR2"; and "complementary determining region 3" or "CDR3" in the art and hereinafter in this specification. Thus, the general structure or sequence of an immunoglobulin variable domain can be shown as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulin variable domain (IVD), particularly the CDRs therein, and even more particularly the CDR3 therein, that confers specificity for an antigen to an antibody by having an antigen-binding site or epitope-binding site. Typically, in a conventional immunoglobulin, 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 both VH and VL contribute (although not necessarily equally) to the antigen-binding site, i.e., a total of six CDRs are involved in the formation of the antigen-binding site. Considering the above definitions, the antigen-binding domains of conventional four-chain antibodies (such as IgG, IgM, IgA, IgD or IgE molecules known in the art), Fab fragments derived from such conventional four-chain antibodies, F(ab’)2 fragments, Fv fragments such as disulfide-linked Fv or scFv fragments, or diabodies (all known in the art) bind to each epitope of an antigen by pairs of (relevant) immunoglobulin domains such as the light and heavy chain variable domains, i.e., by the VH-VL pair of immunoglobulin domains that jointly bind to the epitope of each antigen.

[0036] An "immunoglobulin single variable domain" (abbreviated as "ISVD") is equivalent to the term "single variable domain" and defines a molecule in which the antigen-binding site is present on a single immunoglobulin domain. This sets an immunoglobulin single variable domain apart from "conventional" immunoglobulins or fragments thereof in which two immunoglobulin domains, particularly two variable domains, interact to form an antigen-binding site. An "immunoglobulin single variable domain" (or "ISVD"), as used herein, refers to a protein or peptide having an amino acid sequence that includes four framework regions (FRs) and three complementarity-determining regions (CDRs) in the form 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. Thus, the antigen-binding site of an immunoglobulin single variable domain is formed by three or fewer CDRs. As such, a single variable domain can form a single antigen-binding unit (i.e., a functional antigen-binding unit that consists essentially of a single variable domain and does not need to interact with another variable domain to form a functional antigen-binding unit) and 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. 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, the immunoglobulin single variable domain can be a (single) domain antibody (or an amino acid sequence suitable for use as a (single) domain antibody), the variable domain of the heavy (VH) or light (VL) chain 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 VHHs); or a suitable fragment of any of these.

[0037] In an embodiment, the immunoglobulin single variable domain may be a Nanobody (as defined herein) or a suitable fragment thereof. Note: Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx N.V., a Sanofi Company. For a general description of Nanobodies, see the following further description and the prior art cited herein, such as that described in WO 2008 / 020079. The “VHH domain” is also known as VHH, VHH domain, VHH antibody fragment, and VHH antibody, and was originally described as the antigen-binding immunoglobulin (Ig) (variable) domain of a “heavy chain antibody” (i.e., an antibody lacking a light chain; Hamers-Casterman et al., 1993, Nature 363:446-448). The term “VHH domain” was chosen to distinguish these variable domains from the heavy chain variable domain (referred to herein as the “VH domain”) present in conventional four-chain antibodies, and the light chain variable domain (referred to herein as the “VL domain”) present in conventional four-chain antibodies.For further details on VHHs and Nanobodies, see the review by Muyldermans (2001. Rev Mol Biotechnol 74:277-302), and the following patent applications, which are hereby incorporated by reference 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 documents, Nanobodies (in particular, VHH sequences and partially humanized Nanobodies) can be characterized, in particular, by the presence of one or more "hallmark residues" in one or more of the framework sequences.

[0038] The binding agent or the Sars-CoV binding agent (used interchangeably) according to the invention can, in one aspect, be functionally described by any individual function / embodiment or by any combination of any number of individual functions / embodiments described below, where any number "n" is given in parentheses "(n)". The numerical order of these individual functions is random and does not impose any priority on the individual functions, and similarly, this random numerical order does not impose any priority on any combination of two or more of the individual functions. Since the binding agent or the Sars-CoV binding agent herein exhibits each of these individual functions, such combinations should not be considered to be further optional.

[0039] Thus, the present invention provides a binding agent that specifically binds to sarbecoviruses such as SARS-CoV-2 and SARS-CoV-1, which can also be referred to herein as a sarbecovirus binding agent or a sarbecovirus antibody and antibody fragment, in particular an antibody or an antigen-binding fragment thereof. In certain embodiments, the binding agent (2) does not bind to Middle East respiratory syndrome coronavirus (MERS-CoV).

[0040] "Binding" means any interaction, whether direct or indirect. Direct interaction means contact (e.g., physical or chemical) between two binding partners. Indirect interaction means any interaction in which the interacting partners interact in a complex of more than two molecules. The interaction may be entirely indirect (e.g., two molecules are part of the same complex with the help of one or more cross-linking molecules but do not bind in the absence of the cross-linking molecules). The interaction may be partly direct or partly indirect, and there is still direct contact between the two interacting partners, but such contact is not, for example, stable and is stabilized by interaction with one or more additional molecules.

[0041] "Specificity of binding", "binding specificity", or "specifically binds" refers to the situation where molecule A binds to a target of interest (e.g., a protein) at a particular concentration (e.g., a concentration sufficient to inhibit or neutralize the protein or process of interest) with a higher affinity (e.g., at least 2-fold, 5-fold, or at least 10-fold higher affinity, e.g., at least 20-fold, 50-fold, or 100-fold or more higher affinity) than it can bind to other targets (not the target of interest), if possible. Specific binding does not mean exclusive binding. However, specific binding does mean that the binder has some increased affinity or preference for one or some of its targets. Exclusive binding refers to the situation where the binder binds only to the target of interest. The term "affinity", as used herein, generally refers to the degree to which one molecule (e.g., a ligand, chemical, protein or peptide, antibody or antibody fragment) binds to another molecule (e.g., a (target) protein or peptide) and shifts the equilibrium of the single molecule monomer to a complex formed by the (specific) (non-covalent) binding of two molecules. Non-covalent interactions or bindings between two or more binding partners can involve interactions such as van der Waals interactions, hydrogen bonds, and salt bridges. "Dissociation constant" or "binding constant" (K D ) is commonly used to describe the affinity between two molecules and is often calculated as the ratio of the rate constant for complex formation (referred to as the "k on " value) to the rate constant for dissociation of the complex (the "k off " or "k dis " value). Measurement of the binding affinity of a molecule for another molecule, e.g., an antibody or antibody fragment for an antigen, or a ligand for a receptor, is known to those of skill in the art and includes, for example, real-time, label-free biolayer interferometry assays, e.g., the Octet® RED96 system (ForteBio), or surface plasmon resonance (SPR), e.g., BIACORE™, or solution affinity ELISA.

[0042] The terms "Coronaviridae" and the more common name "Coronavirus" refer to one family of viruses, named after the large spike protein molecules that are present on the surface of the virus and give the virion a crown-like shape. The Coronaviridae family includes the following four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. Coronaviruses represent a diverse family of large enveloped positive-strand RNA viruses that infect a wide range of animals, multiple vertebrate species, and humans. The spike (S) protein of coronaviruses is essential for binding to host receptors and subsequent fusion of the viral membrane with the host cell membrane, resulting in the effective release of the viral nucleocapsid into the cytoplasm of the host cell (Letko et al. (2020) Nat Microbiol 5:562-569).

[0043] Four coronaviruses that are thought to have a zoonotic origin are endemic in humans: HCoV-NL63 and HCoV-229E (alpha-coronaviruses) and HCoV-OC43 and HCoV-HKU1 (beta-coronaviruses). Furthermore, three episodes of severe respiratory disease caused by beta-coronaviruses have occurred since 2000. The severe acute respiratory syndrome virus (SARS) caused by SARS-CoV-1 emerged from a zoonotic origin (through bats as an intermediate species) and disappeared in 2004 (Drosten et al. 2003, N Engl J Med 348:1967–1976). More than 8000 SARS cases were reported, and the mortality rate was approximately 10%. In 2012, Middle East respiratory syndrome (MERS) emerged in the Arabian Peninsula. MERS is caused by MERS-CoV, has been confirmed in more than 2500 cases, and has a case fatality rate of 34% (de Groot et al. 2013, N Engl J Virol 87:7790–7792). Cases of severe community-acquired pneumonia reported in Wuhan City (China) beginning in late 2019 were caused by a novel beta-coronavirus that is now known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) because of its genetic relatedness to SARS-CoV-1 and represents the emergence of the third zoonotic human coronavirus (Chen et al. (2020) Lancet 395:507–513). As with SARS-CoV and MERS-CoV infections, patients presented with symptoms of viral pneumonia, including fever, dyspnea, and in the most severe cases, bilateral lung infiltrates (Gralinski et al. (2020) Viruses 12:135).

[0044] As used herein, the term "sarbecovirus" refers to a subgenus within the genus Betacoronavirus and includes severe acute respiratory syndrome-related coronavirus species (SARS coronavirus, SARS-related coronavirus, and SARSr-CoV or SARS-CoV, also known as severe acute respiratory syndrome coronavirus, which are used interchangeably herein). Non-limiting examples of strains belonging to the SARS-CoV species include SARS-CoV-1 and SARS-CoV-2.

[0045] Based on the first available genomic sequence, the novel human pathogen SARS-CoV-2 was classified into the sarbecovirus subgenus of the Coronaviridae family, the same as the SARS virus. SARS-CoV-2 belongs to the same genus of Betacoronavirus as SARS-CoV (lineage B) and MERS-CoV (lineage C), but genomic analysis revealed a higher 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).

[0046] Among other betacoronaviruses, this virus is characterized by a unique combination of polybasic cleavage sites, which are distinct requirements known to enhance pathogenicity and infectivity. The bat sarbecovirus, Bat CoV RaTG13, sampled from Rhinolophus affinis bats, forms clusters with SARS-CoV-2 in almost all genomic regions and is reported to have approximately 96% genomic sequence identity (more than 93% similarity in the receptor-binding domain (RBD) of the spike protein), suggesting that another mammalian species may act as an intermediate host. One of the suspected intermediate hosts, the Malayan pangolin, harbors a coronavirus that contains mutations thought to facilitate binding to the angiotensin-converting enzyme 2 (ACE2) receptor and shows high similarity to SARS-CoV-2 in the receptor-binding domain, demonstrating 97% amino acid sequence similarity. Both SARS-CoV-1 and -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 distinguishable from SARS-CoV-1 and several SARS-related coronaviruses (SARSr-CoV) such as those reviewed in Abdelrahman et al. (2020. Front Immunol 11:552909).

[0047] SARS-CoV-2 refers to a newly emerged sarbecovirus that originated in Wuhan City, China and was identified as the cause of severe acquired pneumonia that occurred globally. The long-term global spread of SARS-CoV-2, along with the selective pressure of immune escape, has led to the adaptation of the virus to its host and the generation of new SARS-CoV-2 variants. Specifically, multiple mutations in the spike glycoprotein have evolved and are still evolving, including mutations located in the spike S1 subunit. For example, a SARS-CoV-2 variant can 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 in SEQ ID NO: 86). Further non-limiting examples of SARS-CoV-2 variants include SARS-CoV-2 variants containing a mutation at position N501, such as the N501Y variant (e.g., the SARS-CoV-2 alpha variant); SARS-CoV-2 variants containing mutations at positions N501 and E484, such as the N501Y and E484K variants (e.g., the SARS-CoV-2 alpha + E484K variant); SARS-CoV-2 variants containing mutations at positions K417, E484, and N501, such as the K417N, E484K, and N501Y variants (e.g., the SARS-CoV-2 beta variant); SARS-CoV-2 variants containing mutations at positions P384, K417, E484, and N501, such as the P384L, K417N, E484K, and N501Y variants (e.g., the SARS-CoV-2 beta + P384L variant); SARS-CoV-2 variants containing mutations at positions L452 and E484, such as the L452R and E484Q variants (e.g., the SARS-CoV-2 kappa variant); SARS-CoV-2 variants containing mutations at positions L452 and T478, such as the L452R and T478K variants (e.g., the SARS-CoV-2 delta variant); SARS-CoV-2 variants containing a mutation at position L452, such as the L452R variant (e.g., the SARS-CoV-2 epsilon variant);SARS-CoV-2 variants containing mutations at position K417, such as the K417T variant (e.g., SARS-CoV-2 gamma variant); SARS-CoV-2 variants containing mutations at position D614, such as the D614G variant (e.g., SARS-CoV-2 D614G variant, SARS-CoV-2 omicron BA.1 variant or SARS-CoV-2 omicron BA.2 variant); SARS-CoV-2 variants containing mutations at positions K147, W152R, F157, I210, G257, D339, G446 and N460, such as the 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 containing mutations at positions R346, F486 and D1199, such as the R346T, F486S and D1199N variants (e.g., SARS-CoV-2 omicron BA.2.75.2 variant); SARS-CoV-2 variants containing mutations at positions H69, V70, L452 and F486, such as the H69-, V70-, L452R and F486V variants (e.g., SARS-CoV-2 omicron BA.4 / BA.5 variant); SARS-CoV-2 variants containing mutations at positions R346 and N658, such as the R346T and N658S variants (e.g., SARS-CoV-2 omicron BA.4.6 variant); SARS-CoV-2 variants containing mutations at position R346, such as the R346T variant (e.g., SARS-CoV-2 omicron BF.7 variant); SARS-CoV-2 variants containing mutations at positions R346, K444 and N460, such as the R346T, K444T and N460K variants (e.g., SARS-CoV-2 omicron BQ.1.1 variant);SARS-CoV-2 variants containing mutations at positions V83, Y144, H146, Q183, V213, R346, L368, V445, G446, N460, F486, and F490, such as the V83A, Y144-, H146Q, Q183E, V213E, R346T, L368I, V445P, G446S, N460K, F486S, and F490S variants (e.g., the SARS-CoV-2 Omicron XBB variant) or the V83A, Y144-, H146Q, Q183E, V213E, R346T, L368I, V445P, G446S, N460K, F486P, and F490S variants (e.g., the SARS-CoV-2 Omicron XBB.1.5 variant). The Alpha variant of SARS-CoV-2 (also known as lineage B.1.1.1.7) was first detected in the United Kingdom in late 2020 and was one of the first SARS-CoV-2 variants of concern to be reported. It contained several mutations in the spike protein, including the N501Y and D614G mutations. The Omicron variant of SARS-CoV-2 was first identified 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 evolved into at least three lineages, including BA.1, BA.2, and BA.3. Up to 60 mutations have been identified in the BA.1 lineage, 38 of which are in the spike (S) protein, 1 in the envelope (E) protein, 2 in the membrane (M) protein, and 6 in the nucleocapsid (N) protein. The BA.2 lineage has 57 mutations, 31 of which are in the S protein, and the N terminus is significantly different from that of BA.1. The term "SARS-CoV-2" as used herein encompasses both the original strain identified in Wuhan and its variants.;

[0048] Binding agents, in particular antibodies and antibody fragments, specifically bind to or bind to spike proteins of coronaviruses such as (3) SARS-CoV-2 spike protein or SARS-CoV-1 spike protein. In particular, binding agents, in particular antibodies and antibody fragments, specifically bind to or bind to (4) the S2 subunit of the coronavirus spike protein, or a part of the S2 subunit. More specifically, binding agents, in particular antibodies and antibody fragments, specifically bind to or bind to the region of the S2 subunit located from amino acid E1188 to amino acid Y1206 of the SARS-CoV-2 spike protein defined in SEQ ID NO: 86, preferably the region located from amino acid N1192 to amino acid Y1206, or the region located from amino acid E1188 to amino acid L1203, more preferably the region located from amino acid N1192 to amino acid L1203, even more preferably the region located from amino acid N1194 to amino acid L1203, most preferably the region located from amino acid N1194 to amino acid Q1201, or bind within that range. In certain embodiments, binding agents, in particular antibodies and antibody fragments, specifically bind to or bind to (23) the region of the SARS-CoV-2 spike protein defined in SEQ ID NO: 86 from amino acid E1188 to amino acid Y1206, preferably from amino acid N1192 to amino acid Y1206, or from amino acid E1188 to amino acid L1203, more preferably from amino acid N1192 to amino acid L1203, even more preferably from amino acid N1194 to amino acid L1203, most preferably from amino acid N1194 to amino acid Q1201, or bind within that region, or specifically bind to or bind to the spike protein of the coronavirus corresponding to that region or the S2 subunit region of the coronavirus spike protein. More specifically, binding agents, in particular antibodies and antibody fragments, specifically bind to or bind to (5) the heptad repeat 2 (HR2) domain of the coronavirus spike protein (S2 subunit), or a part of the HR2 domain. In certain embodiments, binding agents, in particular antibodies and antibody fragments, specifically bind to or bind to (6) the region of the HR2 domain proximal to the viral membrane.Preferably binds or binds specifically to the region of the SARS-CoV-2 spike protein defined by SEQ ID NO: 86, located from amino acid A1174 to amino acid E1202, more preferably the region located from amino acid I1179 to amino acid E1202, even more preferably the region located from amino acid D1184 to amino acid E1202, still more preferably the region located from amino acid E1188 to amino acid E1202, or the region located from amino acid V1189 to amino acid E1202, yet more preferably the region located from amino acid N1194 to amino acid E1202, most preferably the region located from amino acid N1194 to amino acid Q1201, or binds or binds specifically within that region, or (7) the region of the HR2 domain (or of the S2 subunit) corresponding to the region from amino acid E1188 to amino acid Y1206 of the SARS-CoV-2 spike protein defined by SEQ ID NO: 86, preferably the region of the HR2 domain (or of the S2 subunit) corresponding to the region from amino acid E1188 to amino acid Y1203 of the SARS-CoV-2 spike protein defined by SEQ ID NO: 86, more preferably the region of the HR2 domain (or of the S2 subunit) corresponding to the region from amino acid A1190 to amino acid L1203 of the SARS-CoV-2 spike protein defined by SEQ ID NO: 86, for example, the region of the HR2 domain (or of the S2 subunit) corresponding to the region from amino acid K1191 to amino acid E1202 of the SARS-CoV-2 spike protein defined by SEQ ID NO: 86; or the region of the HR2 domain (or of the S2 subunit) 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 (for example, the region from amino acid N1192 to amino acid Q1201 of the SARS-CoV-2 spike protein defined by SEQ ID NO: 86), even more preferably the region of the HR2 domain (or of the S2 subunit) corresponding to the region from amino acid N1194 to amino acid L1203 of the SARS-CoV-2 spike protein defined by SEQ ID NO: 86, most preferably,The region of the HR2 domain (or the S2 subunit) corresponding to the region from amino acid N1194 to amino acid Q1201 of the SARS-CoV-2 spike protein defined in SEQ ID NO: 86, for example, the region of the HR2 domain (or the S2 subunit) corresponding to the region from amino acid S1196 to amino acid Q1201 of the SARS-CoV-2 spike protein defined in SEQ ID NO: 86, specifically binds to or binds to, or specifically binds to or binds within that region. In certain embodiments, the binding agent, particularly an antibody and antibody fragment, (8) 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 of 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 of 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 of the SARS-CoV-2 spike protein defined in SEQ ID NO: 86, most preferably specifically binds to or binds to at least one or both of the amino acid residues S1196 and Q1201 of the SARS-CoV-2 spike protein defined in SEQ ID NO: 86. In embodiments, the binding agent, particularly an antibody and antibody fragment, (24) 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 spike protein of the sarbecovirus or the S2 subunit or HR2 domain of the sarbecovirus corresponding to the amino acid residues N1192, N1194, S1196, L1197, D1199, L1200, Q1201, and E1202 of 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 of 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 of the SARS-CoV-2 spike protein defined in SEQ ID NO: 86, most preferably, specifically binds to or binds to at least one or both of the amino acid residues corresponding to amino acid residues S1196 and Q1201 of the SARS-CoV-2 spike protein defined in SEQ ID NO: 86. In certain embodiments, the binding agent, particularly an antibody and antibody fragment, (25) specifically binds to or binds to amino acid residues S1196 and Q1201 of the SARS-CoV-2 spike protein defined in SEQ ID NO: 86, or amino acid residues of the spike protein corresponding to said amino acid residues S1196 and Q1201 of the SARS-CoV-2 spike protein defined in SEQ ID NO: 86, optionally, amino acid residues N1194, S1196, D1199 and Q1201 of the SARS-CoV-2 spike protein defined in SEQ ID NO: 86, or amino acid residues of the spike protein corresponding to said amino acid residues N1194, S1196, D1199 and Q1201 of the SARS-CoV-2 spike protein defined in SEQ ID NO: 86.

[0049] In certain embodiments, 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, and Q1201 and E1202 of 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 of 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 of the SARS-CoV-2 spike protein defined in SEQ ID NO: 86, and most preferably at least one or both of the amino acid residues S1196 and Q1201 of the SARS-CoV-2 spike protein defined in SEQ ID NO: 86 are essential for the binding of binders, particularly antibodies and antibody fragments, to the spike protein.In an embodiment, the amino acid residues N1192, N1194, S1196, L1197, D1199, L1200, Q1201, and E1202 of the SARS-CoV-2 spike protein defined by SEQ ID NO: 86, or 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 spike protein of the sarbecovirus corresponding thereto, or the S2 subunit or the HR2 domain of the sarbecovirus spike protein, preferably at least one, at least two, at least three, at least four, or all of the amino acid residues corresponding to the amino acid residues N1194, S1196, D1199, Q1201, and E1202 of the SARS-CoV-2 spike protein defined by SEQ ID NO: 86, more preferably at least one, at least two, at least three, or all of the amino acid residues corresponding to the amino acid residues N1194, S1196, D1199, and Q1201 of the SARS-CoV-2 spike protein defined by SEQ ID NO: 86, and most preferably at least one or both of the amino acid residues corresponding to the amino acid residues S1196 and Q1201 of the SARS-CoV-2 spike protein defined by SEQ ID NO: 86 are essential for the binding of binders to the spike protein, particularly antibodies and antibody fragments. In a particular embodiment, (28) the amino acid residues S1196 and Q1201 of the SARS-CoV-2 spike protein defined by SEQ ID NO: 86, or the amino acid residues of the spike protein corresponding to the amino acid residues S1196 and Q1201 of the SARS-CoV-2 spike protein defined by SEQ ID NO: 86, optionally the amino acid residues N1194, S1196, D1199, and Q1201 of the SARS-CoV-2 spike protein defined by SEQ ID NO: 86, or the amino acid residues of the spike protein corresponding to the amino acid residues N1194, S1196, D1199, and Q1201 of the SARS-CoV-2 spike protein defined by SEQ ID NO: 86 are essential for the binding of binders to the spike protein, particularly antibodies and antibody fragments.

[0050] The evaluation of the binding site can be carried out by determining the crystal structure of a complex of a binder, in particular an antibody or antibody fragment, with a spike protein, or an S2 subunit, or a peptide containing the HR2 domain, for example by applying the crystal structure determination method shown in the examples, and / or by selecting and analyzing viral escape variants / mutants, for example by applying the viral escape selection method shown in the examples, and / or by analyzing hydrogen-deuterium exchange for recombinant spike protein (or S2 subunit or HR2-containing peptide) in the presence and absence of the binder, for example by applying the hydrogen-deuterium exchange method monitored by mass spectrometry (HDX-MS method) shown in the examples.

[0051] Advantageously, these amino acid residues are conserved among simian betacoronaviruses of different clades, particularly among simian betacoronaviruses of clades 1, 2, and 3. In a preferred embodiment, the binder, in particular an antibody or antibody fragment, does not bind to the RBD of the (9) simian betacoronavirus spike protein.

[0052] Binders, particularly antibodies and antibody fragments (29), specifically bind or bind to quaternary epitopes of the spike protein. In particular, binders, particularly antibodies and antibody fragments, specifically bind or bind to (30) the trimeric HR2 domain (or trimeric S2 subunit or trimeric spike protein). In particular, binders, particularly antibodies and antibody fragments, specifically bind or bind to (31) a quaternary epitope within the trimeric HR2 domain (or trimeric S2 subunit or trimeric spike protein). More specifically, binders, particularly antibodies and antibody fragments, specifically bind or bind to (32) a quaternary epitope located within two adjacent HR2 domains or helices. In certain embodiments, binders, particularly antibodies and antibody fragments, specifically bind or bind to (33) a quaternary epitope comprising or consisting of one or more interacting amino acid residues described herein within one HR2 domain or helix and one or more interacting amino acid residues described herein within an adjacent HR2 domain or helix. In certain embodiments, binders, particularly antibodies and antibody fragments, specifically bind or bind to (34) a quaternary epitope within the trimeric spike protein, wherein amino acid residues described herein from at least two, such as two monomers of the trimeric spike protein, particularly one or more interacting amino acid residues, contribute to said quaternary epitope.

