Anti-hemagglutinin antibodies and methods of use thereof

Abstract

Provided herein are monoclonal antibodies or antigen-binding fragments thereof that bind to influenza B hemagglutinin (HA) protein, pharmaceutical compositions comprising the antibodies, and methods of use. The antibodies are useful for inhibiting or neutralizing influenza B viral activity, thus providing a means for treating or preventing influenza infection in humans. Also provided is the use of one or more antibodies that bind to influenza B HA to prevent viral attachment to and / or entry into host cells. The antibodies can be used prophylactically or therapeutically, and can be used alone or in combination with one or more other antiviral agents or vaccines.

Description

[Technical field]

[0001] Government Licensing Rights This invention was made with Government support under Contract HHSO100201700020C awarded by the U.S. Department of Health and Human Services. The Government has certain rights in this invention.

[0002] Sequence Listing An official copy of the Sequence Listing is being submitted electronically via EFS-Web contemporaneously herewith as an ASCII formatted Sequence Listing with the file name "10625WO01_SEQ_LIST_ST25.txt", a creation date of October 28, 2020, and a size of approximately 36 KB. The Sequence Listing contained in this ASCII formatted document is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0003] background Influenza is a highly contagious disease with a long history characterized by waves of pandemics, epidemics, resurgences and outbreaks. Despite annual vaccination efforts, influenza infections cause significant morbidity and mortality.

[0004] Influenza viruses consist of four types: A, B, C, and D. Influenza B causes a significant number of seasonal influenza infections and is associated with severe influenza requiring hospitalization.

[0005] Hemagglutinins are trimeric glycoproteins that contain two structural domains, a globular head domain consisting of a receptor-binding site (i.e., subject to frequent antigenic drift), and a stem region (more conserved among various strains of influenza viruses). The HA protein is synthesized as a precursor (HA0) and undergoes proteolytic processing to generate two subunits (HA1 and HA2), which associate with each other and form a stem / globular head structure. The HA1 peptide is involved in attachment of the virus to the cell surface and is necessary (together with neuraminidase) for viral attachment and entry into the host cell. The HA2 peptide forms a stem-like structure that mediates the fusion of the viral membrane with the cell membrane in the endosome, allowing the release of the ribonucleoprotein complex into the cytoplasm.

[0006] New strains of influenza virus can arise as a result of a phenomenon called antigenic drift, or mutations in the hemagglutinin or neuraminidase molecules that generate new and different epitopes. This results in new vaccines having to be produced each year against viruses predicted to emerge, a process that is not only costly but also highly inefficient. Although technological advances have improved the ability to produce improved influenza antigens for vaccine compositions, there remains a need to provide additional sources of protection against influenza. Summary of the Invention

[0007] overview The present disclosure provides antibodies and antigen-binding fragments thereof that bind to influenza B HA. The antibodies are particularly useful for inhibiting or neutralizing the activity of influenza B HA.

[0008] In a first aspect, the present disclosure provides an isolated, recombinant monoclonal antibody or antigen-binding fragment thereof that specifically binds to influenza B hemagglutinin (HA).

[0009] In some embodiments, the present disclosure provides an isolated, recombinant antibody or antigen-binding fragment thereof that specifically binds to influenza B HA, wherein the antibody has the following characteristics: (a) It is a fully human monoclonal antibody; (b) About 10 -10 EC below M 50 binds influenza B HA; (c) shows an increase in survival of influenza-infected animals; and / or (d) (i) three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO:2; and (ii) three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 10 Includes has one or more of the following:

[0010] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein provide increased protection from influenza B virus in animals (e.g., mammals) when administered either subcutaneously or intravenously and / or before or after infection with influenza B virus.

[0011] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein can reduce the severity of influenza B infection in animals (e.g., mammals) infected with influenza B virus when administered either subcutaneously or intravenously and / or before or after infection with influenza B virus.

[0012] In certain embodiments, the antibodies, or antigen-binding fragments thereof, of the present disclosure, when administered to an animal (e.g., a mammal) exposed to influenza B virus as a single intravenous dose of about 0.5 mg, about 1.0 mg / kg, about 5 mg / kg, about 10 mg / kg, or about 15 mg / kg of the mammal's body weight starting 1, 2, 3, 4, 5, 6, or 7 days prior to infection, provide increased protection from influenza B virus compared to oral administration of an antiviral agent such as, for example, baloxavir marboxil, oseltamivir, zanamivir, or pimodivir.

[0013] In some embodiments, the antibodies, or antigen-binding fragments thereof, provided herein, when administered in an animal (e.g., a mammal) as a single subcutaneous or intravenous dose of 0.5 mg / kg to about 15 mg / kg of body weight of the animal, e.g., about 1 mg / kg, about 5 mg / kg, about 7 mg / kg, about 10 mg / kg, or about 12 mg / kg of body weight of the mammal, provide increased protection from influenza B virus compared to an isotype (negative) control antibody administered to an equivalent animal (e.g., a mammal).

[0014] In some embodiments, the antibodies, or antigen-binding fragments thereof, provided herein, when administered to an animal (e.g., a mammal) as a single intravenous dose of about 0.5 mg, about 1.0 mg / kg, about 5 mg / kg, about 10 mg / kg, or about 15 mg / kg of body weight of the mammal, provide increased protection from influenza B compared to oral administration of an antiviral agent such as baloxavir, marboxil, oseltamivir, zanamivir, or pimodivir. For example, the antiviral agent can be oseltamivir administered at a dose of about 2 mg / kg twice daily for five days.

[0015] In some embodiments, the antibodies, or antigen-binding fragments thereof, provided herein, in a population of animals (e.g., mammals) exposed to and infected with influenza B virus, result in about 100% survival when administered as a single dose of about 0.5 mg / kg, about 1.0 mg / kg, about 5 mg / kg, about 10 mg / kg, or about 15 mg / kg of body weight 24 hours prior to influenza B exposure and infection.

[0016] In some embodiments, the antibodies provided herein, or antigen-binding fragments thereof, provide a 100% survival rate in animals (e.g., mammals) infected with influenza virus when administered as a single intravenous dose of about 0.5 mg / kg, about 1.0 mg / kg, about 5 mg / kg, about 10 mg / kg, or about 15 mg / kg 1, 2, 3, 4, 5, 6, or 7 days prior to influenza B exposure and / or infection.

[0017] In certain embodiments, the antibody, or antigen-binding fragment thereof, when administered to an influenza virus-infected mammal as a single intravenous dose of about 5 mg / kg to about 15 mg / kg beginning on day 1, 2, or 3 post-infection, exhibits increased protection compared to oral administration of an antiviral agent, such as, for example, baloxavir, marboxil, oseltamivir, zanamivir, or pimodivir. For example, the antiviral agent can be oseltamivir administered at a dose of about 2 mg / kg starting on day 1, 2, or 3 post-infection, twice daily for five days.

[0018] In related embodiments, the antibodies, or antigen-binding fragments thereof, provided herein provide increased protection in animals (e.g., mammals) infected with influenza virus when administered either subcutaneously or intravenously and / or when administered prior to or after influenza virus infection.

[0019] In some embodiments, the antibodies provided herein, or antigen-binding fragments thereof, when administered to an infected mammal as a single subcutaneous or intravenous dose of 0.5 mg / kg to about 15 mg / kg, e.g., about 1 mg / kg, about 5 mg / kg, about 7 mg / kg, or about 10 mg / kg, or about 12 mg / kg, show increased protection compared to animals administered an isotype (negative) control antibody.

[0020] In some embodiments, the antibodies, or antigen-binding fragments thereof, provided herein, when administered as a single intravenous dose of about 0.5 mg / kg, about 1.0 mg / kg, about 5 mg / kg, about 10 mg / kg, or about 15 mg / kg to a mammal infected with influenza virus, exhibit increased protection compared to oral administration of an antiviral agent such as baloxavir, marboxil, oseltamivir, zanamivir, or pimodivir. For example, the antiviral agent can be oseltamivir administered at a dose of about 2 mg / kg twice daily for five days.

[0021] In some embodiments, the antibodies, or antigen-binding fragments thereof, provided herein exhibit about a 100% survival rate in a population of animals (e.g., mammals) infected with influenza virus when administered as a single dose of about 0.5 mg, about 1.0 mg / kg, about 5 mg / kg, about 10 mg / kg, or about 15 mg / kg of body weight of the animals at 24 hours or more post-infection.

[0022] In some embodiments, the antibodies, or antigen-binding fragments thereof, provided herein exhibit a 100% survival rate in a population of animals (e.g., mammals) infected with influenza virus when administered as a single intravenous dose of about 5 mg / kg, or about 10 mg / kg, or about 15 mg / kg of body weight of the animals at 24, 48, 72, or 96 hours post-infection.

[0023] In some embodiments, the antibodies, or antigen-binding fragments thereof, provided herein, when administered as a single intravenous dose of about 0.5 mg, about 1.0 mg / kg, about 5 mg / kg, about 10 mg / kg, or about 15 mg / kg of body weight of the animal in a population of animals (e.g., mammals) infected with influenza virus, exhibit a survival rate of about 100% compared to the 80% survival rate observed in a comparable population of animals receiving an antiviral agent, such as, for example, baloxavir, marboxil, oseltamivir, zanamivir, or pimodivir. For example, the antiviral agent can be oseltamivir, administered orally at a dose of about 2 mg / kg twice daily for five days. As described herein, survival can be measured at a fixed time point after administration of an antibody, or antigen-binding fragment thereof, of the present disclosure (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, or more after administration).

[0024] In some embodiments, the antibodies, or antigen-binding fragments thereof, of the present disclosure provide additive protective effects in animals (e.g., mammals) infected with influenza virus when administered an antiviral agent such as baloxavir, marboxil, oseltamivir, zanamivir, or pimodivir. For example, the antiviral agent can be oseltamivir administered more than 48 hours after infection.

[0025] In some embodiments, the antibodies, or antigen-binding fragments thereof, of the present disclosure provide additive protective effects in animals (e.g., mammals) infected with influenza virus when administered an antiviral agent such as baloxavir, marboxil, oseltamivir, zanamivir, or pimodivir. For example, the antiviral agent can be oseltamivir administered 72 hours after infection.

[0026] In some embodiments, the antibodies, or antigen-binding fragments thereof, of the present disclosure, when used in combination with an antiviral agent, provide additive protection when the antibody is administered to an animal (e.g., a mammal) infected with influenza virus as a single intravenous dose in the range of about 7 mg to about 15 mg per kg of the animal's body weight, and an antiviral agent, such as, for example, baloxavir, marboxil, oseltamivir, zanamivir, or pimodivir, is administered orally at a dose of about 2 mg per kg of the animal's body weight twice daily for five days.

[0027] In some embodiments, the antibodies, or antigen-binding fragments thereof, of the disclosure provide additive protection when used in combination with an antiviral agent, such as baloxavir, marboxil, oseltamivir, zanamivir, or pimodivir, 96 hours after influenza virus infection, where the antibody is administered to the animal as a single intravenous dose in the range of about 7 mg to about 15 mg per kg of the animal's body weight, and the antiviral agent, such as oseltamivir, zanamivir, or pimodivir, is administered orally at a dose of about 2 mg per kg of the animal's body weight twice daily for five days.

[0028] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein may be administered intravenously, intranasally, subcutaneously, intradermally, or intramuscularly, and the antiviral agent may be administered orally or intravenously.

[0029] In some embodiments, an anti-viral agent is administered prior to, simultaneously with, or following administration of an antibody provided herein.

[0030] In some embodiments, the antibody, or antigen-binding fragment thereof, and / or antiviral agent, such as baloxavir, marboxil, oseltamivir, zanamivir, or pimodivir, may be administered as a single dose or as multiple doses.

[0031] Exemplary anti-influenza B HA antibodies are provided herein in Tables 1 and 2. Table 1 lists the amino acid sequence identifiers of the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity determining region (HCDR1, HCDR2, and HCDR3), light chain complementarity determining region (LCDR1, LCDR2, and LCDR3), heavy chain (HC), and light chain (LC) of exemplary anti-influenza B HA antibodies. Table 2 lists the nucleic acid sequence identifiers of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HC, and LC of exemplary anti-influenza B HA antibodies.

[0032] Provided herein are antibodies, or antigen-binding fragments thereof, comprising HCVRs comprising an amino acid sequence set forth in Table 1, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity thereto.

[0033] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds influenza B HA comprises the HCVR amino acid sequence of SEQ ID NO:2.

[0034] Provided herein is an antibody, or antigen-binding fragment thereof, comprising an LCVR comprising an amino acid sequence set forth in Table 1, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity thereto.

[0035] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds influenza B HA comprises the LCVR amino acid sequence of SEQ ID NO:10.

[0036] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds influenza B HA comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs:2 / 10.

[0037] In some embodiments, the isolated antibody or antigen-binding fragment comprises: (a) an HCDR1 domain having the amino acid sequence of SEQ ID NO:4; (b) an HCDR2 domain having the amino acid sequence of SEQ ID NO:6; (c) an HCDR3 domain having the amino acid sequence of SEQ ID NO:8; (d) an LCDR1 domain having the amino acid sequence of SEQ ID NO: 12; (e) an LCDR2 domain having the amino acid sequence of SEQ ID NO: 14, and (f) an LCDR3 domain having the amino acid sequence of SEQ ID NO: 16.

[0038] In some embodiments, the isolated antibody or antigen-binding fragment that specifically binds to influenza B HA comprises (a) an HCDR1 of SEQ ID NO: 4, (b) an HCDR2 of SEQ ID NO: 6, (c) an HCDR3 of SEQ ID NO: 8, (d) an LCDR1 of SEQ ID NO: 12, (e) an LCDR2 of SEQ ID NO: 14, and (f) an LCDR3 of SEQ ID NO: 16.

[0039] In some embodiments, the isolated antibody or antigen-binding fragment that specifically binds to influenza B HA comprises (a) an HCDR3 of SEQ ID NO:8 and (b) an LCDR3 of SEQ ID NO:16.

[0040] The present disclosure provides an antibody, or an antigen-binding fragment thereof, comprising a heavy chain CDR1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:4, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity thereto.

[0041] The present disclosure provides an antibody, or an antigen-binding fragment thereof, comprising a heavy chain CDR2 (HCDR2) comprising the amino acid sequence of SEQ ID NO:6, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity thereto.

[0042] The present disclosure provides an antibody, or an antigen-binding fragment thereof, comprising a heavy chain CDR3 (HCDR3) comprising the amino acid sequence of SEQ ID NO:8, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity thereto.

[0043] The present disclosure provides an antibody, or an antigen-binding fragment thereof, comprising a heavy chain CDR1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 12, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity thereto.

[0044] The present disclosure provides an antibody, or an antigen-binding fragment thereof, comprising a heavy chain CDR2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 14, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity thereto.

[0045] The present disclosure provides an antibody, or an antigen-binding fragment thereof, comprising a heavy chain CDR3 (LCDR3) comprising the amino acid sequence of SEQ ID NO: 16, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity thereto.

[0046] The present disclosure provides an antibody, or an antigen-binding fragment thereof, comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) comprising the amino acid sequence of SEQ ID NO: 8 / 16, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity thereto.

[0047] The present invention also provides antibodies, or antigen-binding fragments thereof, that comprise the set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within the HCVR / LCVR amino acid sequences of the exemplary anti-influenza B HA antibodies shown in Table 1. In certain embodiments, the set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences comprises SEQ ID NOs: 4-6-8-12-14-16.

[0048] Methods and techniques for identifying CDRs in HCVR and LCVR amino acid sequences are known in the art and can be used to identify CDRs in a particular HCVR and / or LCVR amino acid sequence disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available to identify CDR sequences within antibodies.

[0049] Further provided herein are antibodies and antigen-binding fragments thereof that comprise the CDRs of an HCVR and the CDRs of an LCVR and that compete with an antibody or antigen-binding fragment thereof for specific binding to influenza B HA, wherein the HCVR and LCVR have amino acid sequences according to the HCVR and LCVR sequences, respectively, listed in Table 1.

[0050] The present invention also provides antibodies and antigen-binding fragments that comprise the CDRs of an HCVR and the CDRs of an LCVR and that cross-compete for binding to influenza B HA or bind to the same epitope on influenza B HA as a reference antibody or antigen-binding fragment thereof, wherein the HCVR and LCVR have amino acid sequences according to the HCVR and LCVR sequences listed in Table 1, respectively.

[0051] The present invention also provides isolated antibodies and antigen-binding fragments thereof that block influenza B HA attachment and / or entry into a host cell.

[0052] In certain embodiments, the antibody or antigen-binding fragment is bispecific, comprising a first binding specificity for a first epitope in influenza B HA and a second binding specificity for another antigen.

[0053] In a second aspect, provided herein are nucleic acid molecules (e.g., DNA or RNA molecules) encoding anti-influenza B HA antibodies or portions thereof. For example, nucleic acid molecules encoding HCVR amino acid sequences are listed in Table 2, and in certain embodiments, the nucleic acid molecule comprises the HCVR polynucleotide sequence of SEQ ID NO: 1, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity thereto. Nucleic acid molecules encoding LCVR amino acid sequences are also listed in Table 2, and in certain embodiments the nucleic acid molecule comprises the LCVR polynucleotide sequence of SEQ ID NO:9, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity thereto.

[0054] Nucleic acid molecules encoding the HCDR1 amino acid sequences listed in Table 1 are also provided herein, and in certain embodiments the nucleic acid molecule comprises an HCDR1 nucleic acid sequence listed in Table 2, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity thereto.

