Anti-metapneumovirus fusion (F) protein antibodies and uses thereof

Abstract

Provided herein are epitope-specific antigen-binding molecules having specificity for human metapneumovirus (hMPV) fusion (F) protein, wherein the antigen-binding molecules comprise a variable domain in contact with the hMPV F protein. The antigen binding molecule having specificity for hMPV F protein comprises a heavy chain and a light chain. Also provided is a method for detecting an antibody specific for hMPV F protein, one method comprising: a.) contacting a biological sample with hMPV F protein; and b.) contacting an epitope specific antigen binding molecule with the hMPV F protein.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 477,092, filed on December 23, 2022, which is incorporated herein by reference in its entirety.

[0003] Incorporation of Sequence Listing by Reference

[0004] This application contains a sequence listing that has been submitted in XML format via EFS - WEB and is hereby incorporated by reference in its entirety. The name of the XML copy created on December 13, 2023 is 061291 - 510001WO_SeqList_ST26.xml, and its size is 55 kilobytes. Technical Field

[0005] The present disclosure generally relates to antibodies against the fusion (F) protein of human metapneumovirus. Background Art

[0006] Human metapneumovirus (hMPV) can cause upper and lower respiratory tract diseases in people of all ages. Symptoms include coughing, fever, nasal congestion, and shortness of breath. In some cases, hMPV infection can progress to severe diseases such as bronchitis or pneumonia.

[0007] hMPV is a single - stranded RNA virus of the paramyxovirus family, which also includes measles, mumps, and other respiratory infections such as respiratory syncytial virus (RSV). hMPV can be divided into two subtypes, A and B, which can be identified by genotyping the G and F genes in the viral genome. The F gene encodes the hMPV fusion glycoprotein (hMPV F protein), which can be used for the treatment or prevention of hMPV infection. However, despite the clinical need, no treatment for human hMPV has been approved to date.

[0008] Therefore, there is an unmet need for the treatment of hMPV. Summary of the Invention

[0009] Provided herein are epitope - specific antigen - binding molecules against the hMPV fusion (F) protein, wherein the antigen - binding molecule comprises variable domains that contact the hMPV F protein at the following positions:

[0010] i.) residues 287, 293, 296, 364, 376, 417, and 419 of the hMPV F protein amino acid sequence shown in SEQ ID NO:1;

[0011] ii.) residues 144, 160, 163, 188, 194, and 199; or

[0012] iii.) residues 44, 45, 49, 150, 156, 160, 229, 232, and 236.

[0013] In some embodiments, an antigen-binding molecule specific for the hMPV F protein comprises a heavy chain and a light chain, wherein the heavy chain comprises H-CDR1, H-CDR2, and H-CDR3, wherein: H-CDR1 comprises the sequence GYTFTSY (SEQ ID NO:3); H-CDR2 comprises the sequence YPGSGS (SEQ ID NO:4); and H-CDR3 comprises the sequence LLRLTFDV (SEQ ID NO:5); and wherein the light chain comprises L-CDR1, L-CDR2, and L-CDR3, wherein: L-CDR1 comprises the sequence RASQDISNYLN (SEQ ID NO:7); L-CDR2 comprises the sequence YTSGLHS (SEQ ID NO:8); and L-CDR3 comprises the sequence QQGNTLPWT (SEQ ID NO:9).

[0014] In some embodiments, the antigen-binding molecule comprises variable domains that contact the hMPV F protein at the following positions:

[0015] i.) residues 287, 293, 296, 364, 376, 417, and 419 of the hMPV F protein amino acid sequence shown in SEQ ID NO:1;

[0016] ii.) residues 144, 160, 163, 188, 194, and 199; or

[0017] iii.) residues 44, 45, 49, 150, 156, 160, 229, 232, and 236.

[0018] In some embodiments, the antigen-binding molecule comprises variable domains that contact the hMPV F protein at any one, any two, any three, any four, any five, any six, or all seven of residues 287, 293, 296, 364, 376, 417, and 419 of the hMPV F protein amino acid sequence shown in SEQ ID NO:1.

[0019] In some embodiments, the heavy chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:2.

[0020] In some embodiments, the light chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:6.

[0021] In some embodiments, the heavy chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:2, and the light chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:6.

[0022] In some embodiments, the antigen-binding molecule comprises a variable heavy (VH) chain domain, wherein the VH domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:50, and a variable light (VL) chain domain, wherein the VL domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:51.

[0023] In some embodiments, the antigen-binding molecule comprises variable domains that contact the hMPV F protein at any one, any two, any three, any four, any five or any six of residues 144, 160, 163, 188, 194 and 199 of the hMPV F protein amino acid sequence shown in SEQ ID NO:1.

[0024] In some embodiments, the antigen-binding molecule comprises a heavy chain and a light chain, wherein the heavy chain comprises H-CDR1, H-CDR2 and H-CDR3, wherein: H-CDR1 comprises the sequence GFTFTDY (SEQ ID NO:11); H-CDR2 comprises the sequence RNKDNGYT (SEQ ID NO:12); and H-CDR3 comprises the sequence YYFGYDGDYFDY (SEQ ID NO:13); and wherein the light chain comprises L-CDR1, L-CDR2 and L-CDR3, wherein: L-CDR1 comprises the sequence SASSSISSNYLH (SEQ ID NO:15); L-CDR2 comprises the sequence RTSNLAS (SEQ ID NO:16); and L-CDR3 comprises the sequence QQGSSLPRT (SEQ ID NO:17).

[0025] In some embodiments, the heavy chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:10.

[0026] In some embodiments, the light chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:14.

[0027] In some embodiments, the heavy chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:10, and the light chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:14.

[0028] In some embodiments, the antigen-binding molecule comprises a variable heavy (VH) chain domain, wherein the VH domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:52, and a variable light (VL) chain domain, wherein the VL domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:53.

[0029] In some embodiments, the antigen-binding molecule comprises variable domains that contact the hMPV F protein at any one, any two, any three, any four, any five, any six, any seven, any eight or any nine of residues 44, 45, 49, 150, 156, 160, 229, 232 and 236 of the hMPV F protein amino acid sequence shown in SEQ ID NO:1.

[0030] In some embodiments, the antigen-binding molecule comprises a heavy chain and a light chain, wherein the heavy chain comprises H-CDR1, H-CDR2, and H-CDR3, wherein: H-CDR1 comprises the sequence GFSLSTFGM (SEQ ID NO:19); H-CDR2 comprises the sequence WWDDD (SEQ ID NO:20); and H-CDR3 comprises the sequence IVKVLEQYFDV (SEQ ID NO:21); and wherein the light chain comprises L-CDR1, L-CDR2, and L-CDR3, wherein: L-CDR1 comprises the sequence KASQDVGTAVA (SEQ ID NO:23); L-CDR2 comprises the sequence WASTRHT (SEQ ID NO:24); and L-CDR3 comprises the sequence QQYTSYPLT (SEQ ID NO:25).

[0031] In some embodiments, the heavy chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:18.

[0032] In some embodiments, the light chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:22.

[0033] In some embodiments, the heavy chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:18, and the light chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:22.

[0034] In some embodiments, the antigen-binding molecule comprises a variable heavy (VH) chain domain, wherein the VH domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:54, and a variable light (VL) chain domain, wherein the VL domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:55.

[0035] In some embodiments, the antigen-binding molecule is an immunoglobulin molecule.

[0036] In some embodiments, the immunoglobulin is an IgG1, IgG2, IgG3, or IgG4 molecule.

[0037] In some embodiments, the immunoglobulin is a humanized antibody.

[0038] Polynucleotides encoding the antigen-binding molecules of the present disclosure are provided.

[0039] A pharmaceutical composition is provided that comprises an antigen-binding molecule of the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient.

[0040] A method for detecting an antibody specific for the hMPV F protein is provided, the method comprising:

[0041] a.) contacting a biological sample with the hMPV F protein; and

[0042] b.) contacting an epitope-specific antigen-binding molecule according to any one of claims 1-19 with the hMPV F protein.

[0043] In some embodiments, the hMPV F protein is coated on a microplate before contacting with the biological sample or the antigen-binding molecule.

[0044] In some embodiments, the half-maximal effective concentration (EC50) of the anti-hMPV F protein antibody in the biological sample is determined by calculating the reciprocal of the dilution of the biological sample at which the binding of the antigen-binding molecule is inhibited by 50%.

[0045] In some embodiments, the biological sample is serum.

[0046] A method for treating or preventing hMPV infection in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of an antigen-binding molecule according to the present disclosure.

[0047] In some embodiments, the subject has an hMPV infection or is at risk of developing an hMPV infection.

[0048] In some embodiments, the subject is a mammal, optionally human.

[0049] In some embodiments, the subject is vulnerable to viral infections.

[0050] In some embodiments, the subject is an elderly subject.

[0051] In some embodiments, the antigen-binding molecule of the present disclosure is administered by intramuscular injection, intravenous injection, or subcutaneous injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1An exemplary hMPV F protein with neutralizing antigenic sites and epitopes for antibodies 17D10, 13E10, and 42C2 is shown. The regions containing the 17D10, 13E10, and 42C2 epitopes are circled, and the antigenic sites for the neutralizing antibodies are labeled II, III, IV, V, DS7, and 66 - 87.

[0053] Figures 2A - 2C is a schematic of the hMPV033 epitopes for 17D10( Figure 2A ), 13E10( Figure 2B ), and 42C2( Figure 2C ).

[0054] Figures 3A - 3C is a graph depicting the binding of neutralizing antibodies (nAbs) 17D10( Figure 3A ), 13E10( Figure 3B ), and 42C2( Figure 3C ) containing mouse (mIgG) and human (hIgG) Fc to hMPV033 (upper table) and DS - Cav1 (RSV control) (lower table). All three nAbs are specific for hMPV F, but not for RSV F or I53 - 50A.

[0055] Figures 4A - 4J is a diagram depicting the interaction of hMPV033 with 17D10_hlgG1. The hMPV033 Protein Data Bank (PDB) structure is colored blue at the epitope site. The blue - colored hMPV033 amino acids correspond to amino acids 287 - 296 (KAAPSCSEKK), amino acids 364 - 376 (SCGRNPISMVALS), and amino acids 417 - 419 (TVT) of the hMPV033 sequence SEQID NO:1. Figures 4A, 4B, 4C, 4D, and 4E show ribbon / surface representations of the front view (Figure 4A), back view (Figure 4B), side view Figure 1 (Figure 4C), side view 2 (Figure 4D), and top view (Figure 4E). Figures 4F, 4G, 4H, 4I, 4J: show the front view (Figure 4F); back view (Figure 4G); side view Figure 1 (Figure 4H); side view 2 (Figure 4I), and top view (Figure 4J) of the ribbon representation.

[0056] Figures 5A - 5JDiagram showing the interaction of hMPV033 with 13E10_hlgG1. The hMPV033 PDB structure is colored blue at the epitope site. The blue-colored hMPV033 amino acids correspond to amino acids 144 - 163 (TN EAVSTLGCGVRVLATAVR) and amino acids 188 - 199 (KMAVSFSQFNRR) of the hMPV033 sequence SEQ ID NO:1. Figures 5A, 5B, 5C, 5D and 5E show the ribbon / surface representations of the front view (Figure 5A), back view (Figure 5B), side view Figure 1 (Figure 5C), side view 2 (Figure 5D) and top view (Figure 5E). Figures 5F, 5G, 5H, 5I and 5J show the front view (Figure 5F); back view (Figure 5G); side view Figure 1 (Figure 5H); side view 2 (Figure 5I) and top view (Figure 5J) of the ribbon representation.

