Antibodies to metapneumovirus fusion (F) protein and uses thereof

Abstract

An epitope-specific antigen-binding molecule specific for the human metapneumovirus (hMPV) fusion (F) protein is provided, which comprises a variable domain that contacts the hMPV F protein. The hMPV F protein-specific antigen-binding molecule comprises a heavy chain and a light chain. Also provided is a method for detecting an antibody specific for the hMPV F protein, comprising: a.) contacting a biological sample with the hMPV F protein; and b.) contacting the epitope-specific antigen-binding molecule with the hMPV F protein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 477,092, filed December 23, 2022, which is incorporated herein by reference in its entirety.

[0002] INCORPORATION-BY-REFERENCE TO SEQUENCE LISTING This application contains a Sequence Listing that has been submitted via EFS-WEB in .XML format and is incorporated herein by reference in its entirety. The .XML copy, created on December 13, 2023, is named 061291-510001WO_SeqList_ST26.xml and is 55 kilobytes in size.

[0003] The present disclosure relates generally to antibodies against metapneumovirus fusion (f) proteins. [Background technology]

[0004] Human metapneumovirus (hMPV) causes upper and lower respiratory tract disease in people of all ages. Symptoms can include cough, fever, nasal congestion, and shortness of breath. In some cases, hMPV infection can progress to severe illnesses such as bronchitis or pneumonia.

[0005] hMPV is a single-stranded RNA virus of the paramyxovirus family, which also includes other respiratory infections such as measles, mumps, and respiratory syncytial virus (RSV). hMPV can be of two subtypes, A and B, identified by genotyping the G and F genes within the viral genome. The F gene encodes the hMPV fusion glycoprotein (hMPV F protein) and can be used to treat or prevent hMPV infection. However, despite the clinical need, there is currently no approved treatment for hMPV in humans.

[0006] Thus, there is an unmet need for treatments for hMPV. Summary of the Invention [Means for solving the problem]

[0007] An epitope-specific antigen-binding molecule specific for the hMPV fusion (F) protein is provided, which comprises a variable domain that contacts the hMPV F protein as follows: i.) residues 287, 293, 296, 364, 376, 417, and 419; ii.) residues 144, 160, 163, 188, 194, and 199; or iii.) Residues 44, 45, 49, 150, 156, 160, 229, 232 and 236.

[0008] In some embodiments, an antigen binding molecule specific for an 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 LLRTFDV (SEQ ID NO: 5); and 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).

[0009] In some embodiments, the antigen binding molecule comprises a variable domain that contacts the hMPV F protein: i.) residues 287, 293, 296, 364, 376, 417, and 419; ii.) residues 144, 160, 163, 188, 194, and 199; or iii.) Residues 44, 45, 49, 150, 156, 160, 229, 232 and 236.

[0010] In some embodiments, the antigen binding molecule comprises a variable domain that contacts the hMPV F protein at any one, any two, any three, any four, any five, any six, or any seven of residues 287, 293, 296, 364, 376, 417, and 419 of the hMPV F protein amino acid sequence set forth in SEQ ID NO:1.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] In some embodiments, the antigen binding molecule comprises a variable domain that contacts 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 set forth in SEQ ID NO:1.

[0016] 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 GYTFTSY (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 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).

[0017] 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.

[0018] 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.

[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: 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.

[0020] 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.

[0021] In some embodiments, the antigen binding molecule comprises a variable domain that contacts 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 set forth in SEQ ID NO:1.

[0022] 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 WDDDD (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).

[0023] 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.

[0024] 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.

[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: 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.

[0026] 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.

[0027] In some embodiments, the antigen-binding molecule is an immunoglobulin molecule.

[0028] In some embodiments, the immunoglobulin is an IgG1, IgG2, IgG3, or IgG4 molecule.

[0029] In some embodiments, the immunoglobulin is a humanized antibody.

[0030] Polynucleotides encoding the antigen-binding molecules of the present disclosure are provided.

[0031] Pharmaceutical compositions comprising an antigen-binding molecule of the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient are provided.

[0032] A method for detecting antibodies specific to an hMPV F protein is provided, the method comprising: a.) contacting the biological sample with an hMPV F protein; and b.) Contacting the epitope-specific antigen-binding molecule of any one of claims 1 to 19 with an MPV F protein.

[0033] In some embodiments, the hMPV F protein is coated onto a microwell plate prior to contacting with the biological sample or antigen-binding molecule.

[0034] In some embodiments, the half-maximal effective concentration (EC50) of an anti-hMPV F protein antibody in a biological sample is determined by calculating the inverse dilution of the biological sample at which binding of the antigen-binding molecule is inhibited by 50%.

[0035] In some embodiments, the biological sample is serum.

[0036] Methods for treating or preventing hMPV infection in a subject in need thereof are provided, comprising administering to the subject an effective amount of an antigen-binding molecule according to the present disclosure.

[0037] In some embodiments, the subject has or is at risk of developing an hMPV infection.

[0038] In some embodiments, the subject is a mammal, optionally a human.

[0039] In some embodiments, the subject is susceptible to a viral infection.

[0040] In some embodiments, the subject is an elderly subject.

[0041] In some embodiments, the antigen binding molecules of the present disclosure are administered by intramuscular, intravenous, or subcutaneous injection. [Brief explanation of the drawings]

[0042] [Figure 1] 1 shows an exemplary hMPV F protein with neutralizing antigenic sites and epitopes for the 17D10, 13E10, and 42C2 antibodies. Regions containing epitopes for 17D10, 13E10, and 42C2, as well as antigenic sites corresponding to neutralizing antibodies designated II, III, IV, V, DS7, and 66-87, are circled.

[0043] [Figure 2A] FIG. 1 is an illustration of hMPV033 epitopes relative to 17D10. [Figure 2B] FIG. 1 is a diagram illustrating hMPV033 epitopes relative to 13E10. [Figure 2C] FIG. 1 is an illustration of hMPV033 epitopes for 42C2.

[0044] [Figure 3A] Graphs showing binding of mouse (mIgG) and human (hIgG) Fc-containing neutralizing antibody (nAb) 17D10 to hMPV033 (top panel) and DS-Cav1 (RSV control) (bottom panel). All three nAbs were specific for hMPV F, but not for RSV F or I53-50A. [Figure 3B] Graphs showing binding of mouse (mIgG) and human (hIgG) Fc-containing neutralizing antibody (nAb) 13E10 to hMPV033 (top panel) and DS-Cav1 (RSV control) (bottom panel). All three nAbs were specific for hMPV F, but not for RSV F or I53-50A. [Figure 3C] Graphs showing the binding of mouse (mIgG) and human (hIgG) Fc-containing neutralizing antibody (nAb) 42C2 to hMPV033 (top panel) and DS-Cav1 (RSV control) (bottom panel). All three nAbs were specific for hMPV F, but not for RSV F or I53-50A.

[0045] [Figure 4]1 shows the interaction between hMPV033 and 17D10_hlgG1. The hMPV033 Protein Data Bank (PDB) structure is colored blue at the epitope site. The hMPV033 amino acids colored blue correspond to amino acids 287-296 (KAAPSCSEKK), 364-376 (SCGRNPISMVALS), and 417-419 (TVT) of SEQ ID NO: 1 of the hMPV033 sequence. A shows a ribbon / surface representation of the front view. B shows a ribbon / surface representation of the back view. C shows a ribbon / surface representation of side view 1. D shows a ribbon / surface representation of side view 2. E shows a ribbon / surface representation of the top view. F shows a ribbon representation of the front view. G shows a ribbon representation of the back view. H shows a ribbon representation of side view 1. I shows a ribbon representation of side view 2. J shows a ribbon representation of the top view.

[0046] [Figure 5] This figure shows the interaction between hMPV033 and 13E10_hgG1. The hMPV033 PDB structure is colored blue at the epitope site. The hMPV033 amino acids colored blue correspond to amino acids 144-163 (TNEAVSTLGCGVRVLATAVR) and 188-199 (KMAVSFSQFNRR) of the hMPV033 sequence (SEQ ID NO: 1). A shows a ribbon / surface representation of the front view. B shows a ribbon / surface representation of the back view. C shows a ribbon / surface representation of side view 1. D shows a ribbon / surface representation of side view 2. E shows a ribbon / surface representation of the top view. F shows a ribbon representation of the front view. G shows a ribbon representation of the back view. H shows a ribbon representation of side view 1. I shows a ribbon representation of side view 2. J shows a ribbon representation of the top view.

