hMPV ANTIBODIES AND THEIR USES

Abstract

Disclosed are antibodies and antigen-binding fragments that specifically bind to human metapneumovirus (hMPV) F protein and neutralize hMPV. 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 hMPV infection or to detect hMPV infection. Human metapneumovirus (hMPV) is a leading cause of morbidity and hospitalization in children worldwide, yet no vaccines or therapeutics are currently available for the prevention and treatment of hMPV disease. The present invention provides a means to solve this problem.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This claims the benefit of U.S. Provisional Application No. 63 / 317,384, filed March 7, 2022, which is incorporated herein by reference.

[0002] Statement of Government Support This invention was made with Government support under Grant Nos. R01 AI143865 and 1K01 OD026569 from the National Institutes of Health. The United States Government has certain rights in this invention.

[0003] Field of Disclosure The present invention relates to the field of metapneumovirus, in particular monoclonal antibodies and antigen-binding fragments thereof that specifically bind to human metapneumovirus F protein.

[0004] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (8618-108103-02 SEQUENCE LISTING.xml; size: 122,880 bytes; creation date: February 20, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0005] background Human metapneumovirus (hMPV) is a leading 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)). First identified in 2001 in 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), including mid- to upper respiratory tract infections that may require hospitalization (Akhras et al., Infect. Dis. Rep. 2, e12 (2010)). Severe disease is seen in immunocompromised patients, e.g., 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 Lett. 379, 100-106 (2016)), as well as patients with HIV (Klein et al., J. Infect. Dis. 201, 297-301 (2010)) and COPD (Kan-o et al., J. Infect. Dis. 215, 1536-1545 (2018)) in patients living with the disease.In contrast to RSV, for which palivizumab (Group and Im, Pediatrics 102, 531-537 (1998)) has been used for many years in certain high-risk infant populations, there is no approved vaccine or specific treatment available for hMPV infection.

[0006] Serological studies have shown that nearly all children are seropositive for hMPV by age 5 years (Edwards et al. N. Engl. J. Med. 368, 633-643 (2013)). hMPV has three surface glycoproteins: the small hydrophobic (SH), binding (G) and fusion (F) proteins. Of these, the hMPV F protein is the only target of neutralizing antibodies (Ulbrandt et al., J. Gen. Virol. 89, 3113-3118 (2008)), which differs from RSV, where both the RSV G and F proteins elicit neutralizing antibodies (Tripp et al., J. Virol. 92, 1-8 (2017)). There is no licensed vaccine that protects against hMPV, but several candidates, including live attenuated viruses, recombinant viruses, vectored vaccines, and recombinant surface proteins, have been tested in animal models (Shafagati and Williams, F1000Res. 7, 135 (2018)). Limited vaccine candidates have advanced into clinical trials, including a live attenuated hMPV vaccine (NCT01255410) and, more recently, an mRNA-based vaccine in combination with parainfluenza virus 3 (NCT03392389, NCT04144348). Similar to the vaccine-enhanced 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 produce enhanced disease 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)). Thus, there remains a need for other therapeutics that target hMPV.

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[0008] Summary of the Disclosure a) SEQ ID NOs: 41 and 55, 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; l) SEQ ID NOs: 89 and 93, respectively; m) SEQ ID NOs: 97 and 101, respectively; n) SEQ ID NOs: 105 and 109, respectively; o) SEQ ID NOs: 113 and 117, respectively; p) SEQ ID NOs: 121 and 125, respectively; q) SEQ ID NOs: 129 and 133, respectively; or r) SEQ ID NOs: 137 and 141, respectively; V, denoted as H and V L heavy chain variable (VV) including heavy chain complementarity determining region (HCDR) 1, HCDR2, and HCDR3 and light chain complementarity determining region (LCDR) 1, LCDR2, and LCDR3 of H ) region and the light chain variable region (V LDisclosed is an isolated monoclonal antibody or antigen-binding fragment thereof comprising:

[0009] In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are a) SEQ ID NOs: 42, 43, 44, 46, 47 and 48, respectively; b) SEQ ID NOs: 10, 11, 12, 14, 15 and 16, respectively; c) SEQ ID NOs: 18, 19, 20, 22, 23 and 24, respectively; d) SEQ ID NOs: 26, 27, 28, 30, 31 and 32, respectively; e) SEQ ID NOs: 34, 35, 36, 38, 39 and 40, respectively; f) SEQ ID NOs: 2, 3, 4, 6, 7 and 8, respectively; g) SEQ ID NOs: 50, 51, 52, 54, 55 and 56, respectively; h) SEQ ID NOs: 58, 59, 60, 62, 63 and 64, respectively; i) SEQ ID NOs: 66, 67, 68, 70, 71 and 72, respectively; j) SEQ ID NOs: 74, 75, 76, 78, 79 and 80, respectively; k) SEQ ID NOs: 82, 83, 84, 86, 87 and 88, respectively; l) SEQ ID NOs: 90, 91, 92, 94, 95 and 96, respectively; m) SEQ ID NOs: 98, 99, 100, 102, 103 and 104, respectively; n) SEQ ID NOs: 106, 107, 108, 110, 111 and 112, respectively; o) SEQ ID NOs: 114, 115, 116, 118, 119 and 120, respectively; p) SEQ ID NOs: 122, 123, 124, 126, 127 and 128, respectively; q) SEQ ID NOs: 130, 131, 132, 134, 135 and 136, respectively; or r) SEQ ID NOs: 138, 139, 140, 142, 143 and 144, respectively. The present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising the amino acid sequence shown as:

[0010] In some embodiments, the antibody or antigen-binding fragment is conjugated to an effector molecule or a detectable marker.

[0011] Bispecific antibodies comprising the antibodies or antigen-binding fragments are also disclosed.

[0012] In further embodiments, the antibody, antigen-binding fragment, or V of the antibody or antigen-binding fragment H Or V L Nucleic acids and vectors encoding

[0013] In more embodiments, a pharmaceutical composition for use in inhibiting hMPV infection is disclosed comprising an effective amount of an antibody, antigen-binding fragment, nucleic acid molecule or vector and a pharma- ceutically acceptable carrier.

[0014] In a further embodiment, a method of producing an antibody or antigen-binding fragment that specifically binds to an hMPV F protein is disclosed.

[0015] In some embodiments, methods for detecting the presence of hMPV in a biological sample from a human subject are also disclosed.

[0016] In a further embodiment, a method for inhibiting hMPV infection in a subject is disclosed, comprising administering to the subject an effective amount of an antibody, antigen-binding fragment, nucleic acid molecule, vector or pharmaceutical composition, wherein the subject is at risk or at risk for hMPV infection.

[0017] The above and other features and advantages of the present invention will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying drawings. [Brief description of the drawings]

[0018] [Figure 1A] Sequence determinants of isolated mAbs. (A) Heavy, kappa and lambda chain gene usage is shown as a percentage of all respective genes from the panel of isolated mAbs. (B) Junction amino acid lengths for heavy and light chains are shown. (C) Percent identity of V genes to predicted germline sequences is shown. [Figure 1B-C] Same as above. [Diagram 2] Epitope mapping of hMPV F-specific mAbs. (A) Epitope binning for mAb binding to hMPV B2 F protein. Data show percent binding of secondary antibodies in the presence of primary antibodies compared to secondary antibodies alone. Cells are colored with a gradient according to annotations shown on the right. (B) Epitopes against control mAbs 101F (site IV), 196 and DS7 (DS7 epitope), MPE8 and MPV364 (site III), and MPV458 (66-87 epitope) were used as secondary mAbs and are labeled according to colors in (A). Selected control mAbs were also used as primary mAbs as positive blocking controls. (C) mAb binding sites for MPE8 and MPV364 (site III), 101F (site IV), DS7, and MPV458 (66-87 epitope) are shown on the surface of monomeric prefusion hMPV F. The epitopes predicted from epitope binning for each mAb are shown. mAbs marked with an asterisk were previously discovered. [Diagram 3] The percent phagocytosis of hMPV F-coated beads by THP-1 cells in the presence of each mAb was assessed using flow cytometry. The relative percent increase in phagocytic cells for each mAb compared to the no mAb control (A) is shown, along with the phagocytosis score (B). Bars indicate the mean of three replicates, and error bars are standard deviations. [Figure 4] Protective efficacy of MPV467 in vivo against hMPV replication. (A) BALB / c mice were treated intraperitoneally with 10 mg / kg mAb MPV467 24 h before (prevention study) or 3 days after (treatment study) intranasal hMPV infection. Viral 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 treatment study (C) were determined by plaque assay. ns=not significant, **P=0.0016, ***P=0.0003-0.0001, ****P<0.0001. Limit of detection (LOD) is indicated by dashed line. ND=not detected. [Diagram 5] MPV467 binds prefusion hMPV F at sites II and V. (A) (Left) Side view of cryo-EM map of the hMPV F-MPV467 Fab complex shown at two different contour levels. The global map is shown as a white transparent map. The particle-subtracted DeepEMhanced map is opaque and a single protomer is identified. (Right) Top-down view of the particle-subtracted DeepEMhanced map. (B) A single protomer of the hMPV F trimer and the MPV467 Fab variable domain are shown as ribbons (hMPV F: blue, Fab: red / orange). (C) Zoomed-in view of the interface between MPV467 and hMPV F. View direction is indicated by boxes and arrows in panel (B). Key residues are shown as sticks. Hydrogen bonds and salt bridges are shown as black dotted lines. Oxygen atoms are colored red and nitrogens are blue. [Figure 6] Neutralization profile of hMPV F protein-specific mAbs. Data show the average from three replicates, error bars are standard deviation. Data are representative of results from at least two independent experiments. [Figure 7]ELISA binding curves of hMPV F protein specific mAbs. ELISA binding curves of isolated mAbs and recombinant hMPV F protein constructs. The pre-fusion RSV F protein (DsCav1) was used to determine whether any mAbs cross-react with RSV F. The previously discovered hMPV F specific mAb MPV196 was used as a negative binding control. Each point represents the average of data from four replicates, with error bars indicating standard deviation. Data are representative of results from at least two independent experiments. [Figure 8] Cryo-EM processing workflow for MPV467. Each step of the cryo-EM data processing workflow is shown, from a representative micrograph to a DeepEMhancer map. The computer programs and algorithms used are labeled for each step. The mask used for particle subtraction is colored as transparent cyan. [Figure 9A-B]Cryo-EM validation of MPV467. (A) (Top) FSC curve for the uniform refinement 3D reconstruction. The horizontal blue line corresponds to an FSC value of 0.143. (Bottom) Viewing distribution plot calculated in cryoSPARC. (B) FSC curve and distribution plot for the particle-subtracted non-uniform refinement 3D reconstruction. (Top) FSC curve for the non-uniform refinement 3D reconstruction. The horizontal blue line corresponds to an FSC value of 0.143. (Bottom) Viewing distribution plot calculated in cryoSPARC. (C) Cryo-EM maps of the uniform refinement (left) and particle-subtracted non-uniform refinement (right) colored by local resolution. The cryo-EM maps are shown as top (top) and side (bottom) views. (D) Cryo-EM maps of DS-CavEs2-IPDS with bound MPV467 are shown in side (left) and top (right) views. Single protomers are shown as transparent colored surfaces with docked 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 maps are shown as transparent surfaces with docked models shown as ribbons and sticks. Same coloring as in D. [Figure 9C-E] Same as above. [Figure 10] Figure 10 is Table 1. Neutralization and binding properties of hMPV F-specific mAbs. Neutralization values ​​were determined using a plaque reduction assay. IC50 corresponds to the mAb concentration at which 50% plaque reduction was observed. EC50 values ​​correspond to the concentration at which a half-maximal signal was obtained in ELISA based on optical density at 405 nm. > indicates that at the highest mAb concentration tested, the binding signal was below 1 μg / mL. 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. [Figure 11] Figure 11 is Table 2. EM Data Collection for MPV467. [Figure 12] Replication and clearance of hMPV in immunosuppressed versus normal cotton rats. Cotton rats immunosuppressed via repeated cyclophosphamide treatment (Immunosuppr) or normal cotton rats (Normal) were challenged with 105 PFU of hMPV per animal. Five, seven or nine days after infection, animals were sacrificed and lung and nasal samples were collected for virus titration by plaque assay followed by immunostaining. Results show geometric mean ± SEM for 4-10 animals per group (data combined from two studies). *p<0.05 when compared to hMPV-infected normal animals sacrificed on the same day. [Figure 13] MPV467 prevention and treatment of hMPV infection in immunosuppressed cotton rats: dose-dependence of antiviral effects. Immunosuppressed S. hispidus were challenged with 105 PFU / animal of hMPV. MPV467 treatment was administered intramuscularly as 0.1, 1 or 10 mg / kg 1 day before (Prophyl) or 3 days after (Therap) hMPV challenge. Five days after infection, animals were sacrificed and lung and nasal samples were collected for hMPV quantification. Results show geometric mean ± SEM for 5 animals per group. *p<0.05 when compared to mock-treated hMPV-infected animals sacrificed 5 days after infection. [Figure 14] Effect of antibody therapy on delayed viral clearance in immunosuppressed cotton rats infected with hMPV. Immunosuppressed and normal S. hispidus were challenged with 105 PFU / animal of hMPV and treated with 10 mg / kg MPV467 3 days (normal) and 3 and 7 days (immunosuppressed) after infection. Five or nine days after infection, animals were sacrificed and lung and nasal samples were collected for hMPV quantification. Results show geometric mean ± SEM for 4-10 animals per group. *p<0.05 when compared to mock-treated hMPV-infected animals sacrificed on the corresponding day. [Figure 15]Effect of antibody therapy on lung histopathology in normal and immunosuppressed cotton rats infected with hMPV. Immunosuppressed and normal S. hispidus were challenged with 105 PFU / animal of hMPV and treated with MPV467 as described in the legend to Figure 14. Lung histopathology was evaluated on hematoxylin-eosin (H&E) slides in each of the following categories: peribronchiolitis (Peribr), perivasculitis (Perivasc), interstitial inflammation (Interst), alveolitis (Alveol) and epithelial hyperplasia (Epith.Hyp.). Results show the mean ± SE for 4-5 animals for each group. [Figure 16] Effect of antibody treatment on pulmonary cytokine / chemokine expression in normal and immunosuppressed cotton rats infected with hMPV. Immunosuppressed and normal S. hispidus were challenged with 105 PFU / animal of hMPV, treated with 10 mg / kg MPV467 or PBS (mock) and sacrificed 9 days after infection. Expression of MIP-1a and IP-10 mRNA was quantified in lung tissue by qPCR and normalized by expression of b-actin in the corresponding organs. Results show the mean ± SE for 4-5 animals for each group. *p<0.05 compared to uninfected (Ctl) or normal animals receiving PBS (mock) or MPV467 (Ab) treatment. #p<0.05 for antibody-treated animals versus mock-treated animals. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS The hMPV F protein is a trimeric class I viral fusion protein with high conservation among viral subgroups (A1, A2, B1, B2) (Huang and Mousa, Front. Immunol. 10, 2778 (2019)). hMPV can infect respiratory epithelial cells in the absence of the hMPV G protein, but hMPV G is required for viral fitness in vivo (Biacchesi et al., J. Virol. 78, 12877-12887 (2004)). The hMPV F protein contains an RGD motif and the receptor is α 5 β 1 It has been hypothesized to be an integrin (Cox et al., J. Virol. 86, 12148-12160 (2012)). Heparan sulfate has also been shown to have a role in hMPV F protein-mediated binding (Chang et al., J. Virol. 86, 3230-3243 (2012)), and direct binding between heparan sulfate and hMPV F protein has recently been demonstrated (Jiachen, H. et al. J. Virol. 95, e00593-21 ​​(2021)). hMPV F induces fusion of the viral membrane with the host cell membrane in a transition from a metastable prefusion state to a postfusion conformation (Poor et al., PNAS 111, 2596-2605 (2014)). 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. J. Virol. 95, e00593-21 ​​(2021);Mas et al. PLoS Pathog. 12, e1005859 (2016)) conformations have been solved, and the protein shares a similar structural topology with the RSV F protein (McLellan et al., Curr. Top. Microbiol. Immunol. 372, 83-104 (2013)).

[0020] There is a paucity of information regarding specific epitopes on the hMPV F protein compared to RSV F. Clear differences in immunological features between RSV F and hMPV F have been identified. For example, pre-fusion and post-fusion hMPV F proteins elicit similar antibody responses, because the majority of neutralizing epitopes are present in both conformations (Jiachen, H. et al. J. Virol. 95, e00593-21 ​​(2021);Battles et al. Nat. Commun. 8, 1528 (2017);Pilaev et al., Vaccine 38, 2122-2127 (2020)), whereas for RSV F, the majority of the most potent neutralizing antibodies target pre-fusion specific epitopes (Huang and Mousa, Front. Immunol. 10, 2778 (2019);Gilman et al., Sci. Immunol. 1, 1-12 (2016);Mousa et al., Nat. Microbiol. 2, 16271 (2017)). (2017)). Known neutralizing epitopes on the hMPV F protein include antigenic sites IV (Mas et al. PLoS Pathog. 12, e1005859 (2016);Mousa et al., PLoS Pathog. 14, e1006837 (2018);Schuster et al., J. Infect. Dis. 211, 1-34 (2014)), III (Corti et al., Nature 501, 439-43 (2013);Wen et al., Nat. Microbiol. 2, 16272 (2017)), and V (Xiao et al., MAbs 11, 1415-1427 (2019)), based on the identification of RSV F mAbs that also neutralize hMPV F. The unique epitope targeted by human mAb DS7 has been structurally defined (Wen et al. Nat. Struc. Mol. Biol. 19, 461-463 (2012)).Additionally, a novel epitope located within the trimer interface of the hMPV F protein, defined by mAb MPV458, was recently identified (Huang and Mousa, PLOS Pathog. 16, e1008942 (2020)). To further understand the neutralizing hMPV F epitopes, a panel of 18 human mAbs against the hMPV F protein was isolated.

[0021] Two mAbs, MPV467 and MPV487, were demonstrated to have potent neutralizing activity, with MPV467 being exceptionally potent. It was also demonstrated that MPV467 can prevent and treat hMPV infection in mice, and using cryo-electron microscopy it was determined that the epitope of MPV467 targets a complex binding site that interfaces with both antigenic sites II and V.

[0022] I. Summary of Terms Unless otherwise indicated, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology can be found in Krebs et al. (eds.), 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 "an antigen" includes single or multiple antigens and can be considered equivalent to the phrase "at least one antigen". As used herein, the term "comprises" means "includes". It is further understood that any and all base or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for descriptive purposes unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, certain suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. Furthermore, the materials, methods and examples are illustrative only and are not intended to be limiting. In order to facilitate review of the various embodiments, the following explanations of terms are provided:

[0023] About: Unless the context indicates otherwise, "about" refers to plus or minus 5% of the reference value. For example, "about" 100 refers to 95 to 105.

[0024] Administration: The introduction of a composition into a subject by a selected route. Administration can be local or systemic. For example, if the selected route is intravenous, the composition (e.g., a composition comprising the disclosed antibody or antigen-binding fragment) is administered by introducing the composition into the subject's vein. Exemplary administration routes include, but are not limited to, oral, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal and intravenous), sublingual, rectal, transdermal (e.g., topical), intranasal, vaginal and inhalation routes.

[0025] Agent: Any substance or combination of substances useful for achieving a purpose or result; for example, a substance or combination of substances useful for inhibiting hMPV infection in a subject. Agents include proteins, nucleic acid molecules, compounds, small molecules, organic compounds, inorganic compounds, or other molecules of interest. Agents may include therapeutic agents (e.g., antiretroviral agents), diagnostic agents, or pharmaceutical agents. In some embodiments, an agent is an antibody that specifically binds to hMPV, optionally in combination with an antiviral agent. Those skilled in the art will understand that a particular agent may be useful for achieving more than one result.

[0026] Amino acid substitution: The replacement of one amino acid in a polypeptide with a different amino acid or with no amino acid (i.e., a deletion). In some instances, an amino acid in a polypeptide is replaced with an amino acid from a homologous polypeptide, for example, an amino acid in a recombinant Group A MPV F polypeptide may be replaced with the corresponding amino acid from a Group B MPV F polypeptide.

[0027] Antibodies and antigen-binding fragments: Immunoglobulins, antigen-binding fragments, or derivatives thereof that specifically bind and recognize an analyte (antigen), e.g., an hMPV F polypeptide. The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments, so long as they exhibit the desired antigen-binding activity.

[0028] Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof that retain binding affinity to an antigen. Examples of antigen-binding fragments include Fv, Fab, Fab', Fab'-SH, F(ab') 2Antibody fragments include, but are not limited to, antigen-binding fragments produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (Eds.), Antibody Engineering, Vols. 1-2, 2002; ... nd ed., Springer-Verlag, 2010).

[0029] Antibodies also include genetically engineered forms such as chimeric antibodies (eg, humanized murine antibodies) and heteroconjugate antibodies (eg, bispecific antibodies).

[0030] An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical or different from each other. For example, naturally occurring immunoglobulins have two identical binding sites, single-chain antibodies or Fab fragments have one binding site, while bispecific or bifunctional antibodies have two different binding sites.

[0031] Typically, naturally occurring immunoglobulins have heavy (H) and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as a myriad of immunoglobulin variable domain genes. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA and IgE.

[0032] Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain). In combination, the heavy and light chain variable regions specifically bind to an antigen.

[0033] "V H References to "VH" or "VH" refer to the variable region of an antibody heavy chain, including antigen-binding fragments such as Fv, scFv, dsFv or Fab. L References to "VL" or "VL" refer to the variable domain of an antibody light chain, including that of an Fv, scFv, dsFv or Fab.

[0034] V H and V L contains a "framework" region interrupted by three hypervariable regions, also called "complementarity determining regions" or "CDRs" (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5 th (See, e.g., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, US Department of Health and Human Services, 1991). The sequences of framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, which is the combined framework regions of the constituent light and heavy chains, functions to position and align the CDRs in three-dimensional space.

[0035] The CDRs are primarily responsible for binding to an epitope of an antigen. The amino acid sequence boundaries of a given CDR can be readily determined using any of several well-known schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, US Department of Health and Human Services, 1991; the "Kabat" numbering scheme), Al-Lazikani et al., ("Standard conformations for the canonical structures of immunoglobulins," J. Mol. Bio., 273(4):927-948, 1997; the "Chothia" numbering scheme), and Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev. Comp. Immunol., 27(1):55-77, 2003; the "IMGT" numbering scheme). The CDRs of each chain are typically referred to (from N-terminus to C-terminus) as CDR1, CDR2, and CDR3, and are also typically identified by the chain in which the particular CDR is located. H CDR3 is the V of the antibody in which it is found H CDR3 derived from V L CDR1 is the V domain of the antibody in which it is found. L The light chain CDRs may be referred to as LCDR1, LCDR2, and LCDR3. The heavy chain CDRs may be referred to as HCDR1, HCDR2, and HCDR3.