[0057] Figures 6A - 6J Diagram showing the interaction of hMPV033 with 42C2_hlgG1. The hMPV033 PDB structure is colored blue at the epitope site. The blue-colored hMPV033 amino acids correspond to amino acids 44 - 49 (YTNVFT), 150 - 160 (TLGCGVRVLAT) and amino acids 229 - 236 (RAISNMPT) of the hMPV033 sequence SEQ ID NO:1. Figures 6A, 6B, 6C, 6D and 6E show the front view (Figure 6A), back view (Figure 6B), side view Figure 1 (Figure 6C), side view 2 (Figure 6D) and top view (Figure 6E) of the ribbon / surface representation. Figures 6F, 6G, 6H, 6I and 6J show the front view (Figure 6F); back view (Figure 6G); side view Figure 1 (Figure 6H); side view 2 (Figure 6I) and top view (Figure 6J) of the ribbon representation.

[0058] Figures 7A - 7D Graph showing the binding of antibodies to pre-fusion and post-fusion hMPV F protein. The binding of post-fusion hMPV F protein-specific antibodies MF1, MF2 and MF3 and pre-fusion hMPV F protein-specific antibody MF10 (control) to pre-fusion hMPV F protein ( Figure 7A ) or post-fusion hMPV F protein ( Figure 7B ) was analyzed by Octet. The binding of hMPV F protein-specific antibodies 17D10, 42C2 and 13E10 to pre-fusion hMPV F protein ( Figure 7C ) or post-fusion hMPV F protein ( Figure 7D) binding. The 17D10 and 42C2 antibodies bind to the pre-fusion and post-fusion conformations of the hMPV F protein; the 13E10 antibody binds only to the pre-fusion hMPV F protein conformation. Detailed Description

[0059] Definitions

[0060] All publications, patents, and patent applications, including any drawings and appendices therein, are hereby incorporated by reference in their entirety for all purposes to the extent that each such individual publication, patent, or patent application, drawing, or appendix is specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred methods and materials are described. For the purposes of this disclosure, the following terms are defined below.

[0062] The term "antigen" refers to a polypeptide or polypeptide complex that includes at least one component intended to elicit an immune response. The term antigen as used herein is not limited to the portion of the polypeptide or polypeptide complex that contains the antigenic epitope.

[0063] The term "infection" refers to both symptomatic and asymptomatic infections.

[0064] The term "linker" refers to a chemical linkage (i.e., a covalent bond or a series of covalent bonds with intervening chemical moieties) or to a polypeptide that is joined at the N-terminus and C-terminus by a peptide bond to produce a fusion protein.

[0065] The term "antigen-binding molecule" refers to a molecule that has binding affinity for a target antigen. It should be understood that the term extends to immunoglobulins, immunoglobulin fragments, and non-immunoglobulin-derived protein frameworks that exhibit antigen-binding activity. Representative antigen-binding molecules useful for practicing the present disclosure include polyclonal and monoclonal antibodies and fragments thereof (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv), and domain antibodies (including, for example, shark and camel antibodies) and fusion proteins comprising an antibody, as well as any other modified configuration of an immunoglobulin molecule that contains an antigen-binding / recognition site. Antibodies include any type of antibody, such as IgG, IgA, or IgM (or subclasses thereof), and antibodies need not belong to any particular class. Immunoglobulins can be designated into different classes based on the amino acid sequence of the constant region of their heavy chains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant regions corresponding to the different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. The subunit structure and three-dimensional configuration of the different classes of immunoglobulins are well known. Antigen-binding molecules also encompass dimeric antibodies and multivalent forms of antibodies. In some embodiments, the antigen-binding molecule is a chimeric antibody, wherein a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, provided that it exhibits the desired biological activity (see, for example, U.S. Patent No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-6855). Humanized antibodies are also contemplated, which are generally produced by transferring the complementarity-determining regions (CDRs) of the heavy and light chain variable regions of a non-human (e.g., rodent, preferably mouse) immunoglobulin into human variable domains. Then, typical residues of the human antibody are substituted into the framework regions of the non-human counterparts. Using the antibody components derived from humanized antibodies avoids potential problems associated with the immunogenicity of non-human constant regions. General techniques for cloning non-human, especially murine, immunoglobulin variable domains are described, for example, by Orlandi et al. (1989, Proc. Natl. Acad. Sci. USA 86:3833).Techniques for generating humanized monoclonal antibodies are described, for example, by Jones et al. (1986, Nature 321:522), Carter et al. (1992, Proc. Natl. Acad. Sci. USA 89:4285), Sandhu (1992, Crit. Rev. Biotech. 12:437), Singer et al. (1993, J. Immun. 150:2844), Sudhir (ed., Antibody Engineering Protocols, Humana Press, Inc. 1995), Kelley (“Engineering Therapeutic Antibodies,” in Protein Engineering: Principles and Practice), Cleland et al. (eds.), pp. 399-434 (John Wiley & Sons, Inc. 1996), and Queen et al., U.S. Patent No. 5,693,762 (1997).

[0066] As used herein, the term "antibody" is used in the broadest sense and specifically encompasses natural antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired immunological interaction. A naturally-occurring "antibody" within its scope encompasses immunoglobulins comprising at least two heavy (H) chains and two light (L) chains that are linked to each other by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of specific CH domains (e.g., CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs). Each VH and VL is composed of three CDRs. An antibody can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), subclass, or a modified form thereof (e.g., an IgG1 isotype that carries the L234A and L235A double mutations (IgG1-LALA)). An antibody can be of any species, chimeric, humanized, or human. In other embodiments, the antibody is a heavy chain-only antibody (e.g., a camelid antibody) that lacks the first constant domain (CH1), but retains the intact heavy chain and is capable of binding an antigen through the antigen-binding domain. Unless otherwise specified, the term "antibody" includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, its derivatives, variants, fragments, and mutant proteins, examples of which are described below. In addition, unless explicitly excluded, antibodies include, respectively, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof. In some embodiments, the term also encompasses peptibodies.

[0067] In certain embodiments, the antibody heavy chain binds an antigen in the absence of the antibody light chain. In certain embodiments, the antibody light chain binds an antigen in the absence of the antibody heavy chain. In certain embodiments, the antibody binding region binds an antigen in the absence of the antibody light chain. In certain embodiments, the antibody binding region binds an antigen in the absence of the antibody heavy chain. In certain embodiments, a single variable region specifically binds an antigen in the absence of other variable regions.

[0068] The term "neutralization" (e.g., "neutralizing antibody") refers to an antibody that prevents infection and / or reduces the level of infection by a pathogen. A neutralizing antibody response can be measured in vitro (e.g., by infecting cells in a culture with a pathogen in the presence of the antibody) or in vivo (e.g., by determining the protective dose of an antibody by administering the antibody to a subject prior to challenge with an infectious dose of the pathogen). A neutralizing antibody can inhibit the infectivity of a pathogen by binding to the pathogen and blocking the molecules required for entry into host cells. A neutralizing antibody can statically interfere with the attachment of a pathogen to a host cell receptor. Without being bound by theory, in the case of a viral infection, a neutralizing antibody can bind to the glycoprotein of an enveloped virus or the capsid protein of a non-enveloped virus and can act by preventing the virus particle from undergoing the structural changes normally required for successful entry into a host cell.

[0069] The term "variable region" or "variable domain" refers to the variable light chain domain (VL) or the variable heavy chain domain (VH). As used herein, a "variable region" or "variable domain" refers to each of the pair of light chain and heavy chain domains that are directly involved in antibody binding to an antigen. The variable light and heavy chain domains have the same general structure, and each domain contains four framework regions (FRs), which have a widely conserved sequence, joined by three CDRs or "hypervariable regions". The FRs adopt a β-sheet conformation, while the CDRs may form loops that connect the β-sheet structures. The CDRs in each chain are held in their three-dimensional structure by the FRs, and together the FRs and CDRs form the antigen-binding site.

[0070] "CDR" or "complementary determining region" (also known as "hypervariable region") refers to the amino acid residues in an antibody that are responsible for antigen binding. As used herein, a CDR refers to the amino acid sequences of the light and heavy chains of an antibody that form a three-dimensional loop structure that contributes to the formation of the antigen-binding site. There are three CDRs in each of the heavy and light chain variable regions of an antibody, referred to as "CDR1", "CDR2", and "CDR3". The term "CDR set" as used herein refers to the set of three CDRs that appear in a single variable region that binds to an antigen. The terms "heavy chain variable region CDR1" and "H-CDR1" are used interchangeably, as are the terms "heavy chain variable region CDR2" and "H-CDR2", "heavy chain variable region CDR3" and "H-CDR3", "light chain variable region CDR1" and "L-CDR1", "light chain variable region CDR2" and "L-CDR2", and "light chain variable region CDR3" and "L-CDR3".

[0071] In certain embodiments, the unambiguous description of the CDRs and the identification of the residues that comprise the antibody binding site are accomplished by resolving the structure of the antibody and / or the structure of the antibody-ligand complex. In certain embodiments, this can be achieved by any of a variety of techniques known to those of skill in the art, such as X-ray crystallography. In certain embodiments, various analytical methods can be employed to identify or roughly estimate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, and the contact definition.

[0072] The exact boundaries of these CDRs are defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991))) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides the precise residue boundaries that define the three CDRs. These CDRs may be referred to as "Kabat CDRs". Chothia and colleagues (Chothia and Lesk, 1987. J. Mol. Biol. 196:901-917; Chothia et al., 1989. Nature 342:877-883) found that although there are large differences at the amino acid sequence level, certain subparts of the Kabat CDRs adopt almost identical peptide backbone conformations. Other boundaries defining CDRs that overlap with the Kabat CDRs were described by Padlan (1995. FASEB J. 9:133-139) and MacCallum (1996. J. Mol. Biol. 262(5):732-745). Other CDR boundary definitions may not strictly follow one of these systems, but still overlap with the Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental results, i.e., specific residues or groups of residues or even entire CDRs do not significantly affect antigen binding.

[0073] "Single-chain variable fragment (scFv)" is a protein chain in which the VL and VH regions pair to form a monovalent molecule (referred to as single-chain Fv (scFv); see, for example, Bird et al., 1988. Science 242:423-426; and Huston et al., 1988. Proc. Natl. Acad. Sci. 85:5879-5883). Although these two domains, VL and VH, are encoded by different genes, they can be joined by a synthetic peptide linker using recombinant methods, which enables them to form a single protein chain. Such single-chain antibodies contain one or more antigen-binding portions. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the utility of the fragments is screened in the same manner as for intact antibodies.

[0074] An antibody that "binds" to an antigen of interest (e.g., the hMPV F protein) is an antibody that binds the antigen with sufficient affinity such that the antibody can be used as a therapeutic agent to target cells or tissues expressing the antigen and does not exhibit significant cross-reactivity with other proteins. In such embodiments, the degree of binding of the antibody to a "non-target" protein will be less than about 10% of the binding of the antibody, oligopeptide, or other organic molecule to its specific target protein, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or radioimmuno-precipitation (RIA). For the binding of an antibody to a target molecule, the terms "specifically binds" or "specifically binds to" or "is specific for" a particular polypeptide or an epitope on a particular polypeptide target mean a binding that is significantly different from non-specific interactions. For example, specific binding can be measured by determining the binding of a molecule compared to the binding of a control molecule, which is typically a molecule having a similar structure but no binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target (e.g., an excess of unlabeled target). In such cases, if the binding of the labeled target to the probe is competitively inhibited by the excess unlabeled target, then specific binding is indicated. The specific region of an antigen that binds to an antibody is commonly referred to as an "epitope". The term "epitope" broadly includes sites on an antigen that are specifically recognized or interact with an antibody or a T cell receptor. Generally, an epitope is an active surface group of a molecule, such as an amino acid or a carbohydrate or a sugar side chain, and typically can have specific three-dimensional structural features as well as specific charge features. Those skilled in the art will understand that virtually any substance to which an antibody can specifically bind can be an epitope.