[0047] [Figure 6]This figure shows the interaction between hMPV033 and 42C2_hgG1. 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 229-236 (RAISMPT) of the hMPV033 sequence (SEQ ID NO: 1). A shows a ribbon / surface representation of the front view. B shows a ribbon / surface representation of the back view. C shows a ribbon / surface representation of side view 1. D shows a ribbon / surface representation of side view 2. E shows a ribbon / surface representation of the top view. F shows a ribbon representation of the front view. G shows a ribbon representation of the back view. H shows a ribbon representation of side view 1. I shows a ribbon representation of side view 2. J shows a ribbon representation of the top view.

[0048] [Figure 7A] Graph showing antibody binding to pre-fusion and post-fusion hMPV F proteins. 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 was analyzed by the Octet method. The 17D10 and 42C2 antibodies bind to both the pre-fusion and post-fusion conformations of the hMPV F protein; 13E10 binds only to the pre-fusion hMPV F protein conformation. [Figure 7B] Graph showing antibody binding to pre-fusion and post-fusion hMPV F proteins. The binding of MF1, MF2, and MF3 post-fusion hMPV F protein-specific antibodies and MF10 pre-fusion hMPV F protein-specific antibody (control) to post-fusion hMPV F protein was analyzed by the Octet method. The 17D10 and 42C2 antibodies bind to both the pre-fusion and post-fusion conformations of the hMPV F protein; 13E10 binds only to the pre-fusion hMPV F protein conformation. [Figure 7C]1 is a graph showing antibody binding to pre-fusion and post-fusion hMPV F proteins. Binding of 17D10, 42C2, and 13E10 hMPV F protein-specific antibodies to the pre-fusion hMPV F protein was analyzed by the Octet method. 17D10 and 42C2 antibodies bind to both the pre-fusion and post-fusion conformations of the hMPV F protein; 13E10 binds only to the pre-fusion hMPV F protein conformation. [Figure 7D] 1 is a graph showing antibody binding to pre-fusion and post-fusion hMPV F proteins. Binding of 17D10, 42C2, and 13E10 hMPV F protein-specific antibodies to post-fusion hMPV F protein was analyzed by the Octet method. 17D10 and 42C2 antibodies bind to both pre-fusion and post-fusion conformations of the hMPV F protein; 13E10 binds only to the pre-fusion hMPV F protein conformation. DETAILED DESCRIPTION OF THE INVENTION

[0049] definition All publications, patents, and patent applications, including any drawings and appendices therein, are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application, drawing, or appendix was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0050] 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 belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, preferred methods and materials are described herein. For purposes of this disclosure, the following terms are defined below.

[0051] The term "antigen" refers to a polypeptide or polypeptide complex that includes at least one component designed to elicit an immune response. As used herein, the term antigen is not limited to the portion of a polypeptide or polypeptide complex that contains an antigenic epitope.

[0052] The term "infection" refers to both symptomatic and asymptomatic infection.

[0053] The term "linker" refers to either a chemical linkage (i.e., a covalent bond or series of covalent bonds with intervening chemical moieties) or to polypeptides linked at their N- and C-termini by peptide bonds to produce a fusion protein.

[0054] The term "antigen-binding molecule" refers to a molecule having binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins, immunoglobulin fragments, and non-immunoglobulin-derived protein frameworks that exhibit antigen-binding activity. Exemplary antigen-binding molecules useful in the practice of the present disclosure include polyclonal and monoclonal antibodies and fragments thereof (Fab, Fab', F(ab')2, Fv, etc.), single-chain (scFv) and domain antibodies (including, for example, shark antibodies and camelid antibodies), as well as fusion proteins comprising antibodies and any other modified configurations of immunoglobulin molecules that contain an antigen-binding / recognition site. Antibodies include any class of antibody (or subclass thereof), such as IgG, IgA, or IgM, and antibodies need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which 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 called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known. Antigen-binding molecules also include dimeric antibodies and multivalent forms of antibodies. In some embodiments, antigen-binding molecules specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (US 4,816,567, and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855).Humanized antibodies are also contemplated, and are generally produced by transferring complementarity-determining regions (CDRs) from the heavy and light variable chains of a non-human (e.g., rodent, preferably murine) immunoglobulin into a human variable domain. Common residues of human antibodies are then substituted into the framework regions of the non-human counterpart. The use of antibody components derived from humanized antibodies eliminates potential problems associated with the immunogenicity of non-human constant regions. General techniques for cloning non-human, particularly murine, immunoglobulin variable domains are described, for example, by Orlandi et al. (1989, Proc. Natl. Acad. Sci. USA 86:3833). Techniques for producing humanized monoclonal antibodies are described, for example, in 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.), pages 399-434 (John Wiley & Sons, Inc. 1996), Queen et al. al., US Pat. No. 5,693,762 (1997).

[0055] As used herein, the term "antibody" is used in the broadest sense and specifically encompasses naturally occurring monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired biological activity. Naturally occurring "antibodies" encompass immunoglobulins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of specific CH domains (e.g., CH1, CH2, and CH3). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs). Each VH and VL is composed of three CDRs. The 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 modified version thereof (e.g., an IgG1 isotype with the L234A and L235A double mutation (IgG1-LALA)). The antibody can be of any species, chimeric, humanized, or human. In other embodiments, the antibody is a homogeneous heavy chain antibody (e.g., a camelid antibody) that lacks the first constant region domain (CH1) but otherwise retains an intact heavy chain and is capable of binding to an antigen via the antigen-binding domain. Unless otherwise indicated, the term "antibody" includes antibodies comprising two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and muteins thereof, examples of which are described below. Additionally, unless expressly excluded, antibody includes 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, respectively. In some embodiments, the term also encompasses peptibodies.

[0056] In certain embodiments, an antibody heavy chain binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody light chain binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an individual variable region specifically binds to an antigen in the absence of other variable regions.

[0057] The term "neutralization" (e.g., "neutralizing antibody") refers to an antibody that prevents infection and / or reduces the level of infection by a pathogen. Neutralizing antibody responses can be measured in either in vitro assays (e.g., infection of cultured cells with a pathogen in the presence of the antibody) or in vivo assays (e.g., determining a protective dose of antibody by administering the antibody to a subject prior to infection with an infectious dose of the pathogen). Neutralizing antibodies can inhibit pathogen infectivity by binding to the pathogen and blocking molecules necessary for host cell invasion. Neutralizing antibodies can statically interfere with the attachment of a pathogen to a host cell receptor. Without being bound by theory, in the case of viral infections, neutralizing antibodies can bind to glycoproteins of enveloped viruses or capsid proteins of non-enveloped viruses and may act by preventing viral particles from undergoing conformational changes often required for successful invasion of host cells.

[0058] The term "variable region" or "variable domain" refers to the variable domain of a light chain (VL) or the variable domain of a heavy chain (VH). As used herein, "variable region" or "variable domain" refers to each of a pair of light and heavy chain domains that are directly involved in binding an antibody to an antigen. Variable light and heavy chain domains have the same general structure, and each domain contains four framework regions (FRs) whose sequences are widely conserved and connected by three CDRs, or "hypervariable regions." The FRs adopt a β-sheet structure, and the CDRs may form loops connecting the β-sheet structure. The CDRs of each chain are held in their three-dimensional structure by the FRs, which, together with the CDRs, form the antigen-binding site.

[0059] "CDR" or "complementarity-determining region" (also referred to as "hypervariable region") refers to the amino acid residues of an antibody responsible for antigen binding. As used herein, CDR refers to the amino acid sequences of the light and heavy chains of an antibody that form the three-dimensional loop structure that contributes to the formation of the antigen-binding site. There are three CDRs, designated "CDR1," "CDR2," and "CDR3," for each of the heavy and light chain variable regions of an antibody. As used herein, the term "CDR set" refers to a group of three CDRs occurring 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 terms such as "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."

[0060] In certain embodiments, the definition of CDRs and the identification of residues comprising the antibody binding site are achieved by solving the structure of the antibody and / or the structure of an antibody-ligand complex. In certain embodiments, this can be achieved by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various analytical methods can be used to identify or approximate 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.

[0061] The exact boundaries of these CDRs are defined differently in 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 antibody variable region, but also provides precise residue boundaries defining the three CDRs. These CDRs are sometimes referred to as "Kabat CDRs." Chothia and colleagues (Chothia and Lek, 1987. J. Mol. Biol. 196:901-917; Chothia et al., 1989. Nature 342:877-883) found that certain small portions of the Kabat CDRs adopt nearly identical peptide backbone conformations despite great diversity at the amino acid sequence level. Other boundaries defining CDRs that overlap with the Kabat CDRs have been described by Padlan (1995. FASEB J. 9:133-139) and MacCallum (1996). J. Mol. Biol. 262(5):732-745). Still other CDR boundary definitions may not strictly adhere to one of these systems, but still overlap with the Kabat CDRs. However, certain residues or groups of residues, or even entire CDRs, may be shortened or lengthened in light of predictions or experimental findings that do not significantly affect antigen binding.