[0036] In some embodiments, the disclosed antibodies comprise a heterologous constant domain, for example, an antibody comprises a constant domain that is different from the native constant domain, for example, a constant domain that comprises one or more modifications to increase half-life (e.g., "LS" mutations).

[0037] A "monoclonal antibody" is an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical and / or bind the same epitope, except for possible variant antibodies that contain, for example, naturally occurring mutations or arise during the production of the monoclonal antibody preparation, and such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin locus, such methods and other exemplary methods for producing monoclonal antibodies are described herein. In some examples, the monoclonal antibody is isolated from a subject. Monoclonal antibodies may 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, 2 nd (see, e.g., New York: Cold Spring Harbor Laboratory Press, 2014).

[0038] A "humanized" antibody or antigen-binding fragment comprises a human framework region and one or more CDRs derived from a non-human (e.g., mouse, rat or synthetic) antibody or antigen-binding fragment. The non-human antibody or antigen-binding fragment providing the CDRs is referred to as the "donor" and the human antibody or antigen-binding fragment providing the framework is referred to as the "acceptor". In one embodiment, all CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if present, can be substantially identical, e.g., at least about 85-90%, e.g., about 95% or more identical, to human immunoglobulin constant regions. Thus, all parts of a humanized antibody or antigen-binding fragment, except possibly the CDRs, are substantially identical to the corresponding parts of a natural human antibody sequence.

[0039] A "chimeric antibody" is an antibody that contains sequences from two different antibodies, typically from different species. In some instances, a chimeric antibody contains one or more CDRs and / or framework regions from one human antibody and the CDRs and / or framework regions from another human antibody.

[0040] A "fully human antibody" or "human antibody" is an antibody that contains sequences from (or derived from) the human genome and does not contain sequences from another species. In some embodiments, a human antibody contains the CDRs, framework regions, and (if present) the Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated using techniques to create antibodies based on sequences from the human genome, for example, by phage display or using transgenic animals (see, for example, Barbas et al. Phage display: A Laboratory Manuel. 1999). stEd. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008).

[0041] MPV-neutralizing antibody or antigen-binding fragment: An antibody or antigen-binding fragment that specifically binds to an hMPV antigen, e.g., hMPV (e.g., F protein), in a manner that inhibits a biological function associated with inhibiting hMPV infection. The antibody can neutralize the activity of hMPV at various points during the pathogen's life cycle.

[0042] Biological sample: sample obtained from subject. Biological sample includes all clinical samples useful for detecting disease or infection (e.g., hMPV infection) in subject, including but not limited to cell, tissue and bodily fluid, such as blood, blood derivatives and fractions (e.g., serum), cerebrospinal fluid; and biopsied or surgically removed tissue, such as unfixed, frozen, or fixed in formalin or paraffin. In a specific example, biological sample is obtained from subject having or suspected of having hMPV infection.

[0043] Bispecific antibodies: recombinant molecules composed of two different antigen-binding domains that result in binding to two different antigenic epitopes. Bispecific antibodies include chemically or genetically linked molecules of two antigen-binding domains. The antigen-binding domains may be linked using a linker. The antigen-binding domains may be monoclonal antibodies, antigen-binding fragments (e.g., Fab, scFv), or combinations thereof. Bispecific antibodies may contain one or more constant domains, but do not necessarily contain a constant domain.

[0044] Conditions sufficient to form an immune complex: conditions that allow an antibody or antigen-binding fragment thereof to bind to its cognate epitope to a degree detectably greater than binding to substantially all other epitopes and / or to the substantial exclusion of binding to substantially all other epitopes. Conditions sufficient to form an immune complex depend on the format of the binding reaction and are typically those utilized in immunoassay protocols or conditions encountered in vivo. For a description of immunoassay formats and conditions, see Harlow & Lane, Antibodies, A Laboratory Manual, 2 nd ed. Cold Spring Harbor Publications, New York (2013). The conditions used in these methods are "physiological conditions," including reference to conditions (e.g., temperature, osmolality, pH) typical inside a living mammal or mammalian cell. Although it is recognized that some organs are exposed to extreme conditions, the intra-organism and intracellular environment is usually at approximately pH 7 (e.g., pH 6.0-pH 8.0, more typically pH 6.5-7.5), contains water as the predominant solvent, and exists at temperatures above 0° C. and below 50° C. The osmolality is within a range that supports cell viability and proliferation.

[0045] Conjugate: A complex of two molecules linked together, e.g., covalently linked together. In one embodiment, an antibody is linked to an effector molecule; e.g., an antibody that specifically binds to hMPV F protein is covalently linked to an effector molecule. Linking can be by chemical or recombinant means. In one embodiment, linking is chemical, where reaction between the antibody moiety and the effector molecule produces a covalent bond formed between the two molecules to form one molecule. A peptide linker (short peptide sequence) can be included between the antibody and the effector molecule, if necessary. Conjugates can be prepared from two molecules with separate functionalities, e.g., an antibody and an effector molecule, and therefore may also be called "chimeric molecules."

[0046] Conservative variant: A "conservative" amino acid substitution is a substitution that does not substantially affect or reduce the function of a protein, such as the ability of the protein to interact with a target protein. For example, an MPV-specific antibody can 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 neutralizing activity. The term conservative variation also includes the use of a substituted amino acid instead of an unsubstituted parent amino acid.

[0047] Individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids (e.g., less than 5%, in some embodiments less than 1%) in an encoded sequence are conservative variations in which the alteration results in the replacement of an amino acid with a chemically similar amino acid.

[0048] The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0049] Non-conservative substitution is a substitution that reduces the activity or function of hMPV specific antibody, such as the ability to specifically bind to hMPV F protein or the ability to neutralize hMPV.For example, if an amino acid residue is essential for the function of a protein, even a conservative substitution in other respects may destroy its activity.Therefore, conservative substitution does not change the basic function of the protein of interest.

[0050] Contacting: placing in direct physical association; includes both in solid form or liquid form, and can occur either in vivo or in vitro. Contacting includes the contact between one molecule and another, for example, the amino acids on the surface of one polypeptide, such as a peptide, that contacts another polypeptide. Contacting can also include contacting cells, for example, by placing a polypeptide in direct physical association with a cell.

[0051] Control: Reference standard. In some embodiments, the control is a negative control sample obtained from a healthy patient. In other embodiments, the control is a positive control sample obtained from a patient diagnosed with MPV infection. In yet other embodiments, the control is a historical control or a standard reference value or range of values ​​(e.g., a previously tested control sample, e.g., a group of MPV patients with known prognosis or outcome, or a group of samples showing baseline or normal values).

[0052] The difference between the test sample and the control can be increased or, conversely, decreased.The difference can be a qualitative difference or a quantitative difference, for example, a statistically significant difference.In some examples, the difference is an increase or decrease of at least about 5%, for example, 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% compared to the control.

[0053] Degenerate variant: In the context of this disclosure, a "degenerate variant" refers to a polynucleotide encoding a polypeptide (e.g., a recombinant MPV F protein or an immunogenic fragment thereof) that comprises a sequence that is degenerate as a result of the genetic code. There are 20 naturally occurring amino acids, most of which are specified by more than one codon. Thus, all degenerate nucleotide sequences that encode a peptide are included, as long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.

[0054] Detectable marker: a detectable molecule (also known as a label) that is directly or indirectly conjugated to a second molecule, such as an antibody, to facilitate detection of the second molecule. For example, a detectable marker may be detectable by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (e.g., CT scan, MRI, ultrasound, fiber optic examination and laparoscopic examination). 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 in the selection of suitable detectable markers for various purposes can be found, for example, in Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4 th 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).

[0055] Detect: To identify the existence, presence, or fact of something.

[0056] DS7 antibody: a neutralizing monoclonal antibody that specifically binds to an epitope on the hMPV F protein present on 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., Nat. Struct. Mol. Biol., 19, 461-463, 2012, which is incorporated 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 are designated as 4DAG_H (DS7 V H ) and 4DAG_L(DS7 V L ), each of which is incorporated by reference herein as present in the database on November 10, 2014. [ka]

[0057] Effective amount: A quantity of a particular substance sufficient to achieve a desired effect in a subject to which it is administered. For example, this may be the amount of an antibody required to inhibit hMPV infection or to measurably alter the outward symptoms of hMPV infection.

[0058] In some embodiments, administration of an effective amount of the disclosed antibodies or antigen-binding fragments that bind to the 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 survival time of infected subjects, or a reduction in symptoms associated with hMPV) by a desired amount, e.g., 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.

[0059] The effective amount of an antibody or antigen-binding fragment that specifically binds to hMPV F protein administered to a subject to inhibit hMPV infection will vary depending on several factors related to the subject, such as the subject's general health and / or weight. Effective amounts can be determined by varying dosages and measuring the resulting response, such as a reduction in pathogen titers. Effective amounts can also be determined via various in vitro, in vivo, or in situ immunoassays.

[0060] Effective amount includes the partial dose that contributes to achieve effective response in combination with previous or subsequent administration.For example, effective amount of drug can be administered in a single dose or in several doses, for example, daily during the course of treatment lasting several days or weeks.However, effective amount can depend on the subject being treated, the severity and type of the condition being treated, and the mode of administration.Unit dosage form of drug can be packaged in a single amount or multiple effective amounts, for example, in a vial (for example, with a pierceable cap) or syringe with sterile components.

[0061] Epitope: Antigenic determinant. These are specific chemical groups or peptide sequences on a molecule that are antigenic so as to elicit a specific immune response, e.g., an epitope is a region of an antigen to which B and / or T cells respond. An antibody can bind to a specific antigenic epitope, e.g., an epitope on the hMPV F protein.

[0062] 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 in some cases are processed to become mRNA. A gene can also be expressed when its mRNA is translated into an amino acid sequence, such as a protein or protein fragment. In certain examples, a heterologous gene is expressed when it is transcribed into RNA. In other examples, a heterologous gene is expressed when its RNA is translated into an amino acid sequence. The term "expression" is used herein to refer to either transcription or translation. Regulation of expression can include control over transcription, translation, RNA transport and processing, degradation of intermediate molecules, such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced.

[0063] Expression control sequence: A nucleic acid sequence that regulates the expression of a heterologous nucleic acid sequence operably linked thereto. An expression control sequence is operably linked to a nucleic acid sequence when the expression control sequence controls and regulates the transcription and optionally the translation of the nucleic acid sequence. Thus, an expression control sequence may include an appropriate promoter, enhancer, transcription terminator, a start codon (ATG) in front of a protein-coding gene, splicing signals for introns, maintaining the correct reading frame of the gene to allow proper translation of mRNA, and a stop codon. The term "control sequence" is intended to include, at a minimum, components whose presence can affect expression, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. An expression control sequence may include a promoter.

[0064] A promoter is a minimal sequence sufficient to direct transcription. Also included are promoter elements sufficient to make promoter-dependent gene expression controllable to be cell type-specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' region of the gene. Both constitutive and inducible promoters are included (see, for example, Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL, plac, ptrp, ptac (ptrp-lac hybrid promoter) of bacteriophage lambda may be used. In one embodiment, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., retroviral long terminal repeat; adenovirus late promoter; vaccinia virus 7.5K promoter) may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide transcription of nucleic acid sequences.

[0065] The polynucleotide may be inserted into an expression vector containing a promoter sequence that facilitates efficient transcription of the inserted gene sequence in the host. Expression vectors typically contain an origin of replication, a promoter, as well as specific nucleic acid sequences that allow for phenotypic selection of transformed cells.

[0066] Expression vector: A vector that contains a recombinant polynucleotide that comprises an expression control sequence operably linked to the nucleotide sequence to be expressed. Expression vectors contain sufficient cis-acting elements for expression; other elements for expression can be provided by host cell or in an in vitro expression system. Expression vectors include all those known in the art that incorporate recombinant polynucleotides, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses).

[0067] Fc region: the constant region of an antibody, excluding 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, N-terminal to these domains, some or all of the flexible hinge. For IgA and IgM, the Fc region may or may not include the tail, and may or may not have the J chain attached. For IgG, the Fc region is typically understood to include the immunoglobulin domains Cγ2 and Cγ3, and optionally the lower part of the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include the residues after C226 or P230, relative to the carboxyl terminus of the Fc, where the numbering is according to Kabat. For IgA, the Fc region includes immunoglobulin domains Cα2 and Cα3, and optionally the lower part of the hinge between Cα1 and Cα2.

[0068] Heterologous: originating from a different genetic source. A nucleic acid molecule heterologous to a cell originating 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, for example, an scFv, is expressed in a cell, for example, 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 acid, including electroporation, lipofection, particle gun acceleration, and homologous recombination.

[0069] Host cell: A cell in which a vector can be propagated and its DNA can be expressed. The cell can be prokaryotic or eukaryotic. This term also includes any progeny of the subject host cell. It is understood that not all progeny are identical to the parent cell, since mutations can occur during replication. However, when the term "host cell" is used, such progeny are included.

[0070] IgG: A polypeptide belonging to the widely recognized class or isotype of antibody substantially encoded by the immunoglobulin gamma gene. In humans, this class is IgG 1 , IgG 2 , IgG 3 and IgG 4 Includes.

[0071] Immune complex: The binding of antibody or antigen-binding fragment (e.g., scFv) to soluble antigen forms immune complex. The formation of immune complex can be detected by conventional methods such as immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (e.g., Western blot), magnetic resonance imaging, CT scan, X-ray examination and affinity chromatography.

[0072] Immune response: A response of a cell of the immune system, e.g., a B cell, T cell, or monocyte, 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, e.g., a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response, resulting in the production of a specific antibody.

[0073] Immunogen: A compound, composition or substance capable of stimulating the production of antibodies or a T-cell response in an animal, including compositions injected or absorbed into an animal, such as hMPV. Immunogens react with the products of specific humoral or cellular immunity.

[0074] Inhibit a disease or condition: Reducing the full onset of a disease or condition in a subject, e.g., reducing the full onset of an MPV infection, e.g., an hMVP infection, in a subject at risk for MPV infection, including neutralizing, antagonizing, inhibiting, suppressing, slowing, destroying, halting, or reversing the progression or severity of a disease or condition.

[0075] Inhibiting a disease or condition can refer to a preventive intervention administered before the onset of a disease or condition (e.g., treatment initiated in a subject at risk of hMPV infection but not infected with hMPV) that reduces the subsequent onset of the disease or condition and / or ameliorates the signs or symptoms of the disease or condition after onset. With respect to inhibiting a disease or condition, the term "ameliorating" refers to any observable beneficial effect of a preventive intervention intended to inhibit a disease or condition. A beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease or condition in a susceptible subject, a reduction in the severity of some or all clinical symptoms of the disease or condition, a slower progression of the disease or condition, an improvement in the general health or well-being of the subject, a reduction in infection, or by other parameters specific to a particular disease or condition.

[0076] In some embodiments, the disclosed hMPV F protein-specific antibodies and antigen-binding fragments inhibit the growth of hMPV in a subject, e.g., the antibodies and antigen-binding fragments inhibit the expansion of hMPV in a subject, resulting in a reduction in pathogen load in the subject compared to a suitable control. For example, the disclosed hMPV F protein-specific antibodies and antigen-binding fragments can inhibit hMPV infection or viral replication in a subject by at least 20%, at least 30%, at least 40%, or at least 50% compared to a suitable control.

[0077] Isolated: A biological component (e.g., a nucleic acid, peptide, protein or protein complex, e.g., an antibody) that is substantially separated, produced away 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 it 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 a host cell, as well as 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.

[0078] Kabat location: Kabat et al. (Sequences of Proteins of Immunological Interest, 5 th The position of the residue in an amino acid sequence according to the numbering conventions described in the National Institutes of Health, Publication No. 91-3242, 1991).

[0079] Linker: A bifunctional molecule that can be used to link two molecules into one continuous molecule, for example, to link an effector molecule to an antibody or to link a detectable marker to an antibody. Non-limiting examples of peptide linkers include glycine-serine linkers.

[0080] The terms "conjugate," "connect," "couple," or "link" can refer to making two molecules into one contiguous molecule; for example, linking two polypeptides into one contiguous polypeptide, or covalently linking an effector molecule or a detectable marker radionuclide or other molecule to a polypeptide, e.g., an scFv. Linking can be by chemical or recombinant means. "Chemical means" refers to a reaction between an antibody moiety and an effector molecule such that a covalent bond is formed between the two molecules to form one molecule.

[0081] Metapneumovirus (MPV): An enveloped, non-segmented, negative single-stranded RNA virus of the Paramyxoviridae family. It is a common cause of lower respiratory tract infections, including bronchiolitis and pneumonia, in children and adults, infecting nearly all humans by age 5. MPV causes recurrent infections, including severe lower respiratory tract disease, that can occur at any age, but especially in the elderly or those with compromised heart, lungs, or immune systems. hMPV can infect humans.

[0082] The MPV genome contains eight genes encoding nine proteins, including glycoproteins SH, G, and F. The F protein mediates fusion, which allows the virus to enter the cell cytoplasm. Two groups of human MPV strains have been described: A and B groups, which are further divided into subgroups A1, A2, B1, and B2. Exemplary MPV strain sequences are known to those skilled in the art. In addition, several models of human MPV infection are available, including model organisms infected with hMPV (see, for example, Herfst et al., J General Virol., 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 upper 50% of the pre-fusion state and the tail is the lower 50% of the pre-fusion state.

[0083] Methods for diagnosing MPV infection are known, including the use of direct fluorescent antibody detection (DFA), rapid antigen detection by chromatography, and detection of viral RNA using RT PCR. 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 treatments include the use of the antiviral drug Ribaviran, as well as 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), which recognize MPV F protein and reduce the incidence of MPV infection and disease in animal models.

[0084] There are several subgroups of MPV in human MPV, including groups A and B, and subgroups A1, A2, B1 and B2. Within the subgroups of MPV, there are individual strains of each subgroup. The sequences of the F proteins from particular MPV strains are known and provided herein (see, e.g., Table 1).

[0085] MPV fusion (F) protein: MPV envelope glycoprotein that facilitates fusion of the viral membrane with the cell membrane. Naturally, the MPV F protein is F 0 It is initially synthesized as a single polypeptide precursor, called F, approximately 540 amino acids in length. 0 contains an N-terminal signal peptide that directs its localization to the endoplasmic reticulum, where it is expressed as a signal peptide (F 0 Approximately the first 18 residues of F are proteolytically cleaved. 0 The residues oligomerize to form trimers, which are then integrated into the protease site (F 0 between approximately positions 102 and 103 of the RQSR 102 (residues 99-102)) to give two disulfide-linked fragments: F 1 and F 2 The smaller of these fragments, F 2 F 0 Originating from the N-terminal portion of the precursor, F 0 The larger of these fragments, F 1 F contains an extracellular / luminal region at the C-terminus (approximately residues 103-490), a transmembrane domain (approximately residues 491-513) and a cytoplasmic domain (approximately residues 514-540). 0 It contains the C-terminal portion of the precursor (approximately residues 103 to 540).

[0086] The three F's 2 -F 1The protomers oligomerize into mature F proteins, which adopt a metastable "pre-fusion" conformation that is induced to undergo a conformational change (to a "post-fusion" conformation) upon contact with the target cell membrane. 1 Located at the N-terminus of the polypeptide, it exposes a hydrophobic sequence known as a fusion peptide, which associates with the host cell membrane and promotes fusion of the viral or infected cell membrane with the target cell membrane.

[0087] The extracellular portion of the MPV F protein is F 2 Protein (approximately positions 20 to 102 of MPV F) and F 1 The MPV F ectodomain comprises the ectodomain (approximately positions 103-490 of MPV F). The MPV F ectodomain trimer comprises a protein complex of three MPV F ectodomains.

[0088] 2A-2C show antigenic sites II, III, IV and V of the F protein based on competitive binding.

[0089] MPV F prefusion conformation: The structural conformation adopted by the MPV F protein prior to the induction of fusogenic events that result in the transition of MPV F to the postfusion conformation and after processing to the mature MPV F protein in the secretion system. The MPV F prefusion conformation is similar in overall structure to the prefusion conformations of the F proteins of other paramyxoviruses (e.g., RSV).

[0090] MPE8 antibody: a neutralizing monoclonal antibody that specifically binds to an epitope on the MPV F protein that is present on the pre-fusion conformation of the MPV F protein but not on the post-fusion conformation. MPE8 antibodies and methods for their 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, and the MPE8 heavy and light chain sequences are provided as AGU13651.1 (MPE8 V H ) and AGU13652.1(MPE8 V L ), each of which is incorporated by reference herein as present in the database as of November 10, 2014. [ka] [ka]

[0091] 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 examples, the infectious agent is a virus. In some examples, an antibody specific for MPV F neutralizes the infectious titer of hMPV. A "broadly neutralizing antibody" is an antibody that binds to and inhibits the function of a related antigen, e.g., an antigen that shares at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the antigenic surface of the antigen. With respect to antigens from pathogens, e.g., viruses, the antibody can bind to antigens from more than one class and / or subclass of the pathogen and inhibit their function. For example, with respect to MPV, the antibody can bind to antigens, e.g., MPV F from more than one group, and inhibit their function.

[0092] Nucleic acid: A polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds. Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs, such as, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "oligonucleotide" typically refers to short polynucleotides, generally no more than about 50 nucleotides. When a nucleotide sequence is shown by a DNA sequence (i.e., A, T, G, C), it is understood that this also includes RNA sequences (i.e., A, U, G, C) in which "U" replaces "T".

[0093] "Nucleotide" includes, but is not limited to, a monomer that contains a base linked to a sugar, e.g., a pyrimidine, a purine or a synthetic analog thereof, or a base linked to an amino acid, such as in a peptide nucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.

[0094] Conventional notation is 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 addition of nucleotides to the nascent RNA transcript in the 5' to 3' direction 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 that DNA and located 5' to the 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 3' to the 3' end of the coding RNA transcript is referred to as the "downstream sequence."

[0095] "cDNA" refers to a DNA complementary or identical to an mRNA, in either single-stranded or double-stranded form.

[0096] "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, either with a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene codes for a protein if transcription and translation of the mRNA produced by the gene produces a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA, can be said to code for the protein or other product of the gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and code for the same amino acid sequence. Protein- and RNA-encoding nucleotide sequences can include introns.

[0097] A first sequence is "antisense" to a second sequence if a polynucleotide whose sequence is a first sequence specifically hybridizes to a polynucleotide whose sequence is a second sequence.

[0098] Operably linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in 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 when the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, if necessary, connect two protein coding regions in the same reading frame.