[0053] As used herein, the term "quaternary epitope" refers to a structural 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 within a single protein (or monomer) of a multimeric complex or may span multiple protomers and be newly formed by their interaction.

[0054] Specific binding or association to a quaternary epitope or multimeric protein can be evaluated by applying, for example, the ELISA assay shown in the examples, by evaluating the binding to monomeric and / or (stabilized) multimeric proteins by an enzyme-linked immunosorbent assay (ELISA). 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. The correlation between the binding to monomeric protein and the density of monomeric protein alone, such as a density of 1.0 ng / mm 2 or higher, preferably 1.2 ng / mm 2 or higher, or 1.5 ng / mm 2 or higher, can indicate specific binding or association to the multimeric structure of the protein. For a given density of monomeric and multimeric proteins, if the binding to the multimeric protein is enhanced compared to the monomeric protein, it can indicate specific binding or association to the multimeric protein.

[0055] While not wishing to be bound by any theory, binding agents described herein, particularly antibodies and antibody fragments, can stabilize the prefusion structure of the spike protein when they bind to the trimeric spike protein, particularly to quaternary epitopes within the trimeric spike protein. More particularly, the binding agent can stabilize or lock the HR2 coiled coil. Thus, the binding agent can prevent the unraveling of the HR2 coiled coil, which is considered an important initial step in the spike-controlled membrane fusion process; or the binding agent can interfere with or block the movement of the HR2 alpha helix to the extended HR1 alpha helix, which is considered an important step in the refolding of the spike protein from the prehairpin intermediate to the postfusion structure; and / or the binding agent can prevent the completion of the six-helix bundle formation, which is considered extremely important for the fusion process. In embodiments, the binding agent, particularly antibodies and antibody fragments (35), can stabilize the prefusion structure of the spike protein of the SARS-CoV virus. In embodiments, the binding agent, particularly antibodies and antibody fragments (36), can stabilize the HR2 coiled coil. SARS-CoV-2 contains, as structural proteins, the spike (S) protein, envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein. Further, 16 non-structural proteins (nsp1-16) have been identified, and these are involved in replication and modification of host defense. The Nsp12 protein corresponds to the RNA-dependent RNA polymerase (RdRp).

[0056] The spike protein or S protein, which is a transmembrane glycoprotein that protrudes from the virus surface and forms homotrimers that give the virus a crown-like appearance, is of particular interest in the present invention. The spike protein has two subunits: S1 and S2.

[0057] The S1 subunit contains an N-terminal domain (NTD), a 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) via the receptor-binding domain (RBD) present in the S1 subunit.

[0058] The S2 subunit is involved in the fusion of the viral and host cell membranes and viral entry, and contains multiple domains: the S2’ protease cleavage site (cleavage by host proteases required for fusion), the fusion peptide (FP), the heptad repeat 1 (HR1) domain, the central helix (CH) domain, the connector domain (CD), the heptad repeat 2 (HR2) domain, the transmembrane (TM) domain, and the cytoplasmic tail (CT) domain (Wang et al. (2020). Front Cell Infect Microbiol 10:587269).

[0059] The S protein usually exists in a prefusion structure. In the prefusion conformation, S1 and S2, which are cleaved at the S1-S2 furin cleavage site during biosynthesis, remain non-covalently bound to each other, different from SARS-CoV in which S1 and S2 remain uncleaved. In the closed state of the S protein (PDB: 6VXX), the three RBD domains in the trimer do not protrude from the trimer, but in the open state (PDB: 6VYB), i.e., the “up” structure, one of the RBDs protrudes from the trimer. The length of the S-trimer ectodomain with a triangular cross-section is approximately 160 angstroms, while the S1 domain is in a V-shape. It seems that 16 out of 22 N-linked glycosylation sites per protomer are glycosylated (Walls et al (2020) Cell 180:281~292).

[0060] The S1 subunit of the S protein binds to ACE2 via its RBD region, promotes the formation of endosomes, and induces viral fusion activity. After S1-ACE2 binding, S is cleaved by intracellular proteases such as transmembrane protease serine subtype 2 (TMPRSS2) or endosomal cathepsin, exposing the fusion peptide (FP) located in the S2 subunit. The FP inserts into the host cell membrane, thereby reducing the distance between the viral membrane and the host cell membrane. The HR1 domain of the S protein is very proximal to the host cell membrane, while the HR2 domain is closer to the viral membrane side. Subsequently, HR2 folds back onto HR1, whereby the two HR domains form a six-helix structure in an antiparallel form of the fusion core. The viral membrane binds tightly while being pulled towards the host cell membrane, and the two membranes fuse to release the viral genome into the host cell (Huang et al. (2020) Acta Pharmalogica Sinica 41: 1141-1149).

[0061] As used herein as synonyms, the terms "spike protein", "S", or "S protein" refer to the spike protein of the sarbecovirus, and can refer to specific S proteins such as the SARS-CoV-2 S protein and the SARS-CoV-1 S protein. The terms "spike protein" and "SARS-CoV-2 spike protein" include protein variants of the sarbecovirus or SARS-CoV-2 spike protein isolated from different sarbecoviruses or SARS-CoV-2 isolates, and recombinant sarbecovirus or SARS-CoV-2 spike proteins, or fragments thereof. The terms also include, for example, a sarbecovirus spike protein or SARS-CoV-2 spike protein connected to a histidine tag, mouse or human Fc, or a signal sequence.

[0062] The sequence of the SARS-CoV-2 spike protein is found under Genbank accession number QHQ82464, version QHQ82464.1, and corresponds to or is defined herein as SEQ ID NO: 86:

Chem.

[0063] Here, the SARS-CoV-2 spike protein HR2 domain corresponds to amino acids 1169-1202 of SEQ ID NO: 86 and is as follows (SEQ ID NO: 87): ISGINASVVNIQKEIDRLNEVAKNLNESLIDLQE (SEQ ID NO: 87) as shown in.

[0064] Here, the SARS-CoV-2 spike protein TM domain corresponds to amino acids 1214-1237 of SEQ ID NO: 86.

[0065] As used herein, the "proximal to the viral membrane" region of the HR2 domain refers to the region within the HR2 domain that is within 40 amino acids of the viral membrane.

[0066] The sequence of the SARS-CoV-1 spike protein can be found under Genbank accession number NP_828851.1 and corresponds to or is defined herein as SEQ ID NO: 111. Here, the SARS-CoV-1 spike protein HR2 domain corresponds to amino acids 1151-1184 of SEQ ID NO: 111 and is shown in SEQ ID NO: 87. The amino acids and amino acid numbering referred to herein are for the SARS-CoV-2 spike protein defined in SEQ ID NO: 86, and the corresponding amino acids in other sarbecovirus spike proteins or spike protein fragments, domains or regions can be readily determined by aligning multiple amino acid sequences.

[0067] As used interchangeably herein, "angiotensin-converting enzyme 2", "ACE2" or "ACE-2" refers to a mammalian protein belonging to the family of dipeptidyl carboxydipeptidases and may be classified as EC:3.4.17.23. The genomic location of the human ACE2 gene is on chrX:15,561,033-15,602,158 (GRCh38 / hg38; minus strand), or chrX:15,579,156-15,620,271 (GRCh37 / hg19; minus strand). ACE2 acts as a receptor for at least the human coronaviruses SARS-CoV, SARS-CoV-2, and NL63 / HCoV-NL63 (also known as the New Haven coronavirus). UniProtKB identifier for the human ACE2 protein: Q9BYF1. Isoform 1 (identifier: Q9BYF1-1) has been selected as the canonical sequence. Reference DNA sequence of the human ACE2 gene in GenBank: NC_000023.11. Reference mRNA sequences of human ACE2 in GenBank NM_001371415.1 and NM_021804.3.

[0068] A further functional feature of the binders described herein, particularly antibodies and antibody fragments, is that they are capable of (10) neutralizing sarbecoviruses, particularly (11) being capable of neutralizing either or both of SARS-CoV-2 and SARS-CoV-1, preferably both.

[0069] As used herein, "neutralizing binder" or "neutralizing antibody" (or "binder or antibody capable of neutralizing sarbecovirus, particularly SARS-CoV-2 and / or SARS-CoV-1") refers to a binder or antibody that binds to sarbecovirus, particularly SARS-CoV-2 and / or SARS-CoV-1, and inhibits or suppresses the ability of sarbecovirus, or SARS-CoV-2 or SARS-CoV-1, to initiate and / or perpetuate infection in a host. A neutralizing binder or antibody can, for example, interfere with binding to a host receptor of sarbecovirus such as SARS-CoV-2 or SARS-CoV-1, particularly ACE2; and / or, for example, interfere with virus entry by inducing shedding of S1 and / or by interfering with virus fusion. Currently, it is not fully clear how the binders and antibodies according to the present invention neutralize, inhibit, block or suppress sarbecovirus infection. In certain embodiments, the binders described herein, particularly antibodies and antibody fragments, do not modulate or interfere with (44) shedding of S1. In certain embodiments, the binders described herein, particularly antibodies and antibody fragments, do not induce (12) shedding of S1. In certain embodiments, the binder, particularly the antibody and antibody fragment, does not prevent (45) shedding of S1. In certain embodiments, the binders described herein, particularly antibodies and antibody fragments, are capable of inhibiting (13) spike-mediated syncytium formation. As a result, the binder, particularly the antibody and antibody fragment, may be capable of inhibiting (14) virus fusion, and without wishing to be bound by any theory, for this reason, it may be possible that the sarbecovirus cannot complete the infection process of the host cell. In certain embodiments, the binders described herein, particularly antibodies and antibody fragments, do not prevent (46) unfolding of HR1. In certain embodiments, the binders described herein, particularly antibodies and antibody fragments, do not interfere with (47) folding of HR1 onto HR2 (such as during the formation of the six-helix bundle of S2). Irrespective of their mechanism of action, the binders according to the present invention, particularly antibodies and antibody fragments, are capable of strongly neutralizing sarbecovirus infection.

[0070] Neutralizing 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 tests. Exemplary methods for performing such neutralization assays are described in the examples herein. Neutralizing activity can also be evaluated by measuring syncytium formation between cells expressing the sarbecovirus spike protein and cells expressing the sarbecovirus receptor ACE2, which are one or more indicators of sarbecovirus, or SARS-CoV-2 or SARS-CoV-1 infection.

[0071] In certain embodiments, the binding agent, particularly an antibody and antibody fragment, has a half-maximal inhibitory concentration or 50% inhibitory concentration (IC 50 ) of 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, and is capable of neutralizing sarbecovirus, particularly SARS-CoV-2 and / or SARS-CoV-1. These concentrations are preferably determined in a sarbecovirus spike protein pseudovirus neutralization assay such as a vesicular stomatitis virus (VSV)-sarbecovirus spike protein pseudovirus neutralization assay, and more preferably 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 spike protein of a sarbecovirus such as the SARS-CoV-2 spike protein, a SARS-CoV-2 variant spike protein, or the SARS-CoV-1 spike protein. As used herein in connection with the neutralizing activity of a binding agent or antibody, "50% inhibitory concentration" or "IC 50" refers to an amount such as the concentration of a binder or antibody required for 50% neutralization of the Salvaco virus.

[0072] In certain embodiments, the binder, particularly an antibody and antibody fragment, is at least one SARS-CoV-2 variant, particularly 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, including mutations at the D614 position (with respect to the SARS-CoV-2 spike amino acid sequence defined in SEQ ID NO: 86), such as the (16)D614G variant, and is preferably capable of neutralizing any one or more, preferably all of them. In certain embodiments, the binder, particularly the antibodies and antibody fragments described herein, preferably has an IC determined in a SARS-CoV-2 variant spike pseudovirus neutralization assay, such as the VSV-SARS-CoV-2 variant spike pseudovirus neutralization assay, of 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. 50and is capable of neutralizing (17) the SARS-CoV-2 alpha variant, (18) the SARS-CoV-2 omicron BA.1 variant, (19) the SARS-CoV-2 omicron BA.2 variant, (37) the SARS-CoV-2 omicron BA.5 variant, (38) the SARS-CoV-2 omicron BA.2.75.2 variant, (39) the SARS-CoV-2 omicron BA.4.6 variant, (40) the SARS-CoV-2 omicron BF.7 variant, (41) the SARS-CoV-2 omicron BQ.1.1 variant, (42) the SARS-CoV-2 omicron XBB variant, and / or (43) the SARS-CoV-2 omicron XBB.1.5 variant.

[0073] The binding agents described herein, particularly antibodies and antibody fragments, are further characterized in that they are capable of inhibiting (14) virus fusion. In certain embodiments, the binding agents described herein, particularly antibodies and antibody fragments, are capable of inhibiting (13) spike-mediated syncytia formation, and more particularly, they are capable of inhibiting syncytia formation between cells expressing sarbecovirus spike proteins such as (20) SARS-CoV-2 and / or SARS-CoV-1 spike proteins and cells expressing sarbecovirus host receptors, particularly the ACE2 receptor.

[0074] As used herein, "viral fusion" refers to the fusion of the viral membrane and 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. As will be apparent to those skilled in the art, complete inhibition is not required, and those skilled in the art can identify binders, antibodies, and antibody fragments that significantly inhibit viral fusion or spike-mediated syncytium formation. Preferably, the binders, particularly antibodies and antibody fragments, described herein can induce at least 50% inhibition, preferably at least 60%, at least 70%, at least 80% or at least 90% inhibition.

[0075] In certain embodiments, some of the functional characteristics of the sarbecovirus binders, particularly sarbecovirus antibodies or antibody fragments, described above herein are combined to characterize such binders, antibodies or antibody fragments, for example, bind or specifically bind to the HR2 domain of the sarbecovirus spike protein and are capable of neutralizing at least one or both of sarbecovirus, particularly SARS-CoV-2 (SARS-CoV-2 Wuhan strain, 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, and preferably, when determined in a vesicular stomatitis virus (VSV)-sarbecovirus spike protein pseudovirus neutralization assay, the 50% inhibitory concentration (IC 50) can neutralize the SARS-CoV at 100 ng / ml or less, preferably 10 ng / ml or less, more preferably 1 ng / ml or less. Such a binder, antibody or antibody fragment can inhibit spike-mediated syncytium formation between cells expressing the SARS-CoV spike protein and cells expressing the angiotensin-converting enzyme 2 (ACE2) receptor, and / or can inhibit virus fusion, and / or can be further characterized by not binding to Middle East respiratory syndrome coronavirus (MERS-CoV).

[0076] The binder described herein can also be structurally defined as a polypeptidic binder (i.e., a binder containing a peptidic, polypeptidic or proteinaceous moiety, or a binder containing a peptide, polypeptide, protein or protein domain) or a polypeptide binder (i.e., the binder is a peptide, polypeptide or protein).

[0077] The terms "protein", "polypeptide", and "peptide" are used interchangeably herein to refer to polymers of amino acid residues, as well as variants and synthetic analogs thereof, and the sequential linear arrangement of amino acids together results in / forms an "amino acid sequence" or "protein sequence". A "peptide" may also be referred to, for example, as a partial amino acid sequence derived from the original protein after enzymatic (e.g., trypsin) digestion. These terms apply not only to naturally occurring amino acid polymers but also to 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. Also included are proteins that include one or more post-translational modifications such as addition of functional groups or covalent attachment to the protein (e.g., glycosylation, phosphorylation, acetylation, ubiquitination, methylation, lipidation, and nitrosylation), or proteolytic processing. Based on the amino acid sequence and modifications, the atomic mass or weight or molecular mass or weight of a polypeptide is expressed in (kilo)daltons (kDa). Further modifications of proteins include the addition of tags such as His tags and sortags. For example, a multi-armed PEG nanobody that neutralizes SARS-CoV2 was constructed by sortagging (sortase-mediated peptide transfer; Popp et al. 2007, Nat Chem Biol 3:707-708) (Moliner-Morro et al. 2020, Biomolecules 10:1661).

[0078] A "protein domain" is a distinct functional and / or structural unit within a protein, or a distinct functional and / or structural part of a protein. Typically, a protein domain is responsible for a specific function or interaction and contributes to the overall (biological) role of the protein. Domains can exist in a variety of biological contexts, and similar domains may be found in different proteins with similar or different functions. A protein domain may have a rigid three-dimensional structure, for example, when constrained by several intramolecular cysteines (such as cysteine knot proteins), or may adopt different 3D structures depending on, for example, the presence or absence of a bound ligand or the presence or absence of, for example, post-translational modifications, or may have a more fluid 3D structure that is less well-defined.

[0079] Amino acids are indicated herein by the defined three-letter or one-letter code nomenclature and are also given as follows in the IUPAC-IUB Joint Commission on Biochemical Nomenclature (Nomenclature and Symbolism for Amino Acids and Peptides. Eur. J. Biochem. 138:9-37 (1984)): 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 Gln), 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).

[0080] More specifically, the binder described herein can be structurally defined as a polypeptide or polypeptide binder that includes a complementarity determining region (CDR) contained in any of the immunoglobulin single variable domains (ISVDs) as defined herein. In preferred embodiments, the polypeptide or polypeptide binder is an (isolated) antibody or antibody fragment.

[0081] In certain embodiments, the binder according to the invention, particularly antibodies and antibody fragments, can be structurally defined as a polypeptide or polypeptide binder, particularly antibodies and antibody fragments, that includes at least CDR3 contained in the immunoglobulin single variable domain (ISVD) as defined herein. In other embodiments, the binder according to the invention, particularly antibodies and antibody fragments, can be structurally defined as a polypeptide or polypeptide binder, particularly antibodies and antibody fragments, that includes at least two (e.g., CDR1 and CDR3, CDR2 and CDR3, CDR1 and CDR2), or all three of CDR1, CDR2, and CDR3 contained in the immunoglobulin single variable domain (ISVD) as defined herein. Such CDRs may be included in any of VHH R3_C4 (defined / described by SEQ ID NO: 1), VHH R4_DC16 (defined / described by SEQ ID NO: 2), VHH R3_DC20 (defined / described by SEQ ID NO: 3), VHH R3_DC2 (defined / described by SEQ ID NO: 4), VHH R4_DC20 (defined / described by SEQ ID NO: 5), VHH R4_DC9 (defined / described by SEQ ID NO: 6), VHH R4_DC6 (defined / described by SEQ ID NO: 7), VHH R3_DC23 (also referred to herein as VHH R3DC23 or R3DC23; defined / described by SEQ ID NO: 8), VHH R3_DC9 (defined / described by SEQ ID NO: 9), or VHH R4_DC13 (defined / described by SEQ ID NO: 10) as shown below in this specification:

[0082] VHH R3_C4:

Chem.

[0083] VHH R4_DC16:

Chem.

[0084] VHH R3_DC20:

Chem.

[0085] VHH R3_DC2:

Chem.

[0086] VHH R4_DC20:

Chem.

[0087] VHH R4_DC9:

Chem.

[0088] VHH R4_DC6:

Chem.

[0089] VHH R3_DC23:

Chem.

[0090] VHH R3_DC9:

Chem.

[0091] VHH R4_DC13:

Chem.

[0092] For the numbering of amino acid residues of any IVD or ISVD, different numbering schemes may be applied. For example, for all heavy chain (VH) and light chain variable domains (VL) given by Honegger & Pluckthun (2001. J Mol Biol 309: 657-70), as applied to the VHH domain of camelids, numbering can be carried out according to the AHo numbering scheme. Another method for numbering the amino acid residues of the VH domain can also be applied to the VHH domain in a similar manner and is known in the art. For example, by using the Kabat numbering system applied to the VHH domain of camelids by Riechmann and Muyldermans (1999. J Immunol Methods 231: 25-38), the FR and CDR sequences can be depicted. As is well known in the art for VH domains and VHH domains, the total number of amino acid residues in each CDR can vary and may not match the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions according to Kabat numbering may not be occupied by the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering). It should be noted that this generally means that 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 domains and VHH domains is usually in the range of 110-120, and is often 112-115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0093] The determination of CDR regions in antibody / immunoglobulin sequences generally depends on the applied algorithm / methodology. For example, the determination of CDR regions can be made according to the contact analysis and binding site topography described in MacCallum et al. (J. Mol. Biol. (1996) 262, pp. 732-745), AbM (AbM is an antibody modeling package of Oxford Molecular Ltd. and is 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; shown at 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 further includes the description of CDR and framework regions (FR) in immunoglobulin domain-containing proteins, and thus is a method and system known to those skilled in the art that can apply these annotations on any antibody / immunoglobulin protein sequence without undue burden. As an example, FIG. 20 illustrates different annotation schemes or methods applied to the amino acid sequence of VHH R3_DC23 (SEQ ID NO: 8).

[0094] Applying different methods to the same antibody / immunoglobulin sequence may result in different CDR amino acid sequences, and the differences may exist in the length of the CDR sequence and / or the depiction of the CDR sequence within the antibody / immunoglobulin / IVD sequence (as exemplified in Figure 20 for VHH R3_DC23). Thus, the CDRs of the ISVD binders described herein, particularly antibodies and antibody fragments, can be described as the CDR sequences present in the ISVD characterized herein. Alternatively, these CDRs can be determined or described as the CDR sequences present in the (herein described) ISVD according to well-known methodologies, for example, according to any one of the Kabat, Martin, Chothia, aHo, MacCallum et al. 1996, AbM, or IMGT numbering schemes or methods, preferably, for example, according to the Martin numbering scheme or method.

[0095] VHHs or Nbs are often classified into different families or even further into superfamilies according to their amino acid sequences, clustering clonally related sequences derived from the same progenitor cells during B cell maturation (Deschaght et al. 2017, Front Immunol 8:420). This classification is often based on the CDR sequences of the VHHs or Nbs. For example, each VHH or Nb family is defined as a cluster of (clonally) related sequences having a sequence identity threshold in the CDR3 region. Within a single VHH family as defined herein, the CDR3 sequences are thus 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, resulting in VHHs or Nbs of the same family that bind to the same binding site and have the same effects such as functional effects.

[0096] As outlined above, there are many systems or methods (Kabat, MacCallum, IMGT, AbM, Chothia, Martin) for numbering amino acids in immunoglobulin protein sequences, including the depiction of CDRs and framework regions (FRs) in these protein sequences. These systems or methods are known to those skilled in the art, and thus, those skilled in the art can apply these systems or methods to any immunoglobulin protein sequence without undue burden (as exemplified in FIG. 20 for VHH R3_DC23).

[0097] In certain embodiments, a binder described herein, particularly an antibody or antibody fragment, more particularly an ISVD, may be characterized by comprising a CDR1 defined by or described in any one of SEQ ID NO: 63, 46, 69, or 77. In certain embodiments, a binder described herein, particularly an antibody or antibody fragment, more particularly an ISVD, may be characterized by comprising a CDR2 defined by or described in any one of SEQ ID NO: 64, 47, 70, 73, or 78. In certain embodiments, a binder described herein, particularly an antibody or antibody fragment, more particularly an ISVD, may be characterized by comprising a CDR3 defined by or described in any one of SEQ ID NO: 48, 67, 74, or 79. In certain embodiments, a binder described herein, particularly an antibody or antibody fragment, more particularly an ISVD, may be characterized by comprising a CDR1 defined by or described in any one of SEQ ID NO: 63, 46, 69, or 77, a CDR2 defined by or described in any one of SEQ ID NO: 64, 47, 70, 73, or 78, and a CDR3 defined by or described in any one of SEQ ID NO: 48, 67, 74, or 79.

[0098] In certain embodiments, a binder described herein, particularly an antibody or antibody fragment, more particularly an ISVD, may comprise: - CDR1 defined by / described in SEQ ID NO: 63; CDR2 defined by / described in SEQ ID NO: 64; and CDR3 defined by / described in SEQ ID NO: 67; or - CDR1 defined by / described in SEQ ID NO: 69; CDR2 defined by / described in SEQ ID NO: 70; and CDR3 defined by / described in SEQ ID NO: 67; or - CDR1 defined by / described in SEQ ID NO: 63; CDR2 defined by / described in SEQ ID NO: 64; and CDR3 defined by / described in SEQ ID NO: 48; or - CDR1 defined by / described in SEQ ID NO: 46; CDR2 defined by / described in SEQ ID NO: 47; and CDR3 defined by / described in SEQ ID NO: 48; or - CDR1 defined by / described in SEQ ID NO: 63; CDR2 defined by / described in SEQ ID NO: 73; and CDR3 defined by / described in SEQ ID NO: 74; or - CDR1 defined by / described in SEQ ID NO: 77; CDR2 defined by / described in SEQ ID NO: 78; and CDR3 defined by / described in SEQ ID NO: 79.