[0055] Also provided herein are nucleic acid molecules encoding the HCDR2 amino acid sequences listed in Table 1, and in certain embodiments the nucleic acid molecule comprises an HCDR2 nucleic acid sequence listed in Table 2, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity thereto.

[0056] Also provided herein are nucleic acid molecules encoding the HCDR3 amino acid sequences listed in Table 1, and in certain embodiments the nucleic acid molecules comprise an HCDR3 nucleic acid sequence listed in Table 2, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity thereto.

[0057] Nucleic acid molecules encoding the LCDR1 amino acid sequences listed in Table 1 are also provided herein, and in certain embodiments, the nucleic acid molecules comprise an LCDR1 nucleic acid sequence listed in Table 2, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity thereto.

[0058] Nucleic acid molecules encoding the LCDR2 amino acid sequences listed in Table 1 are also provided herein, and in certain embodiments, the nucleic acid molecules comprise an LCDR2 nucleic acid sequence listed in Table 2, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity thereto.

[0059] Nucleic acid molecules encoding the LCDR3 amino acid sequences listed in Table 1 are also provided herein, and in certain embodiments, the nucleic acid molecules comprise an LCDR3 nucleic acid sequence listed in Table 2, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity thereto.

[0060] Also provided herein are nucleic acid molecules encoding HCVRs, where the HCVRs comprise a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), and the set of amino acid sequences of HCDR1-HCDR2-HCDR3 are as defined by the exemplary anti-influenza HA antibodies listed in Table 1.

[0061] Also provided herein are nucleic acid molecules encoding LCVRs, wherein the LCVRs comprise a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), wherein the set of amino acid sequences of LCDR1-LCDR2-LCDR3 are as defined by the exemplary anti-influenza HA antibodies listed in Table 1.

[0062] Also provided herein are nucleic acid molecules encoding both an HCVR and an LCVR, where the HCVR comprises the amino acid sequence of SEQ ID NO: 2 and the LCVR comprises the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the nucleic acid molecule comprises an HCVR or LCVR polynucleotide sequence listed in Table 2, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity thereto. In certain embodiments, the nucleic acid molecule encodes an HCVR and an LCVR, where the HCVR and LCVR are both derived from the same anti-influenza B HA antibody listed in Table 1.

[0063] Nucleic acid molecules encoding any of the heavy chain amino acid sequences and / or any of the light chain amino acid sequences listed in Table 1 are also provided.

[0064] In a related aspect, provided herein is a recombinant expression vector capable of expressing a polypeptide comprising a heavy or light chain variable region of an anti-influenza B HA antibody. For example, the vector can comprise a recombinant expression vector comprising any of the nucleic acid molecules described above, i.e., a nucleic acid molecule encoding any of the HCVR, LCVR, and / or CDR sequences as described in Table 1. Host cells into which such vectors have been introduced, as well as methods of producing antibodies or portions thereof by culturing the host cells under conditions that permit the production of the antibodies or antibody fragments, and methods of recovering the antibodies and antibody fragments so produced, are also included within the scope of the disclosure.

[0065] In a third aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one recombinant monoclonal antibody or antigen-binding fragment thereof that specifically binds to influenza B HA and a pharma- ceutically acceptable carrier. In a related aspect, the composition is a combination of an anti-influenza B HA antibody and a second therapeutic agent. In some embodiments, the second therapeutic agent is any agent that is advantageously combined with an anti-influenza B HA antibody. Exemplary agents that can be advantageously combined with an anti-influenza B HA antibody include, but are not limited to, other agents that bind to and / or inhibit influenza HA activity (including other antibodies or antigen-binding fragments thereof, such as antibodies that bind to and / or inhibit influenza A HA, etc.), and / or agents that do not directly bind to influenza HA but nevertheless inhibit viral activity, including infectivity of host cells. In certain embodiments, the pharmaceutical composition comprises (a) a first anti-influenza B HA antibody or antigen-binding fragment thereof, (b) a second anti-influenza B HA antibody or antigen-binding fragment thereof, where the first antibody binds to a first epitope of influenza B HA and the second antibody binds to a second epitope of influenza B HA, and the first and second epitopes are distinct and non-overlapping, and (c) a pharmaceutically acceptable carrier or diluent. In certain embodiments, the pharmaceutical composition comprises (a) a first anti-influenza B HA antibody or antigen-binding fragment thereof, (b) a second anti-influenza B HA antibody or antigen-binding fragment thereof, where the first antibody does not cross-compete with the second antibody for binding to the influenza B HA antibody, and (c) a pharmaceutically acceptable carrier or diluent. In certain embodiments, the pharmaceutical composition comprises (a) a first anti-influenza B HA antibody or antigen-binding fragment thereof; (b) a second anti-influenza antibody or antigen-binding fragment thereof that interacts with a different influenza antigen, where the first antibody binds to an epitope of influenza B HA and the second antibody binds to an epitope of the different influenza antigen; and (c) a pharmaceutically acceptable carrier or diluent.In certain embodiments, the pharmaceutical composition comprises (a) a first anti-influenza B HA antibody or antigen-binding fragment thereof, (b) a second antibody or antigen-binding fragment thereof that interacts with a different viral (non-influenza) antigen, where the first antibody binds to an epitope of the influenza B HA antibody and the second antibody binds to an epitope of a different viral (non-influenza) antigen, and (c) a pharma- ceutically acceptable carrier or diluent. Additional combination therapies and co-formulations involving the anti-influenza B HA antibodies provided herein are disclosed elsewhere herein. In certain embodiments, the pharmaceutical composition comprises (a) an anti-influenza B HA antibody or antigen-binding fragment thereof, and (b) an anti-influenza A HA antibody or antigen-binding fragment thereof.

[0066] In a fourth aspect, provided herein is a therapeutic method for treating a disease or disorder associated with influenza B HA (such as a viral infection in a subject) or at least one symptom associated with a viral infection using an anti-influenza B HA antibody or antigen-binding portion of the antibody provided herein, comprising administering to a subject in need of treatment a therapeutically effective amount of a pharmaceutical composition comprising the antibody or antigen-binding fragment of the antibody. The disorder to be treated is any disease or condition that is ameliorated, ameliorated, suppressed, or prevented by inhibition of influenza HA activity. In certain embodiments, the present disclosure provides a method for preventing, treating, or ameliorating at least one symptom of influenza B infection, comprising administering to a subject in need of treatment a therapeutically effective amount of an anti-influenza HA antibody or antigen-binding fragment thereof.

[0067] In some embodiments, the disclosure provides methods of ameliorating, alleviating or reducing the severity, duration or frequency of occurrence of at least one symptom of influenza infection in a subject by administering an anti-influenza B HA antibody provided herein, wherein the at least one symptom is selected from the group consisting of headache, fever, aches, rhinorrhea (stuffy nose), chills, fatigue, weakness, sore throat, cough, shortness of breath, vomiting, diarrhea, pneumonia, bronchitis, and death.

[0068] In certain embodiments, provided herein is a method of reducing viral load in a subject, comprising administering to the subject an effective amount of an antibody or fragment thereof that binds to influenza B HA and blocks influenza virus binding and / or entry into host cells.

[0069] In certain embodiments, the antibody or antigen-binding fragment thereof may be administered prophylactically or therapeutically to subjects who have, are at risk of, or are susceptible to influenza infection. Subjects at risk include, but are not limited to, immunocompromised individuals, such as those immunocompromised due to autoimmune disease, or those undergoing immunosuppressive therapy (e.g., after organ transplantation), or those suffering from human immunodeficiency syndrome (HIV) or acquired immune deficiency syndrome (AIDS), certain forms of anemia that deplete or destroy white blood cells, those undergoing radiation therapy or chemotherapy, or those suffering from inflammatory disorders. Other subjects at risk of influenza infection include the elderly (over 65 years of age), children under the age of 2, health care workers, and those with underlying diseases such as lung infections, heart disease, or diabetes. Additionally, anyone who has physical contact or close physical proximity to an infected individual is at increased risk of developing influenza virus infection. Additionally, subjects are at risk of contracting influenza infection because of their proximity to disease outbreaks, e.g., living in a densely populated city or in close proximity to subjects with confirmed or suspected influenza virus infection, or because of their occupational choices, e.g., hospital workers, pharmaceutical researchers, travelers to endemic areas, or frequent flyers.

[0070] In certain embodiments, the antibody or antigen-binding fragment thereof is administered to a subject in need thereof in combination with a second therapeutic agent. The second therapeutic agent may be selected from the group consisting of anti-inflammatory drugs (such as corticosteroids and nonsteroidal anti-inflammatory drugs), anti-infective drugs, different antibodies against influenza HA (antibodies against influenza A HA), antibodies against different influenza antigens (e.g., neuraminidase), antiviral drugs, decongestants, antihistamines, influenza vaccines, nutritional supplements such as antioxidants, and any other drug or therapy known in the art useful for improving at least one symptom of influenza infection or reducing the viral load of a patient. In certain embodiments, the second therapeutic agent may be an agent that helps address or alleviate any possible side effects associated with the antibody or antigen-binding fragment thereof provided herein, if such side effects occur. The antibody or fragment thereof may be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intranasally, intramuscularly, or intracranially. In some embodiments, the antibody may be administered as a single intravenous infusion for maximum concentration of the antibody in the subject's serum. The antibody or fragment thereof may be administered at a dose of about 0.01 mg / kg of the subject's body weight to about 100 mg / kg of the subject's body weight. In certain embodiments, the antibodies disclosed herein may be administered in one or more doses, including 50 mg to 5000 mg.

[0071] In a fifth aspect, there is provided herein an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to influenza B hemagglutinin (HA), wherein the antibody has the following characteristics: (a) About 10 -9 EC below M 50 binds influenza B HA; (b) after administration to influenza B infected animals, exhibits an increase in survival of the influenza B infected animals compared to comparable influenza B infected animals without said administration; and / or (c) (i) three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (HCVR) comprising amino acid sequences having at least about 90% sequence identity to the HCVR set forth in SEQ ID NO:2; and (ii) three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light chain variable region (LCVR) comprising an amino acid sequence having at least about 90% sequence identity to the LCVR set forth in SEQ ID NO: 10; Includes has one or more of the following:

[0072] In some embodiments, when administered prophylactically to a mammal as a single intravenous dose of about 5 mg / kg or about 0.5 mg / kg of the mammal's body weight, the isolated antibody or antigen-binding fragment thereof protects the mammal from infection by subsequent exposure to influenza B virus. In some embodiments, when administered prophylactically to a mammal prior to exposure to influenza B virus, the isolated antibody or antigen-binding fragment thereof reduces the risk of influenza infection in the mammal. In some embodiments, when administered to a mammal, the isolated antibody or antigen-binding fragment thereof ameliorates, alleviates, or reduces the severity, duration, or frequency of occurrence of at least one symptom of influenza infection in the mammal. In some embodiments, the at least one symptom is selected from the group consisting of headache, fever, pain, rhinorrhea (nasal congestion), chills, fatigue, weakness, sore throat, cough, shortness of breath, vomiting, diarrhea, pneumonia, bronchitis, and death.

[0073] In some embodiments, when the isolated antibody or antigen-binding fragment thereof is administered to each of a plurality of mammals as a single prophylactic dose of about 0.5 mg / kg, the plurality of mammals has a survival rate of at least about 80% at day 19 after administration. In some embodiments, when the isolated antibody or antigen-binding fragment thereof is administered to each of a plurality of mammals as a single prophylactic dose of about 0.5 mg / kg, the plurality of mammals has a survival rate of at least about 90% at day 19 after administration. In some embodiments, when the isolated antibody or antigen-binding fragment thereof is administered to each of a plurality of mammals as a single prophylactic dose of about 0.5 mg / kg, the plurality of mammals has a survival rate of about 100% at day 19 after administration. In some embodiments, when the isolated antibody or antigen-binding fragment thereof is administered to each of a plurality of mammals as a single prophylactic dose of about 5.0 mg / kg, the plurality of mammals has a survival rate of about 80% at day 19 after administration. In some embodiments, when the isolated antibody or antigen-binding fragment thereof is administered to each of a plurality of mammals as a single prophylactic dose of about 5.0 mg / kg, the plurality of mammals has a survival rate of about 90% at day 19 after administration. In some embodiments, when the isolated antibody or antigen-binding fragment thereof is administered to each of a plurality of mammals as a single prophylactic dose of about 5.0 mg / kg, the plurality of mammals has a survival rate of about 100% at day 19 after administration. In some embodiments, survival is evident at day 13 after administration.

[0074] In some embodiments, the disclosure provides an isolated antibody or antigen-binding fragment thereof as described above, comprising a HCVR having the amino acid sequence of SEQ ID NO: 2. In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a LCVR having the amino acid sequence of SEQ ID NO: 10.

[0075] In a sixth aspect, provided herein is an isolated antibody or antigen-binding fragment thereof, comprising: (a) an HCDR1 domain having the amino acid sequence of SEQ ID NO: 4; (b) an HCDR2 domain having the amino acid sequence of SEQ ID NO: 6; (c) an HCDR3 domain having the amino acid sequence of SEQ ID NO: 8; (d) an LCDR1 domain having the amino acid sequence of SEQ ID NO: 12; (e) an LCDR2 domain having the amino acid sequence of SEQ ID NO: 14; and (f) an LCDR3 domain having the amino acid sequence of SEQ ID NO: 16.

[0076] In some embodiments, the isolated antibody or antigen-binding fragment thereof specifically binds to influenza B hemagglutinin (HA). In some embodiments, the isolated antibody or antigen-binding fragment thereof, when administered prophylactically to a mammal as a single intravenous dose of about 5 mg / kg body weight of the mammal or about 0.5 mg / kg, protects the mammal from infection by subsequent exposure to influenza B virus. In some embodiments, when the isolated antibody or antigen-binding fragment thereof is administered prophylactically to a mammal prior to exposure to influenza B virus, the risk of influenza infection in the mammal is reduced. In some embodiments, when the isolated antibody or antigen-binding fragment thereof is administered to a mammal, the isolated antibody or antigen-binding fragment thereof ameliorates, alleviates, or reduces the severity, duration, or frequency of occurrence of at least one symptom of influenza infection in the mammal. In some embodiments, the at least one symptom is selected from the group consisting of headache, fever, pain, rhinorrhea (nasal congestion), chills, fatigue, weakness, sore throat, cough, shortness of breath, vomiting, diarrhea, pneumonia, bronchitis, and death.

[0077] In some embodiments, when the isolated antibody or antigen-binding fragment thereof is administered to each of a plurality of mammals as a single prophylactic dose of about 0.5 mg / kg, the plurality of mammals has a survival rate of at least about 80% at day 19 after administration. In some embodiments, when the isolated antibody or antigen-binding fragment thereof is administered to each of a plurality of mammals as a single prophylactic dose of about 0.5 mg / kg, the plurality of mammals has a survival rate of at least about 90% at day 19 after administration. In some embodiments, when the isolated antibody or antigen-binding fragment thereof is administered to each of a plurality of mammals as a single prophylactic dose of about 0.5 mg / kg, the plurality of mammals has a survival rate of about 100% at day 19 after administration. In some embodiments, when the isolated antibody or antigen-binding fragment thereof is administered to each of a plurality of mammals as a single prophylactic dose of about 5.0 mg / kg, the plurality of mammals has a survival rate of about 80% at day 19 after administration. In some embodiments, when the isolated antibody or antigen-binding fragment thereof is administered to each of a plurality of mammals as a single prophylactic dose of about 5.0 mg / kg, the plurality of mammals has a survival rate of about 90% at day 19 after administration. In some embodiments, when the isolated antibody or antigen-binding fragment thereof is administered to each of a plurality of mammals as a single prophylactic dose of about 5.0 mg / kg, the plurality of mammals has a survival rate of about 100% at day 19 after administration. In some embodiments, survival is evident at day 13 after administration.

[0078] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises an HCVR having the amino acid sequence of SEQ ID NO:2. In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises an LCVR having the amino acid sequence of SEQ ID NO:10. In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO:2 / 10. In some embodiments, the isolated antibody or antigen-binding fragment thereof is an IgG1 antibody. In some embodiments, the isolated antibody or antigen-binding fragment thereof is an IgG4 antibody. In some embodiments, the isolated antibody or antigen-binding fragment thereof is a bispecific antibody.

[0079] In a seventh aspect, provided herein is a pharmaceutical composition comprising the above-described isolated antibody or antigen-binding fragment thereof and a pharma- ceutically acceptable carrier or diluent.

[0080] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of an antiviral drug, an anti-inflammatory drug, a different antibody that specifically binds to influenza HA, a vaccine for influenza, a nutritional supplement, and another palliative therapy for treating influenza infection. In some embodiments, the anti-inflammatory drug is selected from the group consisting of a corticosteroid and a nonsteroidal anti-inflammatory drug. In some embodiments, the nutritional supplement is an antioxidant. In some embodiments, the antiviral drug is oseltamivir. In some embodiments, the antiviral drug is an anti-influenza A drug. In some embodiments, the anti-influenza A drug is an antibody. In some embodiments, the antibody specifically binds to influenza A HA.

[0081] In some embodiments, the disclosure provides a polynucleotide molecule comprising a polynucleotide sequence encoding the HCVR or LCVR of the above-mentioned antibody. In some embodiments, the disclosure provides a vector comprising the polynucleotide. In some embodiments, the disclosure provides a cell comprising the vector.

[0082] In an eighth aspect, there is provided herein a method of preventing, treating, or ameliorating at least one symptom of influenza infection in a subject infected with influenza, the method comprising administering to the subject an antibody or antigen-binding fragment as described above, or a pharmaceutical composition as described above.

