Anti-hemagglutinin antibodies and methods of use thereof

Abstract

Provided herein are monoclonal antibodies or antigen-binding fragments thereof that bind to an influenza B hemagglutinin (HA) protein, pharmaceutical compositions comprising the antibodies, and methods of use. The antibodies can be used to inhibit or neutralize the activity of an influenza B virus, thereby providing a method of treating or preventing an influenza infection in a human. Also provided is the use of one or more antibodies that bind to the influenza B HA for preventing viral adsorption and / or entry into a host cell. 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

[0001] Government licensing rights

[0002] This invention was completed with government support, pursuant to approval number HHSO100201700020C issued by the U.S. Department of Health and Human Services. The government holds certain rights to this invention.

[0003] sequence list

[0004] An official copy of the sequence list, in ASCII format, was submitted electronically via EFS-Web along with the instruction manual. The file name is “10625WO01_SEQ_LIST_ST25.txt”, created on October 28, 2020, and is approximately 36KB in size. The sequence list contained in this ASCII formatted file is part of the instruction manual and is incorporated herein by reference in its entirety. Background Technology

[0005] Influenza is a highly contagious disease with a long history, characterized by waves of pandemics, epidemics, resurgences, and outbreaks. Despite annual vaccination efforts, influenza infection still causes a significant number of illnesses and deaths.

[0006] There are four types of influenza viruses: A, B, C, and D. Influenza B causes a large number of seasonal flu infections and can be associated with severe influenza requiring hospitalization.

[0007] Hemagglutinin is a trimeric glycoprotein comprising two domains, a globular head domain consisting of a receptor-binding site (where antigenic drift typically occurs) and a stem region (more conserved across various influenza virus strains). The HA protein is synthesized in the form of a precursor (HA0), which is proteolytically processed to produce two subunits (HA1 and HA2), which associate with each other to form the stem / globular head structure. The HA1 peptide is responsible for viral adsorption to the cell surface and is (along with neuraminidase) essential for viral adsorption and entry into the host cell. The HA2 peptide forms the stem structure, which mediates the fusion of the virus and the cell membrane in the endosome, thereby releasing the ribonucleoprotein complex into the cytoplasm.

[0008] Novel influenza virus strains can result from a phenomenon called antigenic drift, or from mutations in hemagglutinin or neuraminidase molecules that produce novel and different epitopes. Therefore, new vaccines must be produced annually against predicted viruses, a process that is not only costly but also extremely inefficient. While technological advancements have improved the ability to generate improved influenza antigens for vaccine compositions, additional sources of protection against influenza remain needed. Summary of the Invention

[0009] This disclosure provides antibodies that bind to influenza B HA and their antigen-binding fragments. The antibodies are particularly useful for inhibiting or neutralizing the activity of influenza B HA.

[0010] In a first aspect, this disclosure provides isolated recombinant monoclonal antibodies or antigen-binding fragments thereof that specifically bind to influenza B hemagglutinin (HA).

[0011] In one embodiment, the present invention provides an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to influenza B HA, wherein the antibody has one or more of the following characteristics:

[0012] (a) is a completely human monoclonal antibody;

[0013] (b) with less than approximately 10 -10 M's EC 50 Influenza B HA;

[0014] (c) Demonstrates increased survival rates in animals infected with influenza; and / or

[0015] (d) comprising (i) three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3), wherein the heavy chain complementarity-determining regions are contained within a heavy chain variable region (HCVR) containing the amino acid sequence shown in SEQ ID NO:2; and (ii) three light chain CDRs (LCDR1, LCDR2, and LCDR3), wherein the light chain CDRs are contained within a light chain variable region (LCVR) containing the amino acid sequence shown in SEQ ID NO:10.

[0016] In some implementations, when administered subcutaneously or intravenously and / or before or after influenza B virus infection, the antibodies or antigen-binding fragments provided herein confer increased protection against influenza B virus in animals (e.g., mammals).

[0017] In some implementations, the antibodies or antigen-binding fragments thereof provided herein may reduce the severity of influenza B infection in animals (e.g., mammals) when administered subcutaneously or intravenously and / or before or after influenza B virus infection.

[0018] In some embodiments, for example, when administered orally to animals (e.g., mammals) exposed to influenza B virus with a single intravenous dose, starting 1, 2, 3, 4, 5, 6, or 7 days prior to infection, the antibody or antigen-binding fragment thereof disclosed herein provides increased protection against influenza B virus, compared to oral administration of antiviral agents such as baloxavir, omabosil, oseltamivir, zanamivir, or pimodiavir. The single intravenous dose is about 0.5 mg / kg, about 1.0 mg / kg, about 5 mg / kg, about 10 mg / kg, or about 15 mg / kg of the mammal's body weight.

[0019] In some embodiments, when administered in a single subcutaneous or intravenous dose, the antibodies or antigen-binding fragments thereof provided herein provide increased protection against influenza B virus to animals (e.g., mammals) compared to isotype (negative) control antibodies already administered to comparable animals (e.g., mammals), said single subcutaneous or intravenous dose ranging from 0.5 mg / kg to about 15 mg / kg of said animal body weight, such as about 1 mg / kg, about 5 mg / kg, about 7 mg / kg, or about 10 mg / kg, or about 12 mg / kg of said animal body weight.

[0020] In some implementations, the antibodies or antigen-binding fragments thereof described herein provide increased protection against influenza B when administered in a single intravenous dose to animals (e.g., mammals) compared to oral administration of antiviral agents such as baloxavir, maposir, osemir, zanamivir, or pimodiavir. The single intravenous dose is approximately 0.5 mg / kg, 1.0 mg / kg, 5 mg / kg, 10 mg / kg, or 15 mg / kg of the animal's body weight. For example, the antiviral agent could be osemir, administered twice daily for 5 days at a dose of approximately 2 mg / kg.

[0021] In some implementations, when administered as a single dose 24 hours prior to exposure to and infection with influenza B, the antibodies or antigen-binding fragments thereof provided herein confer approximately 100% survival in populations of animals (e.g., mammals) exposed to and infected with influenza B virus, said single dose being approximately 0.5 mg / kg, approximately 1.0 mg / kg, approximately 5 mg / kg, approximately 10 mg / kg, or approximately 15 mg / kg body weight.

[0022] In some implementations, when administered in a single intravenous dose 1, 2, 3, 4, 5, 6, or 7 days prior to influenza B exposure and / or infection, the antibodies or antigen-binding fragments thereof provided herein confer 100% survival in animals (e.g., mammals) infected with the influenza virus, said single intravenous dose being about 0.5 mg / kg, about 1.0 mg / kg, about 5 mg / kg, or about 10 mg / kg or about 15 mg / kg.

[0023] In some implementations, for example, antibodies or antigen-binding fragments thereof exhibit increased protection when administered intravenously to mammals infected with influenza virus, starting on day 1, 2, or 3 post-infection, compared to oral administration of antiviral agents such as baloxavir, maposir, osemir, zanamivir, or pimodiavir. The single intravenous dose is approximately 5 mg / kg to approximately 15 mg / kg. For example, the antiviral agent could be osemir, administered twice daily for 5 days starting on day 1, 2, or 3 post-infection, at a dose of approximately 2 mg / kg.

[0024] In one relevant implementation, the antibodies or antigen-binding fragments thereof provided herein confer increased protection to animals (e.g., mammals) infected with influenza virus when administered subcutaneously or intravenously and / or before or after influenza B virus infection.

[0025] In some embodiments, when administered in a single subcutaneous or intravenous dose to infected mammals, the antibodies or antigen-binding fragments provided herein exhibit increased protection compared to animals administered isotype (negative) control antibodies, wherein the single subcutaneous or intravenous dose ranges from 0.5 mg / kg to about 15 mg / kg, for example about 1 mg / kg, about 5 mg / kg, about 7 mg / kg, or about 10 mg / kg or about 12 mg / kg.

[0026] In some implementations, the antibodies or antigen-binding fragments described herein exhibit increased protection when administered intravenously in mammals infected with influenza virus, compared to oral administration of antiviral agents such as baloxavir, maposil, osemir, zanamivir, or pimodiavir, at a single intravenous dose of about 0.5 mg / kg, about 1.0 mg / kg, about 5 mg / kg, about 10 mg / kg, about 10 mg / kg, or about 15 mg / kg. For example, the antiviral agent could be osemir, administered twice daily for 5 days at a dose of about 2 mg / kg.

[0027] In some implementations, when administered as a single dose 24 hours or longer after infection, the antibodies or antigen-binding fragments provided herein exhibit approximately 100% survival in populations of animals (e.g., mammals) infected with influenza virus, said single dose being approximately 0.5 mg / kg, approximately 1.0 mg / kg, approximately 5 mg / kg, approximately 10 mg / kg, or approximately 15 mg / kg of said animal body weight.

[0028] In some embodiments, when administered in a single intravenous dose at 24, 48, 72, or 96 hours post-infection, the antibodies or antigen-binding fragments provided herein exhibit 100% survival in populations of animals (e.g., mammals) infected with influenza virus, said single intravenous dose being about 5 mg / kg, or about 10 mg / kg, or about 15 mg / kg of said animal body weight.

[0029] In some implementations, the antibodies or antigen-binding fragments thereof provided herein exhibit approximately 100% survival in populations of animals (e.g., mammals) infected with influenza virus when administered in a single intravenous dose, compared to the 80% survival observed in comparable animal populations treated with antiviral agents such as baloxavir, maposil, osemisvir, zanamivir, or pimodiavir. The single intravenous dose is approximately 0.5 mg / kg, approximately 1.0 mg / kg, approximately 5 mg / kg, approximately 10 mg / kg, or approximately 15 mg / kg of the animal's body weight. For example, the antiviral agent could be osemisvir, administered orally twice daily for 5 days at a dose of approximately 2 mg / kg. As described herein, survival rates can be measured at fixed points after administration of the antibody or its antigen-binding fragment disclosed herein (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 longer after administration).

[0030] In some embodiments, the antibodies or antigen-binding fragments of this disclosure provide adjuvant protection in animals (e.g., mammals) infected with influenza viruses when an antiviral agent (such as baloxavir, mapossil, osemisvir, zanamivir, or pimodiavir) is administered. For example, the antiviral agent could be osemisvir, administered more than 48 hours after infection.

[0031] In some embodiments, the antibodies or antigen-binding fragments of this disclosure provide adjuvant protection in animals (e.g., mammals) infected with influenza viruses when an antiviral agent (such as baloxavir, mapossil, osemisvir, zanamivir, or pimodiavir) is administered. For example, the antiviral agent could be osemisvir, administered 72 hours after infection.

[0032] In some embodiments, when the antibody is administered in a single intravenous dose to an animal (e.g., a mammal) infected with influenza virus, the antibody of this disclosure or its antigen-binding fragment provides adjuvant protection when used in combination with an antiviral agent, the single intravenous dose ranging from about 7 mg / kg to about 15 mg / kg of the animal's body weight, and the antiviral agent (such as baloxavir, maposil, oseltamivir, zanamivir, or pimodiavir) is administered orally at a dose of about 2 mg / kg of the animal's body weight, twice daily for 5 days.

[0033] In some embodiments, when used in combination with an antiviral agent (such as baloxavir, mapossil, osemir, zanamivir, or pimodiavir) 96 hours after influenza virus infection, the antibody or its antigen-binding fragment of the present disclosure provides adductal protection, wherein the antibody is administered to the animal in a single intravenous dose ranging from about 7 mg / kg to about 15 mg / kg of the animal's body weight, and the antiviral agent (e.g., osemir, zanamivir, or pimodiavir) is administered orally at a dose of about 2 mg / kg of the animal's body weight twice daily for 5 days.

[0034] In some embodiments, the antibodies or antigen-binding fragments provided herein can be administered intravenously, intranasally, subcutaneously, intradermally, or intramuscularly, and the antiviral agents can be administered orally or intravenously.

[0035] In some embodiments, the antiviral agent is administered before, simultaneously with, or after the antibody administration provided herein.

[0036] In some implementations, the antibody or its antigen-binding fragment and / or the antiviral agent (such as baloxavir, maposil, osemir, zanamivir, or pimodiavir) may be administered in a single dose or in multiple doses.

[0037] Exemplary anti-influenza B HA antibodies are provided in Tables 1 and 2 of this document. Table 1 lists the amino acid sequence identifiers of the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3), light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3), heavy chain (HC), and light chain (LC) of the exemplary anti-influenza B HA antibody. Table 2 lists the nucleic acid sequence identifiers of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HC, and LC of the exemplary anti-influenza B HA antibody.

[0038] This document provides antibodies or antigen-binding fragments thereof comprising HCVR, said HCVR comprising an amino acid sequence according to Table 1, or substantially similar sequences 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.

[0039] In some embodiments, the antibody or its antigen-binding fragment specifically binds to influenza B HA, comprising the HCVR amino acid sequence of SEQ ID NO:2.

[0040] This document provides antibodies or antigen-binding fragments thereof comprising LCVRs, said LCVRs comprising amino acid sequences according to Table 1, or substantially similar sequences 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.

[0041] In some embodiments, the antibody or its antigen-binding fragment specifically binds to influenza B HA, comprising the LCVR amino acid sequence of SEQ ID NO:10.

[0042] In some embodiments, the isolated antibody or antigen-binding fragment that specifically binds to influenza B HA comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO:2 / 10.

[0043] In some embodiments, the isolated antibody or antigen-binding fragment comprises:

[0044] (a) The HCDR1 domain, which has the amino acid sequence of SEQ ID NO:4;

[0045] (b) The HCDR2 domain, which has the amino acid sequence of SEQ ID NO:6;

[0046] (c) HCDR3 domain, which has the amino acid sequence of SEQ ID NO:8;

[0047] (d) The LCDR1 domain, which has the amino acid sequence of SEQ ID NO:12;

[0048] (e) The LCDR2 domain, having the amino acid sequence of SEQ ID NO:14; and

[0049] (f) The LCDR3 domain has the amino acid sequence of SEQ ID NO:16.

[0050] In some embodiments, the isolated antibody or antigen-binding fragment that specifically binds to influenza B HA comprises (a) HCDR1 of SEQ ID NO:4, (b) HCDR2 of SEQ ID NO:6, (c) HCDR3 of SEQ ID NO:8, (d) LCDR1 of SEQ ID NO:12, (e) LCDR2 of SEQ ID NO:14, and (f) LCDR3 of SEQ ID NO:16.

[0051] In some embodiments, the isolated antibody or antigen-binding fragment that specifically binds to influenza B HA comprises (a) HCDR3 of SEQ ID NO:8 and (b) LCDR3 of SEQ ID NO:16.

[0052] This disclosure also provides antibodies comprising heavy chain CDR1 (HCDR1) or antigen-binding fragments thereof, said heavy chain CDR1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:4, or substantially similar sequences 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.

[0053] This disclosure also provides antibodies comprising heavy chain CDR2 (HCDR2) or antigen-binding fragments thereof, said heavy chain CDR2 (HCDR2) comprising the amino acid sequence of SEQ ID NO:6, or substantially similar sequences 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.

[0054] This disclosure also provides antibodies comprising heavy chain CDR3 (HCDR3) or antigen-binding fragments thereof, said heavy chain CDR3 (HCDR3) comprising the amino acid sequence of SEQ ID NO:8, or substantially similar sequences 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.

[0055] This disclosure also provides antibodies comprising a light chain CDR1 (LCDR1) or an antigen-binding fragment thereof, said light chain CDR1 (LCDR1) comprising the amino acid sequence of SEQ ID NO:12, or substantially similar sequences 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.

[0056] This disclosure also provides antibodies comprising a light chain CDR2 (LCDR2) or an antigen-binding fragment thereof, said light chain CDR2 (LCDR2) comprising the amino acid sequence of SEQ ID NO:14, or substantially similar sequences 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.

[0057] This disclosure also provides antibodies comprising a light chain CDR3 (LCDR3) or an antigen-binding fragment thereof, said light chain CDR3 (LCDR3) comprising the amino acid sequence of SEQ ID NO:16, or substantially similar sequences 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.

[0058] This disclosure also provides antibodies or antigen-binding fragments thereof comprising HCDR3 and LCDR3 amino acid sequence pairs (HCDR3 / LCDR3), said HCDR3 and LCDR3 amino acid sequence pairs comprising SEQ ID NO:8 / 16, or substantially similar sequences 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.

[0059] This disclosure also provides antibodies or antigen-binding fragments thereof comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3), said CDRs being contained within the HCVR / LCVR amino acid sequences of exemplary anti-influenza B HA antibodies shown in Table 1. In some embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set comprises SEQ ID NO: 4-6-8-12-14-16.

[0060] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally, the Kabat definition is based on sequence differences, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia methods. 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 can also be used to identify CDR sequences within antibodies.

[0061] This document also provides antibodies and their antigen-binding fragments that compete with antibodies or antigen-binding fragments of the CDRs comprising HCVR and LCVR for specific binding to influenza B HA, wherein the HCVR and LCVR each have an amino acid sequence according to the HCVR and LCVR sequences listed in Table 1.

[0062] This disclosure also provides reference antibodies or antigen-binding fragments thereof that cross-compete with the binding of influenza B HA to the CDR comprising HCVR and the CDR comprising LCVR, or antibodies and antigen-binding fragments thereof that bind to the same epitope on influenza B HA, wherein the HCVR and LCVR each have an amino acid sequence according to the HCVR and LCVR sequences listed in Table 1.

[0063] This disclosure also provides isolated antibodies and their antigen-binding fragments that block the adsorption and / or entry of influenza B HA into host cells.

[0064] In some embodiments, the antibody or antigen-binding fragment is bispecific, the bispecificity including a first binding specificity to a first epitope in the influenza B HA and a second binding specificity to another antigen.

[0065] In a second aspect, this document provides nucleic acid molecules (e.g., DNA or RNA molecules) encoding anti-influenza B HA antibodies or portions thereof. For example, said nucleic acid molecules encoding the HCVR amino acid sequence are listed in Table 2; in some embodiments, said nucleic acid molecules comprise the HCVR polynucleotide sequence of SEQ ID NO:1, or substantially similar sequences 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. The nucleic acid molecules encoding the LCVR amino acid sequence are also listed in Table 2; in some embodiments, the nucleic acid molecules comprise the LCVR polynucleotide sequence of SEQ ID NO:9, or substantially similar sequences 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.