[0099]

Table 1-1

[0100]

Table 1-2

[0101] In certain embodiments, the binding agents described herein, particularly antibodies or antibody fragments, more particularly ISVD, may be characterized by comprising a CDR1 defined by or described in any one of SEQ ID NO: 65, 71, 49 or 80. In certain embodiments, the binding agents described herein, particularly antibodies or antibody fragments, more particularly ISVD, may be characterized by comprising a CDR2 defined by or described in any one of SEQ ID NO: 66, 72, 50, 75 or 81. In certain embodiments, the binding agents described herein, particularly antibodies or antibody fragments, more particularly ISVD, may be characterized by comprising a CDR3 defined by or described in any one of SEQ ID NO: 51, 68, 76 or 82. In certain embodiments, the binding agents described herein, particularly antibodies or antibody fragments, more particularly ISVD, may be characterized by comprising a CDR1 defined by or described in any one of SEQ ID NO: 65, 71, 49 or 80, a CDR2 defined by or described in any one of SEQ ID NO: 66, 72, 50, 75 or 81, and a CDR3 defined by or described in any one of SEQ ID NO: 51, 68, 76 or 82.

[0102] In certain embodiments, the binding agents described herein, particularly antibodies or antibody fragments, more particularly ISVD, may comprise: - a CDR1 defined by or described in SEQ ID NO: 65; a CDR2 defined by or described in SEQ ID NO: 66; and a CDR3 defined by or described in SEQ ID NO: 68; or - a CDR1 defined by or described in SEQ ID NO: 71; a CDR2 defined by or described in SEQ ID NO: 72; and a CDR3 defined by or described in SEQ ID NO: 68; or - a CDR1 defined by or described in SEQ ID NO: 65; a CDR2 defined by or described in SEQ ID NO: 66; and a CDR3 defined by or described in SEQ ID NO: 51; or - CDR1 defined by / described in SEQ ID NO:49; CDR2 defined by / described in SEQ ID NO:50; and CDR3 defined by / described in SEQ ID NO:51; or - CDR1 defined by / described in SEQ ID NO:65; CDR2 defined by / described in SEQ ID NO:75; and CDR3 defined by / described in SEQ ID NO:76; or - CDR1 defined by / described in SEQ ID NO:80; CDR2 defined by / described in SEQ ID NO:81; and CDR3 defined by / described in SEQ ID NO:82.

[0103]

Table 2-1

[0104]

Table 2-2

[0105] In certain preferred embodiments, the binding agent or the Zaire ebolavirus binding agent described herein, particularly an antibody or antibody fragment or a Zaire ebolavirus antibody or antibody fragment, more particularly ISVD, may be characterized by comprising a CDR1 present in any of SEQ ID NOs: 1-10, wherein the CDR1 is annotated according to any one of Kabat, MacCallum, IMGT, AbM, Martin or Chothia. In certain preferred embodiments, the binding agent or the Zaire ebolavirus binding agent described herein, particularly an antibody or antibody fragment or a Zaire ebolavirus antibody or antibody fragment, more particularly ISVD, may be characterized by comprising a CDR2 present in any of SEQ ID NOs: 1-10, wherein the CDR2 is annotated according to any one of Kabat, MacCallum, IMGT, AbM, Martin or Chothia. In certain preferred embodiments, the binding agent or the Zaire ebolavirus binding agent described herein, particularly an antibody or antibody fragment or a Zaire ebolavirus antibody or antibody fragment, more particularly ISVD, may be characterized by comprising a CDR3 present in any of SEQ ID NOs: 1-10, wherein the CDR3 is annotated according to any one of Kabat, MacCallum, IMGT, AbM, Martin or Chothia. In certain preferred embodiments, the binding agent or the Zaire ebolavirus binding agent described herein, particularly an antibody or antibody fragment or a Zaire ebolavirus antibody or antibody fragment, more particularly ISVD, may be characterized by comprising CDR1, CDR2 and CDR3 each independently present in any 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.

[0106] In certain preferred embodiments, the binders or Ebola virus binders described herein, particularly antibodies or antibody fragments or Ebola virus antibodies or antibody fragments, and more particularly ISVD, may be characterized by including a combination of CDR1, CDR2, and CDR3, where CDR1, CDR2, and CDR3 are present in a specific one of the sequences set forth in SEQ ID NOs: 1-10, and CDR1, CDR2, and CDR3 are annotated according to any one of Kabat, MacCallum, IMGT, AbM, Martin, or Chothia.

[0107] In certain embodiments, the binders described herein, particularly antibodies or antibody fragments, and more particularly ISVD, may be characterized by including a CDR1 defined by or described in any one of SEQ ID NOs: 52, 53, 54, 11, or 12. In certain embodiments, the binders described herein, particularly antibodies or antibody fragments, and more particularly ISVD, may be characterized by including a CDR2 defined by or described in any one of SEQ ID NOs: 55-62 or 13-20. In certain embodiments, the binders described herein, particularly antibodies or antibody fragments, and more particularly ISVD, may be characterized by including a CDR3 defined by or described in any one of SEQ ID NOs: 21-27. In certain embodiments, the binders described herein, particularly antibodies or antibody fragments, and more particularly ISVD, may be characterized by including a CDR1 defined by or described in any one of SEQ ID NOs: 52, 53, 54, 11, or 12, a CDR2 defined by or described in any one of SEQ ID NOs: 55-62 or 13-20, and a CDR3 defined by or described in any one of SEQ ID NOs: 21-27.

[0108] In certain embodiments, the binders described herein, particularly antibodies or antibody fragments, and more particularly ISVD, may include the following: - CDR1 defined by / described in any one of SEQ ID NOs: 52 to 54; CDR2 defined by / described in any one of SEQ ID NOs: 55 to 62; and CDR3 defined by / described in any one of SEQ ID NOs: 21 to 27; or - CDR1 defined by / described in SEQ ID NO: 11 or 12; CDR2 defined by / described in any one of SEQ ID NOs: 13 to 20; and CDR3 defined by / described in any one of SEQ ID NOs: 21 to 27.

[0109]

Table 3

[0110] For example, a polypeptide or polypeptide binding agent described herein, particularly an antibody or antibody fragment, more particularly an ISVD, can be defined as comprising a complementarity determining region (CDR) present in any one of SEQ ID NOs: 1 to 10, where the CDR is defined according to Kabat. In certain embodiments, a binding agent, particularly an antibody or antibody fragment, more particularly an ISVD, comprises one of the following three sets of complementarity determining regions (CDRs): - CDR1 defined by / described in SEQ ID NO: 11; CDR2 defined by / described in SEQ ID NO: 13; and CDR3 defined by / described in SEQ ID NO: 21; or - CDR1 defined by / described in SEQ ID NO: 11, CDR2 defined by / described in SEQ ID NO: 13, and CDR3 defined by / described in SEQ ID NO: 22; or - CDR1 defined by / described in SEQ ID NO: 11, CDR2 defined by / described in SEQ ID NO: 13, and CDR3 defined by / described in SEQ ID NO: 23; or - CDR1 defined by / described in SEQ ID NO: 11, CDR2 defined by / described in SEQ ID NO: 14, and CDR3 defined by / described in SEQ ID NO: 23; or - CDR1 defined by / described in SEQ ID NO: 11, CDR2 defined by / described in SEQ ID NO: 12, and CDR3 defined by / described in SEQ ID NO: 23; or - CDR1 defined by / described in SEQ ID NO: 11; CDR2 defined by / described in SEQ ID NO: 16; and CDR3 defined by / described in SEQ ID NO: 23; or - CDR1 defined by / described in SEQ ID NO: 11, CDR2 defined by / described in SEQ ID NO: 17, and CDR3 defined by / described in SEQ ID NO: 24; - CDR1 defined by / described in SEQ ID NO: 11, CDR2 defined by / described in SEQ ID NO: 18, and CDR3 defined by / described in SEQ ID NO: 25; - CDR1 defined by / described in SEQ ID NO: 12, CDR2 defined by / described in SEQ ID NO: 19, and CDR3 defined by / described in SEQ ID NO: 26; - CDR1 defined by / described in SEQ ID NO: 11, CDR2 defined by / described in SEQ ID NO: 20, and CDR3 defined by / described in SEQ ID NO: 27.

[0111] In certain embodiments, the polypeptide or polypeptide binding agent described herein, particularly an antibody or antibody fragment, more particularly an ISVD, can be defined as including a complementarity determining region (CDR) present in any one of SEQ ID NOs: 1-10, where the CDR is defined according to Martin. In certain embodiments, the binding agent, particularly an antibody or antibody fragment, more particularly an ISVD, includes one of the following sets of three complementarity determining regions (CDRs): - CDR1 defined by / described in SEQ ID NO: 52; CDR2 defined by / described in SEQ ID NO: 55; and CDR3 defined by / described in SEQ ID NO: 21; or - CDR1 defined by SEQ ID NO:52 / described in SEQ ID NO:52, CDR2 defined by SEQ ID NO:55 / described in SEQ ID NO:55, and CDR3 defined by SEQ ID NO:22 / described in SEQ ID NO:22; or - CDR1 defined by SEQ ID NO:52 / described in SEQ ID NO:52, CDR2 defined by SEQ ID NO:55 / described in SEQ ID NO:55, and CDR3 defined by SEQ ID NO:23 / described in SEQ ID NO:23; or - CDR1 defined by SEQ ID NO:52 / described in SEQ ID NO:52, CDR2 defined by SEQ ID NO:57 / described in SEQ ID NO:57, and CDR3 defined by SEQ ID NO:23 / described in SEQ ID NO:23; or - CDR1 defined by SEQ ID NO:52 / described in SEQ ID NO:52, CDR2 defined by SEQ ID NO:58 / described in SEQ ID NO:58, and CDR3 defined by SEQ ID NO:23 / described in SEQ ID NO:23; or - CDR1 defined by SEQ ID NO:53 / described in SEQ ID NO:53, CDR2 defined by SEQ ID NO:60 / described in SEQ ID NO:60, and CDR3 defined by SEQ ID NO:25 / described in SEQ ID NO:25; or - CDR1 defined by SEQ ID NO:54 / described in SEQ ID NO:54; CDR2 defined by SEQ ID NO:61 / described in SEQ ID NO:61; and CDR3 defined by SEQ ID NO:26 / described in SEQ ID NO:26; - CDR1 defined by SEQ ID NO:53 / described in SEQ ID NO:53, CDR2 defined by SEQ ID NO:62 / described in SEQ ID NO:62, and CDR3 defined by SEQ ID NO:27 / described in SEQ ID NO:27; - CDR1 defined by SEQ ID NO:52 / described in SEQ ID NO:52, CDR2 defined by SEQ ID NO:56 / described in SEQ ID NO:56, and CDR3 defined by SEQ ID NO:23 / described in SEQ ID NO:23; - CDR1 defined by SEQ ID NO:52 / described in SEQ ID NO:52, CDR2 defined by SEQ ID NO:59 / described in SEQ ID NO:59, and CDR3 defined by SEQ ID NO:24 / described in SEQ ID NO:24.

[0112] In a further embodiment, the polypeptide or polypeptide binding agent according to the invention, in particular an antibody and antibody fragment, more particularly an ISVD, may comprise one or more framework regions (FRs) contained in any one of SEQ ID NOs: 1-10, or variants of such FRs. More particularly, such a binding agent, antibody or antibody fragment, or ISVD may each independently comprise at least one, for example one, two, three, or all of the FR1, FR2, FR3, and FR4 regions contained in any one of SEQ ID NOs: 1-10, or variants of such FRs. For example, such a binding agent, antibody or antibody fragment, or ISVD may comprise the FR1 and FR2 regions, FR1 and FR3 regions, FR1 and FR4 regions, FR2 and FR3 regions, FR2 and FR4 regions, FR3 and FR4 regions, FR1, FR2 and FR3 regions, FR1, FR2 and FR4 regions, FR2, FR3 and FR4 regions, or FR1, FR3 and FR4 regions contained in any one of SEQ ID NOs: 1-10, or variants of such FRs. In certain preferred embodiments, such a binding agent, antibody or antibody fragment, or ISVD comprises the FR1 region or FR4 region or FR2 and FR3 regions contained in any one of SEQ ID NOs: 1-10, or variants of such FRs. The description of the FRs in these protein sequences may apply any one of the systems or methods for amino acid numbering in immunoglobulin protein sequences described elsewhere in this specification, exemplified in FIG. 20 for VHH R3_DC23, and known to those skilled in the art. As an example, the sequences of the FRs in certain specific VHHs described herein using the Martin or Kabat methodology are shown in Table 4.

[0113]

Table 4

[0114] The polypeptide or polypeptide binding agent described herein, particularly an antibody or antibody fragment, more particularly an ISVD, may be characterized by comprising a 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 is at least 90% or 95% identical to the FR1 present in any one of SEQ ID NOs: 1-10, or may be characterized by comprising a variant FR1 having a maximum of 3, such as 1, 2 or 3 amino acid substitutions, deletions or additions, such as preferably conservative substitutions and / or humanized substitutions, wherein FR1 is defined according to any one of AbM, Chothia, Martin, Kabat, IMGT or MacCallum.

[0115] The polypeptide or polypeptide binding agent described herein, particularly an antibody or antibody fragment, more particularly an ISVD, may be characterized by comprising a framework region 2 (FR2) 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 is at least 85% or 90% identical to the FR2 present in any one of SEQ ID NOs: 1-10, or may be characterized by comprising a variant FR2 having a maximum of 2, such as 1 or 2 amino acid substitutions, deletions or additions, such as preferably conservative substitutions and / or humanized substitutions, wherein FR2 is defined according to any one of AbM, Chothia, Martin, Kabat, IMGT or MacCallum.

[0116] The polypeptide or polypeptide binding agent described herein, particularly an antibody or antibody fragment, more particularly ISVD, may be characterized by comprising a framework region 3 (FR3) present in any one of SEQ ID NOs: 1-10, wherein the FR3 is defined according to any one of AbM, Chothia, Martin, Kabat, IMGT or MacCallum, or is at least 80%, 85%, 90% or 95% identical to the FR3 present in any one of SEQ ID NOs: 1-10, or may be characterized by comprising a variant FR3 having a maximum of 9, such as 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions or additions, such as preferably conservative substitutions and / or humanized substitutions, wherein the FR3 is defined according to any one of AbM, Chothia, Martin, Kabat, IMGT or MacCallum.

[0117] The polypeptide or polypeptide binding agent described herein, particularly an antibody or antibody fragment, more particularly ISVD, may be characterized by comprising a framework region 4 (FR4) present in any one of SEQ ID NOs: 1-10, wherein the FR4 is defined according to any one of AbM, Chothia, Martin, Kabat, IMGT or MacCallum, or is at least 90% identical to the FR4 present in any one of SEQ ID NOs: 1-10, or may be characterized by comprising a variant FR4 having a maximum of 1 amino acid substitution, deletion or addition, such as preferably conservative substitutions and / or humanized substitutions, wherein the FR4 is defined according to any one of AbM, Chothia, Martin, Kabat, IMGT or MacCallum.

[0118] In a further embodiment, the polypeptide or polypeptide binding agent described herein, particularly an antibody or antibody fragment, more particularly ISVD, may each independently comprise FR1 present in any one of SEQ ID NOs: 1-10 or a variant FR1 as defined above herein; FR2 present in any one of SEQ ID NOs: 1-10 or a variant FR2 as defined above herein; FR3 present in any one of SEQ ID NOs: 1-10 or a variant FR3 as defined above herein; and FR4 present in any one of SEQ ID NOs: 1-10 or a variant FR4 as defined above herein, where FR1, FR2, FR3 and FR4 are defined according to any one of AbM, Chothia, Martin, Kabat, IMGT or MacCallum.

[0119] In certain embodiments, the polypeptide or polypeptide binding agent described herein, particularly an antibody or antibody fragment, more particularly ISVD, may comprise at least one, or a particular combination of two, three or all of the framework regions (FRs) present in any one of SEQ ID NOs: 1-10, or a variant of any of said FRs or FRs as defined above herein, where the FRs are annotated according to any one of Kabat, MacCallum, IMGT, AbM, Martin or Chothia.

[0120] In certain embodiments, the polypeptide or polypeptide binding agent described herein, particularly an antibody or antibody fragment, more particularly ISVD, may comprise at least one, or a particular combination of two, three or all of the framework regions (FRs) present in any one of SEQ ID NOs: 1-10, where the FRs are annotated according to any one of Kabat, MacCallum, IMGT, AbM, Martin or Chothia.

[0121] In a further embodiment, the polypeptide or polypeptide binding agent described herein, particularly an antibody or antibody fragment, more particularly ISVD, 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, where FR1, FR2, FR3, and FR4 are defined according to any one of AbM, Chothia, Martin, Kabat, IMGT, or MacCallum.

[0122] In a further embodiment, the polypeptide or polypeptide binding agent described herein, particularly an antibody or antibody fragment, more particularly ISVD, can be defined as comprising FR1, FR2, FR3, and FR4 present in the same sequence as any of the sequences shown in SEQ ID NOs: 1-10, where FR1, FR2, FR3, and FR4 are defined according to any one of AbM, Chothia, Martin, Kabat, IMGT, or MacCallum.

[0123] In yet a further specific embodiment, the polypeptide or polypeptide binding agent described herein, particularly an antibody or antibody fragment, more particularly ISVD, can be defined as comprising all four framework regions (FRs) present in any one of SEQ ID NOs: 1-10, where the FRs are defined according to any one of AbM, Chothia, Martin, Kabat, IMGT, or MacCallum.

[0124] For example, the polypeptide or polypeptide binding agent described herein, particularly an antibody or antibody fragment, more particularly ISVD, can be defined as comprising a framework region (FR) present in any one of SEQ ID NOs: 1-10, where the FR is defined according to Martin. In certain embodiments, the binding agent, particularly an antibody or antibody fragment, more particularly ISVD, comprises one of the following sets of framework regions (FRs): - FR1 defined by / described in SEQ ID NO: 97; FR2 defined by / described in SEQ ID NO: 33; FR3 defined by / described in SEQ ID NO: 101 and FR4 defined by / described in SEQ ID NO: 45; or - FR1 defined by / described in SEQ ID NO: 97, FR2 defined by / described in SEQ ID NO: 33, FR3 defined by / described in SEQ ID NO: 102, and FR4 defined by / described in SEQ ID NO: 45; or - FR1 defined by / described in SEQ ID NO: 97, FR2 defined by / described in SEQ ID NO: 33, FR3 defined by / described in SEQ ID NO: 103, and FR4 defined by / described in SEQ ID NO: 45; or - FR1 defined by / described in SEQ ID NO: 98, FR2 defined by / described in SEQ ID NO: 33, FR3 defined by / described in SEQ ID NO: 104, and FR4 defined by / described in SEQ ID NO: 45; or - FR1 defined by / described in SEQ ID NO: 98, FR2 defined by / described in SEQ ID NO: 33, FR3 defined by / described in SEQ ID NO: 105, and FR4 defined by / described in SEQ ID NO: 45; or - FR1 defined by / described in SEQ ID NO: 98, FR2 defined by / described in SEQ ID NO: 33, FR3 defined by / described in SEQ ID NO: 106, and FR4 defined by / described in SEQ ID NO: 45; or - FR1 defined by / described in SEQ ID NO: 98, FR2 defined by / described in SEQ ID NO: 33, FR3 defined by / described in SEQ ID NO: 107, and FR4 defined by / described in SEQ ID NO: 45; or - FR1 defined / described by SEQ ID NO:99, FR2 defined / described by SEQ ID NO:34, FR3 defined / described by SEQ ID NO:108, and FR4 defined / described by SEQ ID NO:45; or - FR1 defined / described by SEQ ID NO:99, FR2 defined / described by SEQ ID NO:33, FR3 defined / described by SEQ ID NO:109, and FR4 defined / described by SEQ ID NO:45; or - FR1 defined / described by SEQ ID NO:100, FR2 defined / described by SEQ ID NO:33; FR3 defined / described by SEQ ID NO:110; and FR4 defined / described by SEQ ID NO:29.

[0125] For example, the polypeptide or polypeptide binding agent described herein, particularly an antibody or antibody fragment, more particularly an ISVD, can be defined as including a framework region (FR) present in any one of SEQ ID NOs: 1-10, where the FR is defined according to Kabat. In certain embodiments, the binding agent, particularly an antibody or antibody fragment, more particularly an ISVD, includes one of the following sets of framework regions (FRs): - FR1 defined / described by SEQ ID NO:28; FR2 defined / described by SEQ ID NO:33; FR3 defined / described by SEQ ID NO:35 and FR4 defined / described by SEQ ID NO:45; or - FR1 defined / described by SEQ ID NO:28, FR2 defined / described by SEQ ID NO:33, FR3 defined / described by SEQ ID NO:36, and FR4 defined / described by SEQ ID NO:45; or - FR1 defined / described by SEQ ID NO:28, FR2 defined / described by SEQ ID NO:33, FR3 defined / described by SEQ ID NO:37, and FR4 defined / described by SEQ ID NO:45; or - FR1 defined by / described in SEQ ID NO: 29, FR2 defined by / described in SEQ ID NO: 33, FR3 defined by / described in SEQ ID NO: 38, and FR4 defined by / described in SEQ ID NO: 45; or - FR1 defined by / described in SEQ ID NO: 29, FR2 defined by / described in SEQ ID NO: 33, FR3 defined by / described in SEQ ID NO: 39, and FR4 defined by / described in SEQ ID NO: 45; or - FR1 defined by / described in SEQ ID NO: 29, FR2 defined by / described in SEQ ID NO: 33, FR3 defined by / described in SEQ ID NO: 40, and FR4 defined by / described in SEQ ID NO: 45; or - FR1 defined by / described in SEQ ID NO: 29, FR2 defined by / described in SEQ ID NO: 33, FR3 defined by / described in SEQ ID NO: 41, and FR4 defined by / described in SEQ ID NO: 45; or - FR1 defined by / described in SEQ ID NO: 30, FR2 defined by / described in SEQ ID NO: 34, FR3 defined by / described in SEQ ID NO: 42, and FR4 defined by / described in SEQ ID NO: 45; or - FR1 defined by / described in SEQ ID NO: 31, FR2 defined by / described in SEQ ID NO: 33, FR3 defined by / described in SEQ ID NO: 43, and FR4 defined by / described in SEQ ID NO: 45; or - FR1 defined by / described in SEQ ID NO: 32, FR2 defined by / described in SEQ ID NO: 33; FR3 defined by / described in SEQ ID NO: 44; and FR4 defined by / described in SEQ ID NO: 29.

[0126] In certain embodiments, the polypeptide or polypeptide binder, particularly an antibody or antibody fragment, individually, may be defined by or may comprise one or more ISVDs defined by or described in any one of SEQ ID NOs: 1-10, or may comprise or consist of one or more ISVDs comprising an amino acid sequence selected from the group of SEQ ID NOs: 1-10.

[0127] In a further embodiment, the polypeptide or polypeptide binder, particularly an antibody or antibody fragment, and more particularly the ISVD, comprises or consists of an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group of SEQ ID NOs: 1-10, or having at least 95% identity to an amino acid sequence selected from the group of SEQ ID NOs: 1-10. Such non-identity or variability is preferably limited to non-identity or variability at FR amino acid residues. In particular, such non-identity or variability may be introduced to obtain a humanized variant of the ISVD defined by or described in any one of SEQ ID NOs: 1-10. In particular, such a humanized variant may be a functional ortholog of the original ISVD, where the functional characteristics are one or more of the functional characteristics (1)-(47) broadly outlined above in this specification.

[0128] The terms "wild-type" or "native" refer to a gene or gene product isolated from a naturally occurring source. A wild-type gene is the gene that is most frequently observed in a population and is thus arbitrarily designated as the "normal" or "wild-type" form of the gene or gene product. In contrast, the terms "modified," "variant," "engineered," or "variant" refer to a gene or gene product that, when compared to the wild-type gene or gene product, shows a modification in the sequence (e.g., substitution, mutation or variation, deletion or addition), post-translational modification and / or a change in the biological or functional properties (i.e., a change in characteristics). Note that naturally occurring variants or variants can be isolated; these are identified by the fact that they have changed characteristics when compared to the wild-type gene or gene product. The changed characteristics may only exist at the sequence level or may additionally confer a change in the biological and / or functional properties of the variant or variant compared to the wild-type gene or gene product. Conservative amino acid substitutions can be introduced into a protein or polypeptide such that such substitutions are understood not to have an essential or substantial effect on the activity of the protein. Preferred conservative substitutions meet the criteria defined for acceptable point mutations in Dayhoff et al., Atlas of Protein Sequence and Structure, 5, pp. 345-352 (1978 and Supplements), which are incorporated herein by reference. Examples of conservative substitutions are substitutions that include, but are not limited to, 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(s)" of a protein of interest includes a protein having amino acid substitutions, deletions, and / or insertions relative to the unmodified (e.g., native, wild-type) protein of interest and having biological and functional activities that are essentially or substantially similar to those of the unmodified protein from which it (they) is (are) derived.