[0083] In some embodiments, the at least one symptom is selected from the group consisting of fever, cough, body aches, nasal discharge, shortness of breath, pneumonia, and bronchitis. In some embodiments, the pharmaceutical composition is administered prophylactically to the subject. In some embodiments, the subject is selected from the group consisting of immunocompromised individuals, adults over 65 years of age, health care workers, and people with a medical history or underlying condition. In some embodiments, the underlying condition is selected from the group consisting of heart problems and diabetes. In some embodiments, the pharmaceutical composition is administered in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of antiviral drugs, anti-inflammatory drugs, different antibodies that specifically bind to influenza HA, influenza vaccines, nutritional supplements, and any other palliative therapy for treating influenza infection. In some embodiments, the anti-inflammatory drug is selected from the group consisting of corticosteroids and nonsteroidal anti-inflammatory drugs. In some embodiments, the nutritional supplement is an antioxidant. In some embodiments, the second therapeutic agent is administered via a different route of administration, such as the antibody or antigen-binding fragment thereof. In some embodiments, the second therapeutic agent is administered orally. In some embodiments, the antiviral agent is oseltamivir. In some embodiments, oseltamivir is administered prior to administration of the antibody or antigen-binding fragment thereof. In some embodiments, oseltamivir is administered simultaneously with the antibody or antigen-binding fragment thereof. In some embodiments, oseltamivir is administered after administration of the antibody or antigen-binding fragment thereof. In some embodiments, the antiviral agent is an anti-influenza A agent. In some embodiments, the anti-influenza A agent is an antibody. In some embodiments, the antibody specifically binds to influenza A HA. In some embodiments, the pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intramuscularly, intranasally, or orally.

[0084] The disclosure also includes the use of an anti-influenza B HA antibody, or antigen-binding fragment thereof, to treat a disease or disorder that would benefit from blocking influenza HA binding and / or activity.The disclosure also includes the use of an anti-influenza B HA antibody, or antigen-binding fragment thereof, in the manufacture of a medicament for the treatment of a disease or disorder that would benefit from blocking influenza HA binding and / or activity.

[0085] [The present invention 1001] 1. An isolated, recombinant antibody or antigen-binding fragment thereof that specifically binds to influenza B hemagglutinin (HA), comprising: The antibody has the following characteristics: (a) About 10 -9 EC below M 50 binds influenza B HA; (b) after administration to an influenza B infected animal, exhibits an increase in survival of said influenza B infected animal compared to a comparable influenza B infected animal without said administration; and / or (c) (i) three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (HCVR) comprising amino acid sequences having at least about 90% sequence identity to the HCVR set forth in SEQ ID NO:2; and (ii) three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light chain variable region (LCVR) comprising an amino acid sequence having at least about 90% sequence identity to the LCVR set forth in SEQ ID NO: 10; Includes An isolated recombinant antibody or antigen-binding fragment thereof, comprising one or more of the following: [The present invention 1002] An isolated antibody or antigen-binding fragment thereof of the present invention, which, when administered prophylactically to a mammal as a single intravenous dose of about 5 mg / kg or about 0.5 mg / kg of the mammal's body weight, protects the mammal from infection upon subsequent exposure to influenza B virus. [The present invention 1003] 10. The isolated antibody or antigen-binding fragment thereof of claim 1001 or 1002, which, when administered prophylactically to a mammal prior to exposure to influenza B virus, reduces the risk of influenza infection in said mammal. [The present invention 1004] The isolated antibody or antigen-binding fragment thereof of any of claims 1001 to 1003, wherein when the isolated antibody or antigen-binding fragment thereof is administered to a mammal, the isolated antibody or antigen-binding fragment thereof ameliorates, alleviates, or reduces the severity, duration, or frequency of occurrence of at least one symptom of influenza infection in the mammal. [The present invention 1005] The isolated antibody or antigen-binding fragment thereof of the present invention 1004, wherein the at least one symptom is selected from the group consisting of headache, fever, pain, rhinorrhea (nasal congestion), chills, fatigue, weakness, sore throat, cough, shortness of breath, vomiting, diarrhea, pneumonia, bronchitis, and death. [The present invention 1006] Any of the isolated antibodies or antigen-binding fragments of the present invention 1001 to 1005, which, when administered to each of a plurality of mammals as a single prophylactic dose of about 0.5 mg / kg, has a survival rate of at least about 80% on the 19th day after administration to the plurality of mammals. [The present invention 1007] An isolated antibody or antigen-binding fragment thereof according to any one of claims 1001 to 1006 of the present invention, which, when administered to each of a plurality of mammals as a single prophylactic dose of about 0.5 mg / kg, has a survival rate of at least about 90% on the 19th day after administration to the plurality of mammals. [The present invention 1008] Any of the isolated antibodies or antigen-binding fragments of the present invention 1001 to 1007, which, when administered to each of a plurality of mammals as a single prophylactic dose of about 0.5 mg / kg, has a survival rate of about 100% in the plurality of mammals on the 19th day after administration. [The present invention 1009] Any of the isolated antibodies or antigen-binding fragments of the present invention 1001 to 1005, which, when administered to each of multiple mammals as a single prophylactic dose of about 5.0 mg / kg, has a survival rate of about 80% in the multiple mammals on the 19th day after administration. [The present invention 1010] An isolated antibody or antigen-binding fragment thereof of any of 1001 to 1005 and 1009 of the present invention, which, when administered to each of a plurality of mammals as a single prophylactic dose of about 5.0 mg / kg, the plurality of mammals have a survival rate of about 90% on the 19th day after administration. [The present invention 1011] An isolated antibody or antigen-binding fragment thereof according to any one of claims 1001 to 1005, 1009 and 1010 of the present invention, which, when administered to each of a plurality of mammals as a single prophylactic dose of about 5.0 mg / kg, has a survival rate of about 100% in the plurality of mammals on day 19 after administration. [The present invention 1012] The isolated antibody or antigen-binding fragment thereof of any one of claims 1006 to 1011 of the present invention, wherein the survival rate is evident 13 days after administration. [The present invention 1013] The isolated antibody or antigen-binding fragment thereof according to any one of claims 1001 to 1012, comprising an HCVR having the amino acid sequence of SEQ ID NO:2. [The present invention 1014] An isolated antibody or an antigen-binding fragment thereof according to any one of claims 1001 to 1013 of the present invention, comprising an LCVR having the amino acid sequence of SEQ ID NO:10. [The present invention 1015] (a) an HCDR1 domain having the amino acid sequence of SEQ ID NO:4; (b) an HCDR2 domain having the amino acid sequence of SEQ ID NO:6; (c) an HCDR3 domain having the amino acid sequence of SEQ ID NO:8; (d) an LCDR1 domain having the amino acid sequence of SEQ ID NO: 12; (e) an LCDR2 domain having the amino acid sequence of SEQ ID NO: 14; (f) an LCDR3 domain having the amino acid sequence of SEQ ID NO: 16; 1. An isolated antibody or antigen-binding fragment thereof comprising: [The present invention 1016] 10. An isolated antibody or antigen-binding fragment thereof of the present invention, which specifically binds to influenza B hemagglutinin (HA). [The present invention 1017] An isolated antibody or antigen-binding fragment thereof of the present invention 1015 or 1016, which when administered prophylactically to a mammal as a single intravenous dose of about 5 mg / kg or about 0.5 mg / kg of the mammal's body weight protects the mammal from infection upon subsequent exposure to influenza B virus. [The present invention 1018] An isolated antibody or antigen-binding fragment thereof of any of claims 1015 to 1017, which, when administered prophylactically to a mammal prior to exposure to influenza B virus, reduces the risk of influenza infection in said mammal. [The present invention 1019] The isolated antibody or antigen-binding fragment thereof of any of claims 1015 to 1018, wherein when the isolated antibody or antigen-binding fragment thereof is administered to a mammal, the isolated antibody or antigen-binding fragment thereof ameliorates, alleviates, or reduces the severity, duration, or frequency of occurrence of at least one symptom of influenza infection in the mammal. [The present invention 1020] The isolated antibody or antigen-binding fragment thereof of the present invention, wherein the at least one symptom is selected from the group consisting of headache, fever, pain, rhinorrhea (nasal congestion), chills, fatigue, weakness, sore throat, cough, shortness of breath, vomiting, diarrhea, pneumonia, bronchitis, and death. [The present invention 1021] An isolated antibody or antigen-binding fragment thereof of any of claims 1014 to 1020 of the present invention, which, when administered to each of a plurality of mammals as a single prophylactic dose of about 0.5 mg / kg, said plurality of mammals have a survival rate of at least about 80% on day 19 after administration. [The present invention 1022] Any of the isolated antibodies or antigen-binding fragments of the present invention 1014 to 1021, which, when administered to each of a plurality of mammals as a single prophylactic dose of about 0.5 mg / kg, has a survival rate of at least about 90% on day 19 after administration to the plurality of mammals. [The present invention 1023] An isolated antibody or antigen-binding fragment thereof according to any one of claims 1014 to 1022 of the present invention, which, when administered to each of a plurality of mammals as a single prophylactic dose of about 0.5 mg / kg, has a survival rate of about 100% in the plurality of mammals on day 19 after administration. [The present invention 1024] An isolated antibody or antigen-binding fragment thereof of any of 1014 to 1020 of the present invention, which, when administered to each of a plurality of mammals as a single prophylactic dose of about 5.0 mg / kg, has a survival rate of about 80% in the plurality of mammals on day 19 after administration. [The present invention 1025] An isolated antibody or antigen-binding fragment thereof of any of 1014 to 1020 and 1024 of the present invention, which, when administered to each of a plurality of mammals as a single prophylactic dose of about 5.0 mg / kg, the plurality of mammals have a survival rate of about 90% on the 19th day after administration. [The present invention 1026] An isolated antibody or antigen-binding fragment thereof of any of 1014 to 1020, 1024 and 1025 of the present invention, which, when administered to each of a plurality of mammals as a single prophylactic dose of about 5.0 mg / kg, the plurality of mammals have a survival rate of about 100% on the 19th day after administration. [The present invention 1027] The isolated antibody or antigen-binding fragment thereof of any of claims 1021 to 1026 of the present invention, wherein the survival rate is evident 13 days after administration. [The present invention 1028] An isolated antibody or an antigen-binding fragment thereof according to any one of claims 1014 to 1027 of the present invention, comprising an HCVR having the amino acid sequence of SEQ ID NO:2. [The present invention 1029] An isolated antibody or an antigen-binding fragment thereof according to any one of claims 1014 to 1028 of the present invention, comprising an LCVR having the amino acid sequence of SEQ ID NO:10. [The present invention 1030] An isolated antibody or an antigen-binding fragment thereof according to any one of claims 1001 to 1029 of the present invention, comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 2 / 10. [The present invention 1031] The isolated antibody or antigen-binding fragment thereof according to any one of claims 1001 to 1030, which is an IgG1 antibody. [The present invention 1032] The isolated antibody or antigen-binding fragment thereof according to any one of claims 1001 to 1030, which is an IgG4 antibody. [The present invention 1033] The isolated antibody or antigen-binding fragment thereof according to any one of claims 1001 to 1030, which is a bispecific antibody. [The present invention 1034] A pharmaceutical composition comprising any one of the isolated antibodies or antigen-binding fragments thereof according to the present invention 1001 to 1033 and a pharma- ceutically acceptable carrier or diluent. [The present invention 1035] The pharmaceutical composition of the present invention 1034 further comprising a second therapeutic agent. [The present invention 1036] The pharmaceutical composition of the present invention 1035, wherein the second therapeutic agent is selected from the group consisting of an antiviral drug, an anti-inflammatory drug, a different antibody that specifically binds to influenza HA, a vaccine for influenza, a dietary supplement, and another palliative therapy for treating influenza infection. [The present invention 1037] The pharmaceutical composition of claim 1036, wherein said anti-inflammatory agent is selected from the group consisting of corticosteroids and nonsteroidal anti-inflammatory agents. [The present invention 1038] The pharmaceutical composition of claim 1036, wherein the dietary supplement is an antioxidant. [The present invention 1039] The pharmaceutical composition of the present invention, wherein the antiviral drug is oseltamivir. [The present invention 1040] The pharmaceutical composition of the present invention, wherein said antiviral drug is an anti-influenza A drug. [The present invention 1041] The pharmaceutical composition of the present invention, wherein said anti-influenza A drug is an antibody. [The present invention 1042] The pharmaceutical composition of the present invention 1041, wherein said antibody specifically binds to influenza A HA. [The present invention 1043] A polynucleotide molecule comprising a polynucleotide sequence encoding the HCVR or LCVR of any one of the antibodies of the present invention 1001 to 1033. [The present invention 1044] A vector comprising the polynucleotide of the present invention. [The present invention 1045] A cell comprising the vector of the present invention. [The present invention 1046] 1. A method of preventing, treating, or ameliorating at least one symptom of influenza infection in an influenza-infected subject, comprising: Administering to said subject an antibody or antigen-binding fragment of any one of claims 1001 to 1033 of the present invention, or a pharmaceutical composition of any one of claims 1034 to 1042 of the present invention. A method comprising: [The present invention 1047] The method of claim 1046, wherein said at least one symptom is selected from the group consisting of fever, cough, body aches, nasal discharge, shortness of breath, pneumonia and bronchitis. [The present invention 1048] The method of any one of claims 1046 to 1047, wherein the pharmaceutical composition is administered prophylactically to the subject. [The present invention 1049] The method of any of claims 1046 to 1048, wherein said subject is selected from the group consisting of immunocompromised individuals, adults 65 years of age or older, healthcare workers, and those with a medical history or underlying condition. [The present invention 1050] The method of claim 1049, wherein said underlying disease is selected from the group consisting of heart problems and diabetes. [The present invention 1051] The method of any of claims 1046 to 1050, wherein said pharmaceutical composition is administered in combination with a second therapeutic agent. [The present invention 1052] The method of claim 1051, wherein the second therapeutic agent is selected from the group consisting of an antiviral drug, an anti-inflammatory drug, a different antibody that specifically binds to influenza HA, a vaccine for influenza, a nutritional supplement, and another palliative therapy for treating influenza infection. [The present invention 1053] The method of claim 1052, wherein said anti-inflammatory agent is selected from the group consisting of corticosteroids and nonsteroidal anti-inflammatory agents. [The present invention 1054] The method of claim 1053, wherein the dietary supplement is an antioxidant. [The present invention 1055] The method of any of claims 1051 to 1054, wherein said second therapeutic agent, such as said antibody or antigen-binding fragment thereof, is administered via a different route of administration. [The present invention 1056] The method according to any one of claims 1051 to 1055, wherein said second therapeutic agent is administered orally. [The present invention 1057] The method of claim 1052, wherein said antiviral drug is oseltamivir. [The present invention 1058] The method of claim 1057, wherein said oseltamivir is administered prior to administration of said antibody or antigen-binding fragment thereof. [The present invention 1059] The method of claim 1057, wherein said oseltamivir is administered simultaneously with said antibody or antigen-binding fragment thereof. [The present invention 1060] The method of claim 1057, wherein said oseltamivir is administered after administration of said antibody or antigen-binding fragment thereof. [The present invention 1061] The method of claim 1052, wherein said antiviral agent is an anti-influenza A agent. [The present invention 1062] The method of claim 1061, wherein said anti-influenza A drug is an antibody. [The present invention 1063] The method of claim 1062, wherein said antibody specifically binds to influenza A HA. [The present invention 1064] The method according to any one of claims 1046 to 1063, wherein said pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intramuscularly, intranasally, or orally. Other embodiments will be apparent from consideration of the detailed description that follows. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0086] Detailed Description Before describing the method, it should be understood that the invention is not limited to the specific methods and experimental conditions described herein, as such methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing only specific embodiments, and is not intended to be limiting, since the scope of the present disclosure is limited only by the appended claims.

[0087] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although any method and material similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred method and material are now described.All publications mentioned herein are incorporated herein by reference in their entirety.

[0088] The present disclosure provides antibodies and antigen-binding fragments thereof that bind to influenza B HA. The antibodies of the present invention are particularly useful for inhibiting or neutralizing the activity of influenza B HA.

[0089] The antibodies provided herein may be full-length (e.g., IgG1 or IgG4 full-length antibodies) or may include only an antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments), and may be modified to affect functionality compared to an unmodified antibody, for example, to enhance persistence in the host, or to increase or eliminate residual effector function (Reddy et al., 2000, J. Immunol. 164:1925-1933). In certain embodiments, the antibodies may be bispecific.

[0090] In some embodiments, the antibodies are useful for blocking attachment of influenza virus to host cells and / or preventing influenza virus entry into host cells. In some embodiments, the antibodies function by inhibiting cell-to-cell spread of the virus. In certain embodiments, the antibodies are useful for preventing, treating, or ameliorating at least one symptom of influenza virus infection in a subject. In certain embodiments, the antibodies can be administered prophylactically or therapeutically to a subject having or at risk of contracting an influenza virus infection. In certain embodiments, a composition containing at least one antibody provided herein can be administered to a subject for whom a vaccine is contraindicated or for whom a vaccine is less effective, such as, for example, elderly patients, very young patients, patients who may be allergic to one or more components of the vaccine, or immunocompromised patients who are non-responsive to the vaccine's immunogens. In certain embodiments, a composition containing at least one antibody provided herein can be administered to medical staff, hospitalized patients or elderly care facility residents, or other high-risk patients during an influenza epidemic. In certain embodiments, compositions containing at least one antibody provided herein can be administered to patients as a first line treatment when annual vaccines are predicted to be ineffective or in the event of a pandemic with a strain that has undergone a major antigenic shift.

