Hmpv antibodies and uses thereof

Abstract

Antibodies and antigen-binding fragments that specifically bind to and neutralize human metapneumovirus (hMPV) F protein are disclosed. Nucleic acids, vectors and host cells encoding these antibodies are also provided. The disclosed antibodies, antigen binding fragments, nucleic acids, and vectors can be used, for example, to inhibit or detect hMPV infection.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This article claims the benefit of U.S. Provisional Application No. 63 / 317,384, filed on March 7, 2022, which is incorporated herein by reference.

[0003] Government Support Statement

[0004] This invention was made with government support under Grant Nos. R01AI143865 and 1K01 OD026569 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention. Technical Field

[0005] The present invention relates to the field of human metapneumovirus, and in particular to monoclonal antibodies and antigen-binding fragments thereof that specifically bind to human metapneumovirus F protein.

[0006] References to Electronic Sequence Listings

[0007] The contents of the electronic Sequence Listing (8618-108103-02SEQUENCE LISTING.xml; size: 122,880 bytes; and creation date: February 20, 2023) are incorporated herein by reference in its entirety. Background Art

[0008] Human metapneumovirus (hMPV) is a major cause of respiratory disease in children and the elderly (Panda et al., Int. J. Infect. Dis. 25, 45-52 (2014); Falsey et al., J. Infect. Dis. 187, 785-790 (2003); van den Hoogen et al., J. Infect. Dis. 188, 1571-1577 (2003); Madhi et al., Clin. Infect. Dis. 37, 1705-1710 (2003); Haas et al., Viruses 5, 87-110 (2013)). Initially identified in 2001 from samples collected from children with respiratory tract infections in the Netherlands (van den Hoogen et al., Nat. Med. 7, 719-724 (2001)), the clinical features of hMPV are similar to those of respiratory syncytial virus (RSV) and include middle and upper respiratory tract infections that may require hospitalization (Akhras et al., Infect. Dis. Rep. Dis. Rep. 2, e12 (2010)). Severe disease may occur in immunocompromised patients, such as those undergoing lung transplantation (Larcher et al., J. Hear. Lung Transpl. 24, 1891-1901 (2005)), hematopoietic stem cell transplantation (Cane et al., Bone Marrow Transplant. 31, 309-310 (2003); Englund et al., Ann. Intern. Med. 144, 344-349 (2013); Dokos et al., Transpl. Infect. Dis. 15, 97-101 (2013); Shah et al., Cancer Express. 15, 97-101 (2013); hMPV infection is a rare disease that occurs in infants with a high risk of developing hMPV. In some cases, hMPV infection is caused by a combination of factors, such as HIV (Klein et al., Journal of Infectious Diseases 201, 297-301 (2010)) and COPD (Kan-o et al., Journal of Infectious Diseases 215, 1536-1545 (2018)). In contrast to RSV, there is no approved vaccine or specific treatment for hMPV infection, for which palivizumab (Group and Im, Pediatrics 102, 531-537 (1998)) has been used for many years in specific high-risk infant groups.

[0009] Serological studies have shown that almost all children are seropositive for hMPV at the age of five (Edwards et al., N. Engl. J. Med. 368, 633-643 (2013)). hMPV has three surface glycoproteins - small hydrophobic (SH), attachment (G) and fusion (F) proteins. Of these proteins, the hMPV F protein is the only target for neutralizing antibodies (Ulbrandt et al., J. Gen. Virol. 89, 3113-3118 (2008)), which is different from RSV, in which both RSV G and RSV F proteins can elicit neutralizing antibodies (Tripp et al., J. Virol. 92, 1-8 (2017)). There is currently no licensed vaccine to prevent hMPV, but several candidates have been examined in animal models, including live attenuated viruses, recombinant viruses, vector vaccines, and recombinant surface proteins (Shafagati and Williams, F1000 Res. 7, 135 (2018)). A limited number of vaccine candidates have entered clinical trials, including a live attenuated hMPV vaccine (NCT01255410), and most recently, an mRNA-based vaccine in combination with parainfluenza virus 3 (NCT03392389, NCT04144348). Similar to vaccine enhancement of disease observed with formalin-inactivated RSV (Killikelly et al., Sci. Rep. 6, 34108 (2016); Kim et al., Am. J. Epidemiol. 89, 422-434 (1969); Kapikian et al., Am. J. Epidemiol. 89, 405-421 (1969)), vaccines using formalin-inactivated hMPV resulted in disease enhancement following viral infection in mice, cotton rats, and macaques (Hamelin et al., J. Gen. Virol. 88, 3391-3400 (2007); Yim et al., Vaccine 25, 5034-5040 (2007)). Therefore, there remains a need for other therapeutic agents targeting hMPV. Summary of the invention

[0010] Disclosed is an isolated monoclonal antibody or antigen-binding fragment thereof, wherein the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable (V H ) region and the light chain variable region (V L ), the V H and the V L Including the V H and the V LHeavy chain complementarity determining regions (HCDR) 1, HCDR2 and HCDR3, and light chain complementarity determining regions (LCDR) 1, LCDR2 and LCDR3:

[0011] a) SEQ ID NO: 41 and 55, respectively;

[0012] b) SEQ ID NOs: 9 and 13, respectively;

[0013] c) SEQ ID NOs: 17 and 21, respectively;

[0014] d) SEQ ID NOs: 25 and 29, respectively;

[0015] e) SEQ ID NOs: 33 and 37, respectively;

[0016] f) SEQ ID NOs: 1 and 5, respectively;

[0017] g) SEQ ID NOs: 49 and 53, respectively;

[0018] h) SEQ ID NOs: 57 and 61, respectively;

[0019] i) SEQ ID NOs: 65 and 69, respectively;

[0020] j) SEQ ID NOs: 73 and 77, respectively;

[0021] k) SEQ ID NOs: 81 and 85, respectively;

[0022] 1) are SEQ ID NOs: 89 and 93, respectively;

[0023] m) are SEQ ID NOs: 97 and 101, respectively;

[0024] n) are SEQ ID NOs: 105 and 109, respectively;

[0025] o) are SEQ ID NOs: 113 and 117, respectively;

[0026] p) are SEQ ID NOs: 121 and 125, respectively;

[0027] q) SEQ ID NO: 129 and 133, respectively; or

[0028] r) are SEQ ID NOs: 137 and 141, respectively;

[0029] Wherein the monoclonal antibody specifically binds to human metapneumovirus (hMPV) F protein and neutralizes hMPV. In specific non-limiting examples, CDRs can be identified using the Kabat, Chothia or IMGT numbering schemes.

[0030] In some embodiments, an isolated monoclonal antibody or antigen-binding fragment thereof is disclosed, wherein the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 comprise an amino acid sequence as shown below:

[0031] a) are SEQ ID NOs: 42, 43, 44, 46, 47 and 48, respectively;

[0032] b) SEQ ID NOs: 10, 11, 12, 14, 15 and 16, respectively;

[0033] c) SEQ ID NOs: 18, 19, 20, 22, 23 and 24, respectively;

[0034] d) are SEQ ID NOs: 26, 27, 28, 30, 31 and 32, respectively;

[0035] e) SEQ ID NOs: 34, 35, 36, 38, 39 and 40, respectively;

[0036] f) are SEQ ID NOs: 2, 3, 4, 6, 7 and 8, respectively;

[0037] g) SEQ ID NOs: 50, 51, 52, 54, 55 and 56, respectively;

[0038] h) are SEQ ID NOs: 58, 59, 60, 62, 63 and 64, respectively;

[0039] i) are SEQ ID NOs: 66, 67, 68, 70, 71 and 72, respectively;

[0040] j) are SEQ ID NOs: 74, 75, 76, 78, 79 and 80, respectively;

[0041] k) are SEQ ID NOs: 82, 83, 84, 86, 87 and 88, respectively;

[0042] 1) are SEQ ID NOs: 90, 91, 92, 94, 95 and 96, respectively;

[0043] m) are SEQ ID NOs: 98, 99, 100, 102, 103 and 104, respectively;

[0044] n) are SEQ ID NOs: 106, 107, 108, 110, 111 and 112, respectively;

[0045] o) are SEQ ID NOs: 114, 115, 116, 118, 119 and 120, respectively;

[0046] p) are SEQ ID NOs: 122, 123, 124, 126, 127 and 128, respectively;

[0047] q) are SEQ ID NOs: 130, 131, 132, 134, 135 and 136, respectively; or

[0048] r) are SEQ ID NOs: 138, 139, 140, 142, 143 and 144, respectively.

[0049] In some embodiments, the antibody or antigen-binding fragment is conjugated to an effector molecule or a detectable marker.

[0050] Also disclosed are bispecific antibodies comprising the antibodies or antigen-binding fragments.

[0051] In other embodiments, the nucleic acid and vector encoding the antibody, antigen-binding fragment, or V of the antibody or antigen-binding fragment are H or V L .

[0052] In more embodiments, a pharmaceutical composition for inhibiting hMPV infection is disclosed, wherein the pharmaceutical composition comprises an effective amount of the antibody, the antigen-binding fragment, the nucleic acid molecule or the vector and a pharmaceutically acceptable carrier.

[0053] In additional embodiments, methods of producing antibodies or antigen-binding fragments that specifically bind to hMPV F protein are disclosed.

[0054] In some embodiments, methods for detecting the presence of hMPV in a biological sample from a human subject are also disclosed.

[0055] In another embodiment, a method for inhibiting hMPV infection in a subject is disclosed, the method comprising administering to the subject an effective amount of the antibody, the antigen-binding fragment, the nucleic acid molecule, the vector or the pharmaceutical composition, wherein the subject has hMPV infection or is at risk of hMPV infection.

[0056] The foregoing and other features and advantages of the present invention will become more apparent from the following detailed description of several embodiments with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figures 1A-1C : Sequence determinants of isolated mAbs. (A) The usage of heavy chain, kappa chain and lambda chain genes is shown as the ratio of all corresponding genes from the isolated mAb panel. (B) The amino acid length of the heavy chain and light chain junction is shown. (C) The percent identity of the V gene to the predicted germline sequence is shown.

[0058] Figures 2A-2C : Epitope mapping of hMPV F specific mAbs. (A) Epitope binning of mAbs bound to hMPV B2 F protein. Data indicates the binding percentage of secondary antibodies in the presence of primary antibodies compared to secondary antibodies alone. Cells are colored by gradient according to the legend shown on the right. (B) Epitopes of control mAbs 101F (site IV), 196 and DS7 (DS7 epitope), MPE8 and MPV364 (site III) and MPV458 (66-87 epitope) are used as second mAbs and are marked according to the colors in (A). The control mAbs are also selected to be used as the first mAb as a positive blocking control. (C) mAb binding sites of MPE8 and MPV364 (site III), 101F (site IV), DS7 and MPV458 (66-87 epitope) are shown on the surface of hMPV F before monomer fusion. Estimated epitopes from the epitope binning of each mAb are shown. mAbs with asterisks were previously found.

[0059] Figure 3A-3B : Flow cytometry was used to assess the percentage of phagocytosis of hMPV F-coated beads by THP-1 cells in the presence of each mAb. In addition to the phagocytic score (B), the relative increase in the percentage of phagocytic cells for each mAb relative to the no mAb control (A) is also shown. The bars represent the mean of three replicates, and the error bars are standard deviations.

[0060] Figures 4A-4C : Protective efficacy of MPV467 against hMPV replication in vivo. (A) BALB / c mice were treated intraperitoneally with 10 mg / kg of mAb MPV467 24 hours before (prevention study) or three days after (treatment study) intranasal hMPV infection. Virus titers in lung homogenates of BALB / c mice in each treatment group (n=10 mice / group, 5 males, 5 females) in the prevention study (B) and the treatment study (C) were determined by plaque assay. ns=not significant, **P=0.0016, ***P=0.0003-0.0001, ****P<0.0001. The limit of detection (LOD) is indicated by a dotted line. ND=not detected.

[0061] Figures 5A-5C:MPV467 binds to hMPV F before fusion at sites II and V. (A) (left) Side view of the hMPV F-MPV467 Fab complex cryo-EM map shown at two different contour levels. The global map is shown as a white transparent map. The particle-subtracted depth enhancement map (DeepEMhanced map) is opaque, in which a single promoter (right) particle-subtracted depth enhancement map is identified. (B) The hMPV F trimer and the single promoter of the MPV467Fab variable domain are shown as strips (hMPV F: blue, Fab: red / orange). (C) A magnified view of MPV467 interfacing with hMPV F. The viewing direction is shown in the box and arrow in the inset (B). Important residues are shown as sticks. Hydrogen bonds and salt bridges are depicted as black dashed lines. Oxygen atoms are red, and nitrogen is blue.

[0062] Figure 6 : Neutralization spectra of hMPV F protein-specific mAbs. Data represent the mean from three replicates, and error bars are standard deviations. Data represent the results from at least two independent experiments.

[0063] Figure 7 : ELISA binding curves of hMPV F protein-specific mAbs. ELISA binding curves of isolated mAbs and recombinant hMPV F protein constructs. RSV F protein before fusion (DsCav1) was used to determine whether there were any mAbs that cross-reacted with RSV F. The previously discovered hMPV F-specific mAb MPV196 was used as a negative binding control. Each point represents the mean of the data from four replicates, and the error bars represent the standard deviation. The data represent the results from at least two independent experiments.

[0064] Figure 8 : MPV467 cryo-EM processing workflow. From representative micrographs to DeepEMhancer plots, each step of the cryo-EM data processing workflow is shown. The computational procedures and algorithms used for each step are labeled. Masks used for particle subtraction are colored transparent cyan.

[0065] Figures 9A-9E: MPV467 cryo-EM validation. (A) (Top) FSC curve of homogenously refined 3D reconstruction. The horizontal blue line corresponds to an FSC value of 0.143. (Bottom) View distribution map calculated in cryoSPARC. (B) FSC curve and view distribution map of particle-subtracted inhomogeneous refinement 3D reconstruction. (Top) FSC curve of inhomogeneously refined 3D reconstruction. The horizontal blue line corresponds to an FSC value of 0.143. (Bottom) View distribution map calculated in cryoSPARC. (C) Cryo-EM images of homogenously refined (left) and particle-subtracted inhomogeneous refinement (right) colored by local resolution. Cryo-EM images are shown as top view (top) and side view (bottom). (D) cryo-EM images of DS-CavEs2-IPDS bound by MPV467 are shown in side view (left) and top view (right). Individual promoters are shown as transparent colored surfaces with docking ribbon models (DS-CavEs2-IPDS: blue; MPV467 heavy chain: red, MPV467 light chain: orange). (E) Binding interface of MPV467 heavy chain (left) and light chain (right) with DS-CavEs2-IPDS. Cryo-EM images are shown as transparent surfaces with docking models shown as ribbons and sticks. Coloring is the same as in D.

[0066] Fig.10 Table 1. hMPV F-specific mAb neutralization and binding properties. Neutralization values ​​were determined using a plaque reduction assay, where IC 50 The EC corresponds to the mAb concentration at which a 50% reduction in plaques was observed. Based on the optical density at 405 nm, 50 Values ​​correspond to the concentrations at which half-maximal signals were obtained in the ELISA. > indicates that the binding signal was below 1 μg / mL at the highest mAb concentration tested. Each value is the average of three technical replicates for neutralization experiments and four technical replicates for binding experiments. Each experiment was repeated at least twice independently.

[0067] Fig.11 Table 2. EM data collection for MPV467.

[0068] Fig.12 : hMPV replication and clearance in immunosuppressed versus normal cotton rats. Cotton rats immunosuppressed by repeated cyclophosphamide treatment (immunosuppressed) or normal cotton rats (normal) were treated with 10 5PFU / animal was challenged with hMPV. Animals were sacrificed five, seven or nine days after infection and lung and nasal samples were collected for viral titration by plaque assay followed by immunostaining. Results represent the geometric mean ± SEM of 4-10 animals / group (combined data from two studies). *p<0.05 when compared to normal animals infected with hMPV sacrificed on the same day.

[0069] Fig.13 MPV467 prevents and treats hMPV infection in immunosuppressed cotton rats: dose dependence of antiviral effect. 5 PFU / animal Immunosuppressed cotton rats were challenged with hMPV. MPV467 was administered intramuscularly at 0.1 mg / kg, 1 mg / kg or 10 mg / kg one day before (prevention) or three days after (treatment) hMPV challenge. Five days after infection, animals were sacrificed and lung and nasal samples were collected for hMPV quantification. Results represent the geometric mean ± SEM of 5 animals / group. *p<0.05 when compared to hMPV-infected mock-treated animals sacrificed on the fifth day after infection.

[0070] Fig.14 : Effect of antibody therapy on delayed viral clearance in immunosuppressed cotton rats infected with hMPV. 5 PFU / animal Immunosuppressed and normal cotton rats were challenged with hMPV and treated with 10 mg / kg MPV467. Animals were sacrificed five or nine days after infection and lung and nasal samples were collected for hMPV quantification. Results represent geometric mean ± SEM of 4-10 animals / group. *p<0.05 when compared to hMPV-infected mock-treated animals sacrificed on the corresponding day.

[0071] Fig.15 : Effect of antibody therapy on lung histopathology in normal and immunosuppressed cotton rats infected with hMPV. 5 PFU / animal Immunosuppressed and normal cotton rats were challenged with hMPV and treated with MPV467, as Fig.14 The lung tissue pathology was evaluated in hematoxylin-eosin (H&E) slides for each of the following categories: peribronchiolar inflammation (Peribr), perivascular inflammation (Perivasc), interstitial inflammation (Interst), alveolitis (Alveol), and epithelial hyperplasia (Epith.Hyp.). The results represent the mean ± SE of 4-5 animals / group.

[0072] Fig.16: Effects of antibody therapy on lung cytokine / chemokine expression in normal and immunosuppressed cotton rats infected with hMPV. 5 PFU / animal Immunosuppressed and normal cotton rats were challenged with hMPV, treated with 10 mg / kg MPV467 or PBS (mock) and sacrificed 9 days after infection. Expression of MIP-1a and IP-10 mRNA in lung tissue was quantified by qPCR and normalized by expression of b-actin in the corresponding organs. Results represent mean ± SE of 4-5 animals / group. *p<0.05 compared to normal animals that were not infected (control) or treated with PBS (mock) or MPV467 (Ab). #p<0.05 for antibody-treated animals versus mock-treated animals. DETAILED DESCRIPTION

[0073] The hMPV F protein is a trimeric class I viral fusion protein that is highly conserved between viral subgroups (A1, A2, B1, B2) (Huang and Mousa, Front. Immunol. 10, 2778 (2019)). hMPV can infect respiratory epithelial cells in the absence of hMPV G protein, although hMPV G is required for viral adaptation in vivo (Biacchesi et al., Journal of Virology 78, 12877-12887 (2004)). The hMPV F protein contains the RGD motif, and the hypothesized receptor is α 5 β 1Integrins (Cox et al., Journal of Virology 86, 12148-12160 (2012)). Heparan sulfate has also been shown to play a role in hMPV F protein-mediated attachment (Chang et al., Journal of Virology 86, 3230-3243 (2012)), and direct binding between heparan sulfate and hMPV F protein has recently been demonstrated (Jiachen, H. et al., Journal of Virology 95, e00593-21 ​​(2021)). hMPV F induces fusion of the virus with the host cell membrane in the transition from a metastable prefusion state to a postfusion conformation (Poor et al., Proceedings of the National Academy of Sciences of the United States of America (PNAS) 111, 2596-2605 (2014)). The X-ray crystal structures of the hMPV F protein in pre-fusion (Battles et al., Nat. Commun. 8, 1528 (2017)) and post-fusion (Jiachen, H. et al., Journal of Virology 95, e00593-21 ​​(2021); Más et al., PLoS Pathog. 12, e1005859 (2016)) conformations have been elucidated, and the protein shares a similar structural topology with the RSV F protein (McLellan et al., Curr. Top. Microbiol. Immunol. 372, 83-104 (2013)).

[0074] There is a paucity of information regarding specific epitopes on the hMPV F protein compared to RSV F. Distinct differences in the immunological characteristics between RSV F and hMPV F have been identified. For example, pre- and post-fusion hMPV F proteins elicit similar antibody responses, indicating that most neutralizing epitopes exist in both conformations (Jiachen, H. et al., Journal of Virology 95, e00593-21 ​​(2021); Battles et al., Nature Communications 8, 1528 (2017); Pilaev et al., Vaccine 38, 2122-2127 (2020)), while for RSV F, most and most potent neutralizing antibodies target pre-fusion specific epitopes (Huang and Mousa, Frontiers in Immunology 10, 2778 (2019); Gilman et al., Sci. Immunol. 1, 1-12 (2016); Mousa et al., Nat. Microbiol. 2, 16271 (2017). Known neutralizing epitopes on hMPV F protein include those based on the also neutralizing hMPV Antigenic sites IV (Más et al., PLoS Pathogens 12, e1005859 (2016); Mousa et al., PLoS Pathogens 14, e1006837 (2018); Schuster et al., Journal of Infectious Diseases 211, 1-34 (2014)), III (Corti et al., Nature 501, 439-43 (2013); Wen et al., Nature Microbiology 2, 16272 (2017)) and V (Xiao et al., MAbs 11, 1415-1427 (2019)) identified by RSV F mAbs of F have been identified by human mAbs of F. The unique epitope targeted by DS7 has been structurally defined (Wen et al., Nat. Struc. Mol. Biol. 19, 461-463 (2012)). In addition, a novel epitope located within the trimer interface of the hMPV F protein was recently identified, defined by mAb MPV458 (Huang and Mousa, PLoS Pathogens 16, e1008942 (2020)). To further understand the neutralizing hMPV F epitope, a small group of 18 human mAbs against the hMPV F protein was isolated.

[0075] Two mAbs, MPV467 and MPV487, were shown to have potent neutralizing activity, with MPV467 being exceptionally potent. MPV467 was also shown to prevent and treat hMPV infection in mice, and the epitope targeting of MPV467 was determined to mediate complex binding sites for both antigenic sites II and V using cryo-electron microscopy.

[0076] I. Overview of Terminology

[0077] Unless otherwise stated, technical terms are used according to conventional usage. The definitions of many common terms in molecular biology can be found in Krebs et al. (editor), Lewin's genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms "a / an" and "the" refer to both the singular and the plural, unless the context clearly indicates otherwise. For example, the term "antigen" includes one or more antigens and can be considered to be equivalent to the phrase "at least one antigen". As used herein, the term "comprises" means "includes". It should be further understood that any and all base sizes or amino acid sizes and all molecular weights or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for descriptive purposes, unless otherwise stated. Although many methods and materials similar to or equivalent to the methods and materials described herein can be used, particularly suitable methods and materials are described herein. In the event of a conflict, this specification (including the explanation of the term) shall prevail. In addition, the materials, methods and examples are illustrative only and are not intended to be limiting. To facilitate review of the various embodiments, the following explanations of terms are provided:

[0078] About: Unless the context indicates otherwise, "about" means plus or minus 5% of the reference value. For example, "about" 100 means 95 to 105.

[0079] Administration: Introducing a composition into a subject via a selected route. Administration can be local or systemic. For example, if the selected route is intravenous, the composition (such as a composition comprising a disclosed antibody or antigen-binding fragment) is administered by introducing the composition into a vein of the subject. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (e.g., topical), intranasal, vaginal, and inhalation routes.

[0080] Agent: Any substance or any combination of substances that can be used to achieve a purpose or result; for example, a substance or combination of substances that can be used to inhibit hMPV infection in a subject. Agents include proteins, nucleic acid molecules, compounds, small molecules, organic compounds, inorganic compounds, or other molecules of interest. Agents can include therapeutic agents (such as antiretroviral agents), diagnostic agents, or pharmaceutical agents. In some embodiments, the agent is an antibody that specifically binds to hMPV, optionally in combination with an antiviral agent. Those skilled in the art will appreciate that a particular agent can be used to achieve more than one result.

[0081] Amino acid substitution: Replacement of an amino acid in a polypeptide with a different amino acid or with no amino acid (i.e., deletion). In some examples, an amino acid in a polypeptide is replaced with an amino acid from a homologous polypeptide, for example, and an amino acid in a recombinant Group A MPV F polypeptide can be replaced with the corresponding amino acid from a Group B MPV F polypeptide.

[0082] Antibodies and antigen-binding fragments Immunoglobulins, antigen-binding fragments or derivatives thereof that specifically bind to and recognize an analyte (antigen) such as hMPV F polypeptide. The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including (but not limited to): monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antigen-binding fragments, as long as they exhibit the desired antigen-binding activity.

[0083] Non-limiting examples of antibodies include, for example, intact immunoglobulins that retain binding affinity for an antigen, as well as variants and fragments thereof. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; bifunctional antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed by antibody fragments. Antibody fragments include antigen-binding fragments produced by modifying whole antibodies or synthesized de novo using recombinant DNA methods (see, e.g., Kontermann and Dübel (eds.), Antibody Engineering, Vol. 1-2, 2nd ed., Springer-Verlag, 2010).

[0084] Antibodies also include genetically engineered forms such as chimeric antibodies (eg, humanized murine antibodies) and heteroconjugate antibodies (eg, bispecific antibodies).

[0085] An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to each other or may be different. For example, a naturally occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, and a bispecific or bifunctional antibody has two different binding sites.

[0086] Typically, naturally occurring immunoglobulins have heavy (H) chains and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable domain genes. There are two types of light chains, λ (lambda) and κ (kappa). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE.

[0087] Each heavy chain and light chain contains a constant region (or constant domain) and a variable region (or variable domain). Taken together, the heavy and light chain variable regions specifically bind to an antigen.

[0088] Mention "V H " or "VH" refers to the variable region of an antibody heavy chain, including the variable region of an antigen-binding fragment (e.g., Fv, scFv, dsFv, Fab). References to "V L " or "VL" refers to the variable domain of an antibody light chain, including the variable domains of Fv, scFv, dsFv, and Fab.