[0129] "Percent sequence identity" is calculated by comparing two optimally aligned (amino acid or nucleic acid) sequences over a comparison window, determining the number of positions at which the identical amino acid or nucleotide residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched 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 percent of (amino acid or nucleic acid) sequence identity.

[0130] Immunoglobulin single variable domains such as domain antibodies and Nanobody® (including the VHH domain) can be humanized, i.e., the degree of sequence identity with the closest human germline sequence can be increased. In particular, a humanized immunoglobulin single variable domain such as Nanobody® (including the VHH domain) can be a humanized substitution (as further defined herein) and / or an immunoglobulin single variable domain in which at least one amino acid residue (in particular, at least one framework residue) corresponding thereto is present. Potentially useful humanized substitutions can be identified by comparing the sequence of the framework region of a naturally occurring VHH sequence with the corresponding framework sequences of one or more closely related human VH sequences, and then one or more of the potentially useful humanized substitutions (or combinations thereof) thus determined can be introduced into the VHH sequence (by any method known per se as further described herein), and the resulting humanized VHH sequence can be tested for affinity to the target, stability, ease and level of expression, and / or other desired properties. In this way, with a limited amount of trial and error, other suitable humanized substitutions (or suitable combinations thereof) can be determined by those skilled in the art. Also, based on the foregoing, the (framework region of) an immunoglobulin single variable domain such as Nanobody® (including the VHH domain) can be partially or fully humanized.

[0131] Humanized immunoglobulin single variable domains, particularly Nanobodies™, may have several advantages such as reduced immunogenicity compared to the corresponding naturally occurring VHH domains. Humanization means that the immunogenicity upon administration to human patients is either minimal or non-existent due to mutations. Humanizing substitutions should be selected such that the resulting humanized amino acid sequence and / or ISVD or VHH still retain the favorable properties of the parental (non-humanized) VHH, such as antigen-binding ability. Based on the teachings provided herein, one of ordinary skill in the art will be able to select humanizing substitutions or suitable combinations of humanizing substitutions that optimize or achieve a desired or suitable balance between the favorable properties provided by the humanizing substitutions on the one hand and the favorable properties of the naturally occurring VHH domain on the other hand. Such methods are known to those of ordinary skill in the art. Human consensus sequences can be used as the target sequences for humanization, but other means are also known in the art. One alternative means involves the alignment of several human germline alleles, such as, but not limited to, the alignment of IGHV3 alleles by one of ordinary skill in the art and the use of said alignment for the identification of residues suitable for humanization in the target sequence. Also, a subset of the human germline alleles that are most homologous to the target sequence may be aligned as a starting point for identifying suitable humanizing residues. Alternatively, the VHH is analyzed, the closest homolog in the human alleles is identified, and used in the design of the humanized construct. The humanization techniques applied to camelid VHHs can be carried out by methods that involve replacing certain amino acids either alone or in combination. Said replacements can be selected based on those known from the literature, known humanization efforts, and the human consensus sequence compared to the native VHH sequence, or the human alleles most similar to the VHH sequence of interest. As can be seen from the VHH entropy and VHH variability data given in Tables A-5 to A-8 of WO 08 / 020079, some amino acid residues in the framework region are more conserved between humans and camelids than other amino acid residues.Generally, although not limited to these, any substitution, deletion or insertion (or addition) is preferably made at a position with low conservation. Also generally, amino acid substitutions are preferred over amino acid deletions or insertions. For example, the camelid single domain antibody human-like classification contains hydrophobic FR2 residues typical of conventional antibodies from human or other species, but other substitutions at position 103 that replace the conserved tryptophan residue present in the VH of the bispecific antibody compensate for this loss of hydrophilicity. Thus, peptides belonging to these two classifications show high amino acid sequence homology with the human VH framework region, and the peptides may be administered directly to humans without predicting an undesirable immune response thereby and without adding further humanization. Indeed, some of the camelid VHH sequences show high sequence homology with the human VH framework region, and thus the VHHs can be administered directly to patients without expecting an immune response thereby and without the additional burden or need for humanization.

[0132] Appropriate mutations, particularly substitutions, can be introduced during humanization to generate polypeptides with reduced binding to an existing antibody (see, e.g., WO 2012 / 175741 and WO 2015 / 173325), e.g., at least one of 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 can be suitably humanized with any framework residues, such as one or more hallmark residues (defined below), or one or more other framework residues (i.e., non-hallmark residues), or 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 can also be designed so that one or more sites for post-translational modification (e.g., one or more glycosylation sites) are removed, as is within the ability of one of ordinary skill in the art. Alternatively, the substitution or insertion can be designed to introduce one or more sites for attachment of functional groups (described herein), e.g., to enable site-specific pegylation.

[0133] In some cases, at least one of the typical camelid hallmark residues having hydrophilicity at positions 37, 44, 45 and / or 47 is replaced (see Table A-03 of WO 2008 / 020079). Another example of humanization involves substitution of residues in FR1 such as at positions 1, 5, 11, 14, 16, and / or 28; FR3 such as at positions 73, 74, 75, 76, 78, 79, 82b, 83, 84, 93 and / or 94; and FR4 such as at positions 10, 103, 104, 108, and / or 111 (see Tables A-05 to A08 of WO 2008 / 020079; all numbering according to the Kabat methodology). In certain embodiments, humanized antibodies, particularly humanized ISVDs, include substitution of residues at positions 1, 5, 14, 16, 19, 63, 73, 79, 82c, 83, and / or, preferably, 108 according to Kabat numbering. In other certain embodiments, humanized antibodies, particularly humanized ISVDs, include substitution of residues at positions 1, 5, 14, 16, 19, 63, 73, 79, 83, and / or, preferably, 108 according to Kabat numbering. Humanization typically only relates to substitutions, deletions or additions in the FRs and not in the CDRs, as it can / will affect the binding affinity and / or potency towards the target.

[0134] Certain non-limiting examples of humanized ISVDs described herein include the following:

[0135] Humanized R3_DC23:

Chemical formula

[0136] Humanized R3_C4:

Chemical formula

[0137] Humanized R4_DC20:

Chemical formula

[0138] In certain embodiments, the antibody comprises one or more of the ISVDs described herein (or variants or humanized forms thereof described herein), and the one or more ISVDs (or variants or humanized forms thereof described herein) are bound or fused to the Fc domain.

[0139] As used herein, "Fc domain" refers to the fragment crystallizable region (Fc region) of a conventional antibody, a tail region known to interact with cell surface receptors called Fc receptors and some proteins of the complement system. The Fc domain is composed of two identical protein fragments derived from the second and third constant domains of the two heavy chains of the antibody. All conventional antibodies contain an Fc domain, and thus the Fc domain may be derived from or be a variant of the Fc region of IgG, IgA, and IgD antibodies, and more particularly may be an Fc domain derived from the Fc region of IgG1, IgG2, or IgG4 antibodies. For example, the hinge region of IgG2 may be replaced with the hinge of human IgG1 to yield an ISVD fusion construct, and vice versa. Additionally, Fc variants known to have an extended half-life such as M257Y / S259T / T261E (also known as YTE) or the LS variant (M428L combined with N434S) may be used. These mutations increase the binding of the conventional antibody's Fc domain to the neonatal receptor (FcRn). Preferably, a human Fc domain or humanized Fc domain may be used. Humanized forms include, but are not limited to, IgG humanization variants known in the art such as C-terminal lysine deletions, modifications or cleavages in the hinge region, or LALA (L234A and L235A) or LALAPG (L234A, L235A, and P329G) mutations among other substitutions in the IgG sequence.

[0140] The term "fused to" is used interchangeably herein with "connected to", "conjugated to", "ligated to", and in one aspect refers to, for example, "genetic fusion" by recombinant DNA technology and "chemical and / or enzymatic conjugation" that results in ligation by stable covalent bonds between two nucleic acid molecules. The term "inserted into" is similar, and a fragment of one nucleic acid can be inserted into a second nucleic acid molecule by genetically, enzymatically, or chemically fusing or ligating the two sequences. Peptides or polypeptides can likewise be fused or connected to each other via peptide bonds or by linking one peptide to the side chain of an amino acid in a second peptide.

[0141] Linkers can be used to fuse an ISVD such as the ISVD identified herein (or variants or humanized forms thereof described herein) to an Fc domain such as the human IgG1 Fc domain or its LS variant, or its YTE variant, or the IgG2 Fc domain. Non-limiting examples of linkers include Gly-Ser linkers having n = 1-6 (SEQ ID NO: 120), preferably 2-3 (SEQ ID NOs: 121-122). 4 S) n including, for example.

[0142] In certain embodiments, antibodies comprising one or more ISVDs (or variants or humanized forms thereof described herein) described herein are in "multivalent" and / or "bispecific" forms formed by joining 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.

[0143] Non-limiting examples of multivalent constructs include "bivalent" constructs, "trivalent" constructs, "tetravalent" constructs, etc., each containing two, three, or four ISVDs, respectively. The ISVDs contained within a multivalent construct may be the same or different. The term "multispecific antibody," as used herein, refers in particular to a multivalent antibody in which at least one of 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, any multivalent and multispecific (as defined herein) antibody of the invention may be for two or more different antigens, such as, for example, sarbecovirus, and serum albumin or staphylococcal protein A (SpA) as a half-life extension, and / or for two or more different portions of a particular antigen, such as, for example, two or more different portions, regions, subunits or domains of the sarbecovirus spike protein.

[0144] In certain embodiments, an antibody, particularly a multivalent and / or multispecific antibody, may comprise one or more binding agents such as the ISVD(s) described herein (or variants or humanized forms thereof described herein), and one or more binding agents such as an ISVD capable of binding to the sarbecovirus spike protein receptor binding domain (RBD). Non-limiting examples of ISVDs capable of binding to the sarbecovirus spike protein receptor binding domain (RBD) are described in PCT / EP2021 / 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 sarbecovirus spike protein via interactions in two different regions of the spike protein, particularly the S2 subunit, more particularly the HR2 domain, and the RBD, may result in cross-reactivity and strong inhibition of infection by sarbecovirus, and may further enable a reduction in the risk of emergence of mutant viruses.

[0145] In certain further embodiments, one or more ISVDs capable of binding to the SARS-CoV-2 spike protein RBD can bind to or compete around the VHH72 epitope (or an epitope that undergoes specific binding by VHH72). The VHH72 epitope is described in Wrapp et al. (2020, Cell 184:1004 - 1015; PCT / EP2021 / 052885 and PCT / EP2022 / 062980). The VHH72 epitope as defined herein refers to a structural epitope of the RBD that includes at least one or more of the amino acid residues S371, S375, T376, or C379 described in SEQ ID NO:86, or more specifically, at least one or more of L368, Y369, S371, S375, T376, F377, K378, C379, and Y508 described in SEQ ID NO:86, which is the sequence of the SARS-CoV-2 spike protein. In particular, an ISVD capable of binding to the VHH72 epitope can specifically bind 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 around the VHH72 epitope refers to an ISVD that competes with VHH72 for binding to the spike protein shown in SEQ ID NO:86 or the RBD. "Competing" means that in the presence of an ISVD capable of competing around the VHH72 epitope, the binding of VHH72 to the spike protein shown in SEQ ID NO:86 or the RBD is reduced in intensity by at least 30%, or at least 50%, preferably at least 80%.In particular, an ISVD that can compete with respect to the VHH72 epitope or can compete with the binding of VHH72 to the RBD epitope can specifically bind to an epitope on the spike protein that includes at least 3, at least 4, at least 5, at least 6 or more of the residues L368, Y369, S371, S375, T376, F377, K378, C379 and Y508 of the spike protein of SARS-Cov-2 shown in SEQ ID NO: 86, providing overlapping epitopes. In embodiments, an ISVD that can bind to or compete with respect to the VHH72 epitope may be characterized in that (i) it competes with respect to human receptor (ACE-2 in the case of SARS-CoV-1 and -2) binding upon interaction with the RBD, and / or (ii) it does not compete with an ISVD that can bind to or compete with the VHH3.117 epitope as defined herein. Non-limiting examples of ISVDs that can bind to or compete with respect to 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) described in PCT / EP2021 / 052885 and PCT / EP2022 / 062980, as well as variants including VHH72(S56A) and humanized forms thereof; VHH3.83 family members (including VHH3.83 (also referred to herein as VHH83) (SEQ ID NO: 125)) as well as variants and humanized forms thereof; VHH3.38 family members as well as variants and humanized forms thereof; VHH3.55 family members as well as variants and humanized forms thereof; VHH3.36 family members as well as variants and humanized forms thereof; VHH3.149 family members as well as variants and humanized forms thereof; and VHH3.29 family members as well as variants and humanized forms thereof.

[0146] In certain embodiments, the antibody, particularly a multivalent antibody and / or a multispecific antibody, may comprise one or more of the ISVDs described herein (or variants or humanized forms thereof described herein), as well as an ISVD comprising the CDRs present in SEQ ID NO: 125 or SEQ ID NO: 124, e.g., an ISVD comprising or consisting of the sequences described in SEQ ID NO: 125 (e.g., VHH83) or SEQ ID NO: 124 (e.g., VHH72), or a variant or humanized form thereof, wherein the CDRs are annotated according to Kabat, Martin, MacCallum, IMGT, AbM, or Chothia.

[0147] In certain further embodiments, one or more ISVDs capable of binding to the SARS-CoV-2 spike protein RBD can bind to or compete around the VHH3.117 epitope (or an epitope that undergoes specific binding by VHH3.117). The VHH3.117 epitope is described in PCT / EP2022 / 052919. In particular, an ISVD capable of binding to the VHH3.117 epitope can bind or specifically bind to at least one of the amino acids Asn394 (or alternatively Ser394 in some SARS-CoV-2) of the SARS-CoV-2 spike protein defined in SEQ ID NO: 86, Tyr396, Phe464, Ser514, Glu516, and Arg355, or at least two, at least three, or at least four in order of increasing priority: Optionally, the amino acids Arg357 (or alternatively Lys357 in some SARS-CoV-2) and / or Lys462 (or alternatively Arg462 in some SARS-CoV-2) and / or Glu465 (or alternatively Gly465 in some SARS-CoV-2) and / or Arg466 and / or Leu518 can further bind or specifically bind, for example, at least two of the amino acids Arg357 (or alternatively Lys357 in some SARS-CoV-2) and / or Lys462 (or alternatively Arg462 in some SARS-CoV-2) and / or Glu465 (or alternatively Gly465 in some SARS-CoV-2) and / or Arg466 and / or Leu518, or it may be possible to further bind or specifically bind to all at least three or four in order of increasing priority. An ISVD capable of competing around the VHH3.117 epitope refers to the spike protein shown in SEQ ID NO: 86, or an ISVD that competes with VHH3.117 around binding to the RBD."Competing" means that in the presence of an ISVD capable of competing for the VHH3.117 epitope, the binding 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 intensity. In an embodiment, an ISVD capable of binding to or competing for the VHH3.117 epitope may be characterized in that (i) it does not inhibit the binding of the RBD to the human receptor (ACE-2 in the case of SARS-CoV-1 and -2), i.e., when the ISVD itself binds to the sarbecovirus RBD, it allows the binding of the receptor to the sarbecovirus RBD, or alternatively, the ISVD itself can bind to the sarbecovirus RBD to which the receptor is bound, and / or (ii) it does not compete with an ISVD capable of binding to or competing for the VHH72 epitope as defined herein. Non-limiting examples of ISVDs capable of binding to or competing for the VHH3.117 epitope include VHH3.117 family members (including VHH3.117, 3.42, 3.92, 3.94, 3.180) and their variants and humanized forms (described in PCT / EP2022 / 052919); VHH3.89 family members and their variants and humanized forms (described in PCT / EP2021 / 052885); VHH3_183 family members and their variants and humanized forms; and VHH3C_80 family members and their variants and humanized forms (described in PCT / EP2022 / 062980).

[0148] In certain embodiments, the antibody, particularly a multivalent antibody and / or a multispecific antibody, may comprise one or more ISVDs (or variants or humanized forms thereof described herein), and an ISVD comprising the CDRs present in SEQ ID NO: 127, e.g., an ISVD comprising or consisting of the sequence described in SEQ ID NO: 127 (e.g., VHH3.117), or a variant or humanized form thereof, where the CDRs are annotated according to Kabat, Martin, MacCallum, IMGT, AbM, or Chothia.

[0149] In yet further additional embodiments, the antibody, particularly a multivalent antibody and / or a multispecific antibody, comprises two or more ISVDs capable of binding to the severe acute respiratory syndrome coronavirus spike protein receptor-binding domain (RBD), where at least one ISVD is capable of binding to or competing with respect to the VHH72 epitope as defined herein, and at least one ISVD is capable of binding to or competing with respect to the VHH3.117 epitope as defined herein. Advantageously, a combination of at least two non-competing RBD-targeting ISVDs in the antibody (capable of binding to the RBD of the spike protein by interaction at two different non-competing regions of the RBD) and at least one S2-targeting ISVD results in cross-reactivity and potent inhibition of infection by severe acute respiratory syndrome coronavirus, and advantageously further reduces the risk of mutation escape.

[0150] In certain embodiments, the antibody, particularly a multivalent antibody and / or a multispecific antibody, comprises one or more of the ISVDs described herein (or variants or humanized forms thereof described herein), an ISVD comprising the CDRs present in SEQ ID NO: 125 or SEQ ID NO: 124, such as the sequences described in SEQ ID NO: 125 (e.g., VHH83) or SEQ ID NO: 124 (e.g., VHH72), or an ISVD comprising or consisting of a variant or humanized form thereof, and an ISVD comprising the CDRs present in SEQ ID NO: 126, such as the sequence described in SEQ ID NO: 126 (e.g., VHH3.117), or an ISVD comprising or consisting of a variant or humanized form thereof, where the CDRs are annotated according to Kabat, Martin, MacCallum, IMGT, AbM, or Chothia.

[0151] The multivalent antibodies described herein can be formed, for example, chemically or by recombinant DNA techniques, by directly connecting two or more ISVDs or via a linker, and / or by fusing two or more ISVDs (each) to an Fc domain.

[0152] For example, a single ISVD (or a variant or humanized form thereof) described herein may be fused at its C-terminus to an Fc domain, such as an IgG Fc domain, e.g., a construct comprising the amino acid sequence defined in SEQ ID NO: 96 or SEQ ID NO: 118, resulting in a bivalent form of a chikungunya virus antibody in which two of said ISVDs form an antibody-type molecule of only heavy chains via a disulfide bridge in the hinge region of an Fc moiety such as an IgG Fc moiety.

[0153] In certain embodiments, one or more ISVDs (or variants or humanized forms thereof described herein) described herein are directly, or via a linker, linked, fused or connected to one or more ISVDs capable of binding to the simian virus spike protein as defined herein. Non-limiting examples of suitable linkers for linking to an ISVD include peptide linkers such as (G 4 S) n where n = 1, 2, 3, 4, 5 or 6. Such multispecific binding agents may also be referred to herein as "head-to-tail fusions."

[0154] In a further embodiment, the C-terminus of the head-to-tail fusion described herein can be fused to an Fc domain, for example, by a linker, and this construct forms multivalent antibodies and / or multispecific antibodies via disulfide bridges in the hinge region of the Fc portion upon expression in a host. Thus, in certain embodiments, one or more ISVDs (or variants or humanized forms thereof described herein) described herein are directly or via a linker linked, fused or connected to one or more ISVDs capable of binding to the SARS-CoV-2 spike protein RBD to form a multispecific binding agent or construct, and said multispecific binding agent or construct is fused to an Fc domain. In a preferred embodiment, the antibody comprises a bispecific binding agent or construct fused to an Fc domain, said bispecific binding agent or construct being one ISVD capable of binding to the SARS-CoV-2 spike protein RBD, for example, one ISVD (or a variant or humanized form thereof described herein) directly or via a linker linked, fused or connected to an ISVD capable of binding to or competing with the VHH3.117 epitope described herein. A schematic of such a multispecific antibody, particularly a bispecific antibody, also referred to herein as a "VHH-VHH-Fc fusion", is shown in FIGS. 33A-C. More specific examples of such multispecific antibodies, particularly bispecific antibodies, capable of binding to the HR2 binding site and the VHH3.117 epitope described herein are provided, for example, by SEQ ID NOs: 112-114, or functional variants thereof, or variants having at least 90% identity thereto, or humanized forms thereof, but are not limited thereto. The sequences defined by SEQ ID NOs: 112-114 are also shown below.

Chemical formula

[0155] In certain embodiments, the antibody comprises a trispecific binder or construct fused to an Fc domain, the trispecific binder or construct comprising one ISVD described herein (or a variant or humanized form thereof described herein), one ISVD capable of binding to or competing with the VHH3.117 epitope described herein, and one ISVD capable of binding to or competing with the VHH72 epitope described herein, the ISVDs being linked, fused or connected to each other directly or via a linker, in any order. A schematic diagram of such a multispecific antibody, particularly a trispecific antibody also referred to herein as a "VHH-VHH-VHH-Fc fusion", is shown in FIG. 33F. More specific examples of such multispecific antibodies, particularly trispecific antibodies, capable of binding to the HR2 binding site and the VHH3.117 and VHH72 epitopes described herein are provided, for example, by SEQ ID NO: 117, or a functional variant thereof, or a variant having at least 90% identity thereto, or a humanized form thereof, but are not limited thereto. The sequence defined by SEQ ID NO: 117 is also shown below.

Chemical formula

[0156] 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 RBD of the SARS-CoV spike protein 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 RBD of the SARS-CoV spike protein are fused to the N-terminus of the Fc domain. In a preferred embodiment, the antibody comprises one ISVD described herein (or a variant or humanized form thereof described herein) fused to the N-terminus of the Fc domain, and one ISVD capable of binding to the RBD of the SARS-CoV spike protein, particularly one ISVD capable of binding to or competing with the VHH3.117 epitope described herein fused to the C-terminus of the Fc domain, or one ISVD described herein (or a variant or humanized form thereof described herein) fused to the C-terminus of the Fc domain, and one ISVD capable of binding to the RBD of the SARS-CoV spike protein, particularly one ISVD capable of binding to or competing with the VHH3.117 epitope is fused to the N-terminus of the Fc domain. A schematic diagram of such a trispecific antibody, also referred to herein as a "VHH-Fc-VHH fusion" or a "moonlander", is shown in FIG. 33D. More specific examples of such multispecific antibodies, particularly bispecific antibodies, capable of binding to the HR2 binding site and the VHH3.117 epitope described herein are provided, for example, by SEQ ID NO: 115, or a functional variant thereof, or a variant having at least 90% identity thereto, or a humanized form thereof, but are not limited thereto. The sequence defined by SEQ ID NO: 115 is also shown below.

Chemical Structure

[0157] The multivalent or multispecific antibodies described herein can have (or can be engineered and / or selected for) increased avidity and / or improved selectivity for the desired sarbecovirus interactions, and / or any other desired properties or combinations of desired properties obtainable by use of such multivalent and / or multispecific antibodies.

[0158] In certain embodiments, the binding agents described herein, particularly multivalent and / or multispecific antibodies, more particularly multivalent and / or multispecific antibodies comprising an Fc domain described herein, have antibody-dependent cell cytotoxicity (ADCC) activity. More particularly, the binding agents described herein, particularly multivalent and / or multispecific antibodies, more particularly multivalent and / or multispecific antibodies comprising an Fc domain described herein, are capable of inducing ADCC against target cells expressing the sarbecovirus spike protein. "Antibody-dependent cell cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which an antibody binds to certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages). Secretion of Ig on the Fcγ receptor enables these cytotoxic effector cells to specifically bind to target cells having the antigen and kill the target cells, for example, using cytotoxins. To assess the ADCC activity of the antibody of interest, in vitro, an ADCC assay, for example, the method described in the examples of this application, can be performed.

[0159] Also disclosed herein are other sarbecovirus binding agents that compete with an ISVD defined by an amino acid sequence selected from the group of SEQ ID NOs: 1-10 for binding to a sarbecovirus spike protein or a portion thereof (as described above).