[0062] A "single-chain variable fragment (scFv)" is a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., 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 separate genes, they can be joined using recombinant methods by an artificial peptide linker that allows them to be produced as 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 of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0063] An antibody that "binds" to an antigen of interest (e.g., hMPV F protein) is one that binds to that antigen with sufficient affinity so that it is useful as a therapeutic agent for targeting cells or tissues expressing the antigen and does not significantly cross-react with other polypeptides. In such embodiments, the extent of binding of the antibody to a "non-target" protein is less than about 10% of the binding of the antibody, oligopeptide, or other organic molecule to its particular target protein, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or radioimmunoprecipitation (RIA). With respect to the binding of an antibody to a target molecule, the terms "specific binding" or "specifically binds to" or "is specific for" a particular polypeptide or epitope on a particular polypeptide target refer to binding that is distinct from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is generally a molecule of similar structure that lacks binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, e.g., an excess of unlabeled target. In this case, specific binding is indicated when the binding of the labeled target to the probe is competitively inhibited by excess unlabeled target.The specific region of an antigen to which an antibody binds is typically called an "epitope."The term "epitope" broadly includes the site on an antigen that is specifically recognized by an antibody or T cell receptor, or that interacts with a molecule.An epitope is usually an active surface grouping of molecules, such as amino acids or carbohydrates or sugar side chains, and can usually have specific three-dimensional structural characteristics and specific charge characteristics.As will be understood by those skilled in the art, virtually anything that an antibody can specifically bind to can be an epitope.

[0064] The terms "binding" or "being specific for" or "specifically binding" (used interchangeably herein) of an antibody to a target (e.g., a human metapneumovirus or hMPV protein) are well understood in the art, and methods for determining such specific or preferential binding are also well understood in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell or substance more frequently, more rapidly, for a longer duration, and / or with a higher affinity than with alternative cells or substances. For example, an immunoglobulin that specifically or preferentially binds to thymocytes is an immunoglobulin that binds to thymocytes with higher affinity, avidity, more readily, and / or for a longer duration than it binds 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. In general, reference to binding often, but not necessarily, implies specific binding.

[0065] Throughout this disclosure, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" should be understood to mean the inclusion of the recited step or element, or group of steps or elements, but not the exclusion of other steps or elements, or group of steps or elements. Thus, the use of terms such as "comprising" indicates that the recited elements are required or essential, but that other elements are optional and may or may not be present. "Consisting of" means including and limited to what follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the recited elements are required or essential, and that other elements may not be present. "Consisting essentially of" means including any elements listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or function specified in the disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or essential, but that other elements are optional, and may or may not be present depending on whether they affect the activity or function of the listed elements.

[0066] The term "pharmaceutically acceptable excipient" refers to an excipient that is biologically or pharmacologically compatible for in vivo use in animals or humans, and can mean an excipient that has been approved by a regulatory agency of the U.S. federal or state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia, for use in animals, or more specifically, in humans.

[0067] The term "adjuvant" refers to a pharmaceutically acceptable substance that enhances the immune response to an antigen when administered in combination with an antigen, or when administered before, during, or after administration of the antigen to a subject.

[0068] "Effective amount," in the context of treating a disease or condition, means administering to an individual in need of such treatment or prevention, either as a single dose or as part of multiple doses, an amount of a drug or composition effective to prevent the onset of symptoms, suppress such symptoms, and / or treat existing symptoms of the condition. The effective amount will vary depending on the age, health, and physical condition of the individual being treated, whether symptoms of the disease are apparent, the taxonomic group of the individual being treated, the formulation of the composition, an evaluation of the medical condition, and other relevant factors. Optimal dosing schedules can be calculated from measurements of drug accumulation in the subject's body. Optimal dosages may vary depending on relative efficacy in individual subjects and can generally be estimated based on EC50 values ​​found to be effective in in vitro and in vivo animal models. One of ordinary skill in the art can readily determine optimal dosages, administration methods, and repetition rates. It is expected that the amount will fall within a relatively broad range that can be determined through routine testing.

[0069] As used herein, the term "therapeutically effective amount" or "effective dosage" refers to a dosage or concentration of a drug effective to treat a disease or condition, e.g., with respect to the use of a monoclonal antibody or antigen-binding fragment to treat a viral infection.

[0070] The term "immune response" refers to the elicitation of activity of one or more immune cell types in a subject. Immune responses include, for example, T cell responses and B cell responses.

[0071] The term "humoral immune response" refers to an immune response that results in the generation of plasma or serum antibodies (eg, IgG).

[0072] The term "administering" refers to providing a composition to a subject in a manner that allows the composition to have its intended effect. Administration for vaccination or post-exposure prophylaxis can be by intramuscular injection, intravenous injection, intraperitoneal injection, or any other suitable route.

[0073] The term "subject" refers to a human or non-human animal to which a composition may be administered for vaccination, therapy, 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, llama, or ferret.

[0074] The term "polynucleotide" refers to a polymeric form of nucleotides of more than about 100 nucleotides, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-, double-, 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 nucleotide bases.

[0075] In the context of two or more polynucleotide or polypeptide sequences, the term "identical" or percent "identity" refers to two or more sequences or subsequences that are identical or have an identical specified percentage of amino acid residues or nucleotides (i.e., having at least about 80% identity over a specified region of a reference sequence, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity), when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition also refers to the complement of a test sequence. In some embodiments, identity exists over a region that is at least about 25 amino acids or nucleotides in length, e.g., over a region that is 50, 100, 200, 300, 400 amino acids or nucleotides in length, or over the entire length of the reference sequence.

[0076] For sequence comparison, typically, one sequence serves as a reference sequence, and test sequences are compared with it.When using a sequence comparison algorithm, test sequences and reference sequences are input into a computer, subsequence coordinates are designated as needed, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the program parameters.In some embodiments, the BLAST and BLAST 2.0 algorithms and default parameters are used.

[0077] The terms "treatment," "treating," and the like refer to one or more of alleviating, ameliorating, delaying, reducing, reversing, ameliorating, or managing at least one symptom of a condition in a subject. The term "treating" can also mean one or more of arresting a condition, delaying its onset (i.e., the period before clinical signs of the condition appear), or reducing the risk of its development or worsening.

[0078] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an" used to refer to an "element" means one element or more than one element.

[0079] As used herein, "and / or" refers to and includes all possible combinations of one or more of the associated listed items, as well as the absence of a combination when interpreted alternatively ("or").

[0080] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" can mean, for example, within ±20%, within ±10%, or within ±5%.

[0081] All weight percentages (i.e., "weight %" and "wt.%" and "w / w") referred to herein are measured relative to the total weight of the pharmaceutical composition, unless otherwise specified.

[0082] As used herein, "substantially" or "substantially" refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result. For example, an object that is "substantially" enclosed means that the object is completely enclosed or nearly completely enclosed. The exact degree of acceptable deviation from absolute completeness may, in some cases, depend on the specific context. Generally speaking, however, the proximity of completion is such that the overall result is the same as if absolute and total completion had been achieved. The use of "substantially" is equally applicable when used in the negative sense to refer to the complete or nearly complete absence of an action, characteristic, property, state, structure, item, or result. For example, a composition that is "substantially free" of other active agents is completely devoid of other active agents, or nearly completely devoid of other active agents, so that its effect is the same as if it were completely devoid of other active agents. In other words, a composition that is "substantially free" of a component or element or another active agent may still contain such item as long as there is no measurable effect of that item.

[0083] 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 publication specifically or implicitly referenced is prior art.

[0084] antigen binding molecule An epitope-specific antigen-binding molecule specific for the hMPV fusion (F) protein is provided, the antigen-binding molecule comprising a variable domain that contacts the hMPV F protein as follows: i.) residues 287, 293, 296, 364, 376, 417, and 419; ii.) residues 144, 160, 163, 188, 194, and 199; or iii.) Residues 44, 45, 49, 150, 156, 160, 229, 232 and 236.

[0085] Using the hMPV033 F protein as a reference sequence: (SEQ ID NO: 1)

[0086] 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.

[0087] In some embodiments, an epitope-specific antigen binding molecule specific for an hMPV fusion (F) protein comprises a variable domain that contacts the hMPV F protein at residues 287, 293, 296, 364, 376, 417, and 419 of the hMPV F protein amino acid sequence set forth in SEQ ID NO: 1, and may be referred to as a "17D10" antigen binding molecule.