[0089] V H and V L Contains a "framework" region interrupted by three hypervariable regions, also called a "complementarity determining region" or "CDR" (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, U.S. Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within species. The framework region of an antibody (i.e., the combined framework regions of the constituent light and heavy chains) is used to position and align the CDRs in three-dimensional space.

[0090] The CDR is primarily responsible for binding to the epitope of the antigen. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al., (Protein Sequences of Immunological Significance, 5th Edition, NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, U.S. Department of Health and Human Services, 1991; "Kabat" numbering scheme), Al-Lazikani et al., ("Standard conformations for the canonical structures of immunoglobulins," Journal of Molecular Biology (J.Mol.Bio.), 273(4):927-948, 1997; "Chothia" numbering scheme), and Lefranc et al., ("IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains"). domains), "Dev. Comp. Immunol.," 27(1):55-77, 2003; "IMGT" numbering scheme). The CDRs of each chain are usually referred to as CDR1, CDR2, and CDR3 (from N-terminus to C-terminus), and are also usually identified by the chain in which the particular CDR is located. Thus, V H CDR3 is the V from the antibody in which it was discovered H CDR3, while V L CDR1 is the V from the antibody in which it was discovered L The light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. The heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.

[0091] In some embodiments, the disclosed antibodies comprise a heterologous constant domain. For example, the antibody comprises a constant domain that is different from a native constant domain, such as a constant domain that comprises one or more modifications (such as an "LS" mutation) to increase half-life.

[0092] "Monoclonal antibody" is an antibody obtained from a substantially homogeneous antibody population, i.e., except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody preparations) (such variants are generally present in small amounts), the individual antibodies of the population are identical and / or bound to the same epitope. Compared with polyclonal antibody preparations that generally contain different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates that the characteristic of an antibody is obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring the production of antibodies by any ad hoc method. For example, monoclonal antibodies can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing all or a portion of transgenic animals containing human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein. In some instances, monoclonal antibodies are separated from a subject. Monoclonal antibodies can have conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. (See, e.g., Greenfield (ed.), Antibodies: A Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratory Press, 2014).

[0093] "Humanized" antibodies or antigen-binding fragments comprise human framework regions and one or more CDRs from non-human (such as mouse, rat or synthetic) antibodies or antigen-binding fragments. The non-human antibody or antigen-binding fragment providing the CDR is referred to as a "donor", and the human antibody or antigen-binding fragment providing the framework is referred to as an "acceptor". In one embodiment, all CDRs are from the donor immunoglobulin in the humanized immunoglobulin. Constant regions do not need to be present, but if present, the constant regions may be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Therefore, except for possible CDRs, all parts of the humanized antibody or antigen-binding fragment are substantially identical to the corresponding parts of the natural human antibody sequence.

[0094] A "chimeric antibody" is an antibody comprising sequences derived from two different antibodies, which are generally of different species. In some instances, a chimeric antibody comprises one or more CDRs and / or framework regions from one human antibody and CDRs and / or framework regions from another human antibody.

[0095] "Fully human antibodies" or "human antibodies" are antibodies that contain sequences from (or derived from) the human genome and do not contain sequences from another species. In some embodiments, human antibodies contain CDRs, framework regions, and (if present) Fc regions from (or derived from) the human genome. Human antibodies can be identified and isolated using techniques based on sequences derived from the human genome, such as by phage display or using transgenic animals (see, e.g., Barbas et al., Phage display: A Laboratory Manual, 1st ed. New York: Cold Spring Harbor Laboratory Press, 2004, print; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonenberg, Curr. Opin. Immunol, 20: 450-459, 2008).

[0096] Neutralizing MPV antibodies or antigen-binding fragments are antibodies or antigen-binding fragments that specifically bind to hMPV antigens, such as hMPV (e.g., F protein), in a manner that inhibits biological functions associated with inhibiting hMPV infection. Antibodies can neutralize the activity of hMPV at various points during the pathogen's life cycle.

[0097] Biological sample: A sample obtained from a subject. Biological samples include all clinical samples that can be used to detect a disease or infection (e.g., hMPV infection) in a subject, including but not limited to cells, tissues, and body fluids, such as blood, blood derivatives and fractions (e.g., serum), cerebrospinal fluid; and biopsy or surgically removed tissues, such as unfixed, frozen, or fixed in formalin or paraffin. In a specific example, the biological sample is obtained from a subject suffering from or suspected of suffering from hMPV infection.

[0098] Bispecific antibody: A recombinant molecule composed of two different antigen binding domains that bind to two different antigenic epitopes. Bispecific antibodies comprise molecules in which two antigen binding domains are chemically or genetically linked. The antigen binding domains may be linked using a linker. The antigen binding domains may be a monoclonal antibody, an antigen binding fragment (e.g., Fab, scFv), or a combination thereof. Bispecific antibodies may contain one or more constant domains, but do not necessarily contain constant domains.

[0099] Conditions sufficient to form immune complexes allow the degree of binding of an antibody or its antigen-binding fragment to its cognate epitope to be detectably higher than and / or substantially exclude conditions for the degree of binding to substantially all other epitopes. Conditions sufficient to form immune complexes depend on the format of the binding reaction, and are typically conditions utilized in immunoassay protocols or conditions encountered in vivo. For descriptions of immunoassay formats and conditions, see Harlow and Lane, Antibodies: A Laboratory Manual, 2nd edition, Cold Spring Harbor Publications, New York, (2013). The conditions employed in the method are "physiological conditions", which include typical conditions (e.g., temperature, osmotic pressure, pH) in living mammals or mammalian cells as mentioned. Although it is recognized that some organs are subject to extreme conditions, the intracellular environment in organisms is generally around pH 7 (e.g., pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), containing water as the primary solvent and present at temperatures above 0°C and below 50°C. Osmotic pressure is within the range of supporting cell viability and proliferation.

[0100] Conjugate: A complex of two molecules that are linked together (e.g., linked together by a covalent bond). In one embodiment, an antibody is linked to an effector molecule; for example, an antibody that specifically binds to an hMPV F protein that is covalently linked to an effector molecule. The connection can be chemical or recombinant. In one embodiment, the connection is chemical, wherein the reaction between the antibody portion and the effector molecule has produced a covalent bond formed between the two molecules to form one molecule. A peptide linker (short peptide sequence) may optionally be included between the antibody and the effector molecule. Because conjugates can be prepared from two molecules with separate functions (e.g., an antibody and an effector molecule), the conjugates are sometimes also referred to as "chimeric molecules."

[0101] Conservative variants: "Conservative" amino acid substitutions are those that do not substantially affect or reduce the function of a protein (such as the ability of a protein to interact with a target protein). For example, an MPV-specific antibody may contain up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 conservative substitutions compared to a reference antibody sequence and retain specific binding activity to MPV and / or MPV neutralization activity. The term conservative variation also encompasses the use of substituted amino acids in place of unsubstituted parent amino acids.

[0102] Individual substitutions, deletions or additions that change, add or delete a single amino acid or a small fraction of amino acids (e.g., less than 5%, in some embodiments less than 1%) in the encoded sequence are conservative variations, where the change results in the substitution of an amino acid with a chemically similar amino acid.

[0103] The following six groups are examples of amino acids that are considered conservative substitutions for each other:

[0104] 1) Alanine (A), serine (S), threonine (T);

[0105] 2) Aspartic acid (D), glutamic acid (E);

[0106] 3) Asparagine (N), glutamine (Q);

[0107] 4) Arginine (R), Lysine (K);

[0108] 5) isoleucine (I), leucine (L), methionine (M), valine (V); and

[0109] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0110] Non-conservative substitutions are substitutions that reduce the activity or function of hMPV-specific antibodies (such as the ability to specifically bind to hMPV F protein or neutralize hMPV). For example, if the amino acid residue is essential for the function of the protein, even conservative substitutions may destroy the activity. Therefore, conservative substitutions do not change the basic function of the protein of interest.

[0111] Contact: Placed in direct physical association; includes both solid and liquid forms, and the contact can occur in vivo or in vitro. Contact includes contact between one molecule and another molecule, such as contact of an amino acid on the surface of one polypeptide (such as a peptide) with another polypeptide. Contact can also include contacting a cell, such as by directly physically associating a polypeptide with the cell.

[0112] Control: A reference standard. In some embodiments, a control is a negative control sample obtained from a healthy patient. In other embodiments, a control is a positive control sample obtained from a patient diagnosed with MPV infection. In yet other embodiments, a control is a historical control or a standard reference value or range of values ​​(such as a previously tested control sample, such as a group of MPV patients with a known prognosis or outcome, or a group of samples representing a baseline or normal value).

[0113] The difference between the test sample and the control can be an increase or an opposite reduction. The difference can be a qualitative difference or a quantitative difference, such as a statistically significant difference. In some instances, the difference is to increase or decrease at least about 5% relative to the control, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500% or more than 500%.

[0114] Degenerate variants: In the context of the present disclosure, "degenerate variants" refers to polynucleotides encoding polypeptides (such as recombinant MPV F proteins or immunogenic fragments thereof) that contain sequences that are degenerate due to the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, as long as the amino acid sequence of the peptide encoded by the nucleotide sequence does not change, all degenerate nucleotide sequences encoding the peptide are included.

[0115] Detectable marker: A detectable molecule (also referred to as a label) that is conjugated directly or indirectly to a second molecule (such as an antibody) to facilitate detection of the second molecule. For example, a detectable marker can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques such as CT scans, MRI, ultrasound, fiberoptic inspection, and laparoscopy. Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). Methods for using detectable markers and guidance for selecting detectable markers appropriate for various purposes are discussed, for example, in Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4th ed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. (eds.) (Current Protocols in Molecular Biology, New York: John Wiley and Sons, including supplements, 2017).

[0116] Detect: To determine the existence, presence, or fact of something.

[0117] DS7 antibody: A neutralizing monoclonal antibody that specifically binds to an epitope on the hMPV F protein that is present in the pre-fusion and post-fusion conformations of the hMPV F protein. The DS7 antibody does not specifically bind to hMPV F in its post-fusion conformation. The DS7 antibody and methods for its production are described, for example, in Wen et al., Nature Structural and Molecular Biology, 19, 461-463, 2012, which is incorporated herein by reference in its entirety. The amino acid sequences of the heavy and light variable regions of the DS7 antibody are provided as SEQ ID NOs: 41 and 42, and have been numbered 4DAG_H (DS7 V H ) and 4DAG_L(DS7 V L ) are deposited in the PDB, each of which is incorporated herein by reference as presented in the database on November 10, 2014.

[0118] DS7 V H -EVQLLESGGGLVQPGGSRRLSCAASGFTVSSSYMSWVRQTPGKGLEWISVFYSGGTTYYADAVKGRFSISMDTSKNTLHLQMNSLRVEDTAIYYCARVLSRASGMPDAFDIWGPGTMVTVSS(SEQ ID NO:145)

[0119] DS7 V L -ELALIQPASVSVSPGQTASITCSGDKLGDKYASWYQQKPGQSPVLVIYQDSERPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTAVFGGGTTLTVLGQ(SEQ ID NO:146)

[0120] Effective amount: The amount of a particular substance sufficient to achieve a desired effect in a subject to which the substance is administered. For example, an effective amount may be the amount of antibody necessary to inhibit hMPV infection or to measurably alter the outward symptoms of hMPV infection.

[0121] In some embodiments, administration of an effective amount of a disclosed antibody or antigen-binding fragment that binds to hMPV F protein can reduce or inhibit MPV infection (e.g., as measured by infection of cells, or by the number or percentage of subjects infected with hMPV, or by an increase in the survival time of infected subjects, or a reduction in symptoms associated with hMPV) by a desired amount, such as a reduction or inhibition of at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable hMPV infection) compared to a suitable control.

[0122] The effective amount of an antibody or antigen-binding fragment that specifically binds to the hMPV F protein administered to a subject to inhibit hMPV infection will vary according to many factors related to the subject (e.g., the overall health and / or weight of the subject). The effective amount can be determined by varying the dose and measuring the resulting response (e.g., a decrease in pathogen titer). The effective amount can also be determined by various in vitro, in vivo or in situ immunoassays.

[0123] The effective amount encompasses a partial dose that helps to obtain an effective response in combination with previous or subsequent administration. For example, during a treatment course that lasts for several days or weeks, an effective amount of the medicament can be administered in a single dose or several doses. However, the effective amount can depend on the severity and type of the treated subject, the treated condition, and the mode of administration. The unit dosage form of the medicament can be packaged in the amount of the effective amount or in multiples of the effective amount, for example, in a vial (e.g., with a pierceable lid) or a syringe with a sterile component.

[0124] Epitope: Antigenic determinant. These antigenic determinants are specific chemical groups or peptide sequences on a molecule that are antigenic, such that the antigenic determinant elicits a specific immune response, e.g., an epitope is a region of an antigen to which B and / or T cells respond. Antibodies can bind to specific antigenic epitopes, such as those on the hMPV F protein.

[0125] Expression: Transcription or translation of a nucleic acid sequence. For example, a gene is expressed when its DNA is transcribed into RNA or RNA fragments, which are processed into mRNA in some instances. A gene may also be expressed when its mRNA is translated into an amino acid sequence (such as a protein or protein fragment). In a specific example, a heterologous gene is expressed when its RNA is transcribed into RNA. In another example, a heterologous gene is expressed when its RNA is translated into an amino acid sequence. The term "expression" is used herein to refer to transcription or translation. Regulation of expression may include control of transcription, translation, RNA transport and processing, degradation of intermediate molecules (such as mRNA), or control of activation, inactivation, partitioning or degradation after the production of a specific protein molecule.

[0126] Expression control sequence: a nucleic acid sequence that regulates the expression of a heterologous nucleic acid sequence that is operably connected thereto. When the expression control sequence controls and regulates the transcription and (as the case may be) translation of the nucleic acid sequence, the expression control sequence is operably connected to the nucleic acid sequence. Therefore, the expression control sequence may include a suitable promoter, an enhancer, a transcription terminator, a start codon (ATG) in front of a protein-coding gene, a splicing signal of an intron, the maintenance of the correct reading frame of the gene to allow the correct translation of mRNA, and a stop codon. The term "control sequence" is intended to include at least components whose presence may affect expression, and may also include additional components whose presence is advantageous, such as a leader sequence and a fusion partner sequence. The expression control sequence may include a promoter.

[0127] A promoter is a minimal sequence sufficient to direct transcription. It also includes promoter elements sufficient to make promoter-dependent gene expression cell type-specific, tissue-specific, or induced by external signals or agents; such elements may be located in the 5' or 3' region of the gene. It includes both constitutive and inducible promoters (see, for example, Bitter et al., Methods in Enzymology 153: 516-544, 1987). For example, when cloning in a bacterial system, an inducible promoter can be used, such as pL (ptrp-lac hybrid promoter) of phage λ, plac, ptrp, ptac, etc. In one embodiment, when cloning in a mammalian cell system, a promoter derived from a mammalian cell genome (such as a metallothionein promoter) or a promoter derived from a mammalian virus (such as a retroviral long terminal repeat sequence, adenovirus late promoter, vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques can also be used to provide transcription of nucleic acid sequences.

[0128] The polynucleotide can be inserted into an expression vector containing a promoter sequence that promotes efficient transcription of the inserted gene sequence of the host. The expression vector usually contains a replication origin, a promoter, and a specific nucleic acid sequence that allows phenotypic selection of transformed cells.

[0129] Expression vector: A vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be supplied by a host cell or in an in vitro expression system. Expression vectors include all expression vectors known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating a recombinant polynucleotide.

[0130] Fc region: The constant region of an antibody does not include the first heavy chain constant domain. The Fc region generally refers to the last two heavy chain constant domains of IgA, IgD and IgG, and the last three heavy chain constant domains of IgE and IgM. The Fc region may also include part or all of the flexible hinge N-terminal of these domains. For IgA and IgM, the Fc region may or may not include a tailpiece, and may or may not be bound by a J chain. For IgG, the Fc region is generally understood to include the lower part of the hinge between the immunoglobulin domains Cγ2 and Cγ3 and optionally between Cγ1 and Cγ2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is generally defined as including residues from C226 or P230 to the Fc carboxyl terminus, where numbering is performed according to Kabat. For IgA, the Fc region includes the lower part of the hinge between the immunoglobulin domains Cα2 and Cα3 and optionally between Cα1 and Cα2.

[0131] Heterologous: Derived from a different genetic source. A nucleic acid molecule that is heterologous to a cell is derived from a genetic source other than the cell in which it is expressed. In a specific, non-limiting example, a heterologous nucleic acid molecule encoding a protein (e.g., scFv) is expressed in a cell (e.g., a mammalian cell). Methods for introducing heterologous nucleic acid molecules into cells or organisms are well known in the art, such as transformation with nucleic acids, including electroporation, lipofection, particle gun acceleration, and homologous recombination.

[0132] Host cell: A cell in which a vector can propagate and express its DNA. The cell can be prokaryotic or eukaryotic. The term also encompasses any progeny of the subject host cell. It should be understood that all progeny may not be identical to the parent cell, as mutations may occur during replication. However, when the term "host cell" is used, such progeny are encompassed.

[0133] IgG: A polypeptide belonging to the class or isotype of antibodies encoded essentially by the recognized immunoglobulin gamma genes. In humans, this class includes IgG 1 IgG 2 IgG 3 and IgG 4 .

[0134] Immune complex: The combination of an antibody or antigen-binding fragment (such as scFv) and a soluble antigen forms an immune complex. The formation of immune complexes can be detected by conventional methods, such as immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (e.g., Western blot), magnetic resonance imaging, CT scanning, radiography, and affinity chromatography.

[0135] Immune response: The response of cells of the immune system (such as B cells, T cells or monocytes) to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response"). In one embodiment, the immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response and results in the production of specific antibodies.

[0136] Immunogen: A compound, composition or substance that stimulates antibody production or a T-cell response in an animal, including compositions such as hMPV that are injected or absorbed into an animal. Immunogens react with the products of specific humoral or cellular immunity.

[0137] Inhibit a disease or condition: Reduce the full development of a disease or condition in a subject, for example, reduce the full development of an MPV infection (such as hMVP infection) in a subject at risk of MPV infection. This includes neutralizing, antagonizing, inhibiting, suppressing, slowing, destroying, stopping or reversing the progression or severity of a disease or condition.

[0138] Inhibiting a disease or condition can refer to a preventive and therapeutic intervention administered before the disease or condition begins to develop (e.g., treatment initiated in a subject at risk for hMPV infection but not infected by hMPV), which reduces the subsequent development of the disease or condition and / or improves signs or symptoms after the disease or condition develops. The term "improvement" with respect to inhibiting a disease or condition refers to any observable beneficial effect of a preventive and therapeutic intervention intended to inhibit a disease or condition. The beneficial effect can be demonstrated, for example, by the delayed onset of clinical symptoms of a disease or condition in a susceptible subject, a reduction in the severity of some or all clinical symptoms of a disease or condition, a slowed progression of a disease or condition, an improvement in the overall health or well-being of the subject, a reduction in infection, or by other parameters specific to a particular disease or condition.

[0139] In some embodiments, the disclosed hMPV F protein-specific antibodies and antigen-binding fragments inhibit the growth of hMPV in a subject, for example, the antibodies and antigen-binding fragments inhibit the proliferation of hMPV in a subject compared to a relevant control, thereby reducing the pathogen load in the subject. For example, the disclosed hMPV F protein-specific antibodies and antigen-binding fragments can inhibit hMPV infection in a subject or inhibit viral replication by at least 20%, at least 30%, at least 40%, or at least 50% compared to a suitable control.

[0140] Isolated: A biological component (such as a nucleic acid, peptide, protein or protein complex, such as an antibody) that has been substantially separated, produced separately from, or purified from other biological components (i.e., other chromosomal and extrachromosomal DNA and RNA and proteins) in the cells of the organism in which the component naturally occurs. Thus, isolated nucleic acids, peptides and proteins include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids, peptides and proteins prepared by recombinant expression in host cells and chemically synthesized nucleic acids. An isolated nucleic acid, peptide or protein (e.g., an antibody) can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% pure.

[0141] Kabat position: The position of residues in an amino acid sequence follows the numbering convention outlined by Kabat et al., (Protein Sequences of Immunological Significance, 5th ed., Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, NIH Publication No. 91-3242, 1991).

[0142] Linker: A bifunctional molecule that can be used to link two molecules into one continuous molecule (eg, linking an effector molecule to an antibody, or linking a detectable marker to an antibody). Non-limiting examples of peptide linkers include glycine-serine linkers.

[0143] The terms "conjugated", "joined", "bound" or "linked" can refer to the preparation of two molecules into one continuous molecule; for example, two polypeptides are linked into one continuous polypeptide, or an effector molecule or a detectable marker radionuclide or other molecule is covalently attached to a polypeptide (such as a scFv). The connection can be performed chemically or recombinantly. "Chemical means" refers to the reaction between the antibody portion and the effector molecule, which causes a covalent bond to form between the two molecules to form one molecule.

[0144] Metapneumovirus (MPV): An enveloped, non-segmented, negative-sense, single-stranded RNA virus of the family Paramyxoviridae. The metapneumovirus is a common cause of lower respiratory tract infections in children and adults, including bronchiolitis and pneumonia, and almost all people are infected by the age of five. MPV causes recurrent infections, including severe lower respiratory tract disease, that may occur at any age, especially in the elderly or those with compromised heart, lung, or immune systems. hMPV can infect humans.

[0145] The MPV genome contains eight genes encoding nine proteins (including glycoproteins SH, G and F). The F protein mediates fusion, allowing the virus to enter the cytoplasm. Two groups of human MPV strains have been described, Group A and Group B, which are further divided into subgroups A1, A2, B1 and B2. Exemplary MPV strain sequences are known to those of ordinary skill in the art. Further, several human MPV infection models are available, including model organisms infected with hMPV (see, for example, Herfst et al., Journal of General Virology, 88, 2702-2709, 2007; Bayon et al., Rev. Med. Virol., 2, 15-34, 2013; and Liu et al., Clinical Vaccine Immunol., 20, 1246-1254, 2013). The F protein has a head and a tail; the head is the top 50% of the prefusion state and the tail is the bottom 50% of the prefusion state.

[0146] Methods for diagnosing MPV infection are known, including the use of direct fluorescent antibody detection (DFA), chromatographic rapid antigen detection and the use of RT PCR to detect viral RNA. Quantification of viral load can be determined, for example, by plaque assay, antigen capture enzyme immunoassay (EIA) or PCR. Quantification of antibody levels can be performed by subgroup-specific neutralization assay or ELISA. Current MPV treatment includes the use of antiviral ribavirin (Ribaviran) and passive administration of experimental monoclonal antibodies, such as MPE8 (see, for example, Corti et al., "Nature", 501,439-443,2013) and mAb338 (Medimmune, Inc., see Hamelin et al., "Antiviral Res", 88,31-37,2010), the experimental monoclonal antibodies recognize MPV F protein and reduce the incidence of MPV infection and disease in animal models.

[0147] There are several subgroups of MPV, including group A and group B, and subgroups A1, A2, B1, and B2 in human MPV. Within the subgroups of MPV, separate strains exist for each subgroup. The sequences of the F proteins from specific MPV strains are known and provided herein (see, e.g., Table 1).

[0148] MPV fusion (F) protein: MPV envelope glycoprotein that promotes fusion of viral and cellular membranes. In nature, MPV F protein is initially synthesized as a single polypeptide precursor of approximately 540 amino acids in length, named F 0 . F 0 Contains an N-terminal signal peptide directing localization to the endoplasmic reticulum, wherein the signal peptide (F0 The approximately first 18 residues of F are cleaved by proteolysis. 0 The residues oligomerize to form a trimer which again is located at the protease site (between approximately 0 between positions 102 and 103; for example, RQSR 102 (residues 99-102)) to generate two disulfide-linked fragments, namely F 1 and F 2 The smaller of these fragments F 2 From F 0 The N-terminal portion of the precursor and contains F 0 The larger of these fragments, F 1 Contains F 0 The C-terminal portion of the precursor (approximately residues 103-540) comprises an extracellular / luminal region (approximately residues 103-490), a transmembrane domain (approximately residues 491-513), and a cytoplasmic domain at the C-terminus (approximately residues 514-540).

[0149] Three Fs 2 -F 1 The promoter oligomerizes in the mature F protein, which adopts a metastable "prefusion" conformation that triggers a conformational change (to a "postfusion" conformation) upon contact with the target cell membrane. This conformational change exposes the F 1 A hydrophobic sequence at the N-terminus of the polypeptide, called a fusion peptide, associates with the host cell membrane and promotes fusion of the membrane of the virus or infected cell with the target cell membrane.

[0150] The extracellular portion of the MPV F protein is the MPV F ectodomain, which contains the F 2 protein (approximately positions 20-102 of MPV F) and F 1 Extracellular domain (approximately positions 103-490 of MPV F). MPV F extracellular domain trimer A protein complex containing three MPV F extracellular domains.

[0151] Figures 2A-2C Antigenic sites II, III, IV and V of the F protein based on competitive binding are shown.

[0152] MPV F prefusion conformation: The structural conformation adopted by the MPV F protein before triggering the pro-fusogenic events that lead to the transition of MPV F to the postfusion conformation and after being processed into the mature MPV F protein in the secretion system. The prefusion conformation of MPV F is similar in overall structure to the prefusion conformations of the F proteins of other paramyxoviruses, such as RSV.