[0160] As used herein, the terms "competing" or "cross-competing" refer to a compound or binding agent that shares the ability to bind to a specific region of an antigen and inhibits or blocks the binding of another binding agent. In the present disclosure, a compound or binding agent that is "competitive" or "cross-competitive" has the ability to interfere with the binding of an antibody or antigen-binding fragment described herein, particularly an ISVD defined by an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10, in a competitive binding assay known to those skilled in the art. This term also includes competition between two antibodies or antigen-binding fragments in both directions, i.e., an antibody that binds to a second antibody and blocks the binding of the second antibody, and vice versa. In certain embodiments, a first antigen-binding agent (e.g., an antibody or antigen-binding fragment) and a second antigen-binding agent (e.g., an 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, and one binding may inhibit or block the binding of the second antibody or antigen-binding fragment, for example, through 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 ELISA (enzyme-linked immunosorbent assay) or surface plasmon resonance (SPR). Competition or cross-competition may exist if the binding of an ISVD defined by an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10 to 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, particularly a SARS-CoV-2 S2 subunit or a SARS-CoV-1 S2 subunit, or a portion thereof, and more particularly the SARS-CoV-1 / -2 HR2 domain shown in SEQ ID NO: 87, is reduced by at least 30%, or at least 50%, or preferably at least 80% in the presence of a competing binder. In particular, such other binding agents preferably retain one or more of the functional requirements (1)-(47) broadly outlined above in this specification.

[0161] Thus, the present disclosure also relates to compounds that bind to the SARS-CoV spike protein, particularly the S2 subunit of the SARS-CoV spike protein, and more particularly the SARS-CoV HR2 domain in the SARS-CoV spike protein, and methods for screening for compounds that compete with the ISVD or functional portions thereof (or variants or humanized forms thereof) described herein for binding to the SARS-CoV spike protein, particularly the SARS-CoV S2 subunit, and more particularly the SARS-CoV HR2 domain. Such methods generally include one or more of the following steps: - preparing a compound or a pool of compounds; - contacting the compound or pool of compounds with the SARS-CoV spike protein or the SARS-CoV S2 subunit or the SARS-CoV HR2 domain in the absence of the ISVD or functional portions thereof (or variants or humanized forms thereof) described herein; - contacting the compound or pool of compounds with the SARS-CoV spike protein or the SARS-CoV S2 subunit or the SARS-CoV HR2 domain in the presence of the ISVD or functional portions thereof (or variants or humanized forms thereof) described herein; - measuring, evaluating, determining, assaying whether the compound or pool of compounds is capable of reducing the amount of the ISVD or functional portion thereof bound to the SARS-CoV spike protein or the SARS-CoV S2 subunit or the SARS-CoV HR2 domain; or measuring, evaluating, determining, assaying whether the ISVD or functional portion thereof is capable of reducing the amount of the compound or pool of compounds bound to the SARS-CoV spike protein or the SARS-CoV S2 subunit or the SARS-CoV HR2 domain; - Identifying a compound as a competitor of ISVD or a functional portion thereof for binding to the SARS-CoV spike protein, the SARS-CoV S2 subunit, or the SARS-CoV HR2 domain when the amount of ISVD or a functional portion thereof bound to the SARS-CoV spike protein, the SARS-CoV S2 subunit, or the SARS-CoV HR2 domain is reduced in the presence of the compound; or identifying a compound pool comprising one or more compounds as a competitor of ISVD or a functional portion thereof for binding to the SARS-CoV spike protein, the SARS-CoV S2 subunit, or the SARS-CoV HR2 domain when the amount of ISVD or a functional portion thereof bound to the SARS-CoV spike protein, the SARS-CoV S2 subunit, or the SARS-CoV HR2 domain is reduced in the presence of one or more compounds; or identifying a compound as a competitor of ISVD or a functional portion thereof for binding to the SARS-CoV spike protein, the SARS-CoV S2 subunit, or the SARS-CoV HR2 domain when the amount of a compound that binds to the SARS-CoV spike protein, the SARS-CoV S2 subunit, or the SARS-CoV HR2 domain is reduced in the presence of ISVD or a functional portion thereof; or identifying a compound pool comprising one or more compounds as a competitor of ISVD or a functional portion thereof for binding to the SARS-CoV spike protein, the SARS-CoV S2 subunit, or the SARS-CoV HR2 domain when the amount of a compound pool that binds to the SARS-CoV spike protein, the SARS-CoV S2 subunit, or the SARS-CoV HR2 domain is reduced in the presence of ISVD or a functional portion thereof.

[0162] The terms "compound", "test compound", "candidate compound", or "drug candidate compound", as used herein, refer to molecules, whether natural or synthetic, that are designed, identified, screened, or generated and tested in an assay such as a screening assay or a drug discovery assay, or specifically in a method for identifying compounds that compete with the ISVD described herein (or its variants or humanized forms (such as those described above) for binding to the SARS-CoV spike protein or a portion thereof). Thus, these compounds include organic and inorganic compounds. For high-throughput purposes, test compound libraries such as combinatorial libraries or randomized libraries that provide a sufficient range of diversity may 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, etc. Such compounds may also be referred to as binders, and as referred to herein, they may be "small molecules", which refers to organic compounds of low molecular weight (e.g., <900 Da or <500 Da). Compounds or binders also include chemical substances, polynucleotides, lipids, or hormone analogs characterized by low molecular weight. Other biopolymeric organic test compounds include small peptides or peptidomimetic molecules (peptide mimetics) containing from about 2 to about 40 amino acids, and large polypeptides containing from about 40 to about 500 amino acids such as antibodies, antibody mimetics, antibody fragments, or antibody conjugates.

[0163] As used herein, the terms "determine", "measure", "evaluate", "identify", "screen", and "assay" are used interchangeably and include both quantitative and qualitative determinations.

[0164] In another aspect, the present invention provides an isolated nucleic acid, a (isolated) chimeric gene construct, an expression cassette, etc., comprising a polynucleotide sequence such as a coding sequence encoding the polypeptide portion of the polypeptide or polypeptide-like SVV binder identified herein, particularly an antibody or antibody fragment, more particularly ISVD (or its variant or humanized form) described herein, or a functional portion thereof.

[0165] As used herein, "nucleic acid" or "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, and the sequential linear arrangement of nucleotides together results in / forms a "nucleotide sequence", "DNA sequence" or "RNA sequence". This term refers only to the primary structure of the molecule. Thus, this term includes double-stranded and single-stranded DNA, and RNA. It also includes known types of modifications, such as methylation, "capping", substitution of one or more naturally occurring nucleotides with analogs. Modifications to nucleic acids can be introduced at one or more levels: phosphate linkage modifications (e.g., introduction of one or more of phosphodiester linkages, phosphoramidate linkages or phosphorothioate linkages), sugar modifications (e.g., introduction of one or more of one or more LNA (locked nucleic acid), 2'-O-methyl, 2'-O-methoxy-ethyl, 2'-fluoro, S-constrained ethyl or tricyclo DNA), and / or non-ribose modifications (e.g., introduction of one or more of phosphorodiamidate morpholino or peptide nucleic acid).

[0166] "Nucleic acid construct" means a nucleic acid molecule constructed to contain one or more functional units not found in nature, and thus has a nucleotide sequence not found in nature (non-native nucleotide sequence). Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences derived from lambda phage), viral genomes containing non-native nucleic acid sequences, etc.

[0167] A "coding sequence" is a nucleotide sequence that, when placed under the control of an appropriate (gene) regulatory sequence, can be transcribed into mRNA and / or translated into a polypeptide. The boundaries of the coding sequence are determined by the translation start codon at the 5'-end and the translation stop codon at the 3'-end. The coding sequence includes, but is not limited to, mRNA, cDNA, recombinant nucleotide sequences, or genomic DNA, while introns may also be present under certain circumstances.

[0168] A "chimeric gene" or "chimeric construct" or "chimeric gene construct" interchangeably means a recombinant nucleic acid sequence in which a (gene) promoter or regulatory nucleic acid sequence is operably or functionally linked to, or associated with, a nucleic acid sequence for the purpose of encoding RNA (e.g., a coding sequence, shRNA, etc.), such that the regulatory nucleic acid sequence can regulate the transcription or expression of the nucleic acid of interest. The operable or functional linkage in the chimeric gene between the regulatory nucleic acid sequence and the nucleic acid sequence of interest is not found in nature.

[0169] An "expression cassette" includes any nucleic acid construct capable of directing the expression of a gene / coding sequence of interest operably linked to a (gene) promoter. An expression cassette generally preferably (from 5' to 3' in the transcription direction) is a DNA construct comprising: a (gene) promoter region, a polynucleotide sequence of interest having a transcription start region, and a termination sequence including a stop signal and a polyadenylation signal for RNA polymerase; all of these elements are operably or operationally linked and are meant to be operable (expressed) in a cell when transformed into a cell such as a prokaryotic cell (e.g., bacteria) or a eukaryotic cell (e.g., mammals, yeast, insects, fungi, plants, algae). Preferably, the transcription start region including an RNA polymerase binding site and the promoter region including a polyadenylation signal may be native to the cell to be transformed, may be derived from another source, or may be synthetic as long as it functions in the cell. Such an expression cassette can be constructed, for example, with a "vector" or "expression vector" (linear or circular nucleic acid, plasmid, cosmid, viral vector, phagemid, etc.).

[0170] The present invention also provides a vector into which the above nucleic acid molecule is inserted.

[0171] The terms "vector", "vector construct", "expression vector", "recombinant vector" or "gene transfer vector" as used herein are intended to refer to a nucleic acid molecule capable of carrying another nucleic acid molecule to which it is linked.

[0172] The vector may include a cloning vector or an expression vector, and a delivery vehicle such as a viral vector, a lentiviral vector or an adenoviral vector. The expression vector may include a plasmid, similar to a viral vector, and generally contains an appropriate DNA sequence necessary for the expression of a desired coding sequence and a coding sequence operably linked in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in an in vitro expression system. In particular, the expression vectors described herein may include the nucleic acid molecules described herein, which include a nucleic acid sequence encoding an antibody or antigen-binding fragment described herein operably linked to at least one regulatory sequence. The regulatory sequences are selected to direct the expression of the protein of interest, particularly an antibody or antigen-binding fragment, in a suitable host cell, and include promoters, enhancers, and other expression control elements known to those skilled in the art. Thus, in embodiments, the vector includes a promoter for driving the expression of the nucleic acid of interest, optionally a nucleic acid sequence encoding a signal peptide that secretes an antibody or antigen-binding fragment, and optionally a nucleic acid sequence encoding a terminator. When the expression vector is engineered in a production strain or cell line, the vector may or may not be integrated into the genome of the host cell upon introduction into the host cell. Cloning vectors are generally used to manipulate and amplify a particular desired DNA fragment. Thus, the cloning vector may contain an origin of replication that matches the cell type specified by the cloning vector and may lack the functional sequences necessary for the expression of the desired DNA fragment. Preferably, the vector contains one or more selectable markers. The choice of selectable marker may depend on the host cell to be selected, but this is not critical to the present invention as it is well known to those skilled in the art.The construction of expression vectors for use in cell transfection is also well known in the art and can thus be achieved by standard techniques (e.g., Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.), as well as the Ambion 1998 Catalog (see Ambion, Austin, Tex.).

[0173] More specifically, the vector may include any suitable type known to those skilled in the art, including, but not limited to, plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors, more specifically, lentiviral vectors, adenoviral vectors, AAV vectors or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs). The choice of vector may depend, inter alia, on the nature of the host cell to be selected.

[0174] A further aspect of the present invention provides a host cell comprising an antibody or antigen-binding fragment described herein, for example, an ISVD (or a variant or humanized form thereof) of an antibody or antigen-binding fragment, or a part thereof. Thus, the host cell may contain a nucleic acid molecule encoding the antibody or antigen-binding fragment. The host cell may be a prokaryotic cell or a eukaryotic cell. The host cell may also be a recombinant host cell comprising a cell 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 ISVD include, but are not limited to, bacterial cells, yeast cells, plant cells and animal cells. Bacterial host cells suitable for the production of the antibody or antigen-binding fragment of the present invention include cells of Escherichia spp., Bacillus spp., Streptomyces spp., Erwinia spp., Klebsiella spp., Serratia spp., Pseudomonas spp., Salmonella spp. Yeast host cells suitable for use in the present invention include species within Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia (e.g., Pichia pastoris), Hansenula (e.g., Hansenula polymorpha), Yarowia, Schwaniomyces, Schizosaccharomyces, Zygosaccharomyces, etc. 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 with the present invention include insect cells and mammalian cells (e.g., derived from Chinese hamster (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 Jarvis, Virology Vol. 310 No. 1 May 25, 2003 pp. 1-7). Alternatively, the host cell may be a transgenic animal or plant.

[0175] Introduction of the vector into the host cell can be carried out, for example, by calcium phosphate transfection, viral infection, DEAE-dextran-mediated transfection, transfection with lipofectamine 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.

[0176] A further aspect of the present invention relates to a composition comprising a binder such as an antibody or an antigen-binding fragment thereof comprising one or more ISVDs (or variants or humanized forms thereof) described herein or a part thereof. "Composition", as used herein, refers to a combination of one or more molecules present in a formulation that retains the activity of the binder, particularly in this case HR2 (or S2) binding and simian immunodeficiency virus neutralizing activity, and thus a functional composition. Thus, the composition comprises one or more molecules constituting one or more binders (a binder targeting S2 or a binder targeting the HR2 domain) described herein that specifically binds to the simian immunodeficiency virus spike protein by interaction with its HR2 domain. In certain embodiments, the composition may comprise a bivalent antibody comprising a binder, particularly an ISVD (or variant or humanized form thereof) described herein fused to an Fc domain such as an antibody comprising the amino acid sequence defined in SEQ ID NO: 118. The composition may be a soluble composition or a solid composition.

[0177] In addition to the binder molecule targeting S2, particularly the binder molecule targeting the HR2 domain, the composition may further comprise additional molecules which may be, for example, a buffer component, an adjuvant, or a functional molecule, but are not limited thereto.

[0178] In certain embodiments, the composition may further comprise one or more binders capable of binding to the severe acute respiratory syndrome coronavirus spike protein receptor binding domain (RBD) described elsewhere herein. In certain embodiments, the composition may further comprise one or more (e.g., two, three, four, or more) binders such as an antibody or an antigen-binding fragment thereof comprising one or more ISVDs (or variants or humanized forms thereof) capable of binding to the severe acute respiratory syndrome coronavirus spike protein receptor binding domain (RBD) described elsewhere herein. Thus, the composition may contain at least two binders, wherein one binder specifically binds to the HR2 domain and a second binder specifically binds to the RBD region, such that a composition is obtained in which at least two binders bind to the spike protein non-competitively, and in some cases, simultaneously.

[0179] In a preferred embodiment, the binder capable of binding to the SARS-CoV spike protein RBD is preferably capable of binding to two non-competing binding sites of the RBD via two different ISVDs present in the binder, and the binder may be a bispecific binder or a multispecific binder. More specifically, the binder may comprise one or more ISVDs capable of binding to or competing around the VHH72 epitope as defined herein, and one or more ISVDs capable of binding to or competing around the VHH3.117 epitope as defined herein. Non-limiting examples of binders comprising one or more ISVDs capable of binding to or competing around the VHH72 epitope and one or more ISVDs capable of binding to or competing around the VHH3.117 epitope are described in PCT / EP2022 / 062980.

[0180] In certain embodiments, the composition comprises: (i) a binder, particularly an antibody or an antigen-binding fragment thereof, comprising or consisting of one or more ISVDs, such as those having CDRs present in SEQ ID NO: 8, including the amino acid sequence set forth in SEQ ID NO: 8 (e.g., VHH R3_DC23), or a variant or humanized form thereof; and (ii) a binder, particularly an antibody or an antigen-binding fragment thereof, comprising or consisting of one or more ISVDs, such as those having CDRs present in SEQ ID NO: 125 or SEQ ID NO: 124, including the sequence set forth in SEQ ID NO: 125 (e.g., VHH83) or SEQ ID NO: 124 (e.g., VHH72), or a variant or humanized form thereof, and an ISVD, such as those having CDRs present in SEQ ID NO: 126, including the sequence set forth in SEQ ID NO: 126 (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.In a further specific embodiment, the composition comprises (i) a binding agent, particularly a (bivalent) antibody, comprising or consisting of an ISVD comprising a CDR present in SEQ ID NO: 8, such as an ISVD comprising the amino acid sequence set forth in SEQ ID NO: 8 (e.g., VHH R3_DC23) fused to an Fc domain, such as an antibody comprising the amino acid sequence set forth in SEQ ID NO: 118; and (ii) an ISVD comprising a CDR 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) fused to the N-terminus of the Fc domain defined herein, or a variant or humanized form thereof, and an ISVD comprising a CDR 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 SEQ ID NO: 124 (e.g., VHH72) fused to the N-terminus of the Fc domain, or a variant or humanized form thereof, particularly a bispecific antibody; or an ISVD comprising a CDR 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 may be fused to the N-terminus of the Fc domain, and an ISVD comprising a CDR 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 SEQ ID NO: 124 (e.g., VHH72) or a variant or humanized form thereof may be fused to the C-terminus of the Fc domain, wherein the CDRs are annotated according to Kabat, Martin, MacCallum, IMGT, AbM, or Chothia. In yet 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.

[0181] In an embodiment, the molar ratio of a binder (targeting S2), such as an antibody or an antigen-binding fragment thereof, which comprises one or more ISVDs (or variants or humanized forms thereof) described herein, or a part thereof, to a binder (targeting S1), such as an antibody or an antigen-binding fragment thereof, which comprises one or more ISVDs (or variants or humanized forms thereof) capable of binding to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, in the composition may be in the range of 3:1 to 1:3, preferably 2:1 to 1:2, and more preferably the molar ratio is about 1:1.

[0182] Furthermore, the composition may further contain additional binders or molecules, which may optionally bind to additional binding regions on the same or different epitopes of the spike protein, or to other viral proteins, or target completely unrelated target proteins.

[0183] A further aspect of the invention relates to a medicament or pharmaceutical composition comprising a binder, in particular an antibody or antigen-binding fragment, a nucleic acid encoding it, and / or a (recombinant) vector comprising the nucleic acid, and / or a composition comprising a binder, in particular an antibody or antigen-binding fragment, described herein. 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.

[0184] "Pharmaceutically acceptable" means a substance that is not biologically or otherwise undesirable, i.e., a compound, particularly a salvaco virus binder, more particularly a salvaco virus antibody or antigen-binding fragment, that can be administered to an individual without causing undesirable biological effects or interacting detrimentally with any of the other components of the pharmaceutical composition in which it is contained. A pharmaceutically acceptable carrier is preferably a carrier that is relatively non-toxic and harmless to the patient at a concentration consistent with the effective activity of the active ingredient, and any side effects resulting from the carrier do not nullify the beneficial effects of the active ingredient. Suitable carriers or adjuvants typically include one or more of the compounds contained in the following non-exhaustive list: large macromolecules that are slowly metabolized, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, high molecular weight amino acids, amino acid copolymers, and inactivated virus particles. The term "excipient", as used herein, is intended to include any substance that may be present in a pharmaceutical composition and is not an active ingredient but may contribute, for example, to long-term stability or therapeutic enhancement of the active ingredient (e.g., promotion of drug absorption, reduction of viscosity, or improvement of solubility). Excipients may include, for example, salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), lubricants, thickeners, surfactants, preservatives, emulsifiers, buffering substances, stabilizers, flavorings, or colorants. "Diluents", e.g., particularly "pharmaceutically acceptable diluents", include vehicles such as water, saline, physiological salt solutions, glycerol, ethanol, etc. Auxiliary substances such as wetting or emulsifying agents, pH buffering substances, preservatives, etc. may be included in such vehicles.

[0185] A pharmaceutically effective amount of the binder of the present invention, particularly an antibody or antigen-binding fragment, is preferably an amount that results in or affects the particular condition being treated.

[0186] The pharmaceutical composition of the present invention is lyophilized for storage and can be reconstituted in a suitable carrier before use. When prepared as a lyophilizate or as a liquid, it is necessary to add physiologically acceptable carriers, excipients, and stabilizers to the pharmaceutical composition of the present invention (Remington’s Pharmaceutical Sciences, 22nd Edition, edited by Allen, Loyd V, Jr. (2012)). Preparations containing the pharmaceutical composition of the present invention should be sterilized before injection. This procedure can be carried out using a sterile filtration membrane before and after lyophilization and reconstitution. The pharmaceutical composition can be packaged in a container or vial having a sterile access port, such as an i.v. solution bottle with a rubber stopper - the pharmaceutical composition can be present as a liquid, or the container or vial can be filled with a liquid pharmaceutical composition that is then lyophilized or dried; or it can be packaged in a prefilled syringe.

[0187] A further aspect of the present invention relates to a binder described herein, particularly an antibody or antigen-binding fragment, a nucleic acid encoding the same, a vector containing such a nucleic acid, a composition containing a binder described herein, particularly an antibody or antigen-binding fragment, or a pharmaceutical composition containing a binder, particularly an antibody or antigen-binding fragment, a nucleic acid encoding the same, a (recombinant) vector containing such a nucleic acid, and / or a composition containing a binder described herein, particularly an antibody or antigen-binding fragment, for use as a medicament or pharmaceutical. Alternatively, the use of a binder described herein, particularly an antibody or antigen-binding fragment, a nucleic acid encoding the same, a vector containing such a nucleic acid, or a composition containing a binder described herein, particularly an antibody or antigen-binding fragment, or the use of a pharmaceutical composition containing a binder, particularly an antibody or antigen-binding fragment, a nucleic acid encoding the same, a vector containing such a nucleic acid, and / or a composition containing a binder described herein, particularly an antibody or antigen-binding fragment, in the manufacture of a medicament or pharmaceutical is envisaged.

[0188] In particular, the binders described herein, in particular antibodies or antigen-binding fragments, nucleic acids encoding them, vectors containing such nucleic acids, or compositions containing binders, in particular antibodies or antigen-binding fragments, or pharmaceuticals or pharmaceutical compositions containing binders, in particular antibodies or antigen-binding fragments, nucleic acids encoding them, (recombinant) vectors containing such nucleic acids, and / or compositions containing binders, in particular antibodies or antigen-binding fragments described herein, are for use in passive immunization, for use in treating a subject having a sarbecovirus infection, for use in preventing infection of a subject having a sarbecovirus, or for use in protecting a subject from infection by a sarbecovirus.

[0189] When used for passive immunization, the subject may have an infection by a sarbecovirus (therapeutic passive immunization) or may not have an infection by a sarbecovirus (prophylactic passive immunization).

[0190] Related aspects relate to a method for treating a subject suffering from / having / affected by a sarbecovirus infection, comprising administering to the subject a binder described herein, in particular an antibody or antigen-binding fragment, a nucleic acid encoding it, a (recombinant) vector containing such nucleic acid, or a composition containing a binder, in particular an antibody or antigen-binding fragment, or a pharmaceutical or pharmaceutical composition containing a binder, in particular an antibody or antigen-binding fragment, a nucleic acid encoding it, a (recombinant) vector containing such nucleic acid, and / or a composition containing a binder, in particular an antibody or antigen-binding fragment described herein.

[0191] A further aspect of the invention is a method for protecting a subject from infection by a simian immunodeficiency virus or for preventing infection of a subject by a simian immunodeficiency virus, the method comprising administering to the subject before infection a binding agent described herein, in particular an antibody or antigen-binding fragment, a nucleic acid encoding the same, a (recombinant) vector comprising such a nucleic acid, or a composition comprising a binding agent, in particular an antibody or antigen-binding fragment, or administering to the subject before infection a pharmaceutical or pharmaceutical composition described herein comprising a binding agent, in particular an antibody or antigen-binding fragment, a nucleic acid encoding the same, a (recombinant) vector comprising such a nucleic acid, and / or a composition comprising a binding agent, in particular an antibody or antigen-binding fragment.

[0192] In the above medical aspect, a nucleic acid encoding a binding agent described herein, in particular an antibody or antigen-binding fragment, or a (recombinant) vector comprising such a nucleic acid can be used, for example, in a gene therapy setting. As used herein, "gene therapy" refers to a treatment effected by administration of an expressed or expressible nucleic acid to a subject. In such an application, the nucleic acid molecule or vector described herein enables the production of a binding agent, antibody or antibody fragment intracellularly. A number of methods for gene therapy are available in the art and include, for example, (adeno-associated) virus-mediated gene silencing or virus-mediated gene therapy (see, for example, US Patent Application Publication No. 20040023390; Mendell et al. 2017, N Eng J Med 377:1713-1722). A number of delivery methods are well known to those skilled in the art and include, but are not limited to, virus delivery systems, microinjection of DNA plasmids, gene gun delivery of naked nucleic acids, use of liposomes or artificial exosomes, administration of nucleic acids or vectors formulated into nanoparticles or lipid or lipid-containing particles. In vivo delivery by administration to an individual patient typically occurs by systemic administration (e.g., intravenous, intraperitoneal injection or intracerebral injection; see, for example, Mendell et al. 2017, N Eng J Med 377:1713-1722).