[0099] Pharmaceutically acceptable carriers: The pharma- ceutically acceptable carriers used are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed antibodies and antigen-binding fragments thereof.

[0100] Generally, the nature of the carrier will depend on the particular mode of administration used. For example, parenteral formulations usually contain pharma- ceutically and physiologically acceptable fluids, such as injectable fluids including water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, etc., as vehicles. For solid compositions (e.g., powder, pill, tablet, 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 compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate. In certain embodiments suitable for administration to subjects, the carrier can be sterile and / or can be suspended or otherwise contained in a unit dosage form containing one or more measured doses of the composition. This can also be accompanied by a drug therapy for its use for treatment purposes. The unit dosage form can be, for example, in a sealed vial containing sterile contents or in a syringe for injection into a subject, or can be lyophilized for subsequent solubilization and administration, or can be a solid or controlled release dosage.

[0101] Polypeptide: any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). "Polypeptide" refers to amino acid polymers, including naturally occurring and non-naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are non-natural amino acids, e.g., artificial chemical mimetics of the corresponding naturally occurring amino acids. "Residue" refers to an amino acid or amino acid mimetic incorporated into the polypeptide by an amide bond or amide bond mimetic. A polypeptide has an amino terminus (N-terminus) and a carboxy terminus (C-terminus). "Polypeptide" is used interchangeably with peptide or protein and is used herein to refer to a polymer of amino acid residues.

[0102] Polypeptide Modifications: Polypeptides and peptides, such as the antibodies disclosed herein, can be modified by a variety of chemical techniques to produce derivatives that have essentially the same activity as the unmodified peptide and, if necessary, other desirable properties. For example, the carboxylic acid groups of the protein, whether at the carboxyl terminus or at the side chain, can be provided in the form of a salt of a pharma- ceutically acceptable cation, or can be provided as a C 1 ~C 16 It may be esterified to form an ester, or may be represented by the formula NR 1 R 2 wherein R 1 and R 2 are each independently H or C 1 ~C 16 The amino groups of the peptides, whether amino-terminal or side chain, may be in the form of pharma- ceutically acceptable acid addition salts, such as HCl, HBr, acetate, benzoate, toluenesulfonate, maleate, tartrate and other organic salts, or may be in the form of phenylalanine, ... 1 ~C 16 It may be modified to an alkyl or dialkylamino, or further converted to an amide.

[0103] Hydroxyl groups on peptide side chains are derivatized using well-recognized techniques. 1 ~C 16 Alkoxy or C 1 ~C 16 The phenyl and phenol rings of the peptide side chains can be converted to esters with one or more halogen atoms, e.g., F, Cl, Br, or I, or C 1 ~C 16 Alkyl, C 1 ~C 16 The methylene groups of the peptide side chains may be substituted with the corresponding C 2 ~C 4The thiol can be extended to alkylene. The thiol can be protected with any one of several well-recognized protecting groups, for example, acetamide groups. Those skilled in the art will also recognize methods for introducing cyclic structures into the peptides of the present disclosure to select structures that result in enhanced stability and provide conformational constraints to the structures. For example, C- or N-terminal cysteines can be added to peptides, so that when oxidized, the peptides contain disulfide bonds to generate cyclic peptides. Other peptide cyclization methods include the formation of thioethers and carboxyl- and amino-terminal amides and esters.

[0104] 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 a peptide or protein (e.g., an antibody) is enriched more than the peptide or protein is in its natural environment within a cell. In one embodiment, the preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation.

[0105] Recombinant: A recombinant nucleic acid is a nucleic acid having a sequence that does not occur in nature or that is created by the artificial combination of two otherwise separated segments of sequence. This artificial combination can be achieved by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acid, e.g., by genetic engineering techniques. A recombinant protein is a protein having a sequence that does not occur in nature or that is created by the artificial combination of two otherwise separated segments of sequence. In some embodiments, a recombinant protein is encoded by a heterologous (e.g., recombinant) nucleic acid introduced into a host cell, e.g., a bacterial or eukaryotic cell. The nucleic acid can be introduced, for example, on an expression vector with a signal capable of expressing the protein encoded by the introduced nucleic acid, or the nucleic acid can be integrated into a host cell chromosome.

[0106] Respiratory syncytial virus (RSV): an enveloped, non-segmented, negative single-stranded RNA virus of the Paramyxoviridae family. It is the most common cause of bronchiolitis and pneumonia in children in the first year of life, infecting nearly all children by age 3. RSV also causes repeated infections, including severe lower respiratory tract disease, which can occur at any age, but especially in the elderly or those with compromised heart, lungs, or immune systems. In the United States, RSV bronchiolitis is the leading cause of hospitalization in infants and the leading cause of asthma and wheezing throughout childhood (Shay et al., JAMA, 282, 1440 (1999); Hall et al., N. Engl. J. Med., 360, 588 (2009)). Globally, RSV is responsible for 66,000-199,000 deaths annually in children younger than 5 years of age (Nair et al., Lancet, 375, 1545 (2010)) and 6.7% of deaths in infants aged 1 month to 1 year, more than any other single pathogen except malaria (Lozano et al., Lancet, 380, 2095 (2013)).

[0107] The RSV genome is approximately 15,000 nucleotides in length and contains 10 genes encoding 11 proteins, including glycoproteins SH, G and F. The F protein mediates fusion, which allows the virus to enter the cell cytoplasm and also promotes the formation of syncytia. Based on the difference in antigenicity of the G glycoprotein, two subtypes of human RSV strains have been described: A and B subtypes. RSV strains for other species are also known, including bovine RSV. Exemplary RSV strain sequences are known to those skilled in the art. In addition, several models of human RSV infection are available, including the use of model organisms infected with hRSV, as well as model organisms infected with species-specific RSV, such as bRSV infection in cattle (see, for example, Bern et al., Am J, Physiol. Lung Cell Mol. Physiol., 301: L148-L156, 2011).

[0108] Sequence identity: The similarity between amino acid sequences is expressed in terms of the similarity between sequences, otherwise called sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs, orthologs or variants of a polypeptide share a relatively high degree of sequence identity when aligned using standard methods.

[0109] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

[0110] Once aligned, the number of matches is determined by counting the number of positions where identical nucleotides or amino acid residues occur in both sequences. The percent sequence identity is determined by dividing the number of matches by either the length of the sequence shown in the identified sequence or the length of the link (e.g., 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, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166÷1554). * (100=75.0). Percent sequence identity values ​​are rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 would be rounded to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 would be rounded to 75.2. Length values ​​are always integers.

[0111] 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 on the Internet, for use with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. A description of how to use this program to determine sequence identity is available on the Internet at the NCBI website.

[0112] Polypeptide homologs and variants are typically characterized by possessing at least about 75%, e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full length alignment with the amino acid sequence of interest. Proteins with even greater similarity to the reference sequence will exhibit increasing percentage identity, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity, when assessed by this method. When less than the entire sequence is compared for sequence identity, homologs and variants typically possess at least 80% sequence identity over a short window of 10-20 amino acids, and may possess at least 85% or at least 90% or 95% sequence identity, depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available on the internet at the NCBI website. Those skilled in the art will understand that these sequence identity ranges are provided for guidance only; it is entirely possible that strong and significant homologs may be obtained that fall outside the ranges provided.

[0113] For the sequence comparison of nucleic acid sequence, typically, one sequence acts as a reference sequence, and test sequence is compared to it.When using sequence comparison algorithm, test and reference sequences are input into computer, partial sequence coordinates are designated, and sequence algorithm program parameters are designated as necessary.Default program parameters are used.The method of alignment of sequences for comparison is well known in the art. Optimal alignment of sequences for comparison can be determined, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970, by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4 thed, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013). One example of a useful algorithm is PILEUP. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360, 1987. The method used is similar to that described by Higgins & Sharp, CABIOS 5:151-153, 1989. With PILEUP, a reference sequence is compared to other test sequences to determine percent sequence identity relationships using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res. 12:387-395, 1984).

[0114] Another example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., J. Mol. Biol. 215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402, 1977. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) uses as default a word length (W) of 11, alignment (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLASTP program (for amino acid sequences) uses as defaults a word length (W) of 3, and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989). Oligonucleotides are linear polynucleotide sequences up to about 100 nucleotide bases in length.

[0115] 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.

[0116] Specific Binding: When referring to an antibody or antigen-binding fragment, refers to a binding reaction that determines the presence of a target protein in the presence of a heterogeneous population of proteins and other biologics. Thus, under specified conditions, an antibody will preferentially bind to a particular target protein, peptide or polysaccharide (e.g., an antigen present on the surface of a pathogen, e.g., MPV F protein) and will not bind in significant amounts to other proteins present in the sample or subject. Specific binding may be determined by standard methods. For a description of immunoassay formats and conditions that may be used to determine specific immune reactivity, see Harlow & Lane, Antibodies, A Laboratory Manual, 2 nd ed., Cold Spring Harbor Publications, New York (2013).

[0117] For antibody-antigen complexes, the specific binding between the antigen and the antibody is approximately 10 -7 Less than molar, e.g., about 10 -8 Less than molarity, 10 -9 Less than, or even about 10 -10 Less than molar K D K D refers to the dissociation constant for a given interaction, e.g., a polypeptide-ligand interaction or an antibody-antigen interaction. For example, for a bimolecular interaction between an antibody or antigen-binding fragment and an antigen, this is the concentration of the individual components of the bimolecular interaction divided by the concentration of the complex.

[0118] An antibody that specifically binds to an epitope on hMPV F protein is an antibody that substantially binds to hMPV F protein, including cells or tissues expressing hMPV F protein, substrates to which hMPV F protein is bound, or hMPV F protein in biological samples. Of course, it is recognized that a certain degree of non-specific interaction may occur between an antibody and a non-target (e.g., cells that do not express hMPV F protein). Typically, specific binding results in a much stronger association between an antibody and a protein or cell bearing an antigen than between an antibody and a protein or cell lacking the antigen. Specific binding typically results in a greater than 2-fold, e.g., greater than 5-fold, greater than 10-fold, or greater than 100-fold increase in the amount of antibody bound (per unit time) to a protein containing the epitope or a cell or tissue expressing the target epitope, compared to a protein or cell or tissue lacking this epitope. Specific binding to a protein under such conditions requires an antibody selected for its specificity for a particular protein. A variety of immunoassay formats are appropriate for selecting antibodies or other ligands specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein.

[0119] Subject: A living multi-cellular vertebrate organism, a category that includes humans and non-human mammals. In some examples, the subject is a human. In certain examples, the subject is a newborn infant. In additional examples, a subject is selected that requires inhibition of hMPV infection. For example, the subject is either uninfected with hMPV and at risk of infection, or infected and in need of treatment.

[0120] Therapeutically effective amount: The amount of an agent, such as the disclosed antibody or antigen-binding fragment thereof, sufficient to prevent, treat (including prevent), reduce and / or ameliorate the symptoms and / or underlying causes of a disorder or disease, for example, to prevent, inhibit and / or treat hMPV infection. In some embodiments, a therapeutically effective amount is sufficient to reduce or eliminate the symptoms of a disease, for example, hMPV infection. For example, this may be the amount required to inhibit or prevent viral replication or to measurably change the superficial symptoms of viral infection. Generally, this amount is sufficient to measurably inhibit viral replication or infectivity.

[0121] In one example, the desired response is to inhibit or reduce or prevent hMPV infection.For the method to be effective, MPV infection does not need to be completely eliminated or reduced or prevented.For example, administration of a therapeutically effective amount of an agent can reduce hMPV infection (e.g., measured by infection of cells or by the number or percentage of subjects infected with hMPV) by a desired amount, for example, 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 therapeutically effective amount of the drug can be administered in a single dose or in several doses, for example, daily during the course of treatment (e.g., prime-boost vaccination treatment).However, the therapeutically effective amount can 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 drug can be packaged in a single therapeutic amount or multiple therapeutic amounts, for example, in a vial (e.g., with a pierceable cap) or syringe with sterile components.

[0123] Treating or preventing a disease: For example, inhibiting the full development of a disease or condition in a subject at risk of or having a disease, such as hMPV infection. "Treatment" refers to a therapeutic intervention that ameliorates the signs or symptoms of a disease or pathological condition after it begins to develop. In relation to a disease or pathological condition, the term "ameliorating" refers to any observable beneficial effect of the treatment. A beneficial effect can be evidenced, for example, by 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 slower progression of the disease, a reduction in viral load, an improvement in the overall health or well-being of the subject, or other parameters well known in the art that are specific to a particular disease. A "prophylactic" treatment is a treatment administered to a subject who does not show signs of the disease or who shows only early signs, with the aim of reducing the risk of developing the pathology.

[0124] The term "reduce" is a relative term, such as when the disease or condition is quantitatively reduced after administration of the drug, or when the disease or condition is reduced after administration of the drug, the drug reduces the disease or condition.Similarly, the term "prevent" does not necessarily mean that the drug 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 infection can eliminate infection in the absence of the drug or compared to the reference drug, for example, at least about 50%, for example, at least about 70%, or about 80%, or even about 90%, but does not necessarily have to eliminate it completely, as long as the infection is measurably reduced.

[0125] Transformed: A transformed cell is a cell into which a nucleic acid molecule is 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 by viral vectors, transformation by plasmid vectors, and introduction of DNA by electroporation, lipofection and particle gun acceleration.

[0126] Vector: An entity that contains a nucleic acid molecule (e.g., a DNA or RNA molecule) that is operatively linked to a coding sequence of a protein of interest and that carries a promoter (multiplication) that 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 bacteria or other microorganism that may be replication incompetent, or a virus or bacteria or other microorganism that may be replication competent. A vector may be referred to as a construct. A recombinant DNA vector is a vector that has recombinant DNA. A vector may contain a nucleic acid sequence that allows its replication in a host cell, such as an origin of replication. A vector may also contain one or more selectable marker genes and other genetic elements. A viral vector is a recombinant nucleic acid vector that has at least some nucleic acid sequence derived from one or more viruses. In some embodiments, the viral vector contains a nucleic acid molecule that encodes the disclosed antibody or antigen-binding fragment that specifically binds to hMPV F protein and neutralizes hMPV.

[0127] Under conditions sufficient for: A phrase used to describe any environment that allows for a desired activity.

[0128] II. Description of Several Embodiments Provided are isolated monoclonal antibodies and antigen-binding fragments that specifically bind to 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 that include the antibodies and antigen-binding fragments and a pharma- ceutically acceptable carrier. Also provided are nucleic acids that encode the antibodies or antigen-binding fragments, and expression vectors (e.g., adeno-associated virus (AAV) virus vectors) that include these nucleic acids. The antibodies, antigen-binding fragments, nucleic acid molecules, host cells, and compositions can be used for research, diagnostic, treatment, and prophylactic purposes. For example, the disclosed antibodies and antigen-binding fragments can be used to diagnose a subject with hMPV infection, or can be administered to inhibit hMPV infection in a subject.

[0129] In some embodiments, the disclosed antibodies do not cross-react with the RSV F protein. In other embodiments, these antibodies neutralize both genotypes A and B of hMPV. In further embodiments, the disclosed antibodies bind to site III of the MPV F protein. In yet other embodiments, the disclosed antibodies bind to the pre-fusion F protein with higher affinity than the post-fusion F protein.

[0130] A. Monoclonal antibodies and antigen-binding fragments thereof that specifically bind to the hMPV F protein The following discussion of monoclonal antibodies refers to isolated monoclonal antibodies (or antigen-binding fragments thereof) that contain heavy and / or light chain variable domains that contain CDR1, CDR2 and / or CDR3 according to the IMGT numbering scheme (unless the context indicates otherwise). Various CDR numbering schemes (e.g., Kabat, Chothia or IMGT numbering schemes) can be used to determine the CDR positions. The amino acid sequences of the heavy and light chains and CDRs of the disclosed monoclonal antibodies according to the IMGT numbering scheme are provided in the sequence table, but are merely exemplary.

[0131] In some embodiments, a monoclonal antibody is provided that comprises the heavy and light chain CDRs of any one of the antibodies described herein. In some embodiments, a monoclonal antibody is provided that comprises the heavy and light chain variable regions of any one of the antibodies described herein. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

[0132] a. Monoclonal antibody MPV86 In some embodiments, the antibody or antigen-binding fragment is based on or derived from an MPV86 antibody and specifically binds to an hMPV F protein and neutralizes hMPV.

[0133] In some examples, the antibody or antigen-binding fragment comprises a V domain that contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of the MPV86 antibody (e.g., according to IMGT, Kabat or Chothia). H and VL which specifically binds to the hMPV F protein and neutralizes hMPV.

[0134] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence 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 set forth as SEQ ID NO:1. H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence 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 set forth as SEQ ID NO:5. L and specifically binds to the hMPV F protein and neutralizes hMPV. In additional embodiments, the antibody or antigen-binding fragment comprises a V that independently comprises an amino acid sequence 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 sequences set forth as SEQ ID NOs: 1 and 5, respectively. H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0135] In some embodiments, the antibody or antigen-binding fragment comprises a VHCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 2, 3 and 4, respectively. H and / or V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 6, 7 and 8, respectively. L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0136] In some embodiments, the antibody or antigen-binding fragment comprises a VHCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 2, 3 and 4, respectively. Hand V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 6, 7 and 8, respectively. L Including V H comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1, e.g., an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:1, L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 5, e.g., an amino acid sequence 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 5, and the antibody or antigen-binding fragment specifically binds to hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity is outside of the CDRs.

[0137] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V L In some embodiments, the antibody or antigen-binding fragment comprises a V protein comprising the amino acid sequences shown as SEQ ID NOs: 1 and 5, respectively, which specifically binds to the hMPV F protein and neutralizes hMPV. H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0138] b. Monoclonal antibody MPV414 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 VDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 (e.g., according to IMGT, Kabat or Chothia) of the MPV414 antibody, respectively. H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0139] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence 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 set forth as SEQ ID NO:9. H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence 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 set forth as SEQ ID NO: 13. L and specifically binds to the hMPV F protein and neutralizes hMPV. In additional embodiments, the antibody or antigen-binding fragment comprises a V that independently comprises an amino acid sequence 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 sequences set forth as SEQ ID NOs: 9 and 13, respectively. H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0140] In some embodiments, the antibody or antigen-binding fragment comprises a VHCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 10, 11 and 12, respectively. H and / or V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 14, 15 and 16, respectively. L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0141] In some embodiments, the antibody or antigen-binding fragment comprises a VHCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 10, 11 and 12, respectively. H and V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 14, 15 and 16, respectively. L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO:9, e.g., an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:9, L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 13, e.g., an amino acid sequence 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13, and the antibody or antigen-binding fragment specifically binds to hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity is outside of the CDRs.

[0142] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V L In some embodiments, the antibody or antigen-binding fragment comprises an F protein having an amino acid sequence as set forth as SEQ ID NOs: 9 and 13, respectively, and specifically binds to the hMPV F protein and neutralizes hMPV. H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0143] c. Monoclonal antibody MPV454 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV454 antibody and specifically binds to an hMPV F protein and neutralizes hMPV.

[0144] In some examples, the antibody or antigen-binding fragment comprises a V domain that contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of the MPV454 antibody (e.g., according to IMGT, Kabat or Chothia). H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0145] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence 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 set forth as SEQ ID NO: 17. H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence 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 set forth as SEQ ID NO:21. L and specifically binds to the hMPV F protein and neutralizes hMPV. In additional embodiments, the antibody or antigen-binding fragment comprises a V that independently comprises an amino acid sequence 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 sequences set forth as SEQ ID NOs: 17 and 21, respectively. H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0146] In some embodiments, the antibody or antigen-binding fragment comprises a VHCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 18, 19 and 20, respectively. H and / or V comprising LCDR1, LCDR2 and LCDR3 as set forth as SEQ ID NOs: 22, 23 and 24, respectively. L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0147] In some embodiments, the antibody or antigen-binding fragment comprises a VHCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 18, 19 and 20, respectively. H and V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 22, 23 and 24, respectively. L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO:17, e.g., an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:17, L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 21, e.g., an amino acid sequence 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 21, and the antibody or antigen-binding fragment specifically binds to hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity is outside of the CDRs.

[0148] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V L In some embodiments, the antibody or antigen-binding fragment comprises a V protein comprising the amino acid sequences set forth as SEQ ID NOs: 17 and 21, respectively, which specifically binds to the hMPV F protein and neutralizes hMPV. H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0149] d. Monoclonal antibody MPV456 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV456 antibody and specifically binds to an hMPV F protein and neutralizes hMPV.

[0150] In some examples, the antibody or antigen-binding fragment comprises a V domain that contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of the MPV456 antibody (e.g., according to IMGT, Kabat or Chothia). H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0151] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence 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 set forth as SEQ ID NO:25. H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence 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 set forth as SEQ ID NO:29. L and specifically binds to the hMPV F protein and neutralizes hMPV. In additional embodiments, the antibody or antigen-binding fragment comprises a V that independently comprises an amino acid sequence 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 sequences set forth as SEQ ID NOs: 25 and 29, respectively. H and V L which binds to the hMPV F protein and neutralizes hMPV.

[0152] In some embodiments, the antibody or antigen-binding fragment comprises a V domain comprising HCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 26, 27 and 28, respectively. H and / or V comprising LCDR1, LCDR2 and LCDR3 as set forth as SEQ ID NOs: 30, 31 and 32, respectively. L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0153] In some embodiments, the antibody or antigen-binding fragment comprises a V domain comprising HCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 26, 27 and 28, respectively. H and V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 30, 31 and 32, respectively. L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO:25, e.g., an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25, L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 29, e.g., an amino acid sequence 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 29, and the antibody or antigen-binding fragment specifically binds to hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity is outside of the CDRs.

[0154] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V L In some embodiments, the antibody or antigen-binding fragment comprises a V protein comprising the amino acid sequences set forth as SEQ ID NOs: 25 and 29, respectively, which specifically binds to the hMPV F protein and neutralizes hMPV. H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0155] e. Monoclonal antibody MPV464 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV464 antibody and specifically binds to an hMPV F protein and neutralizes hMPV.

[0156] In some examples, the antibody or antigen-binding fragment comprises a V domain that contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of the MPV464 antibody (e.g., according to IMGT, Kabat or Chothia). H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0157] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence 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 set forth as SEQ ID NO: 33. H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence 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 set forth as SEQ ID NO:37. L and specifically binds to the hMPV F protein and neutralizes hMPV. In additional embodiments, the antibody or antigen-binding fragment comprises a V that independently comprises an amino acid sequence 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 set forth as SEQ ID NOs: 33 and 37, respectively. H and V L which binds to the hMPV F protein and neutralizes hMPV.