[0153] MPE8 antibody: A neutralizing monoclonal antibody that specifically binds to an epitope on the MPV F protein that is present in the pre-fusion conformation of the MPV F protein but not in the post-fusion conformation. The MPE8 antibody and methods for its production are described, for example, in Corti et al., (Nature, 501, 439-443, 2013), which is incorporated herein by reference. MPE8 binds to site III of the MPV F protein; site III can be identified by MPE8 binding. The amino acid sequences of the heavy and light variable regions of the MPE8 antibody used herein are provided as SEQ ID NOs: 43 and 44. The MPE8 heavy and light chain sequences have been given the designation AGU13651.1 (MPE8 V H ) and AGU13652.1(MPE8 V L ) are deposited in GenBank, each of which is incorporated herein by reference as presented in the database on November 10, 2014.

[0154] MPE8 V H- EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISASSSYSDYADSAKGRFTISSRDNAKTSLFLQMNSLRAEDTAIYFCARARATGYSSITPYFDIWGQGTLVTVSS(SEQ ID NO:147)

[0155] MPE8 V L- QSVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYDNNNRPSGVPDRFSASKSGTSASLAITGLQAEDEADYYCQSYDRSLSGVFGTGTKVTVL(SEQ ID NO:148)

[0156] Neutralizing antibody: An antibody that reduces the infectious titer of an infectious agent by binding to a specific antigen on the infectious agent. In some instances, the infectious agent is a virus. In some instances, antibodies specific for MPV F neutralize the infectious titer of hMPV. "Broadly neutralizing antibodies" are antibodies that bind to and inhibit the function of related antigens (such as antigens on the surface of antigens that share at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity of the antigen). For antigens from pathogens (such as viruses), antibodies can bind to and inhibit the function of more than one class and / or subclass of antigens from the pathogen. For example, with respect to MPV, antibodies can bind to and inhibit the function of antigens, such as MPV F from more than one group.

[0157] Nucleic acid: a polymer consisting of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and non-naturally occurring analogs thereof) connected by phosphodiester bonds, related naturally occurring structural variants, and non-naturally occurring analogs thereof. Therefore, the term includes nucleotide polymers, wherein the connection between nucleotides and non-naturally occurring synthetic analogs thereof includes, for example, but not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNA), etc. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "oligonucleotide" generally refers to short polynucleotides, generally no more than about 50 nucleotides. It should be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C), wherein "U" replaces "T".

[0158] "Nucleotide" includes, but is not limited to, monomers such as in peptide nucleic acids (PNA), which include a base linked to a sugar (such as a pyrimidine, purine, or synthetic analogs thereof), or a base linked to an amino acid. A nucleotide is a monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.

[0159] Conventional symbols are used herein to describe nucleotide sequences: the left-hand end of a single-stranded nucleotide sequence is the 5' end; the left-hand direction of a double-stranded nucleotide sequence is referred to as the 5' direction. The direction in which nucleotides are added 5' to 3' to a nascent RNA transcript is referred to as the transcription direction. The DNA strand having the same sequence as the mRNA is referred to as the "coding strand"; the sequence on the DNA strand having the same sequence as the mRNA transcribed from the DNA and located at the 5' to 5' end of the RNA transcript is referred to as the "upstream sequence"; the sequence on the DNA strand having the same sequence as the RNA and being the 3' to 3' end of the coding RNA transcript is referred to as the "downstream sequence".

[0160] "cDNA" refers to DNA that is complementary or identical to mRNA, in either single- or double-stranded form.

[0161] "Coding" refers to the inherent characteristics of the specific sequence of nucleotides in polynucleotides such as genes, cDNA or mRNA, and the sequence is used to serve as a template for synthesizing other polymers and macromolecules in biological processes, and the other polymers and macromolecules have defined nucleotide sequences (that is, rRNA, tRNA and mRNA) or defined amino acid sequences and biological properties produced therefrom. Therefore, if the transcription and translation of the mRNA produced by a gene produce protein in a cell or other biological system, the gene encodes the protein. Both the coding strand (its nucleotide sequence is identical to the mRNA sequence and is generally provided in the sequence table) and the non-coding strand (used as a template for transcription of a gene or cDNA) can be referred to as encoding the protein or other products of the gene or cDNA. Unless otherwise indicated, "nucleotide sequence encoding an amino acid sequence" includes degenerate versions of each other and all nucleotide sequences encoding the same amino acid sequence. The nucleotide sequence of coded proteins and RNA may include introns.

[0162] If a polynucleotide whose sequence is a first sequence specifically hybridizes to a polynucleotide whose sequence is a second sequence, then a first sequence is "antisense" with respect to a second sequence.

[0163] Operably linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed into a functional relationship with the second nucleic acid sequence. For example, a promoter (such as a CMV promoter) is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Typically, operably linked DNA sequences are contiguous and, in the case of two protein coding regions that need to be joined, are in the same reading frame.

[0164] Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers used are conventional. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, PA., 19th ed., 1995, describes compositions and formulations suitable for drug delivery of the disclosed antibodies and antigen-binding fragments thereof.

[0165] In general, the nature of the carrier will depend on the specific mode of administration adopted. For example, parenteral formulations generally include injectable fluids, which include pharmaceutically and physiologically acceptable fluids, such as water, saline, balanced salt solutions, aqueous glucose solutions, glycerol, etc. as vehicles. For solid compositions (e.g., powders, pills, tablets or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grade mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered can contain a small amount of non-toxic auxiliary substances, such as wetting agents or emulsifiers, preservatives and pH buffers, such as sodium acetate or sorbitan monolaurate. In a particular embodiment, the carrier suitable for administration to a subject can be sterile and / or suspended or otherwise included in a unit dosage form containing a composition of one or more measured doses. The carrier may also be accompanied by a drug for its therapeutic purpose. The unit dosage form can be, for example, a sealed vial or syringe containing sterile contents for injection into a subject, or lyophilized for subsequent reconstitution and administration, or as a solid or controlled release dose.

[0166] Polypeptide: Any amino acid chain, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). "Polypeptide" applies to amino acid polymers, including naturally occurring amino acid polymers and non-naturally occurring amino acid polymers, as well as those in which one or more amino acid residues are non-natural amino acids, such as artificial chemical mimetics of the corresponding naturally occurring amino acids. "Residue" refers to an amino acid or amino acid mimetic that is incorporated into a polypeptide via an amide bond or an amide bond mimetic. A polypeptide has an amino terminal (N-terminal) end and a carboxyl terminal (C-terminal) end. "Polypeptide" is used interchangeably with peptide or protein, and is used herein to refer to a polymer of amino acid residues.

[0167] Polypeptide modification: Polypeptides and peptides, such as antibodies disclosed herein, can be modified by a variety of chemical techniques to produce derivatives that have substantially the same activity as the unmodified peptide and optionally have other desired properties. For example, the carboxylic acid group of the protein (either the carboxyl terminus or the side chain) can be provided in the form of a salt of a pharmaceutically acceptable cation, or esterified to form a C 1 -C 16 Ester, or converted to NR 1 R 2 Amide (where R 1 and R 2 Each independently is H or C 1 -C 16alkyl), or combined to form a heterocyclic ring, such as a 5-membered or 6-membered ring. The amino group of the peptide (whether the amino terminal or the side chain) can be in the form of a pharmaceutically acceptable acid addition salt such as HCl, HBr, acetic acid, benzoic acid, toluenesulfonic acid, maleic acid, tartaric acid and other organic salts, or can be modified to C 1 -C 16 Alkyl or dialkylamino groups, or further converted into amides.

[0168] The hydroxyl groups of the peptide side chains can be converted to C using recognized techniques. 1 -C 16 Alkoxy or converted to C 1 -C 16 The phenyl and phenol rings of the peptide side chains may be substituted with one or more halogen atoms (such as F, Cl, Br or I) or with C 1 -C 16 Alkyl, C 1 -C 16 Alkoxy, carboxylic acid and its ester or amide of such carboxylic acid substitution. The methylene group of the peptide side chain can be extended to the homologous C 2 -C 4 Alkylene. Thiols can be protected with any of a number of recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize methods for introducing cyclic structures into the peptides of the present disclosure to select and provide conformational constraints on the structure that result in enhanced stability. For example, a C-terminal or N-terminal cysteine ​​can be added to the peptide so that when oxidized, the peptide will contain a disulfide bond, thereby producing a cyclic peptide. Other peptide cyclization methods include the formation of thioethers and carboxyl-terminal and amino-terminal amides and esters.

[0169] Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein (such as an antibody) is more enriched than the peptide or protein in its natural environment within a cell. In one embodiment, the preparation is purified such that the protein or peptide accounts for at least 50% of the total peptide or protein content of the preparation.

[0170] Recombination: A recombinant nucleic acid is a nucleic acid having a sequence that does not occur naturally or a sequence that is made by artificially combining two otherwise separated sequence segments. This artificial combination can be achieved by chemical synthesis, or more commonly by artificially manipulating the separated nucleic acid segments, such as by genetic engineering techniques. A recombinant protein is a protein having a sequence that does not occur naturally or a sequence that is made by artificially combining two otherwise separated sequence segments. In several embodiments, the recombinant protein is encoded by a heterologous (e.g., recombinant) nucleic acid that has been introduced into a host cell (e.g., a bacterium or a eukaryotic cell). The nucleic acid can be introduced, for example, into an expression vector having a signal that is capable of expressing the protein encoded by the introduced nucleic acid, or the nucleic acid can be integrated into a host cell chromosome.

[0171] Respiratory syncytial virus (RSV): an enveloped, non-segmented, negative-sense, single-stranded RNA virus of the family Paramyxoviridae. The respiratory syncytial virus is the most common cause of bronchiolitis and pneumonia in children in the first year of life, and almost all children are infected by the age of 3. RSV can also cause recurrent infections (including severe lower respiratory tract disease), which may occur at any age, especially in the elderly or people with impaired heart, lung or immune systems. In the United States, RSV bronchiolitis is the leading cause of hospitalization for infants and is also the leading cause of asthma and wheezing throughout childhood (Shay et al., Journal of the American Medical Association (JAMA) 282, 1440 (1999); Hall et al., New England Journal of Medicine, 360, 588 (2009)). Globally, RSV kills 66,000-199,000 children under five years of age each year (Nair et al., Lancet 375, 1545 (2010)) and accounts for 6.7% of deaths among infants aged one month to one year - more than any other single pathogen except malaria (Lozano et al., Lancet 380, 2095 (2013)).

[0172] RSV genome length is about 15,000 nucleotides, and comprises 10 genes encoding 11 kinds of proteins (comprising glycoprotein SH, G and F).F protein mediates fusion, allows virus to enter into cytoplasm and also promotes the formation of syncytium.Based on the difference of G glycoprotein antigenicity, two subtypes of human RSV strains, i.e. A and B subtypes, have been described.RSV strains of other species are also known, comprising bovine RSV.Exemplary RSV strain sequences are known to those of ordinary skill in the art.Further, there are several human RSV infection models available, comprising the model organisms of infecting hRSV, and the model organisms of infecting species-specific RSV, such as the bRSV used in cattle infects (see, for example, Bern et al., " American Journal of Physiology Lung Cell Molecular Physiology (Am J, Physiol. Lung Cell Mol. Physiol.) ", 301: L148-L156, 2011).

[0173] Sequence identity: Similarity between amino acid sequences is expressed as similarity between sequences, otherwise known as sequence identity. Sequence identity is often measured as a percentage of identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs, orthologs or variants of a polypeptide will have a relatively high degree of sequence identity when aligned using standard methods.

[0174] Methods for alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Molecular Biology 48:443, 1970; Pearson and Lipman, PNAS 85:2444, 1988; Higgins and Sharp, Gene, 73:237-44, 1988; Higgins and Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al., Computer Appls. in the Biosciences Biosciences, 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio., 24:307-31, 1994. Altschul et al., J. Molecular Biology, 215:403-10, 1990, present a detailed consideration of sequence alignment methods and homology calculations.

[0175] Once aligned, the number of matches is determined by counting the number of positions where the same nucleotide or amino acid residue exists in the two sequences. The percentage of sequence identity is determined by dividing the number of matches by the sequence length shown in the identified sequence or by the hinge length (such as 100 consecutive nucleotides or amino acid residues from the sequence shown in the identified sequence), and then multiplying the resulting value by 100. For example, when aligned with a test sequence having 1554 amino acids, a peptide sequence with 1166 matches is 75.0% identical to the test sequence (1166 ÷ 1554 * 100 = 75.0). The percentage of sequence identity values ​​are rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. Length values ​​will always be integers.

[0176] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and the Internet for use with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Instructions on how to use this program to determine sequence identity are available on the NCBI website on the Internet.

[0177] Homologues and variants of polypeptides are generally characterized by having at least about 75%, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity in the full-length comparison with the amino acid sequence of interest. When evaluated by this method, proteins with even greater similarity to the reference sequence will show an increased identity percentage, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity. When the sequence identity is less than the entire sequence, homologues and variants will generally have at least 80% sequence identity in a short window of 10-20 amino acids, and may have at least 85% or at least 90% or 95% sequence identity, depending on its identity to the reference sequence. Methods for determining sequence identity in such short windows are available on the NCBI website on the Internet. Those skilled in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible to obtain strong significant homologs outside the ranges provided.

[0178] In order to carry out sequence comparison to nucleotide sequence, usually a sequence serves as a reference sequence for comparison with a test sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are specified (if necessary), and sequence algorithm program parameters are specified. Default program parameters are used. The method for sequence comparison for comparison is well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482, 1981, by the homology alignment algorithm of Needleman and Wunsch, J. Molecular Biology 48:443, 1970, by the similarity search method of Pearson and Lipman, PNAS 85:2444, 1988; computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package at Genetics Computer Group, 575 Science Dr., Madison, WI); or by manual alignment and visual inspection (see, e.g., Sambrook et al., (Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor, New York). York), 2012) and Ausubel et al., ("Contemporary Molecular Biology Experimental Guide", John Wiley & Sons Publishing Company, New York, Supplement 104, 2013). An example of a useful algorithm is PILEUP. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle, "Journal of Molecular Evolution (J.Mol.Evol.)" 35:351-360, 1987. The method used is similar to the method described by Higgins and Sharp, "Application of Computers in Biological Sciences" 5:151-153, 1989. Using PILEUP, the reference sequence is compared with other test sequences to determine the sequence identity percentage relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10) and weighted end gap. PILEUP can be obtained from the GCG sequence analysis software package, such as version 7.0 (Devereaux et al., "Nucleic Acids Research" 12:387-395, 1984).

[0179] Another example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, described in Altschul et al., Journal of Mol. Biol. 215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402, 1977, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both chains as defaults. The BLASTP program (for amino acid sequences) uses a word length (W) of 3, an expectation (E) of 10 as defaults, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989). Oligonucleotides are linear polynucleotide sequences of up to about 100 nucleotide bases in length.

[0180] As used herein, reference to "at least 80% identity" refers to "at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity" to a specified reference sequence. As used herein, reference to "at least 90% identity" refers to "at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity" to a specified reference sequence.

[0181] Specific binding: When referring to an antibody or antigen-binding fragment, it is meant that the binding reaction in the presence of a target protein is determined in the presence of a heterogeneous population of proteins and other biological agents. Thus, under specified conditions, the antibody preferentially binds to a specific target protein, peptide or polysaccharide (such as an antigen present on the surface of a pathogen, e.g., MPV F protein), and does not bind to other proteins present in a sample or subject in significant amounts. Specific binding can be determined by standard methods. See Harlow and Lane, Antibodies: A Laboratory Manual, 2nd ed., Cold Spring Harbor Publications, New York, (2013) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0182] Referring to an antibody-antigen complex, the specific binding of the antigen and the antibody has a specific binding affinity of less than about 10 -7 Mole (e.g. less than about 10 -8 Moore, 10 -9 molar or even less than about 10 -10 molar) K D . K D Refers to the dissociation constant for a given interaction (such as a polypeptide-ligand interaction or an antibody-antigen interaction). For example, for a bimolecular interaction of an antibody or antigen-binding fragment and an antigen, it is the concentration of the individual components of the bimolecular interaction divided by the concentration of the complex.

[0183] The antibody that specifically binds to the epitope on the hMPV F protein is an antibody that is substantially bound to the hMPV F protein (comprising cells or tissues expressing the hMPV F protein, substrates attached to the hMPV F protein, or hMPV F protein in a biological sample). Of course, it has been recognized that a certain degree of non-specific interaction may occur between the antibody and a non-target (such as cells that do not express the hMPV F protein). Generally, specific binding makes the association between the antibody and the protein or cell carrying the antigen much stronger than the association between the antibody and the protein or cell lacking the antigen. Compared with the protein or cell or tissue lacking this epitope, specific binding generally increases the amount (per unit time) of the antibody bound to the protein containing the epitope or the cell or tissue expressing the target epitope by more than 2 times, such as more than 5 times, more than 10 times or more than 100 times. Under such conditions, the specific binding to the protein requires the selection of an antibody that is specific to a specific protein. A variety of immunoassay formats are suitable for selecting antibodies or other ligands that are specifically immunoreactive with a specific protein. For example, solid phase ELISA immunoassays are routinely used to select monoclonal antibodies that are specifically immunoreactive with a protein.

[0184] Subject: A living multicellular vertebrate organism, including the class of humans and non-human mammals. In one example, the subject is a human. In a specific example, the subject is a newborn infant. In another example, a subject is selected in need of inhibiting hMPV infection. For example, the subject is not infected and is at risk of hMPV infection, or is infected and needs treatment.

[0185] Therapeutically effective amount: An amount of an agent (such as a disclosed antibody or antigen-binding fragment thereof) sufficient to prevent, treat (including prevent and treat), reduce and / or ameliorate the symptoms and / or underlying causes of a condition or disease, such as to prevent, inhibit and / or treat an hMPV infection. In some embodiments, a therapeutically effective amount is sufficient to reduce or eliminate symptoms of a disease, such as an hMPV infection. For example, this can be the amount required to inhibit or prevent viral replication or to measurably change the outward symptoms of a viral infection. Generally, this amount is sufficient to measurably inhibit viral replication or infectivity.

[0186] In one example, the desired response is to inhibit or reduce or prevent hMPV infection. In order for the method to be effective, it is not necessary to completely eliminate, reduce or prevent MPV infection. For example, as compared with a suitable control, the administration of a therapeutically effective amount of a medicament can reduce hMPV infection (e.g., as measured by cell infection or by the number or percentage of subjects infected by hMPV) by a desired amount, for example, by reducing at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or even at least 100% (eliminating or preventing detectable hMPV infection).

[0187] The therapeutically effective amount of the medicament can be administered in a single dose or in several doses during the course of treatment (such as primary immunity-boost vaccination treatment), for example, daily. However, the therapeutically effective amount may depend on the subject being treated, the severity and type of the condition being treated, and the mode of administration. The unit dosage form of the medicament can be packaged in a therapeutic amount or in multiples of a therapeutic amount, for example, in a vial (e.g., with a pierceable cap) or a syringe with sterile components.

[0188] Treat or prevent disease: Inhibit the full development of a disease or condition, for example, in a subject who is at risk of or has a disease, such as an hMPV infection. "Treatment" refers to a therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after it has begun to develop. With reference to a disease or pathological condition, the term "improvement" refers to any observable beneficial effect of a treatment. The beneficial effect can be demonstrated, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in the severity of some or all clinical symptoms of the disease, a slowing of the progression of the disease, a reduction in viral load, an improvement in the overall health or well-being of the subject, or by other parameters that are well known in the art and are specific to a particular disease. "Preventive and therapeutic" treatment is a treatment administered to subjects who do not show signs of the disease or only show early signs, with the goal of reducing the risk of developing the pathology.

[0189] The term "reduce" is a relative term, such that an agent reduces a disease or condition if the disease or condition is quantitatively reduced after administration of the agent, or if the disease or condition is reduced after administration of the agent compared to a reference agent. Similarly, the term "prevent" does not necessarily mean that the agent completely eliminates the disease or condition, as long as at least one characteristic of the disease or condition is eliminated. Thus, a composition that reduces or prevents an infection may, but does not necessarily, completely eliminate such an infection, as long as the infection is measurably reduced, for example, by at least about 50%, such as at least about 70%, or about 80%, or even about 90%, in the absence of the agent or compared to a reference agent.

[0190] Transformed: A transformed cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term "transformed" and the like (e.g., transformation, transfection, transduction, etc.) encompasses all techniques by which a nucleic acid molecule can be introduced into such a cell, including transduction with a viral vector, transformation with a plasmid vector, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.

[0191] Vector: An entity containing a nucleic acid molecule (such as a DNA or RNA molecule) carrying a promoter, which is operably connected to the coding sequence of the protein of interest and can express the coding sequence. Non-limiting examples include naked or packaged (lipid and / or protein) DNA, naked or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may not have the ability to replicate, or a virus or bacterium or other microorganism that has the ability to replicate. Vectors are sometimes referred to as constructs. A recombinant DNA vector is a vector with recombinant DNA. The vector may contain a nucleic acid sequence that allows it to replicate in a host cell (such as an origin of replication). The vector may also contain one or more selectable marker genes and other genetic elements. A viral vector is a recombinant nucleic acid vector having at least some nucleic acid sequences derived from one or more viruses. In some embodiments, the viral vector includes a nucleic acid molecule encoding a disclosed antibody or antigen-binding fragment that specifically binds to and neutralizes hMPV with the hMPV F protein.

[0192] Under conditions sufficient to perform the following operations: A phrase used to describe any circumstances that allow the desired activity.

[0193] II. Specific implementation methods

[0194] Provided are isolated monoclonal antibodies and antigen binding fragments that specifically bind to the hMPV F protein. The antibodies and antigen binding fragments can be fully human. The antibodies and antigen binding fragments can neutralize hMPV, for example, the disclosed antibodies can inhibit hMPV infection in vivo, and can be administered before or after infection with hMPV. Also disclosed herein are compositions comprising antibodies and antigen binding fragments and pharmaceutically acceptable carriers. Also provided are nucleic acids encoding antibodies or antigen binding fragments, and expression vectors (such as adeno-associated virus (AAV) viral vectors) comprising these nucleic acids. Antibodies, antigen binding fragments, nucleic acid molecules, host cells and compositions can be used for research, diagnosis, treatment and preventive purposes. For example, the disclosed antibodies and antigen binding fragments can be used to diagnose subjects with hMPV infection or can be administered to inhibit hMPV infection in subjects.

[0195] In some embodiments, the disclosed antibodies do not cross-react with RSV F protein. In other embodiments, these antibodies neutralize both genotypes A and B of hMPV. In additional embodiments, the disclosed antibodies bind to site III of MPV F protein. In yet other embodiments, the disclosed antibodies bind to pre-fusion F protein with a higher affinity than that of post-fusion F protein.

[0196] A. Monoclonal antibodies and antigen-binding fragments thereof that specifically bind to hMPV F protein

[0197] The discussion of monoclonal antibodies below refers to isolated monoclonal antibodies (or antigen-binding fragments thereof) comprising heavy and / or light chain variable domains, including CDR1, CDR2 and / or CDR3 (with reference to the IMGT numbering scheme) (unless otherwise indicated by the context). Various CDR numbering schemes (such as Kabat, Chothia or IMGT numbering schemes) can be used to determine CDR positions. The amino acid sequences and CDRs of the heavy and light chains of the monoclonal antibodies disclosed according to the IMGT numbering scheme are provided in the list of sequences, but these are exemplary only.

[0198] In some embodiments, monoclonal antibodies are provided that include the heavy and light chain CDRs of any of the antibodies described herein. In some embodiments, monoclonal antibodies are provided that include the heavy and light chain variable regions of any of the antibodies described herein.

[0199] Table 1. IMGT CDRs and SEQ ID NOs of antibodies

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208] a. Monoclonal antibody MPV86

[0209] In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV86 antibody and specifically binds to the hMPV F protein and neutralizes hMPV.

[0210] In some examples, the antibody or antigen-binding fragment comprises a V sequence that includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the MPV86 antibody, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0211] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 1. H In further embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 5. L , and specifically binds to hMPV F protein and neutralizes hMPV. In another embodiment, the antibody or antigen-binding fragment comprises a V protein independently comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 1 and 5, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0212] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 2, 3, and 4, respectively. H and / or a V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 6, 7 and 8, respectively. L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0213] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 2, 3, and 4, respectively. H , comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 6, 7 and 8, respectively. L, where V H comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1), and wherein said V L The antibody or antigen-binding fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 5 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 5), and the antibody or antigen-binding fragment specifically binds to the hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity falls outside the CDR.

[0214] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 1. H , and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 5. L , and specifically binds to hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequences shown in SEQ ID NOs: 1 and 5, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0215] b. Monoclonal antibody MPV414

[0216] In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV414 antibody and specifically binds to the hMPV F protein and neutralizes hMPV. In some examples, the antibody or antigen-binding fragment comprises a V protein comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the MPV414 antibody, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0217] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO:9. H, and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 13. L , and specifically binds to hMPV F protein and neutralizes hMPV. In another embodiment, the antibody or antigen-binding fragment comprises a V protein independently comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 9 and 13, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0218] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 10, 11, and 12, respectively. H and / or a V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 14, 15 and 16, respectively. L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0219] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 10, 11, and 12, respectively. H , comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 14, 15 and 16, respectively. L , where V H comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:9 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:9), and wherein said V L The antibody or antigen-binding fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 13 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13), and the antibody or antigen-binding fragment specifically binds to the hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity falls outside the CDR.

[0220] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 9. H, and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 13. L , and specifically binds to hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequences shown in SEQ ID NOs: 9 and 13, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0221] c. Monoclonal antibody MPV454

[0222] In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV454 antibody and specifically binds to the hMPV F protein and neutralizes hMPV.

[0223] In some examples, the antibody or antigen-binding fragment comprises a V sequence that includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the MPV454 antibody, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0224] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 17. H In further embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 21. L , and specifically binds to hMPV F protein and neutralizes hMPV. In another embodiment, the antibody or antigen-binding fragment comprises a V protein independently comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 17 and 21, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0225] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 18, 19, and 20, respectively. H and / or a V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 22, 23 and 24, respectively. L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0226] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 18, 19, and 20, respectively. H , comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 22, 23 and 24, respectively. L , where V H comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 17 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 17), and wherein said V L The antibody or antigen-binding fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 21 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 21), and the antibody or antigen-binding fragment specifically binds to the hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity falls outside the CDR.