[0075] The terms "binds to" or "is specific for" or "specifically binds to" an antibody and a target (e.g., human metapneumovirus or hMPV protein), which are used interchangeably herein, are well-known in the art, and methods for determining such specificity or preferential binding are also well-known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, for a longer duration, and / or with a higher affinity with a particular cell or substance than with an alternative cell or substance. For example, an immunoglobulin that specifically or preferentially binds to thymocytes is an immunoglobulin that binds to thymocytes with a higher affinity, avidity, more readily, and / or for a longer duration than to other cells. An immunoglobulin that specifically binds to a first cell or substance may or may not specifically or preferentially bind to a second cell or substance. Thus, "specific binding" does not necessarily require (although it can include) exclusive binding. Generally but not necessarily, reference to binding means specific binding.

[0076] Throughout this disclosure, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" shall be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, the use of the term "comprising" and the like indicates that the elements listed are required or mandatory, but other elements are optional and may or may not be present. "Consisting of" is intended to include but not be limited to all of the content following the phrase "consisting of". Thus, the phrase "consisting of" indicates that the elements listed are required or mandatory and that no other elements can be present. "Consisting essentially of" is intended to include any elements listed after the phrase and be limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the elements listed are required or mandatory, but other elements are optional and may or may not be present, depending on whether they affect the activity or action of the listed elements.

[0077] The term "pharmaceutically acceptable excipient" refers to an excipient that is biologically or pharmacologically compatible when used in animals or humans, and can mean an excipient that is approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopeia or other recognized pharmacopeias for use in animals (and particularly humans).

[0078] The term "adjuvant" refers to a pharmaceutically acceptable substance that enhances the immune response to an antigen when co-administered with the antigen or administered before, during, or after administration of the antigen to a subject.

[0079] In the context of treating a disease or disorder, "effective amount" means an amount of an agent or composition that, when administered as a single dose or as part of a series, to an individual in need of such treatment or prophylaxis, is effective to prevent the occurrence of symptoms, control such symptoms, and / or treat existing symptoms of the disorder. The effective amount will vary depending on the age, health and physical condition of the individual to be treated, the presence or absence of symptoms of the disease, the taxonomic group of the individual to be treated, the formulation of the composition, the assessment of the medical condition, and other relevant factors. The optimal dosing regimen can be calculated from measurements of drug accumulation in the subject. The optimal dose may vary depending on the relative potency of the individual subject and can generally be estimated based on EC50 values found to be effective in in vitro and in vivo animal models. A person of ordinary skill in the art can readily determine the optimal dose, method of administration, and rate of repetition. The expected amounts will fall within a relatively broad range and can be determined by routine experimentation.

[0080] As used herein, the term "therapeutically effective amount" or "effective dose" refers to the dose or concentration of a drug that is effective in treating a disease or condition. For example, with respect to the use of monoclonal antibodies or antigen-binding fragments to treat viral infections.

[0081] The term "immune response" refers to the activation of one or more types of immune cells in a subject. Immune responses include, for example, T cell and B cell responses.

[0082] The term "humoral immune response" refers to an immune response that results in the production of plasma or serum antibodies (e.g., IgG).

[0083] The term "administer" refers to providing a composition to a subject in a manner that allows the composition to exert its intended effect. Administration for vaccination or post-exposure prophylaxis can be by intramuscular injection, intravenous injection, intraperitoneal injection, or any other suitable route.

[0084] The term "subject" refers to a human or non-human animal to which a composition can be administered for vaccination, treatment, or other purposes. In some embodiments, the non-human animal is a non-human primate: rabbit, hamster, gerbil, pig, cow, sheep, goat, guinea pig, rat, mouse, squirrel, wolf, fox, horse, zebra, giraffe, elephant, cat, dog, camel, or ferret.

[0085] The term "polynucleotide" refers to a polymeric form of nucleotides having more than about 100 nucleotides, which can be ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleobases.

[0086] In the context of two or more polynucleotide or polypeptide sequences, the terms "identical" or percent "identity" refer to two or more sequences or subsequences that are the same or have a specified percentage of identical amino acid residues or nucleotides (i.e., share at least about 80% identity over a specified region with a reference sequence, such as at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity when compared and aligned over a comparison window or specified region using one of the following sequence comparison algorithms or by manual alignment and visual inspection to achieve maximum correspondence). This definition also refers to the complementary sequence of a test sequence. In some embodiments, the identity exists within a region of at least about 25 amino acids or nucleotides, such as within a region of 50, 100, 200, 300, 400 amino acids or nucleotides, or over the full length of the reference sequence.

[0087] For sequence comparison, typically one sequence acts as a reference sequence to be compared with a test sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, (if necessary) specifying subsequence coordinates, and specifying sequence algorithm program parameters. Default program parameters can be used, or alternative parameters can be specified. Then the sequence comparison algorithm calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters. In some embodiments, the BLAST and BLAST 2.0 algorithms and default parameters are used.

[0088] The terms "treatment / treating", etc., refer to alleviating, reducing, delaying, lessening, reversing, improving, or controlling one or more of at least one symptom of a disorder in a subject. The term "treatment" can also mean preventing, delaying the onset of a disorder (i.e., the period before the appearance of clinical manifestations of the disease) or reducing one or more of the risks of the occurrence or worsening of a disorder.

[0089] As used herein, the articles "a" and "an" refer to one or more than one (i.e., at least one) of the grammatical objects of an item. For example, "an element" means one element or more than one element.

[0090] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the case of the absence of a combination when interpreted in the alternative (or).

[0091] The term "about" or "approximately" means that a particular value as determined by a person of ordinary skill in the art is within an acceptable error range, which depends in part on how the value is measured or determined, such as the limitations of the measurement system. For example, "about" can mean within 1 or more standard deviations. Alternatively, "about" can mean a range of plus or minus up to 20%, up to 10%, or up to 5%.

[0092] Unless otherwise indicated, all weight percentages (i.e., "% by weight" and "wt%" and weight / weight) cited herein are measured relative to the total weight of the pharmaceutical composition.

[0093] As used herein, "substantially" or "essentially" means the full or nearly full extent or degree of an action, characteristic, property, state, structure, substance, or result. For example, an object that is "substantially" enclosed means that the object is either fully enclosed or nearly fully enclosed. In some cases, the exact degree of deviation from absolute fullness may depend on the particular circumstances. However, generally, the proximity of completion will be such that the same overall result is obtained as if absolute and complete completion were achieved. When used in a negative sense, the use of "substantially" also applies to mean a complete or nearly complete lack of an action, characteristic, property, state, structure, substance, or result. For example, a composition that is "substantially free" of other active agents either completely lacks other active agents or nearly completely lacks other active agents such that the effect is the same as if it completely lacked other active agents. In other words, a composition that is "substantially free" of a certain ingredient or element or another active agent may still contain such a substance as long as there is no measurable effect thereof.

[0094] The following description includes information that may be useful in understanding the present invention. This is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any specifically or implicitly cited publication is prior art.

[0095] Antigen-binding molecule

[0096] Provided herein are epitope-specific antigen-binding molecules against the hMPV fusion (F) protein, wherein the antigen-binding molecule comprises variable domains that contact the hMPV F protein at the following positions:

[0097] i.) residues 287, 293, 296, 364, 376, 417, and 419 of the hMPV F protein amino acid sequence shown in SEQ ID NO:1;

[0098] ii.) residues 144, 160, 163, 188, 194, and 199; or

[0099] iii.) Residues 44, 45, 49, 150, 156, 160, 229, 232, and 236.

[0100] The hMPV033 F protein is used as a reference sequence:

[0101] MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIEGGGGGGFVLGAIALGVATAAAVTAGVAIAKCIRLESEVTAIKNALKKTNEAVSTLGCGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISKDLMTDAELARAISNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSCGRNPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQCFESIENSQAGSGGSGSGSGGSEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATELE (SEQ ID NO:1)

[0102] In some embodiments, the epitope - specific antigen - binding molecule that is specific for the hMPV fusion (F) protein is an antibody. In some embodiments, the epitope - specific antigen - binding molecule that is specific for the hMPV fusion (F) protein is a neutralizing antibody.

[0103] In some embodiments, an epitope - specific antigen - binding molecule specific for the hMPV fusion (F) protein comprises variable domains that contact the hMPV F protein at residues 287, 293, 296, 364, 376, 417, and 419 of the hMPV F protein amino acid sequence shown in SEQ ID NO:1, and can be referred to as the "17D10" antigen - binding molecule.

[0104] In some embodiments, an epitope - specific antigen - binding molecule specific for the hMPV fusion (F) protein comprises variable domains that contact the hMPV F protein at residues 144, 160, 163, 188, 194, and 199 of the hMPV F protein amino acid sequence shown in SEQ ID NO:1, and can be referred to as the "13E10" antigen - binding molecule.

[0105] In some embodiments, an epitope - specific antigen - binding molecule specific for the hMPV fusion (F) protein comprises variable domains that contact the hMPV F protein at residues 44, 45, 49, 150, 156, 160, 229, 232, and 236 of the hMPV F protein amino acid sequence shown in SEQ ID NO:1, and can be referred to as the "42C2" antigen - binding molecule.

[0106] Table 1 provides exemplary antigen - binding molecule heavy - chain and light - chain sequences.

[0107] Table 1

[0108]

[0109]

[0110]

[0111] Table 2 provides exemplary antigen - binding molecule variable - domain sequences.

[0112] Table 2

[0113]

[0114]

[0115] Table 3 provides exemplary antigen - binding molecule variable - domain CDR sequences.

[0116] Table 3

[0117]

[0118]

[0119] Table 4 provides exemplary antigen-binding molecule variable domain epitope contact residues.

[0120] Table 4

[0121]

[0122] Epitope I-17D10

[0123] The present disclosure encompasses any hMPV F protein antigen-binding molecule that binds to the hMPV F protein, such as a human hMPV F protein in the pre-fusion and post-fusion hMPV F protein conformations. In some embodiments, the hMPV F protein antigen-binding molecule is the 17D10 antibody, which binds to an epitope consisting of at least a portion of the region from amino acid 287 to amino acid 419 of the human hMPV F protein. As Figures 4A - 4J shown, these amino acid residues form a non-linear epitope on the hMPV F protein. The anti-hMPV F protein antigen-binding molecules of the present disclosure that bind the 17D10 epitope bind to hMPV F proteins in both pre-fusion and post-fusion conformations.

[0124] In some embodiments, the epitope-specific antigen-binding molecule specific for the hMPV fusion (F) protein is an antibody. In some embodiments, the epitope-specific antigen-binding molecule specific for the hMPV fusion (F) protein is a neutralizing antibody.

[0125] In some embodiments, the epitope-specific antigen-binding molecule specific for the hMPV fusion (F) protein comprises variable domains that contact the hMPV F protein at residues 287, 293, 296, 364, 376, 417, and 419 of the hMPV F protein amino acid sequence shown in SEQ ID NO:1, and may be referred to as the "17D10" antigen-binding molecule.