[0088] In some embodiments, an epitope-specific antigen binding molecule specific for an hMPV fusion (F) protein comprises a variable domain that contacts the hMPV F protein at residues 144, 160, 163, 188, 194, and 199 of the hMPV F protein amino acid sequence set forth in SEQ ID NO: 1, and may be referred to as a "13E10" antigen binding molecule.

[0089] In some embodiments, an epitope-specific antigen binding molecule specific for an hMPV fusion (F) protein comprises a variable domain that contacts the hMPV F protein at residues 44, 45, 150, 156, 160, 229, 232, and 236 of the hMPV F protein amino acid sequence set forth in SEQ ID NO: 1, and may be referred to as a "42C2" antigen binding molecule.

[0090] The heavy and light chain sequences of exemplary antigen-binding molecules are shown in Table 1. [Table 1-1] [Table 1-2]

[0091] The variable domain sequences of exemplary antigen binding molecules are shown in Table 2. [Table 2-1] [Table 2-2]

[0092] The variable domain CDR sequences of exemplary antigen binding molecules are shown in Table 3. [Table 3]

[0093] Epitope contact residues of exemplary antigen binding molecule variable domains are provided in Table 4. [Table 4]

[0094] Epitope I-17D10 The present disclosure contemplates any hMPV F protein antigen binding molecule that binds to an hMPV F protein, such as a human hMPV F protein, in both pre-fusion and post-fusion hMPV F protein conformations. In some embodiments, the hMPV F protein antigen binding molecule is the 17D10 antibody that binds to an epitope comprising at least a portion of the region from amino acid 287 to amino acid 419 of the human hMPV F protein. As shown in Figures 4A-4J, these amino acid residues form a non-linear epitope on the hMPV F protein. An anti-hMPV F protein antigen binding molecule of the present disclosure that binds to the 17D10 epitope binds to the hMPV F protein in both the pre-fusion and post-fusion conformations.

[0095] 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.

[0096] In some embodiments, an epitope-specific antigen binding molecule specific for an hMPV fusion (F) protein comprises a variable domain that contacts the hMPV F protein at residues 287, 293, 296, 364, 376, 417, and 419 of the hMPV F protein amino acid sequence set forth in SEQ ID NO: 1, and may be referred to as a "17D10" antigen binding molecule.

[0097] In some embodiments, the antigen binding molecule comprises a variable domain that contacts the hMPV F protein at any one, any two, any three, any four, any five, any six, or any seven of residues 287, 293, 296, 364, 376, 417, and 419 of the hMPV F protein amino acid sequence set forth in SEQ ID NO:1.

[0098] In some embodiments, the antigen-binding molecule "17D10" comprises a heavy chain and a light chain, the heavy chain comprising 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 LLRTFDV (SEQ ID NO: 5).

[0099] 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).

[0100] In some embodiments, the antigen binding molecule "17D10" 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 LLRTFDV (SEQ ID NO: 5); and 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).

[0101] 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.

[0102] 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 (SEQ ID NO: 6).

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] Epitope II-13E10 The present disclosure contemplates any hMPV F protein antigen binding molecule that binds to the hMPV F protein, particularly in the pre-fusion hMPV F protein conformation. In some embodiments, the hMPV F protein antigen binding molecule is the 13E10 antibody, which binds to an epitope comprising at least a portion of the region from amino acid 144 to amino acid 199 of the hMPV F protein. As shown in Figures 5A-5J, these amino acid residues form a nonlinear epitope on the hMPV F protein.

[0108] Anti-hMPV F protein antigen binding molecules of the present disclosure that bind to the 13E10 epitope only bind to hMPV F protein in the prefusion conformation.

[0109] 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.

[0110] In some embodiments, an epitope-specific antigen binding molecule specific for an hMPV fusion (F) protein comprises a variable domain that contacts the hMPV F protein at residues 144, 160, 163, 188, 194, and 199 of the hMPV F protein amino acid sequence set forth in SEQ ID NO: 1, and may be referred to as a "13E10" antigen binding molecule.

[0111] In some embodiments, the antigen binding molecule comprises a variable domain that contacts 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 set forth in SEQ ID NO:1.

[0112] 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 GYTFTSY (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 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).

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] Epitope III-42C2 The present disclosure contemplates any hMPV F protein antigen binding molecule that binds to the hMPV F protein in both 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 comprising at least a portion of the region from amino acid 44 to amino acid 236 of the hMPV F protein. As shown in Figures 6A-6J, these amino acid residues form a nonlinear epitope on the hMPV F protein.

[0120] Anti-hMPV F protein antigen binding molecules of the present disclosure that bind to the 42C2 epitope bind to hMPV F protein in both pre-fusion and post-fusion conformations.

[0121] 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.

[0122] In some embodiments, an epitope-specific antigen binding molecule specific for an hMPV fusion (F) protein comprises a variable domain that contacts the hMPV F protein at residues 44, 45, 150, 156, 160, 229, 232, and 236 of the hMPV F protein amino acid sequence set forth in SEQ ID NO: 1, and may be referred to as a "42C2" antigen binding molecule.

[0123] In some embodiments, the antigen binding molecule comprises a variable domain that contacts 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 set forth in SEQ ID NO:1.

[0124] 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 WDDDD (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).

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] In some embodiments, the antigen-binding molecule is an immunoglobulin molecule.

[0132] In some embodiments, the immunoglobulin is an IgG1, IgG2, IgG3, or IgG4 molecule.

[0133] In some embodiments, the immunoglobulin is a humanized antibody.

[0134] In some embodiments, the immunoglobulin is a neutralizing antibody.

[0135] hMPV F protein Human metapneumovirus (hMPV) is a negative-stranded enveloped virus of the Pneumoviridae family. It was discovered in 2001 but had been circulating for at least half a century prior to 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 initially translated as a single polypeptide precursor (F0). Although initially nonfunctional, a proteolytic cleavage event is required to form the F1 and F2 subunits, which are covalently linked by disulfide bonds. The new N-terminus of the F2 polypeptide contains a hydrophobic sequence, which ultimately inserts into the host cell membrane during the process of viral membrane fusion. At some point, either during transport or at the membrane surface, the F protein associates with itself to form a metastable trimer in a structure called the prefusion 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 a dramatic conformational change, extending the fusion peptide into the host cell membrane and then refolding back onto itself to form a six-helix bundle called the post-fusion conformation. The energy difference between the extended intermediate and the post-fusion conformation provides the energy required for membrane fusion.

[0136] The present disclosure provides methods, uses, and compositions comprising hMPV F protein antigen-binding molecules for treating hMPV infection in a subject. The present disclosure also provides methods, uses, and compositions comprising hMPV F protein antigen-binding molecules for treating hMPV.

[0137] The hMPV033 antigen (SEQ ID NO: 1) contains the ectodomain (AA 1-472) of the UT-A CL-28 mutant. The CL-28 mutant was constructed in the A strain sequence with a 6 amino acid Gly linker replacing the F1 / F2 cleavage site. 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 368H. In some embodiments, the mutant contains T127C, N153C, A185P, V231I, L219K, G294E, T365C, V463C, and H368N. The 127C-153C and 365C-463C amino acid bonds form intraprotomer disulfide bonds.

[0138] The hMPV F protein antigen-binding molecule may be a full-length immunoglobulin antibody or an antigen-binding fragment of an intact antibody, representative examples of which include Fab fragments, F(ab')2 fragments, Fd fragments consisting of the VH and CH1 domains, Fv fragments consisting of the VL and VH domains of a single antibody arm, and single-domain antibody (dAb) fragments consisting of the VH domain (Ward et al., 1989. Nature 341:544-546), as well as isolated CDRs. In some embodiments, the hMPV F protein antigen-binding molecule is a chimeric antibody, a humanized antibody, or a human antibody.

[0139] In some embodiments, the hMPV F protein antigen-binding molecule is a humanized antibody. Techniques for producing humanized monoclonal antibodies (mAbs) are well known in the art (see, e.g., 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 or murine monoclonal antibodies can be humanized by introducing murine CDRs from the heavy and light variable chains of a murine immunoglobulin into the corresponding variable domains of a human antibody. The murine framework regions (FRs) of a chimeric monoclonal antibody are also replaced with human FR sequences. Simply introducing murine CDRs into human FRs often reduces or even eliminates the affinity of the antibody, so 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, resulting in an antibody with good binding affinity for the epitope. See, for example, Tempest et al. (1991. Biotechnology 9:266-271) and Verhoeyen et al. (1988, supra). Generally, human FR amino acid residues that, unlike their murine counterparts, are located near or in contact with one or more CDR amino acid residues are candidates for replacement.

[0140] Polynucleotides Polynucleotides encoding the antigen-binding molecules of the present disclosure are provided.