[0227] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 17. H , and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 21. L , and specifically binds to hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequences shown in SEQ ID NOs: 17 and 21, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0228] d. Monoclonal antibody MPV456

[0229] In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV456 antibody and specifically binds to the hMPV F protein and neutralizes hMPV.

[0230] In some examples, the antibody or antigen-binding fragment comprises a V sequence that includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the MPV456 antibody, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0231] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 25. H , and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 29. L , and specifically binds to hMPV F protein and neutralizes hMPV. In another embodiment, the antibody or antigen-binding fragment comprises a V protein independently comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 25 and 29, respectively. H and V L , and binds to hMPV F protein and neutralizes hMPV.

[0232] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 26, 27, and 28, respectively. H and / or a V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 30, 31 and 32, respectively. L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0233] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 26, 27, and 28, respectively. H , comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 30, 31 and 32, respectively. L , where V Hcomprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 25 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 25), and wherein said V L The antibody or antigen-binding fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 29 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 29), and the antibody or antigen-binding fragment specifically binds to the hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity falls outside the CDR.

[0234] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 25. H , and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 29. L , and specifically binds to hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequences shown in SEQ ID NOs: 25 and 29, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0235] e. Monoclonal antibody MPV464

[0236] In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV464 antibody and specifically binds to the hMPV F protein and neutralizes hMPV.

[0237] In some examples, the antibody or antigen-binding fragment comprises a V sequence that includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the MPV464 antibody, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0238] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 33. HIn further embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 37. L , and specifically binds to hMPV F protein and neutralizes hMPV. In another embodiment, the antibody or antigen-binding fragment comprises a V protein independently comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 33 and 37, respectively. H and V L , and binds to hMPV F protein and neutralizes hMPV.

[0239] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 34, 35, and 36, respectively. H and / or a V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 38, 39 and 40, respectively. L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0240] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 34, 35, and 36, respectively. H , comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 38, 39 and 40, respectively. L , where V H comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:33 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:33), and wherein said V L The antibody or antigen-binding fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 37 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 37), and the antibody or antigen-binding fragment specifically binds to the hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity falls outside the CDR.

[0241] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 33. H, and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 37. L , and specifically binds to hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequences shown in SEQ ID NOs: 33 and 37, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0242] f. Monoclonal antibody MPV467

[0243] In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV467 antibody and specifically binds to the hMPV F protein and neutralizes hMPV.

[0244] In some examples, the antibody or antigen-binding fragment comprises a V sequence that includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the MPV467 antibody, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0245] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 41. H , and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 45. L , and specifically binds to hMPV F protein and neutralizes hMPV. In another embodiment, the antibody or antigen-binding fragment comprises a V protein independently comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 41 and 45, respectively. H and V L , and binds to hMPV F protein and neutralizes hMPV.

[0246] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 42, 43, and 44, respectively. H and / or a V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 46, 47 and 48, respectively. L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0247] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 42, 43, and 44, respectively. H , comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 46, 47 and 48, respectively. L , where V H comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:41 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:41), and wherein said V L The antibody or antigen-binding fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 45 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 45), and the antibody or antigen-binding fragment specifically binds to the hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity falls outside the CDR.

[0248] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 41. H , and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 45. L , and specifically binds to hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequences shown in SEQ ID NOs: 41 and 45, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0249] g. Monoclonal antibody MPV477

[0250] In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV477 antibody and specifically binds to the hMPV F protein and neutralizes hMPV.

[0251] In some examples, the antibody or antigen-binding fragment comprises a V sequence that includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the MPV477 antibody, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0252] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 49. H , and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 53. L , and specifically binds to hMPV F protein and neutralizes hMPV. In another embodiment, the antibody or antigen-binding fragment comprises a V protein independently comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 49 and 53, respectively. H and V L , and binds to hMPV F protein and neutralizes hMPV.

[0253] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 50, 51, and 52, respectively. H and / or a V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 54, 55 and 56, respectively. L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0254] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 50, 51, and 52, respectively. H , comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 54, 55 and 56, respectively L , where V Hcomprising an amino acid sequence that is at least 90% identical to SEQ ID NO:49 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:49), and wherein said V L The antibody or antigen-binding fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 53 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 53), and the antibody or antigen-binding fragment specifically binds to the hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity falls outside the CDR.

[0255] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 49. H , and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 53. L , and specifically binds to hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequences shown in SEQ ID NOs: 49 and 53, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0256] h. Monoclonal antibody MPV478

[0257] In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV478 antibody and specifically binds to the hMPV F protein and neutralizes hMPV.

[0258] In some examples, the antibody or antigen-binding fragment comprises a V sequence that includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the MPV478 antibody, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0259] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 57. HIn further embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 61. L , and specifically binds to hMPV F protein and neutralizes hMPV. In another embodiment, the antibody or antigen-binding fragment comprises a V protein independently comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 57 and 61, respectively. H and V L , and binds to hMPV F protein and neutralizes hMPV.

[0260] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 58, 59, and 60, respectively. H and / or a V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 62, 63 and 64, respectively. L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0261] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 58, 59, and 60, respectively. H , comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 62, 63 and 64, respectively. L , where V H comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:57 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:57), and wherein said V L The antibody or antigen-binding fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 61 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 61), and the antibody or antigen-binding fragment specifically binds to the hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity falls outside the CDR.

[0262] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 57. H, and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 61. L , and specifically binds to hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequences shown in SEQ ID NOs: 57 and 61, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0263] i. Monoclonal antibody MPV481

[0264] In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV481 antibody and specifically binds to the hMPV F protein and neutralizes hMPV.

[0265] In some examples, the antibody or antigen-binding fragment comprises a V sequence that includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the MPV481 antibody, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0266] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 65. H , and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 69. L , and specifically binds to hMPV F protein and neutralizes hMPV. In another embodiment, the antibody or antigen-binding fragment comprises a V protein independently comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 65 and 69, respectively. H and V L , and binds to hMPV F protein and neutralizes hMPV.

[0267] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 66, 67, and 68, respectively. H and / or a V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 70, 71 and 72, respectively. L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0268] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 66, 67, and 68, respectively. H , comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 70, 71 and 72, respectively. L , where V H comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 65 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 65), and wherein said V L The antibody or antigen-binding fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 69 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 69), and the antibody or antigen-binding fragment specifically binds to the hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity falls outside the CDR.

[0269] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 65. H , and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 69. L , and specifically binds to hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequences shown in SEQ ID NOs: 65 and 69, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0270] j. Monoclonal antibody MPV482

[0271] In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV482 antibody and specifically binds to the hMPV F protein and neutralizes hMPV.

[0272] In some examples, the antibody or antigen-binding fragment comprises a V sequence that includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the MPV482 antibody, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0273] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 73. H , and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 77. L , and specifically binds to hMPV F protein and neutralizes hMPV. In another embodiment, the antibody or antigen-binding fragment comprises a V protein independently comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 73 and 77, respectively. H and V L , and binds to hMPV F protein and neutralizes hMPV.

[0274] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 74, 75, and 76, respectively. H and / or a V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 78, 79 and 80, respectively. L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0275] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 74, 75, and 76, respectively. H , comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 78, 79 and 80, respectively. L , where V Hcomprising an amino acid sequence that is at least 90% identical to SEQ ID NO:73 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:73), and wherein said V L The antibody or antigen-binding fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 77 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 77), and the antibody or antigen-binding fragment specifically binds to the hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity falls outside the CDR.

[0276] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 73. H , and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 77. L , and specifically binds to hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequences shown in SEQ ID NOs: 73 and 77, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0277] k. Monoclonal antibody MPV483

[0278] In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV483 antibody and specifically binds to the hMPV F protein and neutralizes hMPV.

[0279] In some examples, the antibody or antigen-binding fragment comprises a V sequence that includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the MPV483 antibody, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0280] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 81. H, and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 85. L , and specifically binds to hMPV F protein and neutralizes hMPV. In another embodiment, the antibody or antigen-binding fragment comprises a V protein independently comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 81 and 85, respectively. H and V L , and binds to hMPV F protein and neutralizes hMPV.

[0281] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 82, 83, and 84, respectively. H and / or a V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 86, 87 and 88, respectively. L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0282] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 82, 83, and 84, respectively. H , comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 86, 87 and 88, respectively. L , where V H comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:81 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:81), and wherein said V L The antibody or antigen-binding fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 85 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 85), and the antibody or antigen-binding fragment specifically binds to the hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity falls outside the CDR.

[0283] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 81. H, and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 85. L , and specifically binds to hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequences shown in SEQ ID NOs: 81 and 85, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0284] 1. Monoclonal antibody MPV485

[0285] In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV485 antibody and specifically binds to the hMPV F protein and neutralizes hMPV.

[0286] In some examples, the antibody or antigen-binding fragment comprises a V sequence that includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the MPV485 antibody, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0287] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 89. H In further embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 93. L , and specifically binds to hMPV F protein and neutralizes hMPV. In another embodiment, the antibody or antigen-binding fragment comprises a V protein independently comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 89 and 93, respectively. H and V L , and binds to hMPV F protein and neutralizes hMPV.

[0288] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 90, 91, and 92, respectively. H and / or a V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 94, 95 and 96, respectively. L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0289] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 90, 91, and 92, respectively. H , comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 94, 95 and 96, respectively. L , where V H comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:89 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:89), and wherein said V L The antibody or antigen-binding fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 93 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 93), and the antibody or antigen-binding fragment specifically binds to the hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity falls outside the CDR.

[0290] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 89. H , and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 93. L , and specifically binds to hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequences shown in SEQ ID NOs: 89 and 93, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0291] m. Monoclonal antibody MPV486

[0292] In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV486 antibody and specifically binds to the hMPV F protein and neutralizes hMPV.

[0293] In some examples, the antibody or antigen-binding fragment comprises a V sequence that includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the MPV486 antibody, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0294] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 97. H In further embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 101. L , and specifically binds to hMPV F protein and neutralizes hMPV. In another embodiment, the antibody or antigen-binding fragment comprises a V protein independently comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 97 and 101, respectively. H and V L , and binds to hMPV F protein and neutralizes hMPV.

[0295] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 98, 99, and 100, respectively. H and / or a V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 102, 103 and 104, respectively. L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0296] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 98, 99, and 100, respectively. H , comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 102, 103 and 104, respectively. L , where V Hcomprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 97 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 97), and wherein said V L The antibody or antigen-binding fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 101 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 101), and the antibody or antigen-binding fragment specifically binds to the hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity falls outside the CDR.

[0297] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 97. H , and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 101. L , and specifically binds to hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequences shown in SEQ ID NOs: 97 and 101, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0298] n. Monoclonal antibody MPV487

[0299] In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV487 antibody and specifically binds to the hMPV F protein and neutralizes hMPV.

[0300] In some examples, the antibody or antigen-binding fragment comprises a V sequence that includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the MPV487 antibody, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0301] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 105. HIn further embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 109. L , and specifically binds to hMPV F protein and neutralizes hMPV. In another embodiment, the antibody or antigen-binding fragment comprises a V protein independently comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 105 and 109, respectively. H and V L , and binds to hMPV F protein and neutralizes hMPV.

[0302] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 106, 107, and 108, respectively. H and / or a V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 110, 111 and 112, respectively. L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0303] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 106, 107, and 108, respectively. H , comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 110, 111 and 112, respectively. L , where V H comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 105 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 105), and wherein said V L The antibody or antigen-binding fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 109 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 109), and the antibody or antigen-binding fragment specifically binds to the hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity falls outside the CDR.

[0304] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 105. H, and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 109. L , and specifically binds to hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequences shown in SEQ ID NOs: 105 and 109, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0305] o. Monoclonal antibody MPV488

[0306] In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV488 antibody and specifically binds to the hMPV F protein and neutralizes hMPV.

[0307] In some examples, the antibody or antigen-binding fragment comprises a V sequence that includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the MPV488 antibody, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0308] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 113. H In further embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 117. L , and specifically binds to hMPV F protein and neutralizes hMPV. In another embodiment, the antibody or antigen-binding fragment comprises a V protein independently comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 113 and 117, respectively. H and V L , and binds to hMPV F protein and neutralizes hMPV.

[0309] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 114, 115, and 116, respectively. H and / or a V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 118, 119 and 120, respectively. L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0310] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 114, 115, and 116, respectively. H , comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 118, 119 and 120, respectively. L , where V H comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 113 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 113), and wherein said V L The antibody or antigen-binding fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 117 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 117), and the antibody or antigen-binding fragment specifically binds to the hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity falls outside the CDR.

[0311] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 113. H , and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 117. L , and specifically binds to hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequences shown in SEQ ID NOs: 113 and 117, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0312] p. Monoclonal antibody MPV489

[0313] In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV489 antibody and specifically binds to the hMPV F protein and neutralizes hMPV.

[0314] In some examples, the antibody or antigen-binding fragment comprises a V sequence that includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the MPV489 antibody, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0315] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 121. H In further embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 125. L , and specifically binds to hMPV F protein and neutralizes hMPV. In another embodiment, the antibody or antigen-binding fragment comprises a V protein independently comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 121 and 125, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0316] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 122, 123, and 124, respectively. H and / or a V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 126, 127 and 128, respectively. L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0317] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 122, 123, and 124, respectively. H , comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 126, 127 and 128, respectively. L , where V Hcomprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 121 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 121), and wherein said V L The antibody or antigen-binding fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 125 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 125), and the antibody or antigen-binding fragment specifically binds to the hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity falls outside the CDR.

[0318] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 121. H , and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 125. L , and specifically binds to hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequences shown in SEQ ID NOs: 121 and 125, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0319] q. Monoclonal antibody MPV491

[0320] In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV491 antibody and specifically binds to the hMPV F protein and neutralizes hMPV.

[0321] In some examples, the antibody or antigen-binding fragment comprises a V sequence that includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the MPV491 antibody, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0322] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 129. HIn further embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 133. L , and specifically binds to hMPV F protein and neutralizes hMPV. In another embodiment, the antibody or antigen-binding fragment comprises a V protein independently comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 129 and 133, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0323] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 130, 131, and 132, respectively. H and / or a V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 134, 135 and 136, respectively. L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0324] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 130, 131, and 132, respectively. H , comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 134, 135 and 136, respectively. L , where V H comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 129 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 129), and wherein said V L The antibody or antigen-binding fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 133 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 133), and the antibody or antigen-binding fragment specifically binds to the hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity falls outside the CDR.

[0325] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 129. H, and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 133. L , and specifically binds to hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequences shown in SEQ ID NOs: 129 and 133, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0326] r. Monoclonal antibody MPV503

[0327] In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV503 antibody and specifically binds to the hMPV F protein and neutralizes hMPV.

[0328] In some examples, the antibody or antigen-binding fragment comprises a V sequence that includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the MPV503 antibody, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0329] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 137. H , and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 141. L , and specifically binds to hMPV F protein and neutralizes hMPV. In another embodiment, the antibody or antigen-binding fragment comprises a V protein independently comprising an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequence shown in SEQ ID NO: 137 and 141, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0330] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 138, 139, and 140, respectively. H and / or a V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 142, 143 and 144, respectively. L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0331] In some embodiments, the antibody or antigen-binding fragment comprises a V region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 138, 139, and 140, respectively. H , comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 142, 143 and 144, respectively. L , where V H comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 137 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 137), and wherein said V L The antibody or antigen-binding fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 141 (e.g., 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 141), and the antibody or antigen-binding fragment specifically binds to the hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity falls outside the CDR.

[0332] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 137. H , and specifically binds to hMPV F protein and neutralizes hMPV. In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 141. L , and specifically binds to hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequences shown in SEQ ID NOs: 137 and 141, respectively. H and V L , and specifically binds to hMPV F protein and neutralizes hMPV.

[0333] 1. Additional antibodies that bind to the MP467 epitope on the MPV F protein

[0334] Disclosed herein is MPV467 targeting hMPV F protein, and the antibody binds to the pre-fusion F protein with a higher affinity than the affinity of binding to the post-fusion F protein. The binding posture of MPV467 is disclosed, specifically it binds to a pre-fusion specific epitope overlapping with antigenic sites II and V on a single promoter. The helix-turn-helix (α6-α7) within the antigenic site II bound by MPV467 does not undergo conformational changes between the pre-fusion and post-fusion states. Therefore, in some embodiments, an antibody or antigen-binding fragment that specifically binds to an epitope on the hMPV F protein bound by MPV467 is provided, wherein the antibody does not cross-react with the RSV F protein. In other embodiments, an antibody or antigen-binding fragment that specifically binds to an epitope on the hMPV F protein (bound by any of the disclosed antibodies) is provided.

[0335] In some examples, antibodies that bind to an epitope of interest can be identified based on their ability to cross-compete (e.g., competitively inhibit binding in a statistically significant manner) with an MPV467 antibody provided herein in a binding assay. In other examples, antibodies that bind to an epitope of interest can be identified based on their ability to cross-compete (e.g., competitively inhibit binding in a statistically significant manner) with an MPV467 antibody provided herein or with any of the disclosed antibodies in a binding assay.

[0336] Any suitable method can be used to produce human antibodies that bind to the same epitope on the hMPV F protein to which the MPV467 antibody binds, or to which any of the disclosed antibodies binds. Such antibodies can be prepared, for example, by administering an immunogen to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic stimulation. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus or is present extrachromosomally or randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin loci are typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nature Biotechnology 23: 1117-1125 (2005). See also, for example, description of XENOMOUSE TM U.S. Patent Nos. 6,075,181 and 6,150,584 of the technology; description US Patent No. 5,770,429 describing KM US Patent No. 7,041,870 and the description Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0337] Human antibodies that bind to the same epitope on the hMPV F protein to which the MPV467 antibody binds, or to which any of the disclosed antibodies bind, can also be prepared by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, e.g., Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies produced by human B cell hybridoma technology are also described in Li et al., PNAS, 103:3557-3562 (2006). Additional methods include, for example, methods described in U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies by hybridoma cell lines) and Ni, "Modern Immunology (XiandaiMianyixue)", 26 (4): 265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in: Vollmers and Brandlein, "Histology and Histopathology (Histologyand Histopathology)", 20 (3): 927-937 (2005); and Vollmers and Brandlein, "Methods and Findings in Experimental and Clinical Pharmacology", 27 (3): 185-91 (2005). Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with the desired human constant domains.

[0338] Antibodies and antigen-binding fragments that specifically bind to the same epitope on the hMPV F protein (to which the MPV467 antibody binds) or to the same epitope on the hMPV F protein (to which any of the disclosed antibodies binds) can also be isolated by screening combinatorial libraries for antibodies with the desired binding properties. For example, by generating phage display libraries and screening such libraries for antibodies with the desired binding properties. Such methods are described, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Humana Press, Totowa, NJ). The present invention relates to the synthesis of proteins and their potential applications in the treatment of inflammatory cytokines. The present invention relates to the synthesis of proteins and their potential applications in the treatment of inflammatory cytokines. The present invention relates to the synthesis of proteins and their potential applications in the treatment of inflammatory cytokines. The present invention relates to the synthesis of proteins and their potential applications in the treatment of inflammatory cytokines. The present invention relates to the synthesis of proteins and their potential applications in the treatment of inflammatory cytokines. The present invention relates to the synthesis of proteins and their potential applications in the treatment of inflammatory cytokines. The present invention relates to the synthesis of proteins and their potential applications in the treatment of inflammatory cytokines. =Publishing House, 2003); Sidhu et al., Journal of Molecular Biology 338(2):299-310 (2004); Lee et al., Journal of Molecular Biology 340(5):1073-1093 (2004); Fellouse, Proceedings of the National Academy of Sciences of the United States of America 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0339] In some phage display methods, V H and V LThe phage library can be used to store a reservoir of genes and randomly recombined in a phage library, and then the antigen-binding phage in the phage library can be screened, such as Winter et al., "Ann. Rev. Immunol. ", 12: 433-455 (1994). Phages usually display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immune sources provide high-affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, natural reservoirs (e.g., from humans) can be cloned to provide a single source of antibodies to a wide range of non-self antigens and self-antigens without any immunization, such as Griffiths et al., "EMBO J, ", 12: 725-734 (1993) described. Finally, natural libraries can also be prepared synthetically by cloning unrearranged V-gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and complete rearrangement in vitro, as described by Hoogenboom and Winter, Journal of Mol. Biol. 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373 and U.S. Pat. Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936 and 2009 / 0002360.

[0340] 2. Additional Description of Antibodies and Antigen-Binding Fragments

[0341] The antibody or antigen-binding fragment can be a human antibody or fragment thereof. Chimeric antibodies are also provided. The antibody or antigen-binding fragment can contain any suitable framework region, such as (but not limited to) a human framework region from another source, or an optimized framework region. Alternatively, a heterologous framework region (such as but not limited to a mouse or monkey framework region) can be contained in the heavy chain or light chain of the antibody.

[0342] The antibody can be of any isotype. The antibody can be, for example, an IgM or IgG antibody, such as an IgG 1 IgG 2 IgG 3 or IgG 4 The class of an antibody that specifically binds to hMPV can be switched with another antibody. L or V H The nucleic acid molecule is isolated so that the nucleic acid molecule does not contain any nucleic acid sequence encoding the constant region of the light chain or heavy chain, respectively. Lor V H Nucleic acid molecules encoding C from different classes of immunoglobulin molecules L or C H This can be accomplished, for example, using a nucleic acid sequence comprising C L or C H For example, an antibody that specifically binds to PfCSP that is initially IgG can be class-switched to IgM. Class switching can be used to convert one IgG subclass to another IgG subclass, such as from IgG 1 Convert to IgG 2 IgG 3 or IgG 4 Conversion.

[0343] In some examples, the disclosed antibodies are oligomers of antibodies, such as dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, etc.

[0344] The antibody or antigen binding fragment can be derivatized to another molecule (such as another peptide or protein) or linked thereto. Generally speaking, the antibody or antigen binding fragment is derivatized so that binding to hMPV is not adversely affected by derivatization or labeling. For example, the antibody or antigen binding fragment can be functionally linked (by chemical coupling, genetic fusion, non-covalent association or other means) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a bifunctional antibody), a detectable marker, an effector molecule, or a protein or peptide that can mediate the association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).

[0345] (a) Binding affinity

[0346] In some embodiments, the antibody or antigen-binding fragment is present at a concentration of no more than 1.0 x 10 -8 M, not more than 5.0x 10 - 8 M, not more than 1.0x 10 -9 M, not more than 5.0x 10 -9 M, not more than 1.0x 10 -10 M, not more than 5.0x 10 -10 M or not more than 1.0x 10 -11 The affinity of M (e.g., by K D Measured) specific binding to hMPV F protein. DIt can be measured, for example, by a radiolabeled antigen binding assay (RIA) performed with a Fab version of the antibody of interest and its antigen. In one assay, the solution binding affinity of the Fab for the antigen is measured by ionizing the Fab with a minimal concentration of radiolabeled ( 125 I) Fab was equilibrated with labeled antigen, and then the bound antigen was captured and measured using a plate coated with anti-Fab antibody (see, e.g., Chen et al., Journal of Mol. Biol. 293(4):865-881, 1999). To establish the conditions for the assay, Multiwell plates (Thermo Scientific) were coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and then blocked for two to five hours at room temperature (approximately 23° C.) with PBS containing 2% (w / v) bovine serum albumin. TM Catalog No. 269620), 100 μM or 26 pM [ 125 I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57(20):4593-4599, 1997). The Fab of interest is then incubated overnight; however, the incubation can be continued for a longer period of time (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate is incubated with 0.1% polysorbate 20 When the plate was dry, 150 μl / well of scintillant (MICROSCINT TM -20; PerkinEmler), and place the board on TOPCOUNT TM The concentrations were counted for tens of minutes in a gamma counter (PerkinElmer). The concentration of each Fab that gave less than or equal to 20% of maximal binding was chosen for competitive binding assays.

[0347] In another assay, K D Can be measured using surface plasmon resonance at 25°C -2000 or -3000 (BIAcore, Inc., Piscataway, NJ) was measured using approximately 10 response units (RU) of an immobilized antigen CM5 chip. Briefly, carboxymethylated dextran biosensor chips (CM5, 10 μg / mL) were activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. company( Inc.)). Antigen was diluted to 5 μg / ml (about 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 liters / minute to achieve approximately 10 response units (RU) of coupled protein. After the injection of antigen, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were injected at a flow rate of approximately 25 liters / minute at 25°C into a 10 μL / min column containing 0.05% polysorbate 20 (TWEEN-20 TM ) surfactant (PBST) in PBS. By simultaneously fitting the association sensorgram and the dissociation sensorgram, a simple one-to-one Langmuir binding model ( Evaluation software version 3.2) to calculate the association rate (k on ) and dissociation rate (k off ). Equilibrium dissociation constant (K D ) is calculated as the ratio k off / k on See, e.g., Chen et al., Journal of Molecular Biology 293:865-881 (1999). If the association rate measured by the surface plasmon resonance above exceeds 10 6 M -1s-1 , the association rate can be determined by using a fluorescence quenching technique in the presence of a spectrometer such as a spectrophotometer equipped with a stop flow (Aviv Instruments) or an 8000 series SLM-AMINCO with a stirred cuvette. TM The increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) was measured in the presence of increasing concentrations of antigen as measured in a spectrophotometer (ThermoSpectronic) at 25°C containing 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2).