[0158] In some embodiments, the antibody or antigen-binding fragment comprises a VHCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 34, 35 and 36, respectively. H and / or V comprising LCDR1, LCDR2 and LCDR3 as set forth as SEQ ID NOs: 38, 39 and 40, respectively. L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0159] In some embodiments, the antibody or antigen-binding fragment comprises a VHCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 34, 35 and 36, respectively. H and V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 38, 39 and 40, respectively. L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 33, e.g., an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 33, L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 37, e.g., an amino acid sequence 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 37, and the antibody or antigen-binding fragment specifically binds to hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity is outside of the CDRs.

[0160] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V L In some embodiments, the antibody or antigen-binding fragment comprises a V protein comprising the amino acid sequences set forth as SEQ ID NOs: 33 and 37, respectively, which specifically binds to the hMPV F protein and neutralizes hMPV. H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0161] f. Monoclonal antibody MPV467 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV467 antibody and specifically binds to an hMPV F protein and neutralizes hMPV.

[0162] In some examples, the antibody or antigen-binding fragment comprises a V domain that contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of the MPV467 antibody (e.g., according to IMGT, Kabat or Chothia). H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0163] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence 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 set forth as SEQ ID NO:41. H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence 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 set forth as SEQ ID NO:45. L and specifically binds to the hMPV F protein and neutralizes hMPV. In additional embodiments, the antibody or antigen-binding fragment comprises a V that independently comprises an amino acid sequence 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 set forth as SEQ ID NOs: 41 and 45, respectively. H and V L which binds to the hMPV F protein and neutralizes hMPV.

[0164] In some embodiments, the antibody or antigen-binding fragment comprises a VHCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 42, 43 and 44, respectively. H and / or V comprising LCDR1, LCDR2 and LCDR3 as set forth as SEQ ID NOs: 46, 47 and 48, respectively. L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0165] In some embodiments, the antibody or antigen-binding fragment comprises a VHCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 42, 43 and 44, respectively. H and V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 46, 47 and 48, respectively. L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO:41, e.g., an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:41, L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 45, e.g., an amino acid sequence 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 45, and the antibody or antigen-binding fragment specifically binds to hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity is outside of the CDRs.

[0166] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V L In some embodiments, the antibody or antigen-binding fragment comprises a V protein comprising the amino acid sequences set forth as SEQ ID NOs: 41 and 45, respectively, which specifically binds to the hMPV F protein and neutralizes hMPV. H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0167] g. Monoclonal antibody MPV477 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV477 antibody and specifically binds to an hMPV F protein and neutralizes hMPV.

[0168] In some examples, the antibody or antigen-binding fragment comprises a V domain that contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of the MPV477 antibody (e.g., according to IMGT, Kabat or Chothia). H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0169] In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence 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 set forth as SEQ ID NO:49. H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence 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 set forth as SEQ ID NO:53. L and specifically binds to the hMPV F protein and neutralizes hMPV. In additional embodiments, the antibody or antigen-binding fragment comprises a V that independently comprises an amino acid sequence 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 set forth as SEQ ID NOs: 49 and 53, respectively. H and V L which binds to the hMPV F protein and neutralizes hMPV.

[0170] In some embodiments, the antibody or antigen-binding fragment comprises a V domain comprising HCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 50, 51 and 52, respectively. H and / or V comprising LCDR1, LCDR2 and LCDR3 as set forth as SEQ ID NOs: 54, 55 and 56, respectively. L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0171] In some embodiments, the antibody or antigen-binding fragment comprises a V domain comprising HCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 50, 51 and 52, respectively. H and V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 54, 55 and 56, respectively. L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO:49, e.g., an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:49, L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 53, e.g., an amino acid sequence 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 53, and the antibody or antigen-binding fragment specifically binds to hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity is outside of the CDRs.

[0172] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V L In some embodiments, the antibody or antigen-binding fragment comprises an F protein comprising the amino acid sequence shown as SEQ ID NOs: 49 and 53, respectively, and specifically binds to the hMPV F protein and neutralizes hMPV. H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0173] h. Monoclonal antibody MPV478 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV478 antibody and specifically binds to an hMPV F protein and neutralizes hMPV.

[0174] In some examples, the antibody or antigen-binding fragment comprises a V domain that contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of the MPV478 antibody (e.g., according to IMGT, Kabat or Chothia). H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0175] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence 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 set forth as SEQ ID NO:57. H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence 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 set forth as SEQ ID NO:61. L and specifically binds to the hMPV F protein and neutralizes hMPV. In additional embodiments, the antibody or antigen-binding fragment comprises a V that independently comprises an amino acid sequence 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 set forth as SEQ ID NOs: 57 and 61, respectively. H and V L which binds to the hMPV F protein and neutralizes hMPV.

[0176] In some embodiments, the antibody or antigen-binding fragment comprises a V domain comprising HCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 58, 59 and 60, respectively. H and / or V comprising LCDR1, LCDR2 and LCDR3 as set forth as SEQ ID NOs: 62, 63 and 64, respectively. L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0177] In some embodiments, the antibody or antigen-binding fragment comprises a V domain comprising HCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 58, 59 and 60, respectively. H and V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 62, 63 and 64, respectively. L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO:57, e.g., an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:57, L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 61, e.g., an amino acid sequence 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 61, and the antibody or antigen-binding fragment specifically binds to hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity is outside of the CDRs.

[0178] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V L In some embodiments, the antibody or antigen-binding fragment comprises a V protein comprising the amino acid sequences set forth as SEQ ID NOs: 57 and 61, respectively, which specifically binds to the hMPV F protein and neutralizes hMPV. H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0179] i. Monoclonal antibody MPV481 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV481 antibody and specifically binds to an hMPV F protein and neutralizes hMPV.

[0180] In some examples, the antibody or antigen-binding fragment comprises a V domain that contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of the MPV481 antibody (e.g., according to IMGT, Kabat or Chothia). H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0181] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence 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 set forth as SEQ ID NO:69. L and specifically binds to the hMPV F protein and neutralizes hMPV. In additional embodiments, the antibody or antigen-binding fragment comprises a V that independently comprises an amino acid sequence 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 set forth as SEQ ID NOs: 65 and 69, respectively. H and V L which binds to the hMPV F protein and neutralizes hMPV.

[0182] In some embodiments, the antibody or antigen-binding fragment comprises a VHCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 66, 67 and 68, respectively. H and / or V comprising LCDR1, LCDR2 and LCDR3 as set forth as SEQ ID NOs: 70, 71 and 72, respectively. L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0183] In some embodiments, the antibody or antigen-binding fragment comprises a VHCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 66, 67 and 68, respectively. H and V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 70, 71 and 72, respectively. L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO:65, e.g., an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:65, L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 69, e.g., an amino acid sequence 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 69, and the antibody or antigen-binding fragment specifically binds to hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity is outside of the CDRs.

[0184] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V L In some embodiments, the antibody or antigen-binding fragment comprises a V protein comprising the amino acid sequences set forth as SEQ ID NOs: 65 and 69, respectively, which specifically binds to the hMPV F protein and neutralizes hMPV. H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0185] j. Monoclonal antibody MPV482 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV482 antibody and specifically binds to an hMPV F protein and neutralizes hMPV.

[0186] In some examples, the antibody or antigen-binding fragment comprises a V domain that contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of the MPV482 antibody (e.g., according to IMGT, Kabat or Chothia). H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0187] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence 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 set forth as SEQ ID NO:77. L and specifically binds to the hMPV F protein and neutralizes hMPV. In additional embodiments, the antibody or antigen-binding fragment comprises a V that independently comprises an amino acid sequence 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 set forth as SEQ ID NOs: 73 and 77, respectively. H and V L which binds to the hMPV F protein and neutralizes hMPV.

[0188] In some embodiments, the antibody or antigen-binding fragment comprises a V domain comprising HCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 74, 75 and 76, respectively. H and / or V comprising LCDR1, LCDR2 and LCDR3 as set forth as SEQ ID NOs: 78, 79 and 80, respectively. L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0189] In some embodiments, the antibody or antigen-binding fragment comprises a V domain comprising HCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 74, 75 and 76, respectively. H and V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 78, 79 and 80, respectively. L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 73, e.g., an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 73, L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 77, e.g., an amino acid sequence 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 77, and the antibody or antigen-binding fragment specifically binds to hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity is outside of the CDRs.

[0190] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V L In some embodiments, the antibody or antigen-binding fragment comprises the VF domain comprising the amino acid sequence set forth as SEQ ID NOs: 73 and 77, respectively, and specifically binds to the hMPV F protein and neutralizes hMPV. H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0191] k. Monoclonal antibody MPV483 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV483 antibody and specifically binds to an hMPV F protein and neutralizes hMPV.

[0192] In some examples, the antibody or antigen-binding fragment comprises a V domain that contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of the MPV483 antibody (e.g., according to IMGT, Kabat or Chothia). H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0193] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence 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 set forth as SEQ ID NO:85. L and specifically binds to the hMPV F protein and neutralizes hMPV. In additional embodiments, the antibody or antigen-binding fragment comprises a V that independently comprises an amino acid sequence 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 sequences set forth as SEQ ID NOs: 81 and 85, respectively. H and V L which binds to the hMPV F protein and neutralizes hMPV.

[0194] In some embodiments, the antibody or antigen-binding fragment comprises a VHCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 82, 83 and 84, respectively. H and / or V comprising LCDR1, LCDR2 and LCDR3 as set forth as SEQ ID NOs: 86, 87 and 88, respectively. L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0195] In some embodiments, the antibody or antigen-binding fragment comprises a VHCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 82, 83 and 84, respectively. H and V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 86, 87 and 88, respectively. L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 81, e.g., an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 81, L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 85, e.g., an amino acid sequence 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 85, and the antibody or antigen-binding fragment specifically binds to hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity is outside of the CDRs.

[0196] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V L In some embodiments, the antibody or antigen-binding fragment comprises a V protein comprising the amino acid sequence set forth as SEQ ID NOs: 81 and 85, respectively, which specifically binds to the hMPV F protein and neutralizes hMPV. H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0197] l. Monoclonal antibody MPV485 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV485 antibody and specifically binds to an hMPV F protein and neutralizes hMPV.

[0198] In some examples, the antibody or antigen-binding fragment comprises a V domain that contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of the MPV485 antibody (e.g., according to IMGT, Kabat or Chothia). H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0199] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence 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 set forth as SEQ ID NO:93. L and specifically binds to the hMPV F protein and neutralizes hMPV. In additional embodiments, the antibody or antigen-binding fragment comprises a V that independently comprises an amino acid sequence 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 sequences set forth as SEQ ID NOs: 89 and 93, respectively. H and V L which binds to the hMPV F protein and neutralizes hMPV.

[0200] In some embodiments, the antibody or antigen-binding fragment comprises a V domain comprising HCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 90, 91 and 92, respectively. H and / or V comprising LCDR1, LCDR2 and LCDR3 as set forth as SEQ ID NOs: 94, 95 and 96, respectively. L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0201] In some embodiments, the antibody or antigen-binding fragment comprises a V domain comprising HCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 90, 91 and 92, respectively. H and V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 94, 95 and 96, respectively. L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO:89, e.g., an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:89, L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 93, e.g., an amino acid sequence 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 93, and the antibody or antigen-binding fragment specifically binds to hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity is outside of the CDRs.

[0202] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V L In some embodiments, the antibody or antigen-binding fragment comprises an F protein comprising the amino acid sequence set forth as SEQ ID NOs: 89 and 93, respectively, and specifically binds to the hMPV F protein and neutralizes hMPV. H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0203] m. Monoclonal antibody MPV486 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.

[0204] In some examples, the antibody or antigen-binding fragment comprises a V domain that contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of the MPV486 antibody (e.g., according to IMGT, Kabat or Chothia). H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0205] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence 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 set forth as SEQ ID NO:97. H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence 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 set forth as SEQ ID NO: 101. L and specifically binds to the hMPV F protein and neutralizes hMPV. In additional embodiments, the antibody or antigen-binding fragment comprises a V that independently comprises an amino acid sequence 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 set forth as SEQ ID NOs: 97 and 101, respectively. H and V L which binds to the hMPV F protein and neutralizes hMPV.

[0206] In some embodiments, the antibody or antigen-binding fragment comprises a VHCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 98, 99 and 100, respectively. H and / or V comprising LCDR1, LCDR2 and LCDR3 as set forth as SEQ ID NOs: 102, 103 and 104, respectively. L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0207] In some embodiments, the antibody or antigen-binding fragment comprises a VHCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 98, 99 and 100, respectively. H and V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 102, 103 and 104, respectively. L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 97, e.g., an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 97, L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 101, e.g., an amino acid sequence 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 101, and the antibody or antigen-binding fragment specifically binds to hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity is outside of the CDRs.

[0208] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V L In some embodiments, the antibody or antigen-binding fragment comprises a V protein comprising the amino acid sequence set forth as SEQ ID NOs: 97 and 101, respectively, which specifically binds to the hMPV F protein and neutralizes hMPV. H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0209] n. Monoclonal antibody MPV487 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV487 antibody and specifically binds to an hMPV F protein and neutralizes hMPV.

[0210] In some examples, the antibody or antigen-binding fragment comprises a V domain that contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of the MPV487 antibody (e.g., according to IMGT, Kabat or Chothia). H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0211] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence 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 set forth as SEQ ID NO: 105. H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence 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 set forth as SEQ ID NO: 109. L and specifically binds to the hMPV F protein and neutralizes hMPV. In additional embodiments, the antibody or antigen-binding fragment comprises a V that independently comprises an amino acid sequence 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 sequences set forth as SEQ ID NOs: 105 and 109, respectively. H and V L which binds to the hMPV F protein and neutralizes hMPV.

[0212] In some embodiments, the antibody or antigen-binding fragment comprises a V domain comprising HCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 106, 107 and 108, respectively. H and / or V comprising LCDR1, LCDR2 and LCDR3 as set forth as SEQ ID NOs: 110, 111 and 112, respectively. L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0213] In some embodiments, the antibody or antigen-binding fragment comprises a V domain comprising HCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 106, 107 and 108, respectively. H and V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 110, 111 and 112, respectively. L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 105, e.g., an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 105, L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 109, e.g., an amino acid sequence 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 109, and the antibody or antigen-binding fragment specifically binds to hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity is outside of the CDRs.

[0214] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V L and specifically binds to the hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises the V H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0215] o. Monoclonal antibody MPV488 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV488 antibody and specifically binds to an hMPV F protein and neutralizes hMPV.

[0216] In some examples, the antibody or antigen-binding fragment comprises a V domain that contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of the MPV488 antibody (e.g., according to IMGT, Kabat or Chothia). H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0217] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence 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 set forth as SEQ ID NO:117. L and specifically binds to the hMPV F protein and neutralizes hMPV. In additional embodiments, the antibody or antigen-binding fragment comprises a V that independently comprises an amino acid sequence 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 sequences set forth as SEQ ID NOs: 113 and 117, respectively. H and V L which binds to the hMPV F protein and neutralizes hMPV.

[0218] In some embodiments, the antibody or antigen-binding fragment comprises a VHCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 114, 115 and 116, respectively. H and / or V comprising LCDR1, LCDR2 and LCDR3 as set forth as SEQ ID NOs: 118, 119 and 120, respectively. L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0219] In some embodiments, the antibody or antigen-binding fragment comprises a VHCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 114, 115 and 116, respectively. H and V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 118, 119 and 120, respectively. L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 113, e.g., an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 113, L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 117, e.g., an amino acid sequence 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 117, and the antibody or antigen-binding fragment specifically binds to hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity is outside of the CDRs.

[0220] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V L and specifically binds to the hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises the V H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0221] p. Monoclonal antibody MPV489 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV489 antibody and specifically binds to an hMPV F protein and neutralizes hMPV.

[0222] In some examples, the antibody or antigen-binding fragment comprises a V domain that contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of the MPV489 antibody (e.g., according to IMGT, Kabat or Chothia). H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0223] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence 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 set forth as SEQ ID NO: 125. L and specifically binds to the hMPV F protein and neutralizes hMPV. In additional embodiments, the antibody or antigen-binding fragment comprises a V that independently comprises an amino acid sequence 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 set forth as SEQ ID NOs: 121 and 125, respectively. H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0224] In some embodiments, the antibody or antigen-binding fragment comprises a VHCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 122, 123 and 124, respectively. H and / or V comprising LCDR1, LCDR2 and LCDR3 as set forth as SEQ ID NOs: 126, 127 and 128, respectively. L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0225] In some embodiments, the antibody or antigen-binding fragment comprises a VHCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 122, 123 and 124, respectively. H and V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 126, 127 and 128, respectively. L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 121, e.g., an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 121, L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 125, e.g., an amino acid sequence 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 125, and the antibody or antigen-binding fragment specifically binds to hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity is outside of the CDRs.

[0226] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V L and specifically binds to the hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises the V H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0227] q. Monoclonal antibody MPV491 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV491 antibody and specifically binds to an hMPV F protein and neutralizes hMPV.

[0228] In some examples, the antibody or antigen-binding fragment comprises a V domain that contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of the MPV491 antibody (e.g., according to IMGT, Kabat or Chothia). H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0229] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence 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 set forth as SEQ ID NO: 129. H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence 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 set forth as SEQ ID NO: 133. L and specifically binds to the hMPV F protein and neutralizes hMPV. In additional embodiments, the antibody or antigen-binding fragment comprises a V that independently comprises an amino acid sequence 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 sequences set forth as SEQ ID NOs: 129 and 133, respectively. H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0230] In some embodiments, the antibody or antigen-binding fragment comprises a VHCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 130, 131 and 132, respectively. H and / or V comprising LCDR1, LCDR2 and LCDR3 as set forth as SEQ ID NOs: 134, 135 and 136, respectively. L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0231] In some embodiments, the antibody or antigen-binding fragment comprises a VHCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 130, 131 and 132, respectively. H and V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 134, 135 and 136, respectively. L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 129, e.g., an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 129, L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 133, e.g., an amino acid sequence 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 133, and the antibody or antigen-binding fragment specifically binds to hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity is outside of the CDRs.

[0232] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V L and specifically binds to the hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises the V H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0233] r. Monoclonal antibody MPV503 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the MPV503 antibody and specifically binds to an hMPV F protein and neutralizes hMPV.

[0234] In some examples, the antibody or antigen-binding fragment comprises a V domain that contains the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of the MPV503 antibody (e.g., according to IMGT, Kabat or Chothia). H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0235] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence 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 set forth as SEQ ID NO: 137. H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V protein comprising an amino acid sequence 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 set forth as SEQ ID NO: 141. L and specifically binds to the hMPV F protein and neutralizes hMPV. In additional embodiments, the antibody or antigen-binding fragment comprises a V that independently comprises an amino acid sequence 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 set forth as SEQ ID NOs: 137 and 141, respectively. H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0236] In some embodiments, the antibody or antigen-binding fragment comprises a V domain comprising HCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 138, 139 and 140, respectively. H and / or V comprising LCDR1, LCDR2 and LCDR3 as set forth as SEQ ID NOs: 142, 143 and 144, respectively. L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0237] In some embodiments, the antibody or antigen-binding fragment comprises a V domain comprising HCDR1, HCDR2 and HCDR3 as set forth as SEQ ID NOs: 138, 139 and 140, respectively. H and V comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 142, 143 and 144, respectively. L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 137, e.g., an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 137, L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 141, e.g., an amino acid sequence 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 141, and the antibody or antigen-binding fragment specifically binds to hMPV F protein and neutralizes hMPV. In this embodiment, the variation due to sequence identity is outside of the CDRs.

[0238] In some embodiments, the antibody or antigen-binding fragment comprises a V H and specifically binds to the hMPV F protein and neutralizes hMPV. In more embodiments, the antibody or antigen-binding fragment comprises a V L and specifically binds to the hMPV F protein and neutralizes hMPV. In some embodiments, the antibody or antigen-binding fragment comprises the V H and V L which specifically binds to the hMPV F protein and neutralizes hMPV.

[0239] 1. Additional antibodies that bind to the MP467 epitope on the MPV F protein It is disclosed herein that MPV467 targets the hMPV F protein, and the antibody binds to the pre-fusion F protein with higher affinity than the post-fusion F protein. The binding pose of MPV467 is disclosed, specifically, that it binds to a pre-fusion specific epitope where antigenic sites II and V on a single promoter overlap. The helix-turn-helix (α6-α7) located within antigenic site II to which MPV467 binds does not undergo conformational change between the pre-fusion state and the post-fusion state. Thus, in some embodiments, an antibody or antigen-binding fragment is provided that specifically binds to an epitope on the hMPV F protein to which MPV467 binds, and the antibody does not cross-react with the RSV F protein. In other embodiments, an antibody or antigen-binding fragment is provided that specifically binds to an epitope on the hMPV F protein to which any of the disclosed antibodies bind.

[0240] 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 the binding, in a statistically significant manner) with, for example, 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 the binding, in a statistically significant manner) with, an MPV467 antibody provided herein, or with any of the disclosed antibodies, in a binding assay.

[0241] Human antibodies that bind to the same epitope on the hMPV F protein as the MPV467 antibody or any of the disclosed antibodies can be produced using any suitable method. Such antibodies can be prepared, for example, by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus or is extrachromosomally present or randomly integrated into the animal's chromosome. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology, and U.S. Patent Application Publication No. 2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0242] Human antibodies that bind to the same epitope on the hMPV F protein as the MPV467 antibody or any of the disclosed antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, for example, 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 generated via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and 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 generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains.

[0243] Antibodies and antigen-binding fragments that specifically bind to the same epitope on the hMPV F protein as the MPV467 antibody or that bind to the same epitope on the hMPV F protein as any of the disclosed antibodies can also be isolated by screening combinatorial libraries for antibodies with the desired binding characteristics, e.g., by generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and in, for example, McCafferty et al., Nature 348:552-554;Clackson et al., Nature 352: 624-628 (1991);Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003);Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004);Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

[0244] In one particular phage display method, V is used as a vector for the detection of virions, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). H and V LThe repertoires of genes can be cloned separately by polymerase chain reaction (PCR) and randomly recombined into a phage library, which can then be screened for antigen-binding phage. Phage typically display antibody fragments as either single-chain Fv (scFv) or Fab fragments. Libraries from immunized sources provide high affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences encoding highly variable CDR3 regions to achieve rearrangements in vitro, as described in Hoogenboom and Winter, J. 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. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0245] 2. Additional Description of Antibodies and Antigen-Binding Fragments The antibody or antigen-binding fragment may be a human antibody or a fragment thereof. Chimeric antibodies are also provided. The antibody or antigen-binding fragment may include 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, may be included in the heavy or light chain of the antibody.