[0126] In some embodiments, the antigen-binding molecule comprises variable domains that contact the hMPV F protein at any 1, any 2, any 3, any 4, any 5, any 6, or any 7 of residues 287, 293, 296, 364, 376, 417, and 419 of the hMPV F protein amino acid sequence shown in SEQ ID NO:1.

[0127] In some embodiments, the antigen-binding molecule "17D10" comprises a heavy chain and a light chain, wherein the heavy chain comprises H-CDR1, H-CDR2, and H-CDR3, wherein: H-CDR1 comprises the sequence GYTFTSY (SEQ ID NO:3); H-CDR2 comprises the sequence YPGSGS (SEQ ID NO:4); and H-CDR3 comprises the sequence LLRLTFDV (SEQ ID NO:5).

[0128] In some embodiments, the antigen-binding molecule comprises a light chain comprising L-CDR1, L-CDR2, and L-CDR3, wherein: L-CDR1 comprises the sequence RASQDISNYLN (SEQ ID NO:7); L-CDR2 comprises the sequence YTSGLHS (SEQ ID NO:8); and L-CDR3 comprises the sequence QQGNTLPWT (SEQ ID NO:9).

[0129] In some embodiments, the antigen-binding molecule "17D10" that is specific for the hMPV F protein comprises a heavy chain and a light chain, wherein the heavy chain comprises H-CDR1, H-CDR2, and H-CDR3, wherein: H-CDR1 comprises the sequence GYTFTSY (SEQ ID NO:3); H-CDR2 comprises the sequence YPGSGS (SEQ ID NO:4); and H-CDR3 comprises the sequence LLRLTFDV (SEQ ID NO:5); and wherein the light chain comprises L-CDR1, L-CDR2, and L-CDR3, wherein: L-CDR1 comprises the sequence RASQDISNYLN (SEQ ID NO:7); L-CDR2 comprises the sequence YTSGLHS (SEQ ID NO:8); and L-CDR3 comprises the sequence QQGNTLPWT (SEQ ID NO:9).

[0130] In some embodiments, the heavy chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:2.

[0131] In some embodiments, the light chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:6.

[0132] In some embodiments, the heavy chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:2, and the light chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:6.

[0133] In some embodiments, the antigen-binding molecule comprises a variable heavy (VH) chain domain, wherein the VH domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:50.

[0134] In some embodiments, the antigen-binding molecule comprises a variable light (VL) chain domain, wherein the VL domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:51.

[0135] In some embodiments, the antigen-binding molecule comprises a variable heavy (VH) chain domain, wherein the VH domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:50, and a variable light (VL) chain domain, wherein the VL domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:51.

[0136] Epitope II–13E10

[0137] The present disclosure encompasses any hMPV F protein antigen-binding molecule that binds to the hMPV F protein, particularly to the prefusion hMPV F protein conformation. In some embodiments, the hMPV F protein antigen-binding molecule is the 13E10 antibody, which binds to an epitope consisting of at least a portion of the region of amino acids 144 to 199 of the hMPV F protein. As Figures 5A - 5J shown, these amino acid residues form a non-linear epitope on the hMPV F protein.

[0138] The anti-hMPV F protein antigen-binding molecules of the present disclosure that bind the 13E10 epitope bind the prefusion conformation of the hMPV F protein.

[0139] In some embodiments, the epitope - specific antigen - binding molecule that is specific for the hMPV fusion (F) protein is an antibody. In some embodiments, the epitope - specific antigen - binding molecule that is specific for the hMPV fusion (F) protein is a neutralizing antibody.

[0140] In some embodiments, the epitope - specific antigen - binding molecule that is specific for the hMPV fusion (F) protein comprises variable domains that contact the hMPV F protein at residues 144, 160, 163, 188, 194, and 199 of the hMPV F protein amino acid sequence shown in SEQ ID NO:1 and can be referred to as the "13E10" antigen - binding molecule.

[0141] In some embodiments, the antigen - binding molecule comprises variable domains that contact the hMPV F protein at any 1, any 2, any 3, any 4, any 5, or any 6 of residues 144, 160, 163, 188, 194, and 199 of the hMPV F protein amino acid sequence shown in SEQ ID NO:1.

[0142] In some embodiments, the antigen - binding molecule "13E10" comprises a heavy chain and a light chain, wherein the heavy chain comprises H - CDR1, H - CDR2, and H - CDR3, wherein: H - CDR1 comprises the sequence GFTFTDY (SEQ ID NO:11); H - CDR2 comprises the sequence RNKDNGYT (SEQ ID NO:12); and H - CDR3 comprises the sequence YYFGYDGDYFDY (SEQ ID NO:13); and wherein the light chain comprises L - CDR1, L - CDR2, and L - CDR3, wherein: L - CDR1 comprises the sequence SASSSISSNYLH (SEQ ID NO:15); L - CDR2 comprises the sequence RTSNLAS (SEQ ID NO:16); and L - CDR3 comprises the sequence QQGSSLPRT (SEQ ID NO:17).

[0143] In some embodiments, the heavy chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:10.

[0144] In some embodiments, the light chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:14.

[0145] In some embodiments, the heavy chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:10, and the light chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:14.

[0146] In some embodiments, the antigen-binding molecule comprises a variable heavy (VH) chain domain, wherein the VH domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:52.

[0147] In some embodiments, the antigen-binding molecule comprises a variable light (VL) chain domain, wherein the VL domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:53.

[0148] In some embodiments, the antigen-binding molecule comprises a variable heavy (VH) chain domain, wherein the VH domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:52, and a variable light (VL) chain domain, wherein the VL domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:53.

[0149] Epitope III–42C2

[0150] The present disclosure encompasses any hMPV F protein antigen-binding molecule that binds to the hMPV F protein in the pre-fusion and post-fusion hMPV F protein conformations. In some embodiments, the hMPV F protein antigen-binding molecule is the 42C2 antibody, which binds to an epitope consisting of at least a portion of the region of amino acids 44 to amino acids 236 of the hMPV F protein. As Figures 6A - 6J shown, these amino acid residues form a non-linear epitope on the hMPV F protein.

[0151] The anti-hMPV F protein antibodies of the present disclosure that bind the 42C2 epitope bind to the hMPV F protein in the pre-fusion and post-fusion conformations.

[0152] In some embodiments, the epitope-specific antigen-binding molecule that is specific for the hMPV fusion (F) protein is an antibody. In some embodiments, the epitope-specific antigen-binding molecule that is specific for the hMPV fusion (F) protein is a neutralizing antibody.

[0153] In some embodiments, the epitope-specific antigen-binding molecule that is specific for the hMPV fusion (F) protein comprises variable domains that contact the hMPV F protein at residues 44, 45, 49, 150, 156, 160, 229, 232, and 236 of the hMPV F protein amino acid sequence shown in SEQ ID NO:1, and may be referred to as the "42C2" antigen-binding molecule.

[0154] In some embodiments, the antigen-binding molecule comprises variable domains that contact the hMPV F protein at any 1, any 2, any 3, any 4, any 5, any 6, any 7, any 8, or any 9 of residues 44, 45, 49, 150, 156, 160, 229, 232, and 236 of the hMPV F protein amino acid sequence shown in SEQ ID NO:1.

[0155] In some embodiments, the antigen-binding molecule "42C2" comprises a heavy chain and a light chain, wherein the heavy chain comprises H-CDR1, H-CDR2, and H-CDR3, wherein: H-CDR1 comprises the sequence GFSLSTFGM (SEQ ID NO:19); H-CDR2 comprises the sequence WWDDD (SEQ ID NO:20); and H-CDR3 comprises the sequence IVKVLEQYFDV (SEQ ID NO:21); and wherein the light chain comprises L-CDR1, L-CDR2, and L-CDR3, wherein: L-CDR1 comprises the sequence KASQDVGTAVA (SEQ ID NO:23); L-CDR2 comprises the sequence WASTRHT (SEQ ID NO:24); and L-CDR3 comprises the sequence QQYTSYPLT (SEQ ID NO:25).

[0156] In some embodiments, the heavy chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:18.

[0157] In some embodiments, the light chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:22.

[0158] In some embodiments, the heavy chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:18, and the light chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:22.

[0159] In some embodiments, the antigen-binding molecule comprises a variable heavy (VH) chain domain, wherein the VH domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54.

[0160] In some embodiments, the antigen-binding molecule comprises a variable light (VL) chain domain, wherein the VL domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:55.

[0161] In some embodiments, the antigen-binding molecule comprises a variable heavy (VH) chain domain, wherein the VH domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, and a variable light (VL) chain domain, wherein the VL domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:55.

[0162] In some embodiments, the antigen-binding molecule is an immunoglobulin molecule.

[0163] In some embodiments, the immunoglobulin is an IgG1, IgG2, IgG3 or IgG4 molecule.

[0164] In some embodiments, the immunoglobulin is a humanized antibody.

[0165] In some embodiments, the immunoglobulin is a neutralizing antibody.

[0166] hMPV F protein

[0167] Human metapneumovirus (hMPV) is a negative-sense enveloped virus of the family Pneumoviridae and was discovered in 2001, but had been spreading for at least half a century before its discovery. The hMPV fusion (F) protein is one of three surface glycoproteins encoded by the viral genome. As a class I fusion protein, hMPV F is first translated as a single polypeptide precursor (F0). Initially non-functional, a proteolytic cleavage event is required to form the F1 and F2 subunits covalently linked by disulfide bonds. The new N-terminus of the F2 polypeptide contains a hydrophobic sequence that is inserted into the host cell membrane during the final fusion of the virus and the host cell membrane. In a sense, whether in transit or on the membrane surface, the F protein associates with itself to form a metastable trimer, which is called the pre-fusion conformation. The hMPV fusion protein is cleaved extracellularly by a trypsin-like protease. An unknown triggering event occurs that causes the F protein to undergo significant conformational changes, extending the fusion peptide into the host cell membrane and then folding back on itself to form a six-helix bundle, which is called the post-fusion conformation. The energy difference between the extended intermediate and the post-fusion conformation provides the energy required for membrane fusion.

[0168] The present disclosure provides methods, uses, and compositions for treating hMPV infections in a subject, the compositions comprising an hMPV F protein antigen-binding molecule. The present disclosure also provides methods, uses, and compositions for treating hMPV, the compositions comprising an hMPV F protein antigen-binding molecule.

[0169] The hMPV033 antigen (SEQ ID NO:1) comprises the extracellular domain (1-472AA) of the UT-A CL-28 mutant. The CL-28 mutant strain was constructed in the A strain sequence, replacing the F1 / F2 cleavage site with a 6-amino acid Gly linker. The mutant also contains the following mutations: T127C, N153C, A185P, V231I, L219K, G294E, T365C, and V463C. In some embodiments, the mutant contains 368N. In some embodiments, the mutant contains 368H. In some embodiments, the mutant contains T127C, N153C, A185P, V231I, L219K, G294E, T365C, V463C, and H368N. The 127C-153C amino acid bond and the 365C-463C amino acid bond form in vivo disulfide bonds.