[0348] (b) Multispecific Antibodies

[0349] In some embodiments, there is provided an antibody or antigen binding fragment specifically bound to hMPV as provided herein, or a multispecific antibody, such as a bispecific antibody, of its antigen binding fragment. Any suitable method can be used to design and produce multispecific antibodies, such as cross-linking two or more antibodies, antigen binding fragments of the same type or different types (such as scFv). Exemplary methods for preparing multispecific antibodies include the method described in PCT Publication No. WO2013 / 163427, which is incorporated herein by reference in its entirety. Non-limiting examples of suitable cross-linking agents include heterobifunctional (having two different reactive groups spaced apart by appropriate spacers) (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl suberate) cross-linking agents.

[0350] The multispecific antibody can have any suitable form that allows binding to hMPV F protein by antibodies or antigen-binding fragments as provided herein. The bispecific single-chain antibody can be encoded by a single nucleic acid molecule. Non-limiting examples of bispecific single-chain antibodies and methods for constructing such antibodies are provided below: U.S. Patents Nos. 8,076,459, 8,017,748, 8,007,796, 7,919,089, 7,820,166, 7,635,472, 7,575,923, 7,435,549, 7,332,168, 7,323,440, 7,235,641, 7,229,760, 7,112,324, 6,723,538. Additional examples of bispecific single chain antibodies can be found in: PCT Application No. WO 99 / 54440; Mack et al., Journal of Immunology, 158(8):3965-3970, 1997; Mack et al., Proceedings of the National Academy of Sciences of the United States of America, 92(15):7021-7025, 1995; Kufer et al., Cancer Immunol. Immunother., 45(3-4):193-197, 1997; et al., Blood, 95(6):2098-2103, 2000; and Brühl et al., Journal of Immunology, 166(4):2420-2426, 2001. The generation of bispecific Fab-scFv ("diabody") molecules is described, for example, by Schoonjans et al., (J. Immunology, 165(12):7050-7057, 2000) and Willems et al., (J. Chromatogr. B Analyt. Technol. Biomed Life Sci. 786(1-2):161-176, 2003). For bifunctional antibodies, the scFv molecule can be fused to one of the VL-CL(L) or VH-CH1 chains, for example to generate a bifunctional antibody, one scFv is fused to the C-terminus of the Fab chain.

[0351] (c) Antigen-binding fragment

[0352] The present disclosure encompasses antigen-binding fragments, such as Fab, F(ab') 2 and Fv, the antigen binding fragment comprising a heavy chain and a V L , and specifically binds to the hMPV F protein. These antibody fragments retain the ability to selectively bind to the antigen and are "antigen-binding" fragments. Non-limiting examples of such fragments include:

[0353] (1) Fab, which can be produced by digesting whole antibodies with the enzyme papain to produce a complete light chain and a portion of one heavy chain, containing a monovalent antigen-binding fragment of an antibody molecule;

[0354] (2) Fab′, which can be obtained by treating whole antibody with pepsin followed by reduction to produce an intact light chain and a portion of the heavy chain;

[0355] (3)(Fab') 2 , a fragment of an antibody that can be obtained by treating a whole antibody with the enzyme pepsin without subsequent reduction; F(ab') 2 It is a dimer of two Fab' fragments held together by two disulfide bonds;

[0356] (4) Fv, containing V expressed as two chains L and V L Genetically engineered fragments of

[0357] (5) Single-chain antibodies (e.g., scFv), defined as antibodies containing a V chain linked by a suitable polypeptide linker H and V LThe V in scFv is a genetically engineered molecule as a genetically fused single-chain molecule (see, for example, Ahmad et al., Clin. Dev. Immunol., 2012, doi: 10.1155 / 2012 / 980250; Marbry and Snavely, I Drugs, 13(8): 543-549, 2010). H Domain and V L The intramolecular orientation of the domains is not critical for the provided antibodies (e.g., for the provided multispecific antibodies). Thus, two possible arrangements (V H Domain-Linker Domain-V L Domain; V L Domain-Linker Domain-V H domain).

[0358] (6) Single-chain antibody (scFv 2 ) is defined as a dimer of scFV. This is also called a "minibody".

[0359] Any suitable method for producing the antigen-binding fragments discussed above may be used. Non-limiting examples are provided in Harlow and Lane, Antibodies: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York, 2013.

[0360] Antigen-binding fragments can be prepared by proteolysis of antibodies or by expressing DNA encoding the fragments in host cells, such as E. coli cells. Antigen-binding fragments can be obtained by digesting whole antibodies with pepsin or papain by conventional methods. For example, antigen-binding fragments can be obtained by enzymatic cleavage of antibodies with pepsin to provide a fragment denoted as F(ab'). 2 This fragment can be further cleaved using a thiol reducing agent and, optionally, a blocking group for the sulfhydryl groups generated by cleavage of disulfide linkages to produce 3.5S Fab' monovalent fragments.

[0361] Other methods of cleaving antibodies (such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments or other enzymatic, chemical or genetic techniques) can also be used as long as the fragment binds to the antigen recognized by the intact antibody.

[0362] (d) Variant

[0363] In some embodiments, amino acid sequence variants of antibodies provided herein (such as MPV467 or any of the disclosed antibodies) are provided. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions (into the residues) and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be performed to obtain the final construct, provided that the final construct has the desired properties, for example, antigen binding.

[0364] In certain embodiments, antibody variants with one or more amino acid substitutions are provided. The sites of interest for substitution mutagenesis include CDR and framework regions. Amino acid substitutions can be introduced into the antibody of interest, and the desired activity of the screening product, for example, retained / improved antigen binding, reduced immunogenicity and / or improved ADCC or CDC.

[0365] Variants usually retain the V H With V L The amino acid residues necessary for proper folding and stabilization between the regions will be retained, and the charge characteristics of the residues will be preserved to maintain the low pI and low toxicity of the molecule. H With V L Amino acid substitutions were made in the region to increase yield.

[0366] In some embodiments, the heavy chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 1. In some embodiments, the light chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 5.

[0367] In some embodiments, the heavy chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 9. In some embodiments, the light chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 13.

[0368] In some embodiments, the heavy chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 17. In some embodiments, the light chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 21.

[0369] In some embodiments, the heavy chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 25. In some embodiments, the light chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 29.

[0370] In some embodiments, the heavy chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 33. In some embodiments, the light chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 37.

[0371] In some embodiments, the heavy chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 41. In some embodiments, the light chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 45.

[0372] In some embodiments, the heavy chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 49. In some embodiments, the light chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 53.

[0373] In some embodiments, the heavy chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 57. In some embodiments, the light chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 61.

[0374] In some embodiments, the heavy chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 65. In some embodiments, the light chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 69.

[0375] In some embodiments, the heavy chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 73. In some embodiments, the light chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 77.

[0376] In some embodiments, the heavy chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 81. In some embodiments, the light chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 85.

[0377] In some embodiments, the heavy chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 89. In some embodiments, the light chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 93.

[0378] In some embodiments, the heavy chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 97. In some embodiments, the light chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 101.

[0379] In some embodiments, the heavy chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 105. In some embodiments, the light chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 109.

[0380] In some embodiments, the heavy chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 113. In some embodiments, the light chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 117.

[0381] In some embodiments, the heavy chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 121. In some embodiments, the light chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 125.

[0382] In some embodiments, the heavy chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 129. In some embodiments, the light chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 133.

[0383] In some embodiments, the heavy chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 137. In some embodiments, the light chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence as shown in one of SEQ ID NO: 141.

[0384] In some embodiments, the antibody or antigen-binding fragment may comprise up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) in the framework region of the heavy chain of the antibody, or the light chain of the antibody, or both the heavy and light chains of the antibody, compared to known framework regions, or compared to the framework regions of the following antibodies, and retain specific binding activity to hMPV F protein: MPV86, MPV414, MPV454, MPV456, MPV464, MPV467, MPV477, MPV478, MPV481, MPV482, MPV483, MPV485, MPV486, MPV487, MPV488, MPV489, MPV491, or MPV503.

[0385] In some embodiments, substitutions, insertions or deletions may occur in one or more CDRs, as long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. H and V L In some embodiments of the sequences, each CDR is unchanged, or contains no more than one, two, or three amino acid substitutions.

[0386] To increase the binding affinity of the antibody, V mutations are generated in the HCDR3 region or the LCDR3 region in a process similar to the in vivo somatic mutation process responsible for affinity maturation of antibodies during the natural immune response. L and V H The segments can be randomly mutated. Therefore, in vitro affinity maturation can be performed by amplifying V using PCR primers complementary to HCDR3 or LCDR3, respectively. H and V LIn this process, the primers have been "spike-marked" with a random mixture of four nucleotide bases at certain positions so that the resulting PCR product encodes V H and V L Segment (where random mutations have been introduced into V H and / or V L CDR3 region). These random mutations can be tested H and V L segment to determine the binding affinity to hMPV F protein.

[0387] In some embodiments, the antibody (e.g., MPV86, MPV414, MPV454, MPV456, MPV464, MPV467, MPV477, MPV478, MPV481, MPV482, MPV483, MPV485, MPV486, MPV487, MPV488, MPV489, MPV491, or MPV503) or antigen-binding fragment is altered to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment. Glycosylation sites can be conveniently added or deleted by altering the amino acid sequence so that one or more glycosylation sites are created or removed.

[0388] When the antibody (e.g., MPV86, MPV414, MPV454, MPV456, MPV464, MPV467, MPV477, MPV478, MPV481, MPV482, MPV483, MPV485, MPV486, MPV487, MPV488, MPV489, MPV491, or MPV503) includes an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically include a CH bond to the Fc region, typically via an N-link. 2 The oligosaccharides may be branched biantennary oligosaccharides attached to Asn297 of the 1H domain. See, e.g., Wright et al., Trends Biotechnol. 15(1):26-32, 1997. Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibody may be modified to produce antibody variants with certain improved properties.

[0389] In one embodiment, antibody variants having a carbohydrate structure lacking fucose attached to the Fc region (directly or indirectly) are provided. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain relative to the sum of all sugar structures (e.g., complex, hybrid, and high mannose structures) attached to Asn 297 as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 in the Fc region; however, due to minor sequence variations in antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, US Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US 2004 / 0110282; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki et al., Journal of Molecular Biology, 336(5):1239-1249, 2004; Yamane-Ohnuki et al., Biotechnol. Bioeng. 87(5):614-622, 2004.Examples of cell lines capable of producing defucosylated antibodies include Lec 13 CHO cells lacking protein fucosylation (Ripka et al., Arch. Biochem. Biophys., 249(2):533-545, 1986; U.S. Patent Application Nos. US2003 / 0157108 and WO 2004 / 056312, particularly at Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotechnology and Bioengineering, 87(5):614-622, 2004; Kanda et al., Biotechnology and Bioengineering, 94(4):680-688, 2006; and WO2003 / 085107).

[0390] Antibody variants further provide bisected oligosaccharides, for example, wherein the biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described in, for example, WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharides attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described in, for example, WO 1997 / 30087, WO 1998 / 58964, and WO 1999 / 22764.

[0391] In several embodiments, the constant region of the antibody (such as MPV86, MPV414, MPV454, MPV456, MPV464, MPV467, MPV477, MPV478, MPV481, MPV482, MPV483, MPV485, MPV486, MPV487, MPV488, MPV489, MPV491 or MPV503) includes one or more amino acid substitutions to optimize the in vivo half-life of the antibody. The serum half-life of IgG Ab is regulated by the neonatal Fc receptor (FcRn). Therefore, in several embodiments, the antibody includes an amino acid substitution that increases binding to FcRn. Non-limiting examples of such substitutions include substitutions at T250Q and M428L in the IgG constant region (see, e.g., Hinton et al., Journal of Immunology, 176(1):346-356, 2006); M428L and N434S ("LS" mutations, see, e.g., Zalevsky et al., Nature Biotechnology, 28(2):157-159, 2010); N434A (see, e.g., Petkova et al., Int. Immunol. ol., 18(12):1759-1769, 2006); T307A, E380A and N434A (see, e.g., Petkova et al., Int. Immunol., 18(12):1759-1769, 2006); and M252Y, S254T and T256E (see, e.g., Dall'Acqua et al., J. Biol. Chem., 281(33):23514-23524, 2006). The disclosed antibodies and antigen-binding fragments can be linked to or include an Fc polypeptide comprising any of the substitutions listed above, e.g., an Fc polypeptide can comprise M428L and N434S substitutions.

[0392] In some embodiments, the constant region of the antibody includes one or more amino acid substitutions to optimize ADCC. ADCC is mainly mediated by a collection of closely related Fcγ receptors. In some embodiments, the antibody includes one or more amino acid substitutions that increase binding to FcγRIIIa. Non-limiting examples of such substitutions include IgG constant region S239D and I332E (see, e.g., Lazar et al., Proceedings of the National Academy of Sciences of the United States of America, 103 (11): 4005-4010, 2006); and S239D, A330L and I332E (see, e.g., Lazar et al., Proceedings of the National Academy of Sciences of the United States of America, 103 (11): 4005-4010, 2006) substitutions.

[0393] Combinations of the above substitutions are also included to produce IgG constant regions with increased binding to FcRn and FcγRIIIa. The combination increases antibody half-life and ADCC. For example, such combinations include antibodies with the following amino acid substitutions in the Fc region: (1) S239D / I332E and T250Q / M428L; (2) S239D / I332E and M428L / N434S; (3) S239D / I332E and N434A; (4) S239D / I332E and T307A / E380A / N434A; (5) S239D / I332E and M252Y / S254T / T256E; (6 ) S239D / A330L / I332E and 250Q / M428L; (7) S239D / A330L / I332E and M428L / N434S; (8) S239D / A330L / I332E and N434A; (9) S239D / A330L / I332E and T307A / E380A / N434A; or (10) S239D / A330L / I332E and M252Y / S254T / T256E. In some examples, the antibody or antigen-binding fragment thereof is modified such that it has direct cytotoxicity to infected cells or uses natural defenses such as complement, ADCC, or phagocytosis by macrophages.

[0394] In certain embodiments, the antibody provided herein can be further modified to contain additional non-protein moieties. The derivatized part suitable for the antibody includes but is not limited to water-soluble polymers. The non-limiting examples of water-soluble polymers include but are not limited to polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer) and dextran or poly-(n-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylene polyol (e.g., glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde can have advantages in preparation due to its stability in water. Polymer can have any molecular weight and can be branched or non-branched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, the polymer can be the same or different molecules. In general, the amount and / or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative is used in an application under defined conditions, etc.

[0395] B. Conjugates

[0396] As disclosed herein, antibodies and antigen-binding fragments that specifically bind to the hMPV F protein can be conjugated to an agent such as an effector molecule or a detectable marker. Both covalent and non-covalent attachment methods can be used. Various effector molecules and detectable markers can be used, including but not limited to toxins and radiopharmaceuticals, such as 125 I. 32 P. 14 C. 3 H and 35 S and other labels, targeting moieties and ligands, etc. The choice of a particular effector molecule or detectable marker depends on the particular target molecule or cell and the desired biological effect.

[0397] The procedures used to attach effector molecules or detectable markers to antibodies or antigen-binding fragments vary depending on the chemical structure of the effector. Polypeptides typically contain a variety of functional groups (e.g., carboxyl (-COOH), free amines (-NH 2 ) or sulfhydryl (-SH) group), which functional group can be used to react with a suitable functional group on the polypeptide to produce the binding of the effector molecule or detectable marker. Alternatively, the antibody or antigen binding fragment is derivatized to expose or attach additional reactive functional groups. Derivatization may involve attachment to any suitable linker molecule. The linker is capable of forming a covalent bond with both the antibody or antigen binding fragment and with the effector molecule or detectable marker. Suitable linkers include but are not limited to straight or branched carbon linkers, heterocyclic carbon linkers or peptide linkers. When the antibody or antigen binding fragment and the effector molecule or detectable marker are polypeptides, the linker can be joined to the constitutive amino acids through its side chain (such as through a disulfide bond with cysteine) or alpha carbon, or through the amino and / or carboxyl groups of the terminal amino acids.

[0398] In view of the large number of methods reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), toxins, and other agents to antibodies, an appropriate method for attaching a given agent to an antibody or antigen-binding fragment or other polypeptide can be determined.

[0399] The antibody or antigen-binding fragment can be conjugated to a detectable marker; for example, a detectable marker that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques such as CT, computerized axial tomography (CAT), MRI, magnetic resonance tomography (MTR), ultrasound, fiberoptic inspection, and laparoscopy. Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphorus, and the like. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) are also useful. The antibody or antigen binding fragment can also be conjugated to an enzyme that can be used for detection, such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc. When the antibody or antigen binding fragment is conjugated to a detectable enzyme, it can be detected by adding additional reagents that the enzyme is used to produce a discernible reaction product. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine produces a visually detectable colored reaction product. The antibody or antigen binding fragment can also be conjugated to biotin and detected by indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated to an enzyme or a fluorescent label.

[0400] The antibody or antigen-binding fragment can be conjugated with a paramagnetic agent such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide can also be used as labels. Antibodies can also be conjugated with lanthanides such as europium and dysprosium and manganese. The antibody or antigen-binding fragment can also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter gene such as a leucine zipper pair sequence, a binding site of a secondary antibody, a metal binding domain, an epitope tag.

[0401] The antibody or antigen-binding fragment can also be conjugated to a radiolabeled amino acid, for example, for diagnostic purposes. For example, the radiolabel can be used to detect hMPV by radiography, emission spectroscopy or other diagnostic techniques. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes: 3 H. 14 C. 35 S. 90 Y. 99m Tc, 111 In, 125 I. 131I. Radioactive labels can be detected, for example, using photographic film or a scintillation counter, and fluorescent markers can be detected using a photodetector to detect the emitted illumination. Enzymatic labels are typically detected by providing a substrate to the enzyme and detecting the reaction product resulting from the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

[0402] The average number of effector molecules or detectable marker moieties per antibody or antigen binding fragment in the conjugate can be, for example, in the range of 1 to 20 moieties per antibody or antigen binding fragment. In some embodiments, the average number of effector molecules or detectable marker moieties per antibody or antigen binding fragment in the conjugate is in the range of about 1 to about 2, about 1 to about 3, about 1 to about 8; about 2 to about 6; about 3 to about 5; or about 3 to about 4. The loading (e.g., effector molecule / antibody ratio) of the conjugate can be controlled in different ways, for example by: (i) limiting the molar excess of effector molecule-linker intermediate or linker reagent relative to the antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limiting reducing conditions for cysteine ​​thiol modification, (iv) engineering the amino acid sequence of the antibody by recombinant technology so that the number and position of cysteine ​​residues are modified to control the number or position of linker-effector molecule attachments.

[0403] C. Polynucleotides and Expression

[0404] Nucleic acid molecules (e.g., cDNA or RNA molecules) encoding the amino acid sequences of antibodies, antigen-binding fragments, and conjugates that specifically bind to hMPV as disclosed herein are provided. Nucleic acids encoding these molecules can be readily cloned using the amino acid sequences (e.g., CDR sequences and V H and V L sequences), sequences available in the art (such as framework or constant region sequences), and genetic code generation. In several embodiments, the nucleic acid molecule can encode the V sequence of the disclosed antibody or antigen-binding fragment. H 、V L or V H and V L Both (e.g., in a bicistronic expression vector). In some embodiments, the nucleic acid molecule encodes a scFv. In several embodiments, the nucleic acid molecule can be expressed in a host cell (e.g., a mammalian cell) to produce the disclosed antibodies or antigen-binding fragments.

[0405] The genetic code can be used to construct a variety of functionally equivalent nucleic acid sequences, such as nucleic acids that have different sequences but encode the same antibody sequence, or nucleic acids that encode a V L and / or V H A conjugate of a nucleic acid sequence or a fusion protein of a nucleic acid.

[0406] Nucleic acid molecules encoding antibodies, antigen-binding fragments and conjugates that specifically bind to the hMPV F protein can be prepared by any suitable method, including, for example, cloning appropriate sequences or direct chemical synthesis by standard methods. Chemical synthesis produces single-stranded oligonucleotides. This can be converted into double-stranded DNA by hybridization with complementary sequences or by polymerization with a DNA polymerase using a single strand as a template.

[0407] Exemplary nucleic acids can be prepared by cloning techniques. Examples of suitable cloning and sequencing techniques can be found in, for example, Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4th ed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. (eds.) (Current Molecular Biology Laboratory Manual, New York: John Wiley & Sons, including supplements).

[0408] Nucleic acids can also be prepared by amplification methods, including polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification system (TAS), and self-sustained sequence replication system (3SR).

[0409] Nucleic acid molecules can be expressed in recombinant engineered cells (such as bacteria, plants, yeast, insects and mammalian cells). Antibodies, antigen binding fragments and conjugates can be expressed as V H and / or V L The antibody and antigen-binding fragment may be expressed as a separate protein (linked to an effector molecule or detectable marker as desired), or as a fusion protein. Any suitable method for expressing and purifying antibodies and antigen-binding fragments may be used; non-limiting examples are provided in Al-Rubeai (ed.), Antibody Expression and Production, Dordrecht; New York: Springer, 2011. Immunoadhesins may also be expressed. Thus, in some examples, a V encoding H and V L The nucleic acid sequence may optionally encode a leader sequence.

[0410] To create scFv, V H Coding and V L The coding DNA fragment and another fragment encoding a flexible linker (for example, encoding an amino acid sequence (Gly 4 -Ser) 3 ) are operably linked such that V H and V L The sequence can be expressed as a continuous single-chain protein, where V L and VH The domains are joined by a flexible linker (see, e.g., Bird et al., Science, 242(4877):423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA, 85(16):5879-5883, 1988; McCafferty et al., Nature, 348:552-554, 1990; Kontermann and Dübel (eds.), Antibody Engineering, Vol. 1-2, 2nd ed., Springer-Verlag, 2010; Greenfield (ed.), Antibodies: A Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratory Press, 2014). Optionally, a cleavage site can be included in the linker (e.g., a furin cleavage site).

[0411] If only a single V H and V L , then the single-chain antibody can be monovalent, if two V H and V L , then the single-chain antibody can be bivalent, or if more than two V H and V L , then the single-chain antibody can be multivalent. Bispecific or multivalent antibodies that specifically bind to the hMPV F protein and another antigen can be produced. H and V L Optionally, it may include a H and V L Furin cleavage site between domains.

[0412] One or more DNA sequences encoding antibodies, Fabs or conjugates can be expressed in vitro by transferring the DNA into a suitable host cell. The cell can be prokaryotic or eukaryotic. A variety of expression systems that can be used to express proteins comprising Escherichia coli, other bacterial hosts, yeast and various higher eukaryotic cells (such as COS, CHO, HeLa) and myeloma cell lines can be used to express disclosed antibodies and Fabs. A stable transfer method can be used, which means that the exogenous DNA is continuously maintained in the host. The disclosure also encompasses hybridomas expressing the antibodies of interest.

[0413] The expression of nucleic acid encoding antibodies and antigen-binding fragments described herein can be achieved by operably connecting a DNA or cDNA domain promoter (which is constitutive or inducible) and then incorporating it into an expression cassette. The promoter can be any promoter of interest, including a cytomegalovirus promoter. Optionally, an enhancer (such as a cytomegalovirus enhancer) is included in the construct. The box can be suitable for replication and integration in prokaryotes or eukaryotes. A typical expression cassette contains a specific sequence that can be used to regulate the expression of the DNA encoding the protein. For example, the expression cassette can include a suitable promoter, an enhancer, a transcription and translation terminator, a start sequence, a start codon (i.e., ATG) in front of a protein-coding gene, a splicing signal of an intron, a sequence and a stop codon for maintaining the correct reading frame of the gene to allow the correct translation of mRNA. The vector can encode a selectable marker, such as a marker encoding drug resistance (e.g., ampicillin or tetracycline resistance).

[0414] In order to obtain high-level expression of cloned genes, it is desirable to construct an expression cassette comprising, for example, a strong promoter for instructing transcription, a ribosome binding site (for example, an internal ribosome binding sequence) and a transcription / translation terminator for translation initiation. For Escherichia coli, this can include a promoter (such as T7, trp, lac or lambda promoter), a ribosome binding site, and preferably a transcription termination signal. For eukaryotic cells, control sequences can include promoters and / or enhancers derived from, for example, immunoglobulin genes, HTLV, SV40 or cytomegalovirus, and polyadenylation sequences, and can further include splicing donors and / or acceptor sequences (for example, CMV and / or HTLV splicing acceptors and donor sequences). The cassette can be transferred to a selected host cell by any suitable method, such as conversion or electroporation for Escherichia coli and calcium phosphate treatment, electroporation or lipofection for mammalian cells. The cells transformed by the cassette can be selected by the antibiotic resistance given by the genes contained in the cassette (such as amp, gpt, neo and hyg genes).

[0415] The nucleic acid encoding the polypeptide described herein can be modified without reducing its biological activity. Some modifications can be made to promote the cloning, expression or incorporation of the targeting molecule into the fusion protein. Such modifications include, for example, a stop codon, a sequence for creating a convenient restriction site for localization, and the addition of methionine at the amino terminal to provide a sequence for a start site, or other amino acids (such as poly His) for an auxiliary purification step.

[0416] Once expressed, antibodies, antigen-binding fragments and conjugates can be purified according to standard methods in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, etc. (see generally, Simpson et al. (eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009). Antibodies, antigen-binding fragments and conjugates do not need to be 100% pure. Once purified, partially purified or purified to homogeneity as desired, the polypeptide should be substantially free of endotoxins if used for control.

[0417] Methods for expressing antibodies, antigen-binding fragments and conjugates from mammalian cells and bacteria (such as E. coli) and / or refolding into suitable active forms have been described and are applicable to the antibodies disclosed herein. See, for example, Greenfield (ed.), Antibodies: A Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratory Press, 2014, Simpson et al. (eds.), Basic Methods for Protein Purification and Analysis: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009, and Ward et al., Nature 341(6242):544-546, 1989.