[0246] The antibody can be of any isotype. The antibody can be, for example, an IgM or IgG antibody, e.g., an IgG 1 , IgG 2 , IgG 3 or IgG 4 The classes of antibodies that specifically bind to hMPV can be switched from one to another. L or V H The nucleic acid molecule encoding the V is then isolated so that it is free of any nucleic acid sequences encoding the light or heavy chain constant regions, respectively. L or V H Nucleic acid molecules encoding C L or C H This can be operably linked to a nucleic acid sequence encoding, for example, C L or C H This can be accomplished using vectors or nucleic acid molecules containing the chains. For example, an antibody that specifically binds to PfCSP, originally an IgG, can be class switched to IgM. Class switching can be achieved by transferring one IgG subclass to another subclass, e.g., IgG 1 From IgG 2 , IgG 3 or IgG 4 can be used to convert

[0247] In some examples, the disclosed antibodies are oligomers of antibodies, such as dimers, trimers, tetramers, pentamers, hexamers, septamers, octomers, and the like.

[0248] The antibody or antigen-binding fragment may be derivatized or linked to another molecule (e.g., another peptide or protein). Generally, 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 may be functionally linked (by chemical coupling, genetic fusion, non-covalent association or other methods) to one or more other molecular entities, such as another antibody (e.g., bispecific antibody or diabody), 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 (e.g., a streptavidin core region or a polyhistidine tag).

[0249] (a) Binding affinity In some embodiments, the antibody or antigen-binding fragment is 1.0×10 -8 M or less, 5.0×10 -8 M or less, 1.0×10 -9 M or less, 5.0×10 -9 M or less, 1.0×10 -10 M or less, 5.0×10 -10 M or less or 1.0×10 -11 Affinity below M (e.g., K D ) and specifically binds to the hMPV F protein. D 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 a Fab for an antigen is measured by measuring the binding affinity of the Fab to a minimal concentration of ( 125I) Equilibration of Fab with labeled antigen and then capture of bound antigen with anti-Fab antibody coated plates (see, e.g., Chen et al., J. Mol. Biol. 293(4):865-881, 1999). To establish conditions for the assay, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 μg / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23° C.). In non-adsorbent plates (NUNC™ 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, incubation may continue for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., for 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN®-20) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT™-20; PerkinEmler) is added and the plate is counted in a TOPCOUNT™ gamma counter (PerkinEmler) for 10 minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are selected for use in competitive binding assays.

[0250] In another assay, K Dcan be measured using a surface plasmon resonance assay using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with an antigen CM5 chip immobilized at approximately 10 response units (RU). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE®, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted in 10 mM sodium acetate, pH 4.8 to 5 μg / ml (approximately 0.2 μM) prior to injection at a flow rate of 5 l / min to achieve approximately 10 response units (RU) of coupled protein. After injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) are injected at a flow rate of approximately 25 l / min at 25° C. The association rate (k on ) and dissociation rate (k off The equilibrium dissociation constant (K) is calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. D ) is the ratio k off / k on See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate is 10 6 M -1 s -1If the on-rate exceeds 100%, it can be determined by using a fluorescence quenching technique to measure the increase or decrease in the fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 at 25°C in the presence of increasing concentrations of antigen as measured in a spectrometer, e.g., a stopped-flow equipped spectrophotometer with a stirred cuvette (Aviv Instruments) or an 8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic).

[0251] (b) Multispecific antibody In some embodiments, multispecific antibodies, such as bispecific antibodies or antigen-binding fragments thereof, are provided that comprise an antibody or antigen-binding fragment that specifically binds to hMPV as provided herein. Any suitable method can be used to design and produce multispecific antibodies, such as crosslinking two or more antibodies, antigen-binding fragments (e.g., scFvs) of the same or different types. Exemplary methods for making multispecific antibodies include those described in PCT Publication No. WO2013 / 163427, the entirety of which is incorporated herein by reference. Non-limiting examples of suitable crosslinkers include those that are heterobifunctional (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate), with two separately reactive groups separated by a suitable spacer.

[0252] Multispecific antibodies can have any suitable format that allows the antibody or antigen-binding fragment provided herein to bind to hMPV F protein. Bispecific single chain antibodies can be encoded by a single nucleic acid molecule. Non-limiting examples of bispecific single chain antibodies, as well as methods for constructing such antibodies, are provided in U.S. Patent 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, and 6,723,538. Additional examples of bispecific single chain antibodies can be found in PCT Application No. WO99 / 54440; Mack et al., J. Immunol., 158(8):3965-3970, 1997; Mack et al., Proc. Natl. Acad. Sci. USA, 92(15):7021-7025, 1995; Kufer et al., Cancer Immunol. Immunother., 45(3-4):193-197, 1997; Loffler et al., Blood, 95(6):2098-2103, 2000; and Bruhl et al., J. Immunol., 166(4):2420-2426, 2001. The production of bispecific Fab-scFv ("bibody") molecules has been described, for example, by Schoonjans et al. (J. Immunol., 165(12):7050-7057, 2000) and Willems et al. (J. Chromatogr. B Analyt. Technol. Biomed Life Sci. 786(1-2):161-176, 2003). For bibodies, the scFv molecule can be fused to one of the VL-CL(L) or VH-CH1 chains, for example, to produce a bibody in which one scFv is fused to the C-terminus of the Fab chain.

[0253] (c) Antigen-binding fragment Heavy chain and V Land an antigen-binding fragment that specifically binds to the hMPV F protein, e.g., Fab, F(ab') 2 and Fv are encompassed by the present disclosure. These antibody fragments retain the ability to selectively bind to antigen and are "antigen-binding" fragments. Non-limiting examples of such fragments include: (1) Fab, the fragment containing a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) The fragment of an antibody molecule, Fab', can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; (3) The fragment of an antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction (Fab'). 2 ;F(ab') 2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) V expressed as two chains L and V L Fv, an engineered fragment containing (5) V linked as a single chain molecule genetically fused by a suitable polypeptide linker H and V L Single chain antibodies (e.g., scFv) are defined as genetically engineered molecules that contain a V H -Domain and V L The intramolecular orientation of the V domain is not critical for the provided antibodies (e.g., the provided multispecific antibodies). Thus, both possible configurations (V H - domain - linker domain - V L -Domain;V L - domain - linker domain - V H-domain) can be used. (6) Single-chain antibody dimers, defined as dimers of scFV (scFV 2 ). This is also called a "miniantibody."

[0254] Any suitable method for producing antigen-binding fragments as discussed above may be used. Non-limiting examples include those described in Harlow and Lane, Antibodies: A Laboratory Manual, 2 nd , Cold Spring Harbor Laboratory, New York, 2013.

[0255] Antigen-binding fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in a host cell (e.g., E. coli cells) of DNA encoding the fragment. Antigen-binding fragments can also be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antigen-binding fragments include F(ab') 2 Fab' can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment, called a 3.5S Fab' fragment. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.

[0256] Other methods for cleaving antibodies can also be used, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of the fragments, or other enzymatic, chemical or genetic techniques, so long as the fragments bind to the antigen recognized by the intact antibody.

[0257] (d) Variants In some embodiments, amino acid sequence variants of the antibodies provided herein (e.g., 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. 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 from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding.

[0258] In some embodiments, antibody variants are provided that have one or more amino acid substitutions.The target sites for substitution mutagenesis include CDR and framework regions.Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0259] Variants typically exhibit correct folding and V H Area and V L The amino acid residues necessary for stabilization between the V and V domains are retained, and the charge characteristics of the residues are retained to maintain the low pI and low toxicity of the molecule. H and V L It can be carried out in the region.

[0260] 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 set forth as 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 set forth as SEQ ID NO:5.

[0261] 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 set forth as 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 set forth as SEQ ID NO: 13.

[0262] 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 set forth as 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 set forth as SEQ ID NO: 21.

[0263] 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 set forth as 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 set forth as SEQ ID NO: 29.

[0264] 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 set forth as 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 set forth as SEQ ID NO: 37.

[0265] 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 set forth as 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 set forth as SEQ ID NO: 45.

[0266] 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 set forth as 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 set forth as SEQ ID NO: 53.

[0267] 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 set forth as 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 set forth as SEQ ID NO: 61.

[0268] 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 set forth as 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 set forth as SEQ ID NO: 69.

[0269] 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 set forth as 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 set forth as SEQ ID NO: 77.

[0270] 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 set forth as 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 set forth as SEQ ID NO: 85.

[0271] 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 set forth as 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 set forth as SEQ ID NO: 93.

[0272] 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 set forth as 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 set forth as SEQ ID NO: 101.

[0273] 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 set forth as 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 set forth as SEQ ID NO: 109.

[0274] 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 set forth as 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 set forth as SEQ ID NO: 117.

[0275] 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 set forth as 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 set forth as SEQ ID NO: 125.

[0276] 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 set forth as 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 set forth as SEQ ID NO: 133.

[0277] 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 set forth as SEQ ID NO: 137. 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 set forth as SEQ ID NO: 141.

[0278] In some embodiments, the antibody or antigen-binding fragment may include 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 regions of the antibody heavy chain or the antibody light chain or the antibody heavy and light chains compared to the known framework regions or compared to the framework regions of an MPV86, MPV414, MPV454, MPV456, MPV464, MPV467, MPV477, MPV478, MPV481, MPV482, MPV483, MPV485, MPV486, MPV487, MPV488, MPV489, MPV491, or MPV503 antibody, and maintain specific binding activity for hMPV F protein.

[0279] In some embodiments, substitutions, insertions or deletions may be present in one or more CDRs, so 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. Variant V as provided above H and V L In some embodiments of the sequences, each CDR is either unaltered or contains no more than one, two or three amino acid substitutions.

[0280] To increase the binding affinity of the antibody, V L and V H Segments can be randomly mutated, for example, within the HCDR3 or LCDR3 regions, in a process similar to the in vivo somatic mutation process responsible for affinity maturation of antibodies during natural immune responses. Thus, in vitro affinity maturation can be performed by randomly mutating V H and V L This can be achieved by amplifying the region in which the random mutations occur. H and / or V L V introduced into the CDR3 region Hand V L The primers are "spiked" at certain positions with a random mixture of the four nucleotide bases to encode the segment. These randomly mutated V H and V L The segments can be tested to determine their binding affinity to the hMPV F protein.

[0281] In some embodiments, an 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 extent to which the antibody or antigen-binding fragment is glycosylated. Addition or deletion of glycosylation sites can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0282] If the antibody (e.g., MPV86, MPV414, MPV454, MPV456, MPV464, MPV467, MPV477, MPV478, MPV481, MPV482, MPV483, MPV485, MPV486, MPV487, MPV488, MPV489, MPV491 or MPV503) comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically have a CH of the Fc region. 2The oligosaccharides comprise branched, biantennary oligosaccharides that are typically linked by N-linkage to Asn297 of the domain. See, for example, Wright et al. Trends Biotechnol. 15(1):26-32, 1997. The oligosaccharides may contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose linked to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibody may be made to create antibody variants with certain improved properties.

[0283] In one embodiment, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1%-80%, 1%-65%, 5%-65% or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 compared to the sum of all glycostructures (e.g., complex, hybrid and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, for example, as described in WO2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 in the Fc region; however, Asn297 can also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylation variants can have improved ADCC function. See, e.g., U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.); U.S. Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: U.S. Patent Application Publication No. 2003 / 0157108; WO2000 / 61739; WO2001 / 29246; U.S. Patent Application Publication No. 2003 / 0115614; U.S. Patent Application Publication No. 2002 / 0164328; U.S. Patent Application Publication No. 2004 / 0093621; U.S. Patent Application Publication No. U.S. Patent Application Publication No. 2004 / 0132140; U.S. Patent Application Publication No. 2004 / 0110704; U.S. Patent Application Publication No. 2004 / 0110282; U.S. Patent Application Publication No. 2004 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al., J. Mol. Biol., 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, which are deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249(2):533-545, 1986; U.S. Patent Application Publication Nos. 2003 / 0157108 and WO2004 / 056312, especially Example 11), and knockout cell lines, such as CHO cells in which the alpha-1,6-fucosyltransferase gene FUT8 has been knocked out (see, e.g., Yamane-Ohnuki et al., Biotechnol. Bioeng., 87(5): 614-622, 2004; Kanda et al., Biotechnol. Bioeng., 94(4):680-688, 2006; and WO2003 / 085107).

[0284] The antibody variant further provides a bipartite oligosaccharide, for example, a biantennary oligosaccharide attached to the Fc region of the antibody is bipartite by GlcNAc. Such an antibody variant may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). An antibody variant having at least one galactose residue in the oligosaccharide attached to the Fc region is also provided. Such an antibody variant may have improved CDC function. Such an antibody variant is described, for example, in WO1997 / 30087; WO1998 / 58964; and WO1999 / 22764.

[0285] In some embodiments, the constant region of an antibody (e.g., MPV86, MPV414, MPV454, MPV456, MPV464, MPV467, MPV477, MPV478, MPV481, MPV482, MPV483, MPV485, MPV486, MPV487, MPV488, MPV489, MPV491, or MPV503) comprises one or more amino acid substitutions to optimize the in vivo half-life of the antibody. The serum half-life of IgG Abs is regulated by the neonatal Fc receptor (FcRn). Thus, in some embodiments, the antibody comprises amino acid substitutions that increase binding to FcRn. Non-limiting examples of such substitutions include substitutions in the IgG constant region such as T250Q and M428L (see, e.g., Hinton et al., J Immunol., 176(1):346-356, 2006); M428L and N434S ("LS" mutations, see, e.g., Zalevsky, et al., Nature Biotechnol., 28(2):157-159, 2010); N434A (see, e.g., Petkova et al., Int. Immunol., 18(12):1759-1769, 2006); T307A, E380A, and N434A (see, e.g., Petkova et al., Int. Immunol., 18(12):1759-1769, 2006). 2006); as well as 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 can include an Fc polypeptide that includes any of the substitutions listed above, for example, an Fc polypeptide can include an M428L and an N434S substitution.

[0286] In some embodiments, the constant region of the antibody comprises one or more amino acid substitutions to optimize ADCC. ADCC is mediated primarily through a set of closely related Fcγ receptors. In some embodiments, the antibody comprises one or more amino acid substitutions that increase binding to FcγRIIIa. Non-limiting examples of such substitutions include substitutions S239D and I332E in the IgG constant region (see, e.g., Lazar et al., Proc. Natl., Acad. Sci. USA, 103(11):4005-4010, 2006); and S239D, A330L and I332E (see, e.g., Lazar et al., Proc. Natl., Acad. Sci. USA, 103(11):4005-4010, 2006).

[0287] Combinations of the above substitutions are also included to generate IgG constant regions with increased binding to FcRn and FcγRIIIa. These combinations increase 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 / T2 56E; (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 engineered to be directly cytotoxic to the infected cell or to use natural defenses, such as complement, ADCC, or phagocytosis by macrophages.

[0288] In some embodiments, the antibodies provided herein may be further modified to contain additional non-proteinaceous moieties. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and when more than one polymer is attached, they can be the same molecule or different molecules. In general, the number and / or type of polymers 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 will be used in an application under defined conditions, etc.

[0289] B. Conjugates The antibodies and antigen-binding fragments that specifically bind to the hMPV F protein disclosed herein can be conjugated to agents, such as effector molecules or detectable markers. Both covalent and non-covalent attachment means can be used. Toxins and radioactive agents, such as 125 I, 32 P, 14 C. 3H and 35 A variety of effector molecules and detectable markers can be used, including, but not limited to, S, as well as other labels, targeting moieties and ligands, etc. The choice of a particular effector molecule or detectable marker will depend on the particular target molecule or cell, and the desired biological effect.

[0290] Procedures for attaching effector molecules or detectable markers to antibodies or antigen-binding fragments vary according to the chemical structure of the effector. Polypeptides typically have a variety of functional groups available for reaction with appropriate functional groups on the polypeptide to effect attachment of effector molecules or detectable markers, e.g., carboxyl (-COOH), free amines (-NH 2 ) or sulfhydryl (-SH) groups. Alternatively, the antibody or antigen-binding fragment is derivatized to expose or attach additional reactive functional groups. Derivatization may involve attachment of any suitable linker molecule. The linker is capable of forming covalent bonds to both the antibody or antigen-binding fragment and the effector molecule or detectable marker. Suitable linkers include, but are not limited to, straight or branched chain 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 linkers may be connected to the constituent amino acids through their side chains (e.g., to cysteine ​​via disulfide linkages) or through the alpha carbon, or through the amino and / or carboxyl groups of the terminal amino acids.

[0291] Considering the numerous reported methods for conjugating various radiodiagnostic compounds, radiotherapeutic compounds, labels (e.g., enzymes or fluorescent molecules), toxins and other agents to antibodies, an appropriate method for conjugating a given agent to an antibody or antigen-binding fragment or other polypeptide can be determined.

[0292] The antibody or antigen-binding fragment may be conjugated with a detectable marker; for example, a detectable marker that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (e.g., CT, computed axial tomography (CAT), MRI, magnetic resonance tomography (MTR), ultrasound, fiber optic examination and laparoscopic examination). 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-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent markers such as luciferase, green fluorescent protein (GFP) and yellow fluorescent protein (YFP) are also used. Antibodies or antigen-binding fragments can also be conjugated to enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When an antibody or antigen-binding fragment is conjugated to a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine results in a visually detectable colored reaction product. Antibodies or antigen-binding fragments can also be conjugated to biotin and detected through indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated to an enzyme or a fluorescent label.

[0293] The antibody or antigen-binding fragment may be conjugated with a paramagnetic agent, such as gadolinium. Paramagnetic agents, such as superparamagnetic iron oxide, are also used as labels. The antibody may also be conjugated with lanthanides (e.g., europium and dysprosium) and manganese. The antibody or antigen-binding fragment may also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequence, binding site for secondary antibody, metal binding domain, epitope tag).

[0294] The antibody or antigen-binding fragment may also be conjugated with a radiolabeled amino acid, for example for diagnostic purposes. For example, the radiolabel may be used to detect hMPV by X-ray examination, 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, 131 I. Radioactive labels can be detected, for example, using photographic film or scintillation counters, fluorescent markers can be detected using a photodetector to detect emitted radiation, enzymatic labels are typically detected by providing a substrate for the enzyme and detecting a reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

[0295] The average number of effector molecules or detectable marker moieties per antibody or antigen-binding fragment in the conjugate can range, for example, from 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 ranges from 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 of the conjugate (e.g., the ratio of effector molecules per antibody) can be controlled in different ways, for example, by (i) limiting the molar excess of effector molecule-linker intermediate or linker reagent compared to the antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limited reducing conditions for cysteine ​​thiol modification, (iv) recombinantly engineering the amino acid sequence of the antibody such that the number and position of cysteine ​​residues are modified to control the number or position of linker-effector molecule bonds.

[0296] C. Polynucleotides and Expression Nucleic acid molecules (e.g., cDNA or RNA molecules) encoding the amino acid sequences of the antibodies, antigen-binding fragments, and conjugates that specifically bind to hMPV disclosed herein are provided. The nucleic acids encoding these molecules can be synthesized from the amino acid sequences provided herein (e.g., CDR sequences and V sequences). H and V L In some embodiments, nucleic acid molecules can be readily produced using sequences available in the art (e.g., framework or constant region sequences) and the genetic code. H , V L , or V H and V L In some embodiments, the nucleic acid molecule can encode both an scFv and an scFv (e.g., in a bicistronic expression vector). In some 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.

[0297] The genetic code allows for the creation of a variety of functionally equivalent nucleic acid sequences, e.g., nucleic acids that differ in sequence but encode the same antibody sequence, or L and / or V H It can be used to construct nucleic acids encoding conjugates or fusion proteins comprising the nucleic acid sequence.

[0298] Nucleic acid molecules encoding antibodies, antigen-binding fragments and conjugates that specifically bind to hMPV F protein can be prepared by any suitable method, including, for example, cloning of appropriate sequences, or by 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 DNA polymerase using the single strand as a template.

[0299] Exemplary nucleic acids can be prepared by cloning techniques. Examples of suitable cloning and sequencing techniques can be found, for example, in Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4 th 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).

[0300] Nucleic acids may also be prepared by amplification techniques, including polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification systems (TAS) and self-sustained sequence replication systems (3SR).

[0301] The nucleic acid molecules can be expressed in recombinantly engineered cells, such as bacteria, plant, yeast, insect and mammalian cells. Antibodies, antigen-binding fragments and conjugates can be prepared by the methods described herein. H and / or V L The antibodies may be expressed as individual proteins (optionally linked to an effector molecule or detectable marker) comprising the V, VL or VL2, or as a fusion protein. Any suitable method of 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, V H and V L As well as, nucleic acids encoding the immunoadhesins are provided. The nucleic acid sequence can optionally encode a leader sequence.

[0302] To generate scFv, V H A DNA fragment encoding V L The DNA fragment encoding V H and V L The sequence can be expressed as a contiguous single chain protein, L and V H A flexible linker is encoded, e.g., the amino acid sequence (Gly) 4 -Ser) 3 (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 Dubel (Eds.), Antibody Engineering, Vols. 1-2, 2001; nded., Springer-Verlag, 2010;Greenfield (Ed.), Antibodies: A Laboratory Manual, 2 nd (See, e.g., The Journal of Molecular Biology, ed. New York: Cold Spring Harbor Laboratory Press, 2014.) Optionally, a cleavage site, e.g., a furin cleavage site, can be included in the linker.

[0303] Single-chain antibodies contain a single V H and V L It can be monovalent when only one V is used, and two V H and V L is used, or more than two V H and V L When the encoded V is used, it is multivalent. Bispecific or multivalent antibodies can be generated that specifically bind the hMPV F protein and another antigen. H and V L If necessary, V H Domain and V L A furin cleavage site may be included between the domains.

[0304] One or more DNA sequences encoding antibodies, antigen-binding fragments or conjugates can be expressed in vitro by DNA transfer into suitable host cells. The cells can be prokaryotic or eukaryotic. Numerous expression systems available for protein expression can be used to express the disclosed antibodies and antigen-binding fragments, including E. coli, other bacterial hosts, yeast and various higher eukaryotic cells, such as COS, CHO, HeLa and myeloma cell lines. A method of stable transfer can be used, meaning that the foreign DNA is continuously maintained in the host. Hybridomas expressing the antibody of interest are also encompassed by the present disclosure.