[0141] In one aspect, the present disclosure provides an isolated polynucleotide encoding the antigen-binding molecule of the present disclosure.The isolated polynucleotide sequence can comprise RNA or DNA.As used herein, "isolated nucleic acid" refers to a nucleic acid that is removed from the usual surrounding polynucleotide sequence in genome or cDNA sequence.

[0142] 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 in which a polynucleotide coding region or gene is operably linked to any control sequence capable of effecting expression of the gene product. A "control sequence" operably linked to a polynucleotide sequence of the present disclosure is a polynucleotide sequence capable of effecting expression of the polynucleotide molecule. Control sequences need not be contiguous with the polynucleotide sequence, so long as they function to direct expression of the polynucleotide sequence. Thus, for example, non-translated but transcribed intervening sequences may be present between the promoter sequence and the polynucleotide sequence, and the promoter sequence would still be considered "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, plasmid and viral-based expression vectors. The control sequences used to drive expression of the nucleic acid sequences of the present disclosure in mammalian systems 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 several inducible promoters including, but not limited to, tetracycline, ecdysone, steroid responsive).

[0143] Pharmaceutical Composition Pharmaceutical compositions are provided that include an antigen-binding molecule of the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient. The carrier(s) are "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the composition and not harmful to the recipient (e.g., subject) thereof. Suitable carriers typically include saline or ethanol polyols such as glycerol or propylene glycol.

[0144] The antigen-binding molecule can be formulated in a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups) and are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, maleic acid, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0145] The compositions may be formulated for systemic administration, including intravenous, intramuscular, subcutaneous, or intraperitoneal administration, and conveniently comprise a sterile aqueous solution of the antigen-binding molecule, preferably isotonic with the recipient's blood. Such formulations are typically prepared by dissolving the solid active ingredient in water containing physiologically compatible substances, such as sodium chloride or glycine, to produce an aqueous solution, and adjusting the solution to a buffered pH compatible with physiological conditions, and rendering the solution sterile. These may be prepared in unit-dose or multi-dose containers, such as sealed ampoules or vials.

[0146] The composition may incorporate stabilizers such as polyethylene glycol, proteins, sugars (e.g., trehalose), amino acids, inorganic acids, and mixtures thereof. The stabilizers are used in aqueous solutions at appropriate concentrations and pHs. The pH of the aqueous solutions is adjusted to within the range of 5.0 to 9.0, preferably within the range of 6 to 8. Anti-adsorption agents may also be used when formulating the antigen-binding molecules. Other suitable excipients may typically include antioxidants such as ascorbic acid.

[0147] In certain embodiments, the compositions disclosed herein may be used as a medicament, e.g., for use in treating or preventing an infectious disease in a subject, e.g., a mammal, in need thereof.

[0148] In certain embodiments, the compositions disclosed herein can be used in the manufacture of a medicament for treating or preventing an infectious disease in a subject, e.g., a mammal, in need thereof.

[0149] Therapeutic applications Methods for treating or preventing hMPV infection in a subject in need thereof are provided, comprising administering to the subject an effective amount of an antigen-binding molecule according to the present disclosure.

[0150] In some embodiments, the methods of the present disclosure include therapeutic treatments and prophylactic or preventative measures, the purpose of which is to prevent or delay (alleviate) the targeted pathological condition or disorder. Those in need of treatment include those who already have the disorder, as well as those who are prone to having the disorder or those in need of preventing the disorder. A subject is considered to be successfully "treated" for an infection if, after administering an effective amount of an antigen-binding molecule according to the methods of the present disclosure, the subject shows an observable and / or measurable reduction or elimination of one or more of the following: a reduction in the number of infected cells or the disappearance of infected cells; a reduction in the proportion of total infected cells; some 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 assessing successful treatment and disease improvement can be easily measured by routine procedures familiar to physicians.

[0151] In some embodiments, the subject has or is at risk of developing an hMPV infection.

[0152] In some embodiments, the subject is a mammal, hi some embodiments, the subject is a human.

[0153] In some embodiments, the subject is susceptible to a viral infection (eg, hMPV).

[0154] In some embodiments, the subject is an elderly subject.

[0155] In some embodiments, the antigen binding molecules of the present disclosure are administered by intramuscular, intravenous, or subcutaneous injection.

[0156] Detection Method The antigen-binding molecules of the present disclosure can be used in a variety of detection methods, including those described herein and others known in the art, such as 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 radioimmunoprecipitation (RIA).

[0157] These methods can be performed in vivo, ex vivo, or in vitro. In particular, the step of contacting the antibody with the hMPV F protein or a cell expressing the hMPV F protein on its surface can be performed in vivo, ex vivo, or in vitro. The methods can be performed in cell-based or cell-free systems.

[0158] Potential hMPV F protein antigen-binding molecules can be evaluated in vivo, such as in animal models. In such in vivo models, the effects of the antigen-binding molecules can be evaluated in the circulatory system (e.g., blood) or heart, or in other organs, such as the lungs, liver, kidneys, or brain.

[0159] A method for detecting antibodies specific to an hMPV F protein is provided, the method comprising: a.) contacting a biological sample with a hMPV F protein; and b.) contacting an epitope-specific antigen-binding molecule with an hMPV F protein in accordance with the present disclosure.

[0160] In some embodiments, the hMPV F protein is coated onto a microwell plate prior to contacting with the biological sample or antigen-binding molecule.

[0161] In some embodiments, the half-maximal effective concentration (EC 50) is determined by calculating the inverse dilution of the biological sample at which binding of the antigen-binding molecule is inhibited by 50%.

[0162] In some embodiments, the biological sample is serum.

[0163] For example, a method is provided for detecting the titer of serum anti-hMPV F protein antibodies that compete with biotinylated anti-hMPV F protein antibodies for binding to the hMPV F antigen, the method comprising: a. coating an ELISA plate with hMPV F antigen to prepare an hMPV F antigen-coated ELISA plate; b. Adding serum to an ELISA plate coated with hMPV F antigen; c. adding the biotinylated anti-hMPV F protein antibody to an ELISA plate coated with hMPV F antigen in the presence of serum under conditions effective to allow the biotinylated anti-hMPV F protein antibody to bind to the ELISA plate coated with hMPV F antigen; d. detecting 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 and produces a color that is proportional to the amount of biotinylated anti-hMPV F protein antibody bound to the hMPV F antigen; e. Identifying the titer of serum anti-hMPV F protein antibody that effectively competes with biotinylated anti-hMPV F protein antibody by identifying the reciprocal dilution of serum at which binding of biotinylated anti-hMPV F protein antibody is inhibited by 50%.

[0164] The method contemplates 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 biotinylated 13E10 hMPV F protein epitope antibodies. In some embodiments, the method detects the titer of serum anti-hMPV F protein antibodies that compete with biotinylated 42C2 hMPV F protein epitope antibodies. In some embodiments, the method detects the titer of serum anti-hMPV F protein antibodies that compete with biotinylated 17D10 hMPV F protein epitope antibodies.

[0165] The antigen binding molecules of the present disclosure are contemplated for use in determining the identity of recombinantly produced hMPV F proteins.

[0166] The anti-hMPV F protein antibodies of the present disclosure that bind to the 13E10 epitope only bind to the hMPV F protein in the pre-fusion conformation. Therefore, it is contemplated that the 13E10 epitope anti-hMPV F protein antibodies can be used in the methods of the present invention to evaluate the stability of pre-fusion stabilized hMPV F proteins. In some embodiments, the anti-hMPV F protein antibodies that bind to the 13E10 epitope only bind to the hMPV F protein in the pre-fusion conformation and can therefore be used to determine the conformation of the hMPV F protein.

[0167] Anti-hMPV F protein antibodies of the present disclosure that bind to the 17D10 epitope bind to hMPV F protein in both pre-fusion and post-fusion conformations.

[0168] Anti-hMPV F protein antibodies of the present disclosure that bind to the 42C2 epitope bind only to hMPV F protein in the pre-fusion and post-fusion conformations.

[0169] This method contemplates a binding assay with a 13E10 epitope anti-hMPV F protein antibody and either a 42C2 epitope or a 17D10 epitope anti-hMPV F protein antibody to evaluate 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 antibody does not bind to the post-fusion conformation of the hMPV F protein.

[0170] For example, in a binding assay of a 13E10 epitope anti-hMPV F protein antibody with either a 42C2 epitope or a 17D10 epitope anti-hMPV F protein antibody, the rate of binding to the anti-hMPV F protein antibody can be used to assess the conformation of the hMPV F protein. Without wishing to be bound by theory, a decrease in binding of the 13E10 epitope anti-hMPV F protein antibody but no change in binding of the 42C2 epitope or 17D10 epitope anti-hMPV F protein antibody may indicate a transition from the pre-fusion to the post-fusion conformation of the hMPV F protein.