[0091] definition The term "influenza hemagglutinin", also called "influenza HA", is a trimeric glycoprotein found on the surface of influenza virus particles that mediates virus attachment to host cells (through HA1 binding to α-2,3 sialic acid or α-2,6 sialic acid) and entry (by conformational changes). HA consists of two structural domains, a globular head domain (subject to high frequency antigenic variation) containing the receptor binding site and a stem region (more conserved among various strains of influenza virus). Influenza HA is synthesized as a precursor (HA0) and undergoes proteolytic processing to generate two subunits (HA1 and HA2), which associate with each other to form the stem / globular head structure. The viral HA is the most variable antigen of the virus (18 subtypes can be classified into two groups), whereas the stem (HA2) is highly conserved within each group. The amino acid sequence of full-length influenza B HA is exemplified by the HA from B / Victoria / 2 / 87, SEQ ID NO:21 (GenBank Accession No. AAA43697.1), the HA from B / Nanchang / 3451 / 93, SEQ ID NO:22 (partial sequence, GenBank Accession No. AAD02807.1), the HA from B / Singapore / 11 / 1994, SEQ ID NO:23 (GenBank Accession No. ABN50712.1), and the HA from B / Florida / 4 / 2006, SEQ ID NO:24 (GenBank Accession No. ACA33493.1). The term "influenza-HA" also includes protein variants of influenza HA isolated from other influenza B isolates as well as influenza A isolates. The term "influenza-HA" also includes recombinant influenza HA or fragments thereof. The term also encompasses influenza HA or fragments thereof bound to, for example, a histidine tag, mouse or human Fc, or a signal sequence.

[0092] The term "influenza infection", as used herein, also characterized as "flu", refers to a severe acute respiratory illness caused by an influenza virus. The term includes respiratory infection and symptoms including high fever, headache, generalized aches and pains, fatigue and weakness, in some cases extreme tiredness, stuffy nose, sneezing, sore throat, chest discomfort, coughing, shortness of breath, bronchitis, pneumonia, and in severe cases, death.

[0093] The term "surface plasmon resonance" refers to an optical phenomenon that allows the analysis of biomolecular interactions in real time by detection of changes in protein concentration within a biosensor matrix, for example using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0094] Biolayer interferometry is a label-free technique for measuring biomolecular interactions. It is an optical analysis technique that analyzes the interference pattern of white light reflected from two surfaces: an immobilized protein layer on a biosensor tip and an internal reference layer. Changes in the number of molecules bound to the biosensor tip result in a shift in the interference pattern that can be measured in real time (Abdiche, YN, et al. Analytical Biochemistry, (2008), 377(2), 209-217). In a specific embodiment, the "real-time biolayer interferometer-based biosensor (Octet HTX assay)" was used to evaluate the binding properties of a specific anti-influenza HA antibody.

[0095] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen may have more than two epitopes. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. The term "epitope" also refers to a site on an antigen to which B cells and / or T cells respond. The term also refers to the region of an antigen to which an antibody binds. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have residues that are directly involved in the affinity of the interaction. Epitopes may also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments may have specific three-dimensional structural features, and / or specific charge characteristics.

[0096] As used herein, the term "cross-compete" refers to an antibody or antigen-binding fragment thereof that binds to an antigen and inhibits or blocks the binding of another antibody or antigen-binding fragment thereof. The term also includes bidirectional competition between two antibodies, i.e., a first antibody binds and blocks the binding of a second antibody, and vice versa. In certain embodiments, the first antibody and the second antibody may bind to the same epitope. Alternatively, the first and second antibodies may bind to different but overlapping epitopes, such that the binding of one inhibits or blocks the binding of the second antibody, for example, through steric hindrance. Cross-competition between antibodies can be measured by methods known in the art, for example, real-time label-free biolayer interference assays. To determine whether a test antibody cross-competes with a reference anti-influenza B antibody, the reference antibody is allowed to bind to influenza virus HA or peptide under saturating conditions. The ability of the test antibody to bind to influenza virus HA is then evaluated. If the test antibody is able to bind to influenza virus HA after saturation binding with the reference anti-influenza virus HA antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-influenza virus HA antibody, whereas if the test antibody is unable to bind to influenza virus HA after saturation binding with the reference anti-influenza virus HA antibody, the test antibody is likely to bind to the same epitope as the reference anti-influenza virus HA antibody.

[0097] By the phrase "therapeutically effective amount" is meant the amount that is administered to produce the desired effect. The exact amount will depend on the purpose of the treatment and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0098] As used herein, the term "subject" refers to an animal, e.g., a mammal, such as a human, that is in need of amelioration, prevention, and / or treatment of a disease or disorder, such as a viral infection. The subject may have an influenza infection or may be susceptible to developing an influenza virus infection. A subject that is "susceptible to developing an influenza virus infection" or "may be at high risk of developing an influenza virus infection" is a subject that has an immune deficiency due to an autoimmune disease, a person undergoing immunosuppressive therapy (e.g., after an organ transplant), a person suffering from human immunodeficiency syndrome (HIV) or acquired immune deficiency syndrome (AIDS), a person undergoing certain forms of anemia that deplete or destroy white blood cells, radiation therapy or chemotherapy, or a person suffering from an inflammatory disorder. Additionally, very young or elderly subjects are at increased risk. Anyone who has physical contact or close physical proximity to an infected individual is at increased risk of developing an influenza virus infection. Additionally, subjects are at risk for contracting influenza infection because of their proximity to disease outbreaks, e.g., because they live in a densely populated city or in close proximity to subjects with confirmed or suspected influenza virus infection, or because of their occupational choices, e.g., they are hospital workers, pharmaceutical researchers, travelers to endemic areas, or frequent flyers.

[0099] As used herein, the terms "treat," "treating," or "treatment" refer to the reduction or amelioration of the severity of at least one symptom or sign of influenza infection by administering a therapeutic agent, such as an antibody disclosed herein, to a subject in need of treatment. The term includes the inhibition of disease progression or inhibition of worsening of infection. The term also includes a positive prognosis of the disease, i.e., upon administration of a therapeutic agent, such as an antibody disclosed herein, the subject may not be infected or may have reduced or no viral titers. The therapeutic agent may be administered to the subject in a therapeutic dose.

[0100] The terms "prevent," "preventing," or "prevention" refer to the inhibition of the manifestation of influenza infection, or the inhibition of any symptoms or signs of influenza infection, upon administration of an antibody disclosed herein. This term includes the prevention of the spread of infection in a subject who has been exposed to the virus or is at risk for influenza infection.

[0101] As used herein, a "protective effect" may be demonstrated by any standard procedure known in the art to determine whether an agent, such as an anti-viral agent, or an antibody, such as an anti-influenza-HA antibody disclosed herein, can result in any one or more of, for example, an increase in survival rate following exposure to an infectious agent (compared to a comparable population of subjects untreated and also exposed to the infectious agent), a reduction in viral load (compared to pre-treatment subjects), or an amelioration of at least one symptom associated with the infectious agent (compared to pre-treatment subjects).

[0102] As used herein, the term "antiviral agent" refers to any anti-infective agent or treatment used to treat, prevent, or ameliorate a viral infection in a subject. The term "antiviral agent" includes, but is not limited to, TAMIFLU® (oseltamivir), RELENZA® (zanamivir), baloxavir, marboxil, ribavirin, or interferon alpha 2b. "Antiviral agent" also includes antiviral antibodies. For example, an antiviral agent may be an antibody used to treat, prevent, or ameliorate an influenza infection (e.g., influenza A, influenza B, or both). Such antibodies can target hemagglutinins from influenza A, influenza B, or both. In the present disclosure, the infection being treated is caused by an influenza virus.

[0103] Overview Influenza is an infectious disease caused by RNA viruses (influenza viruses) of the family Orthomyxoviridae. Influenza viruses are classified into four genera, A, B, C, and D, based on the core protein, and further divided into subtypes determined by the viral envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA). Influenza B viruses form a homogenous group that began to diverge in the 1970s into two antigenically distinguishable lineages, B / Victoria / 2 / 87 and B / Yamagata / 16 / 88, now known as the Victoria and Yamagata lineages. Influenza A viruses infect a variety of mammalian and avian species, while infection with types B and C is mainly restricted to humans. Influenza D mainly infects cattle, but not humans. Only types A and B cause human disease of concern.

[0104] The high mutation rate and frequent genetic reassortment of influenza viruses contribute to the large variation in HA and NA antigens. Small point mutations that cause small changes ("antigenic drift") occur relatively frequently. Antigenic drift allows the virus to evade immune recognition, resulting in repeated influenza outbreaks during pandemic periods. Major changes in HA antigens ("antigenic shift") are caused by reassortment of genetic material from various influenza subtypes. Antigenic shift resulting in new pandemic strains is a rare event that occurs through reassortment between animal and human subtypes, for example, in co-infected pigs.

[0105] HA is synthesized as a homotrimeric precursor polypeptide HA0. Each monomer can be independently cleaved post-translationally to form two polypeptides, HA1 and HA2, linked by a single disulfide bond. The larger N-terminal fragment (HAL 320-330 amino acids) forms the membrane-distal globular domain, which contains the receptor-binding site and most of the determinants recognized by virus-neutralizing antibodies. The HA1 polypeptide of HA is involved in attachment of the virus to the cell surface. The smaller C-terminal portion (HA2, about 180 amino acids) forms a stem-like structure that anchors the globular domain to the cell or viral membrane. The HA2 polypeptide mediates fusion of the virus with the cell membrane in the endosome, allowing the release of the ribonucleoprotein complex into the cytoplasm.

[0106] Despite decades of research, there are no commercially available antibodies that broadly neutralize or inhibit influenza B virus infection or reduce disease caused by influenza B virus. Thus, there is a need to identify new antibodies that neutralize multiple subtypes of influenza B virus and can be used as agents for the prevention or treatment of influenza B infection.

[0107] Passive immunotherapy for the prevention or treatment of infectious diseases has been used for over a century, usually in the form of convalescent human serum containing high titers of neutralizing antibodies (Good et al. 1991; Cancer 68:1415-1421). Today, several purified monoclonal antibodies are currently in preclinical and clinical development for use as antimicrobial agents (Marasco et al. 2007; Nature Biotechnology 25:1421-1434).

[0108] The inventors herein described fully human antibodies and antigen-binding fragments thereof that specifically bind influenza hemagglutinin and modulate the interaction of influenza virus with host cells. The anti-influenza B HA antibodies may bind influenza B virus HA with high affinity. In certain embodiments, the antibodies disclosed herein are blocking antibodies, where the antibodies bind to influenza HA and can block the attachment and / or entry of the virus into host cells. In some embodiments, the blocking antibodies can block the binding of influenza virus to cells and thus inhibit or neutralize the viral infection of host cells. In some embodiments, the blocking antibodies may be useful for treating subjects suffering from influenza virus infection. The antibodies, when administered to a subject in need thereof, may reduce infection by viruses such as influenza in the subject. They may be used to reduce the viral load in a subject compared to untreated subjects. They may also be used as adjunctive therapy alone or with other therapeutic moieties or modalities known in the art for treating viral infections. In certain embodiments, these antibodies may bind to an epitope in the stem region of the viral HA. Additionally, the identified antibodies can be used prophylactically (prior to infection) to protect an animal (e.g., a mammal) from infection, or therapeutically (after infection has been established) to ameliorate a previously established infection or to ameliorate at least one symptom associated with the infection.

[0109] Exemplary full-length amino acid sequences of influenza B HAs are set forth in GenBank under accession number AAA43697.1 (from B / Victoria / 2 / 87, see also SEQ ID NO:21), accession number AAD02807.1 (partial sequence from B / Nanchang / 3451 / 93, see also SEQ ID NO:22), accession number ABN50712.1 (from B / Singapore / 11 / 1994, see also SEQ ID NO:23), and accession number ACA33493.1 (from B / Florida / 4 / 2006, see also SEQ ID NO:24).

[0110] In certain embodiments, the antibodies are obtained from mice immunized with a first immunogen, such as full-length influenza B HA, or with a recombinant influenza B HA or fragment thereof, and then immunized with a second immunogen, or with an immunogenically active fragment of influenza B HA. In certain embodiments, the antibodies are obtained from mice immunized with an influenza vaccine composition and then boosted with one or more recombinantly produced HA peptides. For example, the antibodies can be obtained by immunizing mice with B / Victoria / 2 / 87, followed by B / Yamagata / 16 / 88 and then again with B / Victoria / 2 / 87, or by immunizing mice with B / Yamagata / 16 / 88, followed by B / Victoria / 2 / 87 and then again with B / Yamagata / 16 / 88. In some embodiments, the B / Yamagata strain can be replaced with B / Maryland / 03 / 2008, or B / Florida / 4 / 2006, or B / Nanchang / 3451 / 93, or B / Singapore / 11 / 1994. Mice can be boosted with, for example, a mixture of DNA encoding HA from B / Victoria / 2 / 87, B / Yamagata / 16 / 88, B / Maryland / 03 / 2008, B / Nanchang / 3451 / 93, B / Singapore / 11 / 1994, and / or B / Florida / 4 / 2006, e.g., a 1:1 mixture of DNA encoding HA from B / Victoria / 2 / 87 and B / Yamagata / 16 / 88.

[0111] The immunogen may be a biologically active and / or immunogenic fragment of influenza B HA, or DNA encoding the active fragment. The fragment may be derived from the stem region of the HA protein.

[0112] The peptides may be modified to include the addition or substitution of specific residues for tagging or conjugation to a carrier molecule such as KLH. For example, cysteines may be added to either the N-terminus or C-terminus of the peptide, or linker sequences may be added to prepare the peptide for conjugation, e.g., with KLH, for immunization.

[0113] Certain anti-influenza B HA antibodies disclosed herein are capable of binding to and neutralizing the activity of influenza B HA as determined by in vitro or in vivo assays. The ability of an antibody to bind to influenza B HA and neutralize its activity, and thus viral attachment and / or entry into a host cell, and subsequent viral infection, can be measured using any standard method known to one of skill in the art, including the binding assays described herein, or activity assays.

[0114] Non-limiting exemplary in vitro assays for measuring binding activity are known to those skilled in the art. For example, the binding affinity and dissociation constant of anti-influenza B HA antibodies to influenza B HA can be determined by surface plasmon resonance using a Biacore instrument. Neutralization assays can be used to determine the infectivity of various strains of influenza B virus. Antibodies to influenza B HA can mediate complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC) of virus-infected cells in vitro. Exemplary antibodies can neutralize influenza B infection in vivo.

[0115] Antibodies specific for influenza B HA may contain no additional label or moiety, or may contain an N-terminal or C-terminal label or moiety. In some embodiments, the label or moiety is biotin. In binding assays, the position of the label (if any) may determine the orientation of the peptide relative to the surface to which it binds. For example, if the surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C-terminal portion of the peptide is distal to the surface. In some embodiments, the label may be a radionuclide, a fluorescent dye, or an MRI-detectable label. In certain embodiments, such labeled antibodies may be used in diagnostic assays, including imaging assays.

[0116] Antibodies and antigen-binding fragments of antibodies antibody The term "antibody" as used herein is intended to refer to an immunoglobulin molecule (i.e., an "intact antibody molecule") consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain contains a heavy chain variable region ("HCVR" or "V H ") and the heavy chain constant region (C H 1 domain, C H 2 domain and C H Each light chain consists of a light chain variable region ("LCVR" or "V L ") and the light chain constant region (C L ) V H and V L The regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. An amino acid consensus sequence can be defined based on an aligned analysis of two or more CDRs.

[0117] The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies may contain amino acid residues not encoded by human germline immunoglobulin sequences, for example in the CDRs, particularly in CDR3 (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include monoclonal antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) are grafted onto human FR sequences. The term includes antibodies recombinantly produced in non-human mammals or in cells of non-human mammals. The term is not intended to include antibodies isolated from or generated in human subjects.

[0118] The term "recombinant" as used herein refers to an antibody or antigen-binding fragment thereof that is made, expressed, isolated, or obtained by techniques or methods known in the art as recombinant DNA technology, including, for example, DNA splicing and transgenic expression. The term refers to antibodies expressed in a non-human mammal (including, for example, a transgenic non-human mammal such as a transgenic mouse) or cell (e.g., a CHO cell) expression system or isolated from a recombinant combinatorial human antibody library.

[0119] The term "specifically binds" or "specifically binds to" and the like means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1×10 -8 M or less (e.g., a smaller K Dindicates tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, an antibody has been identified in a real-time label-free biolayer interference assay on an Octet® HTX biosensor that specifically binds to influenza-HA. Furthermore, a multispecific antibody that binds to one domain of influenza-HA and one or more additional antigens, or a bispecific antibody that binds to two different regions of influenza-HA, is nevertheless considered an antibody that "specifically binds" as used herein.

[0120] The term "high affinity" antibody refers to an antibody that has an affinity of at least 10 as measured by a real-time label-free biolayer interference assay, such as an Octet® HTX biosensor, or surface plasmon resonance, such as a BIACORE™, or a solution-affinity ELISA. -8 M, at least about 10 -9 M, at least about 10 -10 M, or at least about 10 -11 K of M D The term "antibody" refers to those monoclonal antibodies having binding affinity for influenza-HA, designated as "antibody" in the present specification.