[0305] Expression of nucleic acids encoding the antibodies and antigen-binding fragments described herein can be achieved by operably linking the DNA or cDNA to a promoter (either constitutive or inducible) and then incorporating it into an expression cassette. The promoter can be any promoter of interest, including the cytomegalovirus promoter. Optionally, an enhancer, such as the cytomegalovirus enhancer, is included in the construct. The cassette can be suitable for replication and integration in either prokaryotes or eukaryotes. A typical expression cassette contains certain sequences useful for regulating the expression of DNA encoding a protein. For example, the expression cassette can include a suitable promoter, enhancer, transcription and translation terminator, initiation sequence, a start codon (i.e., ATG) in front of the protein-coding gene, splicing signals for introns, sequences for maintaining the correct reading frame of the gene to allow proper translation of the mRNA, and a stop codon. The vector can encode a selectable marker, such as a marker encoding drug resistance (e.g., ampicillin or tetracycline resistance).

[0306] To obtain high-level expression of the cloned gene, it is desirable to construct an expression cassette that contains, for example, a strong promoter to direct transcription, a ribosome binding site for translation initiation (e.g., an internal ribosome binding sequence), and a transcription / translation terminator. For E. coli, this may include a promoter, for example, T7, trp, lac, or lambda promoter, a ribosome binding site, and preferably, a transcription termination signal. For eukaryotic cells, the control sequences may include, for example, promoters and / or enhancers derived from immunoglobulin genes, HTLV, SV40, or cytomegalovirus, as well as polyadenylation sequences, and may further include splice donor and / or acceptor sequences (e.g., CMV and / or HTLV splice acceptor and donor sequences). The cassette may be transferred into the selected host cell by any suitable method, for example, transformation or electroporation for E. coli, and calcium phosphate treatment, electroporation, or lipofection for mammalian cells. Cells transformed by the cassette can be selected by resistance to antibiotics conferred by genes contained in the cassette, such as the amp, gpt, neo and hyg genes.

[0307] Modifications can be made to the nucleic acid encoding the polypeptide described herein without reducing its biological activity. Some modifications can be made to facilitate cloning, expression, or incorporation of targeting molecules into fusion proteins. Such modifications include, for example, termination codons, sequences for creating conveniently located restriction sites, and sequences for adding methionine to the amino terminus to provide an initiation site, or sequences for adding additional amino acids (e.g., polyHis) to aid in purification steps.

[0308] Once expressed, the antibodies, antigen-binding fragments and conjugates can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see generally Simpson et al. (Eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009). The antibodies, antigen-binding fragments and conjugates need not be 100% pure. Once optionally purified, partially or to homogeneity, if used prophylactically, the polypeptides should be substantially free of endotoxins.

[0309] Methods for the expression of antibodies, antigen-binding fragments and conjugates from mammalian cells and bacteria, such as E. coli, and / or refolding into a suitable active form have been described and are applicable to the antibodies disclosed herein. See, e.g., Greenfield (Ed.), Antibodies: A Laboratory Manual, 2 nd ed. New York: Cold Spring Harbor Laboratory Press, 2014, Simpson et al. (Eds.), Basic methods in 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.

[0310] D. Methods and Compositions 1. Inhibiting hMPV infection Disclosed herein are methods for inhibiting hMPV infection in a subject. These methods include administering to a subject at risk of or having hMPV infection an effective amount (i.e., an amount effective to inhibit hMPV infection in the subject) of the disclosed antibody, antigen-binding fragment, conjugate, or nucleic acid encoding such antibody, antigen-binding fragment, or conjugate. These methods can be used pre-exposure or post-exposure.

[0311] For the method to be effective, hMPV infection does not need to be completely eliminated or inhibited.For example, the method can reduce hMPV infection by a desired amount, for example, 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 with hMPV infection in the absence of treatment.In some embodiments, the subject can also be treated with an effective amount of additional agent, for example, antiviral agent.

[0312] In some embodiments, administration of an effective amount of the disclosed antibodies, antigen-binding fragments, conjugates, or nucleic acid molecules inhibits the establishment and / or subsequent disease progression of hMPV infection in a subject, which may include any statistically significant reduction in hMPV activity (e.g., growth or invasion) or symptoms of hMPV infection in the subject. The antibodies, antigen-binding fragments, conjugates, or nucleic acid molecules may be administered by any route of administration, including systemic or local administration. In one embodiment, administration is intranasal administration. In another embodiment, administration is into the lungs, for example by inhalation. In a further embodiment, administration is intramuscular.

[0313] Disclosed herein are methods for inhibiting hMPV replication in a subject. These methods include administering to a subject at risk of or having hMPV infection an effective amount (i.e., an amount effective to inhibit hMPV replication in the subject) of the disclosed antibody, antigen-binding fragment, conjugate, or nucleic acid encoding such antibody, antigen-binding fragment, or conjugate. These methods can be used pre-exposure or post-exposure.

[0314] Methods for treating hMPV infection in a subject are disclosed. Methods for preventing hMPV infection in a subject are also disclosed. These methods include 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.

[0315] The antibodies and antigen-binding fragments thereof may be administered systemically, for example, by intravenous infusion or intramuscular administration. The antibodies and antigen-binding fragments thereof may be administered intranasally, intramuscularly, or into the lungs, for example, by inhalation. The dose of the antibody or antigen-binding fragment may vary, but generally ranges between about 0.5 mg / kg and about 50 mg / kg, for example, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, or about 50 mg / kg. In some embodiments, the dose of the antibody or antigen-binding fragment may be about 0.5 mg / kg to about 5 mg / kg, for example, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, or about 5 mg / kg. The antibody or antigen-binding fragment is administered according to a dosing schedule determined by a physician. In some examples, the antibody or antigen-binding fragment is administered once a week, every two weeks, every three weeks, or every four weeks.

[0316] 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.

[0317] In some embodiments, the method includes administering a first antibody that specifically binds to the hMPV F protein disclosed herein and a second antibody that also specifically binds to a different epitope of the hMPV F protein, e.g., the 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 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 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 or four, five or six of MPV86, MPV414, MPV454, MPV456, MPV464, MPV467, MPV477, MPV478, MPV481, MPV482, MPV483, MPV485, MPV486, MPV487, MPV488, MPV489, MPV491 or MPV503 may be administered to the subject. In a non-limiting example, the method includes administering an effective amount of MPV467 to the subject. The method may include administering an effective amount of one or more additional antibodies.

[0318] In some embodiments, a subject is administered DNA or RNA encoding the disclosed antibody, for example, to provide in vivo antibody production using the subject's cellular machinery. Any suitable method of nucleic acid administration can 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 of delivery of protein-encoding nucleic acid to an organism. One approach for administration of nucleic acid is direct administration using plasmid DNA, e.g., a mammalian expression plasmid. The nucleotide sequence encoding the disclosed antibody or antigen-binding fragment thereof can be placed under the control of a promoter to increase expression. These methods include liposomal delivery of nucleic acid. Such methods can 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., J. Virol., 79(14), 8828-8834, 2005, incorporated herein by reference).

[0319] In some embodiments, a subject (e.g., a human subject at risk of or having hMPV infection) may be administered an effective amount of an AAV viral vector comprising one or more nucleic acid molecules encoding the disclosed antibodies or antigen-binding fragments. The AAV viral vector is designed for expression of the nucleic acid molecules encoding the disclosed antibodies or antigen-binding fragments, and administration of an effective amount of the AAV viral vector to a subject results in expression of an effective amount of the antibody or antigen-binding fragment in the subject. Non-limiting examples of AAV viral vectors that may be used to express the disclosed antibodies or antigen-binding fragments in a subject include those provided in Johnson et al., Nat. Med., 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.

[0320] In one embodiment, the nucleic acid encoding the disclosed antibody or its antigen-binding fragment is directly introduced into tissue.For example, the nucleic acid can be loaded onto gold microspheres by standard methods and introduced into skin by a device such as Bio-Rad's HELIOS™ Gene Gun.The nucleic acid can be "naked", consisting of a plasmid under the control of a strong promoter.

[0321] Typically, DNA is injected into the muscle, but can also be injected directly into other sites. The dosage for injection is usually approximately 0.5 μg / kg to about 50 mg / kg, typically about 0.005 mg / kg to about 5 mg / kg (see, e.g., U.S. Patent No. 5,589,466).

[0322] Single or multiple administrations of the disclosed hMPV F protein specific antibody, antigen-binding fragment, conjugate, or composition comprising a nucleic acid molecule encoding such a molecule can be administered depending on the dosage and frequency required and tolerated by the patient.Dosages can be administered once, but can be administered periodically until desired results are achieved or until side effects warrant discontinuing treatment.In general, the dosage is sufficient to inhibit hMPV infection without causing unacceptable toxicity to the patient.

[0323] The data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for human use. The dosage is usually sufficient to achieve ED with little or minimal toxicity. 50 The effective dose can be determined from cell culture assays and animal studies.

[0324] hMPV F protein specific antibodies, antigen-binding fragments, bispecific antibodies, conjugates, or nucleic acid molecules encoding such molecules, or compositions comprising such molecules, can be administered to a subject in a variety of ways, including local and systemic administration, for example, by injection, such as subcutaneous, intravenous, intraarterial, intraperitoneal, intramuscular, intradermal, or intrathecal. In some embodiments, antibodies, antigen-binding fragments, bispecific antibodies, conjugates, or nucleic acid molecules encoding such molecules, or compositions comprising such molecules, are administered once daily by a single subcutaneous, intravenous, intraarterial, intraperitoneal, intramuscular, intradermal, or intrathecal injection. Antibodies, antigen-binding fragments, bispecific antibodies, conjugates, or nucleic acid molecules encoding such molecules, or compositions comprising such molecules, can also be administered by direct injection at or near the site of the disease. A further method of administration is by osmotic pumps (e.g., Alzet pumps) or mini-pumps (e.g., Alzet mini-osmotic pumps) that allow for controlled, sustained and / or sustained delivery of antibodies, antigen-binding fragments, conjugates, or nucleic acid molecules encoding such molecules, or compositions comprising such molecules, over a period of time. The osmotic pumps or mini-pumps can be implanted subcutaneously or near the target site.

[0325] 2. Composition Compositions are provided that include 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 pharma- ceutically acceptable carrier. In some embodiments, the compositions include MPV86, MPV414, MPV454, MPV456, MPV464, MPV467, MPV477, MPV478, MPV481, MPV482, MPV483, MPV485, MPV486, MPV487, MPV488, MPV489, MPV491, or MPV503 antibodies or antigen-binding fragments thereof disclosed herein. In some embodiments, the compositions include two, three, four, or more antibodies that specifically bind to hMPV F protein. In a specific, non-limiting example, the antibody is MVP467. The compositions are useful, for example, for inhibiting or detecting hMPV infection. The composition can be prepared in a unit dosage form for administration to a subject.The amount and timing of administration to achieve the desired purpose are at the discretion of the administering physician.Antibody, antigen-binding fragment, conjugate, or nucleic acid molecule encoding such molecule can be formulated for systemic or local administration.In one example, antigen-binding fragment, conjugate, or nucleic acid molecule encoding such molecule is formulated for parenteral administration, for example, intravenous administration.

[0326] 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) macromolecular contaminants, such as other mammalian (e.g., human) proteins.

[0327] The compositions for administration may include a solution of the antibody, antigen-binding fragment, conjugate, or nucleic acid molecule encoding such a molecule, dissolved in a pharma- ceutically acceptable carrier, for example, an aqueous carrier. A variety of aqueous carriers may be used, such as buffered saline, etc. These solutions are sterile and generally free of undesirables. These compositions may be sterilized by any suitable technique. The compositions may contain pharma- ceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the antibody in these formulations may vary widely and is selected primarily based on fluid volumes, viscosities, body weight, etc., according to the particular mode of administration selected and the needs of the subject.

[0328] A typical composition for intravenous administration contains about 0.01 to about 30 mg / kg of the antibody or antigen-binding fragment or conjugate (or the corresponding dose of a conjugate containing the antibody or antigen-binding fragment) per subject per day. Any suitable method may be used to prepare the administrable composition; non-limiting examples are described in Remington: The Science and Practice of Pharmacy, 22 nd ed., London, UK: Pharmaceutical Press, 2013. In some embodiments, the composition may be a liquid formulation comprising one or more antibodies, antigen-binding fragments (e.g., antibodies or antigen-binding fragments that specifically bind to PfCSP) in a concentration range of about 0.1 mg / ml to about 20 mg / ml, or about 0.5 mg / ml to about 20 mg / ml, or about 1 mg / ml to about 20 mg / ml, or about 0.1 mg / ml to about 10 mg / ml, or about 0.5 mg / ml to about 10 mg / ml, or about 1 mg / ml to about 10 mg / ml.

[0329] The antibody or antigen-binding fragment or conjugate thereof or nucleic acid encoding such molecule may be provided in lyophilized form and rehydrated with sterile water prior to administration, but also in a sterile solution of known concentration. The antibody solution, or antigen-binding fragment or nucleic acid encoding such antibody or antigen-binding fragment, may then be added to an infusion bag containing 0.9% sodium chloride, USP, and typically administered at a dosage of 0.5-15 mg / kg body weight. Considerable experience is available in the art in administering antibody drugs that have been marketed in the United States since the approval of Rituximab in 1997. The antibody, antigen-binding fragment, conjugate, or nucleic acid encoding such molecule may be administered by slow infusion rather than intravenous push or bolus. In one example, a higher loading dose is administered, followed by a maintenance dose at a lower level. For example, an initial loading dose of 4 mg / kg may be infused over several 90 minute periods, followed by maintenance doses of 2 mg / kg infused over 30 minute periods once per week for 4 to 8 weeks if the previous dose was well tolerated.

[0330] Controlled release parenteral formulations can be made as implants, oily injections or granular 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. Granular systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres and nanoparticles. Microcapsules contain an active protein drug, such as a cytotoxin or drug, as a central core. In microspheres, the active protein drug is dispersed in the particles. Particles smaller than about 1 μm, microspheres and microcapsules, are generally called nanoparticles, nanospheres and nanocapsules, respectively. Capillaries have a diameter of approximately 5 μm, therefore only nanoparticles are administered intravenously. Microparticles are typically approximately 100 μm in diameter and are administered subcutaneously or intramuscularly. See, e.g., Kreuter, 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.

[0331] Polymers can be used for ion-controlled release of the antibody composition disclosed herein.Any suitable polymer can be used, for example, degradable or non-degradable polymeric matrices designed for use in controlled drug delivery.Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins.In yet another aspect, liposomes are used for controlled release of lipid-encapsulated drugs as well as drug targeting.

[0332] 2. Methods of detection and diagnosis 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 can be detected in a biological sample from a subject and used to identify subjects with hMPV infection. The sample can be any sample, including but not limited to tissue from biopsy, autopsy and pathology specimens. Biological samples also include tissue sections, such as frozen sections obtained for histological purposes. Biological samples further include body fluids, such as blood, serum, plasma, sputum, cerebrospinal fluid or urine. The method of detection can include contacting a cell or sample with an antibody or antigen-binding fragment that specifically binds to hMPV F protein, or a conjugate thereof (e.g., a conjugate that includes a detectable marker) under conditions sufficient to form an immune complex, and detecting the immune complex (e.g., by detecting a detectable marker conjugated to the antibody or antigen-binding fragment).

[0333] In one embodiment, the antibody or antigen-binding fragment is directly labeled with a detectable marker. In another embodiment, the antibody that binds to the hMPV F protein (the primary antibody) is unlabeled, and a secondary antibody or other molecule capable of binding to the primary antibody is utilized for detection. A secondary antibody capable of specifically binding 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 bind to antibodies include, but are not limited to, Protein A and Protein G, both of which are commercially available. Suitable labels for antibodies, antigen-binding fragments, or secondary antibodies are known and described above, and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents, and radioactive materials.

[0334] In some embodiments, the disclosed antibodies or antigen-binding fragments thereof are used to test vaccines, for example, to test whether a vaccine composition comprising an hMPV F protein or a fragment thereof adopts a pre-fusion conformation that includes the epitope of the disclosed antibodies.Thus, a method for testing vaccines is provided herein, which includes contacting a sample containing a vaccine, for example, an hMPV F protein immunogen, with the disclosed antibodies or antigen-binding fragments under conditions sufficient for the formation of an immune complex, and detecting the immune complex in the sample to detect a vaccine that includes the epitope of interest.In one example, detection of the immune complex in the sample indicates that the vaccine component, for example, an hMPV F protein immunogen, adopts a conformation that can bind to the antibody or antigen-binding fragment.

[0335] In view of the many possible embodiments to which the principles of the present invention may be applied, it should be recognized that the illustrated embodiments are merely examples of the invention and should not be considered limitations on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims. EXAMPLES

[0336] Human metapneumovirus (hMPV) is a leading cause of morbidity and hospitalization in children worldwide, yet no vaccines or therapeutics are currently available for the prevention and treatment of hMPV disease. The hMPV fusion (F) protein is the sole target of neutralizing antibodies. To map immunodominant epitopes on the hMPV F protein, 18 human monoclonal antibodies (mAbs) were isolated and these mAbs were evaluated for binding avidity, neutralization potency, and epitope specificity. The majority of mAbs targeted diverse epitopes on the hMPV F protein, and multiple mAb binding approaches were discovered for antigenic site III. The most potent mAb, MPV467, was tested in prophylactic and therapeutic mouse challenge studies, where MPV467 restricted viral replication in mouse lungs when administered 24 hours before or 72 hours after viral infection. Furthermore, this antibody was shown to be effective in a cotton rat (Sigmodon hispidus) model. The structure of MPV467 in complex with hMPV F protein was determined at 3.3 Å resolution using cryo-electron microscopy, revealing a complex prefusion-specific epitope with overlapping antigenic sites II and V on a single protomer. The data revealed new insights into immunodominant antigenic epitopes on the hMPV F protein, identified new mAb therapeutics for the prevention and treatment of hMPV F disease, and provided the discovery of a unique prefusion-specific epitope on the hMPV F protein.

[0337] Example 1 Human monoclonal antibody sequence determinants To further define antigenic epitopes on the hMPV F protein, 18 new human mAbs were isolated using the hMPV B2 F protein (Biacchesi et al., J. Virol. 78, 12877-12887 (2004)), 16 mAbs were generated via human hybridoma technology, while 2 were derived from antigen-specific single B cell sorting (MPV491, 503). The antibody-encoding genes were sequenced, and the results showed the usage of a diverse set of immunoglobulin V genes across the panel (Figure 1A). mAbs utilizing the VH1-69 gene were the most abundant. The VH3 and VH4 gene families constituted the majority of additional mAbs. Diversity was also present in the light chain, with eight and five unique genes utilized for kappa and lambda mAbs, respectively. Kappa isotype mAbs utilized the VK1, VK2, VK3 and VK4 gene families, whereas lambda isotype mAbs used the VL1 and VL3 gene families. The lengths of the heavy and light chain junctions ranged from 14 to 23 amino acids for heavy chains, 8 to 10 amino acids for kappa chains and 9 to 11 amino acids for lambda chains (Figure 1B). The percent identity of the variable genes to the germline sequence ranged from 88 to 97% (average 93.6%) for heavy chains and 90 to 97% (average 93.7%) for light chains (Figure 1C).

[0338] Example 2 mAb binding and functional characterization The neutralizing activity of each mAb was determined by plaque reduction assays using representative viruses from each genotype of hMPV, i.e., hMPV CAN / 97-83 (genotype A) and hMPV TN / 93-32 (genotype B) (Figures 6, 10). All mAbs had neutralizing activity against viruses from both genotypes, with mAbs MPV467, MPV487, MPV454, MPV482 and MPV488 having neutralizing activity below 20 ng / mL against hMPV CAN / 97-83. 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. mAbs MPV86, MPV488, MPV485 and MPV477 had IC values ​​at least 20-fold lower than hMPV TN / 93-32. 50 We demonstrated preferential neutralization of hMPV CAN / 97-83 based on the binding of the mAbs. The binding properties of the mAbs were evaluated using a panel of hMPV F proteins from each subgroup (hMPV A1 F, hMPV A2 F, hMPV B1 F, hMPV B2 F) containing a mixture of pre-fusion and post-fusion hMPV F. mAb binding to additional constructs containing exclusively monomeric pre-fusion hMPV F, post-fusion hMPV F and predominantly trimeric pre-fusion hMPV F (hMPV B2 F GCN4) was also evaluated. Several binding patterns were observed. MPV487, MPV482, MPV503, MPV414, MPV86 and MPV488 had limited binding to the post-fusion F constructs and preferred binding to the pre-fusion constructs. mAbs MPV467, MPV454, MPV477, MPV486 and MPV464 bound to both pre- and post-fusion constructs but had higher binding affinity to the pre-fusion protein. mAbs MPV478, MPV483, MPV481, MPV456, MPV491, MPV489 and MPV485 bound equally to both pre- and post-fusion constructs.

[0339] Example 3 Epitope mapping To determine common binding epitopes for the panel 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., J. Virol. 93, e00342--19 (2019)). Biosensors were loaded with hMPV B2 pre-fusion F protein associated with test mAbs and then exposed to control mAbs with known epitopes to determine competition profiles (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 intratrimer epitope) (Figure 2B). MPV481 and MPV483 were mapped to antigenic site IV, whereas MPV454 competed with both 101F and DS7, suggesting that MPV454 binds to an epitope intermediate between site IV and DS7 (Figure 2B). However, MPV454 did not compete with previously identified MPV196, which competes with DS7. MPV464, MPV491, MPV485 and MPV477 competed with both MPE8 and DS7, similar to our previous results with MPV196, MPV201 and MPV314 (Bar-Peled et al., J. Virol. 93, e00342--19 (2019)). mAbs MPV86, MPV414, MPV482, MPV487 and MPV503 are "MPV364-like" and competed with MPE8 and MPV364 but not with DS7. This differential binding mode at antigenic site III has been previously defined by competition or lack thereof with DS7 (Bar-Peled et al., J. Virol. 93, e00342--19 (2019)).No mAbs were observed to compete with intratrimer targeting MPV458, but intermediate competition was observed between some mAbs and MPV458, suggesting that they may partially block MPV458 binding or limit exposure of the intratrimer epitope centered at amino acids 66-87 to which MPV458 binds. MPV488 and MPV489 had partial competition with MPE8 but not MPV364, whereas MPV467 and MPV456 had partial competition with MPV364 but not MPE8, suggesting that additional epitopes exist near antigenic site III. MPV486 and MPV478 showed partial competition with almost all mAbs and their epitopes could not be defined.

[0340] Example 4 hMPV F-specific mAb enhances antibody-dependent phagocytosis of THP-1 cells Antibodies binding to different hMPV F antigenic sites were selected to assess antibody-dependent phagocytosis activity (Figure 3). All tested mAbs significantly enhanced phagocytosis of THP-1 cells in vitro compared to blank and isotype control mAb (PhtD3, a mAb that binds to Streptococcus pneumoniae) controls. Antibodies binding to the DS7 site showed higher phagocytosis scores overall, while the rest of the mAbs had variable ADP activity. Furthermore, EC 50 / I C 50 No correlation was observed between the Fab-binding epitope and the ADP activity of the mAbs, suggesting that the ADP activity of hMPV F-specific mAbs is independent of the Fab-binding epitope. These data suggest an additional protective mechanism of anti-hMPV F mAbs beyond neutralization.