[0170] The hMPV F protein antigen-binding molecule can be a full-length immunoglobulin antibody or an antigen-binding fragment of a complete antibody. Representative examples thereof include Fab fragments, F(ab′)2 fragments, Fd fragments consisting of VH and CH1 domains, Fv fragments consisting of VL and VH domains of a single arm of an antibody, single-domain antibody (dAb) fragments (Ward et al., 1989. Nature 341:544-546), which consist of a VH domain and isolated CDRs. In some embodiments, the hMPV F protein antigen-binding molecule is a chimeric antibody, a humanized antibody, or a human antibody.

[0171] In some embodiments, the hMPV F protein antigen-binding molecule is a humanized antibody. Techniques for generating humanized monoclonal antibodies (mAbs) are well known in the art (see, for example, Jones et al., 1986. Nature 321:522-525; Riechmann et al. 1988. Nature 332:323-329; Verhoeyen et al., 1988. Science 239:1534-1536; Carter et al., 1992. Proc. Natl. Acad. Sci. USA 89:4285-4289; Sandhu, J.S., 1992. Crit. Rev. Biotech. 12:437-462, and Singer et al., 1993. J. Immunol. 150:2844-2857). Chimeric monoclonal antibodies or murine monoclonal antibodies can be humanized by transferring the murine CDRs of the variable heavy chain and variable light chain from a murine immunoglobulin into the corresponding variable domains of a human antibody. The murine framework regions (FRs) in the chimeric monoclonal antibody are also replaced with human FR sequences. Since simply transferring murine CDRs into human FRs often results in a decrease or even loss of antibody affinity, additional modifications may be required to restore the original affinity of the murine antibody. This can be achieved by replacing one or more human residues in the FR region with their murine counterparts to obtain an antibody with good binding affinity for its epitope. See, for example, Tempest et al. (1991. Biotechnology 9:266-271) and Verhoeyen et al. (1988, supra). Typically, those human FR amino acid residues that are different from their murine counterparts and are located near or adjacent to one or more CDR amino acid residues will be candidates for substitution.

[0172] Polynucleotide

[0173] Polynucleotides encoding the antigen-binding molecules of the present disclosure are provided.

[0174] In one aspect, the present disclosure provides an isolated polynucleotide encoding an antigen-binding molecule of the present disclosure. The isolated polynucleotide sequence can comprise RNA or DNA. As used herein, an "isolated nucleic acid" is one that has been removed from its normal flanking polynucleotide sequences in the genome or in a cDNA sequence.

[0175] In a further aspect, the present disclosure provides a recombinant expression vector comprising an isolated polynucleotide of any embodiment or combination of embodiments of the present disclosure operably linked to a suitable control sequence. A "recombinant expression vector" includes a vector that operably links a polynucleotide coding region or gene to any control sequence capable of influencing the expression of the gene product. A "control sequence" operably linked to a polynucleotide sequence of the present disclosure is a polynucleotide sequence capable of influencing the expression of the polynucleotide molecule. The control sequence need not be adjacent to the polynucleotide sequence so long as it functions to direct its expression. Thus, for example, there may be intervening untranslated but transcribed sequences between a promoter sequence and the polynucleotide sequence, and the promoter sequence can still be considered to be "operably linked" to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such recombinant expression vectors can be of any type known in the art, including but not limited to plasmids and virus-based expression vectors. Control sequences for driving the expression of the disclosed nucleic acid sequences in a mammalian system can be constitutive (driven by any of a variety of promoters, including but not limited to CMV, SV40, RSV, actin, EF) or inducible (driven by any of a variety of inducible promoters, including but not limited to tetracycline, ecdysone, steroid-responsive promoters).

[0176] Pharmaceutical composition

[0177] A pharmaceutical composition is provided that comprises an antigen-binding molecule of the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient. A carrier is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the composition and not harmful to its recipient (e.g., a subject). Suitable carriers typically include saline or ethanol polyols such as glycerol or propylene glycol.

[0178] The antigen-binding molecule can be formulated in a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with a free amino group) formed with inorganic acids such as hydrochloric acid or phosphoric acid or such organic acids as acetic acid, oxalic acid, tartaric acid, and mandelic acid. Salts formed with a free carboxyl group can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, and procaine.

[0179] The composition can be suitably formulated for systemic administration, including intravenous, intramuscular, subcutaneous or intraperitoneal administration, and conveniently comprises a sterile aqueous solution of the antigen-binding molecule, which is preferably isotonic with the recipient's blood. Such formulations are generally prepared by dissolving the solid active ingredient in water containing physiologically compatible substances such as sodium chloride, glycine, etc., and having a buffered pH compatible with physiological conditions to produce an aqueous solution and rendering the solution sterile. These can be prepared in unit dose or multi-dose containers, such as sealed ampoules or vials.

[0180] The composition can include stabilizers such as, for example, polyethylene glycol, proteins, saccharides (such as trehalose), amino acids, inorganic acids and mixtures thereof. The stabilizers are used in the aqueous solution at appropriate concentrations and pH. The pH of the aqueous solution is adjusted to be in the range of 5.0 - 9.0, preferably in the range of 6 - 8. When formulating the antigen-binding molecule, anti-adsorbents can be used. Other suitable excipients can generally include antioxidants such as ascorbic acid.

[0181] In certain embodiments, the compositions disclosed herein can be used as a medicament, for example, for treating or preventing an infection in a subject (such as a mammal) in need thereof.

[0182] In certain embodiments, the compositions disclosed herein can be used in the manufacture of a medicament for treating or preventing an infection in a subject (such as a mammal) in need thereof.

[0183] Therapeutic applications

[0184] A method for treating or preventing hMPV infection in a subject in need thereof is provided, wherein the method comprises administering to the subject an effective amount of an antigen-binding molecule according to the present disclosure.

[0185] In some embodiments, the methods of the present disclosure include therapeutic treatment and prophylactic or preventive measures, wherein the aim is to prevent or slow down (mitigate) a target pathological disorder or condition. Those in need of treatment include those who already have the condition and those who are predisposed to the condition or who are to prevent the condition. If, after receiving an effective amount of the antigen-binding molecule according to the present disclosure, the subject exhibits an observable and / or measurable reduction or disappearance of one or more of the following, then the infection of the subject is successfully "treated": a reduction in the number of infected cells or the disappearance of infected cells; a decrease in the percentage of the total number of infected cells; a certain degree of alleviation of one or more symptoms associated with a particular infection (e.g., symptoms associated with hMPV infection); a reduction in morbidity and mortality, and / or an improvement in quality of life issues. The above parameters for evaluating treatment success and disease improvement can be easily measured by conventional procedures familiar to physicians.

[0186] In some embodiments, the subject has an hMPV infection or is at risk of developing an hMPV infection.

[0187] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0188] In some embodiments, the subject is susceptible to viral infection (e.g., hMPV).

[0189] In some embodiments, the subject is an elderly subject.

[0190] In some embodiments, the antigen-binding molecule of the present disclosure is administered by intramuscular injection, intravenous injection, or subcutaneous injection.

[0191] Detection methods

[0192] The antigen-binding molecules of the present disclosure can be used in a variety of detection methods, including those described herein and other methods known in the art, e.g., methods for detecting antibodies that contact the hMPV F protein or cells expressing the hMPV F protein on their surface. Immunoassays useful in practicing the methods disclosed herein include fluorescence-activated cell sorting (FACS) analysis, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or radioimmuno-precipitation (RIA).

[0193] These methods can be performed in vivo, ex vivo, or in vitro. Specifically, the step of contacting the antibody with the hMPV F protein or with cells expressing the hMPV F protein on their surface can be performed in vivo, ex vivo, or in vitro. The method can be performed in a cell-based or cell-free system.

[0194] Potential hMPV F protein antigen-binding molecules can be evaluated in vivo, such as in an animal model. In such an in vivo model, the effect of the antigen-binding molecule can be evaluated in the circulation (e.g., blood) or heart, or in other organs such as the lung, liver, kidney, or brain.

[0195] A method for detecting an antibody specific for the hMPV F protein is provided, the method comprising:

[0196] a.) contacting a biological sample with the hMPV F protein; and

[0197] b.) contacting an epitope-specific antigen-binding molecule according to the present disclosure with the hMPV F protein.

[0198] In some embodiments, the hMPV F protein is coated on a microplate prior to contacting with the biological sample or the antigen-binding molecule.

[0199] In some embodiments, the half - maximal effective concentration (EC 50 ) of anti - hMPV F protein antibodies in a biological sample is determined by calculating the reciprocal of the dilution of the biological sample at which antigen - binding molecule binding is inhibited by 50%.

[0200] In some embodiments, the biological sample is serum.

[0201] For example, a method for detecting the titer of serum anti - hMPV F protein antibodies that compete with biotinylated anti - hMPV F protein antibodies for binding to hMPV F antigen is provided. The method comprises:

[0202] a. Coating an ELISA plate with hMPV F antigen to prepare an hMPV F antigen - coated ELISA plate;

[0203] b. Adding serum to the hMPV F antigen - coated ELISA plate;

[0204] c. Adding biotinylated anti - hMPV F protein antibody to the hMPV F antigen - coated ELISA plate in the presence of serum under conditions effective to permit binding of the biotinylated anti - hMPV F protein antibody to the hMPV F antigen - coated ELISA plate;

[0205] d. Detecting the binding of the biotinylated anti - hMPV F protein antibody to the hMPV F antigen - coated ELISA plate in the presence of serum using a streptavidin polypeptide that binds to the biotinylated anti - hMPV F protein antibody, wherein the color development induced by the streptavidin polypeptide is proportional to the amount of biotinylated anti - hMPV F protein antibody bound to hMPV F antigen; and

[0206] e. Identifying the titer of serum anti - hMPV F protein antibodies that effectively compete with the biotinylated anti - hMPV F protein antibody by determining the reciprocal serum dilution at which 50% of the binding of the biotinylated anti - hMPV F protein antibody is inhibited.

[0207] The method encompasses any anti - hMPV F protein antibody that binds to the hMPV F protein. In some embodiments, the method detects the titer of serum anti - hMPV F protein antibodies that compete with a biotinylated 13E10 hMPV F protein epitope antibody. In some embodiments, the method detects the titer of serum anti - hMPV F protein antibodies that compete with a biotinylated 42C2 hMPV F protein epitope antibody. In some embodiments, the method detects the titer of serum anti - hMPV F protein antibodies that compete with a biotinylated 17D10 hMPV F protein epitope antibody.

[0208] The antigen-binding molecules of the present disclosure are expected to be used for determining the identity of recombinantly produced hMPV F protein.

[0209] The anti-hMPV F protein antibodies of the present disclosure that bind to the 13E10 epitope bind only to the pre-fusion conformation of the hMPV F protein. Accordingly, the use of 13E10 epitope anti-hMPV F protein antibodies is contemplated in the methods of the invention for assessing the stability of pre-fusion stabilized hMPV F protein. In some embodiments, the anti-hMPV F protein antibodies that bind to the 13E10 epitope can be used to determine the hMPV F protein conformation because the antibodies bind only to the pre-fusion conformation of the hMPV F protein.

[0210] The anti-hMPV F protein antibodies of the present disclosure that bind to the 17D10 epitope bind to both pre-fusion and post-fusion conformations of the hMPV F protein.

[0211] The anti-hMPV F protein antibodies of the present disclosure that bind to the 42C2 epitope bind only to both pre-fusion and post-fusion conformations of the hMPV F protein.

[0212] The methods contemplate the use of 13E10 epitope anti-hMPV F protein antibodies and 42C2 epitope or 17D10 epitope anti-hMPV F protein antibodies in binding assays to assess the post-fusion conformation of the hMPV F protein because the 42C2 epitope and 17D10 epitope anti-hMPV F protein antibodies bind to the post-fusion conformation of the hMPV F protein, but the 13E10 epitope anti-hMPV F protein antibodies do not bind to the post-fusion conformation of the hMPV F protein.