[0193] A "therapeutically active agent" generally refers to any molecule that has, or may have, a therapeutic effect (i.e., a curative or preventive effect) in relation to the treatment of a disease. Preferably, the therapeutically active agent is a disease-modifying therapeutic agent, which may be a cytotoxic agent such as a toxin, or a cytotoxic drug, or an enzyme capable of converting a prodrug into a cytotoxic drug, or a radionuclide, or a cytotoxic cell, or a non-cytotoxic agent. Even more preferably, the therapeutically active agent has a curative effect on the disease. The binder of the present invention, particularly an antibody or antibody fragment, or a pharmaceutical composition, can act as a therapeutically active agent when it is beneficial in treating patients infected with a sarbecovirus such as SARS-CoV-2 or SARS-CoV-1, or patients suffering from COVID-19. The binder, particularly an antibody or antibody fragment, may include a variant of ISVD that binds to the sarbecovirus described herein, preferably an improved variant that binds to the same binding region of the HR2 domain, more preferably a humanized variant thereof, and may contain, or be connected to, additional functional groups that are advantageous when administered to a subject. Examples of such functional groups and techniques for introducing them will be apparent to those skilled in the art and may generally include all functional groups and techniques referred to in the art, as well as functional groups and techniques known per se for the modification of pharmaceutical proteins, particularly the modification of antibodies or antibody fragments. For this, reference is made, for example, to Remington’s Pharmaceutical Sciences, 16th edition, Mack Publishing Co., Easton, PA (1980). Such functional groups may be linked directly (e.g., by covalent bond) to the ISVD or active antibody fragment, or optionally, via a suitable linker or spacer, as will again 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 the attachment of a suitable pharmacologically acceptable polymer such as poly(ethylene glycol) (PEG) or a derivative thereof (e.g., methoxypoly(ethylene glycol) or mPEG).For example, for this purpose, PEG may undergo binding to cysteine residues naturally present in the immunoglobulin single variable domain described herein (or variants or humanized forms thereof described herein), the immunoglobulin single variable domain described herein (or variants or humanized forms thereof described herein) may be modified to preferably introduce one or more cysteine residues for PEG binding, or an amino acid sequence containing one or more cysteine residues for PEG binding may be fused to the N-terminus and / or C-terminus of the ISVD or active antibody fragment described herein (or variants or humanized forms thereof described herein), all of which use techniques of protein manipulation known per se to those skilled in the art. Another, usually less preferred modification includes N-linked or O-linked glycosylation, usually as part of co-translational and / or post-translational modifications, depending on the host cell used to express the antibody or active antibody fragment. Another technique for increasing the half-life of the binding domain, particularly an antibody or antibody fragment, may involve engineering into a bifunctional or bispecific domain (e.g., one ISVD or active antibody fragment against the target sarbecovirus HR2 domain and one against a serum protein such as albumin or staphylococcal protein A (SpA), a surface protein abundant in the lung that helps extend half-life), or an antibody fragment, particularly a fusion of an immunoglobulin single variable domain and a peptide (e.g., a peptide against a serum protein such as albumin). In yet another example, the ISVD described herein (or variants or humanized forms thereof described herein) may be fused to the immunoglobulin Fc domain described elsewhere herein. Examples are further shown in the experimental section and also in the sequence listing. In embodiments, in the above medical aspects, the sarbecovirus is SARS-CoV-2, such as a SARS-CoV-2 variant, or SARS-CoV-1.The SARS-CoV-2 variant may be a variant at position N439, K417, S477, L452, T478, E484, P384, N501 and / or D614 (relative to the SARS-CoV-2 spike amino acid sequence defined in SEQ ID NO: 86), more particularly a variant at position N501 such as the N501Y variant (e.g., the SARS-CoV-2 alpha variant), a variant at positions N501 and E484 such as the N501Y and E484K variants (e.g., the SARS-CoV-2 alpha + E484K variant), a variant at positions K417, E484 and N501 such as the K417N, E484K and N501Y variants (e.g., the SARS-CoV-2 beta variant), a variant at positions P384, K417, E484 and N501 such as the P384L, K417N, E484K and N501Y variants (e.g., the SARS-CoV-2 beta + P384L variant), a variant at positions L452 and E484 such as the L452R and E484Q variants (e.g., the SARS-CoV-2 kappa variant), a variant at positions L452 and T478 such as the L452R and T478K variants (e.g., the SARS-CoV-2 delta variant), a variant at position L452 such as the L452R variant (e.g., the SARS-CoV-2 epsilon variant), a variant at position K417 such as the K417T variant (e.g., the SARS-CoV-2 gamma variant), or a variant at position D614 such as the D614G variant (e.g., the SARS-CoV-2 omicron variant or the SARS-CoV-2 BA.1 variant). In certain embodiments, the sarbecovirus is either or both of SARS-CoV-2 and SARS-CoV-2. In further particular embodiments, SARS-CoV-2 is the SARS-CoV-2 Wuhan strain or an SARS-CoV-2 variant, particularly an 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.

[0194] As used herein, the terms "treatment" or "therapy" refer to the reduction or measurable decrease of one or more symptoms or measurable markers of a disease or disorder, particularly a pathological condition such as a Sarecovirus infection. A measurable decrease includes a statistically significant decline in a measurable symptom or marker. Generally, the term encompasses both curative treatment and treatment aimed at reducing symptoms and / or delaying the progression of a disease. The term encompasses both therapeutic treatment of an already developed pathological condition, particularly a Sarecovirus infection, and preventive or prophylactic measures, the purpose of which is to prevent or reduce the likelihood of the development of a pathological condition, particularly a Sarecovirus infection. Beneficial or desired clinical outcomes include, but are not limited to, prevention of a disease, reduction in the incidence of a disease, alleviation of symptoms associated with a disease, reduction in the severity of a disease, stabilization of a disease, delay or deceleration of the progression of a disease, improvement or alleviation of a disease, or combinations thereof. In certain embodiments, the term may relate to therapeutic treatment. In certain embodiments, the term may relate to prophylactic treatment.

[0195] For example, treatment may refer to passive immunization (therapeutic treatment) of a subject afflicted with a Sarecovirus infection. Prevention of infection by Sarecovirus may be useful in the case of an epidemic or pandemic situation, for example, by prophylactically treating subjects known to be at highest risk of developing severe disease symptoms with a binder described herein, particularly an antibody or antigen-binding fragment, or a nucleic acid encoding the same, or a vector comprising such a nucleic acid, or a composition comprising a binder, particularly an antibody or antigen-binding fragment, to prevent overall infection or prevent the onset or development of severe disease symptoms.

[0196] In embodiments, a therapeutically effective amount of a binder, particularly an antibody or antigen-binding fragment, nucleic acid, vector or pharmaceutical composition is administered to a subject in need thereof. In other embodiments, a prophylactically effective amount of a binder, particularly an antibody or antigen-binding fragment, nucleic acid, vector or pharmaceutical composition is administered to a subject in need thereof. A "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a binder, particularly an antibody or antigen-binding fragment, nucleic acid, vector or pharmaceutical composition that, when administered to a subject, results in a clinically positive response with respect to the therapeutic treatment of a subject afflicted with a SARS-CoV infection, such as curing the infection by SARS-CoV. Similarly, a "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of a binder, particularly an antibody or antigen-binding fragment, nucleic acid, vector or pharmaceutical composition that prevents, inhibits or delays the onset of a SARS-CoV infection, and / or prevents or reduces the risk of clinical symptoms of a SARS-CoV infection, and / or reduces the severity, symptoms and / or duration of a SARS-CoV infection in a subject. To achieve a therapeutic or preventive or prophylactic effect, the binders described herein, particularly antibodies or antigen-binding fragments, or nucleic acids encoding them or vectors containing such nucleic acids, or compositions containing binders, particularly antibodies or antigen-binding fragments, may need to be administered to a subject multiple times, for example, at intervals of one or two weeks; the intervals are defined by the pharmacokinetic behavior or characteristics of the binder, particularly the antibody or antigen-binding fragment, nucleic acid or vector (e.g., half-life or half-life in the circulation of the subject). Alternatively, therapeutic and prophylactic treatments are envisioned in which a single dose of a binder, particularly an antibody or antigen-binding fragment, described herein is administered to a subject. The single dose may be in the range of 0.5 mg / kg to 25 mg / kg.

[0197] As used interchangeably herein, the terms "subject," "individual," or "patient" refer to any organism, such as any mammal, including both vertebrates, particularly humans, and other mammals, for which diagnosis, treatment, or prevention is desired, e.g., animals such as rodents, rabbits, cows, sheep, horses, dogs, cats, llamas, pigs, or non-human primates (e.g., monkeys). Rodents may 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 certain embodiments, the subject is a subject such as a human subject who has or is suspected of having an infection by simian coronavirus, also designated herein as "patient" or "subject." However, it will be understood that the foregoing terms do not imply the presence of symptoms. In certain embodiments, the subject is a mammal susceptible to infection by simian coronavirus, such as a human subject susceptible to infection by SARS-CoV-2, such as a SARS-CoV-2 variant, or SARS-CoV-1.

[0198] The pharmaceutical compositions of the present invention can be administered to any patient according to standard techniques. Administration can be by any suitable mode, including oral, parenteral, topical, nasal, ophthalmic, intrathecal, intraventricular, sublingual, rectal, vaginal, etc. Still other techniques for formulations such as nanotechnology and aerosols and inhalants are also within the scope of the present invention. The dosage and frequency of administration will vary depending on the age, sex, and condition of the patient, co-administration of other drugs, contraindications, and other parameters to be considered by the clinician.

[0199] In certain embodiments of the medical aspects described herein, the binder, particularly an antibody or antigen-binding fragment, nucleic acid, vector, or pharmaceutical composition can be administered to the subject by intravenous injection, subcutaneous injection, or nasally, or alternatively by inhalation or pulmonary delivery.

[0200] A further aspect of the invention relates to a binder as described herein, in particular an antibody or antigen-binding fragment, for use as a diagnostic agent for the diagnosis of Salvethovirus infections. Also contemplated is the use of a Salvethovirus binder as described herein, in particular a nucleic acid encoding a Salvethovirus antibody or antigen-binding fragment, a (recombinant) vector comprising such a nucleic acid, or a composition comprising a Salvethovirus binder as described herein, in particular a Salvethovirus antibody or antigen-binding fragment, for use.

[0201] Also contemplated is the use of a binder as described herein, in particular an antibody or antigen-binding fragment, in the manufacture of an (in vitro) diagnostic agent or diagnostic kit. In particular, a binder as described herein, in particular an antibody or antigen-binding fragment, may be for use in detecting the presence (or absence) of Salvethovirus or a part thereof (e.g., the Salvethovirus spike protein or a part thereof) in a sample, such as a sample obtained from a subject suspected of being infected with Salvethovirus. Also contemplated is the use of a nucleic acid encoding a binder as described herein, in particular an antibody or antigen-binding fragment, a (recombinant) vector comprising such a nucleic acid, or a composition comprising a binder as described herein, in particular an antibody or antigen-binding fragment, in the manufacture of a diagnostic agent or diagnostic kit, such as an in vitro diagnostic agent or kit.

[0202] A further aspect relates to a method for detecting Salvethovirus in a sample, such as a sample obtained from a subject suspected of being infected with Salvethovirus. Such a method typically comprises the steps of obtaining a sample, contacting the sample with a binder as described herein, in particular an antibody or antigen-binding fragment, and detecting, determining, evaluating, assaying, identifying or measuring the binding of the binder, in particular an antibody or antigen-binding fragment, to Salvethovirus or a part thereof (e.g., the Salvethovirus spike protein or a part thereof).

[0203] In certain embodiments of the diagnostic modalities described herein, the sarbecovirus is selected from the group consisting of sarbecoviruses of clades 1a, 1b, 2 and / or clade 3, such as SARS-Cov-2, GD-Pangolin, RaTG13, WIV1, LYRa11, RsSHC014, Rs7327, SARS-CoV-1, Rs4231, Rs4084, Rp3, HKU3-1, or BM48-31 virus, preferably SARS-CoV-2 or a SARS-CoV-1 such as a SARS-CoV-2 variant.

[0204] In embodiments of the diagnostic aspects described herein, the binders described herein, particularly antibodies or antibody fragments, include a detectable moiety fused to, bound to, connected to, conjugated to, complexed with, or chelated to it. A "detectable moiety" generally refers to a moiety that emits a signal or is capable of emitting a signal upon an appropriate stimulus, or a moiety that is detectable by binding or interaction with an additional molecule (such as a tag, e.g., an affinity tag specifically recognized by a labeled antibody), or a moiety that is detectable by any means (preferably non-invasive means if the detection is in vivo / human body). Further, the detectable moiety may enable the synthesis of computer-generated images, and such a detectable moiety may be referred to as an imaging agent. Detectable moieties include, but are not limited to, fluorophores, phosphors, positron emitters, radioluminescent agents, etc., enzymes (capable of measurably converting a substrate), and molecular tags. Examples of radioluminescent agents / 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, 114mIn, 114In, 99mTc, 11C, 32Cl, 33Cl, 34Cl, 123I, 124I, 131I, 186Re, 188Re, 177Lu, 99Tc, 212Bi, 213Bi, 212Pb, 225Ac, 153Sm, and 67Ga. Fluorophores include, but are not limited to, cyanine dyes (e.g., Cy5, Cy5.5, Cy7, Cy7.5), FITC, TRITC, coumarin, indolenine-based dyes, benzoindolenine-based dyes, phenoxazine, 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., 6×His or His6), biotin or streptavidin such as Strep-tag®, Strep-tag II® and Twin-Strep-tag®; solubilizing tags such as thioredoxin (TRX), poly(NANP) and SUMO; chromatography tags such as FLAG tag; epitope tags such as V5 tag, myc tag and HA tag; fluorescent labels or tags (i.e., fluorescent dyes / fluorophores) such as fluorescent proteins (e.g., GFP, YFP, RFP, etc.); luminescent labels or tags such as luciferase, bioluminescent or chemiluminescent compounds (e.g., luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate esters, dioxetanes or GFP and its analogs); phosphorescent labels; metal chelators; and (other) enzyme labels (e.g., peroxidase, alkaline phosphatase, beta-galactosidase, urease, or glucose oxidase).

[0205] Binding agents, particularly antibodies and antibody fragments, containing a detectable moiety as described herein 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") and for in vivo imaging purposes, depending on the selection of a particular label.

[0206] A further aspect relates to a kit comprising a binding agent as described herein, particularly an antibody or antigen-binding fragment, a nucleic acid encoding the same, a vector containing such nucleic acid, or a composition containing a binding agent, particularly an antibody or antigen-binding fragment, or a pharmaceutical composition comprising a binding agent as described herein, particularly an antibody or antigen-binding fragment, a nucleic acid encoding the same, a vector containing such nucleic acid, or a composition containing a binding agent, particularly an antibody or antigen-binding fragment.

[0207] Such a kit may include a container or vial (any suitable container or vial such as a pharmaceutically acceptable container or vial) containing an amount of a binder as described herein, particularly an antibody or antigen-binding fragment, or a nucleic acid encoding the same or a vector containing such a nucleic acid, or a composition containing a binder as described herein, particularly an antibody or antigen-binding fragment, and further include a kit insert such as a medical leaflet or package leaflet containing information regarding, for example, the intended indication (prophylactic or therapeutic treatment of sarbecovirus infection) and potential side effects. The pharmaceutical kit or drug kit may further include, for example, a syringe for administering to a subject an amount of a binder as described herein, particularly an antibody or antigen-binding fragment, a nucleic acid encoding the same, a vector containing such a nucleic acid, or a composition containing a binder, particularly an antibody or antigen-binding fragment.

[0208] Such a kit may also be a diagnostic kit including a container or vial (any suitable container or vial such as a pharmaceutically acceptable container or vial) containing an amount of a binder as described herein, particularly an antibody or antigen-binding fragment, for example, a binder containing a detectable moiety, particularly an antibody or an antigen-binding fragment thereof. Such a diagnostic kit may further include, for example, one or more reagents for detecting the detectable moiety and / or instructions regarding the use of the binder, particularly an antibody or antigen-binding fragment, for detecting sarbecovirus in a sample.

[0209] Although the present invention has been described in conjunction with its specific embodiments, it will be apparent to those skilled in the art that many alternatives, modifications, and variations are obvious in view of the foregoing description. Accordingly, the present invention is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the following claims.

[0210] Certain aspects and embodiments of the present invention are set forth in the following numbered descriptions: (1) An agent capable of neutralizing a sarbecovirus, which is characterized by specifically binding to a region of the heptad repeat 2 (HR2) domain of the spike protein of the sarbecovirus proximal to the viral membrane. (2) The binding agent according to (1), wherein the binding agent specifically binds to a region of the HR2 domain located at or within the amino acids from amino acid I1179 to amino acid E1202, preferably from amino acid D1184 to amino acid E1202, more preferably from amino acid V1189 to amino acid E1202 of the SARS-CoV-2 spike protein defined in SEQ ID NO: 86. (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 in SEQ ID NO: 86. (4) - When determined in a sarbecovirus spike protein pseudovirus neutralization assay such as a vesicular stomatitis virus (VSV)-sarbecovirus spike protein pseudovirus neutralization assay, it can neutralize the sarbecovirus at a 50% inhibitory concentration (IC 50 ) of 100 ng / ml or less, preferably 10 ng / ml or less, more preferably 1 ng / ml or less; - It can neutralize any one or more of SARS-CoV-2, such as SARS-CoV-2 Wuhan strain, SARS-CoV-2 alpha variant, SARS-CoV-2 omicron BA.1 variant, and SARS-CoV-2 omicron BA.2 variant; and / or either or both of SARS-CoV-1; - It can inhibit spike-mediated syncytium formation between cells expressing the sarbecovirus spike protein and cells expressing the angiotensin-converting enzyme 2 (ACE2) receptor; and / or - The binding agent according to any one of (1) to (3), which does not bind to the Middle East respiratory syndrome coronavirus (MERS-CoV). (5) An binder according to any one of (1) to (4), comprising or consisting of an antibody or an antibody fragment. (6) An binder according to any one of (1) to (5), comprising an immunoglobulin single variable domain (ISVD), preferably a VHH. (7) The binder according to (6), wherein the ISVD comprises a complementarity determining region 1 (CDR1) defined by any one of SEQ ID NO: 63, 46, 69 or 77, a complementarity determining region 2 (CDR2) defined by any one of SEQ ID NO: 64, 47, 70, 73 or 78, and a complementarity determining region 3 (CDR3) defined by any one of SEQ ID NO: 48, 67, 74 or 79; preferably, the CDR1 defined by any one of SEQ ID NO: 65, 71, 49 or 80, the CDR2 defined by any one of SEQ ID NO: 66, 72, 50, 75 or 81, and the CDR3 defined by any one of SEQ ID NO: 51, 68, 76 or 82. (8) The binder according to (6) or (7), wherein the ISVD independently comprises CDR1, CDR2 and CDR3 present in any one of SEQ ID NOs: 1 to 10, and CDR1, CDR2 and CDR3 are annotated according to any one of Kabat, MacCallum, IMGT, AbM, Martin or Chothia; preferably, the ISVD comprises a combination of CDR1, CDR2 and CDR3, and CDR1, CDR2 and CDR3 are as present in a specific one of the sequences described in SEQ ID NOs: 1 to 10, and CDR1, CDR2 and CDR3 are annotated according to any one of Kabat, MacCallum, IMGT, AbM, Martin or Chothia. (9) The binder according to any one of (6)-(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. (10) The binder according to any one of (7)-(9), wherein the ISVD comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10. (11) The binder according to any one of (1)-(10), comprising an ISVD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10. (12) The binder according to any one of (1)-(11), which is in a multivalent form and preferably comprises an ISVD fused to an Fc domain. (13) A nucleic acid molecule comprising a polynucleotide sequence encoding the binder according to any one of (1)-(12); a vector comprising the nucleic acid molecule; or a cell expressing the binder according to any one of (1)-(12), or comprising the nucleic acid molecule or the vector. (14) A pharmaceutical composition comprising the binder according to any one of (1)-(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 binder according to any one of (1)-(12). (15) Use in medicine, preferably for the prevention or treatment of Sarecovirus infection in a subject, or for the diagnosis of Sarecovirus infection in a subject, of the binder according to any one of (1)-(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). An in vitro or ex vivo method for detecting SARS-CoV-2 in a sample, comprising: - contacting the sample with the binder according to any one of (1) to (12); and - determining the binding of the binder to SARS-CoV-2 or a part thereof An in vitro or ex vivo method comprising the above steps.

[0211] The aspects and embodiments of the invention disclosed herein are further supported by the following non-limiting examples.

Examples

[0212] Materials and Methods Isolation of SARS-CoV-2 VHH phages To obtain cross-reactive VHHs against SARS-CoV-1 and SARS-CoV-2, llamas pre-immunized with recombinant prefusion-stabilized SARS-CoV-1 and MERS-CoV spike proteins were boost-immunized three times with recombinant SARS-CoV-2 spike protein (S-2P) stabilized in the prefusion conformation (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 the llamas and an immune VHH-display phagemid library was constructed. Phages presenting VHHs specific for SARS-CoV-2 were enriched from this phage library by performing two rounds of biopanning on 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: 1501–1505), which was immobilized onto the wells of microtiter plates (type II, F96 Maxisorp, Nunc) with a coated anti-His antibody in the presence of 10 μg / ml of RBD-SD1-mouse IgG (Sionobiological). Furthermore, 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). Also, in these two additional rounds of the series, biopanning was performed in the presence of 10 μg / ml of RBD-SD1-mouse IgG (Sionobiological). In each round of panning, uncoated wells were used as negative controls.Subsequently, the wells were washed five times with phosphate-buffered saline (PBS) + 0.05% Tween 20, blocked with 4% non-fat milk in PBS SEA BLOCK blocking buffer (Thermo Scientific) in the first panning, Pierce Protein-Free blocking buffer (Thermo Scientific) in the second, SEA BLOCK blocking buffer (Thermo Scientific) in the third, and 1% BSA in the fourth. Non-specifically bound phages were removed by extensive washing with PBS + 0.05% Tween 20. The retained phages were eluted with TEA solution (14% trimethylamine (Sigma), pH 10) and then neutralized with 1 M Tris-HCl, pH 8. The harvested phages were amplified in exponentially growing Escherichia coli (E. coli) TG1 cells, infected with VCS M13 helper phage, and then purified using PEG 8,000 / NaCl precipitation for the next round of selection. The enrichment factor after each panning round was determined by infecting TG1 cells with 10-fold serial dilutions of the harvested phages and then plating the bacteria on LB agar plates containing 100 μg / mL ampicillin and 1% glucose.

[0213] Preparation of periplasmic extract (PE) After 3 or 4 rounds of panning, individual colonies of phage-infected bacteria were randomly selected for further analysis. Individual colonies were inoculated into 2 mL of terrific broth (TB) medium containing 100 μg / mL ampicillin in 24-well deep-well plates. After growing individual colonies at 37 °C for 5 h, isopropyl β-D-1-thiogalactopyranoside (IPTG) (1 mM) was added to induce VHH expression during overnight culture at 37 °C. To prepare the periplasmic extract, bacterial cells were pelleted, resuspended in 250 μL of TES buffer (0.2 M Tris-HCl pH 8, 0.5 mM EDTA, 0.5 M sucrose), and incubated at 4 °C for 30 min. Then, 350 μL of water was added to induce osmotic shock. After incubation at 4 °C for 1 h, the mixture was centrifuged and the periplasmic extract was recovered.

[0214] Periplasmic extract - Enzyme-linked immunosorbent assay Wells of a half-well microtiter plate were coated overnight at 4 °C with 50 ng of recombinant SARS-CoV-2 S-2P protein, SARS-CoV-2 S2 subunit (AcroBiosystems, S2N-C52H5), SARS-CoV-2 RBD (Sinobiologicals), SARS-CoV S, MERS-CoV S, HKU1 S and BSA. The plate was blocked with 5% non-fat dry milk in PBS. The periplasmic extract was diluted 1 / 10 with PBS and added to the blocked wells. Binding of the VHH was detected with a mouse anti-HA antibody (BioLegend 901501, 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 50 μl of 1 M H2SO4 was added to stop the reaction. Absorbance at 450 nm was measured using an iMark Microplate Absorbance Reader (Bio Rad). Curve fitting was performed using non-linear regression (Graphpad 8.0).

[0215] Periplasmic extract - Pseudovirus neutralization assay Pseudoviruses expressing SARS-CoV-2 spike (D614G) were incubated at 37°C for 30 minutes with a 1 / 100 dilution of the periplasmic extract in 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. Next, the incubated pseudoviruses were added to a sub-confluent monolayer of Vero E6 from which the original growth medium had been removed. After 16 hours, the cells were lysed using passive lysis buffer (Promega). Transduction efficiency was quantified by measuring GFP fluorescence in the prepared cell lysates using a Tecan infinite 200 pro plate reader. GFP fluorescence was normalized using the GFP fluorescence of non-infected cells and infected cells treated with PBS.