[0418] D. Methods and Compositions

[0419] 1. Inhibition of hMPV infection

[0420] Disclosed herein is a method for inhibiting hMPV infection in a subject. The method comprises administering to a subject, to a subject at risk of hMPV infection or suffering from hMPV infection, an effective amount (i.e., an amount that effectively inhibits hMPV infection in the subject) of a disclosed antibody, antigen binding fragment, conjugate, or nucleic acid encoding such an antibody, antigen binding fragment, or conjugate. The method can be used before or after exposure.

[0421] In order for the method to be effective, it is not necessary to completely eliminate or inhibit hMPV infection. For example, the method can reduce hMPV infection by a desired amount, such as by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or even at least 100% (eliminating or preventing detectable hMPV infection), compared to hMPV infection in the absence of treatment. In some embodiments, the subject can also be treated with an effective amount of an additional agent (such as an antiviral agent).

[0422] In some embodiments, administration of an effective amount of the disclosed antibodies, Fabs, conjugates or nucleic acid molecules inhibits the establishment of hMPV infection and / or subsequent disease progression of the subject, which can encompass any statistically significant reduction in the hMPV activity (e.g., growth or invasion) of the subject or the symptoms of hMPV infection. Antibodies, Fabs, conjugates or nucleic acid molecules can be administered by any route of administration (including systemic or local administration). In one embodiment, administration is intranasal administration. In another embodiment, administration is such as by inhalation into the lungs. In another embodiment, administration is intramuscular administration.

[0423] Disclosed herein are methods for inhibiting hMPV replication in a subject. The method comprises administering to a subject, to a subject at risk of hMPV infection or suffering from hMPV infection, an effective amount (i.e., an amount that effectively inhibits hMPV replication in the subject) of a disclosed antibody, antigen binding fragment, conjugate, or nucleic acid encoding such an antibody, antigen binding fragment, or conjugate. The method can be used before or after exposure.

[0424] Methods for treating hMPV infection in a subject are disclosed. Methods for preventing hMPV infection in a subject are also disclosed. These methods comprise administering one or more hMPV F protein-specific antibodies, antigen-binding fragments, bispecific antibodies, conjugates, or nucleic acid molecules encoding such molecules or compositions comprising such molecules as disclosed herein.

[0425] Antibodies and Fabs thereof can be administered systemically, such as by intravenous infusion or intramuscular administration. Antibodies and Fabs thereof can be administered intranasally, intramuscularly or as by inhalation into the lungs. The dosage of antibodies or Fabs varies, but generally in the range of about 0.5mg / kg to about 50mg / kg, such as about 1mg / kg, about 5mg / kg, about 10mg / kg, about 20mg / kg, about 30mg / kg, about 40mg / kg or about 50mg / kg. In certain embodiments, the dosage of antibodies or Fabs can be about 0.5mg / kg to about 5mg / kg, such as about 1mg / kg, about 2mg / kg, about 3mg / kg, about 4mg / kg or about 5mg / kg. Antibodies or Fabs are administered according to the dosage regimen determined by a medical practitioner. In some instances, antibodies or Fabs are administered weekly, every two weeks, every three weeks or every four weeks.

[0426] In some embodiments, the method of inhibiting hMPV infection in a subject further comprises administering to the subject one or more additional agents. Additional agents of interest include, but are not limited to, antiviral agents.

[0427] In some embodiments, the method comprises administering a first antibody that specifically binds to a hMPV F protein as disclosed herein and a second antibody that also specifically binds to a hMPV F protein (e.g., a different epitope of a hMPV F protein). In some embodiments, the first antibody is one of: MPV86, MPV414, MPV454, MPV456, MPV464, MPV467, MPV477, MPV478, MPV481, MPV482, MPV483, MPV485, MPV486, MPV487, MPV488, MPV489, MPV491, or MPV503. In further embodiments, the second antibody is DDS7 or MPE8. In more embodiments, the first antibody is one of the following: MPV86, MPV414, MPV454, MPV456, MPV464, MPV467, MPV477, MPV478, MPV481, MPV482, MPV483, MPV485, MPV486, MPV487, MPV488, MPV489, MPV491 or MPV503, and the second antibody is another of the following: MPV86, MPV414, MPV454, MPV456, MPV464, MPV467, MPV477, MPV478, MPV481, MPV482, MPV483, MPV485, MPV486, MPV487, MPV488, MPV489, MPV491 or MPV503. An effective amount of one, two, three, four, five, or six of the following may be administered to the subject: MPV86, MPV414, MPV454, MPV456, MPV464, MPV467, MPV477, MPV478, MPV481, MPV482, MPV483, MPV485, MPV486, MPV487, MPV488, MPV489, MPV491, or MPV503. In a non-limiting example, the method comprises administering to the subject an effective amount of MPV467. The method may comprise administering an effective amount of one or more additional antibodies.

[0428] In some embodiments, DNA or RNA encoding the disclosed antibodies is administered to a subject to provide in vivo antibody production, for example using the subject's cellular machinery. Any suitable method of nucleic acid administration may be used; non-limiting examples are provided in U.S. Pat. No. 5,643,578, U.S. Pat. No. 5,593,972, and U.S. Pat. No. 5,817,637. U.S. Pat. No. 5,880,103 describes several methods for delivering protein-encoding nucleic acids to an organism. One method of administering nucleic acids is direct administration of plasmid DNA, such as administration of a mammalian expression plasmid. The nucleotide sequence encoding the disclosed antibodies or antigen-binding fragments thereof may be placed under the control of a promoter to increase expression. The methods include liposomal delivery of nucleic acids. Such methods may be applied to the production of antibodies or antigen-binding fragments thereof. In some embodiments, the disclosed antibodies or antigen-binding fragments are expressed in a subject using the pVRC8400 vector (described in Barouch et al., Journal of Virology, 79(14), 8828-8834, 2005, which is incorporated herein by reference).

[0429] In several embodiments, an effective amount of an AAV viral vector can be administered to a subject (such as a human subject at risk of hMPV infection or suffering from hMPV infection), wherein the AAV viral vector includes one or more nucleic acid molecules encoding the disclosed antibodies or antigen-binding fragments. The AAV viral vector is designed to express a nucleic acid molecule encoding the disclosed antibodies or antigen-binding fragments, and an effective amount of the AAV viral vector is administered to the subject so that an effective amount of the antibody or antigen-binding fragment is expressed in the subject. Non-limiting examples of AAV viral vectors that can be used to express the disclosed antibodies or antigen-binding fragments in the subject are included in the AAV viral vectors provided below: Johnson et al., Nature Medicine, 15(8):901-906, 2009 and Gardner et al., Nature, 519(7541):87-91, 2015, each of which is incorporated herein by reference in its entirety.

[0430] In one embodiment, nucleic acids encoding the disclosed antibodies or antigen-binding fragments thereof are introduced directly into tissue. For example, nucleic acids can be loaded onto gold microspheres by standard methods and expressed by, for example, the HELIOS TM The nucleic acid can be introduced into the skin by a device such as a gene gun. The nucleic acid can be "naked", consisting of a plasmid under the control of a strong promoter.

[0431] Typically, the DNA is injected into muscle, although the DNA may also be injected directly into other sites. The injection dose is typically around 0.5 μg / kg to about 50 mg / kg, and typically about 0.005 mg / kg to about 5 mg / kg (see, e.g., U.S. Pat. No. 5,589,466).

[0432] Compositions containing the disclosed hMPV F protein-specific antibodies, antigen-binding fragments, conjugates, or nucleic acid molecules encoding such molecules may be administered single or multiple times, depending on the dosage and frequency required and tolerated by the patient. The dose may be administered once, but may be administered regularly until the desired result is achieved or until side effects require discontinuation of therapy. Typically, the dose is sufficient to inhibit hMPV infection without causing unacceptable toxicity to the patient.

[0433] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. Dosages are generally within the range of ED 50 The dosage may vary within this range depending on the dosage form employed and the route of administration utilized. The effective dose may be determined by cell culture assays and animal studies.

[0434] hMPV F protein specific antibody, Fab, bispecific antibody, conjugate or nucleic acid molecule encoding such molecule or composition comprising such molecule can be applied to subject in various ways, and described mode comprises local and systemic administration, for example, by subcutaneous, intravenous, intraarterial, intraperitoneal, intramuscular, intradermal or intrathecal injection.In one embodiment, antibody, Fab, bispecific antibody, conjugate or nucleic acid molecule encoding such molecule or composition comprising such molecule is applied once a day by single subcutaneous, intravenous, intraarterial, intraperitoneal, intramuscular, intradermal or intrathecal injection.Antibody, Fab, bispecific antibody, conjugate or nucleic acid molecule encoding such molecule or composition comprising such molecule can also be applied by direct injection at or near the disease site.Other application method is by osmotic pump (for example, Alzet pump) or miniature pump (for example, Alzet miniature osmotic pump), which allows controlled, continuous and / or sustained release delivery of antibody, Fab, conjugate or nucleic acid molecule encoding such molecule or composition comprising such molecule in a predetermined time period. Osmotic pumps or minipumps can be implanted subcutaneously at or near the target site.

[0435] 2. Composition

[0436] A composition is provided, comprising one or more of the hMPV F protein-specific antibodies, antigen-binding fragments, conjugates, or nucleic acid molecules encoding such molecules disclosed herein in a pharmaceutically acceptable carrier. In some embodiments, the composition includes the following antibodies or antigen-binding fragments thereof disclosed herein: MPV86, MPV414, MPV454, MPV456, MPV464, MPV467, MPV477, MPV478, MPV481, MPV482, MPV483, MPV485, MPV486, MPV487, MPV488, MPV489, MPV491, or MPV503. In some embodiments, the composition includes two, three, four, or more antibodies that specifically bind to the hMPV F protein. In a specific non-limiting example, the antibody is MVP467. The composition can be used, for example, to inhibit or detect hMPV infection. The composition can be prepared in a unit dosage form for administration to a subject. The amount and time of administration are determined by the administering physician to achieve the desired purpose. Antibodies, antigen binding fragments, conjugates, or nucleic acid molecules encoding such molecules can be formulated for systemic or local administration. In one example, antigen binding fragments, conjugates, or nucleic acid molecules encoding such molecules are formulated for parenteral administration, such as intravenous administration.

[0437] In some embodiments, the antibody, antigen-binding fragment, or conjugate thereof in the composition is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) pure. In some embodiments, the composition contains less than 10% (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or even less) of macromolecular contaminants, such as other mammalian (e.g., human) proteins.

[0438] The composition for administration can comprise a solution of an antibody, an Fab, a conjugate or a nucleic acid molecule encoding such a molecule dissolved in a pharmaceutically acceptable carrier (such as an aqueous carrier). A variety of aqueous carriers can be used, such as buffered saline, etc. These solutions are sterile and generally do not contain undesirable substances. These compositions can be sterilized by any suitable technology. The composition can contain pharmaceutically acceptable auxiliary substances required for close physiological conditions, such as pH adjusting agents and buffers, toxicity regulators, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the antibody in these formulations can vary over a wide range, and will be selected based on the specific mode of administration selected and the needs of the subject, mainly based on fluid volume, viscosity, body weight, etc.

[0439] Typical compositions for intravenous administration include about 0.01 to about 30 mg / kg of the antibody or antigen-binding fragment or conjugate / subject / day (or a corresponding dose of a conjugate comprising the antibody or antigen-binding fragment). Any suitable method can be used to prepare an administrable composition; non-limiting examples are provided in such publications as Remington: The Science and Practice of Pharmacy, 22nd edition, London, UK: Pharmaceutical Press, 2013. In some embodiments, the composition can be a liquid formulation comprising one or more antibodies, antigen-binding fragments (such as antibodies or antigen-binding fragments that specifically bind to PfCSP) at a concentration ranging from about 0.1 mg / ml to about 20 mg / ml, or from about 0.5 mg / ml to about 20 mg / ml, or from about 1 mg / ml to about 20 mg / ml, or from about 0.1 mg / ml to about 10 mg / ml, or from about 0.5 mg / ml to about 10 mg / ml, or from about 1 mg / ml to about 10 mg / ml.

[0440] Antibodies or their Fabs or conjugates or nucleic acids encoding such molecules can be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentrations. Antibody solutions or Fabs or nucleic acids encoding such antibodies or Fabs can then be added to an infusion bag containing 0.9% sodium chloride (USP), and are generally administered at a dosage of 0.5 to 15 mg / kg body weight. Since rituximab was approved in 1997, this area has considerable experience in the administration of antibody drugs, which have been listed in the U.S. Antibodies, Fabs, conjugates or nucleic acids encoding such molecules can be administered by slow infusion rather than intravenous push or bolus injection. In one example, a higher loading dose is administered, followed by a maintenance dose administered at a lower level. For example, an initial loading dose of 4 mg / kg can be infused in a time period of about 90 minutes, followed by a weekly maintenance dose of 2 mg / kg infused in a time period of 30 minutes if the previous dose is well tolerated, for 4-8 weeks.

[0441] Controlled release parenteral formulations can be made into implants, oily injections or particle systems. For a broad overview of protein delivery systems, see Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Lancaster, PA: Technomic Publishing Company, Inc., 1995. Microparticle systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres and nanoparticles. Microcapsules contain active protein agents, such as cytotoxins or drugs as central cores. In microspheres, active protein agents are dispersed throughout the particles. Particles, microspheres and microcapsules less than about 1 μm are usually referred to as nanoparticles, nanospheres and nanocapsules, respectively. The diameter of the capillary is about 5 μm, so that nanoparticles can only be administered intravenously. The diameter of microparticles is generally about 100 μm, and is administered subcutaneously or intramuscularly. See, e.g., Colloidal Drug Delivery Systems, J. Kreuter (ed.), New York, NY: Marcel Dekker, Inc., pp. 219-342, 1994; and Tice and Tabibi, Treatise on Controlled Drug Delivery: Fundamentals, Optimization, Applications, A. Kydonieus (ed.), New York, NY: Marcel Dekker, Inc., pp. 315-339, 1992.

[0442] Polymers can be used for the ion controlled release of antibody compositions disclosed herein. Any suitable polymer can be used, such as a degradable or non-degradable polymer matrix designed for controlled drug delivery. Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins. On the other hand, liposomes are used for controlled release of lipid-encapsulated drugs and drug targeting.

[0443] 2. Methods of detection and diagnosis

[0444] Also provided is a method for detecting the presence of hMPV F protein in vitro or in vivo. In one example, the presence of hMPV F protein is detected in a biological sample from a subject, and can be used to identify a subject with hMPV infection. The sample can be any sample, including but not limited to tissues from biopsy, autopsy and pathological samples. The biological sample also includes tissue sections, such as frozen sections obtained for histological purposes. The biological sample further includes body fluids such as blood, serum, plasma, sputum, cerebrospinal fluid or urine. The method of detection can be included in contact with an antibody or antigen-binding fragment or its conjugate (e.g., a conjugate comprising a detectable marker) that specifically binds to a cell or sample and hMPV F protein under conditions sufficient to form an immune complex, and detect the immune complex (e.g., by detecting a detectable marker conjugated to the antibody or antigen-binding fragment).

[0445] In one embodiment, the antibody or Fab is directly labeled with a detectable marker. In another embodiment, the antibody (primary antibody) bound to the hMPV F protein is unlabeled, and a secondary antibody or other molecule that can be bound to the primary antibody is used for detection. A secondary antibody that can be specifically bound to a particular species and class of the first antibody is selected. For example, if the first antibody is human IgG, the secondary antibody can be anti-human IgG. Other molecules that can be bound to the antibody include but are not limited to protein A and protein G, both of which are commercially available. Suitable labels for antibodies, Fabs or secondary antibodies are known and as described above, and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials.

[0446] In some embodiments, the disclosed antibodies or antigen-binding fragments thereof are used to test vaccines. For example, a vaccine composition comprising an hMPV F protein or a fragment thereof is tested to see whether it presents a pre-fusion conformation of an epitope comprising the disclosed antibody. Therefore, a method for testing a vaccine is provided herein, wherein the method includes contacting a sample containing a vaccine (such as an hMPV F protein immunogen) with a disclosed antibody or antigen-binding fragment under conditions sufficient to form an immune complex, and detecting the immune complex to detect a vaccine containing an epitope of interest in the sample. In one example, detection of immune complexes in a sample indicates that a vaccine component (such as an hMPV F protein immunogen) presents a conformation that can bind to an antibody or antigen-binding fragment.

[0447] In view of the many possible embodiments to which the principles of the present invention may be applied, it should be appreciated that the embodiments shown are merely examples of the present invention and should not be viewed as limiting the scope of the present invention. On the contrary, the scope of the present invention is defined by the following claims. Therefore, all contents falling within the scope and spirit of these claims are claimed as the present invention.

[0448] Examples

[0449] Human metapneumovirus (hMPV) is a leading cause of childhood morbidity and hospitalization worldwide, however, there are currently no vaccines or therapeutics available for hMPV disease prevention and treatment. The hMPV fusion (F) protein is the only target for neutralizing antibodies. In order to map the immunodominant epitopes on the hMPV F protein, 18 human monoclonal antibodies (mAbs) were isolated and the binding affinity, neutralization potency, and epitope specificity of the mAbs were evaluated. Most mAbs target different epitopes on the hMPV F protein, and multiple mAb binding methods for antigenic site III were found. The most potent mAb, MPV467, was examined in prophylactic and therapeutic mouse challenge studies, and when administered 24 hours before or 72 hours after viral infection, MPV467 limited viral replication in the lungs of mice. In addition, this antibody has been shown to be effective in the cotton rat (Sigmodon hispidus) model. Using cryo-electron microscopy to The structure of MPV467 in complex with the hMPV F protein was determined at 1.300 nm resolution, revealing a complex prefusion-specific epitope that overlaps with antigenic sites II and V on a single promoter. These data reveal novel insights into immunodominant epitopes on the hMPV F protein, identify novel mAb therapeutics for hMPV F disease prevention and treatment, and provide insights into the discovery of a unique prefusion-specific epitope on the hMPV F protein.

[0450] Example 1

[0451] Human monoclonal antibody sequence determinants

[0452] To further define the antigenic epitopes on the hMPV F protein, 18 novel human mAbs were isolated using the hMPV B2 F protein (Biacchesi et al., Journal of Virology 78, 12877-12887 (2004)), of which sixteen mAbs were generated by human hybridoma technology and two mAbs were derived from antigen-specific single B cell sorting (MPV491, 503). The antibody encoding genes were sequenced and the results indicated that different sets of immunoglobulin V genes were used across the panel ( Figure 1A). mAbs utilizing VH1-69 genes are the most abundant. The VH3 and VH4 gene families comprise the majority of additional mAbs. Diversity also exists in the light chain, with eight and five unique genes for κ and λ mAbs, respectively. κ isotype mAbs utilize the VK1, VK2, VK3, and VK4 gene families, while λ isotype mAbs use the VL1 and VL3 gene families. The lengths of the heavy and light chain joining portions range from 14 to 23 amino acids for the heavy chain; 8 to 10 amino acids for the κ chain; and 9 to 11 amino acids for the λ chain ( Figure 1B The percent identity of the variable genes to the germline sequences ranged from 88-97% (average 93.6%) for the heavy chain and 90-97% (average 93.7%) for the light chain ( Figure 1C ).

[0453] Example 2

[0454] mAb binding and functional properties

[0455] The neutralizing activity of each mAb was determined by plaque reduction assay using representative viruses from each genotype of hMPV, i.e., hMPV CAN / 97-83 (genotype A) and hMPV TN / 93-32 (genotype B) ( Figure 6 , Fig.10 ). All mAbs had neutralizing activity against viruses from both genotypes, with mAbs MPV467, MPV487, MPV454, MPV482, and MPV488 having neutralizing activity against hMPV CAN / 97-83 below 20 ng / mL. mAbs MPV467, MPV487, and MPV454 had the most potent neutralizing activity against both hMPV CAN / 97-83 and hMPV TN / 93-32, with MPV467 reaching picomolar activity (below 1 ng / mL) against hMPV TN / 93-32. Based on IC 50 It is the IC of hMPV TN / 93-32 50At most 1 / 20 of the neutralization of hMPV CAN / 97-83, mAbs MPV86, MPV488, MPV485 and MPV477 demonstrated the preference neutralization of hMPV CAN / 97-83. A small group of hMPV F proteins from each subgroup (hMPV A1 F, hMPV A2 F, hMPV B1 F, hMPVB2 F) of a mixture containing hMPV F before and after fusion was used to evaluate the binding properties of mAbs. The binding of mAbs to other constructs containing only hMPV F before monomer fusion, hMPV F after fusion and hMPV F before major trimer fusion (hMPV B2 F GCN4) was also evaluated. Several binding modes were observed. The binding of MPV487, MPV482, MPV503, MPV414, MPV86 and MPV488 to the F construct after fusion is limited, and tends to bind to the pre-fusion construct. mAbs MPV467, MPV454, MPV477, MPV486 and MPV464 bound to both the pre-fusion and post-fusion constructs, but had a higher binding affinity to the pre-fusion protein. mAbs MPV478, MPV483, MPV481, MPV456, MPV491, MPV489 and MPV485 bound equally to both the pre-fusion and post-fusion constructs.

[0456] Example 3

[0457] Epitope mapping

[0458] To determine the common binding epitopes for a small set of 18 mAbs, epitope binning experiments were performed using biolayer interferometry as previously described (Huang and Mousa, PLOS Pathog. 16, e1008942 (2020); Bar-Peled et al., Journal of Virology 93, e00342--19 (2019)). The biosensor was loaded with hMPV B2 prefusion F protein, associated with the test mAb, and then exposed to a control mAb with a known epitope to determine the competition spectrum ( Figure 2A ). Control mAbs targeting known hMPV epitopes included mAbs MPE8 and MPV364 (site III), DS7 and MPV196 (DS7 epitope), 101F (site IV), and MPV458 (66-87 trimer epitope) ( Figure 2B MPV481 and MPV483 were mapped to antigenic site IV, whereas MPV454 competed with both 101F and DS7, indicating that it binds to an intermediate epitope between site IV and DS7 ( Figure 2B). However, MPV454 did not compete with the previously identified MPV196, but rather with DS7. MPV464, MPV491, MPV485, and MPV477 competed with both MPE8 and DS7, similar to previous results with MPV196, MPV201, and MPV314 (Bar-Peled et al., Journal of Virology 93, e00342--19 (2019)). mAbs MPV86, MPV414, MPV482, MPV487, and MPV503 are "MPV364-like," competing with MPE8 and MPV364, but not DS7. This differential binding mode of antigenic site III has previously been defined by competition with DS7 or lack thereof (Bar-Peled et al., Journal of Virology 93, e00342--19 (2019)). No mAbs were observed to compete with MPV458 targeting within the trimer, although moderate competition was observed between several mAbs and MPV458, suggesting that these mAbs may partially block the binding of MPV458 or limit the exposure of an epitope within the trimer bound by MPV458 centered around amino acids 66-87. MPV488 and MPV489 partially competed with MPE8 but not with MPV364, while MPV467 and MPV456 partially competed with MPV364 but not with MPE8, suggesting the presence of additional epitopes near antigenic site III. MPV486 and MPV478 showed partial competition with almost all mAbs, and their epitopes could not be determined.

[0459] Example 4

[0460] hMPV F-specific mAb enhances antibody-dependent phagocytosis of THP-1 cells

[0461] Antibodies that bind to different hMPV F antigenic sites were selected to evaluate antibody-dependent phagocytic activity (Figure 3). All mAbs tested significantly enhanced phagocytosis of THP-1 cells in vitro compared to blank and isotype control mAb (PhtD3, mAb that binds to Streptococcus pneumoniae). Antibodies that bind to the DS7 site showed an overall higher phagocytic score, while the remaining mAbs differed in ADP activity. In addition, the EC values ​​of the mAbs were significantly higher than those of the controls. 50 / IC 50 No correlation was observed with ADP activity, indicating that the ADP activity of the hMPV F-specific mAbs is not related to the Fab binding epitope. These data suggest that the anti-hMPV F mAbs have additional protective mechanisms besides neutralization.

[0462] Example 5

[0463] Therapeutic efficacy of MPV467

[0464] MPV467 was the most potent mAb in this panel, achieving picomolar neutralization potency against hMPV TN / 93-32 and effectively neutralizing hMPV CAN / 97-83. This mAb has a prefusion bias, as there is a substantial loss of binding to the postfusion hMPV F protein ( Fig.10 ). Based on these data, the protective efficacy of mAb MPV467 was tested in the hMPV infection model of BALB / c mice. In both the preventive and treatment studies, male and female mice were treated with PBS, isotype control human mAb or mAb MPV467 24 hours before hMPV infection or three days after hMPV infection (Figure 4). On the fifth day, the viral titer in the mouse lungs was determined by plaque assay. In any study, no virus was detected in mice treated with MPV467, while viruses were present in both mice treated with PBS and mice treated with isotype mAbs. No differences were observed between MPV467 and uninfected mice, and no differences were observed between PBS and isotype control mice in any study.