[0213] For example, in a binding assay using 13E10 epitope anti-hMPV F protein antibodies and 42C2 epitope or 17D10 epitope anti-hMPV F protein antibodies, the binding ratio to the anti-hMPV F protein antibodies can be used to assess the conformation of the hMPV F protein. Without being bound by theory, a decrease in binding by the 13E10 epitope anti-hMPV F protein antibodies, but no change in binding by the 42C2 epitope or 17D10 epitope anti-hMPV F protein antibodies, can indicate a transition of the hMPV F protein from the pre-fusion conformation to the post-fusion conformation.

[0214] The present method contemplates an assay for evaluating the antigenic conformation of the hMPV F protein vaccine, which comprises contacting the epitope-specific antigen-binding molecule of the present disclosure with the vaccine antigen. For example, the 13E10 epitope anti-hMPV F protein antibody and the 42C2 epitope or 17D10 epitope anti-hMPV F protein antibody can be used in the binding assay of the present disclosure to evaluate the conformation of the hMPV F protein antigen. In other embodiments, when generating the pre-fusion conformation hMPV F protein antigen, the 13E10 epitope anti-hMPV F protein antibody binds only to the pre-fusion conformation of the hMPV F protein, while the 17D10 epitope and 42C2 epitope anti-hMPV F protein antibodies bind to both the pre-fusion and post-fusion conformations of the hMPV F protein. Without being bound by theory, the conformational selectivity of the epitope-specific antibodies of the present disclosure allows for the evaluation of the conformation of the hMPV vaccine antigen.

[0215] In some embodiments, the pre-fusion conformation confers stability to the hMPV protein relative to the post-fusion conformation. Exemplary antibody sequences of the present disclosure are shown in Table 5.

[0216] Table 5: Exemplary Antibody Sequences of the Present Disclosure

[0217]

[0218]

[0219]

[0220]

[0221]

[0222] Examples

[0223] Example 1: Generation of hMPV F Protein Antibodies

[0224] Neutralizing monoclonal antibodies against the hMPV F protein (especially hMPV033) were generated and characterized from mice immunized with an hMPV F protein fragment (hMPV008) fused to I53-50A (as described in WO 2019 / 169120 A1), and antibodies that bind to hMPV033 but not to I53-50A, RSV F protein, or the hexahistidine (his6) tag were screened out.

[0225] Antibodies specifically binding to the hMPV F protein were tested in a virus neutralization assay, and a total of 24 neutralizing antibodies were identified. Three neutralizing antibodies, 17D10 hIgG1, 13E10hIgG1, and 42C2 hIgG1, were humanized, expressed, and purified, and by CovalX TMThe epitopes of each antibody were mapped. These three exemplary antibodies have the following characteristics:

[0226] 1. Bind to the hMPV F protein but not to the RSV F protein

[0227] 2. Neutralizing antibodies

[0228] 3. Bind to non-linear, conformation-dependent epitopes

[0229] 4. Antibodies 17D10 and 42C2 bind to the pre-fusion and post-fusion conformations of the hMPV F protein, while the 13E10 antibody binds only to the pre-fusion conformation of the hMPV F protein.

[0230] The hMPV008 antigen contains the extracellular domain (1-472AA) of the NIH V4-B mutant, which is fused to the virus-like particle I53-50A with a 16-residue GS linker. V4-B was constructed from the B strain sequence and is a single-chain construct in which a 6-amino acid Gly linker replaces the F1 / F2 cleavage site. This mutant also contains mutations of 6 Cys (A63C, A140C, A147C, K188C, K450C, and S470C). Amino acid 63C forms a disulfide bond with the naturally occurring Cys at position 60C, and amino acid 188C forms a disulfide bond with the naturally occurring Cys at position 182C. The 60C-63C bond is an in-protomer bond, while the 182-188 bond is an inter-protomer bond.

[0231] The hMPV033 antigen contains the extracellular domain (1-472AA) of the UT-A CL-28 mutant, which is fused to I53-50A with a 16-residue GS linker. CL-28 was constructed in the A strain sequence, replacing the F1 / F2 cleavage site with a 6-amino acid Gly linker. This mutant also contains the following mutations: T127C, N153C, A185P, V231I, L219K, G294E, T365C, and V463C. In some embodiments, the mutant contains 368N. In some embodiments, the mutant contains T127C, N153C, A185P, V231I, L219K, G294E, T365C, V463C, and H368N. In some embodiments, the mutant contains 368H. 127C-153C and 365C-463C form in-protomer disulfide bonds.

[0232] Production of hMPV008 and hMPV033 antigens

[0233] Expi293 cells in logarithmic growth phase were counted and seeded at 2.5X 10 6Cells were inoculated at a density of 6 cells / mL into four 1-L flasks (total volume 880 mL), 220 mL each. The cells were incubated overnight at 36 °C with shaking (120 rpm). The next day, the cells were counted and diluted to 3×10

[0234] Purification

[0235] The construct was purified directly from conditioned Expi293F expression medium by immobilized metal affinity chromatography (IMAC). The INDIGO Ni-Agarose was washed with 5 column volumes (CV) of water, then with 5 CV of equilibration buffer, and then resuspended in 1–2 CV of equilibration buffer. The supernatant was clarified by centrifugation at 4,000 × g and then filtered using a 0.2 or 0.45 μm vacuum filtration unit. The resin suspension was added to the supernatant such that 2–3 mL of resin was used per liter of supernatant. The supernatant-resin slurry was gently stirred at 4 °C for 2–3 h, then the resin was collected by filtration using a 0.45 μm vacuum filter and transferred to a gravity column using equilibration buffer. The resin was washed with 20 CV of wash buffer and the protein was eluted using 5–10 CV of elution buffer.

[0236] ELISA assay and Octet assay – secondary screening

[0237] To screen the second round of 117 clones against the hMPV F protein, capture ELISA was used. High-binding plates were coated with 100 ng / well of hMPV033 in 100 mM sodium carbonate-sodium bicarbonate buffer (pH 9.6) overnight at 4 °C. The next day, the plates were washed 3 times with wash buffer (1X PBS, 0.05% Tween-20, pH 7.4) and blocked with 150 μL / well of blocking buffer (wash buffer containing 1% BSA) for 1 hour at room temperature. Supernatant samples were thawed at room temperature and then inverted several times to mix. Each sample was diluted 10-fold using blocking buffer as a diluent, and then additionally diluted to 50-fold and finally 500-fold. Samples were loaded at 100 μL / well, and the plates were covered and incubated for 1 hour at room temperature without agitation. The samples were aspirated, the plates were washed 5 times with wash buffer, and then 100 μL / well of goat anti-mouse HRP-conjugated antibody diluted 1:5,000 in blocking buffer was added, and the plates were covered and incubated for 1 hour at room temperature without agitation. The conjugate was aspirated, the plates were washed 5 times with wash buffer, and then 100 μL / well of TMB was applied and incubated for approximately 5 minutes at room temperature in the dark. The reaction was stopped with 100 μL / well of 0.6 N sulfuric acid, and the absorbance at 450 nm was measured within ten minutes. The mAb binding was ranked using the endpoint absorbance values at 50-fold and 500-fold dilutions. Then, the 92 samples with the highest binding were screened by Biolayer Interferometry (BLI). BLI on the Octet Red96 was performed as follows: The anti-penta His biosensor was immersed in assay buffer for 30 seconds to reach baseline. 10 ug / mL of hMPV033 in assay buffer was loaded onto the sensor for 30 seconds, followed by another baseline step. Next, the sensor was immersed in each supernatant for 60 seconds to observe the binding of the antibody to hMPV033. A 120-second offset was used to rank the binding (data not shown).

[0238] Immunization

[0239] Five SJL female mice (4 - 6 weeks old) were immunized with the hMPV008-I53-50A immunogen in the "pre-fusion" conformation. This immunogen contained a C-terminal His6 tag and was produced by a stable miCHO K1 cell line. Serum samples from the immunized mice were collected and tested for titers against hMPV033 to avoid detecting antibodies against I53-50A (as described in WO 2019 / 169120A1). A total of 42 supernatant results evaluated by ELISA showed a positive signal for hMPV033 and a negative signal for I53-50A (data not shown).

[0240] Neutralization assay of the first-round screening materials

[0241] Forty-two supernatants were evaluated for hMPV / A neutralization assays.

[0242] All supernatants were initially diluted 1:8 and then serially diluted two-fold. The neutralizing antibody titer was defined as the final dilution at which a 50% reduction in virus cytopathic effect (CPE) occurred. The final serum dilution was the final dilution of the serum without consideration of the additional four-fold dilution that occurred when the diluted serum (50 μl) was mixed with virus (50 μl) and then cells (100 μl) were added.

[0243] All monoclonal antibodies were at a concentration of 200 μg / ml and were subsequently diluted 1:8 first and then serially diluted two-fold. To calculate the inhibitor concentration 50% (IC50), the final titer needed to be converted to a dilution factor. The original monoclonal antibody concentration was divided by the dilution factor to determine the inhibitor IC50. A further four-fold dilution was used to obtain the final serum dilution for calculating the IC50. Table 6 shows the results of the neutralizing antibody titers (without 4-fold dilution adjustment), the original IC50 (without 4-fold dilution adjustment), and the final IC50 (with 4-fold dilution adjustment).

[0244] For example, the neutralizing antibody titer of monoclonal antibody 37H4 mAB.2mg / mL was 9 log2 and the dilution factor was 512. 200 μg ÷ 512 = 390.6 ng / ml. Due to virus and cell volume dilution, a further four-fold reduction occurred, resulting in a final concentration of 97.7 ng / ml.

[0245] The background signal in the assay was 2.0 log2 and any result of 5.0 log2 or higher was considered positive. Seven antibodies (3G3, 19E9, 8H11, 17D10, 17E10, 13E10, and 18D2) were confirmed to have neutralizing activity higher than 5.0 log2 (Table 6). One of the 7 clones, 18D2, had cross-reactivity and thus was not pursued further. The remaining 6 antibodies were scaled up for antibody production and purification. hMPV / A neutralization assays were performed using the 6 purified antibodies (3G3, 19E9, 8H11, 17D10, 17E10, and 13E10). Data for all 6 antibodies showed neutralizing activity (background 2.0 log2) with titers ranging from 8.0 to 12.5 log2 (data not shown).

[0246] As described above, BLI identified an additional 73 supernatants that bound to hMPV033 but not to I53-50A. The 73 supernatants were evaluated for hMPV / A neutralization assay, showing that the neutralization activities of eighteen antibodies (30G3, 42C2, 30G4, 51D9, 40A9, 37H4, 35F12, 37E1, 27F1, 50B10, 32H7, 39A4, 30D7, 41E5, 56B4, 22B2, 39D8, and 32G3) were higher than 5.0 log2 (background 2.0 log2) (data not shown). Four antibodies (37H4, 42C2, 30G3, and 56B4) were scaled up for antibody production and purification. The hMPV / A neutralization assay was performed using the 4 purified antibodies. The data of all 4 antibodies showed neutralization activities (background 2.0 log2) with titers ranging from 6.5 to 10 log2 (Table 6). Based on these results, recombinant forms of candidate antibodies 19E9, 17D10, 13E10, and 42C2 were generated using human IgG1 Fc.