[0121] The term "slow off rate", "Koff" or "kd" refers to a slow off rate of 1x10 as determined by real-time label-free biolayer interference assays, e.g., Octet® HTX biosensor, or surface plasmon resonance, e.g., BIACORE™. -3 s -1 Less than or equal to 1x10 -4 s -1 The following rate constants are expressed for antibody dissociation from influenza-HA:

[0122] "K D The term "as used herein" is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0123] As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "antigen-binding fragment" of an antibody, or "antibody fragment" refers to one or more fragments of an antibody that retain the ability to bind to influenza HA.

[0124] In certain embodiments, the antibodies or antibody fragments provided herein may be conjugated to a ligand or therapeutic moiety (an "immunoconjugate"), such as an antiviral drug, a second anti-influenza antibody, or any other therapeutic moiety useful for treating infection caused by influenza virus.

[0125] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies (Abs) having different antigen specificities (e.g., an isolated antibody or fragment thereof that specifically binds influenza-HA is substantially free of Abs that specifically bind antigens other than influenza-HA).

[0126] As used herein, a "blocking antibody" or "neutralizing antibody" (or an "antibody that neutralizes influenza-HA activity" or an "antagonist antibody") is intended to refer to an antibody whose binding to influenza-HA results in the inhibition of at least one biological activity of influenza-HA. For example, the antibodies provided herein may prevent or block influenza attachment or entry into a host cell. Furthermore, a "neutralizing antibody" is one that can neutralize, i.e., prevent, inhibit, reduce, hinder or interfere with the ability of a pathogen to initiate and / or perpetuate infection in a host. The terms "neutralizing antibody" and "neutralizing antibody" are used interchangeably herein. These antibodies can be used alone or in appropriate formulations, in combination with other antiviral agents, as prophylactic or therapeutic agents, or in conjunction with active vaccination, or as diagnostic tools.

[0127] antigen-binding fragment Unless specifically stated otherwise, the term "antibody" as used herein is understood to encompass an antibody molecule comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., a "complete antibody molecule") as well as antigen-binding fragments thereof. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "antigen-binding fragment" of an antibody or "antibody fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to influenza B HA. Antibody fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing CDRs, or isolated CDRs. In certain embodiments, the term "antigen-binding fragment" refers to a polypeptide fragment of a multispecific antigen-binding molecule. Antigen-binding fragments of antibodies can be derived from complete antibody molecules using any suitable standard technique, such as, for example, proteolytic or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to place one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0128] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable regions of an antibody (e.g., isolated complementarity determining regions (CDRs), such as CDR3 peptides), or constrained FR3-CDR3-FR4 peptides. Domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein.

[0129] Antigen-binding fragments of antibodies typically contain at least one variable domain. A variable domain may be of any size or amino acid composition and generally contains at least one CDR adjacent to or in frame with one or more framework sequences. L V bound to the domain H In an antigen-binding fragment having a domain, H Domains and V L The domains may be positioned relative to each other in any suitable configuration. For example, the variable region may be a dimer, with the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of the antibody may contain a dimer of monomeric V H or V L It may contain domains.

[0130] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found within the antigen-binding fragments of an antibody disclosed herein include: (i) a V H -C H 1, (ii) V H -C H 2. (iii) V H -C H 3. (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3. (vii) V H -C L、 (viii) V L -C H 1, (ix) V L -C H 2. (x)V L -C H 3. (xi) V L -C H 1-C H 2. (xii) V L -C H 1-C H 2-C H 3. (xiii) V L -C H 2-C H 3, and (xiv) V L -C LIn any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a full or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies disclosed herein may be linked to each other and / or to one or more monomeric V H Or V L The domains may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above in non-covalent association (eg, via disulfide bonds).

[0131] As with intact antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, each capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the examples of bispecific antibody formats disclosed herein, may be adapted for use in conjunction with the antigen-binding fragments of antibodies disclosed herein using routine techniques available in the art.

[0132] Modifications to Antibodies and Antigen-Binding Fragments Thereof In certain embodiments, the framework regions of an antibody (or antigen-binding fragment thereof) may be identical to a human germline sequence, e.g., identical to the sequence of an antibody provided herein, or may be naturally or artificially modified. One or more amino acids in a given framework region (or one or more framework regions) may be substituted, and the substitution may be conservative or non-conservative. Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies have been described in the scientific literature in which one or two CDRs may be omitted for binding. Padlan et al. (1995 FASEB J.9:133-139) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only about 1 / 5 to 1 / 3 of the CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs do not have any amino acids in contact with the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428). Thus, the antibodies provided herein are intended to be useful in determining whether the modified antibody exhibits one or more desirable characteristics, e.g., whether the antibody or antigen-binding fragment thereof exhibits a specific activity that is greater than or equal to about 10 -10 EC below M 50 The modified polypeptides may be effectively modified in the CDR and / or framework regions so long as they maintain their ability to bind to influenza B HA at a specific concentration and / or exhibit an increase in survival of influenza-infected animals following administration to the influenza-infected animals compared to comparable influenza-infected animals not administered the polypeptides.

[0133] Modifications to a given CDR may be made to the CDR sequence from the antibody provided herein, and may include conservative or non-conservative substitutions. Desired substitutions may be determined by molecular modeling and / or empirically. For example, one or more CDR residues may be replaced with an amino acid that occupies the corresponding position in another human antibody sequence or a consensus of such sequences.

[0134] Additionally, the antigen-binding fragment may be an antibody disclosed herein, but modified to omit one or more CDRs and / or one or more framework regions, so long as the modified antibody (aka, antigen-binding fragment) retains binding to influenza B HA.

[0135] CDR residues that do not contact antigen can be identified empirically and / or by molecular modeling from regions of the Kabat CDRs that are outside the Chothia CDRs based on prior studies (e.g., residues H60-H65 in HCDR2 are often not necessary). The antibodies or antigen-binding fragments thereof provided herein can be modified to remove or replace a given CDR, particularly one that does not contact antigen. Light chain CDRs can be replaced, for example, with universal light chain CDRs.

[0136] The fully human anti-influenza-HA monoclonal antibodies provided herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences or compared to the sequences provided herein. Such modifications or mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases, or by comparing the amino acid sequences to the antibodies provided herein, for example, any one of the antibody sequences provided in Table 1.

[0137] The disclosure includes antibodies, and antigen-binding fragments thereof, derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework regions and / or CDRs are modified to provide the engineered antibody with one or more desirable characteristics, e.g., the antibody or antigen-binding fragment thereof has a nucleotide sequence that is at least about 10 times as long as the antibody or antigen-binding fragment ... -10 EC below M 50and / or show an increase in survival of influenza-infected animals following administration to said influenza-infected animals compared to comparable influenza-infected animals not administered said antibody or antigen-binding fragments. Once obtained, antibodies and antigen-binding fragments containing one or more modifications within the framework regions and / or CDRs can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc.

[0138] The present disclosure also includes antibodies, and antigen-binding fragments thereof, derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions have been mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily produce many antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, the V H and / or V LAll of the framework and / or CDR residues in the domain are mutated back to the residue found in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only the mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody is originally derived). Furthermore, the antibodies disclosed herein may contain any combination of two or more germline mutations in the framework and / or CDR regions, for example, certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed by the present disclosure.

[0139] The present disclosure provides antibodies that have "substantial identity" or "substantial similarity" to the sequences provided herein in the CDR or framework regions. Sequence differences, such as differences between the sequences provided in Table 1 or Table 2 and modified sequences based thereon, are indicated by "substantial identity" or "substantial similarity."

[0140] The term "substantial identity" or "substantially identical" when referring to a nucleic acid or a fragment thereof indicates that when optimally aligned with another nucleic acid (or the complementary strand of the other nucleic acid), there is a percent identity of the nucleotide sequence of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99.5%, or 100% of the nucleotide bases as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST, or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0141] As applied to polypeptides, the term "substantial similarity" or "substantially similar" refers to two peptide sequences that share at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99.5%, or 100% sequence identity when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights. In some embodiments, residue positions that are not identical differ only by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other only by conservative substitutions, the percentage or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol.Biol.24:307-331, incorporated herein by reference. Examples of groups of amino acids with side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) basic side chains: lysine, arginine and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443 45. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0142] Sequence identity and / or similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT that can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the region of best overlap between the query sequence and the search sequence (Pearson (2000) supra). Sequences can also be compared using the Smith-Waterman homology search algorithm using an affine gap search with a gap opening penalty of 12, a gap extension penalty of 2, and a BLOSUM matrix of 62. Another preferred algorithm for comparing the sequences disclosed herein with a database containing multiple sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters.See, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25: 3389-3402, each of which is incorporated herein by reference.

[0143] Provided herein are fully human anti-Influenza-HA monoclonal antibodies that include variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more substitutions (e.g., conservative substitutions). For example, the disclosure includes anti-Influenza B-HA antibodies that have HCVR, LCVR, and / or CDR amino acid sequences with, for example, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 amino acid substitution relative to any of the HCVR, LCVR, and / or CDR (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3) amino acid sequences disclosed herein. For example, an anti-Influenza B-HA antibody may contain 20, 19, 18, 17, 16, 15, 14 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution (e.g., conservative amino acid substitution) relative to any of the HCVR, LCVR, and / or CDR (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3) amino acid sequences disclosed herein.

[0144] Exemplarily, the present specification provides an antibody or antigen-binding fragment thereof that specifically binds to influenza B HA comprising an HCVR amino acid sequence that differs from SEQ ID NO: 2 by at least 1 to 50 conservative amino acid substitutions, for example, by at least 10 to 40 conservative amino acid substitutions, or by at least 20 to 50 conservative amino acid substitutions, or by at least 20 to 40 conservative amino acid substitutions. Also provided herein is an antibody or antigen-binding fragment thereof that specifically binds to influenza B HA comprising an LCVR amino acid sequence that differs from SEQ ID NO: 10 by at least 1 to 50 conservative amino acid substitutions, for example, by at least 10 to 40 conservative amino acid substitutions, or by at least 20 to 50 conservative amino acid substitutions, or by at least 20 to 40 conservative amino acid substitutions. Such substitutions may be within the framework region or CDR, and the specificity of the antibody or antigen-binding fragment thereof that binds to influenza B HA can be maintained.

[0145] Also provided herein is an antibody or antigen-binding fragment thereof comprising an HCDR1 having an amino acid sequence that differs from SEQ ID NO:4 by one, two, or three amino acid substitutions (e.g., conservative amino acid substitutions). Provided herein is an antibody or antigen-binding fragment thereof comprising an HCDR2 having an amino acid sequence that differs from SEQ ID NO:6 by one, two, or three amino acid substitutions (e.g., conservative amino acid substitutions). Provided herein is an antibody or antigen-binding fragment thereof comprising an HCDR3 having an amino acid sequence that differs from SEQ ID NO:8 by one, two, or three amino acid substitutions (e.g., conservative amino acid substitutions). Provided herein is an antibody or antigen-binding fragment thereof comprising an LCDR1 having an amino acid sequence that differs from SEQ ID NO:12 by one, two, or three amino acid substitutions (e.g., conservative amino acid substitutions). Provided herein is an antibody or antigen-binding fragment thereof comprising an LCDR2 having an amino acid sequence that differs from SEQ ID NO:14 by one, two, or three amino acid substitutions (e.g., conservative amino acid substitutions). Provided herein are antibodies or antigen-binding fragments thereof that comprise an LCDR3 having an amino acid sequence that differs from SEQ ID NO: 16 by one, or two, or three amino acid substitutions (eg, conservative amino acid substitutions).

[0146] The present disclosure also provides an antibody or antigen-binding fragment thereof comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) that differs from SEQ ID NO: 8 / 16 by at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6 amino acid substitutions (e.g., conservative amino acid substitutions).

[0147] The present invention also provides an antibody, or antigen-binding fragment thereof, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within the HCVR / LCVR amino acid sequence of an exemplary anti-influenza B HA antibody shown in Table 1. In certain embodiments, the aforementioned HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set differs from SEQ ID NOs: 4-6-8-12-14-16 by up to 20 conservative amino acid substitutions, such as up to 1, or up to 2, or up to 3, or up to 3, or up to 4, or up to 5, or up to 6, or up to 7, or up to 8, or up to 9, or up to 10, or up to 11, or up to 12, or up to 13, or up to 14, or up to 15, or up to 16, or up to 17, or up to 18, or up to 19 amino acid conservative amino acid substitutions.

[0148] Provided herein are antibodies, or antigen-binding fragments thereof, comprising an HCVR comprising an amino acid sequence set forth in Table 1, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99.5%, or 100% sequence identity thereto.

[0149] Provided herein is an antibody, or antigen-binding fragment thereof, comprising an LCVR comprising an amino acid sequence set forth in Table 1, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99.5%, or 100% sequence identity thereto.

[0150] Provided herein is an antibody, or antigen-binding fragment thereof, comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NO:2 / 10, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99.5%, or 100% sequence identity thereto.

[0151] The present disclosure provides an antibody, or an antigen-binding fragment thereof, comprising a heavy chain CDR1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:4, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99.5%, or 100% sequence identity thereto.

[0152] The present disclosure provides an antibody, or an antigen-binding fragment thereof, comprising a heavy chain CDR2 (HCDR2) comprising the amino acid sequence of SEQ ID NO:6, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99.5%, or 100% sequence identity thereto.

[0153] The present disclosure provides an antibody, or an antigen-binding fragment thereof, comprising a heavy chain CDR3 (HCDR3) comprising the amino acid sequence of SEQ ID NO:8, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99.5%, or 100% sequence identity thereto.

[0154] The present disclosure provides an antibody, or an antigen-binding fragment thereof, comprising a light chain CDR1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 12, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99.5%, or 100% sequence identity thereto.

[0155] The present disclosure provides an antibody, or an antigen-binding fragment thereof, comprising a light chain CDR2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 14, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99.5%, or 100% sequence identity thereto.

[0156] The present disclosure provides an antibody, or an antigen-binding fragment thereof, comprising a light chain CDR3 (LCDR3) comprising the amino acid sequence of SEQ ID NO: 16, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99.5%, or 100% sequence identity thereto.

[0157] The present disclosure provides an antibody, or an antigen-binding fragment thereof, comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) comprising the amino acid sequence of SEQ ID NO: 8 / 16, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99.5%, or 100% sequence identity thereto.

[0158] Preparation of human antibodies Methods for generating human antibodies in transgenic mice are known in the art. Any such known methods may be used in the context of the present disclosure to generate human antibodies that specifically bind to influenza B HA. An immunogen comprising any one of the following may be used to generate antibodies against influenza B HA. In certain embodiments, antibodies are obtained from mice immunized with full-length native influenza B HA (see, e.g., GenBank Accession Nos. AAA43697.1 or ACA33493.1, or with live attenuated or inactivated virus, or with DNA encoding the protein or a fragment thereof. Alternatively, influenza B HA protein or a fragment thereof may be produced and modified using standard biochemical techniques and used as an immunogen. In some embodiments, the immunogen is a recombinantly produced influenza B HA. In certain embodiments, the immunogen may be an influenza virus vaccine. In certain embodiments, one or more booster injections may be administered. In certain embodiments, the booster injections may include one or more influenza virus strains, or hemagglutinins derived from these strains, for example, B / Victoria / 2 / 87, followed by B / Yamagata / 16 / 88, then B / Victoria / 2 / 87 again, or B / Yamagata / 16 / 88, followed by B / Victoria / 2 / 87, then B / Yamagata / 16 / 88 again. In some aspects, the B / Yamagata strain is replaced with B / Maryland / 03 / 2008 or B / Nanchang / 3451 / 93 or B / Florida / 4 / 2006. All mice can be boosted with a mixture of DNA encoding HA from B / Victoria / 2 / 87, B / Yamagata / 16 / 88, B / Maryland / 03 / 2008, and / or B / Florida / 4 / 2006. In certain embodiments, the booster injection may contain a mixture of influenza strains, or a mixture of hemagglutinins from strains, or DNA encoding HA.In certain embodiments, the immunogen may be a recombinant influenza HA peptide expressed in E. coli, or any other eukaryotic or mammalian cell, such as Chinese Hamster Ovary (CHO) cells, or the influenza virus itself.

[0159] Using VELOCIMMUNE® technology (see, e.g., US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal antibodies, a high affinity chimeric antibody against influenza B HA with a human variable region and a mouse constant region is first isolated. VELOCIMMUNE® technology involves generating a transgenic mouse with a genome that includes a human heavy chain variable region and a human light chain variable region operably linked to an endogenous mouse constant region locus, such that the mouse produces an antibody containing a human variable region and a mouse constant region in response to antigenic stimulation. DNA encoding the variable regions of the antibody's heavy and light chains is isolated and operably linked to DNA encoding the human heavy chain constant region and the human light chain constant region. The DNA is then expressed in a cell capable of expressing a fully human antibody.

[0160] In general, VELOCIMMUNE® mice are loaded with an antigen of interest, and lymphocytes (such as B cells) are collected from the mice that express antibodies. Lymphocytes can be fused with myeloma cell lines to prepare immortalized hybridoma cell lines, which are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the variable regions of the heavy and light chains can be isolated and linked to the desired isotype constant regions of the heavy and light chains. Such antibody proteins can be produced in cells such as CHO cells. Alternatively, DNA encoding the antigen-specific chimeric antibody or the variable domains of the light and heavy chains can be directly isolated from antigen-specific lymphocytes.