[0066] This document also provides nucleic acid molecules encoding the HCDR1 amino acid sequences listed in Table 1; in some embodiments, the nucleic acid molecules comprise the HCDR1 nucleic acid sequences listed in Table 2, or substantially similar sequences 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.

[0067] This document also provides nucleic acid molecules encoding the HCDR2 amino acid sequences listed in Table 1; in some embodiments, the nucleic acid molecules comprise the HCDR2 nucleic acid sequences listed in Table 2, or substantially similar sequences 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.

[0068] This document also provides nucleic acid molecules encoding the HCDR3 amino acid sequences listed in Table 1; in some embodiments, the nucleic acid molecules comprise the HCDR3 nucleic acid sequences listed in Table 2, or substantially similar sequences 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.

[0069] This document also provides nucleic acid molecules encoding the LCDR1 amino acid sequences listed in Table 1; in some embodiments, the nucleic acid molecules comprise the LCDR1 nucleic acid sequences listed in Table 2, or substantially similar sequences 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.

[0070] This document also provides nucleic acid molecules encoding the LCDR2 amino acid sequences listed in Table 1; in some embodiments, the nucleic acid molecules comprise the LCDR2 nucleic acid sequences listed in Table 2, or substantially similar sequences 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.

[0071] This document also provides nucleic acid molecules encoding the LCDR3 amino acid sequences listed in Table 1; in some embodiments, the nucleic acid molecules comprise the LCDR3 nucleic acid sequences listed in Table 2, or substantially similar sequences 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.

[0072] This document also provides a nucleic acid molecule encoding HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), wherein the amino acid sequence set of HCDR1-HCDR2-HCDR3 is defined as that of the exemplary anti-influenza HA antibody listed in Table 1.

[0073] This document also provides a nucleic acid molecule encoding LCVR, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), wherein the amino acid sequence set of LCDR1-LCDR2-LCDR3 is defined as that of the exemplary anti-influenza HA antibody listed in Table 1.

[0074] This document also provides nucleic acid molecules encoding both HCVR and LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO:2, and wherein the LCVR comprises the amino acid sequence of SEQ ID NO:10. In some embodiments, the nucleic acid molecules comprise the HCVR or LCVR polynucleotide sequences listed in Table 2, or substantially similar sequences 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. In some embodiments, the nucleic acid molecules encode HCVR and LCVR, wherein both HCVR and LCVR are derived from the same anti-influenza B HA antibody listed in Table 1.

[0075] Nucleic acid molecules encoding any of the heavy chain amino acid sequences listed in Table 1 and / or any of the light chain amino acid sequences are also provided.

[0076] In one related aspect, this document provides recombinant expression vectors capable of expressing polypeptides containing heavy or light chain variable regions of anti-influenza HA antibodies. For example, the vector may comprise a recombinant expression vector comprising any of the aforementioned nucleic acid molecules, namely, nucleic acid molecules encoding any of the HCVR, LCVR, and / or CDR sequences shown in Table 1. Within the scope of this disclosure also include host cells incorporating such vectors, and methods for producing antibodies or portions thereof by culturing host cells under conditions allowing for the production of antibodies or antibody fragments, and recovering the antibodies and antibody fragments thus produced.

[0077] In a third aspect, this document 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 pharmaceutically acceptable carrier. In a related aspect, the composition may be a combination of an anti-influenza B HA antibody and a second therapeutic agent. In some embodiments, the second therapeutic agent is any agent advantageously combined with an anti-influenza B HA antibody. Exemplary agents that may be advantageously combined with anti-influenza B HA antibodies include, but are not limited to, other agents that bind and / or inhibit influenza HA activity (including other antibodies or antigen-binding fragments thereof, such as antibodies that bind and / or inhibit influenza A HA) and / or agents that do not directly bind to influenza HA but still inhibit viral activity (including infectivity to host cells). In some 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, wherein the first antibody binds to a first epitope on influenza B HA and the second antibody binds to a second epitope on influenza B HA, wherein the first and second epitopes are distinct and non-overlapping; and (c) a pharmaceutically acceptable carrier or diluent. In some embodiments, the pharmaceutical composition comprises: (a) a first anti-influenza B HA antibody or an antigen-binding fragment thereof; (b) a second anti-influenza B HA antibody or an antigen-binding fragment thereof, wherein the first antibody does not cross-competitive with the second antibody for binding to influenza B HA; and (c) a pharmaceutically acceptable carrier or diluent. In some embodiments, the pharmaceutical composition comprises: (a) a first anti-influenza B HA antibody or an antigen-binding fragment thereof; (b) a second anti-influenza antibody or an antigen-binding fragment thereof, which interacts with a different influenza antigen, wherein the first antibody binds to an epitope on influenza B HA, and the second antibody binds to an epitope on a different influenza antigen; and (c) a pharmaceutically acceptable carrier or diluent. In some embodiments, the pharmaceutical composition comprises: (a) a first anti-influenza B HA antibody or an antigen-binding fragment thereof; (b) a second antibody or an antigen-binding fragment thereof, which interacts with a different viral (non-influenza) antigen, wherein the first antibody binds to an epitope on influenza B HA, and the second antibody binds to an epitope on a different viral (non-influenza) antigen; and (c) a pharmaceutically acceptable carrier or diluent. Other combination therapies and co-formulations relating to the anti-influenza B HA antibody provided herein are disclosed elsewhere herein. In some embodiments, the pharmaceutical composition comprises: (a) an anti-influenza B HA antibody or an antigen-binding fragment thereof, and (b) an anti-influenza A HA antibody or an antigen-binding fragment thereof.

[0078] In a fourth aspect, this document provides a treatment method for treating influenza B HA-related diseases or disorders (such as viral infection in a subject) or at least one symptom associated with said viral infection using an anti-influenza B HA antibody or an antigen-binding portion of an antibody provided herein, wherein said treatment method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising an antibody or an antigen-binding fragment of an antibody to a subject in need. The treated condition is any disease or condition that is improved, alleviated, suppressed, or prevented by inhibiting influenza HA activity. In some embodiments, this disclosure provides a method for preventing, treating, or improving at least one symptom of influenza B infection, said method comprising administering a therapeutically effective amount of an anti-influenza HA antibody or an antigen-binding fragment thereof to a subject in need.

[0079] In some embodiments, this disclosure provides a method for improving, alleviating, or reducing the severity, duration, or frequency of at least one symptom of influenza infection in a subject by administering the anti-influenza B HA antibody provided herein, wherein said at least one symptom is selected from the group consisting of: headache, fever, pain, runny nose (nasal congestion), chills, fatigue, weakness, sore throat, cough, shortness of breath, vomiting, diarrhea, pneumonia, bronchitis, and death.

[0080] In some embodiments, this document provides a method for reducing viral load in a subject, the method comprising administering an effective amount of an antibody or a fragment thereof to the subject, the antibody or fragment thereof binding to influenza B HA and blocking influenza virus binding to and / or entry into the host cell.

[0081] In some implementations, antibodies or their antigen-binding fragments may be administered prophylactically or therapeutically to subjects who have influenza infection, are at risk of influenza infection, or are predisposed to influenza infection. Subjects at risk include, but are not limited to, immunocompromised individuals, such as those with autoimmune diseases, those receiving immunosuppressive therapy (e.g., post-organ transplant), those with human immunodeficiency syndrome (HIV) or acquired immunodeficiency syndrome (AIDS), certain forms of anemia that depletes or destroys white blood cells, those receiving radiation or chemotherapy, or those with inflammatory conditions. Other subjects at risk of influenza infection include older adults (65 years or older), children under 2 years of age, healthcare workers, and those with underlying medical conditions (such as lung infections, heart disease, or diabetes). Furthermore, anyone who has physical contact or close physical proximity to an infected individual increases their risk of influenza virus infection. Additionally, subjects are at risk of influenza infection due to proximity to disease outbreaks, such as living in densely populated cities or being very close to subjects diagnosed or suspected of having influenza virus infection, or due to occupational choices such as hospital staff, drug researchers, travel to infected areas, or frequent air travel.

[0082] In some embodiments, the antibody or its antigen-binding fragment is administered in combination with a second therapeutic agent to an individual in need. The second therapeutic agent may be selected from the group consisting of: anti-inflammatory drugs (such as corticosteroids and nonsteroidal anti-inflammatory drugs), anti-infectives, different influenza HA antibodies (e.g., influenza A HA antibody), antibodies against different influenza antigens (e.g., neuraminidase), antiviral drugs, decongestants, antihistamines, influenza vaccines, dietary supplements (such as antioxidants and any other drugs or therapies known in the art that can be used to improve at least one symptom of influenza infection or to reduce viral load in a patient). In some embodiments, the second therapeutic agent may be an agent that helps to counteract or mitigate any possible side effects associated with the antibody or its antigen-binding fragment provided herein. The antibody or its fragment may be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intranasally, intramuscularly, or intracranially. In some embodiments, the antibody may be administered by a single intravenous infusion to maximize the antibody concentration in the subject's serum. The antibody or a fragment thereof may be administered at a dose of about 0.01 mg / kg of subject body weight to about 100 mg / kg of subject body weight. In some embodiments, the antibody disclosed herein may be administered in one or more doses comprising 50 mg to 5000 mg.

[0083] In a fifth aspect, this document provides an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to influenza B hemagglutinin (HA). The antibody described herein has one or more of the following characteristics: (a) less than about 10 -9 M's EC 50 (a) Incorporating into influenza B HA; (b) demonstrating an increased survival rate in influenza B-infected animals after administration compared to comparable influenza B-infected animals that were not administered; and / or (c) comprising (i) three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) containing an amino acid sequence having at least about 90% sequence identity with the HCVR shown in SEQ ID NO:2; and (ii) three light chain CDRs (LCDR1, HCDR2, and HCDR3) contained within a light chain variable region (LCVR) containing an amino acid sequence having at least about 90% sequence identity with the LCVR shown in SEQ ID NO:10.

[0084] In some embodiments, when prophylactically administered to a mammal at a single intravenous dose of about 5 mg / kg of the mammal's body weight or about 0.5 mg / kg, the isolated antibody or its antigen-binding fragment protects the mammal from infection caused by subsequent exposure to influenza B virus. In some embodiments, when prophylactically administered to a mammal prior to exposure to influenza B virus, the isolated antibody or its antigen-binding fragment reduces the mammal's risk of influenza infection. In some embodiments, when the isolated antibody or its antigen-binding fragment is administered to a mammal, the isolated antibody or its antigen-binding fragment improves, alleviates, or reduces the severity, duration, or frequency 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, runny nose (nasal congestion), chills, fatigue, weakness, sore throat, cough, shortness of breath, vomiting, diarrhea, pneumonia, bronchitis, and death.

[0085] In some embodiments, when the isolated antibody or its antigen-binding fragment is administered as a single prophylactic dose of about 0.5 mg / kg to each of a plurality of mammals, the survival rate of the plurality of mammals 19 days after administration is at least about 80%. In some embodiments, when the isolated antibody or its antigen-binding fragment is administered as a single prophylactic dose of about 0.5 mg / kg to each of a plurality of mammals, the survival rate of the plurality of mammals 19 days after administration is at least about 90%. In some embodiments, when the isolated antibody or its antigen-binding fragment is administered as a single prophylactic dose of about 0.5 mg / kg to each of a plurality of mammals, the survival rate of the plurality of mammals 19 days after administration is about 100%. In some embodiments, when the isolated antibody or its antigen-binding fragment is administered as a single prophylactic dose of about 5.0 mg / kg to each of a plurality of mammals, the survival rate of the plurality of mammals 19 days after administration is about 80%. In some embodiments, when the isolated antibody or its antigen-binding fragment is administered as a single prophylactic dose of about 5.0 mg / kg to each of a plurality of mammals, the survival rate of the plurality of mammals is about 90% 19 days after administration. In some embodiments, when the isolated antibody or its antigen-binding fragment is administered as a single prophylactic dose of about 5.0 mg / kg to each of a plurality of mammals, the survival rate of the plurality of mammals is about 100% 19 days after administration. In some embodiments, the survival rate is evident 13 days after administration.

[0086] In some embodiments, this disclosure provides an isolated antibody or antigen-binding fragment thereof as described above, comprising 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.

[0087] In a sixth aspect, this document provides an isolated antibody or an 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.

[0088] In some embodiments, the isolated antibody or its antigen-binding fragment specifically binds to influenza B hemagglutinin (HA). In some embodiments, when the isolated antibody or its antigen-binding fragment is prophylactically administered to a mammal at a single intravenous dose of about 5 mg / kg of the mammal's body weight or about 0.5 mg / kg, the mammal is protected from infection caused by subsequent exposure to influenza B virus. In some embodiments, when the isolated antibody or its antigen-binding fragment is prophylactically administered 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 its antigen-binding fragment is administered to a mammal, the isolated antibody or its antigen-binding fragment improves, alleviates, or reduces the severity, duration, or frequency 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, runny nose (nasal congestion), chills, fatigue, weakness, sore throat, cough, shortness of breath, vomiting, diarrhea, pneumonia, bronchitis, and death.

[0089] In some embodiments, when the isolated antibody or its antigen-binding fragment is administered as a single prophylactic dose of about 0.5 mg / kg to each of a plurality of mammals, the survival rate of the plurality of mammals 19 days after administration is at least about 80%. In some embodiments, when the isolated antibody or its antigen-binding fragment is administered as a single prophylactic dose of about 0.5 mg / kg to each of a plurality of mammals, the survival rate of the plurality of mammals 19 days after administration is at least about 90%. In some embodiments, when the isolated antibody or its antigen-binding fragment is administered as a single prophylactic dose of about 0.5 mg / kg to each of a plurality of mammals, the survival rate of the plurality of mammals 19 days after administration is about 100%. In some embodiments, when the isolated antibody or its antigen-binding fragment is administered as a single prophylactic dose of about 5.0 mg / kg to each of a plurality of mammals, the survival rate of the plurality of mammals 19 days after administration is about 80%. In some embodiments, when the isolated antibody or its antigen-binding fragment is administered as a single prophylactic dose of about 5.0 mg / kg to each of a plurality of mammals, the survival rate of the plurality of mammals is about 90% 19 days after administration. In some embodiments, when the isolated antibody or its antigen-binding fragment is administered as a single prophylactic dose of about 5.0 mg / kg to each of a plurality of mammals, the survival rate of the plurality of mammals is about 100% 19 days after administration. In some embodiments, the survival rate is evident 13 days after administration.

[0090] In some embodiments, the isolated antibody or antigen-binding fragment thereof described above 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 an 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.

[0091] In a seventh aspect, this document provides a pharmaceutical composition comprising the isolated antibody or its antigen-binding fragment described above, and a pharmaceutically acceptable carrier or diluent.

[0092] 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: antiviral drugs, anti-inflammatory drugs, various antibodies that specifically bind to influenza A HA, influenza vaccines, dietary supplements, and other palliative therapies 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 dietary 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.

[0093] In some embodiments, this disclosure provides a polynucleotide molecule comprising a polynucleotide sequence encoding an HCVR or LCVR as described above. In some embodiments, this disclosure provides a vector comprising said polynucleotide. In some embodiments, this disclosure provides a cell comprising said vector.

[0094] In an eighth aspect, this document provides methods for preventing, treating, or improving at least one symptom of influenza infection in a subject, the methods comprising administering an antibody or antigen-binding fragment as described above, or a pharmaceutical composition as described above, to the subject.

[0095] In some embodiments, the at least one symptom is selected from the group consisting of: fever, cough, body aches, runny nose, 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 65 years of age or older, healthcare professionals, and individuals with a history of medical problems or underlying medical conditions. In some embodiments, the underlying medical conditions are selected from the group consisting of: heart disease 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, dietary supplements, and other palliative therapies for 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 dietary supplement is an antioxidant. In some embodiments, the second therapeutic agent is administered via a different route of administration than the antibody or its antigen-binding fragment. In some embodiments, the second therapeutic agent is administered orally. In some embodiments, the antiviral drug is oseltamivir. In some embodiments, oseltamivir is administered prior to the administration of the antibody or its antigen-binding fragment. In some embodiments, oseltamivir is administered simultaneously with the antibody or its antigen-binding fragment. In some embodiments, oseltamivir is administered after the administration of the antibody or its antigen-binding fragment. 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). In some embodiments, the pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intramuscularly, intranasally, or orally.

[0096] This disclosure also includes the use of an anti-influenza B HA antibody or its antigen-binding fragment for the treatment of diseases or disorders that would benefit from blocking influenza HA binding and / or activity. This disclosure also includes the use of an anti-influenza B HA antibody or its antigen-binding fragment in the manufacture of a medicament for the treatment of diseases or disorders that would benefit from blocking influenza HA binding and / or activity.

[0097] Other implementation methods will become apparent upon reading the following detailed description. Detailed Implementation

[0098] Before describing the method of the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of this disclosure will be defined only by the appended claims.

[0099] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, preferred methods and materials are described hereafter. All publications mentioned herein are incorporated herein by reference in their entirety.

[0100] This disclosure provides antibodies that bind to influenza B HA and their antigen-binding fragments. The antibodies are particularly useful for inhibiting or neutralizing the activity of influenza B HA.

[0101] In contrast to unmodified antibodies, the antibodies provided herein may be full-length (e.g., IgG1 or IgG4 antibodies) or may contain only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments), and may be modified to affect function, such as increasing persistence in the host, increasing effector function, or eliminating residual effector function (Reddy et al., 2000, J. Immunol. 164: 1925-1933). In some embodiments, the antibody may be bispecific.