[0465] Example 6

[0466] Structural definition of the hMPV F-MPV467 complex

[0467] Since MPV467 is the most potent hMPV F mAb described to date, capable of protecting against and treating hMPV infection and targeting an undefined epitope, cryo-electron microscopy (cryo-EM) was used to characterize the hMPV F mAb. The structure of MPV467 in complex with pre-fusion hMPV F was determined at global resolution ( Figures 5A-5C The final map for model building was generated by particle subtraction of the flexible Fab constant region and sharpening by DeepEMhancer (Sanchez-Garcia et al., Commun. Biol. 4, 874 (2021)). The structure reveals that MPV467 has an approach angle pointing downward to the viral membrane ( Figure 5A , 5B ). The heavy and light chains cover the hMPV F and The MPV467 epitope spans antigenic sites II and V but also contacts a single residue in antigenic site III, Tyr44. hMPV F site V residue Arg156 makes multiple interactions with MPV467 through hydrogen bonds to both CDRH1 and CDRH3 backbone atoms and salt bridge interactions with CDRH1 Asp31 ( Figure 5C ). In addition, CDRH1 Asp31 also forms a hydrogen bond with site V residue Thr150. The conformationally fixed antigenic site II is bound by MPV467 CDRH3 via two hydrogen bond interactions with hMPV F Asn233 and Thr236. The only specific interaction between the light chain of MPV467 and hMPV F is via a main chain hydrogen bond between CDRL1 Asn30 and hMPV F Ala238.

[0468] Humans have been exposed to hMPV for at least 70 years (van den Hoogen et al., Nature Medicine 7, 719-724 (2001)), yet the major epitopes on the hMPV F protein remain elusive. Variable region sequence analysis showed that the V H and V K / V L Diversity in gene usage. Among these genes, V H 1-69 was shared by 4 of the 18 mAbs (MPV86, MPV414, MPV483, and MPV503), and three of these four mAbs (except MPV483) showed similar binning profiles competing with MPE8 and MPV364 ( Figure 2A , 2B ), indicating that they may bind to the same hMPV F epitope through a similar heavy chain binding mode. A similar correlation between binding epitope and V gene usage was also observed in the light chain. mAbs MPV86 and 487, which share IGKV3-11, and mAbs MPV 414 and 503, which share IGKV3-15, all bind to the MPV364 site, while mAbs MPV464 and 485, which share IGLV3-1, bind to the DS7 site. mAbs MPV414 and MPV503 were identified from two different subjects using two different methods, and they still have the same V H / V K genes, thus suggesting that this pair of genes might be favored by hMPV F MPV364 site-specific mAbs.

[0469] The major antigenic sites on hMPV F were mapped. In addition to the known antigenic sites III, IV, DS7, and 66-87, site V was further characterized and a putative site II was identified. Similar to sites III and IV, the positions of both sites V and II are similar to their counterparts on RSV F, suggesting that the epitopes in these regions share structural features that can be recognized by human antibodies. However, no mAbs were found to bind to the RSV F site on hMPV F. The counterparts of The binding of mAbs to this epitope may be limited by the presence of N-linked glycans on hMPV F at the 37 site (Asn57 and Asn172) (Poor et al., Proc. Natl. Acad. Sci. USA 111, 2596-2605 (2014)).

[0470] The most potent mAb (MPV467) binds to an epitope located across antigenic sites II and V. The structure of hMPV F in its pre- and post-fusion conformations reveals that it has structural homology with the related RSV F protein and it is expected that the neutralizing epitope on RSV F has a counterpart on hMPV F. Structurally, the antigenic sites and V are pre-fusion specific and previously isolated RSV antibodies, and structural studies have shown that antibodies targeting these regions are often highly effective neutralizers (Gilman et al., Science Immunology 1, 1-12 (2016); Mousa et al., Nature Microbiology 2, 16271 (2017)). Here, in the case of the cryo-EM structure of mAb MPV467 in complex with hMPV F, it was determined that this mAb is one of the antibodies targeting site V found for hMPV F and that it does share the properties of potent neutralization. MPV467 binds to a β-hairpin (β3-β4) located within antigenic site V, which undergoes a large conformational change during the transition from pre-fusion to post-fusion. Without being bound by theory, this region of the MPV467 epitope may be responsible for the potency of MPV467. When bound, the fusion protein cannot transition to a post-fusion conformation, which is required for effective viral infection. Additionally, the helix-turn-helix (α6-α7) within antigenic site II bound by MPV467 does not undergo conformational changes between the pre- and post-fusion states. The binding of residues in this region may be why MPV467 is able to bind to both conformational states. Since only this portion of the epitope is present in the post-fusion conformation, a decrease in binding affinity can be seen compared to the pre-fusion conformation, which contains the entire epitope.

[0471] RSV / hMPV F cross-reactive mAb M1C7 (Xiao et al., MAb 11, 1415-1427 (2019)) was previously reported as a potent neutralizing mAb targeting site V. Similarly, RSV F site V-specific mAb (hRSV90) (Mousa et al., Nature Microbiology 2, 16271 (2017)) also has a low IC against RSV 50 (4 ng / mL for RSV A and 10 ng / mL for RSV B), indicating that site V is a vulnerable region favored by ultra-potent neutralizing mAbs against pneumoviruses. The site V epitope of both RSV F and hMPV F is located in the F 1The N-terminus of the subunit is immediately adjacent to the fusion peptide (buried in the center of the trimer pre-fusion F protein). Therefore, conformational changes in the α3 helix and β3-β4 hairpin in site V are critical for exposing the fusion peptide and initiating the fusion process. Antibodies targeting site V may lock the fusion peptide and prevent the formation of the long helical bundle present in the post-fusion conformation. However, based on current findings, the frequency of hMPV site V-specific antibodies is relatively low compared to antibodies targeting sites III, IV, and DS7. Therefore, in vaccines based on hMPV F, antibodies targeting antigenic site V can be boosted.

[0472] Example 7

[0473] Materials and Methods for Examples 1-6

[0474] Blood Drawing and PBMC Isolation: After obtaining informed consent, 90 mL of blood was drawn by venipuncture into 9 heparin-coated tubes, and 10 mL of blood was collected into serum separation tubes. Peripheral blood mononuclear cells (PBMCs) were isolated from human donor blood samples using Ficoll-Histopaque density gradient centrifugation and frozen in liquid nitrogen vapor phase until further use.

[0475] Production and synthesis of recombinant hMPV F proteins: hMPV A1, A2, B1, B2 F and hMPV B2F-GCN4 recombinant proteins were cloned into the pcDNA3.1+ vector The obtained plasmid was synthesized. It was amplified by transformation into DH5α cells resistant to ampicillin (Thermo Fisher Scientific) at 100 ug / ml. The plasmid was purified using the ENZA plasmid large-scale preparation kit (Omega BioTek) according to the manufacturer's instructions. 1 mg of the plasmid was mixed with 4 mg of polyethyleneimine (PEI; PolySciences Inc.) in The DNA-PEI mixture was then added to 10% of Freestyle 293 expression medium (Thermo Fisher Scientific) and incubated for 30 minutes. 6The cells were centrifuged at 6000 g to precipitate the cells. The supernatant was filtered through a 0.45 μm sterile filter. The recombinant protein was directly purified by affinity chromatography, HisTrap Excel column (GE Healthcare Life Sciences). Before the supernatant was loaded onto the column, the column was washed with 5 column volumes (CV) of a wash buffer containing 20 mM Tris-HCl (pH 7.5), 500 mM NaCl and 20 mM imidazole. After the supernatant was passed through the column, the column was washed with the same wash buffer (5 CV) to reduce nonspecific binding, and finally eluted with a buffer containing 20 mM Tris-HCl (pH 7.5), 500 mM NaCl and 250 mM imidazole. After elution, the protein was washed with The cells were concentrated using Ultra-15 centrifugal units (Sigma) with a molecular cut-off of 30 KDa.

[0476] Trypsinization of hMPV F: To obtain trimeric hMPV F, TPCK (L-1-tosylamine-2-phenylethylchloromethylketone)-trypsin (Thermo Fisher Scientific) was dissolved in double distilled water (ddHO) at 2 mg / mL. 2 The obtained hMPV B2 F was incubated with 5 TAME (p-toluenesulfonyl-L-arginine methyl ester) units / mg of TPCK-trypsin at 37°C for 1 hour. The trimer and monomer fractions of hMPV F were separated by size exclusion chromatography. Separation was performed on an S200, 16 / 600 column (GE Healthcare Life Sciences) in column buffer (50 mM Tris (pH 7.5) and 100 mM NaCl). The two fractions were separated by their unique elution profiles. Once separated, they were concentrated as described above. Post-fusion hMPV F was obtained by heating the pooled trimer fractions at 55°C for 20 minutes in a water bath to induce the post-fusion conformation (Jiachen et al., Journal of Virology 95, e00593-21 ​​(2021)).

[0477] Generation of hMPV F-specific hybridomas: For hybridoma generation, 10 million peripheral blood mononuclear cells purified from the blood of human donors were mixed with 8 million previously frozen and gamma-irradiated NIH 3T3 cells modified to express human CD40L, human interleukin-21 (IL-21), and human BAFF (Bar-Peled et al., Journal of Virology 93, e00342-19 (2019)) in 80 mL of Medium A ( Technology Company( Technologies)) containing 6.3 μg / mL CpG (phosphorothioate-modified oligodeoxynucleotide ZOEZOEZZZZOEEZOEZZZT; Invitrogen) and 1 μg / mL cyclosporine (Sigma). The mixture of cells was plated in four 96-well plates at 200 μl / well in StemCell medium A. After 6 days, the culture supernatant was screened by ELISA for binding to recombinant hMPV B2 F protein binding, and cells from positive wells were electrofused as previously described (Bar-Peled et al., Journal of Virology 93, e00342-19 (2019)). Cells from each cuvette were resuspended in 20 mL of 1×HAT (hypoxanthine-aminopterin-thymidine; Sigma-Aldrich), 0.2×HT (hypoxanthine-thymidine; Corning), and 0.3 μg / mL ouabain (Thermo Fisher Scientific). Medium A and plated at 50 μl / well in 384-well plates. After 7 days, cells were fed with 25 μl of StemCell Medium A. After 2 weeks, supernatants from hybridomas were screened by ELISA for antibody production, and cells from wells with reactive supernatants were incubated in 0.5 mL of 1% CO before being screened again by ELISA. Medium E ( Technology Company) to 48-well plates for 1 week. Positive hybridomas were then subjected to single-cell fluorescence-activated sorting to 384-well plates (containing 75% Medium A plus 25% Two weeks after cell sorting, hybridomas were screened by ELISA before further expansion of wells containing hMPV F-specific hybridomas.

[0478] RT-PCR of hybridoma mAb variable γ chain and variable light chain: RNA was isolated from amplified hybridoma cells using ENZA total RNA kit (Omega BioTek) according to the manufacturer's protocol. cDNA synthesis was performed using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Nested PCR was performed using a separate set of three primer mixtures to amplify the variable regions of γ, κ and λ chains (Tiller et al., Journal of Immunological Methods 329, 112-124 (2008)). The product from the second PCR was analyzed by agarose gel electrophoresis, and the purified PCR product (ENZA cycle purification kit; Omega Biotek) was submitted to Genewiz for sequencing. Sequences were analyzed using IMGT / V-Quest (Brochet et al., Nucleic Acids Research 36, 503-508 (2008)).

[0479] Antigen-specific single B cell sorting and expression of recombinant mAbs: 10 million human PBMCs were washed twice with FACS buffer and then resuspended with 1 mL FACS buffer. Cells were incubated with 5% Fc receptor blocker. Treat for 30 minutes and then stain with the following antibodies: human CD19-APC, human IgM-FITC, human IgD-FITC, GHOST DYE TM Red 710 and PE / BV605-streptavidin-conjugated hMPV B2 F. Antigen-specific B cells were gated with CD19+ / IgM- / IgD- / Ghost dye- / PE+ / BV605+ and sorted in 96-well plates at one cell / well in capture buffer B (Qiagen TCL Buffer + 1% beta-mercaptoethanol). The sorted cells were snap frozen and stored at -80°C until they were used for RNA extraction. RNA was isolated using Agencort XP kit, SPRI beads (Beckman Coulter) and immediately The IgG heavy / light chain was reverse transcribed into cDNA by IV synthesis system (Thermo Fisher Scientific). The variable region sequence of IgG heavy / light chain was measured by nested PCR as described above. Based on the use of V / D / J gene alleles, cloned PCR primers were selected to clone PCR with the first PCR product as template. The purified cloned PCR product of heavy / light chain was cloned into expression vector (AbVec-hIgG1, AbVec-hIgκ and Ig-λ expression vector based on pBR322), and plasmid was sent to Genewiz for sequencing. After confirming that all sequences were correct, HC / LC plasmid was transformed into DH5α to carry out large-scale preparation of plasmid. Recombinant mAb was expressed by transfecting 293 cells with HC / LC plasmid, and purified from culture supernatant with protein G column (Cytiva).

[0480] For ELISA with hMPV F protein: 384-well plates for ELISA (Cat. No. 781162; Greiner ) was coated with 2 μg / ml (in PBS) of the recombinant protein (antigen) and incubated overnight at 4°C. This was accompanied by washing the plate once with water, followed by blocking the plate for 1 hour at room temperature with a blocking buffer comprising 2% milk and 0.05% Tween 20 (PBS-T) supplemented with PBS containing 2% goat serum. The plate was washed three more times with PBS-T. 25 μl of the serially diluted primary antibody was added to the wells and incubated for one hour at room temperature, followed by three washes with PBS-T. Next, a goat anti-human IgG Fc secondary antibody (Southern Biotech) diluted in blocking buffer (1:4000) was applied to the wells and incubated again at room temperature for one hour. The plate was washed three more times with PBS-T, and 25 μl of PNPP (p-nitrophenyl phosphate) diluted to a concentration of 1 mg / ml in a buffer (containing 1M Tris base and 0.5 mM magnesium chloride, pH 9.8) was added. The plates were incubated a final time for 1 hour at room temperature before the absorbance was read at 405 nm on a Bio Tek microplate reader. Binding assay data were analyzed on GraphPad Prism using nonlinear regression curve fitting and log(agonist) versus response functions to calculate EC 50 value.

[0481] hMPV plaque neutralization assay: LLC-MK2 cells used in this assay were cultured at 37°C in a CO atmosphere. 2The cells were grown in an incubator in T225 cell culture flasks (Catalog No. 82050-870) in Opti-MEM I (Thermo Fischer Scientific) supplemented with 2% fetal bovine serum. Two days before the start of the neutralization assay, 40,000 cells / well were plated on a 24-well plate. Serially diluted sterile-filtered mAbs isolated from hybridoma supernatants were added to a suspension of either hMPV strain CAN / 97-83 and TN / 93-32 in equal amounts (1:1) and incubated for 1 hour on the day of the experiment. Subsequently, after washing the excess FBS from the OPTI-MEM culture medium three times with PBS, 50 μl of the virus-antibody mixture was added to LLC-MK2 cells. The mixture was incubated at room temperature for one hour with constant shaking. The cells were then incubated with 5 μg / ml trypsin-EDTA and 100 ug / ml CaCl in Opti-MEM I (supplemented with 5 μg / ml trypsin-EDTA and 100 ug / ml CaCl 2 ) covered with 0.75% methylcellulose. The cells were incubated for 4 days and fixed with 10% neutral buffered formalin. The cell monolayer was next blocked for one hour with blocking buffer (comprising 2% skim milk supplemented with PBS-T containing 2% goat serum). Next, the plates were washed three times with water and 200 μl of MPV 364 was added to a final concentration of 1 μg / ml (1:1000 dilution) in blocking solution. The plates were then washed three times with water and 200 μl of goat anti-human IgG HRP secondary antibody (Southern Biotechnology) diluted to a 1:2000 ratio in blocking buffer was added and incubated at room temperature for one hour, followed by one hour of incubation. The plates were washed five more times with water and 200 μl of Peroxidase substrates Add to each well. Incubate the plate for 20-30 minutes until plaques are clearly visible. Plaques are counted manually under a microscope and compared to virus-only controls. GraphPad Prism was used to calculate IC using nonlinear regression curve fitting and log(inhibitor) versus response function. 50 value.

[0482] Epitope binning: After obtaining an initial baseline in running buffer (PBS, 0.5% BSA, 0.05% Tween 20, and 0.04% thimerosal), 100 μg / ml of His-tagged hMPV B2F (untrypsinized) protein was immobilized on an anti-penta-His biosensor tip (Forte'Biosciences) for 120 seconds. The baseline was measured again with the tip immersed in a well containing 100 μg / ml of primary antibody for 300 seconds. The biosensor tip was then immersed in the secondary antibody again for 300 seconds at 100 μg / ml. The binding of the second mAb in the presence of the first mAb was determined by comparing the maximum signal of the second mAb after the addition of the first mAb with the maximum signal of the second mAb alone. A non-competitive mAb is a mAb whose binding is greater than or equal to 70% of the uncompeted binding. Between 30% and 60% is considered intermediate binding, and any binding below 30% is considered to be competing for the same site.

[0483] Antibody-dependent phagocytic activity of mAbs: To measure antibody-dependent phagocytic activity, 2 × 10 9 1-μm neutravidin-coated yellow-green (Invitrogen No. F8776) was resuspended in 1 mL of 0.1% PBS. Centrifuge at 5000 rpm for 15 minutes, remove 900 μL of supernatant, and Resuspend with 900 μL of 0.1% PBS. Repeat this process for a second wash and then Resuspend with 20 μg of biotinylated hMPV B2 F protein and shake end-over-end. Incubate overnight at 4°C in the dark. Next, dilute hMPV F-specific antibody to a final concentration of 1 μg / mL in complete RPMI medium (cRPMI, RPMI + 10% FBS) in a U-bottom 96-well plate. Then, transfer 20 μL of the antibody dilution to a clean F-bottom 96-well plate and add 10 μL of The cells were added together with the antibodies and then incubated at 37°C for 2 h for opsonization. After 1.5 h, THP-1 cells were centrifuged at 200 × g for 5 min, washed once with PBS, and then centrifuged at 5 × 10 5 The cells were resuspended in culture medium (RPMI and 10% FBS) at a concentration of 10 cells / mL. Then, 200 μL of cells were added to each well and incubated at 37°C with 5% CO while shaking. 2Incubate together for 6 hours. Once the incubation is complete, the plate is then centrifuged at 2000 rpm for 5 minutes. Then, 100 μL is removed from each well and replaced with 100 μL of cold 4% paraformaldehyde to fix the cells. The plate is then left at room temperature in the dark for 20 minutes. The plate is then stored at 4°C in the dark. The cells are then washed with Flow cytometric analysis. The percentage of fluorescent beads containing THP-1 cells in each sample (% phagocytosis) was used to calculate the % increase relative to the no mAb control. Phagocytosis scores were calculated as previously described. 50 (Geometric mean intensity - geometric mean intensity of no mAb control) x % phagocytosis.

[0484] Animal studies: BALB / c mice (6 to 8 weeks old; Jackson Laboratory) were randomly selected for each group, each containing 5 male mice and 5 female mice. All mice were pre-bled before the study to verify by ELISA that the mice were not previously exposed to hMPV. Each mouse was intranasally infected with hMPV TN / 93-32 (5x10 5 PFU) and euthanized five days after infection. Mice were injected intraperitoneally with PBS / MPV467 / control antibody (10 mg / kg) 24 hours before infection (prevention) or three days after infection (treatment). At the end point, serum was collected for ELISA to determine the presence of mAbMPV467, and lungs were collected and homogenized to determine viral load by immunostaining as described above.

[0485] Recombinant protein production for cryo-EM studies: The pre-fusion hMPV F construct DS-CavEs2-IPDS (hMPV F A1 NL / 1 / 00, residues 1-490) used for structural studies contained previously described G294E, A185P, L219K, V231I, E453Q and furin cleavage site substitutions (Battles et al., Nature Communications 8, 1528 (2017)). The included disulfide substitutions were L110C / N322C, T127C / N153C, A140C / A147C and T365C / V463C (Hsieh et al., in press) and an inter-promoter disulfide at V84C / A249C (Stewart-Jones et al., Proceedings of the National Academy of Sciences of the United States of America 118, e2106196118 (2021)). DS-CavEs2-IPDS was cloned into the mammalian expression vector pαH, which has a C-terminal "GGGS" linker sequence followed by the T4 minor fiber protein trimerization motif "foldon" (Efimov et al., Journal of Mol. Biol. 242, 470-486 (1994); Miroshnikov et al., Protein Eng. 11, 329-332 (1998)), HRV3C protease site, 8xHis tag and Strep-TagII (Battles et al., Nature Communications 8, 1528 (2017)). FreeStyle 293F cells (Thermo Fisher Scientific) were transiently co-transfected with polyethyleneimine (PEI) at a 4:1 ratio of DS-CavEs2-IPDS: plasmid expressing furin for protein expression. Three hours after transfection, kifunensine and pluronic F-68 (Gibco) were introduced to final concentrations of 5 μM and 0.1% (v / v), respectively. Six days after transfection, STREP- Soluble proteins were purified using agarose resin (IBA) from cell supernatants that had been filtered and buffer exchanged into PBS by tangential flow filtration. Streptococcus-tagged proteins were eluted using a buffer containing 100 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 2.5 mM desthiobiotin. After protein concentration using Ultra-15 centrifugal filter units (Millipore), 6 Increase 10 / 300 column (GE Healthcare) in 2 mM Tris (pH 8.0), 200 mM NaCl and 0.02% NaN 3 The protein was further purified by size exclusion chromatography in running buffer.

[0486] Cryo-EM sample preparation and data collection Purified DS-CavEs2-IPDS was combined with a 1.5-fold molar excess of MPV467 Fab, incubated at room temperature for 10 minutes, and then moved to ice. Immediately before freezing, samples were incubated in 2 mM Tris (pH 8.0), 200 mM NaCl, and 0.02% NaN 3 Buffer was diluted to a concentration of 0.66 mg / mL hMPV F. 1 μL of 0.5% amphipol A8-35 was then mixed with 10 μL of the diluted sample, and 4 μL of this sample was applied to a gold 1.2 / 1.3 300 mesh grid (Protochips Au-Flat) that had been 950 plasma cleaner (Gatan) with 4:1 O 2 / H 2 Ratio plasma cleaning 180 seconds. Use The grids were frozen in a 10°C, 100% humidity chamber using a Mark IV (Thermo Fisher Scientific). The blotting settings were a 5-second wait and then a 4-second blot at -2 force before plunging into nitrogen-cooled liquid ethane. The grids were analyzed using a Falcon 4 direct electron detector (Thermo Fisher Scientific). (Thermo Fisher Scientific) imaged a single grid to collect a total of 1,458 images. / pixel calibrated pixel size and The data are collected based on the total exposure amount. Fig.11 The data collection statistics are listed in .

[0487] Cryo-EM data processing: Micrographs were gain reference corrected and imported into Livev3.2.0 was used for initial data processing: motion correction, defocus estimation, micrograph selection, particle selection and extraction, and particle selection by iterative streaming 2D class averaging. Templates were generated using 2D averages and template-based particle selection was performed. Selected particles were exported to v3.2 for further processing by 2D classification, ab initioreconstruction, heterogeneous refinement, homogeneous refinement, and non-uniform homogeneous refinement using C3 symmetry. Further non-uniform refinement was performed using masking and particle subtraction. Finally, the particle-subtracted inhomogeneous refinement map was sharpened using DeepEMhancer (Sanchez-Garcia et al., Communications Biology 4, 874 (2021); Cianfrocco et al., COSMIC2: A Science Gateway for Cryo-Electron Microscopy Structure Determination, Proceedings of the Practice and Experience in Advanced Research Computing 2017 on Sustainability, Success and Impact (Association for Computing Machinery, 2017). doi:10.1145 / 3093338.3093390). Figure 8 The EM processing workflow is shown in Figure 9, and the EM validation results are shown in Figure 9. For model building, an initial hMPV F model was generated based on PDB ID: 5WB0 and the crystal structure of MPV467 Fab, and the model was docked into the cryoEM map using UCSF ChimeraX (Pettersen et al., Protein Sci. 30, 70-82 (2021)). The model was further constructed and iteratively refined using a combination of Coot (Emsley et al., Acta Crystallogr. D. Biol. Crystallogr. 66, 486-501 (2010)), PHENIX (Liebschner et al., Acta Crystallogr. Sect. D, Struct. Biol. 75, 861-877 (2019)), and ISOLDE (Croll, Acta Crystallogr. Sect. D 74, 519-530 (2018)). Fig.11 Model statistics are shown in .

[0488] Example 8

[0489] Human metapneumovirus (hMPV) infection and MPV467 treatment of immunocompromised cotton rats (Moscicapus truncatus)

[0490] Cotton rat ( Rattus rattus ) is an established model for respiratory viral infection, including infections caused by RSV, influenza virus, adenovirus, parainfluenza virus, rhinovirus, and enterovirus (Blanco et al., J Antivir Antiretrovir 2014;6:40-42; Patent et al., PLoS One 2016;11(11):e0166336). Soon after the discovery of the virus, hMPV infection was modeled in cotton rats, Rattus rattus (Hamelin et al., Journal of Virology 2005; 79(14):8894-903; Williams et al., Journal of Virology 2005; 79(17):10944-51; Wyde et al., Antiviral Research 2005; 66(1):57-66; Yim et al., Vaccine 2007; 25(27):5034-40). Cotton rats, Rattus rattus immunosuppressed by cyclophosphamide, were infected with hMPV, and viral replication and lung inflammation in these animals were compared with those of normal hMPV-infected Rattus rattus. The efficacy of prophylactic and therapeutic administration of the anti-hMPV antibody MPV467 was also evaluated. Compared with normal animals, immunosuppressed animals had higher hMPV lung and nasal titers on day 5 after infection, and a large amount of hMPV was still present in the respiratory tract of immunosuppressed animals on days 7 and 9 after infection, indicating prolonged viral replication. Compared with normal animals, immunosuppression of hMPV-infected cotton rats is accompanied by reduced lung tissue pathology, however, delayed increases in pathology and lung cytokine / chemokine expression were seen in immunosuppressed cotton rats. Preventive and therapeutic MPV467 treatment protects both the upper and lower respiratory tracts from hMPV infection. Therapeutic MPV467 administration reduces lung pathology and lung expression of IP-10 and MIP-1α mRNA. The results are provided below.