[0216] Cell line FreeStyle293F cells (ThermoFisher Scientific) and HEK293-S cells (ThermoFisher Scientific) were cultured at 37°C, 8% CO 2 in FreeStyle 293 expression medium (Life Technologies) with shaking at 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 in the presence of 5% CO 2 at 37°C. ExpiCHO-S cells (GIBCO) were cultured at 37°C, 8% CO 2They were cultured. Vero E6-TMPRSS2 cells that stably express human TMPRSS2 (NIBIOHN, JCRB1819) (Matsuyama et al., PNAS, 2020) were cultured in DMEM containing 10% FBS, penicillin (100 unit / mL), streptomycin (100 μg / mL), and Geneticin (G418) (1 mg / ml). When seeding Vero E6-TMPRSS2 cells for the assay, a medium without Geneticin was used.

[0217] Raji cells and Raji cells stably expressing the SARS-CoV-2 spike protein were cultured at 37 °C, 5% CO 2 in RPMI-1640 medium supplemented with 10% FCS, 0.1 μg / ml puromycin, 1% penicillin, and 1% streptomycin.

[0218] Sotrovimab, cilgavimab, bebtelovimab, and palivizumab Bebtelovimab biosimilar (PX-TA1750), cilgavimab biosimilar (PX-TA1033), and sotrovimab biosimilar (PX-TA1637) were purchased as commercial products from Proteogenix. Clinical-grade palivizumab was obtained from Ghent University hospital.

[0219] Generation of R3_DC23-Fc(YTE) (also referred to herein as huR3DC23-Fc) (SEQ ID NO: 96) The humanized version of R3_DC23 by Kabat numbering (Q1D, Q5V, A14P, D16G, T19R, M63V, S73N, D79Y, T82cL, K83R, and Q108L; in particular, the T82cL modification serves to inactivate glycosylation at the N82a position and may be useful in both humanized and non-humanized versions for expression in mammalian cells) was ordered to be synthesized as a gBlock by IDT to the human IgG1 Fc (EPKSCdel_YTE_K447del) with (G 4 S) 2It was fused via a linker. After arrival, the gBlock was solubilized in ultrapure water at a concentration of 20 ng / μL. The 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 (ThermoFisher). The ORFs of the positive clones were fully sequenced, and the pDNA of the selected clones was prepared using the NucleoBond Xtra Midi kit (Machery-Nagel).

[0220] Transient production of huR3DC23-Fc_LS (also referred to herein as R3_DC23hum-Fc(LS) or XVR013) (SEQ ID NO: 118) 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 CHOExpress™ cells at a cell density of 4.00E+6 cells / ml. The TGE supernatant was recovered by centrifugation and clarified by filtration (0.2 μm) when the cell viability was 10% or less after 10 days. The protein was further purified with Protein A.

[0221] Production of VHH73_S56A, GBP, CB6, and S309 The production of VHH73_S56A, GBP, CB6, and S309 was carried out as described by Schepens et al. (Schepens et al. (2021) Sci. Transl Med. 13: eabi7826).

[0222] HEK S transfection and protein purification protocol; production of YTE variants of VHH-Fc in mammalian cells HEK293-S cells were transfected with a plasmid encoding VHH-Fc(S) using polyethyleneimine (PEI). Briefly, serum-free HEK293-S cells adapted to suspension were seeded at 3×10 6 cells / mL in FreeStyle 293 medium (ThermoFisher Scientific). Next, 4.5 μg of pcDNA3.3-VHH-Fc plasmid DNA was added to the cells and incubated on a shaking platform at 37 °C and 8% CO 2 for 5 minutes. Then, 9 μg of PEI was added to the culture and the cells were incubated for an additional 5 hours, after which an equal culture volume of Ex-Cell-293 (Sigma) was added to the cells. After incubating the transfection for 4 days, the cells were pelleted (10’, 300 g) and the supernatant was filtered and used further. To purify the VHH-Fc protein, the supernatant was loaded onto a 5 mL MAbSelect SuRe column (GE Healthcare). Unbound proteins were washed with McIlvaine buffer pH 7.2 and the bound proteins were eluted using McIlvaine buffer pH 3. Immediately after elution, the fractions containing the protein were neutralized using 30% (v / v) saturated Na 3 PO 4 buffer. Next, these fractions were pooled and loaded onto a HiPrep desalting column to buffer exchange to PBS, pH 7.4.

[0223] Furthermore, huR3DC23-Fc_YTE was expressed in ExpiCHO-S™ cells (ThermoFisher Scientific) according to the manufacturer's protocol. Briefly, 6×10 2 per mL grown at 37 °C, 8% CO 6To 50 mL of a culture of individual cells, 40 μg of pcDNA3.3-VHH72-Fc plasmid DNA was transfected using ExpiFectamine™ CHO reagent. One day after transfection, 300 μL of ExpiCHO™ enhancer and 8 mL of ExpiCHO™ feed were added to the cells, and the culture was further incubated at 32 °C and 5% CO2. A second feeding of cells was performed on day 5 after transfection. The product was harvested when the cell viability dropped below 75%.

[0224] To purify the VHH-Fc protein, the supernatant was loaded onto a 5 mL MAbSelect SuRe column (GE Healthcare). Unbound proteins were washed with McIlvaine buffer pH 7.2, and the bound proteins were eluted using McIlvaine buffer pH 3. Immediately after elution, the fractions containing the protein were neutralized using 30% (v / v) saturated Na3PO4 buffer. These fractions were then pooled and loaded onto a HiPrep desalting column to exchange the buffer to PBS, pH 7.4.

[0225] Fed-batch production at 1 L scale from a stable pool of huR3DC23-Fc_LS The gene encoding huR3DC23-Fc_LS was codon-optimized, synthesized in the ATUM laboratory, and cloned into the pXLG6 backbone vector. It was expanded to a density of approximately 4·10 6 cells / ml and the expression vector and pXLG5 helper vector were co-transfected into parental CHOExpress™ cells. A stable pool was created under a puromycin selection pressure of 50 mg / L (applied daily) and further expanded. On day 14 when the cell viability reached 95%, the stable pool research cell bank was banked.

[0226] Subsequently, the RCB pool was expanded to 1 L scale for protein production and cultured until day 12 (cell density 3.5·10 7per mL, cell viability 96%). The supernatant was recovered by centrifugation and clarified by filtration (0.2 μm). The protein was further purified with MabSelect SuRe LX resin using Protein A. Serial 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 of 100 mM sodium acetate pH 3.5 was neutralized to pH 7.0 by adding 1 M Tris pH 11 (10% v / v). After filter sterilization (0.22 μm), the protein was dispensed at 2 mg / ml.

[0227] Protein Preparation for Biophysical Analysis Prior to biophysical characterization, the protein sample purified with MAbSelect SuRe was equilibrated with Dulbecco's phosphate buffered saline (PBS, Sigma-Aldrich) supplemented with 0.02% sodium azide to prevent microbial growth, or a sample buffer containing 50 mM L-histidine and 150 mM L-arginine (Sigma-Aldrich), 0.02% polysorbate-20, and 0.02% sodium azide, and further purified by size exclusion chromatography (SEC) on a Superdex 200 column (GE-Healthcare) cooled to 12 °C and set to pH 7.0 at 25 °C. After filter sterilization (0.22 μm), an aliquot of 1 mg / ml was snap frozen in a polypropylene tube in liquid nitrogen and stored at -80 °C.

[0228] Sample Composition The purified VHH-Fc samples were characterized by analytical SEC to determine the molecular composition of each sample. After rapid thawing in a 25 °C water bath, centrifugation at 16,000×g for 10 min, and transferring the supernatant to a new tube, 5 μg was injected onto an AdvanceBio SEC column (4.6 × 300 mm) (Agilent) with a porous particle size of 2.7 μm and a pore size of 300 Å calibrated with PBS. Separation was monitored at an absorbance of 280 nm with a bandwidth of 16 nm, and no reference subtraction was performed. For further quality control, proteins were separated by reducing 15% SDS-PAGE using Coomassie staining.

[0229] Generation of a spike protein expression vector for the production of VSVdelG pseudovirus particles expressing spike proteins containing RBD mutations of SARS-CoV-2 variants The pCG1 expression vector for the SARS-CoV-2 spike protein containing the D614G mutation was generated from the pCG1-SARS-2-Sdel18 vector by introducing specific RBD mutations by QuickChange mutagenesis using appropriate primers according to the manufacturer's instructions (Aligent). For the pCG1-SARS-2-Sdel18 expression vector for the Omicron BA.1 variant, the codon-optimized spike protein nucleotide sequence 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, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F) and the adjacent BamHI and SalI restriction sites was ordered from Geneart (Thermo Fischer Scientific) and cloned into the pCG1 vector as a BamHI / SalI fragment. For the pCG1-SARS-2-BA.2 Sdel18 expression vector, the codon-optimized spike protein nucleotide sequence containing the 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 the adjacent BamHI and SalI restriction sites was ordered from Geneart (Thermo Fischer Scientific) and cloned into the pCG1 vector as a BamHI / SalI fragment.For the pCG1-SARS-2-BA.2.75 Sdel18 expression vector, a codon-optimized spike protein nucleotide sequence containing the BA.2.75 mutations (K147E, W152R, F157L, I210V, G257S, D339H, G446S, N460K, R493Q) and the adjacent 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 vector for the Omicron BA.2.75.2 variant was generated from pCG1-SARS-2-BA.2.75 Sdel18 by introducing the R346T, F486S, D1199N mutations using appropriate gBlocks (ordered from IDT) and following the manufacturer's instructions (New England BioLabs) with the Gibson Assembly cloning technique.

[0230] The pCG1 expression vector for the Omicron BA.4 / BA.5 variant was generated from the pCG1-SARS-2-BA.2 Sdel18 vector by introducing the H69-, V70-deletions and the L452R, F486V, R493Q mutations by QuickChange mutagenesis using appropriate primers (ordered from IDT) and following the manufacturer's instructions (Agilent).

[0231] The pCG1 expression vector for the Omicron BA.4.6 variant was generated from pCG1-SARS-2-BA.4 Sdel18 by introducing the R346T and N658S mutations by GQuickChange mutagenesis using appropriate primers (ordered from IDT) and following the manufacturer's instructions (Agilent).

[0232] The pCG1 expression vector for the Omicron BF.7 variant was generated from pCG1-SARS-2-BA.4 Sdel18 by introducing the R346T mutation by QuickChange mutagenesis using appropriate primer k (ordered from IDT) and following the manufacturer's instructions (Agilent).

[0233] The pCG1 expression vector for the Omicron BQ.1.1 variant was generated from the pCG1-SARS-2-BA.5 Sdel18 vector by introducing the R346T, K444T, and N460K mutations via QuickChange mutagenesis using appropriate primers (ordered from IDT) according to the manufacturer's instructions (Agilent).

[0234] The pCG1 expression vector for the Omicron XBB variant was generated from pCG1-SARS-2-BA.2 Sdel18 by introducing the V83A, Y144-, H146Q, Q183E, V213E, D339H, R346T, L368I, V445P, G446S, N460K, F486S, F490S, R493Q mutations via Gibson Assembly cloning technique using appropriate gBlocks (ordered from IDT) according to the manufacturer's instructions (New England BioLabs). The pCG1 expression vector for the Omicron XBB.1.5 variant was generated from pCG1-SARS-2-XBB Sdel18 by introducing the F486P mutation via QuickChange mutagenesis using appropriate primers (ordered from IDT) according to the manufacturer's instructions (Agilent).

[0235] After sequencing, clones containing the correct spike coding sequence were prepared using the Qiagen plasmid Qiagen kit. Prior to use, the spike coding sequence of the prepared pCG1 vector was confirmed by Sanger sequencing.

[0236] Hydrophobic interaction chromatography (HIC) assay The apparent hydrophobicity was evaluated using a hydrophobic interaction chromatography (HIC) assay with a Dionex ProPac HIC-10 column (100 mm × 4.6 mm, Thermo Fisher 063655) containing a stationary phase consisting of a mixed population of ethyl and amide functional groups attached 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 throughout, and the flow rate was 0.8 ml / min. 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. The protein and calibration samples were diluted 1:1 with buffer A and injected onto the column. After holding at B 50% for 5 min, the bound protein was eluted with a linear gradient from B 50% to B 100% over 50 min, followed by holding at B 100% for 5 min. The column was washed with B 100%, then 50 mM ammonium acetate pH 5, and re-equilibrated with B 50% for 10 min prior to the next sample. Separations were monitored at an absorbance of 280 nm with a 16 nm bandwidth, and there was no reference subtraction.

[0237] Mass spectrometry of proteins Intact VHH protein (10 μg) was first reduced with tris(2-carboxyethyl)phosphine (TCEP; 10 mM) at 37 °C for 30 min, and then the reduced protein was separated on an Ultimate 3000 HPLC system (Thermo Fisher Scientific, Bremen, Germany) online-connected to an LTQ Orbitrap XL mass spectrometer (Thermo Fischer Scientific). Briefly, approximately 8 μg of protein was injected onto a Zorbax 300SB-C18 column (5 μm, 300 Å, 1 × 250 mm IDxL; Agilent Technologies) and separated using a 30-min gradient from 5% to 80% solvent B at a flow rate of 100 μl / min (solvent A: water, 0.1% formic acid and 0.05% trifluoroacetic acid; solvent B: acetonitrile, 0.1% formic acid and 0.05% trifluoroacetic acid). The column temperature was maintained at 60 °C. The eluted protein was directly sprayed into the mass spectrometer using an ESI source with the following parameters: spray voltage 4.2 kV, surface-induced dissociation 30 V, capillary temperature 325 °C, capillary voltage 35 V, and sheath gas flow rate 7 (arbitrary units). The mass spectrometer was operated in MS1 mode in profile mode using an Orbitrap analyzer with a resolution of 100,000 (m / z 400) and a mass range of 600 - 4000 m / z. The obtained MS spectra were deconvoluted using the Xtract deconvolution algorithm (isotope-resolved spectra) in BioPharma FinderTM 3.0 software (Thermo Fischer Scientific). The deconvoluted spectra were manually annotated.

[0238] Production of VHH by Escherichia coli For the production of VHH in E. coli, the pMECS vector containing the VHH of interest was transformed into WK6 cells (non-suppressor E. coli strain) and plated on LB plates containing ampicillin. The next day, clones were picked up and grown overnight at 37 °C with shaking at 200 rpm in 2 mL of LB containing 100 μg / ml of ampicillin and 1% glucose. Using 1 ml of this preculture, inoculate 25 ml of TB (Terrific Broth) supplemented with 100 μg / ml of ampicillin, 2 mM MgCl 2 and 0.1% glucose and incubate with shaking (200 - 250 rpm) at 37 °C until the OD600 reaches 0.6 - 0.9. VHH production was induced by adding IPTG to a final concentration of 1 mM. These induced cultures were incubated overnight at 28 °C with shaking at 200 rpm. The produced VHH was extracted from the periplasm and purified as described by Wrapp et al. (2020. Cell 181:1004 - 1015.e15). That is, VHH was purified from the solution using Ni Sepharose beads (GE Healthcare). After elution using 500 mM imidazole, the flow-through fraction containing VHH was buffer-exchanged with PBS using a Vivaspin column (5 kDa cut-off, GE Healthcare). The purified VHH was analyzed by SDS-PAGE and Coomassie staining, as well as intact mass spectrometry.

[0239] Enzyme-linked immunosorbent assay Wells of a microtiter plate (type II, F96 Maxisorp, Nunc) were coated overnight at 4 °C with 100 ng of recombinant SARS-CoV S-6P protein (Hsieh et al. 2020), SARS-CoV-1 S-2P protein (including foldon), His-tagged SARS-CoV-2 RBD (Sinobiologicals), SARS-CoV-2 spike S2 subunit (ACRObiosystems), 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 (ACRObiosystems), mouse Fc-tagged SARS-CoV-2 RBD (Sinobiologicals), or BSA. The coated plates were blocked with 5% non-fat dry milk in PBS. Serial dilutions of VHH or VHH-Fcs or antibodies were added to the wells and incubated for 90 min. After washing, binding was detected by sequentially incubating the plates with HRP-conjugated rabbit anti-camelid VHH antibody (Genscript) or mouse anti-HA antibody (BioLegend 901501, 1 / 2000), followed by anti-mouse IgG-HRP (GE Healthcare, NA931V, 1 / 2000). Binding of VHH-Fcs or conventional human monoclonal antibodies was detected by rabbit anti-human IgG (Sigma), followed by anti-rabbit IgG-HRP (Southern Biotech). After washing, 50 μL of TMB substrate (tetramethylbenzidine, BD OptETA) was added to the plates and 50 μL of 1M H 2 SO 4 was added to stop the reaction. Absorbance at 450 nM was measured using an iMark Microplate Absorbance Reader (Bio Rad). Curve fitting was performed using non-linear regression (Graphpad 8.0).

[0240] Flow Cytometry Analysis of Binding to HEK293 Cells Expressing SARS-CoV Spike Protein To examine the binding of VHHs to spike proteins on the surface of mammalian cells by flow cytometry, the inventors used the pCG1 expression plasmid containing the coding sequence of the SARS-CoV-2 spike protein, from which the C-terminal 18 amino acids were deleted, and introduced the D614G substitution (614G) by QuickChange site-directed mutagenesis (Agilent) according to the manufacturer's instructions. Two days after transfection of HEK293-T cells with a GFP expression plasmid combined with either the spike expression plasmid or the control expression plasmid, the cells were harvested. All subsequent operations were performed on ice. The cells were washed once with PBS and blocked with 1% BSA. The cells were stained with antibody or VHH dilution series for 90 minutes and then washed three times with PBS containing 1% BSA. The binding of VHHs was detected using a goat anti-mouse IgG antibody (Invitrogen) conjugated with AF647 and a mouse anti-His tag antibody (Biorad). The binding of VHH-Fcs or antibodies was detected using a goat anti-human IgG antibody (Invitrogen), and dead cells were stained using Live / Dead staining (Invitrogen). After washing three times with PBS containing 0.5% BSA, the cells were analyzed by flow cytometry using a BD LSRII flow cytometer (BD Biosciences). The binding curves were fitted using non-linear regression (Graphpad 8.0).

[0241] FcRn Binding Affinity Analysis of antibody binding to the purified recombinant human FcRn / FCGRT - B2M protein by surface plasmon resonance (SPR) was performed on a Biacore 8 K+ instrument by FairJourney Biologics (Porto, Portugal). The His-tagged recombinant human FcRn / FCGRT - B2M heterodimer protein was purchased from Acro Biosystems. Bevacizumab biosimilar and human IgG1 isotype control antibody were used as controls for the assay. Briefly, R3_DC23hum - Fc(LS) or the control antibody was immobilized at low density on a CM5 sensor chip (Cytiva) by amine coupling, and the immobilization level was set at 130 - 283 RU. At pH 6.0, the human FcRn / FCGRT - B2M heterodimer protein was injected in a 2-fold dilution series of 8 steps in the range of 1000 nM - 7.8 nM in the control antibody-immobilized channel and 9 steps in the range of 250 nM - 0.97 nM in the R3_DC23hum - Fc(LS)-immobilized channel in a solution of 1.5 μM (anchor point). At pH 7.4, the human FcRn / FCGRT - B2M heterodimer protein was injected in a 2-fold dilution series of 8 steps in the range of 1000 nM - 7.8 nM for all immobilized channels in a solution of 1.5 μM (anchor point). Injection of the analyte was performed for 1 minute at 30 μl / min in the running buffer, including an assay with a concentration of 0 nM as a blank reference. The SPR running buffer contained 1×PBS with 0.05 Tween 20 at pH 6.0 / pH 7.4. A multi-cycle kinetics protocol was applied (off-rate measurement for 90 seconds), and data obtained from antibody or VHH-Fc concentrations above 250 nM were omitted due to bulk effects. After double-reference subtraction, the data were analyzed using the steady-state affinity pre-defined evaluation method of the Biacore Insight Evaluation Software or by fitting a 1:1 binding model in the same software.

[0242] Human Cell Membrane Protein Cell Array Using the Retrogenix Cell Microarray Technology (Charles River Laboratories, UK), specific off-target binding interactions of the test antibodies were screened. First, prescreening was performed to determine the level of background binding of each test antibody to untransfected HEK293 cells and cells overlaid with SARS-CoV-2 FL spike 6-HIS protein. These data were used to evaluate the suitability and optimal concentration for subsequent screening. Second, in library screening, pools of test antibodies were screened for binding to fixed HEK293 cells overexpressing 6,101 full-length human cell membrane proteins, secreted proteins and human secreted proteins tethered to the cell surface, as well as 396 human heterodimers. Library interactions were thereby identified. Finally, in Confirmation screening, all library interactions were reproduced and the test antibodies were probed individually or with control treatments to determine, if any, which interactions were reproducible and specific to each test antibody. This was performed on both fixed and live cells.

[0243] For pre-screening, expression vectors encoding both ZsGreen1 and human CD20 or EGFR were spotted onto slides and used to reverse transfect HEK293 cells. The slides were fixed and then spotted with gelatin + / - SARS-CoV-2 spike protein (SARS-CoV-2 FL spike 6-HIS protein supplied by Peak Proteins, spotted at 0.2 mg / mL). 1, 2.5 or 10 μg / mL of R3_DC23hum-Fc(LS) and 1 μg / mL of rituximab biosimilar or PBS only were added to the above cells / slides after fixation. Binding to target-expressing cells and untransfected cells was evaluated by using an AlexaFluor 647-labeled anti-human IgG Fc (AF647 anti-hIgG Fc) detection antibody, followed by fluorescence imaging. For library screening, 6101 expression vectors encoding both ZsGreen1 and full-length human cell membrane proteins, secreted proteins, or human secreted proteins tethered to the cell surface, and an additional 396 human heterodimers were arrayed in duplicates on cell microarray slides. Subsequently, additional slides were spotted with gelatin + / - SARS-CoV-2 spike protein (0.2 mg / mL). The expression vector (pIRES-hEGFR-IRES-ZsGreen1) was spotted in quadruplicates on all slides and used to confirm that a minimum threshold of transfection efficiency was achieved or exceeded on all slides. Human HEK293 cells were used for reverse transfection / expression. After cell fixation, a pool of test antibodies was added to each slide. Detection of binding was performed using the same fluorescent secondary antibody (AF647 anti-hIgG Fc) as used in the pre-screen. The pool of test antibodies was screened against two replicate slide sets. Fluorescent images were analyzed and quantified (for transfection) using ImageQuant software (GE healthcare, version 8.2). Protein interactions were defined as duplicate spots with increased signal compared to the background level. This was achieved by visual inspection.Interactions were classified as "strong, moderate, weak, or very weak" based on the intensity of the doublet spots. A significant interaction was defined as a signal intensity weaker or greater than that. In the confirmation screening, vectors encoding all interactions identified in the library screening were aligned with control vectors encoding CD20 (positive control) and EGFR (transfection and negative control), and expressed in HEK293 cells on a new slide. The slides for confirmation screening and analysis were performed as library screening either after cell fixation (n = 2) or without fixation (n = 1). Only on the fixed slides, gelatin + / - SARS-CoV-2 spike protein (0.2 mg / mL) was spotted. The slides were treated with 2.5 μg / mL of R3_DC23hum-Fc (LS) or 20 μg / mL of IgG kappa antibody (negative control), 1 μg / mL of rituximab biosimilar (array positive control), or without test molecule (secondary only; negative control). Binding to target-expressing cells and untransfected cells was re-evaluated by fluorescence imaging.

[0244] SARS-CoV pseudovirus neutralization assay To generate replication-deficient VSV pseudotyped viruses, HEK293-T cells transfected with SARS-CoV-1 S or SARS-CoV-2 S were inoculated with a replication-deficient VSV vector 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 for 16 hours in medium supplemented with anti-VSV G mAb (ATCC). The pseudotyped particles were then harvested and clarified by centrifugation.

[0245] For the VSV pseudotype neutralization experiment, the pseudovirus was incubated with different dilutions of purified VHH or VHH-Fc fusion, or GFP-binding protein (GBP: VHH specific for GFP) at 37 °C for 30 min. The incubated pseudovirus was then added to a sub-confluent monolayer of Vero E6 or Vero E6-TMPRSS2 cells. After 16 h, the cells were lysed using passive lysis buffer (Promega). The transduction efficiency was quantified by measuring GFP fluorescence in the prepared cell lysates using a Tecan infinite 200 pro plate reader. GFP fluorescence was normalized using the GFP fluorescence of non-infected cells and infected cells treated with PBS, or either the lowest and highest GFP fluorescence values of each dilution series. IC 50 50 was calculated by non-linear regression curve fitting of log(inhibitor) vs. response (four parameters).