[0102] In some embodiments, the antibody can be used to block the attachment of influenza virus to host cells and / or to prevent influenza virus from entering host cells. In some embodiments, the antibody works by inhibiting intercellular transmission of the virus. In some embodiments, the antibody can be used to prevent, treat, or improve at least one symptom of influenza virus infection in a subject. In some embodiments, the antibody can be administered prophylactically or therapeutically to subjects who have influenza virus infection or are at risk of influenza virus infection. In some embodiments, a composition containing at least one antibody provided herein can be administered to subjects who are contraindicated for vaccination or for whom the vaccine is less effective, such as elderly patients, very young patients, patients who may be allergic to any one or more components of the vaccine, or immunocompromised patients who may not respond to the immunogens in the vaccine. In some embodiments, a composition containing at least one antibody provided herein can be administered during an influenza outbreak to healthcare workers, hospitalized patients, nursing home residents, or other high-risk patients. In some embodiments, a composition containing at least one antibody provided herein can be administered as first-line treatment to patients in cases of predicted annual vaccine ineffectiveness or in the event of a pandemic of strains that have undergone major antigenic shift.

[0103] definition

[0104] The term "influenza hemagglutinin," also known as "influenza HA," is a trimeric glycoprotein present on the surface of influenza virus particles. It mediates viral adsorption (via the binding of HA1 to α-2,3-sialic acid or α-2,6-sialic acid) and entry into host cells (via conformational changes). HA consists of two domains: a globular head domain containing receptor-binding sites (with a high frequency of antigenic mutation) and a stem region (more conserved across various influenza virus strains). Influenza HA is synthesized in the form of a precursor (HA0), which undergoes proteolytic processing to produce two subunits (HA1 and HA2), which associate with each other to form a stem / globular head structure. Viral HA is the most variable antigen on the virus (divided into two groups of 18 subtypes), but the stem (HA2) is highly conserved across the groups. Examples of full-length influenza B HA amino acid sequences are HA from B / Victoria / 2 / 87, SEQ ID NO:21 (GenBank accession number AAA43697.1); HA from B / Nanchang / 3451 / 93, SEQ ID NO:22 (partial sequence; GenBank accession number AAD02807.1); HA from B / Singapore / 11 / 1994, SEQ ID NO:23 (GenBank accession number ABN50712.1); and HA from B / Florida / 4 / 2006, SEQ ID NO:24 (GenBank accession number ACA33493.1). The term "influenza-HA" also includes influenza HA protein variants isolated from other influenza B isolates and influenza A isolates. The term "influenza HA" also includes recombinant influenza HA or fragments thereof. The term also covers influenza HA or fragments thereof coupled to, for example, histidine tags, mouse or human Fc or signal sequences.

[0105] As used herein, the term "influenza infection," also referred to as "influenza," refers to a severe acute respiratory illness caused by the influenza virus. The term encompasses respiratory infection and symptoms including high fever, headache, body aches, fatigue and weakness, in some cases extreme fatigue, nasal congestion, sneezing, sore throat, chest discomfort, cough, shortness of breath, bronchitis, pneumonia, and, in severe cases, death.

[0106] The term "surface plasmon resonance" refers to an optical phenomenon that allows light to pass through the surface of a surface through, for example, the BIACORE. TM The system (Pharmacia Biosensor AB, Uppsala, Sweden, and Piscataway, NJ) detects changes in protein concentration within a biosensor matrix to analyze real-time biomolecular interactions.

[0107] Biolayer interferometry (BIT) is a label-free technique for measuring biomolecular interactions. It is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: a fixed protein layer on the tip of a biosensor and an internal reference layer. Any change in the number of molecules bound to the biosensor tip will cause 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 some embodiments, “biosensors based on real-time biolayer interferometry (Octet HTX assay)” are used to evaluate the binding characteristics of certain anti-influenza HA antibodies.

[0108] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, called a complementary site. A single antigen may have more than one epitope. Therefore, different antibodies can bind to different regions on the antigen and can have different biological effects. The term "epitope" also refers to a site on the antigen that responds to B cells and / or T cells. It also refers to the antigenic region that the antibody binds to. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have those residues that have an affinity that directly promotes the interaction. Epitopes can also be conformational, i.e., composed of nonlinear amino acids. In some embodiments, epitopes may include determinants that are chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some embodiments, may have specific three-dimensional structural features and / or mass-to-charge ratio features.

[0109] As used herein, the term "cross-competition" refers to an antibody or its antigen-binding fragment binding to an antigen and inhibiting or blocking the binding of another antibody or its antigen-binding fragment. The term also includes competition between two antibodies in two directions, i.e., a first antibody binding and blocking the binding of a second antibody, and vice versa. In some embodiments, the first and second antibodies may bind to the same epitope. Alternatively, the first and second antibodies may bind to different but overlapping epitopes, such that binding of one inhibits or blocks binding of the second antibody, for example, by steric hindrance. Cross-competition between antibodies can be measured by methods known in the art, such as by real-time label-free biolayer interferometry. To determine whether a test antibody cross-competes with a reference anti-influenza B antibody, the reference antibody is bound to influenza virus HA or a peptide under saturation conditions. The ability of the test antibody to bind to influenza virus HA is then evaluated. If, after saturation binding with the reference anti-influenza virus HA antibody, the test antibody is able to bind to influenza virus HA, it can be determined that the test antibody binds to a different epitope than the reference anti-influenza virus HA antibody. On the other hand, if the test antibody cannot bind to influenza virus HA after saturation binding with the reference anti-influenza virus HA antibody, then the test antibody can bind to the same epitope as the reference anti-influenza virus HA antibody.

[0110] The phrase “therapeutic effective amount” means the amount that produces the desired effect when administered. The exact amount will depend on the therapeutic purpose and can be determined by a person skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0111] As used herein, the term "subject" refers to an animal, such as a mammal, such as a human, that is in need of improvement, prevention, and / or treatment of a disease or disorder such as a viral infection. Subjects may have influenza infection or be predisposed to influenza virus infection. Subjects "predisposed to influenza virus infection" or "at high risk of influenza virus infection" include those with compromised immune systems due to autoimmune diseases, those receiving immunosuppressive therapy (e.g., after organ transplantation), those with human immunodeficiency syndrome (HIV) or acquired immunodeficiency syndrome (AIDS), certain forms of anemia that depletes or destroys white blood cells, those receiving radiation or chemotherapy, or those with inflammatory conditions. Furthermore, young or elderly subjects are at increased risk. Anyone who has physical contact or close physical proximity to an infected individual increases their risk of influenza virus infection. Additionally, subjects are at risk of influenza infection due to proximity to disease outbreaks, such as living in a densely populated city or being very close to subjects diagnosed or suspected of having influenza virus infection, or due to occupational choices such as hospital staff, drug researchers, travel to infected areas, or frequent air travel.

[0112] As used herein, the term "treatment" refers to a reduction or improvement in the severity of at least one symptom or indication of influenza infection resulting from the administration of a therapeutic agent (such as the antibody disclosed herein) to a subject in need. The term includes suppression of disease progression or worsening of infection. It also includes a positive prognosis, meaning that after administration of a therapeutic agent such as the antibody disclosed herein, the subject may be infection-free or may have a reduced viral titer or a viral titer of zero. The therapeutic agent may be administered to an individual at a therapeutic dose.

[0113] The term "preventing" or "prevention" refers to the suppression of influenza infection or any symptoms or indications of influenza infection following administration of the antibodies disclosed herein. This term includes prevention of the spread of infection in subjects exposed to the virus or at risk of influenza infection.

[0114] As used herein, “protective effect” can be demonstrated by any standard procedure known in the art to determine whether the agents disclosed herein (such as antiviral agents) or antibodies (such as anti-influenza HA antibodies) can provide one or more of the following: for example, increased survival rate in a subject population after exposure to an infectious agent (relative to a comparable untreated subject population that was also exposed to the infectious agent), reduced viral load (relative to subjects before treatment), or improvement in at least one symptom associated with the infectious agent (relative to subjects before treatment).

[0115] As used herein, the term "antiviral drug" means any anti-infective drug or therapy used to treat, prevent, or improve a viral infection in a subject. The term "antiviral drug" includes, but is not limited to, […]. (Osemivir) (Zanamivir), baloxavir, maposil, ribavirin, or interferon-alpha2b. "Antiviral drugs" also include antiviral antibodies. For example, an antiviral drug can be an antibody used to treat, prevent, or improve influenza infection (e.g., influenza A, influenza B, or both). This antibody can target hemagglutinin from influenza A, influenza B, or both. In this disclosure, the infection to be treated is caused by an influenza virus.

[0116] General Instructions

[0117] Influenza is an infectious disease caused by RNA viruses (influenza viruses) belonging to the Orthomyxoviridae family. Influenza viruses are classified into four genera—A, B, C, and D—based on their core proteins. These are further subdivided into subtypes determined by the viral envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA). Type B influenza viruses formed a homologous group, which 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. Type A influenza viruses infect a range of mammalian and avian species, while types B and C primarily infect humans. Type D influenza primarily infects cattle but not humans. Only types A and B cause any human disease of concern.

[0118] The high mutation rate and frequent gene reassortment of influenza viruses lead to significant changes in HA and NA antigens. A small number of point mutations causing minor changes (“antigenic drift”) occur relatively frequently. Antigenic drift allows the virus to evade immune recognition, leading to recurring influenza outbreaks in pandemic years. Major changes in the HA antigen (“antigenic shift”) are caused by recombination of genetic material from different influenza subtypes. Antigenic drift leading to new pandemic virus strains is a rare event, occurring through reassortment between animal and human subtypes, such as in co-infected pigs.

[0119] HA is synthesized as a homotrimeric precursor polypeptide HAO. Each monomer can independently cleave posttranslation to form two polypeptides, HA1 and HA2, linked by a single disulfide bond. The larger N-terminal fragment (HA1, 320-330 amino acids) forms a membrane-side globular domain containing receptor-binding sites and most of the determinants recognized by viral neutralizing antibodies. The HA1 polypeptide of HA is responsible for attaching the virus to the cell surface. The smaller C-terminal portion (HA2, approximately 180 amino acids) forms a stem-like structure that anchors the globular domain to the cell or viral membrane. The HA2 polypeptide mediates the fusion of the virus and cell membrane in endosomes, thereby releasing the ribonucleoprotein complex into the cytoplasm.

[0120] Despite decades of research, no marketed antibodies have been found that broadly neutralize or inhibit influenza B virus infection or attenuate the disease caused by influenza B virus. Therefore, there is a need to identify new antibodies that neutralize multiple influenza B virus subtypes and can be used as drugs for the prevention or treatment of influenza B infection.

[0121] Passive immunotherapy for the prevention or treatment of infectious diseases has been used for over a century, typically in the form of convalescent serum containing high titers of neutralizing antibodies (Good et al. 1991; Cancer 68:1415-1421). Currently, various purified monoclonal antibodies are being used as antimicrobial agents in preclinical and clinical development (Marasco et al. 2007; Nature Biotechnology 25:1421-1434).

[0122] The inventors have described herein fully human antibodies and their antigen-binding fragments that specifically bind to influenza hemagglutinin and modulate the interaction between influenza virus and host cells. Anti-influenza B virus HA antibodies can bind to influenza B virus HA with high affinity. In some embodiments, the antibodies disclosed herein are blocking antibodies, wherein the antibody can bind to influenza HA and block viral adsorption and / or entry into host cells. In some embodiments, blocking antibodies can block the binding of influenza virus to cells, thereby inhibiting or neutralizing viral infectivity of host cells. In some embodiments, blocking antibodies can be used to treat subjects suffering from influenza virus infection. When administered to subjects in need, said antibodies can reduce viral infection such as influenza in the subject. They can be used to reduce viral load in subjects relative to untreated subjects. They can be used alone or as adjunctive therapy in conjunction with other therapeutic components or modalities known in the art to treat viral infections. In some embodiments, these antibodies can bind to epitopes in the stem region of viral HA. In addition, the identified antibodies can be used preventively (before infection) to protect animals (e.g., mammals) from infection, or therapeutically (after infection is established) to improve a previously established infection or improve at least one symptom associated with the infection.

[0123] The full-length amino acid sequences of exemplary influenza B virus HA are shown in GenBank as 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).

[0124] In some embodiments, antibodies are obtained from mice immunized with a primary immunogen (such as full-length influenza B HA) or with a recombinant form of influenza B HA or a fragment thereof, followed by immunization with a secondary immunogen or an immunogenically active fragment of influenza B HA. In some embodiments, antibodies are obtained from mice immunized with an influenza vaccine composition, followed by a booster immunization with one or more recombinantly produced HA peptides. For example, antibodies can be obtained by first immunizing mice with B / Victoria / 2 / 87, then with B / Yamagata / 16 / 88, and then with B / Victoria / 2 / 87; or by first immunizing mice with B / Yamagata / 16 / 88, then with B / Victoria / 2 / 87, and then with B / Yamagata / 16 / 88. In some aspects, the B / Yamagata strain can be replaced by B / Maryland / 03 / 2008, B / Florida / 4 / 2006, B / Nanchang / 3451 / 93, or B / Singapore / 11 / 1994. Mice can be boosted with 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, for example, a 1:1 mixture of DNA encoding HA from B / Victoria / 2 / 87 and B / Yamagata / 16 / 88.

[0125] Immunogens can be biologically active and / or immunogenic fragments of influenza B HA or DNA encoding their active fragments. These fragments can be derived from the stem region of the HA protein.

[0126] Peptides can be modified, including by adding or substituting certain residues, for labeling or conjugation to carrier molecules such as KLH. For example, cysteine ​​residues can be added to the N-terminus or C-terminus of a peptide, or adapter sequences can be added, to prepare peptides conjugated to, for example, KLH, for immunization.

[0127] As determined by in vitro or in vivo assays, certain anti-influenza B HA antibodies disclosed herein are capable of binding to and neutralizing influenza B HA activity. The antibody binding to and neutralizing activity to influenza B HA, and thus the ability of the virus to adsorb and / or enter host cells and subsequently cause viral infection, can be measured using any standard method known to those skilled in the art, including binding assays or activity assays, as described herein.

[0128] 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 using a Biacore instrument via surface plasmon resonance. Neutralization assays can be used to determine the infectivity of various influenza B virus strains. Antibodies against influenza B virus HA can mediate complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC) that can mediate virus infection of cells in vitro. Exemplary antibodies are capable of neutralizing influenza B virus infection in vivo.

[0129] Antibodies specific to influenza B HA may be free of additional labeling or portions, or they may contain N-terminal or C-terminal labels or portions. In some embodiments, the label or portion is biotin. In binding assays, the location of the label (if present) determines the orientation of the peptide relative to the surface to which the peptide binds. For example, if the surface is coated with avidin, a peptide containing 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 some embodiments, antibodies with such labels can be used in diagnostic assays, including imaging analyses.

[0130] Antibodies and antigen-binding fragments of antibodies

[0131] Antibody

[0132] As used herein, the term "antibody" refers to an immunoglobulin molecule consisting of four polypeptide chains—two heavy (H) chains and two light (L) chains—interconnected by disulfide bonds (i.e., a "complete antibody molecule"), and its polymers (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain consists of a heavy chain variable region ("HCVR" or "V"). H ") and heavy chain constant region (by domain C H 1. CH 2 and C H 3. Composition. Each light chain consists of a light chain variable region (“LCVR” or “V”). L ") and light chain constant region (C L ) constitutes. V H and V L The region can be further subdivided into highly variable regions, called complementary determinant regions (CDRs), interspersed with more conservative regions, called framing regions (FRs). Each V H and V L It consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The common amino acid sequence can be defined based on the side-by-side analysis of two or more CDRs.

[0133] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies may include, for example, amino acid residues in the CDR and particularly in CDR3 that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include monoclonal antibodies for which a germline CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human FR sequence. The term includes antibodies produced in or in the cells of non-human mammals in a recombinant manner. The term is not intended to include antibodies isolated from or produced in human individuals.

[0134] As used herein, the term "recombinant" refers to an antibody or antigen-binding fragment thereof produced, 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 also refers to antibodies expressed in non-human mammalian (including transgenic non-human mammalian, such as transgenic mice) or cell (e.g., CHO cell) expression systems, or antibodies isolated from a recombinant human antibody library.

[0135] The terms "specific binding" or "specific binding to" refer to the formation of a relatively stable complex between an antibody or its antigen-binding fragment and the antigen under physiological conditions. Specific binding is characterized by a minimum binding density of approximately 1 × 10⁻⁶. -8 M or a lower equilibrium dissociation constant (e.g., a smaller K) D (Indicating a 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, etc. As described herein, methods have been used... Real-time label-free biolayer interferometry analysis on the HTX biosensor identified antibodies that specifically bind to influenza HA. ​​Furthermore, multispecific antibodies binding to one domain and one or more additional antigens in influenza-HA, or bispecific antibodies binding to two distinct regions of influenza-HA, are still considered "specifically binding" antibodies as used herein.

[0136] The term "high affinity" antibody refers to an antibody with at least 10 affinity for influenza-HA. -8 M; at least about 10 -9 M; at least about 10 -10 M; or at least about 10 -11 Binding affinity of M (with K) D Monoclonal antibodies (represented by) such as those obtained by real-time, label-free biolayer interferometry (e.g.) HTX biosensors), or through surface plasmon resonance (e.g., BIACORE). TM (or measured by solution affinity ELISA).

[0137] The terms "slow dissociation rate," "Koff," or "kd" refer to the rate at which an antibody dissociates at a rate of 1 × 10⁻⁶. -3 s -1 Or smaller, or 1×10 -4 s -1 Or a smaller rate constant dissociates from influenza-HA, such as by real-time, label-free biolayer interferometry (e.g. HTX biosensors) or through surface plasmon resonance (e.g., BIACORE) TM As determined by ).

[0138] As used in this article, the term "K" D "Intended to refer to the equilibrium dissociation constant of a specific antibody-antigen interaction."

[0139] As used herein, the terms “antigen-binding portion” and “antigen-binding fragment” of an antibody include any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the terms “antigen-binding fragment” or “antibody fragment” of an antibody refer to one or more fragments of an antibody that retain the ability to bind to influenza HA.

[0140] In specific implementations, the antibodies or antibody fragments provided herein may be conjugated to portions such as ligands or therapeutic portions (“immunoconjugates”) such as antiviral drugs, secondary anti-influenza antibodies, or any other therapeutic portion used to treat infections caused by influenza viruses.