[0341] Example 5 Therapeutic efficacy of MPV467 MPV467 is the most potent mAb of the panel, reaching picomolar neutralizing potency against hMPV TN / 93-32 and potently neutralizing hMPV CAN / 97-83. This mAb has prefusion-preferred properties, since substantial binding to the hMPV F protein is lost after fusion (FIG. 10). Based on these data, the protective efficacy of mAb MPV467 was tested in a hMPV infection model in BALB / c mice. Male and female mice were treated with PBS, isotype control human mAb or mAb MPV467 24 hours before or 3 days after hMPV infection in both preventive and treatment studies (FIG. 4). On day 5, virus titers in the lungs of mice were determined by plaque assay. In both studies, there was no detectable virus in mice treated with MPV467, but virus was present in both PBS- and isotype-mAb-treated mice. No differences were observed between MPV467 and uninfected mice, nor between PBS and isotype control mice in either study.

[0342] Example 6 Structural definition of the hMPV F-MPV467 complex Since MPV467 is the most potent hMPV F mAb described to date, is protective against and can treat hMPV infection, and targets an undefined epitope, we used cryo-electron microscopy (cryo-EM) to determine the structure of MPV467 in complex with pre-fusion hMPV F to a global resolution of 3.3 Å (Figures 5A-5C). The final map for model building was generated by particle subtraction of the flexible Fab constant region and sharpening via DeepEMhancer (Sanchez-Garcia et al.,. Commun. Biol. 4, 874 (2021)). The structure revealed that MPV467 has an approach angle pointing down towards the viral membrane (Figures 5A, 5B). The heavy and light chains are located on hMPV F at 567 Å, ​​respectively. 2 and 322 Å2 The MPV467 epitope spans antigenic sites II and V, but also contacts a single residue, Tyr44, in antigenic site III. hMPV F site V residue Arg156 makes multiple interactions with MPV467 via hydrogen bonds to main chain atoms of both CDRH1 and CDRH3, as well as a salt bridge interaction with CDRH1 Asp31 (Figure 5C). In addition, CDRH1 Asp31 also forms a hydrogen bond with site V residue Thr150. The conformationally immobile 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 main chain hydrogen bonds between CDRL1 Asn30 and hMPV F Ala238.

[0343] Although humans have been exposed to hMPV for at least 70 years (van den Hoogen et al. Nat. Med. 7, 719-724 (2001)), the predominant epitopes on the hMPV F protein remain poorly understood. H and V K / V L Diversity in gene usage was demonstrated. Among these genes, V H1-69 is shared by 4 of the 18 mAbs (MPV86, MPV414, MPV483 and MPV503), 3 of which (except MPV483) showed similar binning profiles competing with MPE8 and MPV364 (Figures 2A, 2B), indicating that they bind to the same hMPV F epitope, likely via similar binding patterns of the heavy chain. A similar correlation between binding epitopes and V gene usage was also observed for the light chain. mAbs MPV86 and 487, which share IGKV3-11, and mAbs MPV 414 and 503, which share IGKV3-15, both bind to the MPV364 site, whereas mAbs MPV464 and 485, which share IGLV3-1, bind to the DS7 site. mAbs MPV414 and MPV503 were identified from 2 different subjects using 2 different approaches, but V H / V K Both had the same set of genes, suggesting that this pair of genes might be preferred by the hMPV F MPV364 site-specific mAb.

[0344] 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. The locations of both sites V and II, as well as sites III and IV, are similar to their counterparts on RSV F, suggesting that epitopes in these regions share structural features that may be recognized by human antibodies. However, no mAbs were found to bind to the counterpart of RSV F site φ on hMPV F, further suggesting that mAbs against this epitope may be limited due to the N-linked glycans on hMPV F at sites φ (Asn57 and Asn172) (Poor et al., PNAS 111, 2596-2605 (2014)).

[0345] The most potent mAb, MPV467, bound to epitopes located across antigenic sites II and V. The structure of hMPV F in its pre- and post-fusion conformations revealed that it has structural homology with the related RSV F protein, and neutralizing epitopes on RSV F can be expected to have counterparts on hMPV F. Structurally, antigenic sites φ and V are pre-fusion specific, and previously isolated RSV antibodies and structural studies have shown that antibodies targeting these regions tend to be highly potent neutralizers (Gilman et al., Sci. Immunol. 1, 1-12 (2016); Mousa et al., Nat. Microbiol. 2, 16271 (2017)). Here, using the cryo-EM structure of mAb MPV467 in complex with hMPV F, it was determined that this mAb is one of the first site V targeting antibodies discovered for hMPV F and indeed shares the characteristics of potent neutralization. MPV467 binds to a beta hairpin (β3-β4) located within antigenic site V that undergoes a major conformational change during the pre-fusion to post-fusion transition. Without being bound by theory, this region of the MPV467 epitope is likely responsible for MPV467 potency. Even with binding, the fusion protein is unable to transition to a post-fusion conformation that is important for efficient viral infection. Furthermore, the helix-turn-helix (α6-α7) located within antigenic site II to which MPV467 binds does not undergo conformational change between the pre-fusion and post-fusion states. Binding of residues in this region is likely the reason for the observed ability of MPV467 to bind to both conformational states. Because only this portion of the epitope is present in the post-fusion conformation, a reduction in binding affinity was observed compared to the pre-fusion conformation containing the entire epitope.

[0346] The RSV / hMPV F cross-reactive mAb M1C7 (Xiao, et al., MAbs 11, 1415-1427 (2019)) was previously reported as a potent neutralizing mAb targeting site V. Similarly, the RSV F site V-specific mAb, hRSV90 (Mousa et al., Nat. Microbiol. 2, 16271 (2017)), also showed 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 ultrapotent neutralizing mAbs against pneumoviruses. The site V epitope of both RSV F and hMPV F is located immediately after the fusion peptide. 1 It is located on the N-terminus of the subunit and buried in the center of the trimeric pre-fusion F protein. Therefore, the conformational change of the α3 helix and β3-β4 hairpin in site V is essential to expose the fusion peptide and initiate the fusion process. Antibodies targeting site V likely lock the fusion peptide and prevent the formation of the long helical bundle present in the post-fusion conformation. However, based on the latest knowledge, the frequency of hMPV site V-specific antibodies is relatively low compared to those targeting site III, site IV and DS7 sites. Therefore, antigenic site V-targeting antibodies could be boosted in hMPV F-based vaccines.

[0347] Example 7 Materials and Methods for Examples 1-6 Blood collection and PBMC isolation: After obtaining informed consent, 90 mL of blood was collected by venipuncture into nine heparin-coated tubes and 10 mL of blood was collected into serum separator tubes. Peripheral blood mononuclear cells (PBMCs) were isolated from human donor blood samples using Ficoll-Histopaque density gradient centrifugation, and PBMCs were frozen in liquid nitrogen vapor phase until further use.

[0348] Production and synthesis of recombinant hMPV F protein: hMPV A1, A2, B1, B2F and hMPV B2F-GCN4 recombinant proteins were synthesized from GENSCRIPT® derived plasmids cloned into pcDNA3.1+ vector. They were expanded by transformation into DH5α cells to ampicillin (Thermo Scientific) resistance 100ug / ml. Plasmids were purified using the ENZA Plasmid maxiprep kit (Omega BioTek) according to the manufacturer's instructions. 1mg of plasmid was mixed with 4mg of polyethyleneimine (PEI; PolySciences Inc.) in OPTI-MEM® cell culture medium (Thermo Scientific) and incubated for 30 minutes. This was followed by 100μg of PEG in Freestyle 293 expression medium (Thermo Fischer). 6 The DNA-PEI mixture was added to 293 cells at 1000 cells / ml. After 5 days of incubation, the culture was centrifuged at 6000 g to pellet 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, it was washed with 5 column volumes (CV) of 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, it was washed with the same wash buffer (5 CV) to reduce non-specific 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 concentrated in an AMICON® Ultra-15 centrifugation unit (Sigma) with a molecular cut-off of 30 KDa.

[0349] Trypsinization of hMPV F: To obtain trimeric hMPV F, TPCK (L-1-tosylamido-2-phenylethyl chloromethyl ketone)-trypsin (Thermo Scientific) was diluted in double distilled water (ddHO) 2 hMPV B2 F was dissolved at 2mg / mL in 1000mL PBS (pH 7.0). The resulting hMPV B2 F was incubated with 5 TAME (p-toluene-sulfonyl-L-arginine methyl ester) units / mg of TPCK-trypsin at 37°C for 1 hour. Trimeric and monomeric fractions of hMPV F were separated by size exclusion chromatography on a SUPERDEX® S200, 16 / 600 column (GE Healthcare Life Sciences) in column buffer (50mM Tris pH 7.5, and 100mM NaCl). Both fractions were separated by their unique elution profiles. Once separated, they were concentrated as previously mentioned. Post-fusion hMPV F was obtained by heating the pooled trimeric fractions at 55°C on a water bath for 20 minutes to induce the post-fusion conformation (Jiachen et al., J. Virol. 95, e00593-21 ​​(2021)).

[0350] Generation of hMPV F-specific hybridomas: For hybridoma generation, 10 million peripheral blood mononuclear cells purified from human donor blood were cultured in 80 mL of STEMCELL® medium A (STEMCELL® Technologies) containing 6.3 μg / mL CpG (phosphorothioate-modified oligodeoxynucleotide; Invitrogen) and 1 μg / mL cyclosporine (Sigma) with previously frozen, gamma-irradiated NIH 3T3 cells modified to express human CD40L, human interleukin-21 (IL-21), and human BAFF (Bar-Peled et al., J. Virol. 93, e00342-19). (2019)). The mixture of cells was plated in four 96-well plates at 200 μl per well in StemCell medium A. After 6 days, culture supernatants were screened by ELISA for binding to recombinant hMPV B2 F protein, and cells from positive wells were electrofused as previously described (Bar-Peled et al., J. Virol. 93, e00342-19 (2019)). Cells from each cuvette were electrofused in StemCell® medium A containing 1× HAT (hypoxanthine-aminopterin-thymidine; Sigma-Aldrich), 0.2× HT (hypoxanthine-thymidine; Corning), and 0.3 μg / mL ouabain (Thermo Fisher Scientific). The cells were resuspended in 20 mL and plated at 50 μl per well in 384-well plates. After 7 days, the cells were fed with 25 μl of StemCell Medium A. Hybridoma supernatants were screened after 2 weeks for antibody production by ELISA, and cells from wells with reactive supernatants were expanded into 48-well plates in 0.5 mL of STEMCELL® Medium E (STEMCELL® Technologies) for 1 week before being screened again by ELISA.Positive hybridomas were then subjected to single-cell fluorescence-activated sorting into 384-well plates containing 75% STEMCELL® Medium A plus 25% STEMCELL® Medium E. Two weeks after cell sorting, hybridomas were screened by ELISA, after which wells containing hMPV F-specific hybridomas were further expanded.

[0351] RT-PCR for hybridoma mAb variable gamma and variable light chains: RNA was isolated from expanded hybridoma cells using the ENZA total RNA kit (Omega BioTek) according to the manufacturer's protocol. The High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) was used for cDNA synthesis. Three separate sets of primer mixes were used in nested PCR to amplify the variable regions of gamma, kappa, and lambda chains (Tiller et al., J. Immunol. Methods 329, 112-124 (2008)). Products from the second PCR were analyzed by agarose gel electrophoresis, and purified PCR products (ENZA cycle pure kit; Omega Biotek) were submitted to Genewiz for sequencing. Sequences were analyzed using IMGT / V-Quest (Brochet et al., Nucleic Acids Res. 36, 503-508 (2008)).

[0352] Antigen-specific single B cell sorting and expression of recombinant mAb: 10 million human PBMCs were washed twice with FACS buffer and then resuspended in 1 mL of FACS buffer. Cells were treated with 5% Fc receptor blocker (BIOLEGEND®) for 30 minutes and then stained with the following antibodies: human CD19-APC, human IgM-FITC, human IgD-FITC, GHOST DYE™ Red 710 and PE / BV605-streptavidin conjugated hMPV B2 F. Antigen-specific B cells were gated on CD19+ / IgM- / IgD- / Ghost dye- / PE+ / BV605+ and sorted into capture buffer B (Qiagen TCL buffer + 1% beta-mercaptoethanol) at one cell per well in a 96-well plate. Sorted cells were flash frozen and stored at -80°C until they were used for RNA extraction. RNA was extracted using Agencort RNACLEAN® XP kit, SPRI Beads (Beckman Coulter) and immediately reverse transcribed into cDNA using SUPERSCRIPT® IV Synthesis System (ThermoFisher). Variable region sequences of IgG heavy / light chains were determined by nested PCR as described above. Based on the V / D / J gene allele usage, cloning PCR primers were selected for cloning PCR using the first PCR product as template. Purified cloning PCR products of heavy / light chains were cloned into expression vectors (AbVec-hIgG1, AbVec-hIgKappa and pBR322-based Ig-lambda expression vector) and plasmids were sent to Genewiz for sequencing. After confirming that all sequences were correct, HC / LC plasmids were transformed into DH5α for plasmid maxiprep. Recombinant mAbs were expressed by transfecting 293 cells with the HC / LC plasmid and purified from culture supernatants using a protein G column (Cytiva).

[0353] Enzyme-linked immunosorbent assay for binding to hMPV F protein: 384-well plates used for ELISA (catalog no. 781162; Greiner BIO-ONE®) were coated with recombinant protein (antigen) at 2 μg / ml (in PBS) and incubated overnight at 4° C. This involved washing the plates once with water and then blocking them with a blocking buffer consisting of 2% milk supplemented with 2% goat serum in PBS and 0.05% Tween 20 (PBS-T) for 1 h at room temperature. The plates were washed again three times with PBS-T. 25 μl of serially diluted primary antibodies were added to the wells and incubated for 1 h at room temperature, followed by washing three times with PBS-T. Goat anti-human IgG Fc secondary antibody (Southern Biotech) (1:4000) diluted in blocking buffer was then applied to the wells and again incubated for 1 h at room temperature. Plates were washed again three times with PBS-T and 25 μl of PNPP (p-nitrophenyl phosphate) diluted to a concentration of 1 mg / ml in a buffer containing 1 M Tris base and 0.5 mM magnesium chloride with a pH of 9.8 was added. Plates were incubated one last time at room temperature for 1 hour before reading the absorbance at 405 nm on a Bio Tek plate reader. Binding assay data was analyzed using EC 50 Values ​​were analyzed in GraphPad Prism using a nonlinear regression curve fit and log(agonist)-versus-response functions to calculate values.

[0354] hMPV plaque neutralization experiment: LLC-MK2 cells used in this experiment were incubated at 4°C for 1 h at 20°C for 2 h. 2The cells were grown in Opti-MEM I (Thermo Fischer Scientific) supplemented with 2% fetal bovine serum in T225 cell culture flasks (catalogue no. 82050-870) at 37°C in an incubator. 40,000 cells / well were plated on a 24-well plate two days before starting the neutralization assay. Serially diluted sterile filtered mAbs isolated from hybridoma supernatants were added in equal volumes (1:1) to suspensions of either hMPV strains CAN / 97-83 and TN / 93-32 and incubated for 1 hour on the day of the experiment. After this, 50 μl of the virus-antibody mixture was added to the LLC-MK2 cells after washing the excess FBS from the OPTI-MEM medium three times with PBS. The mixture was incubated for 1 hour at room temperature with constant rocking. The cells were then incubated with 5 μg / ml trypsin-EDTA and 100 ug / ml CaCl 2 The plates were overlaid with 0.75% methylcellulose dissolved in Opti-MEM I supplemented with 0.1% PBS-T. The cells were incubated for 4 days and fixed with 10% neutral buffered formalin. The cell monolayer was then blocked for 1 hour with blocking buffer consisting of 2% non-fat milk supplemented with 2% goat serum in PBS-T. The plates were then washed 3 times with water and 200 μl of MPV364 was added to a final concentration of 1 μg / ml (1:1000 dilution) in blocking solution. The plates were then washed 3 times with water and 200 μl of goat anti-human IgG HRP secondary antibody (Southern Biotech) diluted in blocking buffer to a ratio of 1:2000 was added and incubated for 1 hour at room temperature followed by 1 hour of incubation. The plates were again washed 5 times with water and 200 μl of TRUEBLUE® peroxidase substrate (SERACARE®) was added to each well. Plates were incubated for 20-30 min until plaques were clearly visible. Plaques were manually counted under a microscope and compared to virus-only controls. GraphPad Prism was used to calculate IC using nonlinear regression curve fits and log(inhibitor)-versus-response functions. 50 values ​​were calculated.

[0355] Epitope binning: 100 μg / ml his-tagged hMPV B2F (untrypsinized) protein was immobilized on an anti-penta-His biosensor tip (Forte'Biosciences) for 120 seconds after obtaining an initial baseline in running buffer (PBS, 0.5% BSA, 0.05% Tween 20 and 0.04% thimerosal). The baseline was measured again by immersing the tip in a well containing 100 μg / ml primary antibody for 300 seconds. This was followed by immersing the biosensor tip again in 100 μg / ml secondary antibody for 300 seconds. Binding of the second mAb in the presence of the first mAb, determined by comparing the maximum signal of the second mAb after the first mAb, was added to the maximum signal of the second mAb alone. Non-competitive mAbs were mAbs whose binding was greater than or equal to 70% of uncompetitive binding. Between 30% and 60% was considered as intermediate binding, and anything below 30% was considered as competing for the same site.

[0356] Antibody-dependent phagocytic activity of mAb: 2 × 10 91 μm Neutravidin coated yellow-green FLUOSPHERES® (Invitrogen #F8776) were resuspended in 1 mL of 0.1% PBS. The FLUOSPHERES® were then centrifuged at 5000 rpm for 15 minutes, 900 μL of supernatant was removed, and the FLUOSPHERES® were resuspended in 900 μL of 0.1% PBS. This process was repeated for a second wash, and the FLUOSPHERES® were then resuspended with 20 μg of biotinylated hMPV B2 F protein. The FLUOSPHERES® were then incubated overnight at 4° C. with end-to-end rocking, protected from light. Next, hMPV F-specific antibody was diluted in complete RPMI medium (cRPMI, RPMI+10% FBS) to a final concentration of 1 μg / mL in a U-bottom 96-well plate. 20 μL of antibody dilutions were then transferred to a clear F-bottom 96-well plate and 10 μL of FLUOSPHERES® was added along with the antibodies, followed by incubation for 2 hours at 37° C. for opsonization. After 1.5 hours, the THP-1 cells were centrifuged at 200×g for 5 minutes, washed once with PBS, and then diluted with 5×10 5 The cells were resuspended in culture medium (RPMI and 10% FBS) at a concentration of 1000 cells / mL. 200 μL of cells were then added to each well and incubated at 37 °C for 6 h with shaking in 5% CO. 2 The cells were then incubated at 37°C for 10 min at 5°C. Once incubation was complete, the plates were then centrifuged at 2000 rpm for 5 min. 100 μL was then pipetted out of each well and replaced with 100 μL of cold 4% paraformaldehyde to fix the cells. The plates were then placed at room temperature for 20 min, protected from light. The plates were then stored at 4°C in the dark. The cells were then analyzed on a NOVOCYTE® QUANTEON® Flow Cytometer. The percentage of fluorescent beads containing THP-1 cells (% phagocytosis) in each sample was used to calculate the % increase over the no mAb control. Phagocytosis scores were calculated as previously described 50(geometric mean intensity-geometric mean intensity of no mAb control) x % phagocytosis.

[0357] Animal studies: BALB / c mice (6-8 weeks old; The Jackson Laboratory) were randomly selected into groups containing 5 males and 5 females. All mice were pre-bled prior to the study to verify by ELISA that the mice had not been previously exposed to hMPV. Each mouse was injected with hMPV TN / 93-32 (5 × 10 5 Mice were infected intranasally with 10 ...

[0358] Recombinant protein production for cryo-EM studies: The prefusion hMPV F construct DS-CavEs2-IPDS (hMPV F A1 NL / 1 / 00, residues 1-490) used for structural studies contains the previously described G294E, A185P, L219K, V231I, E453Q and furin cleavage site substitutions (Battles et al., Nat. Commun. 8, 1528 (2017)). The disulfide substitutions involved are L110C / N322C, T127C / N153C, A140C / A147C and T365C / V463C (Hsieh et al., in press) as well as an interprotomer disulfide at V84C / A249C (Stewart-Jones et al., Proc. Natl. Acad. Sci. 118, e2106196118 (2021)). DS-CavEs2-IPDS was cloned into the mammalian expression vector pαH with a C-terminal "GGGS" linker sequence followed by the T4 fibritin trimerization motif "foldon" (Efimov et al., J. 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., Nat. Commun. 8, 1528 (2017)). Transient co-transfection of FreeStyle 293F cells (ThermoFisher) with a 4:1 ratio of DS-CavEs2-IPDS:furin expression plasmids with polyethylenimine (PEI) was used for protein expression. Kifunensine and Pluronic® F-68 (Gibco) were introduced 3 hours after transfection to a final concentration of 5 μM and 0.1% (v / v), respectively. Six days after transfection, soluble proteins were purified from cell supernatants that were filtered by tangential flow filtration and buffer exchanged into PBS using STREP-TACTIN® Sepharose resin (IBA).Strep-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 concentrating the proteins using a 30 kDa molecular weight cut-off AMICON® Ultra-15 centrifugal filter unit (Millipore), the proteins were eluted in 2 mM Tris pH 8.0, 200 mM NaCl and 0.02% NaN. 3 The product was further purified by size-exclusion chromatography using a SUPEROSE® 6 Increase 10 / 300 column (GE Healthcare) in running buffer of 1000 ml.

[0359] Cryo-EM sample preparation and data collection: Purified DS-CavEs2-IPDS was combined with a 1.5-fold molar excess of MPV467 Fab and incubated at room temperature for 10 min, then transferred to ice. Immediately prior to freezing, samples were resuspended in 2 mM Tris pH 8.0, 200 mM NaCl and 0.02% NaN 3 The hMPV F concentration was then diluted to 0.66 mg / mL in 0.5% amphipol A8-35. 1 μL of 0.5% amphipol A8-35 was then combined with 10 μL of the diluted sample, and 4 μL of this sample was added to 0.5% amphipol A8-35 in a 4:1 ratio of 0.5% amphipol A8-35. 2 / H 2The grids were plunge-frozen using a VITROBOT® Mark IV (Thermo Fisher) with a 100% humidity chamber at 10°C. Blotting settings were 5 seconds wait, then 4 seconds blotting, -2 force before plunge into nitrogen-cooled liquid ethane. A single grid was imaged using a GLACIOS® (Thermo Scientific) with a Falcon 4 direct electron detector (Thermo Scientific) to collect a total of 1,458 images. Data were analyzed at a magnification of 150,000× corresponding to a calibrated pixel size of 0.94 Å / pix and 40 e - / Å 2 The data were collected at a tilt of 30° with a total exposure of 100 nm. The data collection statistics are listed in FIG.