[0161] First, a high affinity chimeric antibody having a human variable region and a mouse constant region is isolated. The antibody is characterized and selected for desirable characteristics including affinity, selectivity, epitope, etc., as in the following experimental section. The mouse constant region is replaced with the desired human constant region to generate a fully human antibody, e.g., wild-type or modified IgG1 or IgG4. While the constant region selected can vary depending on the particular application, the high affinity antigen binding and target specificity characteristics reside in the variable region.

[0162] biological equivalent The anti-influenza B HA antibodies and antibody fragments disclosed herein include proteins having amino acid sequences that differ from those of the described antibodies but retain the ability to bind influenza HA. ​​Such variant antibodies and antibody fragments contain one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, but exhibit biological activity that is essentially equivalent to the biological activity of the described antibodies. Similarly, DNA sequences encoding the antibodies disclosed herein include sequences that contain one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequences, but encode antibodies or antibody fragments that are essentially biologically equivalent to the antibodies or antibody fragments disclosed herein.

[0163] Two antigen binding proteins or antibodies are considered to be bioequivalent if they are pharmaceutical equivalents or pharmaceutical substitutes and do not show significant differences in their rate and extent of absorption when administered in the same molar dose, either in single or multiple doses, under similar experimental conditions. Some antibodies are considered equivalents, or pharmaceutical substitutes, if the extent of absorption is comparable but the rate of absorption is not, but the antibodies can be considered bioequivalent because the difference in absorption rate is intentional, reflected in the labeling, is not essential, for example, to achieving effective body drug concentrations in chronic use, and is not considered medically significant for the particular pharmaceutical product being tested.

[0164] In some embodiments, two antigen binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, or efficacy.

[0165] In some embodiments, two antigen binding proteins are bioequivalent if a patient can make one or more switches between the reference product and the biological product without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity, or decreased efficacy, as compared to continuous therapy without such switches.

[0166] In some embodiments, two antigen binding proteins are biologically equivalent if they both act through a common mechanism of action to the extent that that mechanism is known for the conditions of use.

[0167] Bioequivalence may be demonstrated by in vivo and / or in vitro methods, including, for example, (a) in vivo tests in humans or other mammals in which the concentration of an antibody or its metabolites is measured in blood, plasma, serum or other biological fluids as a function of time, (b) in vitro tests that correlate with and reasonably predict human in vivo bioavailability data, (c) in vivo tests in humans or other mammals in which the relevant acute pharmacological effects of the antibody (or its target) are measured as a function of time, and (d) well-controlled clinical trials that demonstrate the safety, efficacy, or bioavailability or bioequivalence of an antibody.

[0168] Biologically equivalent variants of antibodies can be constructed, for example, by making various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. In other situations, biologically equivalent antibodies can include antibody variants that contain amino acid changes that alter the glycosylation characteristics of the antibody, for example, mutations that abolish or eliminate glycosylation.

[0169] Anti-influenza HA antibodies, including Fc variants According to certain embodiments disclosed herein, influenza B HA antibodies are provided that include an Fc domain that includes one or more mutations that enhance or decrease antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the present disclosure provides an Fc domain that includes one or more mutations that enhance or decrease antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. H 2 or C HThe present invention includes anti-influenza-HA antibodies that contain mutations in the 3 region that increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in endosomes where the pH ranges from about 5.5 to about 6.0) compared to the same but unmodified antibody. Such mutations can result in an increased serum half-life of the antibody when administered to an animal compared to the same but unmodified antibody. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T), or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F, or Y [N434A, N434W, N434H, N434F, or N434Y]), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In some embodiments, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications, 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications, 433K (e.g., H433K) and 434 (e.g., 434Y) modifications, 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications, 250Q and 428L modifications (e.g., T250Q and M428L), and 307 and / or 308 modifications (e.g., 308F and / or 308P). In yet another embodiment, the modifications include 265A (e.g., D265A) and / or 297A (e.g., N297A) modifications.

[0170] For example, the present disclosure provides an anti-influenza B antibody comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: HA antibodies include: 250Q and 248L (e.g., T250Q and M248L), 252Y, 254T and 256E (e.g., M252Y, S254T and T256E), 428L and 434S (e.g., M428L and N434S), 257I and 311I (e.g., P257I and Q311I), 257I and 434H (e.g., P257I and N434H), 376V and 434H (e.g., D376V and N434H), 307A, 380A and 434A (e.g., T307A, E380A and N434A), and 433K and 434F (e.g., H433K and N434F). All possible combinations of the aforementioned Fc domain mutations, and other mutations in the antibody variable domains disclosed herein, are contemplated to be within the scope disclosed herein.

[0171] As used herein, the chimeric heavy chain constant (C H The present invention provides an anti-influenza B HA antibody comprising the chimeric C H The region is composed of C H For example, the antibody may include a C region derived from a human IgG1 molecule, a human IgG2 molecule, or a human IgG4 molecule. H C from a human IgG1 molecule, a human IgG2 molecule, or a human IgG4 molecule in combination with some or all of the three domains H Chimeric C containing part or all of the 2 domains H According to certain embodiments, the antibody may comprise a chimeric C region having a chimeric hinge region. HFor example, the chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues at positions 216-227 according to EU numbering) derived from a human IgG1, IgG2 or IgG4 hinge region combined with a "lower hinge" sequence (amino acid residues at positions 228-236 according to EU numbering) derived from a human IgG1, IgG2 or IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 upper hinge or a human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. The chimeric hinges described herein may comprise an "upper hinge" amino acid sequence (amino acid residues at positions 216-227 according to EU numbering) derived from a human IgG1, IgG2 or IgG4 hinge region combined with a "lower hinge" sequence (amino acid residues at positions 228-236 according to EU numbering) derived from a human IgG1, IgG2 or IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 upper hinge or a human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. H Antibodies comprising the regions, in certain embodiments, exhibit altered Fc effector functions without negatively affecting the therapeutic or pharmacokinetic properties of the antibody (see, e.g., U.S. Provisional Application No. 61 / 759,578, filed February 1, 2013, the disclosure of which is incorporated herein by reference in its entirety).

[0172] Antibody Biology Generally, the antibodies disclosed herein function by binding to influenza B HA. For example, the present disclosure provides antibodies that bind to influenza B HA with a K of less than 10 nM as measured by surface plasmon resonance on a Biacore instrument or a real-time biolayer interferometer-based biosensor (Octet HTX assay). D In certain embodiments, the antibodies or antigen-binding fragments thereof bind to influenza B HA with a K of less than about 5 nM, less than about 2 nM, less than about 1 nM, less than about 500 pM, less than 250 pM, or less than 100 pM, as measured by surface plasmon resonance, e.g., using an assay format described herein, or a substantially similar assay. D Combine with.

[0173] The present disclosure also includes antibodies and antigen-binding fragments thereof that bind influenza B HA with a dissociation half-life (t1 / 2) of greater than about 75 minutes as measured by surface plasmon resonance at 37° C., e.g., using an assay format as defined herein, or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments disclosed herein bind influenza HA with a t1 / 2 of greater than about 200 minutes, greater than about 300 minutes, greater than about 400 minutes, greater than about 500 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, or greater than about 1000 minutes as measured by surface plasmon resonance at 25° C., e.g., using an assay format as defined herein (e.g., mAb capture format or antigen capture format), or a substantially similar assay.

[0174] The present disclosure also includes antibodies or antigen-binding fragments thereof that neutralize the infectivity of influenza viruses to their host cells. In some embodiments, the antibodies have an IC in the range of about 1 pM to about 800 nM in a microneutralization test assay or a substantially similar assay against various representative influenza viruses, such as B / Victoria / 2 / 87, B / Yamagata / 16 / 88, B / Maryland / 03 / 2008, B / Nanchang / 3451 / 93, and / or B / Florida / 4 / 2006. 50 , for example, in the range of about 1 pM to about 800 pM 50 , IC in the range of about 1 pM to about 10 pM 50 , IC in the range of about 10 pM to about 50 pM 50 , IC in the range of about 1 pM to about 100 pM 50 , IC in the range of about 10 pM to about 100 pM 50 , IC in the range of about 100 pM to about 800 pM 50 , or IC in the range of about 500 pM to about 800 pM 50 IC in the range of about 1 nM to about 10 nM 50 , IC in the range of about 10 nM to about 50 nM 50 , IC in the range of about 1 nM to about 100 nM 50 , IC in the range of about 10 nM to about 100 nM 50, IC in the range of about 100 nM to about 800 nM 50 or an IC in the range of about 500 nM to about 800 nM 50 The neutralizing efficacy of

[0175] The present disclosure also includes antibodies or antigen-binding fragments thereof that bind to influenza B infected cells at sub-nM concentrations and exhibit specific binding to HA (see Example 3).

[0176] The present disclosure also includes anti-influenza B HA antibodies that show increased protection or potent neutralization of influenza B infection in vivo (compared to untreated controls). Certain antibodies show protection when administered prophylactically (pre-infection, see Example 4). In some embodiments, a single dose of 5 mg / kg or 0.5 mg / kg of anti-HA antibody administered 5 days prior to infection resulted in 100% survival of mice when administered prophylactically, compared to mice treated with a human IgG1 isotype control antibody.

[0177] Prophylactic treatment with an anti-influenza B HA antibody or antigen-binding fragment thereof can, for example, protect a mammal from infection by subsequent exposure to influenza virus or reduce the likelihood or risk of infection by subsequent exposure to influenza virus. The protection is selected from the group consisting of ameliorating, alleviating, or reducing the severity, duration, or frequency of occurrence of at least one symptom of influenza infection. In some embodiments, the isolated antibody reduces the risk of influenza infection when administered prophylactically to an animal (e.g., a mammal) prior to exposure to influenza virus (e.g., at least 2 days, at least 3 days, at least 4 days, at least 5 days, or 2-5 days prior to exposure to influenza virus). The at least one symptom can be selected from the group consisting of headache, fever, aches, rhinorrhea (nasal congestion), chills, fatigue, weakness, sore throat, cough, shortness of breath, vomiting, diarrhea, pneumonia, bronchitis, and death.

[0178] In one embodiment, the isolated recombinant antibody or antigen-binding fragment thereof that specifically binds influenza B HA has the following characteristics: (a) It is a fully human monoclonal antibody; (b) About 10 -9 Less than, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 EC below M 50 binds influenza B HA; (c) exhibits an increase in survival of influenza-infected animals compared to comparable influenza-infected animals without treatment with the isolated recombinant antibody or antigen-binding fragment thereof that specifically binds influenza B HA; and / or (d) (i) three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained in a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 2; and (ii) three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 10 Includes has one or more of the following:

[0179] The antibodies disclosed herein may possess one or more of the foregoing biological characteristics, or any combination thereof. Additional biological characteristics of the antibodies disclosed herein will be apparent to those of skill in the art from a consideration of this disclosure, including the Examples herein.

[0180] Epitope mapping and related techniques The present disclosure includes anti-influenza B HA antibodies that interact with one or more amino acids found within one or more domains of the influenza B HA molecule. The epitope to which the antibody binds may consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) amino acids located within the influenza HA molecule (e.g., a linear epitope within a domain). Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) located within the influenza B HA molecule (e.g., a conformational epitope).

[0181] Various techniques known to those skilled in the art can be used to determine whether an antibody "interacts with one or more amino acids" in a polypeptide or protein. Exemplary techniques include routine cross-blocking assays, such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Other methods include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide truncation analysis crystallographic studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer (2000) Prot. Sci. 9:487-496). Another method that can be used to identify amino acids in a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, the hydrogen / deuterium exchange method involves labeling the protein of interest with deuterium, followed by binding of the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, and exchangeable protons in amino acids protected by the antibody complex undergo deuterium to hydrogen back-exchange at a slower rate than exchangeable protons in amino acids that are not part of the interface. As a result, amino acids that form part of the protein / antibody interface may retain deuterium and therefore exhibit a relatively high mass compared to amino acids that are not included in the interface. After dissociation of the antibody, the target protein undergoes protease cleavage and mass spectrometry analysis, thereby revealing deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267:252-259, Engen and Smith (2001) Anal.Chem.73:256A-265A.

[0182] The term "epitope" refers to a site on an antigen to which B cells and / or T cells respond. B cell epitopes can be formed from either contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically comprises at least 3 amino acids, or at least 4 amino acids, e.g., at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids in a unique spatial conformation.

[0183] Modification-assisted profiling (MAP), also known as antigen structure-based antibody profiling (ASAP), is a method for classifying a large number of monoclonal antibodies (mAbs) directed against the same antigen according to the similarity of the binding profile of each antibody to a chemically or enzymatically modified antigen surface (see US2004 / 0101920, specifically incorporated herein by reference in its entirety). Each category may reflect a unique epitope that is distinctly different or partially overlapping with the epitope represented by another category. The technique allows for rapid filtering of genetically identical antibodies, so that characterization can focus on genetically distinct antibodies. When applied to hybridoma screening, MAP may facilitate the identification of rare hybridoma clones that produce monoclonal antibodies with desired properties. MAP can be used to classify the antibodies described herein into groups of antibodies that bind to different epitopes.

[0184] In certain embodiments, the influenza virus A HA antibody or antigen-binding fragment thereof binds to an epitope within one or more regions exemplified by influenza HA, either in its native form or recombinantly produced, or to a fragment thereof.

[0185] The present disclosure includes anti-influenza B HA antibodies that bind to the same epitope, or a portion of the epitope. Similarly, the present disclosure also includes anti-influenza B HA antibodies that compete for binding to influenza B HA or a fragment thereof with any of the specific exemplary antibodies described herein. For example, the present invention includes anti-influenza B HA antibodies that cross-compete for binding to influenza B HA with one or more antibodies derived from the antibodies described in Table 1.

[0186] By using known methods, it can be determined whether an antibody binds to the same epitope as a reference anti-influenza B HA antibody or competes for binding. For example, to determine whether a test antibody binds to the same epitope as a reference anti-influenza B HA antibody, the reference antibody can be bound to influenza B HA or peptide under saturation conditions. The ability of the test antibody to bind to influenza B HA molecules is then evaluated. If the test antibody can bind to influenza B HA after saturation binding with the reference anti-influenza B HA antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-influenza B HA antibody. On the other hand, if the test antibody cannot bind to influenza B HA after saturation binding with the reference anti-influenza B HA antibody, the test antibody may bind to the same epitope as the epitope bound by the reference anti-influenza B HA antibody.

[0187] To determine whether an antibody competes for binding with a reference anti-influenza B HA antibody, the binding methodology described above is performed in two directions: in the first direction, the reference antibody is allowed to bind to influenza B HA under saturating conditions, followed by evaluating the binding of the test antibody to the influenza B HA molecule. In the second direction, the test antibody is allowed to bind to influenza B HA molecules under saturating conditions, followed by evaluating the binding of the reference antibody to the influenza B HA molecule. If in both directions only the first (saturating) antibody can bind to the influenza B HA molecule, it is concluded that the test antibody and the reference antibody compete for binding to influenza B HA. As will be understood by those skilled in the art, an antibody that competes for binding to a reference antibody does not necessarily have to bind to the same epitope as the reference antibody, and may sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0188] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the antigen. That is, a 1, 5, 10, 20, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, for example 75%, 90%, or even 99%, as measured by competitive binding assays (see, for example, Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody.

[0189] Additional routine experiments (e.g., peptide mutations and binding analysis) can be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. This type of experiment can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0190] Immunoconjugates Contemplated herein are human anti-influenza B HA monoclonal antibodies conjugated to a therapeutic moiety such as a toxoid or antiviral drug for treating influenza virus infection ("immunoconjugate"). As used herein, the term "immunoconjugate" refers to an antibody that is chemically or biologically linked to a radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, peptide or protein, or therapeutic agent. The antibody may be linked to a radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, peptide or therapeutic agent anywhere along the molecule as long as it is capable of binding to its target. Examples of immunoconjugates include antibody drug conjugates and antibody-toxin fusion proteins. In one embodiment, the agent may be a second, different antibody against influenza-HA. In certain embodiments, the antibody may be conjugated to an agent specific for virus-infected cells. The type of therapeutic moiety that may be conjugated to the anti-influenza-HA antibody takes into account the condition being treated and the desired therapeutic effect to be achieved. Examples of suitable agents for forming immunoconjugates are known in the art, see, for example, WO05 / 103081.

[0191] multispecific antibodies The antibody disclosed herein may be monospecific, bispecific, or multispecific.Multispecific antibodies may be specific to different epitopes of one target polypeptide, or may contain antigen-binding domains specific to two or more target polypeptides.See, for example, Tutt et al., 1991, J.Immunol.147:60-69; Kufer et al., 2004, Trends Biotechnol.22:238-244.

[0192] Any of the multispecific antigen-binding molecules or variants thereof provided herein can be constructed using standard molecular biology techniques (e.g., recombinant DNA and protein expression techniques) that will be known to those skilled in the art.

[0193] In some embodiments, influenza B HA-specific antibodies are generated in a bispecific format ("bispecific") in which variable regions that bind to separate domains of influenza B HA are linked together to provide dual domain specificity within a single binding molecule. Properly designed bispecifics can enhance overall influenza B HA-protein inhibitory efficacy by increasing both specificity and binding affinity. Variable regions with specificity for individual domains (e.g., segments of the N-terminal domain), or variable regions that can bind to different regions within one domain, are paired on a structural scaffold that allows each region to simultaneously bind separate epitopes, or different regions within one domain. In one example, for bispecificity, a heavy chain variable region (V) from a binding molecule with specificity for one domain is paired with a heavy chain variable region (V H ) is combined with a series of binding-derived light chain variable regions (V L ) and rearrange it into V H Without disturbing the original singularity of the original V H Non-cognate V that can be paired with L Identify your partner. In this way, a single V L Segment (e.g., V L 1) Two different V HDomain (e.g., V H 1 and V H 2) to form two binding "arms" (V H 1-V L 1 and V H 2-V L 1) can be used to generate bispecifics consisting of a single V L The use of segments reduces the complexity of the system, thereby simplifying and improving the efficiency of the cloning, expression, and purification processes used to generate bispecifics (see, e.g., USSN13 / 022759 and US2010 / 0331527).