[0247] Table 6

[0248]

[0249]

[0250] Confirmation of neutralization activity, specificity, binning, and K D of humanized Abs

[0251] The recombinant humanized antibodies (17D10 hIgG1, 13E10 hIgG1, and 42C2 hIgG1) were tested to confirm the specificity, neutralization, binning, and K D estimation of the hMPV F protein. All 3 humanized antibodies were confirmed as potent neutralizing antibodies (Table 6). Octet confirmed that all three humanized antibodies bound to the hMPV F protein but not to the RSV F protein ( Figures 3A - 3C ). To evaluate the affinity of each humanized antibody for hMPV033, I53-50A-hMPV033 was immobilized on an anti-penta His biosensor and then immersed in a dilution series of each humanized neutralizing antibody. The estimated K D values ranged from 4.7 to 30 nM (data not shown).

[0252] Epitope mapping

[0253] Three monoclonal antibodies (17D10 hIgG1, 13E10 hIgG1, and 42C2 hIgG1) were used for epitope mapping. CovalX TMEpitopes recognized by each antibody were identified by cross-linking antigen-antibody complexes, multi-enzyme proteolysis, and nLC-Orbitrap MS-MS analysis, and each antibody was shown to recognize non-linear epitopes( Figures 2A - 2C ). Figure 1 A schematic diagram of the epitopes of neutralizing antibodies against the hMPV F protein and other antigenic sites is shown.

[0254] Two-dimensional class averaging of nsEM images of I53-50A-hMPV033 indicated that the F protein was in the prefusion conformation and was a mixture of compact trimers and open trimers.

[0255] Characterization of the molecular interface

[0256] To determine the epitopes of the hMPV033 / 17D10 hlgG1, hMPV033 / 13E10hlgG1, and hMPV033 / 42C2hlgG1 complexes at high resolution, each protein complex was incubated with a deuterated cross-linker and subjected to multi-enzyme cleavage. After enrichment of the cross-linked peptides, the samples were analyzed by high-resolution mass spectrometry (nLC-Q-Exactive MS), and the data generated were analyzed using XQuest TM and Stavrox TM software.

[0257] In this analysis, nano-liquid chromatography (nLC) and Q-Exactive MS analysis were used in combination, with the following parameters:[[]]

[0258] Ultimate 3000-RSLC

[0259] -A 98 / 02 / 0.1 H2O / ACN / HCOOH v / v / v

[0260] -B 20 / 80 / 0.1 H2O / ACN / HCOOH v / v / v

[0261] -Gradient 4 - 55% B in 33 minutes

[0262] -Injection volume 1 μl

[0263] -Pre-column 300-μm ID x 5-mm C18 PepMapTM

[0264] -Pre-column flow rate 50 μl / min

[0265] -Column 75-μm ID x 15-cm C18 PepMapRSLC

[0266] -Column flow rate 300 nl / min

[0267] Mass spectrometry: Q-Exactive MS analysis

[0268] The Q-Exactive MS analysis was performed with the following parameters:

[0269] - Scan type: Full MS

[0270] - Scan range: 350 - 1600 m / z

[0271] - Resolution: 70,000

[0272] - μ scan: 1

[0273] - Maximum injection time: 100 ms - Spray voltage: 1.7 kV

[0274] - Capillary voltage: 275 °C

[0275] - S-lens RF level: 55.0

[0276] - AGC target: 3e6

[0277] - Default charge state: 2dd-MS 2

[0278] - Resolution: 17,500

[0279] - AGC target: 1e5

[0280] - Ion isolation window: 4 m / z units - Maximum injection time: 50 ms - Normalized collision energy: 30%

[0281] - Cycle count: 5

[0282] - Dynamic exclusion: On - Dynamic exclusion parameters: 30.0 s - Minimum AGC target: 8e3

[0283] - Intensity threshold: 1.6e5

[0284] Reductive alkylation

[0285] Mix 20 μL of the hMPV033 / antibody mixture with 2 μL of DSS d0 / d12 (2 mg / mL; DMF) and incubate for 180 minutes at room temperature. After incubation, terminate the reaction by adding 1 μL of ammonium bicarbonate (final concentration 20 mM) and incubate for 1 hour at room temperature. Then, dry the solution using a vacuum concentrator (speedvac) and add H2O 8M urea suspension (20 μL). After mixing, add 2 μl of DTT (500 mM) to the solution. Then incubate the mixture at 37 °C for 1 hour. After incubation, add 2 μl of iodoacetamide (1 M) and incubate for 1 hour at room temperature in the dark. After incubation, add 80 μl of proteolysis buffer. Trypsin buffer contains 50 mM Ambic pH 8.5, 5% acetonitrile; chymotrypsin buffer contains 100 mM Tris HCl, 10 mM CaCl2 pH 7.8; ASP-N buffer contains 50 MM phosphate buffer pH 7.8; elastase buffer contains 50 mM Tris HCl pH 8.0; and thermolysin buffer contains 50 mM Tris HCl, 0.5 mM CaCl2 pH 9.0.

[0286] Trypsin proteolysis

[0287] Mix 100 μl of the reduced / alkylated hMPV033 / 17D10_hlgG1, hMPV033 / 13E10_hlgG1 or hMPV033 / 42C2_hlgG1 mixture with 1.24 μl of trypsin (Promega) at a ratio of 1 / 100. Incubate the proteolysis mixture overnight at 37 °C.

[0288] Chymotrypsin proteolysis

[0289] Mix 100 μl of the reduced / alkylated hMPV033 / 17D10_hlgG1, hMPV033 / 13E10_hlgG1 or hMPV033 / 42C2_hlgG1 mixture with 0.62 μl of chymotrypsin (Promega) at a ratio of 1 / 200. Incubate the proteolysis mixture overnight at 25 °C.

[0290] ASP-N proteolysis

[0291] Mix 100 μl of the reduced / alkylated hMPV033 / 17D10_hlgG1, hMPV033 / 13E10_hlgG1 or hMPV033 / 42C2_hlgG1 mixture with 0.62 μl of ASP-N (Promega) at a ratio of 1 / 200. Incubate the proteolysis mixture overnight at 37 °C.

[0292] Elastase proteolysis

[0293] Mix 100 μl of the reduced / alkylated hMPV033 / 17D10_hlgG1, hMPV033 / 13E10_hlgG1, or hMPV033 / 42C2_hlgG1 mixture with 1.24 μl of elastase (Promega) at a ratio of 1 / 100. Incubate the proteolysis mixture overnight at 37 °C.

[0294] Thermolysin proteolysis

[0295] Mix 100 μl of the reduced / alkylated hMPV033 / 17D10_hlgG1, hMPV033 / 13E10_hlgG1, or hMPV033 / 42C2_hlgG1 mixture with 2.48 μl of thermolysin (Promega) at a ratio of 1 / 50. Incubate the proteolysis mixture overnight at 70 °C. After digestion, add a final 1% formic acid to the solution.

[0296] Use Xquest TM Version 2.0 and Stavrox TM 3.6 software to analyze crosslinked peptides.

[0297] Results

[0298] hMPV033 / 17D10_hlgG1

[0299] After proteolysis of the protein complex hMPV033 / 17D10_hlgG1 with trypsin, chymotrypsin, ASP-N, elastase, and thermolysin and deuterated d0d12, 12 crosslinked peptides between hMPV033 and 17D10_hlgG1 were detected by nLC-Q-Exactive MS / MS analysis. The molecular interface between hMPV033 and 17D10_hlgG1 was characterized using chemical crosslinking, high-quality MALDI mass spectrometry, and nLC-Q-Exactive mass spectrometry ( Figures 4A - 4J ). Analysis showed that the interaction included the following amino acids on hMPV033: 287, 293, 296, 364, 376, 417, and 419 of the hMPV033 amino acid sequence SEQ ID NO:1.

[0300] hMPV033 / 13E10_hlgG1

[0301] After proteolysis of the protein complex hMPV033 / 13E10_hlgG1 with trypsin, chymotrypsin, ASP-N, elastase, and thermolysin in the presence of deuterated d0d12, 15 cross-linked peptides between hMPV033 and 13E10_hlgG1 were detected by nLC-Q-Exactive MS / MS analysis. The molecular interface between hMPV033 and 13E10_hlgG1 was characterized using chemical cross-linking, high-quality MALDI mass spectrometry, and nLC-Q-Exactive mass spectrometry( Figures 5A - 5J ). Analysis showed that the interaction involved the following amino acids on hMPV033: 144, 160, 163, 188, 194, and 199 of the hMPV033 amino acid sequence SEQ ID NO:1.

[0302] hMPV033 / 42C2_hlgG1

[0303] After proteolysis of the protein complex hMPV033 / 42C2_hlgG1 with trypsin, chymotrypsin, ASP-N, elastase, and thermolysin in the presence of deuterated d0d12, 15 cross-linked peptides between hMPV033 and 42C2_hlgG1 were detected by nLC-Q-Exactive MS / MS analysis. The molecular interface between hMPV033 and 42C2_hlgG1 was characterized using chemical cross-linking, high-quality MALDI mass spectrometry, and nLC-Q-Exactive mass spectrometry( Figures 6A - 6J )。Analysis showed that the interaction involved the following amino acids on hMPV033: 44, 45, 49, 150, 156, 160, 229, 232, and 236 of the hMPV033 amino acid sequence SEQ ID NO:1.

[0304] Example 2: hMPV033 Competitive Antibody ELISA

[0305] A competitive antibody assay was developed to determine the titers of serum antibodies that can compete with mAb 17D10 hIgG1 for binding to the hMPV F protein.

[0306]

[0307]

[0308] Assay Method

[0309] The microtiter plate was coated with hMPV F antigen (dn5B-hMPV033). Serum samples were serially diluted fivefold and then an equal volume of a fixed concentration of biotinylated 17D10 hIgG1 antibody (mAb-Bio) was added. The mixture was added to the microtiter plate and incubated. As controls, "17D10 hIgG1 mAb-Bio only" wells (maximum signal) and "sample dilution buffer only" wells (background signal) were analyzed. After incubation, unbound material was washed out of the wells and HRP-conjugated streptavidin was added to all wells. The wells were then washed again to remove any unbound HRP-conjugated streptavidin. Next, addition of the TMB substrate initiated color development, which was proportional to the amount of 17D10 hIgG1 mAb-Bio bound to the hMPV F antigen. The color development was quenched and the optical density (OD450nm and OD620nm) was measured. The 17D10 hIgG1 mAb-Bio competitive antibody binding titer was expressed as the reciprocal dilution at which 17D10 hIgG1 mAb-Bio binding was inhibited by 50%.

[0310] Assay Optimization

[0311] Combinations of several hMPV F antigen coating concentrations with different concentrations of 17D10 hIgG1 mAb-Bio and different dilutions of HRP-conjugated streptavidin were tested. The maximum binding of 17D10 hIgG1 mAb-Bio to the hMPV F protein resulted in an OD of approximately 2.0 and a low background OD.