[0360] Cryo-EM data processing: Micrographs were corrected for gain reference and imported into CRYOSPARC® Live v3.2.0 for initial data processing: motion correction, defocus estimation, micrograph curation, particle picking and extraction, and particle curation via iterative streaming 2D class averaging. 2D averaging was used to generate templates and template-based particle picking was performed. Curated particles were exported to CRYOSPARC® v3.2 for further processing via rounds of 2D classification, ab initio reconstruction, non-uniform refinement, uniform refinement, and non-uniform uniform refinement using C3 symmetry. Masking and particle subtraction were used for further non-uniform refinement. Finally, the particle-subtracted non-uniform refinement maps were sharpened using DeepEMhancer (Sanchez-Garcia et al,. Commun. Biol. 4, 874 (2021);Cianfrocco, et al., COSMIC2: A Science Gateway for Cryo-Electron Microscopy Structure Determination. in 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). The EM processing workflow is shown in Figure 8 and the EM validation results are shown in Figure 9. For model building, an initial hMPV F model was generated from PDB ID:5WB0 and the crystal structure of MPV467 Fab and used to dock into the cryoEM map using UCSF ChimeraX (Pettersen et al., Protein Sci. 30, 70-82 (2021)).The model was further built 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)). Model statistics are shown in Figure 11.

[0361] Example 8 Human metapneumovirus (hMPV) infection and MPV467 treatment in immunocompromised cotton rats, Sigmodon hispidus The cotton rat, Sigmodon hispidus, is an established model of respiratory viral infections, including those caused by RSV, influenza, adenovirus, parainfluenza, rhinovirus and enterovirus (Blanco et al., J Antivir Antiretrovir 2014; 6:40-42; Patent et al., PLoS One 2016; 11(11):e0166336). hMPV infection was modeled in cotton rats, S. hispidus, shortly after the discovery of the virus (Hamelin et al., J Virol 2005; 79(14):8894-903; Williams et al., J Virol 2005; 79(17):10944-51; Wyde et al., Antiviral Res 2005; 66(1):57-66; Yim et al., Vaccine 2007; 25(27):5034-40). Cyclophosphamide-immunosuppressed cotton rats, S. hispidus, were infected with hMPV, and viral replication and pulmonary inflammation in these animals were compared to those in normal hMPV-infected S. hispidus. The efficacy of prophylactic and therapeutic administration of the anti-hMPV antibody MPV467 was also evaluated. Immunosuppressed animals had higher lung and nasal titers of hMPV 5 days after infection compared to normal animals, and greater amounts of hMPV were still present in the airways of immunosuppressed animals 7 and 9 days after infection, indicating prolonged viral replication. Immunosuppression was accompanied by reduced lung histopathology in hMPV-infected cotton rats compared to normal animals, but a delayed increase in pathology and pulmonary cytokine / chemokine expression was seen in immunosuppressed cotton rats. Prophylactic and therapeutic MPV467 treatment protected both upper and lower airways against hMPV infection. Lung pathology and pulmonary expression of IP-10 and MIP-1α mRNA were reduced by therapeutic MPV467 administration. Results are provided below.

[0362] Immunosuppression results in increased hMPV replication and delayed viral clearance in cotton rats: Immunosuppression may be associated with more severe disease and prolonged replication of respiratory viruses in affected individuals (Welliver, J Pediatr 2003; 143(5 Suppl):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 cyclophosphamide-immunosuppressed cotton rats, animals were infected with cyclophosphamide for 10 min at 4 °C. 5 PFU of hMPV and viral replication was assessed at several time points post-infection. Lung and nasal samples were collected for viral titration at 5 days post-infection, the time of peak viral replication in normal cotton rats, 7 days post-infection, when the virus was cleared from the lungs of infected cotton rats, and at an additional delayed time point, day 9. Comparisons were made to age-matched normal animals infected in parallel with the immunosuppressed animals and sacrificed on the same day. As expected, in normal animals, hMPV was cleared from the lungs and essentially cleared from the nose of infected normal cotton rats by day 7 post-infection, and was undetectable in either the lower or upper respiratory tract by day 9 (Figure 12). In contrast, immunosuppressed cotton rats showed high levels of hMPV in the lungs and nose on all three days. The amount of hMPV present in the lungs and noses of infected animals 5 days post-infection was significantly higher than that detected in normal cotton rats sacrificed at the same time. Overall, these results indicate that hMPV can replicate to higher titers in immunosuppressed cotton rats and that viral clearance is significantly delayed by immunosuppression.

[0363] 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 efficient therapeutic and prophylactic drugs for 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 5 days after infection (Figure 13). Three different doses of MPV467 were tested: 0.1, 1 and 10 mg / kg, 1 day before or 3 days after infection. The results of the study showed that both prophylactic and therapeutic treatment with MPV467 were effective in reducing hMPV replication in the lungs and nose of infected animals. The two highest doses of MPV467 tested, 10 and 1 mg / kg, given either before or after infection, completely protected the lungs of infected animals (undetectable viral replication). The lowest dose of MPV467 tested, 0.1 mg / kg, caused a modest but statistically significant reduction in pulmonary hMPV replication, an effect that was slightly more pronounced for prophylactic treatment. In the nose, significant reductions in hMPV load were obtained by all three doses of MPV467 tested when administered prophylactically, and by the two highest doses when given therapeutically. Overall, these results indicate that MPV467 has strong dose-dependent antiviral activity in immunosuppressed cotton rats.

[0364] MPV467 treatment ameliorates delayed hMPV clearance in immunosuppressed cotton rats: Once the antiviral efficacy of MPV467 was elucidated in immunosuppressed cotton rats by analyzing samples collected at the peak time of viral replication in the lungs, it was determined whether therapeutic administration of the antibody could combat delayed viral clearance in this model. To address this issue, hMPV-infected immunosuppressed animals were treated with 10 mg / kg MPV467 3 and 7 days after infection and sacrificed 9 days after infection for analysis of viral load in the lungs and nose. Normal cotton rats were infected and treated once on day 3. Replication on day 5 was assessed again in hMPV-infected animals (normal and immunosuppressed) treated with 10 mg / kg MPV467 (or mock-treated) 3 days after infection. MPV467 was highly effective in normal cotton rats, reducing viral replication to undetectable levels in day 5 samples (Figure 14). MPV467 was also highly 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, whereas MPV467-treated animals had no detectable hMPV in either the upper or lower respiratory tract on both days of analysis.

[0365] Effect of antibody treatment on lung histopathology and cytokine / chemokine expression in immunosuppressed cotton rats: Lung histopathology, one of the markers of inflammatory response to respiratory infection in the cotton rat model, was used to evaluate differences in the 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 5 and 9 days after infection for analysis of peribronchiolitis, perivasculitis, interstitial inflammation and alveolitis. Normal hMPV-infected animals had a moderate level of pathology characterized primarily by peribronchiolitis and some perivasculitis (Figure 15). The degree of pathology was generally comparable between days 5 and 9. Antibody treatment caused a moderate increase in perivasculitis in normal hMPV-infected cotton rats. Immunosuppressed cotton rats infected with hMPV developed reduced peribronchiolitis compared to normal animals. Lung histopathology in hMPV-infected immunosuppressed animals was slightly higher on day 9 compared to day 5, revealing interstitial inflammation and alveolitis. The effect of therapeutic antibody treatment on lung pathology was evaluated with MPV467 administered at a dose of 10 mg / kg. A reduction in lung histopathology was seen in hMPV-infected antibody-treated immunosuppressed animals 9 days after infection compared to hMPV-infected mock-treated immunosuppressed animals.

[0366] Pulmonary cytokine / chemokine expression is another marker of the pulmonary inflammatory response to infection in the cotton rat model. To determine whether the reduction in interstitial inflammation and alveolitis seen at day 9 in immunosuppressed hMPV-infected animals treated with MPV467 was associated with altered pulmonary cytokine / chemokine expression, we measured the levels of pulmonary MIP-1α and IP-10 (mediators associated with lung injury and immune dysfunction) (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). Immunosuppressed animals had elevated expression of MIP-1α and IP-10 mRNA compared to normal animals ( FIG. 16 ). Expression of both mediators in immunosuppressed animals was significantly reduced by antibody treatment.

[0367] Normal (unmanipulated) cotton rats S. hispidus are susceptible to hMPV infection in the upper and lower respiratory tract, and infection largely resolves within 1 week. 20~21This differs from the persistent hMPV infection reported for normal BALB / c mice, where hMPV replication can 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 resembles self-limited hMPV infection in healthy humans (Moe et al., J Infect Dis 2017; 216(1):110-116; Ebihara et al., J Clin Microbiol 2004; 42:126-32), a valuable feature that allows for the evaluation of potential viral clearance defects that may be caused by immune suppression.

[0368] 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 conditions of suppressed immunity. Delayed viral clearance may generally affect lung function by direct cytopathic effects of prolonged viral replication or by indirect effects on lung inflammation. In this study, it was noted that interstitial inflammation and alveolitis were slightly increased in immunosuppressed cotton rats infected with hMPV at a later stage in infection compared to an earlier time point corresponding to peak viral replication in the lung. At the same time, the expression of pulmonary cytokines / chemokines IP-10 and MIP-1α in immunosuppressed animals at this later stage in infection surpassed that detected in normal cotton rats infected with hMPV. The increase in certain parameters of pulmonary inflammation at the later stage in infection of immunosuppressed animals was similar to that previously seen for RSV, except that cytokines were not measured for the RSV model and no detectable epithelial damage was seen in the lungs of hMPV-infected animals (Boukhvaolova et al., Bone Marrow Transplant 2016; 51(1):119-26). The increased levels of IP-10 and MIP-1α at the later stage in hMPV infection in immunosuppressed animals may have deleterious effects on the lung.Both molecules have been shown to promote the development of lung injury of viral and non-viral origin via 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).

[0369] Therapeutic administration of anti-hMPV antibody MPV467 resulted in the disappearance of hMPV replication in immunosuppressed cotton rats, which also reduced lung pathology and cytokine / chemokine expression in the lungs of immunosuppressed animals. This combined suppression 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 Transplant 2016; 51(1):119-26). For RSV, the ability of therapeutic antibody treatment to reduce pulmonary inflammation in immunosuppressed animals contrasted with a similar lack of efficacy of antiviral antibodies when used alone in normal non-immunosuppressed cotton rats (Prince et al., J Infect Dis 2000; 182:1326-1330; Boukhvalova et al., J Infect Dis 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).

[0370] Example 9 Materials and Methods for Example 8 Reagents: Cyclophosphamide for injection (20 mg / ml USP, Baxter) was obtained from Blue Door Pharma.

[0371] Virus and viral assays: hMPV strain TN / 94-49 / A2 (Williams et al., N Engl J Med 2004; 350(5):443-50; Williams et al., . J Infect Dis 2006; 193(3):387-95), recovered from specimens collected at 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. A single pool of hMPV (3×10 6 pfu / ml) were used in the studies described herein.

[0372] Animals and Animal Studies: Inbred S. hispidus cotton rats were obtained from a colony maintained at Sigmovir Biosystems, Inc. Male and female cotton rats aged 6-8 weeks were used in the study. Animals were housed in large polycarbonate cages and fed a standard diet of rodent chow and water. The colony was monitored for antibodies to adventitious 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, Parvoviruses, Pneumonia virus PVM, RCV / SDA, Sendai virus). hMPV infection in immunocompromised animals and efficacy of MPV467 treatment were examined in two consecutive experiments. A sample size of 4-5 animals per group was selected based on the results of previous experiments to allow detection of statistically significant differences between groups. Comparisons between groups were performed using Student's t-test for unpaired data with unequal variances (KaleidaGraph). Unless indicated, samples were blinded prior to analysis.

[0373] Immunosuppression was induced in cotton rats by repeated treatment with cyclophosphamide (CY) based on a previously published method (Boukhvalova et al., Bone Marrow Transplant 2016; 51(1):119-26). Briefly, 50 mg / kg of CY solution was administered intramuscularly (im) as 250 μl / 100 g animal on a Monday-Wednesday-Friday schedule for 18 days. At the end of this period, whole blood was collected to verify the reduction in total white blood cell and lymphocyte counts. CY treatment was continued until the end of the study. 21 days after the initiation of CY treatment, animals were injected with hMPV (10 5Animals were infected intranasally (in) with 1000 PFU of hMPV. To quantify hMPV load, groups of 5 animals were sacrificed 5 and 7 days (first study) or 5 and 9 days (second study) post-infection for collection of lungs and noses for virus quantification by plaque assay. Groups of age-matched normal cotton rats were infected with hMPV and sacrificed 5, 7, and 9 days post-infection for quantification of hMPV by plaque assay.

[0374] For the assessment of dose-dependent prophylactic and therapeutic efficacy of MPV467, cyclophosphamide immunosuppressed cotton rats were inoculated im with 0.1, 1 or 10 mg / kg MPV467 1 day before or 3 days after hMPV challenge. Control animals were treated with PBS (mock) 1 day before hMPV infection. Animals were sacrificed 5 days after hMPV challenge and lungs and noses were collected for virus titration. For analysis of therapeutic efficacy of MPV467 at the time of delayed clearance (day 9), immunosuppressed animals were infected with hMPV and treated im with 10 mg / kg MPV467 3 days after infection and sacrificed 5 days after infection, or treated with MPV467 3 and 7 days after infection and sacrificed 9 days after infection. Normal animals were treated with MPV467 3 days after infection and sacrificed on day 9. Lungs were collected for histopathology and qPCR analysis, and lungs and noses were collected for virus titration.

[0375] Virus titration: Lung and nasal homogenates were clarified by centrifugation and diluted in EMEM. Confluent LLC-MK-2 monolayers were infected in duplicate in 24-well plates with diluted homogenates. 5% CO 2After 1 hour of incubation at 37°C in an incubator, the wells were overlaid with 0.75% methylcellulose medium. After 7 days of incubation, the overlay was removed and the cells were fixed for 1 hour and air-dried for immunostaining. Upon blocking the wells with 1% BSA in PBS, mouse anti-hMPV-N-protein antibody at 1:1,000 dilution in 1% BSA was added to each well, followed by washing and then incubation with HRP-conjugated rabbit anti-mouse IgG at 1:1,000 dilution in 1% BSA. AEC chromogen detection solution was added to each well and incubated for 2 hours at room temperature. Visible plaques were counted and virus titers were expressed as plaque-forming units per gram of tissue. Viral titers were calculated as the geometric mean ± standard error (SEM) for all animals in a group at a given time point.

[0376] Pulmonary cytokine analysis: Total RNA was extracted from homogenized lung tissue using RNeasy purification kit (QIAGEN). One microgram of total RNA was used to prepare cDNA using oligo 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., J Infect Dis 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:Unit26.6). The signal obtained for each analyzed gene was normalized to the level of β-actin ("housekeeping gene") expressed in the corresponding organ. Cytokine levels were expressed as the geometric mean ± SEM for all animals in a group at a given time point. Differences between groups were assessed by Student's t test for summary data.

[0377] Histopathology Analysis: Lungs were prepared for histopathology analysis as previously described and blindly scored for peribronchiolaritis (inflammatory cells around small airways), perivasculitis (inflammatory cells around small blood vessels), alveolitis (inflammatory cells within the alveolar spaces) and interstitial pneumonitis (inflammatory cell infiltration and alveolar wall thickening) (Prince et al., J Gen Virol 2001;82:2881-8). Each parameter was scored on a scale of 0-4, with 0=0%, 1=5%, 2=25%, 3=75% and 4=100% histological score.

Claims

1. a) SEQ ID NOs: 41 and 45 (MPV467), respectively; b) SEQ ID NOs: 9 and 13 (MPV414), respectively; c) SEQ ID NOs: 17 and 21 (MPV454), respectively; d) SEQ ID NOs: 25 and 29, respectively (MPV456); e) SEQ ID NOs: 33 and 37, respectively (MPV464); f) SEQ ID NOs: 1 and 5 (MPV86), respectively; g) SEQ ID NOs: 49 and 53 (MPV477), respectively; h) SEQ ID NOs: 57 and 61 (MPV478), respectively; i) SEQ ID NOs: 65 and 69 (MPV481), respectively; j) SEQ ID NOs: 73 and 77, respectively (MPV482); k) SEQ ID NOs: 81 and 85 (MPV483), respectively; l) SEQ ID NOs: 89 and 93 (MPV485), respectively; m) SEQ ID NOs: 97 and 101 (MPV486), respectively; n) SEQ ID NOs: 105 and 109 (MPV487), respectively; o) SEQ ID NOs: 113 and 117, respectively (MPV488); p) SEQ ID NOs: 121 and 125 (MPV489), respectively; q) SEQ ID NOs: 129 and 133 (MPV491), respectively; or r) SEQ ID NOs: 137 and 141 (MPV503), respectively; V, shown as Ｈ and V Ｌ heavy chain variable (V) comprising heavy chain complementarity determining regions (HCDR) 1, HCDR2, and HCDR3 and light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3 of Ｈ ) region and the light chain variable region (V Ｌ 1. An isolated monoclonal antibody or antigen-binding fragment thereof comprising:

2. the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 are a) SEQ ID NOs: 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) SEQ ID NOs: 2, 3, 4, 6, 7 and 8, respectively; g) SEQ ID NOs: 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) SEQ ID NOs: 74, 75, 76, 78, 79 (KDK) and 80, respectively; k) SEQ ID NOs: 82, 83, 84, 86, 87 (AAS) and 88, respectively; l) SEQ ID NOs: 90, 91, 92, 94, 95 (QDT) and 96, respectively; m) SEQ ID NOs: 98, 99, 100, 102, 103 (SDN) and 104, respectively; n) SEQ ID NOs: 106, 107, 108, 110, 111 (DAS) and 112, respectively; o) SEQ ID NOs: 114, 115, 116, 118, 119 (WAS) and 120, respectively; p) SEQ ID NOs: 122, 123, 124, 126, 127 and 128, respectively; q) SEQ ID NOs: 130, 131, 132, 134, 135 (RGS) and 136, respectively; or r) SEQ ID NOs: 138, 139, 140, 142, 143 (GAS) and 144, respectively 2. The isolated monoclonal antibody or antigen-binding fragment of claim 1, comprising the amino acid sequence shown as:

3. The V Ｈ and the V Ｌ but, a) SEQ ID NOs: 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; l) SEQ ID NOs: 89 and 93, respectively; m) SEQ ID NOs: 97 and 101, respectively; n) SEQ ID NOs: 105 and 109, respectively; o) SEQ ID NOs: 113 and 117, respectively; p) SEQ ID NOs: 121 and 125, respectively; q) SEQ ID NOs: 129 and 133, respectively; or r) SEQ ID NOs: 137 and 141, respectively 2. The isolated monoclonal antibody or antigen-binding fragment of claim 1, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown as:

4. 2. The isolated monoclonal antibody or antigen-binding fragment of claim 1, comprising a human framework region.

5. The V Ｈ and the V Ｌ but, a) SEQ ID NOs: 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; l) SEQ ID NOs: 89 and 93, respectively; m) SEQ ID NOs: 97 and 101, respectively; n) SEQ ID NOs: 105 and 109, respectively; o) SEQ ID NOs: 113 and 117, respectively; p) SEQ ID NOs: 121 and 125, respectively; q) SEQ ID NOs: 129 and 133, respectively; or r) SEQ ID NOs: 137 and 141, respectively 2. The isolated monoclonal antibody or antigen-binding fragment of claim 1, comprising the amino acid sequence shown as:

6. 2. The isolated monoclonal antibody of claim 1, comprising a human constant domain.

7. The isolated monoclonal antibody of claim 1 , which is a human antibody.

8. 2. The isolated monoclonal antibody of claim 1, which is an IgG.

9. 2. The isolated monoclonal antibody of claim 1, comprising a recombinant constant domain comprising a modification that increases the half-life of the antibody.

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

11. 2. The isolated monoclonal antibody or antigen-binding fragment of claim 1, wherein the antibody neutralizes Group A and Group B hMPV.

12. 2. The isolated monoclonal antibody or antigen-binding fragment of claim 1, wherein the antibody binds to the pre-fusion F protein with higher affinity than to the post-fusion F protein.

13. The isolated monoclonal antigen-binding fragment of claim 1.

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

15. 15. An isolated monoclonal antibody according to any one of claims 1 to 12 or an 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 of any one of claims 1 to 12 or the antigen-binding fragment of claim 13 or claim 14.

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

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

19. 18. The nucleic acid molecule of claim 17 operably linked to a promoter.

20. A vector comprising the nucleic acid molecule of claim 17.

21. A host cell comprising the nucleic acid molecule or vector of claim 17.

22. an effective amount of the monoclonal antibody of any one of claims 1 to 12, the antigen-binding fragment of claim 13 or 14, or a nucleic acid molecule or vector encoding said monoclonal antibody or antigen-binding fragment; and Pharmaceutically acceptable carrier 10. A pharmaceutical composition for use in inhibiting hMPV infection, comprising:

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

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

25. 27. 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 composition comprising a monoclonal antibody described in any one of claims 1 to 12, an antigen-binding fragment described in claim 13 or 14, a bispecific antibody comprising said monoclonal antibody or said antigen-binding fragment, a nucleic acid molecule encoding said monoclonal antibody or said antigen-binding fragment or said bispecific antibody, a vector comprising said nucleic acid molecule, or a pharmaceutical composition comprising said monoclonal antibody or said antigen-binding fragment or said nucleic acid molecule or said vector and a pharmaceutically acceptable carrier, wherein said composition or pharmaceutical composition is for inhibiting hMPV infection in a subject, and said subject is at risk of or in an at-risk state for hMPV infection.

27. ​​A composition comprising a monoclonal antibody described in any one of claims 1 to 12, an antigen-binding fragment described in claim 13 or 14, a bispecific antibody comprising said monoclonal antibody or said antigen-binding fragment, a nucleic acid molecule encoding said monoclonal antibody or said antigen-binding fragment or said bispecific antibody, a vector comprising said nucleic acid molecule, or a pharmaceutical composition comprising said monoclonal antibody or said antigen-binding fragment or said nucleic acid molecule or said vector and a pharmaceutically acceptable carrier, wherein said composition or pharmaceutical composition is for inhibiting hMPV infection in a subject or for detecting the presence of hMPV in a biological sample.

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