[0171] The present method contemplates an assay for assessing the conformation of an hMPV F protein vaccine antigen, which method comprises contacting an epitope-specific antigen-binding molecule of the present disclosure with the vaccine antigen. For example, a 13E10 epitope anti-hMPV F protein antibody and either a 42C2 epitope or a 17D10 epitope anti-hMPV F protein antibody can be used in a binding assay of the present disclosure to assess the conformation of the hMPV F protein antigen. In a further example, in producing a prefusion conformation hMPV F protein antigen, the 13E10 epitope anti-hMPV F protein antibody binds only to the hMPV F protein in the prefusion conformation, whereas the 17D10 epitope and 42C2 epitope anti-hMPV F protein antibodies bind to the hMPV F protein in both the prefusion and postfusion conformations. Without wishing to be bound by theory, the conformational selectivity of the epitope-specific antibodies of the present disclosure allows for assessment of the conformation of the hMPV vaccine antigen.

[0172] In some embodiments, the prefusion conformation confers stability to the hMPV protein compared to the postfusion conformation. Exemplary antibody sequences of the present disclosure are shown in Table 5. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Example]

[0173] Example 1: Generation of hMPV F protein antibodies Neutralizing monoclonal antibodies against the hMPV F protein, specifically hMPV033, were generated and characterized from mice immunized with an hMPV F protein fragment (hMPV008) fused to I53-50A (as described in WO2019 / 169120 A1), and screened for antibodies that bound to hMPV033 but not to I53-50A, the RSV F protein, or the hexahistine (his6) tag.

[0174] Antibodies that specifically bind 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, 13E10 hIgG1, and 42C2 hIgG1, were humanized, expressed, purified, and the epitope of each antibody was mapped using CovalX™. The three exemplary antibodies have the following characteristics: 1. Binds to hMPV F protein but not to RSV F protein 2. Neutralizing antibodies 3. Nonlinear conformational epitope binding 4. Antibodies 17D10 and 42C2 bind to both the pre-fusion and post-fusion conformations of the hMPV F protein, whereas the 13E10 antibody binds only to the pre-fusion conformation of the hMPV F protein.

[0175] The hMPV008 antigen contains the ectodomain (AA 1-472) of the NIH V4-B variant fused to the virus-like particle I53-50A using a 16-residue GS linker. V4-B is a single-chain construct constructed with the B strain sequence and replacing the F1 / F2 cleavage site with a six-amino acid Gly linker. This variant also contains six Cys mutations (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 intraprotomer bond, while the 182-188 bond is an interprotomer bond.

[0176] The hMPV033 antigen contains the ectodomain (AA 1-472) of the UT-A CL-28 mutant fused to I53-50A using a 16-residue GS linker. The CL-28 mutant was constructed in the A strain sequence with a 6-amino acid Gly linker replacing the F1 / F2 cleavage site. 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 intraprotomer disulfide bonds. Generation of hMPV008 and hMPV033 antigens

[0177] Count the Expi293 cells in logarithmic growth phase and distribute 2.5 x 10 cells in 220 ml of each of four 1 L flasks (880 ml total). 6 The cells were then seeded at 3x10 cells / ml. The cells were incubated overnight at 36°C with shaking (120 rpm). The next day, the cells were counted and plated at 3x10 cells / ml in 235 ml per 1 L flask. 6 The cells were transiently transfected as follows: 1000 μg of plasmid DNA was diluted to a final volume of 35 ml with OptiMEM and mixed gently. 2.5 ml of Transporter 5 Transfection Reagent was diluted to a final volume of 35 ml with OptiMEM and mixed gently. The diluted Transporter 5 was added to the diluted DNA, mixed, and incubated at room temperature for 10 minutes, then added dropwise in 17.5 ml volumes to each 1 L flask while gently swirling the flask. The cells were returned to the incubator and shaken for 4 days.

[0178] purification The construct was purified directly from the conditioned Expi293F expression medium by immobilized metal affinity chromatography (IMAC). NDIGO Ni-Agarose was washed with 5 column volumes (CV) of water, followed by 5 CV of equilibration buffer, and then resuspended in 1–2 CV of equilibration buffer. The supernatant was centrifuged at 4,000 x g and then clarified by filtration using a 0.2 or 0.45 μm vacuum filtration unit. The resin suspension was added to the supernatant so 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 hours, and 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.

[0179] ELISA and Octet assays - second screening A capture ELISA was used to screen the 117 clones for the second round against the hMPV F protein. High-binding plates were coated overnight at 4°C with 100 ng / well of hMPV033 in 100 mM sodium carbonate-bicarbonate buffer, pH 9.6. The next day, plates were washed three times with wash buffer (1X PBS, 0.05% Tween®-20, pH 7.4) and blocked with 150 μL / well of block buffer (wash buffer containing 1% BSA) for 1 hour at room temperature. Supernatant samples were thawed at room temperature and then mixed by inversion several times. Each sample was diluted 10-fold using block buffer as the diluent, followed by further final dilutions of 50-fold and 500-fold. Samples were loaded at 100 μL / well and incubated covered at room temperature for 1 hour without agitation. The samples were aspirated, the plate washed five times with wash buffer, and then 100 μL / well of goat anti-mouse HRP-conjugated antibody diluted 1:5,000 in block buffer was added. The plate was covered and incubated at room temperature for 1 hour without agitation. The conjugate was aspirated, the plate washed five times with wash buffer, and then 100 μL / well of TMB was added and incubated in the dark at room temperature for approximately 5 minutes. The reaction progress was stopped with 100 μL / well of 0.6 N sulfuric acid, and absorbance at 450 nm was measured within 10 minutes. mAb binding was ranked using the endpoint absorbance values ​​of 50x and 500x dilutions. The top 92 highest-binding samples were then screened for binding by biolayer interferometry (BLI). BLI with Octet Red 96 was performed as follows: the anti-pentaHis biosensor was immersed in assay buffer for 30 seconds to establish a baseline. The sensor was loaded with 10 μg / mL hMPV033 in assay buffer for 30 seconds, followed by an additional baseline step. The sensor was then immersed in each supernatant for 60 seconds to observe antibody binding to hMPV033. The shift at 120 seconds was used to rank the binding (data not shown).

[0180] immunization Five SJL female mice (4-6 weeks old) were immunized with the hMPV008-I53-50A immunogen in the "prefusion" conformation. The immunogen contained a C-terminal His6 tag and was produced in a stable miCHO K1 cell line. To avoid detection of antibodies against I53-50A, serum samples from immunized mice were collected and tested for titer against hMPV033 (as described in WO2019 / 169120 A1). A total of 42 supernatants evaluated by ELISA yielded positive signals for hMPV033 and negative signals for I53-50A (data not shown).

[0181] Neutralization assay of first screening material Forty-two supernatants were evaluated in the hMPV / A neutralization assay.

[0182] All supernatants were initially diluted 1:8, followed by two-fold serial dilutions. Neutralizing antibody titers were defined as the final dilution at which viral cytopathic effect (CPE) was reduced by 50%. The final serum dilution is the final dilution of serum, not taking into account the additional four-fold dilution that occurs when diluted serum (50 μl) is mixed with virus (50 μl) and then cells (100 μl) are added.

[0183] All monoclonal antibodies were prepared at a concentration of 200 μg / ml and then diluted 1:8 initially, followed by serial 2-fold dilutions. To calculate the 50% inhibitory concentration (IC50), the final titer was converted to a dilution factor. The dilution factor is used to divide the original monoclonal antibody concentration to determine the inhibitor IC50. A further 4-fold dilution is used to obtain the final serum dilution for IC50 calculation. Table 6 shows the results for neutralizing antibody titers (no 4-fold dilution adjustment), original IC50s (no 4-fold dilution adjustment), and final IC50s (with 4-fold dilution adjustment).

[0184] For example, monoclonal antibody 37H4 mAB 0.2 mg / mL exhibits a neutralizing antibody titer of 9 log2, which corresponds to a dilution factor of 512.200 micrograms divided by 512 = 390.6 ng / mL. A further 4-fold reduction to account for virus dilution and cell volume results in a final concentration of 97.7 ng / mL.

[0185] The background signal for this assay was 2.0 log², and results of 5.0 log² or greater were considered positive. Seven antibodies (3G3, 19E9, 8H11, 17D10, 17E10, 13E10, and 18D2) demonstrated neutralizing activity greater than 5.0 log² (Table 6). One of the seven clones, 18D2, exhibited cross-reactivity and was not pursued. The remaining six antibodies were scaled up for antibody production and purification. hMPV / A neutralization assays were performed using six purified antibodies (3G3, 19E9, 8H11, 17D10, 17E10, and 13E10). Data for all six antibodies demonstrated neutralizing activity (background 2.0 log²) with titers ranging from 8.0 to 12.5 log² (data not shown).