[0141] As used herein, “isolated antibody” is intended to refer to an antibody that is substantially free of other antibodies (Abs) that have different antigen specificities (e.g., an isolated antibody or fragment thereof that specifically binds to influenza-HA is substantially free of Abs that specifically bind to antigens other than influenza-HA).

[0142] As used herein, “blocking antibody” or “neutralizing antibody” (or “antibody that neutralizes influenza HA activity” or “antagonist antibody”) is intended to refer to an antibody that binds to influenza HA and results in inhibition of at least one biological activity of influenza HA. ​​For example, the antibodies described herein can prevent or block influenza adsorption or entry into host cells. Additionally, a “neutralizing antibody” is an antibody capable of neutralizing, i.e., preventing, inhibiting, reducing, hindering, or interfering with the ability of a pathogen to initiate and / or persist infection in a host. The terms “neutralizing antibody” and “neutralizing antibody” are used interchangeably herein. These antibodies can be used alone or in combination, as a prophylactic or therapeutic agent in combination with other antiviral agents after appropriate formulation, in combination with an active vaccine, or as a diagnostic tool.

[0143] antigen-binding fragments

[0144] Unless otherwise specifically indicated, as used herein, the term "antibody" should be understood to encompass an antibody molecule comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., a "complete antibody molecule") and its antigen-binding fragment. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., include any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the terms "antigen-binding fragment" or "antibody fragment" of an antibody refer 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 a CDR, or isolated CDRs. In some embodiments, the term "antigen-binding fragment" refers to a polypeptide fragment of a multispecific antigen-binding molecule. Any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding variable and (optionally) constant domains of an antibody, may be used, for example, to derive the antigen-binding fragment of an antibody from a complete antibody molecule. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into suitable conformations, or to introduce codons, generate cysteine ​​residues, modify, add or delete amino acids, etc.

[0145] 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) the smallest recognition unit consisting of amino acid residues mimicking the hypervariable region of an antibody (e.g., a separated complementarity-determining region (CDR) such as a CDR3 peptide) or a restricted FR3-CDR3-FR4 peptide. As used herein, other engineered molecules (such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-transplanted antibodies, dimers, triplets, tetramers, microbodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains) are also included in the description “antigen-binding fragments”.

[0146] Antibody antigen-binding fragments typically contain at least one variable domain. Variable domains can have any size or amino acid composition and generally contain at least one CDR adjacent to or co-framed with one or more frame sequences. In the presence of V... H Domain and V L In the antigen-binding fragment associated with the domain, V H and V L The domains can be positioned relative to each other in any suitable arrangement. For example, the variable region can be a dimer and contain V. H -V H V H -V L or V L -V L Dimer. Alternatively, the antigen-binding fragment of the antibody may contain monomer V. H or V L Structural domain.

[0147] In some embodiments, the antigen-binding fragment of the antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of the variable and constant domains that may be present within the antigen-binding fragments of the antibodies disclosed herein include: (i) 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 H3;(vi)V H -C H 2-C H 3;(vii)V H -C L (viii)V L -C H 1; (ix)V L -CH2;(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 L In any configuration of the variable and constant domains (including any of the exemplary configurations listed above), the variable and constant domains may be directly connected to each other or linked via full-length or partial hinge or linker regions. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that form a flexible or semi-flexible bond between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragments of the antibodies disclosed herein may comprise each other and / or one or more monomers V having any of the variable and constant domain configurations listed above. H or V L The structural domains (e.g., via disulfide bonds) are non-covalently associated homodimers or heterodimers (or other polymers).

[0148] Like complete antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies will typically contain at least two distinct variable domains, each capable of specifically binding to a single antigen or different epitopes on the same antigen. Using conventional techniques available in the art, any form of multispecific antibody (including the exemplary bispecific antibody forms disclosed herein) can be adapted to the context of the antigen-binding fragments of the antibodies disclosed herein.

[0149] Modification of antibodies and their antigen-binding fragments

[0150] In some embodiments, the frame region of the antibody (or its antigen-binding fragment) may be identical to a human germline sequence, such as the sequence of the antibody provided herein, or may be naturally or artificially modified. One or more amino acids in a given frame region (or one or more frame regions) may be substituted, and the substitution may be conserved or non-conserved. It is also possible to substitute one or more CDR residues or omit one or more CDRs. Antibodies have been described in the scientific literature in which one or two CDRs may be assigned for binding. Padlan et al. (1995 FASEB J.9:133-139) analyzed the contact region between the antibody and its antigen based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs do not have amino acids in contact with the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428). Therefore, the antibodies provided herein can be effectively modified in the CDR region and / or frame region, provided that the modified antibody retains one or more desired characteristics, such as the antibody or its antigen-binding fragment having a ratio of less than about 10 -10 M's EC 50 Integrating into influenza B HA; and / or demonstrating an increased survival rate of influenza-infected animals after administration to the influenza-infected animals compared to comparable influenza-infected animals that did not receive the administration.

[0151] A given CDR can be modified relative to the CDR sequence of an antibody provided herein, and such modifications can include conserved or non-conserved substitutions. Desired substitutions can be determined through molecular modeling and / or empirically. For example, one or more CDR residues can be substituted by amino acids occupying corresponding positions in the sequence of another human antibody or by a common sequence of such sequences.

[0152] In addition, their antigen-binding fragments may be antibodies disclosed herein, but modified to omit one or more CDRs and / or one or more frame regions, as long as the modified antibody (aka antigen-binding fragment) remains bound to the influenza B virus HA.

[0153] Based on previous research, non-antigen-contacting CDR residues (e.g., residues H60-H65 in HCDR2 are typically undesirable) can be identified from the Kabat CDR region located outside the Chothia CDR through molecular modeling and / or empirical evidence. The antibodies or their antigen-binding fragments presented herein can be modified to remove or replace a given CDR, particularly a non-antigen-contacting CDR. Light chain CDRs can be replaced with, for example, universal light chain CDRs.

[0154] The fully human anti-influenza HA monoclonal antibody 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 sequence or the sequence provided herein. Such modifications or mutations can be readily determined by comparing the amino acid sequence disclosed herein with germline sequences available from, for example, public antibody sequence databases, or by comparing the amino acid sequence with those sequences of the antibody provided herein (e.g., any of the antibody sequences provided in Table 1).

[0155] This 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 frame regions and / or CDRs are modified, provided that the modified antibody retains one or more desired characteristics, such as the antibody or its antigen-binding fragment having a modifier of less than about 10. -10 M's EC 50 Binding to influenza B HA; and / or demonstrating an increased survival rate in influenza-infected animals after administration to the influenza-infected animals compared to comparable influenza-infected animals that have not received the administration. After obtaining an antibody-antigen binding fragment modified with one or more frame regions and / or CDRs, one or more desired properties of the antibody-antigen binding fragment can be readily tested, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as applicable), reduced immunogenicity, etc.

[0156] This disclosure also includes antibodies and their antigen-binding fragments derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more frames and / or CDR regions are mutated to corresponding residues of the germline sequence yielding the antibody, or corresponding residues of another human germline sequence, or conserved amino acid substitutions of corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). Those skilled in the art can readily generate a variety of antibodies and antigen-binding fragments comprising one or more individual modifications or germline mutations or combinations thereof, starting from the heavy and light chain variable region sequences disclosed herein. In some embodiments, V H and / or V LAll frame and / or CDR residues within the domain are mutated back to residues in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, mutated residues present only in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or mutated residues present only in CDR1, CDR2, or CDR3. In other embodiments, one or more frame and / or one or more CDR residues are mutated to one or more corresponding residues in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies disclosed herein may contain any combination of two or more germline mutations within the frame and / or CDR regions, for example, where certain individual residues are mutated to corresponding residues in a specific germline sequence, while certain other residues different from the original germline sequence are retained or mutated to corresponding residues in a different germline sequence. After obtaining an antibody-antigen binding fragment containing one or more germline mutations, one or more desired properties of the antibody-antigen binding fragment can be readily tested, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibody-antigen binding fragments obtained in this general manner are covered within this disclosure.

[0157] This disclosure provides antibodies that are "substantially identical" or "substantially similar" to sequences in the CDR or frame regions provided herein. Differences in sequences, such as those between sequences provided in Table 1 or Table 2 and modified sequences based thereon, are indicated by "substantially identical" or "substantially similar".

[0158] When referring to nucleic acids or fragments thereof, the terms "substantially identical" or "substantially the same" indicate a percentage of nucleotide sequence identity when best aligned with another nucleic acid (or its complementary strand), for example, 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 sequence identity algorithm such as FASTA, BLAST, or GAP, as discussed below. In some cases, a nucleic acid molecule that is substantially identical to a reference nucleic acid molecule may encode a polypeptide with the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0159] When applied to peptides, the term "substantially similar" or "substantially analogous" means that, when optimally aligned, such as using the procedures GAP or BESTFIT with default gap weights, two peptide sequences share at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence similarity. In some respects, dissimilar residue positions differ due to conserved amino acid substitutions. A "conserved amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced by another amino acid residue containing a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions do not substantially alter the functional properties of a protein. Where two or more amino acid sequences differ from each other due to conserved substitutions, the percentage or degree of similarity can be increased to correct for the conserved nature of the substitutions. The means for making such adjustments are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference. Examples of amino acid groups with similar chemical properties in their side chains 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 acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, 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, which is incorporated herein by reference. A “moderately conservative” substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0160] Sequence identity and / or similarity of peptides are typically measured using sequence analysis software. Protein analysis software uses similarity measures specified to various substitutions, deletions, and other modifications, including conserved amino acid substitutions, to match similar sequences. For example, GCG software contains programs such as GAP and BESTFIT, which, along with default parameters, can be used to determine sequence homology or sequence identity between closely related peptides (e.g., homologous peptides from different biological species) or between wild-type proteins and their mutants. See, for example, GCG version 6.1. Peptide sequences can also be compared using FASTA with default or recommended parameters; FASTA is a program in GCG version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignment of the best overlapping regions between the query and search sequences and a percentage of sequence identity (Pearson (2000) ibid.). Sequences can also be compared using the Smith-Waterman homology search algorithm, which uses an affine vacancy search with a vacancy open penalty of 12, a vacancy extension penalty of 2, and a BLOSUM matrix of 62. When comparing the sequences disclosed herein with databases containing a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST using default parameters, especially BLASTP or TBLASTN. 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.

[0161] This document provides fully human anti-influenza-HA monoclonal antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein, having one or more substitutions (e.g., conserved substitutions). For example, this disclosure includes anti-influenza B-HA antibodies having HCVR, LCVR, and / or CDR amino acid sequences having, for example, 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, 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, the 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., a conserved 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.

[0162] For example, this document provides antibodies or antigen-binding fragments thereof that specifically bind to influenza B HA, comprising an HCVR amino acid sequence different from SEQ ID NO:2, wherein the difference is at least 1 to at most 50 conserved amino acid substitutions, for example, at least 10 to at most 40 conserved amino acid substitutions, or at least 20 to at most 50 conserved amino acid substitutions, or at least 20 to at most 40 conserved amino acid substitutions. Additionally, this document provides antibodies or antigen-binding fragments thereof that specifically bind to influenza B HA, comprising an LCVR amino acid sequence different from SEQ ID NO:10, wherein the difference is at least 1 to at most 50 conserved amino acid substitutions, for example, at least 10 to at most 40 conserved amino acid substitutions, or at least 20 to at most 50 conserved amino acid substitutions, or at least 20 to at most 40 conserved amino acid substitutions. Such substitutions may be within the frame region or CDR and maintain the specificity of the antibody or antigen-binding fragment for binding to influenza B HA.

[0163] This document also provides antibodies or antigen-binding fragments thereof comprising HCDR1 having an amino acid sequence different from that of SEQ ID NO:4, wherein the difference is one, two, or three amino acid substitutions (e.g., conserved amino acid substitutions). This document provides antibodies or antigen-binding fragments thereof comprising HCDR2 having an amino acid sequence different from that of SEQ ID NO:6, wherein the difference is one, two, or three amino acid substitutions (e.g., conserved amino acid substitutions). This document provides antibodies or antigen-binding fragments thereof comprising HCDR3 having an amino acid sequence different from that of SEQ ID NO:8, wherein the difference is one, two, or three amino acid substitutions (e.g., conserved amino acid substitutions). This document provides antibodies or antigen-binding fragments thereof comprising LCDR1 having an amino acid sequence different from that of SEQ ID NO:12, wherein the difference is one, two, or three amino acid substitutions (e.g., conserved amino acid substitutions). This document provides antibodies or antigen-binding fragments thereof comprising LCDR2 having an amino acid sequence different from that of SEQ ID NO:14, wherein the difference is one, two, or three amino acid substitutions (e.g., conserved amino acid substitutions). This document provides an antibody or antigen-binding fragment thereof comprising LCDR3 having an amino acid sequence different from that of SEQ ID NO:16, wherein the difference is one, two, or three amino acid substitutions (e.g., conserved amino acid substitutions).

[0164] This disclosure also provides an antibody or antigen-binding fragment thereof comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) different from SEQ ID NO:8 / 16, wherein the difference is 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., conserved amino acid substitutions).

[0165] This disclosure also provides antibodies or antigen-binding fragments thereof comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3), said CDRs being contained within the HCVR / LCVR amino acid sequences of exemplary anti-influenza B HA antibodies shown in Table 1. In some embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence group differs from SEQ ID NO:4-6-8-12-14-16 in that it has up to 20 conserved amino acid substitutions, for example, up to 1, or up to 2, 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 conserved amino acid substitutions.

[0166] This document provides antibodies or antigen-binding fragments thereof comprising HCVR, said HCVR comprising an amino acid sequence according to Table 1, or substantially similar sequences 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 at least 100% sequence identity.

[0167] This document provides antibodies or antigen-binding fragments thereof comprising LCVRs, said LCVRs comprising amino acid sequences according to Table 1, or substantially similar sequences 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 at least 100% sequence identity.

[0168] This document provides antibodies or antigen-binding fragments thereof containing the HCVR / LCVR amino acid sequence pair of SEQ ID NO:2 / 10, or substantially similar sequences 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 at least 100% sequence identity.

[0169] This disclosure also provides antibodies comprising heavy chain CDR1 (HCDR1) or antigen-binding fragments thereof, said heavy chain CDR1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:4, or substantially similar sequences 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 at least 100% sequence identity.

[0170] This disclosure also provides antibodies comprising heavy chain CDR2 (HCDR2) or antigen-binding fragments thereof, said heavy chain CDR2 (HCDR2) comprising the amino acid sequence of SEQ ID NO:6, or substantially similar sequences 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 at least 100% sequence identity.

[0171] This disclosure also provides antibodies comprising heavy chain CDR3 (HCDR3) or antigen-binding fragments thereof, said heavy chain CDR3 (HCDR3) comprising the amino acid sequence of SEQ ID NO:8, or substantially similar sequences 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 at least 100% sequence identity.

[0172] This disclosure also provides antibodies comprising a light chain CDR1 (LCDR1) or an antigen-binding fragment thereof, said light chain CDR1 (LCDR1) comprising the amino acid sequence of SEQ ID NO:12, or substantially similar sequences 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 at least 100% sequence identity.

[0173] This disclosure also provides antibodies comprising a light chain CDR2 (LCDR2) or an antigen-binding fragment thereof, said light chain CDR2 (LCDR2) comprising the amino acid sequence of SEQ ID NO:14, or substantially similar sequences 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 at least 100% sequence identity.

[0174] This disclosure also provides antibodies comprising a light chain CDR3 (LCDR3) or an antigen-binding fragment thereof, said light chain CDR3 (LCDR3) comprising the amino acid sequence of SEQ ID NO:16, or substantially similar sequences 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 at least 100% sequence identity.

[0175] This disclosure also provides antibodies or antigen-binding fragments thereof comprising the HCDR3 and LCDR3 amino acid sequence pairs (HCDR3 / LCDR3), said HCDR3 and LCDR3 amino acid sequence pairs comprising SEQ ID NO:8 / 16, or substantially similar sequences 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 at least 100% sequence identity.

[0176] Preparation of human antibodies

[0177] Methods for generating human antibodies in transgenic mice are known in the art. Any such known method can be used in the context of this disclosure to prepare human antibodies that specifically bind to influenza B HA. An immunogen comprising any of the following can be used to generate antibodies against influenza B HA. In some embodiments, the antibody is obtained from mice immunized with full-length natural influenza B HA (see, for example, GenBank accession number AAA43697.1 or ACA33493.1), or with live attenuated or inactivated virus, or with DNA encoding a protein or fragment thereof. Alternatively, the influenza B HA protein or fragment thereof can be generated using standard biochemical techniques, modified, and used as an immunogen. In some embodiments, the immunogen can be a recombinantly generated influenza B HA protein or fragment thereof. In some embodiments, the immunogen can be an influenza virus vaccine. In some embodiments, one or more booster injections can be administered. In some implementations, booster shots may contain one or more influenza virus strains, or hemagglutinins derived from these strains, for example, first with B / Victoria / 2 / 87, then with B / Yamagata / 16 / 88, and then with B / Victoria / 2 / 87 again; or B / Yamagata / 16 / 88, then with B / Victoria / 2 / 87, and then with B / Yamagata / 16 / 88 as a booster shot. In some aspects, the B / Yamagata strain is replaced by B / Maryland / 03 / 2008, B / Nanchang / 3451 / 93, or B / Florida / 4 / 2006. All mice may 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 some embodiments, the booster injection may contain a mixture of influenza virus strains, or a mixture of hemagglutinins derived from these strains, or DNA encoding HA. In some embodiments, the immunogen may be a recombinant influenza HA peptide expressed in Escherichia coli or any other eukaryotic or mammalian cell (such as Chinese hamster ovary (CHO) cells), or the influenza virus itself.