[0312] Example 3: Analysis of Antibodies Binding to Pre-Fusion and Post-Fusion hMPV F Proteins

[0313] The CompA-hMPV033 (hMPV F protein) fusion protein, post-fusion hMPV F protein, and mouse antibody of the present disclosure were normalized to a concentration of 10 μg / mL in BLI assay buffer (PBS, 0.5% BSA, 0.05% Tween 20, pH 7.4) and loaded into a black 96-well microplate at 200 μL per well. The Protein G biosensor was hydrated before immersion into the BLI assay buffer and maintained a baseline for 60 seconds in the BLI assay buffer. The biosensor was then immersed into the antibody wells for 120 seconds to immobilize existing but not yet saturated antibodies, followed by another 60-second baseline step. The immobilized antibodies were allowed to associate with the antigen for 120 seconds, after which the biosensor was immersed into the assay buffer for 120 seconds to observe dissociation. MF1, MF2, and MF3 are post-fusion hMPV F protein-specific antibodies, while MF10 is a pre-fusion hMPV F protein-specific antibody and was used as a control. Figure 7A and 7BShows the binding of control antibodies to prefusion and postfusion hMPV F proteins. Binding of the 17D10, 42C2, and 13E10 antibodies to both hMPV F protein conformations was observed, and the 17D10 and 42C2 antibodies bound to both prefusion and postfusion hMPV F proteins, while the 13E10 antibody bound only to the prefusion hMPV F protein, as Figure 7C and 7D shown.

[0314] ****

[0315] Although it has been described in connection with the specific embodiments given herein, it will be understood that further modifications can be made thereto, and this application is intended to cover any variations, uses, or adaptations of the invention, which generally follow the principles of the invention and include departures from the present disclosure that are known or conventional in the practice of the art to which this invention pertains and which can be applied to the essential features given above and within the scope of the appended claims below.

Claims

1. An epitope - specific antigen - binding molecule that is specific for the hMPV fusion (F) protein, wherein the antigen - binding molecule comprises variable domains that contact the hMPV F protein at the following positions: i.) residues 287, 293, 296, 364, 376, 417, and 419 of the hMPV F protein amino acid sequence shown in SEQ ID NO:1; ii.) residues 144, 160, 163, 188, 194, and 199; or iii.) residues 44, 45, 49, 150, 156, 160, 229, 232, and 236.

2. An antigen-binding molecule specific for the hMPV F protein, comprising: a heavy chain and a light chain, wherein the heavy chain comprises H-CDR1, H-CDR2 and H-CDR3, wherein: The H - CDR1 comprises the sequence GYTFTSY (SEQ ID NO:3); the H - CDR2 comprises the sequence YPGSGS (SEQ ID NO:4); and the H - CDR3 comprises the sequence LLRLTFDV (SEQ ID NO:5); and wherein the light chain comprises L - CDR1, L - CDR2, and L - CDR3, wherein: L - CDR1 comprises the sequence RASQDISNYLN (SEQ ID NO:7); L - CDR2 comprises the sequence YTSGLHS (SEQ ID NO:8); and L - CDR3 comprises the sequence QQGNTLPWT (SEQ ID NO:9).

3. The antigen - binding molecule according to claim 2, wherein the antigen - binding molecule comprises variable domains that contact the hMPV F protein at the following positions: i.) residues 287, 293, 296, 364, 376, 417, and 419 of the hMPV F protein amino acid sequence shown in SEQ ID NO:1; ii.) residues 144, 160, 163, 188, 194, and 199; or iii.) residues 44, 45, 49, 150, 156, 160, 229, 232, and 236.

4. The antigen - binding molecule according to claim 1, wherein the antigen - binding molecule comprises variable domains that contact the hMPV F protein at the following positions: any 1, any 2, any 3, any 4, any 5, any 6, or any 7 of residues 287, 293, 296, 364, 376, 417, and 419 of the hMPV F protein amino acid sequence shown in SEQ ID NO:

1.

5. The antigen-binding molecule according to claim 4, comprising: a heavy chain and a light chain, wherein the heavy chain comprises H-CDR1, H-CDR2, and H-CDR3, wherein: The H - CDR1 comprises the sequence GYTFTSY (SEQ ID NO:3); the H - CDR2 comprises the sequence YPGSGS (SEQ ID NO:4); and the H - CDR3 comprises the sequence LLRLTFDV (SEQ ID NO:5); and wherein the light chain comprises L - CDR1, L - CDR2, and L - CDR3, wherein: L - CDR1 comprises the sequence RASQDISNYLN (SEQ ID NO:7); L - CDR2 comprises the sequence YTSGLHS (SEQ ID NO:8); and L - CDR3 comprises the sequence QQGNTLPWT (SEQ ID NO:9).

6. The antigen-binding molecule according to any one of claims 1-5, wherein the heavy chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

2.

7. The antigen-binding molecule according to any one of claims 1-5, wherein the light chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

6.

8. The antigen-binding molecule according to any one of claims 1-7, wherein the heavy chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:2, and wherein the light chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

6.

9. The antigen-binding molecule according to any one of claims 1-8, which comprises a variable heavy (VH) chain domain, wherein the VH domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:50, and a variable light (VL) chain domain, wherein the VL domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

51.

10. The antigen-binding molecule according to claim 1, wherein the antigen-binding molecule comprises variable domains that contact the hMPV F protein at any one, any two, any three, any four, any five or any six of residues 144, 160, 163, 188, 194 and 199 of the hMPV F protein amino acid sequence shown in SEQ ID NO:

1.

11. The antigen-binding molecule according to claim 10, comprising: a heavy chain and a light chain, wherein the heavy chain comprises H-CDR1, H-CDR2, and H-CDR3, wherein: H-CDR1 comprises the sequence GFTFTDY (SEQ ID NO:11); H-CDR2 comprises the sequence RNKDNGYT (SEQ ID NO:12); and H-CDR3 comprises the sequence YYFGYDGDYFDY (SEQ ID NO:13); and wherein the light chain comprises L-CDR1, L-CDR2 and L-CDR3, wherein: L-CDR1 comprises the sequence SASSSISSNYLH (SEQ ID NO:15); L-CDR2 comprises the sequence RTSNLAS (SEQ ID NO:16); and L-CDR3 comprises the sequence QQGSSLPRT (SEQ ID NO:17).

12. The antigen-binding molecule according to any one of claims 10-11, wherein the heavy chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

10.

13. The antigen-binding molecule according to any one of claims 10-11, wherein the light chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

14.

14. The antigen-binding molecule according to any one of claims 10-13, wherein the heavy chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 10, and wherein the light chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

14.

15. The antigen-binding molecule according to any one of claims 10-14, which comprises a variable heavy (VH) chain domain, wherein the VH domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 52, and a variable light (VL) chain domain, wherein the VL domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

53.

16. The antigen-binding molecule according to claim 1, wherein the antigen-binding molecule comprises variable domains that contact the hMPV F protein at any one, any two, any three, any four, any five, any six, any seven, any eight or any nine of residues 44, 45, 49, 150, 156, 160, 229, 232 and 236 of the hMPV F protein amino acid sequence shown in SEQ ID NO:

1.

17. The antigen-binding molecule according to claim 16, comprising: a heavy chain and a light chain, wherein the heavy chain comprises H-CDR1, H-CDR2 and H-CDR3, wherein: H-CDR1 comprises the sequence GFSLSTFGM (SEQ ID NO: 19); H-CDR2 comprises the sequence WWDDD (SEQ ID NO: 20); and H-CDR3 comprises the sequence IVKVLEQYFDV (SEQ ID NO: 21); and The light chain comprises L-CDR1, L-CDR2 and L-CDR3, wherein: L-CDR1 comprises the sequence KASQDVGTAVA (SEQ ID NO:23); L-CDR2 comprises the sequence WASTRHT (SEQ ID NO:24); and L-CDR3 comprises the sequence QQYTSYPLT (SEQ ID NO:25).

18. The antigen-binding molecule according to any one of claims 16-17, wherein the heavy chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

18.

19. The antigen-binding molecule according to any one of claims 16-17, wherein the light chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

22.

20. The antigen-binding molecule according to any one of claims 16-19, wherein the heavy chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:18, and wherein the light chain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

22.

21. The antigen-binding molecule according to any one of claims 16-20, which comprises a variable heavy (VH) chain domain, wherein the VH domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, and a variable light (VL) chain domain, wherein the VL domain comprises a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

55.

22. The antigen-binding molecule according to any one of claims 1-21, wherein the antigen-binding molecule is an immunoglobulin molecule.

23. The antigen-binding molecule according to claim 22, wherein the immunoglobulin is an IgG1, IgG2, IgG3 or IgG4 molecule.

24. The antigen-binding molecule according to claim 22, wherein the immunoglobulin is a humanized antibody.

25. The antigen-binding molecule according to claim 22, wherein the immunoglobulin is a neutralizing antibody.

26. The antigen-binding molecule according to any one of claims 1-25, wherein the antigen-binding molecule specifically binds to the pre-fusion conformation of the hMPV F protein.

27. The antigen-binding molecule according to any one of claims 1-25, wherein the antigen-binding molecule binds to the hMPV F protein in the pre-fusion or post-fusion conformation.

28. A polynucleotide encoding the antigen-binding molecule according to any one of claims 1-27.

29. A pharmaceutical composition or vaccine comprising the antigen-binding molecule according to any one of claims 1-27, and a pharmaceutically acceptable carrier, diluent or excipient.

30. A method for detecting an antibody specific for the hMPV F protein, the method comprising: a.) contacting a biological sample with the hMPV F protein; and b.) contacting an epitope-specific antigen-binding molecule according to any one of claims 1-27 with the hMPV F protein.

31. The method according to claim 30, wherein the hMPV F protein is coated on a microtiter plate prior to contacting with the biological sample or the antigen-binding molecule.

32. The method according to claim 31, which comprises determining the half maximal effective concentration (EC 50 ) of the anti-hMPV F protein antibody in the biological sample by determining the reciprocal dilution of the biological sample at which the binding of the antigen-binding molecule is inhibited by 50%.

33. The method according to any one of claims 30-32, wherein the antigen-binding molecule comprises variable domains that contact the hMPV F protein at the following positions: i.) residues 287, 293, 296, 364, 376, 417 and 419 of the hMPV F protein amino acid sequence shown in SEQ ID NO:1; ii.) residues 144, 160, 163, 188, 194 and 199; or iii.) residues 44, 45, 49, 150, 156, 160, 229, 232 and 236.

34. The method according to any one of claims 30-33, wherein the biological sample is serum.

35. The method according to any one of claims 30-34, wherein the method specifically detects the hMPV F protein in the pre-fusion conformation.

36. The method according to any one of claims 30-34, wherein the method detects the hMPV F protein in the pre-fusion or post-fusion conformation.

37. A method for treating or preventing hMPV infection in a subject in need thereof, the method comprising administering to the subject an effective amount of the antigen-binding molecule according to any one of claims 1-27 or the pharmaceutical composition or vaccine according to claim 29.

38. The method according to claim 37, wherein the subject has an hMPV infection or is at risk of developing an hMPV infection.

39. The method according to claim 37 or claim 38, wherein the subject is a mammal, optionally a human.

40. The method according to any one of claims 37-39, wherein the subject is susceptible to viral infection.

41. The method according to any one of claims 37-39, wherein the subject is an elderly subject.

42. The method according to any one of claims 37-41, wherein the antigen-binding molecule, pharmaceutical composition or vaccine is administered by intramuscular injection, intravenous injection or subcutaneous injection.

43. A method for measuring the immunogenicity of an antigen, wherein the method comprises detecting the ratio of the pre-fusion conformation hMPV F protein antigen-binding molecule to the post-fusion conformation hMPV F protein antigen-binding molecule produced after immunization with the hMPV F protein vaccine as described in claim 29.

44. A method for measuring the stability of an antigen composition, wherein the method comprises detecting the ratio of the pre-fusion conformation hMPV F protein antigen-binding molecule to the post-fusion conformation hMPV F protein antigen-binding molecule that binds to the antigen composition.

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