[0194] Alternatively, two or more domains and antibodies that bind to a second target, such as, but not limited to, a second different anti-influenza B HA antibody, may be prepared in a bispecific format using the techniques described herein or other techniques known to those skilled in the art. An antibody variable region that binds to a separate region can be linked to a variable region that binds to a relevant site on the influenza virus, for example, to provide dual antigen specificity within a single binding molecule. This property of properly designed bispecificity serves a dual function. A variable region with specificity for the extracellular domain is combined with a variable region with specificity for the outside of the extracellular domain, and paired on a structural scaffold that allows each variable region to bind to a separate antigen.

[0195] An exemplary bispecific antibody format that can be used in the context disclosed herein is a first immunoglobulin (Ig) C H 3 domain and second Ig C H 3 domains, in which the first and second Ig C H The three domains differ from each other by at least one amino acid, and in this case, the at least one amino acid difference reduces binding of the bispecific antibody to Protein A compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig C H The 3 domain binds to protein A and the second Ig CH The third domain contains a mutation that reduces or eliminates Protein A binding, such as the H95R (according to IMGT exon numbering; H435R in EU numbering) modification. H 3 may further include a Y96F modification (Y436F in EU according to IMGT). H Additional modifications that may be found in 3 include: D16E, L18M, N44S, K52N, V57M, and V82I for IgG1 antibodies (D356E, L358M, N384S, K392N, V397M, and V422I in EU by IMGT), N44S, K52N, and V82I for IgG2 antibodies (N384S, K392N, and V422I in IMGT, EU), and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I for IgG4 antibodies (Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I in EU by IMGT). Variations on the bispecific antibody formats described above are also contemplated within the scope of the present disclosure.

[0196] Other exemplary bispecific formats that may be used in the context disclosed herein include, but are not limited to, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadromas, knobs-into-holes, common light chains (e.g., common light chains with knobs-into-holes), CrossMab, CrossFab, (SEED) bodies, leucine zippers, duobodies, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab 2Bispecific formats include (for a review of the aforementioned formats, see, for example, Klein et al. 2012, mAbs 4:6,1-11, and references cited therein). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, for example using unnatural amino acids with orthogonal chemical reactivity to generate site-specific antibody-oligonucleotide conjugates that then self-assemble into multimeric complexes with defined composition, valency and structure. (See, for example, Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

[0197] Therapeutic Administration and Formulations Provided herein is a therapeutic composition comprising an anti-influenza B HA antibody or an antigen-binding fragment thereof. The therapeutic composition according to the present disclosure is administered with pharma- ceutically acceptable carriers, excipients, and other agents that are incorporated into the formulation to provide improved transport, delivery, tolerability, and the like. Numerous suitable formulations can be found in a formulary known to every pharmacist: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipids (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0198] The dose of the antibody may vary depending on the age and size of the subject to be administered, the target disease, condition, route of administration, etc. When the antibodies disclosed herein are used to treat a disease or disorder in an adult patient or to prevent such a disease, it is advantageous to administer the antibodies disclosed herein in a single dose, typically about 0.1 to about 60 mg per kg of body weight, or about 5 to about 60 mg, about 10 to about 50 mg, or about 20 to about 50 mg per kg of body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. In certain embodiments, the antibody or antigen-binding fragment thereof may be administered as an initial dose of at least about 0.1 mg to about 5000 mg, about 1 to about 2000 mg, about 5 to about 1000 mg, or about 10 to about 500 mg, about 100 mg, or about 50 mg. In certain embodiments, the initial dose may be followed by administration of a second or multiple subsequent doses of the antibody or antigen-binding fragment thereof in an amount that may be approximately the same as the initial dose or a smaller dose than the initial dose, where the subsequent doses are separated by at least 1 to 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 12 weeks, or at least 14 weeks.

[0199] A variety of delivery systems are known and can be used to administer pharmaceutical compositions, including, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis, and the like (see, for example, Wu et al. (1987) J.Biol.Chem.262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (such as, for example, oral mucosa, rectal mucosa, and intestinal mucosa), and can be administered together with other biologically active agents. Administration can be systemic or local. Pharmaceutical compositions can be delivered in vesicles, in particular liposomes (see, eg, Langer (1990) Science 249:1527-1533).

[0200] The use of nanoparticles to deliver the antibodies disclosed herein is also contemplated herein. Antibody-conjugated nanoparticles can be used for both therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and preparation and use methods are described in detail in Arruebo, M., et al. 2009 ("Antibody-conjugated nanoparticles for biomedical applications" in J.Nanomat.Volume 2009,Article ID 439389,24 pages,doi:10.1155 / 2009 / 439389), which is incorporated herein by reference. Nanoparticles may be developed and conjugated to antibodies contained in pharmaceutical compositions to target virus-infected cells. Nanoparticles for drug delivery are also described, for example, in US8257740, or US8246995, each of which is incorporated herein in its entirety.

[0201] In certain circumstances, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, a polymeric material may be used. In yet another embodiment, the controlled release system may be placed in the vicinity of the target of the composition, thus requiring only a fraction of the systemic dose.

[0202] The injectable preparations may include administration forms such as intravenous, subcutaneous, intradermal, intracranial, intraperitoneal and intramuscular injections, infusions, etc. These injectable preparations may be prepared by known methods. For example, the injectable preparations may be prepared by dissolving, suspending, or emulsifying the above-mentioned antibody or its salt in a sterile aqueous or oily medium conventionally used for injections. The aqueous medium for injection may be, for example, physiological saline, an isotonic solution containing glucose, and other auxiliary agents, which may be combined with a suitable solubilizing agent such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. The oily medium may be, for example, sesame oil, soybean oil, etc., which may be combined with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection solution thus prepared is preferably filled into a suitable ampoule.

[0203] The pharmaceutical compositions disclosed herein can be delivered subcutaneously or intravenously with a standard needle and syringe. Furthermore, for subcutaneous delivery, a pen delivery device facilitates the use in delivering the pharmaceutical compositions disclosed herein. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition inside the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device is sold pre-filled with the pharmaceutical composition held in a reservoir inside the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0204] Numerous reusable pen and autoinjector delivery devices find use in the subcutaneous delivery of the pharmaceutical compositions disclosed herein. Examples include the AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN™, to name just a few. Examples of disposable pen-type delivery devices that find use in the subcutaneous delivery of pharmaceutical compositions disclosed herein include, but are in no way limited to, SOLOSTAR Pen (Sanofi-Aventis), FLEXPEN (Novo Nordisk), and KWIKPEN (Eli Lilly), SURECLICK Autoinjector (Amgen, Thousand Oaks, CA), PENLET (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP) and HUMIRA Pen (Abbott Labs, Abbott Park, IL), to name just a few.

[0205] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared in dosage forms with unit doses suitable for adapting the dose of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of antibody contained is generally about 5 to about 5000 mg per dosage form in unit dose, and particularly in the form of injection, the antibody is preferably contained in about 5 to about 500 mg, and for other dosage forms, it is preferably contained in about 10 to about 250 mg.

[0206] Therapeutic uses of antibodies The antibodies disclosed herein are useful for treating and / or preventing a disease or disorder or condition associated with influenza B virus infection and / or ameliorating at least one symptom associated with such disease, disorder or condition.

[0207] In certain embodiments, the antibodies provided herein are useful for treating subjects suffering from severe and acute respiratory infections caused by influenza B virus. In some embodiments, the antibodies are useful for reducing viral titers or reducing viral load in a host. In one embodiment, the antibodies or antigen-binding fragments of the present invention may be administered in therapeutic doses to patients with influenza B virus infection.

[0208] For example, one or more antibodies disclosed herein can be administered to alleviate or prevent or reduce the severity of one or more symptoms or conditions of a disease or disorder compared to an untreated but similarly situated subject. The antibodies can be used to ameliorate or reduce the severity of at least one symptom of influenza virus infection, including, but not limited to, fever, cough, sore throat, headache, body aches, fatigue, exhaustion, shortness of breath, bronchitis, pneumonia, and death.

[0209] It is also contemplated that one or more of the antibodies disclosed herein may be used prophylactically in subjects at risk of developing an influenza virus infection, such as immunocompromised individuals, the elderly (over 65 years of age), children under the age of 2, healthcare workers, close family members of patients with influenza virus infection, and patients with a medical history (e.g., increased risk of lung infections, heart disease, or diabetes).

[0210] In a further embodiment, the antibody is used in the preparation of a pharmaceutical composition for treating a patient suffering from an influenza virus infection, hi another embodiment, the antibody is used as an adjunct therapy with any other agent or treatment known to one of skill in the art useful for treating or ameliorating an influenza virus infection.

[0211] Combination therapy Combination therapy can include anti-influenza B HA antibodies and any additional therapeutic agent that can be advantageously combined with the antibody or biologically active fragment of the antibody. The antibodies disclosed herein can be synergistically combined with one or more drugs or agents (e.g., antiviral agents) used to treat influenza viruses.

[0212] For example, exemplary antiviral agents include, for example, vaccines, neuraminidase inhibitors, or nucleoside analogs. Other exemplary antiviral agents that may be used in combination with the antibodies provided herein include, for example, zidovudine, gangcyclovir, vidarabine, idoxuridine, trifluridine, foscarnet, acyclovir, ribavirin, amantadine, remantidine, saquinavir, indinavir, ritonavir, alpha-interferon and other interferons, neuraminidase inhibitors (e.g., zanamivir (RELENZA®), oseltamivir (TAMIFLU®), laninamivir, peramivir), or rimantadine.

[0213] Other exemplary antiviral drugs include, but are not limited to, HA inhibitors, sialic acid inhibitors, and M2 ion channel inhibitors. In one embodiment, the M2 ion channel inhibitor is amantadine or rimantadine.

[0214] In some embodiments, the antibodies provided herein may be combined with a second therapeutic agent to reduce the viral load in a patient with an influenza virus infection or to ameliorate one or more symptoms of the infection.

[0215] The antibodies provided herein may be used in combination with anti-inflammatory drugs (e.g., corticosteroids, and nonsteroidal anti-inflammatory drugs), decongestants, antihistamines, anti-infective drugs, different antibodies against influenza virus, antiviral drugs, vaccines against influenza virus such as FLUMIST® or FLUVIRIN®, nutritional supplements such as antioxidants, or any other palliative therapy to treat influenza virus infection. Palliative therapy includes treating a subject to make the subject more comfortable and / or reduce the severity of the symptoms of influenza B infection.

[0216] In certain embodiments, the second therapeutic agent is another antibody against influenza. In certain embodiments, the second therapeutic agent is another antibody against influenza hemagglutinin (e.g., influenza A hemagglutinin). In certain embodiments, the second therapeutic agent is another antibody against a different influenza protein, such as neuraminidase, or tetrameric ectodomain of matrix protein 2 (M2e protein). In certain embodiments, the second therapeutic agent is an antibody against a different protein, such as host transmembrane protease, serine 2 (TMPRSS2). The second antibody may be specific for one or more different influenza virus proteins from different subtypes or strains of the virus. It is contemplated herein to use a combination ("cocktail") of antibodies that have broad neutralizing or inhibitory activity against influenza viruses. In some embodiments, non-competing antibodies can be combined and administered to a subject in need thereof to reduce the ability of influenza viruses to escape due to rapid mutation as a result of selection pressure. In some embodiments, the antibodies comprising the combination bind to distinct, non-overlapping epitopes on the HA protein. The antibodies, including combinations, may block viral attachment and / or entry into and / or fusion with host cells. The antibodies may interact with a hemagglutinin selected from any one or more of the influenza B strains, and when used alone or in combination with any one or more of the agents described above, may neutralize any one or more of the influenza B strains, including B / Victoria / 2 / 87, B / Yamagata / 16 / 88, B / Maryland / 03 / 2008, B / Nanchang / 3451 / 93, and / or B / Florida / 4 / 2006.

[0217] Also contemplated herein is the use of combinations of anti-influenza HA antibodies in addition to the anti-influenza B HA antibodies disclosed herein, where the combination comprises one or more antibodies that do not cross-compete, and in some embodiments the combination comprises a first antibody with broad neutralizing activity and a second antibody that has activity against a narrow spectrum of isolates and does not cross-compete with the first antibody.

[0218] As used herein, the term "in combination with" refers to an additional therapeutically active ingredient that may be administered before, simultaneously with, or after administration of an anti-influenza B HA antibody of the invention. The term "in combination with" also includes sequential or simultaneous administration of an anti-influenza-HA antibody and a second therapeutic agent.

[0219] The additional therapeutically active component may be administered to the subject prior to administration of the anti-influenza B HA antibody disclosed herein. For example, the first component may be considered to be administered "before" the second component if the first component is administered 1 week, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or less than 1 minute before administration of the second component. In other embodiments, the additional therapeutically active component may be administered to the subject after administration of the anti-influenza B HA antibody disclosed herein. For example, a first component may be considered to be administered "after" a second component if the first component is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours after administration of the second component. In yet other embodiments, an additional therapeutically active component may be administered to a subject simultaneously with administration of an anti-influenza B HA antibody disclosed herein. For purposes of this disclosure, "concurrent" administration includes, for example, administration of an anti-influenza B HA antibody and an additional therapeutically active component to a subject in a single dosage form or in separate dosage forms that are administered to the subject within about 30 minutes of each other. When administered in separate dosage forms, each dosage form can be administered via the same route (e.g., both the anti-influenza B HA antibody and the additional therapeutically active ingredient can be administered intravenously), or alternatively, each dosage form can be administered via a different route (e.g., the anti-influenza B HA antibody can be administered intravenously and the additional therapeutically active ingredient can be administered orally). In either case, administration of the ingredients in a single dosage form, separate dosage forms via the same route, or separate dosage forms via different routes, are all considered "co-administration" for purposes of this disclosure. For purposes of this disclosure, administration of an anti-influenza B HA antibody "before," "concurrently" or "after" administration of the additional therapeutically active ingredient (as these terms are defined above) is considered administration of the anti-influenza B HA antibody "in combination" with the additional therapeutically active ingredient.

[0220] The present disclosure includes pharmaceutical compositions in which the anti-influenza B HA antibodies disclosed herein are formulated together with one or more additional therapeutically active ingredients as described elsewhere herein.

[0221] Dosing regimen According to certain embodiments, a single dose of the anti-influenza B HA antibody or antigen-binding fragment thereof provided herein (or a pharmaceutical composition comprising a combination of the anti-influenza HA antibody and any of the additional therapeutically active agents mentioned herein) can be administered to a subject in need thereof. According to certain embodiments disclosed herein, multiple doses of the anti-influenza B HA antibody (or a pharmaceutical composition comprising a combination of the anti-influenza B HA antibody and any of the additional therapeutically active agents mentioned herein) can be administered to a subject over a defined time course. The method according to this aspect of the disclosure includes sequentially administering multiple doses of the anti-influenza B HA antibody to the subject. As used herein, "sequentially administering" means that each dose of the anti-influenza B HA antibody is administered to the subject at different times, such as, for example, on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present disclosure includes a method including sequentially administering to a patient a single initial dose of the anti-influenza B HA antibody, followed by one or more secondary doses of the anti-influenza B HA antibody, and then optionally one or more tertiary doses of the anti-influenza B HA.

[0222] The terms "initial dose", "secondary dose", and "tertiary dose" refer to the temporal order of administration of the anti-influenza B HA antibody of the present invention. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"), a "secondary dose" is a dose administered after the initial dose, and a "tertiary dose" is a dose administered after the secondary dose. The initial dose, secondary dose, and tertiary dose may all contain the same amount of anti-influenza-HA antibody, but generally may differ from each other in terms of frequency of administration. However, in certain embodiments, the amount of anti-influenza B HA antibody contained in the initial dose, secondary dose, and / or tertiary dose varies from each other (e.g., adjusted up or down) during the course of treatment. In certain embodiments, two or more doses (e.g., 2, 3, 4, or 5) are administered as "loading doses" at the beginning of a treatment regimen, with subsequent doses administered on a less frequent basis (e.g., "maintenance doses").

[0223] In certain exemplary embodiments, each of the secondary and / or tertiary doses is administered 1 to 48 hours (e.g., 1 hour, 1 1 / 2 hours, 2 hours, 2 1 / 2 hours, 3 hours, 3 1 / 2 hours, 4 hours, 4 1 / 2 hours, 5 hours, 5 1 / 2 hours, 6 hours, 6 1 / 2 hours, 7 hours, 7 1 / 2 hours, 8 hours, 8 1 / 2 hours, 9 hours, 9 1 / 2 hours, 10 hours, 10 1 / 2 hours, 11 hours, 11 1 / 2 hours, 12 hours, 12 1 / 2 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 81 hours, 82 hours, 83 hours, 84 hours, 85 hours, 86 The phrase "immediately preceding dose" as used herein refers to a dose of anti-influenza B HA antibody administered to a patient in a multiple administration sequence with no intervening dose prior to administration of the next dose in the sequence.

[0224] The method according to this aspect of the invention may include administering any number of secondary and / or tertiary doses of anti-influenza B HA antibodies to the patient. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0225] In certain embodiments, the frequency at which the second and / or third doses are administered to a patient may vary during the course of the treatment regimen. The frequency of administration may be adjusted during the course of treatment by a physician depending on the needs of an individual patient after clinical testing.