[0186] As described above, 73 additional supernatants that bound to hMPV033 but not I53-50A were identified by BLI. The 73 supernatants were evaluated in hMPV / A neutralization assays, and 18 Abs (30G3, 42C2, 30G4, 51D9, 40A9, 37H4, 35F12, 37E1, 27F1, 50B10, 32H7, 39A4, 30D7, 41E5, 56B4, 22B2, 39D8, and 32G3) demonstrated neutralizing activity greater 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. hMPV / A neutralization assays were performed using these four purified antibodies. Data for all four antibodies demonstrated neutralizing activity (background 2.0 log2) at titers of 6.5-10 log2 (Table 6). Following these results, recombinant versions of antibody candidates 19E9, 17D10, 13E10, and 42C2 were generated with human IgG1 Fc. [Table 6]

[0187] Neutralizing activity, specificity, bin, and K of humanized Abs D Confirmation Recombinant humanized antibodies (17D10 hIgG1, 13E10 hIgG1, and 42C2 hIgG1) were tested to assess hMPV F protein specificity, neutralization, binning, and K D The estimated K was confirmed. All three humanized antibodies were confirmed to be potent neutralizing antibodies (Table 6). Binding to the hMPV F protein, but not the RSV F protein, was confirmed by Octet for all three humanized antibodies (Figures 3A-3C). To estimate the affinity of each humanized antibody for hMPV033, I53-50A-hMPV033 was immobilized on an anti-pentaHis 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).

[0188] Epitope mapping Three monoclonal antibodies (17D10 hIgG1, 13E10 hIgG1, and 42C2 hIgG1) were used for epitope mapping. CovalX™ identified the epitope recognized by each antibody by cross-linking of the antigen-antibody complex, multienzyme proteolysis, and subsequent nLC-Orbitrap MS-MS analysis, demonstrating that each antibody recognizes a nonlinear epitope (Figures 2A-2C). A graphical representation of the epitopes for neutralizing antibodies against the hMPV F protein, as well as other antigenic sites, is shown in Figure 1.

[0189] 2D class averaging of nsEM images of I53-50A-hMPV033 showed that the F protein was in the prefusion conformation and in a mixture of compact and open trimers.

[0190] Characterization of molecular interfaces To determine the epitopes of the hMPV033 / 17D10 hlgG1, hMPV033 / 13E10 hlgG1, and hMPV033 / 42C2 hlgG1 complexes at high resolution, each protein complex was incubated with a deuterated cross-linker and subjected to multiple enzymatic cleavage. After enrichment of the cross-linked peptides, the samples were analyzed by high-resolution mass spectrometry (nLC-Q-Exactive MS), and the generated data were analyzed using XQuest™ and Stavrox™ software.

[0191] For this analysis, nano-liquid chromatography (nLC) coupled with Q-Exactive MS analysis was used with the following parameters: Ultimate 3000-RSLC -A 98 / 02 / 0.1 H2O / ACN / HCOOH v / v / v -B 20 / 80 / 0.1 H2O / ACN / HCOOH v / v / v - Gradient from 4 to 55% B in 33 min Injection volume 1 μl -Pre-column 300μm ID x 5mm C18 PepMap(TM) -Pre-column flow rate 50 μl / min -Column 75μm ID x 15cm C18 PepMapRSLC -Column flow rate 300nl / min Mass spectrometry: Q-Exactive MS analysis Q-Exactive MS analysis was performed using the following parameters: -Scan type: Full MS -Scan range: 350~1600m / z -Resolution: 70,000 -Microscan: 1 -Maximum injection time: 100ms -Spray voltage: 1.7 kV -Capillary voltage: 275℃ -S Lens RF Level: 55.0 -AGC target: 3e6 - Default charging state: 2 dd-MS 2 -Resolution: 17,500 -AGC target: 1e5 -Ion isolation window: 4 m / z units -Maximum injection time: 50ms -Normalized collision energy: 30% Loop count: 5 -Dynamic Exclusion: On - Dynamic Exclusion Parameter: 30.0 seconds -Min AGC target: 8e3 -Intensity Threshold: 1.6e5

[0192] Reductive alkylation 20 μL of the hMPV033 / antibody mixture was mixed with 2 μL of DSS d0 / d12 (2 mg / mL; DMF) and incubated for 180 minutes at room temperature. After incubation, the reaction was stopped by adding 1 μL of ammonium bicarbonate (final concentration 20 mM) within a 1-hour incubation period at room temperature. The solution was then dried in a speed vac and subsequently treated with a suspension of HO and 8 M urea (20 μL). After mixing, 2 μL of DTT (500 mM) was added to the solution. The mixture was then incubated for 1 hour at 37°C. After incubation, 2 μL of iodoacetamide (1 M) was added and incubated for 1 hour at room temperature in the dark. After incubation, 80 μL of proteolysis buffer was added. The trypsin buffer contains 50 mM ambic (pH 8.5), 5% acetonitrile; the chymotrypsin buffer contains 100 mM Tris-HCl, 10 mM CaCl2 (pH 7.8); the ASP-N buffer contains 50 mM phosphate buffer (pH 7.8); the elastase buffer contains 50 mM Tris-HCl, pH 8.0; and the thermolysin buffer contains 50 mM Tris-HCl, 0.5 mM CaCl2 (pH 9.0).

[0193] Trypsin proteolysis 100 μl of reduced / alkylated hMPV033 / 17D10_hlgG1, hMPV033 / 13E10_hlgG1, or hMPV033 / 42C2_hlgG1 mixture was mixed with 1.24 μl of trypsin (Promega) at a ratio of 1 / 100. The proteolysis mixture was incubated overnight at 37°C.

[0194] Chymotrypsin proteolysis 100 μl of reduced / alkylated hMPV033 / 17D10_hlgG1, hMPV033 / 13E10_hlgG1, or hMPV033 / 42C2_hlgG1 mixture was mixed with 0.62 μl of chymotrypsin (Promega) at a ratio of 1 / 200. The proteolysis mixture was incubated overnight at 25°C.

[0195] Protein degradation by ASP-N 100 μl of reduced / alkylated hMPV033 / 17D10_hlgG1, hMPV033 / 13E10_hlgG1, or hMPV033 / 42C2_hlgG1 mixture was mixed with 0.62 μl of ASP-N (Promega) at a ratio of 1 / 200. The proteolysis mixture was incubated overnight at 37°C.

[0196] Elastase proteolysis 100 μl of reduced / alkylated hMPV033 / 17D10_hlgG1, hMPV033 / 13E10_hlgG1, or hMPV033 / 42C2_hlgG1 mixture was mixed with 1.24 μl of elastase (Promega) at a ratio of 1 / 100. The proteolysis mixture was incubated overnight at 37°C.

[0197] Thermolysin proteolysis 100 μl of reduced / alkylated hMPV033 / 17D10_hlgG1, hMPV033 / 13E10_hlgG1, or hMPV033 / 42C2_hlgG1 mixture was mixed with 2.48 μl of thermolysin (Promega) at a ratio of 1:50. The proteolysis mixture was incubated at 70°C overnight. After digestion, formic acid was added to the solution to a final concentration of 1%.

[0198] Cross-linked peptides were analyzed using Xquest™ version 2.0 and Stavrox™ 3.6 software.

[0199] result hMPV033 / 17D10_hlgG1 After proteolysis of the hMPV033 / 17D10_hlgG1 protein complex with deuterated d0d12 by trypsin, chymotrypsin, ASP-N, elastase, and thermolysin, nLC-Q-Exactive MS / MS analysis detected 12 cross-linked peptides between hMPV033 and 17D10_hlgG1. Using chemical cross-linking, high-mass MALDI mass spectrometry, and nLC-Q-Exactive mass spectrometry, we characterized the molecular interface between hMPV033 and 17D10_hlgG1 (Figures 4A-4J). The analysis indicated that the interaction involves the following amino acids on hMPV033: 287, 293, 296, 364, 376, 417, and 419 of the hMPV033 amino acid sequence (SEQ ID NO: 1).

[0200] hMPV033 / 13E10_hlgG1 After proteolysis of the hMPV033 / 13E10_hlgG1 protein complex with deuterated d0d12 by trypsin, chymotrypsin, ASP-N, elastase, and thermolysin, nLC-Q-Exactive MS / MS analysis detected 15 cross-linked peptides between hMPV033 and 17D10_hlgG1. Using chemical cross-linking, high-mass MALDI mass spectrometry, and nLC-Q-Exactive mass spectrometry, we characterized the molecular interface between hMPV033 and 13E10_hlgG1 (Figures 5A-5J). The analysis indicated that the interaction involves the following amino acids on hMPV033: 144, 160, 163, 188, 194, and 199 of the hMPV033 amino acid sequence (SEQ ID NO: 1).