[0178] use Technology (see, for example, US 6,596,541, Regeneron Pharmaceuticals,) (or any other known method for generating monoclonal antibodies) can first isolate a high-affinity influenza B HA chimeric antibody with both human variable and mouse constant regions. The technology involves generating transgenic mice having a genome containing human heavy and light chain variable regions operatively linked to endogenous mouse constant region loci, such that the mice produce antibodies containing human variable regions and mouse constant regions in response to antigen stimulation. DNA encoding the heavy and light chain variable regions of the antibodies is isolated and operatively linked to DNA encoding the human heavy and light chain constant regions. Subsequently, the DNA is expressed in cells capable of expressing fully human antibodies.

[0179] Generally, attack with the antigen of interest Mice are used, and lymphocytes (such as B cells) are recovered from mice expressing antibodies. Lymphocytes can be fused with myeloma cell lines to prepare indefinitely proliferating hybridoma cell lines, and these hybridoma cell lines can be 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 antigen-specific chimeric antibodies or variable regions of the light and heavy chains can be directly isolated from antigen-specific lymphocytes.

[0180] First, high-affinity chimeric antibodies containing both human variable regions and mouse constant regions are isolated. As described in the Experimental Section below, antibodies are characterized and selected for desired characteristics, including affinity, selectivity, epitopes, etc. The mouse constant region is replaced with the desired human constant region to generate fully human antibodies, such as wild-type or modified IgG1 or IgG4. While the selected constant region can vary depending on the specific application, high-affinity antigen binding and target-specific characteristics are present in the variable region.

[0181] bioequivalent

[0182] The anti-influenza B HA antibodies and antibody fragments disclosed herein encompass proteins whose amino acid sequences differ from those of the described antibodies but retain the ability to bind influenza HA. ​​When compared with parental sequences, such variant antibodies and antibody fragments contain one or more additions, deletions, or substitutions of amino acids but exhibit biological activities substantially equivalent to those of the described antibodies. Similarly, the DNA sequences encoding antibodies disclosed herein encompass sequences containing one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequences, but encoding antibodies or antibody fragments substantially bioequivalent to those disclosed herein.

[0183] Two antigen-binding proteins or antibodies are considered bioequivalent if, for example, they are drug equivalents or drug substitutes that do not show significant differences in absorption rate and extent when administered at the same molar dose in a single or multiple doses under similar experimental conditions. If some antibodies are equivalent in extent of absorption but not in rate of absorption, they are considered equivalents or drug substitutes, but because such differences in absorption rate are intentional and reflected in the labeling, they can be considered bioequivalent. These antibodies are not necessary, for example, to achieve effective in vivo drug concentrations with prolonged use, and are considered not clinically significant for the specific drug being studied.

[0184] In some implementations, two antigen-binding proteins are considered bioequivalent if there are no clinically significant differences in their safety, purity, or potency.

[0185] In some implementations, two antigen-binding proteins are bioequivalent if a patient can make one or more such switches compared to continuous therapy without switching between a reference product and a biological product, without an expected increase in the risk of adverse reactions, including clinically significant changes in immunogenicity or reduced effectiveness.

[0186] In some implementations, the two antigen-binding proteins are bioequivalent if they function through one or more co-operating mechanisms targeting one or more conditions of use to the extent that such mechanisms are known.

[0187] Bioequivalence can be demonstrated by in vivo and / or in vitro methods. Bioequivalence measures include, for example, (a) in vivo testing in humans or other mammals in which the concentration of the antibody or its metabolites in blood, plasma, serum or other biological fluids over time is measured; (b) in vitro testing that is correlated with and can reasonably predict in vivo bioavailability data; (c) in vivo testing in humans or other mammals in which an appropriate acute pharmacological effect of the antibody (or its target) over time is measured; and (d) clinical trials that establish a well-controlled safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0188] Bioequivalent variants of antibodies can be constructed, for example, by various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. For instance, cysteine ​​residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unwanted or incorrect intramolecular disulfide bridges during renaturation. In other cases, bioequivalent antibodies can include antibody variants that incorporate amino acid changes that modify the antibody's glycosylation characteristics, such as mutations that eliminate or remove glycosylation.

[0189] Anti-influenza-HA antibodies containing Fc variants

[0190] According to certain embodiments disclosed herein, an anti-influenza B HA antibody is provided, the anti-influenza B HA antibody comprising an Fc domain, the Fc domain containing one or more mutations that, for example, enhance or reduce the binding of the antibody to the FcRn receptor at an acidic pH compared to a neutral pH. For example, this disclosure includes an anti-influenza-HA antibody, the anti-influenza-HA antibody containing a C-terminus in the Fc domain. H 2 or C H Region 3 contains mutations in which one or more mutations increase the affinity of the Fc domain for FcRn in acidic environments (e.g., in endosomes with a pH range of about 5.5 to about 6.0), relative to the same but unmodified antibody. When administered to animals, such mutations can lead to an increased serum half-life of the antibody relative 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 modifications at position 434. In some embodiments, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S); 428L, 259I (e.g., V259I) and 308F (e.g., V308F); 433K (e.g., H433K) and 434 (e.g., 434Y); 252, 254 and 256 (e.g., 252Y, 254T and 256E); 250Q and 428L (e.g., T250Q and M428L); and 307 and / or 308 (e.g., 308F or 308P). In yet another embodiment, the modifications include 265A (e.g., D265A) and / or 297A (e.g., N297A).

[0191] For example, this disclosure includes an anti-influenza B HA antibody, said anti-influenza B HA antibody comprising an Fc domain, said Fc domain comprising one or more pairs or one or more groups of mutations selected from the following groups: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M42... 8L 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 within the antibody variable domains disclosed herein are covered within the scope of this disclosure.

[0192] This article provides information on chimeric heavy chain constancy (C) H Anti-influenza B HA antibodies in the region ) wherein the chimeric C H The region contains C derived from more than one immunoglobulin isotype. H The segment of the region. For example, an antibody may contain chimeric C. H The region, comprising C derived from human IgG1, human IgG2, or human IgG4 molecules. H 2. Part or all of the structural domain, which is related to the C-terminal molecule derived from human IgG1, human IgG2, or human IgG4. H 3. A combination of some or all of the three domains. According to certain embodiments, the antibody comprises a chimeric C-domain having a chimeric hinge region. H The chimeric hinge region, for example, may comprise an “upper hinge” amino acid sequence (amino acid residues 216 to 227 according to EU numbering) derived from the hinge region of human IgG1, human IgG2, or human IgG4, combined with a “lower hinge” sequence (amino acid residues 228 to 236 according to EU numbering) derived from the hinge region of human IgG1, human IgG2, or human IgG4. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from the upper hinge of human IgG1 or human IgG4 and amino acid residues derived from the lower hinge of human IgG2. Containing chimeric C as described herein. H Antibodies in certain embodiments may exhibit modified Fc effector function without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, for example, U.S. Provisional Application No. 61 / 759,578, filed February 1, 2013, the disclosure of which is incorporated herein by reference in its entirety.)

[0193] Biological characteristics of antibodies

[0194] Generally, the antibodies disclosed herein exert their effects by binding to influenza B HA. For example, this disclosure includes an antibody and an antigen-binding fragment of the antibody that binds to influenza B HA at a KD of less than 10 nM. 例如 (At 25°C or 37°C), as measured in a Biacore instrument by surface plasmon resonance, or by a biosensor based on a real-time biolayer interferometer (OctetHTX assay). In some embodiments, the antibody or its antigen-binding fragment is expressed at a Kc concentration 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. D Combined with influenza B HA, as measured by surface plasmon resonance, for example using a assay form as described herein or a substantially similar assay.

[0195] This disclosure also includes antibodies binding to influenza B virus HA and antigen-binding fragments thereof, having a dissociation half-life (t1 / 2) greater than about 75 minutes, as measured by surface plasmon resonance at 37°C, for example, using an assay format as defined herein, or a substantially similar assay. In some embodiments, the antibodies or antigen-binding fragments disclosed herein bind to influenza HA with a t1 / 2 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, for example, using an assay format as defined herein (e.g., mAb capture or antigen capture format) or a substantially similar assay.

[0196] This 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 exhibit neutralizing potency against a variety of 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, in a micro-neutralization assay or substantially similar assay, IC50 50 The range is from about 1pM to about 800nM, for example, IC 50 The range is from approximately 1 pM to approximately 800 pM, IC 50 The range is from approximately 1 pM to approximately 10 pM, IC 50 The range is from approximately 10 pM to approximately 50 pM, IC 50 The range is from approximately 1 pM to approximately 100 pM, IC 50The range is from approximately 10 pM to approximately 100 pM, and the range of IC50 is from 100 pM to approximately 800 pM, or IC 50 The range is from approximately 500pM to approximately 800pM, IC 50 The range is from about 1 nM to about 10 nM, IC 50 The range is from approximately 10 nm to approximately 50 nm, IC 50 The range is from about 1 nM to about 100 nM, IC 50 The range is from approximately 10 nm to approximately 100 nm, IC 50 The range is from approximately 100 nm to approximately 800 nm, or IC. 50 The range is from approximately 500 nM to approximately 800 nM.

[0197] This disclosure also includes antibodies or antigen-binding fragments thereof that bind to influenza B-infected cells at sub-nanomolar / liter concentrations and exhibit HA-specific binding (see Example 3).

[0198] This disclosure also includes anti-influenza B HA antibodies that demonstrate increased protection (relative to untreated subjects) or effective neutralization of influenza B infection in the body. Some antibodies demonstrate protection upon prophylactic administration (before infection; see Example 4). In some embodiments, a single dose of 5 mg / kg or 0.5 mg / kg anti-HA antibody administered 5 days prior to infection resulted in 100% survival in mice upon prophylactic administration, compared to mice treated with a human IgG1 isotype control antibody.

[0199] For example, prophylactic treatment with anti-influenza B HA antibodies or their antigen-binding fragments can protect mammals from subsequent exposure to influenza virus infection, or reduce the probability or risk of subsequent exposure to influenza virus infection. The protection is selected from the group consisting of: improvement, relief, or reduction in the severity, duration, or frequency of at least one symptom of influenza infection. In some aspects, when prophylactic administration is given to animals (e.g., mammals) prior to exposure to influenza (e.g., at least 2 days, at least 3 days, at least 4 days, at least 5 days, or 2 to 5 days prior to exposure to influenza virus), the risk of influenza infection is reduced. At least one symptom can be selected from the group consisting of: headache, fever, pain, runny nose (nasal congestion), chills, fatigue, weakness, sore throat, cough, shortness of breath, vomiting, diarrhea, pneumonia, bronchitis, and death.

[0200] In one embodiment, the isolated recombinant antibody or its antigen-binding fragment that specifically binds to influenza B HA has one or more of the following characteristics: (a) it is a fully human monoclonal antibody; (b) it has a specific binding concentration of less than about 10 ppm. -9 Less than approximately 10 - 10 M, less than approximately 10 -11M, less than approximately 10 -12 M or less than approximately 10 -13 M's EC 50 (c) showing increased survival of influenza-infected animals compared to comparable influenza-infected animals not treated with isolated recombinant antibodies or their antigen-binding fragments that specifically bind to influenza B HA; and / or (d) comprising (i) three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) containing the amino acid sequence shown in SEQ ID NO:2; and (ii) three light chain CDRs (LCDR1, HCDR2, and HCDR3) contained within a light chain variable region (LCVR) containing the amino acid sequence shown in SEQ ID NO:10.

[0201] The antibodies disclosed herein may have one or more of the aforementioned biological characteristics, or any combination thereof. Other biological characteristics of the antibodies disclosed herein will be apparent to those skilled in the art from this review of the present disclosure, including the examples herein.

[0202] Epitope plotting and related techniques

[0203] This disclosure includes an anti-influenza B HA antibody that interacts with one or more amino acids located within one or more domains of an influenza B HA molecule. The epitope to which the antibody binds may consist of a single adjacent sequence (e.g., a linear epitope within a domain) of three or more amino acids located within the influenza HA molecule. Alternatively, the epitope may consist of multiple non-adjacent amino acids (or amino acid sequences) located within the influenza B HA molecule (e.g., conformational epitopes).

[0204] Various techniques known to those skilled in the art can be used to determine whether an antibody “interacts with one or more amino acids” within a peptide or protein. Exemplary techniques include, for example, conventional cross-blocking analyses, 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 blotting analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide cleavage analysis, crystallographic studies, and NMR analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be employed (Tomer (2000) Prot. Sci. 9:487-496). Another method that can be used to identify amino acids within a peptide interacting with an antibody is hydrogen / deuterium exchange detected by mass spectrometry. Generally, hydrogen / deuterium exchange involves deuteration labeling of the protein of interest, followed by the binding of the antibody to the deuterated protein. Next, the protein / antibody complex is transferred to water. Exchangeable protons within the amino acids protected by the antibody complex undergo deuterium reverse exchange at a slower rate than exchangeable protons within amino acids not part of the interface. Therefore, amino acids forming part of the protein / antibody interface may retain deuterium and thus exhibit relatively higher mass compared to amino acids not included in the interface. After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry analysis to reveal deuterium-tagged residues corresponding to specific amino acids that interact with the antibody. See, for example, Ehring (1999) Analytical Biochemistry 267:252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0205] The term "epitope" refers to a site on an antigen that responds to B cells and / or T cells. B cell epitopes can be formed from consecutive amino acids or from discontinuous amino acids juxtaposed through the ternary folding of a protein. Epitopes formed from consecutive amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed through ternary folding are generally lost upon treatment with denaturing solvents. Epitopes typically comprise at least three amino acids in a unique spatial conformation, or at least four amino acids, for example, at least five, six, seven, eight, nine, or ten amino acids.

[0206] 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) against the same antigen based on the similarity of the binding profiles of each antibody to a chemically or enzymatically modified antigen surface (see US 2004 / 0101920, specifically incorporated herein by reference in its entirety). Each category can reflect a unique epitope that is distinctly different from or partially overlaps with the epitopes represented by another category. This technique allows for rapid filtering of genetically identical antibodies, thus enabling characterization to focus on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce monoclonal antibodies with the desired characteristics. MAP can be used to classify the antibodies presented in this article into groups of antibodies that bind to different epitopes.

[0207] In some implementations, an influenza A virus HA antibody or its antigen-binding fragment binds to an epitope or fragment thereof within any one or more example regions of influenza HA, said epitope being naturally formed or generated in a recombinant manner.

[0208] This disclosure includes anti-influenza B HA antibodies that bind to the same epitope or a portion of an epitope. Similarly, this disclosure also includes anti-influenza B HA antibodies that compete with any of the specific exemplary antibodies described herein for binding to influenza B HA or fragments thereof. For example, this disclosure includes anti-influenza B HA antibodies that cross-compete with one or more antibodies obtained from those antibodies described in Table 1 for binding to influenza B HA.

[0209] Whether an antibody binds to the same epitope as a reference anti-influenza B HA antibody or competitively binds to a reference anti-influenza B HA antibody can be determined using known methods. For example, to determine whether a test antibody binds to the same epitope as a reference anti-influenza B HA antibody, the reference antibody is saturated with influenza B HA or a peptide. The ability of the test antibody to bind to the influenza B HA molecule is then evaluated. If the test antibody binds 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. Conversely, if the test antibody does not 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 reference anti-influenza B HA antibody.

[0210] To determine whether an antibody competes with a reference anti-influenza B HA antibody for binding, the binding method described above is performed in two directions: In the first direction, the reference antibody is bound to the influenza B HA molecule under saturation conditions, and then the binding of the test antibody to the influenza B HA molecule is evaluated. In the second direction, the test antibody is bound to the influenza B HA molecule under saturation conditions, and then the binding of the reference antibody to the influenza B HA molecule is evaluated. If only the first (saturated) antibody can bind to the influenza B HA molecule in both directions, it can be concluded that the test antibody and the reference antibody compete for binding to the influenza B HA molecule. As will be understood by those skilled in the art, an antibody competing with the reference antibody does not necessarily bind to the same epitope as the reference antibody, but can spatially block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0211] If two antibodies competitively inhibit (block) the binding of another antibody to an antigen, then both antibodies bind to the same or overlapping epitopes. That is, as measured in a competitive binding assay, an excess of 1, 5, 10, 20, or 100 times that of one antibody inhibits the binding of the other antibody by at least 50%, such as 75%, 90%, or even 99% (see, for example, Junghans et al., Cancer Res. 199050:1495-1502). Alternatively, if substantially 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, then the two antibodies have the same epitope. If some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody, then the two antibodies have overlapping epitopes.

[0212] Further routine experiments (e.g., peptide mutation and binding assays) can then be performed to confirm whether the observed lack of binding to the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether steric hindrance (or another phenomenon) is the cause of the observed lack of binding. Such experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0213] Immunoconjugates

[0214] This document envisions human anti-influenza B HA monoclonal antibodies (“immunoconjugates”), such as toxoids or antiviral drugs, conjugated with a therapeutic portion to treat influenza virus infection. As used herein, the term “immunoconjugate” refers to an antibody chemically or biologically linked to a radioactive agent, cytokine, interferon, target or reporter portion, enzyme, peptide or protein, or therapeutic agent. An antibody may be conjugated to a radioactive agent, cytokine, interferon, target or reporter portion, enzyme, peptide, or therapeutic agent at any position along the molecule, as long as it can bind to its target. Examples of immunoconjugates include antibody-pharmaceutical conjugates and antibody-toxin fusion proteins. In one embodiment, the agent may be a second, different antibody against influenza HA. ​​In some embodiments, the antibody may be conjugated to an agent specifically targeting virus-infected cells. The type of therapeutic portion that may be conjugated to an anti-influenza HA antibody will take into account the condition to be treated and the desired therapeutic effect to be achieved. Examples of suitable agents for forming immunoconjugates are known in the art; see, for example, WO 05 / 103081.

[0215] Multispecific antibodies

[0216] The antibodies disclosed herein can be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific to different epitopes of a single target polypeptide, or may contain antigen-binding domains specific to more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244.

[0217] 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) as known to those skilled in the art.