[0246] Alternatively, a dilution series 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 from the original isolate. It should be noted that during the propagation of this virus clone (S1-10a) in Vero E6 cells, the furin cleavage site mutated and became inactivated itself (Koenig et al. (2021) Science 371: eabe6230). After incubation at 37 °C for 30 min, the virus-antibody mixture was added to a monolayer of Vero E6 cells and allowed to infect and replicate for 3 days.

[0247] In all neutralization assays using pseudotyped VSV virus particles, 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 was used to prepare the VHH or antibody-virus mixture. The mixture was added to the cells from which the original growth medium had been removed.

[0248] SARS-CoV-2 plaque reduction neutralization test (PRNT) The plaque reduction assay using the authentic virus was performed with 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 virus growth was carried out in Vero E6-TMPRSS2 cells.

[0249] Both viruses were titrated using the plaque assay, in which monolayers of Vero E6-TMPRSS2 cells were infected in duplicate with 2 vials of each virus in dilution series prepared in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 2% fetal bovine serum (FBS). Two hours after infection, avicel was added to a final concentration of 0.3% (w / v).

[0250] The dose-dependent neutralization of individual 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 mix was then added to the 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, and the cells were fixed with 3.7% paraformaldehyde (PFA) and stained with 0.5% crystal violet. The maximum half dose of neutralizing titer (PRNT 50 ) was defined as the VHH-Fc concentration at which plaques were reduced by 50% on two independent plates.

[0251] Live virus assay The live virus assay was performed using SARS-CoV-2 viruses belonging to different strains (614G, Delta, Omicron BA.1, Omicron BA.2, and Omicron BA.5) isolated from nasopharyngeal swabs collected from patients / travelers between January 2020 and July 2022.

[0252]

Table 5

[0253] Dose-dependent neutralization of test items, positive controls (bevetelovimab biosimilar, cilgavimab biosimilar, sotrovimab biosimilar), and negative controls (isotype control) was evaluated in the live virus neutralization assay. HuR3DC23-Fc_LS produced from transiently transfected cells was used in all assays testing live D614G, Delta, BA.1, and BA.2 SARS-CoV-2. HuR3DC23_Fc_LS produced from stable cell pools was used in all assays testing SARS-CoV-2 BA.5. For each variant, three independent runs were performed. The assay included different sets of controls: cells only (media only), virus only, and an internal positive control (human serum). Briefly, 5-fold or 7-fold dilutions of test items and controls were incubated with a fixed amount of virus at room temperature for 1 hour.

[0254]

Table 6

[0255] The Vero-E6 cell monolayer was inoculated with the virus antibody mixture at 37°C for 1 hour. In the next step, the inoculum was removed and incubated at 37°C with infection medium (minimum essential medium (MEM) supplemented with 2 mM L-glutamine, 1× non-essential amino acids, 25 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid), 1% heat-inactivated fetal bovine serum (FBS) and 1× antibiotic-antimycotic (Gibco)) (until 18 - 24 hours post-infection). Subsequently, the SARS-CoV2-infected cells were fixed and immunostained with a SARS-CoV nucleocapsid antibody (Sinobiological, catalog number: 40143-MM05), followed by a goat anti-mouse IgG (H+L) secondary antibody conjugated with HRP (Invitrogen, catalog number A16072). Spots (infected cells) were counted using an ImmunoSpot® Analyzer S6 Ultimate (CTL). For each antibody / construct, the concentration (IC 50 ) showing a 50% reduction in infection was calculated based on the Zielinska method (Zielinska et al. (2005) Virology Journal 2:84). The geometric mean was calculated based on 3 independent runs.

[0256] S1 shedding assay Antibodies or VHHs were added at a final concentration of 10 μg / ml to 1 million Raji cells without spike or expressing the SARS-CoV-2 spike. The antibody / VHH-cell mixture was incubated at 37°C, 5% CO 2It was then incubated for 30 minutes or 1 hour. After incubation, the cells were pelleted by centrifugation, the supernatant was transferred to a new tube, and the cell pellet was lysed with RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 1% NP-40). 20 μl samples of the supernatant and the lysate were separated on an 8% SDS-PAGE gel and electroblotted onto a nitrocellulose membrane. The membrane was blocked with 4% milk and stained with rabbit anti-SARS-S1 antibody (1 / 1000, Sino biologics, 40591-T62), followed by anti-rabbit IgG-HRP (1 / 2000, GE Healthcare, NA934V), and developed using Pierce™ ECL Western Blotting Substrate (Thermofisher Scientific).

[0257] Fusion inhibition assay using a replication-competent GFP reporter VSV virus pseudotyped with Wuhan SARS-CoV-2 spike (del-18) Vero E6-TMPRSS2 cells were infected with 40 PFU of a SARS-CoV-2 spike GFP-expressing replication-competent VSV-GFP virus (Koenig et al. 2021). After 2 hours, the indicated monoclonal antibodies or VHHs were added. Uninfected cells were used as a negative control. The cells were infected overnight and imaged with a fluorescence microscope. GFP fluorescence was measured with a fluorometer. It should be noted that, unlike the clones of the 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 as confirmed by Sanger sequencing.

[0258] Fusion inhibition assay using spike-expressing Vero E6 cells Vero E6 cells were transfected with a GFP expression vector combined with either a control expression vector (without spike) or a SARS-CoV-2 spike expression vector using Fugene. Two hours after transfection, PBS, monoclonal antibody or VHH was added to a final concentration of 10 μg / ml. Twenty-two hours after transfection, the cells were fixed with 3.7% paraformaldehyde, washed with PBS, and then imaged with a fluorescence microscope.

[0259] Virus escape selection A monolayer of Vero E6-TMPRSS2 cells seeded in a 96-well plate was 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 of VHH.R3_DC23 was added. No VHH was added to one well as a control.

[0260] The growth medium of the wells that showed syncytium formation or virus replication in the presence of VHH.R3_DC23 was collected and used to isolate single plaques of escape virus in the presence of 2 μg / ml VHH.R3_DC23 by limiting dilution. Using this growth medium, the virus was grown on a monolayer of Vero E6-TMPRSS2 cells seeded in 6-well plates in the presence of VHH.R3_DC23. From these infected cells, RNA was prepared using the NucleoSpin RNA Virus kit (Macherey Nagel Bioanalysis), and cDNA was generated using random hexamer primers. Using this cDNA, the coding sequence of spike S2 was amplified by PCR. These PCR fragments were purified and sequenced using Sanger sequencing. The resulting nucleotide sequences were analyzed and aligned to the spike proteins of WT SARS-CoV-2 and sarbecoviruses of clades 1, 2, and 3 (Letko et al. (2020) Nature Microbiology 5:562 - 569) using CLC Main Workbench 20.0.4. Mutations were visualized using pymol on a model of the full-length glycosylated spike protein obtained from Charmm-gui.org (PDB: 6VXX_1_1_1 model) or the SARS-CoV-2 HR2 coiled coil determined by NMR (PDB: 3FXP).

[0261] Growth kinetics of virus escape variants Vero E6 cells seeded in 96-well plates were infected with 50 PFU of GFP-expressing replication-competent VSV virus particles pseudotyped with the SARS-CoV-2 spike protein obtained during escape selection. GFP expression was monitored every hour using an Incucyte Zoom device and analyzed with the accompanying software.

[0262] HDX-MS epitope mapping The 3.33 μM SARS-CoV-2 S-2P trimer was incubated overnight at 37 °C. The protein was then diluted to 1.66 μM trimer in 1× PBS (pH 7.4, Sigma-Aldrich P4417) in the presence or absence of 6.25 μM R3DC23. To initiate exchange, the protein was diluted 10-fold into a temperature-equilibrated deuterated buffer made by lyophilizing 1× PBS and resuspending it in D2O (Sigma-Aldrich 151882). At each time point (15 s, 3 min, 30 min, 3 h), samples were quenched by mixing 60 ul of the exchange reaction with 60 ul of ice-cold 2× quench buffer (3.6 M guanidinium chloride, 500 mM TCEP, 200 mM glycine pH 2.4). The quenched samples were incubated on ice for 1 min, 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).

[0263] Expression and purification of HR2 For structural biology purposes, the HR2 protein was expressed in a bacterial expression system. Thus, a synthetic gene encoding residues H1159-K1211 of the HR2 protein was cloned into the pFloat-SUMO vector to create a His-tagged SUMO-HR2 fusion protein. This construct also contained a 3C protease cleavage site for removal of 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 LB culture supplemented with 100 μg / ml kanamycin and grown overnight at 37 °C. Then, 20 ml of this preculture was inoculated into 1 L of LB culture and grown at 37 °C until OD 600It was grown until it reached 0.8. At this point, protein expression was induced using 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The cells were further incubated overnight at 20 °C and then harvested by centrifugation (Beckman rotor 8.1000, 5000 rpm, 15 min, 4 °C). The pellet was resuspended in PBS, 500 mM NaCl, 10 mM imidazole, 5 mM β-mercaptoethanol, 0.1 mg / mL 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), 1 μg / mL leupeptin, 50 μg / mL DNaseI, 20 mM MgCl2. The cells were lysed at 20 kpsi 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 with 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. Fractions containing His-SUMO-HR2 protein were pooled, dialyzed overnight at 4 °C against 20 mM Tris-HCl pH 7.5, 150 mM NaCl, and then incubated with 3C protease for 2 h at room temperature. The cleaved sample was loaded again onto a Ni-Sepharose FF HiLoad column equilibrated with the same buffer. The flow-through containing HR2 protein was concentrated and applied to a BioRad Enrich70 10 / 30 size exclusion column (SEC) equilibrated with 20 mM Tris-HCl pH 7.5, 150 mM NaCl. The SEC fractions containing HR2 were pooled.

[0264] Crystallization, X-ray data collection, processing, and structure determination of R3DC23-HR2 For crystallization, R3DC23 was added to HR2 in a 1.2-fold molar excess and concentrated to 19 mg / ml using an Amicon Ultra 3 kDa cut-off centrifugal filter device. 0.1 μl of R3DC23-HR2 and 0.2 μl of the bottom solution were mixed, and crystallization screening was set up using the sitting-drop vapor diffusion technique. Crystals were grown from a 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, the crystals were flash-frozen in liquid nitrogen. X-ray data were collected at the i24 beamline of the Diamond Light Source synchrotron facility (Didcot, UK). The X-ray data were processed using autoPROC+Staraniso (Vonrhin et al. 2011 Acta Crystallogr D Biol Crystallogr 67:293-302; Vonrhein et al. 2023 Acta Crystallographica Section A: Foundations and Advances). The structure of the R3DC23-HR2 complex was analyzed using the automated molecular replacement workflow of the CCP4 cloud (Krissinel et al. 2022 Acta Cryst D 78:1079-1089). The initial model was further built manually 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) of the Phenix crystallography software suite (Adams et al. 2010 Acta Crystallogr D Biol Crystallogr 66:213-221). The data collection parameters, as well as the processing and refinement statistics, are shown in Table 6.

[0265]

Table 7

[0266] Viral load in vivo in K18-hACE2 mice K18-hACE2 mice: B6.Cg-Tg(K18-ACE2)2Prlmn / J (7 - 9 weeks old) were purchased from The Jackson Laboratory and bred under specific pathogen-free (SPF) conditions. SARS-CoV-2 infection was performed under biosafety level 3 (BSL3) conditions. Treatment with antibodies was performed by intraperitoneal injection using a volume of 100 μl. Animals were anesthetized by isoflurane inhalation, and 3 × 10 2 PFU of the SARS-CoV-2 614G variant virus (SARS-CoV-2 / human / FRA / 702 / 2020, obtained from the European Virus Archive) was administered by intratracheal injection. Animals were monitored daily by blinded observers, body weight changes were measured, and scored for humane endpoints: hunched back (1 point), piloerection (1 point), little movement even when the cage was opened (1 point), no movement when touched (2 points), neurological symptoms (tremors, balance, 3 points), rapid breathing (3 points). Mice that lost more than 25% of their initial body weight or reached the humane endpoint with a score of 5 points were euthanized.

[0267] Titration of SARS-CoV-2 virus titer in mouse lung homogenates After dissection, the right lung lobe was stored at -80 °C. To quantify the lung virus titer, lung samples were homogenized using a Precellys Evolution tissue homogenizer (Bertin-technologies). The lung homogenate was clarified by centrifugation (1,000×g) at 4 °C for 15 minutes, and the virus titer was determined by a plaque assay on VeroE6-TMPRSS2 cells in duplicate using 12-well plates. After adding the lung homogenate dilution series to the cells, the plates were incubated at 37 °C for 2 hours. Then, the cells were washed twice and incubated in medium containing 0.3% (w / v) Avicel. 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 dye to visualize the virus plaques.

[0268] To quantify the RNA level of the SARS-CoV-2 virus, RNA was prepared from the lung homogenate and analyzed by qRT-PCR. cDNA was prepared using the iScript™ cDNA Synthesis Kit and random hexamer primers. qPCR was performed using the SARS-CoV-2 Research Use Only (RUO) qPCR Primers & Probes Kit (IDT) according to the manufacturer's instructions.

[0269] Load model of Syrian golden hamsters

[0270] Male Syrian golden hamsters (Mesocricetus auratus) aged 9 - 10 weeks and weighing 89.8 g to 132.3 g were obtained from Janvier (France). Six hamsters per group were nasally infected with 10^2.0 50% tissue culture infectious dose (TCID50) / dose of SARS-CoV-2 (Wuhan strain) in a total volume of 100 μl equally divided into both nostrils. XVR012 (a cocktail of XVR014 and XVR013), XVR013, or XVR014, palivizumab (10 mg / kg), or bevoterozumab (10 mg / kg) at the indicated doses were administered by intraperitoneal injection either 4 hours after SARS-CoV2 load (therapeutic setting) or approximately 24 hours before infection (prophylactic setting). An irrelevant antibody, palivizumab (Synagis, anti-RSV antibody), was used as a negative control, and bevoterozumab was used as a positive control. The huR3DC23-Fc_LS used in the hamster study was produced from a stable cell pool. Hamsters were monitored daily for behavior, appearance, and body weight. Animals were euthanized on day 4 post-infection. At necropsy, gross pathological examination was performed and abnormalities were recorded. Samples were collected from the right lung lobe and frozen for virological analysis. To determine the virus titer, quadruplicate 10-fold serial dilutions were used on a confluent layer of Vero E6 cells. For this purpose, serial dilutions of the sample (lung tissue homogenate) were incubated with the Vero E6 monolayer at 37°C for 1 hour. The Vero E6 monolayer was 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) using the viability marker WST8 (colorimetric readout). The virus titer (Log10 TCID50 / g) was calculated using the Spearman-Karber method (Karber 1931 Archiv F.Experiment.Pathol.u.Pharmakol. 162:480 - 483). In the virus titration, the lower limit of detection (LLOD) was in the range of 1.1 - 1.3 log10 TCID / g. Lung tissue homogenate was used to detect viral RNA. RNA was isolated and Taqman PCR was performed. The copy number (Log10 CP / g) in different samples was calculated relative to the standards included in each run.For viral RNA in lung tissue, the LLOD was 3.5 Log10 CP / g. Blood samples were collected for pharmacokinetic analysis at -2 days before the start of the study (approximately 200 μl of blood was collected for serum under isoflurane anesthesia) and at necropsy on day 4 post-infection (p.i.). Blood samples for serum were immediately transferred to appropriate tubes containing a clot activator. Serum was collected and stored frozen. To inactivate any potential infectious substances and enable 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.

[0271] ADCC assay / FcγRIIIa reporter assay ADCC evaluation was performed by Antibody Analytics using a SARS CoV-2 Spike Protein target cell line expressing CHO-K1 as the target cell and Jurkat FcγRIIIa (CD16) V176-NFAT-RE Luc as the reporter cell.

[0272] Biolayer interferometry (BLI) Biolayer interferometry was performed using an Octet RED96 system (ForteBio). Streptavidin (SA) biosensors (Sartorius) were immersed in 1× kinetics buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.1 mg / ml bovine serum albumin, 0.02% Tween-20, 0.02% sodium azide) for 20 minutes before use. The trimeric S-2P-foldon-His-Strep was thawed from -20 °C, incubated at 37 °C for 24 hours, and then at 30 °C for 30 minutes, after which it was immobilized on the SA biosensor at 36.5 μg / ml (80 nM) for 450 seconds to set the signal at 1.5 - 1.7 nm. Binding (120 seconds) and dissociation (480 seconds) of a tw...

Claims

1. A binder capable of neutralizing salvecovirus, characterized by specifically binding to the region of the salvecovirus spike protein or within the region corresponding to the region of the SARS-CoV-2 spike protein from amino acid E1188 to amino acid Y1206 as defined in SEQ ID NO:

86.

2. The binder according to claim 1, which specifically binds to a region of the spike protein or within a region corresponding to the region of the SARS-CoV-2 spike protein defined in Sequence ID No. 86, from amino acid E1188 to amino acid L1203, or preferably from amino acid E1188 to amino acid E1202.

3. The binder according to claim 1 or 2, which specifically binds to a region of the spike protein or within a region corresponding to the region of the SARS-CoV-2 spike protein defined in Sequence ID No. 86, from amino acid N1194 to amino acid L1203, or preferably from amino acid N1194 to amino acid E1202.

4. The binder according to claim 1 or 2, which specifically binds to the region of the heptad repeat 2 (HR2) domain of the Salvecovirus spike protein proximal to the viral membrane.

5. A binder according to claim 1 or 2, which specifically binds to a quaternary epitope in a spike protein trimer.

6. The binder 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 binder according to claim 1 or 2, wherein it specifically binds to at least one amino acid residue of the spike protein corresponding to amino acid residues N1194, S1196, D1199, or Q1201 of the SARS-CoV-2 spike protein as defined in Sequence ID No. 86; or at least one amino acid residue of the spike protein corresponding to amino acid residues N1194, S1196, D1199, or Q1201 of the SARS-CoV-2 spike protein as defined in Sequence ID No. 86 is essential for the binding of the binder to the spike protein.

8. The binder according to claim 1 or 2, wherein it specifically binds to at least one amino acid residue of the spike protein corresponding to amino acid residue S1196 or Q1201 of the SARS-CoV-2 spike protein as defined in SEQ ID NO: 86; or at least one amino acid residue of the spike protein corresponding to amino acid residue S1196 or Q1201 of the SARS-CoV-2 spike protein as defined in SEQ ID NO: 86 is essential for the binding of the binder to the spike protein.

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

10. A binder according to claim 1 or 2, which stabilizes the prefusion conformation of a spike protein, for example, stabilizing the HR2 coiled coil of a spike protein.

11. - Vesicular stomatitis virus (VSV) - Salvecovirus spike protein pseudovirus neutralization assay, etc., when determined by a Salvecovirus spike protein pseudovirus neutralization assay, the 50% inhibitory concentration (IC) is 100 ng / ml or less, preferably 10 ng / ml or less, more preferably 1 ng / ml or less. ５０ ) can neutralize the Salvecovirus; - SARS-CoV-2 Wuhan strain, SARS-CoV-2 alpha variant, SARS-CoV-2 Omicron BA.1 variant, and 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. It is possible to neutralize one or more of the 1.5 variants of SARS-CoV-2, as well as one or both of SARS-CoV-1; - It is possible to inhibit spike-mediated syncytia formation between cells expressing the Salvecovirus spike protein and cells expressing the angiotensin-converting enzyme 2 (ACE2) receptor; and / or - The binder according to claim 1 or 2, which does not bind to Middle East Respiratory Syndrome Coronavirus (MERS-CoV).

12. The binder according to claim 1 or 2, comprising or consisting of an antibody or antibody fragment.

13. The binder according to claim 1, comprising an immunoglobulin monovariate domain (ISVD), preferably VHH.

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

15. The binder according to claim 13 or 14, wherein the ISVD each independently comprises CDR1, CDR2, and CDR3 present in any of sequence numbers 1 to 10, and CDR1, CDR2, and CDR3 are annotated according to one of Kabat, MacCallum, IMGT, AbM, Martin, or Chothia; preferably, the ISVD comprises a combination of CDR1, CDR2, and CDR3, wherein CDR1, CDR2, and CDR3 are present in any one of the sequences described in sequence numbers 1 to 10, and CDR1, CDR2, and CDR3 are annotated according to one of Kabat, MacCallum, IMGT, AbM, Martin, or Chothia.

16. The binder according to claim 13 or 14, wherein the ISVD comprises CDR1 as defined by SEQ ID NO: 63, CDR2 as defined by SEQ ID NO: 64, and CDR3 as defined by SEQ ID NO: 48; preferably CDR1 as defined by SEQ ID NO: 65, CDR2 as defined by SEQ ID NO: 66, and CDR3 as defined by SEQ ID NO: 51; more preferably CDR1 as defined by any one of SEQ ID NOs: 52 to 54, CDR2 as defined by any one of SEQ ID NOs: 55 to 62, and CDR3 as defined by any one of SEQ ID NOs: 21 to 27.

17. The binder according to claim 14, wherein the ISVD comprises an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 10 and 127 to 129.

18. The binder according to claim 1 or 2, comprising an ISVD containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 10 and 127 to 129.

19. The binder according to claim 1 or 2, which is in a polyvalent form and preferably comprises ISVD fused to an Fc domain.

20. The binder according to claim 1 or 2, comprising an amino acid sequence defined by any one of sequence numbers 96, 118, 130, or 131.

21. A multispecific binder comprising a binder as defined in claim 1, further comprising a binder that specifically binds to the salvecovirus spike protein receptor-binding domain (RBD).

22. The multispecific binder according to claim 21, wherein the binder that specifically binds to the RBD includes at least one ISVD that can bind to or compete for an epitope that receives specific binding by VHH72 as defined by SEQ ID NO: 124, such as an ISVD containing the sequence described in SEQ ID NO: 125 or SEQ ID NO: 124 or a humanized form thereof.

23. The multispecific binder according to claim 21 or 22, wherein the binder that specifically binds to the RBD includes at least one ISVD capable of binding to or competing for an epitope that receives specific binding by VHH3.117 as defined by SEQ ID NO: 127, such as an ISVD containing the sequence described in SEQ ID NO: 127 or a humanized form thereof.

24. The multispecific binder according to claim 21 or 22, comprising an amino acid sequence defined by any one of sequence numbers 112 to 117.

25. A nucleic acid molecule comprising a polynucleotide sequence encoding the binder described in claim 1; a vector comprising the nucleic acid molecule; or a cell expressing the binder described in claim 1, or comprising the nucleic acid molecule or the vector.

26. A composition comprising a binder as defined in claim 1, further comprising a binder that specifically binds to the salvecovirus spike protein receptor-binding domain (RBD).

27. The composition according to claim 26, wherein the binder that specifically binds to the RBD comprises at least one ISVD capable of binding to or competing for an epitope that receives specific binding by VHH72 as defined by SEQ ID NO: 124, and at least one ISVD capable of binding to or competing for an epitope that receives specific binding by VHH3.117 as defined by SEQ ID NO: 127, such as the binder defined by SEQ ID NO:

119.

28. The composition according to claim 26, comprising a binder defined by SEQ ID NO: 118 and a binder defined by SEQ ID NO:

119.

29. A pharmaceutical composition comprising the binder according to claim 1 or 2, the multispecific binder according to claim 21 or 22, the nucleic acid molecule according to claim 25, the vector according to claim 25, or the composition according to any one of claims 26 to 28, and a pharmaceutically acceptable carrier, or a kit such as a diagnostic kit comprising the binder according to claim 1 or 2, the multispecific binder according to claim 21 or 22, or the composition according to any one of claims 26 to 28.

30. A binder according to claim 1 or 2, a multispecific binder according to claim 21 or 22, 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, for use in pharmaceuticals, preferably for use in the prevention or treatment of salvecovirus infection in a subject, or for use in the diagnosis of salvecovirus infection in a subject.

31. The pharmaceutical composition according to claim 29 for use in pharmaceuticals, preferably for use in the prevention or treatment of salvecovirus infection in a subject, or for use in the diagnosis of salvecovirus infection in a subject.

32. The kit according to claim 29 for use in pharmaceuticals, preferably for use in the prevention or treatment of salvecovirus infection in a subject, or for use in the diagnosis of salvecovirus infection in a subject.

33. An in vitro or ex vivo method for detecting salvecovirus in a sample, - The step of contacting the sample with the binder described in claim 1 or 2 or the multispecific binder described in claim 21 or 22, - A step of determining the binding of the binder to the salvecovirus or a part thereof. In vitro or ex vivo methods, including [specific methods].

Images