[0201] hMPV033 / 42C2_hlgG1 After proteolysis of the hMPV033 / 42C2_hlgG1 protein complex with deuterated d0d12 using trypsin, chymotrypsin, ASP-N, elastase, and thermolysin, nLC-Q-Exactive MS / MS analysis detected 15 cross-linked peptides between hMPV033 and 42C2_hlgG1. Using chemical cross-linking, high-mass MALDI mass spectrometry, and nLC-Q-Exactive mass spectrometry, we characterized the molecular interface between hMPV033 and 42C2_hlgG1 (Figures 6A-6J). The analysis indicated that the interaction involves 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).

[0202] Example 2: hMPV033 competitive antibody ELISA A competitive antibody assay was developed to determine the titer of serum antibodies that could compete with mAb 17D10 hIgG1 for binding to HMPV F protein. [Table 7]

[0203] Assay Method Microtiter plates were coated with hMPV F antigen (dn5B-hMPV033). Serum samples were serially diluted 5-fold, followed by the addition of an equal volume of a fixed concentration of biotinylated 17D10 hIgG1 antibody (mAb-Bio). The mixture was added to the microtiter plate and incubated. As controls, wells containing "17D10 hIgG1 mAb-Bio only" (maximum signal) and "sample dilution buffer only" (background signal) were analyzed. After incubation, unbound material was washed from the wells, and HRP-labeled streptavidin was added to all wells. The wells were then washed again to remove unbound HRP-labeled streptavidin. Next, the addition of TMB substrate induced color development proportional to the amount of 17D10 hIgG1 mAb-Bio bound to the hMPV F antigen. The color development was quenched, and the optical density (OD 450 nm and OD 620 nm) was measured. The binding titer of the 17D10 hIgG1 mAb-Bio competitor antibody is expressed as the reciprocal dilution at which 50% of the binding of 17D10 hIgG1 mAb-Bio is inhibited.

[0204] Assay optimization Several hMPV F antigen coating concentrations are tested in combination with different concentrations of 17D10 hIgG1 mAb-Bio and different dilutions of HRP-labeled streptavidin. Maximum binding of 17D10 hIgG1 mAb-Bio to the hMPV F protein results in an OD of approximately 2.0, with a low background OD.

[0205] Example 3: Analysis of antibody binding to pre-fusion and post-fusion hMPV F proteins The CompA-hMPV033 (hMPV F protein) fusion protein of the present disclosure, post-fusion hMPV F protein, and mouse antibodies 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 at 200 μL per well of a black 96-well microplate. Protein G biosensors were hydrated and then immersed in BLI assay buffer and baselined for 60 seconds. The biosensors were then immersed in antibody wells for 120 seconds to immobilize the antibodies present there without reaching saturation, followed by an additional 60-second baseline step. After allowing the immobilized antibodies to associate with the antigen for 120 seconds, the biosensors were again immersed in assay buffer for 120 seconds to observe dissociation. MF1, MF2, and MF3 are post-fusion hMPV F protein-specific antibodies, and MF10 is a pre-fusion hMPV F protein-specific antibody used as a control. Binding of control antibodies to pre-fusion and post-fusion hMPV F proteins is shown in Figures 7A and 7B. As shown in Figures 7C and 7D, binding of the 17D10, 42C2, and 13E10 antibodies to both hMPV F protein conformations was observed, with the 17D10 and 42C2 antibodies binding to both pre-fusion and post-fusion hMPV F proteins and the 13E10 antibody binding only to the pre-fusion hMPV F protein. * * * *

[0206] While the invention has been described in relation to specific suggested embodiments thereof, it is to be understood that further modifications are possible, and this application is intended to cover any variations, uses, or applications of the invention which generally follow from the principles of the invention, including such departures from the present disclosure as come within known or customary practice in the art to which this invention pertains and which may apply to the essential features described above and fall within the scope of the appended claims.

Claims

1. An epitope-specific antigen-binding molecule specific for hMPV fusion (F) protein, wherein the antigen-binding molecule has the amino acid sequence of hMPV F protein set forth in SEQ ID NO:

1. i.) residues 287, 293, 296, 364, 376, 417, and 419; ii.) residues 144, 160, 163, 188, 194, and 199; or iii.) Residues 44, 45, 49, 150, 156, 160, 229, 232, and 236 The epitope-specific antigen-binding molecule comprises a variable domain that contacts the hMPV F protein at

2. An antigen binding molecule specific for hMPV F protein, 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 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); 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 has the amino acid sequence of hMPV F protein set forth in SEQ ID NO:

1. i.) residues 287, 293, 296, 364, 376, 417, and 419; ii.) residues 144, 160, 163, 188, 194, and 199; or iii.) Residues 44, 45, 49, 150, 156, 160, 229, 232, and 236 The antigen-binding molecule of claim 2, comprising a variable domain that contacts the hMPV F protein at

4. The antigen-binding molecule of claim 1, wherein the antigen-binding molecule comprises a variable domain that contacts the hMPV F protein at any one, any two, any three, any four, any five, any six, or any seven of residues 287, 293, 296, 364, 376, 417, and 419 of the hMPV F protein amino acid sequence set forth in SEQ ID NO:

1.

5. a heavy chain and a light chain, the heavy chain comprising 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); The antigen-binding molecule of claim 4, 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 of any one of claims 1 to 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 of any one of claims 1 to 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 (SEQ ID NO: 6).

8. 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; The antigen-binding molecule of any one of claims 1 to 7, 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. 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 The antigen-binding molecule of any one of claims 1 to 8, comprising 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 of claim 1, wherein the antigen-binding molecule comprises a variable domain that contacts 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 set forth in SEQ ID NO:

1.

11. a heavy chain and a light chain, the heavy chain comprising 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); The antigen-binding molecule of claim 10, wherein the light chain comprises L-CDR1, L-CDR2, and L-CDR3, wherein L-CDR1 comprises the sequence SASSISSNYLH (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 of any one of claims 10 to 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 of any one of claims 10 to 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 (SEQ ID NO: 14).

14. 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; The antigen-binding molecule of any one of claims 10 to 13, 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. 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 The antigen-binding molecule of any one of claims 10 to 14, comprising 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 of claim 1, wherein the antigen-binding molecule comprises a variable domain that contacts 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 set forth in SEQ ID NO:

1.

17. 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); The antigen-binding molecule of claim 16, 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).

18. The antigen-binding molecule of any one of claims 16 to 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 of any one of claims 16 to 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 (SEQ ID NO: 22).

20. 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 antigen-binding molecule of any one of claims 16 to 19, 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. 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 The antigen-binding molecule of any one of claims 16 to 20, comprising 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 of any one of claims 1 to 21, wherein the antigen-binding molecule is an immunoglobulin molecule.

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

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

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

26. The antigen-binding molecule of any one of claims 1 to 25, wherein the antigen-binding molecule specifically binds to hMPV F protein in a prefusion conformation.

27. The antigen-binding molecule of any one of claims 1 to 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 of any one of claims 1 to 27.

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

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

31. 31. The method of claim 30, wherein the hMPV F protein is coated onto a microwell plate before contacting with the biological sample or antigen-binding molecule.

32. The half-maximal effective concentration (EC) of the anti-hMPV F protein antibody in the biological sample was determined by determining the inverse dilution of the biological sample at which 50% of the binding of the antigen-binding molecule was inhibited. ５０ 32. The method of claim 31 , comprising determining:

33. The antigen-binding molecule has the amino acid sequence of hMPV F protein set forth in SEQ ID NO:

1. i.) residues 287, 293, 296, 364, 376, 417, and 419; ii.) residues 144, 160, 163, 188, 194, and 199; or iii.) Residues 44, 45, 49, 150, 156, 160, 229, 232, and 236 33. The method of any one of claims 30 to 32, wherein the variable domain contacts the hMPV F protein at

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

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

36. 35. The method of any one of claims 30 to 34, wherein the method detects 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 of any one of claims 1 to 27 or the pharmaceutical composition or vaccine of claim 29.

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

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

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

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

42. The method of any one of claims 37 to 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, comprising detecting the ratio of pre-fusion conformation hMPV F protein antigen binding molecules to post-fusion conformation hMPV F protein antigen binding molecules produced after immunization with the hMPV F protein vaccine of claim 29.

44. A method for measuring the stability of an antigen composition, comprising detecting the ratio of pre-fusion conformation hMPV F protein antigen binding molecules to post-fusion conformation hMPV F protein antigen binding molecules bound to the antigen composition.

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