[0218] In some implementations, influenza B HA-specific antibodies are generated in a bispecific form (“bi-specific” or “bispecific”), wherein variable regions that bind to different domains of influenza B HA are linked together to confer bidomain specificity within a single binding molecule. Properly designed bispecificity can enhance the overall inhibitory efficacy of the influenza B HA protein by increasing specificity and binding affinity. Variable regions specific to individual domains (e.g., segments of the N-terminal domain), or variable regions that can bind to different regions within a domain, are paired on a structural backbone that allows each region to bind to a single epitope simultaneously, or to different regions within a domain. In one example of bispecificity, a heavy chain variable region (V... H) and the light chain variable region (V) from a series of conjugates specific to the second domain L Recombined to determine which can be combined with the original V H Pairing without destroying the V H The original specific non-homologous V L Matching elements. In this way, a single V L Section (e.g., V) L 1) Can be used with two different V H Domain (e.g., V) H 1 and V H 2) Combining to produce a combination of two combined "groups" (V) H 1-V L 1 and V H 2-V L 1) The dual specificity of the structure. Using a single V L The segments reduce the complexity of the system, thereby simplifying and improving the efficiency of the cloning, expression, and purification processes used to generate bispecific clones (see, for example, USSN13 / 022759 and US2010 / 0331527).

[0219] Alternatively, antibodies binding to more than one domain and a second target, such as, but not limited to, a second distinct anti-influenza B HA antibody, can be prepared in a bispecific form using the techniques described herein or other techniques known to those skilled in the art. The antibody variable region binding to different regions can be linked to a variable region binding to an associated site, for example, on the influenza virus, to confer dual antigen specificity within a single binding molecule. Such bispecificity, appropriately designed, has a dual function. Variable regions specific to the extracellular domain are combined with variable regions specific to areas outside the extracellular domain and paired on a structural backbone, enabling each variable region to bind to a separate antigen.

[0220] Exemplary bispecific antibody forms that may be used in the context of this disclosure relate to first immunoglobulin (Ig) C H 3 structural domains and second Ig C H 3. Use of structural domains, wherein the first and second Ig C H At least one amino acid in the three domains differs from each other, and this difference in at least one amino acid reduces the binding of the bispecific antibody to protein A compared to bispecific antibodies lacking this difference. In one embodiment, the first Ig C H 3-domain binding to protein A and second IgC H Domain 3 contains mutations that reduce or eliminate protein A binding, such as H95R modification (based on IMGT exon numbering; H435R based on EU numbering). Second C H3 may also include Y96F modifications (according to IMGT; Y436F according to EU). In the second C H Other modifications that may be found within 3 include: in the case of IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V422I according to EU); in the case of IgG2 antibodies, N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I according to EU); and in the case of IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I according to EU). Variations of the bispecific antibody forms described above are contemplated within the scope of this disclosure.

[0221] Other exemplary bispecific forms that may be used in the context disclosed herein include, but are not limited to, scFv-based or bispecific forms, IgG-scFv fusions, dual variable domain (DVD)-Ig, tetrageneous hybridomas, buttonholes, common light chains (e.g., common light chains with buttonholes, etc.), CrossMab, CrossFab, (SEED) bodies, leucine zippers, Duobody, IgG1 / IgG2, dual-action Fab (DAF)-IgG, and Mab2 bispecific forms (for a review of the foregoing forms, see, for example, Klein et al., 2012, mAbs 4:6, 1-11, and the references cited therein). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugations, for example, where site-specific antibody-oligonucleotide conjugations are generated using non-natural amino acids with orthogonal chemical reactivity, and then self-assembled into multimeric complexes with defined composition, valence, and geometry. (See, for example, Kazane et al., J. Am. Chem. Soc. [Epub: December 4, 2012]).

[0222] Therapeutic application and formulation

[0223] This document provides therapeutic compositions comprising an anti-influenza B HA antibody or an antigen-binding fragment thereof. The therapeutic compositions according to this disclosure are to be administered together with pharmaceutically acceptable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerability, etc. Numerous suitable formulations can be found in all formularies known to pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, and lipid-containing (cationic or anionic lipid) vesicles (such as LIPOFECTIN). TM DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol 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.

[0224] The antibody dosage may vary depending on the age and physical condition of the individual to be administered, the target disease, symptoms, route of administration, etc. When the antibodies disclosed herein are used to treat or prevent diseases or disorders in adult patients, it is generally advantageous to administer the antibodies disclosed herein in a single dose of about 0.1 to about 60 mg / kg body weight, or about 5 to about 60, about 10 to about 50, or about 20 to about 50 mg / kg body weight. The frequency and duration of treatment may be adjusted depending on the severity of the condition. In some embodiments, the antibody or its antigen-binding fragment may be administered at 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 some embodiments, a second or more subsequent doses of the antibody or its antigen-binding fragment may be administered after the initial dose, the amount of which may be substantially equal to or less than the amount of the initial dose, wherein the interval between the subsequent doses is at least 1 to 3 days; at least one 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.

[0225] Various delivery systems are known and can be used to administer pharmaceutical compositions, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, for example, Wu et al., (1987) J. Biol. Chem. 262:4429-4432). Methods of administration include (but are not limited to): intradermal, percutaneous, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered via any convenient route, such as by infusion or bolus injection, by absorption through the epithelial or mucosal inner lining of the skin (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered with other bioactive agents. Administration can be systemic or local. Pharmaceutical compositions can also be delivered in vesicles, especially liposomes (see, for example, Langer (1990) Science 249:1527-1533).

[0226] This document also considers the use of nanoparticles to deliver the antibodies disclosed herein. Antibody-conjugated nanoparticles can be used for therapeutic and diagnostic applications. Antibody-conjugated nanoparticles, as well as methods of their preparation and use, are described in detail by Arruebo, M. et al., 2009 (“Antibody-conjugated nanoparticles for biomedical applications”, J. Nanomat. Vol. 2009, Article No. 439389, p. 24, doi:10.1155 / 2009 / 439389), which is incorporated herein by reference. Nanoparticles can be developed and conjugated with antibodies contained in pharmaceutical compositions to target virus-infected cells. Nanoparticles for drug delivery have also been described, for example, in US 8257740 or US 8246995, each of which is incorporated herein by reference in its entirety.

[0227] In some cases, a controlled-release system can be used to deliver a drug composition. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be used. In yet another embodiment, the controlled-release system can be placed near the target of the composition, thereby requiring only a portion of the systemic dose.

[0228] Injectable formulations can include dosage forms for intravenous, subcutaneous, intradermal, intracranial, intraperitoneal, and intramuscular injection, infusion, etc. These injectable formulations can be prepared by well-known methods. For example, injectable formulations can be prepared by dissolving, suspending, or emulsifying, for instance, the antibodies or their salts described above in a sterile aqueous or oily medium conventionally used for injection. As an aqueous medium for injection, there are isotonic solutions such as physiological saline, glucose, and other adjuvants, which can be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (hydrogenated castor oil polyoxyethylene (50 mol) adduct)]. As an oily medium, sesame oil, soybean oil, etc., are used, which can be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, etc. The resulting injection is preferably filled in a suitable ampoule.

[0229] The disclosed pharmaceutical compositions can be delivered subcutaneously or intravenously using standard needles and syringes. Furthermore, for subcutaneous delivery, pen-type delivery devices are readily applicable for delivering the disclosed pharmaceutical compositions. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all the pharmaceutical composition in 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-type delivery device can then be reused. In disposable pen-type delivery devices, there is no replaceable cartridge. In fact, disposable pen-type delivery devices have a reservoir pre-filled with the pharmaceutical composition within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0230] Various reusable pen-based delivery devices and auto-injector delivery devices are used for subcutaneous delivery of the pharmaceutical compositions disclosed herein. Examples include, but are not limited to: AUTOPEN TM (Owen Mumford, Woodstock, UK); DISETRONIC TM Pen (Disetronic Medical Systems, Burghdorf, Switzerland); HUMALOG MIX 75 / 25 TM Pen; HUMALOG TM Pen; HUMALIN 70 / 30 TM Pen (Eli Lilly and Co., Indianapolis, IN); NOVOPEN TMI, II, and III (Novo Nordisk, Copenhagen, Denmark); NOVOPEN JUNIOR TM (NovoNordisk in Copenhagen, Denmark); BD TM Pen (Becton Dickinson, Franklin Lakes, NJ); OPTIPEN TM OPTIPEN PRO TM ;OPTIPEN STARLET TM And OPTICLIK TM (e.g., Sanofi-Aventis in Frankfurt, Germany). Examples of disposable pen delivery devices that can be used for subcutaneous delivery of the pharmaceutical compositions disclosed herein include, but are not limited to, SOLOSTAR. TM Pen (Sanofi-Aventis), FLEXPEN TM (Novo Nordisk) and KWIKPEN TM (Eli Lilly), SURECLICK TM Automatic injector (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA) TM Pen (Abbott Labs, Abbott Park, IL), etc.

[0231] Advantageously, the pharmaceutical compositions described above for oral or non-enteric use are prepared into dosage forms suitable for unit doses corresponding to the dosage of the active ingredient. Such unit-dose dosage forms include, for example, tablets, pills, capsules, ampoules, suppositories, etc. In a unit dose, each dosage form typically contains an antibody amount of about 5 to about 5000 mg; particularly in injectable forms, it is preferred that the antibody content is about 5 to about 500 mg, and in other dosage forms about 10 to about 250 mg.

[0232] Therapeutic uses of antibodies

[0233] The antibodies disclosed herein may be used to treat and / or prevent diseases, disorders or conditions associated with influenza B virus infection and / or to improve at least one symptom associated with such diseases, disorders or conditions.

[0234] In some embodiments, the antibodies provided herein can be used to treat subjects suffering from severe and acute respiratory infections caused by influenza B virus. In some embodiments, the antibodies can be used to reduce viral titers or reduce viral load in the host. In one embodiment, the antibody or its antigen-binding fragment can be administered at a therapeutic dose to a patient infected with influenza B virus.

[0235] Compared to untreated but similarly localized subjects, 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. The antibodies can be used to improve 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, extreme exhaustion, shortness of breath, bronchitis, pneumonia, and death.

[0236] This paper also envisions the prophylactic use of one or more antibodies disclosed herein in subjects at risk of developing influenza virus infection, such as immunocompromised individuals, older adults (65 years or older), children under 2 years of age, healthcare workers, family members who have been in close contact with patients with influenza virus infection, and patients with a history of illness (e.g., increased risk of lung infection, heart disease, or diabetes).

[0237] In another embodiment, the antibody of the present invention is used to prepare a pharmaceutical composition for treating a patient suffering from an influenza virus infection. In another embodiment, the antibody is used as an adjunct therapy in conjunction with any other agent or therapy known to those skilled in the art for treating or improving influenza virus infection.

[0238] Combination therapy

[0239] Combination therapies may include anti-influenza B HA antibodies and any other therapeutic agents that can be advantageously combined with the antibody or a biologically active fragment of the antibody. The antibodies disclosed herein may be synergistically combined with one or more drugs or agents used to treat influenza viruses (e.g., antiviral agents).

[0240] For example, exemplary antiviral agents include, for instance, vaccines, neuraminidase inhibitors, or nucleoside analogs. Other exemplary antiviral agents that can be used in combination with the antibodies provided herein may include, for instance, zidovudine, ganciclovir, vidarabine, iodouridine, trifluuridine, phosphonoformic acid, acyclovir, ribavirin, amantadine, remantidine, saquinavir, indinavir, ritonavir, alpha-interferon and other interferons, and neuraminidase inhibitors (e.g., zanamivir). Osemivir Laninavir, peramivir, or amantadine.

[0241] 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.

[0242] In some implementations, the antibodies described herein can be combined with a second therapeutic agent to reduce the viral load in patients with influenza virus infection or to improve one or more symptoms of the infection.

[0243] The antibodies disclosed in this article can be used with anti-inflammatory drugs (e.g., corticosteroids and nonsteroidal anti-inflammatory drugs), decongestants, antihistamines, anti-infectives, different influenza virus antibodies, antiviral drugs, and influenza virus vaccines (such as...). or It can be used in combination with dietary supplements (such as antioxidants) or another palliative therapy for treating influenza virus infection. Palliative therapy includes treating the subject to make the subject more comfortable and / or reduce the severity of influenza B infection symptoms.

[0244] In some embodiments, the second therapeutic agent is another influenza antibody. In some embodiments, the second therapeutic agent is another antibody targeting influenza hemagglutinin (e.g., influenza A hemagglutinin). In some embodiments, the second therapeutic agent is another antibody targeting the tetrameric extracellular domain of a different influenza protein, such as neuraminidase or matrix protein 2 (M2e protein). In some embodiments, the second therapeutic agent is an antibody targeting a different protein, such as host transmembrane protease serine 2 (TMPRSS2). The second antibody may specifically target one or more different influenza virus proteins from different subtypes or strains. The use of antibody combinations (“mixtures”) with broad neutralizing or inhibitory activity against influenza viruses is considered herein. In some embodiments, non-competitive antibody combinations may be combined and administered to subjects in need to reduce the ability of influenza viruses to rapidly mutate and evade due to selective pressure. In some embodiments, the antibody comprising the combination binds to different non-overlapping epitopes on the HA protein. The antibody comprising the combination may prevent the virus from attaching to and / or entering and / or fusing with host cells. The antibody can interact with hemagglutinin selected from any one or more influenza B virus strains, and when used alone or in combination with any one or more of the above-mentioned agents, it can neutralize any one or more influenza B virus strains, including B / Victoria / 2 / 87, B / Yamagata / 16 / 88, B / Maryland / 03 / 2008, B / Nanchang / 3451 / 93 and / or B / Florida / 4 / 2006.

[0245] In addition to the anti-influenza B HA antibodies disclosed herein, combinations of anti-influenza HA antibodies are also considered, wherein the combination comprises one or more non-competitive antibodies; in some embodiments, the combination comprises a first antibody having broad neutralizing activity and a second antibody having activity against narrow-spectrum isolates and not cross-competing with the first antibody.

[0246] As used herein, the term "in combination with" means that one or more additional therapeutically active components may be administered before, simultaneously with, or after the administration of the anti-influenza B HA antibody disclosed herein. The term "in combination with" also includes sequential or simultaneous administration of the anti-influenza HA antibody and a second therapeutic agent.

[0247] Prior to administering the anti-influenza B HA antibody disclosed herein, one or more additional therapeutically active components may be administered to the subject. For example, 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 administering the second component, then the first component can be considered to have been administered "before" the second component. In other embodiments, after administering the anti-influenza B HA antibody disclosed herein, one or more additional therapeutically active components may be administered to the subject. For example, 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, or 72 hours after the administration of the second component, then the first component can be considered to be administered "after" the second component. In other embodiments, one or more additional therapeutically active components may be administered to the subject concurrently with the administration of the anti-influenza B HA antibody disclosed herein. For the purposes of this disclosure, "concurrent" administration includes, for example, administering the anti-influenza B HA antibody and the additional therapeutically active component to the subject in a single dosage form or in separate dosage forms approximately 30 minutes or less apart. If administered in separate dosage forms, each dosage form may be administered via the same route (e.g., the anti-influenza B HA antibody and the additional therapeutic active component may both be administered intravenously); or each dosage form may be administered via different routes (e.g., the anti-influenza B HA antibody may be administered intravenously, while the additional therapeutic active component may be administered orally). In any case, for the purposes of this disclosure, administration of the components in a single dosage form, in a single dosage form via the same route, or in a single dosage form via different routes is considered "simultaneous administration." For the purposes of this disclosure, administration of the additional therapeutic active component "before," "simultaneously with," or "after" administration of the anti-influenza B HA antibody (those terms as defined above) is considered administration of the anti-influenza B HA antibody in "combination" with the additional therapeutic active component.

[0248] This disclosure includes pharmaceutical compositions wherein the anti-influenza B HA antibody disclosed herein is co-formulated with one or more additional therapeutically active components as described elsewhere herein.

[0249] Application plan

[0250] According to certain implementation schemes, the single-dose anti-influenza B HA antibody or its antigen-binding fragment (or...) provided herein... Pharmaceutical compositions comprising such anti-influenza HA antibodies or fragments in combination with any other therapeutically active agents mentioned herein can be administered to subjects in need. According to certain embodiments disclosed herein, multiple doses of anti-influenza B HA antibodies (or pharmaceutical compositions comprising anti-influenza B HA antibodies in combination with any of the other therapeutically active agents mentioned herein) can be administered to subjects within a defined timeframe. Methods according to this aspect of the disclosure include sequentially administering multiple doses of anti-influenza B HA antibodies to a subject. As used herein, “sequentially administering” means administering each dose of anti-influenza B HA antibody at different time points, for example, on different days separated by predetermined intervals (e.g., hours, days, weeks, or months). The disclosure includes methods that sequentially administer a single initial dose of anti-influenza B HA antibody, followed by one or more second doses of anti-influenza B HA antibody, and optionally subsequently one or more third doses of anti-influenza B HA antibody to a patient.

[0251] The terms "initial dose," "second dose," and "third dose" refer to the temporal sequence of administration of anti-influenza B HA antibodies. Therefore, the "initial dose" is the dose administered at the start of the treatment regimen (also known as the "basal dose"); the "secondary dose" is the dose administered after the initial dose; and the "tertiary dose" is the dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of anti-influenza HA antibodies, but they can generally differ in their frequency of administration. However, in some embodiments, the amount of anti-influenza B HA antibodies contained in the initial, second, and / or third doses may differ from each other during treatment (e.g., appropriately up- or down-adjusted). In some embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the start of the treatment regimen as a "loading dose," followed by subsequent doses (e.g., "maintenance doses") at a lower frequency.