[0226] Diagnostic Uses of Antibodies Anti-influenza B HA antibodies may be used to detect and / or measure influenza B HA in a sample, for example, for diagnostic purposes. Some embodiments contemplate the use of one or more antibodies in an assay to detect a disease or disorder, such as a viral infection. An exemplary diagnostic assay for influenza B HA includes, for example, contacting a sample obtained from a patient with an anti-influenza B HA antibody of the invention, where the anti-influenza B HA antibody is labeled with a detectable label or reporter molecule or is used as a capture ligand to selectively isolate influenza B HA from the patient sample. Alternatively, an unlabeled anti-influenza B HA antibody can be used for diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be, for example, a marker that is capable of detecting influenza B HA. 3 H, 14 C. 32 P, 35 S, or 125The immunoreactive agent may be a radioisotope such as I, a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine, or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure influenza B HA in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).

[0227] Samples that can be used in influenza B HA diagnostic assays include any tissue or fluid sample that can be obtained from a patient that contains a detectable amount of influenza B HA, or any of its fragments, under normal or pathological conditions.Generally, the level of influenza B HA in a particular sample obtained from a healthy patient (e.g., a patient not suffering from influenza-related disease) is measured to first establish a baseline or standard influenza B HA level.This baseline level of influenza B HA can then be compared with the level of influenza B HA measured in a sample obtained from an individual suspected of having influenza B HA-related symptoms or symptoms associated with such conditions.

[0228] Antibodies specific for influenza B HA may contain no additional label or moiety, or may contain N-terminal or C-terminal label or moiety. In one embodiment, the label or moiety is biotin. In binding assays, the position of the label (if any) may determine the orientation of the peptide relative to the surface to which it binds. For example, if the surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C-terminal portion of the peptide is distal from the surface. EXAMPLES

[0229] The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the methods and compositions disclosed herein, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, room temperature is about 25°C, and pressure is at or near atmospheric pressure.

[0230] Example 1: Generation of human antibodies against influenza B hemagglutinin (HA) Human antibodies against influenza hemagglutinin were generated in VELOCIMMUNE® mice containing DNA encoding human immunoglobulin heavy chain variable regions and kappa light chain variable regions. Mice were immunized with a combination of vectors expressing influenza A and B hemagglutinins, followed by infection and recovery with influenza A and B strains, followed by a booster with a mixture of recombinant hemagglutinin proteins. Antibody immune responses were monitored by influenza HA-specific immunoassays. Anti-influenza B HA antibodies were isolated directly from antigen-positive mouse B cells without fusion with myeloma cells, as described in U.S. Pat. No. 7,582,298, which is expressly incorporated herein by reference in its entirety. Using this method, fully human anti-influenza HA antibodies (i.e., antibodies possessing human variable and human constant domains) were obtained.

[0231] The exemplary antibody described herein is designated mAb 35490. The biological properties of the exemplary antibodies generated according to the methods of this example are described in detail in the Examples set forth below.

[0232] Example 2: Amino acid and nucleotide sequences of the heavy and light chain variable regions Table 1 lists the heavy and light chain variable regions and CDRs of exemplary anti-influenza B HA antibodies, as well as amino acid sequence identifiers for the heavy and light chain sequences. The corresponding nucleic acid sequence identifiers are listed in Table 2.

[0233] Table 1: Amino acid sequence identifiers TIFF0007611908000001.tif21166

[0234] Table 2: Nucleic acid sequence identifiers TIFF0007611908000002.tif21166

[0235] The antibodies disclosed herein have fully human variable regions, but can have mouse constant regions (e.g., mouse IgG1 Fc or mouse IgG2 Fc (a or b isotypes)) or human constant regions (e.g., human IgG1 Fc or human IgG4 Fc). As will be appreciated by one of skill in the art, an antibody having a particular Fc isotype can be converted to an antibody having a different Fc isotype (e.g., an antibody having a mouse IgG1 Fc can be converted to an antibody having a human IgG4, etc.), but in either case, the variable domains (including the CDRs) indicated by the numerical identifiers shown in Tables 1 and 2 will remain the same, and the binding characteristics to the antigen are expected to be the same or substantially similar regardless of the nature of the Fc domain.

[0236] Example 3: Anti-hemagglutinin antibodies bind to influenza-infected cells MDCK London cells were seeded at 40,000 cells / well in 50 μL of infection medium (DMEM containing 1% sodium pyruvate, 0.21% low IgG BSA solution, and 0.5% gentamicin) in 96-well plates. The cells were incubated at 37°C with 5% CO2 for 4 hours. The plates were then infected with 50 μL of influenza B / Florida / 4 / 2006 virus (vaccine strain of B / Yamagata lineage) at a dilution of 10E-2.5, tapped, and placed back at 37°C with 5% CO2 for 20 hours. The plates were then washed once with PBS, fixed with 50 μL of 4% PFA in PBS, and incubated at room temperature for 15 minutes. The plates were washed three times with PBS and blocked with 300 μL of StartingBlock™ (PBS) blocking buffer (Thermo Fisher Scientific) for 1 hour at room temperature. Influenza B anti-HA antibody mAb35490P (formatted with human IgG1 Fc) and human IgG1 isotype control antibody were diluted in blocking buffer to a starting concentration of 100 μg / mL and titrated 1:4 to a final concentration of 6.1E-3 μg / mL. After incubation, blocking buffer was removed from the plate and diluted antibody was added onto the cells at 75 μL / well. Plates were incubated at room temperature for 1 hour. After incubation, plates were washed 3 times with wash buffer (imidazole buffered saline and Tween® 20 diluted 1X with Milli-Q water) and overlaid with 75 μL / well of secondary antibody (Peroxidase AffiniPure donkey anti-human IgG, Jackson ImmunoResearch) diluted 1:2000 in blocking buffer. Secondary solution was incubated on the plate at room temperature for 1 hour. Plates were then washed 3 times with wash buffer and 75 μL / well of colorimetric ELISA Pico Substrate (prepared 1:1) was added. Plates were immediately read for luminescence on a Molecular Devices Spectramax i3x plate reader. EC 50 The data are shown in Table 3.

[0237] (Table 3) FluB infected cells anti-HA binding ELISA TIFF0007611908000003.tif15148

[0238] As can be seen in Table 3, mAb35490 binds to influenza B infected cells at sub-nM concentrations and demonstrates specific binding to HA.

[0239] Example 4: Prevention of influenza B-induced mortality by prophylactic antibody administration Balb / C Elite mice were treated with 5 mg / kg or 0.5 mg / kg mAb35490 (formatted with human IgG1 Fc) or 5 mg / kg human IgG1 isotype control antibody diluted in PBS by subcutaneous injection into the neck scrape 5 days prior to infection. On the day of infection, influenza B / Florida / 4 / 2006 virus stock was thawed on ice and diluted in PBS from a 2.05E08 pfu / mL stock to contain 4000 pfu virus in 20 μL of PBS (2.0E05 pfu / mL). Virus was kept on ice at all times. Each mouse was injected with a cocktail of 2.4 mg / kg ketamine and 0.1 mg / kg xylazine and allowed to rest for approximately 10-15 min until completely asleep. Each mouse was then administered 20 μL of virus (4000 pfu) intranasally to allow complete inhalation of the virus. Mice were then monitored for weight loss and morbidity for two weeks post-infection. Mice were euthanized if they lost more than 25% of their initial weight at the time of infection. The results are shown in Table 4.

[0240] Table 4. Mouse survival after administration of mAb35490 or isotype control TIFF0007611908000004.tif28148

[0241] All animals treated with the human IgG1 isotype control antibody succumbed to infection and had to be euthanized by day 8 post-infection. All animals treated with both 5 mg / kg and 0.5 mg / kg mAb35490 survived infection, demonstrating the efficacy of the FluB anti-HA antibody mAb35490.

Claims

1. 1. An isolated and recombinant antibody or antigen-binding fragment thereof that specifically binds to influenza B hemagglutinin (HA), comprising: (i) a heavy chain variable region (HCVR) comprising a heavy chain complementarity determining region (HCDR) 1 domain comprising the amino acid sequence of SEQ ID NO: 4, a HCDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a HCDR3 domain comprising the amino acid sequence of SEQ ID NO: 8; and (ii) a light chain variable region (LCVR) comprising a light chain complementarity determining region (LCDR) 1 domain comprising the amino acid sequence of SEQ ID NO: 12, a LCDR2 domain comprising the amino acid sequence of SEQ ID NO: 14, and a LCDR3 domain comprising the amino acid sequence of SEQ ID NO:

16.

1. An isolated recombinant antibody or antigen-binding fragment thereof comprising:

2. An isolated recombinant antibody or antigen-binding fragment thereof described in claim 1, wherein HCVR comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, and LCVR comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

10.

3. The antibody or antigen-binding fragment thereof has the following characteristics: (a)10 －９ EC below M ５０ and / or binds to influenza B HA at (b) When administered prophylactically to animals 5 days prior to influenza B infection, it shows an increase in survival compared to untreated animals similarly exposed to influenza B.

3. The isolated antibody or antigen-binding fragment thereof of claim 1 or 2, having one or both of the following:

4. 4. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein when the isolated antibody or antigen-binding fragment thereof is administered to a mammal, the isolated antibody or antigen-binding fragment thereof ameliorates, alleviates, or reduces the severity, duration, or frequency of occurrence of at least one symptom of influenza infection in the mammal.

5. 5. The isolated antibody or antigen-binding fragment thereof of claim 4, wherein the at least one symptom is selected from the group consisting of headache, fever, pain, rhinorrhea (nasal congestion), chills, fatigue, weakness, sore throat, cough, shortness of breath, vomiting, diarrhea, pneumonia, bronchitis, and death.

6. An isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 5, which, when administered to each of a plurality of mammals as a single prophylactic dose of 0.5 mg / kg, said plurality of mammals have a survival rate of at least 80% on the 19th day after administration.

7. An isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 6, which, when administered to each of a plurality of mammals as a single prophylactic dose of 0.5 mg / kg, said plurality of mammals have a survival rate of at least 90% on the 19th day after administration.

8. An isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 7, which, when administered to each of a plurality of mammals as a single prophylactic dose of 0.5 mg / kg, said plurality of mammals have a 100% survival rate 19 days after administration.

9. An isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 5, which, when administered to each of a plurality of mammals as a single prophylactic dose of 5.0 mg / kg, said plurality of mammals have a survival rate of at least 80% on the 19th day after administration.

10. An isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 5 and 9, which, when administered to each of a plurality of mammals as a single prophylactic dose of 5.0 mg / kg, said plurality of mammals have a survival rate of at least 90% on the 19th day after administration.

11. An isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 5, 9 and 10, which, when administered to each of a plurality of mammals as a single prophylactic dose of 5.0 mg / kg, said plurality of mammals have a 100% survival rate 19 days after administration.

12. 12. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, comprising an HCVR comprising the amino acid sequence of SEQ ID NO:

2.

13. 13. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, comprising an LCVR comprising the amino acid sequence of SEQ ID NO:

10.

14. 12. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NO:2 / 10.

15. An isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 14, wherein the antibody is an IgG1 antibody.

16. An isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 14, wherein the antibody is an IgG4 antibody.

17. 17. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 16, which is a bispecific antibody.

18. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 17 and a pharma- ceutically acceptable carrier or diluent.

19. 20. The pharmaceutical composition of claim 18, further comprising a second therapeutic agent.

20. 20. The pharmaceutical composition of claim 19, wherein the second therapeutic agent is selected from the group consisting of an antiviral drug, an anti-inflammatory drug, a different antibody that specifically binds to influenza HA, a vaccine for influenza, a dietary supplement, and another palliative therapy for treating an influenza infection.

21. 21. The pharmaceutical composition of claim 20, wherein the anti-inflammatory agent is selected from the group consisting of corticosteroids and nonsteroidal anti-inflammatory agents.

22. 21. The pharmaceutical composition of claim 20, wherein the dietary supplement is an antioxidant.

23. 21. The pharmaceutical composition of claim 20, wherein the antiviral drug is oseltamivir.

24. 21. The pharmaceutical composition of claim 20, wherein the antiviral agent is an anti-influenza A agent.

25. 25. The pharmaceutical composition of claim 24, wherein the anti-influenza A drug is an antibody.

26. 26. The pharmaceutical composition of claim 25, wherein the antibody specifically binds to influenza A HA.

27. A polynucleotide molecule comprising a polynucleotide sequence encoding the HCVR and LCVR of the antibody of any one of claims 1 to 17.

28. A vector comprising the polynucleotide molecule of claim 27.

29. A cell comprising the vector of claim 28.

30. A method for producing an antibody or antigen-binding fragment thereof that specifically binds to influenza B hemagglutinin (HA), comprising culturing a cell described in claim 29 under conditions that permit production of the antibody or antigen-binding fragment thereof, and recovering the produced antibody or antigen-binding fragment thereof.

31. A polynucleotide molecule comprising a polynucleotide sequence encoding the HCVR of an antibody described in any one of claims 1 to 17.

32. A vector comprising the polynucleotide molecule described in claim 31.

33. A cell comprising the vector described in claim 32.

34. A first nucleic acid molecule comprising a polynucleotide sequence encoding the HCVR of the antibody of any one of claims 1 to 17; and A second nucleic acid molecule comprising a polynucleotide sequence encoding the LCVR of said antibody. A composition comprising:

35. A first nucleic acid molecule comprising a polynucleotide sequence encoding the HCVR of the antibody of any one of claims 1 to 17; and A second nucleic acid molecule comprising a polynucleotide sequence encoding the LCVR of said antibody. comprising a cell.

36. A method for producing an antibody or antigen-binding fragment thereof that specifically binds to influenza B hemagglutinin (HA), comprising culturing a cell described in claim 35 under conditions that permit production of the antibody or antigen-binding fragment thereof, and recovering the produced antibody or antigen-binding fragment thereof.

37. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 17, or the pharmaceutical composition of any one of claims 18 to 26, for use in a method for preventing, treating or ameliorating at least one symptom of influenza infection in an influenza-infected subject.

38. 38. The pharmaceutical composition of claim 37, wherein the at least one symptom is selected from the group consisting of fever, cough, body aches, nasal discharge, shortness of breath, pneumonia and bronchitis.

39. 39. The pharmaceutical composition of claim 37 or 38, formulated for prophylactic use.

40. The pharmaceutical composition of any one of claims 37 to 39, wherein the subject is selected from the group consisting of immunocompromised individuals, adults aged 65 years or older, healthcare workers, and those with a medical history or underlying condition.

41. 41. The pharmaceutical composition of claim 40, wherein the underlying disease is selected from the group consisting of heart failure and diabetes.

42. The pharmaceutical composition of any one of claims 37 to 41 in combination with a second therapeutic agent.

43. 43. The pharmaceutical composition of claim 42, wherein the second therapeutic agent is selected from the group consisting of an antiviral drug, an anti-inflammatory drug, a different antibody that specifically binds to influenza HA, a vaccine for influenza, a dietary supplement, and another palliative therapy for treating an influenza infection.

44. 44. The pharmaceutical composition of claim 43, wherein the anti-inflammatory agent is selected from the group consisting of corticosteroids and nonsteroidal anti-inflammatory agents.

45. 44. The pharmaceutical composition of claim 43, wherein the dietary supplement is an antioxidant.

46. The pharmaceutical composition of any one of claims 42-45, wherein the second therapeutic agent is formulated for a different route of administration than the antibody or antigen-binding fragment thereof.

47. The pharmaceutical composition of any one of claims 42-46, wherein the second therapeutic agent is formulated for oral use.

48. 44. The pharmaceutical composition of claim 43, wherein the antiviral agent is oseltamivir.

49. 49. The pharmaceutical composition of claim 48, wherein the oseltamivir is administered prior to the antibody or antigen-binding fragment thereof.

50. 49. The pharmaceutical composition of claim 48, wherein the oseltamivir is used simultaneously with the antibody or antigen-binding fragment thereof.

51. 49. The pharmaceutical composition of claim 48, wherein the oseltamivir is administered after the antibody or antigen-binding fragment thereof.

52. 44. The pharmaceutical composition of claim 43, wherein the antiviral agent is an anti-influenza A agent.

53. 53. The pharmaceutical composition of claim 52, wherein the anti-influenza A drug is an antibody.

54. 54. The pharmaceutical composition of claim 53, wherein the antibody specifically binds to influenza A HA.

55. 55. The pharmaceutical composition of any one of claims 37 to 54, formulated for subcutaneous, intravenous, intradermal, intramuscular, intranasal, or oral use.

56. The pharmaceutical composition of any one of claims 37 to 55, wherein the antibody or antigen-binding fragment thereof protects the mammal from infection upon subsequent exposure to influenza B virus.

57. A pharmaceutical composition comprising an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to influenza B hemagglutinin (HA), comprising: The antibody or antigen-binding fragment thereof, (i) a HCVR comprising an HCDR1 domain comprising the amino acid sequence of SEQ ID NO: 4, an HCDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and an HCDR3 domain comprising the amino acid sequence of SEQ ID NO: 8; and (ii) an LCDR comprising an LCDR1 domain comprising the amino acid sequence of SEQ ID NO: 12, an LCDR2 domain comprising the amino acid sequence of SEQ ID NO: 14, and an LCDR3 domain comprising the amino acid sequence of SEQ ID NO: 16; Including, The pharmaceutical composition is formulated for systemic administration. Pharmaceutical compositions.

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