[0491] Immunosuppression leads to increased hMPV replication and delayed viral clearance in cotton rats: Immunosuppression may be associated with more severe disease and prolonged respiratory viral replication in affected individuals (Welliver, J Pediatr 2003;143(Suppl 5):S112-7; Lion, Clin Microbiol Rev 2014;27(3):441-62; Hijano et al., Front Microbiol 2018;9:3097). To assess hMPV replication and clearance in cotton rats immunosuppressed with cyclophosphamide, 10 5 Animals were infected with PFU of hMPV and viral replication was assessed at several time points after infection. Lung and nasal samples were collected for viral titration on days 5 (time when viral replication in normal cotton rats peaks), 7 (time when virus is cleared from the lungs of infected cotton rats), and 9 (additional delayed time points) after infection. Normal age-matched animals that were infected in parallel with immunosuppressed animals and sacrificed on the same day were compared. As expected, in normal animals, hMPV was cleared from the lungs of infected normal cotton rats on day 7 after infection, and was largely cleared from the nose, and was undetectable in the lower or upper respiratory tract on day 9 ( Fig.12 ). In contrast, immunosuppressed cotton rats showed high levels of hMPV in the lungs and noses on all three days. On day 5 post-infection, the amount of hMPV present in the lungs and noses of infected animals was significantly higher than that detected in normal cotton rats sacrificed at the same time. Taken together, these results indicate that hMPV can replicate to higher titers in immunosuppressed cotton rats and that clearance of the virus is significantly delayed by immunosuppression.

[0492] Prophylactic or therapeutic treatment with anti-hMPV antibodies reduces hMPV load in immunosuppressed cotton rats in a dose-dependent manner: Monoclonal antibodies targeting viral surface proteins are the most effective therapeutic and prophylactic drugs against viral infections used today (Pantaleo et al., Nature Reviews Drug Discovery 2022; 21: 676-696). The prophylactic and therapeutic efficacy of MPV467 was evaluated in normal and immunosuppressed cotton rats infected with hMPV and sacrificed at day 5 post-infection ( Fig.13). The following three different doses of MPV467 were tested one day before infection or three days after infection: 0.1 mg / kg, 1 mg / kg and 10 mg / kg. The test results show that both prophylactic and therapeutic treatments with MPV467 effectively reduced hMPV replication in the lungs and noses of infected animals. The two highest doses of MPV467 (10 mg / kg and 1 mg / kg) administered before or after infection completely protected the lungs of infected animals (no viral replication was detectable). The lowest dose of MPV467 tested, 0.1 mg / kg, caused a modest but statistically significant reduction in hMPV replication in the lungs, with the effect being slightly more pronounced for prophylactic treatment. In the nose, prophylactic administration of all three doses tested and the two highest doses of MPV467 administered therapeutically significantly reduced the hMPV load. In summary, these results indicate that MPV467 has a potent, dose-dependent antiviral activity in immunosuppressed cotton rats.

[0493] MPV467 therapy improves delayed hMPV clearance in immunosuppressed cotton rats: Once the antiviral efficacy of MPV467 in immunosuppressed cotton rats was determined by analyzing samples collected in the lungs at the peak time of viral replication, it was determined whether therapeutic administration of the antibody could combat delayed viral clearance in the model. To address this issue, hMPV-infected immunosuppressed animals were treated with 10 mg / kg MPV467 three and seven days after infection and sacrificed on the ninth day after infection to analyze viral loads in the lungs and nose. Normal cotton rats were infected and treated once on the third day. Replication on the fifth day was evaluated again in hMPV-infected animals (normal and immunosuppressed animals) treated with 10 mg / kg MPV467 (or mock-treated) three days after infection. MPV467 was very effective in normal cotton rats, reducing viral replication to undetectable levels in samples on the fifth day ( Fig.14 MPV467 was also very effective in immunosuppressed animals. Mock-treated hMPV-infected immunosuppressed animals had comparable amounts of detectable hMPV in the lungs and nose on days 5 and 9, while animals treated with MPV467 had no detectable hMPV in the upper or lower respiratory tract on both days of analysis.

[0494] Effect of antibody therapy on lung histopathology and cytokine / chemokine expression in immunosuppressed cotton rats: Lung histopathology is one of the markers of the inflammatory response to respiratory infection in the cotton rat model and was used to evaluate the differences in lung response to hMPV in normal and immunosuppressed animals in the presence or absence of antibody treatment. Cotton rats were infected with hMPV and sacrificed on days 5 and 9 post infection to analyze peribronchiolar inflammation, perivasculitis, interstitial inflammation, and alveolitis. Normal hMPV-infected animals had a moderate level of pathology, characterized primarily by peribronchiolar inflammation and some perivasculitis ( Fig.15 ). The extent of pathology between days five and nine was largely comparable. Antibody treatment resulted in a modest increase in perivascular inflammation in normal hMPV-infected cotton rats. The incidence of peribronchiolar inflammation was reduced in immunosuppressed cotton rats infected with hMPV compared to normal animals. Lung histopathology was slightly higher in hMPV-infected immunosuppressed animals on day nine compared to day five, with interstitial inflammation and alveolitis becoming evident. The effects of therapeutic antibody treatment of MPV467 administered at a dose of 10 mg / kg on lung pathology were evaluated. A reduction in lung histopathology was seen in hMPV-infected antibody-treated immunosuppressed animals on day nine after infection compared to hMPV-infected mock-treated immunosuppressed animals.

[0495] Pulmonary cytokine / chemokine expression is another marker of the pulmonary response to infection in the cotton rat model. To determine whether the reduction in interstitial inflammation and alveolitis seen on day nine in immunosuppressed hMPV-infected animals treated with MPV467 was associated with altered lung cytokine / chemokine expression, levels of lung MIP-1α and IP-10 (mediators associated with lung injury and immune dysfunction) were measured (Ichikawa et al., Am J Respir Crit Care Med 2013; 187(1): 65-77; Shanley et al., J Immunol 1995; 154(9): 4793-802; Kameda et al., PLoS One 2020; 15(11): e0241719; Smith et al., J Immunol 1994; 153(10): 4704-12). Compared with normal animals, immunosuppressed animals had elevated MIP-1α and IP-10 mRNA expression ( Fig.16 ). Antibody treatment significantly reduced the expression of both mediators in immunosuppressed animals.

[0496] Normal (unmanipulated) cotton rats, Rattus rigescens, are susceptible to respiratory and lower respiratory tract hMPV infections, with the infections largely resolved within a week. 20-21 This is different from the reported persistent hMPV infection of conventional BALB / c mice, in which hMPV replication can sometimes persist for weeks to months (Alvarez et al., J. Virol. 2004; 78: 14003-14011; Alvarez et al., J. Virol. 2005; 79(10): 5971-8; Moe et al., J. Infect. Dis. 2017; 216(1): 110-116). Self-limited hMPV infection in unmanipulated cotton rats is similar to that in healthy humans (Moe et al., J. Infect. Dis. 2017; 216(1): 110-116; Ebihara et al., J. Clin Microbiol. 2004; 42: 126-32), and this is a useful feature that allows for the evaluation of potential defects in viral clearance that may be caused by immunosuppression.

[0497] Similar to what has been reported in immunocompromised humans (Spahr et al., Open Forum Infect Dis 2018; 5(5):ofy077; Chu et al., J Pediatric Infect Dis Soc 2014; 3(4):286-93; Debiaggi et al., J Infect Dis 2006; 194(4):474-8; Debiaggi et al., New Microbiol 2007; 30(3):255-8), hMPV replication was significantly prolonged in immunosuppressed cotton rats, confirming delayed viral clearance under immunosuppressive conditions. In general, delayed viral clearance may affect lung function through direct cytopathic effects of prolonged viral replication or through indirect effects on lung inflammation. In this work, a slight increase in interstitial inflammation and alveolitis was noted in immunosuppressed hMPV-infected cotton rats late in infection compared to the early time corresponding to peak viral replication in the lungs. At the same time, at this late stage of infection, the expression of pulmonary cytokines / chemokines IP-10 and MIP-1α in immunosuppressed animals exceeded that detected in normal cotton rats infected with hMPV. The increase in specific parameters of lung inflammation in immunosuppressed animals at late stages of infection was similar to those seen previously for RSV (Boukhvaolova et al., Bone Marrow Transplantation 2016; 51(1): 119-26), except that cytokines were not detected in the RSV model and no detectable epithelial damage was seen in the lungs of animals infected with hMPV. In late stages of hMPV infection, the increased IP-10 and MIP-1α levels in immunosuppressed animals may have deleterious effects on the lungs. Both molecules have been shown to promote the development of lung injury of both viral and non-viral origin through neutrophil-mediated mechanisms (Ichikawa et al., Am J Respir Crit Care Med 2013;187(1):65-77; Shanley et al., J Immunol 1995;154(9):4793-802); and both molecules have been associated with the development of autoimmunity and pulmonary fibrosis (Kameda et al., PLoS One 2020;15(11):e0241719; Smith et al., J Immunol 1994;153(10):4704-12).

[0498] Therapeutic administration of the anti-hMPV antibody MPV467 resulted in ablation of hMPV replication in immunosuppressed cotton rats and also reduced lung pathology and cytokine / chemokine expression in the lungs of immunosuppressed animals. This combined inhibition of viral replication and lung inflammation by therapeutically administered antiviral antibodies is similar to the effects seen in RSV-infected immunosuppressed cotton rats treated with anti-RSV Ig (Boukhvaolova et al., Bone Marrow Transplantation 2016; 51(1): 119-26). For RSV, the ability of therapeutic antibody treatment to reduce lung inflammation in immunosuppressed animals contrasts with the lack of similar efficacy of antiviral antibodies when used alone in normal, non-immunosuppressed cotton rats (Prince et al., J Infect. 2000; 182: 1326-1330; Boukhvalova et al., J Infect. 2007; 195(4): 511-8) or humans (Rodriguez et al., Pediatrics 1997; 100(6): 937-42; Rodriguez et al., Pediatrics 1997; 99(3): 454-61).

[0499] Example 9

[0500] Materials and Methods for Example 8

[0501] Reagents: Cyclophosphamide for injection (20 mg / ml USP, Baxter) was obtained from Blue Door Pharma.

[0502] Viruses and Virus Assays: hMPV strain TN / 94-49 / A2 (Williams et al., NEJM 2004;350(5):443-50; Williams et al., J Infect. 2006;193(3):387-95) recovered from samples collected from the Vanderbilt Vaccine Clinic was grown on LLC-MK2 cells in minimal essential medium supplemented with 0.2% glucose, 0.1% bovine serum albumin, 0.0002% trypsin, and 1% gentamicin. Individual pools of hMPV (3 x 10 6 pfu / ml) were used for the studies described in this article.

[0503] Animals and Animal Studies: Inbred cotton rats (Haired Rat) were obtained from a colony maintained at Sigmovir Biosystems. Male and female cotton rats aged six to eight weeks were used for the study. Animals were housed in large polycarbonate cages and fed a standard diet of rodent food and water. The colonies were monitored for antibodies to exogenous respiratory viruses and other common rodent pathogens, and no such antibodies were found (VRL Test 80221-RAT 1 Ab profile: Carbacillus CARB, Toolan's H-1 virus, Kilham rat virus (KRV), Mycoplasma pulmonis, Parvovirus, Pneumovirus PVM, RCV / SDA, Sendai virus). The efficacy of hMPV infection and MPV467 treatment of immunocompromised animals was verified in two consecutive experiments. Based on the results of previous experiments, a sample size of four to five animals / group was selected to allow statistically significant differences between each test group. For unpaired data with unequal variances, Student t-test was used to run comparisons between groups (KaleidaGraph). Unless otherwise stated, samples were not blinded before analysis.

[0504] Based on previously published methods, immunosuppression was induced in cotton rats by repeated treatment with cyclophosphamide (CY) (Boukhvalova et al., MMT 2016;51(1):119-26). Briefly, a 50 mg / kg CY solution was administered intramuscularly (im) at 250 μl / 100 g of animal on a Monday-Wednesday-Friday schedule for 18 days. At the end of this period, whole blood was collected to verify the decrease in total leukocyte and lymphocyte counts. CY treatment was continued until the end of the study. Twenty-one days after the start of CY treatment, animals were infected intranasally (in) with hMPV (10 5 PFU / animal). To quantify hMPV load, 5 animals from each group were sacrificed on days 5 and 7 post infection (first study) or days 5 and 9 post infection (second study) to collect lungs and noses for virus quantification by plaque assay. A group of normal, age-matched cotton rats were infected with hMPV and sacrificed on days 5, 7, and 9 post infection for hMPV quantification by plaque assay.

[0505] In order to evaluate the dose-dependency of the preventive and therapeutic efficacy of MPV467, cotton rats immunosuppressed with cyclophosphamide were inoculated intramuscularly with 0.1mg / kg, 1mg / kg or 10mg / kg MPV467 one day before or three days after hMPV stimulation. The control animals were treated with PBS (simulants) one day before hMPV infection. The animals were killed 5 days after hMPV stimulation, and the lungs and noses were collected for virus titration. In order to analyze the therapeutic efficacy of MPV467 when delayed clearance (9th day), immunosuppressed animals infected with hMPV were treated intramuscularly with 10mg / kg MPV467 on the 3rd day and killed on the 5th day after infection, or treated with MPV467 on the 3rd and 7th days and killed on the 9th day after infection. Normal animals were treated with MPV467 3 days after infection and killed on the 9th day. Lungs were collected for histopathology and qPCR analysis. Lungs and noses were collected for virus titration.

[0506] Virus titration: Lung and nasal homogenates were clarified by centrifugation and diluted in EMEM. Confluent LLC-MK-2 monolayers were infected with the diluted homogenates in duplicate in 24-well plates. The cells were incubated at 37°C in 5% CO 2 After one hour of incubation in the incubator, the wells were covered with 0.75% methylcellulose medium. After 7 days of incubation, the cover was removed, the cells were fixed for one hour and air-dried for immunostaining. After the wells were blocked with PBS containing 1% BSA, mouse anti-hMPV-N-protein antibodies diluted at a dilution of 1:1,000 in 1% BSA were added to each well, followed by washing, and then incubated with rabbit anti-mouse IgG conjugated to HRP at a dilution of 1:1,000 in 1% BSA. AEC chromogen detection solution was added to each well and incubated at room temperature for 2 hours. Visible plaques were counted and the virus titer was expressed as plaque forming units / gram of tissue. The virus titer of all animals in a group was calculated as the geometric mean ± standard error (SEM) at a given time.

[0507] Lung cytokine analysis: Total RNA was extracted from homogenized lung tissue using the RNeasy purification kit (Qiagen). One microgram of total RNA was used to prepare cDNA using oligonucleotide dT primers and Super Script II RT (Invitrogen). Cotton rat cytokine cDNA was analyzed by qPCR using primers and conditions previously described (Blanco et al., Journal of Infectious Diseases 2002; 185: 1780-5; Blanco et al., J Interferon Cytokine Res 2004; 24: 21-8; Baukhvalova et al., Curr Protoc Cell Biol 2010; Chapter 26: Unit 26.6). The signal obtained for each analyzed gene was normalized to the level of β-actin ("housekeeping gene") expressed in the corresponding organ. Cytokine levels for all animals in a group at a given time are expressed as geometric mean ± SEM. Differences between groups were assessed by Student's t test for pooled data.

[0508] Histopathological analysis: Lungs were prepared for histopathological analysis as previously described and scored blindly for peribronchiolar inflammation (inflammatory cells around small airways), perivasculitis (inflammatory cells around small blood vessels), alveolitis (inflammatory cells within the alveolar spaces), and interstitial pneumonia (inflammatory cell infiltration and alveolar wall thickening) (Prince et al., Journal of General Virology 2001; 82: 2881-8). Each parameter was scored on a 0-4 scale, with 0 = 0%, 1 = 5%, 2 = 25%, 3 = 75%, and 4 = 100% histological score.

Claims

1. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain variable (V H ) region and the light chain variable region (V L ), the V H and the V L Including the V H and the V L Heavy chain complementarity determining regions (HCDR) 1, HCDR2 and HCDR3, and light chain complementarity determining regions (LCDR) 1, LCDR2 and LCDR3: a) SEQ ID NO: 41 and 45 (MPV467), respectively; b) SEQ ID NO: 9 and 13 (MPV414), respectively; c) SEQ ID NOs: 17 and 21, respectively (MPV454); d) SEQ ID NO: 25 and 29 (MPV456), respectively; e) SEQ ID NO: 33 and 37, respectively (MPV464); f) SEQ ID NO: 1 and 5 (MPV86), respectively; g) SEQ ID NO: 49 and 53, respectively (MPV477); h) SEQ ID NO: 57 and 61 (MPV478), respectively; i) SEQ ID NO: 65 and 69 (MPV481), respectively; j) SEQ ID NO: 73 and 77, respectively (MPV482); k) SEQ ID NO: 81 and 85 (MPV483), respectively; 1) SEQ ID NO: 89 and 93 (MPV485), respectively; m) are SEQ ID NOs: 97 and 101 (MPV486), respectively; n) are SEQ ID NOs: 105 and 109 (MPV487), respectively; o) SEQ ID NO: 113 and 117 (MPV488), respectively; p) are SEQ ID NOs: 121 and 125 (MPV489), respectively; q) SEQ ID NO: 129 and 133 (MPV491), respectively; or r) are SEQ ID NOs: 137 and 141 (MPV503), respectively; The monoclonal antibody specifically binds to the F protein of human metapneumovirus (hMPV) and neutralizes hMPV.

2. The isolated monoclonal antibody or antigen-binding fragment of claim 1, wherein the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 comprise the amino acid sequence shown below: a) SEQ ID NO: 42, 43, 44, 46, 47 (GEN) and 48, respectively; b) SEQ ID NOs: 10, 11, 12, 14, 15 (GAS) and 16, respectively; c) SEQ ID NOs: 18, 19, 20, 22, 23 (VAS) and 24, respectively; d) SEQ ID NOs: 26, 27, 28, 30, 31 (KTS) and 32, respectively; e) SEQ ID NOs: 34, 35, 36, 38, 39 (QDN) and 40, respectively; f) are SEQ ID NOs: 2, 3, 4, 6, 7 and 8, respectively; g) SEQ ID NO: 50, 51, 52, 54, 55 (DNS) and 56, respectively; h) SEQ ID NOs: 58, 59, 60, 62, 63 (WAS) and 64, respectively; i) SEQ ID NOs: 66, 67, 68, 70, 71 (GAS) and 72, respectively; j) are SEQ ID NOs: 74, 75, 76, 78, 79 (KDK) and 80, respectively; k) are SEQ ID NOs: 82, 83, 84, 86, 87 (AAS) and 88, respectively; 1) are SEQ ID NOs: 90, 91, 92, 94, 95 (QDT) and 96, respectively; m) are SEQ ID NOs: 98, 99, 100, 102, 103 (SDN) and 104, respectively; n) are SEQ ID NOs: 106, 107, 108, 110, 111 (DAS) and 112, respectively; o) are SEQ ID NOs: 114, 115, 116, 118, 119 (WAS) and 120, respectively; p) are SEQ ID NOs: 122, 123, 124, 126, 127 and 128, respectively; q) SEQ ID NO: 130, 131, 132, 134, 135 (RGS) and 136, respectively; or r) are SEQ ID NOs: 138, 139, 140, 142, 143 (GAS) and 144, respectively.

3. The isolated monoclonal antibody or antigen-binding fragment according to claim 1 or claim 2, wherein the V H and the V L comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown below: a) SEQ ID NO: 41 and 45, respectively; b) SEQ ID NOs: 9 and 13, respectively; c) SEQ ID NOs: 17 and 21, respectively; d) SEQ ID NOs: 25 and 29, respectively; e) SEQ ID NOs: 33 and 37, respectively; f) SEQ ID NOs: 1 and 5, respectively; g) SEQ ID NOs: 49 and 53, respectively; h) SEQ ID NOs: 57 and 61, respectively; i) SEQ ID NOs: 65 and 69, respectively; j) SEQ ID NOs: 73 and 77, respectively; k) SEQ ID NOs: 81 and 85, respectively; 1) are SEQ ID NOs: 89 and 93, respectively; m) are SEQ ID NOs: 97 and 101, respectively; n) are SEQ ID NOs: 105 and 109, respectively; o) are SEQ ID NOs: 113 and 117, respectively; p) are SEQ ID NOs: 121 and 125, respectively; q) SEQ ID NO: 129 and 133, respectively; or r) are SEQ ID NOs: 137 and 141, respectively.

4. The isolated monoclonal antibody or antigen-binding fragment of any one of claims 1 to 3, comprising a human framework region.

5. The isolated monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 4, wherein the V H and the V L Includes the amino acid sequence shown below: a) SEQ ID NO: 41 and 45, respectively; b) SEQ ID NOs: 9 and 13, respectively; c) SEQ ID NOs: 17 and 21, respectively; d) SEQ ID NOs: 25 and 29, respectively; e) SEQ ID NOs: 33 and 37, respectively; f) SEQ ID NOs: 1 and 5, respectively; g) SEQ ID NOs: 49 and 53, respectively; h) SEQ ID NOs: 57 and 61, respectively; i) SEQ ID NOs: 65 and 69, respectively; j) SEQ ID NOs: 73 and 77, respectively; k) SEQ ID NOs: 81 and 85, respectively; 1) are SEQ ID NOs: 89 and 93, respectively; m) are SEQ ID NOs: 97 and 101, respectively; n) are SEQ ID NOs: 105 and 109, respectively; o) are SEQ ID NOs: 113 and 117, respectively; p) are SEQ ID NOs: 121 and 125, respectively; q) SEQ ID NO: 129 and 133, respectively; or r) are SEQ ID NOs: 137 and 141, respectively.

6. The isolated monoclonal antibody of any one of claims 1 to 5, wherein the antibody comprises a human constant domain.

7. The isolated monoclonal antibody according to any one of claims 1 to 6, wherein the antibody is a human antibody.

8. The isolated monoclonal antibody according to any one of claims 1 to 7, wherein the antibody is IgG.

9. The isolated monoclonal antibody according to any one of claims 1 to 8, comprising a recombinant constant domain comprising a modification that increases the half-life of the antibody.

10. The isolated monoclonal antibody of claim 9, wherein the modification increases binding to the neonatal Fc receptor.

11. The isolated monoclonal antibody or antigen-binding fragment of any one of claims 1 to 10, wherein the antibody neutralizes Group A and Group B hMPVs.

12. The isolated monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 11, wherein the antibody binds to the pre-fusion F protein with a higher affinity than the post-fusion F protein.

13. An isolated monoclonal antigen-binding fragment of any one of claims 1 to 5 or 11 to 12.

14. The isolated monoclonal antigen-binding fragment of claim 13, wherein the antigen-binding fragment is a Fv, Fab, F(ab')2, scFV or scFV2 ​​fragment.

15. The isolated monoclonal antibody according to any one of claims 1 to 12 or the antigen-binding fragment according to claim 13 or claim 14, conjugated to an effector molecule or a detectable marker.

16. A bispecific antibody comprising the monoclonal antibody according to any one of claims 1 to 12 or the antigen-binding fragment according to claim 13 or claim 14.

17. An isolated nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 12, or the antigen-binding fragment according to any one of claims 13 or 14, or the V sequence of the monoclonal antibody or the antigen-binding fragment. H or V L .

18. The nucleic acid molecule of claim 17, wherein the nucleic acid molecule is a cDNA sequence.

19. A nucleic acid molecule according to claim 17 or claim 18, operably linked to a promoter.

20. A vector comprising the nucleic acid molecule according to any one of claims 17 to 19.

21. A host cell comprising a nucleic acid molecule or a vector according to any one of claims 17 to 20.

22. A pharmaceutical composition for inhibiting hMPV infection, comprising an effective amount of the monoclonal antibody according to any one of claims 1 to 12, the antigen-binding fragment according to claim 13 or 14, or a nucleic acid molecule or vector encoding the monoclonal antibody or the antigen-binding fragment; and A pharmaceutically acceptable carrier.

23. A method for producing a monoclonal antibody or antigen-binding fragment that specifically binds to hMPV F protein, the method comprising: expressing one or more nucleic acid molecules encoding the monoclonal antibody according to any one of claims 1 to 12, the antigen-binding fragment according to claim 13 or claim 14, or the bispecific antibody according to claim 16 in a host cell; and The antibody or antigen-binding fragment is purified.

24. A method of detecting the presence of hMPV in a biological sample from a human subject, the method comprising: contacting the biological sample with an effective amount of the monoclonal antibody according to any one of claims 1 to 12 or 15, the antigen-binding fragment according to any one of claims 13 to 15, or the bispecific antibody according to claim 16 under conditions sufficient to form an immune complex; and The presence of the immune complex in the biological sample is detected, wherein the presence of the immune complex in the biological sample indicates the presence of the hMPV in the sample.

25. The method of claim 26, wherein detecting the presence of the immune complex in the biological sample indicates that the subject has an hMVP infection.

26. A method for inhibiting hMPV infection in a subject, the method comprising administering to the subject an effective amount of the monoclonal antibody according to any one of claims 1 to 12, the antigen binding fragment according to claim 13 or 14, the bispecific antibody according to claim 15, a nucleic acid molecule encoding the monoclonal antibody, the antigen binding fragment or the bispecific antibody, a vector comprising the nucleic acid molecule, or the pharmaceutical composition according to claim 22, wherein the subject has hMPV infection or is at risk of hMPV infection.

27. A monoclonal antibody according to any one of claims 1 to 12, an antigen-binding fragment according to claim 13 or 14, a bispecific antibody according to claim 15, a nucleic acid molecule encoding the monoclonal antibody, the antigen-binding fragment or the bispecific antibody, a vector comprising the nucleic acid molecule, or Use of the pharmaceutical composition according to claim 22, for inhibiting hMPV infection in a subject or detecting the presence of hMPV in a biological sample.

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