[0252] In some exemplary embodiments, each second and / or third dose is administered 1 to 48 hours immediately following the previous dose (e.g., 1, 1...). 1 / 2、2、2 1 / 2, 3, 3 1 / 2、4、4 1 / 2, 5, 5 1 / 2, 6, 6 1 / 2、7、7 1 / 2、8、8 1 / 2, 9, 9 1 / 2, 10, 10 1 / 2, 11, 11 1 / 2, 12, 12 1 / 2, 13, 13 1 / 2, 14, 141 / 2, 15, 15 1 / 2, 16, 16 1 / 2, 17, 17 1 / 2, 18, 18 1 / 2, 19, 19 1 / 2, 20, 20 1 / 2、21、21 1 / 2、22、22 1 / 2、23、23 1 / 2、24、24 1 / 2、25、25 1 / 2, 26, 26 1 (2 hours or longer) Administered. As used herein, the phrase “immediately following the previous dose” means that in a sequence of multiple administrations, the dose of the anti-influenza B HA antibody is administered to the patient before the next dose in the sequence, without intermediate doses.

[0253] Methods according to this aspect may include administering any number of second and / or third doses of anti-influenza B HA antibody to a patient. For example, in some 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 some 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.

[0254] In some implementation schemes, the frequency of administering the second and / or third dose to the patient during the treatment regimen can vary. The physician may also adjust the administration frequency based on the individual patient's needs following clinical examination.

[0255] Diagnostic uses of antibodies

[0256] Anti-influenza B HA antibodies can be used to detect and / or measure influenza B HA in samples, for example, for diagnostic purposes. Some embodiments consider using one or more antibodies in assays to detect diseases or disorders such as viral infections. Exemplary diagnostic assays for influenza B HA may include, for example, contacting a sample obtained from a patient with an anti-influenza B HA antibody, wherein the anti-influenza B HA antibody is labeled with a detectable marker or reporter molecule or used as a capture ligand to selectively isolate influenza B virus HA from the patient sample. Alternatively, unlabeled anti-influenza B HA antibodies may be combined with a secondary antibody that is itself detectably labeled for diagnostic applications. The detectable marker or reporter molecule may be a radioactive isotope, such as... 3 H, 14 C 32 P,35 S or 125 I; a fluorescent or chemiluminescent component, 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).

[0257] Samples that can be used in influenza B HA diagnostic assays include any tissue or fluid sample obtainable from a patient that contains detectable amounts of influenza B HA or fragments thereof under normal or pathological conditions. Typically, the level of influenza B HA is measured in a specific sample obtained from a healthy patient (e.g., a patient without influenza-related illness) to initially establish a baseline or standard level of influenza B HA. This baseline level of influenza B HA can then be compared with the level of influenza B HA measured in samples obtained from individuals suspected of having influenza B HA-related illness or symptoms associated with such illness.

[0258] Antibodies specific to influenza B HA may be free of additional labels or portions, or they may contain N-terminal or C-terminal labels or portions. In one embodiment, the label or portion is biotin. In binding assays, the position of the label (if present) determines the orientation of the peptide relative to the surface to which the peptide binds. For example, if the surface is coated with avidin, peptides containing N-terminal biotin will be oriented such that the C-terminal portion of the peptide is distal to the surface.

[0259] Example

[0260] The following examples are provided to provide a complete disclosure and description of how to prepare and use the methods and compositions disclosed herein to those skilled in the art, and are not intended to limit the scope of the disclosure as the inventors believe. Efforts have been made to ensure the accuracy of the figures used (e.g., amounts, temperatures, etc.), but some experimental errors and biases should be taken into account. Unless otherwise specified, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, room temperature is about 25°C, and pressure is atmospheric pressure or close to atmospheric pressure.

[0261] Example 1: Production of human antibodies against influenza B hemagglutinin (HA)

[0262] exist Human antibodies against influenza hemagglutinin were generated in mice, containing DNA encoding the variable regions of the heavy and κ light chains of human immunoglobulins. Mice were immunized with a combination of vectors expressing influenza A and B hemagglutinin, then infected and recovered with influenza A and B virus strains, followed by administration of a booster containing a mixture of recombinant hemagglutinin proteins. The antibody immune response was monitored by an influenza HA-specific immunoassay. As described in U.S. Patent 7,582,298, anti-influenza B HA antibodies were isolated directly from antigen-positive mouse B cells that were not fused with myeloma cells, which is specifically incorporated herein by reference in its entirety. Using this method, fully human anti-influenza HA antibodies (i.e., antibodies having human variable and human constant domains) were obtained.

[0263] The exemplary antibody described herein is referred to as mAb 35490. The biological characteristics of the exemplary antibody produced according to the method of this embodiment are described in detail in the embodiments set forth below.

[0264] Example 2: Amino acid and nucleotide sequences of the variable regions of the heavy and light chains

[0265] Table 1 lists the heavy and light chain variable regions and CDRs, as well as the amino acid sequence identifiers of the heavy and light chain sequences of exemplary anti-influenza B HA antibodies. The corresponding nucleic acid sequence identifiers are listed in Table 2.

[0266] Table 1: Amino Acid Sequence Identifiers

[0267]

[0268] Table 2: Nucleic Acid Sequence Identifiers

[0269]

[0270] The antibodies disclosed herein have a fully human variable region, but may have a mouse constant region (e.g., mouse IgG1 Fc or mouse IgG2 Fc (a or b isotype)) or a human constant region (e.g., human IgG1 Fc or human IgG4 Fc). As will be understood by those skilled in the art, antibodies with a particular Fc isotype can be converted into antibodies with a different Fc isotype (e.g., an antibody with mouse IgG1 Fc can be converted into an antibody with human IgG4, etc.), but in any case, the variable domains (including CDRs)—represented by the numerical identifiers shown in Tables 1 and 2—will remain unchanged, and the binding properties with the antigen are expected to be the same or substantially similar, regardless of the nature of the Fc domains.

[0271] Example 3: Antihemagglutinin antibodies binding to influenza-infected cells

[0272] 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. Cells were incubated at 37°C and 5% CO2 for four hours. The plates were then infected with 50 μL of 10E-2.5 dilution of influenza B / Florida / 4 / 2006 virus (a vaccine strain of the B / Yamagata lineage), gently tapped, and returned to 37°C and 5% CO2 for 20 hours. The plates were then washed once with PBS and fixed with 50 μL of 4% PFA in PBS solution, and incubated at room temperature for 15 minutes. The plates were washed three times with PBS and then incubated with 300 μL of StartingBlock solution. TM Block with (PBS) blocking buffer (Thermo Fisher Scientific) for one hour at room temperature. Influenza B anti-HA antibody mAb35490P (formulated with human IgG1 Fc) and human IgG1 isotype control antibody were diluted in blocking buffer to an initial concentration of 100 μg / mL, and titrated 1:4 to a final concentration of 6.1E-3 μg / mL. After incubation, the blocking buffer was removed from the plates, and the diluted antibody was added to the cells at 75 μL / well. The plates were incubated at room temperature for one hour. After incubation, the plates were washed with wash buffer (imidazole-buffered saline and... 20. Wash plates three times with a solution diluted to 1X in Milli-Q water and cover with 75 μL / well of secondary antibody (peroxidase AffiniPure donkey anti-human IgG, Jackson Immuno Research) (diluted 1:2000 in blocking buffer). Incubate the second solution on the plates at room temperature for one hour. Then, wash the plates three times with washing buffer and add 75 μL / well of the developing substrate ELISA Pico substrate (prepared 1:1). Immediately read the luminescence of the plates on a Molecular Devices Spectramax i3x reader. EC 50 The data is shown in Table 3.

[0273] Table 3: Anti-HA binding ELISA of FluB-infected cells

[0274] Antibody <![CDATA[EC 50 log[M]]]> Human IgG1 isotype control Do not combine mAb35490 7.610E-10

[0275] As shown in Table 3, mAb35490 bound to influenza B-infected cells at sub-nanomoles / liter concentrations, demonstrating specific binding to HA.

[0276] Example 4: Prevention of death caused by influenza B through prophylactic antibody administration

[0277] Five days prior to infection, Balb / C Elite mice were treated with a subcutaneous injection into the nape of the neck with either 5 mg / kg or 0.5 mg / kg mAb35490 (formulated with human IgG1 Fc) or 5 mg / kg human IgG1 isotype control antibody (diluted in PBS). On the day of infection, the influenza B / Florida / 4 / 2006 virus stock solution was thawed on ice and diluted in PBS from 2.05E08 pfu / mL to 20 μL PBS (2.0E05 pfu / mL) containing 4000 pfu of virus. The virus was kept on ice. Each mouse was injected with a mixture of 2.4 mg / kg chlorpheniramine and 0.1 mg / kg toluidine and allowed to rest for approximately 10–15 minutes until they were fully asleep. Then, each mouse was given 20 μL of virus (4000 pfu) intranasally for complete inhalation. Weight loss and morbidity were then monitored in the mice two weeks post-infection. A weight loss of 25% or more compared to the initial weight at the time of infection necessitates euthanasia. The results are shown in Table 4.

[0278] Table 4: Survival rate of mice after administration of mAb35490 or isotype control

[0279]

[0280] All animals treated with the human IgG1 isotype control antibody died from the infection and required euthanasia eight days after infection. All animals treated with 5 mg / kg and 0.5 mg / kg mAb35490 survived the infection, demonstrating the efficacy of the FluB anti-HA antibody mAb35490. sequence list <110> Regeneron Pharmaceuticals, Inc. Alina A. Baum Christos, Kyratsous <120> Antihemagglutinin antibodies and their usage <130> 070860-8082CN01 <140> TBD <141> 2020-10-28 <150> 62 / 926,914 <151> 2019-10-28 <150> 63 / 094,170 <151> 2020-10-20 <160> twenty four <170> PatentIn Version 3.5<…><210> 1<…><211> 351<…><212> DNA<…><213> Artificial Sequence<…><220><…><223> Synthetic<…><400> 1[[ID=1S]]<…>gaggtgcagc tggtggagtc tggaggagac ttggtccagc ctggggggtc cctgagactc 60<…>tcctgtgcag cctctggatt caccgtcagt agcaactata tgagttgggt ccgccaggtt 120<…>ccagggaagg ggctggactg ggtctcagtt acttatagcg gtggtaacac atactacgca 180[[ID=Z1]]<…>gactccgtga aaggccgatt caccatttcc agacacaatt ccaagaacac gctatatctt 240<…>caaatgaaca gcctgagaat tgaggacacg gccgtttatt actgtgcgac cgtaccctcg 300<…>tttcacggta tggacgtctg gggccaaggg accacggtca ccgtctcctc a 351<…><210> 2<…><211> irl<…><212> PRT<…><Z13> Artificial Sequence<…><220><…><223> Synthetic<…><400> 2<…>Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly<…>1 5 10 15<…>Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn 20 25 30 Tyr Met Ser Trp Val Arg Gln Val Pro Gly Lys Gly Leu Asp Trp Val 35 40 45 Ser Val Thr Tyr Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg His Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ile Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Thr Val Pro Ser Phe His Gly Met Asp Val Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser [[ID=2⑥]]115 <210> 3 [[ID=③0]]<211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 3 ggattcaccg tcagtagcaa ctat 24 <2①0> 4 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 4 Gly Phe Thr Val Ser Ser Asn Tyr 1 5 <210> 5 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> synthesis <400> 5 acttatagcg gtggtaacac a 21 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 6 Thr Tyr Ser Gly Gly Asn Thr 1 5 <210> 7 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> synthesis <400> 7 gcgaccgtac cctcgtttca cggtatggac gtc 33 <210> 8 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 8 Ala Thr Val Pro Ser Phe His Gly Met Asp Val 1 5 10 <210> 9 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 9 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccgtca 180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg caacttacta ctgtcaacag agttacagta cccctccgat caccttcggc 300 caagggacac gactggagat taaa 324[[ID=二十一]] [[ID=二十二]]<210> 10[[ID=二十三]] [[ID=二十四]]<211> 108[[ID=二十五]] [[ID=二十六]]<212> PRT[[ID=二十七]]<二十四> [[ID=二十八]]<213> Artificial Sequence[[ID=二十九]] [[ID=三十]]<220>[[ID=三十一]] [[ID=三十二]]<223> Synthetic[[ID=三十三]]<00009>[[ID=三十四]]<400> 10[[ID=三十五]] [[ID=三十六]]Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly[[ID=三十七]]<0000九> [[ID=三十八]]1 5 10 15[[ID=三十九]]<000090三> [[ID=四十]]Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr[[ID=四十一]]<000090四> [[ID=四十二]]20 25 30[[ID=四十三]]<000090五>Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> synthesis <400> 11 cagagcatta gcagctat 18 <210> 12 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 12 Gln Ser Ile Ser Ser Tyr 1 5 <210> 13 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> synthesis <400> 13 gctgcatcc 9 <210> 14 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 14 Ala Ala Ser 1 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> synthesis <400> 15 caacagagtt acagtacccc tccgatcacc 30 <210> 16 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 16 Gln Gln Ser Tyr Ser Thr Pro Pro Ile Thr 1 5 10 <210> 17 <211> 1344 <212> DNA <213> Artificial Sequence <220> <223> synthesis <400> 17 gaggtgcagc tggtggagtc tggagaggagac ttggtccagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt caccgtcagt agcaactata tgagttgggt ccgccaggtt 120 ccagggaagg ggctggactg ggtctcagtt acttatagcg gtggtaacac atactacgca 180 gactccgtga aaggccgatt caccatttcc agacacaatt ccaagaacac gctatatctt 240 aaatgaaca gcctgagaat tgaggacacg gccgtttatt actgtgcgac cgtaccctcg 300 tttcacggta tggacgtctg gggccaaggg accacggtca ccgtctctc agcctccacc 360 aagggcccat cggtcttcc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 420 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 480 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 540 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 600 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 660 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 720 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 780 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 840 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 960 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1020 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 1080 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1140 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1260 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagtcc 1320 ctctccctgt ctccgggtaa atga 1344 <210> 18 <211> 447 ​​​​​​​​​​Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn 20 25 30 Tyr Met Ser Trp Val Arg Gln Val Pro Gly Lys Gly Leu Asp Trp Val 35 40 45 Ser Val Thr Tyr Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg His Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ile Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Thr Val Pro Ser Phe His Gly Met Asp Val Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu ​​​​​​​​​​​​​​​​​​​​gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccgtca 180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg caacttacta ctgtcaacag agttacagta cccctccgat caccttcggc 300 caagggacac gactggagat taaacgaact gtggctgcac catctgtctt catcttcccg 360 ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420 tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480 caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540.<000107>acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600 ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgttag 648 <210> 20 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 20 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 21 <211> 585 <212> PRT <213> Artificial Sequence <220> <223> AAA43697.1 HA from B / Victoria / 2 / 87 <400> 21 Met Lys Ala Ile Ile Val Leu Leu Met Val Val Thr Ser Asn Ala Asp 1 5 10 15 Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val Lys 20 25 30 Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu Thr 35 40 45 Thr Thr Pro Thr Lys Ser His Phe Ala Asn Leu Lys Gly Thr Lys Thr 50 55 60 Arg Gly Lys Leu Cys Pro Lys Cys Leu Asn Cys Thr Asp Leu Asp Val 65 70 75 80 Ala Leu Ala Arg Pro Lys Cys Met Gly Thr Ile Pro Ser Ala Lys Ala 85 90 95 Ser Ile Leu His Glu Val Lys Pro Val Thr Ser Gly Cys Phe Pro Ile 100 105 110 Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg Gly 115 120 125 Tyr Glu His Ile Arg Leu Ser Thr His Asn Val Ile Asn Ala Glu Thr 130 135 140 Ala Pro Gly Gly Pro Tyr Lys Val Gly Thr Ser Gly Ser Cys Pro Asn 145 150 155 160 Val Thr Asn Gly Asn Gly Phe Phe Ala Thr Met Ala Trp Ala Val Pro 165 170 175 Lys Asn Asp Asn Asn Lys Thr Ala Thr Asn Pro Leu Thr Val Glu Val 180 185 190 Pro Tyr Ile Cys Thr Glu Gly Glu Asp Gln Ile Thr Val Trp Gly Phe 195 200 205 His Ser Asp Ser Glu Thr Gln Met Val Lys Leu Tyr Gly Asp Ser Lys 210 215 220 Pro Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val 225 230 235 240 Ser Gln Ile Gly Gly Phe Pro Asn Gln Ala Glu Asp Gly Gly Leu Pro 245 250 255 Gln Ser Gly Arg Ile Val Val Asp Tyr Met Val Gln Lys Ser Gly Lys 260 265 270 Thr Gly Thr Ile Thr Tyr Gln Arg Gly Ile Leu Leu Pro Gln Lys Val 275 280 285 Trp Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu 290 295 300 Ile Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys 305 310 315 320 Ser Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys 325 330 335 Pro Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr 340 345 350 Arg Pro Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile 355 360 365 Ala Gly Phe Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His 370 375 380 Gly Tyr Thr Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu 385 390 395 400 Lys Ser Thr Gln Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser 405 410 415 Leu Ser Glu Leu Glu Val Lys Asn Leu Gln Arg Leu Ser Gly Ala Met 420 425 430 Asp Glu Leu His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp 435 440 445 Leu Arg Ala Asp Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu Leu 450 455 460 Ser Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu 465 470 475 480 Glu Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Glu Ile Gly 485 490 495 Asn Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gln Thr Cys Leu Asp 500 505 510 Arg Ile Ala Ala Gly Thr Phe Asn Ala Gly Glu Phe Ser Leu Pro Thr 515 520 525 Phe Asp Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu 530 535 540 Asp Asn His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu 545 550 555 560 Ala Val Thr Leu Met Ile Ala Ile Phe Ile Val Tyr Met Val Ser Arg 565 570 575 Asp Asn Val Ser Cys Ser Ile Cys Leu 580 585 <210> 22 <211> 346 <212> PRT <213> Artificial Sequence <220> <223> AAD02807.1 HA from B / Nanchang / 3451 / 93 <400> 22 Asp Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val 1 5 10 15 Lys Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu 20 25 30 Thr Thr Thr Pro Thr Lys Ser His Phe Ala Asn Leu Lys Gly Thr Lys 35 40 45 Thr Arg Gly Lys Leu Cys Pro Asn Cys Leu Asn Cys Thr Asp Leu Asp 50 55 60 Val Ala Leu Gly Arg Pro Met Cys Val Gly Thr Thr Pro Ser Ala Lys 65 70 75 80 Ala Ser Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe Pro 85 90 95 Ile Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg 100 105 110 Gly Tyr Glu Asn Ile Arg Leu Ser Thr Gln Asn Val Ile Asn Ala Glu 115 120 125 Lys Ala Pro Gly Gly Pro Tyr Arg Leu Gly Thr Ser Gly Ser Cys Pro 130 135 140 Asn Ala Thr Ser Arg Ser Gly Phe Phe Ala Thr Met Ala Trp Ala Val 145 150 155 160 Pro Gly Asp Asn Asn Lys Thr Ala Thr Gly Pro Leu Thr Val Glu Val 165 170 175 Pro Tyr Ile Cys Thr Lys Gly Glu Asp Gln Ile Thr Val Trp Gly Phe 180 185 190 His Ser Asp Ser Lys Thr Arg Met Arg Ser Leu Tyr Gly Asp Ser Asn 195 200 205 Pro Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val 210 215 220 Ser Gln Ile Gly Gly Phe Pro Asp Gln Thr Glu Asp Gly Gly Leu Pro 225 230 235 240 Gln Ser Gly Arg Ile Val Val Asp Tyr Met Val Gln Lys Pro Gly Lys 245 250 255 Thr Gly Thr Ile Val Tyr Gln Arg Gly Val Leu Leu Pro Gln Lys Val 260 265 270 Trp Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu 275 280 285 Ile Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys 290 295 300 Ser Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys 305 310 315 320 Pro Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr 325 330 335 Arg Pro Pro Ala Lys Leu Leu Lys Glu Arg 340 345 <210> 23 <211> 584 <212> PRT <213> Artificial Sequence <220> <223> ABN50712.1 HA from B / Singapore / 11 / 1994 <400> 23 Met Lys Ala Ile Ile Val Leu Leu Met Val Val Thr Ser Asn Ala Asp 1 5 10 15 Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val Lys 20 25 30 Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu Thr 35 40 45 Thr Thr Pro Thr Lys Ser His Phe Ala Asn Leu Lys Gly Thr Lys Thr 50 55 60 Arg Gly Lys Leu Cys Pro Asn Cys Leu Asn Cys Thr Asp Leu Asp Val 65 70 75 80 Ala Leu Gly Arg Pro Met Cys Val Gly Thr Thr Pro Ser Ala Lys Ala 85 90 95 Ser Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe Pro Ile 100 105 110 Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg Gly 115 120 125 Tyr Glu Asn Ile Arg Leu Ser Thr Gln Asn Val Ile Asn Ala Glu Lys 130 135 140 Ala Pro Gly Gly Pro Tyr Arg Leu Gly Thr Ser Gly Ser Cys Pro Asn 145 150 155 160 Ala Thr Ser Arg Ser Gly Phe Phe Ala Thr Met Ala Trp Ala Val Pro 165 170 175 Arg Asp Asn Asn Lys Thr Ala Thr Asn Pro Leu Thr Val Glu Val Pro 180 185 190 Tyr Val Cys Thr Glu Gly Glu Asp Gln Ile Thr Val Trp Gly Phe His 195 200 205 Ser Asp Asn Lys Thr Gln Met Lys Asn Leu Tyr Gly Asp Ser Asn Pro 210 215 220 Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val Ser 225 230 235 240 Gln Ile Gly Gly Phe Pro Asp Gln Thr Glu Asp Gly Gly Leu Pro Gln 245 250 255 Ser Gly Arg Ile Val Val Asp Tyr Met Val Gln Lys Pro Gly Lys Thr 260 265 270 Gly Thr Ile Val Tyr Gln Arg Gly Val Leu Leu Pro Gln Lys Val Trp 275 280 285 Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu Ile 290 295 300 Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys Ser 305 310 315 320 Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys Pro 325 330 335 Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr Arg 340 345 350 Pro Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile Ala 355 360 365 Gly Phe Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His Gly 370 375 380 Tyr Thr Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu Lys 385 390 395 400 Ser Thr Gln Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser Leu 405 410 415 Ser Glu Leu Glu Val Lys Asn Leu Gln Arg Leu Ser Gly Ala Met Asp 420 425 430 Glu Leu His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp Leu 435 440 445 Arg Ala Asp Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu Leu Ser 450 455 460 Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu Glu 465 470 475 480 Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Asp Ile Gly Asn 485 490 495 Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gln Thr Cys Leu Asp Arg 500 505 510 Ile Ala Ala Gly Thr Phe Asn Ala Gly Glu Phe Ser Leu Pro Thr Phe 515 520 525 Asp Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu Asp 530 535 540 Asn His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu Ala 545 550 555 560 Val Thr Leu Met Ile Ala Ile Phe Ile Val Tyr Met Val Ser Arg Asp 565 570 575 Asn Val Ser Cys Ser Ile Cys Leu 580 <210> 24 <211> 584 <212> PRT <213> Artificial Sequence <220> <223> ACA33493.1 HA from B / Florida / 4 / 2006 <400> 24 Met Lys Ala Ile Ile Val Leu Leu Met Val Val Thr Ser Asn Ala Asp 1 5 10 15 Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val Lys 20 25 30 Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu Thr 35 40 45 Thr Thr Pro Thr Lys Ser Tyr Phe Ala Asn Leu Lys Gly Thr Arg Thr 50 55 60 Arg Gly Lys Leu Cys Pro Asp Cys Leu Asn Cys Thr Asp Leu Asp Val 65 70 75 80 Ala Leu Gly Arg Pro Met Cys Val Gly Thr Thr Pro Ser Ala Lys Ala 85 90 95 Ser Ile Leu His Glu Val Lys Pro Val Thr Ser Gly Cys Phe Pro Ile 100 105 110 Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg Gly 115 120 125 Tyr Glu Asn Ile Arg Leu Ser Thr Gln Asn Val Ile Asp Ala Glu Lys 130 135 140 Ala Pro Gly Gly Pro Tyr Arg Leu Gly Thr Ser Gly Ser Cys Pro Asn 145 150 155 160 Ala Thr Ser Lys Ser Gly Phe Phe Ala Thr Met Ala Trp Ala Val Pro 165 170 175 Lys Asp Asn Asn Lys Asn Ala Thr Asn Pro Leu Thr Val Glu Val Pro 180 185 190 Tyr Ile Cys Thr Glu Gly Glu Asp Gln Ile Thr Val Trp Gly Phe His 195 200 205 Ser Asp Asp Lys Thr Gln Met Lys Asn Leu Tyr Gly Asp Ser Asn Pro 210 215 220 Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val Ser 225 230 235 240 Gln Ile Gly Ser Phe Pro Asp Gln Thr Glu Asp Gly Gly Leu Pro Gln 245 250 255 Ser Gly Arg Ile Val Val Asp Tyr Met Met Gln Lys Pro Gly Lys Thr 260 265 270 Gly Thr Ile Val Tyr Gln Arg Gly Val Leu Leu Pro Gln Lys Val Trp 275 280 285 Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu Ile 290 295 300 Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys Ser 305 310 315 320 Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys Pro 325 330 335 Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr Arg 340 345 350 Pro Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile Ala 355 360 365 Gly Phe Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His Gly 370 375 380 Tyr Thr Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu Lys 385 390 395 400 Ser Thr Gln Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser Leu 405 410 415 Ser Glu Leu Glu Val Lys Asn Leu Gln Arg Leu Ser Gly Ala Met Asp 420 425 430 Glu Leu His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp Leu 435 440 445 Arg Ala Asp Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu Leu Ser 450 455 460 Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu Glu 465 470 475 480 Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Glu Ile Gly Asn 485 490 495 Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gln Thr Cys Leu Asp Arg 500 505 510 Ile Ala Ala Gly Thr Phe Asn Ala Gly Glu Phe Ser Leu Pro Thr Phe 515 520 525 Asp Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu Asp 530 535 540 Asn His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu Ala 545 550 555 560 Val Thr Leu Met Leu Ala Ile Phe Ile Val Tyr Met Val Ser Arg Asp 565 570 575 Asn Val Ser Cys Ser Ile Cys Leu 580

Claims

1. An isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to influenza B HA, wherein the antibody or antigen-binding fragment comprises: (i) a heavy chain variable region HCVR, the HCVR comprising an HCDR1 domain as shown in SEQ ID NO: 4, an HCDR2 domain as shown in SEQ ID NO: 6, and an HCDR3 domain as shown in SEQ ID NO: 8, and (ii) a light chain variable region LCVR, the LCVR comprising an LCDR1 domain as shown in SEQ ID NO: 12, an LCDR2 domain as shown in SEQ ID NO: 14, and an LCDR3 domain as shown in SEQ ID NO:

16.

2. The isolated antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the antibody or antigen-binding fragment thereof has one or both of the following characteristics: (a) with less than 10 -9 M's EC 50 Infection with influenza B HA; and / or (b) Demonstrates that, after administration to animals infected with influenza B, the survival rate of the animals infected with influenza B increased compared to comparable animals infected with influenza B that were not administered.

3. The isolated antibody or its antigen-binding fragment as claimed in claim 1, wherein when prophylactically administered to a mammal at a dose of 5 mg / kg of the mammal’s body weight or a single intravenous dose of 0.5 mg / kg, the mammal is protected from subsequent exposure to influenza B virus infection.

4. The isolated antibody or antigen-binding fragment thereof as claimed in claim 1, wherein when prophylactically administered to a mammal prior to exposure to influenza B virus, it reduces the risk of influenza infection in the mammal.

5. The isolated antibody or antigen-binding fragment thereof as claimed in claim 1, wherein when the isolated antibody or antigen-binding fragment thereof is administered to a mammal, the isolated antibody or antigen-binding fragment thereof improves, alleviates or reduces the severity, duration or frequency of at least one symptom of influenza infection in the mammal.

6. The isolated antibody or antigen-binding fragment thereof as claimed in claim 5, wherein the at least one symptom is selected from the group consisting of: fever, pain, runny nose, chills, fatigue, weakness, cough, shortness of breath, vomiting, diarrhea, pneumonia, bronchitis, and death.

7. The isolated antibody or antigen-binding fragment thereof as claimed in claim 1, wherein when administered at a single prophylactic dose of 0.5 mg / kg to each of a plurality of mammals, the survival rate of the plurality of mammals is at least 80% 19 days after administration.

8. The isolated antibody or antigen-binding fragment thereof as claimed in claim 1, wherein when administered at a single prophylactic dose of 0.5 mg / kg to each of a plurality of mammals, the survival rate of the plurality of mammals is at least 90% 19 days after administration.

9. The isolated antibody or antigen-binding fragment thereof as claimed in claim 1, wherein when administered at a single prophylactic dose of 0.5 mg / kg to each of a plurality of mammals, the survival rate of the plurality of mammals is 100% 19 days after administration.

10. The isolated antibody or antigen-binding fragment thereof as claimed in claim 1, wherein when administered at a single prophylactic dose of 5.0 mg / kg to each of a plurality of mammals, the survival rate of the plurality of mammals is 80% 19 days after administration.

11. The isolated antibody or antigen-binding fragment thereof as claimed in claim 1, wherein when administered at a single prophylactic dose of 5.0 mg / kg to each of a plurality of mammals, the survival rate of the plurality of mammals is 90% 19 days after administration.

12. The isolated antibody or antigen-binding fragment thereof as claimed in claim 1, wherein when administered at a single prophylactic dose of 5.0 mg / kg to each of a plurality of mammals, the survival rate of the plurality of mammals is 100% 19 days after administration.

13. The isolated antibody or its antigen-binding fragment as described in claim 1, wherein the survival rate is evident 13 days after administration.

14. The isolated antibody or antigen-binding fragment thereof as claimed in claim 1, comprising HCVR having the amino acid sequence of SEQ ID NO:

2.

15. The isolated antibody or antigen-binding fragment thereof as claimed in claim 1, comprising an LCVR having the amino acid sequence of SEQ ID NO:

10.

16. The isolated antibody or its antigen-binding fragment as described in claim 1, comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 2 / 10.

17. The isolated antibody or its antigen-binding fragment as described in claim 1, wherein it is an IgG1 antibody.

18. The isolated antibody or its antigen-binding fragment as described in claim 1, wherein it is an IgG4 antibody.

19. The isolated antibody or its antigen-binding fragment as described in claim 1, wherein it is a bispecific antibody.

20. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof as described in claim 1, and a pharmaceutically acceptable carrier or diluent.

21. The pharmaceutical composition of claim 20, wherein the pharmaceutical composition further comprises a second therapeutic agent.

22. The pharmaceutical composition of claim 21, wherein the second therapeutic agent is selected from the group consisting of: antiviral drugs, anti-inflammatory drugs, influenza vaccines, and dietary supplements.

23. The pharmaceutical composition of claim 22, wherein the anti-inflammatory agent is selected from the group consisting of corticosteroids and nonsteroidal anti-inflammatory drugs.

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

25. The pharmaceutical composition of claim 22, wherein the antiviral agent is oseltamivir.

26. The pharmaceutical composition of claim 22, wherein the antiviral drug is an anti-influenza A drug.

27. The pharmaceutical composition of claim 26, wherein the anti-influenza A drug is an antibody.

28. The pharmaceutical composition of claim 27, wherein the antibody specifically binds to influenza A (HA).

29. A polynucleotide molecule comprising polynucleotide sequences encoding HCVR and LCVR of an antibody or antigen-binding fragment thereof as described in claim 1.

30. A vector comprising the polynucleotide sequence as described in claim 29.

31. A cell that expresses the vector as described in claim 30.

32. A method for generating an antibody or antigen-binding fragment thereof that specifically binds to influenza HA, the method comprising culturing the cells of claim 31 under conditions that allow for the generation of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof thus generated.

33. A vector comprising: (i) a polynucleotide molecule, said polynucleotide molecule including a polynucleotide molecule encoding a polynucleotide sequence encoding an HCVR of the antibody isolated as claimed in claim 1 or an antigen-binding fragment thereof, and (ii) a polynucleotide molecule, said polynucleotide molecule including a polynucleotide sequence encoding an LCVR of the antibody isolated as claimed in claim 1 or an antigen-binding fragment thereof.

34. A cell comprising the carrier of claim 33.

35. A composition comprising a first nucleotide molecule and a second nucleotide molecule, the first nucleotide molecule comprising a polynucleotide sequence encoding an HCVR of the isolated antibody or antigen-binding fragment thereof as claimed in claim 1, and the second nucleotide molecule comprising a polynucleotide sequence encoding an LCVR of the isolated antibody or antigen-binding fragment thereof as claimed in claim 1.

36. A cell comprising a first nucleotide molecule and a second nucleotide molecule, the first nucleotide molecule comprising a polynucleotide sequence encoding an HCVR of an antibody isolated as claimed in claim 1 or an antigen-binding fragment thereof, and the second nucleotide molecule comprising a polynucleotide sequence encoding an LCVR of an antibody isolated as claimed in claim 1 or an antigen-binding fragment thereof.

37. A method for generating an antibody or antigen-binding fragment thereof that specifically binds to influenza HA, the method comprising culturing the cells of claim 36 under conditions that allow for the generation of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof thus generated.

38. Use of the isolated antibody or antigen-binding fragment thereof as described in any one of claims 1-19, or the pharmaceutical composition as described in any one of claims 20-28, in a medicament for a method of preparing at least one symptom of influenza infection in a subject, the method comprising administering the medicament to the subject.

39. The use as claimed in claim 38, wherein the at least one symptom is selected from the group consisting of: fever, cough, body aches, runny nose, shortness of breath, pneumonia, and bronchitis.

40. The use as described in claim 38, wherein the pharmaceutical composition is administered prophylactically to the subject.

41. The use as claimed in claim 38, wherein the subject is selected from the group consisting of: individuals with weakened immune function, adults aged 65 years or older, healthcare professionals, and persons with a history of medical problems or potential medical conditions.

42. The use as claimed in claim 41, wherein the potential medical condition is selected from the group consisting of: heart disease and diabetes.

43. The use as claimed in claim 38, wherein the pharmaceutical composition is administered in combination with a second therapeutic agent.

44. The use as claimed in claim 43, wherein the second therapeutic agent is selected from the group consisting of: antiviral drugs, anti-inflammatory drugs, influenza vaccines, and dietary supplements.

45. The use as claimed in claim 44, wherein the anti-inflammatory agent is selected from the group consisting of corticosteroids and nonsteroidal anti-inflammatory drugs.

46. ​​The use as described in claim 44, wherein the dietary supplement is an antioxidant.

47. The use as claimed in claim 43, wherein the second therapeutic agent is administered via a route of administration different from that of the antibody or its antigen-binding fragment.

48. The use as described in claim 43, wherein the second therapeutic agent is administered orally.

49. The use as described in claim 44, wherein the antiviral drug is oseltamivir, and wherein: (a) Osemivir is administered prior to the administration of the antibody or its antigen-binding fragment; (b) Osemivir is administered concurrently with the antibody or its antigen-binding fragment; or (c) Osemivir is administered after the administration of the antibody or its antigen-binding fragment.

50. The use as described in claim 44, wherein the antiviral drug is an anti-influenza A drug.

51. The use as described in claim 50, wherein the anti-influenza A drug is an antibody.

52. The use as described in claim 51, wherein the antibody specifically binds to influenza A (HA).

53. The use as described in claim 38, wherein the pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intramuscularly, intranasally, or orally.

54. The use as described in claim 38, wherein the isolated antibody or its antigen-binding fragment protects the subject from subsequent exposure to influenza B virus infection.

55. Use of an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to influenza B HA in a medicament for preparing a method of preventing, treating or improving at least one symptom of influenza infection in a subject infected with influenza, wherein the isolated recombinant antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable region HCVR, the HCVR comprising an HCDR1 domain as shown in SEQ ID NO: 4, an HCDR2 domain as shown in SEQ ID NO: 6, and an HCDR3 domain as shown in SEQ ID NO: 8, and (ii) a light chain variable region LCVR, the LCVR comprising an LCDR1 domain as shown in SEQ ID NO: 12, an LCDR2 domain as shown in SEQ ID NO: 14, and an LCDR3 domain as shown in SEQ ID NO: 16, the medicament being formulated for systemic administration.

56. The isolated antibody or its antigen-binding fragment as described in claim 6, wherein the pain includes headache and sore throat.

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